Laure Guillou

Bénédicte Charrier

C Reinhartii Chlamydomonas

I Ohad

Keywords: phytoplankton, dinoflagellate, coccolithophore, diatom, chlorophyte, photosynthesis, cyclic electron transfer, photosystem, viral infection, mesocosm

J'ai souvent eu du mal à choisir la voie que j'avais envie de suivre. Mais quelque fois, le chemin s'est tracé naturellement, et c'est de cette manière que je suis arrivée à l'UMR7141 pour commencer ce doctorat sous la direction de Benjamin. En commençant une thèse, je n'imaginais pas que ce serait une expérience tant humaine que scientifique. Et je tiens à remercier en tout premier lieu Benjamin, mon directeur de thèse et la personne avec qui j'ai entretenu la relation la plus intense de ces trois dernières années. J'ai adoré nos discussions scientifiques et je suis contente d'avoir été encadrée par un aussi bon « mentor ». Ça n'a pas été facile tous les jours mais c'est un beau voyage que nous avons fait. Et j'espère sincèrement que l'on continuera d'entretenir une belle relation par la suite. Je tiens aussi à remercier Francis-André, dont le bureau magique a été pour moi le lieu de confidences et de conseils précieux. Merci d'avoir cru en moi et d'avoir su trouver les mots qui parlent quand j'en avais besoin. En arrivant à l'UMR7141, j'ai aussi trouvé une famille et je tiens à remercier tous les membres du labo, présents et passés pour cet environnement bienveillant que chacun contribue à entretenir. Alexandra, Domitille, je suis contente d'avoir fait ce voyage avec vous ! Je tiens aussi à remercier tous ceux qui ont contribué à mon travail de thèse. Ils sont nombreux, mais j'aimerais remercier en particulier Pascal Campagne, sans qui mon chapitre 3 n'aurait pas fière allure, Kim Thamatrakoln, Kay Bidle et Assaf Vardi qui m'ont permis de participer aux campagnes de terrain en Norvège, Laure Guillou, qui nous a accueilli à Roscoff pour des manips de terrain, Michael Schroda qui m'a invité à étudier le phénotype du mutant deg1, Sandrine sans qui mes cultures seraient encore bactériennes, la collection de cultures de Roscoff et en particulier Martin pour leurs efforts administratifs, mon comité de

coup de pouce secret qui m'a bien aidé ! Florian Pellet, Pierre Crozet et Gauthier Vilmart pour leur écoute, soutien et propositions constructives. Et tous ceux qui m'ont relevé quand j'ai craqué ! Ces trois années ont aussi été marqués par une vie hors labo riche et épanouissante. Et j'ai envie de remercier ici tous ceux qui ont contribué à nourrir et qui nourrissent encore mes besoins de partage, d'amour et de joie. Dans les semaines qui ont suivi mon arrivée à Paris, il y a 5 ans, j'ai rencontré Lorna et Chawee. Depuis, ils sont le socle d'amour de ma vie parisienne et je ressens beaucoup de gratitude pour leur présence dans ma vie. Ces 5 années ont aussi été rythmées par les moments avec le Chaos par UpperCuite, avec qui on est sûrs de ne pas manquer de joie et de partage (parfois plus qu'on aimerait), par les sorties avec la Banda Festayre, les Capellous et la Band'arnac qui ne sont pas les plus fins mais qui ont bien d'autres qualités. Merci à Claude et Alain Trautmann qui m'ont offert un refuge, une présence bienveillante et de beaux moments d'échange pendant ces cinq années parisiennes. Merci à tous les amis, aux cousins, aux oncles et tantes, qui me nourrissent de l'essentiel. Merci à Artur, qui était là pour le début et que j'ai aimé de tout mon coeur.

Ces remerciements sont aussi l'occasion de dire ma reconnaissance à tous ceux qui ont croisé mon chemin, et en particulier ceux qui m'ont ouvert des portes et marqué ma construction intérieure. Ceux qui ont nourri mon goût pour l'abstraction et la connaissance, parmi eux, Christine Palandjian, Eric Mestre, Sophie Paradis. Ceux qui m'ont ouvert les portes de la musique, Cylette Monginous qui m'a initiée et Gérard Luck qui, par son insistance, m'a fait entrer dans le monde des bandas. Ceux qui m'ont ouvert les portes du sport, Bertrand Diviès pour l'escalade, Jean-Claude Pélissier, pour la course à pied, José Machado pour le rugby. Et enfin, ceux qui ont régulièrement ravivé mon goût pour l'agriculture, Samuel Marguet, Ase et Sigurd Avdem, Marie-Eve et Julien Lasalle et ceux qui m'ont appris le travail du bois dont Even et Cédric Linon.

Je remercie aussi mes 10 cm 2 de terminaisons nerveuses cutanées, mon ménisque et mon petit bout de couenne du pouce que j'ai perdu en chemin. Votre perte m'aura beaucoup appris sur la nécessité de s'écouter. A Badr et son Ekipe, à Antoine Gerometta qui ont sauvé mon genou (et par là même, ma santé psychologique).

Enfin je voudrais dire merci à ma famille qui est là quoiqu'il arrive. A mes frères et soeurs qui ont enveloppé mon enfance d'un amour bienveillant et de beaucoup de joie. A Colombe, que j'aime depuis le premier jour et qui est un des piliers de ma vie. A Catherine et Denis, mes parents, qui m'ont aimé et qui ont contribué à mon développement par leurs merveilleuses ressources personnelles et en me poussant à chercher plus en dehors du cercle familial.

Tables

Suzanne FERTE -Doctoral Thesis -

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Foreword

When I arrived at the laboratory Biologie du Chloroplaste et Perception de la Lumière chez les micro-algues (at that time Physiologie membranaire et moléculaire du chloroplaste), my PhD topic was "Competition for nutrients within phytoplankton". This is to say how much the progression of my thesis was not linear. Benjamin Bailleul, my supervisor, has been able to listen to my interests and my desires and this manuscript reflects my journey guided by two main calls: that of the marine world, one of the last terra incognita, by far the least-understood ecosystem on Earth. However, it shelters a photosynthetic diversity who is just beginning to be apprehended although of global importance. And by a growing interest for methodological development. In the end, my thesis can be seen as an attempt to explore photosynthetic diversity in microalgae using as an object of interest two protein complexes: photosystems I and II. After all, all marine life, oxygen, fossil fuels, food webs are dependent on the conversion of light energy by these two fascinating molecular machines.

Résumé (français)

Les micro-algues sont à l'origine de l'immense majorité des chaines trophiques océaniques et participent à presque la moitié de la photosynthèse sur Terre. Si leur diversité génétique est aujourd'hui bien connue, leur diversité fonctionnelle reste encore peu explorée. La voie principale de la photosynthèse, le flux linéaire d'électrons depuis l'eau vers le CO2 semble bien conservé dans la phylogénie. Il fait intervenir deux photosystèmes en série, les photosystèmes II (PSII) et I (PSI). Chez les plantes et les algues vertes, il existe un flux cyclique d'électrons qui ne fait intervenir que le PSI auquel on attribue plusieurs rôles cruciaux dans la régulation de la photosynthèse. L'étude de ce flux cyclique est freinée par l'absence de méthodologie robuste pour le mesurer. Nous avons montré que la méthode la plus utilisée pour estimer l'activité du PSI n'est pas fiable. Sur cette base, nous proposons une méthode alternative pour détecter la présence de ce flux cyclique et pour étudier sa dépendance envers le flux linéaire. Cela a permis de montrer des comportements très différents entre espèces de micro-algues. Enfin, l'étude d'une efflorescence de la coccolithophore Emiliania huxleyi en mésocosme nous a permis de mettre en évidence une signature de l'infection des cellules par un virus qui pourrait faire intervenir le flux cyclique autour du PSI.

Mots clés: phytoplancton, dinoflagellé, coccolithophore, diatomée, chlorophycée, photosynthèse, transfert d'électron cyclique, photosystème, infection virale, mésocosme

Résumé long (français)

La quasi-totalité de l'énergie circulant dans la biosphère provient initialement de l'énergie solaire, convertie en énergie chimique par des organismes dits phototrophes. Chez tous ces organismes, la conversion énergétique est permise par une seule et même famille de complexes protéiques : les centres réactionnels. Au cours de l'évolution, un groupe de Nous avons pu mettre en évidence que les méthodes P700 et ECS divergent lorsque certains P700 ne sont pas oxydés par le pulse de lumière saturante. Nous avons montré que cette discordance est liée à un biais de la méthode P700 et nous proposons un modèle expliquant ce biais. Lorsque plusieurs séparations de charge ont lieu pendant le pulse utilisé par la méthode P700, alors des accepteurs PSI initialement oxydés se réduisent. Et lorsque le nombre d'accepteurs PSI est plus faible que le nombre de donneurs, certains P700 initialement oxydables ne le sont pourtant pas à la fin du pulse parce que leurs accepteurs ont été réduits entre temps. Cette erreur n'est pas négligeable. Dans nos données, la méthode P700 sousestime la fraction de PSI ouverts jusqu'à un facteur 10. Dans la suite de nos travaux, nous avons donc privilégié la méthode ECS pour estimer l'activité des PSI.

La méthode P700 peut néanmoins être corrigée. Nous proposons un modèle permettant de prendre en compte la fraction d'accepteurs réduits pendant le pulse qui est soutenu par nos données empiriques. On peut donc proposer une correction des données obtenue avec la méthode P700. Cette correction ouvre la porte à la ré-interprétation des données publiées dans la littérature depuis plus de 25 ans utilisant la méthode P700. Nous avons commencé ce travail et pu montrer par exemple que le phénotype du mutant pgr5, potentiellement impliqué dans le FCE, pourrait être en partie dû au biais de la méthode P700. This introduction attempts to introduce the importance of the study of photosynthesis in the context of the exploration of the marine world, insisting on the concepts necessary for the understanding of chapter III "field measurements". In a second time the photosynthetic process will be presented, emphasizing the two photosystems and their relative functions. [START_REF] Rusch | The Sorcerer II global ocean sampling expedition: northwest Atlantic through eastern tropical Pacific[END_REF] and the Malaspina expedition (Pesant et al., 2015). And one of the first question that was addressed in the light of this new set of data is: who is living in the ocean? And how diverse are the organisms? The circumglobal Tara ocean expedition collected billions of organisms but is it representative of the whole ocean diversity? To tackle this issue, a commonly used technique is called rarefaction and estimates the number of new species that would be found by sampling more organisms. From Tara samples, about 500 million of ribosomal DNA were sequenced and grouped in operational taxonomic unit (OTU), i.e. closely related organisms. Figure 1-1 shows rarefaction curves plotting the number of OTU described as a function of the number of sequencing since the beginning. The slope of the curves, representing the number of new OTU per read, decreases to reach really low values meaning that more sequencing would not allow to discover much new species (de Vargas et al., 2015). We now know that more than 95% of the weight of living matter in the ocean is made of microscopic organisms, more than whales and fishes put together, and most of it is plankton. [0.8-5µm]; 'nano', [5-20µm]; 'micro', [20-180µm]; 'meso', [180-2,000µm]). Sequences were clustered in operational taxonomy units (OTU). Proximity to saturation is indicated by weak slopes at the end of each rarefaction curve (e.g., 1.2/100,000 means 1.2 novel metabarcodes obtained every 100 000 rDNA reads sequenced). Shannon index has been calculated for each size fraction as an indicator of genetic diversity.

Marine ecosystems, phytoplankton and photosynthesis

The word "plankton" comes from the Greek planktos "errant" and includes diverse organisms based on a common characteristic: their limited ability to move, meaning that they drift with the water current. From the virus of less than one micron to the siphonophore over ten meters long, their size varies greatly but most of them are microscopic. They cannot be detected by naked eye and this is why their importance have long been underestimated. Among the planktonic organisms, phytoplankton are the first link in aquatic food webs.

2. Phytoplankton plays a crucial role in fluxes of matter and energy in the ocean

Defined as autotrophic planktonic organisms [START_REF] Haeckel | Plankton-studien[END_REF], these organisms account for less than 1% of the global primary biomass of primary producers and are nonetheless responsible for 45% of annual net primary production at the surface of the earth [START_REF] Falkowski | The evolution of modern eukaryotic phytoplankton[END_REF], Field et al., 1998) and more than 75% of primary productivity at the ocean scale.

Phytoplankton fix more than a hundred million tons of carbon every day [START_REF] Behrenfeld | Biospheric primary production during an ENSO transition[END_REF][START_REF] Falkowski | Biogeochemical controls and feedbacks on ocean primary production[END_REF]. Their primary production is estimated about 50 Pg year -1 [START_REF] Behrenfeld | Biospheric primary production during an ENSO transition[END_REF][START_REF] Falkowski | Biogeochemical controls and feedbacks on ocean primary production[END_REF].

Like terrestrial plants, phytoplankton performs photosynthesis: using solar light energy that is captured with pigments (mainly chlorophylls), it transforms CO2 and water into carbohydrates while producing oxygen. And this is virtually the only gateway for energy and carbon input in the marine biosphere. Phytoplankton plays therefore a crucial role in feeding the trophic chains, producing half the O2 that we breathe and storing an important amount of atmospheric CO2 through the biological pump, playing an important -but still not fully resolved-role of buffer of global climate changes.

Besides the rather regular seasonal patterns at global scale, some species are capable of explosive growth over a few days or weeks reaching high concentrations (up to tens of thousands of cells per milliliter). And although it is microscopic, phytoplankton become visible to the naked eye during those phenomena known as blooms (Figure 1-2). Density becomes so high that phytoplankton pigments and cell wall properties induce changes of the water color. Blooms can occur on a relatively small scale or cover hundreds of square kilometers of the ocean's surface, becoming visible from space. Among the huge diversity of phytoplankton, only few species have the capacities to bloom. One of them is the coccolithophore Emiliania huxleyi whose blooms can be visualized from satellites because it possesses microscopic calcite shells that diffuses light and can give water a chalky color (Figure 1-2A). The third chapter of my thesis will focus on the photo-physiology of this species, a model organism for coccolithophores (e.g. Tyrell and Merico, 2004;[START_REF] Wilson | Complete genome sequence and lytic phase transcription profile of a Coccolithovirus[END_REF]Bidle and Vardi, 2011). Historically, and by analogy with land marine food webs, primary consumers were thought to be the zooplankton, serving as a crucial link between primary producers and the rest of the trophic chain including higher consumers such as large fish and ultimately top predators (Figure 1-3). And it is important to note that this concerns directly human survival since in 2015, according to the Food and Agriculture Organization of the United Nations report, 17%

of animal proteins consumed in the world came from fish. However, this classical food web view has been challenged by the discovery of abundant heterotrophic prokaryotes. It is now commonly assumed that net zooplankton are not metabolically dominant and that a large proportion of the flux of matter and energy in the marine food web passes through bacteria in the form of dissolved organic matter (DOM), a concept named the microbial loop. The microbial loop is the process by which DOM derived from primary producers such as phytoplankton is foraged and recycled by heterotrophic bacteria that use DOM as a food source [START_REF] Azam | The ecological role of water-column microbes[END_REF][START_REF] Hobbie | A study of the distribution and activity of microorganisms in ocean water 1[END_REF][START_REF] Pomeroy | The ocean's food web, a changing paradigm[END_REF]. These bacteria are

Chapter 1: General introduction

Suzanne FERTE -Doctoral Thesis -2019 consumed by zooplankton, and the link to the traditional food web is established (Figure 1-3). Marine bacteria hold a significant influence in this loop, as they allow for an energy pathway that may have otherwise been lost. Viruses, by infecting and lysing marine bacteria and phytoplankton, are also vital players in the fixation and cycling of key elements, such as carbon, nitrogen, and phosphorus [START_REF] Wilhelm | Viruses and nutrient cycles in the sea: viruses play critical roles in the structure and function of aquatic food webs[END_REF]. Through the viral shunt, viral lysis of microbial cells releases dissolved organic matter and particulate organic matter back into the microbial loop. By doing so, viruses drive carbon and nutrients of these cells away from grazers, and redirects it to other microorganisms through the form of DOM (Figure 1-3). It has been estimated that ocean viruses might turn over as much as 150 gigatons of carbon per year [START_REF] Suttle | Marine viruses-major players in the global ecosystem[END_REF].

Figure 1-3: Bacterial transformation of phytoplankton-derived organic matter

The marine carbon cycle includes a number of processes, several of which are mediated by microorganisms. Key processes of the marine carbon cycle include the conversion of inorganic carbon (such as CO2) to organic carbon by photosynthetic phytoplankton species (step 1); the release of both dissolved organic matter (DOM; which includes dissolved organic carbon (DOC), dissolved organic nitrogen (DON) and dissolved organic phosphorous (DOP)) and particulate organic matter (POM; which includes particulate organic carbon (POC), particulate organic nitrogen (PON) and particulate organic phosphorous (POP)) from phytoplankton (step 2); the consumption of phytoplankton biomass by zooplankton grazers (step 3) and the mineralization (that is the release of CO2 via respiration during the catabolism of organic matter) and recycling of organic matter by diverse heterotrophic bacteria (which is known as the microbial loop; step 4). A fraction of the heterotrophic bacteria is consumed by zooplankton, and the carbon is further transferred up the food web. Heterotrophic bacteria also contribute to the remineralization of organic nutrients, including DON and DOP, to inorganic forms, which are then available for use by phytoplankton. The microbial carbon pump (step 5) refers to the transformation of organic carbon into recalcitrant DOC that resists further degradation and is sequestered in the ocean for thousands of years. The biological pump (step 6) refers to the export of phytoplankton-derived POM from the surface oceans to deeper depths via sinking. Finally, the viral shunt (step 7) describes the contributions of viral-mediated cell lysis to the release of dissolved and particulate matter from both the phytoplankton and the bacterial pool. From [START_REF] Buchan | Master recyclers: features and functions of bacteria associated with phytoplankton blooms[END_REF].

Chapter 1: General introduction

Suzanne FERTE -Doctoral Thesis -2019

3. Origin and taxonomic diversity of phytoplankton

Who are the phytoplankton organisms? Are they part of the same phylogenetic family as terrestrial plants? We also find marine phototrophs within Alveolata, among which we find dinoflagellates (Dinophyceae), Stramenopila which comprises diatoms, Haptophyta which includes coccolithophores, but also Rhizaria, Discoba or Cryptista (Obornik, 2019).

The ability to perform oxygen photosynthesis is also found in some species of bacteria (not shown on this tree), called cyanobacteria. A question that arises with regard to this tree is:

how to explain the presence of photosynthetic organisms within such diverse phylogenetic groups? Has the ability to achieve photosynthesis appeared several times during evolution by evolutionary convergence? Several arguments led to propose a chimeric origin of photosynthetic eukaryotes. In 1970, based on morphological arguments (the resemblance between chloroplasts and cyanobacteria), Margulis proposed that the chloroplasts of eukaryotic cells come from the incorporation of a cyanobacterium by a heterotrophic unicellular [START_REF] Margulis | Origin of eukaryotic cells: evidence and research implications for a theory of the origin and evolution of microbial, plant, and animal cells on the Precambrian earth[END_REF]: this is the endosymbiotic theory. Since then, many arguments have accumulated to support this theory, one of the most robust of which is phylogenetic: chloroplast DNA analyses show the close kinship between all chloroplast and cyanobacteria [START_REF] Rodríguez-Ezpeleta | Monophyly of primary photosynthetic eukaryotes: green plants, red algae, and glaucophytes[END_REF].

Nowaday, it is well admitted that the photosynthetic organelle of algae and plants (the plastid) traces its origin to a primary endosymbiotic event in which a previously nonphotosynthetic protist engulfed and enslaved a cyanobacterium (Figure 1 -5). This eukaryote then gave rise to the red, green and glaucophyte algae [START_REF] Bhattacharya | Photosynthetic eukaryotes unite: endosymbiosis connects the dots[END_REF]. However, many algal lineages, such as the chlorophyll c-containing stramenopiles, have a more complicated evolutionary history involving a secondary endosymbiotic event, in which a protist engulfed an existing eukaryotic alga, in this case, a red alga; the additional engulfment of a green alga was suggested [START_REF] Bhattacharya | Photosynthetic eukaryotes unite: endosymbiosis connects the dots[END_REF][START_REF] Prihoda | Chloroplastmitochondria cross-talk in diatoms[END_REF], Moustafa et al., 2009). Another algal group, the dinoflagellates, has undergone tertiary (engulfment of a secondary plastid) and even quaternary endosymbiosis (Figure 1 -5).

2. Reactions and mechanisms involved in eukaryotic photosynthesis

In this second part of the introduction, after presenting the diversity of photosynthetic types, we will detail the major reaction steps involved in oxygenic photosynthesis as well as the molecular mechanisms allowing the transformation of light energy into chemical energy.

2. Different types of photosynthesis

Among the three fundamental domains of living organisms, bacteria, archaea and eukaryotes [START_REF] Woese | Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya[END_REF], we find photosynthetic organisms within bacteria and eukaryotes only. It is to note that another mechanism of conversion of light energy into chemical energy exists which is fundamentally different from the chlorophyll-based photosynthesis. Halophilic archaea possess bacteriorhodopsins, retinal-binding integral membrane proteins, which can use light to pump protons, generate an electrochemical proton gradient and produce adenosine triphosphate (ATP) from adenosine diphosphate (ADP) through this process [START_REF] Hayashi | Molecular dynamics simulation of bacteriorhodopsin's photoisomerization using ab initio forces for the excited chromophore[END_REF]. Later, metagenomics approaches revealed widespread retinal-binding rhodopsin proteins, called proteorhodopsins in marine bacteria (Beja et al, 2000). It also exists in eukaryotes [START_REF] Bieszke | The nop-1 gene of Neurospora crassa encodes a seven transmembrane helix retinal-binding protein homologous to archaeal rhodopsins[END_REF]. These discoveries have challenged the notion that only chlorophyll-based photosynthesis allows the conversion of solar energy into the marine ecosystem (Beja et al, 2000). The importance of proteorhodopsins in the global conversion of light into chemical energy and biomass is still a matter of debate [START_REF] Fuhrman | Proteorhodopsins: an array of physiological roles?[END_REF]. Here, we will not consider the light-driven ATP synthesis catalyzed by bacteriorhodopsins and proteorhodopsins, and the term "photosynthesis" will be used in its historical meaning, i.e.

for the conversion of light into chemical energy based on chlorophylls or bacteriochlorophylls.

The use of chlorophyll or bacteriochlorophyll also distinguishes two different types of photosynthesis, whose general principles are the same but which depend on different sources of electrons. Oxygenic photosynthesis uses water as a source of electrons, a reaction that results in the release of molecular oxygen, and those electrons can be transferred through two photosystems to reduce carbon dioxide. But some photosynthetic bacteria can

Chapter 1: General introduction

Suzanne FERTE -Doctoral Thesis -2019 use light to extract electrons from other molecules than water. They use instead inorganic compounds, like H2 or H2S, or inorganic compounds like malate. Because they do not evolve O2, their photosynthesis is named anoxygenic photosynthesis. Another difference with oxygenic phototrophs is that they possess only one type of reaction center: a pheophytinquinone (type II) reaction center, or an iron-sulfur (type I) one. Among anoxygenic phototrophs using type-II reaction centers are purple bacteria and green filamentous bacteria. On the contrary, green sulfur bacteria and heliobacteria use type-I reaction center [START_REF] Blankenship | Origin and early evolution of photosynthesis[END_REF].

In this work, we will only consider oxygenic photosynthesis, which exists in cyanobacteria and in photosynthetic eukaryotes. A major difference between these two groups of organisms is structural: in photosynthetic eukaryotes, photosynthesis occurs in specialized organelles, the plastids, while cyanobacteria do not possess intracellular organelles. In the latter, photosynthesis occurs in membrane invaginations. The following paragraph therefore only concerns eukaryotes.

2. 2. The chloroplast: an organelle hosting photosynthesis

The site of photosynthesis in eukaryotes is the plastid, a membranous subcellular organelle ( Staehelin, 1986;[START_REF] Falkowski | Aquatic photosynthesis[END_REF]. The inner membrane encloses the stroma, a concentrated solution of enzymes that also contains the DNA, RNA and ribosomes involved in the synthesis of several chloroplast proteins. Inside the chloroplast, membrane folds form bags called thylakoids which delimit an internal space, the lumen. In higher plants, thylakoids consist of stacks of disk-like sacs named grana which are interconnected by unstacked stroma lamellae whereas in green algae, the grana organization typical of plants is replaced by a mix of stacked and unstacked thylakoid membranes that still allow a lateral heterogeneity in protein content of these membranes (Staehelin, 1986, Figure 1-6). In secondary phototrophs (photosynthetic species deriving from the second endosymbiosis), the organelle where photosynthesis takes place is not properly speaking a chloroplast, since this term was historically used for chlorophyll a and b containing plastids.

Secondary endosymbionts contain chlorophyll a and c, and we use the generic term plastid [START_REF] Gould | Plastid evolution[END_REF]. Electron micrographs show no thylakoid subdomains in secondary plastids, only loose stacks of two, three or four thylakoids (most of the time 3, Pyszniak and Gibbs, 1992) which still lead to a marked difference in protein content between the outer membrane of a stack and its inner membranes.

The main role of chloroplasts/plastids is the photosynthetic metabolism, but it also hosts nitrate and sulfate reduction, as well as synthesis of chlorophylls, carotenoids, haems, amino acids and fatty acids [START_REF] Jensen | Chloroplast evolution, structure and functions[END_REF].

Chapter 1: General introduction

Suzanne FERTE -Doctoral Thesis -2019 It is now well understood that photosynthesis is enabled by two steps: the production of reducing power in the form of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and chemical energy in the form of ATP that occurs in the membrane photosynthetic chain thanks to light energy. Carbon fixation then uses products of the photosynthetic chain in the stromal compartment. The result of the "light" phase of photosynthesis is the conversion of light energy into chemical energy, stored in molecules with high energy potential, ATP and NADPH. In the "dark" phase of photosynthesis, ATP and NADPH are used together to catalyze all of the carbon assimilation reactions used to make carbohydrates, called the Benson-Calvin cycle. When the final carbon molecule is glucose, the entire process described so far leads to the following reaction:

12 H2O + 6 CO2 + hν → 6 O2 + C6H12O6 + 6 H2O

In the following two sections, I will describe first the Calvin Benson Bassham cycle where reduction of carbon dioxyde occurs (the biosynthetic, or "dark", phase of photosynthesis), and then the photochemical phase which occurs in the thylakoid membranes (also called "light" phase of photosynthesis). Benson [START_REF] Bassham | Isotope studies in photosynthesis[END_REF]. The basic experimental strategy they used was to expose growing cultures of the green alga Chlorella to radioactive labelled 14 CO2 for varying times and under different illumination conditions and then to drop the cells into boiling alcohol so as to disrupt them while preserving their labeling pattern. The radioactive products were then separated and identified by a two-dimensional paper chromatography coupled to autoradiography. The overall pathway is presented in Figure 1-7 and shows that the reduction of CO2 requires an energy supply in the form of adenosine triphosphate (ATP)

and an input of reducing power provided by a coenzyme, the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH). These two substrates are used with a ratio of 3 ATP for 2 NADPH. Although other carbon fixation pathways have been found in autotroph bacteria and archae, CBB cycle is the only carbon fixation pathway operating in eukaryotes (Hügler and Sievert, 2011). The first step along the reductive pathway of CO2 is catalyzed by the Ribulose-1,5bisphophaste Carboxylase Oxygenase, RuBisCO, a big protein complex. RuBisCO has a low affinity for CO2 and catalyzes also a reaction of ribulose-1,5-bisphosphate and molecular oxygen (O2) that is competing with carbon fixation. Carbon fixation rate is thus depending on the ratio of CO2 concentration over O2 concentration in the surrounding environment of RuBisCO [START_REF] Delgado | Species variation in Rubisco specificity factor[END_REF].

The way to deal with CO2 is obviously very different for terrestrial organisms (plants) and for aquatic organisms. CO2 can dissolve into water and its concentration in surface Ocean ranges from 10 to 30 µM [START_REF] Reinfelder | Carbon concentrating mechanisms in eukaryotic marine phytoplankton[END_REF] The ionic forms do not contribute to the vapor pressure of the gaseous form, thus the concentration of the sum of all dissolved inorganic carbon can greatly exceed the atmosphere/water equilibrium concentration of gaseous CO2. In the surface ocean, total dissolved inorganic carbon is approximately 200 times higher than dissolved CO2 concentration [START_REF] Falkowski | Aquatic photosynthesis[END_REF]. Nevertheless, transient patches of CO2-depleted surface seawater may persist for several days to weeks [START_REF] Takahashi | Climatological mean and decadal change in surface ocean pCO2, and net sea-air CO2 flux over the global oceans[END_REF] because CO2(d) diffusion rate and bicarbonate dehydration to CO2 are low processes regarding potential consumption by photosynthetic reactions [START_REF] Falkowski | Aquatic photosynthesis[END_REF].

In all marine phytoplankton, the intrinsic Michaelis constant (Km) of the Rubisco is much higher than the apparent photosynthetic requirement for extracellular dissolved CO2 (K1/2) [START_REF] Kaplan | CO2 concentrating mechanisms in photosynthetic microorganisms[END_REF]. This is attributed to carbon concentration mechanisms, CCM, that increase locally the CO2 concentration. The biophysics and biochemistry of CCMs vary within and among marine phytoplankton clades such as dinoflagellates, haptophytes and diatoms [START_REF] Reinfelder | Carbon concentrating mechanisms in eukaryotic marine phytoplankton[END_REF]. They may involve the activity of external or intracellular carbonic anhydrase and active HCO3 transport. CCM have thus a cost of energy and nutrient: for enzymes production and for active transport of bicarbonate. In general, coccolithophores have low-efficiency CCM, and diatoms and the haptophyte genus Phaeocystis have highefficiency CCM while dinoflagellates appear to possess moderately efficient CCM [START_REF] Takahashi | Climatological mean and decadal change in surface ocean pCO2, and net sea-air CO2 flux over the global oceans[END_REF].

2. 4. The photosynthetic electron transfer chain uses light energy to supply the CBB cycle

Calvin Benson cycle reactions involve consumption of reducing power in the form of NADPH and chemical energy in the form of ATP high energy bond. This is supplied by the photosynthetic electron transfer chain whose mechanistic details have been resolved in the second part of the 20 th century. Resolution of the pathway allowing electron transfer from water to NADP+ has first been investigated with chemical tools. Thus, the global pattern of the pathways has been resolved before understanding the molecules and protein complexes that were involved. a) Oxidoreduction reactions involved in the photosynthetic electron transport

 Resolution of the photosynthetic electron transport pathway

In 1953, the groups of Robert Hill working with isolated chloroplast and the one of Cornelius van Niel working with bacteria could show that light energy was used for endergonic electron transfer and not for photolysis of CO2 [START_REF] Van Niel | On the photochemical reduction of nitrate by algae[END_REF]. Arnon and collaborators then highlighted that isolated chloroplasts are capable of ATP synthesis (Arnon, Allen and Whatley, 1954). This is suggesting that light driven electron transfer is followed by a downhill exergonic reaction coupled to ATP synthesis. Cyclic electron transfer has first been proposed as the pathway of photophosphorylation. But in 1958, Arnon's group could show that ATP synthesis was coupled to a linear electron pathway inducing a donor oxidation and a final acceptor reduction [START_REF] Arnon | Triphosphopyridine nucleotide as a catalyst of photosynthetic phosphorylation[END_REF]. This raised a thermodynamic problem: it was difficult to imagine a downhill electron transport that could be coupled to the endergonic ATP synthesis.

Emerson, Chalmers and Cederstrand, revealed the existence of two site of photochemistry, based on the spectral dependence of the photosynthetic process [START_REF] Emerson | Some factors influencing the long-wave limit of photosynthesis[END_REF]. Those two sites were called photosystem I and photosystem II. Later, two cytochromes were discovered in the chloroplast, named cytochrome b6 and cytochrome f, and Hill and Bendall proposed that those cytochromes allow the electron transfer between the two photosystems and their operation in series [START_REF] Hill | Function of the two cytochrome components in chloroplasts: a working hypothesis[END_REF]. In agreement with the redox potentials of the two cytochromes and the two reaction centers, the general Z scheme or diagram of the photosynthetic electron transfer, was elucidated [START_REF] Hill | Function of the two cytochrome components in chloroplasts: a working hypothesis[END_REF]. In addition to the electron transfer chain between PSII

Exploring the diversity of cyclic electron flow around photosystem I in microalgae species and PSI, involving cytochromes b6 and f, this included a set of reactions allowing the transfer of electrons from water to the donor side of the PSII, and some electron transfer steps on the acceptor side of PSI, where NADPH is produced before being exported for carbon assimilation. Figure 1-8 shows the Z-scheme completed thanks to the well-established characterization of the cofactors involved in electron transfer and the determination of their redox potential.

 Electron transfer chain and redox potentials

Describing the Z-scheme of photosynthesis requires the use of oxidoreduction principles.

"Oxidation" and "reduction" reactions, refer to the loss or gain of one or more electrons, respectively. The redox potential is a scale (in volts) that has been developed to predict the tendency of a redox reaction. The molecules with the lowest redox potential will tend to be oxidized by molecules of higher redox potential.

When these considerations are applied to the photosynthetic electron transfer chain, it is clear that linear electron transfer from water to NADP + is unfavorable. Two photochemical steps that occur in photosystem II (PSII) and photosystem I (PSI) are crucial: the light catalyzed electron transfer from the high redox potential special pairs P680 and P700 towards the low redox potential acceptors, QA and iron-sulfur centers (FX, FA and FB), respectively [START_REF] Nelson | Structure and function of photosystems I and II[END_REF]. Those are highly endergonic (i.e. thermodynamically unfavorable) reactions. Those two steps are put in series thanks to exergonic (i.e.

thermodynamically favorable) electron transfer from PSII to PSI via the membrane plastoquinones (PQ), the cytochrome b6f complex (cyt. b6f) and the luminal plastocyanins (PC) that reduce the PSI primary donor, P700. At the donor side of PSII, Oxygen Evolving Complex (OEC), a metallic center (Mn)4Ca catalyzes transfer of electrons from water to a tyrosine residue, YZ that in turn reduces P680 + . At the acceptor side of PSI, FB reduces ferredoxin (Fd) that in turn reduces NADP + .

Chapter 1: General introduction

Suzanne FERTE -Doctoral Thesis -2019 The principle of the coupling between the exergonic electron transfer from PSII acceptors to PSI donors and ATP production has been proposed by Peter Mitchell in 1968[START_REF] Mitchell | Chemiosmotic coupling and energy transduction[END_REF]. The previous assumption was that ADP was converted into ATP by direct transfer of high-energy phosphoryl groups from some other intermediate, as was known to occur during glycolysis. Thus, it was postulated that high-energy intermediates were produced as a result of electron transfer reactions and that these intermediates then drove ATP synthesis by phosphoryl group transfer. However, no such intermediates were found and phosphorylation of ADP was rather observed to be closely associated to the membrane and dependent of its integrity. These findings led Peter Mitchell, in 1961, to formulate the chemiosmotic hypothesis in which he postulated that the proton electrochemical gradient across the membrane was the driving force for ATP synthesis [START_REF] Mitchell | Coupling of phosphorylation to electron and hydrogen transfer by a chemiosmotic type of mechanism[END_REF]. After long and intense debates during the 1960s and 70s, Mitchell's chemiosmotic hypothesis -which became known instead as the chemiosmotic theory -became a paradigm in the intellectual framework of bioenergetics and he won a Chemistry Nobel Prize in 1978.

The electrochemical proton gradient, H + , has two components: a chemical one that depend on the pH difference between lumen and stroma, pH, and an electric component that is a function of the electric field across the membrane,  :

H + = F. -2.3 RT.pH, where F is the Faraday constant, R the gas constant and T, the temperature.

The photosynthetic electron flow builds an electrochemical proton gradient and we will describe the mechanisms coupling proton movements to electron transfer along the thylakoid hereafter, when describing the molecular actors of the electron transfer.

 Proton liberation and oxygen evolution

On the PSII donor side, the Oxygen Evolving Complex (OEC), also known as the watersplitting complex plays a crucial role. It contains a metalloenzyme core containing 4 atoms of manganese and one atom of calcium, (Mn)4Ca. The OEC allows the transfer of electrons liberated by the oxidation of a water molecule, according to 2 H2O → 4 H + + 4 e -+ O2 up to a tyrosine Z residue involved in the reduction of P680 + . The great works of Joliot andKok, between 1968 and1975, based on the observation of oscillations of period 4 in the emission of oxygen upon short saturating flashes demonstrated the role of of the manganese complex in the electron accumulation [START_REF] Kok | Cooperation of charges in photosynthetic O2 evolution-I. A linear four step mechanism[END_REF]. The OEC is in its most oxidized form every four photochemical events (the manganese atoms are oxidized) and then reacts with a water molecule to produce molecular oxygen O2. This process liberates 4

protons while fueling 4 electrons into the electron transfer chain.

 Cytochrome b6f and Q-cycle

The electron transfer from water to the plastoquinone pool in the thylakoid membrane is made possible thanks to PSII; PSI allows electron transfer from plastocyanin (or cytochrome c6) to NADPH. The missing piece, which allows the functioning in series of the two organisms performing oxygenic photosynthesis i.e. cyanobacteria and its derivatives from a primary and secondary endosymbiotic event. Four major subunits constitute the heart of the complex: cytochrome b 6 , the subunit IV, the Rieske protein and the cytochrome f (Figure 123456789). The complex forms a homodimer [START_REF] Huang | Characterization of the chloroplast cytochrome b6f complex as a structural and functional dimer[END_REF] as confirmed by electron microscopy and X-ray crystallography [START_REF] Stroebel | An atypical haem in the cytochrome b6f complex[END_REF]. In total seven prosthetic groups are contained within a cytochrome b6f monomer. Two b haems (bL and bH) are axially ligated to the cytochrome b6 and a haem ci also is linked to cytochrome b6 but its function is not clear yet. One c haem is covalently attached to the extramembrane lumenal domain of cytochrome f and one Fe2S2 cluster is present in the extramembraner lumenal domain of the Rieske protein. In addition, one chlorophyll a and one β-carotene are found in the complex, whose roles are still not fully understood [START_REF] Stroebel | An atypical haem in the cytochrome b6f complex[END_REF]. Electron transfer from plastoquinols to plastocyanins is exergonic and is coupled at the cytochrome b6f site to proton translocation from the stroma to the lumen against the electrochemical proton gradient. Two mechanisms are involved. In the first mechanism, a plastoquinone is reduced by two electrons at the acceptor side of PSII, recruiting two protons from the stroma, and is reoxidized at the lumenal QO site of cytochrome b6f, thus releasing 2 protons inside the lumen. The second mechanism is called the Q-cycle for "Quinone cycle" and was first described by Mitchell in 1975(Mitchell, 1975). The two electrons released at the Qo site follow two different pathways: one is directed to the cytochrome f and will reduce a PC while the other one is transferred via bL and bHhaems to the stromal Qi site where a plastoquinone fixed. Hence, oxidation of two plastoquinols at the Qo site induce the reduction of one plastoquinone at the Qi site thanks to two stromal protons (Figure 1-10). All in all, 4 H + are translocated to the lumen every 2 electrons transferred through the cytochrome b6f. After many debates whether the Q-cycle is facultative or not [START_REF] Berry | Proton to electron stoichiometry in electron transport of spinach thylakoids[END_REF], the Q-cycle is now considered to be constitutive (Sacksteder et al., 2000).

c) ATP synthase

As predicted by Mitchell in the chemiosmotic theory, the chloroplast (CF0-F1) ATP synthase is located inside the thylakoid membrane, in the non-appressed regions in plants. This large complex is able to use the H + to produce ATP from ADP and inorganic phosphate, Pi. The mechanism involved in ATP synthesis has been resolved by John Walker and Paul Boyer who shared a Noble Prize for Chemistry in 1997. As shown in Figure 1-11, the enzyme has two major domains: the three-fold CF1 catalytic domain that protrudes at the stromal side and that is linked to the membrane domain CF0 thanks to a peripheral stalk.

The F1 domain consists of three  heterodimers, which define the catalytic site, and of the central  and  subunits which are attached to the c-ring (polymer of c subunits arranged in a ring). The F0 domain, the motor of the enzyme, is composed of three subunits with a stoichiometry a1b2c9-12. Figure 1-11 presents the model of rotation generation of CF0 induced by proton translocation from lumen to stroma. This movement induces rotation of the central stalk and causes conformational changes in the  heterodimers, resulting in the synthesis of ATP. According to this mechanism, one c-ring rotation results in three ATP molecules. Hence, the number of protons needed to synthesize one ATP molecule would not depend on the extent of the ΔμH + but only on the number of c subunits composing the c-ring (Hahn et al., 2018). It is of note that the ATP synthase is a reversible enzyme, the sense of the reaction being only determined by thermodynamics, i.e. the electrochemical proton gradient and the ratio ATP over ADP+Pi. 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 [START_REF] Jensen | Chloroplast evolution, structure and functions[END_REF]. 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).

2. 5. From light absorption to energy storage a) 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) [START_REF] Förster | Zwischenmolekulare energiewanderung und fluoreszenz[END_REF], ( 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. 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 [START_REF] Emerson | The photochemical reaction in photosynthesis[END_REF]. 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 [START_REF] Croce | Natural strategies for photosynthetic light harvesting[END_REF].

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 [START_REF] Caffarri | A comparison between plant photosystem I and photosystem II architecture and functioning[END_REF]. 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 [START_REF] Dekker | Supramolecular organization of thylakoid membrane proteins in green plants[END_REF]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 [START_REF] Amunts | Structure determination and improved model of plant photosystem I[END_REF].

c) 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. The PSII core is a multisubunit complex composed of approximately 25-30 subunits [START_REF] Guskov | Cyanobacterial photosystem II at 2.9-Å resolution and the role of quinones, lipids, channels and chloride[END_REF]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 [START_REF] Jensen | Structure, function and regulation of plant photosystem I[END_REF]. 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.

 Electron transfer chains within photosystems

In PSII and PSI, the cofactors involved in electron transfer (ET) reactions are organized into two parallel chains (or branches) which are perpendicular to the plane of the membrane (Figure 1-13B). This configuration allows the transfer of electrons through the membrane and the formation of an electrochemical potential generated by photochemistry (reviewed in Nelson and[START_REF] Nelson | Structure and function of photosystems I and II[END_REF][START_REF] Caffarri | A comparison between plant photosystem I and photosystem II architecture and functioning[END_REF].

All the structural models of photosynthetic reaction center (RC) with sufficient resolution show a similar pattern in the cofactors involved in electron transfer reactions: they are organized into two chains located at the interface of the two main subunits of the reaction centers. Thus, each monomer of the reaction centers, PsaA and PsaB for PSI, PsbA (D1) and PsbD (D2) for PSII, contains a similar set of cofactors (Figure 1-13B): a group of at least six chlorines, which are the pigments from which the primary charge separation is initiated, and two quinone type molecules, the plastoquinones (PQ) in the PSII and the phylloquinones (PhQ) in the PSI, which act as successive electron acceptors. Other cofactors are specific to each electron transfer chain. In the case of PSI, the 6 chlorines involved in the separation of the initial charges are chl a while in PSII four are chl a and the others are pheophytins. Of the six chlorine pigments, two molecules of chl a form a dimer face to face (each coordinated by one of the two RC subunits). These have been frequently identified as the sites for primary charge separation (primary donor, P), and for this reason are also called "special pairs" and named P680 for PSII and P700 for PSI (names indicate their maximum absorption change upon photo-oxidation in the red region). The exact charge separation mechanisms in both PSI and PSII are still a subject of debate.

In the case of PSII, only one of the two electron transfer branches is used for photochemistry (branch A, Figure 1-13B, [START_REF] Diner | Structure, dynamics, and energy conversion efficiency in photosystem II[END_REF]reviewed in Nelson and[START_REF] Nelson | Structure and function of photosystems I and II[END_REF][START_REF] Caffarri | A comparison between plant photosystem I and photosystem II architecture and functioning[END_REF] and therefore only one of the two Pheo molecules (linked to the D1 subunit) is photochemically reduced. On the other hand, in PSI, the two electron transfer branches are used after charge separation (branches A and B, Figure 1-13B;Santabarbara et al., 2010). For PSII and PSI, models are extracted from the structure of cyanobacteria and pea, respectively. P680 is the "special pair" of PSII. Electron extracted from water in the Mn center of the OEC goes through a Tyrosine (Tyrz) to the P680. From excited P680*, electrons follow the "A branch" by reducing in sequence the ChlD1, the PheoD1 (pheophytin), the QA (quinone) and finally the QB (which gets double reduced and detaches from PSII when protonated). An iron atom (Fe) is in between the two quinones. P700 is the "special pair" of PSI. From excited P700*, electrons follow both the "A branch" and "B branch" to reduce an accessory Chl eC2A/eC2B, a second Chl A0A/A0B and then a PhQ A1A/1B (phylloquinone). Electrons from both branches converge on the FX acceptor and exit from PSI by passing two other 4Fe-4S centers, FA and FB. See text for further discussion on ET mechanism and reaction sequence.

2. 6. Monitoring photosynthesis with chlorophyll fluorescence

We saw previously that when a chlorophyll is excited after absorbing a photon, one of the relaxation pathway involves emission of a photon of lower energy (fluorescence). This signal can be used to probe PSII activity and the principle of the method is presented hereafter.

 Theoretical principle of chl a fluorescence

Chlorophyll fluorescence is now widely used for physiology and ecophysiology studies. The principle is based on a model for photochemistry in PSII where all excited chlorophyll relaxation pathway compete with each-other [START_REF] Butler | Energy distribution in the photochemical apparatus of photosynthesis[END_REF]. When a chlorophyll is excited after absorbing a photon, there are three pathways (see Figure 1-14) that allow its relaxation to its fundamental energy level:

(1) photochemistry, which corresponds to the transfer of an electron from the special pair of chlorophyll, P680 to its primary acceptor, a pheophytin and from there to the first stable acceptor QA,

(2) the non-radiative relaxation, which consists in the dissipation of energy in the form of heat,

(3) the radiative relaxation, which is the re-emission of a lower energy photon (i.e. at a longer wavelength).

Chapter 1: General introduction

Suzanne FERTE -Doctoral Thesis -2019 Based on these parameters, several systems have been proposed, which vary greatly depending on the model of energetic connection between PSIIs, i.e. whether a "puddle" model (all PSII are isolated with their own antenna) or a "lake" model (where the exciton is shared by all PSII centers) is used. Some parameters are more robust than others and prove valid in both models. It is the case of the (Fm-F0)/Fm, generally written Fv/Fm, the maximal quantum yield of PSII when it is dark-adapted, or its equivalent in the light, Y(II) = (Fm' -F')/ Fm', the quantum efficiency of PSII (Genty, 1989;Lazar, 2015). and Joliot, 1964).

This behavior was confirmed with fluorescence measurements. When PSII is inhibited by an inhibitor allowing only a single charge separation per PSII, e.g. orthophenantroline, we expect that the kinetics of fluorescence rise follow an exponential law in the case of nonconnected PSIIs. Instead the fluorescence rise showed an initial sigmoidicity confirming that the closure of centers is not followed by an equivalent decrease in photosynthetic activity

(Figure 1-16).
In the absence of connectivity, i.e. in the "puddle" model, the fraction of open PSII centers can be calculated with the term qP which is the ratio of the quantum yield to the maximal quantum yield (when all QA are oxidized): qP = Y(II)/(Fv'/Fm') (Lazar, 2015) where Fv'/Fm' = (Fm' -F0')/Fm' is the maximal quantum yield measured after a short dark adaptation (typically few tens of seconds) of the illuminated sample.

An extreme case of connectivity is the "lake" model where an exciton can be shared by all PSII. A parameter qL has been calculated to estimate the fraction of open PSII centers in this extreme case scenario: qL = Y(II)/(Fv'/Fm') .F0'/F' (Kramer, 2004).

The amplitude of connectivity has been well studied in plants [START_REF] Stirbet | Excitonic connectivity between photosystem II units: what is it, and how to measure it?[END_REF] and in some green algae like Chlorella [START_REF] Joliot | Etude cinétique de la réaction photochimique libérant l'oxygène au cours de la photosynthèse[END_REF][START_REF] Lavorel | A connected model of the photosynthetic unit[END_REF] but not much is known about connectivity in marine microalgae. A conservative approach consists in estimating the fraction of open PSII centers based on the two extreme models, "puddle" and "lake" which provide upper and lower limits to the estimation, respectively.

3. Regulation of the photosynthetic process and acclimation to changing environments

In the previous section, we discussed the stoichiometry of the two products of the "light phase" of photosynthesis, ATP and NADPH, which needs to be adjusted to the metabolic requirements downstream PSI. Another constraint on the photosynthetic physiology concerns the substrates of the photosynthetic electron transfer chain, the light intensity. The photosynthetic process balances the absorption of photons to the capacity of the metabolic pathways after PSI, through a combination of regulatory mechanisms. In this section, I will present the various regulatory mechanisms allowing the photosynthetic chain to adjust electron and proton circuits to the availability of its substrate and the metabolism demand for its products.

3. Light stress and photo-inhibition

The These include also the probability of internal conversion to form triplet state of chlorophylls [START_REF] Krieger-Liszkay | Singlet oxygen production in photosynthesis[END_REF], which is not one of the main pathways of chlorophyll deexcitation and is not shown in Figure 1-17.However, the formation of triplet states can also be enhanced because of an increased probability that charge separation between P680 + and Pheo -recombines (i.e. reverses, the transfer from Pheo -to P680 + being thermodynamically favorable) when oxidized QA is not available to move the electron further [START_REF] Krieger-Liszkay | Singlet oxygen production in photosynthesis[END_REF]. The formation of triplets is dangerous because chlorophylls in a triplet state can react with O2 to produce singlet oxygen, 1 O 2* , a very toxic molecule which can degrade lipids, pigments or proteins at the vicinity of PSII. In particular, singlet oxygen can degrade the D1 protein (Nixon et al., 2010). This leads to photo-inhibition (Figure 1-17), together with other processes like the limitation in electron transfer from the donor-side of PSII (Nixon et al., 2010).

( 3 Chl * ) (Krieger
It is not clear yet if the degradation of the D1 protein is a side-damage or if it is an advantageous process, involved in protection of PSII (Nixon et al., 2010, Keren and[START_REF] Keren | Photoinhibition: molecular mechanisms and physiological significance[END_REF]. In any case, there is a PSII-repair mechanism, which appears as a regulation process and operates in three phases: (i) disassembly of the PSII, (ii) the replacement of the damaged subunit specifically by a newly synthetized subunit D1, sometimes D2, [START_REF] Ohad | Membrane protein damage and repair: removal and replacement of inactivated 32-kilodalton polypeptides in chloroplast membranes[END_REF], and (iii) PSII reassembly (Nixon, 2010). Photo-inhibition occurs when the rate of damage of PSII outcompetes the rate of its repair. In contrast to PSII, PSI is very rarely damaged (Sonoike, 2011;[START_REF] Scheller | Photoinhibition of photosystem I[END_REF] but, when this happens, the photo-inhibition of PSI is very fast and the damage is practically irreversible [START_REF] Tikkanen | Photosystem II photoinhibition-repair cycle protects Photosystem I from irreversible damage[END_REF]. Damage on PSI occurs in conditions of acceptor side limitation (when electrons accumulate after PSI), which favors reduction of O2 into superoxide anion radicals, hydrogen peroxide and hydroxyl radicals which destroy the ironsulfur centers involved in the electron transfer (Sonoike et al. 1995). The process is almost irreversible because there is no known repair mechanism for PSI (Sonoike, 2011).

3. 2. Protecting photosystem I and II from light stress

Efficient photosynthesis must meet several criteria -we have listed 3 of them so far:

providing enough ATP for the downstream metabolism, protecting PSII from high light stress and protecting PSI from acceptor side limitation. How does the photosynthetic machinery achieve proper function in such a constrained situation? As discussed in this section, a master regulator of the photosynthetic electron transfer chain allows this, which is the electrochemical proton gradient across the thylakoid. Indeed, ΔµH + plays the dual role of driving ATP [START_REF] Mitchell | Chemiosmotic coupling in oxidative and photosynthetic phosphorylation[END_REF] and regulating photo-protection mechanisms through its osmotic component, the ΔpH (Kramer, 2003).

 Cytochrome b6f activity is regulated by the pH across the thylakoid membrane

When the capacity of reoxidation of PSI acceptors is low, putting PSI at high risk of photoinhibition, a process called "photosynthetic control" allows to relax the reducing pressure on the iron-sulfur centers of PSI. This is achieved by making cyt. b6f the actual rate-limiting step in photosynthesis (Rumberg and Siggel, 1969;[START_REF] Laisk | Control of cytochrome b6f at low and high light intensity and cyclic electron transport in leaves[END_REF]. In conditions of limitation of photosynthesis downstream PSI, the ATP/ADP ratio and the ΔpH increase, making the H + release thermodynamically more difficult during the oxidation of PQH2 in the cytochrome b6f [START_REF] Laisk | Control of cytochrome b6f at low and high light intensity and cyclic electron transport in leaves[END_REF] The consequent slowdown of cytochrome b6f electron transfer results in oxidized cytochrome f, PC and P700 (Rumberg and Siggel, 1969). The involvement of pH is involved in this regulation as demonstrated by the effect of nigericin, a H + /K + antiporter (Joliot and Johnson, 2011). Nigericin dissipates the pH without significantly affecting the electric field Ψ and thereby maintaining ATP synthesis. In the presence of nigericin, P700 is more reduced and more PSI photoinhibition occurs, indicating that "photosynthetic control" is arrested.

 pH-dependent -quenching of excited chorophylls

The osmotic component of the electrochemical proton gradient, the ΔpH, also regulates PSII photo-protective mechanisms (Demmig-Adams, Gilmore & Adams 1996; [START_REF] Holt | Toward an understanding of the mechanism of nonphotochemical quenching in green plants[END_REF][START_REF] Holt | Toward an understanding of the mechanism of nonphotochemical quenching in green plants[END_REF]) via the qE process, an energy-dependent quenching of excited chlorophylls in the antenna. This process harmlessly dissipates excess light in the form of heat [START_REF] Holt | Toward an understanding of the mechanism of nonphotochemical quenching in green plants[END_REF] and can be visualized in vivo through the non-photochemical quenching (NPQ) of chlorophyll fluorescence. In saturating light, when photochemistry is low, the heat and fluorescence pathways compete with each other. Therefore, the development of the qE mechanism is accompanied by a decrease of the fluorescence. It is to note that photo-inhibition of PSII, sometimes called qI, also results in a decrease of the fluorescence, but, as mentioned earlier, the two processes can be distinguished based on their relaxation kinetics: qE relaxes in the timescale of minutes whereas the recovery from qI is in the timescale of tens of minutes or hours [START_REF] Holt | Toward an understanding of the mechanism of nonphotochemical quenching in green plants[END_REF]).

The exact mechanism by which heat dissipation is regulated is still debated, but the molecular actors of the process are known, as well as their dependence upon pH. The qE mechanism is present in most photosynthetic organisms and involves changes of pigments (the xanthophyll cycle) and/or conformational changes of specific PSII subunits [START_REF] Goss | Biodiversity of NPQ[END_REF].

 Xanthophyll cycle: role of carotenoids

In plants and green algae or brown algae, the development of qE, the rapidly reversible NPQ, is associated to the xanthophyll cycle. It consists in the reversible conversion of a carotenoid pigment, violaxanthine (Vx) into a de-epoxidized form, zeaxanthine (Zx) via the intermediate form antheraxanthin [START_REF] Yamamoto | Studies on the light and dark interconversions of leaf xanthophylls[END_REF] Another xanthophyll cycle is found in diatoms, haptophytes and dinoflagellates [START_REF] Brown | Diurnal changes in photochemical efficiency and xanthophyll concentrations in shallow water reef corals: evidence for photoinhibition and photoprotection[END_REF][START_REF] Hager | Das Carotinoidmuster und die Verbreitung des lichtinduzierten Xanthophyllcyclus in verschiedenen Algenklassen[END_REF][START_REF] Hager | Das Carotinoidmuster und die Verbreitung des lichtinduzierten Xanthophyllcyclus in verschiedenen Algenklassen[END_REF], which consists in a one-step reaction from diadinoxanthin (DD) to its de-epoxidized form diatoxanthin (DT).

De-epoxidation of Vx (or DD) into Zx (or DT) is catalyzed by the violaxanthin de-epoxidase (VDE) soluble in the lumen and active at low pH (optimum at pH around 5 for the violaxanthin de-epoxidase). The zeaxanthin epoxidase (ZEP) catalyzes the reverse reaction, from Zx (DT) to Vx (DD). The main working model is that under high light, photosynthetic chain is actively translocating protons into the lumen, thus decreasing the luminal pH, activating VDE which increases the concentration of Zx and heat dissipation.

 PsbS/LHCSR3/LHCX

PsbS is a PSII subunit that is involved in NPQ mechanism (Li et al., 2000) and that displays several acidic residues in the lumen, playing the role of pH sensors and inducing some conformational changes in PSII favoring heat dissipation processes. In Chlamydomonas reinhardtii reversible NPQ is highly affected in mutants lacking LHCSR3, a protein that is part of PSII antennae (Peers et al., 2009). LHCSR proteins are not expressed constitutively but only in specific conditions, e.g. transition from dark to light (Savard, Richard and Guertin,

Chapter 1: General introduction

Suzanne FERTE -Doctoral Thesis -2019 1996) or sulfur deprivation (Zhang et al., 2004). Like for PsbS, it contains acidic residues that can sense the luminal pH and generate conformational changes and heat dissipation (Bonente et al., 2011). In diatoms, LHCX proteins are part of the LI818/Lhcsr family to whom LHCSR3 belongs (Koziol et al., 2007) and also participate to the modulation of NPQ (Bailleul & Rogato et al., 2010;[START_REF] Zhu | Photoprotection in the diatom Thalassiosira pseudonana: role of LI818-like proteins in response to high light stress[END_REF][START_REF] Buck | Lhcx proteins provide photoprotection via thermal dissipation of absorbed light in the diatom Phaeodactylum tricornutum[END_REF].

 Other photosynthetic acclimation mechanisms

Even though ΔµH + plays a major role in the regulation of photosynthesis, other important modes of regulation do not rely on it. Higher plants and green algae are able to adjust light excitation energy between the two photosystems, a process called state transition [START_REF] Bonaventura | Fluorescence and oxygen evolution from Chlorella pyrenoidosa[END_REF]. The current admitted model is that state transitions are mediated by the reversible displacement of a fraction of the chlb-containing light harvesting proteins LHCII, between PSII and PSI (Figure 1 -18). This redistribution of light harvesting between the two photosystems is regulated by the redox state of the PQ pool and cytochrome b6f complex. In a situation where PSII activity exceeds PSI activity, the reduction of these intersystem electron carriers activates the STT7/STN7 kinase which in turn phosphorylates the LHCII leading to their lateral movement towards PSI [START_REF] Wollman | State transitions reveal the dynamics and flexibility of the photosynthetic apparatus[END_REF].

The opposite situation -where the intersystem electron carrier would be highly oxidizedleads to the deactivation of the kinase. Then, a phosphatase dephosphorylates the LHCII which re-associate with PSII [START_REF] Wollman | State transitions reveal the dynamics and flexibility of the photosynthetic apparatus[END_REF]. In Chlamydomonas reinhardtii, this process can modify the absorption cross-section of PSII by up to 40% in (Nawrocki et al., 2016). This process seems to be crucial in response to changing environment. Stn7 Arabidopsis mutant growth is impaired under changing light conditions (Bellaflore et al., 2005).

3. 3. Photosynthetic alternative pathways

In photosynthetic organisms, besides the linear electron flow from water to CO2, there are alternative electron transfers, that is to say transfers of electrons along the electron transfer chain, which do not lead to the reduction of CO2 (Eberhard, Finazzi and Wollman, 2008).

These alternative pathways allow the establishment of an electrochemical gradient of protons (and thus the synthesis of ATP) regardless of the activity of the CBB cycle and they allow an increase of the ATP/NADPH ratio produced by the photosynthetic chain.

We mentioned previously that the limitation of the electron flow downstream PSI can have dramatic consequences, causing the photoinhibition of photosystems, and especially PSI.

Moreover, we have shown that the protective mechanisms allowing the downregulation of the cyt. b6f activity and the increase in light energy dissipation as heat depend on the implementation of an electrochemical proton gradient. Alternative electron transfers therefore represent a major evolutionary advantage: not only they produce extra ATP but they increase the ΔμH + and therefore the protection of both photosystems.

Several alternative pathways have been described in the literature, that we will briefly review here. Figure 1-19 summarizes the different alternative electron pathways. Alternative electron flows (AEF) can be classified according to their final electron acceptor. A first type of AEF corresponds to the water to water cycle: the electrons extracted from water at the PSII level are used by oxidases to reduce molecular oxygen and regenerate water.

Several oxidases have been identified among which most of them use electrons downstream PSI. We can mention the flavodiiron proteins identified in Chlamydomonas reinhartdii (Chaux et al., 2017), in moss [START_REF] Gerotto | Flavodiiron proteins act as safety valve for electrons in Physcomitrella patens[END_REF] and in cyanobacteria (Zhang et al., 2009) but the gene coding for these proteins is absent in angiosperms (Yamamoto et al. 2016). This alternative pathway would be mainly involved under changing light conditions, as a transitory sink for electrons during the acclimation period (Chaux et al., 2017, Allahverdiyeva et al., 2013).

Another water-to-water route concerns the Mehler reactions, or superoxide dismutase pathway which transfers electrons from the acceptor side of PSI to O2. The superoxide formed is detoxified by a superoxide dismutase and ascorbate peroxidase, consuming NADPH (Asada et al., 2000). The superoxide dismutase has been identified in plants (Asada et al., 2000), green algae [START_REF] Chen | The iron-containing superoxide dismutaseencoding gene from Chlamydomonas reinhardtii obtained by direct and inverse PCR[END_REF], cyanobacteria [START_REF] Asada | Superoxide dismutases from a blue-green alga, Plectonema boryanum[END_REF], diatoms [START_REF] Wolfe-Simon | Localization and role of manganese superoxide dismutase in a marine diatom[END_REF] and dinoflagellates where it is said to be the main alternative electron flow (Roberty et al., 2014). This reaction occurs in isolated thylakoids [START_REF] Asada | The water-water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons[END_REF] but its rate in vivo is not clear yet because of sources of confusion with other water to water cycles, especially the one involving flavodiiron proteins.

Finally, the electrons can be rerouted to the mitochondrial chain after PSI. The existence of such a flow has been shown in green algae (Lemaire, Wollman and Bennoun, 1988), diatoms (Bailleul et al., 2015) or in plants [START_REF] Scheibe | Malate valves to balance cellular energy supply[END_REF] and other observations suggest strong energetic exchanges between organelles in the green lineage [START_REF] Cardol | The onset of NPQ and ΔμH+ upon illumination of tobacco plants studied through the influence of mitochondrial electron transport[END_REF][START_REF] Cardol | Impaired respiration discloses the physiological significance of state transitions in Chlamydomonas[END_REF]. Such a pathway can be considered like a "meta"-water to water cycle. This pathway requires the diffusion of reducing power from the plastid to the mitochondria that is enabled by malate shuttles in plants [START_REF] Scheibe | Malate valves to balance cellular energy supply[END_REF] but with potentially slow rates (Scheibe et al., 2005). In diatoms, it would play an important role with about 10% of electrons constitutively rerouted from the chloroplast to the mitochondrion (Bailleul et al., 2015).

A particular water to water pathway is that of the Plastid Terminal oxidase (PTOX) which redirects the electrons from the plastoquinol to molecular oxygen, thus not involving PSI.

This pathway has been found in several phylogenetic clades such as plants [START_REF] Wetzel | Nuclear-organelle interactions: the immutans variegation mutant of Arabidopsis is plastid autonomous and impaired in carotenoid biosynthesis[END_REF][START_REF] Carol | Mutations in the Arabidopsis gene IMMUTANS cause a variegated phenotype by inactivating a chloroplast terminal oxidase associated with phytoene desaturation[END_REF] or cyanobacteria (Cardol et al., 2008;[START_REF] Bailey | Alternative photosynthetic electron flow to oxygen in marine Synechococcus[END_REF]. But under PTOX for 100 PSII, [START_REF] Lennon | Location, expression and orientation of the putative chlororespiratory enzymes, Ndh and IMMUTANS, in higher-plant plastids[END_REF] and should not play a crucial role in photosynthetic regulation (Nawrocki et al, 2016;Nawrocki et al., 2019c). This path seems to play only a role in very specific stress conditions, as in the case of microalgae deprived of iron (Cardol et al., 2008;[START_REF] Bailey | Alternative photosynthetic electron flow to oxygen in marine Synechococcus[END_REF] grown at low temperature and high irradiance [START_REF] Ivanov | Implications of alternative electron sinks in increased resistance of PSII and PSI photochemistry to high light stress in cold-acclimated Arabidopsis thaliana[END_REF] or in alpine plants that constitutively express high levels of PTOX [START_REF] Streb | Evidence for alternative electron sinks to photosynthetic carbon assimilation in the high mountain plant species Ranunculus glacialis[END_REF]. A model has been proposed to explain the apparently low activities of PTOX in vivo and its high turnover rate in vitro. PTOX would bind to the membrane only in conditions of high light and alkaline stromal pH. It would then play a role of safety valve protecting the PQ pool from over-reduction (Krieger-Liszkay and Feilke, 2016).

Other alternative pathways ultimately reduce other molecules than O2 and can take place in anoxia. This is the case of the hydrogenase pathway, only expressed after several tens of minutes of anoxia in Chlamydomonas reinhardtii (Godaux et al., 2015) in Chlorella fusca but not in Chlorella variabilis [START_REF] Winkler | Isolation and molecular characterization of the [Fe]-hydrogenase from the unicellular green alga Chlorella fusca[END_REF] and which uses electrons from the acceptor side of the PSI to reduce protons into dihydrogen.

We can also mention the rerouting of photosynthetic electrons for nitrate reduction followed by nitrite excretion that has been described in some cyanobacteria [START_REF] Reyes | Effect of glucose utilization on nitrite excretion by the unicellular cyanobacterium Synechocystis sp. strain PCC 6803[END_REF]. In cyanobacteria, an artificial alternative pathway was engineered via a sucrose sink [START_REF] Abramson | Increased photochemical efficiency in cyanobacteria via an engineered sucrose sink[END_REF].

Finally, there is an alternative electron flow that does not have any final product since it reroutes the electrons from acceptor side of the PSI back to the inter-system chain, upstream cytochrome b6f, giving rise to a cyclic electron flow around PSI (CEF). In plant and green algae, cyclic electron flow is considered essential for photosynthesis and would be of crucial importance for photosynthetic response to changing environment. In the following section, we will present the state of the art concerning this pathway, i.e. the proteins involved in the re-introduction of the reducing power from PSI acceptor side back to the chain, and the conditions favoring it.

3. 4. CEF, a crucial regulative pathway remaining mysterious a) CEF would be crucial under stress conditions

As an alternative electron flow, three main roles have been proposed for CEF. The first one is to participate to the photosynthetic control in conditions where metabolic pathway downstream PSI are limiting the linear electron flow. The second one is to protect PSII by activating pH dependent heat dissipation processes. The last one is to adjust the ATP/NADPH ratio produced by the light phase of photosynthesis to the metabolic pathways requirement.

All these roles could be involved in response to changing environment, when CEF has been proposed to play a crucial role.

It would be crucial under several light regimes: under high light in plants (Joliot and Johnson, 2011;Huang and Zhang, 2012;Miyake et al., 2005a;[START_REF] Rumeau | Chlororespiration and cyclic electron flow around PSI during photosynthesis and plant stress response[END_REF], under fluctuating light (Suorsa et al., 2012), at light onset (Joliot and Joliot 2002).

But CEF would also be involved in dealing with fluctuating gas availability: its rate would change depending on carbon availability in green algae (Lucker & Kramer, 2013) and in plants (Miyake et al., 2005b) to respond to the higher ATP demand associated to CCMs.I It would also be enhanced during anoxia in plants [START_REF] Kukuczka | Proton gradient regulation5-like1-mediated cyclic electron flow is crucial for acclimation to anoxia and complementary to nonphotochemical quenching in stress adaptation[END_REF] and green algae (Alric, 2010) or as a starter at light onset in anoxia (Godaux et al., 2015). CEF could also be involved in coping with high O2 concentration under high light [START_REF] Walker | The response of cyclic electron flow around photosystem I to changes in photorespiration and nitrate assimilation[END_REF]. CEF has been proposed to be important in response to changing temperatures in dinoflagellates [START_REF] Aihara | Heat induction of cyclic electron flow around photosystem I in the symbiotic dinoflagellate Symbiodinium[END_REF] and plants [START_REF] Huang | Stimulation of cyclic electron flow during recovery after chilling-induced photoinhibition of PSII[END_REF]Yamori et al, 2011) and its rate would depend on life stages as during leaf senescence in plants (Junker and Ensminger 2016).

Although CEF seems very important in response to environmental factors, CEF remains widely mysterious. One of the first points to emphasize is that there is no clear consensus regarding the enzymes involved in the reinjection step from PSI acceptors, back into the chain, that we will call hereafter the "cycling step". Three main pathways have been proposed in the literature, none of them excluding the other ones. And I will present them in the next section. In 2002, the PGR5 protein has been proposed to catalyze a major pathway for CEF.

Munekage et al isolated a pgr5 Arabidopsis thaliana mutant that was affected in qE implementation during high light illumination. Isolated chloroplasts were affected in nonphotochemical reduction of the PQ pool [START_REF] Munekage | PGR5 is involved in cyclic electron flow around photosystem I and is essential for photoprotection in Arabidopsis[END_REF]. Since no redox cofactor has been identified in the PGR5 protein it has been proposed to catalyse CEF. In 2008, DalCorso et al. isolated a PGRL1 mutant presenting the same phenotype as PGR5 mutant and showed that PGRL1 and PGR5 could form heterodimer complexes (DalCorso et al., 2008). PGRL1 is supposed to house a Fe containing cofactor (Hertle et al., 2013) and might be involved in PSI/b6f supercomplexes performing an enclosed cyclic electron flow (Iwai et al., 2010). Nevertheless, the effective implication of PGRL1-PGR5 proteins in CEF is more and more debated. Several studies suggest that the CEF amplitude is not affected in pgrl1 or pgr5 mutants. In the transition from dark to light the pool of electrons accumulated in the dark allows transitory high CEF in PSII inhibited samples. In these conditions, CEF is easily assessable since linear electron flow is not active. During transition from dark to light, the Arabidopsis pgr5 mutant and Chlamydomonas pgrl1 mutant have no phenotype and display the same maximal CEF as their respective wild types (Nandha et al., 2007;Nawrocki et al., 2019a).

 NDH / Nda2 pathway

Cyanobacteria and higher plants express a membrane type I NADPH dehydrogenase complex commonly called NDH (Mastubayashi et al., 1987;[START_REF] Peltier | NDH-1 and NDH-2 plastoquinone reductases in oxygenic photosynthesis[END_REF]. This complex is partly homologous to bacterial and mitochondrial complex I, a NADH:ubiquinone oxidoreductase. By analogy, it has been proposed that the chloroplast NDH is involved in the transfer of electrons from NADPH to the plastoquinone pool, thus enabling CEF. NDH expression is low under non stress conditions [START_REF] Livingston | An Arabidopsis mutant with high cyclic electron flow around photosystem I (hcef) involving the NADPH dehydrogenase complex[END_REF] but mutants overexpressing NDH1 show a phenotype compatible with an increased CEF compared to the WT [START_REF] Livingston | An Arabidopsis mutant with high cyclic electron flow around photosystem I (hcef) involving the NADPH dehydrogenase complex[END_REF]. Moreover, by analogy with bacterial and mitochondrial complex I, , it has been proposed that the chloroplast NDH1 is able to move 2 protons from the stroma to the lumen per electron transferred, thus increasing the ATP production of CEF (Strand et al, 2017). It is to note NDH1 lacks an NADH binding site and most likely uses ferredoxin as a substrate (Yamamoto, Peng, Fukao, & Shikanai, 2011).

Some green algae including Chlamydomonas reinhardtii (Maul et al., 2002) and conifers are lacking NDH1 (Wakasugi et al., 1994). But Chlamydomonas reinhardtii have another protein performing a similar function, Nda2, which is not a transmembrane protein and does not couple electron transfer to proton translocation [START_REF] Jans | A type II NAD (P) H dehydrogenase mediates light-independent plastoquinone reduction in the chloroplast of Chlamydomonas[END_REF]. Nevertheless, its maximal rate is expected to be low by comparison with the linear electron flow (few electrons per second, Nawrocki et al, 2019b).

 Ci haem from the cytochrome b6f complex

Cytochrome b6f houses a haem ci whose role has not been understood yet. Figure 1-20b

shows that it is located at the stromal side of the complex, near the Qi site where PQ is reduced by the b haems. Moreover, Figure 1-20a shows a crystal structures of a cytochrome b6f monomer showing that haem ci also would be accessible for a reduction from a stromal protein (Kurisu, Zhang, Smith, & Cramer, 2003;[START_REF] Stroebel | An atypical haem in the cytochrome b6f complex[END_REF]. On Figure 1-20a, we can see that Fd could attach on the stromal side of the cytochrome b6f and its distance to the ci haem would enable an electron transfer [START_REF] Stroebel | An atypical haem in the cytochrome b6f complex[END_REF]. The structure of the cytochrome b6f gave support to an idea originally proposed by Mitchell (Mitchell, 1975): a CEF pathway involving transfer to haem ci through a ferredoxin oxidase enzyme such as ferredoxin:NADP + reductase, FNR, or directly from the ferredoxin (Iwai et al., 2010;Nawrocki et al., 2019b). In such a pathway the concentration of the enzyme involved in the "cycling step" (ie the cyt. b6f itself) is in a stoichiometric concentration with PSI (from 0.5 to 1.25 in plants, [START_REF] Anderson | Cytochrome b 6 f complex: dynamic molecular organization, function and acclimation[END_REF], which theoretically enables high rates of CEF.

 Summerizing our understanding of the CEF pathway

Investigation of the pathway(s) involved in CEF has mainly been driven by a genetic approach. It is important to note that that until now, we donnot know of any mutant that would perform no CEF. Neither NDH, nor pgrl1 or pgr5 mutants show a total absence of CEF.

This suggests that either the different pathways compensate each other, or that we have not identified the principal pathway yet or that the total absence of CEF would be lethal.

A main limitation of this strategy is that it is not an "all other things being equal" approach.

Indeed, any mutation of an intermediate of the photosynthetic chain would change intermediate redox equilibriums and potentially CEF even if it is not directly involved in CEF (Kramer & Evans, 2011). For example, it has been suggested that the lack of qE phenotype observed in the pgr5 mutant [START_REF] Munekage | PGR5 is involved in cyclic electron flow around photosystem I and is essential for photoprotection in Arabidopsis[END_REF] is related to changes in ATP synthase permeability more than to CEF involvement (Avenson et al., 2005). Thus, it seems crucial to understand the CEF dependence on linear electron flow and on cell physiology to better interpret the phenotype observed in putative CEF mutants.

4. Thesis outline

The recent awareness of the importance of marine photosynthesis to global biogeochemical processes resulted in unprecedented efforts to study phytoplankton. While we now roughly assessed the whole ocean diversity at a genomic level, the main challenge is to understand the ocean diversity at the functional level. Investigating the diversity of cyclic electron flow within photosynthetic organisms is the main objective of my PhD. Our approach was driven by methodological considerations. We first determined the reliable tools that were available to probe CEF and then defined the biological questions we could address with these tools. I hope it will participate to the global effort on understanding the different microalgae assets and strategies to deal with environment constraints.

This thesis is divided in three chapters.

Chapter 2: Since cyclic electron flow is only involving photosystem I, investigating the former requires the accurate measurement of the latter. The first chapter of this manuscript aims at determining the best method to measure PSI quantum yield, and from there the electron flow through PSI. The most commonly used method is based on the absorbance changes of the PSI special pair, P700, depending on its redox state. Proposed 25 years ago, this method is based on a strong assumption that has never been tested. In order to test the domain of validity of this method, we describe an alternative method for measuring the activity of PSI which is based on probing the electric field generated across the thylakoid by PSI photochemistry.

Chapter 3: The second chapter is divided into two parts. First, I propose a simple and robust method based on the outputs of chapter 2 to highlight the presence of CEF, and if so, its relationship with LEF. Applying this method to several microalgae species, I could show three typical and distinct CEF behaviors. When CEF is independent of LEF, the measurement of its amplitude is simple: it is the residual activity when the LEF is inhibited. But when CEF depends on LEF, as it is the case for Chlamydomonas reinhardtii, CEF measurement is more complex and requires a more sophisticated approach. Thus, I performed an in depth analysis of LEF and CEF in the latter species using more complicated (and less robust)

How to probe photosystem I with absorption spectroscopy?

To investigate Cyclic Electron Flow around PSI in vivo in microalgae, the most powerful approach consists in comparing the activities of photosystem II, which participates in linear electron flow only, and photosystem I, involved in both linear and cyclic electron flows (Fan et al., 2016). It is also important to have robust measurement of the state of PSI to visualize the accumulation of stromal reductants downstream PSI or to get insights into PSI photoinhibition. In this first chapter, I will present the methodological work I have done to determine the best way to investigate PSI state and activity, through absorption difference spectroscopy.

Photosynthetic organisms absorb sunlight with an absorption spectrum that depends on the pigments that compose them. This absorption spectrum is slightly modified when it is transferred from darkness to light, i.e. whether photosynthetic activity is taking place or not.

These light-induced absorption changes are mostly due to the oxidation/reduction of some photosynthetic electron carriers and/or to the trans-thylakoidal electric field generated by photosynthesis, and can be used to our advantage for probing the photosynthetic process.

In this section, I will compare two methods based on absorption difference spectroscopy to measure the PSI conversion efficiency, or quantum yield. Since the maximal quantum yield of photosystem I is close to 1 (Nelson 2009), measuring the quantum yield of PSI is equivalent to measuring the fraction of PSI open centers. The aim is to determine the most accurate and robust approach to investigate PSI yield before we perform comparisons of PSII and PSI activities in chapter 3.

Some reminders about photosystem I

Photosystem I (PSI) operates at the end of the electron transfer chain and is a plastocyaninferredoxin oxydo-reductase: it uses the light energy to catalyze the endergonic reduction of the ferredoxins by the plastocyanins [START_REF] Nelson | Structure and function of photosystems I and II[END_REF][START_REF] Scheller | In vitro cyclic electron transport in barley thylakoids follows two independent pathways[END_REF]. PSI reaction center is a multiple subunit complex (Figure 2-1A) that contains non-proteic electron carriers: P700, A0 , A1, Fx, FA and FB (Figure 2-1A and B) that were first identified spectroscopically during the last half-century [START_REF] Brettel | Electron transfer and arrangement of the redox cofactors in photosystem I[END_REF]. Light energy enables photochemistry, which consists in the oxidation of P700, a pair of chlorophyll a called "special pair" (whose redox potential E'm = +430 mV; Kok, 1961;Sétif and Mathis, 1980), and the reduction of A0, a chlorophyll a monomer. Alternative models propose that P700 is not involved in the charge separation directly but instead in its stabilization (Holtzwarth et al., 2006;Li et al., 2006). The electron is then transferred to A1, a phylloquinone in most organisms, FX an interpolypeptide iron-sulfur center (4Fe-4S) (E'm ∼ -705 mV; Golbeck and Bryant, 1991), and finally to FA (E'm -520 mV; Golbeck, 1993) and FB (E'm -580 mV; Golbeck, 1993), which are (4Fe-4S) clusters bound to PsaC, an extrinsic PSI subunit. Oxidized ferredoxin can dock at the stromal side of PSI and get an electron from FB, thus stabilizing the charge separation.

Hence, a turnover of PSI results in three main consequences: the oxidation of P700, the reduction of iron-sulfur proteins and the transfer of a net positive charge from the stroma to the lumen. These changes, occurring after a photochemical event in PSI, also determine the signals potentially available to study the activity of this photosystem.

The reduction of iron-sulfur proteins and especially of the soluble ferredoxin could therefore be used to probe PSI activity but the absorption changes related to ferredoxin oxidation/reduction occurs in the near infra-red region, where they are difficult to deconvolute from other signals, mainly iron-sulfur clusters FA, FB and FX, plastocyanin and P700 contributions (Klughammer and Schreiber, 1991). Thanks to recent advances in the deconvolution of those signals [START_REF] Schreiber | Redox changes of ferredoxin, P700, and plastocyanin measured simultaneously in intact leaves[END_REF], in vivo measurements of ferredoxin redox state might be soon possible, but the methodology is not yet completely mature.

The other two available signals have been routinely used for decades and will be exploited in this chapter: (i) the oxidation of P700 can be assessed by measuring absorption changes in the near far red region and (ii) the electric field generated across the thylakoid membrane by PSI turnovers can be probed through Electrochromic Shift (ECS) signals. Those two methods are described in more detail below.

Chapter 2: Probing PSI activity

Suzanne FERTE -Doctoral Thesis -2019 Chlamydomonas reinhardtii [START_REF] Webber | Sitedirected mutations affecting the spectroscopic characteristics and midpoint potential of the primary donor in photosystem I[END_REF].

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 [START_REF] Drop | Light-harvesting complex II (LHCII) and its supramolecular organization in Chlamydomonas reinhardtii[END_REF]Hiyama and Ke 1972;[START_REF] Webber | Sitedirected mutations affecting the spectroscopic characteristics and midpoint potential of the primary donor in photosystem I[END_REF].

b) Signal deconvolution

We should note that the absorption difference signals in the near infrared region are not only due to P700 absorption changes. They also contain contributions from plastocyanin oxidation (which has a relatively flat spectrum), some local electric field indicating changes, ferredoxin reduction as well as iron-sulfur centers (Klughammer and Schreiber 1991). On top of that, an important fluorescence of chlorophylls can be artefactually measured in the near infra-red.

For this reason, obtaining the sole contribution related to P700 redox changes requires some deconvolution step. Two procedures are commonly used to deconvolute the P700 contribution from others.

-the first one is favored in plants and measures the difference between the absorption changes at 820 and 870 nm (e.g. [START_REF] Joliot | Cyclic electron flow in C3 plants[END_REF]Klughammer and Schreiber, 1994). This is the procedure used in the Walz systems, like the Dual-PAM-100 measuring system (Walz, Germany).

Chapter 2: Probing PSI activity

Suzanne FERTE -Doctoral Thesis -2019

-the second one is used in microalgae and measures the difference of absorbance changes at 705 and at 735/740 nm (e.g. Godaux et al. 2015;Nawrocki et al. 2019). This is the procedure used by default in the Joliot-type spectrometers JTS-10 or JTS-100 (Bio-Logic, Grenoble, France).

It is to note that the first procedure gives smaller signals but also minimizes the artefactual contribution of chlorophyll fluorescence. In this chapter, I always worked in conditions of fully inhibited PSII; the variable fluorescence from PSII is abolished and this is why I used 705-735 nm instead (Alric et al. 2010b).

c) Probing P700 redox state Regardless of the combination of wavelengths used to determine the absorption changes related to P700 oxidation/reduction, the determination of its absolute redox state also requires two absorption references: the absorption level when P700 is fully reduced and the absorption level when P700 is fully oxidized.

The maximum absorbance, when P700 is completely reduced, is obtained in the dark, when all the photosynthetic intermediates are in thermodynamic equilibrium with the ambient redox potential of the cell (P700 being a very strong oxidant, it is virtually fully reduced). At the opposite, reaching the situation where 100% of P700 are in the oxidized state is a lot more difficult, because this situation is way off equilibrium. This situation is usually attained when two requirements are met, (i) P700 oxidation rate is very high, under high light illumination and

(ii) the electron transfer towards PSI is slowed-down, making P700 reduction as slow as possible. This is usually obtained by inhibiting PSII with DCMU or by using specific PSI excitation (far red in plants). As mentioned in the manuscript and in the discussion of this chapter, the measurement of the absorption references corresponding to fully oxidized P700 is a difficult task. The fraction of [P700 + ] centers is noted Y(ND) hereafter.

-[P700] centers with a reduced primary donor, described as open centers (i.e. ready to transfer an electron).

The PSI yield, which is the fraction of absorbed photons converted into stable charge separation, was calculated as the fraction of open PSI, [P700] as shown in Figure 2-3 [START_REF] Foyer | Control of the quantum efficiencies of photosystems I and II, electron flow, and enzyme activation following dark-to-light transitions in pea leaves: relationship between NADP/NADPH ratios and NADP-malate dehydrogenase activation state[END_REF]Harbinson and Foyer 1991).

In 1994, Klughammer and Schreiber suggested that in some conditions, [P700] centers can be closed because their acceptor, called A hereafter and corresponding to all the PSI iron-suflur proteins [START_REF] Malkin | Primary reactions of photosynthesis: photoreduction of a bound chloroplast ferredoxin at low temperature as detected by EPR spectroscopy[END_REF]Oh-Oka et al., 1991), is reduced (Klughammer and Schreiber 1994). These The most straightforward method would be to use a single turnover laser flash, i.e. a flash of light high enough so that all PSI receive at least a photon and short enough so that all PSI cannot perform more than one charge separation. But because P700 reduction by plastocyanin (PC) is fast (compared to the time resolution of commercial devices) and falls in the same kinetics as charge recombination, the use of a single turnover laser flash to probe the fraction of open PSI is not possible. Indeed, the electron transfer from PC to P700 can occur with halftimes of ~5µs or ~200µs following P700 oxidation, if the plastocyanin is docked to PSI or needs to diffuse in the lumen, respectively (Hippler et al. 1997). This falls in the same timerange as the charge recombination between P700 + and A1 - (Warren et al. 1990). It is thus intrinsically impossible to discriminate between [P700 A] and [P700 A -] by the sole measurement of P700 absorption changes after a single turnover laser flash stimulation.

Klughammer and Schreiber suggested to use a few milliseconds saturating pulse of light instead (Klughammer and Schreiber, 1994). This pulse, whose intensity must be high enough to allow oxidation of all PSI secondary donors before a b6f turnover, is supposed to oxidize all P700 from [P700 A] centers (Figure 2-3). But this method is assuming a strong hypothesis: that

Electrochromic Shift: an internal voltmeter

As discussed before, the photochemistry of PSI generates an electric field across the thylakoid membrane because an electron is transferred from the luminal side (P700 / plastocyanin) to the stromal side (iron-sulfur centers / ferredoxin). Therefore, measuring the electric field across the thylakoid could, in principle, probe the activity of PSI. Such a measurement of the trans-thylakoid electric field exists and is based on the Stark effect on photosynthetic pigments. It is commonly called Electrochromic Shift (ECS) signal (Witt 1979). The principle of the method based on this signal is presented hereafter. When the photosynthetic chain is at work, photosystems transfer electrons from the lumen to the stroma and the electron transfer from water to NADPH is coupled to a translocation of protons from the stroma to the lumen. This results in a proton motive force which includes an electric field, , between the inside and the outside of the thylakoid. Some photosynthetic pigments embedded in the membrane are sensitive to this electric field.

As shown in the Figure 2-4A, in the presence of an electric field, the energy levels of the fundamental and excited states of a pigment can be modified. As a result, the energy of the photon which can be absorbed (whose energy is equal to the difference between excited and fundamental energy levels) by the pigment will be modified and the absorption spectrum of the latter will be shifted. This phenomenon is known as the Stark effect (Stark 1913). The spectrum of the "field indicating absorption change" corresponds to the difference between the absorption of the pigment in the presence of the electric field and the one in its absence, and typically displays a "double-wave" spectrum (Figure 2-4B). Such "field indicating absorption changes" were first reported in photosynthetic systems by Junge and Witt [START_REF] Junge | On the ion transport system of photosynthesis-Investigations on a molecular level[END_REF]) and give information about the electric field across photosynthetic membranes (Witt 1979).

The electrochromic shift can depend on the electric field in two different manners: it can increase linearly with the electric field (this is the case for a pigment with a permanent dipolar moment) or quadratically with the electric field (this is basically the case for a pigment with no permanent dipolar moment but a polarizability, i.e. its dipolar moment will be induced by the electric field itself). Such a quadratic electric field has been described in mutants of green algae [START_REF] Joliot | Characterization of linear and quadratic electrochromic probes in Chlorella sorokiniana and Chlamydomonas reinhardtii[END_REF], in diatoms (Bailleul et al., 2015) and other stramenopiles [START_REF] Berne | The peculiar NPQ regulation in the stramenopile Phaeomonas sp. challenges the xanthophyll cycle dogma[END_REF]. In Chlamydomonas reinhardtii, the species used in this chapter, the electrochromic signal increases linearly with the electric field generated across the photosynthetic membrane [START_REF] Joliot | Characterization of linear and quadratic electrochromic probes in Chlorella sorokiniana and Chlamydomonas reinhardtii[END_REF]. The amplitude of this signal will therefore directly measure the amplitude of the electric field created by the photosynthetic activity. Most photosynthetic eukaryotes possess ECS signals, and this is also true for cyanobacteria even though the signals are very small in the latter (Viola et al., 2019). However, the ECS spectra differ depending on the phylogenetic clade, indicating that the pigments sensitive to the electric field are different (Bailleul et al., 2010). The presence of ECS signals in most eukaryotic photosynthetic organisms make the approach followed in this chapter applicable to other species, as we will see in Chapter 3.

b) ECS signal deconvolution

The ECS spectrum of Chlamydomonas reinhartdii is presented in Figure 2345. ECS signal shows a maximum in the green region and we should note that ECS signal is not the only one contribution to light-induced absorption changes in this spectral region. There is also the oxidation/reduction of c-type cytochromes (like cytochrome f or cytochrome c6). Practically, in green algae like Chlamydomonas reinhardtii, as well as in plants, the ECS signal is measured as the difference between the absorption change at 520 nm and the one at 546 nm (e.g. [START_REF] Bujaldon | Functional accumulation of antenna proteins in chlorophyll b-less mutants of Chlamydomonas reinhardtii[END_REF]Buchert et al., 2017). The electrochromic signal allows many applications for the study of photosynthesis (reviewed in Bailleul et al., 2010). In this chapter, I will mostly focus on the flash-induced ECS signals, i.e. the measurement of the ECS signal after a single turnover laser flash. The first observations of ECS signals were performed with single turnover flashes [START_REF] Junge | On the ion transport system of photosynthesis-Investigations on a molecular level[END_REF]) and the ECS kinetics following the flash was thoroughly investigated first in green algae (Joliot and Delosme, 1974). Let's underline the role of the photosynthetic complexes in the implementation and dissipation of the electric field. The chloroplastic F1-F0 ATP synthase (CF1-F0) is the only photosynthetic complex which dissipates the electric field by transferring protons from the stroma to the lumen. The three other photosynthetic complexes, involved in the transfer of electrons from water to NADPH, set up the electric field with a yield of one positive charge transferred to the lumen per turnover:

-the activity of the photosystem II leads to the oxidation of water molecules and the release of a proton in the lumen per electron transferred (and the uptake of a proton on the stromal side to reduce a plastoquinone into plastoquinol); -the photosystem I transfers one electron from the luminal plastocyanins to the stromal ferredoxins;

Chapter 2: Probing PSI activity Suzanne FERTE -Doctoral Thesis -2019 -the cytochrome b6f translocates two protons in the lumen per electron transferred, assuming the Q cycle is always operating (Sacksteder et al., 2000). His net contribution is therefore one positive charge transferred to the lumen per turnover.

The photosystems are the sites of photochemistry and their activities are immediately modified by light perturbations whereas the activities of cytochrome b6f and ATP synthase, that are biochemical steps, are modified later after the light disturbance. Hence, after a single turnover flash on a dark-adapted sample, following the kinetics of ECS is a very powerful way to obtain a lot of information on the photosynthetic complexes (Joliot and Delosme, 1974).

The open PS will perform a charge separation right after the flash, giving rise to a first increase of the ECS called "a phase" (Figure 2-6B). At the end of this "a phase", a PSI donor has been oxidized and an electron has been transferred to the plastoquinol pool. This out-ofequilibrium situation is followed by the turnover of the cytochrome b6f, which transfers an electron from a plastoquinol to PSI donors and pumps protons concomitantly. This b6f contribution induces a second ECS rise known as "b phase". Finally, the electric field generated by those two phases is dissipated by ATP synthase until dark adapted state is reached again, and this third phase is known as "c phase". Given the aim of this chapter, we need to distinguish between the contributions of PSII and PSI. PSII can be inhibited by the addition of two inhibitors. The first one is 3-(3,4dichlorophenyl)-1,1-dimethylurea (DCMU) which competes with PQ for the QB pocket of PSII and inhibits electron transfer from QA to the plastoquinone pool. In the presence of DCMU, one charge separation can still occur in PSII (from P680 to QA) and this is why we add hydroxylamine, an effective P680 electron donor that blocks irreversibly PSII in a [P680 QA -] state, unable to perform any charge separation. This can be easily checked by using chlorophyll fluorescence: there should not be any variable fluorescence left once PSII is fully inhibited with DCMU and HA. In these conditions, only PSI participates to the generation of electric field and the fraction of open PSI centers can be calculated as discussed before.

The "a phase" can also be used to calculate the ratio between the functional PSII and PSI and the principle is schematized in Figure 2

Article: Critical reappraisal of methods to measure photosystem I activity

Introduction

In oxygenic photosynthesis, the photosynthetic electron transfer chain comprises two photosystems. Photosystem II (PSII) and photosystem I (PSI) work in series to allow linear transfer of electrons generated from the oxidation of water in PSII to reduce NADP + into nicotinamide adenine dinucleotide phosphate (NADPH) downstream of PSI. The electron transfer from PSII to PSI involves the plastoquinone pool diffusing in the thylakoid membrane, the lumen-soluble plastocyanins (or cytochrome c6) and a plastoquinol-plastocyanin oxidoreductase called cytochrome b6f (cyt b6f). The electron transfer is coupled to the translocation of protons across the thylakoid membrane, giving rise to an electrochemical proton gradient or proton motive force (pmf). The pmf comprises both an electric (Δψ) and an osmotic (ΔpH) component and fuels the synthesis of adenosine triphosphate (ATP) by the CF1-F0 ATP synthase. The methods used to measure the linear electron flow from water to NADPH mostly rely on measurements of PSII because it is the most convenient photosynthetic complex to probe. The PSII chlorophyll fluorescence yield is high enough to allow the energy conversion in PSII to be calculated (Kautsky et al., 1960;Duysens, 1963;reviewed in Baker, 2008). The oxidation of water, the final electron donor, occurs in the oxygen evolving complex of PSII so it is also possible to measure O2 evolution as a powerful alternative assay.

Fluorescence and oxygen-based methodologies have been cross-validated (Genty et al., 1992) and are now well established and widely used by the community of photosynthesis researchers. However, measuring PSII activity alone is not sufficient to view the complexity of photosynthetic function and regulatory flexibility. For instance, in addition to the linear electron flow involving the two photosystems, several modes of alternative electron flow can play an important role in providing some flexibility in terms of ATP/NADPH ratios (Eberhard et al., 2008). In cyanobacteria, terrestrial plants and green algae, it has been shown that electron transfer from reduced PSI acceptors back to the inter-photosystem chain at the plastoquinone pool or at the b6f level can occur, allowing PSI to operate independently from PSII [START_REF] Arnon | Assimilatory power in photosynthesis[END_REF]; reviewed thoroughly in Bendall andManasse, 1995, andin Shikanai, 2007). This gives rise to a cyclic electron flow (CEF) around PSI, which is thought to play a crucial role in photosynthetic regulation by increasing the ATP/NADPH balance (Allen, 2002).

Since CEF does not produce any quantifiable net product, the most accurate way to measure CEF in physiological conditions is to compare PSII and PSI activities (Fan et al., 2016). The CEF participates, together with other alternative electron flows, in the protection of both photosystems under excess light by regulating cyt b6f and PSII activities (Endo and Asada, 2008;Chaux et al., 2015). The over-reduction of PSI acceptors when the Calvin-Benson-Bassham cycle performs poorly (as in conditions like chilling or low CO2 availability) results in severe photo-inhibition of PSI (Havaux and Davaud, 1994;Terashima et al., 1994;reviewed in Sonoike, 2011 and[START_REF] Scheller | In vitro cyclic electron transport in barley thylakoids follows two independent pathways[END_REF][START_REF] Scheller | Photoinhibition of photosystem I[END_REF] through the oxidative destruction of the iron-sulfur clusters (Sonoike et al., 1995). Photosynthetic organisms can respond to this harmful situation by slowing down cyt b6f activity, through "photosynthetic control" (Rumberg and Siggel, 1969;Foyer et al, 2012;Joliot and Johnson, 2011). To fully understand the complexity of photosynthetic regulation by investigating this important process requires measurements of the PSI energy conversion efficiency, and of its acceptor-side and donorside limitations, i.e. the loss of PSI efficiency due to lack of PSI acceptors or donors, respectively. However, measurements of PSI activity are not as straightforward as for PSII.

Measuring PSI yield with pulse-induced P700 absorption changes PSI is a protein complex embedded in the thylakoid membrane which catalyzes the electron transfer from the special pair P700 to the iron-sulfur [4Fe-4S] centers and to the stromal soluble electron carrier [2Fe-2S] ferredoxin [START_REF] Golbeck | Photosystem I: the light-driven plastocyanin: ferredoxin oxidoreductase[END_REF]. In 1972, Hiyama and Ke showed in vitro that the absorption spectrum of P700 changes depending on its redox state (Hiyama and Ke, 1972). The same is true in vivo and absorption variation at 705 and 820 nm are mainly due to P700 redox state changes (Harbinson and Woodward, 1987). Historically, this property has been used to differentiate between the fractions of PSI with a reduced or oxidized P700 that were originally considered as open and closed centers, respectively (Harbinson et al., 1989;Harbinson and Foyer, 1991). Because of the high maximal quantum yield of PSI, close to 1 (Nelson, 2009), the fraction of open PSI centers can be considered as the quantum yield of PSI. In 1994, Klughammer and Schreiber suggested that in some conditions, PSI centers may be closed even though they have a reduced P700, because of over-reduction of the PSI acceptor. In these conditions, the fraction of open PSI measured with the Harbinson and Foyer method was overestimated. An advanced method was thus proposed based on P700 absorption changes in response to a multiple turnover light pulse (Klughammer and Schreiber, 1994). The rationale behind this method is based on the fact that PSI can be found in three different redox states. Two PSI cofactors are involved in the first stable charge separation: P700 and the first stable acceptor, called A hereafter, which corresponds to the PSI iron-suflur proteins [START_REF] Malkin | Primary reactions of photosynthesis: photoreduction of a bound chloroplast ferredoxin at low temperature as detected by EPR spectroscopy[END_REF]Oh-Oka et al., 1991). To perform a stable charge separation, defined here as transferring an electron from P700 to A, a PSI center needs P700 to be reduced and A to be oxidized. These [P700 A] PSI centers are thus considered to be open as they are ready to perform a charge separation as soon as the next photon is absorbed.

Accordingly, centers in which both P700 and A are oxidized, denoted [P700 + A], or in which both P700 and A are reduced, [P700 A -], are considered to be closed, as they will not be able to

An internal contradiction in the P700 pulse method

For all photo-oxidizable P700 to be oxidised despite being rapidly reduced by plastocyanin or other secondary donors like cytochrome c6 (t1/2 ≈ 5 µs if the secondary donor is already fixed, otherwise t1/2 ≈ 200 µs, Hippler et al., 1997), according to Klughammer and Schreiber "the light pulses must be sufficiently long to allow several PSI turnovers, such that the immediate donor pool is emptied during a pulse". When all secondary donors are oxidized, the P700 reduction rate does indeed become limited by the rate of electron transfer through cyt b6f (t1/2 ≈ 10 ms), slowing down the re-reduction of P700 by 2 to 3 orders of magnitude. A sufficiently strong light pulse would therefore allow all photo-oxidizable P700 to be probed.

Chapter 2: Probing PSI activity

Suzanne FERTE -Doctoral Thesis -2019 However, an explicit assumption of this method is that "at least during the early phase of a saturation pulse, the oxidation of the donor side is more pronounced than the reduction at the acceptor side. For this reason, it appears unlikely that a reaction center in the state P700 A is converted into the state P700 A -during the first milliseconds of a saturating light pulse". These two hypotheses are clearly contradictory. PSI photochemistry and charge conservation dictate that electrons extracted on the donor side are transferred to the acceptor side. If several photochemical events are allowed to oxidize several secondary donors, then several electrons will be transferred through PSI, modifying the redox state of the acceptors. For this reason, we are concerned that the pulse-method could overestimate the acceptor-side limitation, Y(NA)P700 > Y(NA), and underestimate the fraction of open PSI centers Y(I)P700 < Y(I), when the pool of PSI donors is large.

Electro-chromic shift as an alternative method to measure PSI yield

Why not use a single turnover flash instead of a multiple turnover pulse? The single turnover flash, which can be produced with a laser, is routinely favored in photophysiology studies (e.g. Junesch and Graber, 1987;Joliot and Delosme, 1974). It would have the advantage of allowing all open PSI to perform one -and only one -charge separation without perturbing the state of the acceptor side. Unfortunately, the charge recombination between P700 + and A -, when A (the pool of iron sulfur clusters) is reduced, has the same kinetics as the electron transfer from a secondary donor to P700 + (Warren et al., 1990). Both processes will give the same output in terms of the amplitude of the flash-induced P700 oxidation and/or its relaxation kinetics, making P700 unsuited as a variable for distinguishing between open centers and acceptor-side limited centers after a single turnover flash. However, there is at least one clear difference between a charge separation stabilized by the electron transfer from secondary donors to P700 and a charge separation followed by a charge recombination: in the first case, a positive charge remains in the lumen, but in the second case the charge is cancelled. The electric field across the thylakoid can be probed in vivo by monitoring the electro-chromic shift (ECS) of the photosynthetic pigments (Witt, 1979;Bailleul et al., 2010). This phenomenon which is a special case of the Stark effect (Stark, 1914) Despite the inherent contradiction mentioned earlier, the P700 pulse method has been widely used to investigate many aspects of photosynthesis, e.g. PSI photo-inhibition (e.g. Brestic et al., 2015;[START_REF] Zivcak | Repetitive light pulseinduced photoinhibition of photosystem I severely affects CO2 assimilation and photoprotection in wheat leaves[END_REF], photosynthetic control (e.g. Weis et al., 1987), ferredoxin oxidation pathways (e.g. Voss et al., 2008), responses to cold temperatures (e.g. Suzuki et al., 2011;Wang et al., 2013) or cyclic electron flow (e.g. [START_REF] Laisk | Fast cyclic electron transport around photosystem I in leaves under far-red light: a proton-uncoupled pathway?[END_REF]Talts et al., 2007;DalCorso et al., 2008;[START_REF] Kono | Roles of the cyclic electron flow around PSI (CEF-PSI) and O2dependent alternative pathways in regulation of the photosynthetic electron flow in short-term fluctuating light in Arabidopsis thaliana[END_REF][START_REF] Kou | Partially dissecting the steady-state electron fluxes in Photosystem I in wild-type and pgr5 and ndh mutants of Arabidopsis[END_REF]. Commercial measurement devices have been developed on this basis, like the Dual-Pam distributed by Walz (Germany) and the P700 pulse method has become the standard methodology to probe PSI activity. The authors of the original paper acknowledged that since "an alternative, proven method for ΦI determination is lacking, only indirect evidence for the correctness and general applicability of the proposed approach can presently be given" (Klughammer and Schreiber, 1994). We believe that measuring the stable electric field (stabilized charge separation) induced by a single turnover flash with ECS is such an alternative method to probe the fraction of open centers. It has the advantage of clearly distinguishing charge recombination events occurring in acceptor-side limited centers (the electric field relaxes in hundreds of µs) and stabilized charge separation in open centers (the electric field remains stable). In this work, we have compared estimates of the fraction of PSI open centers by measuring P700 oxidation after a multiple turnover pulse and ECS generated by a single turnover flash. We observed that whenever a significant Y(NA)P700 is measured at the end of the pulse, the P700 pulse method overestimates the acceptor-side limitation and therefore underestimates the fraction of PSI open centers by up to ten-fold in our dataset. We show that the overestimation is proportional to (1-Y(NA)P700) as expected based on theoretical calculations, and a simple formula can therefore be applied to the P700 pulse method to correct measurements of Y(I). We propose that potentially erroneous literature on photosynthetic regulation is reappraised and corrected.

Material and methods

Strains, growth and sampling. Chlamydomonas reinhardtii wild type laboratory strain T222 and the pc3.1 + mutant strain were obtained from the Chlamystation (http://chlamystation.free.fr/). Arabidopsis thaliana Col-0 was a kind gift from Dr Fredy Barneche and Menuu Singla (Ecole Normale Supérieure, Paris, France). The dinoflagellate Amphidinium carterae (RCC1522) was obtained from the Roscoff Culture Collection (http://roscoff-culture-collection.org/). Chlamydomonas cells were grown in Tris-acetate-

Chapter 2: Probing PSI activity

Suzanne FERTE -Doctoral Thesis -2019 phosphate (TAP) medium (Harris, 2009) at 25°C under continuous light provided by white LEDs and were harvested when the cell density was between 2 and 6 million cells per mL. The light irradiance was 30 µmol photons m -2 s -1 for the wild type and 10 µmol photons m -2 s -1 for the light-sensitive pc3.1 + . A. thaliana was grown at 22°C with an 8-hr light/16-hr dark photoperiod at a light intensity of 100 µmol photons m -2 s -1 . Leaves were harvested at the rosette stage ( 5weeks after germination). Am. carterae was grown in enriched seawater/artificial water (Berges, Franklin, and Harrison 2001) at 19°C under a 12-hr photoperiod at a light intensity of 30 µE m -2 s -1 using fluorescent tubes. Cells were harvested in the exponential growth phase when the cell density was between 80 000 and 200 000 cells per mL.

For photosynthetic measurements, liquid cultures of C. reinhardtii or Am. carterae were concentrated by centrifugation (4 min at 3500 rpm) and resuspended in their supernatant to a density of ~10 7 or 10 6 cells mL -1 , respectively. Ficoll® was added at 5 to 10 % (volume-tovolume) to prevent drifts in the absorption measurements due to sedimentation of the cells. Concentrated samples were then stirred at 350 rpm under 10 µE m -2 s -1 white light for at least 30 min before starting measurements.

Inhibitors/chemicals. Hydroxylamine (HA), (3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), methylviologen (MV), dibromo-6-isopropyl-3-methyl-1,4-benzoquinone (DBMIB), glucose and glucose oxidase were obtained from Sigma Aldrich. DCMU and DBMIB were diluted in ethanol.

HA, MV, glucose and glucose oxidase were diluted in distilled water. PSII was inhibited with 10 µM DCMU and 100 to 500 µM HA (for each experiment, the minimal concentration to reach maximal fluorescence in the dark was determined and used). When necessary, anoxic conditions were induced by addition of 20 mM glucose and glucose oxidase (~280 U mL -1 ) in brimful, tightly closed cuvettes. MV at a concentration of 4 mM was added to the microalgae sample or infiltrated into leaves 4 min before measurements to allow it to fully infiltrate the stroma. To compare leaf samples with and without MV, we used two pieces of the same leaf, sampled symmetrically across the leaf axis. MV did not seem to release acceptor-side limitation in the dinoflagellate Am. carterae.

In vivo spectroscopy. All spectroscopic measurements were performed using a Joliot Type Spectrophotometer (JTS10, Biologic, Grenoble, France) so fluorescence and absorption difference were measured on the same sample. The actinic continuous light and the saturating pulse (5000 µmol photons m -2 s -1 , corresponding to ~2200 photons per PSI per second) were provided by orange LEDs (630 nm) and the saturating laser flash was provided by a dye laser (690 nm) pumped with a Nd:YAG laser (6 ns duration, 532 nm).

For fluorescence, detection pulses were provided by a white LED with a blue filter (470 nm).

Reference and measuring photodiodes were protected from actinic light with a BG39 Schott filter (Mainz, Germany) and an LPF650+RG665 Schott filter, respectively. PSII maximal quantum yield was calculated as Fv/Fm = (Fm -F0)/Fm, where F0 is the fluorescence level in the dark-adapted (for 1 min) sample and Fm is the maximal fluorescence levels obtained after a saturating light pulse in dark-adapted sample. These measurements were performed to verify that after DCMU and HA treatment, all PSII centers were closed, i.e. Fv/Fm = 0.

To correct for artefacts, all data shown for absorption change measurements (both P700 and ECS) are the differences between the measurement in the presence of the actinic light (laser flash, background actinic light or saturating pulse) and the measurement in the absence of actinic light. P700 redox changes were measured as the difference in the absorbance changes at 705 and 735 nm to correct for contributions from scattering and plastocyanin absorption changes. Usually, the 810-870 nm region is preferred for probing P700 to minimize artefacts due to changes in the fluorescence yields. This was not the case for the protocol used here as we used DCMU and HA to fully close all PSII centers. In these conditions, we found that 705-735 nm and 810-870 nm gave similar results (not shown) but we chose the near far-red region because the signal-to-noise ratios are better. The light-detecting diodes were protected from scattered actinic light with an RG695 Schott filter (Mainz, Germany). We calculated Y(I)P700, Y(NA)P700 and Y(ND) as described in Klughammer and Schreiber (1994). To normalize the output, we realized that the maximal absorption change corresponding to the oxidation of 100% of P700 was not reached with common protocols described in the literature (i.e. using far-red light or a saturating DCMU concentration). The absorption change corresponding to 100% P700 oxidation was reached by adding a saturating laser flash to a few ms of a saturating pulse in the presence of the cyt b6f inhibitor DBMIB (1 µM) and the PSI electron acceptor MV.

Electro-chromic shift (ECS) signals were measured as the difference in the absorbance changes at 520 and 546 nm for C. reinhardtii and A. thaliana or at 563 nm for Am. carterae. These wavelengths were chosen to eliminate absorption changes related to c-type cytochromes. The detection wavelengths were provided by a white LED with appropriate interferential filters (10 nm full width at half-maximum, Edmund Optics). The measuring and reference photodiodes were protected from scattered and transmitted actinic light by a BG39 Schott filter (Mainz, Germany). All ECS data were normalized to the ECS increase produced by a

Results

We aimed to test the validity of the P700 pulse method by comparing it to an alternative ECSbased method. The principles by which the two methods can be used to estimate the fraction of open PSI centers are presented in Fig. 1A (see also Methods). For initial validation studies, we decided to use the green alga Chlamydomonas reinhardtii instead of higher plant leaves, because it is easier to use chemicals with liquid cell cultures as samples are more homogeneous and results readily reproducible. In all experiments, (3,4-dichlorophenyl)-1,1dimethylurea (DCMU) and hydroxylamine (HA) were added to the cells in order to fully inhibit PSII. In these conditions the flash-induced ECS only reflects the contribution from PSI. We first varied the fraction of open PSI centers, or PSI yield, by modulating the light intensity. In these conditions, the PSI yield is determined by the rate of photon absorption by PSI and the rate of the CEF around PSI. After photosynthesis reached its steady-state rate, we measured the PSI yield with the ECS-based method, Y(I)ECS, and with the P700 pulse method, Y(I)P700. As expected, the PSI yield measured with both methods (see Methods) decreased with increasing light irradiance, reflecting the fact that CEF saturates under relatively low irradiance, but not in the first two irradiance conditions for Y(I)P700 (Fig. 1 B-C).

Discrepancy between ECS and P700 based measurements of PSI yield

Results from the two methods were plotted against each other (Figure 2A). In most light conditions, the two methods produce the same results. The notable exceptions are the first two irradiance conditions, i.e. in the dark (see also Figure 1A intensities of 26 µmol photons m -2 s -1 and higher, no discrepancy was measured once a steady state was achieved, i.e. after at least 3 minutes of illumination (Figure 2A). However, even under light intensities for which the two methods produce similar results in the steady state, a discrepancy was observed at the dark-to-light transition. Figure 2C shows how Y(I)ECS and Y(I)P700, initially as in the dark-adapted situation as described above, change at the onset of irradiance at 800 µmol photons m -2 s -1 . During the transition from dark to light, the PSI yield measured by P700 is always lower than the one measured with ECS (Figure 2C).

The discrepancy increases with the level of reduction of PSI acceptors at the end of the pulse

In the absence of acceptor-side limited centers, a situation which can be reached by using the artificial PSI acceptor methylviologen (MV), all reduced P700 are photo-oxidizable. In these conditions, Y(I) should be equal to the fraction of reduced P700 and should be correctly assessed with the P700 pulse method. As expected, in the presence of MV the two methods give the same results (Figure 1A and Figure 2B). In the presence of MV, since electrons are efficiently rerouted towards O2 on the acceptor side of PSI (Izawa, 1980), CEF is significantly slowed down (Fan et al., 2007). This results in a similar PSI yield for a light intensity 10 times lower (compare middle and right panels in Figure 1A). The agreement between P700 and ECS estimations of PSI yield in the absence of acceptor-side limitation was also observed recently in cyanobacteria using phenazine methosulfate as a PSI electron acceptor (Figure 3 in Viola et al., 2019). This consistency between results strongly suggests that there is no intrinsic error or artefact in the methods used, and that the discrepancy in dark and low light or during dark to light transitions is related to the presence of reduced PSI acceptors. To further confirm the causal link between the discrepancy and the level of reduced PSI acceptors, we took the opposite approach by creating conditions in which the acceptor-side limitation is known to be especially high. In anoxic conditions, the reductants produced by glycolysis and the Krebs cycle cannot be re-oxidized by the mitochondrial respiration chain. This reducing power diffuses in the cell via membrane shuttles and results in a reducing pressure in the chloroplast stroma (Hoefnagel et al., 1998). What is more, chloroplast oxidases, like plastid terminal oxidase (Bennoun, 1983;Houilles-Verne et al., 2011;Nawroki et al., 2019) or flavodirion proteins (Chaux et al., 2017), cannot deal with this reducing pressure in the absence of their substrate O2. Consequently, anoxia leads to enhanced reducing pressure on PSI acceptors. In dark-adapted anoxic cells, the discrepancy was even higher than in oxic conditions as the Y(I)ECS remained equal to 1 (Figure 3A andC) but the Y(I)P700 decreased to a value of ~0. 3 (Fig 3A and 3D). Again, we compared Y(I)ECS and Y(I)P700 in 3 as given by the P700 pulse method, or 1 as ECS indicates? We can answer this question using positive apagogy, or reductio ad absurdum. Imagine that the correct value is the one given by Y(I)P700 and that ECS method overestimates the PSI yield due to an artefact (e.g. charge recombination not being finished at the time of detection). In that case, after a saturating laser flash, only 30% of PSI will perform a stable charge separation, which will in turn oxidize 0.3 PSI donors (plastocyanin or cytochrome f). After the charge separation is complete, the subsequent turnover of cyt b6f will reduce the PSI donors, giving rise to a second ECS rise, the b phase (Joliot and Delosme, 1974;Bailleul et al., 2010). As cyt b6f cannot transfer more electrons than the number of oxidized luminal carriers (i.e. 0.3 electrons), the b phase cannot exceed the ECS increase induced by PSI photochemistry. Figure 3C shows the flash-induced ECS kinetics in C. reinhardtii, with the characteristic a phase (PSI photochemistry here), b phase (cyt b6f contribution) and decay phase (due to pmf consumption by CF1F0 ATP synthase). The value of the ECS at the end of the b phase is 2 ± 0.1 charge separations per PSI, very far from the expected value (~0.6) if only 30% of PSI were open. So this scenario is incompatible with our initial hypothesis. If the initial hypothesis is wrong, we conclude that the P700 based method does not give a good estimate of the PSI yield.

Moreover, an amplitude of 1 for the b phase is exactly what is expected if all PSI are open and perform a stable charge separation after a saturating laser flash as indicated by the ECS-based method. It is to note that such a 1:1 ratio between a and b phases is met in anoxic conditions because (i) the reducing pressure in the chloroplast ensures that the plastoquinone pool is almost fully reduced [START_REF] Nawrocki | The plastid terminal oxidase: its elusive function points to multiple contributions to plastid physiology[END_REF] providing enough substrate for cyt b6f, and (ii) the activity of the CF1F0 ATPase is slowed down sufficiently to ensure that the b phase is not covered by the CF1F0 related ECS decay (Buchert et al., 2017). Finally, the b phase was suppressed in the presence of the cyt b6f inhibitor DBMIB, confirming that this second rise in ECS does indeed correspond to the b6f -catalyzed pumping of protons. We conclude that it is not the Y(I)ECS which over-estimates the fraction of open PSI centers, but that Y(I)P700 underestimates it. The underestimation by the P700 pulse method stems from the use of the multiple turnover pulse The P700 pulse method underestimates PSI yield at the expense of Y(NA)P700 which indicates that the open [P700 A] and acceptor-side limited [P700 A -] centers are not distinguished. As postulated in the Introduction, this might be the result of using a multiple turnover pulse. If several charge separations are necessary before reaching the maximal oxidation of P700, then several electrons will be transferred to the PSI acceptor side. The fraction of closed PSI centers due to reduction of the acceptor, [P700 A -], at the end of the pulse will be overestimated compared to before the pulse. Therefore, the measured Y(I)P700 will be lower than the actual PSI yield, Y(I). The number of charge separations can be measured by monitoring the electric field or ECS generated during the multiple turnover pulse and comparing it to the one generated by one saturating laser flash. In dark-adapted oxic Chlamydomonas, 4 PSI turnovers occur during the pulse before maximal P700 oxidation is reached (Figure 4B) which corresponds to 50% of P700 (Figure 4A andB). The 4 electrons transferred might have reduced the acceptor side of PSI and generated some [P700 A -] centers, preventing the full oxidation of P700. When tested in the presence of MV, one more charge separation can occur (Figure 4C) and this allows almost all P700 to be oxidized during the pulse (Figure 4A andB). As a control, we used a plastocyanin mutant which can only perform one charge separation because its P700 cannot be reduced. In this case, the pulse-induced ECS increase confirms that only one charge separation occurs (Figure 4C), that all P700 is oxidized during the pulse (Figure 4A andB), and that the ECS and P700 methods give the same result (Figure 4A). This demonstrates that the P700 pulse bias comes from the use of a multiple turnover pulse, which makes the fraction of [P700 A -] at the end of the pulse higher than that before the pulse.

We investigated whether the discrepancy measured in C. reinhardtii is also observed in other photosynthetic species. In the dark, the fraction of open PSI centers should not depend on the activity of other photosynthetic complexes, so we could compare different strains and species, independently modifying the donor and acceptor pool size by adding chemicals. We measured Y(I)ECS and Y(I)P700 in dark-adapted leaves of Arabidopsis thaliana and in the dinoflagellate Amphidinium carterae. In both cases, we observed the same trend: Y(I)ECS was higher than Y(I)P700. Like in C. reinhardtii, there was no discrepancy in A. thaliana in the presence of MV (Figure 4). Equivalent data with MV in the dinoflagellate Am. carterae was not obtained because we did not detect any effect of MV in this species. there is a good agreement between the two methods (darker blue points), whereas all the situations with a discrepancy between the two methods correspond to high Y(NA)P700 (darker red points). This is true for the measurements in aerobic steady-state illumination (Supplementary Figure 1A), where only the dark and 3 µmol photons m -2 s -1 points show a significant Y(NA)P700 and a discrepancy between the two methods. When light is switched on, both in oxic and anoxic conditions, the discrepancy decreases with time concomitant with a decrease of Y(NA)P700 (Supplementary Figure 2A and3). This strongly supports our initial concern that the multiple turnover pulse over-reduces PSI acceptors during the pulse, leading to an overestimation of the acceptor-side limitation at the expense of the PSI yield. In agreement with this, both the Y(NA)P700 and the discrepancy are annulled with the addition of MV (Supplementary Fig 1B and2B).

Rationalizing the PSI yield underestimation by the P700 pulse method

Can we rationalize the degree by which PSI yield is underestimated when a multiple turnover pulse is used in the P700 method and hence correct for the inherent bias? For this, it is necessary to return to the internal contradiction enounced in the Introduction. In the original description of the K&S method, the authors state that a multiple turnover pulse is necessary to oxidize all PSI secondary donors and P700 (Klughammer and Schreiber, 1994). What happens during the pulse? The hypothesis was that all initially open centers become donor-side limited during the pulse, i.e. no PSI are closed on the acceptor side during the pulse. This is very unlikely in conditions where the activity of PSI is strongly limited by a downhill step, i.e. when the initial pool of acceptors is small and/or the initial pool of PSI donors is large. As demonstrated before, this assumption proves wrong. Another extreme case scenario is to consider that the pulse generates only acceptor-side limited centers, i.e. the amount of donorside limited centers is unchanged. This would be extremely unlikely in conditions where the activity of PSI is strongly limited by an uphill step, i.e. the donor-side limitation [P700 + A] is high.

So what kind of reasonable assumption can one make regarding the amount of acceptor-side and donor-side limited centers that the pulse will generate? We propose that the most parsimonious hypothesis is to consider that the ratio of acceptor-side to donor-side limited centers at the end of the pulse will be equal to the ratio preceding the pulse.

Discussion

Comparing the PSI parameters estimated based on the P700 pulse method with an alternative approach based on ECS allowed us to demonstrate that the P700 pulse method underestimates the PSI yield when a significant Y(NA)P700 is measured, i.e. when there is an acceptor-side limitation at the end of the pulse. The underestimation went as high as a factor 10 in our dataset (Figure 5B). When a multiple turnover pulse is used and the initial pool of PSI donors is large, several electrons will be transferred to the PSI acceptors during the pulse. The amount by which the PSI yield is underestimated is proportional to Y(NA)P700, a dependency that we were able to predict with the following simple assumption. The multiple turnover pulse generates both acceptor-side and donor-side limited PSI centers at a ratio which remains the same as before the pulse. This assumption simply means that the "capacity" for oxidation of PSI acceptors and the "capacity" for reduction of PSI donors do not change within the few ms of the pulse.

Early approaches to assessing PSI yield based on P700 measurements considered that all PSI with reduced P700 were open (Harbinson and Foyer, 1991). The P700 pulse method was developed as an improvement on those approaches, allowing open and acceptor-side limited PSI centers to be distinguished. In conditions where all P700 are photo-oxidizable, i.e. Y(NA) is 0, both approaches prove right. As soon as there is a fraction of acceptor-side limited centers, both methods prove wrong and the K&S method cannot be considered as an improvement on the Harbinson and Foyer (H&F) method. This can be clearly visualized in Figure 5D, which displays the relative error of Y(I) estimated by the P700 pulse method and by the H&F method, as a function of the true acceptor-side limitation, i.e. the fraction of [P700 A -] centers or Y(NA).

In both cases, the relative error is proportional to the acceptor-side limitation, but the H&F method naturally overestimates Y(I) whereas the K&S method underestimates it. The slope depends on the (true) PSI yield. For a Y(I) of 0.5, the relative errors made by the H&F and K&S methods mirror each other. However, a major difference is that the dependency between the error and the acceptor-side limitation increases with Y(I) in the case of K&S method (the slope is (1-Y(I)) -1 , see Appendix 1), but decreases with Y(I) in the case of H&F method (the slope is Y(I) -1 , see Appendix 1).

The good news is that as the underestimation of the PSI yield with the P700 pulse method is proportional to the Y(NA)P700 a correction can be made. The correct value for Y(I) can be calculated from the parameters derived from the P700 pulse method: Y(I) = Y(I)P700 / (1-Y(NA)P700). When such correction is done, the comparison between the corrected PSI yield 4), except for the data points for which the Y(NA)P700 is higher than 0.8. For those data points, the correction is very prone to small errors as both Y(I)P700 and (1-Y(NA)P700) are small numbers.

Revisiting 25 years of research base on the P700 pule method, covering many aspects of photosynthesis, will be a challenge. The values of PSI yield measured in dark-adapted photosynthetic samples are good examples of the errors that have been recorded and propagated in the literature. It is clear that given the ambient redox potential in a darkadapted chloroplast, P700 should be fully reduced (the redox potential of P700 being 430 mV, Kok, 1961;Sétif and Mathis, 1980) and A should be fully oxidized (the redox potential of ironsulfur centers being in the -520 to -705 mV range, (Golbeck and Bryant, 1991;Golbeck, 1993).

Therefore the PSI yield in the dark should be equal to 1. This is not what the P700 pulse method shows. We obtained Y(I)P700 values of ~0.8 in A. thaliana leaves, ~0.5 in C. reinhardtii and ~0.6 in Am. carterae. In the literature, Y(I)P700 is 0.8 in dark-adapted A. thaliana leaves (Kono et al., 2018), 0.8-0.9 in sunflower leaves (Klughammer and Schreiber, 1994;[START_REF] Sejima | Repetitive short-pulse light mainly inactivates photosystem I in sunflower leaves[END_REF], 0.7 in barley leaves [START_REF] Pfündel | Monitoring the effects of reduced PS II antenna size on quantum yields of photosystems I and II using the Dual-PAM-100 measuring system[END_REF], 0.5 in pea leaves [START_REF] Schansker | Characterization of the 820nm transmission signal paralleling the chlorophyll a fluorescence rise (OJIP) in pea leaves[END_REF], 0.8 in wheat leaves (Brestic et al., 2015;[START_REF] Zivcak | Repetitive light pulseinduced photoinhibition of photosystem I severely affects CO2 assimilation and photoprotection in wheat leaves[END_REF] The literature regarding CEF, and especially the deduced role of the A. thaliana proton gradient regulator 5 (PGR5) and proton gradient regulator-like 1 (PGRL1) proteins in this process, might have also been strongly affected by the errors inherent in the P700 pulse method. Indeed, one of the main arguments in favor of a role of these proteins in CEF comes from the measurement of a lower Y(I)P700 and a higher Y(NA)P700 in the pgrl1 and pgr5 mutants compared to the wild type (e.g. DalCorso et al., 2008;[START_REF] Kono | Roles of the cyclic electron flow around PSI (CEF-PSI) and O2dependent alternative pathways in regulation of the photosynthetic electron flow in short-term fluctuating light in Arabidopsis thaliana[END_REF][START_REF] Kou | Partially dissecting the steady-state electron fluxes in Photosystem I in wild-type and pgr5 and ndh mutants of Arabidopsis[END_REF].

These observations have been interpreted as the signature of a lower CEF rate, leading to acceptor-side limitation. The limitations of the K&S method demonstrated here invalidate this conclusion. It would be wise to reappraise the literature on CEF in a timely way and correct any relevant data as we propose here. Going forward this would increase the robustness and precision of experimental data and give a more accurate view of how the light phase of photosynthesis is regulated in all its diversity.

Chapter 2: Probing PSI activity

Suzanne FERTE -Doctoral Thesis -2019

Discussion

In the manuscript presented above, we have shown evidences that the P700 pulse method developed by Klughammer and Schreiber 25 years ago is not valid, and we proposed an alternative calculation for PSI parameters using the same experimental parameters.

Here, I would like first to summarize our findings, add some important points that were not addressed because of space limitation and give supplementary support to our conclusions.

Second, I would like to discuss the difficult technical aspects of the P700 method. Indeed, assuming that the correction we propose is valid and gives correct estimates of PSI parameters, it is nevertheless necessary to have good measurements of the experimental parameters related to PSI state. I will come back to some important methodological aspects.

Third, all the data shown in this manuscript were obtained in conditions of PSII inhibition for the simple reason that it was convenient to measure an ECS signal which depends only on PSI.

One could wonder if this induces a bias; it is important to show that the correction we propose still holds when PSII is active because it is the case in most of the publications using the P700 pulse method in the literature. This will naturally lead us to discuss the consequences of the error in the P700 method in the literature of the last 25 years in photosynthesis. Using data presented in the manuscript and some chosen representative publications using the P700 pulse method, I will try to establish to what extent and in which conditions can the P700 method be at the origin of wrong interpretations.

The P700 pulse method underestimates Y(I) because of reduction of PSI acceptors during the multiple turnover pulse a) All PSI centers are open in the dark

One of the strong arguments to demonstrate that the P700 pulse method underestimates the fraction of open PSI centers comes from the results in dark adapted samples.

In the article presented above, we demonstrated that in Chlamydomonas reinhardtii, in dark and anoxic conditions, the ECS generated by a single turnover is equal to the one measured in oxic conditions in the presence of MV, when all PSI are open according to both methods. This means that all PSI centers are able to perform a stable charge separation if receiving a photon (Article, Figure 3C). In these conditions however, the P700 pulse method measures that only 30% of PSI centers are open. Moreover, in oxic dark conditions, all PSI can perform 4 charge separations (Article, Figure 4C), meaning that they have at least, in average, 4 oxidized acceptors. This is hard to reconcile with the conclusion based on P700 pulse method, which gives only 50% of PSI centers open.

In addition to those experimental evidences, there is also a thermodynamical argument in dark-adapted samples. In dark conditions, all photosynthetic intermediates are at the thermodynamic equilibrium with the ambient redox potential. In dark-adapted

Chlamydomonas reinhardtii cells, the ambient redox potential is not far from the redox potential of the plastoquinol/plastoquinone couple, i.e. in the -100 to +100 mV range. It has been shown that the plastoquinone pool is ~70% oxidized in oxic conditions and fully reduced in anoxic conditions (Houille-Vernes et al., 2011). In anoxia, it becomes closer to the redox potential of QA/QA -, i.e. -140mV to -145mV in Chlamydomonas reinhardtii (Shibamoto et al., 2009;Brinkert et al., 2016). Figure 2-7 shows a scheme where the pool of each intermediates is represented as a flared reservoir and where areas reflect their nernstian redox buffering.

Under these conditions and at thermodynamic equilibrium, P700 should be completely reduced (its redox potential is E'm = +430 mV, Kok, 1961;Sétif and Mathis, 1980) and the iron-sulfur proteins should be completely oxidized (the redox potential of iron-sulfur centers being in the -520 to -705 mV range, Golbeck and Bryant, 1991;Golbeck, 1993) 2) is larger than the pool of PSI donors (1). In this situation, both ECS and P700 based methods will give a correct result.

In the second one (bottom), P700 and one plastocyanin are reduced and only one PSI acceptor is oxidized before the measurement, i.e. the pool of PSI donors ( 2) is larger than the pool of PSI acceptors (1). In this situation, the ECS method will rightly measure that this PSI center is open. The multiple turnover pulse will reduce all PSI acceptors before oxidizing all PSI donors and therefore the P700 pulse method will wrongly consider that this PSI center was initially closed. The figure present the behavior of two open PSI during a multiple turnover saturating light pulse. P700 represents the PSI primary donor, the special pair of chlorophyll; PC refers to plastocyanin, the PSI secondary donor (PC and PC + being its reduced and oxidized form, respectively); A represents PSI primary acceptor (A and A -being its oxidized and reduced form, respectively). In example 1, the number of PSI donors (1 P700) is higher than the number of PSI acceptors (2). In example 2, PSI has two donors (1 PC and 1 P700) and only one acceptor.

Technical considerations regarding P700 measurements

The P700 pulse method remains an easy measurement, provided that correct estimations of P700 parameters are used:

-P0, the P700 absorption when all P700 are reduced -Pstat, the P700 absorption under conditions of interest -Pm', the P700 absorption after a saturating light pulse -Pm, the P700 absorption when all P700 are oxidized It does not require the use of a laser flash (an expensive setup) and is provided by several commercial instruments. However, even with the correction we provide in the manuscript, we became aware of some technical problems in the practical application of the P700 method.

I feel it is important to discuss them here.

An important aspect of the P700 method is the normalization, i.e. the measurement of the absorption difference corresponding to the change from 100% reduced (P0) to 100% oxidized (Pm) P700. Obviously, the redox state of P700 depends on the ratio of the oxidation and reduction rates of P700. Figure 2-9 shows the main P700 oxidation and reduction pathways as well as the action of the chemicals used in the following part. The full oxidation of P700 is a particularly difficult situation to achieve. Indeed, its redox potential is the highest of the inter-system chain. Hence, from a thermodynamic point of view, it is the last intermediate of the chain to be oxidized. In order to oxidize all P700, the principle is to: (i) increase the rate of oxidation of P700 by using a saturating light, and (ii) decrease the reduction rate of P700 and it is this point that we will discuss hereafter.

In the literature, two methods are commonly used to decrease the reduction rate of P700. The first one, commonly used in microalgae, is the inhibition of PSII by using a specific inhibitor (DCMU) suppressing the linear flow of electrons. The second one, used in plants, takes advantage of the different absorption spectra between PSII and PSI. When shining far red light, thanks to low energy chlorophylls, PSI is much more excited than PSII which causes oxidation of carriers between PSII and PSI [START_REF] Gobets | Energy transfer and trapping in photosystem I[END_REF]. In both cases, the oxidation of the inter-system chain would be complete if there was no alternative electron input in the chain (Figure 23456789). As presented in the introduction, there is at least one electron input pathway in the interchain which is the one used by cyclic electron flow (Nda2 or PGRL1/PGR5 pathway). For this reason, we could show that the DCMU treatment could not fully oxidize P700. In the presence of DBMIB, a cytochrome b6f inhibitor, both the linear and the cyclic electron flow are arrested (Figure 23456789). We could show that the reduction of P700 by the CEF is not negligible since the addition of DBMIB increases the measured Pm by about 10%. Even in the presence of DBMIB, the additional use of the artificial PSI electrons acceptor methylviologen (MV) increased slightly (by 2 to 5 %) the measured Pm, which probably reflects the fact that DBMIB is a leaky inhibitor. If the acceptor side of PSI becomes reduced during the pulse, then even weak electron fluxes from DBMIB-treated cytochrome b6f could be enough to keep some P700 reduced. For these reasons, the protocol we used to reach the full oxidation of P700 was to superimpose a strong pulse of light (about 2000 photons s -1 PSI -1 ) and a saturating laser flash, in the presence of 1µM DBMIB and 4mM MV (incubated for at least 4 min). b) Reaching the "P700 fully reduced" reference P0 P700 being a high oxidant (Em  0.430 Volts, Kok, 1961;Sétif and Mathis, 1980), it is thus fully reduced in dark conditions, when all photosynthetic intermediates are at the thermodynamic equilibrium with the ambient redox potential. Measuring the absorption level when all P700 are reduced seems straightforward: in the absence of actinic light, 100% of P700 should be Chapter 2: Probing PSI activity Suzanne FERTE -Doctoral Thesis -2019 reduced. However, even this simple measurement requires some precautions. Indeed, the absorbance in the dark is probed by small pulses of light (detecting light). Although they are short and weak, these pulses lead to the oxidation of a small fraction of P700. This actinic effect of the detecting pulses is small and can be neglected in most conditions. But in the conditions mentioned above (i.e. DBMIB and MV) leading to a very slow re-reduction of P700, if the frequency and/or intensity of the detectors is too high, then the cumulative effect causes a significant oxidation of P700. For this reason, the sequence we used starts in the dark and performs only one detection per second to measure the absorbance in the dark.

c) Potential errors on normalization values (Pm -P0)

To illustrate the error that can be made on the absorption changes corresponding to the oxidation of all P700, we compared the values obtained with the protocol we used in the manuscript and the ones obtained by classical method used in the literature, i.e. adding the sole inhibitor DCMU and detecting every 100ms in dark conditions. In average, the Pm-P0 value obtained with the standard protocol is about 25% lower than the one measured with our protocol (Figure 2 Pm was measured as the difference between absorbance in the dark and after a multiple turnover saturating pulse of light (12ms duration). In the standard protocol (light red), only PSII were inhibited by saturating concentration of DCMU and the light sequence provided detection pulses every 100ms in the dark. In our improved protocol (dark red), cytochrome b6f was inhibited by saturating concentrations of DBMIB (1µM) and methylviologen (4mM incubated for at least 4 minutes), a PSI exogenic acceptor, was added in saturating concentrations. Moreover, the sequence provided detection pulses every seconds in dark conditions. Data with both protocols were normalized to the value obtained with the improved protocol and represent the average of three biological samples of Chlamydomonas reinhardtii in oxic conditions (SD) Another possible source of error comes from changes in the number of functional PSI through the course of an experiment, since absorption change depends on the concentration of the absorbing molecule (here P700). Such change can originate from neo-synthesis of PSI in the sample or from PSI photoinhibition which is the destruction of PSI proteins at the stromal side of PSI and the degradation of P700 which occurs almost concomitantly (Terashima et al, 1994;[START_REF] Sonoike | Mechanism of photosystem-I photoinhibition in leaves of Cucumis sativus L[END_REF][START_REF] Sonoike | Degradation of psaB gene product, the reaction center subunit of photosystem I, is caused during photoinhibition of photosystem I: possible involvement of active oxygen species[END_REF]. In the case of photoinhibition, one should therefore be cautious when interpreting the decrease in Pm' measurement, i.e. the absorption change after the saturating pulse: it can be due to an accumulation of electrons on the acceptor side of PSI, or it can be due to the destruction of some P700 through photoinhibition. We will illustrate this issue with an example from the literature [START_REF] Sejima | Repetitive short-pulse light mainly inactivates photosystem I in sunflower leaves[END_REF]. In this work, the authors incubated sunflower leaves in the dark and subjected them to repetitive short pulses of saturating light. They compared the physiological responses under atmospheric conditions or low CO2 concentration.

At the end of the treatment and under atmospheric conditions, the Pm decreased by 90% (Figure 2-11B). This means that 90% of the PSI centers are photoinhibited. If this is the case, then the increase in Y(NA) (that is the proportion of the PSI centers of which P700 cannot be oxidized) up to 0.9 does not mean that the chain is reduced downstream of the PSI, as suggested by the authors, but simply that 90% of PSI centers are inactive/absent (degraded or irreversibly blocked at the iron-sulfur centers). Here, the decrease in Pm is used by the authors both as a measure of the number of photoinhibited PSI and as a measure of the physiological state downstream of the PSI. This leads them to propose that the accumulation of electrons downstream of the PSI leads to PSI photoinhibition, which is not supported by the data. To obtain information on the physiology downstream of the PSI, it is necessary to measure the Y(NA) after normalization on all the active PSI at the time of the measurement, therefore recalculate the Pm at the time of the measurement.

Because the maximal quantum yield of PSI is about 1, we will consider that Y(I)=FOC(I).

Therefore, we can use equation ( 1) to estimate Y(I) from Y(total), measured by ECS, and FOC(II), provided that we can estimate accurately the value of the latter. Fluorescence analysis upon saturation pulse can be used to derive several useful photosynthetic parameters. Among However, it has been shown that closed PSII centers can transfer their exciton to an adjacent open center, a process known as connectivity [START_REF] Joliot | Etude cinétique de la réaction photochimique libérant l'oxygène au cours de la photosynthèse[END_REF]. More realistic models are therefore preferred to the "puddle" model where PSII reaction centers are connected by shared antenna, and are called 'lake' model or 'connected units' model. Kramer and coworkers (Kramer et al., 2004) Moreover, the correction of the P700 data that we proposed in the manuscript allows better concordance between ECS and P700 estimations (Figure 2-12B). As expected, the correction does not modify the results obtained in the presence of MV, since no significant Y(NA)P700 was measured in those conditions. These results confirm that the conclusions of the article remain true in the presence of functional PSII: the ECS and P700 methods concord when Y(NA)P700 is negligible, i.e. when MV is added. In the other cases, the proposed correction on P700 method holds true.

It is to note here that results in MV are compatible with an intermediate PSII connectivity between the puddle and the lake model. Indeed, in these conditions both Y(I)P700 and Y(I)ECS are supposed to be correct, and the concordance line (Y(I)P700 = Y(I)P700) is right in between the values obtained with the two extreme models.

Revisiting the literature based on the P700 pulse method

In the article presented above, we demonstrated that when P700 method measures some reduced PSI acceptors at the end of the pulse (Y(NA)P700 is different from 0), it always overestimates the real acceptor side limitation. This leads in turn to an underestimation of the yield of PSI. The questions that automatically rise are:

-In what environmental and physiological conditions is P700 method underestimating the PSI yield? a) Dark to light transition During dark to light transition, stromal reactions and photosynthetic chain reactions adjust to each other and reach an equilibrium. In the literature, several works use P700 pulse method to evaluate the dynamic of activation of the different stromal pathways, e.g. CBB cycle, CEF or Mehler reactions. In this part, we show that (i) since this process is characterized by transient accumulation of reduced PSI acceptors, the P700 method generates important errors, and (ii) these errors led to underestimate the role of CEF during the transition from dark to light.

In this section, I will first come back to the dark-to-light transitions we used in the article manuscript (figure 2C and3B) and then show an example from the literature.

In Figure 2-13, data from figures 2C and 3B of the manuscript are plotted as a function of time after light onset (one of the three replicates is shown). It compares: the P700 parameters (Y(I)P700 and Y(NA)P700, red curves), the same estimations after correction (Y(I)P700,corr and Y(NA)P700,corr, green curves), as well as their estimations by the ECS method (Y(I)ECS and Y(NA)ECS, blue curves). As stated in the manuscript, PSII were inhibited and samples were placed in oxic conditions at 800 µmol photons m -2 s -1 (left panels: A, B, C) or in anoxic conditions at 135 µmol photons m -2 s -1 (right panels: D, E, F).

When steady state is reached (~450 ms and ~60 s for oxic and anoxic conditions, respectively), all methods provide similar estimation of PSI parameters. However, during the transition period, P700 method underestimates the fraction of open PSI centers: Y(I)P700 is always lower than Y(I)ECS, by a factor up to 4 in oxic conditions and up to 10 in anoxic conditions. It is to note that for all data, when Y(NA)P700 is lower than 0.8, the P700 correction is reliable: corrected P700-based and ECS-based parameters are very close. At the contrary, when Y(NA) is higher than 0.8 (e.g. during the first 10 ms after light onset in oxic conditions, Figure 2-13F), the P700 correction is not reliable (as established already in the discussion of the article).

We could estimate the number of PSI stable charge separation occurring during the transitory phase and approximate the ones effected through CEF. An integration of the PSI electron transfer rate, ETR(I), over time until the steady-state is achieved yields the number of stable charge separations performed by PSI, hereafter noted N (Nawrocki et al., 2019). We estimated ETR(I) as: We estimated I . I through the initial slope of the ECS increase at the onset of light in conditions where PSII is inhibited (see chapter 3 for more details) and we assumed that it was constant over time. PSI was absorbing 118 ± 17 photons s -1 and 509 ± 43 photons s -1 at 135 µmol photons m -2 s -1 and 800 µmol photons m -2 s -1 , respectively. Can we go further and determine the PSI charge separations through CEF pathway? In this data, PSII is inhibited. Then, the number of electrons transferred through PSI is the sum of two processes: (i) the oxidation of the initial pool of electrons in the photosynthetic chain in the dark, which represents approximately 10 electrons (Nawrocki et al., 2019) and (ii) the cyclic electron transfer around PSI that reinjects reducing power from the PSI acceptors into the inter-system chain. We calculated the number of charge separation through CEF as N-10 and estimated an average CEF during transitory phase. ECS and corrected P700 data gave an average CEF of ~75 electrons PSI -1 s -1 and ~50 electrons PSI -1 s -1 for the oxic and anoxic conditions, respectively, close to the recent estimation of the maximal cyclic electron flow in Chlamydomonas reinhardtii, i.e. ~60 electrons PSI -1 s -1 (Nawrocki et al., 2019). But P700 data largely underestimated the CEF during the transition phase with ~25 electrons PSI -1 s -1 and ~20 electrons PSI -1 s -1 for the oxic and anoxic conditions, respectively. (E and F, respectively). PSII was inhibited by saturating concentrations of DCMU (10µM) and hydoxylamine (saturating concentration, see material and methods from article) and anoxia was induced by glucose (20mM) and glucose oxidase (280U.mL -1 ) addition. Data were obtained from P700 method (red), ECS method (blue) or correction of the P700 method (green) (see introduction of section 2.3.4 for calculation details). The number of charge separation during the oxic (C) and anoxic (F) transition from dark to light were calculated as the integrated electron transfer rate over time until the steady state is reached. The first 450 ms and first 60 s were considered for oxic and anoxic conditions, respectively. Data corresponds to one representative sample of Chlamydomonas reinhardtii wild type T222 + .

In the literature, measurements of PSI activity through the P700 pulse method at the onset of light often show a transiently very high acceptor side limitation. Figure 2-14 is taken from a publication on sunflower leaves (Klughammer and Schreiber, 1994). When light is turned on, quantum yields of PSI (here noted ΦI) and PSII (here noted ΦII) are very low and later increase concomitantly with time, what is often interpreted as a lag phase before CBB cycle pathway activation (e.g. [START_REF] Schansker | Methylviologen and dibromothymoquinone treatments of pea leaves reveal the role of photosystem I in the Chl a fluorescence rise OJIP[END_REF][START_REF] Schansker | Dark recovery of the Chl a fluorescence transient (OJIP) after light adaptation: the qT-component of non-photochemical quenching is related to an activated photosystem I acceptor side[END_REF]. Most importantly, ΦI and ΦII are equal throughout the kinetics, which was interpreted by the authors as an evidence for a pure linear electron flow.

In the light of what we have learnt from the critical analysis of the P700 pulse method, it is reasonable to question this conclusion. In these data, "red. P700" represents the fraction of reduced P700 and Y(NA)P700 is easily calculated as Y(NA) = red.P700 -ΦI. At the light onset, Y(NA)P700 decreases from 0.9 initially to 0.1 after 3 minutes of illumination. According to our correction, during those 3 first minutes, ΦI is therefore largely underestimated (by the factor 1-Y(NA)P700). This suggests that a transient CEF probably occurs during the first minutes after light onset, which could not be apprehended with the P700 pulse method. CEF has often been proposed to be a crucial starter for photosynthesis: when stromal sinks

are not yet activated in the dark, CEF would build some ATP and a pmf [START_REF] Joliot | Cyclic electron flow in C3 plants[END_REF] which helps protecting both photosystems (Endo and Asada, 2008;Chaux et al., 2015). The considerations above illustrate how much the error made by the P700 pulse method can lead to wrong conclusions: it overestimates the acceptor side limitation at the onset of light and largely underestimates the efficiency of the transitory CEF. It is especially true in anoxic conditions where it wrongly measures no CEF during the first seconds following the onset of light.

b) Under steady state low light, PSI yield is underestimated In this section and in the following ones, we will focus on steady-state situations.  When PSII is active, P700 method underestimates PSI activity under low light I believe that such underestimation of PSI activity under low light is common in the literature. photoinhibition of PSI [START_REF] Zivcak | Repetitive light pulseinduced photoinhibition of photosystem I severely affects CO2 assimilation and photoprotection in wheat leaves[END_REF] in wheat leaves. They show that under low light (<500µmol m -2 s -1 ), Y(NA)P700 is high and PSI yield shows a local drop. This kind of drop of PSI yield under low light is common in the literature and can be found for example in wheat again (Brestic et al., 2015), and for 2 species of fern, Microsorum punctatum and Paraleptochillus decurrens (Wang et al., 2013). It is reasonable, in the light of our results, to question the meaning of this drop under low light. Because this drop of PSI yield corresponds to a significant increase of Y(NA)P700, I propose that this is a bias due to the overestimation of the acceptor side limitation at the end of the multiple turnover pulse. , 21, 30, 45, 61, 78, 103, 134, 174, 224, 281, 347, 438, 539, 668, 833, 1036, 1295, 1602, and 1930 μmol photons m -2 s -1 ); the duration of each interval with a given light intensity was 30s. Data corresponds to the average values from six plants (± SD).

 When PSII is inhibited, P700 method leads to misunderstanding of PSI activity regulation under low light

c) Role of Antimycin A and PGR5 in CEF

Antimycin A is proposed to be an inhibitor of one of the cyclic electron flow pathways, the PGR5-PGRL1 route [START_REF] Tagawa | Role of chloroplast ferredoxin in the energy conversion process of photosynthesis[END_REF]. Here we would like to suggest that the bias of P700 method could sometimes lead to misunderstanding the role of antimycin A in CEF. We will discuss this based on one example.

Figure 2-17 shows data from an article which is about dissecting electron flows in Arabidopsis thaliana [START_REF] Kou | Partially dissecting the steady-state electron fluxes in Photosystem I in wild-type and pgr5 and ndh mutants of Arabidopsis[END_REF]. After we shared with the authors our questions regarding the P700 pulse method, the authors kindly sent us the raw data regarding PSI parameters so we could apply the P700 correction we propose in the paper. After correction, conclusions are reversed: Y(I)P700,corr appears higher in antimycin A treated samples. This would result in a higher ETR(I). ETR(II) being similar between treated and non-treated (see data in [START_REF] Kou | Partially dissecting the steady-state electron fluxes in Photosystem I in wild-type and pgr5 and ndh mutants of Arabidopsis[END_REF], Δflux would be higher in antimycin A treated samples than on control leaves, indicating a higher CEF! If this increase of CEF is true (and not due to an artifact related to protocols of normalization for example, i.e. an underestimated Pm leading to a correction bias) we can suggest that this phenotype is due to an inhibition of the respiratory chain, a well-known effect of antimycin A (first proposed by [START_REF] Ahmad | Studies on the biological action of antimycin A[END_REF], reviewed in Slater et al., 1973). Indeed, it has been shown that inhibition of the respiration chain results in modification of the photosynthetic activity (e.g. Larosa et al., 2018[START_REF] Cardol | Photosynthesis and state transitions in mitochondrial mutants of Chlamydomonas reinhardtii affected in respiration[END_REF]. Moreover, we can hypothesize that the lack of ATP production by respiration results in an enhancement of CEF as it has been proposed in the literature (e.g. Alric et al., 2010b;Fisher et al., 2019).

PGR5 is a thylakoid enzyme that have been proposed to be involved [START_REF] Munekage | PGR5 is involved in cyclic electron flow around photosystem I and is essential for photoprotection in Arabidopsis[END_REF] together with PGRL1 (Hertle et al. 2013), in cyclic electron flow by reinjecting electrons from acceptor side of PSI into the chain. Here we show that the bias of P700 method might also lead to misunderstanding the pgr5 mutant phenotype.

Figure 2-17G shows PSI parameters measured in Arabidopsis thaliana pgr5 mutant [START_REF] Kou | Partially dissecting the steady-state electron fluxes in Photosystem I in wild-type and pgr5 and ndh mutants of Arabidopsis[END_REF]. Y(I)P700 is always lower in pgr5 mutant than in the WT (Figure 2-17A and G) because of a higher Y(NA)P700 and it exhibits a similar trend as antimycin A treated samples. As presented above, authors calculated a Δflux much lower in pgr5 mutant than in the WT, thus concluding in a lower CEF occurrence and proposing PGR5 as an important pathway for CEF (Figure 2-17I).

As previously, we calculated PSI parameters with the correction proposed in the article.

Y(I)P700,corr appears much higher in pgr5 mutant than in the WT. This would result in a higher ETR(I). ETR(II) being similar between WT and pgr5 mutant (see data in [START_REF] Kou | Partially dissecting the steady-state electron fluxes in Photosystem I in wild-type and pgr5 and ndh mutants of Arabidopsis[END_REF], Δflux should be much higher in pgr5 mutant than in the WT, indicating a much higher CEF! It is to note that our correction gives a Y(I) evolution with light intensity that is not expected: it remains rather stable, meaning that PSI activity does not saturate, even under high light. We have no clear explanation. It might be because under high light (higher than 580 µmol photons m -2 s -1 ) Y(NA) is higher than 0.7 and the correction might be biased in the sense of overestimate of Y(I). But it could also be because of a low PSI/PSII stoichiometry in the PGR5 mutant, as mentioned several times (e.g. [START_REF] Munekage | PGR5 is involved in cyclic electron flow around photosystem I and is essential for photoprotection in Arabidopsis[END_REF].. If there are much less PSI than PSII in the pgrl1 mutant compared to the wild type, then, a higher ETR per PSI does not mean that the flow through all PSI is higher than the flow through all PSII. And Δflux would not reflect the amplitude of CEF.

But still, here, pgr5 mutant would not show a lower CEF than in WT And it is to note that similar data were obtained in Arabidopsis thaliana pgr5 mutant in another work (Huang and Zhang, 2012). In the recent literature, the mechanistic role of the PGR5-PGRL1 heterodimer has been questioned, and a regulatory role is now proposed instead. Indeed, the maximal CEF measured in Chlamydomonas reinhardtii was the same, ~60 electrons PSI -1 s -1 , in the wild type and in the pgrl1 mutant (Nawrocki et al., 2019).

Partial conclusions and transition

Measuring the quantum efficiency of PSI is very important and P700 measurement is probably the easiest and the most accessible way to do so. Indeed, such measurement does not require an expensive laser, the signal over noise is very satisfactory and in organisms with weak ECS signals (e.g. cyanobacteria), it is the only simple way to assess the PSI activity.

However, we have shown that the method proposed by Klughammer and Schreiber and based on P700 measurements present a bias: it systematically underestimates the PSI yield proportionally to the acceptor side limitation at the end of the pulse, i.e. the parameter Y(NA)P700. We propose a correction of the data allowing to correctly estimate the quantum yield of PSI with the same set of parameters: the correction does not need any additional measurement. By this mean, already produced data can be revisited. Through a short literature review, we could show that, in many physiological conditions, P700 method lead to misunderstanding the physiology and in particular, CEF amplitude and variations. A more careful and exhaustive reinterpretation of the literature data might enable advances in the understanding of the pathways and mechanisms of regulation of the CEF.

Nevertheless, for the moment, the correction we propose is not fully satisfying: when Y(ND) is very small and/or Y(NA) very large, the correction fails, considering the ECS measurement as a reference. I think it would be very useful to continue using cross test between ECS and P700 methods and to try to find a better correction giving an agreement between the two measurements in all conditions. A third alternative to measure PSI yield could come from photoacoustic measurements, and this could be a powerful way to decide what is the most robust and accurate measurement of PSI yield based on P700 measurements.

For the moment, we think that the most reliable method is the ECS. This is why I will favor its use in future chapters.

Introduction: CEF, a still mysterious alternative pathway

The existence of cyclic phosphorylation in chloroplasts has first been proposed by Arnon and his collaborators in 1954 (Arnon, Allen and Whatley, 1954). It is now well established that in plants and green algae, a cyclic electron flow (CEF) around PSI can generate ATP. As already mentioned in the Introduction to the thesis (Chapter 1), genetic analyzes have elucidated mainly two independent pathways of CEF in plants, a pathway involving the proteins PGR5 and PGRL1 [START_REF] Munekage | PGR5 is involved in cyclic electron flow around photosystem I and is essential for photoprotection in Arabidopsis[END_REF]Hertle et al, 2013), and another one using the chloroplast complex NADPH dehydrogenase, NDH [START_REF] Munekage | PGR5 is involved in cyclic electron flow around photosystem I and is essential for photoprotection in Arabidopsis[END_REF]. Structures of the cytochrome b6f [START_REF] Stroebel | An atypical haem in the cytochrome b6f complex[END_REF] have revived a third possibility proposed by Mitchell (Mitchell, 1975) and reiterated afterwards [START_REF] Joliot | Cyclic electron flow under saturating excitation of darkadapted Arabidopsis leaves[END_REF]Nawrocki et al, 2019a) which involves the electron transfer from ferredoxins directly to the Qi site of the cyt b6f complex.

The mechanistic aspects of CEF and LEF, the important question of the proton-to-electron coupling in both LEF and CEF modes and the photo-protection mechanisms have been discussed in the general introduction (chapter 1), and are only briefly recalled in this chapter. In the introduction of this chapter, I will focus on the proposed roles of CEF (3.1.1), on its possible modes of regulation (3.1.2) and on the methodological limitations of its measurement (3.1.3).

Role of CEF in ATP/NADPH adjustment and photo-protection

Two main roles have been proposed for CEF. Hereafter, we present arguments that led to highlight those roles and discuss them. it takes 14 H + to synthesize 3 ATP (Hahn et al., 2018). Moreover, 6 H + are moved into the lumen for 2 electrons transferred through the linear chain to reduce 1 NADP + into NADPH.

All in all, the linear transport chain produces about 1.3 ATP per NADPH (1.286 from the computation above).

Chapter 3: Diversity of cyclic electron flow in microalgae

Suzanne FERTE -Doctoral Thesis -2019

It is well admitted that CBB cycle uses 1.5 ATP per NADPH but this ratio can increase to 1.65 depending on CO2 concentration (Kramer & Evans, 2011). Another metabolic process that depends on CO2 concentration and on photosynthetic activity is the carbon concentrating mechanisms (CCM), the active transport of inorganic carbon inside the chloroplast (see chapter 1 -general introduction for more details), which requires additional ATP. All in all, the minimal requirement of carbon assimilation is 1.5 ATP per NADPH but this ratio may increase depending on environmental conditions.

It is therefore widely assumed that in plants and green algae, the ATP/NADPH produced by LEF is insufficient. CEF has been proposed as a complementary pathway to increase ATP production by the photosynthetic chain (Allen, 2002). Indeed, the latter flow induces an electron transfer through the cytochrome b6f, and thus proton pumping and synthesis of ATP/NADPH ratio produced by the photosynthetic chain can thus be calculated as:

(1) ATP/NADPH = (9/14 LEF + 6/14 CEF) / (0.5 LEF) = 18/14 + 12/14 CEF/LEF Therefore, one can calculate that the minimal CEF/LEF ratio which allows CEF to fulfill the ATP shortfall. When the minimal value of 1.5 ATP per NADPH in the CBB cycle is considered, equation ( 1) indicates that a CEF/LEF of 0.25 (20% of PSI turnovers are used for CEF) is enough to fuel CO2 assimilation.

b) Role of CEF in photoprotection

We have discussed the risk of photo-destruction of both photosystem I and II in conditions of excess light in the general introduction (chapter 1). The CEF can reduce the risk of photodestruction of photosystems by playing a role in two main photo-protection mechanisms. The first is the slowdown of cytochrome b6f via a regulation called "photosynthetic control": by increasing the l proton gradient (ΔpH), the CEF decreases the turnover of cytochrome b6f, of which kinetically limiting step is the release of protons at the Q0 site (Tikonov et al., 2014). This moves the limiting step of the electron transfer from downhill PSI to uphill PSI, releasing the reducing pressure on the acceptor side of the PSI.

The second consequence of the ΔpH generated by CEF is to promote non-photochemical quenching of PSII fluorescence, qE, a mechanism that increases the light energy dissipation as heat [START_REF] Golding | Reduction of the thylakoid electron transport chain by stromal reductants-evidence for activation of cyclic electron transport upon dark adaptation or under drought[END_REF][START_REF] Heber | Concerning a dual function of coupled cyclic electron transport in leaves[END_REF][START_REF] Kramer | Dynamic flexibility in the light reactions of photosynthesis governed by both electron and proton transfer reactions[END_REF]Li et al., 2000;[START_REF] Müller | Non-photochemical quenching. A response to excess light energy[END_REF]. We should note here that some studies show qE implementation under high light without the intervention of CEF (Avenson, Cruz, Kanazawa, & Kramer, 2005;[START_REF] Ishikawa | Electron transport activities of Arabidopsis thaliana mutants with impaired chloroplastic NAD (P) H dehydrogenase[END_REF].

c) What about other alternative electron flows?

Several other alternative pathways have been proposed to increase the ATP/NADPH production by the photosynthetic chain and to play a role in photosystems photoprotection.

The main ones are grouped as water to water cycles which use electrons from water at the Oxygen evolving complex and reroute them to an oxidase. Several oxidases associated to water to water cycles have been identified, including the flavodiiron proteins (Chaux et al., 2017;[START_REF] Allahverdiyeva | Flavodiiron proteins Flv1 and Flv3 enable cyanobacterial growth and photosynthesis under fluctuating light[END_REF], and the Mehler reaction, or superoxide dismutase pathway (Asada, Allen, Foyer, & Matthijs, 2000), which both transfer electrons to O2

downhill PSI (Nawrocki et al., 2019). The plastoquinone terminal oxidase (PTOX) reroutes the electrons from plastoquinol towards molecular oxygen, but seems to play a role only in some very specific cases, like in iron deprivated algae [START_REF] Bailey | Alternative photosynthetic electron flow to oxygen in marine Synechococcus[END_REF]Cardol et al, 2008;[START_REF] Zehr | Photosynthesis in the open ocean[END_REF], Nawrocki et al., 2019b) or in alpine plants [START_REF] Streb | Evidence for alternative electron sinks to photosynthetic carbon assimilation in the high mountain plant species Ranunculus glacialis[END_REF], Lareau et al., 2013). Last, the rerouting of reducing power from the stroma towards the mitochondrion, which can take place in green algae (Lemaire, Wollman, Bennoun, & Plant, 1988), in diatoms (Bailleul et al., 2015) or in plants [START_REF] Scheibe | Malate valves to balance cellular energy supply[END_REF]) can be considered as a "meta" water to water cycle. There are also alternative pathways that reroute electrons to other molecules than molecular oxygen. This is the case of the hydrogenase pathway which concentration (i.e. the redox state of the PQ pool and the redox state of PSI acceptors, ferredoxin and NADPH) or physical separation of photosystems and other photosynthetic complexes involved in the two processes. The CEF rate could be simply determined passively by the concentrations of its substrates. In a model where electrons are reintroduced in the inter-system chain at the level of the plastoquinone pool, from ferredoxin (the FQR pathway), then the rate of CEF would be determined by the redox state of the PQ pool and the redox state of ferredoxins (Allen, 2003;[START_REF] Alric | Redox and ATP control of photosynthetic cyclic electron flow in Chlamydomonas reinhardtii (I) aerobic conditions[END_REF]. The ATP shortfall for CO2 assimilation in the CBB cycle would lead to an over-reduction of PSI acceptors. The increase in the concentration of the latter, substrates of CEF would in turn favor CEF, increasing ATP production.

However, another mode of regulation is possible, where the kinetic constant of the electron re-injection step is regulated. Kramer and collaborators proposed that the both ferredoxinquinone reductase (FQR) and the NDH pathway are inhibited by the ATP level itself (Fisher et al., 2019). Again, the ATP shortfall would at the contrary favor the CEF, producing more ATP as a response.

Another mode of regulation of the CEF and LEF rates comes from the segregation of LEF and CEF chains. Several works propose the formation of super-complexes involving cytochrome b6f, PSI and enzymes involved specifically in CEF such as PGRL1 and PGR5 (Iwai et al., 2010[START_REF] Steinbeck | Structure of a PSI-LHCI-cyt b6f supercomplex in Chlamydomonas reinhardtii promoting cyclic electron flow under anaerobic conditions[END_REF], Kouril et al., 2014). If such super-complexes exist in significant concentration -which has not been demonstrated yet-they would constrain the electron carriers in a cyclic mode by reducing the distance between the intermediates [START_REF] Steinbeck | Structure of a PSI-LHCI-cyt b6f supercomplex in Chlamydomonas reinhardtii promoting cyclic electron flow under anaerobic conditions[END_REF]. It has also been proposed that under certain physiological conditions, the FNR could move from the PSI vicinity towards a docking site on the b6f causing an increase of the affinity of ferredoxins for the CEF pathway (Joliot and Johnson, 2011;[START_REF] Zhang | Photosynthetic physiology of the coccolithophore Emiliania huxleyi Major environmental determinants[END_REF]. Another mode of structuration has been proposed which involved a fine coupling between CEF/LEF ratio, and state transitions [START_REF] Finazzi | Involvement of state transitions in the switch between linear and cyclic electron flow in Chlamydomonas reinhardtii[END_REF] but is not considered any more after several works demonstrated that there is no causal link between the two processes [START_REF] Takahashi | Cyclic electron flow is redox-controlled but independent of state transition[END_REF].

Another important issue in the regulation of CEF rate is the one of its limiting step. This issue is rarely discussed explicitly but has a tremendous importance regarding the potential role and mode of regulation of CEF. Under high light, the limiting step of the electron transfer from plastoquinols to PSI acceptors, which is common to both LEF and CEF, is the cytochrome b6f. Is the cytochrome b6f also the limiting step of CEF under high light? Or is it CEF has no net product. Thus, its rate cannot be probed thanks to the evolution of a product, like LEF with molecular oxygen for example. In addition, there is no clear consensus regarding the pathway of the "cycling step" (through PGRL1/PGR5, through the NDH or directly via the QCO2 site of cytochrome b6f) and no specific spectroscopic signal associated to them. In order to reduce the complexity of the system, a simple approach is to inhibit the PSII with chemicals like DCMU (Fan et al., 2007;Joliot and Joliot, 2002;Nawrocki et al., 2019c) or to diminish its activity by using PSI specific light quality (far red in plants, [START_REF] Joliot | Quantification of cyclic and linear flows in plants[END_REF]. Then, the PSI activity, and more generally photosynthetic activity, can only be attributed to CEF. However, if LEF and CEF are interacting, in competitions for a substrate for example, then the flux measured when the PSII is inhibited is not representative of the actual flux under physiological conditions (i.e. when the PSII is active) (Fan et al 2007). If one wants to understand the physiological role of the CEF, it seems important to first understand the relationship between CEF and LEF before discussing the results obtained when the PSII is inactive.

The only remaining approach is then to estimate the CEF rate as the difference between the electron transfer rate through PSI, the photosystem involved in both CEF and LEF, and the one through photosystem II, involved in LEF only (Fan et al, 2016). The electron transfer rate From equation ( 2), it appears that a correct measurement of CEF requires accurate measurements of both PSI and PSII yields and absorption cross-sections, and the photosystems stoichiometry. Each error on the measurement of one of those parameters will sum up in the error on the contribution of CEF to the overall photosynthetic activity. To test whether CEF can represent 20% of LEF, a relative error or noise-to-signal ratio of 10%

for each of those parameters would be already prohibitive.

Chapter 3: Diversity of cyclic electron flow in microalgae

Suzanne FERTE -Doctoral Thesis -2019

Chapter outlines

CEF capacity is conserved among plants and green algae [START_REF] Alric | Alternative electron transport pathways in photosynthesis: a confluence of regulation[END_REF], where most of the studies have been conducted. Therefore, our knowledge is mainly based on results obtained in higher plants and green algae. The model systems Arabidospis thaliana, for plants, and Chlamydomonas reinhardtii, for green algae, played a major role because genetics approaches are available [START_REF] Grossman | Chlamydomonas reinhardtii and photosynthesis: genetics to genomics[END_REF]. The aim of this chapter is to investigate the occurrence and if so, the diversity of CEF characteristics within eukaryotic photosynthetic organisms. The percentage of publications was estimated by searching on the Pubmed database the number of publications mentioning "Photosynthesis" AND "cyclic electron flow" AND a photosynthetic clade such as "Plant", "Cyanobacteria", ["Green algae" OR "Chlorophytes"], ["Diatom" OR "Stramenopiles" OR "Brown algae"], "Red algae", "Dinoflagellates" and "Haptophytes".

When considering the amount of work done in the last 50 years to progress on understanding the mechanisms and regulations of the CEF in plants and green algae, one can be pessimistic about the possibility of reaching the same level of knowledge in all the phytoplankton groups before tens of years. Some studies have been performed on phytoplankton groups (e.g. Bailleul et al, 2015;Roberty et al, 2014;[START_REF] Meneghesso | Photoacclimation of photosynthesis in the Eustigmatophycean Nannochloropsis gaditana[END_REF], showing low CEF rates and concluding that CEF was not sufficient to regulate the ATP/NADPH ratio. But most of those studies were performed in DCMU conditions and could have under-estimated the CEF rate under physiological (non-treated) conditions. Genetic approaches are not yet developed for most phytoplankton groups (transformation techniques are now routinely used in diatoms only). And we have listed all the methodological difficulties related to the measurement of CEF. This is why it seems reasonable to take a step back and think about a simple and robust method usable for all species and that still gives basic important information about CEF.

In The results of this chapter are divided in two parts:

-I first determined a robust and simple protocol to assess CEF in vivo, while minimizing sources of errors or noise. With this, we could investigate whether CEF exists or not in several microalgae and, when it is present to determine its relationship with LEF. This approach revealed three different CEF behaviors, provided CEF/LEF ratios for two of the three cases, but failed at giving absolute rates of CEF (3.2. Results (I): Exploring CEF diversity).

-The second part can be seen as a more ambitious attempt to measure absolute rates of CEF and LEF. Here, I estimated the PSII and PSI absorption cross-sections and stoichiometry, and from there I could calculate CEF and LEF absolute rates. A significant fraction of PSI activity is associated to CEF in Chlamydomonas reinhardtii and this ratio remains the same at all light intensities. This is consistent with a role in ATP/NADPH regulation, and the extent of CEF seems largely sufficient to provide the extra ATP needed for C assimilation (3.3. Results (II): Measuring absolute rates of LEF and CEF in Chlamydomonas reinhardtii).

Marine" laboratory of the Roscoff Biological Station. More than 600 cells were counted and the percentage of each species was calculated.

Chemicals

Hydroxylamine (HA) and (3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) were obtained from Sigma Aldrich (Saint Louis, Missouri, USA). DCMU was diluted in ethanol. HA was diluted in distilled water.

FCCP was diluted in dimethylsulfoxide. PSII was inhibited with 10 µM DCMU and 100 to 500 µM HA (for each experiment, the minimal concentration to reach maximal fluorescence in the dark was determined and used).

In vivo spectroscopy

All spectroscopic measurements were performed using a Joliot Type Spectrophotometer (JTS10, Biologic, Grenoble, France), which allowed to perform fluorescence and absorption difference measurements on the same sample. The actinic continuous light and the saturating pulse (5000 µmol photons m -2 s -1 ) were provided by orange leds (630 nm) and the saturating laser flash was provided by a dye laser (690 nm) pumped with a Nd:YAG laser (6 ns duration, 532 nm).

For fluorescence, the detecting pulses were provided by a white led filtered by a blue filter (470 nm). The reference and measuring photodiodes were protected from actinic light by a BG39 Schott filter (Mainz, Germany) and a LPF650+RG665 Schott filter, respectively.

PSII quantum yield in the light were measured as Y(II) = (Fm'-F')/Fm', respectively. F' is the fluorescence level under actinic light illumination, Fm' is the maximal fluorescence level obtained after a saturating light pulse in light-adapted sample. The maximal quantum yield of PSII was measured in dark-adapted cells (at least 1 minutes in the dark) and calculated as Fv/Fm = (Fm -F0)/ Fm, where Fm is the maximal value of fluorescence, measured after a saturating light pulse and F0 is the minimal value of fluorescence in a dark adapted sample.

For Electro-chromic Shift (ECS) measurements, all data shown are the difference between the measurement in the presence of the actinic light (laser flash, background actinic light or saturating pulse) and the one in the absence of actinic light, to correct for artifacts. ECS signals were measured as the difference of the absorbance changes at 520 and 546 nm for Chlamydomonas reinhardtii and for the Chaetoceros dominated sample from the field; at 563 nm for Amphidinium carterae and for Symbiodinium sp. Those wavelength or combinations of wavelength were chosen to eliminate absorption changes related to c-type cytochromes. The detecting wavelength were provided by a white led filtered with interferential filters at the wavelength of interest (10 nm full width at half-maximum, Edmund Optics). The measuring and reference photodiodes were protected from scattered and transmitted actinic light by a BG39 Schott filter (Mainz, Germany).

The fraction of open PSI centers, Y(I)ECS, was calculated as the ratio between the saturating flash induced ECS increase in the condition of interest and the one in dark-adapted samples.

The stoichiometry between photosystems was calculated as n2/n1 where n2 and n1 (1-n2) are the fraction of photosystem II and photosystem I to the total photosystems, respectively. n2

was calculated as the ratio between the saturating flash induced ECS in conditions of PSII inhibition (DCMU and HA) and the one in control conditions (Bailleul et al, 2010).

The photochemical rate was calculated as in (Bailleul et al, 2010). In brief, the method uses "dark window" (Joliot and Joliot, 2002) or "dark interval relaxation kinetics" (DIRK, Sacksteder et al., 2000). The principle is the following: the steady state value of the electric field (and ECS) is the sum of the activities of the two photosystems, and other photosynthetic complexes or processes involved in the movement of charges across the thylakoid membrane (cytochrome b6f, ATP synthase, proton pumping NDH, ion channels…).

When the light is turned off, only the two photosystems stop instantaneously (they are the only ones using light as substrate) and therefore the initial slope of the ECS decay measures the rate at which the two photosystems were moving charges before the light offset. The slope of the ECS decay was normalized to the flash induced ECS increase in the dark-adapted sample, to express the photochemical rate in charge separations per PS per second.

ECS spectra and linearity with electric field

Symbiodinium sp ECS spectrum was obtained from a previous work published in 2014 (Roberty et al., 2014) and its linearity over the electric field was verified in the same publication. To determine Amphidinium carterae ECS spectrum, a concentrated sample was treated with an uncoupler (FCCP) with concentration necessary and sufficient to decrease the electric field in the dark. Under these conditions, the low concentration of uncoupler does not prevent the light-induced establishment of an electric field, but the electric field in the dark is suppressed and the activity of the plastid ATP synthase is slowed down [START_REF] Joliot | Quantification of the electrochemical proton gradient and activation of ATP synthase in leaves[END_REF]

Absorption cross section assessment

The total absorption cross section per PS, σtotal, as well as the absorption cross section of PSI, σI were estimated by a method based on the ECS. σtotal. I and σI. I were calculated as the slope of ECS at light onset in the absence and in the presence of DCMU and HA, respectively.

The measurement time has been determined so that the ECS creation does not exceed 0.5 charge separation per PS and per PS1, respectively. Figure 3-8A is an exception used to demonstrate the linearity of ECS with time.

The measurements of σtotal. I and σI .I were performed at several intensities (56, 135, 340, 800, 1500 μmol photons m -2 s -1 ). The curves σtotal. I = f (I) and σI. I = f (I) were fitted by a linear function forcing the intercept to 0, the slope giving σtotal and σI, respectively.

Absorption cross section of PSII, σII, was estimated by two different methods. It was calculated from σtotal and σI thanks to the equation σtotal = n1.σI + n2.Fv/Fm. σII. σII was also estimated using the kinetics of fluorescence rise at the onset of light in the presence of saturating DCMU concentration (10 µM). The curves were fitted by an exponential fucntion (y = y0 + A1.exp (-t / τ)) and σII.I was estimated as 1/ τ. The estimation of σII.I was carried out at several light irradiances (as was done with ECS for σtotal and σI) and the curves σII. I = f (I)

were fitted by a linear function, forcing the intercept to 0, the slope giving σII.

Which observables?

At this stage, we still need to explain how ETR(total) can be calculated with ECS. There are two ways to do this. The first one measures the photochemical rate of PSII and PSI by measuring the slope of the ECS at the offset of light (see Methods). The second one postulates that, by analogy with ETR(II) and ETR(I), we can calculate the total electron transfer rate through the fraction of open photosystems (PSI + PSII), that we will call Y(total)

hereafter, and the total absorption cross-sections of PSII and PSI, that we will call total (m 2 ) from now. This gives:

(4) ETR(total) (electrons per PS per second)= total . Y(total) . I.

Combining equations ( 3) and ( 4) gives: Y(total) respectively. And we can follow the protocol presented in the previous section (3.3.1) to highlight CEF and investigate its dependence upon LEF. It is important to note that equation Y(total) is not a weighted average of the yield of the two photosystems, and that the equation ( 4) cannot be derived mathematically from the expressions of ETR(II) and ETR(I). We will test experimentally the validity of this calculation of ETR(total) as the sum of activities of the two photosystems in part 3.4.1 and discuss its theoretical validity in the discussion (part 3.5.2). The meaning of total will also be discussed later (part 3.4.1).

Finally, the protocol I propose is to follow the relative changes of Y(total) and Y(II) during a DCMU titration. Such a protocol does not require the measurement of PSII an total absorption cross-sections and requires only the measurements of the quantum yield of photosystem II and the Y(total). The PSII quantum yield is routinely assessed through chlorophyll fluorescence measurements (Harbinson, Genty, & Baker, 1989;Kramer et al., 2004). This method has been cross validated with O2 evolution (Genty et al., 1992) and is well established in the community. Moreover, the PSII quantum yield Y(II) is valid whatever the level of energetic connectivity we consider (i.e. in both extreme "puddle" and "lake" models). Y(total) can be calculated through the "a phase" of the ECS signal generated by a saturating laser flash (see chapter 2). We are working on organisms that do not perform any state transition, i.e. there is no movement of antenna between photosystems. State transitions are absent in diatoms [START_REF] Owens | Light-harvesting function in the diatom Phaeodactylum tricornutum: II. Distribution of excitation energy between the photosystems[END_REF], there is no report, to my knowledge of state transitions in dinoflagellates. And we used a mutant of state transitions for

Chlamydomonas reinhardtii (the stt7 mutant; [START_REF] Depège | Role of chloroplast protein kinase Stt7 in LHCII phosphorylation and state transition in Chlamydomonas[END_REF]. Thus, II and total are constant throughout the experiment. Yields measured in the initial conditions were not exactly the same between replicates (< 10% variation), but the normalization to the initial values of Y(total) and Y(II) allows to get rid of small biological variation between replicates.

In the introduction, we have shown that most of the questions regarding CEF role, regulation or pathways would benefit from a better understanding of the relationship between CEF and LEF. CEF has mostly been investigated in higher plants and green algae. Thus, all the models regarding CEF mechanisms and regulations have been elaborated from a very small sample of the phylogenetic diversity. With such a protocol, I voluntarily give up the possibility to give absolute rates of CEF. But it allows to test models of CEF behavior, like situations where no CEF is occurring, others where CEF is present but is not influenced by LEF or CEF is occurring and influenced by LEF. We decided to apply this protocol on species from various photosynthetic groups. In this work, we present the results obtained in three species, two dinoflagellates, Amphidinium carterae and Symbiodinium sp, that originates from secondary endosymbiosis and a green alga Chlamydomonas reinhardtii, which is used here as a model for the green lineage and where the existence of CEF is well established (e.g. Joliot and Johnson, 2011;[START_REF] Shikanai | Central role of cyclic electron transport around photosystem I in the regulation of photosynthesis[END_REF]Alric, 2010;Chaux, 2015). Results obtained on natural population of diatoms will be shown also (see 3.5.1.c). I also investigated CEF in a haptophyte, Isochrysis galbana, and in Euglena gracilis, but those results are not shown (because the ECS signals were too small and giving low signal-to-noise or because independent replicates showed a too high variability to conclude).

CEF is not essential to photosynthesis in the dinoflagellate

Amphidinium carterae

In the dinoflagellate Amphidinium carterae, the Y(total) and the photosystem II quantum yield Y(II) decrease at the same rate when titrating with DCMU (Figure 3 function of Y(II) for three biological replicates, we get an affine relationship: linear regression gives a very good correlation coefficient (R 2 =0.995). Contrary to the case of the previous dinoflagellate, the linear fit of the experimental points does not go through the origin of axes. This data is compatible with our model of cyclic electron flow functionally independent on LEF.

Linear regression estimates a y-intercept of 0.19 ± 0.01. This means that about 19% of initial photosystems (PSI + PSII) activity in the absence of DCMU was involved in CEF. Before normalization the Y(total) in the absence of DCMU was 0.87 ± 0.11, meaning that 87% of photosystems were initially open. From there, we can conclude that ~ 16.5% of the photosystems are involved in CEF in these light conditions. We did not estimate the PSII/PSI ratio because Symbiodinium sp. is hardly sensitive to inhibitors. But, assuming that PSII/PSI = 1 (as it is the case in most of the Symbiodinium strains, (Roberty et al, 2014), then, we can calculate that about 33% of PSI were initially involved in CEF. separation, which could be due to lateral segregation of the photosystems or to the confinement of some PSI in super-complexes (see discussion, in 3.5.1.b).

Chlamydomonas reinhardtii displays a CEF which is dependent on LEF

When titrating Y(total) and Y(II) with DCMU in Chlamydomonas reinhardtii, the behavior is a lot more complex than for the two dinoflagellates (Figure 34567). Y(total) and Y(II) do not follow each other. When plotting Y(total) as a function of Y(II) for seven biological replicates, we obtain a non-affine relationship: the amplitude of CEF is not constant when PSII activity is changing. Hence, CEF is depending on PSII activity, that is to say, on LEF. We see two different phases. At low DCMU concentrations, Y(total) decreases more slowly than Y(II) and, as discussed before, this most probably indicates the increase of CEF. The situation at low DCMU concentration can be better understood by considering an extreme case, where the activity of PSI is constant, i.e. the decrease of LEF with increasing DCMU is compensated by an increase of CEF. In such a situation, the Y(total) would decrease linearly with Y(II) with a yintercept at 0.5 (the fraction of PS corresponding to PSI given the stoichiometry PSII/PSI close to 1 in Chlamydomonas reinhardtii). In the Figure 3-7, the red dashed line represents this extreme case scenario. At low concentrations of DCMU (inhibition of Y(II) lower than 20%), experimental data is compatible with this simulation. We can therefore assume that at low DCMU concentration, the CEF compensates the decrease of LEF at the level of the PSI.

At high concentration of DCMU however, the opposite behavior takes place: Y(total) decreases faster than Y(II). This means that CEF decreases dramatically with decreasing PSII activity. We attribute this effect to the need of electrons from PSII to compensate for the electron loss in the CEF process due to leaks downhill PSI (to CBB or other metabolic sinks).

The fact that CEF in saturating DCMU concentration is lower than its maximal value due to those leaks is well documented in Chlamydomonas reinhardtii (Nawrocki et al. 2019c;Joliot and Johnson, 2011). In summary, the Y(total) relationship to Y(II) leads us to make three conclusions:

-The observation that Y(total), reflecting activities by both photosystems, and Y(II), reflecting the activity of PSII alone, are not proportional to each other during DCMU titration necessarily implies some activity of CEF in Chlamydomonas reinhardtii.

-Under low DCMU concentrations, PSI activity seems to remain constant despite LEF inhibition, which indicates that CEF activity replaces LEF activity in PSI when PSII is inhibited, i.e. a competition between LEF and CEF.

-Under high DCMU conditions, when PSII activity becomes very low, the total activity diminishes faster than PSII activity, which reveals that CEF decreases together with LEF. We propose that such a dependency of CEF on LEF stems from the need for electrons from PSII to compensate the electron leak from CEF.

Chapter 3: Diversity of cyclic electron flow in microalgae

Suzanne FERTE -Doctoral Thesis -2019

Partial conclusion on CEF diversity

Despite its proposed crucial role for photosynthesis regulation, the CEF in many photosynthetic groups is hardly investigated. In plants and green algae, although CEF has been investigated for almost 70 years, models of regulation remain unclear. I believe that the approach proposed in this section will make it possible to study further the diversity of CEF and to understand better its relationship with LEF. The approach used here is robust and reliable because: (i) it relies on quantum yield measurements only, whose reliability have been established by cross validation (Genty et al, 1992), (ii) it does not require the assessment of absorption cross-sections, which are prone to bias, and (iii) it can distinguish between the three main possibilities: the absence of CEF, the presence of CEF independent from LEF and the inter-dependent CEF and LEF.

We have shown that several models of relationship between CEF and LEF occurs depending on the species suggesting that CEF regulation models can differ between species. It is important here to highlight that the three behaviors we have identified are not signatures of the species, since the CEF behavior has been investigated only in one specific condition (see discussion, part 3.5.1.d).

This approach allows to highlight CEF and to study its relationship with LEF. But it can also be used to calculate absolute rates of CEF in two of the three cases. When CEF is independent on LEF, we can easily calculate the ratio CEF/LEF since CEF under saturating DCMU is then equal to CEF under physiological conditions (before addition of DCMU, when LEF is occurring). It is the case in the two dinoflagellates, Amphidinium carterae and Symbiodinium sp. In the case of A. carterae, it is an easy situation where one can conclude that there was no CEF in the conditions before DCMU was added. In Symbiodinium case, we calculated that one third of the PSI activity was a cyclic activity, assuming a PSII/PSI stoichiometry of 1.

When LEF is dependent on CEF though, physiological rate of CEF is not accessible through this method. The assessment of CEF in saturating DCMU conditions is not an option since by definition CEF is modified by DCMU. Then, a full picture of the CEF when CEF and LEF are inter-dependent cannot be obtained by the robust but simple protocol proposed in this chapter. This is one of the limitations of the method. Then, measurements of absolute rates, ETR(total) and ETR(II), are necessary, which themselves require the measurements of absorption cross-sections (σII and σtotal). This is the task I am going to work on in the next section, on the example of Chlamydomonas reinhardtii.

Results (II): Evaluating CEF and LEF absolute rates as a function of light irradiance in the green alga

Chlamydomonas reinhardtii

In the previous section (3.3), we proposed a method to test for the presence of CEF and to investigate the relationship between CEF and LEF. We could show that CEF behavior is not conserved among phylogeny suggesting that CEF regulation mechanisms are not universal.

The method proposed is sufficient to fully characterize CEF in two of the three types of situations presented. When there is no CEF, no further investigation is needed. If CEF is occurring independently of LEF, then, by nature, CEF rate measured when PSII is fully inhibited (saturating concentration of DCMU) is the same as the one occurring in physiological conditions. This enables easy access to CEF rate and the calculation of the CEF/LEF ratio is straightforward. This was the case in Symbiodinium sp in the conditions we used. It was unexpected on the basis of our knowledge on CEF in plants and green algae.

When CEF is dependent on LEF, it is not possible to assess CEF rate in physiological conditions with the method proposed in the previous section. We need to measure absolute LEF and CEF rates and this is the aim of this section.

We decided to use Chlamydomonas reinhardtii as a working organism. As shown in the previous section (3.3), in this microalga CEF is dependent on LEF and the method we proposed is not sufficient to assess CEF in physiological conditions. In addition, it is a model organism for photosynthesis investigation in the green lineage and many controls have already been carried out on spectroscopic methods for the study of photosynthetic physiology. Moreover, it is the only microalgae for which we have mutants of proteins involved in CEF, PGRL1, PGR5 (Alric 2014), or of the kinase involved in state transitions (stt7, [START_REF] Depège | Role of chloroplast protein kinase Stt7 in LHCII phosphorylation and state transition in Chlamydomonas[END_REF]. Moreover, it does not perform any qE when grown under low light (Peers, Nature, 2009). Single mutants can be crossed allowing easy establishment of double mutants.

As you will see, obtaining LEF and CEF absolute values requires many different measurements and controls. It is not a rapid screening protocol like the one developed in the previous section. We divided this work into two parts: the first one is dedicated to testing the reliability of the methods for assessment of absorption cross-sections (3.4.1 and 3.4.2) and the second part aims to estimate absolute values of CEF under different conditions of light irradiance (3.4.3).

Chapter 3: Diversity of cyclic electron flow in microalgae

Suzanne FERTE -Doctoral Thesis -2019

ECS-based estimations of the PSI and PSII absorption cross sections

Y(total) is accurately measured by ECS and Y(II) is assessable with fluorescence measurement. The PSII/PSI stoichiometry measured by flash-induced ECS (see Methods) is also a routine measurement. Here, the methodological challenge is to estimate the PSI, PSII and total absorption cross-sections. In a first time, we will expose the method we used to estimate those parameters with ECS measurements (paragraph 3.4.1). The approach is similar to what was used in (Roberty el al, 2014). Then, we will test the results using two cross validation approaches (paragraph 3.4.2).

Measurement of photochemical rate of both photosystems at light onset is assessable thanks to ECS. When the light is switched on, the PS start working immediately while the activation of the b6f and ATPase, that are non-photochemical steps, is slower. In the first ms after light onset, we assume that the increase of the electric field is only due to the activity of the PS. This activity depends on light irradiance, absorption cross-section and the fraction of open photosystems. If all PS are open, then the slope is proportional to the product of light irradiance and absorption cross-section. Absorption cross-sections should therefore be measured in conditions where the photochemistry is not limited by the availability of photosystems donors and acceptors. It is the case in dark-adapted samples; then, all the complexes of the chain are at the thermodynamic equilibrium with the ambient redox potential: the PSII and PSI primary donors, P680 and P700, are reduced and the primary acceptors, QA and iron-sulfur proteins, are oxidized.

Let's start with the measurement of PSI absorption cross-section. It is possible to inhibit the participation of PSII to the electric field creation by the addition of two inhibitors, DCMU and hydroxylamine (HA). In these conditions, ECS increase at light onset is only due to PSI charge separation and PSI photochemistry only depends on the light irradiance and the absorption cross-section (and its maximal quantum yield, close to 1 for PSI). The ECS increase at light onset is therefore a good measurement of the product of the light irradiance and the absorption cross-section of PSI, σI. The situation is more complex for the measurements in the absence of PSII inhibitors. Of course, the slope of ECS increase then reflects the sum of PSII and PSI photochemistry. But it cannot be considered as the product of light irradiance and the sum of PSI + PSII absorption cross-sections. This is because the maximal quantum yield of PSII, Fv/Fm, is significantly different from 1. Therefore the slope is proportional to I.σtotal, where σtotal is not strictly speaking the sum of PSI and PSII absorption cross-sections.

Instead:

(6) σtotal = σI + Fv/Fm σII The photochemical rate of PSII is simply the difference between the total photochemical rate and the PSI photochemical rate. The absorption cross-section of PSII can then be directly calculated from equation ( 6) as:σII = (σtotal -σI) / (Fv/Fm)

Validation of the method for measurement of absorption cross sections

a) Cross-validation of the two ECS-based methods to estimate ETR(total)

In order to test the reliability of absorption cross-section measurements, we propose to compare the values of ETR(total) measured with the two methods mentioned in 3.3.1.

Indeed ETR(total) can be calculated as the product of σtotal, I and Y(total) but also with an alternative ECS-based method which does not require measurements of absorption crosssections. The latter is the measurement of the photochemical rate of PSI and PSII through the initial slope of ECS at the offset of light (see Methods). Figure 3-9 shows ECS decay after light offset that were previously set at 135 (A) and 1500 (B) µmol photons m -2 s -1 . Data has been fitted by an exponential decay in the first 16 ms (that we expect if b6f and ATPase activity do not change during the time of measurement) and initial slope has been drawn in red. As expected, the initial slope is higher at 1500 µmol photons m -2 s -1 than at 135 µmol photons m -2 s -1 , reflecting a higher photochemical rate at higher light irradiance. (Alric, 2010) affecting the estimation of Y(total). With our spectroscopic tools, it is not possible to discriminate charge recombinations occurring faster than the hundreds of microsecond timescale, i.e. between P700 and the phylloquinone.

The centers giving rise to such unstable charge recombinations will therefore be counted as closed. But we observed that under high light, the ECS decay after the saturating laser flash was biphasic with a first phase taking place in between 0.3 and 1.2 ms. This early phase was attributed to slower charge recombination, i.e. to ECS creation that was not due to a stable charge separation. In order to remove this contribution, we followed a modified protocol. In a background light irradiance, we followed the increase of ECS upon a laser flash and then followed the relaxation of this ECS during a short (22 ms) period of darkness. We fitted the ECS decay after rejecting the first point (300 µs after the flash) by a mono-exponential decay and extrapolated the curve to get the amplitude of ECS increase after the laser flash that was due to stable charge recombination only. Thus, the fitted Y(total) diverged from the flash-induced ECS increase. The Y(total) obtained with this modified protocol is plotted as a function of light irradiance in proportionality between the two methods but fluorescence based estimation is 33% higher than ECS based one. The reason for this discrepancy can be the known underestimation of the PSII absorption cross-section due to the small reduction of QA (the fluorescence value in the dark is slightly higher than the true F0) in the presence of DCMU (Tian el at, 2019).

In the next section and for the sake of clarity, we will present the reasoning steps with the results obtained with ECS-based σII. But the figures on which the discussion is based will also show the results obtained from σII estimated by fluorescence. It is important to note that here, ETR(total) and ETR(II) are not expressed in the same unit, e.g. ETR(II) is expressed per PSII and ETR(total) is expressed per total PS. If we want to compare those flows and calculate ETR(I) from them, one needs to express them in the same unit. Here, we will normalize all the ETR per total PS. For this purpose, we multiply ETR2 by the ratio n2 which is the fraction of PSII (number of PSII/number of PS). Here, the raw data is the same as in Figure 3-13 but ETR(total) and ETR(II) are both normalized per total PS. ETR(I) was calculated as the difference between ETR(total) and ETR(II) (being normalized per total PS).

panel C). This indicates that the ATP/NADPH ratio produced by the light phase of photosynthesis is ~2.25 at all irradiances, significantly higher than the ratio required for carbon assimilation (1.5) even the higher value of 1.65 expected in low CO2 concentration (Kramer and Evans, 2011). The ATP overage is used for other metabolic pathways such as carbon concentrating mechanisms. Indeed, it is known that CEF is increasing in conditions where inorganic carbon is lowly available (Lucker & Kramer, 2013b), strongly suggesting that Calculations of ATP/NADPH ratio produced by the chain (Figure 3-16, closed circles) shows a lower -but still independent of light -between 1.5 and 1.8. This means that the photosynthetic chain ATP production at least equals the CBB cycle even when the highest estimation of the PSII absorption cross-section is used. In this chapter, we first developed a simple and robust method based on the measurements of only two parameters, the quantum yield of PSII by fluorometry, and the "averaged" quantum yield of photosystems probed with ECS. Titrating these parameters with an inhibitor affecting specifically LEF (DCMU) enables (i) to test the existence of CEF in a given species and in a given condition and (ii) in case CEF is occurring, to characterize the relationship between CEF and LEF.

This method seems particularly convenient for screening CEF diversity. Chlorophyll fluorescence is a signal available in all organisms performing oxygenic photosynthesis. ECS is also present in most of the eukaryotic photosynthetic organisms (Bailleul, Cardol, Breyton, & Finazzi, 2010), including diatoms (Bailleul et al, 2015), dinoflagellates (Roberty et al, 2013), eustigmatophytes [START_REF] Meneghesso | Photoacclimation of photosynthesis in the Eustigmatophycean Nannochloropsis gaditana[END_REF] and cyanobacteria (Viola et al., 2019).

chloroplast ATP synthase in Amphidinium carterae possess 12 (or less) c subunits. In that case only 12H + would be needed to produce 3 ATP molecules and the ratio of ATP/NADPH produced by LEF would be 3/2, reaching exactly the CBB cycle requirement. Is this a credible hypothesis? Phylogenetic diversity of the chloroplast ATP synthase structure has scarcely been investigated until now but it seems that the number of c subunits of ATP synthases may vary according to genetic or physiological determinants. Structural data show that the stoichiometry of c subunits per (C)F1 in spinach chloroplast CF0-CF1 is 14 [START_REF] Seelert | Structural biology: proton-powered turbine of a plant motor[END_REF] while it ranges between 13 and 15 in cyanobacteria (Pogoryelov et al., 2007) but is only 10 yeast mitochondria [START_REF] Stock | The rotary mechanism of ATP synthase[END_REF]. The number of c subunits could also depend on environmental determinants like carbon source as shown in E. coli [START_REF] Schemidt | Effects of carbon source on expression of Fo genes and on the stoichiometry of the c subunit in the F1Fo ATPase of Escherichia coli[END_REF]. It is to note that changes in the number of c subunits number induce a change the Gibbs free energy per proton translocation needed to synthesize ATP: if there are less fold, the minimal energy per proton required would increase. The second possibility for Amphidinium carterae is to replace the CEF by other alternative pathways, like water to water cycles. By definition, the alternative pathways cause rerouting of high energy electrons produced by the photosynthetic chain to other pathways than carbon reduction.

These alternative routes therefore enable to increase the ATP/NADPH ratio available for the CBB cycle. They also generate an extra pmf which would play a role in PSI and PSII photoprotection (Eberhard, Finazzi and Wollman, 2008).

Can we say that Amphidinium carterae does never perform CEF? This would be the case if the genes coding for enzymes required for CEF were absent from the genome, or if some structural differences in the structure of the b6f disallowed direct transfer to Qi site of b6f, for examples. Without surprise, the answer is no. Results obtained with our protocol is valid in one species/ one condition. This is illustrated by the following experiment.

In order to understand better the determinants of CEF and thus to better discriminate between genetic capacity to perform CEF and physiology influence on CEF behavior, I tested CEF behavior in Amphidinium carterae in different environmental conditions. Amphidinium carterae was cultivated in minimum media with 10% nitrogen concentration. The stationary phase occurred before the one in complete media (Figure 3 rereduction rate (Alric, 2010).

In our case, we calculated that roughly a third of PSI was involved in CEF. From equation (5) this gives an ATP/NADPH ratio of ~1.7, which is largely sufficient to rule out a shortfall of ATP for downhill metabolic processes. These considerations are true for the physiological conditions used in the experiment and the light irradiance that Symbiodinium experiences during in the experiment.

The independence between CEF and LEF suggests a functional isolation of the two pathways [START_REF] Anderson | Insights into the consequences of grana stacking of thylakoid membranes in vascular plants: a personal perspective[END_REF]. PSII are mostly localized into the grana regions while PSI are mostly concentrated into lamellae [START_REF] Anderson | Insights into the consequences of grana stacking of thylakoid membranes in vascular plants: a personal perspective[END_REF]. If plastoquinone or plastocyanin diffusion from one region to the other one is limiting, then regions are functionally isolated (at least partly). One can imagine that one third of the PSI are physically separated from PSII, then it would explain the CEF behavior in Symbiodinium. However, the ultrastructure of thylakoids in secondary plastids is very homogeneous, showing no grana/lamella structuration (Pyszniak and Gibbs, 1992;[START_REF] Bedoshvili | Chloroplast structure of diatoms of different classes[END_REF]. But this does not mean that segregation between photosystems is absent; for example PSII can be organized in patches in cyanobacteria [START_REF] Folea | Domain organization of photosystem II in membranes of the cyanobacterium Synechocystis PCC6803 investigated by electron microscopy[END_REF]. Another possibility would be that CEF are isolated from LEF by protein arrangement. Several works propose that CEF occur in super-complexes containing PSI, cytochrome b6f and proteins involved in reducing power reintroduction into the chain (Iwai et al, 2010). In supercomplexes, the distance between PSI first acceptors and cytochrome b6f is reduced, which would contribute to reduce drastically the competition with LEF, thus highly favoring CEF rather than electron leak to other pathway.

What does a model of independence between CEF and LEF mean regarding the putative role of CEF in ATP/NADPH ratio equilibration? To my opinion, there are two possibilities: if the limiting step of the electron transfer process in the physically separated CEF and LEF chains are the same (e.g. cytochrome b6f) then the light dependence of the CEF and LEF will be the same. Therefore, one can imagine that the ATP/NADPH ratio remains the one we calculated (i.e. ~1.7) regardless of light irradiance. But if this is not the case, if the electron flow in CEF chains saturates at lower intensities than the electron flow in LEF chain, then the problem of the ATP shortfall arises again under high light. Symbiodinium species have been shown to possess efficient alternative electron flows under high light conditions, which were attributed to Mehler reaction (Roberty et al, 2014). Understanding the photosynthetic regulation in Symbiodinium will therefore require a more sophisticated approach to consider both CEF and water to water cycles.

c) The DCMU-titration method in the field?

One question I wanted to address is whether this method can be applied in the field. In June 2017, I had the pleasure of being welcomed by Laure Guillou, within the UMR "Adaptation and Diversity in Marine" of the biological station of Roscoff (France). Near the station, the Penzé River flows into the sea and thanks to the high nutrient content of the water [START_REF] Maguer | Nitrogen and phosphorus requirements of an Alexandrium minutum bloom in the Penzé Estuary, France[END_REF], its estuary is the site of massive microalgae development in the spring [START_REF] Chambouvet | Control of toxic marine dinoflagellate blooms by serial parasitic killers[END_REF]. In June 2017, we were able to collect a sample rich in diatoms that we concentrated and purified by filtration on a 20μm filter. We kept the retentate and the cytological analysis revealed a composition largely dominated by one species, Chaetoceros (more than 94% of the phytoplankton cells, Figure 3 This experience shows that CEF measurement in the field is possible and this paves the way of the study of environmental determinants of CEF, in situ. However, it should be noted that certain conditions are necessary to use this method. First, algae concentrations must be important in order to get a signal-to-noise high enough. This is particularly important for the ECS absorbance changes measurements. In the laboratory, the minimum concentrations of microalgae required to have a noise of less than 10% of the signal vary between 10 6 and 10 7 cells/mL depending on the species. In the field, microalgae concentrations of 10 5 are reached anecdotally (e.g. Vardi et al., 2004[START_REF] Lefebvre | Long-term hydrological and phytoplankton monitoring (1992-2007) of three potentially eutrophic systems in the eastern English Channel and the Southern Bight of the North Sea[END_REF] and would make it possible to obtain satisfactory concentrations by means of concentration processes. But the average phytoplankton concentration in the ocean are often lower (estimated between 10 and 5.10 3 cells/mL, Pesant et al., 2015). Another major constraint is that monospecific samples are required. Indeed, in a mixture of species, measurements reflect the signal emitted by all the species present in the sample. An average signal is then measured which does not make it possible to determine the specificities of each species. is depending on LEF rate. Small perturbations of photosynthesis by adding small amounts of DCMU revealed that CEF increases and compensates for the loss of LEF. This behavior is a signature of a competition between the two processes, which could occur at the PQ pool, at the b6f level or on the PSI acceptor side (Figure 3-17 panel F). This will be discussed further in part 3.5.3.

Limitations of the DCMU titration method

The protocol used to explore the CEF diversity is based on the hypothesis that throughout the DCMU titration, relative variations of Y(II) and Y(total) are equal to relative variations of ETR(total) and ETR(II), respectively. This remains true only if:

-the PSII and total absorption cross-sections, σII and σII, remain constant during the experiment. Here, all the strains used do not perform state transitions and this criterion can be considered met.

-the stoichiometry of the two photosystems does not change. It is reasonable to suppose that it is the case given the relatively short duration of the experiment.

-the expression ETR(total) = Y(total). σtotal. I is indeed a good measurement of the total photosystem activity which is the weighted average of ETR(I) and ETR(II), i.e. the photochemical rate.

A priori, the expression ETR(total) = Y(total). σtotal. I suffers of several approximations. As stated previously, it cannot be derived mathematically from the sum of ETR(II) and ETR(I) (see general discussion). However the comparison between ETR(total) and the photochemical rate gave a very good agreement (part 3.4.2, Figure 3-11D). It would however be interesting to follow the same protocol using the photochemical rate (measured as the ECS initial slope at light offset) instead of the ETR(total).

The most important in the DCMU method is that the conclusions of the experiments are not affected by these approximations; the demonstration of the validity of the Y(total) versus Y(II) titration method is demonstrated in the case of a "puddle" model (see appendix 1).

Chapter 3: Diversity of cyclic electron flow in microalgae

Suzanne FERTE -Doctoral Thesis -2019

Another possibility would be to use other treatments than DCMU to modify the LEF, and study the modification of the CEF/LEF ratio in these treatments. LEF and CEF share the electron transfer chain from the PQ pool to PSI acceptors, so the two possibilities to affect specifically LEF are (i) using PSII inhibitors, i.e inhibiting LEF uphill the PQ pool (that is the approach we used here) or (ii) inhibiting the LEF downhill PSI acceptors. We can imagine two ways to achieve the second possibility -there are more.

One would be to play with the substrate of carbon assimilation, CO2; this is very similar to the approach used by Lucker and Kramer (Lucker and Kramer, 2013), who demonstrated that the CO2 status modifies the CEF/LEF. The authors conclude that CEF is needed to provide extra ATP for CBB cycle and CCM to operate. In the light of the work of Lucker and

Kramer the high level of CEF in our samples suggests that in our conditions Chlamydomonas reinhardtii was CO2 limited; this is likely since our sample was only manually mixed every minute which might be insufficient to replenish the sample with CO2. We expect the CEF/LEF ratio to decreases if CO2 is bubbled in the cuvette.

An alternative way to affect the metabolic pathways downhill PSI would be to modify temperature. Temperature modifies the kinetics all physico-chemical processes but will especially affect the fate of electrons downhill PSI, which require diffusion of molecules.

Despite a complex relationship, CEF and LEF remain proportional at all light intensities in Chlamydomonas reinhardtii

The increase of the CEF when LEF is inhibited indicates a competition between these two tracks. Since the two processes share the same components except the "cycling step" the competition most likely takes place at the level of the substrates of this step. If we consider the FQR pathway or the NDH pathway (here NDA2 in Chlamydomonas reinhardtii, [START_REF] Peltier | Chlororespiration[END_REF], this means that the sites of competition could be the PQ pool or the ferredoxin pool. This is compatible both with the model proposed by Allen in which he states that CEF depends mainly on PQ redox state (Allen, 2003), and with the model proposed by

Alric and co-workers of a CEF regulation at the level of the PSI acceptors, ferredoxin or NADPH (Alric, 2010).

The measurement of the absolute rates of CEF and LEF at different light irradiances revealed that the CEF/LEF ratio was very important (0.5 or 1.5 depending on the mode of estimation of the absorption cross-section of PSII). It is not easy to find a model which can explain both the concomitant changes of CEF and LEF with light irradiance, but their inter-dependence during DCMU titration. The parallel increase of CEF and LEF with light is not compatible with the regulative model proposed by Allen (Allen, 2003) in which PQ redox state would be the main determinant of CEF amplitude. Indeed, from low light to high light, because cytochrome b6f becomes limiting, the plastoquinone pool gets reduced (almost fully). Hence, under this hypothesis, CEF should decrease with light irradiance while LEF is increasing and CEF/LEF would not decline. Regulative process compatible with this behavior will be discussed more deeply in the overall discussion. Another surprising observation which must be taken into account in the building of a model of the CEF-to-LEF regulation is the fact that the LEF saturates at 100-125 electrons per second per PSII, which would often be interpreted as a sign of photosynthetic control. However, the PSI activity saturates at 200-250 electrons per second per PSI, which means that the cytochrome b6f, which is stoichiometric to PSI in Chlamydomonas, transfers electrons with a maximal turnover of ~5ms or less, close to the maximal turnover rate of the cytochrome b6f and therefore incompatible compatible with a photosynthetic control.

Finally, one important point to discuss is the difference of ETR amplitudes between the three replicates shown in section . In Figure 3-21, we represented the fraction of PSI involved in CEF in the three replicates that is rather stable at all light irradiance. However, its average amplitude varies depending on the replicate. This difference in CEF amplitude can be attributed to difference in physiological conditions, for example the CO2 availability, which is not well controlled in our set up, and that is known to influence the CEF amplitude (Lucker and Kramer, 2013). But if the biological variability prevents us from giving a precise value of the CEF rate, this variability also has one advantage. Indeed, the rather stable CEF/LEF ratio in all replicates, even if the amplitude is different, is a strong argument in favor of a fine tuning of the CEF/LEF ratio. (Joliot, 1964), following a similar observation by Vredenberg and Duysens in a purple photosynthetic bacterium [START_REF] Vredenberg | Transfer of energy from bacteriochlorophyll to a reaction center during bacterial photosynthesis[END_REF]. Because of the connectivity between PSII centers, the fluorescence rise is sigmoidal instead of exponential in plants and Chlorella [START_REF] Strasser | The fluorescence transient as a tool to characterize and screen photosynthetic samples[END_REF]. The reason why the sigmoidicity does not always appear clearly is because in DCMU conditions, a fraction of QA is reduced in the dark. The area above the fluorescence induction curve corresponds to the average time between two photo absorption, assuming that the initial state is [P 680 , Q A ] (open PSII reaction centers) and the steady-state is [P 680 , Q A -] (fully closed PSII). We know that at least one of these conditions is not met. This is because the semi quinone QB -is at the equilibrium with QA and when DCMU replace QB, part of the QA remains reduced (Lazár, 1999). It can be seen in Figure 3-12A

where the initial value at the beginning of the fluorescence rise is slightly higher than the true F0 measured in the absence of DCMU. This experimental default may explain the divergence between the σII measured with fluorescence or with ECS. A recent work suggests that the underestimation of the area above the curve, and thus the underestimation of the σII can reach up to 36% when F0 were increased by a factor 1.5 [START_REF] Tian | pH dependence, kinetics and light-harvesting regulation of nonphotochemical quenching in Chlamydomonas[END_REF].

Considering the sigmoidicity of the fluorescence rise in the fluorescence-based estimation of the PSII absorption cross-section might reconcile its estimations by fluorescence and ECS, and strengthen the conclusions we have drawn from the calculations of the light dependence of absolute rates of LEF and CEF.

After studying CEF in the laboratory, where conditions are well controlled, I wanted to investigate this process in the field and had two opportunities to do so. The first one was the study of the phytoplankton community sampled in Penzé estuary (Brittany, France) which allowed me to perform a study of the dependency of CEF on LEF in a natural population of diatoms (presented in 3.5.1). The second attempt to do so was in two field campaigns that took place in the Bergen marine station (Norway), the goals of which were to study the rapid development (i.e. bloom) and demise of a population of the coccolithophore Emiliania huxleyi.

At the risk of spoiling the suspense too rapidly, this second attempt failed. This chapter will therefore seem a little aside of the main topic of my thesis at first sight: it aims to understand the determinants of the photosynthetic physiology of the coccolithophore during the course of the bloom. We thus attempted to determine the major environmental (biotic and abiotic)

factors influencing photosynthesis and more specifically the linear electron transfer. However, questions about the CEF amplitude will emerge from this study.

Introduction

The coccolithophore Emiliania huxleyi is one of the species that forms the largest ocean blooms (Tyrrell and Merico, 2004). Reaching areas of several square kilometers, these bodies of water containing up to tens of thousands of cells per milliliter are visible from space as whitish areas in the ocean (Tyrrell and Merico, 2004). Emiliania huxleyi blooms, which develop in time scales of the order of the week, then collapse in a few days giving rise to a massive release of organic matter feeding the trophic chain, and calcitic shells that surround them can sediment and give rise to carbon storage in the form of calcareous sediments [START_REF] Bramlette | Significance of coccolithophorids in calcium-carbonate deposition[END_REF][START_REF] Milliman | Production and accumulation of calcium carbonate in the ocean: budget of a nonsteady state[END_REF]Westbroek et al., 1993). The parameters determining the birth and death of a bloom are still an unresolved research topic. The causes of Emiliania huxleyi's bloom demise are most often attributed to a specific virus of this species, Emiliania huxleyi virus (Ehv), that is part of the DNA double stranded nucleocytoplasmic, viruses family of Phycodnaviridae [START_REF] Wilson | The Phycodnaviridae: the story of how tiny giants rule the world[END_REF]. However, it has recently been shown that some strains of Emiliania huxleyi are resistant to viruses and that bacteria could also be involved in bloom demise [START_REF] Barak-Gavish | Bacterial virulence against an oceanic bloom-forming phytoplankter is mediated by algal DMSP[END_REF].

In order to study the dynamics of the birth and death of a bloom, an intermediate solution between the laboratory reductionism and the open sea oceanography surveys, more random because of the difficulty of predicting blooms, is the use of mesocosm [START_REF] Odum | The mesocosm[END_REF]. During campaign was part of a collaboration with several international laboratories, each of them bringing a particular expertise (see material and methods of this chapter, section 4.2.1) for a better understanding of the ecological determinants of an Emiliania huxleyi bloom and demise. Within this consortium, we were responsible for following the evolution of photosynthetic physiology. Emiliania huxleyi is a photosynthetic species and the vast majority of its anabolic pathways are provided by the products of photosynthesis. When investigating the reasons of its growth, measuring the efficiency of its photosynthesis is crucial. Moreover, in such organisms, the study of photosynthesis is a unique tool to address the physiological state of whole cells and to investigate the result of abiotic stresses such as nutrient deficiency (e.g. [START_REF] Cullen | Nutrient limitation of marine photosynthesis[END_REF][START_REF] Kolber | Effects of growth irradiance and nitrogen limitation on photosynthetic energy conversion in photosystem II[END_REF][START_REF] Berges | Differential effects of nitrogen limitation on photosynthetic efficiency of photosystems I and II in microalgae[END_REF];[START_REF] Jiang | Photosynthetic performance, lipid production and biomass composition in response to nitrogen limitation in marine microalgae[END_REF]), iron deficiency [START_REF] Muggli | Effects of nitrogen source on the physiology and metal nutrition of Emiliania huxleyi grown under different iron and light conditions[END_REF], light or temperature stress [START_REF] Loebl | Physiological basis for high resistance to photoinhibition under nitrogen depletion in Emiliania huxleyi[END_REF][START_REF] Trimborn | Effect of varying calcium concentrations and light intensities on calcification and photosynthesis in Emiliania huxleyi[END_REF][START_REF] Teoh | Response of Antarctic, temperate, and tropical microalgae to temperature stress[END_REF].

We had several objectives, some more ambitious than others. The first one was to follow the photosynthetic physiology of Emiliania huxleyi and to identify the environmental parameters which determine the efficiency of photosynthesis. We had two other objectives whose attainment were more uncertain. The first one was directly related to CEF: we hoped that the cell concentration reached at the top of the bloom would be high enough to test our CEF methods in the field, on Emiliania huxleyi dominated samples. But even at the top of the bloom and with gentle cell concentration, signals-to-noise of the absorption difference spectroscopy approaches were too low and this objective was abandoned. The other one was to identify a photosynthetic signature of the viral infection (if a viral infection happened), which seems to be successful as we will see in the following. -the connectivity between PSII, p, that explains the shape of the induction curve (sigmoidal versus exponential). where ETRmax is the maximal PSII electron transfer rate when light is saturating, Ek is the light saturation parameter. In order to exclude light intensity that were much higher than the saturating light (for which the measurement noises are important), two consecutive fits were performed. The first one enabled a preliminary estimation of Ek from the whole dataset and the second one used only data below 3*Ek intensities. ETRmax and Ek were taken from the second fit and the initial slope of the fitted curve was noted α and was calculated as α=ETRmax/Ek. Data presented in this graph were measured on day 14 and on bag 3.

The complete measurement protocol for photosynthetic physiology assessment consisted of a first measurement right after sampling that measured the maximal fluorescence in situ, Fm'.

A second fluorescence induction was performed after one minute of dark adaptation to estimate Fv/Fm in situ. Then, an incubation of 15 minutes at low light intensity (10 to 20 μmol photons m -2 s -1 ) allowed a relaxation of the mechanisms of dissipation of excess light in place in situ (qE, fast reversible NPQ). The time required for this relaxation to be complete was tested daily around 10 am and never exceeded 10 minutes (data not shown). The samples were then subjected to a series of increasing light intensities and a fluorescence induction measurement was performed after acclimation to each light step. The time required for full acclimation to each light step was tested at the beginning of the experiment (and checked every day) and a measurement program was written specifically for our project by the miniFIRe designer, Max Goburnov (FPRO-ut for FIRe Programme -Utile Time) and was calibrated to ensure that steady state photosynthesis was reached at each light intensity.

Statistical analyses

All statistical analyses were performed using R version 3. Between-Class Analyses (BCA) were performed using the package ade4.

Principal Component Analysis (PCA) is a mathematical algorithm that reduces the number of dimensions of a dataset while retaining most of its variation. It achieves this reduction by identifying directions, called principal components, along which the data variation is maximum. Samples can then be plotted, along those axes allowing a visual assessment of similarities and differences between samples and whether samples can be grouped together [START_REF] Hotelling | Analysis of a complex of statistical variables into principal components[END_REF][START_REF] Bibliography Abdi | Principal component analysis[END_REF]. Here, based on a first PCA applied to a numerical matrix of variables, we applied a BCA that operates another set of rotations to maximize contrasts among previously defined groups. Instead of maximizing the projection of variance bound to individual observations such as done with a PCA, the BCA maximizes the projected variance of group barycentres. d) Partial least square regression (PLS) Partial Least Square regression is generally used to explain or predict a response variable Y as a function of a set of predictors (dependent variables, X). It is generally used (i) when the number of predictors is high as compared to the number of observations and (ii) when the predictors are intercorrelated (multicollinearity) as it is often the case in environmental variables. More usual statistical approaches such as the multiple regression may not estimate correctly variable coefficients when the predictors are multicolinear. Here we use PLS regression of type I (or PLS1), that is a mode of PLS regression where the response is univariate.

Instead of directly relating the Y variable to the predictors X, PLS regression makes uses of latent variables (i.e., multivariate axes) to relate those. More precisely, PLS regression determines orthogonal components in the matrix X of predictors that maximize their correlation with the Y variable. The analysis then reconstitutes the regression coefficients of the original predictors based on their association with the multivariate components.

It may therefore be interpreted in a similar manner to multiple regression: (i) a coefficient of determination R2 represents the amount of variation in Y explained by X, (ii) the sign and values of the regression coefficients provide information on the effect of a predictor on the response variable. Note that predictors which are not well represented on the multivariate axes will tend to be characterized by coefficients close to 0.

We performed the PLS regression using the R package (pls). The optimal number of components used in the analysis was determined based on a cross validation procedure ("leave-one-out"): it estimates the number of components minimizing prediction error while avoiding over-fitting, an issue that may arise when the model fits to closely to specificities of a given sample. The significance of PLS regression coefficients was assessed with a standard Jacknife method (i.e., a re-sampling procedure close to Bootstrap) that is provided in the pls package: 95% confidence intervals were calculate to test whether the regression coefficients significantly differed from 0.

Photosynthetic physiology a) Nine parameters to describe photosynthesis

One of the main objectives of our study was to evaluate the effect of abiotic environmental parameters on the photosynthetic physiology of microalgae (hoping that the population would be dominated by Emiliania huxleyi). One of the major constraints in the field is the fact that the environmental parameters, and in particular those having a circadian rhythm such as the light intensity and the temperature, vary rapidly over time. In order to minimize the drift between bags, we have developed a protocol to collect as much information as possible in the shortest possible time, i.e. not more than 2 hours between the first and the last measurement.

We controlled that the photosynthetic parameters were not changing significantly during those 2 hours by measuring the photosynthetic physiology of samples from open water (water sampled from the fjord, outside the bags) at the beginning and the end of the set of measurements. The photosynthetic parameters were similar for the two measurements (see one example for Fm in Figure 4-8A) indicating that the physiology was quite constant during the two hours of measurement.

In order to measure physiology in situ, we installed our fluorometer directly on the raft, which allowed the first measurement to be performed ~10s after sampling. The principle of our protocol is based on three steps.

First, we measured photosynthetic parameters in situ, just after sampling:

-The maximum efficiency of the PSII, in situ Fv/Fm (Figure 4-8H). In situ Fv/Fm is clearly lower in bags 1 to 4 than in bags 5 to 7 for days 9 to 14. These days were marked by a particularly high intensity of light and follow the stop of nutrient addition (Figure 4-8C).

-The in situ NPQ, mechanism of dissipation of excess light energy in the form of heat, that is occurring in situ and that is reversible in the minute time scale, (Figure 4-8I).

Then, after a relaxation period under low light to dissipate any fast reversible photoprotection mechanisms, we measured dark-adapted PSII parameters. We have initially tested that a low light incubation for 15 min was sufficient, whatever the intensity of light in situ (data not shown). We measured:

-The PSII maximal efficiency, Fv/Fm, i. -A second parameter is the characteristic saturation intensity of photosynthesis, Ek (Figure 4-8E) which gives information on the optimal intensity of operation of the photosynthetic chain.

-Thirdly, the α parameter (Figure 4 This protocol was carried out every day of the campaign. We hypothesized that environmental parameters play a role in photosynthetic physiology but how to evaluate the contributions of environmental parameters and stochastic effects on the variation of the photosynthetic parameters? By a statistical approach, we can assess the relative contribution to the variance of the photosynthetic parameters due to environmental parameters (which should be the same for all bags) and the variability between bags (which reflect technical noise, bias in the bag filling at day 0, stochastic effects). The fraction of common variability between the bags (repeatability score) estimates the amount of variability between days in the total variability of photosynthetic physiology. Overall, it appears that much of the variability in photosynthetic physiology is explained by external control (environmental parameters) as evidenced by high RS values (greater than 0.6 for all except in situ parameters and α, up to at 0.86 for ETRmax). In noise since the measurement was done "as quickly as possible after sampling" which does not exclude differences depending on the efficiency of the sampler.

Evolution of environmental parameters during the experiment

We have shown above that the variation of photosynthetic physiology appeared mainly due to environmental and / or population evolution parameters. The signals being mainly emitted by the nanoeukaryotes and mainly coming from Emiliania huxleyi from day 9 indicates that physiology is rather dictated by the environmental parameters.

Here we made the initial bet that abiotic factors would be the most important. Is that the case? What are the most important parameters? The principle of our approach is based on statistical model design explaining the variance of photosynthetic physiology using environmental parameters. In order to optimize our model, we used all the available environmental parameters that are known to influence the photosynthetic physiology in the laboratory: the temperature of air and water, the concentration of nutrients in their most abundant dissolved form such as nitrogen (NO3 -, NO2 -, NH4 + ) phosphorus (PO4 3-) and silica (SiO4 4-) and the light intensity at the time of measurement. The evolution of these parameters over time are presented in Figure 4-14. reflects the fact that inorganic nitrogen and phosphorous are increasing due to enrichment; from day 4-5 to 10, the evolution reflects mostly the decrease of inorganic nitrogen and phosphorous. From day 10 to 16, environmental parameters are rather stable. The evolution from day 16 to 21 indicate mostly that air temperature, water temperature and light decrease; finally, from day 21 to 23, the same parameters increase again.

It can be noted that several non-consecutive days cluster together (indicating that they are characterized by similar environmental parameters): days 1, 8, 14, 16; days 0, 10, 11, 12; days 9, 13 and 15 and days 17, 22, 23. These observations will be important for the robustness of the statistical model. Indeed, if the environmental parameters actually determine the photosynthetic physiology, then these days should have similar phenotypes.

Environmental determinants of E. huxleyi photosynthesis a) Pairwise correlations

How to highlight correlations between environmental variables, taxonomic distributions and photosynthetic physiology parameters? The simplest approach that I initially applied is to test for pairwise correlations between all those parameters. Correlation matrix is presented in Figure 4-16 where the correlation coefficient is indicated by a color from dark red (-1) to dark blue (+1). The level of significance is represented by the size of the disk and the crosses indicate non-significant correlations. Strong correlations can be highlighted such as between air temperature and Fv/Fm, ETRmax and connectivity or between inorganic nitrogen concentration and Fv/Fm, ETRmax, Ek and NPQ. The in situ Fv/Fm and in situ NPQ are strongly anti-correlated, which makes sense: the development of heat dissipation mechanisms in PSII should decrease its maximal efficiency. However, this type of approach seems limited since only pairwise correlations are tested and this could not highlight correlations if a photosynthetic variable depends on several environmental factors. To illustrate this, air temperature and light intensity are positively correlated, which is just a random effect (cloudy days from day 16 to 21 were also cold days). Then, is the positive correlation between ETRmax and light irradiance meaningful, or just an artifact due to the random correlation between air temperature and light irradiance?

b) Multivariate model

When several explanatory variables can be involved in the variations of a parameter, a multivariate approach is necessary. In order to understand better the mathematical concepts of this approach, I followed a multivariate modeling course organized by Agroparitech Partial least square analysis was performed on Fv/Fm, ETRmax and NPQ using 9 abiotic parameters (water and air temperature, SiO4 2-, PO4 3-, NO3 -, NO2 -, total inorganic nitrogen, NH4 + and light intensity). The percentage of the variance explained by the model is 75, 47 and 37% for Fv/Fm, ETRmax and NPQ, respectively. Value of the regression coefficients for each of the environmental variables are indicated in the horizontal axis. Error bars delimit 95% confidence intervals. In grey, blue and orange, the non-significant, positive and negative standardized coefficients, respectively.

Influence of nutrients on photosynthesis

The concentration of dissolved silica seems to be significantly correlated with Fv/Fm and ETRmax. This could be due to an artifact. The main variations of the silica concentration concerns a decrease during the first 3 days of the experiment (due to the development of diatoms in the samples seen by flow cam data, not shown), and is concomitant with an increase of Fv/Fm and ETRmax that might be related to the initial addition of nutrients.

Four parameters related to N availability are used in the model. The two most significant ones are the total dissolved inorganic nitrogen and nitrate concentration, whereas nitrite and ammonium show less clear behaviors. Moreover, nitrate and total nitrogen concentrations have the virtually the same influence on the three photosynthetic parameters considered here. This can be explained easily: nitrate is the most abundant form, and contributes to >90% of dissolved nitrogen. NH4 + represented a negligible fraction of the total nitrogen in the first 10 days, and only after day 10, its contribution to total nitrogen became significant and rather constant. Its apparent effect on ETRmax and NPQ could therefore be biased since the first 10

A photosynthetic signature of viral infection?

In the previous section, we have seen that an important part of the variance of our photosynthetic parameters can be explained by environmental parameters. This is especially true for ETRmax (75%), and to a lesser extent for Fv/Fm (47%). In this section, we will focus on the fraction of the variance which is not explained by the environmental parameters considered. Part of this unexplained variance comes from noise and stochastic effects, all the more important as the field experiments are subject to the vagaries of an outdoor laboratory. Some of it can stem from the fact that some of the environmental parameters are not well chosen; for example, we considered the light intensity at the time of sampling but maybe a most relevant parameter is the average intensity experiences over the last 24h, as it was shown for the green alga Chlamydomonas reinhardtii [START_REF] Nawrocki | Chlamydomonas reinhardtii exhibits de facto constitutive NPQ capacity in physiologically relevant conditions[END_REF]. Also, the parameters used for the nitrogen status are the concentration of dissolved nitrogen in the water at the moment of sampling. The dissolved inorganic nitrogen concentration is virtually null in the bags at days 10 to 12 and at days 15 to 17 (except for bag 7), even though for the second period, nutrient addition were performed daily. Therefore, it is possible that considering the concentration of dissolved nitrogen at the moment of the addition would be more relevant to assess the nutrient status of phytoplankton.

But it is also possible that some of the unexplained variance comes from a factor which has not been taken into consideration, which could correspond to biotic interactions for example. This possibility can be addressed by studying the remaining variance of a photosynthetic parameter, i.e. the residuals of the PSL model. The residuals correspond to the difference between the value predicted by the PSL model and the experimental value. If their distribution is stochastic, then it suggests that most of the unexplained variance stems from technical noise. If, on the contrary, a significant abnormality has a good chance to reveal a relevant predictor of the considered photosynthetic parameter. The residues calculated on the Fv/Fm are presented in Figure 4-19A. A generally similar residual variance is observed between the bags (RS = 0.71), and residues are small, except for four events: day 3 for bag 5, day 9 for bag 4, days 16-17 for bag 7 and days 18-19 for bag 4. The first event will not be considered here; it occurs before Emiliania huxleyi dominates the signal and therefore is difficult to attribute.

Those deviations can be highlighted by another means -less satisfactory from the point of view of statistics-which is to calculate, for each day, the difference between the Fv/Fm of a given bag and the median of all bags. The results are presented in Figure 4-19C and show that the decrease in Fv/Fm observed for the above-mentioned abnormalities correspond to a deviation of 25-30% from the median value. The same analysis was carried out on the ETRmax and does not display significant residues for the days concerned by the fall of Fv/Fm (note that overall ETRmax variance is much more important than Fv/Fm variance, see Figure 45678). The same analysis will be done in the near future on the other parameters of photosynthetic physiology. From the evolution of photosynthetic parameters (see the days concerned by the decrease of Fv/Fm do not show any particular phenotype concerning non-photochemical quenching (NPQ). But a drop appears for the two parameters which are expected to depend on the efficiency of PSII, the  parameter that characterizes the efficiency of photosynthesis at low light intensity and the in situ Fv/Fm on the particular days concerned by the negative residues of Fv/Fm (Figure 45678).

Conclusion and discussion

In this chapter, we followed the evolution of the photosynthetic physiology during a bloom of Emiliania huxleyi followed in seven mesocosms. The fluorescence measurements used to describe the photosynthetic physiology of the phytoplankton mainly reflected the signals emitted by the nanoeukaryotes and in particular by E. huxleyi, except during its growth lag phase from day 0 to day 9. A multivariate statistical approach allowed us to develop a model correlating the environmental parameters and the photosynthetic physiology of E. huxleyi explaining almost half of the total variance. This approach allowed us to highlight the main determinants of E. huxleyi photosynthesis. Most of the variations stems from the availability of nitrogen, air temperature and light intensity. Dissolved nitrogen and air temperature influence positively the maximal efficiency of PSII and the maximal rate of photosynthesis, and negatively the photoprotection mechanisms. Light intensity correlates positively with the three photosynthetic parameters. The statistical analysis did not reveal a significant influence of water temperature on photosynthesis of the coccolithophore, which suggests that there is no clear photosynthetic long-term acclimation to the temperature experienced in the mesocosm.

Once the contribution of the environmental parameters was "substracted" from the experimental values of PSII maximal efficiency and maximal photosynthetic rate, some abnormalities appeared. Around day 16-17 in bag 7 and day 18-19 in bag 4, a specific decrease of the PSII efficiency appeared (without significant modification of the maximal rate of photosynthesis). Those transitory drops strongly correlate to a bundle of evidences for a viral infection, in those bags only: an increase of viral genes, the detection of intra-cellular viruses in E. huxleyi single cells, the apparition of specific sphingolipids whose biosynthesis pathway is encoded in the viral genome, and finally growth arrest. A question immediately arises: has this specific photosynthesis phenotype already been described as a response of viral infection in the laboratory?

Several laboratory studies described such a decrease in Fv/Fm, concomitant with the increase in the concentration of viral particles and the decrease in the concentration of Emiliania huxleyi cells [START_REF] Evans | Viral infection of Emiliania huxleyi (Prymnesiophyceae) leads to elevated production of reactive oxygen species 1[END_REF][START_REF] Bidle | Viral activation and recruitment of metacaspases in the unicellular coccolithophore, Emiliania huxleyi[END_REF][START_REF] Gilg | Differential gene expression is tied to photochemical efficiency reduction in virally infected Emiliania huxleyi[END_REF]. In a mesocosm, a decrease of less than 10% of Fv/Fm was observed, which occured both during the bloom of Emiliania huxleyi and its demise (Kimmance and 2014). In this work, the possible environmental 2007). During the infection of Emiliania huxleyi by its coccolithovirus, the activity of PSII decreases. One could imagine that the maintenance of an active mitochondria throughout the infection progression [START_REF] Ku | Tracking infection dynamics at single-cell level reveals highly resolved expression programs of a large virus infecting algal blooms[END_REF] Another example relates to the measurement of Pm, the maximum variation of P700 absorbance between its completely reduced and completely oxidized state. In the presence of MV, when we expect the P700 pulse method to be reliable, we initially obtained a linear relationship between Y(I)P700 and Y(I)ECS. However, the slope of the relationship was not 1 but ~1.15, indicating an overestimation of Y(I)P700. This discrepancy led us to understand that we underestimated Pm, the normalization factor in the calculation of Y(I)P700. Indeed, under saturating concentration of DCMU and in strong light, we did not reach 100% of oxidized P700

because the reduction of P700 by the CEF became significant. We then modified the way we measured Pm by adding DBMIB which inhibits most of the CEF.

These changes in experimental procedures, however minor they may look at first sight, are of paramount importance since they radically change the results. When, as it is the case for the CEF investigation, several measurement methods are used while we try to measure potentially small values or variations, these details are particularly important. I have learned from my thesis to be cautious about the way one measures the CEF and LEF in

Chlamydomonas. Indeed, one cross-validation attempt failed during my thesis and remains to be solved: the discrepancy between the measurements of PSII absorption cross-section by the method based on ECS and on fluorescence measurements. As we have seen, this might stem from not considering correctly energetic connectivity between PSII centers, drawing the path for the next step of improvement. The principle of the method we proposed in chapter 3 (section 3.3) for the screening of CEF behaviors in different microalgae is to compare the changes of electron flow through PSII (i.e. the LEF) and the sum of PSI and PSII activities and to test the relationship models between LEF and CEF. In chapter 3 section 3.4, we showed that, when light intensity is varied, the DIRK method, and the method based on the estimation of Y(total), and the total absorption cross section, σtotal agreed with each other. However, the choice I have made to use the second method instead of the DIRK one is a priori questionable because the DIRK method is easy to use and does not require any strong assumptions. At the contrary, the validity of the ETR(total) as a measurement of the sum of PSII and PSI activities is not mathematically correct and it is necessary to question its window of validity. I will come back to this, but first I would like to insist on the importance of the work performed in chapter 1 to invalidate P700 based methods for the use of CEF.

a) Questioning the choice of ECS versus P700 to investigate CEF At the beginning of my PhD, we used a different protocol to investigate CEF. We used the P700 method to estimate the evolution of the flow through PSI and fluorescence for the one through PSII, as suggested in a recent review (Fan et al, 2016). The results obtained on a representative sample of Chlamydomonas reinhardtii are presented in These observations led us to question the P700 method, which gave birth to chapter 2 of my thesis. At the beginning, we compared the results obtained with ECS and P700 method, Y(I)P700 and Y(I)ECS respectively in order to understand better the conditions when P700 was not reliable. Thus, when we understood that Y(I)P700 was not reliable, we decided to study the CEF and LEF relationship by comparing the evolutions of Y(II) and Y(total) calculated from ECS signals (see Figure 5-2, black line). We therefore continued with the same single turnover flash method and did not consider other methods to estimate the total electron flow changes such as the photochemical rate. The choice of the ECS-based method we used in chapter 3 comes from this and deserves critical thinking.

Since n1.ETR(I) = n2.ETR(II) + n1.CEF, and σtotal = (n1.σI + n2.Fv/Fm.σII) equation ( 2) can be rewritten to express ETR(total) as a function of ETR(II) and CEF: In part 3.4, in order to calculate the absolute values of ETR(total), using this expression would have not been correct, despite the good agreement between the absolute values of ETR(total) and the photochemical rates obtained with the DIRK method. Instead, we used the latter method for this part of the chapter 3. Regarding the part 3.3, where we titrated the Y(total) and Y(II) to investigate the behavior of CEF, the use of ETR(total) is correct, assuming a "puddle" model. The demonstration of the validity of this approach is demonstrated in Appendix 1.

1. 3. Studying CEF and its abiotic and biotic determinants in the field:

an accessible project?

In the field, the complexity of the systems studied is much greater than in the laboratory: for example, there are mixtures of several species, microalgae are generally present in low concentrations and most of the time, several environmental parameters vary concomitantly. This is the interest of the field approach: we study the microalgae in their real life, which is the platform for new discoveries. But it has a price: the interpretation of the experimental results is much more complex than in the lab. When going into the field, one of my dreams was to measure CEF in situ and to study the biotic and abiotic determinants of its amplitude in the field. In this section, I would like to come back to the three constraints that we were able to identify concerning the study of CEF in situ and to discuss the ways we could overcome them in the future. If one wants to understand the biotic and abiotic determinants of CEF in the field, some conditions are required: we generally work at cell concentrations of the order of 10 7 cells/mL (which measure 3 to 20 µm in diameter). In the field, the concentrations of algae are always much lower. For example, in mesocosms experiments and after adding nutrients, the concentrations of Emiliania huxleyi can reach 10 5 cells/ mL (data from Chapter 4;Catsberg et al., 2001) but are generally lower than 10 3 cells/mL (e.g. Rochelle-Newall et al., 2004;Martinez-Martinez et al., 2006). During blooms in the open sea, the concentration of Emiliania huxleyi varies between 10 3 and 10 5 cells/mL (reviewed in Tyrell and Merico, 2004) and the picoeukaryote blooms reach similar cell densities (e.g. Not et al., 2005;[START_REF] Thyssen | Onset of the spring bloom in the northwestern Mediterranean Sea: influence of Chapter 5: General discussion Suzanne FERTE -Doctoral Thesis -2019 environmental pulse events on the in situ hourly-scale dynamics of the phytoplankton community structure[END_REF][START_REF] Zhao | Variations of picoplankton abundances during blooms in the East China Sea[END_REF].

From our own experience, it is difficult to concentrate without modifying the physiology. In the current state of biophysical devices, it therefore seems difficult to measure CEF in situ in field experiments.

A way to overcome this constraint would be to increase the amplitude of the absorption spectroscopy signals. This is presently on its way in the laboratory were a prototype sample cuvette which is based on an integrating sphere principle has recently been produced. It In the laboratory, we can show the effect of a biotic or abiotic parameter on photosynthetic physiology using a reductionist approach: we modify this parameter everything else being equal and we measure possible phenotypic changes. For example, the CEF amplitude is known to depend on light irradiance and CO2 availability. Is it so in the field as well? Are some parameters more important than others? Can we highlight signatures of a particular environmental parameter? Very few studies deal with photosynthetic physiology in situ by taking into account all the environmental parameters known to influence photosynthetic activity.

This was the aim of our study depicted in Chapter 4. We could show that during a 24-days campaign, the parameters describing photosynthetic physiology varied significantly. In order to explain their variance, we measured abiotic parameters such as dissolved nutrient concentrations, temperature, and light intensity and could build a statistical model explaining most of the photo-physiology variance (75%, 47% and 37% of the variance explained by the model for ETRmax, Fv/Fm and NPQ, respectively). We have shown that because of the inevitable partial correlation between abiotic parameters, a correct statistical treatment requires a multivariate approach and that the PLS (partial least square) regression is particularly suitable under conditions where abiotic parameters are partially correlated the possible ways of utilization of those compounds (CBB cycle, CCM, other pathways). But these calculations are based on strong assumptions such as:

1. the conservation of the structure of the chloroplast ATP synthase within the phylogeny;

2. the conservation of the Q-cycle mechanism;

3. the absence of alternative electron flows other than CEF.

Hereafter, I propose to discuss those hypotheses and to estimate the possible estimation errors we did.

 About variation of the number of c-subunits composing ATP synthases

If the number of ATP synthase c subunits was greater than 14, as counted in plants, then the number of ATP produced per proton transferred would be lower than what we estimated and we would have overestimated the ATP/NADPH ratio produced by the chain. The highest number of c subunits mentioned in the literature is 15 in cyanobacteria (Pogoryelov et al., 2007). If this was the case in the organisms studied, then the ATP/NADPH ratios would be overestimated by 7%.

If the number of c subunits of ATPase was less than 14, then the number of ATP produced per proton transferred would be greater and we would have underestimated the ATP/NADPH ratio produced by the chain. In other cyanobacteria, down to 13 c subunits were counted in F-ATP synthase (Pogoryelov et al., 2007) and the mitochondrial ATP synthase of Saccharomyces cerevisiae counts only 10 c subunits [START_REF] Stock | The rotary mechanism of ATP synthase[END_REF]. If there was only 13 c subunits per chloroplastic ATP synthase in the organisms studied, then the ATP/NADPH ratios would be underestimated by 8%.

 Occurrence of the Q-cycle

We can show that the Q-cycle is occurring in the organisms studied in chapter 3. Indeed, after a single turnover laser flash in the dark, we observe a first phase of ECS increase in time scales of the order of 100 µs which is attributed to the activity of PS and then a second phase of ECS increase in the time scales of the order of ms and which is attributed to the activity of cyt. b6f which is therefore electrogenic (data not shown). If the Q-cycle did not take place, the cyt. b6f activity would not modify the electric field across the thylakoid membrane: b6f turnovers would result in 1 electron (negative charge) transferred from the membrane PQ to the lumen per proton (positive charge) transferred from the stroma to the lumen.

LEF would induce a transient lack of ATP and therefore (i) the suppression of competitive inhibition of the cycling step enzymes and the increase in CEF until reaching the amplitude of CEF for which the ATP/NADPH ratio is adjusted to the needs of the CBB cycle or (ii) a limitation of the CBB cycle and the reduction of PSI acceptors and thioredoxin system that would activate the cycling step enzymes. Here again the CEF would increase until reaching an amplitude for which the ATP/NADPH ratio would be adjusted to the needs of the CBB cycle. In these 2 models, the CEF would follow changes of the LEF and the models are compatible with a CEF always proportional to the LEF. However, the results obtained by DCMU titration show that when the LEF is decreased by inhibition of the PSII, the CEF does not decrease in proportion to the LEF (instead, it increases or remains constant). For the same reasons, model ( 2) is not compatible with the results obtained in Symbiodinium sp and Chaetoceros sp.

Another regulatory model compatible with the results obtained in Chlamydomonas as a function of light intensity is model (3), that is to say the segregation of linear and cyclic electron transfer chains. In this case, we expect a proportional increase in CEF and LEF activity depending on the light intensity (at constant antenna size, as was the case in our experiments). But the supercomplex model is not compatible with the amplitude of CEF measured in Chlamydomonas reinhardtii. I could estimate an average of 35% or 55% of PSI involved in CEF, (results obtained with the antenna sizes measured by ECS or by fluorescence, respectively). If the amount of CEF was determined by the amount of PSI/cyt.

b6f supercomplexes, then at least 35% of PSI should be included in such supercomplexes.

And this is not what is suggested by studies aimed at purifying supercomplexes. The latter either do not succeed [START_REF] Buchert | The labile interactions of cyclic electron flow effector proteins[END_REF] either isolate only a few percent of PSI involved in supercomplexes (Iwai et al., 2010). There is no work aiming at isolating supercomplexes in Symbiodinium sp. or Chaetoceros sp., we thus cannot discuss their results. In addition, the model of segregation of the chains is also difficult to reconcile with the results obtained when titrating the LEF with DCMU. The specific inhibition of PSII (and therefore of the linear chains) should not affect the amplitude of the CEF. However, the results in Chlamydomonas reinhardtii are compatible with a CEF which increases when LEF is inhibited. It is to note that in a model of segregated linear and cyclic chains, the proportionality of LEF and CEF at all light irradiances is not automatic: this requires that the limiting step of photosynthesis under high light is the same in the two types of chains. This would strongly suggest that the limiting step of the cyclic chains is the cytochrome b6f.

Chapter 5: General discussion

Suzanne FERTE -Doctoral Thesis -2019

Model ( 1) is more difficult to discuss because two parameters are to be considered (the redox state of PQ and Fd) and because these parameters are difficult to measure by direct methods. After eliminating the first two models, this one remains our favorite but it is difficult to go further for the moment.

Finally, the differences in CEF behavior observed in the different organisms studied in Chapter 3 seem particularly interesting for discussing the diversity of regulatory modes of CEF. Are there different modes of regulation between species? Are those results related to differences in enzymes involved in the cycling step(s)? Are the differences only due to differences in physiological state? To answer these questions, it seems interesting to test more experimental conditions in these three organisms.

3. Three years were too short for…

3. 1. Revisiting literature using P700 method

In Chapter 2, we have shown that the P700 method is not reliable under conditions where the PSI acceptor side is reduced during the pulse, which corresponds to conditions where the PSI acceptor pool is small compared to the PSI donor pool. The induced errors result in underestimation of the flux through PSI by a factor up to 10 (in the dataset gathered. This method was proposed in 1994, 25 years ago, and has been used in many articles (cited 175 times according to Google scholar). It therefore seems important to revisit the literature.

This task will benefit from the correction to the P700 method we proposed which is applicable without requiring new measurements. I started doing so in the discussion of Chapter 2 but it might be interesting to continue this work.

3. 2. Exploring cyclic electron flow using our methods

In Chapter 3, Section 3.3, I presented a robust method to:

-highlight the presence or absence of CEF in a given species and given experimental conditions.

-test the relationship between CEF and LEF (if CEF is present)

-but also test the evolution of CEF according to the conditions applied to the sample.

The development and testing of the method has been long. It now seems reliable and ready to be used to answer to more biological questions. I did not have time to go further during my thesis but I would like to propose here some tracks for complementary investigations.

One idea could be to test the presence of CEF and its relation with LEF in the different clades of photosynthetic species. I started this screen but preferred not to show the results which were too preliminary. However, they suggest that the presence of CEF and its relation with LEF is not preserved within the same clade (e.g. the two species of dinoflagellates, Amphidinium carterae and Symbiodinium sp).

Another way is to test the evolution of the CEF according to environmental conditions such as the availability of nutrients, gas, the effect of temperature, or of biotic interactions.

Thirdly, we have shown that the method we propose may be used in the field, provided the presence of monospecific samples and sufficient concentrations to obtain significant signals.

In Chapter 4, we have shown that a statistical approach enables to highlight abiotic and biotic factors potentially involved in the changes of photosynthetic physiology. Following the same approach, we could propose to study the determinants of CEF in the field.

3. 3. Harness all data collected in the field

Chapter 4 shows some preliminary conclusions on the effect of viral infection on photosynthesis, which require the support of data from other teams with whom we collaborated. Not all the analyses have been performed yet, and the multivariate analysis will soon benefit from data from meta-genomics, meta-transcriptomics and metabolomics.

In addition, our dataset could be further exploited. For example, we could study deviations from the explanatory model for all the parameters of photosynthetic physiology to look for

4. 3 5 : 1 . 5 . 2 . 5 . 3 .

 3515253 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? .......................................................................... General discussion ..............................................................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? ........ 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 .............................................................................................................. 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 ........................................................................................ .................................................................................................................

Figure 1 1 :

 11 Figure 1 1: V9 rDNA Operational taxonomy unit rarefaction curves and overall diversity (Shannon index, inset) for each plankton organismal size fraction .......................................................

Figure 1 2 :

 2 Figure 1 2: Examples of phytoplankton blooms and microscopic observation of the species involved .

Figure 1 3 :

 3 Figure 1 3: Bacterial transformation of phytoplankton-derived organic matter .....................................

Figure 1 4 :

 4 Figure 1 4: Diversity of eukaryotic phototrophs .....................................................................................

Figure 1 5 :

 5 Figure 1 5: Origins of plastids via A: primary, B: secondary, and C: tertiary endosymbiosis. ..................

Figure 1 6

 1 Figure 1 6: Diversity of chloroplast structures .........................................................................................

Figure 1 7

 1 Figure 1 7: Schematic representation of the balanced equation of the Calvin Benson Bassham cycle (CBB cycle) ..............................................................................................................................

Figure 1 8

 1 Figure 1 8: Z-scheme of the electron transfer along photosynthetic chain intermediates .....................

Figure 1 9

 1 Figure 1 9: Structure of a dimer of cytochrome b6f .................................................................................

Figure 1 10:

 1 Figure 1 10: The cytochrome b6f complex drives the Q-cycle .................................................................

Figure 1

 1 Figure 1 11: ATP synthase structure and mechanism involved in the rotation of the CF0 domain .........

Figure 1

 1 Figure 1 12: A scheme of energetic levels (the so-called Jablonski diagram) of a single molecule showing routes of formation and deactivation of the energetic levels.................................

Figure 1

 1 Figure 1 13: Structure of plant photosystem II (PSII) and photosystem I (PSI) ........................................

Figure 1

 1 Figure 1 14: Schematic representation of the main pathways for excitonic energy dissipation ............

Figure 1

 1 Figure 1 15: The irradiance dependence of chlorophyll fluorescence yields in a marine diatom, P. tricornutum ............................................................................................................................

Figure 1 16:

 1 Figure 1 16: Evidence for excitonic connectivity between PSII units ......................................................

Figure 1 17:

 1 Figure 1 17: Efficiency of light absorption and utilization by photosynthetic chain ...............................

Figure 1

 1 Figure 1 18: A quantitative schematic representation of photosystem and antenna distribution heterogeneity, and of its change during state transitions, in C. reinhardtii ..........................

Figure 1 19:

 1 Figure 1 19: Schematic representation of the diversity of alternative electron pathways .....................

Figure 1

 1 Figure 1 20: Electron and plastoquinone transfer within cyt. b6f ............................................................

Figure 2 1 :

 1 Figure 2 1: Structure of the photosystem I ..............................................................................................

Figure 2 2 :

 2 Figure 2 2: P700 oxidized minus reduced difference spectrum .................................................................

Figure 2 3 :

 3 Figure 2 3: Principle of the P700-based methods for the determination of the fraction of open PSI centers, Y(I) ............................................................................................................................

Figure 2 4 :

 4 Figure 2 4: Electrochromic Shift principle ................................................................................................

Figure 2 5 :

 5 Figure 2 5: Chlamydomonas reinhardtii electrochromic shift spectrum .................................................

Figure 2 6

 2 Figure 2 6: Kinetic of the ECS after excitation with a single turnover laser flash ....................................

Figure 2 7

 2 Figure 2 7: A representation of the Z-scheme with flared reservoirs, where areas reflect the nernstian redox buffering of the redox components ..........................................................................

Figure 2 8

 2 Figure 2 8: Schematic model explaining the error made by the P700 pulse method ..............................

Figure 2 9

 2 Figure 2 9: P700 oxidation and reduction pathways (blue arrows) and action of the chemicals that are used in our Pm measurement protocol (red crosses or arrows) .........................................

Figure 2

 2 Figure 2 10: Maximal P700 absorption change, Pm, measured with the standard protocol or with our improved protocol ...............................................................................................................

Figure 2 11:

 2 Figure 2 11: Effects of repetitive shorts pulses treatment on PSI from sunflower leaves in atmospheric conditions (air) or in low oxygen concentration (low O2) ....................................................

Figure 2 12:

 2 Figure 2 12: Comparison of the fraction of open PSI centers in C. reinhardtii estimated with the ECS and P700 methods under different light irradiances ....................................................................

Figure 2

 2 Figure 2 13: PSI parameters measured by ECS and P700 method during light to dark transition when PSII is inhibited ............................................................................................................................

Figure 2

 2 Figure 2 14: Comparison of time-dependent changes of reduced P700 (red. P700), Y(I) and Y(II) (here noted ΦI and ΦII, respectively) during a dark-light induction period of a sunflower leaf in air .....................................................................................................................................

Figure 2

 2 Figure 2 15: Light dependence of PSI parameters, Y(I) (left panel), Y(NA) (middle panel) and Y(ND) (right panel), measured by ECS and P700 method when PSII is inhibited. ............................

Figure 2 16:

 2 Figure 2 16: Light dependence of P700 parameters measured before PSI inactivation treatment (Control) and 2 days after PSI inactivation (Inactivated) on wheat leaves. ........................................

Figure 2 17:

 2 Figure 2 17: Light irradiance dependency of Arabidopsis thaliana PSI parameters in WT (A and B), WT with antimycin A (C and D) and pgr5 mutant (E and F). ......................................................

Figure 2 18:

 2 Figure 2 18: Evolution of PSI parameters and CEF estimations during sugar maple leaf senescence ..

Figure 3 1 :

 1 Figure 3 1: Percentage of the publications about cyclic electron flow among the various oxygenic photosynthetic clades ..........................................................................................................

Figure 3 2 :Figure 3 3 :

 233 Figure 3 2: ECS spectrum of Amphidinium carterae (A.) and Symbiodinium sp (B.) ..............................

Figure 3 4 :

 4 Figure 3 4: Simulations of the relative changes of ETR(II) (blue) and ETR(total) (black) when titrating LEF by DCMU ..............................................................................................................................

Figure 3 5 :

 5 Figure 3 5: Relative changes of the fraction of open PS, Y(total), and the quantum yield of PSII, Y(II), when titrating LEF by DCMU in the dinoflagellate Amphidinium carterae .........................

Figure 3 6

 3 Figure 3 6: Relative changes of the fraction of open PS, Y(total), and the quantum yield of PSII, Y(II), when titrating LEF by DCMU in the dinoflagellate Symbiodinium sp ..................................

Figure 3 7

 3 Figure 3 7: Relative changes of the fraction of open PS, Y(total), and the quantum yield of PSII, Y(II), when titrating LEF by DCMU in the green alga Chlamydomonas reinhardtii ......................

Figure 3 8

 3 Figure 3 8: Estimation of the fraction of light absorbed by all PS and by PSI with an ECS-based method .................................................................................................................................

Figure 3 9

 3 Figure 3 9: Kinetics of ECS decrease after light offset at two different light irradiances ......................

Figure 3

 3 Figure 3 10: ECS decrease after a single turnover laser flash superimposed to two different light irradiances ............................................................................................................................

Figure 3 11:

 3 Figure 3 11: Comparison of the total electron transfer rate, ETR(total), estimated by the DIRK method and by the yield method ......................................................................................................

Figure 3 12:

 3 Figure 3 12: Estimation of the PSII photon absorption rate by fluorescence and comparison with the ECS-based method ...............................................................................................................

Figure 3

 3 Figure 3 13: ETR(total) (black) and ETR(II) (blue) as a function of light irradiance in three biological replicates (shown in panels A, B and C) ...............................................................................

Figure 3

 3 Figure 3 14: ETR(total) (black), ETR(II) (blue) and ETR(I) (red) as a function of light irradiance in three biological replicates (shown in panels A, B and C) ..............................................................

Figure 3

 3 Figure 3 15: ETR(total) (black), ETR(II) (blue), ETR(I) (red, open circles) and ETR(I)CEF (red, closed circles) as a function of light irradiance in three biological replicates (shown in panels A, B and C) ...................................................................................................................................

Figure 3 16:

 3 Figure 3 16: Light dependency of the ratios ETR(I)CEF:ETR(I), CEF:LEF and ATP/NADPH produced by the photosynthetic chain ...........................................................................................................

Figure 3 17:

 3 Figure 3 17: Relative changes of Y(total) and Y(II) when titrating LEF by DCMU in (A) Amphidinium carterae (dinoflagellate), (B) Symbiodinium sp. (dinoflagellate) and (C) Chlamydomonas reinhardtii (green alga) ........................................................................................................

Figure 3

 3 Figure 3 18: Relative changes of Y(total) and Y(II) when titrating LEF by DCMU in nitrogen depleted Amphidinium carterae (dinoflagellate) ................................................................................

Figure 3

 3 Figure 3 19: Testing CEF behaviour on a Chaetoceros sp. dominated sample from the field ...............

Figure 3

 3 Figure 3 20: Evolution of Y(total) (black) and Y(II) (blue) when temperature is decreasing (phase 1) and increasing (phase 2). ............................................................................................................

Figure 3

 3 Figure 3 21 : Fraction of PSI involved in CEF as a function of light intensity for the three replicates used in section 3.4.3 ..................................................................................................................

Figure 4 1 :

 1 Figure 4 1: Gates used for flow cytometry measurements ...................................................................

Figure 4 2 :Figure 4 3 :

 243 Figure 4 2: MiniFIRe is a hand luggage transportable device that measures chlorophyll fluorescence

Figure 4 4 :

 4 Figure 4 4: Location of the experiment and mesocosms setting ...........................................................

Figure 4 5 :

 5 Figure 4 5: Estimation of the chorophyll concentration in the mesocosms water along the campaign ..............................................................................................................................

Figure 4 6

 4 Figure 4 6: Evolution of the concentration of the different groups of phytoplankton from day 0 to day 24 of the campaign ..............................................................................................................

Figure 4 7

 4 Figure 4 7: Measurement of Non Photochemical quenching (NPQ) measured in steady state at 500 µmol photons m -2 s -1 .(A) and its reversible (qE, fully reversed in 2-3 minutes) and irreversible (qI) components (B and C, respectively) ...........................................................

Figure 4 8

 4 Figure 4 8: Evolution of fluorescence parameters indicative of photosynthetic physiology from day 0 to day 23 of the campaign ........................................................................................................

Figure 4 9

 4 Figure 4 9: Between Class Analysis (BCA) on the photosynthetic parameters ......................................

Figure 4

 4 Figure 4 10: Maximal fluorescence, Fm, as a function of the concentration of nanoeukaryotes (A), picoeukaryotes (B) and Synechococcus cells (C) .................................................................

Figure 4

 4 Figure 4 11: Comparison of the water color between day 0 (A) and day 21 of the campaign (B) for the bag 3. ...................................................................................................................................

Figure 4 12:

 4 Figure 4 12: Electron microscopy observation of a sample harvested in bag 4, day 16 of the campaign ..............................................................................................................................

Figure 4 13:

 4 Figure 4 13: Estimation of the fraction of the fluorescence signal emitted by Emiliania huxleyi .........

Figure 4 14:

 4 Figure 4 14: Evolution of abiotic parameters as a function of time ......................................................

Figure 4

 4 Figure 4 15: Between Class Analysis (BCA) on environmental parameters ...........................................

Figure 4 Figure 4

 44 Figure 4 16: Correlation matrix containing photosynthetic variables and abiotic environmental parameters ...........................................................................................................................

Figure 4

 4 Figure 4 18: NPQ at 500 µmol photons m -2 s -1 as a function of light intensity ......................................

Figure 4 19:

 4 Figure 4 19: Estimation of the deviation to the model of the maximal PSII efficiency, Fv/Fm (A, B) and the maximal electron transfer rate, ETRmax (C, D). ...............................................................

Figure 4 20:

 4 Figure 4 20: Evolution of the number of mcp gene copies per mL along the experiment in each bag.

Figure 5 1 :

 1 Figure 5 1: Importance of considering charge recombinations when measuring the fraction of PS open centers. ................................................................................................................................

Figure 5 2 :

 2 Figure 5 2: DCMU titration of Y(total), Y(II) and Y(I)P700 in Chlamydomonas reinhardtii stt7 mutant. ..

Figure 5 3 :

 3 Figure 5 3: Example of ECS spectra measured in mono-cultures and in co-culture. .............................

Figure 5 4 :

 4 Figure 5 4: Expected versus measured ECS signals in 3 mixtures of microalgae. ..................................

Chapitre 2 :

 2 phototrophes a remporté un succès planétaire : ceux qui pratiquent la photosynthèse oxygénique grâce à deux sous familles de centres réactionnels, les photosystèmes II (PSII) et photosystèmes I (PSI). Les travaux réalisés chez les plantes supérieures et les algues vertes ont permis de montrer que les PSII et PSI peuvent fonctionner en série pour oxyder l'eau et réduire le CO2, produisant ainsi du dioxygène et des molécules organiques contenant du carbone réduit. Chez les eucaryotes, ces réactions se produisent dans le chloroplaste, plus précisément dans les thylacoïdes qui sont des vésicules aplaties structurées par deux membranes qui délimitent le lumen, compartiment interne, et le stroma, à l'extérieur. Le CO2 est réduit dans le stroma par le cycle de Calvin-Benson-Bassham qui consomme de l'énergie et du pouvoir réducteur sous la forme d'ATP et de NADPH. Ces composés sont produits par la chaine photosynthétique du thylacoïde : le PSII oxyde l'eau et réduit des plastoquinones membranaires, les électrons extraits de la molécule d'eau sont ensuite transférés au côté donneur du PSI via un complexe protéique membranaire, le cytochrome b6f, puis via une protéine luménale, la plastocyanine (PC). Le PSI transfère ensuite les électrons depuis les PC vers des ferrédoxines et le NADPH de l'autre côté de la membrane. L'ensemble de ces réactions d'oxydo-réduction correspond à ce que l'on appelle le flux linéaire d'électrons (FLE). Ce flux permet aussi la synthèse d'ATP : en accord avec la théorie chimio-osmotique de Mitchell, le flux d'électrons à travers le b6f est couplé à la translocation de protons depuis le stroma vers le lumen mettant en place un gradient électrochimique utilisé par une ATP synthétase membranaire pour former de l'ATP. Outre le FLE, chez les plantes et les algues vertes, on sait que le PSI peut fonctionner de manière indépendante du PSII : les électrons issus du PSI peuvent être réinjectés dans la chaîne donnant lieu, via le cytochrome b6f, à un flux cyclique d'électrons autour du PSI (FCE). Celui-ci serait essentiel pour ajuster les productions d'ATP et de NADPH aux besoins des voies métaboliques en aval du PSI, notamment le cycle de Calvin, puisqu'il permet la mise en place d'un gradient électrochimique de protons et donc la synthèse d'ATP sans produire de pouvoir réducteur. Le FCE serait aussi impliqué dans les mécanismes de photoprotection en conditions où le cycle de Calvin limite

(

  [2010][2011] [START_REF] Laursen | Spain's ship come in[END_REF] carried out global surveys of prokaryotic metagenomes from the ocean's surface and bathypelagic layer (>1,000 m), respectively. The Tara Oceans expedition (2009-2013) complemented these surveys by collecting a wide variety of planktonic organisms (from viruses to fish larvae) from the ocean's surface (0-200 m) and mesopelagic zone (200-1 000 m) at a global scale. Tara ocean surveyed 210 ecosystems in 20 biogeographic provinces, collecting over 40 000 samples of seawater and plankton

Figure 1 - 1 :

 11 Figure 1-1: V9 rDNA Operational taxonomy unit rarefaction curves and overall diversity (Shannon index, inset) for each plankton organismal size fraction From de Vargas et al., 2015. 334 plankton samples were collected during the circumglobal Tara Oceans expedition. Hypervariable V9 rDNA was amplified by PCR and sequenced by illumine method in different size fractions of plankton ('pico-nano',[0.8-5µm]; 'nano',[5-20µm];'micro', [20-180µm]; 'meso', [180-2,000µm]). Sequences were clustered in operational taxonomy units (OTU). Proximity to saturation is indicated by weak slopes at the end of each rarefaction curve (e.g., 1.2/100,000 means 1.2 novel metabarcodes obtained every 100 000 rDNA reads sequenced). Shannon index has been calculated for each size fraction as an indicator of genetic diversity.

Figure 1 - 2 :

 12 Figure 1-2: Examples of phytoplankton blooms and microscopic observation of the species involved A. An exceptionally dense bloom of the dinoflagellate Alexandrium monilatum in lower Chesapeake Bay along the north shore of the York River between Sarah's Creek and the Perrin River on 8/17/2015. Credit: © W. Vogelbein/VIMS. B. Alexandrium monilatum observation through an optic microscope. Source: Center for applied aquatic ecology C. The Florida "red tide" dinoflagellate Karenia brevis), along the Gulf Coast of the United States. Source: National Oceanic and Atmospheric Administration (NOAA). D. Karenia brevis observation through an optic microscope. Source: Schrope, Nature 2008 E. South Atlantic Ocean image from space, December 2, 2014. The aquamarine stripes and swirls are likely coccolithophores. The various shades of green are probably a mix of diatoms, dinoflagellates and other species. Source: NASA images by Norman Kuring, NASA's Ocean Color Group, using the Visible Infrared Imaging Radiometer Suite (VIIRS) data from the Suomi National Polar-orbiting Partnership. The coccolithophore Emiliania huxleyi (F) and the dinoflagellate Prorocentrum sp. (G) were previously observed in the region during ship-based studies. Observation through optic microscope (F) and transmission electronic microscope (G). Source: Roscoff culture collection (RCC 2563 and RCC 1212, respectively).

Figure 1 -

 1 Figure 1-4 presents a phylogenetic tree of eukaryotes based on the analysis of the gene encoding the RNA 18s cells. Photosynthetic organisms are indicated on the colored branches. On this tree, we see that terrestrial plants belong to a single monophyletic family belonging to Chlorophyta. Green algae are close relatives of terrestrial plants and also belong to the Chlorophyta clade. On the other hand, marine photosynthetic eukaryotes are distributed in many branches of the phylogenetic tree of eukaryotes such as Glaucophyta and Rhodophyta, which group together with Chlorophyta in the Archaeplastida (Figure 1-4).

Figure 1 - 4 :

 14 Figure 1-4: Diversity of eukaryotic phototrophs Endosymbiotic events are shown in the hypothetical tree : P-primary endosymbiosis ; C-complex endosymbiosis; S-secondary endosymbiosis. Losses of photosynthetic ability (white rectangles) or losses of the entire plastid (black rectangles) are indicated. SAR: Stramenopila + Alveolata + Rhizaria. From Oborník, 2019

Figure 1 - 5 :

 15 Figure 1-5: Origins of plastids via A: primary, B: secondary, and C: tertiary endosymbiosis.Gene transfer from the endosymbiont to the host nucleus is shown with the arrows and the colored chromosomes. The mitochondrion has been omitted from these figures. The remnant algal nucleus (nucleomorph) in secondary and tertiary endosymbioses is shown. This genome has been lost in all algae but the cryptophytes (member of the chromalveolates) and chlorarachniophytes (marked with asterisks). From[START_REF] Bhattacharya | Photosynthetic eukaryotes unite: endosymbiosis connects the dots[END_REF]

Figure 1 -

 1 . Chloroplast numbers per cell vary considerably in plants, from 1 to 1000 depending on species, life stage and environment. Microalgae generally possess one or a few chloroplasts delimited by 2, 3 or 4 membranes(Staehelin, 1986; Falkowski and Raven,

Figure 1 - 6 :

 16 Figure 1-6: Diversity of chloroplast structures A. Originally published by Ohad et al., 1967). Transmission electron micrograph (TEM) of a Chlamydomonas reinhardtii cell. B. From Salomé and Merchant, 2019. Drawing of a C. reinhardtii cell based on the TEM image in (A). C. From Buma et al., 2000. TEM micrographs cell of Emiliania huxleyi. N, nucleus; P, pyrenoid; C, chloroplast. Scale varies; bars represent 1 μm. D. From Dodge, 1968. Transmission electronic micrographs of cell of the dinoflagellate Aureodinium pigmentosum. A median section through a chloroplast to show the large number of more or less parallel lamellae composed of 2-4 thylakoids, the granular stroma with dark lipid droplets and the thick chloroplast envelope. x 70000.

Chapter 1 :

 1 General introduction Suzanne FERTE -Doctoral Thesis -2019 b) Discovery and chemical equation of the CBB cycle The metabolic pathway by which most of the autotrophs incorporate CO2 into carbohydrates was elucidated between 1946 and 1953 by Melvin Calvin, James Bassham and Andrew

Figure 1 - 7 :

 17 Figure 1-7: Schematic representation of the balanced equation of the Calvin Benson Bassham cycle (CBB cycle) A form I RuBisCO is represented by a model of its structure. Large subunits are shown in red and green and small subunits are shown in yellow and orange.

Figure 1 -

 1 Figure 1-8: Z-scheme of the electron transfer along photosynthetic chain intermediates Adapted from Govindjee and Björn, 2017.The redox carriers were placed according to the accepted midpoint redox potentials (pH 7). OEC: oxygen evolving complex, Z: electron donor to photosystem II (PSII), P680: reaction center chlorophyll (Chl) a of PSII, Ph: pheophytin, Q: quinone, Cyt: cytochrome, FeSR: Reiske iron-sulfur protein, Pc: plastocyanin, P700: reaction center Chl a of photosystem I (PSI), A0 and A1: primary electron acceptors of PSI, FeSX, FeSB, and FeSA: bound iron-sulfur protein acceptors of PSI, Fd: soluble ferredoxin, Fp: flavoprotein (ferredoxin-NADP reductase). In this diagram, electron transfer through the redox components of plastoquinone pool and Cyt b6f complex is not shown.

Chapter 1 :

 1 photosystems and involves the two cytochromes discovered by Hill and Bendal is a quinol:plastocyanin (cytochrome c 6 ) oxidoreductase: the cytochrome b 6 f complex. The transfer from water to NADPH permitted by the combined activities of the two photosystems and the cytochrome b6f is called linear electron flow (LEF). It is found in all

Figure 1 - 9 :

 19 Figure 1-9: Structure of a dimer of cytochrome b6f From de Vitry and Kuras 2009. Helices for cytochrome b6 are colored in cyan, helices for subunit IV are shown in blue, the Rieske subunit is shown in orange, cytochrome f is shown in red, the PetG, PetL, PetM and PetN subunits are shown in green. Shown as wireframes are the β-carotenes colored in yellow, the chlorophylls colored in green and the hemes colored either in pink (b-type hemes) or in red (c-type hemes). The Fe2S2 centers are shown as a ball-and-stick model (yellow and orange).

Figure 1 -

 1 Figure 1-10: The cytochrome b6f complex drives the Q-cycle From Dumas et al., 2016. Electron transfer (solid black arrows) is coupled to proton translocation (blue arrow) across the thylakoid membrane, establishing the ΔμH + . PQH2 oxidation occurs at the Qo site and PQ reduction at the Qi site. Electrons are transferred to the low-potential chain (haems bL, bH and ci) and to the high-potential chain (Fe-S, cyt f, PC, PSI) upon PQH2 oxidation. Movement of the Rieske subunit between proximal and distal positions is necessary for electron transfer from PQH2 to cyt f. Dashed black arrows represent the movement of quinone species between the PQ pool and the two cyt b6f catalytic sites.

Figure 1 -

 1 Figure 1-11: ATP synthase structure and mechanism involved in the rotation of the CF0 domain A. Adapted from Hahn et al., 2018. High-resolution cryo-EM map of the spinach chloroplast cF1FoATP synthase. Surface representation of the overall structure of the spinach chloroplast cF1FoATP synthase showing α subunits (dark green), β subunits (light green), γ (blue); δ (orange), ε (purple), a (light blue), b (violet), b′ (pink), c14 (pale yellow), ATP (red). cF0 (abb′c14) is embedded in the thylakoid membrane (grey), whereas cF1 (a3b3ged) extends into the stroma. B. Adapted from Stock et al., 2000. Model of the generation of rotation by movement of protons through the F0 domain of ATP synthase. Two half channels across the interface between the a subunit (green) and the c ring (blue) are linked by rotation of the c ring.

Figure 1 -

 1 Figure 1-12: A scheme of energetic levels (the so-called Jablonski diagram) of a single molecule showing routes of formation and deactivation of the energetic levels From Lazar et al., 2015. The thin horizontal lines show vibrational energy levels which are superimposed on electronic energy levels of the ground state (S0), excited singlet states (S1 and S2) and excited triplet state (T1) shown by thick horizontal lines. Vertical thick arrows symbolically show orientation of spin; when the arrows in the ground and exited states are in the opposite/same directions, the singlet/triplet excited state is realized. Vertical wavy arrows indicate radiative transitions by means of absorption of light (A) of different wavelengths (shorter wavelength (blue) means higher energy whereas longer wavelength (orange) means lower energy) and by means of emission of light by fluorescence (F; S1-S0 transition) and by phosphorescence (P; T1-S0 transition). Thin vertical or skew arrows indicate non-radiative transitions by means of vibrational relaxation (VR), internal conversion (IC), and inter-system crossing (ISC) which all represent a loss of energy by heat dissipation. For the case of a photosynthetic pigment molecule, additional deactivations of the excited states not shown in the figure can occur: via energy transfer to another molecule and via photochemistry (the primary charge separation).



  General structure of the PS

Figure 1 -

 1 Figure 1-13: Structure of plant photosystem II (PSII) and photosystem I (PSI) From Caffarri et al., 2014. A. Lateral (upper diagrams) and lumenal (lower diagrams) views of the PSII-LHCII supercomplex and of the PSI-LHCII supercomplex (as found in State II conditions). PSII-LHCII model has been constructed from a crystal structure of the cyanobacterial PSII core, and from the spinach LHC structures. PSI model is based on the structure from pea. In the upper diagram of the two monomers composing the dimeric core of PSII, the D1 and D2 reaction center subunits and the internal antenna proteins of the core complex CP43 and CP47 are indicated in color and other core proteins are shown in transparent grey. Peripheral antenna proteins are also indicated: CP24, CP26 and CP29 are the monomeric minor LHCb proteins and LHCII-S, -M and -L are the trimeric LHCII. In the PSI model the PsaA and PsaB core subunits are indicated, as well as the Lhca antenna complexes and other subunits visible from the lumenal surface. The position of Chls and carotenoids is also shown. In the case of the Chls, for simplicity they are represented by only four atoms: the central magnesium and the 4 nitrogen atoms. In PSII and LHCII, Chla is indicated in blue and Chlb in green. For PSI, the low resolution of the crystal structure in the Lhca region, where Chlb molecules are, does not allow assigning their position and thus all Chls are indicated only in blue. Carotenoids are drawn using the following colors: -carotene, brown; lutein, orange; violaxanthin, violet; neoxanthin, yellow. The special pairs (P680 in PSII and P700 in PSI) are highlighted with dotted ovals. ChlZ in PSII RC is also indicated. The FeS center acceptors are only shown for the lateral view of PSI. B. Cofactors involved in electron transfer reactions of PSII (left) and PSI (right). For PSII and PSI, models are extracted from the structure of cyanobacteria and pea, respectively. P680 is the "special pair" of PSII. Electron

Figure 1 -

 1 Figure 1-14: Schematic representation of the main pathways for excitonic energy dissipation From Lazar, 2015. KP, Kf, KD and KNPQ represent degenerate kinetics constant of photochemistry, fluorescence, and basal and regulated heat dissipation, respectively. As these three de-excitation pathways compete with each other, photochemistry, i.e. the electron transfer from P680 + to QA quenches fluorescence (photochemical quenching), as does an increase in the rate of non-radiative relaxation (heat loss) (non-photochemical quenching). The accurate measurement of the photochemical yield of PSII therefore requires the measurement of both photochemical and non-photochemical quenching. Two extreme cases can be drawn: in the dark, QA is completely oxidized and PSII centers are open: the photochemical yield is maximal and the fluorescence yield is minimal, termed F0. At the opposite, when applying a brief saturating pulse of light which reduces all QA on a darkadapted sample, photochemical yield drops to zero and the fluorescence yield is maximal (Fm). A photosynthetic sample exposed to a continuous actinic illumination will have a fluorescence yield (F'), which is intermediate between these two values. If this actinic illumination is strong, the fluorescence yield of this illuminated sample after a brief saturating pulse of light (Fm') can be different from Fm, if light-induced heat dissipation processes (see Figure 1-15) are activated.

Figure 1 -

 1 Figure1-15 shows the light dependence of photosynthetic parameters F0', F' and Fm'. Under high light, the maximal fluorescence (after the saturating pulse is applied, i.e when the contribution of photochemistry is almost null) decreases, reflecting the activation of light induced regulatory heat dissipation mechanism (see. The efficiency of lightinduced heat dissipation process can be probed with the parameter NPQ = (Fm-Fm')/Fm(Bilger and Björkman, 1990) The NPQ must be used cautiously: it has two components, the qE process which is reversible in the minute timescale, and the photoinhibition qI process which relaxes very slowly, in the tens of minutes to hours timescale. The two processes can therefore be distinguished based on their different relaxation characteristics.

Figure 1 -

 1 Figure 1-15: The irradiance dependence of chlorophyll fluorescence yields in a marine diatom, P. tricornutum From MiniFIRe guide, Max Gorbunov (DMCS, Rutgers University, NJ, USA). Fo and Fm are the minimum (open reaction centers) and maximum (closed centers) fluorescence yields, respectively, measured in dark-adapted cells. Fo' and Fm' are the minimum (open centers) and maximum (closed centers) fluorescence yields, respectively, measured in a light adapted state. Fo' is measured in lightadapted cells following a brief (~ 1s) exposure to darkness. F' is the actual fluorescence yield measured under ambient light. PQ and NPQ are photochemical quenching and non-photochemical quenching, respectively.

Figure 1 -

 1 Figure 1-16: Evidence for excitonic connectivity between PSII units From Stirbet, 2013. a. Chlorophyll a fluorescence induction kinetics: experimental data on Chlorella cells (continuous red line) and theoretical transients (dashed lines) for different values of the connectivity constant p. b. Rates of oxygen evolution (diamonds) in Chlorella cells as a function of the calculated photochemical complex concentration (i.e., fraction of active photosystem II units, q). The figure was redrawn from the original figures (Figs 2,3) of[START_REF] Joliot | Etude cinétique de la réaction photochimique libérant l'oxygène au cours de la photosynthèse[END_REF].

  photosynthesis is proportional to light intensity -light is limiting photosynthesis, (ii) under high light, the photosynthetic process becomes limited by chemical reactions (cytochrome b6f, CBB cycle) instead of photo-chemical ones (Figure1-17), and (iii) under very high light, a drop in photosynthetic rate might appear, which reflects the photo-destruction of the photosynthetic machinery (Figure 1-17). Chlorophylls of the photosynthetic antenna absorb light and reach an excited singlet state ( 1 Chl * ). The exciton can be transferred by Förster resonance energy transfer towards the special pair of chlorophyll performing photochemistry. In a low light regime, most of PSII are open and therefore the photochemical yield is high. But in high light conditions, photosynthesis saturates, indicating that one of the light independent electron transfer reactions becomes the limiting step and that the excitation flux exceeds the assimilation capacity (Figure 1-17) leading to an overreduction of the PQ pool and QA. When QA is reduced, the photochemical yield decreases and the yield of other relaxation pathways increase in a complementary way (Figure 1-12).

Figure 1 -

 1 Figure 1-17: Efficiency of light absorption and utilization by photosynthetic chain

Figure 1 -

 1 Figure 1-18: A quantitative schematic representation of photosystem and antenna distribution heterogeneity, and of its change during state transitions, in C. reinhardtii From Nawrocki et al., 2016.In state I, when the intersystem electron carriers are oxidized, PSII-LHCII supercomplexes form group in the stacked regions of the thylakoids, whereas PSI form supercomplexes with nine LHCa proteins mostly in the unstacked regions. Upon reduction of the intersystem electron carriers, phosphorylation of PSII core and antenna occurs, leading to electrostatic repulsion from the stacked regions of the membrane and the antenna migration to the unstacked regions, loosening the overall stacking. In state II, the PSI-LHCa supercomplex further increases its size by about 40% (inNawrocki et al., 2016) by attaching two LHCII trimers and a monomeric antenna.

Figure 1 -

 1 Figure 1-19: Schematic representation of the diversity of alternative electron pathwaysArrows represent linear electron flow from water to CO2 (blue), the alternative pathways using both PSII and PSI, e.g. Mehler reactions, flavodiiron proteins or hydrogenase pathway (green), the ones using only PSII, PTOX pathway (purple), and the ones using only PSI, the cyclic electron flow around PSI (orange).

Chapter 1 :

 1 General introductionSuzanne FERTE -Doctoral Thesis -2019 non-stressed conditions, PTOX is found at low concentrations in plants thylakoids (about 1

Chapter 1 :

 1 General introductionSuzanne FERTE -Doctoral Thesis -2019 b) Three main CEF pathways are proposed in the literature  PGRL1-PGR5 dependent pathway

Chapter 1 :Figure 1 -

 11 Figure 1-20: Electron and plastoquinone transfer within cyt. b6f From Nawrocki et al., 2019b a. A scheme of possible cyclic electron transfer within cyt.b6f. A side view of the cyt.b6f monomer from the dimerization surface perspective. The stromal and lumenal surfaces of the thylakoid membrane are depicted in grey. Each subunit of the monomer is colored as indicated on the left side of the figure. The electron transfer pathways are shown as yellow arrows. Ferredoxin in its putative binding mode is shown in a red cartoon form.b. PQ/PQH2 transfer within cyt.b6f dimer. Side view of the complex. One cyt.b6f monomer is in orange, while the other one is blue. Possible route for the quinone/quinol between the Qo and Qi side are represented in green for transfer in the same monomer, which implies crossing the narrow portion of the crevice, or in green/orange for transfer in the opposite monomer, which uses a broader and shorter path.

Figure 2 - 1 :

 21 Figure 2-1: Structure of the photosystem I From (Nelson and Yocum 2006). A. side view of the structure of plant photosystem I. The cofactors involved in light-induced electron transport, i.e., P700, A0, A1, FX, FA, and FB, are shown along with the backbones of the 16 subunits. The positions of the proteic subunits, PsaB, PsaC, PsaD, PsaE, and PsaF are shown. In the electron transport chain, the P700 Chl (red) and the other Chl (green) are indicated. The quinones (blue) and, in the iron-sulfur clusters, the iron (red) and the sulfur (yellow) are also indicated. B. Structural model of the pathway for light-induced electron transport from plastocyanin (PC) to ferredoxin (Fd) in photosystem I. Chlorophylls (blue), phylloquinones (black), the copper atom of PC (blue), and the iron (red balls) and sulfur (green balls) of the three Fe4-S4 clusters and the Fd Fe2-S2 are depicted. Two tryptophan residues (light-blue and light-pink space-filling structures) that might be involved in electron transport from PC to P700 are also shown in the context of their secondary structural environment.

Figure 2 - 2 :

 22 Figure 2-2: P700 oxidized minus reduced difference spectrum From Hiyama and Ke, 1972. A short flash (20 µs duration) was applied on a reaction mixture containing: D-144 particles (i.e. a fraction of lysed chloroplasts enriched in PSI, see Anderson and Boardman, 1966 for protocol details). All Δε values were calculated from ΔA based on Δε701nm = 64 mequiv -1 .cm -1 .

  d) PSI yield and P700 redox state Since the 1980s, changes in P700 absorption, reflecting its redox state, have been used to study the quantum yield of PSI and the most recent method has been proposed by Klughammer and Schreiber in 1994. Let's first explain the link between the redox state of P700 and the yield of PSI, i.e. the fraction of open PSI centers. By definition, an open center will perform a charge separation if it receives a photon. In the beginning of the 1990s, Harbinson and Foyer used P700 absorption changes to differentiate two types of PSI centers: -[P700 + ] centers that are closed because their primary donor is oxidized. Those centers are called donor side limited because PSI donor does not have any electron to transfer.

  [P700 A -] centers are called acceptor side limited centers because the acceptor cannot accept any electron. The fraction of [P700 A -] and of [P700 A] centers are noted Y(NA) and Y(I), respectively. Klughammer and Schreiber proposed a method to discriminate between open PSI centers, [P700 A] and acceptor side limited centers, [P700 A -]. This method is based on the fact that among [P700] centers, only P700 from open centers are oxidizable if they receive enough light.

Chapter 2 :

 2 Probing PSI activitySuzanne FERTE -Doctoral Thesis -2019 it is possible to transfer several negative charges to the stromal side without modifying the fraction of [P700 A -] centers. In this chapter, I tested the reliability of this hypothesis.

Figure 2 - 3 :

 23 Figure 2-3: Principle of the P700-based methods for the determination of the fraction of open PSI centers, Y(I) In both Habinson & Foyer and Klughammer & Schreiber methods, the fraction of donor side limited centers, Y(ND) is estimated as the fraction of P700 that is oxidized under the given light conditions. Harbinson & Foyer method consider Y(I) as the fraction of reduced P700 while Klughammer & Schreiber method estimates Y(I) as the fraction of P700 that can be oxidized during a multiple turnover saturating light pulse (SP). Black, white and red horizontal bars correspond to darkness, light and multiple turnover saturating light pulse, respectively.

  of the ECS signal

Chapter 2 :

 2 Probing PSI activitySuzanne FERTE -Doctoral Thesis -2019

Figure 2 - 4 :

 24 Figure 2-4: Electrochromic Shift principle A: Modification of the energy levels of the pigment in the presence of an electric field. B: Shift of the absorption spectrum (orange versus green) of the pigment and the resulting absorption difference (open black circles) spectrum. From Bailleul et al. 2010.

Figure 2 - 5 :

 25 Figure 2-5: Chlamydomonas reinhardtii electrochromic shift spectrumAbsorption change was measured 100 µs after a single turnover saturating laser flash in the Chlamydomonas reinhardtii T222 + wild type. Cultures were centrifugated, resuspended at the concentration of 10 7 cells.mL -1 in minimum medium and dark-adapted for 20 min while vigorously stirred. Adapted from[START_REF] Bujaldon | Functional accumulation of antenna proteins in chlorophyll b-less mutants of Chlamydomonas reinhardtii[END_REF]

  photon. After a single turnover flash, only open centers will perform a charge separation and generate an electric field: the amplitude of the a phase is proportional to the number of open centers. By normalizing this signal by the amplitude of the a phase when all the centers are open, i.e. in the same sample once dark-adapted, we can calculate a fraction of open centers.

Figure 2 - 6 :

 26 photosystems being PSII and PSI, respectively:

  perform a charge separation. [P700 + A] centers are called donor-side limited centers because the oxidation of P700 in the light reflects a bottleneck in the electron transfer upstream of PSI (on the donor side). Under a given light irradiance, the donor-side limited fraction can be measured straightforwardly with absorption difference spectroscopy as the fraction of oxidized P700, called Y(ND). It should be noted that the centers in which P700 is oxidized and A is reduced, [P700 + A -] would also be considered to be donor-side limited. The difficulty stems from the distinction between the fraction of open centers, called Y(I) or ΦI, and the fraction of [P700 A -] centers, called Y(NA), because both have a reduced P700. The latter are called acceptor-side limited centers because the reduction of both cofactors in the light reflects a bottleneck in the electron transfer downstream of PSI (on the acceptor side). To avoid confusion, we will use Y(I)P700 and Y(NA)P700 hereafter when referring to estimations of the PSI yield and acceptorside limitation with the P700 pulse method, while reserving the notation Y(I) and Y(NA) for the true values of the PSI yield and acceptor-side limitation, respectively. Klughammer and Schreiber reasoned that the acceptor-side limited centers cannot perform charge separation whatever the light intensity and therefore calculated Y(NA)P700 as the fraction of centers whose P700 is not photo-oxidizable by a multiple turnover pulse of very intense light. By deduction, open centers are therefore defined as centers with reduced but photo-oxidizable P700, i.e. Y(I)P700 = 1 -Y(NA)P700 -Y(ND).

  stems from the shift in the absorption spectrum of some of the photosynthetic pigments embedded in the thylakoid membrane when photosynthetic activity generates an electric field (a component of the pmf) across the thylakoid. ECS could in principle indicate the proportion of PSI centers that are open by quantifying the stable electric field generated by a saturating single turnover flash.

Chapter 2 :

 2 Probing PSI activitySuzanne FERTE -Doctoral Thesis -2019 single-turnover saturating flash in the presence of DCMU and HA in dark-adapted cells, and results are therefore expressed as charge separation per PSI. The fraction of open PSI centers, Y(I)ECS, was calculated as the ratio between the ECS increase induced by the saturating flash in the condition of interest and that in dark-adapted samples.

Figure 1

 1 Figure 1 Light dependency of the fraction of open PSI centers in C. reinhardtii estimated with ECS (blue) and P700 (red) methods Experiments were carried out in oxic conditions and PSII was inhibited with DCMU (10 µM) and HA (saturating concentrations, see Material and methods). (A) Examples of ECS (blue) and P700 (red) kinetics. Experiments were carried out in the dark or on top of a background light. A single turnover laser flash (for ECS) or a multiple turnover saturating pulse of light (for P700) was applied after steady state was reached and the flash-induced absorption change (ECS) or the maximal absorption change during the pulse (P700) was used to estimate the fraction of open PSI centers (Y(I)ECS or Y(I)P700). Blue arrows represent the single turnover laser flash. White, black and red bars represent light, dark and multiple turnover saturating pulse of light, respectively. The acceptor and donor side limitation, Y(NA) and Y(ND), are calculated as in (Klughammer and Schreiber, 1994) and shown on the panels. Left: in dark-adapted cells, without MV. Middle: under a 26 µmol photons m -2 s -1 bakground light without MV. Right: in the presence of MV (4 mM), under a 3 µmol photons m -2 s -1 background light. (B and C) Light dependency of Y(I)P700 (B) and Y(I)ECS (C) under steady-state illumination. Data of B and C are the average (± SD) of 3 biological replicates.

  right) and at 3 µmol photons m - Chapter 2: Probing PSI activity Suzanne FERTE -Doctoral Thesis -2019 equivalent to ~100% PSI centers being open. The Y(I)P700 is ~0.5 in these two conditions, meaning that the P700 pulse method measures only 50% of PSI centers as being open. At light

Figure 2

 2 Figure 2 Comparison of the fractions of PSI open centers estimated with ECS and P700 methods in C. reinhardtii in oxic conditions PSII were inhibited with DCMU (10µM) and HA (saturating concentration, see Material and methods). Experiments were carried out in the absence (A and C) or presence (B and D) of MV (4 mM). (A and B) Y(I)P700 plotted as a function of Y(I)ECS at steady state of light for different light intensities. Data points correspond to those shown in Figure 1B and 1C. (C and D) Y(I)P700 plotted as a function of Y(I)ECS during the dark-to-light transition at the onset of a 800 µmol photons m -2 s -1 light irradiance. Datapoints correspond to measurements at different times during the 700 ms following light onset. Data corresponds to the average (± SD) of 3 biological replicates (one for panel D).

Chapter 2 :

 2 Probing PSI activitySuzanne FERTE -Doctoral Thesis -2019 transitory situations at different times after the onset of different light irradiances. For both methods, the estimated fraction of open PSI centers was very low in the first seconds after the onset of light but increased to reach steady-state values in 1 or 2 minutes. In the steady state the two values were the same but during the transitory phase, Y(I)ECS was systematically higher than Y(I)P700 (Figure3B). The discrepancy at the very onset of light was very strong with initial values of Y(I)ECS ~0.3-0.4 whereas Y(I)P700 was very close to 0 (Figure3B). It should be noted that assays with MV cannot be done in anoxic conditions, because O2 is the final electron acceptor in the MV-mediated electron transfer from PSI.The P700 pulse method underestimates PSI yieldWith these results, it is clear that one of the estimations of the PSI yield is wrong. Either Y(I)P700 underestimates the fraction of open PSI centers or Y(I)ECS overestimates it. As reasoned in the introduction, the discrepancy could arise from an underestimation of Y(I)P700 due to the use of a multiple turnover pulse. However, at this stage, we need to demonstrate that Y(I)P700 underestimates the fraction of open PSI centers. Looking more closely at the data obtained in dark-adapted anoxic conditions, the two methods give strikingly different results. Is the fraction of open PSI centers 0.

Figure 3

 3 Figure 3 Comparison of the fractions of open PSI centers estimated with ECS and P700 methods in C. reinhardtii in anoxic conditions Anoxia was induced by adding glucose (20 mM) and glucose oxidase (280 U.mL -1 ) (see Material and methods). PSII were inhibited with DCMU (10 µM) and HA (saturating concentration, see material and methods). (A) PSI yield was measured with the two methods in dark-adapted anoxic conditions (B) Y(I)P700 as a function of Y(I)ECS during the dark-to-light transition in anoxic conditions at different times during the first 3 minutes following the onset of light corresponding to 26 (diamonds), 56 (circles) or 135 (squares) µmol photons m -2 s -1 . (C) Flashinduced ECS kinetics was measured from dark adapted anoxic conditions in the absence (open circles) or presence (closed circles) of DBMIB (5 µM), a cyt. b6f inhibitor. Blue arrow corresponds to the single turnover laser flash. (D) Kinetics of P700 redox changes (open circles). In this panel, oxic conditions (closed circles) are shown as a control. Black and red horizontal bars correspond to darkness and multiple turnover saturating pulse of light, respectively. For all panels, data corresponds to the average (± SD) of 3 biological replicates.

Chapter 2 :

 2 Probing PSI activitySuzanne FERTE -Doctoral Thesis -2019

Figure 4

 4 Figure 4 Comparison of the fractions of open PSI centers estimated with ECS and P700 methods in dark-adapted conditions in different photosynthetic organisms PSII were inhibited with DCMU (10 µM) and HA (saturating concentration, see Material and methods). When needed, anoxia was induced by adding glucose (20 mM) and glucose oxidase (280 U.mL -1 ) (see Material and methods). (A) ECS and P700 based PSI yield in the green alga Chlamydomonas reinhardtii, the higher plant Arabidopsis thaliana and the dinoflagellate Amphidinium carterae. T222, wild type; pcy, plastocyanin mutant; +MV (4 mM), methylviologen. (B and C) Kinetics of P700 (B) and ECS (C) during and after the multiple turnover saturating light pulse in C. reinhardtii wild-type, with (closed circles) and without (open circles) MV (4 mM) and in the plastocyanin mutant without MV (triangles). Black and red horizontal bars correspond to darkness and multiple saturating light pulses, respectively. For all panels, data corresponds to the average (± SD) of 3 biological replicates.

Figure

  Figure 5A sums up all the data presented in Figures 1, 2 and 3 where the ECS and P700 methods were compared. We used a color code based on the Y(NA)P700 parameter from the Klughammer and Schreiber (K&S) method, which in their original work was intended to measure the acceptor-side limited centers. In all the situations where the Y(NA)P700 is low

  From there, one can calculate the true Y(I) from the P700 pulse measurements and estimate the degree of underestimation due to the assumption that all open PSI centers become donor-side limited during the pulse (see Appendix 1 for calculations). If our simple assumption holds true, then Y(I)P700 should underestimate the true PSI yield proportionally to Y(NA)P700. We obtain the following equation: Y(I)P700/Y(I) = 1-Y(NA)P700 (equation 4 in Appendix 1). This is exactly the relationship that we obtained experimentally by plotting the ratio between the PSI yield measured by the two methods, i.e. Y(I)P700/Y(I)ECS, as a function of the Y(NA)P700 (Fig 5B). This validates our hypothesis that the error made by the P700 pulse method originates from the erroneous hypothesis that the pulse only generates donor-side limited centers, regardless of the physiological conditions before the pulse.

Figure 5

 5 Figure 5 Comparison of the fractions of open PSI centers estimated with ECS and P700 methods in C. reinhardtii For (A), (B) and (C), data from Figures 1, 2 and 3 were used. The color scale on the right represents Y(NA)P700 values (i.e. the fraction of P700 that are not oxidized during the multiple turnover saturating light pulse). (A) Y(I)P700 is plotted as a function of Y(I)ECS, (B) The ratio between Y(I)P700 and Y(I)ECS is plotted as a function of Y(NA)P700. The dashed grey line corresponds to Y(I)P700/Y(I)ECS = [1-Y(NA)P700]. Color scale as in A. (C) The corrected PSI yield Y(I)P700 = Y(I)P700 / [1-Y(NA)P700] is plotted as a function of Y(I)ECS. The color scale on the right represents Y(NA)P700 values (D) Expected relative error of the Y(I) based on the P700 pulse method (black) and the Harbinson and Foyer method (red) as a function of the real acceptor side limitation, Y(NA). In each case the expected relationships for Y(I) values of 0.25, 0.5 and 0.75 are shown (see text and Appendix 1).

  and Y(I)ECS are in very close agreement (Fig 5C), i.e. the ratio becomes 1 as expected (Sup Fig

  , 0.3-0.42 in Jatropha curcas L (Ranjan et al. 2014), 0.65 in Microsorum punctatum and 0.55 in Paraleptochillus decurrens (Wang et al. 2013). Similar low values of Y(I)P700 in dark-adapted photosynthetic organisms have been documented in the angiosperm Ipomoea nil and in the cyanobacterium Synechococcus elongatus PCC 7942 (Shimakawa et al., 2019).

Figure S3 :

 S3 Figure S3: Comparison of the fractions of PSI open centers estimated with ECS and P700 methods in C. reinhardtii in oxic conditions PSII were inhibited with DCMU (10 µM) and HA (saturating concentrations, see Material and methods) while glucose (20 mM) and glucose oxidase (280 U.mL -1 ) were used to produce anoxia (see Methods). Y(I)P700 and Y(I)ECS were measured at different times after the onset of light (26, 56 or 135 µmol photons m -2 s -1 ). Data points correspond to those shown in Figure 3B and color code stands for Y(NA)P700 values (i.e. the fraction of P700 that are not oxidized during a multiple turnover saturating light pulse).

  . As a consequence, virtually all PSI should be fully open. b) Schematic model explaining the error made by the P700 pulse method In the manuscript, we proposed that P700 method underestimates Y(I) yield because (i) the multiple turnover pulse allows several charge separations to occur and (ii) the transfer of several electrons on the acceptor side of PSI will change the redox state of PSI acceptors. In order to better visualize this, we propose a scheme (Figure 2.8) representing what happens during a multiple turnover in a dark-adapted sample for an open PSI center. Two situations are considered: in the first one (top), P700 is reduced but plastocyanins are oxidized, as well as two PSI acceptors, i.e. the pool of PSI acceptors (

Figure 2 - 8 :

 28 Figure 2-8: Schematic model explaining the error made by the P700 pulse methodThe figure present the behavior of two open PSI during a multiple turnover saturating light pulse. P700 represents the PSI primary donor, the special pair of chlorophyll; PC refers to plastocyanin, the PSI secondary donor (PC and PC + being its reduced and oxidized form, respectively); A represents PSI primary acceptor (A and A -being its oxidized and reduced form, respectively). In example 1, the number of PSI donors (1 P700) is higher than the number of PSI acceptors (2). In example 2, PSI has two donors (1 PC and 1 P700) and only one acceptor.

Figure 2 - 9 :

 29 Figure 2-9: P700 oxidation and reduction pathways (dashed and solid blue arrows, respectively) and action of the chemicals that are used in our Pm measurement protocol (red crosses or arrows) Black arrows represent photosynthetic electron flows upstream and downstream PSI. DCMU, DBMIB, MV stand for 3-(3,4-dichlorophenyl)-1,1-dimethylurea, dibromo-6-isopropyl-3-methyl-1,4-benzoquinone and methylviologen, respectively.

  -10). Of course, a 25% error on the normalization results in a 25% error on the values of Y(I) and Y(ND) but has even more important consequences on the estimate of Y(NA)P700.

Figure 2 -

 2 Figure 2-10: Maximal P700 absorption change, Pm, measured with the standard protocol or with our improved protocolPm was measured as the difference between absorbance in the dark and after a multiple turnover saturating pulse of light (12ms duration). In the standard protocol (light red), only PSII were inhibited by saturating concentration of DCMU and the light sequence provided detection pulses every 100ms in the dark. In our improved protocol (dark red), cytochrome b6f was inhibited by saturating concentrations of DBMIB (1µM) and methylviologen (4mM incubated for at least 4 minutes), a PSI exogenic acceptor, was added in saturating concentrations. Moreover, the sequence provided detection pulses every seconds in dark conditions. Data with both protocols were normalized to the value obtained with the improved protocol and represent the average of three biological samples of Chlamydomonas reinhardtii in oxic conditions(SD)

  them, qP is a good estimation of the fraction of PSII open centers, when a pure 'puddle' antenna model is considered. In such a model, each PSII center possesses its own

  proposed another estimation of the fraction of open PSII centers: the parameter qL, which is more appropriate to species (like terrestrial plants) with a high connectivity of PSII units. connectivity between PSII centers can vary between species and can also depend on physiological conditions. That's the reason why we considered a conservative approach where the fraction of open PSII centers is intermediate between the values obtained with the two extreme models: puddle and lake. two extreme estimations of the fraction of open PSII from the Y(total), we could calculate a minimal and maximal value for Y(I) ECS . Figure 2-12A shows comparison of Y(I)P700 and Y(I)ECS measured under several light intensities in the absence of PSII inhibitors. Y(I)ECS was shown as a bar whose extremities correspond to its estimations using the puddle or lake model (lower and higher estimations, respectively). In these conditions, Y(NA)P700 is high and, as expected, Y(I)P700 underestimates the fraction of open PSI regardless of the model we used for Y(II) estimation. In the presence of MV, like in PSII treated samples, the Y(NA)P700 is suppressed and the P700 and ECS methods concord (Figure 2-12C).

Chapter 2 :

 2 Probing PSI activitySuzanne FERTE -Doctoral Thesis -2019

Figure 2 -

 2 Figure 2-12: Comparison of the fraction of open PSI centers in C. reinhardtii estimated with the ECS and P700 methods under different light irradiances The fraction of open PSI centers was estimated with the ECS and P700 methods (Y(I)P700 and Y(I)ECS, respectively). The right and left extremity for Y(I)ECS correspond to Y(total) -qL and Y(total) -qP respectively. Experiments were carried out in oxic conditions at 0, 56 and 340 µmol photons m -2 s -1 in the absence (A) or presence (C) of MV. Panels B and D show the same raw data as panels A and C, respectively, but the value on the ordinate axis is the corrected value Y(I)P700 / (1-Y(NA)). Data correspond to the average (± S.D.) of 3 independent biological samples.

  ETR(I) = Y(I) . I . Iwhere I . I is the rate of photon absorption by PSI at the light irradiance I.

Chapter 2 :

 2 Probing PSI activitySuzanne FERTE -Doctoral Thesis -2019

Figure 2 -

 2 Figure 2-13C and F present the number of stable PSI charge separations performed before steady state is reached in oxic high light and anoxic medium light conditions, respectively. In both conditions, the P700 bias induces an underestimation by a factor ~2 of the number of PSI stable charge separations.

Figure 2 -

 2 Figure 2-13: PSI parameters measured by ECS and P700 method during light to dark transition when PSII is inhibitedThe fraction of open PSI centers (Y(I), A/D), and acceptor side limited centers (Y(NA), B/E) were measured at different times after light onset of at 800 µmol photons.m -2 .s -1 in oxic conditions (A and B, respectively) and under 135 µmol photons.m -2 .s -1 in anoxic conditions (E and F, respectively). PSII was inhibited by saturating concentrations of DCMU (10µM) and hydoxylamine (saturating concentration, see material and methods from article) and anoxia was induced by glucose (20mM) and glucose oxidase (280U.mL -1 ) addition. Data were obtained from P700 method (red), ECS method (blue) or correction of the P700 method (green) (see introduction of section 2.3.4 for calculation details). The number of charge separation during the oxic (C) and anoxic (F) transition from dark to light were calculated as the integrated electron transfer rate over time until the steady state is reached. The first 450 ms and first 60 s were considered for oxic and anoxic conditions, respectively. Data corresponds to one representative sample of Chlamydomonas reinhardtii wild type T222 + .

Figure 2 -

 2 Figure 2-14: Comparison of time-dependent changes of reduced P700 (red. P700), Y(I) and Y(II) (here noted ΦI and ΦII, respectively) during a dark-light induction period of a sunflower leaf in air From Klughammer and Schreiber 1994.Fluorescence parameters and P700 absorption changes were recorded simultaneously. Actinic light (800 μmol photon m -2 s -1 ) was turned on at time 0.

Figure 2 -

 2 Figure2-15 shows data already presented in the article (Figure2A) but here, PSI parameters are presented as a function of light irradiance. We represented data obtained with P700 method (red), with computation of ECS and fluorescence data (blue) and correcting P700 method (green). PSII were inhibited and, as previously discussed, P700, corrected-P700 and ECS methods concord on data from moderate to high light (> 26 µmol photons m -2 s -1 ). But under low light, Y(I)P700 is much lower than Y(I)ECS and Y(I)P700,corr, up to a factor 2. This leads to underestimating the electron transfer rate through PSI under low light.

Figure 2 -

 2 Figure 2-15: Light dependence of PSI parameters, Y(I) (left panel), Y(NA) (middle panel) and Y(ND) (right panel), measured by ECS and P700 method when PSII is inhibited.PSI parameters were measured under steady state light (after a minimum of 2 minutes acclimation) and in oxic conditions. PSII was inhibited by saturating concentrations of DCMU (10µM) and hydroxylamine (saturating concentration, see material and methods of the manuscript). Data were obtained from P700 method (red), ECS method (blue) or correction of P700 method (green) (see introduction of section 2.3.4 for calculation details). Data are the average of three independent biological samples (±SD) of the Chlamydomonas reinhardtii wild type T222 + and correspond to data presented in Figure1B/C of the manuscript.

Figure 2 -

 2  shows results from a study on the effect of short pulses in the dark to

Figure 2 -

 2 Figure 2-16: Light dependence of P700 parameters measured before PSI inactivation treatment (Control) and 2 days after PSI inactivation (Inactivated) on wheat leaves. From Zivcak et al., 2015.The fraction of open PSI, here noted ΦPSI (A), the fraction of donor side limited centers, here noted Φ(ND) (B), and the fraction of acceptor side limited centers, here noted Φ(NA) (C) were estimated using the P700 method under different light intensities. The apparent electron transport rate in PSI (ETRPSI) was estimated based on ΦPSI values calculated using initial Pm. The rapid light curves were obtained after previous acclimation to moderatelight (light intensities 14, 21, 30, 45, 61, 78, 103, 134, 174, 224, 281, 347, 438, 539, 668, 833, 1036, 1295, 1602, and 1930 μmol photons m -2 s -1 ); the duration of each interval with a given light intensity was 30s. Data corresponds to the average values from six plants (± SD).

Figure 2 -

 2 17A and B show PSI parameters measured in Arabidopsis thaliana wild type in the absence (A) and presence (D) of antimycin A. Y(I)P700 is always lower in the presence of antimycin A because of higher Y(NA)P700. Authors then use Y(I)P700 to calculate an electron flow through PSI that they compare to the electron flow through PSII, LEFO2 in order to calculate a Δflux indicating the amplitude of CEF (Figure 2-17C). Based on this observation, the authors concluded that the CEF is reduced in the presence of Antimycin A.

Figure 2 -

 2 Figure 2-17: Light irradiance dependency of Arabidopsis thaliana PSI parameters in WT (A and B), WT with antimycin A (C and D) and pgr5 mutant (E and F). Raw data from Kou et al. 2015 was kindly sent by the authors. Fraction of open PSI,Y(I) and fraction of acceptor side limited PSI, Y(NA), were estimated with the P700 method (left panels, A, C and E) and with the correction proposed in our article (right panels, B, D and F). Each leaf disk was exposed to increasing light irradiance steps using white halogen light filtered through neutral density filters. The leaf disk was maintained under each irradiance for about 10 min to reach steady-state photosynthesis. Measurements were performed at 25•C under air conditions. Values are means of 8, ND, and 16 leaf disks for A-B, C-D, and E-F, respectively.

  a) Role of CEF in ATP/NADPH ratio equilibration It has long been debated whether the ATP/NADPH ratio produced by LEF is equivalent to the ATP/NADPH ratio consumed in the CBB cycle and other metabolic processes. According to the most recent cryo-electron microscopy works on plant chloroplast ATP synthase (CF1-F0),

(

  photons m -2 s -1 ), the absorption cross section  (m 2 ) and the quantum yield of the photosystem i.e. the probability that a photon gives rise to a charge separation, Y (without unit). We can write: ETR(I) (electrons/PSI/s) = I. I. Y(I) and ETR(II) (electrons/PSII/s) = I. II. Y(II), which, once the fractions n2 and n1 of PSII and PSI are considered, leads to:

Figure 3 -

 3 photosynthetic organisms. Figure3-1 displays the fraction of publication dedicated to CEF in the different groups of photosynthetic organisms. The proportion of the studies focusing on diatoms, dinoflagellates and haptophytes is very small; only 2.5% of the publications on the subject concern species which are not cyanobacteria or part of the green lineage.

Figure 3 - 1 :

 31 Figure 3-1: Percentage of the publications about cyclic electron flow among the various oxygenic photosynthetic cladesThe percentage of publications was estimated by searching on the Pubmed database the number of publications mentioning "Photosynthesis" AND "cyclic electron flow" AND a photosynthetic clade such as "Plant", "Cyanobacteria", ["Green algae" OR "Chlorophytes"], ["Diatom" OR "Stramenopiles" OR "Brown algae"], "Red algae", "Dinoflagellates" and "Haptophytes".

Chapter 3 :

 3 Diversity of cyclic electron flow in microalgaeSuzanne FERTE -Doctoral Thesis -2019

  . Hence, the long-lived ECS signals are no longer contaminated by other light-induced absorption changes (e.g. cytochrome oxidation-reduction signals). A strong pulse of light (~ 2000 photons s -1 PS1 -1 ) allowed the generation of electric field and ECS signals were measured from 50 ms after the end of the pulse. The homothetic decay of the ECS spectrum of Amphidinium carterae indicates that there cannot be both a linear and a quadratic component of ECS. The fact that the ECS induced by a saturating laser flash does not depend on the pre-existing electric field demonstrates the linearity of the ECS signal as a function of the electric field (data not shown here). The ECS spectra of Amphidinium carterae (A) and Symbiodinium sp. (B) are shown in Figure 3-2.

Figure 3 - 2 :

 32 Figure 3-2: ECS spectrum of Amphidinium carterae (A.) and Symbiodinium sp (B.) A. Amphidinium carterae ECS spectrum was obtained by Benjamin Bailleul before I started my phD and have been measured as the absorption changes induced by a saturating pulse of light and remaining after 50 ms of dark (see Methods). B. Symbiodinium sp. ECS spectrum was obtained from Roberty and co-workers (Roberty et al, 2014) and correspond to the absorption changes induced by a saturating pulse of light in anoxia.

Figure 3 - 4 :

 34 Figure 3-4: Simulations of the relative changes of ETR(II) (blue) and ETR(total) (black) when titrating LEF by DCMU Simulations correspond to different proportions of total photosystems (I and II not differentiated) initially involved in CEF (0%, 10% and 20%). A. shows evolutions of ETRT(II) and ETR(total) as a function of DCMU concentration. Note that when there is no cyclic electron flow, ETR(total) and ETR(II) evolutions are confounded. B. shows the same simulations as in A but ETR(total) is plotted as a function of ETR(II).

( 5 ) 3 :

 53 photosystems stoichiometry. In this work, in order to increase the robustness of our approach, we don't measure any absolute values of electron flows but only the quantum yield Y(II) and Y(total), of photosystems. If II and total are constant, relative changes in electron flows, ETR(II) and ETR(total), are proportional to relative changes in Y(II) and

  -5A). The sum of the activities of the two photosystems follows the activity of PSII alone, whatever the DCMU concentration, suggesting that PSII activity is the only determinant of PSI activity. When plotting Y(total) as a function of Y(II) for three biological replicates, we get a linear relationship: the data was fitted by an affine function, the correlation coefficient is high (R 2 = 0.997) and the intercept corresponds to the origin of the axis (y-intercept = 0.02 ± 0.01) (Figure3-5B). This behavior is compatible with the model of absence of cyclic electron flow we drew before.

Figure 3 - 6 : 3 :

 363 Figure 3-6: Relative changes of the fraction of open PS, Y(total), and the quantum yield of PSII, Y(II), when titrating LEF by DCMU in the dinoflagellate Symbiodinium sp All measurements were performed under steady state illumination at 26 µmol photons m -2 s -1 . Y(total) and Y(II) were normalized to their initial values, when no DCMU was added. A. Y(total) (black) and Y(II) (blue) as a function of DCMU concentration obtained in one representative biological replicate (green points in panel B). Note that DCMU concentrations are expressed in an arbitrary unit the full inhibitor penetration until the QB site is a very long process in this species. B. Y(total) as a function of Y(II). Data corresponds to 3 independent biological replicates. The dashed grey line shows the y=x line, corresponding to the situation where there is no CEF. The dashed black line shows the linear regression of the experimental data (y = 0.80 x + 0.19 with an adjusted correlation coefficient of 0.995).

Figure 3 - 7 :

 37 Figure 3-7: Relative changes of the fraction of open PS, Y(total), and the quantum yield of PSII, Y(II), when titrating LEF by DCMU in the green alga Chlamydomonas reinhardtii All measurements were performed under steady state illumination at 135 µmol photons.m -2 .s -1 . Y(total) and Y(II) were normalized to their initial values, when no DCMU was added. A. Y(total) (black) and Y(II) (blue) as a function of DCMU concentration obtained in one representative biological replicate (pink points in panel B). B. Y(total) as a function of Y(II). Data corresponds to 7 independent biological replicates. The dashed grey line shows the y=x line, corresponding to the situation where there is no CEF. The dashed red line shows the y=0.5*(1+x) line, corresponding to the situation where PSI activity remains constant regardless PSII activity. The dashed black line corresponds to polynomial fit of the experimental data.

Figure 3 -

 3 Figure 3-8A/B shows measurements of ECS increase in the presence and absence of PSII inhibitors after light onset at different light intensities and at different time after light onset. As expected, the lowest is the light irradiance, the slowest is the ECS increase. And the slope of ECS increase is proportional to light irradiance. The observation that ECS increases linearly with time in this range indicates that the PS remain open. The plot of the slopes of the ECS increase as a function of light irradiance directly gives σtotal and σI in the absence (Figure 3-8D) and presence (Figure 3-8C) of PSII inhibitors, respectively.

Figure 3 - 9 : 3 :

 393 Figure 3-9: Kinetics of ECS decrease after light offset at two different light irradiances Light was turned off at time 0 and he ECS decrease from 1.3 ms to 16 ms was fitted by an exponential decay function (blue line). The initial slope is calculated from the result of the fit (red line) and estimates the total electron transfer rate, ETR(total). Measurements were performed for two different light intensities: 135 µmol photons m -2 s -1 (A) and 1500 µmol photons m -2 s -1 (B). White and black horizontal bars correspond to light and darkness, respectively. Each panel corresponds to one representative measurement performed on a same sample of the Chlamydomonas reinhardtii stt7-9 mutant.

Figure 3 -

 3 10 shows that under low light (135 µmol photons m -2 s -1 , Figure3-10A), ECS increase measured after a single turnover laser flash equals the Y(total) fitted from the dark relaxation. At the contrary, under high light (1500 µmol photons m -2 s -1 , Figure3-10B), ECS decrease after a single turnover laser flash shows a biphasic relaxation, the first phase being attributed to fast charge recombination.

Figure 3 -

 3 Figure 3-10: ECS decrease after a single turnover laser flash superimposed to two different light irradiances A single turnover laser flash was superimposed to a beam of actinic light of 135 and 1500 μmol photons m -2 s -1for panels A and B, respectively. The subsequent ECS decrease in the dark was measured and fitted by an exponential function excluding the first point of the kinetics and extrapolated to the time of "a phase" measurement (i.e. 300µs). White and black horizontal bars correspond to light and darkness, respectively. Red arrows represent the single turnover laser flash that was applied at time 0. Each panel shows one representative measurement performed on a same sample of the Chlamydomonas reinhardtii stt7-9 mutant.

Figure 3 -

 3 Figure 3-11B. As expected, Y(total) decreases with light irradiance because PS activity becomes limited by the rate of chemical steps (cytochrome b6f or CBB cycle). Figure 3-11C shows the ETR(total) calculated as the product Y(total), I and σtotal. The results are very similar to the ones obtained with the DIRK method (Figure 3-11C).

Figure 3 -

 3  shows the relationship between the photochemical rate obtained with the DIRK method as a function of the ETR(total) based on yield and absorption cross-section.Both methods concord at all light intensities (linear relationship with a factor of proportionality of 1.02 and a correlation coefficient of 0.995). This is very reinsuring: the concordance between the two methods, which rely on the same signal (ECS) but very different measurements, strongly suggests that the methods we use for measurements of photochemical rate, yields and absorption cross-sections are correct.

Figure 3 -

 3 Figure 3-13: ETR(total) (black) and ETR(II) (blue) as a function of light irradiance in three biological replicates (shown in panels A, B and C) ETR(total) and ETR(II) are normalized per total PS and per PSII, respectively. ETR(total) was assessed with the DIRK method and ETR(II) has been calculated from ECS-based PSII photon absorption rate, σII . I. Data was obtained on three independent biological samples of the Chlamydomonas reinhardtii stt7-9 mutant.

Figure 3 -

 3 14 shows ETR(total) and ETR(II) expressed per total PS measured in three biological replicates (same experimental data as in Figure 3-13), with additional calculation of ETR(I), also expressed in electrons per second per PS. The latter value was calculated as: ETR(I) = ETR(total) -ETR(II).

Figure 3 -

 3 Figure 3-14: ETR(total) (black), ETR(II) (blue) and ETR(I) (red) as a function of light irradiance in three biological replicates (shown in panels A, B and C)Here, the raw data is the same as in Figure3-13 but ETR(total) and ETR(II) are both normalized per total PS. ETR(I) was calculated as the difference between ETR(total) and ETR(II) (being normalized per total PS).

  photosynthetic chain production and metabolic pathways requirements.However, results obtained with ECS-based σII show a smaller ratio between CEF and LEF (~0.5, panel B) meaning that about a third of the PSI are involved in CEF (panel A).

3. 5 Discussion and future perspectives 3 . 5 . 1 A

 5351 simple and robust protocol to investigate CEF diversity highlights three different behaviors CEF has been proposed as an alternative electron pathway about 70 years ago and, while linear electron flow is now rather well understood, many questions regarding CEF remain unclear, especially: is CEF occurring in all photosynthetic organisms? How are the rates of LEF and CEF regulated?

  -18A) indicating that cells were suffering nitrogen limitation which limited their growth. At the beginning of stationary phase, roughly one or two days after cells started experiencing nitrogen limitation, we harvested cells and titrated DCMU while measuring Y(total) and Y(II) evolution (Figure 3-18B). Results are very different from the ones in non-stressful conditions. The relationship simply assessed in conditions of saturating DCMU concentration. The y-intercept corresponds here to the yield of PSI which can be multiplied by PSI absorption cross-section and light irradiance to give the CEF rate. But other classical methods ae used in routine to measure CEF in saturating DCMU conditions: photochemical rate from ECS or P700

Figure 3 -

 3 . The first possibility invokes a physical separation of the LEF and CEF chains, i.e. a regionalization of CEF at the plastid scale. This could be a lateral segregation of PSII and PSI. In plants and green algae, PSII and PSI are not homogeneously distributed into the cell[START_REF] Anderson | Insights into the consequences of grana stacking of thylakoid membranes in vascular plants: a personal perspective[END_REF]. PSII are mostly localized into the grana regions

  photosystems (PSI + PSII) activity in the absence of DCMU was involved in CEF. Before normalization the Y(total) in the absence of DCMU was 0.85, meaning that 85% of photosystems were initially open.

Figure 3 -Chapter 3 :

 33 Figure 3-19: Testing CEF behaviour on a Chaetoceros sp. dominated sample from the field A. Water has been sampled in the Penzé River (Roscoff, Britany, France) and the percentage of Chaetoceros sp (diatom), other diatoms, dinoflagellates and other cells (NA) were determined by morphological identification of more than 600 cells. B. Normalized values of Y(total) and Y(II), measured at different DCMU concentrations in the Chaetoceros sp. dominated sample, are plotted one against the other. Measurements were performed under steady state light at 135 µmol photons m -2 s -1 . The dashed grey line shows the y=x line, corresponding to the situation where there is no CEF. The black dashed line shows the linear regression of the experimental data (y = 0.89 x + 0.11 with an adjusted correlation coefficient of 0.999). Data correspond to one sample measurement.

Figure 3 -

 3  is not meant to represent robust data but can be useful to illustrate this possibility (it is a very preliminary experiment performed only once). When the temperature of a Chlamydomonas (stt7 mutant) sample was decreased (phase 1 in Figure3-20), the Y(total) decreased less than the Y(II) indicating an activation of CEF. This response was reversible when the temperature was increased again. Developing such a protocol would help understanding how the photosynthetic physiology, in terms of CEF/LEF regulation, responds to temperature changes.

Figure 3 -

 3 Figure 3-20: Evolution of Y(total) (black) and Y(II) (blue) when temperature is decreasing (phase 1) and increasing (phase 2). Y(total) and Y(II) were normalized to 1 at the beginning of the experiment (i.e. time 0 and T°C = 20 °C). Temperature change was induced by a water bath system surrounding the sample cuvette. Temperature within the sample varied from 20°C to approximately 10°C. Data correspond to measurements done in one biological sample.

Chapter 3 :

 3 Diversity of cyclic electron flow in microalgaeSuzanne FERTE -Doctoral Thesis -2019

Figure 3 -

 3 Figure 3-21 : Fraction of PSI involved in CEF as a function of light intensity for the three replicates used in section 3.4.3 The three colors correspond to the three replicates shown in Figure 3-13, Figure 3-14 and Figure 3-15 (purple, orange and red, being the replicate shown in the left, middle and right panels, respectively). Here, only the data obtained with ECS-based estimation of the photon absorption rate (σII . I) is shown. Purple, orange and red dashed lines correspond to the average ETR(I)CEF/ETR(I) for each replicate, ETR(I) being the electron transfer rate through the PSI. Black dashed line correspond to the ratio ETR(I)CEF/ETR(I) required for the CBB cycle to function.

  my thesis, I had the pleasure of participating in two field on the coast of Raunefjorden near Bergen in Norway, in the spring of 2017 and 2018. In 2017, the Mesohux campaign was organized by Bidle and Thamatrakoln labs (Rutgers, USA). In this chapter, I present the results from the most spectacular and verified bloom of Emiliania huxleyi, obtained in 2018 as part of a scientific campaign organized by the Vardi group (Weizmann Institute, Israel). This

  Photophysiology by Fast Induction and Relaxation fluorometry (FIRe)Photosynthesis physiology was measured for each bag independently every day between 8 am and 10 am. The photosynthetic physiology was measured directly after sampling, the fluorometer being installed on the raft adjacent to the mesocosms. We used a miniFIRE (Miniaturized Fluorescence Induction and Relaxation System, Figure 4-2A). Our MiniFire was built by Maxim Gorbunov, from Paul Falkowski's laboratory (Department of Marine Sciences, Rutgers University, NJ, USA). MiniFIRe records fluorescence induced by a series of subsaturating flashes that cumulatively saturate PSII within 100-200 s (Kolber et al. 1998). A background actinic light could be added (blue LED). A typical induction curve is presented in Figure 4-2B. MiniFIRe analysis program fits induction curve with a mathematical model that estimate 4 parameters:-the initial fluorescence level in the conditions of interest (extrapolated at time 0). This level is called F' if measured in the presence of background light, and F0 if measured on dark adapted sample, -the maximal fluorescence yield at the end of the induction. It is called Fm or Fm' in dark adapted or illuminated samples, respectively -the absorption cross section, II, that depends on the kinetics of F0 (or F') to Fm (or Fm') fluorescence rise. We only use this value from dark-adapted samples.

Figure 4 - 3 :

 43 Figure 4-3: Three parameters are estimated out of the ETR(II) versus intensity curves ETR(II) was measured under steady state at different light irradiances (blue points). Data was fitted by the exponential function: ETR(II) = ETRmax * exp(-I/Ek) (orange line)where ETRmax is the maximal PSII electron transfer rate when light is saturating, Ek is the light saturation parameter. In order to exclude light intensity that were much higher than the saturating light (for which the measurement noises are important), two consecutive fits were performed. The first one enabled a preliminary estimation of Ek from the whole dataset and the second one used only data below 3*Ek intensities. ETRmax and Ek were taken from the second fit and the initial slope of the fitted curve was noted α and was calculated as α=ETRmax/Ek. Data presented in this graph were measured on day 14 and on bag 3.

  5.2 in collaboration with Pascal Campagne, research engineer in the group STATS of the Institut Pasteur, Paris. He helped also in the preparation of the plots related to the statistical analyses. a) RS: function repeatability Variance observed in each photosynthetic and environmental variable was partitioned to assess the proportion of variance observed within each bag across days (RS, a repeatability score) and the proportion of variance (1 -RS) that could be attributed to differences among bags (within a day). Note that any arbitrary distribution of observations into groups (bags, days) results in a non-null amount of variance among groups, due to random sampling fluctuation. We used ad hoc functions (R package heritability) that correct for such a random noise so that effects due to contrasts across days only (RS) are properly estimated. b) Correlation matrix Pairwise Pearson's correlation coefficients were computed and plotted using both packages psych and corrplot. The corresponding p values were further adjusted to account for multiple testing using the Benjamini-Yekutieli procedure. c) Between-Class Analysis (BCA)

Figure 4 - 4 :

 44 Figure 4-4: Location of the experiment and mesocosms setting A. Map of Norway. Red arrow points to the location of the University of Bergen mesocosm center (Googlemaps). B. Emiliania huxleyi is naturally blooming on the Norwegian coast nearby Bergen. Picture was taken from satellite. Red arrow points to the location the University of Bergen mesocosm center. Image provided by the SeaWiFS Project, NASA/Goddard Space Flight Center and GeoEye. C. Satellite view of the Bergen's Marine Biological Station (Googlemaps). Yellow arrow points to the raft where the mesocosms were setand red arrow points to the laboratory. D. and E. Set up consisted in four uncovered mesocosms (D.) and 3 covered ones with a polyethylene plastic sheeting (E.) (Courtesy of Vardi lab)

Chapter 4 :

 4 photochemistry when the photosystem II is open (Figure 4-8A), -The PSII absorption cross section, II, which provides information on the ability of PSII to capture light energy (Figure 4-8B)

  photosynthesis versus intensity curve, informs about the efficiency of photosynthesis under low light.A last parameter concerns the capacity of the chain to set up mechanisms for the dissipation of light energy in the form of heat, NPQ (Figure4-8G). The level of NPQ depends on the light irradiance in a non-linear manner; here, we used the amplitude of the NPQ at the maximum intensity tested (500 µmol photons m -2 s -1 ). The daily measurement of the relaxation of this NPQ under low light intensity on pooled samples from bags 1 to 4 made it possible to demonstrate that the measured NPQ was mainly reversible after 2-3 minutes of low light (Figure4-7). It can therefore be attributed to regulative mechanisms (qE) and not to photoinhibition of PSII (qI).

Figure 4 - 7 :

 47 Figure 4-7: Measurement of Non Photochemical quenching (NPQ) measured in steady state at 500 µmol photons m -2 s -1 .(A) and its reversible (qE, fully reversed in 2-3 minutes) and irreversible (qI) components (B and C, respectively)Measurements were performed around 10.30 am on a sample pooled from bags 1 to 4. Non photochemical quenching, NPQ, was measured under steady state at 500 µmol photons m -2 s -1 each day of the campaign (A). NPQ relaxation was monitored until reaching a pseudo steady state (from 1 to 5 minutes of relaxation) and the reversible NPQ was noted qE (B) while the remaining NPQ was noted qI (C).

  situ data have lower RS. We can propose two explanations: covered bags received slightly less light than open bags because of the plastic cover covering them, causing different light stress. Secondly, we have to highlight that those measurements were subjected to more technical Chapter 4: Photosynthetic physiology of the coccolithophore Emiliania huxleyi Suzanne FERTE -Doctoral Thesis -2019

Figure 4 - 8 :

 48 Figure 4-8: Evolution of fluorescence parameters indicative of photosynthetic physiology from day 0 to day 23 of the campaignEach day of the campaign, 9 parameters were estimated from fluorescence measurement data: the maximal PSII quantum yield, Fv/Fm (A), the absorption cross section, II (B), the connectivity between PSII (C), the maximal PSII electron transfer rate, ETRmax (D), the light saturation parameter, Ek (E), the initial slope of the photosynthesis versus intensity curve, α (F), the non-photochemical quenching, NPQ (G), the maximal PSII quantum yield measured directly after sampling, In situ Fv/Fm (H) and the non-photochemical quenching in place in the bags (before sampling), In situ NPQ (I). The repeatability score, RS, that is the fraction of common variability between the bags was estimated for all variables. For Fv/Fm, II, and p, RS values are 0.707, 0.712 and 0.77, respectively. For ETRmax, Ek and , RS values are 0.864, 0.695 and 0.474, respectively. For NPQ, in situ Fv/Fm and in situ NPQ, RS values are 0.626, 0.485 and 0.551, respectively. Bags 1 to 4 were uncovered, bags 5 to 7 were covered by a polyethylene plastic sheeting.

Figure 4 -

 4 Figure 4-13: Estimation of the fraction of the fluorescence signal emitted by Emiliania huxleyi From day 9 of the campaign, a sample pooled from bags 1 to 4 was filtrated on a 3 µm filter and the retentate was resuspended in the same volume of sea water filtrated on a 22µm. A. The maximal fluorescence emitted by the retentate (> 3µm fraction) is expressed as a proportion of the maximal fluorescence emitted by the initial sample (before filtration). B. The relative abundance of the 4 groups of phytoplankton was measured by flow cytometry in the retentate (> 3 µm fraction) (color code of the species on the right). Analysis of panel B was performed by F. Vincent and A. Vardi.

Figure 4 -

 4 Figure 4-15: Between Class Analysis (BCA) on environmental parameters BCA analysis was performed on the environmental parameters dataset from the whole campaign. A. For each day of the campaign, the barycenter of the 7 bags was projected on the plan made of the two first axes of the BCA (BCA1 and BCA2) and is denoted by a number. Data from each bag has also been projected on the plan and are represented by a grey point connected to the barycenter with a grey line. B. correlation circle of the environmental parameters. Variables whose arrows are close to the correlation circle of radius 1 tend to be fully represented on the two first axes.

Figure 4 -

 4 Figure 4-16: Correlation matrix containing photosynthetic variables and abiotic environmental parametersData from all bags and all days of the campaign were used to calculate pairwise correlations. The correlation coefficient, R 2 , is presented as a color code (legend on the right). Crosses represent correlations that are not significant (p<0.5).

(

  National School of Agronomy) and the University of Paris Saclay and received a great support from Pascal Campagne, research engineer in the Institut Pasteur Institute (Paris), an expert in statistics applied to experimental results in biology. Because the explanatory variables of the model (i.e. the environmental parameters) are partially correlated, the regression-based approaches cannot be used (one of its fundamental hypotheses is the independence of the

Figure 4 -

 4 Figure 4-17: Contribution of environmental variables to the explanatory model for maximal PSII efficiency, Fv/Fm (A), maximal PSII electron transfer rate, ETRmax (B) and non-photochemical quenching, NPQ (C)Partial least square analysis was performed on Fv/Fm, ETRmax and NPQ using 9 abiotic parameters (water and air temperature, SiO4 2-, PO4 3-, NO3 -, NO2 -, total inorganic nitrogen, NH4 + and light intensity). The percentage of the variance explained by the model is 75, 47 and 37% for Fv/Fm, ETRmax and NPQ, respectively. Value of the regression coefficients for each of the environmental variables are indicated in the horizontal axis. Error bars delimit 95% confidence intervals. In grey, blue and orange, the non-significant, positive and negative standardized coefficients, respectively.

Figure 4 -

 4 Figure 4-19: Estimation of the deviation to the model of the maximal PSII efficiency, Fv/Fm (A, B) and the maximal electron transfer rate, ETRmax (C, D). A. and C. Data from bag 1 to 7 was fitted to a partial least square model (see results in Figure 4-17) and the residual variance was calculated for each bag and each day for Fv/Fm and ETRmax, respectively. B. and D. represent the relative deviation to the daily median value of all bags for Fv/Fm and ETRmax. Color codes correspond to different bags as shown on the right-hand side of figure.

  is sufficient to provide the high demand in reducing power and energy required for the viral replication. However, the number of viral particles released at the moment of the cell lysis phase depends on the light irradiance during the infection (Tamatrakoln, 2018). Such a light dependency of the production of viral particles in conditions of PSII inhibition suggests that a light-dependent PSII-independent process might be involved, making it plausible that cyclic electron flow around PSI plays a role during Emiliania huxleyi infection. Interestingly, what we suggest here corresponds to the behavior observed in Chlamydomonas: when PSII is inhibited, PSI can compensate the loss of activity related to linear electron flow by increasing CEF. Such a response during E. huxleyi infection would provide a precious supplementary source of ATP. With the protocol developed in the chapter 3 (part 3.3), one could test such a model where the inhibition of PSII (due to the arrest of plastid genome expression and of PSII repair) is compensated by PSI-driven CEF, supplying the high energy demanding virus infection process with ATP.

5 . 2 .

 52 Viral serine palmitoyltransferase induces metabolic switch in sphingolipid biosynthesis and is required for infection of a marine alga. Proceedings of the National Academy ofSciences, 113(13), E1907-E1916. Chapter 5: General discussion Summary 5. 1. Methodological overview ......................................................................................... 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? ..... 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 ........................................................................................................... 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 ............................................................................................................. range of a few hundreds of µs which affects the ECS decay at light offset and affects the DIRK measurements of photochemical rate as well. Realizing the disagreement between the two methods allowed me to identify an unanticipated problem and to correct it. After correction (we eliminated the contribution of charge recombination by extrapolating the decay kinetics in the first 1.2 ms where charge recombination occurs), the two methods agreed (Figure 5-1).

Figure 5 - 1 : 5 . 1 . 2 .

 51512 Photochemical rate

Figure 5 - 2 .

 52 When adding DCMU, Y(II) (blue line) decreases as expected. But the evolution of Y(I)P700 (red line) is spectacular. When Y(II) decreases by 30% (4 th point of the titration), Y(I) is almost multiplied by 3 which, when interpreted blindly, would indicate that the CEF has increased tremendously. The electron transfer rate through PSI would have increased by almost afactor 3, which is at least difficult to explain. In addition, the activity of the PSI when all PSII are inhibited (i.e. when there is only CEF) appears to be equal to the initial activity. This would suggest that the CEF in conditions when the PSII is inhibited is equivalent to or higher than LEF under initial conditions, against all the literature on CEF in Chlamydomonas under saturating DCMU. The fluxes measured in saturating DCMU are much lower than untreated LEF flows measured through the PSII (e.g.Alric 2010; Theis et al., 2019).

  n2.σtotal 2 /(σI.(Fv/Fm.σII)) and β = n1.σtotal/σI. Note that when the maximal photochemical rates of PSI (σI.I) and PSII (Fv/Fm.σII.I) are equal, which means that σtotal.I = σI.I = Fv/Fm.σII.I, then ETR(total) = n2.ETR(II) + n1.CEF, i.e the correct expression. In all other cases, ETR(total) is not a proper expression of the total PS activity.

Chapter 5 :

 5 General discussionSuzanne FERTE -Doctoral Thesis -2019 -samples need to be concentrated enough to obtain an appropriate signal-to-noise ratio; -the signals emitted by the species of interest need to be distinguished from other signals; -the photosynthetic physiology should be carefully assessed in order to determine a statistical model that allows one to highlight the influence of each environmental parameter. a) Measuring absorption changes on field samples In the field, most of the studies of photosynthetic physiology are based on the analysis of fluorescence parameters as a means to study the electron flow through PSII. Fluorescence signals are high enough to be measured in a single cell. However, as we pointed out in Chapter 3, the methods used to investigate CEF are based on the measurement of absorption changes that are signals of much lower amplitude. For example in the laboratory,

  aims at increasing the optical path, and thus the absorption signals. It consists in a hollow spherical cavity with its interior covered with a diffuse white reflective coating, with small holes for optical paths. Its relevant property is a uniform scattering or diffusing effect. Light rays incident on any point on the inner surface are, by multiple scattering reflections, distributed equally to all other points. The first tests on this prototype are highly promising with an optical path increased by 10-100 fold. b) Tackling the issue of the species mixture in the field In the field, we measured photosynthetic activity resulting from a mix of several species. It is thus difficult -if not impossible-to study the physiology of each microalga independently. This constraint can be alleviated when we are faced with rather monospecific samples as for the Chaetoceros dominated sample from the Penzé (Roscoff, France) on which we have shown the existence of a CEF independent of LEF (chapter 3) or for photosynthetic physiology measurements on a population dominated by Emiliania huxleyi (chapter 4). Still in most in situ situations the study of photosynthetic physiology is hampered by this constraint.I worked together with Alexandra Peltekis, another PhD student, and Benjamin Bailleul on the development of an innovative method to deconvolute the ECS signals emitted by each species in a mixture. The principle of the method is based on the differences in ECS spectrum between species of different clades (see some ECS spectrum examples inBailleul et al., 2010). Within this project, I developed a Matlab script for the deconvolution of ECS signals and participated to the proof of methods. Figure 5-3 shows the ECS spectra obtained 300 μs after a single turnover laser flash in a homogenous sample of the haptophyte Isochrysis galbana (panel A), in a homogenous sample of the dictiophyte Pseudopedinella sp (panel B) and in the mixture of these two species (1/1 ratio, volume-to-volume) (panel C, black curve). The expected spectrum of the mixture (average of the spectra obtained in isolated samples, panels A and B) is shown in blue in panel C and is almost superimposed to the experimental spectrum of the mixture. Here, the ECS signal of the mixture is equal to the sum of the signals emitted by each species.

Figure 5 - 4 :

 54 Figure 5-4: Expected versus measured ECS signals in 3 mixtures of microalgae. ECS signals were measured during the ECS decay after light offset in isolated samples and mixed samples of the dinoflagellate Amphidinium carterae and the eustigmatophyte Nannochloropsis salina (A), Amphidinium carterae and the haptophyte Isochrysis galbana (B) and the dictophyte Pseudopedinella sp and Isochrysis galbana (C). The ECS signal in the mixture was plotted as a function of the expected ECS signal based on the sum of the signals emitted in isolated samples in the same conditions. Grey line represents the expected y=x line. Data correspond to measurements obtained in one representative experiment in each panel.

  And finally, in Chapter 4, we were able to show that the viral infection of Emiliania huxleyi induces the loss of activity of a significant fraction of PSII. The consequences of such changes in the structure of the photosynthetic chain remains unexplored. Viral multiplication requires a significant energy input. In addition, a recent study shows that the dynamics of viral lysis is light dependent(Thamatrakoln et al., 2019) suggesting the dependence of the efficiency of viral infection on photosynthetic activity. It would be interesting to test the presence of CEF during the viral infection and, if necessary, the evolution of its amplitude with time of infection and loss of PSII efficiency (like what was done in chapter 3 with DCMU titration).

Chapitre 3 : Diversité du FCE au sein de la phylogénie

  Afin d'explorer la diversité du FCE, nous avons proposé une méthode simple, robuste et utilisable dans la plupart groupes photosynthétiques qui permet de mettre en évidence l'existence de FCE chez une espèce et de tester sa relation avec le FLE.Le principe est de suivre l'évolution du FCE (ou d'une fonction du FCE) lorsque l'on inhibe progressivement et spécifiquement le FLE par un inhibiteur du PSII, le DCMU. On peut modéliser simplement les résultats attendus s'il n'y a pas de CEF ou si le CEF est indépendant du LEF et tester les résultats obtenus chez différentes espèces. Cette méthode présente un avantage majeur : en travaillant avec des organismes pour lesquels la section efficace de capture () est constante, suivre l'évolution des rendements quantiques revient à suivre l'évolution des valeurs absolues de flux. On peut alors éviter la mesure du flux de photons absorbés par les PS, source majeure d'approximations. Nous avons estimé le rendement

quantique du PSII par la mesure de sa fluorescence et la fraction de centres (PSI + PSII) ouverts par mesure de signaux ECS (méthode présentée dans la section précédente).

Un premier résultat inattendu est que le FCE n'est pas absolument essentiel à la photosynthèse puisqu'un dinoflagellé, Amphidinium carterae, ne présente pas de FCE dans les conditions que nous avons testées. Lorsqu'un FCE a été mis en évidence, nous avons montré deux types de relation fonctionnelle avec le FLE. Il peut se dérouler de manière indépendante du FLE, comme c'est le cas chez le dinoflagellé Symbiodinium sp. Dans ce cas, la mesure de FCE est simple : l'activité résiduelle mesurée par ECS lorsque tous les PSII sont inhibés est identique à l'activité cyclique lorsque tous les PS fonctionnent. Dans un troisième cas de figure, nous avons montré que le FCE peut être influencé par les variations de FLE comme c'est le cas chez l'algue verte modèle, Chlamydomonas reinhardtii. Ces deux comportements sontils le résultat de mécanismes de régulation différents ? De différences d'enzyme(s) impliquée(s) dans le CEF ? Ou simplement d'un état physiologique différent ? Ces questions restent à approfondir. Dans le cas de Chlamydomonas reinhardtii, puisque le FCE est dépendant du FLE, la mesure du FCE nécessite la mesure de valeurs absolues de flux PSII et de flux totaux dans les conditions que l'on souhaite étudier. Pour cela, nous avons commencé un travail de développement et de validation de méthodes de mesure des flux de photons parvenant aux PSII et à tous les PS. Nos résultats sont encourageants même si une incertitude reste quant à l'amplitude des flux à travers le PSII. Nous avons pu montrer que le FCE est proportionnel au FLE quelle que soit l'intensité de lumière, ce qui constitue un argument fort dans le sens d'un rôle du FCE dans l'ajustement des ratios ATP/NADPH produits par la chaine photosynthétique aux besoins du cycle de Calvin. De plus, en utilisant les valeurs les plus extrêmes de flux à travers le PSII, nous montrons que le FCE permet de produire suffisamment d'ATP pour subvenir aux besoins du cycle de Calvin…et sûrement plus. Quels mécanismes régulateurs permettent d'assurer une proportionnalité entre les FCE et FLE ? Qu'est-ce qui détermine l'amplitude du CEF ? Certaines conditions entraînent-elles une modification de cette relation ? Ces questions restent des pistes de travail pour la suite de ce projet. Chapitre 4 :

Étude sur le terrain des déterminants environnementaux de la physiologie photosynthétique de l'haptophyte Emiliania huxleyi

  Une approche plus holistique pour étudier la diversité des mécanismes photosynthétiques est l'étude de populations prélevées sur le terrain. Au printemps 2018, nous avons été invités à participer à une campagne scientifique sur la côte norvégienne dont l'objectif était d'étudier Ce travail de thèse est fondé sur deux axes de recherche: l'exploration de la diversité du FCE chez les microalgues et l'étude de la physiologie photosynthétique sur le terrain. La convergence de ces deux axes a seulement été initiée et une perspective générale de ce travail serait d'explorer les déterminants, les rôles et les mécanismes de régulation du FCE in situ.Pour cela, je propose des pistes d'améliorations en vue de la mesure de FCE sur le terrain

les déterminants environnementaux biotiques et abiotiques qui sont en jeu lors d'une efflorescence de l'haptophyte Emiliania huxleyi. En participant à cette campagne, nous avions l'espoir de tester notre méthode de mesure du FCE développée au chapitre 3 sur des échantillons prélevés directement sur le terrain. Cependant, en raison de la faible concentration des microalgues étudiées in situ, les signaux de spectroscopie d'absorption (par exemple les mesures d'ECS et de P700 nécessaires pour sonder le FCE) étaient trop faibles pour être exploitables. Les signaux de fluorescence étant plus importants, nous avons pu suivre l'évolution de paramètres liés au PSII et au LEF au cours de la campagne et mettre en évidence l'influence de facteurs environnementaux déterminant la physiologie photosynthétique. La campagne s'est déroulée à la station marine de Bergen en Norvège et a duré 24 jours. De grands sacs en plastique transparents ont été remplis d'environ 11 m 3 d'eau prélevée directement dans le fjord et ont été fixés sur un radeau à quelques dizaines de mètres de la côte. L'ajout de nutriments a permis de provoquer le développement spécifique d'Emiliania huxleyi et nous avons pu montrer qu'à partir du jour 9, les signaux de fluorescence mesurés étaient principalement ceux d'Emiliania huxleyi. En utilisant une approche de statistique multivariée, nous avons pu étudier les facteurs environnementaux influençant la physiologie photosynthétique d'Emiliania huxleyi. Notre modèle statistique permet de prédire les résultats expérimentaux avec une bonne fiabilité et de montrer les principaux facteurs environnementaux déterminant la physiologie photosynthétique. Mais l'intérêt principal de cette approche s'est révélé dans l'étude des jours et des sacs pour lesquels les paramètres photosynthétiques dévient par rapport au modèle. Nous avons ainsi pu identifier une diminution du rendement quantique maximal du PSII de l'ordre de 25 à 30% uniquement certains jours et pour certains réplicats. Même si les résultats sont encore préliminaires, ces jours semblent parfaitement corrélés à des évènements d'infection intracellulaire d'Emiliania huxleyi par son virus spécifique. L'inhibition spécifique du PSII pose une question immédiate : le FCE est-il stimulé pendant l'infection virale, puisqu'il pourrait fournir de l'ATP pour un processus infectieux qui est énergétiquement coûteux ? Chapter 1: General introduction Suzanne FERTE -Doctoral Thesis -2019

1. 1. 1. Most of the genetic diversity in the Ocean is known

The ocean is a single, continuous and global entity. It covers 71% of the Earth surface but remains little explored in comparison to land environment. Within the past decades, ocean ecosystems exploration flourished thanks to new technologies and revealed the complexity and particularity of marine ecosystems. Over many centuries, global expeditions have led to major scientific breakthroughs, notably with the early voyages of the H.M.S. Beagle (1831-1836) and the H.M.S. Challenger (1872-1876). But because microbiology was not yet mature, those expeditions missed most of the ocean diversity. Last century's biology was marked by laboratories studies and molecular scale tools were developed. This enabled a fine understanding of molecular mechanisms of cell division, organization and metabolism. Last decades were also widely influenced by genomics development. And reinforced by those new tools and knowledges, recent global expeditions discovered a new facet of ocean ecosystems: the planktonic world. Recently, the

Sorcerer II expeditions (2003-2010)

1. 2. 3. The Calvin-Benson-Bassham cycle synthesizes organic carbon from CO2

	a) Oxygenic photosynthesis, a two-step reaction
	In the first decade of the 20 th century, it was generally assumed that light, absorbed by
	photosynthetic pigments, directly reduced CO2 which in turn combined with water to form
	carbohydrates. And under this hypothesis, CO2 was considered as the source of O2
	generated by photosynthesis. But in 1931, based on work on purple sulfur bacteria, Cornelis
	van Niel proposed that photosynthesis is a two-stage process in which light energy is used to
	oxidize H2O into O2 (van Niel and Muller, 1931). The validity of van Niel's hypothesis was

established unequivocally by two experiments. In 1937, Robert Hill discovered that when isolated chloroplasts that lack CO2 are illuminated in the presence of an artificial electron acceptor, O2 evolved with concomitant reduction of the acceptor. This Hill reaction demonstrates that CO2 does not participate directly in the O2 producing reaction. In 1941, when the oxygen isotope 18 O became available, Samuel Ruben and Martin Kamen directly demonstrated that the source of O2 in photosynthesis is H2O.

, Gibbons, C., Arechaga, I., Leslie, A. G., & Walker, J. E. (2000). The

  (2010). Bidirectional electron transfer in the reaction centre of photosystem I. Journal of integrative plant biology, 52(8),735-749. (2005). Modelling of the electron transfer reactions in Photosystem I by electron tunnelling theory: the phylloquinones bound to the PsaA and the PsaB reaction centre subunits of PS I are almost isoenergetic to the iron-sulfur cluster FX.

	Biochimica et
	Biophysica Acta (BBA)-Bioenergetics, 1708(3), 283-310.
	Savard, F., Richard, C., & Guertin, M. (1996). The Chlamydomonas reinhardtii LI818 gene represents
	a distant relative of the cabI/II genes that is regulated during the cell cycle and in response to
	illumination. Plant molecular biology, 32(3), 461-473.
	Scheibe, R. (2004). Malate valves to balance cellular energy supply. Physiologia plantarum, 120(1),
	21-26.
	Scheibe, R., Backhausen, J. E., Emmerlich, V., & Holtgrefe, S. (2005). Strategies to maintain redox
	homeostasis during photosynthesis under changing conditions. Journal of experimental botany,
	56(416), 1481-1489.
	Streb, P., Josse, E. M., Gallouet, E., Baptist, F., Kuntz, M., & Cornic, G. (2005). Evidence for
	alternative electron sinks to photosynthetic carbon assimilation in the high mountain plant species
	Ranunculus glacialis. Plant, Cell & Environment, 28(9), 1123-1135.
	Stroebel, D., Choquet, Y., Popot, J. L., & Picot, D. (2003). An atypical haem in the cytochrome b6f
	complex. Nature, 426(6965), 413.

Chapter 1: General introduction Suzanne FERTE -Doctoral Thesis -2019 Santabarbara, S., Galuppini, L., & Casazza, A. P. Santabarbara, S., Heathcote, P., & Evans, M. C. Scheller, H. V., & Haldrup, A. (2005). Photoinhibition of photosystem I. Planta, 221(1), 5-8. Schrope, M. (2008). Oceanography: Red tide rising. Nature News, 452(7183), 24-26. Sonoike, K. (2011). Photoinhibition of photosystem I. Physiologia Plantarum, 142(1), 56-64 Sonoike, K., Terashima, I., Iwaki, M., & Itoh, S. (1995). Destruction of photosystem I iron-sulfur centers in leaves of Cucumis sativus L. by weak illumination at chilling temperatures. FEBS letters, 362(2), 235-238. Staehelin, L. A. (1986). Chloroplast structure and supramolecular organization of photosynthetic membranes. In Photosynthesis III (pp. 1-84). Springer, Berlin, Heidelberg. Stirbet, A. (2013). Excitonic connectivity between photosystem II units: what is it, and how to measure it? Photosynthesis research, 116(2-3), 189-214. Stock, D.rotary mechanism of ATP synthase. Current opinion in structural biology, 10(6), 672-679. Strand, D. D., Fisher, N., & Kramer, D. M. (2017). The higher plant plastid NAD (P) H dehydrogenaselike complex (NDH) is a high efficiency proton pump that increases ATP production by cyclic electron flow. Journal of Biological Chemistry, 292(28), 11850-11860. Stransky, H. T., & Hager, A. (1970). Das Carotinoidmuster und die Verbreitung des lichtinduzierten Xanthophyllcyclus in verschiedenen Algenklassen. Archiv für Mikrobiologie, 71(2), 164-190. Suorsa, M., Järvi, S., Grieco, M., Nurmi, M., Pietrzykowska, M., Rantala, M., ... & Aro, E. M. (2012). Proton gradient regulation 5 is essential for proper acclimation of Arabidopsis photosystem I to naturally and artificially fluctuating light conditions. The Plant Cell, 24(7), 2934-2948.

  Rules for the direction of the mind, Descartes says : "Sur les objets proposés à notre étude il faut chercher, non ce que d'autres ont pensé ou ce que nous-même nous conjecturons, mais ce dont nous pouvons avoir l'intuition claire et évidente ou ce que nous pouvons déduire avec certitude : car ce n'est pas autrement que la science s'acquiert », traduced in english by "As regards any subject we propose to investigate, we must inquire not what other people have thought, or what we ourselves conjecture, but what we can clearly and manifestly perceive by intuition or deduce with certainty. For there is no other way of acquiring knowledge." We will follow this advice and spend some time determining what can reasonably be done regarding CEF measurements, before starting a "screening" of CEF in the phytoplankton groups.

Résumé long (français)Suzanne FERTE -Doctoral Thesis -2019

Suzanne FERTE -Doctoral Thesis -2019

Chapter 1: General introductionSuzanne FERTE -Doctoral Thesis -2019 measurements. These approaches lead me to discuss the relative amplitude of the CEF and the LEF as well as their modes of regulation.Chapter 4: The chapter is not immediately connected to the CEF topic. It deals with results obtained in the field during a mesocom study campaign of a bloom of Emiliania huxleyi, a haptophyte whose ecological importance we mentioned in the introduction. By participating in this project, my goal was to test the method developed in Chapter 3 on field samples. This part of the project did not succeed because the signals related to PSI activity were far too low. However, thanks to measurements based on PSII fluorescence, I followed the photosynthetic physiology of Emiliania huxleyi during the campaign and in collaboration with a statistician of the Pasteur Institute, I developed a model to explain the variation of photosynthetic parameters from environmental data ... and you will see that this approach finally made it possible to address questions related to CEF.

Chapter 2: Probing PSI activitySuzanne FERTE -Doctoral Thesis -2019

s -1 , the lowest irradiance tested which corresponds to ~2 photons per PSI per second (see Methods). In these two conditions, the PSI yield based on ECS, Y(I)ECS, is close to 1 which is

Chapter 3: Diversity of cyclic electron flow in microalgaeSuzanne FERTE -Doctoral Thesis -2019

Chapter 4: Photosynthetic physiology of the coccolithophore Emiliania huxleyi Suzanne FERTE -Doctoral Thesis -2019

and

4;. Nutrients were added on days 1 to 7 and 13 to 17 (see Methods).

Remerciements

Acknowledgements

We would like to thank Fredy Barnèche and Meenu Singla for their technical help growing Arabidopsis thaliana. We would also like to thank Stefano Santabarbara, Helmut Kirchhoff, Anja Krieger-Liszkay and Pierre Sétif for fruitful discussions and Anja Krieger-Liszkay for access to the Dual-PAM. B.B. and S.F. acknowledge financial support from the European Research Council (ERC) under the European Union Horizon 2020 research and innovation program (grant agreement no. 715579). We also thank the Roscoff culture collection for providing the strain of Am. carterae.

Witt, H. T. (1979). Energy conversion in the functional membrane of photosynthesis. Analysis by light pulse and electric pulse methods: The central role of the electric field. Biochimica et Biophysica Acta (BBA)-Reviews on Bioenergetics, 505 (3)(4), 355-427.

Zivcak, M., Brestic, M., Kunderlikova, K., Sytar, O., Allakhverdiev, S. I. (2015). Repetitive light pulse-induced photoinhibition of photosystem I severely affects CO 2 assimilation and photoprotection in wheat leaves. Photosynthesis research, 126 (2)(3), 449-463. 2)

What is the relationship between the true Y(I) and Y(NA) and the values calculated with the P700 method? We propose a parsimonious hypothesis in which the ratio of acceptor-side limited to donorside limited centers at the end of the pulse is equal to the ratio before the pulse, i.e. [P700 A -]p / [P700 + A]p = [P700 A -] / [P700 + A], which gives: Y(NA)P700 / (Y(ND) + Y(I)P700) = Y(NA) / (Y(ND) (Equation 3)

Combining equations ( 1), ( 2) and ( 3 Ordinate axes correspond to the standard redox potential of the intermediates (mV). The overall areas of QA and P700 correspond to one electron. The author considered that the photosynthetic unit contain 1 PSII, 1 PSI and approximately the same number of electron carriers at the donor and acceptor side of PSI: PSI secondary electron donors (2 plastocyanins; 1 cytochrome f, 1 Rieske; about 6 quinones representing about 12 electrons), and PSI secondary electron acceptors (5 electrons on ferredoxin and ferredoxin NADP-reductase; and 12 on NADP+/NADPH). A. and B. represent the redox state of the photosynthetic chain intermediates in the dark, in oxic and anoxic conditions, respectively. Note that the midpoint potential of QA is incorrect: it is indicated as -20 mV instead of -140 to -145 mV (Shibamoto et al., 2009;Brinkert et al., 2016).

Inversely, the measurement of the "b phase" of the flash-induced ECS kinetics demonstrates that the "a phase" of ECS accurately estimates the fraction of open centers. It is important here to discuss the limit of validity of the measurement of PSI yield with ECS. Indeed, in situation of effective acceptor side limitation and high light, ECS can overestimate Y(I) by counting charge separations before they recombine (Alric 2014), a bias which can be taken into account. These aspects will be further discussed in the chapter 3; in particular I will demonstrate that the photochemical rates based on calculations of ECS-based photosystem yields to the photochemical rates based on dark interval ECS relaxation kinetic (Joliot and Joliot 2002;Sacksteder and Kramer 2001) show a perfect match, which is a strong argument for the reliability of Y(I)ECS. Sunflower leaves were submitted to 300ms pulses of 20,000 μmol photons m -2 s -1 applied every 10s in dark conditions. (A) The fraction of acceptor side limited centers, Y(NA) was measured after different time of the treatment. (B) The maximal P700 absorbance change, Pm, was measured at the end of the experiment (240 min after the beginning of the treatment) and is expressed as a percentage of the initial Pm. In both graphs, results obtained in atmospheric conditions or in low O2 concentration are shown.

Generalization to the case of an active photosystem II

All the article data were obtained in PSII inhibited samples. From a theoretical point of view, the inhibition of the PSII does not change the conclusions that we drew from our experiments.

The P700 method error originates from the fact that the multiple turnover pulse potentially induces a modification of the redox state of the PSI acceptors. Because of the short pulse duration and its high intensity, no electron transfer through the cytochrome b6f should occur: the changes induced by the pulse involve only lumenal PSI donors after cytochrome b6f and PSII activity is not involved. However, one may wonder if the conclusions drawn remain true when the PSII is functioning, and we decided to address this question experimentally. In order to answer those questions, we propose to re-interpret the data shown in figure 2A, 2C and 3B of the article with a more physiological point of view and to highlight the misunderstanding induced by P700 bias. We also provide a short review of the literature using P700 method using to our advantage the correction to the P700 pulse method that we propose, which allows us to recalculate the PSI parameters from the erroneous ones.

In this part, we use the P700 data correction proposed in the article (discussion part) to reassess PSI parameters. The corrected PSI yield, Y(I)P700,corr, and the acceptor side limitation, Y(NA)P700,corr, were calculated as:

We will consider in the following that the correct values for the PSI yield and acceptor side limitation are the ones estimated with ECS estimation of the fraction of open centers and P700 estimation of the donor-side limited centers, Y(ND).

In the following, we will discuss the consequences of the bias of the P700 pulse method, both during dark-to-light transition and during steady state photosynthesis. The P700 pulse method was first developed -and is still mostly used-for investigations of the cyclic electron flow around PSI through the comparison of PSII and PSI yields. Most of the discussion in the following will focus on how the P700 pulse method may have drawn erroneous conclusions regarding CEF. This part is not meant to be an exhaustive review of the literature using the P700 pulse method, but presents some conditions in which the P700 pulse method can lead to misunderstand the photosynthetic physiology.

Chapter 2: Probing PSI activity

Suzanne FERTE -Doctoral Thesis -2019 d) CEF estimation during leaf senescence Figure 2-18 shows results from an article on sugar maple leaves physiology during senescence (Junker and Ensminger 2016). From green summer to green autumn, yellow, orange and red leaves, the quantum yield of PSI, Y(I)P700 (here referred as ΦPSI) shows a rather stable trend compared to ΦPSII which decreases a lot more. The authors then estimate the evolution of cyclic electron flow during leaf senescence. In this purpose, they estimate the electron flow through photosystem II (JPSII), and through photosystem I (JPSI) and propose that the JPSI/JPSII ratio is an indicator of CEF (Figure 2-18B).

But do we get the same results if we consider the error induced by the P700 method? From green summer leaf to red leaf, Y(NA)P700 goes from 0.1 to 0.7. Thus, the underestimation of Y(I) by the P700 method (calculated as 1/(1-Y(NA)P700) goes from a factor 1.1 in the green summer leaves to a factor 3.3 in the red leaves. This means that the evolution of the CEF, if correctly estimated by JPSI/JPSII ratio, during leaf senescence is underestimated by a factor 3: instead of increasing by a factor ~4, it might actually increase by a factor ~12. A. PSI parameters were measured using P700 method in green sugar maple leaves sampled in summer, and in green, yellow, orange and red leaves sampled in autumn. ΦPSI, ΦND and ΦNA stands for the fraction of open, acceptor side limited and donor side limited centers, respectively. B. The ratio of electron transport at PSI to electron transport at PSII, JPSI JPSII -1 was used as an indicator for cyclic electron transport. Data represents the average five to six leaves sampled from three trees in two consecutive years (±SD) taken at a light intensity of 1200 µmol photons m -2 s -1 . Significant differences (p < 0.05 using linear-mixed-effect modeling followed by Tukey's multiple comparison, see article for more details) are indicated by different letters.

Chapter 3: Diversity of cyclic electron flow in microalgae

Suzanne FERTE -Doctoral Thesis -2019 occurs mostly in anoxia [START_REF] Ghirardi | Hydrogenases and hydrogen photoproduction in oxygenic photosynthetic organisms[END_REF] and transfers electrons from the acceptor side of the PSI to catalyze the reduction of protons into dihydrogen (see chapter 1 for more information on water to water cycles).

All those alternative electron flows also involve the photosystem II and (except for PTOX) the cytochrome b6f, favoring extra proton pumping (Eberhard, Wollman and Finazzi, 2008). By doing so, they participate to the ΔpH dependent processes, qE and photosynthetic control.

They also promote ATP production, without producing any reducing power, increasing the ATP/NADPH produced by the overall photosynthetic activity. This must be consider when discussing the role of CEF in ATP/NADPH adjustment or in the ΔpH related photo-protective mechanisms. It might be a naïve view to assume that CEF alone regulates those photosynthetic regulations, and a more cautious point of view would be to consider a priori that they are ensured by all these alternative electron flows.

Regulation of CEF rate

In the first section, we have discussed the several roles of CEF: adjusting the ATP/NADPH produced by the photosynthetic chain to the metabolic needs, increasing the proton motive force (and especially its osmotic component ΔpH) to protect photosystem II (through the qE mechanism) and photosystem I (through the photosynthetic control). We have also mentioned in the general discussion (chapter 1) that the main molecular actors involved in the electron transfer step allowing the recycling of electrons from PSI acceptors back to the inter-system chain are not yet fully determined. A third important aspect of CEF that needs to be discussed in order to complete the view about this process is the mode of regulation.

What does the rate of CEF depend on? It is clear that CEF rates need to be adjusted to LEF rates to fulfill its roles. It can be involved efficiently in photo-protection if -and only if-its rate modifies significantly the pmf under high light. It can provide the extra ATP/NADPH needed for CO2 assimilation if -and only if-the rate of its ATP production matches with the CBB associated ATP shortfall. This means its rate must be at least a given fraction (~20%, see 3.1.1) of the LEF rate, regardless of the irradiance. How does the photosynthetic chain regulate the relative rates of LEF and CEF? Determinants of CEF rate remain unclear and models are still strongly debated. Three major modes have been proposed for CEF-to-LEF regulation that are not mutually exclusive: a modulation of kinetic constants of enzymes involved in CEF, a regulation by substrate

Chapter 3: Diversity of cyclic electron flow in microalgae

Suzanne FERTE -Doctoral Thesis -2019 instead the "cycling step", i.e. the electron transfer step from PSI acceptors to the intersystem chain closing the cycle? Many studies suggest that CEF rates are low in comparison to maximal LEF rates under nonstressful conditions. Values range from 3% in wild type Chlamydomonas to 10% in a FNR mutant of the same green alga (Alric, 2010), whereas it is less than 14% of the maximal LEF rate in plants [START_REF] Laisk | Rates and roles of cyclic and alternative electron flow in potato leaves[END_REF][START_REF] Livingston | An Arabidopsis mutant with high cyclic electron flow around photosystem I (hcef) involving the NADPH dehydrogenase complex[END_REF]Fan et al., 2007). This has been an argument to propose that the CEF limiting step is slower than the LEF limiting step, most likely because the kinetics of reinjection of the reducing power into the chain would be limiting for CEF (Alric 2010). In that case, the CEF/LEF ratio is expected to decrease with light irradiance, and a role of CEF under high light is doubtful. But many of the measurements in steady state illumination were performed in the presence of DCMU, which might not provide a good measurement of the "physiological" CEF (Fan et al, 2016) (see 3.1.3). On the other hand, measurements of CEF during dark to light transition in the presence of DCMU give a different view. In these conditions, CEF rate reaches 60 electrons.s -1 .PSI -1 in Chlamydomonas reinhardtii (Nawrocki et al., 2019c) and up to 120 electrons.s -1 .PSI -1 in plant leaves (Joliot and Johnson, 2011;[START_REF] Joliot | Cyclic electron flow under saturating excitation of darkadapted Arabidopsis leaves[END_REF]. The latter value is close to the maximal LEF rate in plant leaves, which would mean that in physiological conditions, CEF might be limited by the turnover of the cytochrome b6f. If CEF and LEF have the same limited step, it is plausible that the CEF/LEF remains constant at all light irradiances, making CEF a likely candidate for ATP/NADPH regulation.

Measuring CEF is a methodological challenge

There are still many doubts as to the physiological significance of CEF, its amplitude in stationary illumination regime, or even its existence in many groups of photosynthetic organisms. The main reason for the remaining doubts and open questions regarding CEF, after half a century of research, is the absence of a definitive method for measuring the CEF in vivo under physiological conditions [START_REF] Shikanai | Central role of cyclic electron transport around photosystem I in the regulation of photosynthesis[END_REF][START_REF] Johnson | Cyclic electron transport in C3 plants: fact or artefact[END_REF]. In this chapter, I aim to set the basis of an exploration of the diversity of the CEF in phytoplankton, where very little is known. This task is obviously endless and there are numerous traps when one wants to measure CEF, as highlighted in chapter 2. That is the reason I will review here the limitations in CEF measurements.

Chapter 3: Diversity of cyclic electron flow in microalgae

Suzanne FERTE -Doctoral Thesis -2019

Material and methods

Strains, growth and sampling

Chlamydomonas reinhardtii stt7-9 mutant were obtained from the Chlamystation (http://chlamystation.free.fr/). The dinoflagellates Amphidinium carterae (RCC1522) was obtained from the Roscoff Culture Collection (http://roscoff-culture-collection.org/) and Symbiodinium sp. strain FlAp1 isolated from Aiptasia pallida (clade B1, see Roberty et al, 2014) was obtained from M. A. Coffroth (the BURR Culture Collection, University at Buffalo, NY, USA).

Chlamydomonas cells were grown in Tris/Acetate/Phosphate (TAP) medium (Harris, 1989), at 25°C and under continuous light provided by white LEDs and were harvested between 2 and 6 million cells per mL. The irradiance was 30 µmol photons m -2 s -1 . Amphidinium carterae and Symbiodinium sp were grown in ESAW medium (Berges et al, 2001) or ESAW with only 10% if the nitrogen indicated in the recipe (Figure 3-18) at 19°C and under 12h photoperiod at a light irradiance of 30 µmol photons.m -2 .s -1 using fluorescent tubes. It was harvested in exponential phase between 80 000 and 200 000 cells per mL.

For photosynthetic measurements, liquid cultures of Chlamydomonas reinhardtii, Amphidinium carterae and Symbiodinium sp were concentrated by centrifugation (4 minutes at 3500 rpm) and resuspended in their supernatant to reach ~10 7 cells.mL -1 . Ficoll® was added to avoid drifts in the absorption measurements due to fast sedimentation of the cells. Concentrated samples were then stirred at 350 rpm under low light (~10 µmol photons.m - 2 .s -1 white light) for at least 30 minutes before starting measurements.

Chaetoceros dominated sample from the field was collected in the Penzé estuary (Roscoff, France). Water was sampled from surface brackish water (salinity: 16.3g.L -1 , water temperature: 16 °C) and then kept for 24 hours in a temperature controlled chamber (set at 16°C) with a 14h light/10h dark photoperiod. The sample was then purified and concentrated by filtration with a 20μm polycarbonate filter and preservation of the retentate, later measured. A fraction of the sample was fixed by addition of iodine and its specific composition was determined later. We sedimented 10 mL of the sample using a combined plate chamber [START_REF] Utermöhl | Neue Wege in der quantitativen Erfassung des Plankton.(Mit besonderer Berücksichtigung des Ultraplanktons.) Mit 4 Abbildungen im Text[END_REF]). The sample was then observed under an inverted light microscope and cells species were identified using a phytoplankton species determination guide provided by Laure Guillou from the "Adaptation and Diversity in

Results (I): Exploring CEF diversity

In this first part of the results, I will present the work I have done to explore the CEF diversity in freshwater and marine microalgae. For that, the first step consisted in elaborating a robust, simple and accurate protocol to extract basic information about CEF. I first determined the best spectroscopic tools that we possess to assess CEF in vivo. In order to get the most reliable measurements, we propose to probe CEF using the less parameters measurement as possible in order to reduce the technical noise and/or errors associated with each parameter measurement. The following two sections will present the reasoning behind the choice of the protocol we used to investigate CEF in different microalgae.

DCMU titration reveals CEF behavior

We have written before that the CEF rate can be calculated as:

(2) CEF = ETR(I) -n2/n1. ETR (II) This equation indicates that calculations of CEF through the comparison of PSII and PSI activities require accurate and non-biased measurement of both PSI and PSII yields and absorption cross-sections. The most widely used method to assess PSI quantum yield is based on P700 absorption change (Klughammer & Schreiber, 1994), but we have seen in chapter 2 that this method induces a bias that cannot be perfectly corrected (Ferté et al, unpublished). Based on this, we eliminated P700 absorption changes to probe PSI yield, Y(I).

We have seen in chapter 2 that the PSI yield can be obtained thanks to ECS if PSII is inhibited. But in conditions where PSII is functioning normally, ECS probes the activity of both photosystems. If we call ETR(total) the sum of PSI and PSII activities, i.e. ETR(total) (electrons per PS per second) = n2. ETR(II) + n1. ETR(I), then equation (2) becomes:

(3) ETR(total) = 2. n2. ETR(II) + n1. CEF

The protocol we used is the following: under steady state light, we titrated both the ETR(II) and ETR(total) with a PSII inhibitor, the DCMU. In order to better understand our approach, I represented the expected DCMU dependence of ETR(II) (blue curve in Figure 3-3A) and simulated the evolution of ETR(total). Let's start with a situation where CEF is independent of the linear electron flow, i.e. does not change with DCMU concentrations. The activity of PSI involved in LEF should be, by definition equal to the one of PSII. And the contribution of Once normalized to the initial value (untreated situation), the evolutions of ETR(II) (blue curve) and ETR(total) (black curves for different CEF scenarios) as a function of DCMU are shown in Figure 3-4A. If one is plotted against the other (Figure 3-4B), this gives a plot which is easy to interpret. If there is no CEF, we then expect a relationship of proportionality between ETR(total) and ETR(II). If CEF exists but is not dependent of LEF variation, then we expect ETR(total) and ETR(II) to follow an affine relationship. The y-intersect reflects the fraction of total activity corresponding to CEF. And by elimination, if CEF depends on LEF, we expect a more complex relationship between ETR(total) and ETR(II) which depends on the relationship between the two modes of electron flow and is not shown here.

Chapter 3: Diversity of cyclic electron flow in microalgae

Suzanne FERTE -Doctoral Thesis -2019 The most parsimonious conclusion is that, in the conditions we use here, CEF is not essential to photosynthesis in Amphidinium carterae. In plants and green algae, CEF has been proposed to play a crucial role in equilibration of ATP/NADPH production and requirement and in the photo-protection under saturating light. The case of Amphidinium carterae shows that it is not absolutely necessary in all photosynthetic species. Amphidinium carterae might set up other strategies to fulfill the ATP/NADPH shortfall (see discussion in 3.5.1.a).

CEF is independent on LEF photosynthesis in the dinoflagellate

Symbiodinium sp.

When titrating photosynthesis with DCMU in Symbiodinium sp., Y(total) decreases more slowly than Y(II) (Figure 3-6A) meaning that total electron flow decreases more slowly than PSII electron flow. It is to note that DCMU concentrations are expressed in arbitrary units because the full inhibitor penetration until the QB site is a very long process in this species (~45 minutes) and measurements of total and PSII yields were performed before the effect of the given concentration of DCMU was steady, i.e. during the kinetics of penetration. This does not affect the relationship between Y(total) and Y(II) since both measurements were made at the same time. It is to note that despite its slow penetration kinetics, DCMU inhibited more than 95% of PSII at the end of the experiments. When plotting Y(total) as a Absorption cross-section measurement through ECS probe has been scarcely used in the literature (Roberty et al, 2014) and never validated to my knowledge. We thus propose to test its reliability through two cross validation approaches.

Chapter 3: Diversity of cyclic electron flow in microalgae

Suzanne FERTE -Doctoral Thesis -2019

Evaluating CEF absolute rates from ETR(total) and ETR(II)

In the previous part, I introduced the methods to measure the different parameters to calculate total and PSII yields and absorption cross-sections. In this part, we will use these parameters to calculate CEF.

In this section, we will calculate ETR(II) as:

, where σII is calculated based on the ECS method described in the previous section and Y(II) is the quantum yield of PSII based on variable fluorescence measurements. For ETR(total), we have seen in the previous section that the two ECS-based methods give similar methods. Here we will use the photochemical rate based on DIRK method to calculate ETR(total). and ETR(II) are expressed per total PS and per PSII, respectively. The 3 biological replicates differ in terms of ETR amplitudes. This will be addressed in the discussion of the results.

Calculations of the ATP/NADPH ratio produced by the photosynthetic chain

With such high rates of CEF, one can wonder if it is sufficient to provide the missing ATP for We could show that a dinoflagellate, Amphidinium carterae, is not displaying CEF under steady state light (Figure 3-17, A and D), indicating that a pure linear electron flow takes place in this species in the conditions we used. This means that CEF is not mandatory for photosynthesis. In plants and green algae, CEF has been proposed to play a crucial role in equilibration of ATP/NADPH production and requirement and in the photoprotection under saturating light. The stromal CO2 reduction pathway being conserved in all photosynthetic eukaryotes (Hügler & Sievert, 2011), suggesting that the shortfall of ATP for CO2 assimilation is a common feature for organisms performing oxygenic photosynthesis. The case of Amphidinium carterare shows that it is possible to fully depend on other strategies to respond to this common constraint.

Very little is known about Amphidinium carterae's photosynthetic physiology. We can imagine two possible strategies for ATP/NAPDH ratio equilibration. The first one is an increase of the ATP/NADPH ratio produced by linear electron flow. It is possible if the between Y(total) and Y(II) is similar to the one in Chlamydomonas, indicating that there is some CEF at play and that there is an interplay between CEF and LEF. This clearly shows that Amphidinium carterae is not species which cannot perform CEF. It possesses the ability to perform CEF but does not do it constitutively, in non-stressful conditions. Here, a very striking peculiarity shoud be further discussed: in these conditions, Amphidinium carterae performs CEF but in saturating DCMU conditions, there was absolutely no sign of CEF. This is a very clear evidence that measurements made in saturating DCMU conditions should be interpreted with a lot of care! We also pointed out that another dinoflagellate, Symbiodinium sp., was performing CEF that remained constant as LEF was increasingly inhibited. In this case, physiological and absolute CEF measurement can be extracted from the experiment. First, the CEF/LEF ratio can be obtained straightforwardly by looking at the ratio between the photosystems activity involved in CEF (the y-intercept, at saturating DCMU concentration) to the initial photosystems activity. Since CEF does not depend on LEF, the absolute rate of CEF can be

In the case of Chlamydomonas, the protocol we used cannot provide a full characterization of the CEF regulation and extent. We cannot provide an absolute rate of CEF or a CEF/LEF ratio; this is one of the limitations of the protocol. Another limitation is that we cannot exclude the occurrence of two concomitant CEF mechanisms in Chlamydomonas reinhardtii:

one being independent of LEF and another one being dependent. In order to better investigate the relationship between CEF and LEF and to get estimation of the amplitude of CEF, this simple protocol was not sufficient. We developed, tested and applied a method requiring measurements of PSI and PSII absorption cross-sections and monitor CEF and LEF absolute rates as a function of light irradiance. This is the topic of the section (3.5.3). Before that, I would like to list some questions which could be addressed with this simple protocol and to propose variants of the DCMU titration protocol which could help investigating the effect of environmental parameters on the regulation of CEF. 3.5.3 Future directions for CEF investigations

The methods developed in this work pave the way for tackling many questions regarding CEF pathway, role, regulation and diversity. We detail below some research tracks that could be followed in the future. The results we presented above were obtained in one environmental and physiological condition. But we don't know if the different behaviors observed are due to genetic differences between species (presence / absence of enzymes involved in CEF), or to physiological differences (difference in redox state of the photosynthetic intermediates for example). Thus, it is difficult to question our results with a phylogenetic point of view. In order to understand better the determinants of CEF and thus to better discriminate between genetic capacity to perform CEF and physiology influence on CEF behavior, we tested CEF evolution in different environmental conditions. We will discuss this in the section 3. Hereafter, only material and methods of the data that we used will be described.

Flow cytometry/ qPCR

Phytoplankton composition and abundance were assessed by analyses of fresh samples on an Eclipse (iCyt) FACS flow cytometer. Samples for flow cytometric counts were taken twice a day, morning (7 am) and evening (8-9 pm) using 50 mL centrifugal tubes and following filtration using a 50 μm cell strainer to remove chains of diatoms. (25 µmol photons m -2 s -1 ) because it was faster than in dark conditions (data not shown) and therefore allowed a better discrimination between qE and qI. State transitions were not considered since genes coding for the kinase involved in this process has not been found in other clades than the green lineage [START_REF] Grouneva | Phylogenetic viewpoints on regulation of light harvesting and electron transport in eukaryotic photosynthetic organisms[END_REF].

ETR(II) was measured under 25, 50, 100, 200, 300, 400 and 500 µmol photons m -2 s -1 (blue light). ETR(II) vs I curves were fitted by an exponential function: ETR(II) = ETRmax (1 -exp(-I/Ek)) [START_REF] Platt | Photoinhibition of photosynthesis in natural assemblages of marine phytoplankton[END_REF] using non-linear least square method. Three parameters were estimated:

the maximal electron transport rate, ETRmax, the light saturation parameter, Ek, and the initial slope,  = ETRmax/Ek (see .

Chapter 4: Photosynthetic physiology of the coccolithophore Emiliania huxleyi

Suzanne FERTE -Doctoral Thesis -2019

Results

Emiliania huxleyi is a model microalga for the study of the establishment and collapse of blooms, and especially for the virus-induced ending of blooms (Bidle and Vardi, 2011). As with all photosynthetic organisms, one of the determining parameters of its growth is the efficiency of its photosynthesis. The collapse of Emiliania huxleyi's blooms is often attributed to infection of cells by its specific coccolithovirus, Ehv, and laboratory studies show that the infection may have an influence on photosynthetic physiology [START_REF] Evans | Viral infection of Emiliania huxleyi (Prymnesiophyceae) leads to elevated production of reactive oxygen species 1[END_REF][START_REF] Bidle | Viral activation and recruitment of metacaspases in the unicellular coccolithophore, Emiliania huxleyi[END_REF][START_REF] Gilg | Differential gene expression is tied to photochemical efficiency reduction in virally infected Emiliania huxleyi[END_REF]Thamatrakoln et al., 2018). From lab experiments, we know that photosynthetic physiology is influenced by environmental parameters like light, nitrogen, iron, phosphorus or temperature [START_REF] Loebl | Physiological basis for high resistance to photoinhibition under nitrogen depletion in Emiliania huxleyi[END_REF][START_REF] Trimborn | Effect of varying calcium concentrations and light intensities on calcification and photosynthesis in Emiliania huxleyi[END_REF][START_REF] Muggli | Effects of nitrogen source on the physiology and metal nutrition of Emiliania huxleyi grown under different iron and light conditions[END_REF][START_REF] Paasche | Enhanced calcification in the coccolithophorid Emiliania huxleyi (Haptophyceae) under phosphorus limitation[END_REF][START_REF] Feng | Interactive effects of increased pCO2, temperature and irradiance on the marine coccolithophore Emiliania huxleyi (Prymnesiophyceae)[END_REF]. A particularity of Emiliania huxleyi is to be highly sensitive to pH, CO2, and calcium since it possesses coccoliths, which are cell walls made of carbonate [START_REF] Bach | Dissecting the impact of CO2 and pH on the mechanisms of photosynthesis and calcification in the coccolithophore Emiliania huxleyi[END_REF][START_REF] Trimborn | Effect of varying calcium concentrations and light intensities on calcification and photosynthesis in Emiliania huxleyi[END_REF][START_REF] Balch | Calcification, photosynthesis and growth of the bloom-forming coccolithophore, Emiliania huxleyi[END_REF][START_REF] Feng | Interactive effects of increased pCO2, temperature and irradiance on the marine coccolithophore Emiliania huxleyi (Prymnesiophyceae)[END_REF]. The questions we addressed in this project where the following: can we identify the relative influence of each environmental parameters on E. huxleyi photosynthesis in the field? If so, can we "substract" the effects of those abiotic parameters to highlight abnormalities? Can we relate those abnormalities to the more subtile influence of other factors like biotic interactions (allelopathy, viral infection)?

In order to tackle those issues, we used University of Bergen mesocosm facilities that are part of the Aquacosm european network of mesoscosms and that are particularly adapted for the study of Emiliania huxleyi. This biological station is located in the Raunefjorden, near Bergen The structure of the populations of microorganisms present in the mesocosms as well as their dynamics of growth could be followed more precisely thanks to flow cytometry. Here, phytoplankton was divided into three classes, nanoeucaryotes (Neuks) that display high chlorophyll signal, picoeucaryotes (Peuks) cells that show lower chlorophyll signal and a group corresponding to the cyanobacterium Synechococcus that is easily distinguishable by its high phycoerythrin content and was by far the most abundant cyanobacterium (based on 16S, data not shown). Neuks have been divided into two subclasses: those that scatter the light in a particularly important way, they are called high side scattering nanoeukaryotes (Neuks HSS) while the the others, which scatter less the light are called low side scattering nanoeukaryotes (Neuks LSS) (see Methods for discrimination of populations). Emiliania huxleyi displays two life forms, a diploid calcified one which participates to the high-scattering population and a haploid non-calcified one which participates to the low scattering population [START_REF] Frada | The "Cheshire Cat" escape strategy of the coccolithophore Emiliania huxleyi in response to viral infection[END_REF][START_REF] Green | The life-cycle of Emiliania huxleyi: A brief review and a study of relative ploidy levels analysed by flow cytometry[END_REF]. The composition of the phytoplankton population was measured every day of the campaign and the results are presented in Figure 4-6. At day 0, the composition of the bags and the fjord are similar but from day 2, the populations diverge with a significant growth of phytoplankton in the mesocosms.

The evolution of the calcified Emiliania huxleyi population follows a similar evolution in all bags. Their concentration becomes detectable on day 9, after a lag phase. It then develops and reaches a peak of growth rate between days 14 and 17 (almost a doubling per day) reaching a maximum concentration of 4 to 9.10 4 cells per mL for bags 1 to 6. Note nevertheless that the maximum growth phase is not observed in the bag 7 and that the maximum concentration reached in the bag 4 is lower than those reached in the other uncovered replicates. From day 18 to the end of the campaign, the population collapses. Waves of demise and growths are observed on flow cytometry data but could be due to artifacts (Flora Vincent, Assaf Vardi, personal communication). Indeed, those waves are not detected in chlorophyll or Fm measurements. It is important to note that from day 10 to the end of the campaign, the phytoplankton population was dominated by nanoeukaryotes in terms of biomass volume.

Suzanne FERTE -Doctoral Thesis -2019 Here, the BCA explains more than 77% of the variance with two axes, which means that little variance is not represented by these two axes. From day 2 to day 8, the parameters remain relatively stable and correlated with a strong ETRmax, Fv/Fm, in situ Fv/Fm and a low NPQ. The third phase, from day 10 to day 23, shows globally a decrease in ETRmax, Fv/Fm and an increase in NPQ. These last two phases correspond to the pre-bloom and bloom phases of Emiliania huxleyi defined based on flow cytometry data. We can hypothesize that the microalgae species that developed before Emiliania huxleyi (mainly nanoeukaryotes) had greater photosynthetic efficiency than the latter. 9 marking the beginning of the bloom. We regularly filtered the water samples on a 3μm polycarbonate filter in order to separate the cells smaller than 3μm (in theory mainly the picoeukaryotes and Synechococcus) and the cells larger than 3μm (in theory mainly nanoeukaryotes including E. huxleyi). We then measured the maximum fluorescence in the initial sample and in the fraction greater than 3 μm and calculated a percentage of the signal emitted by the fraction greater than 3 μm (Figure 4-13A). From day 9, the signal emitted by this fraction is greater than 60% and reaches 90% of the signal between days 15 and 21. We also estimated the composition of the population in the fraction higher than 3µm by flow cytometry and it shows that from day 15, the Neuks HSS (most probably E. huxleyi) dominate the fraction higher than 3μm. It should also be noted that this estimation is probably an underestimation for two reasons. First, a fraction of the low scattering nanoeukaryotes may be non-calcified E. huxleyi, and some E. huxleyi cells have been found in the fraction less than 3 μm (some cells might be also lost on the filter). A more complete view of the contribution of E. huxleyi will be available soon (sequencing of ribosomal genes and analysis of photosynthetic pigments from our samples are planned). In the following, we will therefore assume that the fluorescence signals measured from to the end day 9 mainly informs mostly the photosynthetic physiology of Emiliania huxleyi. We performed the same BCA analysis used for photosynthetic parameters, on the environmental parameters. The first two dimensions resume about 65% of the total variance of the dataset meaning that most of the variations are represented by these two axes. Some variables are partially correlated as it is the case for air temperature and inorganic nitrogen (mostly due to the increase of air temperature during the period of nutrient enrichment from day 0 to day 5), air temperature and light intensity (mostly due to cloudy and cold days from day 17 to 22) or dissolved inorganic nitrogen and phosphorus (mostly because they were added in a joint way from day 0 to day 7).Without surprise, the temperature of the air and water seem to be only slightly correlated. Right panel of Figure 4-15 shows that the temperature of the water follows the evolutions of the temperature of the air with a delay of 3 days. This is easily explained by the high thermal capacity of a 10 000 L water body: it takes time to heat up such a volume.

Left panel of Figure 4-15 shows the projection of environmental data day by day on the axes of the BCA. Several phases can be described: the evolution from day 0 to day 4-5 mostly Chapter 4: Photosynthetic physiology of the coccolithophore Emiliania huxleyi Suzanne FERTE -Doctoral Thesis -2019 explanatory variables). With the help of Pascal, we followed a different approach based on Partial Least Square (PLS) regression, which is generally used to explain or predict a response variable as a function of a set of predictors (dependent variables).

At first, we decided to focus our analyses on the three most descriptive parameters of the photosynthetic physiology: the maximum efficiency of PSII (Fv/Fm) which is an indicator of the "physiological state" of photosystem II, the maximum electron transfer rate of the photosynthetic chain (ETRmax) and the ability to set up mechanisms for the dissipation of excess light energy by heat dissipation (NPQ). The results of the PLS analysis are presented in Figure 4-17. For each environmental parameter, the amplitude of the bar represents the regression coefficient of this predictor. Negative, positive, and non-significant coefficients are colored in orange, blue, and grey, respectively. The analysis gave very satisfying outputs, the PLS models explaining a significant part of the variance of the photosynthetic parameters. For ETRmax, 2 orthogonal components in the environment parameters matrix explain 75% of the variance (Figure 4-17B). We have seen previously that ~14% of the ETRmax variance was due to noise and/or stochastic effects (calculated as 1-RS). This means that most of the remaining variance is "captured" by the 2 orthogonal components used. For Fv/Fm and NPQ, for which the contribution of stochastic effects and noise was higher (29 and 37% of the variance respectively), one component could still explain 47 and 37% of the variance, respectively.

Chapter 4: Photosynthetic physiology of the coccolithophore Emiliania huxleyi

Suzanne FERTE -Doctoral Thesis -2019 days also correspond to high temperature and presence of NO3 -in the water, parameters that are correlated to high ETRmax and low NPQ. The analysis did not catch any significant influence of phosphate concentration on photosynthesis, except for a negative correlation with NPQ.

The nutrient data show that from day 10, the dissolved nitrogen is close to 0 whereas there is a significant amount of phosphate (and silicate) throughout the mesocosm. The N/P ratio is way below the Redfield ratio, which is the consistent nitrogen to phosphorus ratio found in marine phytoplankton [START_REF] Redfield | On the proportions of organic derivatives in sea water and their relation to the composition of plankton[END_REF]. This simply means that the growth of phytoplankton is nitrogen-limited.

Influence of temperature on photosynthesis

The relative influences of water and air temperatures on photosynthesis are strikingly different. There is no significant effect of water temperature on Fv/Fm or NPQ and only a small negative correlation with ETRmax. In contrast, the influence of air temperature seems to be very significant on those three parameters. The PLS analysis suggests that ETRmax and Fv/Fm are positively correlated with air temperature. It is important to remember here that the average temperature phytoplankton cells experience is the one of the water; however, they were sample at the surface where the strong mixing due to pumping allows fast equilibration of air and surface water temperatures. Moreover, the measurement was made at air temperature. Those results might imply that (i) photosynthetic rate and PSII maximal quantum yield are dependent on the immediate temperature at the moment of sampling/measurement, and (ii) there is no significant long-term acclimation of the photosynthetic physiology to the temperature in the range of temperature experiences in this mesocosm.

The correlation between air temperature and ETRmax can easily be explained using the Arrhenius law, which describes the relationship between the kinetic constant of a chemical reaction, k, and the temperature, T: k = A.exp(

), where A is a constant, Ea is the activation energy of the reaction and R is the universal constant of the perfect gases. As previously explained, the ETRmax depends on the speed of a chemical step and the measurements were made directly on the raft, at the temperature of the air; such a relation between the ETRmax and the air temperature is therefore expected. The very strong influence of temperature on Fv/Fm could reflect the temperature-dependent process of PSII reparation (see general introduction, chapter 1).

Influence of light intensity

Light irradiance is the only environmental parameter which correlates positively and significantly with the three photosynthetic parameters considered here. This is probably due to light acclimation processes. Indeed, it is known that some proteins involved in the establishment of NPQ (i.e. LHCX, Bailleul et al, 2010;[START_REF] Zhu | Photoprotection in the diatom Thalassiosira pseudonana: role of LI818-like proteins in response to high light stress[END_REF]or LHCSR3 in green algae, Peers et al, 2009) can be induced by high light and give rise to a greater capacity to set up NPQ mechanisms. Note that the PLS model is best adapted to catch linear relationships between variables while the expected relationship between NPQ and acclimation light intensity is not linear [START_REF] Serôdio | A method for the rapid generation of nonsequential light-response curves of chlorophyll fluorescence[END_REF]. If we plot the NPQ according to the intensity of light from our dataset, we get a non-linear relationship (Figure 4-18). For low intensities, the implementation of NPQ is weak and increases from a threshold intensity (here ~450 μmol photons m -2 s -1 of solar light). It can be proposed that the induction of the proteins involved in the NPQ only takes place when the acclimation intensities exceed this threshold. Let's focus on the three parameters which appear to be significant in all explanatory models: the air temperature, the light intensity at the time of sampling and the concentration of inorganic nitrogen (which follows nitrate concentration, see correlation matrix, Figure 45678910111213141516).

The trend is here very clear: low nitrogen availability or low temperature decrease the photosynthetic efficiency (Fv/Fm, ETRmax), and light intensity is positively correlated to those parameters. Since the NPQ is a process which takes place when light absorption exceeds the capacity of the photosynthetic chain, it is not surprising that it positively correlates with light intensity and negatively correlates to the environmental factors that favour high photosynthetic activity, i.e. temperature and nitrogen availability. Chapter 4: Photosynthetic physiology of the coccolithophore Emiliania huxleyi Suzanne FERTE -Doctoral Thesis -2019 parameters variation has not been considered. Thamatrakoln and coworkers showed recently that the maximum rate of photosynthetic electron transfer was maintained during the viral infection in the laboratory (Thamatrakoln et al, 2018). However, the mechanisms underlying the specific decrease of Fv/Fm during infection is not yet explored.

I would like to propose a model to explain such a phenotype during viral infection, which is based on recent studies in the laboratory of Assaf Vardi [START_REF] Rosenwasser | Rewiring host lipid metabolism by large viruses determines the fate of Emiliania huxleyi, a bloom-forming alga in the ocean[END_REF][START_REF] Gilg | Differential gene expression is tied to photochemical efficiency reduction in virally infected Emiliania huxleyi[END_REF][START_REF] Ku | Tracking infection dynamics at single-cell level reveals highly resolved expression programs of a large virus infecting algal blooms[END_REF]. The analysis of the single-cell transcriptome of Emiliania huxleyi during its infection by its specific coccolithovirus reveals that the expression of nuclear genome of the host is arrested at the early stage of the infection, while plastid and mitochondrial transcriptomes were completely arrested at later stages [START_REF] Ku | Tracking infection dynamics at single-cell level reveals highly resolved expression programs of a large virus infecting algal blooms[END_REF].

It is well known that the D1 protein, a subunit of PSII, has a faster turnover than the other proteins involved in the photosynthetic chain [START_REF] Lindahl | The thylakoid FtsH protease plays a role in the light-induced turnover of the photosystem II D1 protein[END_REF]Aro et al., 1993). For example, the turnover of the D1 pool is ~20 min in high light stressed Dunaliella salina (Kim et al., 1993a). D1 being encoded by the PsbA gene in the plastid genome, blocking protein expression from the chloroplast genome by specific inhibitors, like chloramphenicol or lincomycin, therefore leads to a decrease in the D1 protein of photosytem II in plants, as well as in Emiliania huxleyi [START_REF] Tyystjärvi | The rate constant of photoinhibition, measured in lincomycintreated leaves, is directly proportional to light intensity[END_REF][START_REF] Loebl | Physiological basis for high resistance to photoinhibition under nitrogen depletion in Emiliania huxleyi[END_REF]. However, this treatment affects the abundance of other proteins of the photosynthetic machinery only at later stages.

During early stages, one therefore expects the repair of PSII to be hampered, whereas the other photosynthetic protein complexes remain unaffected. This would explain why viral infection leads to a specific decrease of the maximal PSII quantum efficiency without modifying the maximal rate of photosynthesis, as measured when light (and thus PSII efficiency) is not limiting electron transfer. Such a specific drop of Fv/Fm has energetic consequences for the efficiency of viral infection.

Viral replication requires the production of new viral particles from energy and organic matter [START_REF] Ku | Tracking infection dynamics at single-cell level reveals highly resolved expression programs of a large virus infecting algal blooms[END_REF]. In photosynthetic cells, the vast majority of anabolic pathways are provided by the activity of the photosynthetic chain. Maintaining, at least initially, a photosynthetic activity of the host cell thus seems particularly important for the effectiveness of the viral multiplication. Some cyanophages are known to carry in their genome homologues of genes encoding the D1 protein [START_REF] Mann | Phages of the marine cyanobacterial picophytoplankton[END_REF][START_REF] Dammeyer | Efficient phage-mediated pigment biosynthesis in oceanic cyanobacteria[END_REF] to maintain maximum efficiency of the PSII. In addition, some viruses such as Micromonas pusilla virus SP1 may even increase the maximum efficiency of PSII at certain critical stages of infection (Brown et al., This thesis work has two main axes: the exploration of CEF in microalgae and the study of determinants of photosynthesis in the field. In this discussion, I first suggest some methodological conclusions in a more general context and some research tracks that could be developed to enable CEF measurement in situ. In a second time, I discuss the putative roles of CEF and propose an additional function related to the response to specific inhibition of PSII that occurs in several conditions in the field. I also discuss the CEF regulation mechanisms compatible with our results. Finally, I present some lines of research that I would have liked to continue if the PhD thesis was lasting more than three years.

1. Methodological overview

The work I have presented partly consists in a methodological exploration around two central themes: the CEF and the study of photosynthesis in the field. I would first like to take a step back on the methodological approaches used in my work before proposing some methodological tracks to progress in the study of CEF in the field.

1. 1. The importance of cross validations of methods

Are we measuring what we believe we are measuring? Of course, this question is fundamental when trying to answer a biological question. In this thesis, a systematic verification of the methods used has been performed, often relying on cross-validations. This approach allowed to (i) show that the P700 method, commonly used for 25 years to estimate the activity of PSI, is unreliable under many particularly interesting physiological conditions (chapter 2), but also (ii) to question and refine protocols (chapter 2 and 3).

The interest of cross-validations for refining methods was mentioned in several places along this manuscript but I would like to come back to it through few examples. When we compared the ETR(total) values measured with the DIRK method or with the flash-induced method, we initially measured discrepancies at high light irradiances (Figure 5-1). By questioning the possible causes of this discrepancy, we were able to highlight that this was the consequences of charge recombination: the "a phase" measured in ECS that is thought to estimate stable charge separations (Bailleul et al., 2010) wrongly counted unstable charge separations. Those unstable charge separations lead to charge recombination in the time We then checked the relationship between the expected and measured ECS in this mixture and other mixtures of different species (Figure 5-4). We used the ECS kinetics at the light This approach could be used to measure CEF amplitude of each species in a mixture. Indeed, by inhibiting PSII, we could use ECS signals to measure PSI activity for each species, that would only be due to CEF.

Chapter 5: General discussion

Suzanne FERTE -Doctoral Thesis -2019 (e.g. temperature and intensity). This approach makes it possible to highlight potential causal relationships between an abiotic parameter and photosynthetic physiology. If we had been able to measure CEF in the field (which was not feasible) on the same population and to follow its evolution overtime, we would be have been able to discuss the major environmental parameters that determine CEF in situ.

Despite these present limitations in the study of photosynthetic physiology, we could show that the residues of the PLS model (remaining variance) could be used as a powerful tool for determining the influence of other parameters, such as biotic interactions. If the residuals to the model are not stochastically distributed along time, then we can highlight "abnormalities", i.e. the effect of a parameter which is not considered by the model. In our study, we were able to show anecdotal strong deviations from the model explaining the maximum efficiency of PSII, Fv/Fm. And those deviations seem to correlate with viral infection of cells. More generally, this approach could be used to highlight anecdotal effects of biotic parameters leading to a deviation from the model, such as the effects of pollutants or the allelopathic effects of a minority species on an algal population. Provided that a high frequency coverage is used, it could be also applied to disentangle light responses from circadian rhythms for example.

2. Roles of CEF and mechanisms of the regulation of the CEF and LEF

2. 1. ATP:NADPH ratio equilibration

In Chapter 3, we calculated ATP/NADPH ratios produced by the photosynthetic chain using the formula:

(1) ATP/NADPH = 18/14 + 12/14 CEF/LEF

We estimated that the photosynthetic chain produced ATP and NADPH in ratios of ~1.3 in Amphidinium carterae, ~1.8 in Symbiodinium sp and ~1.8 or ~2.5 in Chlamydomonas reinhardtii (depending on the method used to calculate the photon fluxes at PSII). We then used the estimated ratio of ATP per NADPH produced by the photosynthetic chain to discuss Can we estimate the extent of AEF in the three species we studied in chapter 3?

In the literature, there is no work aiming at measuring AEF in Amphidinium carterae. In Symbiodinium sp, the amplitude of Mehler reactions were studied by Roberty and collaborators (Roberty et al., 2014) and they estimated that up to 50% of the electrons transferred through PSII ultimately reduce molecular oxygen which means that the ATP/NADPH ratios calculated could be largely underestimated. In Chlamydomonas reinhardtii, AEF would be negligible under steady state light. Flavodiiron protein pathway would be mainly active at light onset (Chaux et al., 2017), super oxide dismutase involved in Mehler reaction would be down regulated in light [START_REF] Shao | Photosynthetic electron flow affects H 2 O 2 signaling by inactivation of catalase in Chlamydomonas reinhardtii[END_REF] and PTOX pathway would be negligible under steady state light regarding the LEF amplitude with a maximal rate of about 6 electrons s -1 PSII -1 (Houille-Vernes et al., 2011).

All in all, it is more likely that we underestimated rather than overestimated the ATP/NADPH ratios produced by the photosynthetic electron transport chain. It would be particularly interesting to quantify AEF and in particular the water to water cycle reactions in order to discuss more significantly the ATP/NADPH ratios produced by the photosynthetic chain. For example, we could couple spectroscopic measurements as we performed in chapter 3 and oxygen exchange measurement using [ 18 O]-labeled O2 and a membrane inlet mass spectrometer (as used for example in Chaux et al., 2017).

Chapter 5: General discussion

Suzanne FERTE -Doctoral Thesis -2019 repairing PSII. Indeed, the total inhibition of the linear flow by DCMU only decreases the speed of repair of the PSII while the addition of DCMU and a membrane uncoupler (preventing the establishment of an electrochemical gradient of protons on either side of the membrane) almost completely inhibits repair mechanisms [START_REF] Allakhverdiev | Systematic analysis of the relation of electron transport and ATP synthesis to the photodamage and repair of photosystem II in Synechocystis[END_REF].

In the case of PSII destruction induced by allelopathic molecules or viral infection, the role of CEF has never been explored.

We have seen that other AEF than CEF can fulfill the roles of photoprotection in high light or adjustment of the ATP/NADPH ratio proposed for the CEF. But only the CEF enables the production of ATP in the absence of PSII activity. It thus seems particularly interesting to explore the role of CEF in these conditions in order to better understand the evolutionary advantages linked to the ability to perform a CEF.

2. 3. CEF and LEF regulation

There is no consensus regarding the regulation of the relative extent of CEF and LEF in the photosynthesis community even in plants and green algae, where the role of CEF is established and where CEF has been investigated for decades. Several mechanisms have been proposed to adjust CEF and LEF:

(1) a regulation by the concentration of CEF substrates that are reduced Fd and oxidized PQ (e.g. Allen, 2003;Alric, 2010;Alric 2014);

(2) a regulation by changing the kinetic constants of the enzymes involved in the cycling step (i.e. the NDH/Nda2 or the PGRL1/PGR5). Propositions include the thioredxin-mediated redox activation/inactivation of those enzymes putatively involved in the cycling step [START_REF] Strand | Redox regulation of the antimycin A sensitive pathway of cyclic electron flow around photosystem I in higher plant thylakoids[END_REF] or competitive inhibition by ATP (Fisher et al., 2019);

(3) the segregation of CEF and LEF chains and the regulation of the fraction of PSI involved in each of them. This separation could be related to establishment of supercomplexes associating the intermediates involved in the CEF (Joliot and Joliot, 2002;Iwai et al., 2010), or structural segregation of the PSII and PSI centers in the appressed and nonappressed regions, respectively, and by restricted diffusion of the PQ (Joliot and Joliot, 2002).

The results I obtained on Chlamydomonas reinhardtii in Chapter 3 allow me to discuss some of these models. In Chlamydomonas reinhardtii, when we increased light irradiance, CEF was always proportional to LEF. This behavior is compatible with most of the regulatory modes presented above. For example, in model ( 2) when the light intensity is raised, the increase in signs of exceptional events such as population changes, allelopathic effects, or other surprises.

In addition, we have collected other data that have not been shown in this thesis but that could be further developed. For example, in collaboration with the Vardi lab, we tested the allelopathic effect of species present in mesocosms on a lab strain of Emiliania huxleyi. For this, we sampled water in the bags at different times of the campaign and put them in coculture with a strain of Emiliania huxleyi in an apparatus allowing to separate the two populations while making possible the diffusion of the chemical molecules. The Vardi lab team followed the evolution of the concentration of Emiliania huxleyi in co-culture and monoculture while we were comparing the photosynthetic activity of these two populations.

We have been able to highlight potential allelopathic interactions targeting photosynthesis.

Strains were isolated on site from the bags by collaborators from Station Biologique de Roscoff (France) and their effect on the growth of Emiliania huxleyi has been tested by the

Vardi lab team and appears to show allelopathic effects. It might be interesting to test the allelopathic effect of these strains on the photosynthesis of Emiliania huxleyi.

In addition, we followed the photosynthetic physiology of a diatom population present in the Fjord during a diel cycle and a collaborative team measured the storage of carbohydrates in the cells. The results show a high consumption of carbohydrate reserves in the morning, while photosynthesis was activated. The exploration of these data seems promising but is very preliminary for the moment.