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Several phytoplankton species produce the metabolites dimethylsulfoniopropionate (DMSP) and dimethylsulfoxide (DMSO) but their intracellular roles need to be better understood. To improve the understanding of the DMSP antioxidant function suggested by Sunda et al. (2002), we exposed the diatom Skeletonema costatum, the Prymnesiophyceae Phaeocystis globosa and the dinoflagellate Heterocapsa triquetra to experimental treatments known to cause potential oxidative stress (high light intensities (HL); HL with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU); menadione sodium bisulfite (MSB)). DMSP and DMSO concentrations decreased after 6 h in all treatments indicating an interaction with Reactive Oxygen Species (ROS) produced. DMSP and DMSO-to-cell ratios in control conditions were higher for H. triquetra, while being unable to grow under HL. DMSP and DMSO-to-cell carbon were the highest for P. globosa, while the other species had similar values. During long-term treatment, these ratios were not increased in high-light grown cells of P. globosa and S. costatum. Overall, this illustrates that (1) the DMSP- and DMSO-to-cell or carbon seems to be not indicative of the capability of the species to tolerate an oxidative stress, (2) these molecules could react with ROS and lower their cellular concentration, but no clues demonstrated that these molecules are part of the antioxidant response of the cell.

Increasing industrial and anthropogenic activities are producing and releasing more and more pollutants in the environment. Among them, toxic metals are one of the major threats for human health and natural ecosystems. Because photosynthetic organisms play a critical role in primary productivity and pollution management, investigating their response to metal toxicity is of major interest. Here, the green microalga Chlamydomonas (Chlamydomonas reinhardtii) was subjected to short (3 d) or chronic (6 months) exposure to 50 µM cadmium (Cd), and the recovery from chronic exposure was also examined. An extensive phenotypic characterization and transcriptomic analysis showed that the impact of Cd on biomass production of short-term (ST) exposed cells was almost entirely abolished by long-term (LT) acclimation. The underlying mechanisms were initiated at ST and further amplified after LT exposure resulting in a reversible equilibrium allowing biomass production similar to control condition. This included modification of cell wall-related gene expression and biofilm-like structure formation, dynamics of metal ion uptake and homeostasis, photosynthesis efficiency recovery and Cd acclimation through metal homeostasis adjustment. The contribution of the identified coordination of phosphorus and iron homeostasis (partly) mediated by the main phosphorus homeostasis regulator, Phosphate Starvation Response 1, and a basic Helix-Loop-Helix transcription factor (Cre05.g241636) was further investigated. The study reveals the highly dynamic physiological plasticity enabling algal cell growth in an extreme environment.

The mutualistic relationship existing between scleractinian corals and their photosynthetic endosymbionts involves a complex integration of the metabolic pathways within the holobiont. Respiration and photosynthesis are the most important of these processes and although they have been extensively studied, our understanding of their interactions and regulatory mechanisms is still limited. In this work we performed chlorophyll-a fluorescence, oxygen exchange and time-resolved absorption spectroscopy measurements on small and thin fragments (0.3 cm2) of the coral Stylophora pistillata. We showed that the capacity of mitochondrial alternative oxidase accounted for ca. 25% of total coral respiration, and that the high-light dependent oxygen uptake, commonly present in isolated Symbiodiniaceae, was negligible. The ratio between photosystem I (PSI) and photosystem II (PSII) active centers as well as their respective electron transport rates, indicated that PSI cyclic electron flow occurred in high light in S. pistillata and in some branching and lamellar coral species freshly collected in the field. Altogether, these results show the potential of applying advanced biophysical and spectroscopic methods on small coral fragments to understand the complex mechanisms of coral photosynthesis and respiration and their responses to environmental changes.

Mitochondrial F

The coupling between mitochondrial respiration and photosynthesis plays an important role in the energetic physiology of green plants and some secondary-red photosynthetic eukaryotes (diatoms), allowing an efficient CO

Dimethylsulfonopropionate (DMSP) and dimethylsulfoxide (DMSO) are two compounds involved in the carbon and sulfur cycle and are the precursors of the climate cooling gas dimethylsulfide (DMS). Despite decades of research, their role as osmoregulator, cryoprotector or antioxidant within the phytoplankton cells remains uncertain in some part. Since the antioxidant cascade system from the DMSP reported by Sunda & al. (2002), more investigation need to be conducted to confirm or accurate these interactions. This study aims to improve the knowledge about DMSP and DMSO and their hypothetic role of antioxidant on three different classes of phytoplankton (Dinophyceae – Prymnesiophyceae – diatom) and one diatom no-DMSP producer Chaetoceros sp. as negative control. Laboratory cultures were submitted to three oxidative stress to produce Reactive Oxygen Species (ROS) with (1) increasing light intensity from 100 to 600 and up to 1200 µmole/m²s for a global and natural oxidative stress; (2) using the menadone bisulfite (MSB) to generate ·O2 and (3) using 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) to inhibit the photosystem II (PSII). The PSII activity, the Chlorophyll a concentration (Chl a), the lipidic peroxidation (LOP), the ROS production and the pigment variation were analysed after 6h of incubation and related to the evolution of the DMSP and DMSO concentrations to better understand the cellular oxidative stress and its impact on the phytoplankton cell and DMSP and DMSO production.

Mitochondrial F1FO-ATP synthase of the chlorophycean algae Polytomella sp. can be isolated as a highly stable dimeric complex of 1600kDa. It is composed of eight highly conserved orthodox subunits (α, β, γ, δ, e, OSCP, a, and c) and nine subunits (Asa1-9) that are exclusive of chlorophycean algae. The Asa subunits replace those that build up the peripheral stalk and the dimerization domains of the ATP synthase in other organisms. Little is known about the disposition of subunits Asa6, Asa8 and Asa9, that are predicted to have transmembrane stretches and that along with subunit a and a ring of c-subunits, seem to constitute the membrane-embedded Fo domain of the algal ATP synthase. Here, we over-expressed and purified the three Asa hydrophobic subunits and explored their interactions in vitro using a combination of immunochemical techniques, affinity chromatography, and an in vivo yeast-two hybrid assays. The results obtained suggest the following interactions Asa6-Asa6, Asa6-Asa8, Asa6-Asa9, Asa8-Asa8 and Asa8-Asa9. Cross-linking experiments carried out with the intact enzyme corroborated some of these interactions. Based on these results, we propose a model of the disposition of these hydrophobic subunits in the membrane-embedded sector of the algal ATP synthase. We also propose based on sequence analysis and hydrophobicity plots, that the algal subunit a is atypical in as much it lacks the first transmembrane stretch, exhibiting only four hydrophobic, tilted alpha helices.

Mitochondrial respiratory-chain complexes from Euglenozoa comprise classical subunits described in other eukaryotes (i.e. mammals and fungi) and subunits that are restricted to Euglenozoa (e.g. Euglena gracilis and Trypanosoma brucei). Here we studied the mitochondrial F1FO-ATP synthase (or Complex V) from the photosynthetic eukaryote E. gracilis in detail. The enzyme was purified by a two-step chromatographic procedure and its subunit composition was resolved by a three-dimensional gel electrophoresis (BN/SDS/SDS). Twenty-two different subunits were identified by mass-spectrometry analyses among which the canonical α, β, γ, δ, ε, and OSCP subunits, and at least seven subunits previously found in Trypanosoma. The ADP/ATP carrier was also associated to the ATP synthase into a dimeric ATP synthasome. Single-particle analysis by transmission electron microscopy of the dimeric ATP synthase indicated that the structures of both the catalytic and central rotor parts are conserved while other structural features are original. These new features include a large membrane-spanning region joining the monomers, an external peripheral stalk and a structure that goes through the membrane and reaches the inter membrane space below the c-ring, the latter having not been reported for any mitochondrial F-ATPase.

International audience; Cyclic electron flow (CEF) is defined as a return of the reductants from the acceptor side of Photosystem I (PSI) to the pool of its donors via the cytochrome b6f. It is described to be complementary to the linear electron flow and essential for photosynthesis. However, despite many efforts aimed to characterize CEF, its pathway and its regulation modes remain equivocal, and its physiological significance is still not clear. Here we use novel spectroscopic to measure the rate of CEF at the onset of light in the green alga Chlamydomonas reinhardtii. The initial redox state of the photosynthetic chain or the oxygen concentration do not modify the initial maximal rate of CEF (60 electrons per second per PSI) but rather strongly influence its duration. Neither the maximal rate nor the duration of CEF are different in the pgrl1 mutant compared to the wild type, disqualifying PGRL1 as the ferredoxin-plastoquinone reductase involved in the CEF mechanism.

Apart from the canonical light-driven linear electron flow (LEF) from water to CO2, numerous regulatory and alternative electron transfer pathways exist in chloroplasts. One of them is the cyclic electron flow around Photosystem I (CEF), contributing to photoprotection of both Photosystem I and II (PSI, PSII) and supplying extra ATP to fix atmospheric carbon. Nonetheless, CEF remains an enigma in the field of functional photosynthesis as we lack understanding of its pathway. Here, we address the discrepancies between functional and genetic/biochemical data in the literature and formulate novel hypotheses about the pathway and regulation of CEF based on recent structural and kinetic information.

A Correction to this paper has been published: 10.1007/s10863-021-09890-8

The algae Chlamydomonas reinhardtii and Polytomella sp., a green and a colorless member of the chlorophycean lineage respectively, exhibit a highly-stable dimeric mitochondrial F1Fo-ATP synthase (complex V), with a molecular mass of 1600 kDa. Polytomella, lacking both chloroplasts and a cell wall, has greatly facilitated the purification of the algal ATP-synthase. Each monomer of the enzyme has 17 polypeptides, eight of which are the conserved, main functional components, and nine polypeptides (Asa1 to Asa9) unique to chlorophycean algae. These atypical subunits form the two robust peripheral stalks observed in the highly-stable dimer of the algal ATP synthase in several electron-microscopy studies. The topological disposition of the components of the enzyme has been addressed with cross-linking experiments in the isolated complex; generation of subcomplexes by limited dissociation of complex V; detection of subunit-subunit interactions using recombinant subunits; in vitro reconstitution of subcomplexes; silencing of the expression of Asa subunits; and modeling of the overall structural features of the complex by EM image reconstruction. Here, we report that the amphipathic polymer Amphipol A8-35 partially dissociates the enzyme, giving rise to two discrete dimeric subcomplexes, whose compositions were characterized. An updated model for the topological disposition of the 17 polypeptides that constitute the algal enzyme is suggested. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

Mitochondrial F1FO-ATP synthase of chlorophycean algae is dimeric. It contains eight orthodox subunits (alpha, beta, gamma, delta, epsilon, OSCP, a and c) and nine atypical subunits (Asa1 to 9). These subunits build the peripheral stalk of the enzyme and stabilize its dimeric structure. The location of the 66.1kDa subunit Asa1 has been debated. On one hand, it was found in a transient subcomplex that contained membrane-bound subunits Asa1/Asa3/Asa5/Asa8/a (Atp6)/c (Atp9). On the other hand, Asa1 was proposed to form the bulky structure of the peripheral stalk that contacts the OSCP subunit in the F1 sector. Here, we overexpressed and purified the recombinant proteins Asa1 and OSCP and explored their interactions in vitro, using immunochemical techniques and affinity chromatography. Asa1 and OSCP interact strongly, and the carboxy-terminal half of OSCP seems to be instrumental for this association. In addition, the algal ATP synthase was partially dissociated at relatively high detergent concentrations, and an Asa1/Asa3/Asa5/Asa8/a/c10 subcomplex was identified. Furthermore, Far-Western analysis suggests an Asa1-Asa8 interaction. Based on these results, a model is proposed in which Asa1 spans the whole peripheral arm of the enzyme, from a region close to the matrix-exposed side of the mitochondrial inner membrane to the F1 region where OSCP is located. 3D models show elongated, helix-rich structures for chlorophycean Asa1 subunits. Asa1 subunit probably plays a scaffolding role in the peripheral stalk analogous to the one of subunit b in orthodox mitochondrial enzymes.

The unicellular green alga Chlamydomonas reinhardtii is a model organism for studying energetic metabolism. Most mitochondrial respiratory-deficient mutants characterized to date have been isolated on the basis of their reduced ability to grow in heterotrophic conditions. Mitochondrial deficiencies are usually partly compensated by adjustment of photosynthetic activity and more particularly by transition to state 2. In this work, we explored the opportunity to select mutants impaired in respiration and/or altered in dark metabolism by measuring maximum photosynthetic efficiency by chlorophyll fluorescence analyses (FV/FM). Out of about 2900 hygromycin-resistant insertional mutants generated from wild type or from a mutant strain deficient in state transitions (stt7 strain), 22 were found to grow slowly in heterotrophic conditions and 8 of them also showed a lower FV/FM value. Several disrupted coding sequences were identified, including genes coding for three different subunits of respiratory-chain complex I (NUO9, NUOA9, NUOP4) or for isocitrate lyase (ICL1). Overall, the comparison of respiratory mutants obtained in wild-type or stt7 genetic backgrounds indicated that the FV/FM value can be used to isolate mutants severely impaired in dark metabolism.

The proposal that the respiratory complexes can associate with each other in larger structures named supercomplexes (SC) is generally accepted. In the last decades most of the data about this association came from studies in yeasts, mammals and plants, and information is scarce in other lineages. Here we studied the supramolecular association of the F1FO-ATP synthase (complex V) and the respiratory complexes I, III and IV of the colorless alga Polytomella sp. with an approach that involves solubilization using mild detergents, n-dodecyl-β-D-maltoside (DDM) or digitonin, followed by separation of native protein complexes by electrophoresis (BN-PAGE), after which we identified oligomeric forms of complex V (mainly V2 and V4) and different respiratory supercomplexes (I/IV6, I/III4, I/IV). In addition, purification/reconstitution of the supercomplexes by anion exchange chromatography was also performed. The data show that these complexes have the ability to strongly associate with each other and form DDM-stable macromolecular structures. The stable V4 ATPase oligomer was observed by electron-microscopy and the association of the respiratory complexes in the so-called "respirasome" was able to perform in-vitro oxygen consumption.

During the last decades, coral reefs have been affected by several large-scale bleaching events, and such phenomena are expected to increase in frequency and severity in the future, thus compromising their survival. High sea surface temperature accompanied by high levels of solar irradiance has been found to be responsible for the induction of oxidative stress ultimately ending with the disruption of the symbiosis between cnidarians and Symbiodinium. For two decades, many studies have pointed to the water–water cycle (WWC) as being one of the primary mediators of this phenomenon, but the impacts of environmental stress on the O2 reduction by PSI and the associated reactive oxygen species (ROS)-detoxifying enzymes remain to be determined. In this study, we analyzed the impacts of acute thermal and light stress on the WWC in the model Symbiodinium strain A1. We observed that the high light treatment at 26 °C resulted in the up-regulation of superoxide dismutase, ascorbate peroxidase, and glutathione reductase activities and an increased production of ROS with no significant change in O2-dependent electron transport. Under high light and at 33 °C, O2-dependent electron transport was significantly increased relative to total electron transport. This increase was concomitant with a twofold increase in ROS generation compared with the treatment at 26 °C, while enzymes involved in the WWC were largely inactivated. These data show for the first time that combined heat and light stress inactivate antioxidant capacities of the WWC and suggests that its photoprotective functions are overwhelmed under these conditions. This study also indicates that cnidarians may be more prone to bleach if they harbor Symbiodinium cells having a highly active Mehler-type electron transport, unless they are able to quickly up-regulate their antioxidant capacities.

The model green microalga Chlamydomonas reinhardtii is frequently subject to periods of dark and anoxia in its natural environment. Here, by resorting to mutants defective in the maturation of the chloroplastic oxygen-sensitive hydrogenases or in Proton-Gradient Regulation-Like1 (PGRL1)-dependent cyclic electron flow around photosystem I (PSI-CEF), we demonstrate the sequential contribution of these alternative electron flows (AEFs) in the reactivation of photosynthetic carbon fixation during a shift from dark anoxia to light. At light onset, hydrogenase activity sustains a linear electron flow from photosystem II, which is followed by a transient PSI-CEF in the wild type. By promoting ATP synthesis without net generation of photosynthetic reductants, the two AEF are critical for restoration of the capacity for carbon dioxide fixation in the light. Our data also suggest that the decrease in hydrogen evolution with time of illumination might be due to competition for reduced ferredoxins between ferredoxin-NADP(+) oxidoreductase and hydrogenases, rather than due to the sensitivity of hydrogenase activity to oxygen. Finally, the absence of the two alternative pathways in a double mutant pgrl1 hydrogenase maturation factor G-2 is detrimental for photosynthesis and growth and cannot be compensated by any other AEF or anoxic metabolic responses. This highlights the role of hydrogenase activity and PSI-CEF in the ecological success of microalgae in low-oxygen environments.

Cyclic electron flow (CEF), one of the major alternative electron transport pathways to the primary linear electron flow (LEF) in chloroplasts has been discovered in the middle of the last century. It is defined as a return of the reductants from the acceptor side of the Photosystem I (PSI) to the pool of its donors via the cytochromeb6f, and has proven essential for photosynthesis. However, despite many efforts aimed at its characterisation, the pathway and regulation of CEF remain equivocal, and its physiological significance remains to be properly defined. Here we use novel spectroscopic approaches to measure CEF in transitory conditions in the green algaChlamydomonas reinhardtii.We show that CEF operates at the same maximal rate regardless of the oxygen concentration, and that the latter influences LEF, rather than CEF in vivo, which questions the recent hypotheses about the CEF supercomplex formation. We further reveal that the pathways proposed for CEF in the literature are inconsistent with the kinetic information provided by our measurements. We finally provide cues on the regulation of CEF by light.

Mitochondrion is the site where the Krebs cycle and oxidative phosphorylation (OXPHOS) take place. After a brief overview of Krebs cycle and acetate metabolism in Chlamydomonas, this chapter focuses on OXPHOS components. OXPHOS is composed of five major multiprotein complexes: NADH:ubiquinone oxidoreductase (complex I), succinate dehydrogenase (complex II), ubiquinone:cytochrome c oxidoreductase (complex III), cytochrome c oxidase (complex IV), and ATP synthase. Three complexes (complexes I, III, and IV) pump protons from the matrix to the intermembrane space (IMS) and build a gradient which is used by ATP synthase to produce ATP. In Chlamydomonas and other eukaryotes, proteins forming these complexes have a dual genetic origin. A few proteins, mostly hydrophobic polypeptides, are mitochondrion-encoded, while the vast majority are nucleus-encoded and imported from the cytoplasm. Here we will review our current knowledge about these complexes.

Understanding of the molecular mechanisms of photosynthetic electron and proton transports and their regulation in plants and algae in response to changes in environmental conditions is an important issue for fundamental research on photosynthesis, and may extend even to practical applications by identifying important sites for improvement of photosynthesis. The significance and often centrality of regulatory mechanisms of photosynthetic electron transport is well established for processes in plant acclimation. In recent years, significant advancements have been achieved in understanding of regulatory processes such as dissipation of excess energy in the antenna systems, state transitions, cyclic electron flow, oxygen reduction by flavodiiron enzymes and many others.

In Chlamydomonas, unlike in flowering plants, genes coding for Nd7 (NAD7/49 kDa) and Nd9 (NAD9/30 kDa) core subunits of mitochondrial respiratory-chain complex I are nucleus-encoded. Both genes possess all the features that facilitate their expression and proper import of the polypeptides in mitochondria. By inactivating their expression by RNA interference or insertional mutagenesis, we show that both subunits are required for complex I assembly and activity. Inactivation of complex I impairs the cell growth rate, reduces the respiratory rate, leads to lower intracellular ROS production and lower expression of ROS scavenging enzymes, and is associated to a diminished capacity to concentrate CO2 without compromising photosynthetic capacity.

The mitochondrion is an essential organelle for the production of cellular ATP in most eukaryotic cells. It is extensively studied, including in parasitic organisms such as trypanosomes, as a potential therapeutic target. Recently, numerous additional subunits of the respiratory-chain complexes have been described in Trypanosoma brucei and Trypanosoma cruzi. Since these subunits had apparently no counterparts in other organisms, they were interpreted as potentially associated with the parasitic trypanosome lifestyle. Here we used two complementary approaches to characterise the subunit composition of respiratory complexes in Euglena gracilis, a non-parasitic secondary green alga related to trypanosomes. First, we developed a phylogenetic pipeline aimed at mining sequence databases for identifying homologues to known respiratory-complex subunits with high confidence. Second, we used MS/MS proteomics after two-dimensional separation of the respiratory complexes by Blue Native- and SDS-PAGE both to confirm in silico predictions and to identify further additional subunits. Altogether, we identified 41 subunits that are restricted to E. gracilis, T. brucei and T. cruzi, along with 48 classical subunits described in other eukaryotes (i.e. plants, mammals and fungi). This moreover demonstrates that at least half of the subunits recently reported in T. brucei and T. cruzi are actually not specific to Trypanosomatidae, but extend at least to other Euglenozoa, and that their origin and function are thus not specifically associated with the parasitic lifestyle. Furthermore, preliminary biochemical analyses suggest that some of these additional subunits underlie the peculiarities of the respiratory chain observed in Euglenozoa.

Genetic manipulation of the unicellular green alga Chlamydomonas reinhardtii is straightforward. Nuclear genes can be interrupted by insertional mutagenesis or targeted by RNA interference whereas random or site-directed mutagenesis allows the introduction of mutations in the mitochondrial genome. This, combined with a screen that easily allows discriminating respiratory-deficient mutants, makes Chlamydomonas a model system of choice to study mitochondria biology in photosynthetic organisms. Since the first description of Chlamydomonas respiratory-deficient mutants in 1977 by random mutagenesis, many other mutants affected in mitochondrial components have been characterized. These respiratory-deficient mutants increased our knowledge on function and assembly of the respiratory enzyme complexes. More recently some of these mutants allowed the study of mitochondrial gene expression processes poorly understood in Chlamydomonas. In this review, we update the data concerning the respiratory components with a special focus on the assembly factors identified on other organisms. In addition, we make an inventory of different mitochondrial respiratory mutants that are inactivated either on mitochondrial or nuclear genes.

Isocitrate lyase is a key enzyme of the glyoxylate cycle. This cycle plays an essential role in cell growth on acetate, and is important for gluconeogenesis as it bypasses the two oxidative steps of the tricarboxylic acid (TCA) cycle in which CO₂ is evolved. In this paper, a null icl mutant of the green microalga Chlamydomonas reinhardtii is described. Our data show that isocitrate lyase is required for growth in darkness on acetate (heterotrophic conditions), as well as for efficient growth in the light when acetate is supplied (mixotrophic conditions). Under these latter conditions, reduced acetate assimilation and concomitant reduced respiration occur, and biomass composition analysis reveals an increase in total fatty acid content, including neutral lipids and free fatty acids. Quantitative proteomic analysis by ¹⁴N/¹⁵N labelling was performed, and more than 1600 proteins were identified. These analyses reveal a strong decrease in the amounts of enzymes of the glyoxylate cycle and gluconeogenesis in parallel with a shift of the TCA cycle towards amino acid synthesis, accompanied by an increase in free amino acids. The decrease of the glyoxylate cycle and gluconeogenesis, as well as the decrease in enzymes involved in β-oxidation of fatty acids in the icl mutant are probably major factors that contribute to remodelling of lipids in the icl mutant. These modifications are probably responsible for the elevation of the response to oxidative stress, with significantly augmented levels and activities of superoxide dismutase and ascorbate peroxidase, and increased resistance to paraquat.

In Chlamydomonas reinhardtii, prolonged anaerobiosis leads to the expression of enzymes belonging to various fermentative pathways. Among them, oxygen-sensitive hydrogenases (HydA1/2) catalyze the synthesis of molecular hydrogen from protons and reduced ferredoxin in the stroma. In this work, by analyzing wild type and mutants affected in H2 production, we show that maximal PSII photosynthetic electron transfer during the first seconds of illumination after a prolonged dark-anaerobiosis period is linearly related to hydrogenase capacity. Based on the specific chlorophyll fluorescence induction kinetics typical of hydrogenase-deficient mutants, we set up an in vivo fluorescence imaging screening protocol allowing to isolate mutants impaired in hydrogenase expression or activity, as well as mutants altered in related metabolic pathways required for energy production in anaerobiosis. Compared to previously described screens for mutants impaired in H2 production, our screening method is remarkably fast, sensitive and non-invasive. Out of 3000 clones from a small-sized insertional mutant library, five mutants were isolated and the most affected one was analyzed and shown to be defective for the hydrogenase HydG assembly factor.

International audience; Unicellular algae are characterized by an extreme flexibility with respect to their responses to environmental constraints. This flexibility probably explains why microalgae show a very high biomass yield, constitute one of the major contributors to primary productivity in the oceans and are considered a promising choice for biotechnological applications. Flexibility results from a combination of several factors including fast changes in the light-harvesting apparatus and a strong interaction between different metabolic processes (e.g. respiration and photosynthesis), which all take place within the same cell. Microalgae are also capable of modifying their photosynthetic electron flow capacity, by changing its maximum rate and/or by diverting photogenerated electrons towards different sinks depending on their growth status. In this review, we will focus on the occurrence and regulation of alternative electron flows in unicellular algae and compare data obtained in these systems with those available in vascular plants. This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts.

Photosynthetic organisms are exposed to drastic changes in light conditions, which can affect their photosynthetic efficiency and induce photodamage. To face these changes, they have developed a series of acclimation mechanisms. In this work, we have studied the acclimation strategies of Chlamydomonas reinhardtii, a model green alga that can grow using various carbon sources and is thus an excellent system in which to study photosynthesis. Like other photosynthetic algae, it has evolved inducible mechanisms to adapt to conditions where carbon supply is limiting. We have analyzed how the carbon availability influences the composition and organization of the photosynthetic apparatus and the capacity of the cells to acclimate to different light conditions. Using electron microscopy, biochemical, and fluorescence measurements, we show that differences in CO2 availability not only have a strong effect on the induction of the carbon-concentrating mechanisms but also change the acclimation strategy of the cells to light. For example, while cells in limiting CO2 maintain a large antenna even in high light and switch on energy-dissipative mechanisms, cells in high CO2 reduce the amount of pigments per cell and the antenna size. Our results show the high plasticity of the photosynthetic apparatus of C. reinhardtii. This alga is able to use various photoacclimation strategies, and the choice of which to activate strongly depends on the carbon availability.

Type-II NAD(P)H dehydrogenases form a multigene family that comprise six members in the green microalga Chlamydomonas. To date, only one enzyme (Nda2) located in the chloroplast has been characterized in this alga and demonstrated to participate in the reduction of the plastoquinone pool. We present here the functional characterization of Nda1. The enzyme is located on the inner face of the inner mitochondrial membrane. Its downregulation leads to a slight decrease of NADH:ferricyanide activity and of dark whole cell respiration. To determine whether the reduction of Nda1 combined with the lack of complex I would affect mitochondrial processes, double mutants affected in both Nda1 and complex I were isolated. Respiration and growth rates in heterotrophic conditions were significantly altered in the double mutants investigated, suggesting that Nda1 plays a role in the oxidation of matrix NADH in the absence of complex I.

Mitochondrial F1Fo-ATP synthase (complex V) is an oligomeric complex that exhibits a molecular mass around 550 kDa. The F1Fo-ATP synthase of the green chlorophycean alga Chlamydomonas reinhardtii and of its close colorless relative Polytomella sp. is isolated as a highly-stable dimer of 1600 kDa after solubilization of algal mitochondria with lauryl-maltoside. Each monomer of the enzyme is constituted by 17 polypeptides, eight of which are the conserved subunits alpha, beta, gamma, delta, epsilon, a (Atp6), c (Atp9), and OSCP, plus nine atypical polypeptides, named Asa1 to Asa9, that seem to be present only in the chlorophycean lineage. The Asa subunits (named after ATP Synthase Associated subunits) form the very robust peripheral stalk that has been observed in several electron-microscope studies of the algal enzyme. We have addressed the close-neighbor relationships of the ASA subunits and their interactions with the orthodox, conserved subunits. For this purpose, we have generated sub-complexes after partial dissociation of the dimeric ATP synthase with high detergent concentrations or with amphiphilic polymers; detected several subunit-subunit interactions based on cross-linking experiments; reconstituted various sub-complexes of the peripheral arm using recombinant subunits; and silenced the synthesis of certain subunits in vivo. Recently, an electron cryo-microscopy map of the enzyme was obtained. Putting together all the structural data available to date, we suggest a model of the complete topological disposition of the subunits that constitute the atypical, algal mitochondrial ATP synthase. The presence of such a stout peripheral stalk in the ATP synthase of chlorophycean alga, seems to have more than the necessary stiffness to counteract the rotation of the central stalk, but will probably make less twisting motions, and thus will store less transient elastic energy.

The COX3 gene encodes a core subunit of mitochondrial cytochrome c oxidase (complex IV) whereas the COX17 gene encodes a chaperone delivering copper to the enzyme. Mutants of these two genes were isolated by RNA interference in the microalga Chlamydomonas. The COX3 mRNA was completely lacking in the cox3-RNAi mutant and no activity and assembly of complex IV were detected. The cox17-RNAi mutant presented a reduced level of COX17 mRNA, a reduced activity of the cytochrome c oxidase but no modification of its amount. The cox3-RNAi mutant had only 40% of the wild-type rate of dark respiration which was cyanide-insensitive. The mutant presented a 60% decrease of H(2)O(2) production in the dark compared to wild type, which probably accounts for a reduced electron leakage by respiratory complexes III and IV. In contrast, the cox17-RNAi mutant showed no modification of respiration and of H(2)O(2) production in the dark but a two to threefold increase of H(2)O(2) in the light compared to wild type and the cox3-RNAi mutant. The cox17-RNAi mutant was more sensitive to cadmium than the wild-type and cox3-RNAi strains. This suggested that besides its role in complex IV assembly, Cox17 could have additional functions in the cell such as metal detoxification or Reactive Oxygen Species protection or signaling. Concerning Cox3, its role in Chlamydomonas complex IV is similar to that of other eukaryotes although this subunit is encoded in the nuclear genome in the alga contrary to the situation found in all other organisms.

International audience; In photosynthesis, electron transfer along the photosynthetic chain results in a vectorial transfer of protons from the stroma to the lumenal space of the thylakoids. This promotes the generation of an electrochemical proton gradient (Δμ(H)(+)), which comprises a gradient of electric potential (ΔΨ) and of proton concentration (ΔpH). The Δμ(H)(+) has a central role in the photosynthetic process, providing the energy source for ATP synthesis. It is also involved in many regulatory mechanisms. The ΔpH modulates the rate of electron transfer and triggers deexcitation of excess energy within the light harvesting complexes. The ΔΨ is required for metabolite and protein transport across the membranes. Its presence also induces a shift in the absorption spectra of some photosynthetic pigments, resulting in the so-called ElectroChromic Shift (ECS). In this review, we discuss the characteristic features of the ECS, and illustrate possible applications for the study of photosynthetic processes in vivo.

In the present work, we have isolated by RNA interference and characterized at the functional and the proteomic levels a Chlamydomonas reinhardtii strain devoid of the mitochondrial alternative oxidase 1 (AOX1). The AOX1-deficient strain displays a remarkable doubling of the cell volume and biomass without alteration of the generation time or change in total respiratory rate, with a significantly higher ROS production. To identify the molecular adaptation underlying these observations, we have carried out a comparative study of both the mitochondrial and the cellular soluble proteomes. Our results indicate a strong up-regulation of the ROS scavenging systems and important quantitative modifications of proteins involved in the primary metabolism, namely an increase of enzymes involved in anabolic pathways and a concomitant general down-regulation of enzymes of the main catabolic pathways.

In yeast, mammals, and land plants, mitochondrial F(1)F(O)-ATP synthase (complex V) is a remarkable enzymatic machinery that comprises about 15 conserved subunits. Peculiar among eukaryotes, complex V from Chlamydomonadales algae (order of chlorophycean class) has an atypical subunit composition of its peripheral stator and dimerization module, with nine subunits of unknown evolutionary origin (Asa subunits). In vitro, this enzyme exhibits an increased stability of its dimeric form, and in vivo, Chlamydomonas reinhardtii cells are insensitive to oligomycins, which are potent inhibitors of proton translocation through the F(O) moiety. In this work, we showed that the atypical features of the Chlamydomonadales complex V enzyme are shared by the other chlorophycean orders. By biochemical and in silico analyses, we detected several atypical Asa subunits in Scenedesmus obliquus (Sphaeropleales) and Chlorococcum ellipsoideum (Chlorococcales). In contrast, complex V has a canonical subunit composition in other classes of Chlorophytes (Trebouxiophyceae, Prasinophyceae, and Ulvophyceae) as well as in Streptophytes (land plants), and in Rhodophytes (red algae). Growth, respiration, and ATP levels in Chlorophyceae were also barely affected by oligomycin concentrations that affect representatives of the other classes of Chlorophytes. We finally studied the function of the Asa7 atypical subunit by using RNA interference in C. reinhardtii. Although the loss of Asa7 subunit has no impact on cell bioenergetics or mitochondrial structures, it destabilizes in vitro the enzyme dimeric form and renders growth, respiration, and ATP level sensitive to oligomycins. Altogether, our results suggest that the loss of canonical components of the complex V stator happened at the root of chlorophycean lineage and was accompanied by the recruitment of novel polypeptides. Such a massive modification of complex V stator features might have conferred novel properties, including the stabilization of the enzyme dimeric form and the shielding of the proton channel. In these respects, we discuss an evolutionary scenario for F(1)F(O)-ATP synthase in the whole green lineage (i.e., Chlorophyta and Streptophyta).

The relationship between the development of photoprotective mechanisms (non-photochemical quenching, NPQ), the generation of the electrochemical proton gradient in the chloroplast and the capacity to assimilate CO2 was studied in tobacco dark-adapted leaves at the onset of illumination with low light. These conditions induce the generation of a transient NPQ, which relaxes in the light in parallel with the activation of the Calvin cycle. Wild-type plants were compared with a CMSII mitochondrial mutant, which lacks the respiratory complex I and shows a delayed activation of photosynthesis. In the mutant, a slower onset of photosynthesis was mirrored by a decreased capacity to develop NPQ. This correlates with a reduced efficiency to reroute electrons at the PSI reducing side towards cyclic electron flow around PSI and/or other alternative acceptor pools, and with a smaller ability to generate a proton motive force in the light. Altogether, these data illustrate the tight relationship existing between the capacity to evacuate excess electrons accumulated in the intersystem carriers and the capacity to dissipate excess photons during a dark to light transition. These data also underline the essential role of respiration in modulating the photoprotective response in dark-adapted leaves, by poising the cellular redox state.