APX2 Is an Ascorbate Peroxidase-Related Protein that Regulates the Levels of Plastocyanin in Chlamydomonas.

The function of ascorbate peroxidase-related (APX-R) proteins, present in all green photosynthetic eukaryotes, remains unclear. This study focuses on APX-R from Chlamydomonas reinhardtii, namely, ascorbate peroxidase 2 (APX2). We showed that apx2 mutants exhibited a faster oxidation of the photosystem I primary electron donor, P700, upon sudden light increase and a slower re-reduction rate compared to the wild type, pointing to a limitation of plastocyanin. Spectroscopic, proteomic and immunoblot analyses confirmed that the phenotype was a result of lower levels of plastocyanin in the apx2 mutants. The redox state of P700 did not differ between wild type and apx2 mutants when the loss of function in plastocyanin was nutritionally complemented by growing apx2 mutants under copper deficiency. In this case, cytochrome c6 functionally replaces plastocyanin, confirming that lower levels of plastocyanin were the primary defect caused by the absence of APX2. Overall, the results presented here shed light on an unexpected regulation of plastocyanin level under copper-replete conditions, induced by APX2 in Chlamydomonas.

Induction of photosynthesis under anoxic condition in Thalassiosira pseudonana and Euglena gracilis: interactions between fermentation and photosynthesis.

Introduction: In their natural environment, microalgae can be transiently exposed to hypoxic or anoxic environments. Whereas fermentative pathways and their interactions with photosynthesis are relatively well characterized in the green alga model Chlamydomonas reinhardtii, little information is available in other groups of photosynthetic micro-eukaryotes. In C. reinhardtii cyclic electron flow (CEF) around photosystem (PS) I, and light-dependent oxygen-sensitive hydrogenase activity both contribute to restoring photosynthetic linear electron flow (LEF) in anoxic conditions. Methods: Here we analyzed photosynthetic electron transfer after incubation in dark anoxic conditions (up to 24 h) in two secondary microalgae: the marine diatom Thalassiosira pseudonana and the excavate Euglena gracilis. Results: Both species showed sustained abilities to prevent over-reduction of photosynthetic electron carriers and to restore LEF. A high and transient CEF around PSI was also observed specifically in anoxic conditions at light onset in both species. In contrast, at variance with C. reinhardtii, no sustained hydrogenase activity was detected in anoxic conditions in both species. Discussion: Altogether our results suggest that another fermentative pathway might contribute, along with CEF around PSI, to restore photosynthetic activity in anoxic conditions in E. gracilis and T. pseudonana. We discuss the possible implication of the dissimilatory nitrate reduction to ammonium (DNRA) in T. pseudonana and the wax ester fermentation in E. gracilis.

Weak acids produced during anaerobic respiration suppress both photosynthesis and aerobic respiration.

While photosynthesis transforms sunlight energy into sugar, aerobic and anaerobic respiration (fermentation) catabolizes sugars to fuel cellular activities. These processes take place within one cell across several compartments, however it remains largely unexplored how they interact with one another. Here we report that the weak acids produced during fermentation down-regulate both photosynthesis and aerobic respiration. This effect is mechanistically explained with an "ion trapping" model, in which the lipid bilayer selectively traps protons that effectively acidify subcellular compartments with smaller buffer capacities - such as the thylakoid lumen. Physiologically, we propose that under certain conditions, e.g., dim light at dawn, tuning down the photosynthetic light reaction could mitigate the pressure on its electron transport chains, while suppression of respiration could accelerate the net oxygen evolution, thus speeding up the recovery from hypoxia. Since we show that this effect is conserved across photosynthetic phyla, these results indicate that fermentation metabolites exert widespread feedback control over photosynthesis and aerobic respiration. This likely allows algae to better cope with changing environmental conditions.

Natural variation of nutrient homeostasis among laboratory and field strains of Chlamydomonas reinhardtii.

Natural variation among individuals and populations exists in all species, playing key roles in response to environmental stress and adaptation. Micro- and macronutrients have a wide range of functions in photosynthetic organisms, and mineral nutrition thus plays a sizable role in biomass production. To maintain nutrient concentrations inside the cell within physiological limits and prevent the detrimental effects of deficiency or excess, complex homeostatic networks have evolved in photosynthetic cells. The microalga Chlamydomonas reinhardtii (Chlamydomonas) is a unicellular eukaryotic model for studying such mechanisms. In this work, 24 Chlamydomonas strains, comprising field isolates and laboratory strains, were examined for intraspecific differences in nutrient homeostasis. Growth and mineral content were quantified in mixotrophy, as full nutrition control, and compared with autotrophy and nine deficiency conditions for macronutrients (-Ca, -Mg, -N, -P, and -S) and micronutrients (-Cu, -Fe, -Mn, and -Zn). Growth differences among strains were relatively limited. However, similar growth was accompanied by highly divergent mineral accumulation among strains. The expression of nutrient status marker genes and photosynthesis were scored in pairs of contrasting field strains, revealing distinct transcriptional regulation and nutrient requirements. Leveraging this natural variation should enable a better understanding of nutrient homeostasis in Chlamydomonas.

Euglena International Network (EIN): Driving euglenoid biotechnology for the benefit of a challenged world.

Euglenoids (Euglenida) are unicellular flagellates possessing exceptionally wide geographical and ecological distribution. Euglenoids combine a biotechnological potential with a unique position in the eukaryotic tree of life. In large part these microbes owe this success to diverse genetics including secondary endosymbiosis and likely additional sources of genes. Multiple euglenoid species have translational applications and show great promise in production of biofuels, nutraceuticals, bioremediation, cancer treatments and more exotically as robotics design simulators. An absence of reference genomes currently limits these applications, including development of efficient tools for identification of critical factors in regulation, growth or optimization of metabolic pathways. The Euglena International Network (EIN) seeks to provide a forum to overcome these challenges. EIN has agreed specific goals, mobilized scientists, established a clear roadmap (Grand Challenges), connected academic and industry stakeholders and is currently formulating policy and partnership principles to propel these efforts in a coordinated and efficient manner.

Cell adaptation of the extremophilic red microalga Galdieria sulphuraria to the availability of carbon sources.

Global energy demand and fossil fuels impact on climate can be partially managed by an increase in the use of biofuels for transports and industries. Biodiesel production is generally preceded by a transesterification process of the green biomass triacylglycerols that generates large amounts of glycerol as a by-product. In this study, the extremophilic red microalga Galdieria sulphuraria 074W was cultivated in heterotrophy. The microalgal growth parameters and biomass composition were compared when grown on an equivalent molar concentration of carbon of either glucose or glycerol as unique carbon source. The maximal biomass reached in these two conditions was not significantly different (∼2.5 g.L-1). Fatty acid profile, protein and storage carbohydrate contents were also statistically similar, irrespectively of the metabolized carbon source. We also observed that the pigment content of G. sulphuraria cells decreased during heterotrophic growth compared to photoautotrophic cultivated cells, and that this diminution was more important in the presence of glucose than glycerol: cells were yellowish in the presence of glucose and green in the presence of glycerol. The pigmentation was restored when glucose was totally consumed in the medium, suggesting that the presence of glucose repressed pigment synthesis. Based on this observation, a transcriptome analysis was performed in order to better understand the mechanisms involved in the loss of color mediated by darkness and by glucose in G. sulphuraria. Three conditions were analyzed: heterotrophy with glycerol or glucose and phototrophy. This allowed us to understand the transcriptional response of cells to light and dark environments both at the nuclear and chloroplast levels, and to show that transcription of gene families, acquired by horizontal gene transfer, such as sugar, amino acid, or acetate transporters, were involved in the response to the availability of different (in)organic sources.

Automated Open-Hardware Multiwell Imaging Station for Microorganisms Observation.

Bright field microscopes are particularly useful tools for biologists for cell and tissue observation, phenotyping, cell counting, and so on. Direct cell observation provides a wealth of information on cells' nature and physiological condition. Microscopic analyses are, however, time-consuming and usually not easy to parallelize. We describe the fabrication of a stand-alone microscope able to automatically collect samples with 3D printed pumps, and capture images at up to 50× optical magnification with a digital camera at a good throughput (up to 24 different samples can be collected and scanned in less than 10 min). Furthermore, the proposed device can store and analyze pictures using computer vision algorithms running on a low power integrated single board computer. Our device can perform a large set of tasks, with minimal human intervention, that no single commercially available machine can perform. The proposed open-hardware device has a modular design and can be freely reproduced at a very competitive price with the use of widely documented and user-friendly components such as Arduino, Raspberry pi, and 3D printers.

Beyond being an energy supplier, ATP synthase is a sculptor of mitochondrial cristae.

Mitochondrial F1FO-ATP synthase plays a key role in cellular bioenergetics; this enzyme is present in all eukaryotic linages except in amitochondriate organisms. Despite its ancestral origin, traceable to the alpha proteobacterial endosymbiotic event, the actual structural diversity of these complexes, due to large differences in their polypeptide composition, reflects an important evolutionary divergence between eukaryotic lineages. We discuss the effect of these structural differences on the oligomerization of the complex and the shape of mitochondrial cristae.

Long-term acclimation to cadmium exposure reveals extensive phenotypic plasticity in Chlamydomonas.

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.

Trophic state alters the mechanism whereby energetic coupling between photosynthesis and respiration occurs in Euglena gracilis.

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 CO2 assimilation and optimal growth. Using the flagellate Euglena gracilis, we first tested if photosynthesis-respiration coupling occurs in this species harbouring secondary green plastids (i.e. originated from an endosymbiosis between a green alga and a phagotrophic euglenozoan). Second, we tested how the trophic state (mixotrophy and photoautotrophy) of the cell alters the mechanisms involved in the photosynthesis-respiration coupling. Energetic coupling between photosynthesis and respiration was determined by testing the effect of respiratory inhibitors on photosynthesis, and measuring the simultaneous variation of photosynthesis and respiration rates as a function of temperature (i.e. thermal response curves). The mechanism involved in the photosynthesis-respiration coupling was assessed by combining proteomics, biophysical and cytological analyses. Our work shows that there is photosynthesis-respiration coupling and membrane contacts between mitochondria and chloroplasts in E. gracilis. However, whereas in mixotrophy adjustment of the chloroplast ATP/NADPH ratio drives the interaction, in photoautotrophy the coupling is conditioned by CO2 limitation and photorespiration. This indicates that maintenance of photosynthesis-respiration coupling, through plastic metabolic responses, is key to E. gracilis functioning under changing environmental conditions.

De Novo Transcriptome Meta-Assembly of the Mixotrophic Freshwater Microalga Euglena gracilis.

Euglena gracilis is a well-known photosynthetic microeukaryote considered as the product of a secondary endosymbiosis between a green alga and a phagotrophic unicellular belonging to the same eukaryotic phylum as the parasitic trypanosomatids. As its nuclear genome has proven difficult to sequence, reliable transcriptomes are important for functional studies. In this work, we assembled a new consensus transcriptome by combining sequencing reads from five independent studies. Based on a detailed comparison with two previously released transcriptomes, our consensus transcriptome appears to be the most complete so far. Remapping the reads on it allowed us to compare the expression of the transcripts across multiple culture conditions at once and to infer a functionally annotated network of co-expressed genes. Although the emergence of meaningful gene clusters indicates that some biological signal lies in gene expression levels, our analyses confirm that gene regulation in euglenozoans is not primarily controlled at the transcriptional level. Regarding the origin of E. gracilis, we observe a heavily mixed gene ancestry, as previously reported, and rule out sequence contamination as a possible explanation for these observations. Instead, they indicate that this complex alga has evolved through a convoluted process involving much more than two partners.

Open-hardware wireless controller and 3D-printed pumps for efficient liquid manipulation.

Many routines in biological experiments require the precise handling of liquid volumes in the range of microliters up to liters. In this paper, we describe a new wireless controller that is adapted to liquid manipulation tasks, in particular when combined with the proposed 3D-printed pumps. It can be built from widely available electronic components and managed with open-source software. The use of peristaltic pumps enables to move volumes from milliliters to liters with a relative error below 1% or a syringe pump capable of injecting volumes in the range of milliliters with microliter accuracy. The system is remotely controllable over WiFi and easily automated using the MQTT communication protocol. The programming of the microcontroller is performed on the Arduino IDE. The WiFi settings and the calibration value can be easily modified, stored and exported in the form of a JSON file to create a user friendly, plug and play and easily scalable device. Additional sensors or actuators can be added, allowing the system to adapt to various usages. Finally, in addition to its low manufacturing cost and its capability to fit a large variety of tasks involving liquid handling, our system has been specifically designed for research environments where adaptability and repeatability of experiments is essential.

In vivo assessment of mitochondrial respiratory alternative oxidase activity and cyclic electron flow around photosystem I on small coral fragments.

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.

Different levels of energetic coupling between photosynthesis and respiration do not determine the occurrence of adaptive responses of Symbiodiniaceae to global warming.

Disentangling the metabolic functioning of corals' endosymbionts (Symbiodiniaceae) is relevant to understanding the response of coral reefs to warming oceans. In this work, we first question whether there is an energetic coupling between photosynthesis and respiration in Symbiodiniaceae (Symbiodinium, Durusdinium and Effrenium), and second, how different levels of energetic coupling will affect their adaptive responses to global warming. Coupling between photosynthesis and respiration was established by determining the variation of metabolic rates during thermal response curves, and how inhibition of respiration affects photosynthesis. Adaptive (irreversible) responses were studied by exposing two Symbiodinium species with different levels of photosynthesis-respiration interaction to high temperature conditions (32°C) for 1 yr. We found that some Symbiodiniaceae have a high level of energetic coupling; that is, photosynthesis and respiration have the same temperature dependency, and photosynthesis is negatively affected when respiration is inhibited. Conversely, photosynthesis and respiration are not coupled in other species. In any case, prolonged exposure to high temperature caused adjustments in both photosynthesis and respiration, but these changes were fully reversible. We conclude that energetic coupling between photosynthesis and respiration exhibits wide variation amongst Symbiodiniaceae and does not determine the occurrence of adaptive responses in Symbiodiniaceae to temperature increase.

Photosynthetic capacity of the endosymbiotic dinoflagellate Cladocopium sp. is preserved during digestion of its jellyfish host Mastigias papua by the anemone Entacmaea medusivora.

The sea anemone Entacmaea medusivora (Actiniaria, Anthozoa) commonly feeds on the golden jellyfish Mastigias papua (Rhizostomeae, Scyphozoa) which harbours an endosymbiotic dinoflagellate of the genus Cladocopium (Symbiodiniaceae). In this study, we monitored the photosynthetic activity of the endosymbiotic microalgae while their host jellyfish were ingested and digested by starved medusivorous anemones. By analyzing the photosynthetic yield of photosystem II, we observed that Cladocopium cells remain photosynthetically competent during the whole digestion process, thus confirming the exceptional resistance of Symbiodiniaceae to digestive enzymes. In the gastric cavity of E. medusivora, Cladocopium cells release oxygen, which could broadly stimulate the gastric microbiotic flora of the sea anemone. Ultimately, E. medusivora is not able to retain Cladocopium cells more than few days and physiologically-unaltered cells are therefore expelled in faecal pellets. The potential contribution of E. medusivora to maintain a reservoir of Cladocopium symbionts and its role in the life cycle of M. papua is discussed.

Oxidative phosphorylation supercomplexes and respirasome reconstitution of the colorless alga Polytomella sp.

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-ß-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.

In vivo chlorophyll fluorescence screening allows the isolation of a Chlamydomonas mutant defective for NDUFAF3, an assembly factor involved in mitochondrial complex I assembly.

The qualitative screening method used to select complex I mutants in the microalga Chlamydomonas, based on reduced growth under heterotrophic conditions, is not suitable for high-throughput screening. In order to develop a fast screening method based on measurements of chlorophyll fluorescence, we first demonstrated that complex I mutants displayed decreased photosystem II efficiency in the genetic background of a photosynthetic mutation leading to reduced formation of the electrochemical proton gradient in the chloroplast (pgrl1 mutation). In contrast, single mutants (complex I and pgrl1 mutants) could not be distinguished from the wild type by their photosystem II efficiency under the conditions tested. We next performed insertional mutagenesis on the pgrl1 mutant. Out of about 3000 hygromycin-resistant insertional transformants, 46 had decreased photosystem II efficiency and three were complex I mutants. One of the mutants was tagged and whole genome sequencing identified the resistance cassette in NDUFAF3, a homolog of the human NDUFAF3 gene, encoding for an assembly factor involved in complex I assembly. Complemented strains showed restored complex I activity and assembly. Overall, we describe here a screening method which is fast and particularly suited for the identification of Chlamydomonas complex I mutants.

Near-neighbor interactions of the membrane-embedded subunits of the mitochondrial ATP synthase of a chlorophycean alga.

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, ß, γ, δ, ε, 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.

Atypical composition and structure of the mitochondrial dimeric ATP synthase from Euglena gracilis.

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, ß, 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.