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 homologs 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 to both 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.Liège, february 06 th 2014Dear Editor, Please find the fully revised version of our manuscript "The mitochondrial respiratory chain of the secondary green alga Euglena gracilis shares many additional subunits with parasitic Trypanosomatidae."We are grateful to you for having encouraged us to resubmit a modified manuscript. We have addressed all the comments raised by the reviewers on our first submission. A point to point answer has been submitted along with the manuscript. Main changes are : (i) additional explanations about the phylogenic/bioinformatic strategy including some phylogenetic trees (supplemental figures 1 and 2); (ii) all data that were previously not shown (mass spectrometry data, in vivo respiratory assays) are now included in the manuscript as supplemental figures/tables.We hope that we have now significantly improved our manuscript so you'll find it suitable for publication in the special issue of Mitochondrion dedicated to "Plant Mitochondria Biology". R: Primary MS data are now given in Supplemental Table 3 for each polypeptide for which the score was > 60. (BLAST alignments, Evalues, etc.) are actually presented in the paper to support the inference that a particular E. gracilis protein is a true homolog of a given kinetoplastid protein. The most important conclusion of t...

Acetate can be efficiently metabolized by the green microalga Chlamydomonas reinhardtii. The regular concentration is 17 mM, although higher concentrations are reported to increase starch and fatty acid content. To understand the responses to higher acetate concentrations, Chlamydomonas cells were cultivated in batch mode in the light at 17, 31, 44, and 57 mM acetate. Metabolic analyses show that cells grown at 57 mM acetate possess increased contents of all components analyzed (starch, chlorophylls, fatty acids, and proteins), with a three-fold increased volumetric biomass yield compared to cells cultivated at 17 mM acetate at the entry of stationary phase. Physiological analyses highlight the importance of photosynthesis for the low-acetate and exponential-phase samples. The stationary phase is reached when acetate is depleted, except for the cells grown at 57 mM acetate, which still divide until ammonium exhaustion. Surprisal analysis of the transcriptomics data supports the biological significance of our experiments. This allows the establishment of a model for acetate assimilation, its transcriptional regulation and the identification of candidates for genetic engineering of this metabolic pathway. Altogether, our analyses suggest that growing at high-acetate concentrations could increase biomass productivities in low-light and CO2-limiting air-bubbled medium for biotechnology.

Mitochondrial F1FO ATP synthase (Complex V) catalyses ATP synthesis from ADP and inorganic phosphate using the proton-motive force generated by the substrate-driven electron transfer chain. In this work, we investigated the impact of the loss of activity of the mitochondrial enzyme in a photosynthetic organism. In this purpose, we inactivated by RNA interference the expression of the ATP2 gene, coding for the catalytic subunit beta, in the green alga Chlamydomonas reinhardtii. We demonstrate that in the absence of beta subunit, complex V is not assembled, respiratory rate is decreased by half and ATP synthesis coupled to the respiratory activity is fully impaired. Lack of ATP synthase also affects the morphology of mitochondria which are deprived of cristae. We also show that mutants are obligate phototrophs and that rearrangements of the photosynthetic apparatus occur in the chloroplast as a response to ATP synthase deficiency in mitochondria. Altogether, our results contribute to the understanding of the yet poorly studied bioenergetic interactions between organelles in photosynthetic organisms.

The usual cultivation mode of the green microalga Chlamydomonas is liquid medium and light. However, the microalga can also be grown on agar plates and in darkness. Our aim is to analyze and compare gene expression of cells cultivated in these different conditions. For that purpose, RNA-seq data are obtained from Chlamydomonas samples of two different labs grown in four environmental conditions (agar@light, agar@dark, liquid@light, liquid@dark). The RNA seq data are analyzed by surprisal analysis, which allows the simultaneous meta-analysis of all the samples. First we identify a balance state, which defines a state where the expression levels are similar in all the samples irrespectively of their growth conditions, or lab origin. In addition our analysis identifies additional constraints needed to quantify the deviation with respect to the balance state. The first constraint differentiates the agar samples versus the liquid ones; the second constraint the dark samples versus the light ones. The two constraints are almost of equal importance. Pathways involved in stress responses are found in the agar phenotype while the liquid phenotype comprises ATP and NADH production pathways. Remodeling of membrane is suggested in the dark phenotype while photosynthetic pathways characterize the light phenotype. The same trends are also present when performing purely statistical analysis such as K-means clustering and differentially expressed genes.

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.

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 2 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 CO 2 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.

In this work, we first show that mitochondrial F1FO ATP synthase from chlorophycean green algae has an atypical subunit composition of its peripheral stator and dimerization module, with 9 subunits of unknown evolutionary origin (Asa subunits). In contrast, other classes of green algae possess a canonical subunit composition. In addition, respiration, ATP levels and growth of Chlorophyceae are also barely affected by oligomycin concentrations that affect representatives of the other classes of Chlorophytes.

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