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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - BEAL (Bioenergetics in microalgae : regulation modes of mitochondrial respiration, photosynthesis, and fermentative pathways, and their interactions in secondary algae)

Teaser

Summary

During the course of eukaryote evolution, photosynthesis was propagated from primary eukaryotic algae to non-photosynthetic organisms through multiple secondary endosymbiotic events. Collectively referred to as â€œsecondary algaeâ€, these photosynthetic organisms account for only 1-2% of the total global biomass, but produce a far larger part of the global annual fixation of carbon on Earth.
ATP is the universal chemical energy carrier in living cells. In photosynthetic eukaryotes, it is produced by two major cellular processes: photosynthesis and respiration taking place in chloroplasts and mitochondria, respectively. Both processes support the production of biomass and govern gas (O2 and CO2) exchanges at earth scale. On the other hand, anaerobic fermentative enzymes have also been identified in several primary and secondary algae. The regulation modes and interactions of respiration, photosynthesis and fermentation are fairly well understood in primary green algae. Conversely, the complex evolutionary history of secondary algae implies a great variety of original regulatory mechanisms that have been barely investigated to date.
The overall objectives of the project are to (i) characterize and compare the photosynthetic regulation modes by biophysical approaches, (ii) use genetic and biochemical approaches to gain fundamental knowledge on aerobic respiration and anaerobic fermentative pathways, and (iii) investigate and compare interconnections between respiration, photosynthesis, and fermentation in organisms resulting from distinct evolutionary scenarios. On a long term, these developments will be instrumental to unravel bioenergetics constraints on growth in microalgae, a required knowledge to exploit the microalgal diversity in a biotechnological perspective.

Work performed

Mitochondria is the essential organelle for the production of cellular ATP in most eukaryotic cells, whether photosynthetic or not. It is widely studied, including in non-photosynthetic parasites such as trypanosomes, as a potential therapeutic target. We determined that many subunits of the respiratory complexes of Trypanosomes are shared with a non-paratitic photosynthetic species, Euglena gracilis. In this respect Euglena ancestor acquired its chloroplast by a secondary endosymbiotic event from a green primary eukaryotic alga. Structural analyzes have further highlighted unusual protein extra-structure associated with various respiratory complexes.
Euglena gracilis is an historical microalga, being one the first protist described by microbiologists. Its photosynthesis has been broadly studied during the last century. One striking example is that Euglena was the organism studied by Melvin Calvin when he discovered the biochemical cycle which allow fixation of carbon dioxide into organic matter. In the frame of this project, we determined that most of the regulatory mechanisms of photosynthetic electron flow described so far in plants model organisms (Arabidopsis, Chlamydomonas) were not retained in Euglena. We then showed that original mechanisms (e.g., in terms of antennae composition or in terms of regulation of ATP/NADPH ratio) have been developed.

Diatoms (Bacillariophyceae, Stramenopiles, secondary red-algae) and Dinoflagellattes are two of the most ecologically successful classes of photosynthetic marine eukaryotes in contemporary oceans.
Photo-protection mechanisms have been studied in a closely related species of diatoms, Phaeomonas sp (pinguiophyceae, Stramenopiles) We have revealed that the accumulation of photo-protective pigments, usually observed only at high light intensity in green algae, also occurs during periods of darkness in Phaeomonas sp .. This strategy allows to cope with rapid and significant fluctuations in light intensity.
We then studied various strains of dinoflagellates originating not only from reef-building corals but also from soft octocorals, sea anemones, or even from giant clam from distant geographic locations (e.g. Hawai, Panama, Red sea, Japan). We determined the existence of three phenotyping groups with respect to regulatory mechanisms of photosynthetic electron transfer. This may have important ecological consequences due to the critical role of these processes on cellular energetic balance and on the photoprotection capacity of cells.

Many microalgae can be exposed to hypoxia or even anoxia in their environment. The green primary alga Chlamydomonas reinhardtii is known for its unusual richness of fermentation pathways and the vast remodeling of its metabolism in anoxic conditions. The prolonged anoxia leads in particular to the expression of oxygen-sensitive hydrogenases which catalyze the synthesis of molecular hydrogen from protons and electrons coming from the photosynthetic electron transfer chain. One of the cofactors of photosynthetic electron transfer chain is a phylloquinone derivative (vitamin K). We isolated mutants from the vitamin K biosynthetic pathway in Chlamydomonas and showed that in the absence of vitamin K, electron transfer was specifically blocked in anoxia, even in the presence of an active hydrogenase.

Final results

In general, it will be necessary to continue the study of the complex energy metabolism of the primary and secondary algae responsible for a large part of terrestrial carbon dioxide fixation. To do this, a multidisciplinary approach (genetic transformation of algae, biochemistry of organelles, study of photosynthesis by fast absorption spectroscopy, phylogeny of chimeric metabolisms) will be implemented.

The first part of the rest of the project will therefore be dedicated to the detailed study of the regulation of photosynthesis in other secondary algae, and in particular the response of photosynthesis to changing light. Among the species for which genomic data are available, several species already grown in our laboratory will be selected from the different lines (Excavates, Alveolates, Haptophyta, etc.). Photosynthetic electron transfers photoprotection mechanisms will be determined by methods already implemented in the laboratory. In particular, their amplitude in response to light intensity will be studied with a chlorophyll fluorescence imaging system and correlated with putative changes in the composition of the pigments. These functional results will then be compared with the presence / absence of modes of regulation of photosynthesis predicted by the exploitation of genomic databases and the phylogenetic study of the corresponding proteins.

The second component will aim to deepen our knowledge of the role of chloroplast-mitochondria interactions in the optimization of photosynthesis. The objective will be, in certain chimeric species chosen among those studied in the first part, to determine to what extent the mitochondrial respiration and the interactions between the organelles are similar between the primary and secondary algae or between the secondary algae.

Finally, in a third part, we will determine the extent and diversity of anaerobic fermentation pathways in microalgae. These anaerobic fermentation routes make it possible to tolerate hypoxic or anoxic environments for a limited period of time. Paradoxically, different microalgae carrying out oxygenic photosynthesis (that is to say, releasing oxygen) have a set of genes coding for various anaerobic fermentative pathways, and in particular two secondary algae: Thalassiosira pseudonana, a marine diatom which inhabits the coastal, pelagic and benthic environments, and Euglena gracilis, a freshwater excavator that lives in rich or polluted waters. In order to discover the diversity of end products and fermentation pathways in anoxia, we will compare proteomic and metabolite compositions between oxic and anoxic conditions. In primary green alga, the involvement of anaerobic fermentative enzymes in the reactivation of photosynthesis provides direct evidence of the interconnection between anaerobic fermentation and photosynthesis. Through biophysical approaches, we will determine the impact of anoxia on the reactivation of photosynthesis in E. gracilis and T. pseudonana. At the same time, we will identify deficient strains in the fermentation process and / or in the reactivation of photosynthesis in anoxia. The acquisition, compilation and comparison of all biochemical, molecular and bioinformatic data should provide a better understanding of the diversity and functional role of fermentation pathways in microalgae.

In the long term, these developments will contribute to understanding the complexity of cellular energy systems and to achieving the knowledge required to exploit biodiversity from a biotechnological perspective.