Environmentally-informed functional characterisation of the secondary red chloroplast proteome

Project Information

ChloroMosaic

Grant agreement ID: 101039760

DOI

10.3030/101039760

EC signature date 4 July 2022

Start date 1 January 2023

End date 31 August 2028

Funded under

• European Research Council (ERC)

Total cost

€ 1 498 352,00

EU contribution

€ 1 485 852,00

Coordinated by

CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE CNRS
France

Periodic Reporting for period 1 - ChloroMosaic (Environmentally-informed functional characterisation of the secondary red chloroplast proteome)

Reporting period: 2023-01-01 to 2025-06-30

Summary of the context and overall objectives of the project

Half of the oxygen that we breathe is produced by aquatic algae. These organisms are evolutionarily diverse, ranging from tiny single-celled cyanobacteria to giant multicellular seaweeds, and are spread across all marine and freshwater habitats, ranging from the polar ice-caps threatened by melting to tropical oceans impacted by increasingly recurrent heatwaves as a result of anthropogenic climate change. The most abundant algae within these envieonments come from groups diatoms, haptophytes, dinoflagellates) that possess chloroplasts derived from the secondary or higher endosymbiotic uptake of a red alga, itself possessing a nucleus and chloroplasts. These algae and their chloroplasts are very distantly related to plants, and are supported by novel proteins that come from a « mosaic » of different sources but may be essential to their functions in the wild.

ChloroMosaic seeks to understand the dark matter of the secondary red chloroplast proteome, its evolutionary origins, its cellular functions, and its environmental relevance to a heating global ocean. First, we are profiling the chloroplast proteome of dinoflagellate algae, essential symbionts of coral reefs, with distinctive cell biology very different from that of other algal groups. Next, we are using combined evolutionary and cell biology techniques to characterise individual chloroplast proteins found across secondary red algal plastids but not associated with plants, focussing in particular on diatoms, the single most abundant of these algal groups. Finally, we are considering at a more conceptual level how mosaic algal genomes form and function, focussing in particular on the distantly related algae native to the Arctic, and to one algal group, haptophytes, who are spread across a heating global ocean.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

To explore the chloroplast proteome of dinoflagellates, we are using the model species Amphidinium, whose chloroplast genome is directly transformable. We aim to generate synthetic Amphidinium lines in which we can either tag and sequence unknown chloroplast proteins, or suppress their expression and measure defects in dinoflagellate physiology. Alongside this, we are assembling a compendium of dinoflagellate chloroplast proteins from genome data, focussing in particular on the group Kareniaceae, which are a model for understanding how secondary red chloroplasts evolve. An important early result from ChloroMosaic is that Kareniaceae have repeatedly acquired their chloroplasts from one single algal group, the haptophytes, with different proteins evolving to support this symbiosis in each case.

To understand novel chloroplast proteins in diatoms, we are using combined environmental data from the Tara Oceans expedition and the model transformable species Phaeodactylum, testing carbon metabolism, transporter proteins, and a novel protein related to ATP synthase. We are particularly interested to make convergent predictions on protein function using environmental and experimental techniques. In one early project we show, for example, that diatom chloroplast lower-half glycolysis is most highly expressed in subpolar oceans, characterised by long days and low temperatures, and Phaeodactylum mutant lines for these proteins show perturbed physiology in response both to illumination and cold stress. We are now moving beyond working strictly on diatoms, testing some of our proteins in the model green alga Chlamydomonas and in heterotrophic eukaryotes.

Finally, to understand how mosaic evolution connects to the environment, we are sequencing nearly 50 new algal genomes, with a particular concentration on the Arctic Ocean. Early in ChloroMosaic, we showed that a non-chloroplast protein family, ice-binding proteins, shows as a geographically structured mosaic history, with Arctic species possessing distinct isoforms to closely related species from the Antarctic Southern Ocean. We are now exploring how these proteins, and algal chloroplast metabolism in general, respond to heating and salinity stress, as will become increasingly prevalent in a global ocean impacted by anthropogenic climate change.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

ChloroMosaic seeks to give new insights into the dark matter of algal chloroplasts, provide fundamental mechanistics for the evolutionary complexification of algal life, and help calibrate predictive models of algal resilience and fragility to anthropogenic climate change. Resolving the chloroplast proteome of dinoflagellates, which remain due to their unusual cell structure and internal biology by far the least understood of the major eukaryotic algal groups, will provide important insights into their functions in the world ocean, including as essential photosynthetic symbionts of coral reefs, and as toxic agents of harmful red tides. The elaboration of Kareniaceae, as a model for the endosymbiotic acquisition of chloroplasts, gives us a new window into what cellular and ecological factors underpin the repeated evolution of photosynthetic capacity across the algal tree of life.

Characterising novel diatom chloroplast proteins may provide us with explanations for their striking success in the world ocean, and identify new gene targets for synthetic engineering of increased primary production and metabolic activity in transformable model algal species. In particular, the complementary use of experimental and environmental data allows us to develop new strategies for inferring protein function, insofar as we can now use meta-genomics to « predict » protein phenotypes that can be validated at the bench. We are now using these techniques to identify protein functions that have never previously been associated with chloroplasts, e.g the probable transport of novel metal substrates, and use of a novel strategy for ATP management in algal chloroplasts.

Finally, by sequencing and understanding the mosaic fomration of Arctic algal genomes will provide us with unprecedented insight into this biologically unique and fragile ocean biome, alongside providing us with clues about how algal chloroplast metabolism underpins species responses to ocean heating. We are now using these data with globally distributed haptophyte algae to develop whole-cell models of how haptophyte chloropalst activity changes with temperature, which aim to use these to refine our predictions of algal fitness and chloroplast function in the future ocean.

Diversity of eukaryotic life, with algal groups shown in coloured boxes

Schematic diagram of a eukaryotic cell with a secondary red chloroplast and mosaic proteome