Periodic Reporting for period 1 - INSPirAUTOR (Understanding how Inositol Polyphosphates regulate autophagy and lipid body formation in photosynthetic organisms: crosstalk with TOR signaling.)
Berichtszeitraum: 2018-03-01 bis 2020-02-29
Increasing concentrations of CO2 are accumulating in the atmosphere in the latest century causing the global warming effect in the planet. Mitigation of CO2 is one of the most important problems that governments are facing in the last decade. In fact, European Union is proposing a 40% reduction of emissions by the end of 2030 in the latest Climate and Energy framework (https://ec.europa.eu/clima/policies/strategies). In this sense, biological CO2 capturing, photosynthesis and its molecular regulation is an ancient process that needs to be revisited in order to help in the reduction of this greenhouse gas. In this sense, microalgae are considered the most efficient organisms to perform this process due to their high growth rate, robust photosynthetic activity and carbon storage capacity. In green algae, fixed CO2 is normally redirected to two different fates, cell growth (proteins) and carbon storage, mainly lipids and carbohydrates in the form of starch. In this sense, the use of these microorganisms for the production of biofuels is a good alternative to land crops because it lacks the main ethical implications on food or feed market and land use. Thus, the understanding of the intracellular regulation of CO2 capturing and partitioning in green cells is fundamental in order to optimize them. In this sense, we have previously found an intersection between two signaling pathways that govern these aspects in these microorganisms. The well-conserved Target of Rapamycin (TOR) and the signaling molecules Inositol polyphosphates (InsPs) coordinate to integrate external signals within photosynthetic cells and target carbon metabolism and storage. However the molecular mechanisms that they use to communicate with each other and their common targets are still unclear. During this action, we used the unicellular green alga Chlamydomonas reinhardtii to elucidate the molecular interaction between these two pathways. First, we evaluated InsPs fluctuations after TOR inhibition and the impact of autophagy activation and lipid body formation. Second, we identified novel targets of this interaction by using proteomics approaches. Finally, we monitored InsPs levels and TOR activity under different nutrient stress conditions. Overall, our data add a new level of complexity in the understanding of carbon assimilation in green organisms that goes beyond PTMs and most likely includes protein-molecule interactions that has not been reported so far.
We proposed 3 different Aims for this project:
1. Determine the relationship between TOR kinase, InsPs, autophagy and lipid storage using TOR inhibitors. We worked on the dissection of the TOR/InsPs regulatory network using analytical approaches (mainly InsPs and lipids determination). Additionally, we analyzed the connection between TOR signaling and P metabolism in Chlamydomonas Part of these results was included in a publication of the host lab in The Plant Cell (Couso et al. 2020).
2. Proteomic analysis of InsPs-deficient mutant to identify C metabolism/partitioning networks. In this study, we found several well-known TOR targets to be differentially regulated in the mutant. This indicates a possible interaction between these two signaling pathways that share common targets. Overall, these data are very relevant for the field of photosynthetic organisms because we found a new level of regulation that has never been reported.
3. Determine InsPs and TOR interaction under nutritional stress. N and P starvation were used to monitored autophagy activation in the green alga Chlamydomonas reinhardtii and these results were used to validate the TOR activity read out in this alga and to investigate a new mutant lst8-1 that is defective in one of the core proteins of the TOR complex 1. These results helped us to elucidate the mechanism by which TOR is involved in the regulation of P limitation and are part of one publication of the host lab in The Plant Cell (Couso et al., 2020).
In order to communicate the results obtained during this action, we have participated in three different international conferences, one as a poster presentation (EMBO conference on” TOR signaling in photosynthetic organisms” in France), one invited talk in “The 18th International Conference on the Cell and Molecular Biology of Chlamydomonas” in USA) and an oral communication (“Autophagy Virtual Days” virtual conference organized in Austria). After completing this project, we have submitted a paper that includes the results obtained after analyzing proteomics and TOR activity in a InsPs deficient mutant called “Inositol pyrophosphates and TOR signaling coordinate a new level of complexity in the regulation of the phosphoproteome in Chlamydomonas reinhardtii” that is currently under evaluation. We also participated in an article in the journal “The Plant Cell” (https://doi.org/10.1105/tpc.19.00179) that got a special mention of a science editor in the same journal (https://doi.org/10.1105/tpc.19.00888). Additionally, we have participated in different outreach activities in three different events: oral communications at the European Researchers´ night in 2018 and 2020 and one at the International Day of Women in Science in 2020.
1. Determine the relationship between TOR kinase, InsPs, autophagy and lipid storage using TOR inhibitors. We worked on the dissection of the TOR/InsPs regulatory network using analytical approaches (mainly InsPs and lipids determination). Additionally, we analyzed the connection between TOR signaling and P metabolism in Chlamydomonas Part of these results was included in a publication of the host lab in The Plant Cell (Couso et al. 2020).
2. Proteomic analysis of InsPs-deficient mutant to identify C metabolism/partitioning networks. In this study, we found several well-known TOR targets to be differentially regulated in the mutant. This indicates a possible interaction between these two signaling pathways that share common targets. Overall, these data are very relevant for the field of photosynthetic organisms because we found a new level of regulation that has never been reported.
3. Determine InsPs and TOR interaction under nutritional stress. N and P starvation were used to monitored autophagy activation in the green alga Chlamydomonas reinhardtii and these results were used to validate the TOR activity read out in this alga and to investigate a new mutant lst8-1 that is defective in one of the core proteins of the TOR complex 1. These results helped us to elucidate the mechanism by which TOR is involved in the regulation of P limitation and are part of one publication of the host lab in The Plant Cell (Couso et al., 2020).
In order to communicate the results obtained during this action, we have participated in three different international conferences, one as a poster presentation (EMBO conference on” TOR signaling in photosynthetic organisms” in France), one invited talk in “The 18th International Conference on the Cell and Molecular Biology of Chlamydomonas” in USA) and an oral communication (“Autophagy Virtual Days” virtual conference organized in Austria). After completing this project, we have submitted a paper that includes the results obtained after analyzing proteomics and TOR activity in a InsPs deficient mutant called “Inositol pyrophosphates and TOR signaling coordinate a new level of complexity in the regulation of the phosphoproteome in Chlamydomonas reinhardtii” that is currently under evaluation. We also participated in an article in the journal “The Plant Cell” (https://doi.org/10.1105/tpc.19.00179) that got a special mention of a science editor in the same journal (https://doi.org/10.1105/tpc.19.00888). Additionally, we have participated in different outreach activities in three different events: oral communications at the European Researchers´ night in 2018 and 2020 and one at the International Day of Women in Science in 2020.
During this action, we investigated the possible implications of InsPs in carbon assimilation in green organisms and furthermore how these signaling molecules are coordinated with the well-conserved master regulator of growth TOR. We found that these two signaling pathways are controlling carbon assimilation by sharing common targets. Beyond these, we also validate several targets that uncovered a complex regulation of the carbon uptake and metabolism that has not been studied in any green organism so far. This information will be very valuable in the use of algae in CO2 mitigation, which is of high-environmental importance especially for the EU climate-neutral strategy and opens the possibility to genetically modify this pathway in order to improve carbon capturing in these microorganisms.