Final Report Summary - EPISYSBIOL (Epigenetic regulation of the adaptative response of C.reinhardtii to development and environmental stresses: Characterization of .Histone PTMS, gene regulation and p
In our project, we investigated the biology of the responsive mechanisms to cold and nitrogen deprivation stresses in Chlamydomonas reinhardtii, and showed the importance of integrative and non-directed -omics experiments for discovering new biological phenomena in this model algae.
Our work builds on the experience gathered in our laboratory after many year of working with high resolution mass spectrometers in the field of proteomics and metabolomics, and in the research experience in molecular biology of the fellow. We depicted the molecular changes induced by target abiotic stresses at different levels (proteomic, metabolomics, and transcriptomic). This approach gave us the opportunity of gather a comprehensive understanding of what is really happening during the stress adaption and recovery processes, in contrast to most of the bibliographic references that focus only on particular mechanisms.
Using this approach, we have defined how central metabolism dynamics are linked to a cold-stress-dependent sugar sensing and autophagy mechanisms controlled by novel genes in such as CKIN1, CKIN2 and hitherto unknown protein kinase named CKIN3. Cold stress affects extensively the physiology and the organisation of the cell, as it is summarised in the left image. Gluconeogenesis and starch biosynthesis pathways are activated leading to a pronounced remodelling of metabolism and starch and sugar accumulation. At the same time a decrease in photosynthesis is observed. Quantitative lipid profiles also indicate a sharp decrease in the lipophilic fraction and an increase in polyunsaturated fatty acids suggesting this as a mechanism of maintaining membrane fluidity. The proteome is also remodelled: changes at the ribosome and the spliceosome may mediate the biosynthesis of protein isoforms important for adaptation to low temperatures and specific proteasome degradation may be mediated by the observed cold-specific changes in the ubiquinilation system. Twenty percent of the proteins responsive to cold are uncharacterised proteins, this representing a considerable resource for new discoveries in cold stress biology in alga and plants.
The experience gained during the cold stress allowed us to improve the methodologies for the nitrogen depletion and recovery experiment. These improvements were translated into a more accurate datasets reducing the noise and artefacts, increasing the quality of the analysis. Nitrogen starvation led to profound physiological changes, as it can be seen in the PCA plot below, that affect multiple biochemical pathways. A clear consequence of this metabolic remodelling is the accumulation of lipid bodies (yellow dots in the image below) just after 24 h, with the maximum accumulation after 72 h of hat can be used as a basis for biodiesel production.
Despite the biotechnological importance of this process, the signal transduction mechanisms that lead to the activation of the lipids biosynthesis and its accumulation are still poorly understood. Our combined approach allowed us to determine some novel transcription factors and non-yet characterised proteins that have specific dynamics during nitrogen starvation and recovery. Some of these proteins have been characterised using homology searches and validated studying its role in the different biochemical networks. For example CKIN3, a protein discovered in the cold stress assay but confirmed here, is responsive to this stress. Potential targets are a broad family of transcription factors, responsible of the regulation of the central metabolism. These results opened the door to the engineering of cell lines that over express or silence this target proteins with the aim of developing a hyper productive strain that will increase the efficiency of biodiesel production in green algae.
The development of new analytical methodologies, and the characterisation and deep and comprehensive explanation of the responsive processes to cold and nitrogen deprivation stresses, both with important biotechnological implications because of the accumulation of sugars (bioethanol) and lipids (biodiesel), respectively. Furthermore, we have gathered sufficient experimental evidences for some proteins as key-regulators of these processes including biomass production, lipid production, photosynthetic activity and starch production. In the figure below the complex network interaction of these components with the proteins is shown.
These proteins are priority candidates for the biotechnological improvement of the biofuel production and CO2 trapping by microalgae cultures. These are tangible contributions that will not only strength the European position in the fields of microalgae and systems biology research, but also will give new tools for moving further to develop better CO2 trapping strategies and also sustainable production of biofuels.
Our work builds on the experience gathered in our laboratory after many year of working with high resolution mass spectrometers in the field of proteomics and metabolomics, and in the research experience in molecular biology of the fellow. We depicted the molecular changes induced by target abiotic stresses at different levels (proteomic, metabolomics, and transcriptomic). This approach gave us the opportunity of gather a comprehensive understanding of what is really happening during the stress adaption and recovery processes, in contrast to most of the bibliographic references that focus only on particular mechanisms.
Using this approach, we have defined how central metabolism dynamics are linked to a cold-stress-dependent sugar sensing and autophagy mechanisms controlled by novel genes in such as CKIN1, CKIN2 and hitherto unknown protein kinase named CKIN3. Cold stress affects extensively the physiology and the organisation of the cell, as it is summarised in the left image. Gluconeogenesis and starch biosynthesis pathways are activated leading to a pronounced remodelling of metabolism and starch and sugar accumulation. At the same time a decrease in photosynthesis is observed. Quantitative lipid profiles also indicate a sharp decrease in the lipophilic fraction and an increase in polyunsaturated fatty acids suggesting this as a mechanism of maintaining membrane fluidity. The proteome is also remodelled: changes at the ribosome and the spliceosome may mediate the biosynthesis of protein isoforms important for adaptation to low temperatures and specific proteasome degradation may be mediated by the observed cold-specific changes in the ubiquinilation system. Twenty percent of the proteins responsive to cold are uncharacterised proteins, this representing a considerable resource for new discoveries in cold stress biology in alga and plants.
The experience gained during the cold stress allowed us to improve the methodologies for the nitrogen depletion and recovery experiment. These improvements were translated into a more accurate datasets reducing the noise and artefacts, increasing the quality of the analysis. Nitrogen starvation led to profound physiological changes, as it can be seen in the PCA plot below, that affect multiple biochemical pathways. A clear consequence of this metabolic remodelling is the accumulation of lipid bodies (yellow dots in the image below) just after 24 h, with the maximum accumulation after 72 h of hat can be used as a basis for biodiesel production.
Despite the biotechnological importance of this process, the signal transduction mechanisms that lead to the activation of the lipids biosynthesis and its accumulation are still poorly understood. Our combined approach allowed us to determine some novel transcription factors and non-yet characterised proteins that have specific dynamics during nitrogen starvation and recovery. Some of these proteins have been characterised using homology searches and validated studying its role in the different biochemical networks. For example CKIN3, a protein discovered in the cold stress assay but confirmed here, is responsive to this stress. Potential targets are a broad family of transcription factors, responsible of the regulation of the central metabolism. These results opened the door to the engineering of cell lines that over express or silence this target proteins with the aim of developing a hyper productive strain that will increase the efficiency of biodiesel production in green algae.
The development of new analytical methodologies, and the characterisation and deep and comprehensive explanation of the responsive processes to cold and nitrogen deprivation stresses, both with important biotechnological implications because of the accumulation of sugars (bioethanol) and lipids (biodiesel), respectively. Furthermore, we have gathered sufficient experimental evidences for some proteins as key-regulators of these processes including biomass production, lipid production, photosynthetic activity and starch production. In the figure below the complex network interaction of these components with the proteins is shown.
These proteins are priority candidates for the biotechnological improvement of the biofuel production and CO2 trapping by microalgae cultures. These are tangible contributions that will not only strength the European position in the fields of microalgae and systems biology research, but also will give new tools for moving further to develop better CO2 trapping strategies and also sustainable production of biofuels.