Periodic Reporting for period 1 - CHROMATOPHORES (From endosymbiont to organelle: Mechanisms of cellular integration between Paulinella and its chromatophores)
Période du rapport: 2023-05-01 au 2025-04-30
Ancient partnerships between organisms have shaped the evolution of life. Endosymbiosis is the most intimate form of species interaction as one partner physically lives inside the cells of the other partner. Endosymbiosis can, on occasion, lead to extreme integration and the loss of independence; which is what occurred during the formation of the energy-converting organelles, the chloroplast and mitochondria. The evolution of these intimate partnerships is difficult to study because many of our present-day examples are no longer in the key transition phases. A eukaryotic microbe, called Paulinella, provides an important ‘missing-link’ in this evolutionary process. Paulinella has ancient cyanobacterial endosymbionts (called chromatophores) that act like the chloroplasts of plants and algae. Studying Paulinella, therefore, provides a unique window into how an endosymbiotic partnership can evolve to become an organelle. Previous work has described the genetics of Paulinella and its cyanobacterial-derived chromatophores, however, little is known about the integration of the chromatophores in terms of their cellular physiology.
This project, CHROMATOPHORES, aimed to address this knowledge-gap by exploring this unique association at the metabolomic, transcriptomic and proteomic levels. The first aim was to characterise which metabolites were exchanged between the cell and chromatophores (objective 1). The second to study how the Paulinella cell and the chromatophores coordinate their response to light, which for a photosynthetic organism is the most important environmental factor (objective 2). The third, and final, aim was to perform a long-term evolution experiment to test whether the light response of Paulinella could still adapt to a new light environment (objective 3). The results provide insight into the molecular mechanisms that facilitate the integration of the chromatophores within Paulinella. More broadly, these results improve our understanding of how photosynthetic endosymbionts are integrated, and how the evolutionary trajectory from endosymbiont to organelle can occur.
This project, CHROMATOPHORES, aimed to address this knowledge-gap by exploring this unique association at the metabolomic, transcriptomic and proteomic levels. The first aim was to characterise which metabolites were exchanged between the cell and chromatophores (objective 1). The second to study how the Paulinella cell and the chromatophores coordinate their response to light, which for a photosynthetic organism is the most important environmental factor (objective 2). The third, and final, aim was to perform a long-term evolution experiment to test whether the light response of Paulinella could still adapt to a new light environment (objective 3). The results provide insight into the molecular mechanisms that facilitate the integration of the chromatophores within Paulinella. More broadly, these results improve our understanding of how photosynthetic endosymbionts are integrated, and how the evolutionary trajectory from endosymbiont to organelle can occur.
To achieve the aim of this project the following activities were conducted:
Objective 1: To identify the metabolites exchanged between the cell and chromatophores
Activities: In collaboration with the metabolomics facility, I optimised a metabolomics extraction protocol for Paulinella. This is the first time this methodology has been established in this organism. The process involved both optimising a harvesting method, which needed to pause the metabolism in a natural state as quickly as possible, and a metabolite extraction step, which needed to make the metabolites available for the mass spectrometry identification. The optimisation process took longer than originally envisioned. Once established the investigation into Paulinella could be conducted.
Outcome: An important outcome of objective 1 was to establish the metabolite extractions protocol, which can now be utilised for future investigations. I then utilised this protocol and performed the first untargeted metabolomic experiment in Paulinella. The results reveal the highly integrated nature of chromatophore and Paulinella metabolic pathways.
Objective 2: To characterise the mechanisms of light regulation at the transcriptomic, metabolic, and proteomic levels
Activities: I conducted a dual-transcriptomic and proteomic experiment with multiple time points over the day-night cycle. This study is the first time that a transcriptomic study was conducted that covered both the host transcripts and the chromatophore transcripts, and so could study the coordination between the two. (The metabolic aspect had to be omitted owing to the extra time needed for the protocol optimisation). In addition to this main experiment, follow-up experiments were conducted to test the hypotheses that arose.
Outcome: The main experiment demonstrated the highly level of integration between both Paulinella ‘host’ and chromatophore on both the transcriptional and protein level. In particular, the results revealed the role of the circadian clock in coordinating the response.
Objective 3: To test whether the light regulation can evolve in response to new light environments using experimental evolution
Activities: I performed a year-long evolution experiment to test whether Paulinella could adapt its light regulation and increase its tolerance to a moderately higher light intensity. I conducted a range of physiological assays at the start and end of the experiment to document any changes, and took care to separate between acclimation and adaptation responses.
Outcome: The experiment was successful conducted, and Paulinella’s growth did improve in a moderately higher light. The changes in growth rate appear to be driven by both adaptation and acclimation responses. Interestingly, there were both similarities and differences between the responses of the continuous high light and fluctuating light condition, and the adaptations to the moderately high condition did not aid in tolerating even higher light conditions.
Objective 1: To identify the metabolites exchanged between the cell and chromatophores
Activities: In collaboration with the metabolomics facility, I optimised a metabolomics extraction protocol for Paulinella. This is the first time this methodology has been established in this organism. The process involved both optimising a harvesting method, which needed to pause the metabolism in a natural state as quickly as possible, and a metabolite extraction step, which needed to make the metabolites available for the mass spectrometry identification. The optimisation process took longer than originally envisioned. Once established the investigation into Paulinella could be conducted.
Outcome: An important outcome of objective 1 was to establish the metabolite extractions protocol, which can now be utilised for future investigations. I then utilised this protocol and performed the first untargeted metabolomic experiment in Paulinella. The results reveal the highly integrated nature of chromatophore and Paulinella metabolic pathways.
Objective 2: To characterise the mechanisms of light regulation at the transcriptomic, metabolic, and proteomic levels
Activities: I conducted a dual-transcriptomic and proteomic experiment with multiple time points over the day-night cycle. This study is the first time that a transcriptomic study was conducted that covered both the host transcripts and the chromatophore transcripts, and so could study the coordination between the two. (The metabolic aspect had to be omitted owing to the extra time needed for the protocol optimisation). In addition to this main experiment, follow-up experiments were conducted to test the hypotheses that arose.
Outcome: The main experiment demonstrated the highly level of integration between both Paulinella ‘host’ and chromatophore on both the transcriptional and protein level. In particular, the results revealed the role of the circadian clock in coordinating the response.
Objective 3: To test whether the light regulation can evolve in response to new light environments using experimental evolution
Activities: I performed a year-long evolution experiment to test whether Paulinella could adapt its light regulation and increase its tolerance to a moderately higher light intensity. I conducted a range of physiological assays at the start and end of the experiment to document any changes, and took care to separate between acclimation and adaptation responses.
Outcome: The experiment was successful conducted, and Paulinella’s growth did improve in a moderately higher light. The changes in growth rate appear to be driven by both adaptation and acclimation responses. Interestingly, there were both similarities and differences between the responses of the continuous high light and fluctuating light condition, and the adaptations to the moderately high condition did not aid in tolerating even higher light conditions.
This project has expanded our knowledge of Paulinella and its chromatophores into new directions. It has highlighted the degree of tight integration that occurs at the transcript, protein and metabolite level, and how these coordinated pathways can be modulated in response to light on both the short and long-term. As a result of which valuable insights have been gained into how the molecular interactions shape the environmental niche of Paulinella, which is thought to be largely determined by light. Overall, the results of this project indicate that, despite the relatively short duration of their partnership, the integration between the Paulinella cell and its chromatophores is already tightly coordinated at every level of their biology. As such, this project adds important contributions to our understanding of the evolutionary trajectory from endosymbiont to organelle.