Periodic Reporting for period 1 - PlastidOrigin (Testing the paradigm of a single plastid origin in eukaryotes)
Berichtszeitraum: 2023-01-01 bis 2025-06-30
Photosynthesis was acquired by eukaryotes some 2 billion years ago through the merger of two cells, one inside another, a process called endosymbiosis. Similar to the origin of mitochondria, this process resulted in new cellular organelles that in this case are called the plastids. There exists different kinds of plastids, perhaps the best known are the chloroplasts. From the origin of plastids by endosymbiosis between early eukaryotes and cyanobacteria evolved the first eukaryotic algae, giving rise to land plants but also triggering the evolution of most photosynthetic eukaryotes by subsequent endosymbioses between these first algae and other eukaryotes. Thus, the origin of plastids profoundly changed the course of eukaryotic life by being the launching point that shaped the biological diversity of most primary producers, and allowing life as we know it to evolve. Despite this importance, our understanding of how plastids originated remains largely uncertain. The current paradigm describes this transformative event as a single primary endosymbiosis, but we argue in PlastidOrigin that critical data is lacking, notably from the vast hidden environmental diversity of microbes, to adequately test this hypothesis. In this project, we propose to gain insight into the origin of plastids by addressing four main questions: 1) What is the currently hidden diversity of high-ranked microbial taxa, similar to better known ‘kingdoms’ of life such as animals or plants, related to known photosynthetic lineages? 2) What can we find out about these cells, for example do they have plastids? 3) Are some lineages genetically predisposed to establish plastids from the acquisition of foreign genes by lateral gene transfers? 4) What was the composition, size, and origin of the ancestral primary plastid proteomes? To answer these questions, we will link environmental sequencing (notably using long-read sequencing), single-cell transcriptomics, and genomics to cell structure and behavior of novel key lineages related to known algae, and produce crucially missing plastid proteomes to allow comprehensive comparative proteomic analysis. This project will not only have immediate implications on our understanding of the origin of plastids and more generally the fundamental process of endosymbiosis, but the approaches developed will be a test bed for future global studies aimed at understanding the evolution and ecology of the microbial majority of complex life.
We are now in the meat of the project, and have made headways in all main aspects. After an initial period of acclimatation, the trainees in the project are now up to speed and performing well. We have identified several potential new major environmental lineages related to algal groups, and build a phylogenetic framework for all of them. These are the very groups we initially hypothesised existed and are important to study further the evolution of plastids. From the short sequence fragments that we know for these groups, we have designed probes that (will) allow us to visualise these cells for the first time, getting a glimpse at species that have escaped detection until now. We are also testing different combinations of dyes and antibodies that will allow us to better determine the morphology of these enigmatic cells. We have generated single-cell transcriptomic and genomic data for many of our target candidates, including the first transcriptomic data for Picozoa. One example of novel diversity at high taxonomic level is our newly discovered algal group that we called leptophytes. This is a remarkable discovery because totally novel algal diversity is only very rarely reported, similar to the discovery of a new animal group. All this data is currently being analysed in our comprehensive phylogenomic framwork. Excitingly, we have established a culture for a close relative to photosynthetic Paulinella (the first culture for a non-photosynthetic Paulinella), and are now in the process of obtaining high-quality DNA for long-read sequencing to obtain a reference-quality genome. For the spatial proteomic part of the project, we have generated very nice cellular fractions for two red algal species and have done a couple of preliminary mass spectrometry runs. To complement this data, we are currently generating RNA-seq data and volume microscopy with FIB-SEM for the same species. The results are highly encouraging, with already about 5000 proteins identified.
It is still early in the project for identifying results beyond state of the art, but I will mention two aspects. First, our culture of heterotrophic Paulinella is not to be under-evaluated. More than 100 years after the discovery of Paulinella, and more than 20 years after the realisation of the importance of this group to study plastid evolution, to our knowledge our culture represents the first culture available for a close sister group of the better known photosynthetic species. Because the culture is growing well, we anticipate that we will be able to optain a high-quality reference genome, perhaps telomere-to-telomere, which will tremendously help to address outstanding questions related to the acquisition of the plastid in this group. For anyone interested in plastid evolution and Paulinella in particular, this will be invaluable data. The second aspect is our attempt to generate single-cell transcriptomes for cells sorted directly from the environment. Automated single-cell sorting with flow cytometry has been used to obtain single-cell genomes following genome-amplification, but to our knowledge it has never been attempted for single-cell transcriptomics for pico-sized marine cells. Our preliminary analyses indicate that this approach has worked well at least for some of the sorted cells, which represents an exciting avenue for future work as complementary or to replace single-cell genomes.