Multi-omics analysis of the picoeukaryote green alga Picochlorum sp. SENEW3 and its response to broad-spectrum herbicides
Photosynthetic picoeukaryotes (PPEs) are a globally distributed group of ‘pico’ sized (1 – 3 μm) algae. They are found throughout marine and some inland waters, but appear to be most abundant in coastal environments, where they play important roles in marine ecosystems and primary production. Most PPEs contain small genomes and have recently gained attention as minimal photosynthetic eukaryotic model organisms. Within PPEs, the green algae Picochlorum have been of particular interest due to their robust growth characteristics and potential applications in aquaculture, and biotechnology. Ongoing anthropogenic stressors such as the intensive application of herbicides are, however placing increasing pressure on primary producing aquatic organisms like PPEs. Despite this, very few studies have examined the molecular impacts of herbicides on algae, and tom our knowledge, none on PPEs. Using the isolate Picochlorum sp. SENEW3 as a model, this thesis provides an annotated chromosomal assembly of its genome, examines its genomic structure, and provides detailed photophysiological, transcriptomic and proteomic analysis of P. SENEW3’s response to the herbicides atrazine and glyphosate.
The P. SENEW3 genome was sequenced and assembled via a combination of short-read and long-read sequencing. Genomic analysis highlighted a compact genome with a reduced set of selenoproteins, numerous transporters, and the gene complement for CAM and C4 photosynthesis. In addition, Hi-C analysis revealed an organised Rabl-like chromatin structure, centromeric regions and possible large-scale genomic translocations. This high-quality assembly provides detailed insights into the structural organisation of the P. SENEW3 genome and its unique environmental adaptations.
Photophysiological and transcriptomic impacts of sublethal atrazine (0.25 μM) and glyphosate (2.5 mM) were assessed via pulsed amplitude modulation (PAM) fluorescence and RNA-Seq. Photophysiological measurements indicated significant photosynthetic stress in response to atrazine (decrease in Fv/Fm) and an increase in non-photochemical quenching (NPQ, decrease in Fm) response to glyphosate. Transcriptomic analysis revealed increased transcription of genes related to translation and carotenoid biosynthesis, coupled with reduced transcription of cell cycle and redox related genes under atrazine treatment. Similarly, treatment with glyphosate resulted in increased transcription of zeaxanthin and NPQ related genes and decreased transcription of protein and amino acid synthesis genes.
Subsequent analysis assessed the proteomic response of P. SENEW3 to atrazine and glyphosate via normalised spectral abundance factors (NSAFs). At the protein level, both herbicides induced greater abundances of proteolytic and reactive oxygen stress (ROS) related proteins. In the case of atrazine, these included peroxiredoxin, glutaredoxin and lycopene biosynthesis, whereas treatment with glyphosate largely increased proteins involved in the ascorbate-glutathione cycle. While analysis of both herbicides indicated a shift in photosynthetic carbon fixation towards photorespiration, chlorophyll biosynthesis proteins were increased in abundance under atrazine exposure but decreased in abundance when treated with glyphosate.
Gene-to-gene transcriptomic and proteomic profiles displayed a high degree of complementarity, together uncovering the largely ROS-related molecular profiles by which P. SENEW3 responds to atrazine- and glyphosate-induced stress. Taken together, this thesis provides a template for the assessment of anthropogenic stressors on PPEs and indicates additional avenues of investigation including characterisation of centromeres, PPE C4 photosynthesis and highly transcribed novel proteins.
ARC Centre of Excellence in Synthetic Biology
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