Project description:The sea-ice dwelling diatom Fragilariopsis Cylindrus was cultured for 4 months under dark or light exposed conditions to mimic the effects of Antarctic winter growth conditions. Cells were harvested periodically and LFQ proteomics used to investigate the molecular mechanisms of dark survival.
Project description:Fragilariopsis cylindrus is an abundant diatom species in the Southern Ocean, where low iron and manganese availability constrain microbial growth and biogeochemical cycles. The molecular mechanisms used by polar diatoms including F. cylindrus to cope with trace metal limitations remain largely unexplored. Here we present phenotypic characterizations and high resolution proteomics profiles of F. cylindrus grown under controlled iron (low, medium, high) and manganese (low, high) conditions that reflect those observed in the Southern Ocean. Using data-independent acquisition mass spectrometry, we quantified over 8,000 unique proteins with high reproducibility. We captured diverse metabolic responses related to photosynthesis, elemental transport and intracellular trafficking, and protein synthesis. We show that several canonical iron stress response proteins (e.g., phytotransferrin) are consistently identified under low iron conditions, and identify additional useful iron and manganese stress biomarkers that could be explored in field measurements. Our data also support the notion that one flavodoxin copy in F. cylindrus is iron responsive and one is not. This dataset is a valuable resource for understanding trace metal physiology in polar diatoms, and provides a way for connecting molecular responses with expressed phenotypes and biogeochemical activity in the ocean.
Project description:We performed RNA-sequencing experiments to examine the differential regulation of genes in the genome of the Southern Ocean diatom Fragilariopsis cylindrus including diverged alleles. RNA-seq was performed on three replicate samples for each experimental condition. Phytoplankton cells were grown under six different experimental conditions including (1) optimal growth, (2) freezing temperatures, (3) elevated temperature, (4) elevated carbon dioxide concentrations, (5) low iron concentrations and (6) prolonged darkness. Total RNA was extracted using a guanidinium thiocyanate-phenol-chloroform extraction protocol, followed by DNase I treatment and RNA purification (Quiagen). First strand cDNA synthesis was performed using random hexamers. Library preparation was performed using the RNA-seq Sample Prep Kit (Illumina) and sequencing was conducted according to the TruSeq RNA sequencing protocol (Illumina) All samples were sequenced together in one flowcell on one lane.
Project description:The Southern Ocean diatom Fragilariopsis cylindrus was grown under half-saturating concentrations of silicate (0.3 uM) and in the blue (480 - 540 nm) and red (550 - 700 nm) light spectrum to investigate gene expression responses using RNA-seq relative to optimal growth conditions (deposited under accession E-MTAB-5024). RNA-seq was performed on three replicate samples for each experimental condition. Total RNA was extracted using a guanidinium thiocyanate-phenol-chloroform extraction protocol, followed by DNase I treatment and RNA purification (Quiagen). First strand cDNA synthesis was performed using random hexamers. Library preparation was performed using the RNA-seq Sample Prep Kit (Illumina) and sequencing was conducted according to the TruSeq RNA sequencing protocol (Illumina) All samples were sequenced together in one flowcell on one lane.
