Deciphering stress resilience and programed cell death mechanisms in marine diatoms

Oceanic diatoms are unicellular algae, contributing ~20% of global photosynthesis. They form large-scale blooms which impact global nutrient recycling. Bloom dynamics include exponential cell proliferation, followed by a synchronized demise attributed to different stressors as nutrient deprivation, viruses and grazers. Diatom mortality has some characteristics of programmed cell death (PCD), yet, cellular machinery mediating unicellular organisms PCD as diatoms are understudied. We characterize novel genes involved in stress resilience and PCD in the model diatom Phaeodactylum tricornutum. Using CRISPR-Cas9, we knocked-out three metacaspase genes presumably involved in PCD due to structure similarity to caspase, a key protease in animal PCD. Mutants grew slowly than WT, and showed higher mortality rate after heat and H₂O₂ stress, mimicking environmental conditions which induce oxidative stress. Thus, metacaspases have a possible unexpected vital role involving protein aggregates disposal, as opposed to their common suggested role in algal PCD. Furthermore, to expand candidate genes involved in stress acclimation and death, we investigated a RNA-sequencing dataset from H₂O₂-treated P.tricornutum, to study the role of genes upregulated in subpopulations with two contrasting cell fates, death and survival. We then used an available mutant library of the model Chlamydomonas reinhardtii to validate mutant phenotypes in selected orthologues upregulated in each subpopulation. Ascorbate-peroxidase knockout lines died more than WT under H₂O₂ stress, while Cathepsin-X knockouts died less. Finally, candidate genes were knocked-out in P.tricornutum. Their phenotypes are currently being characterized. By combining targeted and untargeted genetic approaches, we aim to reveal cellular pathways mediating phytoplankton death in the ocean.