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Programmed Cell Death (PCD) in eukaryotes is a regulated process occurring during development, cell differentiation and aging. Apoptosis is a particularly well studied morphotype of PCD, only observed in animal cells (metazoan). Its most definitive hallmark is the formation and release of membrane-enclosed extracellular vesicles called Apoptotic Bodies (ABs). Although apoptotic-like features have been described in plants, yeast, protozoa and phytoplankton, the production of ABs has been thought to be limited to multicellular animals. Here we report the production and release of extracellular ABs in a non-metazoan unicellular eukaryote, the cryptophyte alga Guillardia theta. Morphologies of G. theta cells during aging and pharmacologically-induced cell death confirm the presence of ABs and apoptosis in phytoplankton. G. theta ABs have similar composition to metazoan ABs, carrying DNA, proteins, lipids, carbohydrates, fragments of organelles and cytosol portions. Our results demonstrate that G. theta, a microalga that arose from secondary endosymbiosis, experiences apoptotic cell death in physiological conditions, similar to animal cells. Since secondary endosymbiosis occurred prior to the origin of multicellularity, our discovery questions the evolutionary origin of PCD.

Metacaspases are cysteine proteases present in plants, fungi and protists. While the association of metacaspases with cell death is studied in a range of organisms, their native substrates are largely unknown. Here, we explored the in vivo proteolytic landscape of the two metacaspases, CrMCA-I and CrMCA-II, present in the green freshwater alga Chlamydomonas reinhardtii, using mass spectrometry-based degradomics approach, during control conditions and salt stress. Comparison between the cleavage events of CrMCA-I and CrMCA-II in metacaspase mutants revealed unique cleavage preferences and substrate specificity. Degradome analysis demonstrated the relevance of the predicted metacaspase substrates to the physiology of C. reinhardtii cells and its adaptation during salt stress. Functional enrichment analysis indicated an involvement of CrMCA-I in the catabolism of carboxylic acids, while CrMCA-II plays an important role in photosynthesis and translation. Altogether, our findings suggest distinct cellular functions of the two metacaspases in C. reinhardtii during salt stress response.