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Metacaspases are proteolytic enzymes that share structural homology with caspases, the executioners of cell death in animals. These enzymes are conserved across the tree of life, tracing back to ancient lineages such as cyanobacteria and microalgae – organisms that, together, produce most of the oxygen we breathe. In this thesis, the function of metacaspases was explored in microalgae, some of the planet's oldest and most essential life forms, under conditions of stress and cell death.

Papers I and II focused on the cell death process in Guillardia theta (G. theta), an evolutionarily unique cryptophyte microalga. During natural aging, G. theta cells underwent a form of cell death analogous to animal apoptosis, marked by apoptotic features at morphological and biochemical levels. The most striking apoptotic characteristic, however, was the fragmentation of G. theta cells into apoptotic bodies, a novel finding in the unicellular world. Biochemical characterization revealed that apoptotic bodies resemble their animal counterparts in composition. Furthermore, alterations in the apoptotic proteome indicated that the underlying pathway is mitochondria-dependent and involves proteins homologous to components of the metazoan apoptotic cascade. Finally, the contribution of metacaspases to this cascade was studied at the transcript, protein, and catalytic activity levels. The varying behavior of G. theta metacaspases pointed to multilayered regulation of their function.

In Paper III, the role of metacaspases in the salt-triggered stress response of Chlamydomonas reinhardtii was investigated. Single metacaspase mutants, each lacking one of the two metacaspases encoded in its genome, displayed resistance to salt stress, evidenced by delayed decline in culture viability. Mapping native metacaspase substrates further revealed their involvement in cellular stress adaptation mechanisms.

Overall, this thesis demonstrates the diverse roles of metacaspases in the survival and death of microalgae and challenges existing views on the evolutionary origins of regulated cell death.