How shall we measure programmed cell death in eukaryotic microalgae?

Abstract

Abstract Programmed cell death (PCD) plays a key role in unicellular microalgal ecology. However, the methodologies for detecting PCD are problematic. Clearly, to interpret the empirical data, clarity on how to measure microalgal PCD is essential. Here, we critically review the current measurements of PCD and provide suggestions for future methodological developments and interpretations. We review the traditional measures of PCD and associated cellular responses in microalgae and provide assessments of their frequencies of use and true positive rates. Traditional physiological measurements of photosynthetic activity, change in gene regulation, measurements of reactive oxygen species and terminal deoxynucleotidyl transferase dUTP nick end labelling are highly sensitive assays. They provide important measures of cellular physiological responses but are not unique to PCD. Both caspase-like and metacaspase activity reveal useful information about stress responses and demonstrate high (94% and 100%, respectively) positivity rates, however, they can play a role in cell activities other than death. Furthermore, the controversy surrounding positive caspase assays, even though microalgae encode metacaspases rather than orthologous caspases, is highlighted. DNA laddering had the lowest true positive rate (64%) and was not reported in diatoms while phosphatidylserine externalization was consistently positive in all taxa except dinoflagellates. These data illustrate the limitations of some PCD markers across different taxa. Ultrastructural alterations (transmission electron microscopy) were highly correlated with PCD across all microalgal taxa (true positive rate of 94%) and seem essential for the initial assessments of whether a cell is dying in an organized, ‘programmed’ way. However, in the face of the complexity of PCD phenotypes and the non-specific nature of the methodologies, no single indicator can be used to diagnose PCD. Here, we highlight the importance of employing a time-sensitive multi-assay approach to detect PCD in the eukaryotic microalgae before any ecological or evolutionary interpretations can be made. Highlights Measurements of PCD have different specificities and sensitivities. TEM appears essential as part of an initial investigation. Complementary markers provide information about cell stress and death responses.