Background

PCD is an irreversible, genetically-controlled form of cell suicide which is a fundamental feature of prokaryotic and eukaryotic microbial life essential for the regulation of cellular and tissue homeostasis in metazoans (Aravind et al., 1999). In phytoplankton, PCD has been defined as a form of autocatalytic cell suicide in which an endogenous biochemical pathway leads to morphological changes and ultimately, cellular dissolution (Bidle and Falkowski, 2004). PCD has been demonstrated in diverse evolutionary lineages of prokaryotic and eukaryotic phytoplankton including cyanobacteria, coccolithophores, diatoms, and dinoflagellates (Vardi et al., 1999; Segovia et al., 2003; Berman-Frank et al., 2004; Bidle and Falkowski, 2004; Bidle and Bender, 2008). In phytoplankton, environmental stresses, such as cell age, nutrient stress, excessive salt concentrations or oxidative stress and viral attacks may initiate PCD (Berman-Frank et al., 2004; Bidle and Falkowski, 2004; Spungin et al., 2019). This process is accompanied by de novo protein synthesis of a family of proteases-the caspases, (cysteine aspartic proteases) cleaving specifically at Asp residues at the P1 position. Caspases are often used as a diagnostic marker of PCD. Classic caspases are unique in metazoans and in several viruses (Minina et al., 2017). Higher plants, unicellular protists, fungi, and bacteria lack true caspases but contain metacaspases (Uren et al., 2000), cysteine-proteases that share structural properties, specifically a histidine-cysteine catalytic dyad in the predicted active site (Tsiatsiani et al., 2011). Metacaspases are widespread among prokaryotic and eukaryotic phytoplankton genomes. It has been indicated that prokaryotic phytoplankton, as well as evolving eukaryotic lineages, not only express PCD that involves caspase-like activity, but also a core set of proteins that are orthologues of metazoan caspases (Bidle and Falkowski, 2004; Bidle, 2016; Koonin and Aravind 2002).

Although phytoplankton do not contain true caspases, upon PCD induction caspase proteolytic activity is demonstrated when synthetic fluorogenic activity substrates are applied with an Asp at the P1 position (Berman-Frank et al., 2004 and 2007; Bidle and Bender, 2008; Thamatrakoln et al., 2012; Bar-Zeev et al., 2013). Unlike caspases, metacaspases lack Asp specificity and cleave their peptide substrates after Arg or Lys in the P1 position (Tsiatsiani et al., 2011). This implies that caspase specific substrates are not indicative of metacaspase catalytic activity. Thus, it has been suggested to use substrates with Arg or Lys residues at the P1 position to detect metacaspase activities in cellular extracts (Tsiatsiani et al., 2011; Klemenčič et al., 2015). Recently developed substrates applied to investigate the activity of specific metacaspases (Tsiatsiani et al., 2011; Klemenčič et al., 2015) have enabled further examination into the roles of metacaspases in organisms possessing metacaspase genes.

The method we detail herewith specifically tests direct metacaspase activity in phytoplankton by examining the cleavage of the fluorogenic metacaspase substrate Ac-VRPR-AMC (Ac-Val-Arg-Pro-Arg-AMC). Metacaspase activity tests and method calibrations were performed specifically in the marine cyanobacteria Trichodesmium under conditions that induce PCD (e.g., iron starvation and oxidative stress) and in samples collected from the South West Pacific Ocean (Spungin et al., 2018; Spungin et al., 2019). In this method, Trichodesmium cell lysate was tested for protease activity by the addition of a metacaspase specific peptide that is conjugated to the fluorescent reporter molecule, for example, 7-amino-4-methyl coumarin (AMC). The cleavage of the peptide by the metacaspase releases the fluorochrome that, when excited by light at 380 nm wavelength, emits fluorescence at 460 nm. Enzymatic activity in the cell lysate is directly proportional to the fluorescence signal detected with a fluorimeter or a fluorescent microplate reader.