INHIBITION OF CASPASE-LIKE ACTIVITIES PREVENTS THE APPEARANCE OF REACTIVE OXYGEN SPECIES AND DARK-INDUCED APOPTOSIS IN THE UNICELLULAR CHLOROPHYTE DUNALIELLA TERTIOLECTA 1 Marı´a Segovia 2 Department of Ecology, Faculty of Sciences, University of Ma ´laga, Bulevar Louis Pasteur s ⁄ n, 29071-Ma ´laga, Spain and John A. Berges Department of Biological Sciences, University of Wisconsin-Milwaukee, P.O. Box 413, Milwaukee, Wisconsin 53201, USA When the chlorophyte alga Dunaliella tertiolecta Butcher is placed in darkness, a form of pro- grammed cell death with many similarities to apoptosis is induced, including the induction of cas- pase-like proteases. Many uncertainties about the regulation and mediators that participate in the pro- cess remain. To examine the relationship between caspase-like activities and different apoptotic events (i.e., phosphatidylserine [PS] translocation), increases in membrane permeability and numbers of dead cells revealed by SYTOX-green staining, and the generation of reactive oxygen species (ROS), we used the broad-range caspase inhibitor Boc-D-FMK to block the activity of the whole class of caspase-like proteins simultaneously. In the pres- ence of the inhibitor, ROS were not produced, and cells did not die. Loss of membrane asymmetry, indicated by external labeling of PS by annexin V, was apparent at midstages of light deprivation, although it did not conform to the typical pattern for PS exposure observed in metazoans or vascular plants, which occurs at early stages of the apoptotic event. Thus, we have evidence for a link between ROS and cell death involving caspase-like enzymes in an alga. The fact that caspase-like inhibitors pre- vent not only cell death, but also ROS and loss of cell membrane integrity and asymmetry, suggests that caspase-like proteases might have regulatory roles early in cell death, in addition to dismantling functions. Key index words: apoptosis inhibition; caspase-like activities; cell death; cell viability; Dunaliella tertiolecta; phosphatidylserine; phytoplankton; reactive oxygen species; unicellular chlorophyte Abbreviations: PCD, programmed cell death; PS, phosphatidylserine; ROS, reactive oxygen species Apoptosis is a ubiquitous physiological process, essential for the proper growth and development of metazoans (Kerr et al. 1972). It is typically defined as a type of programmed cell death (PCD) that is dependent on the activity of specific proteases called caspases (cysteinyl aspartate-specific proteases (Thornberry 1999) and is accompanied by highly conserved morphological changes, including chro- matin condensation and margination and ordered DNA cleavage, while the cytoplasm and organelles remain unaffected (Cohen 1997). Recent studies have demonstrated that unicellular organisms, including chlorophytes (Berges and Falkowski 1998, Segovia et al. 2003, Segovia and Berges 2005), dinoflagellates (Vardi et al. 1999, Franklin and Berges 2004, Segovia 2007), diatoms (Casotti et al. 2005, Bidle and Bender 2008), coccolithophorids (Bidle et al. 2007), yeast (Frohlich and Madeo 2000, Burhans et al. 2003), and bacteria (Cairns 2002, Rice and Bayles 2003), including cyanobacteria (Ning et al. 2002a,b, Berman-Frank et al. 2004), undergo PCD. The factors that cause cell death in unicells, its roles, and the details of the apoptotic process remain unclear (Franklin et al. 2006, Deponte 2008), but model species, such as D. tertiolecta, are proving useful in examining common apoptotic features. When placed in darkness, this unicellular chlorophyte undergoes a form of cell death reminiscent of apoptosis in metazoans. Many mor- phological criteria of apoptotic cell death are met, including an increase chromatin margination, deg- radation of the nucleus, and DNA fragmentation (Segovia et al. 2003). Assays using caspase-specific fluorogenic substrates demonstrate that activities increase with time in darkness, in parallel with the morphological changes. Furthermore, these activi- ties are inhibited by caspase-specific inhibitors, and antibodies raised against mammalian caspases cross- reacted with proteins in the alga. The pattern of expression of these immunologically reactive pro- teins was correlated with the enzymatic activity mea- sured, with the onset of cell death (Segovia et al. 1 Received 31 July 2008. Accepted 16 March 2009. 2 Author for correspondence: e-mail segovia@uma.es. J. Phycol. 45, 1116–1126 (2009) Ó 2009 Phycological Society of America DOI: 10.1111/j.1529-8817.2009.00733.x 1116