Different ways to die: cell death modes of the unicellular chlorophyte Dunaliella viridis exposed to various environmental stresses are mediated by the caspase-like activity DEVDase

Abstract

Programmed cell death is necessary for homeostasis in multicellular organisms and it is also widely recognized to occur in unicellular organisms. However, the mechanisms through which it occurs in unicells, and the enzymes involved within the final response is still the subject of heated debate. It is shown here that exposure of the unicellular microalga Dunaliella viridis to several environmental stresses, induced different cell death morphotypes, depending on the stimulus received. Senescent cells demonstrated classical and unambiguous apoptotic-like characteristics such as chromatin condensation, DNA fragmentation, intact organelles, and blebbing of the cell membrane. Acute heat shock caused general swelling and altered plasma membrane, but the presence of chromatin clusters and DNA strand breaks suggested a necrotic-like event. UV irradiated cells presented changes typical for necrosis, together with apoptotic characteristics resembling an intermediate cell-death phenotype termed aponecrosis-like. Cells subjected to hyperosmotic shock revealed chromatin spotting without DNA fragmentation, and extensive cytoplasmic swelling and vacuolization, comparable to a paraptotic-like cell death phenotype. Nitrogen-starved cells showed pyknosis, blebbing, and cytoplasmic consumption, indicating a similarity to autophagic/vacuolar-like cell death. The caspase-like activity DEVDase was measured by using the fluorescent substrate Ac-DEVD-AMC and antibodies against the human caspase-3 active enzyme cross-reacted with bands, the intensity of which paralleled the activity. All the environmental stresses tested produced a substantial increase in both DEVDase activity and protein levels. The irreversible caspase-3 inhibitor Z-DEVD-FMK completely inhibited the enzymatic activity whereas serine and aspartyl proteases inhibitors did not. These results show that cell death in D. viridis does not conform to a single pattern and that environmental stimuli may produce different types of cell death depending on the type and intensity of the stimulus, all of which help to understand the cell death-dependent and cell death-independent functions of caspase-like proteins. Hence, these data support the theory that alternative, non-apoptotic programmed cell death (PCDs), exist either in parallel or in an independent manner with apoptosis and were already present in single-celled organisms that evolved some 1.2-1.6 billion years ago.

Fig. 1.

Representative transmission electron micrographs showing the morphological changes in Dunaliella viridis subjected to different lethal stress treatments. C, chloroplast; Fi, flagellar insertion; G, Golgi, M, mitochondria; Mb, plasmalemma; N, nucleus; P, pyrenoid; S, starch. Arrows indicate the alterations indicated in the text. (A) Normal vegetative cells grown in PAR. (B) Cells After 3 h of lethal hyperosmotic shock (5.5 M NaCl). Note chromatin condensation together with extensive cytoplasmic swelling and vacuolation (arrow 1) in the absence of nuclear fragmentation and cellular blebbing (arrow 2). (C) Cells after 4 h of UV radiation. Characteristic cell swelling, organelle membranes are disrupted, mitochondria are condensed and cytoplasmic blebs appear (arrow 1). Cell membrane is intact and shows blebbing (Arrow 2). (D) Cells after 2 h of heat shock. Cells experience nuclear edema and swelling, mitochondrial rupture, disrupted organelle membranes and eventually altered plasma membrane (arrow 1), some chromatin clusters are observed (arrow 2). (E) Cells after 7 d of nitrogen starvation. Note pyknosis (arrow 1) but not margination of chromatin, cell membrane with blebbing (arrow 2), and cytoplasmic consumption (arrow 3). (F) Senescent cells after 12 d. Nucleus suffers margination in the cell, chromatin is condensed (arrow 1), clumped and marginated in the nucleus, while cell membrane is intact with blebbing (arrow 2) and undamaged organelles. Picture augmentation was ×25 000.

Fig. 2.

Cell death judged by Evans Blue mortal staining of cells under light microscopy with lethal stress treatments. The dashed horizontal line represents live control cells in PAR at t=0. (A) Cells after osmotic shock. (B) Cells after UV radiation. (C) Cells after heat shock. (D) Cells under nitrogen starvation. (E) Culture senescence.

Fig. 3.

DNA condensation in Dunaliella viridis revealed by DAPI staining and confocal laser microscopy. (A) Control cells in PAR. (B) Cells after osmotic shock (5.5 M NaCl). (C) Cells after 4 h of UV radiation. (D) Cells after 2 h of heat shock. (E) Cells after 7 d of nitrogen starvation. (F) Culture senescence. Horizontal bar is 1 μm. (This figure is available in colour at JXB online.)

Fig. 4.

DNA fragmentation in Dunaliella tertiolecta after different environmental stresses revealed by TUNEL staining. (A) Control cells in PAR. (B) Cells after 3 h of osmotic shock. (C) Cells after 4 h of UV radiation. (D) Cells after 3 h of heat shock. (E) Cells after 7 d of nitrogen starvation. (F) Senescence after 12 d. Horizontal bar is 20 μm. (This figure is available in colour at JXB online.)

Fig. 5.

DEVDase activity in Dunaliella viridis following exposure to various stresses. Changes in enzymatic activity were measured as hydrolysis of 7-amino-4-fluoromethyl coumarin-labelled specific substrate DEVD, in all stress treatments. (A) Osmotic shock. (B) UV radiation. (C) Heat shock. Lethal conditions of stress factors are represented by black bars and sub-lethal conditions by white bars. (D) Nitrogen starvation. (E) Senescent cultures. (F) Inhibition of the enzymatic activity by using the irreversible caspase-3-inhibitor Z-DEVD-FMK after 4 h of UV lethal stress. The highest point of DEVDase activity for the rest of the treatments was inhibited with the highest concentration of the inhibitor (200 μM) and any of them showed activity. Statistical differences (P <0.05) during the time-course for the lethal stress are marked with letters. Same letter means no differences.

Fig. 6.

Western blot showing cross-reactions of protein extracts from Dunaliella viridis with antibodies raised against human caspase 3. Lanes correspond to the hours or days exposed to each particular stress treatment. (A) Osmotic shock. (B) UV radiation. (C) Heat shock. (D) Nitrogen starvation. (E) Culture senescence.