Table 1 A brief account of major hallmarks of animal apoptosis analysed in plant models for AL-PCD
Hallmark of apoptosis | Experimental model/detection method | Reference | Controversy |
|---|---|---|---|
Impermeable PM | 52 °C HS of Arabidopsis thaliana cell culture/Trypan blue staining Victorin-treated oat (Avena sativa) seedlings/TMRM staining | [51] [52] | At least half of the cells with shrunken protoplasts were Trypan blue-positive already 30 min post-HS. Therefore, PM integrity is not a reliable hallmark for this cell death. TMRM is a cell permeant mitochondrial dye and cannot be used for the analysis of PM integrity. |
ATP requirement | Benzyladenosine-treated tobacco (Nicotiana tabacum) BY-2 cell culture/ATP bioluminiscence assay Ceramide-treated rice (Oryza sativa) protoplasts/ATP bioluminiscence assay | [53] [54] | Drop of ATP detected in both studies, whereas apoptosis is ATP-dependent process. |
Cytochrome c release from mitochondria | 55 °C HS of cucumber (Cucumis sativus) cotyledons/Western blotting 55 °C HS of BY-2 cell culture/Western blotting | [48] [55] | Cytochrome c translocation during apoptosis is required for apoptosome-mediated activation of caspases [56]. The mechanistic role of cytosolic cytochrome c in plant cell death remains unknown. |
Activity of caspases | Salt-stressed Thellungiella halophila cell culture/Caspase substrates and inhibitors 45 °C HS- or victorin-treated oat (Avena sativa) seedlings/Caspase substrates and inhibitors | [57] [58] | Increased DEVDase activity detected; protease(s) responsible for the activity is unknown. Increased DEVDase and VADase activities caused by subtilisin-like serine proteases. In both cases, biological targets of the proteases with caspase-like activities are unknown undermining conclusions on similarities with caspase-dependent signaling and execution of apoptosis. |
DNA fragmentation | Sanguinarine-treated onion (Allium cepa) roots/TUNEL staining Arabidopsis protoplasts subjected to ultraviolet-C/TUNEL staining | [59] [60] | DNA fragmentation is not specific to apoptosis and can occur under various types of cell death [11]. |
PM blebbing | 55 °C HS of carrot (Daucus carota) cells cultured at low density/TEM Microspore degeneration in the female flowers of Actinidia deliciosa/TEM | [61] [62] | No evidence for impermeability of PM and bleb formation outwards on the cell surface [45] in both studies. |
Formation of apoptotic bodies | 52 °C HS of tobacco (N. tabacum) cell culture/TUNEL staining Mycotoxin fumonisin B1-treated tomato (Solanum lycopersicum) seedlings/PI and TUNEL staining | [63] [64] | No extracellular vesicles revealed. Instead, nuclear pyknosis characteristic for both apoptosis and necrosis in animal models [65] was detected in both studies. |
Phosphatidylserine exposure | Chlamydomonas reinhardtii cells subjected to ultraviolet-C/Annexin-V binding assay, fluorescent microscopy Campothecin-treated tobacco (Nicotiana plumbaginifolia) protoplasts/Annexin-V binding assay, flow cytometry | [66] [67] | Phosphatidylserine exposure during apoptosis constitutes “eat me” signal for phagocytes which are absent in plants. Furthermore, phosphatidylserine exposure is not exclusive to apoptosis and also occurs in regulated forms of necrosis [68]. |