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Table 1 A brief account of major hallmarks of animal apoptosis analysed in plant models for AL-PCD

From: Apoptosis is not conserved in plants as revealed by critical examination of a model for plant apoptosis-like cell death

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
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
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
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
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
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
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
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
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].
  1. In addition to the above-listed apoptotic hallmarks, most of those studies reported protoplast shrinkage. Since this is not a review article, we are not providing an exhausting list of relevant publications, but only representative studies illustrating controversies within the field of plant AL-PCD. More comprehensive account of previous works describing the occurrence of AL-PCD in plants can be found elsewhere [38,39,40,41, 69, 70]. HS, heat shock; PI, propidium iodide; PM, plasma membrane; TEM, transmission electron microscopy; TMRM, tetramethylrhodamine methyl ester; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling.