Heat shock treatments have been found to alter cell metabolism, disrupt mitochondria, and result in an increase in ROS [23],[25]. Depending on the severity, heat shock will result in cell death. In the 55°C treatment, anthocyanin disappearance was apparent immediately after treatment. It is suspected by the authors, that a spike in ROS may have played a role in anthocyanin disappearance, although anthocyanin is also a heat sensitive pigment [26]. In addition to anthocyanin disappearance, there was a marked increase in the number of vesicles within the cell. The characteristic increase in vesicles, the appearance of organelles in the vacuole, and an increased volume of the central vacuole, up until tonoplast collapse, provides evidence for both macro-, and mega-autophagy. The retraction of the PM from the cell wall after tonoplast collapse resembles the PM retraction observed during lace plant developmental PCD, however the ultrastructure of the PM at this point was not investigated. Although, it should be noted that the cell corpse in developmental PCD exhibits a more condensed morphology in comparison. Similarly, PM shrinkage has been shown in heat shock experiments using lace plant protoplasts [23]. Surprisingly, the protoplasts were less susceptible to heat shock at 55°C than the in situ cells in our experiment, with the protoplasts undergoing PCD after 20 min while the in situ cells underwent PCD after 10 min. The severity of the 65°C treatment resulted in cell death before completion of the treatment. The 65°C cell death morphology appeared remarkably different compared to the 55°C treatment, lacking PM retraction and with a loss of chlorophyll from the chloroplasts. The textured appearance along the periphery of the cell is believed to be the remains of cellular debris. Membranes within the cell are not believed to have retained their integrity. The subsequent morphology of the 65°C treatment is characteristic of what is commonly considered necrotic cell death [15],[16].
In the 100 mM NaCl treatment, there was a dramatic slowing in cytoplasmic streaming. Sodium chloride stress has been implicated in an increase in cytoplasmic Ca2+, which can arrest cytoplasmic streaming, by Na+ displacing Ca2+ from the PM, and from liberating Ca2+ from internal stores [27]. However, there is little research that assesses the effects of salinity on cytoplasmic streaming [28]. In the 100 mM and 400 mM NaCl treatments, the chloroplasts took on a wrinkled appearance. This wrinkled effect on chloroplast ultrastructure has similarly been observed in TEM images of tomato cells grown in a medium containing 100 mM NaCl [29]. Chloroplasts appeared swollen in the 400 mM NaCl treatment, but this effect was not observed in the 2 M NaCl experiments. In potato cultivars, electron microscopy showed that although the structural integrity of cells appeared intact, the chloroplasts appeared swollen when plants were irrigated with 100 mM, and 200 mM NaCl solution, respectively [30]. Swollen chloroplasts have also been seen in wheat and sweet potato leaves under salt stress [30],[31].
Interestingly, the vacuole appeared to increase in size in the 100 mM NaCl treatment, which occurs similarly in LPCD cells during leaf perforation developmental PCD. This rapid increase in vacuole size, in response to saline conditions, has been demonstrated in suspension-cultured of mangrove cells and barley root meristematic cells [32]. Na+ accumulation in the central vacuole and subsequent increase in vacuolar volumes has been shown to be an active process and is believed to be one strategy employed by the cell in response to salt stress [32]. Vesicle formation occurred in the 400 mM and 2 M NaCl treatments, suggesting an increase in macro-autophagy, perhaps to recycle damaged intracellular components [33]. High salt solutions have been shown to elicit autophagy in Arabidopsis thaliana by up-regulating autophagy related genes [33].The initial retraction of the PM from the cell wall in the 400 mM and 2 M NaCl treatments, in contrast to the late-stage PM retraction seen in developmental PCD, is plasmolysis and is due to changes in osmotic pressure. The filamentous structures between the PM and the cell wall observed during plasmolysis are speculated to be hechtian strands ([34]; Additional files 6, and 7). Interestingly, there were many more of these strands in the 400 mM NaCal compared to the 2 M NaCl treatment. At the final stages of death there was a contrast between these two treatments; cell treated with 400 mM NaCl exhibited tonoplast rupture and no PM retraction, whereas the 2 M NaCl treatment had a significant PM retraction. The authors speculate that the numerous strands connecting to the cell wall in the 400 mM NaCl treatment group played a role in negating the PM retraction which occurred after treatment at the higher concentration.
The most striking characteristic of the 12 M HCl treatment is the surprising dramatic retraction of the PM from the cell wall. The PM retraction in the 12 M HCl treatment resembled PM retraction seen at the end of developmentally regulated PCD in lace plant perforation formation. Although this retraction appears to be morphologically similar, this cell death lies in contrast to PCD in perforation formation, which typically takes several days. The cell death process in the 12 M HCl treatment was rapid and although the ultrastructural changes to the PM are unknown, the authors suspect this is a passive process. In the 3 mM HCl treatment, cytoplasmic streaming slowed, which may have been a result of either a change in cytoplasmic pH or an increase in cytosolic Ca2+. The vacuole swelled extensively, which may have been a cellular response to extracellular acidity by increasing the volume of the vacuole, as it is generally more acidic than the cytoplasm under normal conditions. The swelling of the vacuole in this treatment resembled swelling in LPCD cells during developmental PCD. The observation of vesicles was similar to the salt stress treatments, and may be indicative of an increase in macro-autophagy. Cell death occurred with the permeabilization of the tonoplast. In the 30 mM NaOH treatment, there was a considerable increase in vesicles compared to other treatments, which may also indicate an increase in macro-autophagy. The change in colour of the vacuole from pink to blue/green immediately before cell death suggests that the vacuolar pH was dramatically raised to near alkalinity as anthocyanin’s visible colour shifts.
Comparison between induced cell death and its developmental counterpart revealed that there are several common characteristics, including cessation of cytoplasmic streaming and tonoplast collapse (Table 1). Vacuolar dynamics appear to be consistent among the developmental and induced cell death videos (Table 1), and occupying the majority of a plant cell, it is likely to make a substantial contribution to cell death processes. Perinuclear chloroplast formations only occurred during developmental cell death. Likewise, the 12 M HCl treatment was the only cell death without anthocyanin disappearance, which is likely due to the response of the pigment to the low pH of the solution. In the NaOH treatments, nuclear condensation was not observed, in contrast to all other cell death types. Vesicle formation was a common characteristic of all cell death types except those that resulted in very rapid cell death, such as the 12 M HCL and 1 M NaOH treatments. PM retraction was seen in perforation formation, the 55°C, 2 M NaCl and 12 M HCl treatment groups. Interestingly, in all cases where cell death was observed, the vacuole played a central role, specifically seen with tonoplast collapse occurring in all cell death types. The results of this comparative study are summarized in Table 1.
In animal cells, there exists a morphologic classification system of cell death types, with three categories: apoptosis, autophagic cell death, and necrosis. Among the most apopotic-like characteristic seen in the lace plant is retraction of the PM due to a reduction in cell volume observed during leaf perforation developmental PCD. A similar morphology can be seen in the 55°C treatment, 2 M NaCl and the 12 M HCl treatment. Regarding autophagic cell death, an increase in vacuolar swelling and vesicle formation was observed in heat, salt, and most pH treatments. Notably, an increase in vesicles was not observed in the most extreme pH treatments (12 M HCl, 1 M NaOH). In most cell death morphologies induced by less severe stressors, such as in 30 mM NaOH treatment in leaf sheath tissue, whole organelles encapsulated by a vesicle were seen to fuse with the vacuole prior to tonoplast collapse. Recently, a dual role of autophagy as either an initiator of PCD during the HR (hypersensitive response), or a downstream executioner during developmental PCD has been proposed by Minina et al. [36]. The current authors believe that the examples of autophagy shown here are acting downstream, perhaps through the activation of some components similar to the lace plant leaf perforation developmental pathway. Interestingly, the high number of spherical opaque bodies which formed in the 2 M NaCl treatment fused with others that were in close proximity, and either disappeared or shrunk before PM retraction and cell death, but more research is needed to determine their composition and function.
Necrotic features such as early rupture of the PM, were typically seen in the most extreme treatments. A reduction of cellular volume, along with the active retraction of the PM is typically associated with a slower, more internally regulated form of cell death; however, PM retraction was seen in the most extreme acid treatment (12 M HCl) and took place within minutes. While the PM retractions observed in the 55°C and 2 M NaCl treatments were morphologically similar to the 12 M HCl treatment, the timeframe for cell death to occur was much longer in comparison. Considering the relatively slow timeframe for cell death from the 55°C and 2 M NaCl treatments (5.57 ± 0.21 h and 4.25 ± 0.38 h, respectively), the authors hypothesize that the PM retraction is an active process, whereas the 12 M HCl treatment represents a necrotic collapse. Further investigation is required, however, to determine whether or not the various induced cell death morphologies shown here are forms of PCD.
In 2000, Fukuda proposed the existence of three PCD categories in plants: apoptotic-like, leaf senescence, and one in which the vacuole plays a central role [7]. Since then, there have been several proposed classification systems for plant PCD, but currently none are unanimously accepted. Proposed classifications have often centered on characteristics of a particular organelle, notably the vacuole, or PM. The PM is commonly used due to its conspicuous appearance when retracted from the cell wall. This retraction, along with a reduction of cellular volume are characteristics present in apoptosis but absent in necrosis in animal models. Emphasis on the vacuole is likely due to its expansive nature in plant cells, often occupying up to 90 % of cellular volume which is unlike any autolytic organelle found in animal cells. Also, the vacuole is known for its autolytic properties, participating in cellular processes such as autophagy, which are associated to cell death events. Macro-autophagy occurs when cellular components are sequestered to the vacuole via double membrane vesicles. Vacuolar swelling followed by tonoplast collapse, known as mega-autophagy, is common in plant cell death processes, as was observed in this study. It is very possible that future cell death classifications will center on the role of the vacuole in plant cell death, as has been seen in previous classification proposals. Our data indicates that individual stressors typically result in different cell death morphologies amongst differing stressor intensities, despite being within a single system. Other researchers are encouraged to consider the means by which induced PCD studies are carried out. Although the experiments in this paper used isolated stressors, it has been found that more damage occurs to plants when multiple stressors occur simultaneously [35], and therefore it may be considered that treatments should replicate combined stressors that would occur naturally.