An improved method for monitoring cell death in cell suspension and leaf disc assays using evans blue
01 Oct 1994-Plant Cell Tissue and Organ Culture (Kluwer Academic Publishers)-Vol. 39, Iss: 1, pp 7-12
TL;DR: This work has used Evans blue stain to develop a spectrophotometric procedure that allows rapid, reproducible quantification of the stain retained by dead cells, and was used to compare plant/bacteria interactions involving either soybean/Pseudomonas syringae pv.
Abstract: Cell viability or cell death is an important variable to monitor in many studies of host/pathogen interactions. However for studies that focus on events within the first few hours of the interaction, many of the viability assays currently being used are either too laborious and time consuming or measure the cell's temporary metabolic state rather than irreversible cell death. Evans blue has proven over the years to be a dependable stain for microscopic determination of cell death. We have used this stain to develop a spectrophotometric procedure that allows rapid, reproducible quantification of the stain retained by dead cells. This spectrophotometric procedure was used to compare plant/bacteria interactions involving either soybean/Pseudomonas syringae pv. glycinea or tobacco/P. syringae pv. syringae. Relative increases in cell death during these interactions in suspension cell systems were measured by both the spectrophotometric and microscopic technique and found to be similar. The spectrophotometric procedure was also adapted for leaf disc assays.
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TL;DR: In conclusion, Cd did not cause necrotic injury in root tips but appeared to expedite differentiation, thus leading to accelerated aging and may have triggered the developmental program leading to xylogenesis.
Abstract: To investigate whether Cd induces common plant defense pathways or unspecific necrosis, the temporal sequence of physiological reactions, including hydrogen peroxide (H(2)O(2)) production, changes in ascorbate-glutathione-related antioxidant systems, secondary metabolism (peroxidases, phenolics, and lignification), and developmental changes, was characterized in roots of hydroponically grown Scots pine (Pinus sylvestris) seedlings. Cd (50 microM, 6 h) initially increased superoxide dismutase, inhibited the systems involved in H(2)O(2) removal (glutathione/glutathione reductase, catalase [CAT], and ascorbate peroxidase [APX]), and caused H(2)O(2) accumulation. Elongation of the roots was completely inhibited within 12 h. After 24 h, glutathione reductase activities recovered to control levels; APX and CAT were stimulated by factors of 5.5 and 1.5. Cell death was increased. After 48 h, nonspecific peroxidases and lignification were increased, and APX and CAT activities were decreased. Histochemical analysis showed that soluble phenolics accumulated in the cytosol of Cd-treated roots but lignification was confined to newly formed protoxylem elements, which were found in the region of the root tip that normally constitutes the elongation zone. Roots exposed to 5 microM Cd showed less pronounced responses and only a small decrease in the elongation rate. These results suggest that in cells challenged by Cd at concentrations exceeding the detoxification capacity, H(2)O(2) accumulated because of an imbalance of redox systems. This, in turn, may have triggered the developmental program leading to xylogenesis. In conclusion, Cd did not cause necrotic injury in root tips but appeared to expedite differentiation, thus leading to accelerated aging.
720 citations
TL;DR: Pea (Pisum sativum) roots were treated with aluminum in a calcium solution, and lipid peroxidation was investigated histochemically and biochemically, as well as other events caused by aluminum exposure, finding it to be a relatively early symptom induced by the accumulation of aluminum.
Abstract: Pea (Pisum sativum) roots were treated with aluminum in a calcium solution, and lipid peroxidation was investigated histochemically and biochemically, as well as other events caused by aluminum exposure. Histochemical stainings were observed to distribute similarly on the entire surface of the root apex for three events (aluminum accumulation, lipid peroxidation, and callose production), but the loss of plasma membrane integrity (detected by Evans blue uptake) was localized exclusively at the periphery of the cracks on the surface of root apex. The enhancement of four events (aluminum accumulation, lipid peroxidation, callose production, and root elongation inhibition) displayed similar aluminum dose dependencies and occurred by 4 h. The loss of membrane integrity, however, was enhanced at lower aluminum concentrations and after longer aluminum exposure (8 h). The addition of butylated hydroxyanisole (a lipophilic antioxidant) during aluminum treatment completely prevented lipid peroxidation and callose production by 40%, but did not prevent or slow the other events. Thus lipid peroxidation is a relatively early symptom induced by the accumulation of aluminum and appears to cause, in part, callose production, but not the root elongation inhibition; by comparison, the loss of plasma membrane integrity is a relatively late symptom caused by cracks in the root due to the inhibition of root elongation.
599 citations
TL;DR: A novel mutation in Arabidopsis, hlm1, which causes aberrant regulation of cell death, manifested by a lesion-mimic phenotype and an altered HR, segregated as a single recessive allele exhibited increased resistance to a virulent strain of Pseudomonas syringae pv tomato.
Abstract: The hypersensitive response (HR) in plants is a programmed cell death that is commonly associated with disease resistance. A novel mutation in Arabidopsis, hlm1, which causes aberrant regulation of cell death, manifested by a lesion-mimic phenotype and an altered HR, segregated as a single recessive allele. Broad-spectrum defense mechanisms remained functional or were constitutive in the mutant plants, which also exhibited increased resistance to a virulent strain of Pseudomonas syringae pv tomato. In response to avirulent strains of the same pathogen, the hlm1 mutant showed differential abilities to restrict bacterial growth, depending on the avirulence gene expressed by the pathogen. The HLM1 gene encodes a cyclic nucleotide–gated channel, CNGC4. Preliminary study of the HLM1/CNGC4 gene pro-duct in Xenopus oocytes (inside-out patch-clamp technique) showed that CNGC4 is permeable to both K+ and Na+ and is activated by both cGMP and cAMP. HLM1 gene expression is induced in response to pathogen infection and some pathogen-related signals. Thus, HLM1 might constitute a common downstream component of the signaling pathways leading to HR/resistance.
350 citations
Cites background or methods from "An improved method for monitoring c..."
...25%) by leaf discs from hlm1-1 and Ws-4 inoculated or healthy plants (Baker and Mock, 1994)....
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...%) by leaf discs from hlm1-1 and Ws-4 inoculated or healthy plants (Baker and Mock, 1994)....
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...response to avirulent pathogens in the mutant, cell death was assessed using the Evans blue leaf disc assay (Baker and Mock, 1994)....
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...(D) Observation of leaves with a fluorescence microscope (Zeiss Axiophot using an FT510 filter) shows autofluorescence corresponding to the lesions observed in (B). response to avirulent pathogens in the mutant, cell death was assessed using the Evans blue leaf disc assay (Baker and Mock, 1994)....
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TL;DR: It is shown that Sclerotinia spp.-secreted OA is an elicitor of PCD in plants and is responsible for induction of apoptotic-like features in the plant during disease development, which is essential for fungal pathogenicity and involves ROS.
Abstract: Accumulating evidence supports the idea that necrotrophic plant pathogens interact with their hosts by controlling cell death. Sclerotinia sclerotiorum is a necrotrophic ascomycete fungus with a broad host range (>400 species). Previously, we established that oxalic acid (OA) is an important pathogenicity determinant of this fungus. In this report, we describe a mechanism by which oxalate contributes to the pathogenic success of this fungus; namely, that OA induces a programmed cell death (PCD) response in plant tissue that is required for disease development. This response exhibits features associated with mammalian apoptosis, including DNA laddering and TUNEL reactive cells. Fungal mutants deficient in OA production are nonpathogenic, and apoptotic-like characteristics are not observed following plant inoculation. The induction of PCD by OA is independent of the pH-reducing abilities of this organic acid, which is required for sclerotial development. Moreover, oxalate also induces increased reactive oxygen species (ROS) levels in the plant, which correlate to PCD. When ROS induction is inhibited, apoptotic-like cell death induced by OA does not occur. Taken together, we show that Sclerotinia spp.-secreted OA is an elicitor of PCD in plants and is responsible for induction of apoptotic-like features in the plant during disease development. This PCD is essential for fungal pathogenicity and involves ROS. Thus, OA appears to function by triggering in the plant pathways responsible for PCD. Further, OA secretion by Sclerotinia spp. is not directly toxic but, more subtly, may function as a signaling molecule.
327 citations
Cites background from "An improved method for monitoring c..."
...Cell membranes are permeable and, thus, stained when cell death occurs (Baker and Mock 1994)....
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TL;DR: In this article, the effects of graphene on root and shoot growth, biomass, shape, cell death, and reactive oxygen species (ROS) of cabbage, tomato, red spinach, and lettuce, were investigated using a concentration range from 500 to 2000 mg/L.
326 citations
Cites methods from "An improved method for monitoring c..."
...Evaluation of cell death The cell death of the selected plant roots was evaluated by the method previously described by Baker and Mock [31] using Evans blue (0.025% v/v) for 2 h after 20 days of exposure to different concentrations (0, 500, 1000, and 2000 mg/L) of graphene....
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...Evaluation of cell death The cell death of the selected plant roots was evaluated by the method previously described by Baker and Mock [31] using Evans blue (0....
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References
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TL;DR: The most suitable dyes tested were fluorescein diacetate for viable cells and phenosafranine for dead cells and these dyes stained specifically whether the cells were from cultures of different ages, of different species or if the cells had been treated in various, often toxic, ways.
Abstract: Various stains were tested with suspension cell cultures of Nicotiana tabacum (tobacco), Oryza sativa (rice), Glycine max (soybean), Daucus carota (carrot) and Lycopersicon esculentum (tomato). A drop of the culture medium containing each dye was mixed with a drop of cells on a microscope slide and viewed after 5 min. The most suitable dyes tested were fluorescein diacetate for viable cells and phenosafranine for dead cells. These dyes stained specifically whether the cells were from cultures of different ages, of different species or if the cells had been treated in various, often toxic, ways. The viability of the cells was confirmed by observing cyclosis and plasmolysis, and whether the cells could grow further when cultured.
1,396 citations
349 citations
TL;DR: It is concluded that increased plasmalemma Ca(2+) influx is required for the K(+)/H(+) and hypersensitive responses in tobacco.
Abstract: An early event in the hypersensitive response of tobacco to Pseudomonas syringae pv syringae is the initiation of a K+/H+ response characterized by specific plasma membrane K+ efflux, extracellular alkalinization, and intracellular acidification. We investigated the role of calcium in induction of these host responses. Suspension-cultured tobacco cells exhibited a baseline Ca2+ influx of 0.02 to 0.06 micromole per gram per hour as determined from 45Ca2+ uptake. Following bacterial inoculation, uptake rates began to increase coincidently with onset of the K+/H+ response. Rates increased steadily for 2 to 3 hours, reaching 0.5 to 1 micromole per gram per hour. This increased Ca2+ influx was prevented by EGTA and calcium channel blockers such as La3+, Co2+, and Cd2+ but not by verapamil and nifedipine. Lanthanum, cobalt, cadmium, and EGTA inhibited the K+/H+ response in both suspension-cultured cells and leaf discs and prevented hypersensitive cell death in leaf discs. We conclude that increased plasmalemma Ca2+ influx is required for the K+/H+ and hypersensitive responses in tobacco.
186 citations
"An improved method for monitoring c..." refers methods in this paper
...This spectrophotometric method for quantifying the Evans blue retained by plant tissue has proven to be ideal for studies involving pathogens that cause cell death ( Atkinson et al. 1990; Baker et al. 1993)....
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...Our method is a spectrophotometric assay based on the exclusion of Evans blue stain from intact cells and is a modification of a technique used in earlier studies ! ( Atkinson et al. 1990; Baker et al. 1990)....
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TL;DR: Modifications of the neutral red vital staining technique of Drgsel and co-workers to sort live and dead marine copepods have made possible live-dead determinations of most components of estuarine zooplankton.
Abstract: Modifications of the neutral red vital staining technique of Drgsel and co-workers to sort live and dead marine copepods have made possible live-dead determinations of most components of estuarine ...
122 citations
102 citations
"An improved method for monitoring c..." refers background in this paper
...In general, a cell is considered viable if it has the capability to live, grow and develop (Palta et al. 1978; Pegg 1989 )....
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