JXB Advance Access originally published online on February 14, 2005
Journal of Experimental Botany 2005 56(413):997-1006; doi:10.1093/jxb/eri093
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RESEARCH PAPER |
Involvement of the mitochondrial KATP+ channel in H2O2- or NO-induced programmed death of soybean suspension cell cultures
áková *
Section of Plant Biology, Department of Biology and Agro-Industrial Economics, University of Udine, via Cotonificio 108, I-33100 Udine, Italy
To whom correspondence should be addressed. Fax: +39 0432 558784. E-mail: biolveg{at}dbea.uniud.it
Received 6 July 2004; Accepted 28 November 2004
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Abstract |
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Soybean suspension cell cultures were treated by H2O2 or nitric oxide (NO), to assess the mechanism leading to programmed cell death (PCD). Hydrogen peroxide (5 mM) induced PCD. Cells become necrotic at 20 mM H2O2, with cells exhibiting intermediate hallmarks before that (necrapoptotic cells). The level of ATP and of glucose-6-phosphate remained constant in cells undergoing PCD, while it decreased significantly in the necrotic ones. Mitochondria, isolated from 5 mM H2O2-treated (apoptotic) cells, showed that succinate-dependent oxygen consumption was slightly uncoupled, and the electrical potential difference (
) weakly decreased. The addition of KCl to the formed determined a partial dissipation, which was higher than the dissipation observed in mitochondria from control cells. The addition of cyclosporin A (CsA) to de-energized mitochondria also induced formation, due to a K+ efflux from the matrix, which was decreased in mitochondria from treated cells. The same pattern of response was also observed in mitochondria isolated from 1 mM sodium nitroprusside (NO)-treated cells, exhibiting apoptotic symptoms. In mitochondria isolated from 20 mM H2O2-treated (necrotic) cells, succinate-dependent oxygen consumption was completely uncoupled, generation significantly inhibited, and CsA-dependent formation prevented. In addition, mitochondria isolated from control cells still underwent swelling, which was partially or completely prevented in mitochondria isolated from apoptotic or necrotic cells, respectively. The moderate swelling was accompanied by a slight rupture of the outer membrane and by a release of cytochrome c. These results point to the involvement of a 
channel during the manifestation of PCD induced by H2O2 or NO in plants.
Key words:
Hydrogen peroxide, channel, mitochondria, nitric oxide, programmed cell death, soybean cells
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Introduction |
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Programmed cell death (PCD) is a well-recognized process in plants, which occurs during development and in the response of plants to environmental cues. In particular, PCD fulfils several functions related to aleurone and root cap cell elimination, somatic embryogenesis, leaf and petal senescence, xylogenesis, reproduction (Greenberg, 1996
; Pennell and Lamb, 1997), aerenchyma formation (Drew et al., 2000) and the response of the cell to biotic and abiotic stresses (Greenberg, 1996; Pennell and Lamb, 1997; Beers and McDowell, 2001; Lam et al., 2001). Plant PCD is accompanied by some morphological changes that appear to be similar to those described during apoptosis (a form of PCD) of animal cells (Danon et al., 2000). The main hallmarks concern chromatin condensation, cytoplasm shrinkage, and DNA cleavage, while other PCD markers (e.g. apoptotic bodies) have not been described. In addition, plant PCD is characterized, in some instances (e.g. xylogenesis), by the disruption of the tonoplast and the subsequent release of hydrolases sequestered in the vacuole (Jones, 2000). These symptoms are differentially expressed depending on the purposes of controlled cell death that occurs in plant cells (Lam, 2004).
Programmed death is commonly distinguished from the necrotic (oncotic) death, although these forms of cell death are considered as the two extremes of the same phenomenon, named ‘necrapoptosis’ (Lemasters, 1999). At least in animal cells, the life-or-death switch is controlled by a delicate equilibrium between anti- and pro-apoptotic factors. In this context, a major role is played by proteins of the Bcl-2 family (Tsujimoto and Shimuzu, 2000). From a mechanistic point of view, cell death can be subdivided into three phases: initiation, decision, and degradation (Kroemer and Reed, 2000). During the first phase, cells accumulate effector molecules, which directly and selectively alter the permeability of the mitochondrial membranes. This determines the release of mitochondrial apoptogenic factors (i.e. cytochrome c, apoptosis-inducing factors, smac-diablo, etc.) that trigger the degradation phase. The last phase implies the activation of specific cysteine proteases (caspases) and then endonucleases, which are responsible for the typical degradation products of PCD. In this scenario a major role is performed by mitochondria, which perform a crucial step during the manifestation of mammalian PCD (Desagher and Martinou, 2000; Kroemer and Reed, 2000; Bernardi et al., 2001).
Programmed death is also under control in plant cells, where phytohormones, lipid and membrane-associated signals, as well as oxidative stress can act as regulators (Lam, 2004). The involvement of plant mitochondria in PCD has recently been suggested (Jones, 2000). Although mounting evidence supports this view (Christensen et al., 2002; Maxwell et al., 2002; Tiwari et al., 2002; Yao et al., 2002), their possible function in controlling the cell death execution phase remains to be elucidated (Lam, 2004). In addition, no genes for Bcl-2 proteins or caspases have been identified in plants yet (Watanabe and Lam, 2004). Nevertheless, a vast amount of indirect evidence suggests the existence of caspase-like activity (Woltering et al., 2002).
The most common mitochondrial mechanistic feature between mammalian and plant PCD is the release of cytochrome c, which has been found in different plant cells, in which death was induced by heat (Balk et al., 1999), menadione (Sun et al., 1999), PET1-cytoplasmic male sterility mutation (Balk and Leaver, 2001), or oxidative stress (Tiwari et al., 2002). In mammalian cells, this release has been explained by mechanisms implying the involvement of both mitochondrial swelling and non-swelling (Desagher and Martinou, 2000; Kroemer and Reed, 2000; Bernardi et al., 2001). In the first case a major role is played by the so-called permeability transition pore (PTP) on the inner membrane, responsible for a high-amplitude swelling. This mitochondrial swelling determines the outer membrane rupture and the consequent release of cytochrome c. The non-swelling mechanism is based on the voltage-dependent anion channel (VDAC) or porin, located on the outer membrane. The VDAC interacts with pro-apoptotic proteins of the Bcl-2 family to form channels through which the apoptogenic factors are released.
A swelling mechanism, involving cyclosporin A-sensitive and -insensitive PTP, has also been claimed to be responsible for the cytochrome c release from plant mitochondria during anoxia (Arpagaus et al., 2002; Virolainen et al., 2002). However, this release can be explained by a different mechanism, involving a channel, also located on the inner mitochondrial membrane (Chiandussi et al., 2002). In addition, a non-swelling mechanism has been suggested (Lam et al., 1999) on the basis of experiments with tobacco cells, in which a pro-apoptotic protein (Bax) was expressed (Lacomme and Santa Cruz, 1999). The Bax, forming a pore with VDAC, would allow the release of apoptogenic proteins.
The hypersensitive response (HR) is a form of a localized cell death induced by an invading pathogen. This response is considered a form of PCD (Lam et al., 2001). An increase of two signal molecules (H2O2 and NO), generated by the host cell, is typically induced by the invading pathogen or by elicitors (oxidative and nitrosative burst, respectively) (Beers and McDowell, 2001; Delledonne et al., 2002; Neill et al., 2002).
Therefore, in this work, the PCD was induced in soybean suspension cell cultures by H2O2 or NO: (i) to assess the conditions leading to programmed or necrotic death; (ii) to determine the involvement of plant mitochondria in this phenomenon; and (iii) to clarify the mechanism of cytochrome c release from the latter organelles.
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Materials and methods |
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Plant material and growth conditions
Two cell lines derived from callus induced on the sterile embryonal axes of Glycine max (L.) Merr., var. Nikir (Pioneer) were maintained in GB5 (Gamborg et al., 1968
) liquid medium, supplemented with 30 g l–1 sucrose and 1 mg l–1 2,4-dichlorophenoxyacetic acid at 22 °C under a 16/8 h light/dark regime, with agitation at 100 rpm. Cells were subcultured at 7 d intervals by transferring suspension aliquots, which represent 8 ml of packed cell volume, into 180 ml of fresh medium.
Chemicals and treatments
All the compounds added to cell cultures (hydrogen peroxide, sodium nitroprusside (SNP) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (Carboxy-PTIO)) and used for the experiments (except for the ATP assay) were purchased from Sigma-Aldrich. In the case of experiments with sodium nitroprusside, a stock solution in TRIS–HCl buffer was freshly made on the day of the experiment and all the cell cultures were kept in the dark.
Cell density and viability
To evaluate cell density, aliquots of the cell culture were either filtered, centrifuged, and weighed during the course of the experiment, or the total weight of the culture was estimated by filtration through a 50 µm nylon mesh after 5, 24 or 144 h.
Cell death was estimated by Evan's blue staining (0.4% w/v, final concentration) as described by Baker and Mock (1994). Stained (apoptotic and necrotic cells) and unstained cells were observed by a Leitz Fluovert microscope (100x magnification) and counted in 10 fields containing at least 50 cells per field.
Nuclear morphology
Cells were washed and resuspended in 50 mM TRIS–HCl (pH 7.2), fixed with 2% (v/v) formaldehyde for at least 30 min and then washed twice with the resuspending medium. Cells were incubated in 0.1% (v/v) Triton X-100 and 1 µg ml–1 4,6-diamino-2-phenylindole (DAPI) for 20 min. Nuclei were visualized using a Leitz Fluovert microscope with an excitation filter of 360 nm and emission filter of 420 nm.
Measurement of cellular glucose-6-phosphate
Aliquots of soybean cells were filtered and centrifuged at 480 g (3000 rpm) for 2 min by a Sorvall (HB-4). Cells were collected, weighed, and resuspended in 50 mM TRIS–HCl (pH 7.5), 0.05% (v/v) Triton X-100, and boiled for 2 min. After a brief centrifugation, the supernatants were collected and used for the fluorimetric determination of cytosolic glucose-6-phosphate.
Glucose-6-phosphate was monitored as an increase of fluorescence of ß-NADPH by a Perkin Elmer LS5B spectrofluorimeter. Wavelengths at 429 nm and 460 nm for excitation and emission were used, respectively (Bergmeyer et al., 1974). The incubation mixture in TRIS–HCl contained 0.125 mM ß-NADP+ and an aliquot (100 µl) of the supernatant. The reaction was started with 0.5 IU ml–1 of glucose-6-P dehydrogenase.
Measurement of cellular ATP
Supernatants prepared as above were used for total cellular ATP determination, by means of the luciferin–luciferase luminometric assay, except for the addition of 5 mM EDTA in lysis buffer. Aliquots (200 µl) of supernatant were mixed with 150 µl of luciferine–luciferase buffer (50 mM glycine, pH 8.0, 7.5 mM DTE, 1 mM EDTA, 2 mM MgSO4, 20 µM luciferin, and 5 µg ml–1 luciferase) in 1 ml of 50 mM TRIS–HCl (pH 7.5). Both reagents were purchased from Biaffin GmbH & Co KG. The signals were detected by means of a luminometer (LKB-Wallac, model 1250). The actual ATP concentration of each experiment was calculated from an ATP calibration curve (8–100 nM) constructed with commercially purchased ATP.
Isolation of mitochondria
Cells were filtered through 50 µm nylon mesh and weighed. Approximately 20 g of fresh cells were taken and used for mitochondria isolation. Cells were homogenized at 4 °C in a mortar with 140 ml of extraction medium composed of 20 mM HEPES-TRIS (pH 7.5), 0.3 M sucrose, 5 mM Na-EDTA, 1 mM DTE, 0.3% (w/v) BSA, and 0.6% (w/v) PVPP. The filtrate was centrifuged at 2500 g (SS34 Sorvall rotor) for 3 min, the supernatant was taken and centrifuged at 28 000 g for 5 min. The pellet was resuspended in the isolation buffer, without PVPP, by a Potter homogenizer and then centrifuged at 2500 g for 3 min. The supernatant was finally centrifuged at 28 000 g for 5 min and the final pellet, resuspended in c. 0.5 ml of 20 mM HEPES-TRIS (pH 7.5), 0.3 M sucrose, and 0.1% (w/v) BSA, constituted the mitochondrial fraction.
Membrane electrical potential () measurement
Safranin O was used to estimate changes, as previously described (Petrussa et al., 2001). Variations of fluorescence (F), expressed in arbitrary units (AU), were evaluated. The incubation mixture was made up to 2 ml (final volume) of resuspending medium to which mitochondria (0.1 mg protein ml–1) and 5 mM succinic acid (to energize mitochondria), were added.
Mitochondrial swelling
Swelling experiments were performed as described by Pastore et al. (1999). Absorbance changes at 540 nm of the mitochondrial suspension (0.3 mg protein ml–1) in 0.15 M KCl and 20 mM HEPES-TRIS (pH 7.2) were monitored at 25 °C by a Perkin-Elmer 
15 spectrophotometer.
Cytochrome c release determination
Mitochondrial proteins, isolated from living (control) and H2O2- or NO-treated cells (showing PCD hallmarks), were separated by SDS-PAGE (15% acrylamide/4% acrylamide stacker), to detect the presence of cytochrome c according to the method described by Mather and Rottenberg (2001), with minor changes. The resolved polypeptides were electroblotted to a nitrocellulose membrane in 25 mM TRIS–0.2 M glycine–20% (v/v) methanol transfer buffer for 90 min at 15 V. The blots were saturated for 1 h in a blocking buffer (5% skimmed milk in TRIS-saline buffer) and then incubated overnight at 4°C with anti-cytochrome c monoclonal antibody (7H8.2C12), as primary antibody (1/500). After washing, the membrane was incubated with anti-mouse peroxidase-conjugated IgG (1/80 000) for 1 h at room temperature. Labelling was detected using the ‘SuperSignal West Dura’ substrate, chemiluminescence reagent, according to the supplier's manual (Pierce). The membrane was then exposed to an X-ray film (X-OMAT, East Man Kodak, Rochester, NY, USA) for 5 min.
Oxygen consumption determination
Oxygen consumption was monitored at 25 °C by a Clark-type oxygen electrode. The incubation medium and substrates of mitochondria were the same as for measurements.
Protein determination
The mitochondrial protein was determined by the Bradford method (Bradford, 1976), using the BioRad protein assay.
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Results |
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Programmed (apoptotic) versus unprogrammed (necrotic) death in suspension cell cultures of soybean
The first set of experiments was aimed at distinguishing cells undergoing necrotic or programmed (apoptotic) death (McCabe and Leaver, 2000
). Figure 1 shows the time-dependent death in soybean suspension cell cultures (evaluated by Evan's blue), which had been treated with 5 mM H2O2 (Fig. 1A) or 1 mM SNP (NO) (Fig. 1B). The starting cultures exhibited per se a certain number of dead cells. This level, however, remained constant in control samples, while it increased after 5 h and 24 h in H2O2-treated cells. The same pattern was also obtained after 144 h of incubation with NO. In this specific case, a known NO scavenger, carboxy-PTIO, prevented the NO-induced death.
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A fine parameter to distinguish apoptosis from necrosis in mammalian cells is the ATP level (Lemasters, 1999
). In apoptotic cells ATP has to be maintained high to allow the formation of the apoptosome (Desagher and Martinou, 2000; Kroemer and Reed, 2000), while in the necrotic cells it is significantly lowered. As can be seen in Fig. 2A, ATP concentration in soybean suspension cells was slightly decreased or significantly lowered by 5 or 20 mM H2O2 treatments, after 5 h incubation, respectively. Cell death and ATP concentration after treatment with 20% (v/v) ethanol were comparable to those obtained after 20 mM H2O2 treatment. The level of glucose-6-phosphate, a related parameter, was also determined (Fig. 2B). Its concentration remained high, after 5 h incubation, in 5 mM H2O2-treated cells, whereas it greatly decreased in 20 mM H2O2- or 20% ethanol-treated cells. Microscopic observations of the same soybean cells, stained by Evan's blue (Fig. 3), showed that, when compared with control cells (Fig. 3a), the cytoplasm of 5 mM H2O2-treated cells was shrunk (Fig. 3b, apoptotic cells). Treatment by 20 mM H2O2 caused their partial cell lysis (necrapoptotic cells, Fig. 3c), while 20% ethanol determined their complete cell lysis (necrotic cells, Fig. 3d). After 144 h incubation, NO caused a cytoplasmic shrinkage (not shown) suggesting that even these cells underwent a programmed death. In agreement, the nuclei of control cells, stained by DAPI, exhibited an orthodox conformation (Fig. 4a), while 5 mM H2O2- or 1 mM SNP-treated cells showed a clear-cut chromatin condensation (Fig. 4b, c). Therefore, cells exhibiting programmed death still had high levels of ATP and glucose-6-phosphate. Conversely, in the early stages of necrotic death, the concentration of ATP and glucose-6-phosphate decreased significantly. These results show that ATP and glucose-6-phosphate are two valuable parameters for distinguishing programmed (apoptotic) from necrotic death in plant cells. In addition, they show that, in the population of soybean suspension cultures, are present cells exhibiting apoptotic, necrotic or necrapoptotic hallmarks.
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Features of mitochondria isolated from suspension cell cultures of soybean under programmed or necrotic death
Crude mitochondria were isolated by conventional techniques from control and 5 mM H2O2-treated suspension cell cultures, in which programmed cell death was occurring. The isolation was performed when the dead cells reached c. 30–40% of the total. Purified mitochondria were not used, because this procedure would have resulted in the elimination of mitochondria that could be damaged by treatments (de Virville et al., 1994
).
Organelles from control samples showed a succinate-dependent and ADP-stimulated oxygen consumption (Fig. 5A, trace a). The respiratory control ratio (RCR) was approximately 3±0.4, thus indicating that mitochondria were coupled. During state 4 respiration, KCN demonstrated c. 40% inhibition, which was completely blocked after diamide addition. The resulting KCN-insensitive O2 uptake (alternative oxidase capacity) was c. 59%. Mitochondria, isolated from H2O2-treated cells (trace b), were partially uncoupled (RCR=c. 2), with an alternative oxidase capacity comparable to that of the control. Alternative oxidase was probably not at its maximal capacity, because it was evaluated in the absence of reducing agents (activators) or keto-acids (allosteric effectors). The succinate-dependent generation (Fig. 5B, trace a) was also partially decreased (trace b). The addition of KCl, caused a more significant dissipation in mitochondria from H2O2-treated cells than in those from control cells. The residual completely collapsed with FCCP.
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Hence, mitochondria from H2O2-treated cells possess the known
channel (Pastore et al., 1999) in a more opened conformation, thus allowing the entry of K+ into the mitochondrial matrix. The opening of this channel can be induced by the well-known immunosuppressive agent, cyclosporin A (CsA) (Petrussa et al., 2001). Its activity can, therefore, be easily followed as a CsA-induced generation in de-energized mitochondria, resuspended in a low-salt medium, as a consequence of an efflux of K+ from the mitochondrial matrix. While CsA built up in mitochondria from control cells, this was partially inhibited in mitochondria from 5 mM H2O2-treated cells (Fig. 5C, traces a and b). Therefore, the channel seems to operate mainly in an inwardly rectifying fashion, in mitochondria from treated cells.
The same pattern of responses could also be followed in mitochondria isolated from NO-treated cells. When compared with control mitochondria, oxygen consumption was slightly uncoupled (Fig. 6A) and accompanied by a mild inhibition of succinate-energized formation (Fig. 6B) and CsA-dependent formation (Fig. 6C). These results, therefore, show that 5 mM H2O2 or 1 mM SNP (NO) treatments of soybean cells caused a loss of mitochondrial functionality and a minimal rupture of the outer membrane (78% and 72% of integrity in mitochondria from control and treated cells, respectively).
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Mitochondria were also extracted from 20 mM H2O2-treated soybean cells, undergoing mainly a necrotic death (c. 30–40% of dead cells). The succinate-dependent O2 uptake was completely uncoupled in mitochondria from treated cells (RCR=c. 1) and the alternative oxidase capacity was 59% of O2 uptake in control mitochondria against 33% in those from treated-cells (Fig. 7A, traces a and b). Accordingly, the succinate-dependent
generation was significantly inhibited in mitochondria from H2O2-treated cells (Fig. 7B, traces a and b). The dissipation of by KCl was comparable in mitochondria from both control and H2O2-treated cells. The addition of CsA determined the generation of a typical in control mitochondria (Fig. 7C, trace a), while it was completely suppressed in organelles from treated cells (Fig. 7C, trace b). These effects were accompanied by a pronounced rupture of the outer membrane (78% and 39% integrity in mitochondria from control and treated cells, respectively).
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The picture emerging in mitochondria isolated from cells where necrotic or programmed death was occurring, is different. In mitochondria from necrotic cells, the functionality of these organelles was dramatically compromised; in the other case, mitochondria were still functioning and had an open
channel with an inward-rectifying activity. The channel determined the entry of K+ from the cytoplasm to the mitochondrial matrix, resulting in partial dissipation of and in a mitochondrial swelling. This interpretation was confirmed by the observation that isolated mitochondria from control cells, resuspended in KCl medium (Fig. 8A, trace a), underwent swelling. Conversely, in organelles from 5 or 20 mM H2O2-treated cells the swelling was slightly (Fig. 8A, trace b) or completely prevented (Fig. 8A, trace c), respectively. This suggests that mitochondria from treated cells were already partially swollen or completely disrupted, before isolation, depending of the type of treatment.
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The most widespread feature that relates animal and plant mitochondria to programmed cell death is the release of cytochrome c (Desagher and Martinou, 2000
; Kroemer and Reed, 2000). Accordingly, the mitochondrial fraction, isolated from H2O2- or NO-treated cells contained less cytochrome c than control cells (Fig. 8B). This suggests that both treatments induced its release, through a moderate swelling-dependent mechanism, which seems to be mediated by the channel. |
Discussion |
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It is known that some cells, in suspension cultures, die during the normal course of their proliferation. The death is accelerated by the addition of specific inducers, which determine a necrotic or a programmed death, depending on cell suspension density, the physiological state, or the level of the stressors (McCabe et al., 1997
, Lo Schiavo et al., 2000; McCabe and Leaver, 2000). According to others (Beers and McDowell, 2001; Delledonne et al., 2002; Neill et al., 2002) and in this work, it was observed that cell death in soybean suspension cultures could also be induced by H2O2 and NO alone. As already seen (Houot et al., 2001), the concentration of H2O2 was crucial to determine the type (necrotic or apoptotic) of cell death. In any case, the phenomenon appears to be the same, because either cells exhibiting necrotic symptoms (at high concentration of H2O2) or apoptotic symptoms (at low concentration of H2O2) were found, with some cells showing intermediate hallmarks. Therefore, as suggested for animal cells (Lemasters, 1999), necrotic and programmed death in plant cells can also be considered as the two extremes of the same phenomenon, named necrapoptosis.
In mammals, the ATP level is a valuable parameter with which to distinguish necrotic from apoptotic cells (Desagher and Martinou, 2000; Kroemer and Reed, 2000; Bernardi et al., 2001). ATP concentration is maintained constant in apoptotic cells, while it decreases in necrotic cells. A similar response has now been observed in soybean suspension cell cultures, although an ATP depletion has been associated with the manifestation of PCD in tobacco (BY-2) cells, treated by benzyladenosine (Mlejnek et al., 2003), or in Arabidopsis thaliana cells subjected to oxidative stress (Tiwari et al., 2002). In addition, the concentration of glucose-6-phosphate exhibited the same trend. Therefore, necrotic and apoptotic cells can be distinguished on the basis of either morphological (cytoplasmic shrinkage, chromatin condensation, etc.) or biochemical features (ATP or glucose-6-phosphate levels, release of cytochrome c, etc.). Among the biochemical hallmarks, glucose-6-phosphate appears to be a valuable additional parameter.
The function of plant mitochondria in programmed cell death is still controversial, although their involvement has been claimed in plant cells undergoing programmed death, triggered by several inducers (Greenberg, 1996; Pennell and Lamb, 1997; Beers and McDowell, 2001; Lam et al., 2001). The results presented here confirm the critical role of these organelles, through the release of the apoptogenic factor, cytochrome c, which is the most common biochemical feature linking plant and mammalian cells subjected to programmed death.
At least in plant cells, the morphological hallmarks linked to programmed cell death can be distinguished into two types. In the hypersensitive response the disruption of the vacuole takes place at a late stage of cell death after chromatin condensation, whereas during tracheary differentiation this happens before DNA fragmentation (Lam, 2004). The role of mitochondria may be different in these two situations. In the hypersensitive response, mitochondria can have an active role in death, while in the tracheary differentiation they appear to undergo autolysis, similar to other cell components. While mitochondria are likely involved in plant PCD, their contribution could be explained by mechanisms implying the release of intermembrane space proteins, such as cytochrome c (Balk et al., 1999; Sun et al., 1999; Balk and Leaver, 2001; Tiwari et al., 2002) or a recently identified Mg2+-dependent nuclease (Balk et al., 2003).
The release of cytochrome c from animal mitochondria has been explained in different ways involving both swelling and non-swelling mechanisms (Desagher and Martinou, 2000; Kroemer and Reed, 2000; Bernardi et al., 2001). Current knowledge in plant cells is still scarce. Cytochrome c could be released through VDAC (non-swelling mechanism) (Lacomme and Santa Cruz, 1999; Lam et al., 1999) or through of CsA-sensitive or -insensitive PTP (swelling mechanism) (Arpagaus et al., 2002; Fortes et al., 2001; Virolainen et al., 2002). This evidence, however, appears to be indirect only (Balk et al., 1999; Sun et al., 1999; Balk and Leaver, 2001; Tiwari et al., 2002). In addition, plant mitochondria possess a CsA-stimulated channel (Petrussa et al., 2001), regulated by H2O2 and NO (Chiandussi et al., 2002), and is responsible for a low-amplitude permeability transition, which determines, in isolated mitochondria, a moderate swelling (Petrussa et al., 2004). The results presented here suggest that the channel could be involved during mitochondrial swelling and release of cytochrome c, induced in H2O2- or NO-treated cells. These effects seem to be caused by the inwardly rectifying conformation of the channel and the consequent K+ influx electrically coupled to an electrogenic H+ efflux (Garlid and Paucek, 2003). This mechanism significantly resembles that already described in human mitochondria, where a Bcl-2-sensitive K+ accumulation is responsible for both swelling dependent- and independent-permeabilization of the outer membrane (Eliseev et al., 2002). This moderate swelling is regulated in an opposite manner by pro-(truncated Bid) and anti-(Bcl-2) apoptotic proteins, belonging to the Bcl-2 family (Eliseev et al., 2003). Although no evidence for the presence of genes/proteins of the Bcl-2 family is available in plant genomic databases (Watanabe and Lam, 2004), the K+ accumulation shown in human mitochondria corroborate this interpretation of the involvement of the plant mitochondrial channel in the manifestation of PCD.
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Acknowledgements |
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This research was supported by the ‘Ministero dell’ Istruzione, dell'Università e della Ricerca' (Cofin 2001–2002) in the frame of the program entitled: ‘Improvement of plant resistance to pathogens by the induction of endogenous mechanisms of defence’ and by Regione Friuli-Venezia Giulia (LR 3/1998).
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Footnotes |
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* Permanent address: Forest Research Institute, TG Masaryka, 22, 960 92 Zvolen, Slovakia.
Abbreviations: NO, nitric oxide; PCD, programmed cell death; , electrical potential difference; CsA, cyclosporin A; PTP, permeability transition pore; VDAC, voltage-dependent anion channel; HR, hypersensitive response; SNP, sodium nitroprusside; Carboxy-PTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; DAPI, 4,6-diamino-2-phenylindole; DTE, dithioerythritol; PVPP, polyvinylpolypyrrolidone; F, variations of fluorescence; RCR, respiratory control ratio; FCCP, carbonyl cyanide 4-(trifluoromethoxyphenylhydrazone).
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