AOBPreview originally published online on September 4, 2002
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Annals of Botany 90: 509-516, 2002
© 2002 Annals of Botany Company
Ca2+-induced High Amplitude Swelling and Cytochrome c Release From Wheat (Triticum aestivum L.) Mitochondria Under Anoxic Stress
1 Department of Biosciences, Division of Plant Physiology, PO Box 56, FIN-00014 Helsinki University, Finland
* For correspondence. Fax +358 9 19159552, e-mail kurt.fagerstedt{at}helsinki.fi
Received: 1 October 2002; Returned for revision: 12 February 2002; Accepted: 12 June 2002 Published electronically: 4 September 2002
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ABSTRACT |
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Under stress conditions, mitochondria sense metabolic changes, e.g. in pH, cytoplasmic Ca2+, energy status, and reactive oxygen species (ROS), and respond by induction of the permeability transition pore (PTP) and by releasing cytochrome c, thus initiating the programmed cell death (PCD) cascade in animal cells. In plant cells, the presence of all the components of the cascade has not yet been shown. In wheat (Triticum aestivum L.) root mitochondria, the onset of anoxia caused rapid dissipation of the inner membrane potential, initial shrinkage of the mitochondrial matrix and the release of previously accumulated Ca2+. Ca2+ uptake by mitochondria was dependent on the presence of inorganic phosphate. Treatment of mitochondria with high micromolar and millimolar Ca2+ (but not Mg2+) concentrations induced high amplitude swelling, indicative of PTP opening. Alterations in mitochondrial volume were confirmed by transmission electron microscopy. Mitochondrial swelling was not sensitive to cyclosporin A (CsA)—an inhibitor of mammalian PTP. The release of cytochrome c was monitored under lack of oxygen. Anoxia alone failed to induce cytochrome c release from mitochondria. Oxygen deprivation and Ca2+ ions together caused cytochrome c release in a CsA-insensitive manner. This process correlated positively with Ca2+ concentration and required Ca2+ localization in the mitochondrial matrix. Functional characteristics of wheat root mitochondria, such as membrane potential, Ca2+ transport, swelling, and cytochrome c release under lack of oxygen are discussed in relation to PCD.
Key words: Anoxia, apoptosis, Ca2+, calcium, oxygen deprivation, permeability transition, plant mitochondria, programmed cell death (PCD), reactive oxygen species (ROS), Triticum aestivum, wheat.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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During the last few years a putative role for plant mitochondria as cellular stress sensors and as central organelles in programmed cell death (PCD) has attracted increasing interest. Though several characteristic features of animal cell PCD have not been found as such in plant cell PCD, e.g. morphology of the dying cell and activation of caspases (Jones, 2000), increasing evidence exists for the involvement of plant mitochondria in stress sensing and in the cell death pathway. Caspase-like cysteine proteases have been detected in Arabidopsis thaliana root tissue (Safadi et al., 1997) and in Zea mays roots (Subbaiah et al., 2000).
The role of mitochondria in animal cell death has been studied for a number of years during which the involvement of opening of the mitochondrial permeability transition pore (PTP) has been suggested to be a factor in cell damage. The involvement of mitochondria in apoptosis was further confirmed when mitochondrial proteins such as pro- and anti-apoptotic members of the BCL-2 family, apoptosis-inducing factor (AIF) and cytochrome c were identified to be modulators and participants in the execution phase of apoptosis (reviewed by Bernardi et al., 1999). Mito chondria, together with the endoplasmic reticulum (ER), are major Ca2+ stores, having a central role both in Ca2+-signalling and in cellular Ca2+ homeostasis (reviewed by Smaili et al., 2000). Matrix-localized Ca2+-ions are one of the factors that favour the open conformation of the PTP in animal mitochondria (Bernardi, 1999). A common event preceding apoptosis in animal cells is the accumulation of Ca2+-ions into the mitochondrial matrix that induces opening of the PTP and leads to the loss of inner membrane potential, to the swelling of the mitochondrial matrix and to the release of cytochrome c from the intermembrane space to the cytosol (Bernardi et al., 1999; Smaili et al., 2000).
Earlier studies on Ca2+ transport in plant mitochondria suggest that mitochondria isolated from different plant species vary to a great extent in their ability to take up Ca2+ (Chen and Lehninger, 1973). In a study by Martins and Vercesi (1985), mitochondria isolated from corn coleoptiles were able to transport Ca2+-ions while those of white cabbage leaves did not take up Ca2+-ions. In maize suspension-cultured cells, mitochondria seem to participate in Ca2+ signalling of anoxic stress (Subbaiah et al., 1998). Since results of Ca2+-transport studies on plant mitochondria are still non-uniform, the role of the organelle in Ca2+ homeostasis and Ca2+ signalling calls for further investigation.
Data on the existence of PTP in plant mitochondria have been presented in several articles (Fortes et al., 2001; Arpagaus et al., 2002; Curtis and Wolpert, 2002; Tiwari et al., 2002) and PTP in plant mitochondria has been shown to be sensitive to cyclosporin A (CsA) in some plant species (Arpagaus et al., 2002; Tiwari et al., 2002; Yu et al., 2002), but not others (Fortes et al., 2001; Curtis and Wolpert, 2002).
PCD in plant cells has been described during several developmental stages, such as in leaf senescence and in xylogenesis, and in defence reactions when pathogen attack induces the hypersensitive response (HR) (Jones, 2001). Several abiotic stresses induce PCD in plant cells such as oxygen deprivation stress (Drew et al., 2000), heat stress (Balk et al., 1999) and salt-induced stress (Katsuhara and Kawasaki, 1996). Cytochrome c release from mitochondria, the key event in animal cell PCD, has been shown to occur in several experiments on plant cell PCD indicating the involvement of mitochondria (Yu et al., 2002). Moreover, in a recent paper by Tiwari et al. (2002), the entire cell death pathway from the induction of PCD to the final stage, cell death, has been shown to take place in arabidopsis cells. According to their results, oxidative stress, induced by hydrogen peroxide, enhanced formation of ROS in mitochondria leading to PTP opening, cytochrome c release, ATP depletion and, finally, to cell death (Tiwari et al., 2002).
We have monitored the functional and morphological changes that take place in isolated wheat (Triticum aestivum L. ‘Leningradka’) root mitochondria subjected to high calcium concentrations and oxygen deprivation stress. We have also studied membrane potential and mitochondrial matrix volume changes in the presence and absence of CsA, Ca2+ transport and cytochrome c release from wheat root mitochondria. To confirm the spectrophotometric data, ultrastructural changes induced by high calcium levels and anoxia were studied. According to our results, wheat root mitochondria subjected to known permeability transition inducers in animal cells, e.g. high calcium concentration and phosphate ions, accumulate calcium ions into the mitochondrial matrix. This is followed by high amplitude swelling and cytochrome c release, indicating PTP opening.
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MATERIALS AND METHODS |
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Plant material
Wheat seeds (Triticum aestivum) were soaked for 15 min in tap water containing a small amount of commercial detergent and rinsed in tap water. Imbibition took place for 1 h in warm (40 °C) tap water. Seeds were then planted in plastic trays containing Knop’s nutrient medium and placed in growth chambers. Seeds were grown in the dark (to prevent chloroplast formation) for 6–7 d at a constant temperature (24 °C) with continuous aeration of the nutrient medium.
Isolation of root mitochondria
Roots of the etiolated wheat seedlings were washed several times in tap water and three times in distilled water and kept thereafter at 0–4 °C. Roots were homogenized first with scissors and then using a Waring-Blendor (2 x 3 s at low speed) in extraction medium containing 0·25 M sucrose, 5 mM EDTA, 1 mM EGTA, 1 mM dithioerythritol (DTE), 0·1 % (w/v) BSA, 2 % (w/v) Polyclar AT and 20 mM HEPES–Tris (pH 7·4). The homogenate was filtered through Miracloth. The pH of the homogenate was raised to 7·4 using 1 M Tris during homogenization and after filtration. The filtrate was centrifuged at 12 000 g for 17 min in a Sorvall (Wilmington, USA) RC5C centrifuge (rotor SS-34 fixed angle) at 4 °C. Pellets were re-suspended in approx. 48 ml washing medium containing 0·25 M sucrose, 5 mM EDTA, 1 mM EGTA, 0·1 % (w/v) BSA and 10 mM HEPES–Tris (pH 7·4) and were homogenized using a Potter-Elvehjelm-type glass homogenizer with a loose fitting pestle. This fraction was centrifuged at 6000 g for 5 min and the supernatant was centrifuged at 12 000 g for 10 min. The pellet (washed mitochondrial fraction) was re-suspended in 6 ml washing medium and homogenized using the glass homogenizer.
Washed mitochondria were further purified by layering portions of mitochondria (3 ml) on top of self-generating Percoll density gradient [0·25 M sucrose, 5 mM EDTA, 1 mM EGTA, 0·1% (w/v) BSA, 10 mM HEPES–Tris (pH 7·4) and 28 % (v/v) Percoll], and centrifuged at 40 000 g for 35 min. The layer of purified mitochondria was removed using a pipette and diluted 10–15-fold with a medium containing 0·25 M sucrose, 10 µM EGTA and 10 mM HEPES–Tris (pH 7·4). The diluted mitochondria were centrifuged at 12 000 g for 10 min and the pellets (purified mitochondria) were homogenized and re-suspended in a small volume of washing medium. All steps in the procedure were carried out at 0–4 °C.
Determination of membrane potential changes (
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Membrane potential was measured using safranine O as an indicator of membrane potential changes () recorded by a dual beam UV-Visible spectrophotometer (Hewlett-Packard 8452A; Avondale, USA) using 511 and 533 nm as the measuring and reference wavelengths, respectively (Moore and Bonner, 1982). Measurements were carried out at 25 °C in 1 ml medium containing 0·25 M sucrose, 10 µM EGTA, 5·3 µM safranine O, 10 mM HEPES–Tris (pH 7·4) and 0·2 mg mitochondrial protein.
Determination of Ca2+ transport across the inner mitochondrial membrane
Ca2+ transport of mitochondria was assayed with a metallochromic indicator Arsenazo III (Scarpa, 1979) using the wavelength pair 665 and 685 nm as measuring and reference wavelengths, respectively, recorded by a dual beam UV-Visible spectrophotometer (Hewlett-Packard 8452A). Measurements were carried out at 25 °C in medium (1 ml) containing 0·25 M sucrose, 25 µM EGTA (to chelate Ca2+ originating from impurities in the reagents), 25 µM Arsenazo III, 10 mM HEPES–Tris (pH 7·4) and 0·2 mg mitochondrial protein.
Mitochondrial swelling and determination of released proteins
Mitochondrial volume changes were monitored using a spectrophotometer (UV-Visible Recording Spectrophoto meter, UV-200; Shimadzu, Columbia, USA) at 540 nm (Scorrano et al., 1997). Measurements of swelling were carried out at 25 °C in an incubation medium (1 ml) consisting of 0·25 M sucrose, 10 µM EGTA, 10 mM HEPES–Tris (pH 7·4) and 0·2 mg mitochondrial protein. The duration of each measurement was 30 min. At the end of each measurement samples were centrifuged at 12 000 g for 14 min. Supernatants containing the released proteins were transferred into new tubes, and ice-cold acetone (50 %, v/v) was used to precipitate proteins at –20 °C overnight. After centrifugation, pellets were washed twice with 100 % acetone and twice with 80 % acetone, dried under vacuum and dissolved in SDS buffer (50 mM Tris–HCl, pH 8·5, 2 % SDS, 2 % 2-mercaptoethanol, 20 % glycerol). Samples were boiled for 5 min and centrifuged at full speed for 15 min at 4 °C in a microcentrifuge (Beckman Microfuge ‘E’, Beckman Instruments Inc., Glenrothes, UK). Five microlitres of 0·05 % bromophenol blue with 20 % glycerol was added to the samples.
SDS-PAGE and Western Blot Analysis
Samples containing the released mitochondrial proteins were subjected to 15 % SDS-PAGE using a Bio-Rad (Hercules, USA) Mini-PROTEAN 3 Cell. After electrophoresis, proteins in the gel were visualized using silver staining (O’Connell and Stults, 1997) or transferred onto a nitrocellulose membrane (0·45 µm, Bio-Rad) in a transfer buffer [0·03 M Tris–acetate, pH 8·3, 0·2 M glycine, 20 % (v/v) glycerol] using a Bio-Rad Mini Trans-Blot. Membranes were stained with Ponceau Red to confirm electrophoretic transfer of the proteins. Blocking of membranes was performed using a solution of 3 % (w/v) bovine serum albumin, 0·13 NaCl, 7 mM Na2HPO4, 3 mM NaH2PO4, pH 7·3 (PBS) for 1 h or overnight. A mouse anti-cytochrome c monoclonal antibody (clone 7H8.2C12; BD Pharmingen Biosciences, San Diego, USA) was used as a primary antibody at 1 : 1000 dilution. A goat anti-mouse IgG-conjugated horseradish peroxidase (BD Transduction Laboratories, San Diego, USA) was used as a secondary antibody at a dilution of 1 : 600. The blots were incubated for 2 h at room temperature with the primary and secondary antibodies. Blots were stained with a solution (0·003 % H2O2, 10 mM guaiacol in 0·1 M citrate-phosphate buffer, pH 5·0) to visualize cytochrome c through the peroxidase reaction (Vares et al., 1995).
Transmission electron microscopy (TEM)
Wheat root mitochondria were prepared for TEM at three different stages during the swelling experiments: before swelling of mitochondria had started; during the contraction stage of mitochondria; and at the end of the swelling experiment when the mitochondria were most swollen. The incubation medium consisted of 0·25 M sucrose, 10 µM EGTA, 10 mM HEPES–Tris pH 7·4 and 0·2 mg mitochondrial protein, and was fixed as such with 2·5 % glutaraldehyde for at least 1 h. After fixation, samples were centrifuged at 12 000 g for 14 min and further processed for TEM.
Oxygen consumption by mitochondria
Oxygen consumption of mitochondria was monitored using a Clark-type oxygen electrode (Hansatech Instruments Ltd, Kings Lynn, UK) at 25 °C. The incubation medium (1 ml) contained 0·25 M sucrose, 25 µM EGTA, 100 µM MgCl2, 10 mM HEPES–Tris (pH 7·4) and 0·2 mg mitochondrial protein.
Mitochondrial integrity experiments
Integrity of the isolated wheat root mitochondria was evaluated by measuring the integrity of the outer membrane of the mitochondria by following the reduction of exogenously added cytochrome c by succinate:cytochrome c oxidoreductase at 550 nm in experimental conditions modified after Gualberto et al. (1995). Integrity of the outer membrane of wheat mitochondria was 93·9 % (mean value from three different mitochondrial isolations).
Protein assay
Mitochondrial protein was determined using the Bradford method (Bradford, 1976) using the Bio-Rad Protein Assay (Bio-Rad Laboratories) with bovine serum albumin as a standard.
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RESULTS |
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Monitoring of basic mitochondrial functions, such as the inner membrane potential, oxygen consumption, and corresponding matrix volume changes, were performed under normoxic conditions and after the onset of anoxia. When mitochondria were fed with a respiratory substrate, NADH, and inorganic phosphate (Pi), membrane potential was created rapidly. Simultaneously with the developing membrane potential, the matrix volume of mitochondria increased, i.e. physiological swelling took place, indicated by a decrease in absorbance at 540 nm. Mitochondria energized with NADH (and Pi) consumed all the oxygen in the cuvette in approx. 10–15 min. Addition of Ca2+ increased the respiration rate, and hence oxygen disappeared from the cuvette faster (data not shown). At the onset of anoxia the membrane potential of mitochondria collapsed and simultaneous shrinkage of the mitochondrial matrix occurred (Fig. 1)
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Ca2+ fluxes to and from mitochondria were monitored during normoxic and anoxic conditions (Fig. 2). Initially, energized and respiring mitochondria accumulated Ca2+ rapidly. Once the reaction medium turned anoxic and the membrane potential decreased (Fig. 1), mitochondria began to gradually release the accumulated Ca2+.
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Figure 3 shows the energy-dependent Ca2+ uptake of wheat root mitochondria. The addition of 100 µM Ca2+ caused a rapid increase in absorbance followed by a decrease, indicating Ca2+ uptake by the mitochondria from the reaction medium. Addition of the Ca2+-specific ionophore A23187 caused release of the accumulated Ca2+ from the matrix space.
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High amplitude swelling of mitochondria was observed after treatment with 2·5 mM Ca2+ (Fig. 4, trace 1) and 0·5 mM Ca2+, but not with 100 µM Ca2+ (data not shown). In animal cells, changes in the mitochondrial volume in response to Ca2+ treatment are due to PTP opening and are the prerequisite for PCD. In our experiments, application of cyclosporin A, a specific PTP inhibitor, had no effect on mitochondrial swelling (Fig. 4, trace 2). Swelling after Ca2+ addition is Ca2+-specific, since the substitution of Ca2+ by Mg2+ at the same concentration completely abolished volume changes (Fig. 4, trace 3).
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To visualize ultrastructural changes and to assess the integrity of mitochondrial membranes under Ca2+ treatment and during cytochrome c release, samples were taken for electron microscopy at the times indicated in Fig. 4. At the beginning of the experiment (Fig. 5A), mitochondria were characterized by an electron dense matrix, intact membranes and distinct cristae. No significant changes in mitochondrial ultrastructure were detected during the contraction phase (Fig. 5B). The sample was taken when the amplitude of contraction was maximal (approx. 5 min after addition of 2·5 mM Ca2+). The decrease in absorbance of Ca2+-treated mitochondria at 540 nm was due to profound swelling characterized by volume enlargement, a decrease in matrix density and disintegration of cristae (Fig. 5C and D). Note that the inner mitochondrial membrane remained intact (Fig. 5D).
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To elucidate a specific role for Ca2+ in mitochondria-associated PCD, cytochrome c release was investigated by immunoassay under different Ca2+ concentrations and in the presence/absence of CsA. In potato tuber mitochondria, cyclosporin A has been shown to inhibit PTP only in the presence of a reductant, dithioerythritol (DTE) (Arpagaus et al., 2002), hence, in our experiments, DTE was present in the incubation medium. An intensive band of cytochrome c was detected in the lane where proteins extracted from osmotically and mechanically broken mitochondria were separated (Fig. 6A). In isolated mitochondria, the integrity of the outer membrane was lost continuously during the 30 min of the experiment (from 93·4 % integrity to 84·2 % in the case of non-treated actively respiring mitochondria); hence minor cytochrome c release can occur in an uncontrolled manner. In our experiments such a release was almost below the level of sensitivity of the immunoassay (Fig. 6B). It is important to point out that the experiments on cytochrome c release were performed on mitochondria undergoing oxygen deprivation stress. Anoxia alone did not cause cytochrome c release (Fig. 6B); only combination with Ca2+ treatment in the millimolar range resulted in significant release of cytochrome c (Fig. 6E–H). The presence of 1·5 µM CsA and 1 mM DTE in the incubation medium to inhibit PTP did not affect cytochrome c release from Ca2+-treated mitochondria (cf. Fig. 6E and F–H).
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Cytochrome c bands were detected in samples treated with high concentrations of Ca2+ (0·5 and 2·5 mM; Fig. 6E and G). Treatment of mitochondria with 100 µM Ca2+ did not cause such a pronounced release of cytochrome c (Fig. 6D), neither did it cause any high amplitude swelling (data not shown). If Pi was excluded from the incubation medium to prevent Ca2+ uptake into mitochondria, the release of cytochrome c was less pronounced (Fig. 6I). Substitution of 2·5 mM Ca2+ with 2·5 mM Mg2+ resulted in significant inhibition of cytochrome c release (Fig. 6J).
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DISCUSSION |
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In the sequence of events leading to a PCD in animal cells, such factors as PTP opening, mitochondrial swelling, and cytochrome c release are the checkpoints of the PCD process. PTP opening is favoured by depolarization of the inner mitochondrial membrane, high matrix Ca2+ level, and high matrix pH (Bernardi et al., 1999). Permeability transition results in swelling of mitochondria which is considered to be one of the mechanisms of cytochrome c release (Martinou et al., 2000). Under normal physiological conditions, wheat mitochondria respond with energization-dependent matrix volume changes of an osmotic nature (Fig. 1). Under anoxic stress, dissipation of the membrane potential caused passive leakage of matrix ions resulting in concurrent water efflux and reduction of mitochondrial volume (Fig. 1). In wheat root mitochondria, the dissipation of the membrane potential not only affects the matrix volume but also results in the release of previously accumulated Ca2+ from the matrix (Fig. 2), a phenomenon that has been described as one of the prerequisites for PCD. The appearance of Ca2+ in close proximity to mitochondria under anoxic shock, and Ca2+ uptake upon re-oxygenation, have been observed in maize cells (Subbaiah et al., 1998). According to earlier investigations not all plant mitochondria are able to take up Ca2+ from the medium (Chen and Lehninger, 1973; Fortes et al., 2001). Wheat root mitochondria, however, were able to take up Ca2+ from the incubation medium and released it upon application of the Ca-ionophore A23187, thus verifying matrix localization of Ca2+ (Fig. 3). Application of a millimolar concentration of Ca2+ caused high amplitude swelling of mitochondria (Fig. 4). The effect was specific to Ca2; Mg2+ in the same concentration did not cause any changes in mitochondrial volume (Fig. 4, trace 3). Similar results have been demonstrated by Arpagaus et al. (2002). These findings support the involvement of both a highly specific Ca2+ uptake mechanism and successive permeability transition. Application of a PTP-inhibitor, CsA, did not prevent high amplitude swelling (Fig. 4, trace 2). However, an inhibitory effect of CsA under the same experimental conditions has been shown in potato tuber mitochondria (Arpagaus et al., 2002). In contrast, in another potato cultivar, Bintje, CsA-insensitive permeabilization has been detected under de-energized conditions (Fortes et al., 2001).
In our experiments, wheat root mitochondria were able to take up Ca2+, swell, and release cytochrome c in a Ca2+-dependent and CsA-insensitive manner. Indeed, if Ca2+ was not present in the medium (Fig. 6B), or was substituted by Mg2+ (Fig. 6J), no significant release of cytochrome c was detected. Likewise, the exclusion of Pi (required to sustain Ca2+ uptake into mitochondria) from the incubation medium resulted in a considerable decrease in cytochrome c release (Fig. 6I). Therefore, Ca2+ has to act from the matrix side of plant mitochondria to provide the release of cytochrome c, possibly via PTP-induction. The amount of cytochrome c released correlated positively with Ca2+ concentration, i.e. at low Ca2+ concentrations (100 µM, Fig. 6D) the release of cytochrome c was negligible as compared with that under 0·5 mM Ca2+ (Fig. 6E) or 2·5 mM Ca2+ (Fig. 6G) treatments. This in vitro finding may reflect the in vivo situation when mitochondria act as cytoplasmic Ca2+-sensors: small amounts of Ca2+ are taken up (cytoplasmic clearance) while high cytoplasmic Ca2+ triggers PCD via PTP opening. In the experiments of He and Lemasters (2002), the concentration of Ca2+ was shown to affect the sensitivity of mitochondrial swelling to CsA: 100 and 500 µM Ca2+ induced CsA-sensitive swelling, while 1 mM Ca2+ induced swelling that was not blocked by CsA. This behaviour has been attributed to the two-mode PTP operation: regulated CsA-sensitive, and unregulated CsA-insensitive (He and Lemasters, 2002).
Cytochrome c release from plant mitochondria has been detected in earlier experiments where permeability transition and PTP opening has been induced (Arpagaus et al., 2002; Curtis and Wolpert, 2002; Tiwari et al., 2002). Cytochrome c release from plant mitochondria to the cytosol has also been detected in plant cells during PCD, such as in cucumber seedlings exposed to heat (Balk et al., 1999), tobacco protoplasts exposed to menadione treatment (Sun et al., 1999), maize suspension-cultured cells exposed to mannose (Stein and Hansen, 1999) and in anther cells of a sterile sunflower (Balk and Leaver, 2001). According to the results, cytochrome c release is considered to be an essential step in plant cell PCD as it is in animal PCD. However, no cytochrome c release was detected from mitochondria due to PCD in pollination-induced senescence of petunia petals (Xu and Hanson, 2000). Different approaches have been used to reveal common steps between animal and plant cell PCD. In tobacco leaves expressing mammalian Bax, a pro-apoptotic member of the BCL-2 family of proteins, cell death and defence reactions were induced suggesting that mammalian Bax activated the cell death pathway in tobacco cells (Lacomme and Santa Cruz, 1999). Similar results have been obtained with tobacco overexpressing animal-derived anti-apoptotic proteins, BCL-XL and Ced-9, which inhibited cell death in tobacco cells (Mitsuhara et al., 1999). Moreover, in the experiments of Zhao et al. (1999), animal-derived cytochrome c added to carrot cell cytosol led to the activation of caspase-like proteins, which in turn induced apoptosis in mouse liver cells. These reports suggest that plant and animal cells share—at least to some extent—a conserved evolutionary pathway for the cell death cascade.
Our demonstration of CsA-insensitive, Ca2+-dependent high amplitude swelling and cytochrome c release in wheat root mitochondria suggests PTP opening. Insensitivity of high amplitude swelling to CsA may be an inherent property of wheat PTP or may reflect the fact that under these particular experimental conditions sensitivity was not induced. In animal tissues under particular conditions, treatment of mitochondria by butylated hydroxytoluene, the signal peptide mastoparan, the hormone thyroxine, and palmitic acid elicit a CsA-insensitive permeability transition (Sultan and Sokolove, 2001). A differently regulated, CsA-insensitive PTP has been described in yeast mitochondria (Jung et al., 1997). Together these findings indicate that the induction and regulation of PTP (He and Lemasters, 2002) and the release of cytochrome c (Andreyev et al., 1998; Martinou et al., 2000) can occur via several mechanisms and require specific conditions in both plant and animal tissues.
During the transition from normoxia to anoxia, which gradually leads to lowered adenylate energy charge (Hanhijärvi and Fagerstedt, 1995), cytoplasmic acidosis, elevation of cytoplasmic Ca2+ (Subbaiah et al., 1998) and increased probability of ROS formation (Blokhina et al., 2001), plant mitochondria undergo high amplitude swelling, releasing Ca2+ and cytochrome c in a regulated manner. In animals this controlled sequence of reactions is characteristic of PCD. It is noteworthy that in wheat root mitochondria a diminished O2 concentration alone fails to induce these changes, unless high micromolar Ca2+ (but not Mg2+) is present. The process has to be tightly regulated since transient Ca2+ uptake into the mitochondrial matrix is necessary to trigger the cascade of above-mentioned reactions under oxygen deprivation. In turn, lack of O2 sets limits for Ca2+ uptake due to the dissipation of inner membrane potential. For the survival of the whole organism under hypoxic stress, the ability to induce PCD can be beneficial (e.g. aerenchyma formation, Drew et al., 2000; elimination of ROS-overproducing cells).
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ACKNOWLEDGEMENTS |
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We wish to acknowledge the technical support of the Electron Microscopy Unit of the Institute of Biotechnology, University of Helsinki. This work was funded by the Academy of Finland and the Finnish Ministry of Education as part of the Center of Excellence on Plant Biology (project no. 164346).
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