Journal of Experimental Botany, Vol. 52, No. 361, pp. 1721-1730,
August 1, 2001
© 2001 Oxford University Press
Original Papers |
Hydrogen peroxide induces programmed cell death features in cultured tobacco BY-2 cells, in a dose-dependent manner
UMR INRA-Université de Bourgogne, Phytopharmacie et Biochimie des Interactions Cellulaires, INRA BP 86510, 21065 Dijon cedex, France
Received 12 January 2001; Accepted 22 March 2001
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Abstract |
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Active oxygen species (AOS), especially hydrogen peroxide, play a critical role in the defence of plants against invading pathogens and in the hypersensitive response (HR). This is characterized by the induction of a massive production of AOS and the rapid appearance of necrotic lesions is considered as a programmed cell death (PCD) process during which a limited number of cells die at the site of infection. This work was aimed at investigating the mode of cell death observed in cultures of BY-2 tobacco cells exposed to H2O2. It was shown that H2O2 is able to induce various morphological cell death features in cultured tobacco BY-2 cells. The hallmarks of cell death observed with fluorescent and electron microscopy differed greatly with the amount of H2O2 added to the cell culture. The appearance of nuclear fragmentation similar to ‘apoptotic bodies’ associated with a fragmentation of the nuclear DNA into small fragments appear for almost 18% of the cells treated with 12.5 mM H2O2. The early stages of the induction of this PCD process consisted in cell shrinkage and chromatin condensation at the periphery of the nucleus. Above 50 mM, H2O2 induces high necrotic cell death. These data suggest that H2O2-induced cell damage is associated with the induction of various cell death processes that could be involved differently in plant defence reactions.
Key words: Apoptosis, DNA fragmentation, hydrogen peroxide, hypersensitive response, nuclear fragmentation, tobacco BY-2 cells, programmed cell death.
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Introduction |
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Plants have developed elaborate mechanisms to protect themselves from invading pathogens and aggressors. Complementary to pre-existing physical and chemical barriers, various defence responses are induced after pathogen attack leading to the induction of plant resistance (for review see Kombrink and Somssich, 1995
). In recent years, natural plant defence reactions gained high attention both in ‘reasoned agriculture’ and fundamental research. Such studies were focused on plant–pathogen recognition, signal transduction and induction of resistance such as systemic acquired resistance (SAR). The activation of resistance in plants is generally initiated by host recognition of elicitors directly or indirectly released from the invading pathogen. Despite their various chemical composition, many elicitors of plant defence reactions share a signalling pathway that co-ordinates the plant defences (Ebel and Cosio, 1994; Ebel and Mithöfer, 1998). The main common defence reaction is the hypersensitive response (HR), characterized by the induction of rapid cell death at the site of the attempted attack by either an avirulent strain of a pathogen or a non-pathogen (for review see Dangl et al., 1996). HR includes some physiological perturbations such as ion fluxes across the plasma membrane (namely Ca2+ influx and K+, Cl- efflux), pH changes, plasma membrane depolarization, and an oxidative burst, i.e. the consumption of molecular O2 and its reduction to components collectively named Active Oxygen Species (AOS), including 
(superoxide radical), H2O2 (hydrogen peroxide) and HO (hydroxyl radical). HR is accompanied by a set of defence reactions, including activation of defence genes (Ward et al., 1991; Zhu et al., 1996) and the onset of systemic acquired resistance correlated with the systemic induction of genes, especially genes encoding pathogenesis-related proteins (Lamb et al., 1989; Ryals et al., 1996). In addition, H2O2 could play a key role in plant HR (Doke, 1983; Alvarez et al., 1998). Death of infected and stressed cells in order to prevent systemic spread of a pathogen appears to be a conserved strategy in both plants and animals and recent studies might indicate that HR cell death could be considered as programmed cell death (PCD) (Jabs et al., 1996; Naton et al., 1996; Lamb and Dixon, 1997) during which a limited number of cells die at the site of infection (Greenberg et al., 1994; Greenberg, 1997; Pontier et al., 1998). The biological significance of PCD in plants, especially its implication in plant defence reactions, has attracted considerable interest (Lam et al., 1999). AOS play key roles in the initiation of PCD and analogies were observed concerning cell death programmes in animals and in plants (Mittler et al., 1997, 1999; Desikan et al., 1998). The most commonly studied PCD in animals is apoptosis, which was defined as changes in the cell structure culminating with the appearance of apoptotic bodies and DNA fragmentation. PCD inducible by various stimuli was a question of central interest in the elucidation of HR and systemic acquired resistance processes.
In many systems, sublethal oxidative stress was found to be involved either directly or indirectly in PCD processes (for review see Payne et al., 1995; Lamb and Dixon, 1997). PCD was observed in plants but not always with all apoptotic hallmarks as reviewed earlier (Buckner et al., 2000). Now there is a consensus that PCD in plants rarely follows the same apoptotic processes as in animals.
It was shown previously that H2O2 induces PCD in soybean and Arabidopsis thaliana cell cultures (Levine et al., 1994; Desikan et al., 1998; Solomon et al., 1999), but recent evidence also suggests that cell death and the induction of defence genes are activated by the same signal but are regulated by separate mechanisms (for review see Richberg et al., 1998).
In the present work, the events leading to the death of cultured tobacco BY-2 cells treated with various doses of hydrogen peroxide have been investigated with light and electron microscopy. It is thus shown that H2O2, a signal involved in HR and SAR, can induce PCD by a process similar to apoptosis (cell shrinkage, chromatin condensation, vesicularization of the plasmic and nuclear membranes, formation of micronuclei similar to ‘apoptotic bodies’, DNA fragmentation), but only in a dose-dependent manner.
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Materials and methods |
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Tobacco cells
Tobacco (Nicotiana tabacum L. cv. Bright Yellow, BY-2) cell suspensions were grown in liquid Murashige and Skoog medium with constant shaking (150 rpm) at 25 °C, and subcultured as previously described (Hemmerlin and Bach, 1998
). Cells in the exponential growth phase were exposed to H2O2 for various times under culture conditions. H2O2 was added to the cell suspension at final concentrations between 0 and 100 mM. Control and treated cells were collected by filtration at various times, washed, frozen in liquid nitrogen and stored at -80 °C before genomic DNA extraction, or immediatly treated for microscopy. All experiments were carried out in sterile conditions.
Cytological methods
The viability of cells was measured by the addition of erythrosin B (Sigma; 4 mg ml-1) that only penetrates dead cells and leads to a red staining of the cellular contents, or by addition of fluorescein diacetate (Sigma; 100 µg ml-1) that is specific to living cells (Widholm, 1972). The non-polar molecule enters the living cells where esterase cleaves off the acetate residues leaving fluorescein which then accumulates and, under UV light, fluoresces leading to a yellow-green fluorescence-staining of the cytoplasm. Cells were observed under white or fluorescent light. For epifluorescence microscopy, 4 ml of tobacco cells in suspension were treated by adding 20 µl of Tritonx100 (10% w/w) and 60 µl formaldehyde (37% w/w) whereby fixed tissues are stable within 1 month. Hoechst 33258 (Sigma, 10 µg ml-1) was used to observe nuclear morphology by epifluorescent microscopy using 365 nm as excitation and 410 nm as barrier. Five hundred cells were observed in each preparation and the numbers of each morphological change were expressed as a percentage of the total cells.
For transmission electron microscopy, tobacco cells were collected at various times after treatment with H2O2, fixed with 2% glutaraldehyde and 3% formaldehyde in 0.1 M phosphate buffer pH 7.4 for 4 h, at room temperature. Cells were post-fixed with 0.5% osmium tetroxide in phosphate buffer for 1 h at 4 °C, washed and treated with tannic acid. The samples were then dehydrated in a graded ethanol series and embedded in Epon 812 (TAAB, England). Ultra-thin sections (90 nm) were cut with an ultramicrotome (Reichert, Ultracut E), stained with 3% uranyl acetate in ethanol for 30 min and in lead citrate for 10 min before observation (Hitachi 600 transmission electron microscope operating at 75 kV).
Isolation of genomic DNA and gel electrophoresis of DNA
Total genomic DNA was isolated from control or treated cells using the DNeasy Plant Mini kit (Qiagen France). Briefly, the frozen tobacco cells were first mechanically disrupted with a mortar and pestle in the presence of liquid nitrogen, then lysed at 65 °C. RNase in the lysis buffer digests the RNA. After lysis, proteins and polysaccharides are salt-precipitated and removed by centrifugation through the QIAshredder column. DNA binds to the membrane in the presence of binding buffer and ethanol, whereas the contaminants are washed. Pure DNA was eluted in a small volume of low-salt buffer or water. Electrophoresis was carried out on 1% agarose gel in TAE buffer in the presence of ethidium bromide. The gels were observed with a BioImager (Biorad).
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Results |
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Effect of H2O2 on viability of cultured tobacco BY-2 cells
As shown in Fig. 1
, the exposure of cultured tobacco BY-2 cells to exogenous H2O2 for 4, 10 or 18 h resulted in a dose-dependent increase of death rate compared with control cells (time 0).
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), 10 h (
) or 18 h (•) after the treatment, stained either with fluorescein diacetate (specific for living cells) or with erythrosin B (specific for dead cells) and counted. Each point represents the mean of three independent experiments.This experiment provided evidence of high cytotoxicity of exogenous H2O2 only above 50 mM. At 50 and 100 mM cell death increased with time of treatment: within 4 h after the addition of 100 mM H2O2, 40% of cells are dead whereas 18 h after the treatment, 50% (50 mM) or 90% (100 mM) of the cells were dead and exhibited high cellular shrinkage, as observed with erythrosin B. The low percentage of surviving cells was confirmed by fluorescein diacetate staining. Above 100 mM, cell death occurred more quickly (1–3 h), with dramatic swelling and total cell disorganization. The amount of H2O2 added appears high, but it is important to note that H2O2 is quickly degraded in the culture medium. It was observed that almost 80% H2O2 (measured by luminol) was destroyed within 1 min when added to the culture medium cleared from cells by filtration (not shown). Thus the amount of H2O2 actually entering the cell was greatly decreased.
Characterization of structural changes in tobacco BY-2 cells treated with H2O2
To determine more precisely the effect of exogenous H2O2, tobacco BY-2 cells were treated with 0–100 mM H2O2 for various times, harvested, fixed, stained with Hoechst 33258 and observed with epifluorescence microscopy.
As shown in Fig. 2, H2O2-treated tobacco cells undergo various morphological changes depending principally on the amount of H2O2. Some structural changes were observable 10 h after the treatment. The first observable event was a condensation of chromatin manifesting as hyperchromasia of DNA stained with Hoechst 33258. Compared with control cells (Fig. 2A), cells treated with H2O2 showed chromatin condensed at the periphery of the nucleus (Fig. 2B: ‘pre-apoptotic nuclei’), or the appearance of micronuclei (Fig. 2C, D: ‘apoptotic-like nuclei’) resembling those observed during the induction of apoptosis in animal cells, for example, in hematopoietic cells such as HL60 (Solary et al., 1994). As can be seen using mixed light, H2O2 also induces high cell content shrinkage on these apoptotic-like cells (Fig. 2E, F). This cell shrinkage was confirmed by erythrosin B (not shown). In some cases, a loss of nuclear architecture (‘lace’ chromatin appearance and ‘stretched nuclei’: Fig. 2G) or empty cells with completely lysed nuclei (Fig. 2H) were also observed.
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Figure 3
shows the proportion of the different types of morphological modifications related to the concentration of exogenous H2O2 applied to cultured tobacco BY-2 cells. Above 20 mM of H2O2, cells with ‘stretched’ and ‘lace’ nuclei predominate. Moreover, the highest percentage of ‘apoptotic-like’ nuclei was obtained with 12.5 mM H2O2. The frequency of apoptotic-like cells progressively increased 10 h after treatment with H2O2, reaching a maximum (almost 18%) between 18 h and 20 h, especially for cells treated with 12.5 mM H2O2 (final concentration). The cells presenting apoptotic-like bodies are still alive, as observed with erythrosin B and with fluorescein diacetate, effective cell death taking place later (by 30 h). Conversely, cells showing ‘lace nuclei’ or high ‘stretched nuclei’ were dead within 18 h. At high concentrations in H2O2 (100 mM and higher) dead cells were observed 4 h after the start of treatment.
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Characterization of ultrastructural changes in tobacco BY-2 cells treated with H2O2
The ultrastructural study was performed on control tobacco BY-2 cells and at various times after exposure to H2O2. Longitudinal sections through tobacco BY-2 cells, fixed 10, 15 or 20 h after the addition of H2O2 to the suspension culture, were observed by conventional electron microscopy and attention was focused on tobacco cells treated with 12.5 mM H2O2. As previously observed, 18–20% of tobacco cells undergo nucleus fragmentation within 20 h after the addition of H2O2. Figure 4 shows various stages of the ultrastructural changes induced by H2O2 in tobacco cells. Thus, Fig. 4A, B and C shows untreated cultured cells with an intact nuclear envelope and weakly condensed chromatin. Significant alterations occur in tobacco cells by 10–15 h after the treatment with H2O2. Firstly, an apparent condensation of nuclear material was observed throughout the nucleus (Fig. 4D, E, F), as well as a condensation of the cytoplasm with shrinkage of the cell contents, while the cell walls appear unaltered. The appearance of invaginations of the nuclear envelope was also observed. Figure 4G, H and I shows a progressive amplification of these changes 10–20 h after the treatment, with a high condensation of chromatin and cytoplasm, and invaginations of the nuclear and plasma membranes with the formation of vesicles containing highly condensed material attached to their interior surface (Fig. 4J, K, L). Favourable images of the nuclear bulge show that these membrane profiles originate from the invaginations of the nuclear envelope. These morphological features correspond to those of apoptosis but the vesicularization appear slightly different to those observed in animal cells undergoing apoptosis, probably due to the presence of a cell wall. At these late stages the nucleus shows a high condensation of the remaining chromatin and numerous vesicles (Fig. 4M, N, O). The cells with ‘lace’ and ‘stretched’ nuclei, classed as dead cells when observed with erythrosin B or fluorescein diacetate, appear to be highly collapsed. These cells, presenting anarchic structural changes resembling explosive necrosis, are weak and very difficult to observe. Hence, H2O2 (5–100 mM) appears to induce necrosis and apoptotic-like cell death.
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Internucleosomal DNA fragmentation in cultured tobacco BY-2 cells treated with H2O2
Internucleosomal cleavage of nuclear DNA into nucleosomal fragments, which are multiples of 180 bp, is usually identified by agarose gel electrophoresis as DNA ladders in apoptotic animals cells. Similarly, DNA laddering was also detected in plants (Gao and Showalter, 1999) but detection of such a laddering could be difficult depending on the percentage of affected cells in the total population.
Total DNA was extracted from control cells and from cells treated with increased amount of H2O2 within 20 h. The integrity of DNA was monitored by electrophoresis in a 1.5% agarose gel (Fig. 5). DNA isolated from control cells migrated as an unresolved high molecular weight band of more than 10 kbp in length. In contrast, DNA from cells treated with 12.5 mM H2O2 showed a fragmentation-forming ladder as observed previously in animal cells undergoing apoptosis and in some plant cells (Koukalova et al., 1997; Gao and Showalter, 1999). The bands 170–190 bp apart are characteristic of internucleosomal DNA fragmentation. Such a fragmentation was not observable using lower or higher concentrations of H2O2 (not shown).
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Fragmentation of DNA, occurring within 18–20 h after the treatment and concerning a small amount of the genomic DNA could correspond to the appearance of the ‘apoptotic-like bodies’ in almost 20% of tobacco cells. Taken together, the two features are consistent with a PCD response resembling the apoptotic pathway.
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Discussion |
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In this study, it was shown that exogenous H2O2 applied to BY-2 tobacco cell suspensions cultures, induced various cell death processes, amongst which was an apoptotic-like process that was closely dependent on the amount of applied H2O2. The most important result obtained in this work is the observation of all the major hallmarks of apoptosis following the treatment of tobacco BY-2 cells with 12.5 mM H2O2. Cell content shrinkage with a high condensation of the cytoplasm, chromatin condensation first at the periphery of the nucleus and then as apoptotic like-bodies seen with epifluorescence microscopy and electron microscopy, and cleavage of nuclear DNA into small fragments of almost 170–190 bp typical of internucleosomal DNA fragments were all observed. Moreover, above 50 mM, H2O2 induces necrotic cell death with cell disorganization but normal chromatin appearance. H2O2 between 12.5 and 50 mM was shown to induce various cell death features not entirely typical of apoptosis or necrosis.
The results obtained in the present study support the following conclusions:
- (a) H2O2 plays an important role in the cell death signalling pathway in plant cells leading to various types of cell death.
- (b) Exogenous H2O2 could initiate an apoptotic-like cell death process in cultured tobacco BY-2 cells only at a given concentration, suggesting that there are complex processes in PCD regulation.
- (b) Exogenous H2O2 could initiate an apoptotic-like cell death process in cultured tobacco BY-2 cells only at a given concentration, suggesting that there are complex processes in PCD regulation.
Similar to the data reported here on the features of cell death in tobacco BY-2 cells treated with H2O2, O'Brien and coworkers showed that camptothecin, okaidic and salicylic acid, or hydrogen peroxide and the calcium ionophore A23187 induced chromatin condensation and DNA fragmentation in tobacco protoplasts (O'Brien et al., 1998). Exposure of cells to a low concentration or removal of the chemical agent resulted in an initial phase of chromatin condensation followed by its reversal. These results are not easily transposable to cultured tobacco cells because protoplasts could be considered as ‘stressed’ cells. However, in this work it was observed that tobacco cells treated with 12.5 mM H2O2 and showing ‘apoptotic like bodies’ are still alive while cells showing high condensation of cytoplasm are dead.
These data concur with results obtained on cultures of mouse distal–proximal straight tubule cells where H2O2 induced a dose-dependent decrease in cell viability (Takeda et al., 1999). Such cells showed features of necrosis, apoptosis, oncosis, and apoptotic necrosis. These data are suggestive of the possible involvement of H2O2, in whole plants, for the induction of various PCD pathways depending on the amounts of H2O2 (and/or other AOS) accumulated in given cells. The mechanism of plant cell death could be very similar to apoptosis described in animals, as all the phenomena (nuclear condensation, cell shrinkage, DNA fragmentation, protease activity, etc.) were previously observed even if not in the same model. Thus, when studying some of the morphological and biochemical events associated with PCD during the hypersensitive response of tobacco plants infected with tobacco mosaic virus, Mittler et al. observed a condensation and vacuolization of the cytoplasm and cleavage of nuclear DNA to 50 kb large fragments (Mittler et al., 1997). In contrast, internucleosomal fragmentation, condensation of the chromatin at the nuclear periphery, and apoptotic bodies were not observed. The authors concluded that their findings suggest that certain aspects of PCD may have been conserved during the evolution of plants and animals. In the present study, the application of 12.5 mM H2O2 to cultured tobacco BY-2 cells allowed the induction of cell death with the hallmarks of apoptosis as previously described in mammalian cells, especially the appearance of nuclear fragmentation similar to ‘apoptotic bodies’ associated with the cleavage of part of the nuclear DNA into small fragments. The presence of a cell wall in plant cells could prevent the development of the same events as in animal cells, especially the appearance of typical apoptotic bodies and phagocytosis.
The concentration of H2O2 applied exogenously to tobacco cells was high (12.5 mM), but H2O2 (at least 80%) was very rapidly destroyed by plant cell cultures (due to the presence of catalases and peroxidases) and the amount of H2O2 that penetrates in the cells was not easily measurable. Such amounts of H2O2 (5–100 mM) were previously used to induce cell death in plant cell suspension cultures (Levine et al., 1994; Desikan et al., 1998). Low levels of H2O2 may be sufficient for the induction of defence-related genes, but higher levels are needed for the induction of cell death (Levine et al., 1994). For example, after the addition of elicitors of defence reactions in tobacco cell suspensions, the production of AOS, as measured by the luminol assay, was almost 50–100 µM Eq H2O2 (not shown). According to these observations it could be possible to retain a biological significance to treatment with H2O2 at high concentrations (between 5–100 mM). Moreover, in tobacco leaves treated by infiltration of H2O2 it was observed in the infiltrating area cells presenting necrotic hallmarks (swelling and disruption) or apoptotic hallmarks such as chromatin condensation, nuclear blebbing and cell shrinkage (not shown).
It has previously been reported that tcI 7 a gene encoding a ß-subunit of proteasome was up-regulated in tobacco cells and leaves treated with either elicitins, salicylic acid or H2O2, and the 5' flanking region of tcI 7 was cloned by PCR gene walking (Petitot et al., 1997; Etienne et al., 2000). By sequence analysis various putative regulatory boxes were localized such as myb boxes (regulated by salicylic acid) and a NF-
B regulatory sequence. In animals, it was known that NF-B transcription factors respond to H2O2 (Schreck et al., 1991), and H2O2 is an intracellular molecular signal activating NF-B in the induction of inflammatory, immune and stress responses. Moreover, recent papers place proteasomes at the top of the apoptotic machinery in animals, and it is possible that the ubiquitin proteasome system integrates various stimuli for or against apoptotic processes, generating a decision which is executed by the proteolysis of key proteins (for review see Wojcik, 1999). The possible implication of the ubiquitin-proteasome-dependent protein degradation responses of plants to various stresses has been shown (Conrath et al., 1998; Ito et al., 1999). Moreover, using epidermal cells of hypocotyls from etiolated cucumber seedlings and specific inhibitors of proteasome, the implication of the ubiquitin/proteasome system as a process that switches on the signalling pathway for diverse plant responses has been shown (Becker et al., 2000). It has also been suggested that signalling for H2O2 elicitation (Tenhaken and Rübel, 1998) and the HR (Boyes et al., 1998) may involve proteins with short half-lives. Then, it could be suggested as a working hypothesis that genes encoding various subunits of proteasome amongst which is tcI 7 activated by H2O2, pathogens or elicitors could be involved in plant defence mechanisms via the induction of various PCD processes.
In a recent review on cell death in animals, it was concluded that there are multiple pathways leading to cell death (Fiers et al., 1999). Furthermore, different pathways can co-exist in the same cell and are switched on by specific stimuli. Apoptotic cell death and necrotic cell death could be two extremes of various more complex real situations. In plants, AOS could play a capital role in atypical forms of PCD, named apoptotic/necrotic cell death, as reported in mammalian cells treated with 1 mM H2O2 (Anderson et al., 1999). In a recent work, it was examined if two early molecular markers of HR cell death (HIN1 and HSR203J) as well as a senescence marker (SAG12) are co-ordinately induced during the cell death processes (Pontier et al., 1999). The results indicated evidence of some cross-talk between both cell death pathways with distinct spatial and temporal characteristics of gene activation during HR and senescence. It was also revealed that a senescence-like process seems to be triggered at the periphery of the HR lesion. The authors suggested that cells committed to die during the HR might release a signal able to induce senescence in the neighbouring cells corresponding to the establishment of a second barrier against pathogens. The results of this study indicate the possible induction of various PCD processes in plant cells.
Moreover, it was previously observed that elicitins able to induce high production of reactive oxygen species and high foliar necrosis are also able to induce the highest protection (for review see Ponchet et al., 1999). This protection depends on a complex signalling network including the production of active oxygen species (especially hydrogen peroxide) and specific lipid peroxidation (Rustérucci et al., 1996). The lipoxygenase-dependent peroxidative pathway responsible for tissue necrosis appears as being one of the features of hypersensitive cell death. The results and hypothesis presented here agree in part with the results and hypothesis recently described earlier (Sasabe et al., 2000) which characterized pharmacologically the hypersensitive cell death of tobacco BY-2 cells treated with INF1, the major secreted elicitin of late blight pathogen P. infestans. INF1 elicitin treatment resulted in the fragmentation of tobacco DNA as early as 3 h post-treatment. The authors showed that the cell death observed following INF1 treatment was likely to depend on proteases and Ca2+, but they did not observe apoptotic bodies previously observed in tomato protoplasts treated with AAL toxin secreted by Alternaria alternata f.sp lycopersici (Wang et al., 1996). The authors suggest that PCD in plants may result in different forms depending on the inducing stimulus.
It is important to optimize the study of morphological and molecular hallmarks of PCD in order to characterize more precisely the various pathways leading to PCD in plants.
Based on the results from this work, it could be suggested that only cells accumulating a given concentration of H2O2 and/or polyunsaturated fatty acid hydroperoxides, and thus undergoing apoptosis-like processes, could play an important role in the induction of systemic acquired resistance. However, when applied to tobacco leaves, elicitins are able to initiate apoptotic-like cell death occurring within 15–22 h after treatment, but only in discrete cells as observed under an epifluorescence microscope (not shown). Hence, ongoing work using methods able to show precisely the induction of cell death, cell per cell, in plants will provide a better understanding of the key processes leading to systemic acquired resistance in plants.
Moreover, these data show that H2O2 cause tobacco BY-2 cells to die with PCD processes that depend on the AOS concentration. It is suggested that the HR response in planta, characterized by the production of AOS and the appearance of cell death, could involve various cell death processes including apoptotic-like cell death, necrotic cell death and intermediary programmed cell death processes. The implication of the occurrence of these various cell death process remains unknown and further studies should be performed to identify them.
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Acknowledgments |
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We are grateful to Professor E Solary for helpful discussions on cell death, especially on apoptotic processes. We thank Jeanine Lherminier for help in electron microscopy and Spencer Brown for the generous gift of tobacco BY-2 cells. We are also thankful to Dr Ochatt for critical reading of the English manuscript and helpful discussions. This work is supported by INRA and by the ‘Conseil Régional de Bourgogne’. Ph Etienne is supported by a grant from the ‘Ministère de l'Education Nationale, de la Recherche et la Technologie’.
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Notes |
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1 To whom correspondence should be addressed. Fax: +33 3 80 69 32 65. E-mail: suty{at}epoisses.inra.fr
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Alvarez ME, Pennell RI, Meijer PJ, Ishikawa A, Dixon RA, Lamb C. 1998. Reactive oxygen intermediates mediate a systemic signal network in the establishment of plant immunity. Cell 92, 773–778.[Web of Science][Medline]
Anderson KM, Seed T, Ou D, Harris JE. 1999. Free radicals and reactive oxygen species in programmed cell death. Medical Hypotheses 52, 457–463.
Becker J, Kempf R, Jeblick W, Kauss H. 2000. Induction of competence for elicitation of defence responses in cucumber hypocotyls requires proteasome activity. The Plant Journal 21, 311–316.[Web of Science][Medline]
Boyes DC, Nam J, Dangl JL. 1998. The Arabidopsis thaliana RPM1 disease resistance gene product is a peripheral plasma membrane protein that is degraded coincident with the hypersensitive response. Proceedings of National Academy of Sciences, USA 95, 15849–15854.
Buckner B, Johal GS, Janick-Buckner D. 2000. Cell death in maize. Physiologia Plantarum 108, 231–239.
Conrath U, Klessig DF, Bachmair A. 1998. Tobacco plants perturbed in the ubiquitin-dependent protein degradation system accumulate callose, salicylic acid and pathogenesis-related protein1. Plant Cell Reports17, 876–880.
Dangl JL, Dietrich RA, Richberg MH. 1996. Death don't have no mercy: cell death programs in plant–microbe interaction. The Plant Cell 8, 1793–1807.[Web of Science][Medline]
Desikan R, Reynolds A, Hancock JJ, Neill SJ. 1998. Harpin and hydrogen peroxide both initiate programmed cell death but have differential effects on defence gene expression in Arabidopsis suspension cultures. The Biochemical Journal 330, 115–120.
Doke N. 1983. Involvement of superoxide anion generation in the hypersensitive response of potato tuber tissues to infection with an incompatible race of Phytophthora infestans and to hyphal wall components. Physiology of Plant Patholology 23, 359–367.
Ebel J, Cosio E. 1994. Elicitors of plant defence responses. International Review of Cytology 148, 1–36.
Ebel J, Mithöfer A. 1998. Early events in the elicitation of plant defence. Planta 206, 335–348.[Web of Science]
Etienne P, Petitot A-S, Houot V, Blein J-P, Suty L. 2000. Induction of tcI 7, a gene encoding a ß-subunit of proteasome, in tobacco plants treated with elicitins, salicylic acid or hydrogen peroxide. FEBS Letters 466, 213–218.[Web of Science][Medline]
Fiers W, Beyaert R, Declercq W, Vandenabeele P. 1999. More than one way to die: apoptosis, necrosis and reactive oxygen damage. Oncogene 18, 7719–7730.[Web of Science][Medline]
Gao M, Showalter AM. 1999. Yariv reagent treatment induces programmed cell death in Arabidopsis cell cultures and implicates arabinogalactan protein involvement. The Plant Journal 19, 321–331.[Web of Science][Medline]
Greenberg JT. 1997. Programmed cell death in plant–pathogen interaction. Annual Review of Plant Physiology and Plant Molecular Biology 48, 525–545.[Web of Science]
Greenberg JT, Ailan G, Klessig D, Ausubel FM. 1994. Programmed cell death in plants: a pathogen-triggered response activated coordinately with multiple defence functions. Cell 77, 551–563.[Web of Science][Medline]
Hemmerlin A, Bach TJ. 1998. Effects of mevinolin on cell cycle progression and viability of tobacco BY-2 cells. The Plant Journal 14, 65–74.[Web of Science][Medline]
Ito N, Seo S, Ohtsubo N, Nakagawa H, Ohashi Y. 1999. Involvement of proteasome-ubiquitin system in wound-signalling in tobacco plants. Plant Cell Physiology 40, 355–360.
Jabs T, Dietrich R, Dangl JL. 1996. Initiation of runaway cell death in an Arabidopsis mutant by extracellular superoxide. Science 273, 1853–1856.
Kombrink E, Somssich IE. 1995. Defence responses of plants to pathogens. Advances in Botanical Research 21, 1–34.
Koukalova B, Kovarik A, Fajkus J, Siroky J. 1997. Chromatin fragmentation associated with apoptotic changes in tobacco cells exposed to cold stress. FEBS Letters 414, 289–292.[Web of Science][Medline]
Lam E, Pontier D, del Pozo O. 1999. Die and let live-programmed cell death in plants. Current Opinion in Plant Biology 2, 502–507.[Web of Science][Medline]
Lamb C, Dixon RA. 1997. The oxidative burst in plant disease resistance. Annual Review of Plant Physiology and Plant Molecular Biology 48, 251–275.[Web of Science]
Lamb CJ, Lawton MA, Dron M, Dixon RA. 1989. Signals and transduction mechanisms for activation of plant defences against microbial attack. Cell 56, 215–224.[Web of Science][Medline]
Levine A, Tenhaken R, Dixon RA, Lamb C. 1994. H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell 79, 583–593.[Web of Science][Medline]
Mittler R, Herr EK, Orvar BL, van Camp W, Willekens H, Inzé D, Ellis B. 1999. Transgenic tobacco plants with reduced capability to detoxify reactive oxygen intermediates are hyper-responsive to pathogen infection. Proceeding of the National Academy of Sciences, USA 96, 14165–14170.
Mittler R, Simon L, Lam E. 1997. Pathogen-induced programmed cell death. Journal of Cell Science 110, 1333–1344.[Abstract]
Naton B, Hahlbrock K, Schmelzer E. 1996. Correlation of rapid cell death with the metabolic changes in fungus-infected, cultured parsley cells. Plant Physiology 112, 433–444.[Abstract]
O'Brien I, Baguley B, Murray B, Morris B, Ferguson I. 1998. Early stages of the apoptotic pathway in plant cells are reversible. The Plant Journal 13, 803–814.[Web of Science]
Payne CM, Bernstein C, Bernstein M. 1995. Apoptosis overview emphasizing the role of oxidative stress, DNA damage and signal transduction pathways. Leukemia and Lymphoma 19, 43–93.
Petitot AS, Blein JP, Pugin A, Suty L. 1997. Cloning of two plant cDNAs encoding a ß-type proteasome subunit and a transformer-2-like SR-related protein: early induction of the corresponding genes in tobacco cells treated with cryptogein. Plant Molecular Biology 35, 261–269.[Web of Science][Medline]
Ponchet M, Panabières F, Milat M-L, Mikes V, Montillet J-L, Suty L, Triantaphylides C, Tirilly Y, Blein J-P. 1999. Are elicitins cryptograms in plant–Oomycete communications? Cellular and Molecular Life Sciences 56, 1020–1047.[Web of Science][Medline]
Pontier D, Tronchet M, Rogowsky P, Lam E, Roby D. 1998. Activation of hsr 203, a plant gene expressed during incompatible plant–pathogen interactions, is correlated with programmed cell death. Molecular Plant–Microbe Interaction 11, 544–554.
Pontier D, Gan S, Amasino RM, Roby D, Lam E. 1999. Markers for hypersensitive response and senescence show distinct patterns of expression. Plant Molecular Biology 39, 1243–1255.[Web of Science][Medline]
Richberg MH, Aviv DH, Dangl JL. 1998. Dead cells do tell tales. Current Opinion in Plant Biology 1, 480–485.[Web of Science][Medline]
Rustérucci C, Stallaert V, Milat ML, Pugin A, Ricci P, Blein JP. 1996. Relationship between active oxygen species, lipid peroxidation, necrosis and phytoalexin production induced by elicitins in Nicotiana. Plant Physiology 111, 885–891.[Abstract]
Ryals JA, Neuenschwander UH, Willits MG, Molina A, Steiner HY, Hunt MD. 1996. Systemic acquired resistance. The Plant Cell 8, 1809–1819.[Web of Science][Medline]
Sasabe M, Takeuchi K, Kamoun S, Ichinose Y, Govers F, Toyoda K, Shiraishi T, Yamada T. 2000. Independent pathways leading to apoptotic cell death, oxidative burst and defence gene expression in response to elicitin in tobacco cell suspension culture. European Journal of Biochemistry 15, 5005–5013.
Schreck R, Rieber P, Bauerle PA. 1991. Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-B transcription factor and HIV-1. The EMBO Journal 10, 2247–2258.[Web of Science][Medline]
Solary E, Bertrand R, Pommier Y. 1994. Apoptosis of human leukemic HL-60 cells induced to differentiate by phorbol ester treatment. Leukemia 8, 792–797.[Web of Science][Medline]
Solomon M, Belenghi B, Delledone M, Menachem E, Levine A. 1999. The involvement of cystein-proteases and protease inhibitor genes in the regulation of programmed cell death in plants. The Plant Cell 11, 431–443.
Takeda M, Shirato I, Kobayashi M, Endou H. 1999. Hydrogen peroxide induces necrosis, apoptosis, oncosis and apoptotic oncosis of mouse terminal proximal straight tubule cells. Nephron 81, 234–238.[Web of Science][Medline]
Tenhaken R, Rübel C. 1998. Induction of alkalinization and an oxidative burst by low doses of cycloheximide in soybean cells. Planta 206, 666–672.
Wang H, Li J, Bostok RM, Gilchrist DG. 1996. Apoptosis: a functional paradigm for programmed plant cell death induced by a host-selective phytotoxin and invoked during development. The Plant Cell 8, 375–391.[Abstract]
Ward ER, Uknes SJ, Williams SC, Dincher SS, Wiederhol DL, Alexander DC, Ahl-Goy P, Métraux JP, Ryals J. 1991. Coordinate gene activity in response to agents that induce systemic acquired resistance. The Plant Cell 3, 1085–1094.
Widholm JM. 1972. The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells. Stain Technology 47, 189–194.[Web of Science][Medline]
Wojcik C. 1999. Proteasomes in apoptosis: villains or guardians? Cellular and Molecular Life Science 56, 908–917.[Web of Science][Medline]
Zhu Q, Masond S, Dixon RE, Lamb C. 1996. Transcriptional activation of plant defence genes. Current Opinion in Genetical Development 6, 624–634.
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