Introduction

Programmed cell death (PCD) is a common and almost universal feature of life. This process has been extensively studied in animals, where it is called apoptosis (Desagher and Martinou 2000; Strasser et al. 2000; Kaufmann and Hengartner 2001), but there is much less knowledge about PCD in plants (Danon et al. 2000; Lam 2004). Plant PCD and animal apoptosis seem to share some morphological and biochemical features, such as cell shrinkage, nuclear DNA degradation, loss of mitochondrial membrane potential or the activation of specific proteases (Lam et al. 1999, 2001; Danon et al. 2000; Woltering et al. 2002; Sanmartín et al. 2005). PCD in plants seems to play a critical role in a number of development events and in response to environmental biotic and abiotic stresses (Balk et al. 1999; Lam et al. 2001; Lam 2004). In addition, PCD has been identified as part of the plant response to pathogen invasion, in a process known as the hypersensitive response (Heath 2000).

It is well established that oxidative stress is involved in plant PCD processes (Lamb and Dixon 1997; Mittler 2002), and several investigators have shown that H2O2 induces PCD in suspension cultures of plant cells (Desikan et al. 1998; de Pinto et al. 2006). High levels of the vegetal hormone salicylic acid (SA) have also been shown to promote plant cell death by enhancing H2O2 production, lipid peroxidation and oxidative damage (Rao and Davis 1999; Mazel and Levine 2001; Brodersen et al. 2005).

Acetylsalicylic acid (ASA), the acetylated derivate of SA, is a well-known non-steroidal anti-rheumatic agent. ASA can behave as an apoptosis-inducing agent in animal cancer cells by acting as an inhibitor of cyclooxygenases (COXs) through the acetylation of a specific serine in the protein active site (Loll et al. 1995). COXs are enzymes linked with the synthesis of prostaglandins, which are related to inflammation, pain or angiogenesis (Méric et al. 2006). In plants, however, the function of COXs in prostaglandin synthesis is mimicked by a different pathway, which leads to the synthesis of two prostanoid-like fatty acids, 12-oxo-phytodienoic acid (12-oxo-PDA) and jasmonic acid (JA) (Harms et al. 1998; Pan et al. 1998; Pierpoint 2002).

Earlier work on the effects of salicylates on plants indicated that exogenous applications of ASA and SA blocked plant response to wounds (Harms et al. 1998). It has also been shown that salicylates inhibit JA formation and the subsequent JA-induced expression of defense-related genes, probably through the inhibition of allene oxide synthase (AOS), one of the enzymes involved in JA synthesis, which is irreversibly inhibited by the acetylation of serine residues (Pan et al. 1998; Pierpoint 2002). Such inhibition process is similar to the ASA-mediated inhibition of COX in animal cells (Méric et al. 2006).

In this work, we demonstrate that ASA can act as an efficient PCD-inducing agent in cell cultures of the model plant Arabidopsis thaliana (hereafter Arabidopsis), in a way similar to the well-established PCD-inducing agent H2O2 and more efficiently than the related vegetal hormone SA. Thus, ASA treatment of Arabidopsis cells induces typical PCD-linked morphological and biochemical changes. We also report that the addition of JA in part inhibits the effect of ASA as PCD-inducing agent, thus suggesting additional similarities between animal apoptosis and plant PCD.

Materials and methods

Cell cultures and PCD induction

Arabidopsis thaliana cell line MM2d suspensions (ecotype Landsberg erecta, Bayer Bioscience N.V., Ghent, Belgium) were grown under continuous irradiation in a rotary shaker at 100 rpm and 25°C in modified MS basal medium (Murashige and Skoog 1962), supplemented with 3% sucrose, 50 μg mL−1 kinetin, 500 μg mL−1 naphthaleneacetic acid, 0.2 mg mL−1 cefotaxime and 0.2 mg mL−1 penicillin. The cells were subcultured every 7 days, during the exponential growth phase, by making a 1:10 dilution in fresh medium. Cell cultures with a chlorophyll concentration of 1.5–2 μg mL−1 were used for the experiments 4 days after subculturing. Chlorophyll concentration in Arabidopsis cultures was determined by measuring the absorbance values at 665 nm, in methanol-extracted samples according to the method described by MacKinney (1941).

For the induction of PCD, minimal volumes of stock solutions of H2O2, ASA or SA were added to fresh cultures of Arabidopsis cells. In the case of H2O2, a final concentration of 35 mM in the culture was used, according to Desikan et al. (1998). For the induction of necrosis, the cells were subjected to a heat shock at 70°C for 10 min.

Cell viability was monitored after 5 min of incubation in a 1:1 diluted solution of cells and Trypan Blue 0.04% (Sigma). Cells were then monitored in an Olympus BX-60 light microscope and at least 1,000 cells were counted.

Analysis of cell morphology changes by microscopy

Changes of cell morphology were observed and recorded with an Olympus BX-60 light microscope. For the fluorescence microscopy analysis of cell survival by DAPI staining (Bethke et al. 1999), 1 mL of untreated or treated Arabidopsis cells suspensions were incubated with 5 μg mL−1 of 4′,6-diamidino-2-phenylindole (DAPI) at room temperature. After 5 min, fluorescence emission at 461 nm while exciting at 358 nm was recorded.

DNA extraction and agarose gel electrophoresis

For DNA extraction, cells from 5 mL of culture were harvested by decantation and further centrifuged at 2,000g for 5 min. DNA extraction from pelleted cells was carried out as described by Murray and Thompson (1980).

For agarose gel electrophoresis, samples were previously treated with 1 μg mL−1 RNAse for 1 h at 37°C. Electrophoresis was run at 50 V for 2 h in 1.5% agarose gels supplemented with 0.4 μg mL−1 of ethidium bromide.

TUNEL assay

DNA fragmentation in Arabidopsis cells along the PCD process was determined by using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay. Cells were collected at the indicated times, centrifuged at 1,500g for 5 min, resuspended in 1 mL of fixing solution (PBS 1X, paraformaldehyde 4%) and gently stirred for 1 h at room temperature. Cell walls were then digested by the addition of 1% cellulase and 0.1% pectinase and further incubated for 2 h at 30°C.

The TUNEL assay was performed with the In Situ Cell Death Detection Kit, Fluorescein (Roche Diagnostic), as indicated by the manufacturer. The samples were visualized using a Leica DMRE confocal microscopy, using an excitation wavelength of 488 nm and a detection wavelength within the range of 515–565 nm.

Preparation of cellular fractions and Western-blot analysis

Arabidopsis cytosolic fractions were obtained as described by Krause and Durner (2004), except that the filtration step after cell homogenization was substituted by a centrifugation at 5,000g for 2 min. The resulting pellets were used as starting material for the mitochondrial fraction separation. To obtain cytosolic samples, the supernatants were centrifuged once again at 12,000g for 10 min, in order to eliminate any cellular debris, and finally frozen in liquid N2 and stored at −80°C.

For obtaining mitochondrial extracts, the pellets from the first centrifugation were resuspended in 1 mL of cold lysis buffer A (20 mM Hepes, pH 7,5, 0.2 M sucrose, 20 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM DTT) and sonicated for 25 s at 20W. The samples were then centrifuged at 4°C for 15 min at 12,000g, and the supernatants were considered as the mitochondrial fraction. Total protein concentrations of the different fractions were determined by the Lowry method (Lowry et al. 1951) using bovine seroalbumine (BSA) as standard. Activity measurements of the mitochondrial malate dehydrogenase (MDH) enzyme were carried out to discard mitochondrial contamination in the cytosolic extract. The MDH activity in samples containing 10 μg of protein was measured as described by Gómez et al. (2002).

Antibodies against Arabidopsis cytochrome c were obtained at the Centro de Producción y Experimentación Animal (University of Seville, Spain) by inoculating the purified protein (Rodríguez-Roldán et al. 2006) into rabbits. The antibodies extracted from the resulting serum blood were specific for the purified cytochrome c (data not shown).

For the Western-blot analyses, 100 μg of protein extracts were loaded into 12% polyacrylamide gels, running at 100 V for 2 h, and finally transferred at 2.5 mA cm−2 for 1 h to Immuno-Blot PVDF membranes (Bio-Rad). The blots were blocked overnight at 4°C with 5% non-fat dry milk in PBS (blocking buffer), and immunoblotted with the primary antibody at a dilution of 1:500 for 90 min in blocking buffer. The membranes were then washed three times in PBS buffer containing 0.1% Tween 20 (washing buffer), incubated with the anti-rabbit IgG-HRP conjugate (Sigma) (dilution 1:10,000) in blocking buffer for 90 min and washed three times with PBS buffer. Labelled proteins were detected by adding the ECL Western Blotting Solution (Amersham), developing for 1 min and finally exposing in an X-ray film.

Measurement of caspase-like activity

Caspase 3-like activities were measured in total extracts of Arabidopsis cells in order to test specific protease activity along the PCD process. Samples of 5 mL from untreated and treated cultures were centrifuged at 3,000g for 3 min. Pelleted cells were resuspended in 500 μL of cold lysis buffer B (20 mM Hepes-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM PMSF) supplemented with 1 μL of plant Protease Inhibitor Cocktail (Sigma), sonicated for 10 s at 20 W, centrifuged at 4°C for 15 min at 12,0000g to eliminate cell debris, frozen in liquid N2 and stored at −80°C. Protein concentration of the different fractions was determined as mentioned above (Lowry et al. 1951).

Measurements of caspase 3-like activity were carried out in samples containing 250 μg of protein from the previous fractions, by following the method described by Clarke et al. (2000), but incubating with 75 μM of Ac-DEVD-AMC, a fluorogenic substrate for caspase 3. The relative fluorescence intensity at 460 nm of the samples while exciting at 360 nm was measured at pH 5 to enhance caspase 3-like activity (Woltering et al. 2002).

For the caspase-like activity inhibition assays, the samples were preincubated for 1 h at 30°C in the presence of 50 μM Ac-DEVD-CHO, a reversible inhibitor for caspase 3, before adding the Ac-DEVD-AMC substrate.

Determination of mitochondrial membrane potential

Changes in mitochondrial membrane potential (ΔΨm) were monitored by using the JC-1 dye (Fluka) as a probe, following the fluorescence emission shift from green to red (Krause and Durner 2004). A volume of 200 μL of Arabidopsis cells suspension was centrifuged at 2,000g for 5 min, and pelleted cells were resuspended in 1 mL of fresh medium. The cells were then stained with the JC-1 dye (final concentration, 5 μg mL−1) for 20 min in the dark under gentle stirring. The samples were washed with fresh medium twice by centrifuging at 2,000g for 5 min, and finally resuspended in 1 mL of fresh medium. Fluorescence emission intensities were measured both at 590 and 527 nm, with an excitation wavelength of 490 nm.

Treatment with JA

ASA-treated cells were collected 11 h after addition of the PCD inducer, washed twice with PBS and resuspended in the same volume of modified MS basal medium supplemented with 5 μM JA. As a control, non-PCD-induced cells were also treated with JA. Samples were collected at different times in order to determine cell viability and chlorophyll and protein concentrations.

Statistical analysis

All the data herein presented are the mean values of at least three independent experiments ± the standard deviation.

Results

ASA induces cell death in Arabidopsis

To compare the effect of ASA as PCD trigger with a well-established cell death-inducing agent in Arabidopsis cell cultures, 35 mM H2O2 was used as standard, according to the results of Desikan et al. (1998). Cell death was thus monitored both as the decay of chlorophyll content in the cultures (see below) and DAPI staining of dead cells (not shown) (Bethke et al. 1999) after the addition of H2O2. Different concentrations of ASA were added to Arabidopsis cultures (Fig. 1), and a pattern of cell death similar to that previously obtained with H2O2 was observed after addition of 2 mM ASA, which was thus selected as the standard concentration of this cell death-inducing agent. The same concentration of SA was also used for comparative purposes (Fig. 1). After 36–48 h of treatment with ASA or H2O2, but not with SA, a general process of cell death was observed (see below).

Fig. 1
figure 1

Changes in the viability of Arabidopsis cells on addition of ASA (left) or SA (right) at varying concentrations. Cell viability was monitored at the indicated times after addition of the PCD-inducing agent, as described under “Materials and methods”

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ASA induces morphological and growth changes linked to PCD

In plants, the PCD process is characterized by some different features as compared with animal apoptosis. Morphologically, the rigid plant cell wall does not allow the formation of apoptotic bodies like in animal cells, and as a consequence plant cells undergoing PCD suffer a collapse of their cytoplasm. The addition of 2 mM ASA to Arabidopsis liquid cultures promotes similar morphological changes than the addition of H2O2. As shown in Fig. 2, these changes include organelle degradation, vacuolization and loss of swelling. An intense vacuolization of cells could be observed upon addition of both substances to different cultures at the same state of growth, with the maximum vacuolization level being reached after 8–12 h of cell death induction (Fig. 2b, c). Vacuolization was concomitant with the loss of chloroplasts (Fig. 2e, h). In addition, some cells showed a collapse of their cytoplasm, another feature of plant PCD, 24 h after the addition of either ASA or H2O2 (Fig. 2c).

Fig. 2
figure 2

Effect of ASA on Arabidopsis cells in liquid cultures. The morphological changes were observed by light bright field microscopy in untreated cells (a), in treated cells after 8 h (b) or 24 h (c) of 2 mM ASA addition, and in necrotic cells (d). The loss of chloroplast integrity was visualized by fluorescence (e, f) and light bright field (g, h) microscopy, in untreated (e, g) and in treated cells after 24 h of ASA addition (f, h). In c, the arrows point to the loss of swelling and plasmatic membrane separation from the cell wall

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However, the addition of 2 mM SA to Arabidopsis cells liquid cultures promoted comparatively different effects. Thus, although SA induced the initial formation of vacuoles inside the cells after 4 h, the cells did not show any collapse of their cytoplasm and cultures did not totally die until at least 72 h after the addition of SA (data not shown). As a control, necrotic cells obtained after a high temperature shock did not show vacuolization or collapse of the cytoplasm (Fig. 2d).

Besides the morphological changes, the treatment of the cells with H2O2, ASA or SA promotes alterations in the culture growth, as can be monitored by following the time course of chlorophyll concentration (Fig. 3). Actually, a similar initial decrease in chlorophyll content could be observed upon addition of any of these three compounds. However, further decay of the chlorophyll concentration at longer culture time was slower in cells treated with SA as compared to those treated with H2O2 and ASA. Similar decays in protein content were also observed (not shown). Unlike cells undergoing PCD, necrotic cells showed a rapid fall in chlorophyll and protein concentrations. The chlorophyll and protein decays could reflect some effect on cell division, but by themselves do not prove the occurrence of a PCD process. Notwithstanding, these findings are compatible with PCD when linked to the observed morphological changes in cells treated with both ASA and the well-established PCD-inducing agent H2O2. The effect of ASA is observed at much lower concentration than that of H2O2; in addition, SA, used at the same concentration than ASA, is a comparatively poor PCD-inducing agent.

Fig. 3
figure 3

Changes in chlorophyll concentration in Arabidopsis cultures induced by different cell death treatments. The indicated cell death agents were added to the cultures (chlorophyll concentration around 1.5 μg mL−1) at the following concentrations: ASA 2 mM; SA 2 mM; H2O2 35 mM. Necrosis was induced by heat shock at 70°C for 10 min. Other conditions were as described in “Materials and methods”

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DNA degradation

DNA damage and degradation are other hallmarks of PCD. In order to analyse DNA integrity along the PCD process of Arabidopsis cells, total DNA was extracted from untreated and ASA-, H2O2- or SA-treated cells, and the samples were subjected to agarose gel electrophoresis. Although the typical DNA fragmentation pattern during apoptosis (DNA ladder) was not observed in cells with either H2O2 or ASA, a gradual DNA degradation started 24 h after treatment (data not shown). However, cells treated with SA only showed similar levels of DNA degradation at a longer time after the hormone addition (24–48 h).

One of the last stages of PCD is the fragmentation of nuclear chromatin. In fact, PCD-induced DNA degradation generates DNA strands with exposed 3′-hydroxyl ends, which can be fluorescently labelled with the TUNEL (Fig. 4). In our case, untreated cells showed low unspecific TUNEL staining (Fig. 4c), whereas positive TUNEL labelling was observed 12 h after treatment of cells with either H2O2 or ASA (Fig. 4a, b), indicating DNA fragmentation associated with PCD. We did not observe any positive TUNEL staining in necrotic cells (Fig. 4d), as expected from the total and rapid degradation of nuclear DNA under such conditions.

Fig. 4
figure 4

DNA fragmentation as determined by TUNEL staining 12 h after inducing PCD in Arabidopsis cultures. The cells were treated with either 35 mM H2O2 (a) or 2 mM ASA (b), untreated (c), or necrotic (d). The arrows point to positive TUNEL staining. Other experimental conditions were as in Fig.3. See “Materials and methods” for further details

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ASA induces cytochrome c release and changes in mitochondrial membrane potential

A key step in animal apoptosis is the release of cytochrome c from mitochondria to the cytosol, where it activates the caspases route. However, a similar role of cytochrome c in the induction of plant PCD is not established yet, mainly because of the late release of the heme-protein when it has been observed (Yu et al. 2002; Krause and Durner 2004; Zuppini et al. 2006). Here, we have studied the time course of cytochrome c release from the mitochondria in cells treated with H2O2 or ASA as compared to cells treated with SA, along with the decrease in mitochondrial membrane potential (ΔΨm) through the PCD process, another typical PCD hallmark (Fig. 5).

Fig. 5
figure 5

Cytochrome c release and changes in mitochondrial membrane potential. (Upper) Time course of cytochrome c release from mitochondria as determined by immunoblotting in Arabidopsis cultures treated with different cell death inducing agents. C cytoplasmic extract; M mitochondrial extract. Purified Arabidopsis cytochrome c (Cyt), 20 ng, was used as a control. (Lower) Time course of changes in the mitochondrial membrane potential (ΔΨm) value as measured by the JC-1 fluorescence shift in untreated (filled circle) and necrotic (inverted triangle) cells, as well as in cells treated with 35 mM H2O2 (square), 2 mM ASA (filled triangle) and 2 mM SA (open circle). Other experimental conditions were as in Fig. 3

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Treatment of cells with H2O2, ASA or SA induced cytochrome c release from mitochondria to cytosol, as determined by Western blotting (Fig. 5, upper). In the case of H2O2 and ASA, the cytochrome c release started ca. 3 h after inducer addition and was complete 5 h later. However, in cells treated with SA, the release of cytochrome c is delayed for 12 h after PCD induction. In necrotic cells treated with a heat shock, cytochrome c was released from the mitochondria almost immediately, as a result of the disruption of cellular membranes (Fig. 5, upper). In order to discard unspecific cytochrome c detection in the cytosolic fractions coming from mitochondrial contaminations, malate dehydrogenase (MDH) activity (MDH is a mitochondrial soluble enzyme from the Krebs cycle) was measured to check mitochondrial integrity. No significant MDH activities were detected in the cytosolic fractions, whereas mitochondrial extracts showed high levels of such enzyme activity (not shown).

Fluorescence changes associated with the JC-1 dye are widely used to monitor mitochondrial ΔΨm modifications (Krause and Durner 2004). JC-1 undergoes a reversible change in fluorescence emission from green to red, as mitochondrial membrane potential increases, promoting the formation of dye aggregates. Therefore, the red/green fluorescence ratio allows measuring mitochondrial membrane depolarization (Fig. 5, lower). Whereas control cells exhibited a stable ΔΨm value along the time course of culture growth, necrotic cells showed a rapid and almost complete collapse of ΔΨm, as a consequence of the disorganization of cellular membranes after the heat shock, in agreement with the rapid release of cytochrome c to the cytosol (Fig. 5).

In cells treated with H2O2 or ASA, a continuous decline of ΔΨm was observed, the fluorescence signal decreasing up to 50% of the initial levels. These results agree with previously observed data for PCD induced by oxidative burst in Arabidopsis cells (Krause and Durner 2004). In contrast, the ΔΨm value in SA-treated cells experienced a similar initial decay, followed by a partial recovery so as to finally decrease again to reach the same levels as in H2O2 or ASA treated cells (Fig. 5, lower). Thus, the ΔΨm decline basically follows the same time course as cytochrome c release in the cells treated with any of the three PCD-inducing agents (Fig. 5).

Caspase-like activities

Caspase activity is a specific characteristic of apoptosis in animal systems. Although there are no evident caspases homologues in plants, previous studies have demonstrated the occurrence of specific caspase-like activities (reviewed in Sanmartín et al. 2005), in some cases associated with cytochrome c release from mitochondria (Sun et al. 1999; Zuppini et al. 2006). Caspase-like protease activation in ASA-, H2O2- or SA-treated cells is investigated here by using Ac-DEVD-AMC, a specific cleavage synthetic substrate for caspase 3 (Fig. 6). Whereas untreated cells did not show any specific cleavage activity for this substrate, ASA, H2O2 or SA treatment induced activation of caspase-like activities (Fig. 6). A similar activation pattern was observed upon addition of ASA or H2O2, with caspase-like activity levels significantly increasing between 4 and 8 h after PCD induction to reach a plateau at 24 h (Fig. 6). However, the levels of caspase-like activity in the cells treated with SA did not experience any significant increase until 12–24 h after hormone addition, although the levels of the caspase-like activity attained 24 h after PCD induction were similar with the three inducing agents (Fig. 6). As expected from a specific caspase activity, pre-incubation of the extracts with Ac-DEVD-CHO, a specific inhibitor of caspase 3, makes caspase-like activities decrease nearly to control levels (Fig. 6).

Fig. 6
figure 6

Time course of caspase 3-like activity in Arabidopsis cells treated with the indicated cell death inducing agents in the absence (white bars) or presence (black bars) of the caspase 3 inhibitor Ac-DEVD-CHO. The 100% values correspond to the caspase activity level before addition of the cell death inducing agent. Other experimental conditions were as in Fig. 3. See “Materials and methods” for further details

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Effect of JA on PCD-induced cells

It has been previously reported that ASA inhibits both COX-2, an enzyme involved in the synthesis of prostaglandins in animal cells (Méric et al. 2006), and AOS, a key enzyme in the synthesis of JA in plants (Harms et al. 1998; Pan et al. 1998). Thus, ASA could promote plant PCD by inhibiting JA formation. In order to check such a hypothesis, we studied the effect of the addition of JA to cultures of ASA-dependent PCD-induced cells. As can be seen in Fig. 7, whereas no effects of JA were observed in control cells, there were significant differences between ASA-treated cells in the absence and in the presence of JA, the latter showing much higher viability (up to 50%) than the former. In agreement with these data, the cells treated with ASA and JA also showed higher chlorophyll and protein concentrations at longer times as compared with the cells treated only with ASA (not shown). In addition, JA did not exhibit any effect in control experiments with H2O2 as a PCD inducer (not shown). The greater error of experimental data for cells treated with ASA + JA, as compared to cells just treated with ASA (Fig. 7), could in part be ascribed to the JA-mediated inhibition of cell division (Ueda and Kato 1982; Swiatek et al. 2002), thus making the lower number of cells increase the uncertainty in viability determination. Taking together, these data thus suggest a possible role for ASA as inhibitor of the JA synthesis pathway linked to PCD.

Fig. 7
figure 7

Effect of JA on the viability of ASA-dependent PCD-induced cells. After treatment with 2 mM ASA, the viability of the cell cultures in the presence or absence of 5 μM JA was monitored at the indicated times. Other experimental conditions were as described under “Materials and methods”

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Discussion

Our results clearly indicate that aspirin (ASA), a well-known drug with antitumoral and anti-inflammatory properties, induces a typical PCD process in Arabidopsis cells, similar to that observed when treating cells with H2O2, a well-known PCD-inducing agent (Desikan et al. 1998; de Pinto et al. 2006). First, both morphological (organelle degradation, vacuolization and loss of swelling, collapse of the cytoplasm) and biochemical changes (DNA degradation, changes in protein and chlorophyll contents) are compatible with a PCD process. Second, ASA treatment induces the typical features of PCD concerning the plant mitochondria, namely loss of mitochondrial membrane potential and cytochrome c release to cytosol.

Caspase activity is another specific characteristic of apoptosis in animal systems. Although there are no clear caspases homologues in plants, two groups of plant proteases have been postulated to possess caspase-like activities: the vacuolar processing enzymes and metacaspases (Woltering et al. 2002; Sanmartín et al. 2005). Both groups of enzymes are related to animal caspases and have been associated with plant PCD processes (Sanmartín et al. 2005). Our results demonstrate that ASA treatment promotes a specific caspase-like activity induction, as expected from a characteristic PCD process.

Parallel PCD features are observed when comparing ASA and H2O2 effects, with the PCD process mainly taking place within the first 12 h after addition of the triggering agents. In this sense, both ASA and H2O2 behaved in a similar way, acting as a more efficient PCD-inducing agents than SA. The effect of ASA is thus similar to the effect of an array of other death-inducing chemicals and stresses, generally explained in terms of a common cell death mechanism involving phospholipid signalling, ROS production, ethylene action and caspase-like proteases activation (Desikan et al. 1998; Sun et al. 1999; Mazel and Levine 2001; Danon et al. 2004; Brodersen et al. 2005).

On the other hand, it is also known that ASA inhibits both COX-2, an animal enzyme involved in the synthesis of prostaglandins (Méric et al. 2006), and AOS, a plant enzyme involved in the synthesis of JA (Harms et al. 1998; Pan et al. 1998). It has also been shown that SA can promote plant PCD inhibiting JA formation by blocking the AOS enzyme (Harms et al. 1998). COX-2 has an important role in cancer cells by acting over proteins like Akt, Rac and MMP (Méric et al. 2006), which all have homologues in the Arabidopsis genome (Zhang et al. 1994; Maidment et al. 1999; Winge et al. 2000). As AOS and COX-2 have an equivalent physiological function (Pan et al. 1998) and animal prostaglandins and plant JA-derived hormones are structurally related compounds, the effect of ASA as PCD-inducing agent in Arabidopsis cells could be ascribed to inhibition of JA production. However, although our results suggest that ASA may induce PCD by blocking JA formation, as it has been previously proposed for SA, this possibility deserves further investigation.

Finally, the role of ASA acting both as an apoptosis and PCD-triggering agent, respectively, in animal and plant cells, clearly indicates the existence of common control points in the animal and plant processes leading to cell death.