Involvement of endogenous salicylic acid content, lipoxygenase and antioxidant enzyme activities in the response of tomato cell suspension cultures to NaCl

Authors

  • Anabel Molina,

    1. Departamento de Bioquímica, Biología Celular y Molecular de Plantas. Estación Experimental del Zaidín, CSIC, Apartado 419, 18008-Granada, Spain
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  • Pablo Bueno,

    1. Departamento de Bioquímica, Biología Celular y Molecular de Plantas. Estación Experimental del Zaidín, CSIC, Apartado 419, 18008-Granada, Spain
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  • María Carmen Marín,

    1. Departamento de Bioquímica, Biología Celular y Molecular de Plantas. Estación Experimental del Zaidín, CSIC, Apartado 419, 18008-Granada, Spain
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  • María Pilar Rodríguez-Rosales,

    1. Departamento de Bioquímica, Biología Celular y Molecular de Plantas. Estación Experimental del Zaidín, CSIC, Apartado 419, 18008-Granada, Spain
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  • Andrés Belver,

    1. Departamento de Bioquímica, Biología Celular y Molecular de Plantas. Estación Experimental del Zaidín, CSIC, Apartado 419, 18008-Granada, Spain
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  • Kees Venema,

    1. Departamento de Bioquímica, Biología Celular y Molecular de Plantas. Estación Experimental del Zaidín, CSIC, Apartado 419, 18008-Granada, Spain
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  • Juan Pedro Donaire

    Corresponding author
    1. Departamento de Bioquímica, Biología Celular y Molecular de Plantas. Estación Experimental del Zaidín, CSIC, Apartado 419, 18008-Granada, Spain
      Author for correspondence: Juan Pedro Donaire Tel: +34 958 121011 Fax: +34 958 129600 Email: juanpedro.donaire@eez.csic.es
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Author for correspondence: Juan Pedro Donaire Tel: +34 958 121011 Fax: +34 958 129600 Email: juanpedro.donaire@eez.csic.es

Summary

  • •   The effects of salt stress and adaptation on salicylic acid (SA) content and on antioxidant and lipoxygenase (LOX) enzyme activities were studied in tomato (Lycopersicon esculentum cv. Pera) cells.
  • •   NaCl-adapted cells were obtained from calli adapted to 100 mm NaCl by successive subcultures in medium supplemented with 100 mm NaCl. Salt stress treatments consisted of the addition of 100 mm NaCl to cells.
  • •   Adapted cells contained a lower concentration of SA than unadapted cells. The lower manganese-containing superoxide dismutase (Mn-SOD) and LOX activities as well as the higher glutathione reductase (GR) and ascorbate peroxidase (APX) activities in adapted cells than in unadapted cells could be correlated with the development of salt adaptation. Salt stress increased APX and LOX activities as well as lipid peroxidation in unadapted cells and increased Mn-SOD activity in both types of cells. Application of 200 µm SA + 100 mm NaCl inhibited APX activity in both unadapted and adapted cells, induced the Mn-SOD in adapted cells and increased lipid peroxidation in unadapted cells.
  • •   Our data indicate that adaptation of tomato cells to NaCl results in a higher tolerance to NaCl-induced oxidative stress and suggest a role for SA in this response.

Introduction

Plants exposed to salt stress adapt their metabolism in order to cope with the changed environment. Survival under these stressful conditions depends on the plant's ability to perceive the stimulus, generate and transmit signals and instigate biochemical changes that adjust the metabolism accordingly (Hasegawa et al., 2000). High concentrations of NaCl cause ion imbalance and hyperosmotic stress in plants. To maintain ionic homeostasis and water potential in the cytosol, plants under NaCl stress need to accumulate various compatible osmolytes in the cytosol, thus lowering the osmotic potential to sustain water uptake from saline solutions (Zhu et al., 1997). As a consequence of these primary effects, secondary stresses such as oxidative damage often arise (Scandalios, 1993; Zhu, 2001). Plant cells contain a range of protective and repair systems, which, under normal circumstances, minimize the occurrence of oxidative damage. These are systems which react with active forms of oxygen and keep them at a low level (superoxide dismutase (SOD), catalase and peroxidases) and systems which regenerate oxidized antioxidants (glutathione reductase (GR), mono- and dehydroascorbate reductases) (Allen, 1995). In this connection, Hernández et al. (1993) demonstrated that NaCl treatments decreased manganese-containing SOD (Mn-SOD) activity in mitochondria from NaCl-sensitive pea, but induced this isozyme activity in NaCl-tolerant plants; Gossett et al. (1996) indicated that NaCl-tolerant cell lines of cotton exhibited a more active ascorbate–gluthatione cycle than unadapted cell lines. Similarly, Bueno et al. (1998) reported the induction of SOD genes in tobacco cells exposed to 200 mm NaCl. More recently, it has been demonstrated that Arabidopsis pst 1 mutant plants, which have a mutation in an unknown putative negative regulator of oxidative stress responses, are more resistant to high salt concentrations and show an increased capacity to tolerate oxidative stress (Tsugane et al., 1999).

Various agents such as Ca2+, ethylene, jasmonic acid and salicylic acid (SA) have been proposed as signal transducers in plant responses to biotic and abiotic stresses (Enyedi et al., 1992; Klessig & Malamy, 1994). Salicylic acid has received particular attention because of its involvement in plant defense against pathogen attack (Yu et al., 1997). A number of studies have shown that SA can potentiate several responses to pathogens, including the oxidative burst and hypersensitive cell death (Draper, 1997), which suggest that SA may operate at different positions in the signal transduction pathway, leading to disease resistance. Rao et al. (1997) proposed that plants respond similarly to SA and to pathogen infection, by increasing the production of reactive oxygen species (ROS) such as superoxide radical (O2 · ) that are dismutated to hydrogen peroxide (H2O2) by the activation of SOD enzyme. The mechanisms by which SA enhances H2O2 content are not clear, but they could involve inactivation of H2O2-removing enzymes by SA (Durner & Klessig, 1995; Durner et al., 1997). A recent study has shown the responses to different abiotic stress conditions of wild-type Arabidopsis and a SA-deficient transgenic line expressing NahG, a salicylate hydroxylase gene (Borsani et al., 2001). Thus, wild-type plants germinated in media supplemented with NaCl or mannitol showed extensive necrosis in the shoot, whereas NahG plants germinated under the same conditions remained green and developed true leaves, suggesting that SA potentiates the generation of ROS in photosynthetic tissues during salt and osmotic stresses. Polyunsaturated fatty acid hydroperoxides resulting from the action of lipoxygenase (LOX) undergo a variety of reactions, including the generation of free radicals, which provoke changes in mem-brane properties, ultimately leading to disfunction of the lipid bilayer, membrane deterioration and senescence (Gardner, 1991). It is also known that LOX, a nonheme iron containing dioxygenase, can generate superoxide via oxidation of pyridine nucleotides and may therefore significantly contribute to oxidative stress in plant cells (van Gestelen et al., 1997).

The aim of this study was to investigate the contribution of the endogenous content of SA as well as LOX and antioxidant enzyme activities to the mechanisms of salt adaptation and salt stress in tomato cell suspension cultures.

Materials and Methods

Cell suspension cultures and selection of NaCl-adapted cells

Tomato (Lycopersicon esculentum Mill. cv. Pera) hypocotyl calli were obtained from in vitro germinated seeds, kindly donated by Dr J. Cuartero (Estación Experimental La Mayora, CSIC, Málaga, Spain) and cell suspension cultures were prepared from these calli as described previously (Rodríguez-Rosales et al., 1999; Kripkyy et al., 2001). The cultures were renewed every 11 d by transferring 12 g of cells into 100 ml fresh medium of the same composition as used for calli multiplication (Rodríguez-Rosales et al., 1999), but without agar, polyvinylpyrrolidone and ascorbic acid (standard culture medium). Cells were incubated in the dark at 27°C and under continuous shaking at 120 r.p.m. The NaCl-adapted cells were obtained by disaggregating calli adapted to 100 mm NaCl in standard culture medium supplemented with 100 mm NaCl. These cells were used for experiments after at least six successive subcultures in the standard medium supplemented with 100 mM NaCl.

Preparation of cell suspensions for experiments

All experiments were performed with 6-d-old unadapted and 100 mm NaCl-adapted cell suspension cultures (corresponding to the middle of the exponential growth phase). Treatment of unadapted and 100 mm NaCl-adapted cells with NaCl (salt stress) or SA plus NaCl was performed in 25 ml Erlenmeyer flasks. The cells were collected from each flask by filtration through a Whatman No. 4 filter paper and the f. wt was determined at the indicated times after the treatments. Finally, the cells were frozen in liquid nitrogen and stored at −80°C.

Quantification of free and conjugated SA

Free SA was extracted and quantified essentially as described by Malamy et al. (1992). One gram of frozen cells was ground in 3 ml of 90% (v : v) methanol containing 50 ng ml−1 of 2-methoxybenzoic acid as an internal standard. The internal standard was added after confirming that its endogenous level was similar in unadapted and NaCl-adapted cell suspensions and after different cell treatments. The ground material was centrifuged at 6000 g for 15 min and the pellet was re-extracted with 3 ml of 100% (v/v) methanol and centrifuged. Methanol extracts were combined, centrifuged at 6000 g for 10 min, and dried at 40°C under vacuum. Extracts were then resuspended in 3.5 ml of water at 80°C, an equal volume of 0.2 m sodium acetate, pH 5.0, was added, and the pH was adjusted to 1.5–2.0 with HCl before extraction. Free SA was extracted two times into two volumes of cyclopentane–ethylacetate–isopropanol (50 : 50 : 1 by volume) and the organic extract was dried under nitrogen, resuspended in methanol and analysed by high-pressure liquid chromatography (HPLC).

The SA conjugates were indirectly quantified by β-glucosidase digestion, as described by Southerton & Deverall (1990). For each sample, the dried methanol extract was resuspended in 3 ml of water at 80°C, and then an equal volume of 0.2 m acetate, pH 5.0, containing 0.1 mg ml−1β-glucosidase (22 U mg−1) was added and the solution incubated at 37°C overnight. After digestion, samples were acidified to pH 1.5–2.0 with HCl and extracted two times with two volumes of cyclopentane–ethyl acetate–isopropanol (50 : 50 : 1 by volume). The organic extracts were dried under vacuum, resuspended in 500 µl methanol and analysed by HPLC. Levels of free and total (free plus conjugated) SA and 2-methoxybenzoic acid were determined by HPLC performed with a reverse-phase column (Lithosphere C18, 150 × 4.6 mm, 5 µm; Varian, CA, USA) linked to a fluorescence photometer (Prostar 363 equipped with a 10-µl flow cell; Varian). Excitation and emission wavelengths were 313 nm and 405 nm, respectively, and separation was performed at 40°C using a flow rate of 1 ml min−1. Elution was carried out with methanol–acetic acid–H2O2 (30 : 1 : 68 by volume) for 20 min and equilibration with the same solvent system for 10 min before the next run. All data were corrected for SA recovery, which ranged from 45 to 65%. The identity of the SA molecule isolated from tomato cell suspensions was confirmed by mass spectrometry. The lowest concentration of SA that could be quantified by this procedure was 5 ng g−1 f. wt.

Enzyme assays

Lipoxygenase (EC 1.13.11.12) activity was assayed spectrophotometrically at 234 nm in a Shimadzu UV-160 spectrophotometer (Shimadzu Corporation, Kyoto, Japan) according to Axelrod et al. (1981). Superoxide dismutase (EC 1.15.1.1) activity was determined by the ferricytochrome c method using xanthine–xanthine oxidase as the source of superoxide radicals according to McCord & Fridovich (1969). Ascorbate peroxidase (APX; EC 1.11.1.11) was determined following the method described by Amako et al. (1994), and GR (EC 1.6.4.2) according to Kang et al. (1996).

Lipid peroxidation was estimated by determining the concentration of malondialdehyde with thiobarbituric acid, according to Buege & Aust (1978).

Protein quantification

Protein content was determined by the method of Lowry et al. (1951), using bovine serum albumin (BSA) fraction V as a standard.

Chemicals

All chemicals were of the purest grade commercially available. β-Glucosidase, SA and 2-methoxybenzoic acid were obtained from Sigma-Aldrich Fine Chemicals, Madrid, Spain.

Statistics

Each experiment was carried out in duplicate or triplicate and standard errors are indicated in the figures and tables. Where indicated variance analysis (Duncan test) was carried out.

Results

A comparison of growth between tomato cell suspension cultures continuously maintained in the absence of NaCl (unadapted cells) or in the presence of 100 mm NaCl for about 20 subculture cycles (salt-adapted cells) is shown in Fig. 1. The growth of 100 mm NaCl-adapted cells in the presence of 100 mm NaCl in the culture medium was similar to that of unadapted cells in the absence of NaCl. The growth curve of both types of cells showed a lag period of 2–3 d, an exponential phase of 4 d and a stationary phase. The f. and d. wts of NaCl-adapted cells decreased less with NaCl stress than those of the unadapted cells (Table 1). Since the f. and d. wts of the unadapted and NaCl-adapted cells were similarly affected by treatments with NaCl, the results were expressed only on f. wt basis.

Figure 1.

Growth curves of unadapted and 100 mm NaCl-adapted tomato cell suspension cultures. Closed circles, NaCl-unadapted cells in the standard culture medium; open circles, NaCl-adapted cells in medium supplemented with 100 mm NaCl. Data are means ± SE of four independent experiments with three replicates per experiment.

Table 1.  Fresh weight and levels of free and conjugated salicylic acid (SA) in unadapted and 100 mm NaCl-adapted tomato cell suspension cultures
TreatmentF. wt (g)D. wt (g)SA (µg g−1 f. wt)
Free SAConjugated SA
  1. Cells were incubated for 1 h or 4 h with 100 mm NaCl or for 2 h in 200 µm SA following 4 h with 100 mM NaCl. Mean values ± SE of three independent experiments are shown. Values followed by different superscript letters in each column differ significantly (Duncan's test, P = 0.05).

Unadapted cells12.00 ± 0.14a0.66 ± 0.06a  0.49 ± 0.11c  5.36 ± 0.61d
+1 h 100 mm NaCl  4.56 ± 1.16c0.23 ± 0.01d  0.60 ± 0.15c  7.02 ± 0.82c
+4 h 100 mm NaCl  3.28 ± 0.71c0.15 ± 0.00e  0.74 ± 0.14c  7.26 ± 0.74c
+2 h 200 µm SA + 4 h 100 mm NaCl  3.43 ± 0.29c0.14 ± 0.01e15.08 ± 1.90a18.90 ± 2.06a
100 mM NaCl-adapted cells12.06 ± 0.17a0.54 ± 0.05ab  0.10 ± 0.03d  0.28 ± 0.08e
+1 h 100 mm NaCl10.55 ± 1.07ab0.52 ± 0.02b  0.12 ± 0.06d  0.40 ± 0.10e
+4 h 100 mm NaCl  9.96 ± 0.91b0.45 ± 0.01c  0.09 ± 0.05d  0.31 ± 0.09e
+2 h 200 µm SA + 4 h 100 mm NaCl  9.63 ± 1.05b0.44 ± 0.02c  2.49 ± 0.32b  8.93 ± 1.01bc

Our data also showed that Mn-SOD and LOX activities were higher, while GR and APX activities were lower in unadapted cells compared with 100 mm NaCl-adapted cells (Tables 2 and 3). Moreover, these enzyme activities were differently affected in unadapted and NaCl-adapted cells as a result of a stress with 100 mm NaCl. After 4-h of treatment with 100 mm NaCl, APX and LOX activities were increased in unadapted cells, GR was slightly enhanced in NaCl-adapted cells and Mn-SOD was significantly increased in both types of cells.

Table 2.  Antioxidant enzyme activities in unadapted and 100 mm NaCl-adapted tomato cell suspension cultures
TreatmentMn-SOD (U mg−1 protein)APX (µmol mg−1 protein)GR (µmol mg−1 protein)
  1. Cells were incubated for 4 h with 100 mM NaCl or for 2 h in 200 µm SA following 4 h with 100 mM NaCl. Each value is the mean ± SE of two independent experiments with three replicates of cell extracts. Values followed by different superscript letters in each column differ significantly (Duncan's test, P = 0.05); n.d., not detected; Mn-SOD, manganese-containing superoxide dismutase; APX, ascorbate peroxidase; GR, glutathione reductase.

NaCl-unadapted cells  73.21 ± 6.18b1.30 ± 0.09c3.21 ± 0.76c
+4 h 100 mm NaCl114.20 ± 9.60a2.70 ± 0.19b3.08 ± 0.57c
+2 h 200 µm SA + 4 h 100 mM NaCl130.81 ± 8.71an.d.3.19 ± 0.80bc
100 mM NaCl-adapted cells  40.10 ± 4.31d6.74 ± 0.81a5.03 ± 1.01b
+4 h 100 mm NaCl  56.18 ± 5.14c7.24 ± 0.49a7.91 ± 0.98a
+2 h 200 µm SA + 4 h 100 mM NaCl126.13 ± 9.17an.d.7.01 ± 1.01a
Table 3.  Lipoxygenase (LOX) activity and lipid peroxidation, measured as content of thiobarbituric acid-reactive substances (TBARS), in NaCl-unadapted and 100 mM NaCl-adapted tomato cell suspension cultures
TreatmentLOX (mmol mg−1 protein)TBARS (µmol g−1 f. wt)
  1. Cells were incubated for 4 h with 100 mM NaCl or for 2 h in 200 µM salicylic acid (SA) following 4 h with 100 mM NaCl. Each value is the mean ± SE of two independent experiments with four replicates of cell extracts. Values followed by different superscript letters in each column differ significantly (Duncan's test, P = 0.05).

NaCl-unadapted cells18.11 ± 2.01b  3.11 ± 0.37d
+4 h 100 mm NaCl25.90 ± 2.05a  5.39 ± 0.60bc
+2 h 200 µm SA + 4 h 100 mm NaCl27.14 ± 2.21a12.73 ± 1.37c
100 mM NaCl-adapted cells10.37 ± 0.78a  4.01 ± 0.46c
+4 h 100 mm NaCl12.83 ± 1.17c  4.87 ± 0.52c
+2 h 200 µm SA + 4 h 100 mm NaCl13.88 ± 1.61c  3.92 ± 0.39c

To study whether salt adaptation induced changes in the endogenous SA content we analysed the levels of free and conjugated SA in unadapted and 100 mm NaCl-adapted cells (Table 1). The results indicate that SA is present in tomato cells as a free acid or conjugated to another compound(s). Treatments with β-glucosidase resulted in a significant increase in the amount of SA in the cell extracts, indicating that most of the SA is present in the conjugated form. Unadapted cells showed high endogenous levels of both free and conjugated SA, whereas a significant decrease in both SA forms occurred in response to salt adaptation. To assess the role of SA in salt-stress response we treated unadapted and NaCl-adapted cells with 100 mm NaCl and determined the f. and d. wts as well as the levels of free and conjugated SA contents after 1 h and 4 h of salt treatment (Table 1). Stress with 100 mm NaCl provoked a slight increase in conjugated SA contents in unadapted but not in NaCl-adapted cells. The preincubation of cells for 2 h in 200 µm SA followed by 4 h with 100 µm NaCl affected neither the f. wt nor the d. wt of tomato cells, but increased the level of conjugated SA (3.5-fold) and, above all, of free SA (30-fold) in unadapted cells (Table 1). However, in 100 mm NaCl-adapted cells this treatment increased the levels of conjugated and free SA similarly (25-fold and 30-fold, respectively). The exogenous application of SA during the 2-h pretreatment affected neither the f. wt nor the d. wt of tomato cells (data not shown).

The antioxidant and LOX enzyme activities were also differently affected in both types of cells by treatment with 200 µm SA followed by stress with 100 mM NaCl. When unadapted and NaCl-adapted cells were preincubated for 2 h in 200 µm SA followed by 4 h of stress with 100 mm NaCl, no differences in GR and LOX activities and a total inhibition in APX activity were detected, while a significant increase in Mn-SOD activity was detected in 100 mm NaCl-adapted cells (Tables 2 and 3).

To determine whether salt-adaptation or salt-stress caused oxidative damages in tomato cells, we monitored changes in lipid peroxidation by measuring the levels of thiobarbituric acid-reactive substances (TBARS). The results in Table 3 showed no significant differences in lipid peroxidation between unadapted and NaCl-adapted cells. However, stress with 100 mm NaCl during 4 h and, above all, treatments for 2 h with 200 µm SA followed by 4 h stress with 100 mm NaCl increased the peroxidation of lipids in unadapted cells while no differences were detected in NaCl-adapted cells.

Discussion

In this work we studied some of the mechanisms of salt adaptation in tomato cell suspensions. The nature of the damage caused by NaCl stress at the plant cellular level is not entirely clear. It is known that NaCl stress causes oxidative damage (Hernández et al., 1993, 1995; Gossett et al., 1994, 1996; Borsani et al., 2001). Drought, salt and cold stress all induce the accumulation of ROS such as O2 · –, H2O2 and hydroxyl radicals (Hasegawa et al., 2000). These ROS may be signals inducing ROS scavengers and other protective mechanisms, as well as damaging agents contributing to stress injury in plants (Prasad et al., 1994). Plants subjected to stress display complex molecular responses including the production of stress proteins and compatible osmolytes (Zhu et al., 1997). Many of the osmolytes and stress proteins with unknown functions probably detoxify plants by scavenging ROS or prevent them from damaging cellular structures. This is the case of transgenic plants overexpressing enzymes involved in oxidative protection, such as glutathione peroxidase, SOD, ascorbate peroxidases and GRs (Allen et al., 1997). A large body of evidence has accumulated from various plant systems showing that drought and salt stress alter the amounts and the activities of enzymes involved in scavenging oxygen radicals. Activities of cytosolic and chloroplastic copper (Cu)- and/or zinc (Zn)-SOD and cytosolic APX were increased by drought treatment of pea plants (Mittler & Zilinska, 1994). Similarly, osmotic stress induced the increase of Mn-SOD transcript abundance in maize (Zhu & Scandalios, 1994), and increased activities of the respective mitochondrial and chloroplastic SOD and APX were observed in pea plants under salt stress (Hernández et al., 1993).

The significant growth reduction brought about by salt stress in unadapted tomato cells compared with NaCl-adapted cells (Table 1) indicated a higher salt tolerance of the adapted cell line. Our study also indicates that the higher APX and GR activities in salt-adapted compared with unadapted cells (Table 2) could be related to a greater ability of adapted cells to decompose H2O2 and to control the reduced state of the glutathione pool, and thus be a key for the adaptation of tomato cell growth under saline conditions. In this respect, transgenic tobacco plants overexpressing GR had elevated levels of reduced glutathione and increased tolerance to oxidative stress in leaves (Broadbent et al., 1995). However, the fact that unadapted cells showed significantly higher Mn-SOD and LOX activities than NaCl-adapted cells (Tables 2 and 3) indicates that unadapted cells have a greater capacity for scavenging and dismutating O2 · – to H2O2 and for oxidizing polyunsaturated fatty acids.

If the salt adaptation process causes an increase in the capacity of tomato cells to protect themselves against oxidative damage, then it is possible that adaptation will also increase tolerance to additional salt-stress induced oxidative damage. We tested this hypothesis by comparing the resistance of unadapted and 100 mm NaCl adapted cells to 100 mm NaCl stress. When unadapted cells were incubated for 4 h with 100 mm NaCl, significant increases of Mn-SOD, APX (Table 2) and LOX (Table 3) activities were detected. In this way, a strong decrease in the mitochondrial Mn-SOD activity was detected in salt-sensitive cultivars of Pisum sativum under salt stress, whereas NaCl-tolerant cultivars appear to have a protection mechanism against salt-induced O2 · – production through induction of Mn-SOD activity (Hernández et al., 1993). Similarly, an increase in APX occurred in both Lycopersicon esculentum and Lycopersicon chilense subjected to water deficit (Smirnoff, 1993), and a recent study suggests that high levels of antioxidants and an active ascorbate–glutathione cycle are associated with salt-tolerance of cotton (Ashraf, 2002). In addition, Gossett et al. (1994) indicated that calli of the salt-tolerant cultivar of cotton grown at 150 mm NaCl had a higher capacity for scavenging and dismutating O2 · – and increased ability to decompose H2O2 when grown in media amended with NaCl. By contrast, many of the oxygenated products of fatty acids produced by LOX action have proven roles in growth, development, senescence and response to external stresses (Siedow, 1991). The results obtained by Roy et al. (1994) and van Gestelen et al. (1997) suggest that LOX can generate not only lipid hydroperoxides but also superoxide radicals via oxidation of pyridine nucleotides and therefore may significantly contribute to oxidative stress in plant cells. Similarly, it has been established that plant defense mechanisms such as membrane lipid deterioration or later reactions that coincide with the formation of lipid-derived phytotoxic substances may be challenged by LOX (Croft et al., 1993).

Various agents, such as Ca2+, ethylene, jasmonic acid and SA, have been proposed as signal transducers in plants (Klessig & Malamy, 1994). In many plant species, including tomato, soybean and rice, basal SA levels far exceed the high levels associated with systemic acquired resistance in tobacco and Arabidopsis without apparent deleterious biological effects (Coquoz et al., 1995; Hammond-Kosack et al., 1996). Because SA is involved in the oxidative response of plant tissues during salt and osmotic stress (Borsani et al., 2001), in the present work we have studied the role of SA in the adaptation to NaCl and in the oxidative stress response induced by NaCl stress in tomato cell suspensions. To our knowledge, this is the first study on the effect of SA content on salt adaptation and salt stress in plant cell suspensions. The lower amount of free and conjugated SA in tomato cells adapted to 100 mm NaCl compared with unadapted cells (Table 1) is consistent with the possibility that salt adaptation could be related to the concentration of SA in the cells. These results, together with the higher lipid peroxidation and the higher endogenous levels of conjugated SA in unadapted than in NaCl-adapted cells after a 4 h incubation with 100 mm NaCl (Tables 1 and 3), suggest that SA and lipid peroxidation could be involved in the pathway leading to the oxidative response induced by NaCl stress in tomato cells. Recently, Borsani et al. (2001) reported on the responses of wild-type Arabidopsis and a SA-deficient transgenic line expressing NahG, a salicylate hydroxylase coding gene, to different abiotic stress conditions. Their data suggest that SA is involved in the pathway leading to the induction of oxidative response triggered by salt stress. Similarly, it was found that SA is required for ozone tolerance by maintaining the cellular redox state and allowing defense responses (Sharma et al., 1996). However, by using an Arabidopsis genotype that accumulated high levels of SA, it was shown that SA activates an oxidative burst and cell death pathway leading to ozone sensitivity (Rao & Davis, 1999).

The total inhibition of APX and the stimulation of Mn-SOD observed in unadapted and NaCl-adapted cells preincubated in 200 µm SA followed by 4 h treatment with 100 mM NaCl (Table 2), suggest that the oxidative damage provoked by SA in tomato cells under salt stress could be caused by H2O2 accumulation. The ability of SA to block catalase and APX activities in tobacco (Durner & Klessig, 1995; Durner et al., 1997), supports the hypothesis that SA-induced defense response to tobacco mosaic virus is mediated, in part, through elevated H2O2 concentrations, providing the first indication of the existence of a link between SA and the oxidative stress in plants. Similarly, spraying mustard seedlings with SA significantly improved their tolerance to a subsequent heat shock at 55°C for 1.5 h (Dat et al., 1998), suggesting that thermoprotection obtained either by SA or by heat acclimatization may be achieved by a common signal transduction pathway involving an early increase in H2O2.

The present results indicate that the acquisition of salt-adaptation by tomato cells could be a consequence of a higher tolerance to oxidative stress, due to the regulation of LOX and antioxidant enzyme activities as well as the endogenous levels of SA. Thus, the increased activity of APX and GR as well as the reduced LOX activity and SA content in salt-adapted cells compared with unadapted cells may contribute to salt tolerance. Similarly, the increased LOX activity, lipid peroxidation and endogenous levels of conjugated SA in unadapted cells under 100 mm NaCl or under a treatment with SA followed by stress with 100 mm NaCl may contribute to the development of an oxidative stress response induced by NaCl.

Acknowledgements

The authors thank Conchi Santiago for help with the tomato cell suspension cultures. This work was supported by grants from the Spanish Dirección General de Investigación Científica y Técnica (PB 97-1266), the European Union FEDER Program (FD 97-0496-C03-02), and the Plan Andaluz de Investigación (PAI, Junta de Andalucía, Spain). AM and MCM were supported by FEDER Program fellowships; KV is recipient of a Ramón y Cajal research contract from the Spanish MCyT.

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