ABSTRACT

To gain better insights into the generation of oxidativestress by NaCl stress, we compared the effects of toxic, sub-lethal concentrations of NaCl (150 mM) with those ofCuSO4 (100 µM) on the antioxidant defence responses ofNicotiana plumbaginifolia L. plants grown in hydroponicculture. Short- and long-term adaptive responses wereinvestigated as well as the effects of root cutting to allowrapid absorption of ions. Our results show that photosyn-thesis is already affected after 12 h of both treatments andthat photoinhibition is induced. Furthermore, the twotreatments showed distinct patterns of induction of thegenes of the antioxidant systems. Principally, NaCl stimu-lated catalase activity through activation of the Cat2 andCat3genes, whereas with CuSO4, transcript levels, proteinlevels, and enzymatic activities of glutathione peroxidase,ascorbate peroxidase, and superoxide dismutase were dif-ferentially induced. The antioxidants ascorbate and glu-tathione were found to be largely oxidized after 12 h,suggesting an inability of the recycling process to cope withthe stress. In addition, both NaCl and CuSO4 treatmentsstimulated the accumulation of the osmolyte proline.Therefore, we conclude that the uptake and accumulationof different types of ions lead to the generation of distinctantioxidant responses in N. plumbaginifolia.

Key-words: Nicotiana plumbaginifolia L.; ascorbate–glu-tathione cycle; copper stress; oxidative stress; salt stress.

INTRODUCTION

Environmental stresses, such as drought, air pollutants, her-bicides, extreme temperature, or ultraviolet (UV) light gen-erate reactive oxygen species (ROS) in plant cells (Bowler,Van Montagu & Inzé 1992; Foyer & Harbinson 1994).Superoxide and hydrogen peroxide are generated by an

impairment of the electron transport in chloroplasts andmitochondria. Although superoxide- or hydrogen peroxide-induced radicals are generally not harmful at physiologicalconcentrations, they can be converted in a reaction catal-ysed by metals, such as Fe3+ or Cu2+ into one of the mostreactive molecules known in nature, the hydroxyl radical(OH·) (Halliwell & Gutteridge 1989). Hydroxyl radicalstrigger self-propagated peroxide reactions that lead tosevere membrane peroxidation, protein denaturation, andDNA damage. To avoid the production of these reactivemolecules, aerobic organisms have developed a number ofenzymatic mechanisms to deal with ROS. The dismutationof superoxide is catalysed by the superoxide dismutases(SODs). SODs are classified according to their metal cofac-tors and to date, four cDNAs have been characterized inNicotiana plumbaginifolia: SodCcand SodCpcoding forthe Cu/ZnSODs present in the cytosol and the chloroplast,respectively, SodAcoding for the mitochondrial MnSOD,and SodBcoding for the FeSOD located in the chloroplast(Inzé & Van Montagu 1995). Hydrogen peroxides areremoved by the H2O2-scavenging catalases (CATs) and per-oxidases. In N. plumbaginifolia, three different CATisozymes have been identified and shown to be differen-tially expressed according to the type of stress (Willekenset al. 1995). CAT1 is involved in removing H2O2 duringphotorespiration in the peroxisomes, CAT2 may play a rolein abiotic stress protection, and CAT3 is mainly associatedwith fatty acid degradation during glyoxysomal processes.Moreover, glutathione peroxidase (GPx), considered as oneof the key enzymes involved in scavenging oxyradicals inanimals, has recently been identified in plants [for a review,see Eshdat et al. (1997)]. GPx catalyses the reduction ofH2O2 and of organic and lipid hydroperoxides byglutathione (GSH). Ascorbate peroxidases (APx) usingascorbate (L-AsA) as an electron donor are one of the mostimportant H2O2 scavengers and are present in both thecytosol and the chloroplasts (Inzé & Van Montagu 1995).

High salinity also has a significant impact on plant cellmetabolism, resulting in reduced plant productivity by pro-voking osmotic stress and ion toxicity (Serrano & Gaxiola1994). Adaptation of the plant cell to high salinity mayinvolve osmotic adjustment and compartmentation of toxicions, but an increasing body of evidence suggests that high

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NaCl and CuSO 4 treatments trigger distinct oxidative defence

mechanisms in Nicotiana plumbaginifolia L.

A. SAVOURÉ,1* D. THORIN,1 M. DAVEY,1 XUE-JUN HUA,1 S. MAURO,2 M. VAN MONTAGU,1 D. INZÉ3 &N. VERBRUGGEN1

1Laboratorium voor Genetica, Departement Plantengenetica, Vlaams Interuniversitair Instituut voor Biotechnologie (VIB),Universiteit Gent, K.L. Ledeganckstraat 35, B-9000 Gent, 2 Laboratoire de Physiologie et Agrotechnologies Végétales, UniversitéLibre de Bruxelles, B-1050 Bruxelles, and 3 Laboratoire Associé de l’Institut National de la Recherche Agronomique (France),Universiteit Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium

ORIGINAL ARTICLE OA 220 EN

Correspondence: M. Van Montagu. Fax: 32-9-2645349; E-mail:mamon@gengenp.rug.ac.be

*Present address: Centre de Ressources Régionales en BiologieMoléculaire, Université de Picardie Jules Verne, F-80039 AmiensCedex, France.

387

salinity also induces oxidative stress, leading to structuraldamage to both membranes and the photosynthetic appara-tus. Depending on the plant species studied, differentenzymes of the antioxidant system are involved in theresponse to NaCl stress. NaCl was found to enhance SODs,CAT, APx, and GPx activities in Gossypium hirsutumcal-lus tissue (Gossett et al. 1994) and in citrus cells (Piqueraset al. 1996), whereas in NaCl-tolerant cells of Pisumsativum, two Cu/ZnSOD isoforms were induced whencompared with NaCl-sensitive cells (Olmos et al. 1994).An increase of Gpx transcript levels was also observed incitrus treated by NaCl (Holland et al. 1993). Recently,Willekens et al. (1997) demonstrated that CAT1-deficientplants are hypersensitive to NaCl stress.

In addition to these enzymatic systems, plants also con-tain non-enzymatic antioxidant systems involving low-molecular weight compounds, such as GSH and L-AsA,and are closely related through the Halliwell–Asada or L-AsA–GSH cycle (Asada & Takahashi 1987; Halliwell &Gutteridge 1989). Certain compatible solutes, such as sor-bitol, mannitol, and proline that accumulate in plant cellsmainly in response to osmotic stress and other environ-mental stresses, also show hydroxyl radical scavengingactivity in vitro (Smirnoff & Cumbes 1989). Proline con-tent is enhanced by an excess of Cu in the Cu-tolerantArmeria maritima(Farago & Mehra 1992) and a Lemnaminor (Bassi & Sharma 1993).

While Cu is an essential microelement for the growth ofplants, at higher doses it is toxic, triggering strong lipidperoxidation, and causing denaturation of both membranesand chlorophyll (Sandmann & Böger 1980; Luna,González & Trippi 1994). This metal ion is also an efficientcatalyst in the formation of harmful ROS and free radicals(Halliwel & Gutteridge 1984; Aust, Morehouse & Thomas1985; Girotti 1985). Adaptation to Cu excess involves adifferential activation of the plastid-localized SOD in N.tabacum (Kurepa, Van Montagu & Inzé 1997). ACu/ZnSOD was also shown to be induced by Cu in soy-bean root (Chongpraditnun, Mori & Chino 1992).

Comparison of NaCl-induced antioxidant defenceresponses of N. plumbaginifoliaplants grown in hydro-ponic culture to toxic, sublethal concentrations of CuSO4

may give better insight into the effects of NaCl in the gen-eration of oxidative stress. The hydroponic system pro-vides a suitable model system to analyse both short- andlong-term adaptive stress responses of N. plumbaginifoliaand to gain better knowledge into the timing of the antioxi-dant response. We present evidence that NaCl and CuSO4

trigger distinct oxidative stress responses, involving differ-ent protection mechanisms with different transcriptionaland post-transcriptional controls.

MATERIALS AND METHODS

Plant material and growth conditions

Seeds of N. plumbaginifoliaL. were germinated on moist-ened vermiculite according to Kampfenkel, Van Montagu

& Inzé (1995). Ten-day-old seedlings were then transferredto a hydroponic system and grown in a phytotron with alight intensity of 130µmol m–2s–1 on a 16 h light/8 h darkcycle at 24–26 °C. Salt and copper stresses were induced byplacing 7–8-week-old plants in fresh nutritive solution con-taining either 150 mM NaCl or 100µM CuSO4. In parallel,control plants were placed in a fresh nutritive solution. Thesame treatments were also applied to half of the plants fromwhich two-thirds of the roots had been removed beforetreatment, as described by Kampfenkel et al.(1995).

Young fully expanded leaves were collected 12 h and120 h after the beginning of the treatment. The leaves wereweighed, frozen in liquid nitrogen, and stored at – 80 °Cprior to analysis.

Analysis of the photosynthesis capacity

Measurements of photosynthesis capacity were carried outaccording to the method of Seaton & Walker (1990). Leafdiscs (9·6 cm2) were placed in an O2 electrode (HansatechLtd, Norfolk, UK) that had been modified to allow simulta-neous detection of chlorophyll and fluorescence. To followO2 evolution by polarography, the leaf chamber had to beused as a closed system. A capillary matting moistened withcarbonate buffer was used to permit high rates of photosyn-thesis. Chlorophylls were excited repeatedly with light-emitting diodes equipped with a 600 nm filter. Thelight-emitting diodes emitted a pulse of red light at 1·6 kHzor 100 kHz. Although the intensity of this light was insuffi-cient to start fluorescence induction, it enabled the measure-ment of the initial fluorescence level F0. Then, a shortsaturated white light of 4000µmol m–2 s–1 was used todetermine the maximum fluorescence intensity of the dark-adapted state (Fm). The sample was then adapted to anactinic non-modulated white light (200–2000 µmol m–2 s–1)to induce the kinetics of the chlorophyll fluorescence.Under this light, fluorescence induction reached a steadystate characterized by the intensity Fs. The maximum fluo-rescence intensity of the light-adapted state (Fm′) wasmeasured by adding a flash of saturated light. The initialintensity of the light-adapted state F0′ was obtained byputting the actinic light off and simultaneously applying asecond pulse of far-red light. Light intensity was measuredwith a LI-188B photometer (Li-Cor, Lincoln, NE, USA).

Gene expression analysis

Extraction of total RNA from frozen tissue and sequen-tial hybridization by using DNA or RNA probes wereperformed as previously described (Savouré et al. 1994).For RNA gel blot analysis, 10µg of total RNA was sepa-rated in 1·2% formaldehyde gels, transferred, and cross-linked to a Nylon Hybond-N membrane (Amersham,Aylesbury, UK). Prehybridization and hybridizationwere performed at 65 °C according to the method ofChurch & Gilbert (1984).

DNAprobes were [32P]-labelled using the random primerlabelling Megaprime system (Amersham). The cDNAs of

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N. plumbaginifoliaused were fragments of Cat1 (EcoRI-BamHI fragment of pCat1A; Willekens et al. 1994a), Cat2(BamHI fragment of pCat2A; Willekens et al. 1994a), Cat3(BamHI fragment of pCat3A; Willekens et al. 1994a),SodA(PstI fragment of pSOD1; Bowler et al. 1991), SodB(PstI fragment of pSOD2; Van Camp et al. 1990), SodCc(PstI fragment of pSOD3; Tsang et al. 1991), and Gpx(EcoRI fragment of 6P229; Criqui et al. 1992).

To obtain Apx- and SodCp-specific signals, [32P]-labelled RNA probes were generated with Arabidopsis Apx(linearized pAPXBLU vector with PstI and T3 RNA poly-merase; S. Kushnir, unpublished results, Universiteit Gent,Belgium) and N. plumbaginifolia SodCp(linearizedpGSOD4 vector with AccI and T7 RNA polymerase; Tsanget al. 1991).

The membranes were stained with methylene blue as acontrol for equal RNA loading (Sambrook, Fritsch &Maniatis 1989).

Enzymatic activities

Approximately 0·4–0·6 g of frozen leaf material wasground in liquid nitrogen. The powder was resuspendedin 1 cm3 of 50 mM N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulphonic acid) (pH 7·4), 5 mM MgCl2, 1 mM

ethylenediaminetetraacetic acid (EDTA), 1 mM ethyleneglycol-bis (β-aminoethyl ether) N,N,N′,N′-tetraaceticacid, 0·1% (v/v) Triton X-100. One tablet of the proteaseinhibitor cocktail (Complete; Boehringer, Mannheim,Germany) was added to 25 cm3 of the buffer solution. Todetermine APx activity, ascorbic acid was also includedin the buffer at a final concentration of 1 mM. Aliquots of100 mm3 were frozen in liquid nitrogen until used formeasurements.

For determination of SOD activity, 30µg of leaf proteinswere assayed by the ferricytochrome c reduction spec-trophotometric test (McCord & Fridovich 1969; Flohé &Otting 1984). Briefly, xanthine oxidase was added to atotal of 3 cm3 of assay buffer containing 50 mM potassiumphosphate, pH 7·8, 0·1 mM EDTA, 0·01 mM cytochrome c,and 0·05 mM xanthine; the reduction rate of cytochrome c(decreased by SOD) was monitored as an increase inabsorbance at 550 nm for 2 min after the addition of xan-thine oxidase. Assays were also performed using KCN andH2O2 that inhibit Cu/ZnSOD and FeSOD activities,respectively. Spectrophotometric determination of theCAT activity was performed according to the method ofClare et al. (1984).

APx activity was determined by monitoring the H2O2-dependent oxidation of ascorbic acid (Amako, Chen &Asada 1994). Protein extracts were desalted using aSephadex G-25 column (Pharmacia, Uppsala, Sweden)equilibrated in 50 mM potassium phosphate (pH 7·0),1 mM ascorbic acid, and 1 mM EDTA, and then concen-trated using a microcon-10 concentrator (Amicon,Beverly, MA, USA). Protein extract (15µg) was thenadded to the APx reaction assay containing 50 mM potas-sium phosphate (pH 7·0), 1 mM ascorbic acid, and 0·5 mM

H2O2. Measurements of the rate of the H2O2-dependentoxidation of ascorbic acid were established by monitoringthe decrease in absorbance at 290 nm. Measurements werealso performed in the presence of 200µM of the APxinhibitor p-chloromercuribenzoate (pCMB). To establishwhich of the plastidic or cytosolic APx isoforms wereinvolved in the activity, APx sensitivity to L-AsA wasinvestigated in the presence or the absence of 1 mM L-AsAin the desalting buffer.

Western blot analysis

Leaf proteins (15µg) were separated by sodium dodecylsulphate–polyacrylamide gel electrophoresis (SDS–PAGE)on a 12·5% gel and transferred to a nitrocellulose Hybond-C super membrane (Amersham) using a semidry blotter II(Biozym, Landgraaf, The Netherlands). The membraneswere blocked overnight with 5% skim milk and 0·1% poly-oxyethylenesorbitan (Tween-20) in phosphate-bufferedsaline (PBS) solution at 4 °C. After washing with PBS and0·1% Tween-20 for 50 min, the membranes were exposedfor 2 h to either anti-APx antibodies raised against a maizecytosolic APx (Koshiba 1993) or to anti-GPx antibodiesraised against a N. sylvestrisGPx (unpublished results). Themembranes were further washed with a 0·1% PBS–Tweensolution and then exposed for 2 h to horseradish peroxi-dase-linked antirabbit antibodies. After washing with a0·1% PBS–Tween solution, detection was performed usingan enhanced chemiluminescence (ECL) kit and ECL-Hyperfilm (Amersham).

Quantitation of proline, lipid peroxidation, L-AsA,and GSH

Proline determination was carried out according to themethod of Bates, Waldren & Teare (1973). To measure thedegree of lipid peroxidation, ≈ 0·1 g of leaves was groundin 20 mM Tris (pH 7·5) and the homogenate centrifugedfor 10 min at 4 °C. From the supernatant, the malonalde-hyde concentration was determined using the LPO-586 kitwith a standard curve of malonaldehyde (Bioxytech,Bonneuil-sur-Marne, France).

L-AsA and GSH were analysed by high-performanceliquid chromatography (HPLC) (Davey et al. in prepara-tion). Briefly, samples of 50–200 mg of leaf tissue wereweighed, snap-frozen in liquid nitrogen, and then thor-oughly crushed with liquid nitrogen using a precooledpestle and mortar. Extraction solution (0·4 cm3 of 3%metaphosphoric acid containing 1 mM EDTA) was addedand the samples were homogenized with frozen extractionbuffer until the mixture thawed. The samples were thentransferred to an Eppendorf tube and centrifuged. After asecond extraction, the supernatants were combined andfiltered for HPLC analysis. Analyses were carried outusing a reverse-phase HPLC column with simultaneousphotodiode array and electrochemical detection.Duplicate analyses of 10–20 mm3 of extracts were per-formed and oxidized L-AsA and GSH were calculated by

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subtraction after reduction of samples with dithiothreitoland repeat HPLC analysis. The concentrations were calcu-lated from standard curves prepared using pure standardsin extraction buffer.

RESULTS

Seven- to 8-week-old N. plumbaginifoliaplants treatedwith either 150 mM NaCl or 100µM CuSO4 were analysedafter 12 h or 120 h. For half of the plants, two-thirds of theroots were removed to permit rapid uptake of ions throughthe transpiration stream. As a 12-h treatment of hydroponi-cally grown plants without root cutting had only minoreffects on the oxidative stress system, probably because ofthe limited absorption of ions (Kampfenkel et al.1995; ourdata), this experimental condition was not developed in thepresent study.

Effect of NaCl and CuSO 4 treatments onphotosynthetic capacity and lipid peroxidation

The photosynthetic capacity of plants exposed to NaCl orCuSO4 was analysed by the Fm/F0 fluorescence parameterand measurement of O2 production (Table 1). Comparedwith the control, 12 h of NaCl or CuSO4 treatmentsdecreased the Fm/F0 value by 21 and 28%, respectively,indicating that the leaves had been photoinhibited. Bothtreatments also reduced the photosynthetic O2 production.This effect was more pronounced at low-light than at high-light intensities.

Lipid peroxidation, as estimated from the levels of mal-onaldehyde, was found to increase with NaCl treatmentand to a greater extent with CuSO4 treatment (Fig. 1). Thelipid peroxidation values from only one experiment areindicated in Fig. 1, because the baseline levels variedslightly between different experiments. However, in all thedifferent experiments performed, similar ratios of increasewere found between the control, NaCl, and CuSO4.Furthermore, we observed that an increase of the exposuretime to NaCl or CuSO4 from 12 h to 120 h alwaysenhanced the degree of lipid peroxidation.

Differential expression of Gpx, Apx , Sod, and Catgenes in response to NaCl and CuSO 4

The effects of 12-h and 120-h treatments on the expres-sion of antioxidant genes are shown in Fig. 2. With the

exception of SodCp, the steady-state transcript levels ofGpx, Apx, SodA, SodB, and SodCcincreased after 12 h ofNaCl treatment (Fig. 2a). CuSO4 treatment was found toonly trigger the accumulation of Gpx and SodCctran-scripts and to strongly downregulate SodB. Interestingly,Gpx displayed an expression pattern that was similar tothat of SodCcwhatever the stress. The 120-h NaCl treat-ment had little or no effect on the transcript levels of thevarious antioxidant genes except that Apxand SodBwereenhanced in plants with partially removed roots, and thatSodAand SodCptranscript levels were slightly increasedin plants with intact roots. In contrast, CuSO4 treatmentenhanced the steady-state transcript level of SodAanddownregulated Apx and SodB. SodCcand SodCptran-script levels were very low and seemingly unaffected byCuSO4 treatment. Apart from Gpxand SodB, there was nosignificant difference in the transcript levels of theantioxidant genes analysed between control plants withintact or partially cut roots.

The Cat genes also showed different patterns ofexpression according to the treatment. In control plants,the Cat1 transcript level was higher after 120 h than 12 h(Fig. 2b). However, Cat1 was downregulated by CuSO4

and unaffected by NaCl, whatever the duration of thetreatment. Cat2and Cat3showed similar expression pat-terns, with expression of both transcripts being enhanced

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O2 (µmol m–2 h–1)

Light intensity (µmol m–2 s–1)Samples Fm/F0 2000 1000 500 200

Control 4·47 ± 0·27 38·20 ± 2·59 29·72 ± 1·21 25·15 ± 1·32 6·86 ± 1·98NaCl 3·52 ± 0·55 23·33 ± 2·31 16·81 ± 2·87 15·87 ± 1·96 5·45 ± 1·81CuSO4 3·22 ± 0·47 22·66 ± 2·50 14·45 ± 0·89 14·45 ± 0·89 1·48 ± 1·04

Table 1. Evolution of the Fm/F0

fluorescence parameter and O2 rateaccording to the light intensity in control andin NaCl- and CuSO4-treated leaves

Figure 1. Lipid peroxidation in leaves after NaCl or CuSO4

treatments. Seven- to 8-week-old plants were treated with eithernutritive solution alone or supplemented with 150 mM NaCl or100 µM CuSO4 for 12 h and 120 h. Half of the plants had two-thirds of their roots removed (root cutting). The values shown arefrom one experiment and are expressed as malonaldehydemolecule produced per g fresh weight calculated by usingstandard curves prepared from pure standard.

by NaCl after only 12 h of treatment and to a higherextent by CuSO4 after 120 h of treatment.

Effects of NaCl or CuSO 4 treatments on SOD,CAT, APx, and GPx enzymes

The activities of SOD, CAT, and APx in leaves are pre-sented in Fig. 3. After 12 h, both NaCl and CuSO4 treat-ments decreased total SOD activity (Fig. 3a). After 120 hhowever, CuSO4 treatment was found to strongly stimulateSOD activity by an average of 3·6-fold in the two types ofplants, whereas SOD activity remained lower than in con-trol plants in the NaCl treatments. No large differenceswere found in plants with intact or cut roots. In order toidentify the SOD isozymes induced by CuSO4 after 120 h,the effects of different inhibitors of the activity of the dif-ferent isozymes were tested. In the presence of KCN,which inhibits Cu/ZnSOD activity, the CuSO4-inducedSOD activity was dramatically suppressed to levels com-parable with the control plant (data not shown). HigherCu/ZnSOD activity was also observed on SOD activitygels (data not shown). Therefore, the increase in SODactivity triggered by CuSO4 is mainly due to an increase inthe levels of the Cu/ZnSOD isoforms.

CAT activity was stimulated by 12-h and 120-h NaCltreatments whereas CuSO4 treatment repressed it (Fig. 3b).In the control plants after 120 h of treatment, higher CATactivity was detected in the leaves from plants with intactroots compared with plants with cut roots.

A decrease in APx activity was observed after 12 h oftreatment with either NaCl or CuSO4. After 120 h of treat-ment, CuSO4 stimulated APx activity 6-fold whereas NaClhad no effect. These results were similar in intact plants orin plants with partially cut roots, whatever the conditions(Fig. 3c). It is documented that the chloroplastic APx iso-form has a high specificity for L-AsA and as a result, theenzyme becomes extremely labile when L-AsA is absent(Chen & Asada 1989). In contrast, the cytosolic isoform isless sensitive to L-AsA depletion in pea (Mittler &Zilinskas 1991). Using an L-AsA-depleted medium, 50%of the total APx activity was inactivated (data not shown),indicating that the CuSO4-induced APx activity is due toan increase of both the cytosolic and the chloroplastic iso-forms. This enhanced APx activity resulted partly from anincrease of the cytosolic APx protein content as detectedon Western blot (Fig. 4a).

Because guaiacol peroxidases can also utilize L-AsA asan electron donor, we performed a specific assay usingpCMB, an inhibitor of APx activity, to determine theinvolvement of these enzymes in the observed increase inAPx activity. pCMB completely abolished APx activity,demonstrating that the measured APx activity was not dueto any guaiacol peroxidase activity.

Already after 12 h of treatment, CuSO4 induced the pro-tein content of GPx, which tended to be higher after 120 h,whereas NaCl treatment had little or no inductive effect onthe GPx protein content in plants with intact or cut roots,respectively (Fig. 4b).

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Figure 2. Differential expression of antioxidant genes afterNaCl or CuSO4 treatments in Nicotiana plumbaginifolia.Experimental conditions are as indicated in Fig. 1. Northern blotsof total RNA samples isolated from leaf samples were hybridizedwith Gpx, Apx, SodA, SodB, SodCc, and SodCp(a) or with Cat1,Cat2, and Cat3 (b). For RNA control loading, the gels werestained with methylene blue.

NaCl and CuSO 4 treatments trigger prolineaccumulation

After 12 h of treatment, proline levels were induced by2·9-fold and 1·9-fold in NaCl- and in Cu-treated plants,respectively, relative to the control plants (Fig. 5). After120 h, the proline content of treated plants had increaseda further 3-fold to reach levels 10-fold higher than thoseof control plants. Statistically, the differences between

proline levels after CuSO4 or NaCl treatments were notsignificant. The removal of the roots had apparently noeffect on the proline content in the control plants regard-less of the experimental conditions.

NaCl and CuSO 4 stimulate non-enzymaticantioxidant defence mechanisms

In addition to enzymatic activities, we also determined theeffects of NaCl or CuSO4 on the levels of reduced andoxidized forms of the major cellular antioxidants, L-AsAand GSH (Fig. 6). After 12 h of treatment, both NaCl andCuSO4 decreased the levels of L-AsA with a correspondingincrease in the levels of the oxidized form (dehydroascor-bic acid (DHAA)). The GSH level was enhanced byCuSO4 with a parallel decrease of the oxidized form(GSSG). NaCl only decreased GSSG levels. After 120 h oftreatment, the level of L-AsA was almost undetectable inboth NaCl and CuSO4 treatments, whereas the levels of theoxidized (DHAA) form were high. In contrast, NaCl andCuSO4 both stimulated GSH accumulation to the detrimentof the GSSG levels, which were very low. A very highGSH level was observed in intact plants treated withCuSO4.

DISCUSSION

Here we present evidence that both NaCl and CuSO4

treatments generate the production of powerful oxidantsin N. plumbaginifolia. A comparison of these two differ-ent types of stresses has brought new insights into thedifferent signal transduction pathways involved in theoxidative stress response.

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Figure 3. Antioxidant enzyme activities after NaCl or CuSO4

treatments. Experimental conditions are as indicated in Fig. 1.Superoxide dismutase (SOD; a), catalase (CAT; b), and ascorbateperoxidase (APx; c) activities are expressed as µmol min–1 mg–1

protein. Bars represent the standard deviation from threeindependent experiments.

Figure 4. Ascorbate peroxidase (APx) and glutathione peroxidase(GPx) protein contents in leaves. Immunodetection of APx (a) andGPx (b) in leaves treated with either the nutritive solution alone orsupplemented with NaCl or CuSO4 for 12 and 120 h.

The measurement of fluorescence and O2 productionshowed that after only 12 h of stress, photosynthesis isaffected by NaCl and, to a greater extent, by CuSO4 treat-ment, although the effects on the metabolism are small.CuSO4 had a dramatic effect on the photosynthetic capac-ity of the cells, possibly by impairing the electron transportcapacity, leading to the production of hydroxyl radicals.Lipids are very sensitive to hydroxyl radicals and the pres-ence of lipid peroxides is one of the first signs of oxidativestress. NaCl and, to a greater extent, CuSO4 stimulate thelevels of lipid peroxidation in leaves and similarly thesteady-state transcript level of Gpx. Furthermore, CuSO4treatment had a strong enhancing effect on the amount ofGPx proteins, indicating that the accumulation of GPx isdependent on the severity of the stress. NaCl and CuSO4

had a similar inductive effect on Gpx expression, as haspreviously been observed with O3, sulphur dioxide, andUV-B treatments (Willekens et al. 1994b). Our results fur-ther strengthen the proposition that GPx may play animportant role in the rapid defence responses of plant cellsto lipid peroxidation (Willekens et al. 1994c).

Our results show that genes coding for enzymesinvolved in the removal of ROS are regulated at the tran-scriptional and post-transcriptional levels according to thetype of stress. NaCl enhanced the steady-state transcriptlevels of SodA, SodB, and SodCc, although the total SODactivity actually diminished. This effect was not due to adecrease in the activity of a particular SOD isozyme (deter-mined by isoelectrofocusing isoform separation; data notshown). The discrepancy between transcript levels andenzyme activities during NaCl treatment may, therefore,result from a higher turnover of SODs and/or an increase oftheir inactivation by the H2O2 end product (Scandalios1993). On the other hand, CuSO4 increased the steady-state transcript level of SodCcresulting in an enhancedactivity of the cytosolic Cu/ZnSOD, in agreement withprevious observations in soybean root (Chongpraditnunet al. 1992). However, SodCpexpression was unaffectedby CuSO4, although chloroplastic Cu/ZnSOD activity

increased (determined by isoelectrofocusing isoform separa-tion; data not shown). The increase in SOD activities wouldreflect an enhanced production of superoxide in the chloro-plasts and in the cytoplasm, probably because of impairmentof electron transport in the photosystems and Cu-catalysedauto-oxidation of cellular components, respectively. SODactivity may depend not only on transcriptional but also onpost-transcriptional regulation of Sodgenes.

L-AsA and GSH are among the most important non-enzymatic antioxidant defence compounds. The ratio ofthe reduced:oxidized forms is considered an importantindicator of the redox state of the cell and the degree ofoxidative stress experienced. After 12 h of treatment, asubstantial proportion of both GSH and L-AsA was still inthe reduced form, although both stress treatments resultedin higher proportions of GSSG and DHAA than in the con-trols. The fact that the plants maintained levels of L-AsAand GSH after 12 h is indicative of an ability to regenerateL-AsA and GSH, presumably through the L-AsA–GSHcycle. Both the proportion and the levels of DHAA presentsuggest that the severity of the stress increases in the order:control < NaCl < CuSO4. The increase in total L-AsA (L-AsA + DHAA) levels also suggests that L-AsA biosynthe-sis has been stimulated to deal with the stress conditions.

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Figure 5. Proline accumulation in leaves after NaCl andCuSO4 treatments. Experimental conditions are as indicated inFig. 1. Bars represent the standard deviation from threeindependent experiments.

Figure 6. Endogenous levels of ascorbate (L-AsA), DHAA,glutathione (GSH), and GSSG in leaves after 12 h and 120 h ofNaCl or CuSO4 treatment. The L-AsA and GSH levels weredetermined by high-performance liquid chromatography analyseswith simultaneous ultraviolet (UV) and electrochemical detection.Experimental conditions are as indicated in Fig. 1. Duplicateextractions of the same tissue generally yielded L-AsA/GSHvalues that agreed within 5–10% of each other, while replicateanalyses of the same extract agreed within ≈ 3–5% of each other.Because of this, and limitations in the amount of tissue available,only one set of extractions was performed.

The results after 120 h of treatment are more difficult tointerpret. In none of the plants examined was there a signif-icant amount of L-AsA present, with all the ascorbate beingpresent in the oxidized form (i.e. DHAA). Moreover, thetotal L-AsA levels were lower, suggesting disruption of thebiosynthetic processes or an ‘uncoupling’ of the L-AsA–GSH pathway. Surprisingly, we could not detect anysignificant GSSG levels at 120 h. The accumulation ofGSH may also correspond to an increase of the glutathionereductase activity, the overproduction of which has beenshown to partially protect the L-AsA pool from oxidation intobacco (Foyer et al. 1991). In either case, it is clear thatafter 120 h of treatment, aspects of the antioxidant defencesystem are severely disrupted.

APxs play a key role in the removal of H2O2 in thechloroplast and in the cytosol. Long-term treatment withCuSO4 stimulated both cytosolic and chloroplastic APxactivity and APx protein levels by 5–7-fold, although NaClhad no significant effect on APx activity and only a limitedenhancing effect on APx protein level. The Apx transcriptlevel, however, was found to be repressed by the CuSO4

treatment. These contradictory results suggest that theenhanced APx protein is encoded by a gene that remainedundetected in the RNA gel blot analysis. In strong supportof this hypothesis, several genes coding for APx have beencloned in Arabidopsis that had not previously beendetected by RNA gel blot (Santos et al. 1996).

CATs are associated with the removal of H2O2 in peroxi-somes [for a review see Willekens et al. (1995)]. NaCl andCuSO4 triggered differential alteration in the transcript lev-els of the different Cat genes. The NaCl induction of Cattranscripts was correlated with increased CAT activity. Incontrast, accumulation of Cat transcripts with CuSO4 didnot lead to a corresponding increase in CAT activity. Thismay be due to a decrease in the levels of the Cat1transcriptby CuSO4 because Cat1 is the major transcript present inphotosynthesizing cells of N. plumbaginifolia(Willekenset al. 1994b). However, the NaCl-induced Cat transcriptlevels and CAT activity suggest that the peroxisomes arevery sensitive to NaCl stress. It is still not clear whetherthis CAT response is due to the presence of toxic concen-trations of ions or due to osmotic stress.

The decrease observed in CAT activity with CuSO4

may, therefore, be due to photoinhibition of photosystemII preventing H2O2 production both in the chloroplastsand in the peroxisomes (Volk & Feierabend 1989;Feierabend, Schaan & Hertwig 1992). At the same time,an increase in APx activity may compensate for the lossof CAT activity.

Both CuSO4 and NaCl treatments (i.e. non-osmotic andosmotic stress, respectively) triggered an accumulation ofproline. We also found that proline accumulated to a higherlevel and more rapidly in young leaves of NaCl-treatedthan in Cu-treated plants where it accumulated homoge-nously in all leaves, but to a lower level (data not shown).Proline may play a role in the antioxidant adaptation ofthe plant cells against the presence of hydroxyl radicals, ashas been previously demonstrated by in vitro assays

(Smirnoff & Cumbes 1989). However, the induction ofoxidative stress by iron does not lead to significant prolineaccumulation in N. plumbaginifolia(unpublished results).These results suggest that proline may play different rolesin the two stresses, that proline accumulation is not coupledto oxygen radical production, and that it is independent ofthe oxidative stress signal transduction.

Both NaCl and CuSO4 treatments stimulated the prolinebiosynthesis pathway and the antioxidant capacity of theplant cell (Fig. 7). However, a comparison of these twostresses demonstrates the dramatic ability of CuSO4 togenerate severe oxidative damage in the cell, while NaClonly induced a limited antioxidant response. NaCl seemsto stimulate the plastid-located antioxidant systems; theincreased sensitivity of CAT1-deficient plants to NaClstress provides proof for the role of H2O2 as an importantmediator of cellular toxicity (Willekens et al. 1997). H2O2

may be produced in the chloroplasts and by various oxi-dases in the peroxisomes during NaCl treatment. This is incontrast to CuSO4, which has a major impact on thecytosolic antioxidant system mainly by inducing SOD andAPx activities as well as GPx protein content.

Together these results suggest that ROS were generatedin different compartments of the cell and that specific andcommon transduction cascades are stimulated according tothe type of treatment, resulting in a fine regulation of theantioxidant system at transcriptional and post-transcrip-tional levels for the adaptive benefit of the plant.

ACKNOWLEDGMENTS

The authors are grateful to Dr K. Kampfenkel for helpfuladvice and discussion and thank Dr L. Lepiniec for GPxantibodies, Dr T. Koshiba for APx antibodies, K. Spruyt forthe photographic work, and Dr M. De Cock for help inpreparing the manuscript. This work was supported bygrants from the Vlaams Actieprogramma Biotechnologie(ETC 002), the International Human Frontier ScienceProgram (IHFSP RG-434/94 M) and the Fund for ScientificResearch (Flanders) (G.0047.96). A.S. is indebted to the

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Figure 7. Effects of NaCl and CuSO4 on both short- and long-termadaptative antioxidant responses in Nicotiana plumbaginifolia.

European Union for a Human Capital and Mobility post-doctoral fellowship (ERBCHBICT-941824). D.I. is aResearch Director of the Institut National de la RechercheAgronomique (France).

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Received 17 June 1998; received in revised form 28 September1998; accepted for publication 5 October 1998

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