PHYSIOLOGIA PLANTARUM 109: 435–442. 2000

Copyright © Physiologia Plantarum 2000 ISSN 0031-9317

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Differential response of antioxidant compounds to salinity stress in salt-tolerant and salt-sensitive seedlings of foxtail millet (Setaria italica) N. Sreenivasulua,b, B. Grimma, U. Wobusa and W. Weschkea,* a

Institut fu¨r Pflanzengenetik und Kulturpflanzenforschung, D-06466 Gatersleben, Germany Department of Botany, S.K. Uni6ersity, Anantapur-515 003, India *Corresponding author, e-mail: [email protected]

b

Received 20 August 1999; revised 30 November 1999; in final form 20 March 2000

The modulation of antioxidant components was comparatively analysed in a salt-tolerant (cv. Prasad) and salt-sensitive (cv. Lepakshi) cultivar of foxtail millet (Setaria italica L.) under different NaCl concentrations. Under conditions of salt stress, the salt-tolerant cultivar exhibited increased total superoxide dismutase (SOD) and ascorbate peroxidase (APX) activity, whereas both enzyme activities decreased in acutely saltstressed seedlings of the sensitive cultivar. At 200 mM NaCl, the tolerant foxtail millet cultivar responded with induction of cytosolic Cu/Zn-SOD and the Mn-SOD isoform at the protein level. The induced accumulation of the cytosolic Cu/Zn-SOD

protein/activity is positively correlated with an elevated level of the cytosolic APX gene activity. The elevated cytosolic Cu/ZnSOD and cytosolic APX activity correlates with an induced accumulation of their transcripts. Tolerant 5-day-old seedlings grown during high salinity treatment (200 mM NaCl) contained a lower amount of Na + ions and showed a lower electrolyte leakage than sensitive seedlings. In conclusion, our comparative studies indicate that salt-induced oxidative tolerance is conferred by an enhanced compartment-specific activity of the antioxidant enzymes in response to compartment-specific signals.

Introduction The progressive salinisation of irrigated land is a major environmental threat for crop production. Therefore, the selection and characterisation of salt-resistant species are important to ensure future productivity of the arid agricultural regions. High exogenous salt concentrations cause an imbalance of the cellular ions resulting in ion toxicity, osmotic stress and production of active oxygen species (Cheeseman 1988, Navari-Izzo et al. 1988, Cramer et al. 1994). Plants develop defence strategies against salt stress based on the activation of the ion transport system, osmotic adjustment and induction of antioxidant enzymes (Gibson et al. 1984, Dionisio-Sese and Tobita 1998). High salt concentrations normally impair the cellular electron transport within the different subcellular compartments and lead to the generation of reactive oxygen species (ROS) such as singlet oxygen, superoxide, hydrogen peroxide and hydroxyl radicals (Elstner 1982, Hernandez et al. 1993, 1995). Excess of ROS triggers phytotoxic reactions such as lipid peroxidation, protein degradation and DNA

mutation (Stewart and Bewley 1980, Fridovich 1986, Davies 1987, Smirnoff 1993). To overcome salt-mediated oxidative stress, plants detoxify ROS by upregulating antioxidative enzymes (superoxide dismutase [SOD], ascorbate peroxidase [APX], glutathione cycle enzymes, etc.) and producing low molecular mass antioxidants (mainly flavonones, anthocyanins, a-tocopherol, ascorbate, glutathione, and polyphenolic compounds). A main protective role is attributed to SOD in catalysing the dismutation of superoxide anions to dioxygen and hydrogen peroxide (H2O2). The increased production of H2O2 is subsequently counteracted by ascorbate peroxidase or catalase detoxification (Nakano and Asada 1981). A number of studies indicated that the degree of oxidative cellular damage in plants exposed to abiotic stress is controlled by the capacity of the antioxidative systems (Dhindsa 1991, Perl-Treves and Galun 1991, Zhang and Kirkham 1994, Zhu and Scandalios 1994, McKersie et al. 1996, Noctor and Foyer 1998). A correlation between the antioxidant capacity and NaCl tolerance

Abbre6iations – APX, ascorbate peroxidase; EL, electrolyte leakage; H2O2, hydrogen peroxide; NBT, nitro blue tetrazolium; ROS, reactive oxygen species; SOD, superoxide dismutase. Physiol. Plant. 109, 2000

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was demonstrated in several plant species, e.g. Gossypium hirsutum cultivars (Gossett et al. 1994) and Oryza sati6a (Dionisio-Sese and Tobita 1998). Bueno et al. (1998) showed the upregulation of antioxidants, superoxide dismutase and ascorbate peroxidase in response to salt stress at the transcriptional and translational level. However, comparative analyses of NaCl-dependent antioxidant modulation between tolerant and sensitive cultivars of the same plant species are still quite rare. In a previous paper, we presented data on qualitative and quantitative changes in peroxidase activity upon NaCl incubation in 5-day-old seedlings of two Setaria italica L. genotypes contrasting in salt tolerance. The tolerant seedlings responded to NaCl with an increase of total peroxidase activity and induction of acidic guaicol peroxidase, pI 5.5 (Sreenivasulu et al. 1999). Using the same experimental system, we compared physiological, biochemical and molecular parameters such as growth rate, electrolyte leakage, Na + content, steady-state transcripts and protein levels of different compounds of the antioxidative system upon different NaCl concentrations. We propose that the lower endogenous Na + concentration measured under salt stress in cells of the tolerant cultivar, as well as the increased enzymatic capacity and inducibility of cytosolic Cu/Zn-SOD, Mn-SOD and cytosolic APX, significantly contributes to the tolerance against salt-induced oxidative stress.

Materials and methods Plant material and salinity treatments Seeds of foxtail millet (Setaria italica L.) cv. Prasad (salt-tolerant) and cv. Lepakshi (salt-sensitive) were provided from Andhra Pradesh Agricultural Experimental Station, Anantapur, India. Seeds were surface-sterilised in 0.1% (w/v) sodium hypochlorite for 5 min, thoroughly rinsed with distilled water and germinated on filter paper in Petri dishes. Seeds were incubated in Hoagland medium with 0 (control), 50, 100, 150 and 200 mM NaCl. The Petri dishes were kept at 25°C under aseptic conditions for 5 days in the dark. Growth measurements The length of the primary shoot was measured for 100 seedlings (5-day-old) from each cultivar grown with 0 (control), 50, 100, 150 and 200 mM NaCl in 3 independent experiments. The percentage of relative shoot growth inhibition was calculated from the mean shoot length measurements. Determination of sodium content Sodium concentrations were determined as described by Dionisio-Sese and Tobita (1998). Dried seedlings (10 mg) were cut into pieces of 5 mm length and placed in test tubes containing 20 ml distilled deionised water. The tubes were incubated in boiling water for 1 h, autoclaved at 121°C for 20 min and cooled. The sodium concentration was determined by using an atomic absorption spectrophotometer (AA-660, Shimadzu, Tokyo, Japan). 436

Electrolyte leakage Electrolyte leakage was determined as described by DionisioSese and Tobita (1998). Fresh seedlings (200 mg) were cut into pieces of 5 mm length and placed in test tubes containing 10 ml distilled deionised water. The tubes were incubated in a water bath at 32°C for 2 h and the initial electrical conductivity of the medium (EC1) was measured. The samples were autoclaved at 121°C for 20 min to release all electrolytes, cooled to 25°C and the final electrical conductivity (EC2) was measured. The electrolyte leakage (EL) was calculated by using the formula EL = EC1/EC2 × 100. Determination of ascorbate content Ascorbate content was determined according to the modified method of Law et al. (1983). Seedlings (100 mg) were homogenised in 1 ml 5% (w/v) sulphosalicylic acid. After centrifugation, the supernatant was adjusted to pH 5.5–6.5 by adding 800 ml 150 mM phosphate buffer (pH 6.4) and 33 ml 5 M NaOH. The aliquots of samples and blank were then sequentially mixed with 100 ml H2O, 200 ml 10% (w/v) tri-chloro acetic acid, 200 ml 44% (v/v) H3PO4, 200 ml 4% (w/v) bipyridyl (dissolved in 70% ethanol) and 100 ml of 3% (w/v) FeCl3. After 60 min incubation at 30°C, the absorbance of the solution was measured at 525 nm. For measuring the content of total ascorbate, the oxidised fraction was reduced by adding 50 ml of 10 mM DTT. The solution was incubated for 15 min at room temperature and the surplus DTT subsequently inactivated by adding 50 ml of 0.5% (w/v) N-ethylmaleimide. The concentrations of ascorbate and oxidised ascorbate were determined by using a calibration curve of ascorbate and dehydroascorbate standards, respectively. The determination of total and reduced ascorbate was 9-fold repeated for each independent sample. Superoxide dismutase activity and activity pattern of SOD isoforms Five-day-old seedlings (100 mg) of both cultivars were ground to a fine powder in liquid nitrogen and homogenised in 50 mM potassium phosphate buffer (pH 7.8) containing 5 mM sodium ascorbate, 7 mM b-mercaptoethanol and 0.2% (v/v) Triton X-100 and centrifuged at 9000 g for 10 min at 4°C. Total SOD activity of the samples was assayed by measuring its ability to inhibit the photochemical reduction of nitro blue tetrazolium (NBT), according to Stewart and Bewley (1980). The reaction mixture (3 ml) contained 13 mM methionine, 75 mM NBT, 100 mM EDTA, 50 ml of enzyme extract within 50 mM phosphate buffer (pH 7.8). The reaction was started with 2 mM riboflavin by exposing the cuvette to a 15-W fluorescent tube for 10 min. The absorbance of each reaction mixture was measured at 560 nm. One unit of SOD activity was defined as the amount of enzyme which causes 50% inhibition of the photochemical reduction of NBT. For the separation of SOD isozymes, 25 mg protein extract was applied to native PAGE using a 5% stacking and a 13% separating gel. After electrophoresis, the gel was stained according to the method of Beauchamp and Fridovich (1971) to identify each SOD isoform. Four bands Physiol. Plant. 109, 2000

of different intensity were visible. According to the pattern of SOD isoforms in native gels described for tobacco leaves (Van Camp et al. 1994, Slooten et al. 1995), the first (from the top of the gel) very sharp band should correspond to the Mn-SOD. At the second position, a highly intensive and broad band was seen followed by two bands of lower intensity. The latter bands were assigned to two different isoforms of the plastidic Cu/Zn-SOD. The four different bands specific for millet seedlings were quantified by using the software package Bio 1D of an imaging system (Vilber Lourmat, France). A second set of protein extracts separated on a native gel was stained with a final concentration of 2 mM KCN/5 mM H2O2 in the reaction mixture. Only the first very sharp Mn-SOD band was visible in the same intensity, indicating that the other activity bands correspond to the cytosolic and the two plastidic isoform(s) of the Cu/Zn-SOD, respectively. Contrary to the results reported for tobacco leaves (Van Camp et al. 1994, Slooten et al. 1995), no activity of a Fe-SOD was detectable in millet seedlings. Ascorbate peroxidase assay Ascorbate peroxidase activity was measured according to Aono et al. (1995). Seedlings (100 mg) were homogenised in 1 ml of 50 mM phosphate buffer (pH 7.8) containing 5 mM ascorbate, 5 mM DTT, 5 mM EDTA, 100 mM NaCl and 2% (w/v) polyvinyl pyrrolidone (PVP). The homogenised material was centrifuged at 15 000 g for 15 min at 4°C. The reaction was initiated by adding H2O2 to a final concentration of 44 mM as described by Nakano and Asada (1981). The reaction rate was monitored by the decrease in absorbance at 290 nm. The rate constant was calculated using the extinction coefficient of 2.8 mM − 1 cm − 1 and corrected for the rate obtained prior to the addition of H2O2. Extraction of proteins and western blot analysis Five-day-old seedlings of both cultivars were homogenised in 50 mM Tris-HCl buffer, pH 7.4, at 4°C. The homogenate was centrifuged at 9000 g for 20 min at 4°C in a refrigerated high speed centrifuge (Himac Centrifuge, Hitachi Medical Systems, Du¨sseldorf, Germany). The resultant supernatants were assayed for protein content by the method of Bradford (1976) using bovine serum albumin (BSA) as the standard. Equal amounts of protein (25 mg per lane) were resolved on a 12% SDS-PAGE. Western blot analysis was performed as described by Kruse et al. (1995). Membranes were blocked with 3% (w/v) BSA and treated with spinach cytosolic Cu/Zn-SOD antibody (kindly provided by Dr K. Asada, Fukuyama, Japan; see Kanematsu and Asada 1990, Ogawa et al. 1997 for detailed information on the specificity of the cytosolic Cu/Zn-SOD antibody), tobacco Mn-SOD antibody (kindly provided by Dr D. Inze´, Gent, Belgium; see Bowler et al. 1989, 1991 for detailed information on the specificity of the Mn-SOD antibody) and an antiserum against spinach cytosolic ascorbate peroxidase (kindly provided by Dr Saji, Onagawa, Tsukuba, Japan; see Mock et al. 1998 for information on the antibody). The bound Physiol. Plant. 109, 2000

antibodies were detected with an anti-rabbit IgG-peroxidase conjugate using the ECL reaction according to the manufacturer’s protocol (Amersham Pharmacia Biotech UK Ltd., Little Chalfont, UK). RNA isolation and RNA gel blot analysis RNA was extracted from frozen seedlings as described by Heim et al. (1993). Total RNA (15 mg per lane) was separated on 1% agarose-formaldehyde gels and blotted on Hybond N + membranes (Amersham Pharmacia) according to the manufacturer’s protocol. Filters were hybridised at 55°C according to the method of Church and Gilbert (1984) to 32P-labelled fragments specific for SODs and APX from tobacco and cucumber, respectively. The mitochondrial, cytosolic, plastidic SOD and APX cDNA clones were kindly provided by Dr D. Inze´ (University of Gent, Belgium; see Bueno et al. 1998 and Mock et al. 1998 for detailed information on the cDNA fragments). The hybridisation signals were quantified using the Bio Image Analyser (BAS 2000, Fuji Photo Film Co., Tokyo, Japan). To check the amount of total RNA loaded on each lane, filters were rehybridised with a 32P-labelled 26S rDNA fragment of potato. Statistical evaluation In general, mean values were examined statistically by using the one-way analysis of variance (ANOVA) at a significance level of PB0.05 followed by the Tukey-Kramer multiple comparison test.

Results As shown in Fig. 1, increasing NaCl concentration gradually inhibited the growth rate of both cultivars. The growth of the salt-sensitive cultivar was extensively inhibited already at salt concentrations lower than 200 mM NaCl. In contrast, the salt-tolerant cultivar still developed shoots upon 250 mM NaCl, but growth was almost completely inhibited at 300 mM NaCl. The percentage of relative growth inhibition of the shoots upon increasing NaCl concentrations is indicated in the insert of Fig. 1. As shown in Fig. 2, seedlings of the two foxtail millet cultivars exhibited a remarkable difference in the endogenous Na + concentration upon increasing NaCl concentration in the medium, which indicates the difference in the adaptive response to salinity of both cultivars. In the saltsensitive cultivar, Na + accumulated progressively with increasing amounts of exogenous salt. In the tolerant cultivar, the endogenous Na + content increased only at 200 mM NaCl. The extent of membrane damage was assessed by an indirect measurement of the solute leakage of tolerant and sensitive seedlings. The ion leakage of seedlings correlated with increasing NaCl concentrations. While the sensitive cultivar showed the highest electrolyte leakage already after incubation with 150 mM NaCl, the salt-tolerant cultivar displayed a 1/3 lower leakage than the sensitive cultivar (Fig. 3), which is consistent with a lower impairment of 437

Fig. 1. Differences in root and shoot length of 5-day-old seedlings of a salt-tolerant (P – Prasad) and a salt-sensitive (L – Lepakshi) foxtail millet cultivar grown under control conditions (CP – control Prasad; CL – control Lepakshi) and at different NaCl concentrations (SP – salt-treated Prasad; SL – salt-treated Lepakshi). Percentages of relative shoot growth inhibition are shown in the insert. Seedlings were grown at 25°C in Hoagland medium without NaCl (control) and with different NaCl concentrations (150, 200, 250 and 300 mM).

membrane integrity in the tolerant in comparison with the sensitive cultivar. We analysed pools of total and reduced ascorbate in tolerant and sensitive foxtail millet seedlings (Fig. 4). A significant increase in the total ascorbate content (PB 0.001) was found in tolerant seedlings at 50 mM NaCl in comparison with control medium without NaCl. Upon the same conditions, no increase of total ascorbate was found in seedlings of the sensitive cultivar. Increasing salt concentrations (100 and 150 mM NaCl) led to a reduction of the ascorbate content in sensitive seedlings. At 200 mM NaCl, the amount of both total and reduced ascorbate is reduced in general, but to a significantly higher extent in seedlings of the sensitive cultivar (P B0.001). The effect of increasing NaCl concentrations on the total SOD activity of the two cultivars was measured (Fig. 5). The SOD activity increased gradually in the salt-tolerant cultivar upon increasing NaCl concentrations. In comparison with the control (100%), the SOD activity reached a maximum value of 134% at 200 mM NaCl in the salt-tolerant cultivar. In contrast, seedlings of the salt-sensitive cultivar exhibited a gradual decline in SOD activity. At 200 mM NaCl in the medium, SOD activity dropped down to 73% of the control. The activities of the SOD isoforms were analysed in extracts of control and NaCl (200 mM) treated seedlings of both cultivars after 13% native PAGE (Fig. 6a). Initial inhibition experiments permitted to assign the activity of bands to specific SOD isoforms (see Materials and methods: ‘Superoxide dismutase activity and activity pattern of SOD isoforms’). The activity of cytosolic Cu/Zn-SOD and MnSOD activity increased in the tolerant cultivar upon salt incubation and comprised the major portion of the total SOD activity. 438

The steady-state levels of the cytosolic Cu/Zn-SOD and the Mn-SOD isoform were determined in protein extracts of both foxtail millet cultivars by western blot analysis. The highest amount of cytosolic Cu/Zn-SOD (17 kDa) was found in seedlings of the tolerant cultivar grown upon 200 mM NaCl (Fig. 6b, Cu/Zn-SOD). A similar expression profile was found for the 22 kDa Mn-SOD (Fig. 6b, MnSOD). The accumulation of both SOD isoforms in the salt-tolerant cultivar at 200 mM NaCl treatment correlates with their enhanced enzymatic activity. The steady-state transcript levels of cytosolic Cu/ZnSOD, plastidic Cu/Zn-SOD and mitochondrial Mn-SOD mRNA were subsequently examined in seedlings of the salt-tolerant and the salt-sensitive cultivar under control conditions and upon salinity stress (200 mM NaCl) to correlate them with the protein/activity levels. As shown in

Fig. 2. Na + accumulation in 5-day-old seedlings of the tolerant and sensitive foxtail millet cultivar grown at different NaCl concentrations. Data are the average of 3 independent sets of experiments. Physiol. Plant. 109, 2000

Fig. 3. Electrolyte leakage rate measured in cells of 5-day-old seedlings of the tolerant and sensitive foxtail millet cultivars grown at different salt concentrations. Data are the average of 3 independent sets of experiments.

Fig. 6c, the transcript level, especially of the cytosolic Cu/ Zn-SOD, was significantly increased in the salt-tolerant cultivar upon salt treatment. The induced accumulation of the cytosolic Cu/Zn-SOD mRNA correlated with increase in protein level and enzyme activity (see Fig. 6b and a, respectively). In the tolerant cultivar, the mRNA content of plastidic Cu/Zn-SOD slightly increased in response to salt stress, but did not affect the enzyme activity, whereas in the sensitive cultivar the mRNA content/enzyme activity remained low in both control and salt-treated seedlings. In the tolerant cultivar, Mn-SOD mRNA was slightly more abundant in the control than after salt treatment, and its transcript levels were higher in the tolerant than in the sensitive cultivar. However, only in the tolerant cultivar the protein as well as the activity level of Mn-SOD was significantly increased under salt stress. Enzyme extracts from both cultivars were assayed for total APX activity after exposure to different NaCl concentrations (Fig. 7a). In seedlings of the salt-sensitive cultivar,

Fig. 5. Total SOD activity in 5-day-old seedlings of the tolerant and sensitive cultivars grown at different NaCl concentrations. Data are the average of 3 independent sets of experiments.

APX activity showed nearly the same profile up to 150 mM NaCl. However, at 200 mM NaCl, APX activity declined to about 60% of the control, whereas in tolerant seedlings a significantly higher APX activity (142% of the control) was measured. The steady-state protein levels of the cytosolic APX isoform (28 kDa) were determined in protein extracts of the foxtail millet cultivars using an antiserum against spinach cytosolic APX (Fig. 7b). The highest amount of APX protein was found in the tolerant cultivar upon 200 mM NaCl, while the amount of the APX protein was lower in the control and in the sensitive cultivar. To examine the effect of salinity on the level of APX mRNAs, we examined the two cultivars under control conditions and upon 200 mM NaCl. Steady-state levels of APX mRNA increased 2-fold in the tolerant cultivar incubated with 200 mM NaCl in comparison with the control and were not significantly affected in the sensitive one (Fig. 7c).

Discussion

Fig. 4. Content of total and reduced ascorbate in 5-day-old seedlings of the tolerant and sensitive foxtail millet cultivars grown at different NaCl concentrations. Data are the average of 9 independent sets of experiments. Physiol. Plant. 109, 2000

Seed germination and seedling growth are normally limited by increasing concentration of NaCl. Accordingly, 5-day-old seedlings of the tolerant and the sensitive foxtail millet cultivar grown upon high amounts of NaCl (up to 250 mM) showed differences in their growth pattern (root and shoot length). Seedlings of the tolerant cultivar were able to grow normally even at 200 mM, whereas germination of seedlings of the sensitive cultivar was strictly inhibited already at 150 mM NaCl (Fig. 1). The analysis of the Na + content in 5-day-old seedlings of the two foxtail millet cultivars showed a high concentration of Na + in the sensitive and a lower one in the tolerant cultivar even at high NaCl concentrations in the medium (Fig. 2). The growth of the salt-tolerant foxtail millet cultivar is certainly favoured by the maintenance of a constant Na + -ion level in the plant at high external NaCl concentration. This is consistent with efficient ion transport activity preventing an imbalance of the cellular ion content. In this context, it is important to mention that 439

an antiserum raised against proteins of the salt-tolerant cultivar recognised a putative Na + /H + antiporter, which is highly enriched in the tolerant foxtail millet seedlings (N. Sreenivasulu, unpublished results). The contribution of the Na + /H + antiporter gene to salt tolerance will be investigated in future. Our results are in accordance with previous

Fig. 7. Total APX activity (a), cytosolic APX protein level (b) and cytosolic APX steady-state mRNA level (c) in 5-day-old seedlings of the tolerant and sensitive foxtail millet cultivar grown in Hoagland medium without NaCl or containing 200 mM NaCl. The intensity of northern blot signals was quantified by using the BioImage Analyser and the signal intensity is given in percentages of the most intensive signal (100%). Standard deviations were calculated from 3 independent northern blot experiments. Fig. 6. Activity, protein amount and steady-state mRNA level of different SOD isoforms in 5-day-old seedlings of the tolerant and sensitive foxtail millet cultivar grown in Hoagland medium or in medium containing 200 mM NaCl. (a) Enzyme activity of individual SOD isoforms. SOD activity is given in percentages regarding the most intensive signal (100%). (b) Estimation of the protein levels of cytosolic Cu/Zn-SOD and Mn-SOD by western blotting. Positions of molecular mass of marker proteins (94.0, 67.0, 43.0, 30.0, 20.1, 14.4 kDa) are indicated. (c) The intensity of northern blot signals was quantified by using the BioImage Analyser (see Materials and methods). Standard deviations were calculated from 3 independent northern blot experiments. The northern blot signal intensity is given in percentages of the most intensive signal (100%).

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reports of an inverse relationship between endogenous Na + concentrations and salt tolerance in different organs of rice (Yeo and Flowers 1983, Dioniso-Sese and Tobita 1998). Furthermore, salinity causes stronger electrolyte leakage of sensitive seedlings (Fig. 3). Consistent with our results, strong differences in electrolyte leakage between tolerant and sensitive cultivars were described for 3-week-old rice seedlings grown under salinity stress (Dionisio-Sese and Tobita 1998). In our previous paper, we found an increase in MDA content in sensitive seedlings during 150 mM NaCl Physiol. Plant. 109, 2000

treatment (Sreenivasulu et al. 1999). The constant value of electrolyte leakage in combination with a low content of MDA (Sreenivasulu et al. 1999) obtained with the tolerant seedlings, provides some evidence for lower lipid peroxidation and less affected membrane integrity. Salt tolerance also seems to be conferred by an increased antioxidative capacity to detoxify reactive oxygen species (ROS). Higher antioxidant levels and activities of ROS-scavenging enzymes have shown to correlate with salt-induced oxidative stress tolerance and were found to be characteristic of tolerant varieties. Seedlings of the tolerant cultivar grown upon high amounts of NaCl showed an upregulation of the total SOD activity, whereas the SOD activity remained lower in sensitive seedlings (Fig. 5). Our results corroborate previous reports indicating an increase in the activity of antioxidant enzymes in response to high salinity (Bowler et al. 1992, Hernandez et al. 1993, 1995, Gossett et al. 1994, Olmos et al. 1994, Sehmer et al. 1995). The mechanisms regulating the expression and activity of different SOD isoforms are complex and the genes respond differentially to environmental signals (Sen Gupta et al. 1993, for review see Noctor and Foyer 1998). It is apparently important for the tolerance mechanism that SOD isoforms are compartment-specifically induced in the tolerant foxtail millet seedlings in response to salt stress. Increased cytoplasmic Cu/Zn-SOD activities found in the salinised tolerant cultivar can most likely be explained by induced gene activities, which correlate with the protein/enzymatic activity of the gene product. Bueno et al. (1998) observed an increase in the steady-state level of cytosolic Cu/Zn-SOD transcripts during salt treatment but not during drought stress imposed by polyethylene glycol (PEG) treatment. Furthermore, comparative studies of NaCl tolerant and NaCl sensitive pea cell lines have shown the induction of two Cu/Zn-SODs only in the tolerant calli (Olmos et al. 1994). The increased activities of Mn-SOD found in the salinised tolerant foxtail millet cultivar are in accordance with observations of Hernandez et al. (1993, 1995). The higher Mn-SOD activity found in the tolerant foxtail millet cultivar directly corresponds to the increased protein level, but not to increased transcript levels. The present findings of possible requirements of translational/post-translational processes for the upregulation of mitochondrial Mn-SOD under high salt treatment are supported by similar results from Bueno et al. (1998). In conclusion, our results confirmed that salt tolerance is conferred by enhanced cytosolic Cu/Zn-SOD and Mn-SOD activity in response to compartment-specific signals. SOD converts the relatively less toxic O2’ − radical to the more toxic H2O2. Therefore, an increase in H2O2-scavenging capacity is required to enable rapid removal of H2O2 (Foyer et al. 1994). During NaCl treatment, the salt-tolerant foxtail millet cultivar exhibited a higher activity of the H2O2-detoxifying enzyme APX (Fig. 7a). The increase of both SOD and APX activity shown in the present work is in agreement with the induction of both activities in a salt-treated Arabidopsis mutant line (PS1) with photoautotrophic salt tolerance (Tsugane et al. 1999) and in salt-tolerant peas treated with NaCl (Hernandez et al. 1995). The parallel increase of the cytoplasmic APX steady-state mRNA level corresponds Physiol. Plant. 109, 2000

to an increased protein amount, which reflects transcript based regulation. In summary, the present work shows substantial differences in the cellular reactivity between the salt-tolerant and salt-sensitive foxtail millet cultivars in response to salinity stress. Upon high NaCl concentrations, seedlings of the salt-sensitive cultivar exhibit high Na + concentration, resulting in symptoms of oxidative damage which are accompanied by a decrease in SOD and APX activity. The process leads to electrolyte leakage and ultimately to cell death. Grown upon the same NaCl concentration, seedlings of the tolerant cultivar show maintenance of cellular intactness and display a significantly lower Na + accumulation. The increase of antioxidative enzyme activity induced in parallel at low intracellular Na + concentration represents a second part of the complex salt tolerance mechanism. In combination with a lower Na + uptake or/and an increased efflux of ions the resulting higher capacity for oxygen radical scavenging could explain the ability of the tolerant foxtail millet cultivar to grow at higher NaCl concentrations than the sensitive one. Acknowledgements – This work was supported by grants from Deutscher Akademischer Austauschdienst (Award No. A/98/ 00061). We thank Professor Dr D. Inze´ (Gent, Belgium) for providing cDNA clones and Mn-SOD antisera. We also thank Dr K. Asada (Fukuyama, Japan) and Dr H. Saji (Onagawa, Tsukuba, Japan) for their kind gift of cytosolic Cu/Zn-SOD and ascorbate peroxidase antisera, respectively.

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Physiol. Plant. 109, 2000