Comparative lipid peroxidation, antioxidant defense systems and proline content in roots of two rice cultivars differing in salt tolerance

Environmental and Experimental Botany 53 (2005) 247–257

Comparative lipid peroxidation, antioxidant defense systems and proline content in roots of two rice cultivars differing in salt tolerance ˙ Tijen Demiral, Ismail Türkan∗ Department of Biology, Science Faculty, Ege University, 35100 Bornova-I˙ zmir, Turkey Accepted 23 March 2004

Abstract The changes in the activity of antioxidant enzymes such as superoxide dismutase (SOD: EC 1.15.1.1), catalase (CAT: EC 1.11.1.6), peroxidase (POX: EC 1.11.1.7), ascorbate peroxidase (APOX: EC 1.11.1.11) and glutathione reductase (GR: EC 1.6.4.2), free proline content, and the rate of lipid peroxidation level in terms of malondialdehyde (MDA) in roots of two rice cultivars (cvs.) differing in salt tolerance were investigated. Plants were subjected to three salt treatments, 0, 60, and 120 mol m−3 NaCl for 7 days. The results showed that activated oxygen species may play a role in cellular toxicity of NaCl and indicated differences in activation of antioxidant defense systems between the two cvs. The roots of both cultivars showed a decrease in GR activity with increase in salinity. CAT and APOX activities increased with increasing salt stress in roots of salt-tolerant cultivar Pokkali but decreased and showed no change, respectively, in roots of IR-28 cultivar. POX activity decreased with increasing NaCl concentrations in salt-tolerant Pokkali but increased in IR-28. SOD activity showed no change in roots of both cultivars under increasing salinity. MDA level in the roots increased under salt stress in sensitive IR-28 but showed no change in Pokkali. IR-28 produced higher amount of proline under salt stress than in Pokkali. Increasing NaCl concentration caused a reduction in root fresh weight of Pokkali and root dry weight of IR-28. The results indicate that improved tolerance to salt stress in root tissues of rice plants may be accomplished by increased capacity of antioxidative system. © 2004 Elsevier B.V. All rights reserved. Keywords: Antioxidant enzymes; Malondialdehyde; Proline; Rice; Salt stress

1. Introduction

Abbreviations: AOS, active oxygen species; MDA, malondialdehyde; EDTA, ethylenediaminetetraacetic acid; GSSG, oxidized glutathione; GSH, reduced glutathione; MDHA, monodehydroascorbate; DW, dry weight; FW, fresh weight ∗ Corresponding author. Tel.: +90-232-3884000x2443; fax: +90-232-3881036. ˙ Türkan). E-mail address: [email protected] (I.

Increasing salinity of agricultural irrigation water together with progressive salinization of agricultural land is of increasing importance to agriculture because it limits the distribution of plants in certain natural habitats and induces a wide range of adverse metabolic responses in higher plants. Rice (Oryza sativa L.) is the primary stable food for over two billion people in Asia, Africa, and Latin America (Salekdeh et al.,

0098-8472/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2004.03.017

248

T. Demiral, I˙ . Türkan / Environmental and Experimental Botany 53 (2005) 247–257

2002); salinity is becoming a serious problem in many coastal, arid, and irrigated rice production systems. High concentrations of salts disrupt homeostasis in water potential and ion distribution in plants. Altered water status most likely brings about initial growth reduction (Dash and Panda, 2001). Crucial changes in ion and water homeostasis lead to molecular damage, growth arrest, and even death. Specific effects of salt stress on plant metabolism, especially on leaf senescence, have been related to the accumulation of toxic ions (Na+ and Cl− ) or to K+ and Ca2+ depletion (Kingsburry and Epstein, 1986; Rengel, 1992; Pérez-Alfocea et al., 1996; Al-Karaki, 2000). As a consequence of ion imbalance and hyperosmotic stresses, which are primary effects of salt stress, secondary stresses such as oxidative damage may occur. Limited CO2 fixation due to stress conditions leads to a decrease in carbon reduction by the Calvin cycle and to a decrease in oxidized NADP+ to serve as an electron acceptor in photosynthesis. When ferrodoxin is overreduced during photosynthetic electron transfer, electrons may be transferred from PS-I to oxygen to form superoxide radicals (O2 •− ) by the process called Mehler reaction (Heldt, 1997, pp. 60–104; Hsu and Kao, 2003), which triggers chain reactions that generate more aggressive oxygen radicals. It is already known that these cytotoxic active oxygen species (AOS), which are also generated during metabolic processes in the mitochondria and peroxisomes, can destroy normal metabolism through oxidative damage of lipids, proteins, and nucleic acids (McCord, 2000). Lipid peroxidation, induced by free radicals, is also important in membrane deterioration (Halliwell, 1987; McCord, 2000). To scavenge AOS, plants have evolved specific defense tactics involving both enzymatic and non-enzymatic antioxidant mechanisms. Several antioxidant enzymes participate in the detoxification of AOS. Superoxide dismutases react with superoxide radicals (O2 •− ) to produce H2 O2 (Bowler et al., 1992). In the absence of natural scavengers such as catalase and peroxidase, H2 O2 accumulates in tissues to high levels. Ascorbate peroxidase (APOX), together with monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase, and glutathione reductase (GR), removes H2 O2 through the Halliwell–Asada pathway (Foyer and Halliwell, 1976; Halliwell, 1987). In plants, APOX, which reduces H2 O2 to water at

the expense of oxidizing ascorbate to monodehydroascorbate (MDHA) (Foyer, 1996; Asada, 1997), plays a key role in the ascorbate–glutathione cycle. MDHA is then reduced to ascorbate by the action of MDHAR. Nonetheless, two molecules of MDHA can also be converted non-enzymatically to MDHA and dehydroascorbate, which in turn is reduced to ascorbate via the dehydroascorbate reductase and GR cycle (Foyer and Halliwell, 1976; Noctor et al., 2002). In this cycle, reduced glutathione (GSH) is oxidized to oxidized glutathione (GSSG) by the action of GR. Rapid accumulation of free proline is a typical response to salt stress. When exposed to drought or a high salt content in the soil (both leading to water stress), many plants accumulate high amounts of proline, in some cases several times the sum of all the other amino acids (Mansour, 2000). Proline has been found to protect cell membranes of onion against salt injury (Mansour, 1998). Sultana et al. (1999) have suggested that proline accumulation in both salinized leaves and grains of rice plants is implicated in osmotic adjustment to salinity. In contrast, Lutts et al. (1996) argued that proline accumulated in salt-stressed calluses had a negligible effect on osmotic adjustment and did not play a role in salt resistance in rice callus cultures. Thus, the protective role of proline as an osmoticum in drought and salt stress is still debatable. Studies have shown that salt tolerance may be improved if the free radicals formed during the accompanying activated oxygen damage are scavenged by an enhanced antioxidative defense system (Alscher et al., 2002; Shigeoka et al., 2002). There is good evidence that the alleviation of oxidative damage and increased resistance to salinity and other environmental stresses is often correlated with an efficient antioxidative system (Scandalios, 1993; Cakmak et al., 1993; Hasegawa et al., 2000; Acar et al., 2001; Sato et al., 2001; Bor et al., 2003). Dionisio-Sese and Tobita (1998) studied the activities of SOD and POX enzymes under NaCl stress in the leaves of four cultivars of rice exhibiting different sensitivities to NaCl. Their results have indicated that salt tolerance capacity of salt-tolerant species is closely related with the maintenance of specific activity of antioxidant enzymes studied (Dionisio-Sese and Tobita, 1998). Comparison of antioxidant defense systems, lipid peroxidation, and proline contents in roots of rice cultivars differing in salt tolerance may be helpful in

T. Demiral, I˙ . Türkan / Environmental and Experimental Botany 53 (2005) 247–257

developing a better understanding of tolerance mechanisms to salt stress. We hypothesize that higher constitutive or induced activity of antioxidant enzymes in roots and photosynthetic tissues of rice plants provides a mechanism of tolerance to short-term salt stress. The present study was conducted to evaluate the mechanism of adaptation to salt stress in the roots of two rice cvs. differing in salt tolerance. Comparative effects of salt stress were investigated on growth, lipid peroxidation levels, antioxidant enzyme activities, and proline contents in roots of two contrasting cultivars of rice seedlings under salt stress.

249

week. Water lost by evapotranspiration was compensated for by daily addition of deionized water. Fe-stock solution (10%) was sprayed on the leaves of rice plants three times a week to provide adequate Fe during their growth. Three weeks after placement, salt stress treatment was imposed by adding 60 or 120 mol m−3 NaCl to the full-strength nutrient solution. Nutrient solution without NaCl addition (0 mol m−3 NaCl) served as the control group. At harvest, roots were washed quickly with distilled water and blotted dried on filter paper and then stored at −20 ◦ C for enzyme analyses. 2.2. Growth parameters

2. Materials and methods 2.1. Plant material and salt stress applications Seeds of Oryza sativa L., cultivars Pokkali (salt-resistant) and IR-28 (salt-sensitive), were obtained from the IRRI (International Rice Research Institute, Los Baños, Philippines). IRGC accessions for FAO-designated germplasm supplied by INGER (acc) were 8948 for Pokkali and 30411 for IR-28. They were surface sterilized with 0.1% sodium dodecyl sulphate (SDS) solution on a magnetic stirrer for 20 min and thoroughly washed with deionized water. Seeds of each cultivar were then soaked in sterile deionized water at 28 ◦ C for 6 h and then transferred to two sheets of sterile filter paper moistened with deionized water. Seeds were placed in plastic trays for germination at 28 ◦ C for 72 h in the dark. Germinated seeds were sown into holes of styrofoam boards in deionized water, and grown hydroponically in the growth room for 3 weeks under fluorescent and incandescent lights. The temperature of the growth room was maintained at 27 ± 2 ◦ C, and the level of photosynthetically active radiation was 350 ␮mol m−2 s−1 . Daytime humidity was between 60 and 70%. Seven and 14 days after placement of the rice seeds in deionized water (on the filter paper), the deionized water was replaced by one-half and full-strength nutrient solution, respectively. The nutrient solution used was that of Yoshida et al. (1976) and the nutrients contained in mg/L were 40 N, 10 P, 40 K, 40 Ca, 40 Mg, 0.05 Mo, 0.2 B, 0.5 Mn, and 0.01 Zn. The pH of the nutrient solution was adjusted to 5.2 by adding HCl or NaOH, and nutrient solution was replaced twice a

After 0 and 7 days of NaCl treatment, 20 plants for each group were taken at random and divided into separate leaf and root fractions. The fresh weights of leaves and roots were weighed, and root lengths were measured. The samples were then dried in a forced draft oven at 70 ◦ C for 72 h, and the dry weights were determined. 2.3. Lipid peroxidation Lipid peroxidation was determined by measuring malondialdehyde (MDA) formation using the thiobarbituric acid method described by Madhava Rao and Sresty (2000). For MDA extraction, 0.5 g of root samples was homogenized with 2.5 mL of 0.1% trichloroacetic acid (TCA). The homogenate was centrifuged for 10 min at 10,000 × g. For every 1 mL of the aliquot, 4 mL of 20% TCA containing 0.5% thiobarbituric acid (TBA) was added. The mixture was heated at 95 ◦ C for 30 min and then cooled quickly on an ice bath. Afterwards, the mixture was centrifuged for 15 min at 10,000 × g and the absorbance of the supernatant was measured at 532 nm. Measurements were corrected for unspecific turbidity by substracting the absorbance at 600 nm. The concentration of MDA was calculated by using an extinction coefficient of 155 mM−1 cm−1 . 2.4. Enzyme extractions and essays For protein and enzyme extractions, 0.5 g of root samples were homogenized with 50 mM sodium phosphate buffer (pH 6.8) containing 1 mM EDTA·Na2 and 2% (w/v) polyvinylpolypyrrolidone (PVPP). The

250

T. Demiral, I˙ . Türkan / Environmental and Experimental Botany 53 (2005) 247–257

whole extraction procedure was carried out at 4 ◦ C. Homogenates were then centrifuged at 4 ◦ C for 40 min at 13,000 × g, and supernatants were used for determination of enzyme activity. Protein concentration was determined according to Bradford (1976), using bovine serum albumin as a standard. Ascorbate peroxidase (APOX, EC 1.11.1.11) activity was determined according to the method of Nakano and Asada (1981). The reaction mixture contained 0.05 M Na–phosphate buffer (pH 7), 0.5 mM ascorbate, 0.1 mM EDTA·Na2 , 1.2 mM H2 O2 , and 0.1 mL enzyme extract in a final assay volume of 1 mL. Ascorbate oxidation was followed at 290 nm. The concentration of oxidized ascorbate was calculated using extinction coefficient (ε = 2.8 mM−1 cm−1 ). One unit of APOX was defined as 1 mmol mL−1 ascorbate oxidized per minute. Glutathione reductase (GR; EC 1.6.4.2) activity was measured according to Foyer and Halliwell (1976). The assay medium contained 0.025 mM Na–phosphate buffer (pH 7.8), 0.5 mM GSSG, 0.12 mM NADPH.Na4 , and 0.1 mL enzyme of extract in a final assay volume of 1 mL. NADPH oxidation was determined at 340 nm. Activity was calculated using the extinction coefficient (ε = 6.2 mM−1 cm−1 ) for GSSG. One unit of GR was defined as 1 mmol mL−1 GSSG reduced per minute. Catalase (CAT, EC 1.11.1.6) activity was assayed by measuring the initial rate of disappearance of H2 O2 (Bergmeyer, 1970). The reaction mixture contained 3% H2 O2 and 0.1 mM EDTA in 0.05 M Na–phosphate buffer (pH 7). The decrease in H2 O2 was measured as a decline in optical density at 240 nm, and activity was calculated as ␮mol H2 O2 consumed per minute. Superoxide dismutase (SOD, EC 1.15.1.1) activity was assayed spectrophotometrically as the inhibition of photochemical reduction of nitro-blue tetrazolium (NBT) at 560 nm (Beauchamp and Fridovich, 1971). The reaction mixture contained 33 ␮M NBT, 10 mM l-methionine, 0.66 mM EDTA·Na2 , and 0.0033 mM riboflavin in 0.05 M Na–phosphate buffer (pH 7.8). The riboflavin was added last. The test tubes containing reaction mixture were shaken and waited for 10 min under 300 ␮mol m−2 s−1 irradiance at room temperature. The reaction mixture with no enzyme developed maximum color due to maximum reduction of NBT. A non-irradiated reaction mixture did not develop color and served as the control. The reduction

of NBT was inversely proportional to the SOD activity. One unit of SOD was defined as the amount of enzyme that inhibits 50% NBT photoreduction. Peroxidase (POX; EC 1.11.1.7) activity was determined according to Herzog and Fahimi (1973). The reaction mixture contained 3,3 -diaminobenzidine-tetrahydrochloride dihydrate (DAB) solution containing 50% (w/v) gelatine and 0.15 M Na–phosphate–citrate buffer (pH 4.4) and 0.6% H2 O2 . The increase in absorbance was recorded at 465 nm through 3 min. A unit of peroxidase activity was expressed as ␮mol mL−1 H2 O2 decomposed per minute. The specific enzyme activity for all enzymes was expressed as units mg−1 protein g−1 FW. 2.5. Proline level Determination of free proline content was done according to Bates et al. (1973). Root samples (0.5 g) from each group were homogenized in 3% (w/v) sulphosalycylic acid and homogenate filtered through filter paper. After addition of acid ninhydrin and glacial acetic acid, resulting mixture was heated at 100 ◦ C for 1 h in water bath. Reaction was then stopped by using ice bath. The mixture was extracted with toluene, and the absorbance of fraction with toluene aspired from liquid phase was read at 520 nm. Proline concentration was determined using calibration curve and expressed as ␮mol proline g−1 FW. 2.6. Statistical analysis The experiments were repeated two times independently, and each data point was the mean of three replicates (n = 6) except for root lengths of rice seedlings (n = 20). All data obtained were subjected to a one-way analysis of variance (ANOVA), and the mean differences were compared by lowest standard deviations (LSD) test. Comparisons with P < 0.05 were considered significantly different. In all the figures, the spread of values is shown as error bars representing standard errors of the means. 3. Results 3.1. Growth parameters Root growth was followed by measuring fresh weight (FW), dry weight (DW), and root length.

T. Demiral, I˙ . Türkan / Environmental and Experimental Botany 53 (2005) 247–257

251

Table 1 Effect of salt stress on the root length (cm) of two rice cultivars Cultivar

Day 7: NaCl concentration (mol m−3 )

Day 0

11.4 ± 3.3 a 14.6 ± 3.2 a

Pokkali IR-28

0

60

120

14.7 ± 2.6 b 16.3 ± 3.4 a

14.6 ± 3.7 b 14.8 ± 4.0 a

14.8 ± 3.7 b 14.2 ± 3.5 a

Each value indicated is the mean of 20 (n = 20) replicates recorded ± S.E. Means followed by the same letters are not significantly different at P < 0.05 within the same cultivar.

Table 2 Effect of salt stress on the fresh weight (FW) and dry weight (DW) of roots (mg) in two rice cultivars Cultivar

Parameter

Day 0

Day 7: NaCl concentration (mol m−3 ) 0

60

120

Pokkali

FW DW

44.0 ± 1.0 a 3.2 ± 0.2 a

80.0 ± 2.2 b 4.3 ± 1.0 b

72.0 ± 3.6 b 4.4 ± 0.4 b

46.0 ± 2.5 a 4.1 ± 0.6 ab

IR-28

FW DW

26.0 ± 0.9 a 2.7 ± 0.1 a

28.0 ± 0.8 a 3.9 ± 0.2 b

27.0 ± 0.4 a 3.1 ± 0.2 a

24.0 ± 0.4 a 3.1 ± 0.1 a

Each value indicated is the means of six replicates recorded ± S.E. Means followed by the same letters are not significantly different at P < 0.05 within the same cultivar.

Table 1 shows the effect of NaCl on root length of rice seedlings. Although a decrease was seen in root length of IR-28 under increasing salinity, there was no significant effect of salinity on root length of both cultivars (Table 3). Overall, there was a significant decrease by 43% in FW under severe salt stress (120 mol m−3 NaCl) for Pokkali (Tables 2 and 3). However, no significant change was found in FW of IR-28 roots. There were remarkable decreases by 21% in DW under both 60 and 120 mol m−3 NaCl concentrations for IR-28, but no significant change occurred in DW of Pokkali roots.

3.3. Antioxidant enzyme activities There were striking differences in antioxidant enzyme activities between the two cultivars with increasing NaCl concentration (Table 3). SOD activity Table 3 Results of two-way analysis of variance (ANOVA) of cultivar (cv.), NaCl concentrations (NaCl) and their interaction (cv. × NaCl) for root length, root fresh, and dry weights, lipid peroxidation (MDA), SOD, CAT, POX, APOX, and GR activities, and proline content Dependent variable

Independent variable cv.

3.2. Lipid peroxidation Lipid peroxidation levels in roots of the two rice cultivars, measured as the content of MDA, are given in Fig. 1. In roots of both cultivars growing under normal growth conditions, a small ‘age-dependent’ increase in lipid peroxidation level became apparent after 7 days, which was statistically insignificant for Pokkali; however, under salinity stress, there was a gradual increase in lipid peroxidation level of IR-28 (Fig. 1). In contrast to IR-28, roots of Pokkali showed no significant change in lipid peroxidation with increased salinity.

Root length Root fresh weight Root dry weight MDA content SOD activity CAT activity POX activity APOX activity GR activity Proline content

0.85ns

152.45∗∗∗ 3.98ns 8.40∗ 2.35ns 27.42∗∗∗ 6.86∗ 4.48∗ 1.06ns 25.53∗∗∗

NaCl

cv. × NaCl

114.94∗∗∗

0.50ns 5.70∗∗ 1.31ns 2.55ns 3.48∗ 8.22∗∗ 32.11∗∗∗ 1.04ns 0.91ns 11.13∗∗∗

43.28∗∗∗ 20.82∗∗∗ 99.86∗∗∗ 81.09∗∗∗ 52.24∗∗∗ 230.22∗∗∗ 47.56∗∗∗ 27.19∗∗∗ 139.78∗∗∗

Numbers represent F values at 5% level: ns, not significant. ∗ P < 0.05. ∗∗ P < 0.01. ∗∗∗ P < 0.001.

252

T. Demiral, I˙ . Türkan / Environmental and Experimental Botany 53 (2005) 247–257

Fig. 1. Salt stress-induced changes in the malondialdehyde (MDA) content (nmol g−1 FW) in roots of Pokkali (A) and IR-28 (B). Values are the means of six replicates ± S.E. Bars with different letters are significantly different at P < 0.05.

in the roots of both cultivars showed no remarkable change with increased salinity. However, SOD activity in Pokkali roots under 120 mol m−3 NaCl stress was higher than that of IR-28 roots at both NaCl concentrations after 7 days of salt treatment (Fig. 2). CAT activity showed an increase in roots of Pokkali and a decrease in roots of IR-28 with increased salinity (Fig. 3). The basal level of CAT activity was also higher in Pokkali than in IR-28. POX activity decreased with increased NaCl concentration in salt-tolerant Pokkali but increased in IR-28 (Fig. 4). APOX activity increased slightly in roots of Pokkali but did not change significantly in

roots of IR-28 after 7 days of exposure to increased salinity (Fig. 5). GR activity significantly decreased in roots of both cultivars with increasing salinity. At both 60 and 120 mol m−3 NaCl, salt-tolerant Pokkali showed a lesser decline in GR activity than in salt-sensitive IR-28 (Fig. 6). 3.4. Proline accumulation Free proline levels were higher in IR-28 than in Pokkali irrespective of experimental conditions (Fig. 7). Free proline content did not change significantly in roots of Pokkali with increasing NaCl

Fig. 2. Superoxide dismutase (SOD) activity in roots of Pokkali (A) and IR-28 (B) before and after exposure to salt stress. Values are mean ± S.E. based on six replicates. Bars with different letters are significantly different at P < 0.05.

T. Demiral, I˙ . Türkan / Environmental and Experimental Botany 53 (2005) 247–257

253

Fig. 3. Catalase (CAT) activity in roots of Pokkali (A) and IR-28 (B) under saline stress. Values are mean ± S.E. based on six replicates. Bars with different letters are significantly different at P < 0.05.

Fig. 4. Peroxidase (POX) activity in roots of Pokkali (A) and IR-28 (B) after 0 and 7 days under increasing concentrations of NaCl solutions. Values are mean ± S.E. based on six replicates. Bars with different letters are significantly different at P < 0.05.

Fig. 5. Ascorbate peroxidase (APOX) activity in roots of Pokkali (A) and IR-28 (B) after 0 and 7 days under increasing concentrations of NaCl solutions. Values are mean ± S.E. based on six replicates. Bars with different letters are significantly different at P < 0.05.

254

T. Demiral, I˙ . Türkan / Environmental and Experimental Botany 53 (2005) 247–257

Fig. 6. Glutathione reductase (GR) activity in roots of Pokkali (A) and IR-28 (B) under salt stress. Values are mean ± S.E. based on six replicates. Bars with different letters are significantly different at P < 0.05.

Fig. 7. Free proline content (␮mol g−1 FW) in roots of Pokkali (A) and IR-28 (B) treated for 7 days at two NaCl levels. Values are the means of six replicates ± S.E. Bars with different letters are significantly different at P < 0.05.

concentrations, but increased markedly in roots of IR-28 particularly at 120 mol m−3 NaCl.

4. Discussion Since salt stress can cause membrane damage, reduced uptake of CO2 as a result of stomatal closure, decreased hydrolytic enzyme activity and increased lipid peroxidation level, it may stimulate formation of AOS such as superoxide, hydrogen peroxide, and hydroxyl radicals. Among AOS, superoxide is converted by SOD enzyme into H2 O2 , which is further scav-

enged by CAT and various peroxidases. APOX and GR also play a key role by reducing H2 O2 to water through the Halliwell–Asada pathway (Noctor and Foyer, 1998). In the present study, short-term salt stress did not show remarkable effect on the root lengths of both cultivars (Table 1). However, if based on root DW we can say that root growth of IR-28 was more inhibited by salt stress than that of Pokkali (Table 2). Lin and Kao (2001) have showed that not only increased concentration of NaCl from 50 to 150 mM but also exogenous application of H2 O2 increased the level of endogenous H2 O2 in roots of rice seedlings

T. Demiral, I˙ . Türkan / Environmental and Experimental Botany 53 (2005) 247–257

and caused the reduction of root growth (based on FW and DW). Hence, salt-dependent growth reduction in roots of IR-28 might have resulted from the increase in the level of H2 O2 in roots induced by NaCl. Proline is generally assumed to serve as a physiologically compatible solute that increases as needed to maintain a favorable osmotic potential between the cell and its surroundings (Pollard and Wyn Jones, 1979). Nevertheless, the concentrations of proline in roots of both Pokkali and IR-28 under salt stress were not so high to obtain a significant degree of protection (Fig. 7). Lin and Kao (1996) have found that proline accumulation is considered as a contributor to NaCl-inhibited root growth. Higher level of proline accumulation in salt-stressed roots of IR-28 than of Pokkali might have caused a reduction in DW accumulation in IR-28 roots, as the concentration of proline accumulated was not so high to provide osmotic adjustment and thus salt tolerance. It is already known that free radical-induced peroxidation of lipid membranes is a reflection of stress-induced damage at the cellular level (Jain et al., 2001). Therefore, the level of MDA, produced during peroxidation of membrane lipids, is often used as an indicator of oxidative damage. The lower level of lipid peroxidation in roots of Pokkali than of IR-28 suggests that it may have better protection against oxidative damage under salt stress. The improved protection in Pokkali may reflect a more efficient antioxidative system as evidenced by a higher activity of SOD (under 120 mol m−3 NaCl), CAT, and APOX enzymes (Figs. 2, 3 and 5). However, significant increase in MDA level in roots of IR-28 appeared to be correlated with a decrease in activity of CAT and GR and a non-induced activity of SOD and APOX enzymes under salt stress. Antioxidative responses of these two cultivars to salinity have already been investigated at the shoot level. Dionisio-Sese and Tobita (1998) have reported that slightly increased and decreased activity of SOD is found in leaves of Pokkali and IR-28 under increased salt concentrations, respectively. Vaidyanathan et al. (2003) have found elevated activity of CAT, APOX, MDHAR, dehydroascorbate reductase, and GR enzymes but a slight increase in SOD activity in leaves of Pokkali. We found only a slight increase in SOD activity in roots of Pokkali under 120 mol m−3 NaCl in comparison to salt-stressed roots of IR-28 at both salt levels. But this does not im-

255

ply that Pokkali is experiencing little or no oxidative stress (Vaidyanathan et al., 2003), since upon exposure to salinity, roots of Pokkali showed increased activity of CAT and APOX enzymes. As suggested by Vaidyanathan et al. (2003), Pokkali might have employed non-enzymatic routes for conversion of O2 •− to H2 O2 using antioxidants like GSH and ascorbate. Considering the fact that Pokkali roots have a higher activity of CAT and APOX enzymes (both inherent and salt-induced), we suggest that APOX and CAT enzymes have equal importance in detoxification of H2 O2 in roots of Pokkali. These results are in good agreement with those of Shalata et al. (2001) who found that SOD and CAT activities decreased in roots of a salt-sensitive tomato cultivar but increased in roots of a salt-tolerant tomato cultivar under salt stress. Singha and Choudhuri (1990) reported that H2 O2 accumulation in the leaves of Vigna and Oryza seedlings under salinity stress was related to a decrease in CAT activity. Decreased CAT activities, in turn, might have promoted H2 O2 accumulation in IR-28 roots (Fig. 3), which could result in a Haber–Weiss reaction to form hydroxyl radicals (Bowler et al., 1992). Since OH• radicals are known to damage biological membranes and react with most compounds present in biological systems (Halliwell and Gutteridge, 1989), they might have hastened lipid peroxidation and membrane damage in the salt-sensitive cultivar IR-28. Since both SOD and CAT are inactivated by singlet oxygen and peroxyl radicals (Escobar et al., 1996), these enzymes might have been deactivated in roots of IR-28 by the increased levels of AOS. Peroxidases are involved not only in scavenging of H2 O2 produced in chloroplasts but also in growth and developmental processes (Dionisio-Sese and Tobita, 1998). Mittal and Dubey (1991) compared two sets of rice cultivars differing in salt tolerance to determine a possible correlation between peroxidase activity and the degree of salt tolerance in rice; they found a negative correlation between peroxidase activity and salt tolerance of rice cvs. Their results are consistent with ours in that with increased salinity, there was an increase in peroxidase activity in roots of salt-sensitive IR-28 but a decrease in roots of salt-tolerant Pokkali (Fig. 4). Lin and Kao (2001) demonstrated that reduction of root growth with increasing NaCl concentrations was correlated with an increase in ionically bound cell wall POX activity; thus, increased POX

256

T. Demiral, I˙ . Türkan / Environmental and Experimental Botany 53 (2005) 247–257

activity by NaCl might have been involved in the reduction in root growth of IR-28 in our study (based on dry weight). Conversely, the enhanced salt tolerance of Pokkali roots may be correlated with a reduction in POX activity. Increased GR activity in leaves of sugar beet plants has been reported to be closely related with salt tolerance capacity of these plants (Bor et al., 2003). However, in our study, GR activity decreased in the roots of both cultivars; this decline was slightly greater in sensitive IR-28 than in salt-tolerant Pokkali (Fig. 6). Constitutive levels of GR were higher in Pokkali than in IR-28. Consistent with our results, Shalata and Tal (1998) observed a decrease in GR activity in leaves of both salt-tolerant and salt-sensitive tomato cvs. under salt stress. Since decreased GR activity enhances stress sensitivity (Aono et al., 1995), more severe decrease in GR activity in roots of IR-28 than in roots of Pokkali may result in more sensitive structural arrangement in roots of IR-28 against stress. In our study, opposite patterns of response to NaCl stress were obtained as judged by activities of SOD, CAT, and POX enzymes. Higher free radicalscavenging capacity and protection mechanism of Pokkali against salt stress was also revealed by lower level of lipid peroxidation. In conclusion, our hypothesis was confirmed by the results of this study which showed that significant cultivar differences in response to salt stress in rice is closely related to differences in the activities of antioxidant enzymes. Acknowledgements The authors wish to thank Digna R. Izon for kind and helpful efforts to provide rice seeds from IRRI, Philippines, and Dr. Melike Bor for statistical analysis in order to evaluate the results of this study, and also Prof. Dr. Filiz Özdemir and M.Sc A. Hediye Sekmen for their excellent technical assistance to complete this research.

References Acar, O., Türkan, I., Özdemir, F., 2001. Superoxide dismutase and peroxidase activites in drought sensitive and resistant barley (Hordeum vulgare L.) cultivars. Acta Physiol. Plant. 23 (3), 351–356.

Al-Karaki, G.N., 2000. Growth, water use efficiency, and sodium and potassium acquisition by tomato cultivars grown under salt stress. J. Plant Nutr. 23 (1), 1–8. Alscher, R.G., Erturk, N., Heath, L.S., 2002. Role of superoxide dismutases (SODs) in controlling oxidative stress in plants. J. Exp. Bot. 53 (372), 1331–1341. Aono, M., Saji, H., Fujiyama, K., Sugita, M., Kondo, N., Tanaka, K., 1995. Decrease in activity of glutathione reductase enhances paraquat sensitivity in transgenic Nicotiana tabacum. Plant Physiol. 107, 645–648. Asada, K., 1997. The role of ascorbate peroxidase and monodehydroascorbate reductase in H2 O2 scavenging in plants. In: Scandalios, J.G. (Ed.), Oxidative and the Molecular Biology of Antioxidant Defenses. Cold Spring Harbor Laboratory Press, New York, pp. 715–735. Bates, L.S., Waldren, R.P., Teare, I.D., 1973. Rapid determination of free proline for water-stress studies. Plant Soil 39, 205–207. Beauchamp, C., Fridovich, I., 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44, 276–287. Bergmeyer, N., 1970. Methoden der enzymatischen, Analyse, vol. 1. Akademie Verlag, Berlin, pp. 636–647. Bor, M., Özdemir, F., Türkan, I., 2003. The effect of salt stress on lipid peroxidation and antioxidants in leaves of sugar beet Beta vulgaris L. and wild beet Beta maritima L. Plant Sci. 164, 77–84. Bowler, C., Montagu, M.V., Inzè, D., 1992. Superoxide dismutase and stress tolerance. Annu. Rev. Plant Physiol. Plant Mol. Biol. 43, 83–116. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Cakmak, I., Strbac, D., Marschner, H., 1993. Activities of hydrogen peroxide-scavenging enzymes in germinating wheat seeds. J. Exp. Bot. 44 (258), 127–132. Dash, M., Panda, S.K., 2001. Salt stress induced changes in growth and enzyme activities in germinating Phaseolus mungo seeds. Biol. Plant. 44 (4), 587–589. Dionisio-Sese, M.L., Tobita, S., 1998. Antioxidant responses of rice seedlings to salinity stress. Plant Sci. 135, 1–9. Escobar, J.A., Rubio, M.A., Lissi, E.A., 1996. SOD and catalase inactivation by singlet oxygen and peroxyl radicals. Free Radic. Biol. Med. 20 (3), 285–290. Foyer, C.H., 1996. Free radical processes in plants. Biochem. Soc. Trans. 24, 427–434. Foyer, C.H., Halliwell, B., 1976. The presence of glutathione and glutathione reductase in chloroplasts: a proposed role in ascorbic acid metabolism. Planta 133, 21–25. Halliwell, B., 1987. Oxidative damage, lipid peroxidation, and antioxidant protection in chloroplasts. Chem. Phys. Lipids 44, 327–340. Halliwell, B., Gutteridge, J.M.C., 1989. Protection against oxidants in biological systems: the super oxide theory of oxygen toxicity. In: Halliwell, B., Gutteridge, J.M.C. (Eds.), Free Radicals in Biology and Medicine. Clarendon Press, Oxford, pp. 86–123. Hasegawa, P.M., Bressan, R.A., Zhu, J.K., Bohnert, H.J., 2000. Plant cellular and molecular responses to high salinity.

T. Demiral, I˙ . Türkan / Environmental and Experimental Botany 53 (2005) 247–257 Annu. Rev. Plant Physiol. Plant Mol. Biol. 51, 463– 499. Heldt, H.W., 1997. Plant Biochemistry and Molecular Biology. Oxford University Press, New York. Herzog, V., Fahimi, H., 1973. Determination of the activity of peroxidase. Anal. Biochem. 55, 554–562. Hsu, S.Y., Kao, C.H., 2003. Differential effect of sorbitol and polyethylene glycol on antioxidant enzymes in rice leaves. Plant Growth Reg. 39, 83–90. Jain, M., Mathur, G., Koul, S., Sarin, N.B., 2001. Ameliorative effects of proline on salt stress-induced lipid peroxidation in cell lines of groundnut (Arachis hypogea L.). Plant Cell Rep. 20, 463–468. Kingsburry, R.W., Epstein, E., 1986. Salt sensitivity in wheat; a case for specific ion toxicity. Plant Physiol. 80, 651–654. Lin, C.C., Kao, C.H., 1996. Proline accumulation is associated with inhibition of rice seedling root growth caused by NaCl. Plant Sci. 114, 121–128. Lin, C.C., Kao, C.H., 2001. Cell wall peroxidase activity, hydrogen peroxide level and NaCl-inhibited root growth of rice seedlings. Plant Soil 230, 135–143. Lutts, S., Kinet, J.M., Bouharmont, J., 1996. Effects of various salts and of mannitol on ion and proline accumulation in relation to osmotic adjustment in rice (Oryza sativa L.) callus cultures. J. Plant Physiol. 149, 186–195. Madhava Rao, K.V., Sresty, T.V.S., 2000. Antioxidative parameters in the seedlings of pigeonpea (Cajanus cajan L. Millspaugh) in response to Zn and Ni stresses. Plant Sci. 157, 113– 128. Mansour, M.M.F., 1998. Protection of plasma membrane of onion epidermal cells by glycinebetaine and proline against NaCl stress. Plant Physiol. Biochem. 36 (10), 767–772. Mansour, M.M.F., 2000. Nitrogen containing compounds and adaptation of plants to salinity stress. Biol. Plant. 43 (4), 491– 500. McCord, J.M., 2000. The evolution of free radicals and oxidative stress. Am. J. Med. 108, 652–659. Mittal, R., Dubey, R.S., 1991. Behaviour of peroxidases in rice: changes in enzyme activity and isoforms in relation to salt tolerance. Plant Physiol. Biochem. 29 (1), 31–40. Nakano, Y., Asada, K., 1981. Hydrogen peroxide is scavenged by ascorbate-spesific peroxidase in spinach chloroplasts. Plant Cell Physiol. 22 (5), 867–880. Noctor, G., Foyer, C.H., 1998. Ascorbate and glutathione: Keeping active oxygen under control. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 249–279. Noctor, G., Gomez, L., Vanacker, H., Foyer, C.H., 2002. Interactions between biosynthesis, compartmentation and

257

transport in the control of glutathione homeostasis and signaling. J. Exp. Bot. 53 (372), 1283–1304. Pérez-Alfocea, F., Balibrea, M.E., Santa Cruz, A., Estañ, M.T., 1996. Agronomical and physiological characterization of salinity tolerance in a commercial tomato hybrid. Plant Soil 180, 251–257. Pollard, A., Wyn Jones, R.G., 1979. Enzyme activities in concentrated solutions of glycinebetaine and other solutes. Planta 144, 291–298. Rengel, Z., 1992. The role of calcium in salt toxicity. Plant Cell Environ. 15, 625–632. Salekdeh, G.H., Siopongco, J., Wade, L.J., Ghareyazie, B., Bennett, J., 2002. A proteomic approach to analyzing drought- and salt-responsiveness in rice. Field Crops Res. 76, 199–219. Sato, Y., Murakami, T., Funatsuki, H., Matsuba, S., Saruyama, H., Tanida, M., 2001. Heat shock-mediated APX gene expression and protection against chilling injury in rice seedlings. J. Exp. Bot. 52 (354), 145–151. Scandalios, J.G., 1993. Oxygen stress and superoxide dismutases. Plant Physiol. 101, 7–12. Shalata, A., Mittova, V., Volokita, M., Guy, M., Tal, M., 2001. Response of the cultivated tomato and its wild salt-tolerant relative Lycopersicon pennellii to salt-dependent oxidative stress: the root antioxidative system. Physiol. Plant. 112, 487– 494. Shalata, A., Tal, M., 1998. The effect of salt stress on lipid peroxidation and antioxidants in the leaf of the cultivated tomato and its wild salt-tolerant relative Lycopersicon pennellii. Physiol. Plant. 104, 169–174. Shigeoka, S., Ishikawa, T., Tamoi, M., Miyagawa, Y., Takeda, T., Yabuta, Y., Yoshimura, K., 2002. Regulation and function of ascorbate peroxidase isoenzymes. J. Exp. Bot. 53 (372), 1305– 1319. Singha, S., Choudhuri, M.A., 1990. Effect of salinity (NaCl) stress on H2 O2 metabolism in Vigna and Oryza seedlings. Biochem. Physiol. Pflanzen 186, 69–74. Sultana, N., Ikeda, T., Itoh, R., 1999. Effect of NaCl salinity on photosynthesis and dry matter accumulation in developing rice grains. Environ. Exp. Bot. 42, 211–220. Vaidyanathan, H., Sivakumar, P., Chakrabarty, R., Thomas, G., 2003. Scavenging of reactive oxygen species in NaCl-stressed rice (Oryza sativa L.)—differential response in salt-tolerant and sensitive varieties. Plant Sci. 165, 1411–1418. Yoshida, S., Forno, A.D., Cook, J.H., Gomes, K.A., 1976. Routine procedure for growing rice plants in culture solution. In: Laboratory Manual for Physiological Studies of Rice, third ed. The International Rice Research Institute, Los Baños, Laguna, Philippines, pp. 61–65.