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Archives of Agronomy and Soil Science

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Differential response of rice seedlings to salt stress in relation to antioxidant enzyme activity and membrane stability index
P. Senguttuvel
c a a b

, C. Vijayalakshmi , K. Thiyagarajan , R.
d a b

Sritharan , S. Geetha , J.R. KannanBapu & B.C. Viraktamath

Centre for Plant Breeding and Genetics, Tamil Nadu Agricultural University, Coimbatore, India
b c

Directorate of Rice Research, Rajendranagar, India

Department of Plant Physiology, Tamil Nadu Agricultural University, Coimbatore, India

d

Department of Plant Breeding and Genetics, Anbil Dharmalingam Agricultural College, Trichy, TN, India Version of record first published: 05 Oct 2012.

To cite this article: P. Senguttuvel, C. Vijayalakshmi, K. Thiyagarajan, R. Sritharan, S. Geetha, J.R. KannanBapu & B.C. Viraktamath (): Differential response of rice seedlings to salt stress in relation to antioxidant enzyme activity and membrane stability index, Archives of Agronomy and Soil Science, DOI:10.1080/03650340.2012.724170 To link to this article: http://dx.doi.org/10.1080/03650340.2012.724170

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Archives of Agronomy and Soil Science 2012, 113, iFirst article

Dierential response of rice seedlings to salt stress in relation to antioxidant enzyme activity and membrane stability index
P. Senguttuvela,b*, C. Vijayalakshmic, K. Thiyagarajana, R. Sritharanc, S. Geethad, J.R. KannanBapua and B.C. Viraktamathb
a Centre for Plant Breeding and Genetics, Tamil Nadu Agricultural University, Coimbatore, India; bDirectorate of Rice Research, Rajendranagar, India; cDepartment of Plant Physiology, Tamil Nadu Agricultural University, Coimbatore, India; dDepartment of Plant Breeding and Genetics, Anbil Dharmalingam Agricultural College, Trichy, TN, India

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(Received 25 June 2011; nal version received 15 August 2012) Three rice genotypes, IR 74802, IR 73104 and IR 72593, along with FL 478 and IR 29 as resistant and susceptible controls, respectively, were subjected to 21 days salinity stress at the seedling stage in modied yoshida solution with two salt levels (60 and 120 mM NaCl). The results indicated that there was a profound increase in proline and ascorbic acid levels, and in the activity of nitrate reductase and antioxidant enzymes, i.e. catalase, peroxidase and ascorbate peroxidase, as well as malondialdhyde and membrane stability index, which were associated with salt tolerance. Salt stress had a signicant and drastic eect on all parameters when the salinity level increased to 120 mM NaCl. The increased enzyme activity was directly related to an increased membrane stability index, as in IR 72593, which is identied as the most tolerant among the genotypes tested. It is clearly conrmed that predicting tolerance at the early seedling stage is the best way to assess the salinity tolerance level by utilizing physiological parameters, especially antioxidant enzyme activities which are found to be closely associated with salinity tolerance. Physiological adaptation of the plant to NaCl salt stress resulted in enhanced activity of stress-related enzymes and low sodium uptake in tolerant genotypes. Keywords: rice; antioxidants; peroxidase; APX; MDA; membrane stability index

Introduction Salinity is one of the most serious abiotic stresses, aecting *7% of the worlds total land area and limiting crop production. In India, 4 8.6 6 106 ha of land are aected by salt, constituting a major portion of problem soils in India. Rice is moderately tolerant to salinity, especially during germination, active tillering and the latter part of maturity, although it is sensitive at the early seedling and reproductive stages (Lati et al. 2004). High salinity limits plant growth and causes ion toxicity, osmotic stress, mineral deciencies, physio-biochemical perturbation and combinations of these factors (Munns 1993). Salt tolerance involves the accumulation of compatible osmolytes that regulate additional water uptake, thus buering against water

*Corresponding author. Email: senguttuvel@gmail.com

ISSN 0365-0340 print/ISSN 1476-3567 online 2012 Taylor & Francis http://dx.doi.org/10.1080/03650340.2012.724170 http://www.tandfonline.com

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shortage within the plant (Misra and Gupta 2005). Nitrogen assimilation in plants is regulated by the enzyme nitrate reductase (NR); NR activity plays a constructive role in nitrogen utilization by plants through nitrogen metabolism and NR is very sensitive to NaCl (Gouia et al.1994). Proline (Pro) is an a-amino acid that accumulates in large amounts in salt-stressed plants when compared with all other amino acids (Ali et al.1999). In addition to proline accumulation, stressrelated enzymes are involved in upregulating redox potentials as hydroxyl radical scavengers. Ascorbic acid (AsA) is an important primary metabolite in plants, functioning as an antioxidant, an enzyme cofactor and a cell-signaling modulator in a wide array of crucial physiological processes, including biosynthesis of the cell wall, photoprotection, cell division and growth (Wolucka et al. 2005). The predominant peroxidase is ascorbate peroxidase (APX), which catalyses oxidation of ascorbate by H2O2, generating dehydroascorbate radicals (Hideg 1999); APX increases signicantly during salt stress. A correlation between antioxidant capacity and NaCl tolerance has been demonstrated in some plant species (Dionisio-Sese and Tobita 1998). If there is a serious imbalance in any cell compartment between the production of reactive oxygen species (ROS) and antioxidant defense, oxidative stress and damage occur (Mittler 2002). Several studies have pointed out that salt-tolerant species increase their antioxidant enzyme activities and antioxidant contents in response to salt treatment, whereas salt-sensitive species fail to do so (Demiral and Turkan 2005). To minimize the eect of oxidative stress, plant cells have evolved a complex antioxidant system composed of low molecular mass antioxidants (glutathione, ascorbate and carotenoids) and ROS-scavenging enzymes such as catalase (CAT), APX, glutathione peroxidase (GPX) and glutathione reductase (GR) (Apel and Hirt 2004; Singh MP et al. 2007). A decrease in malondialdehyde (MDA) concentrations after long-term exposure to the highest intensity stress was seen only in salt-sensitive cultivars and this can be linked to an overall inhibition of plant metabolism (Lutts et al. 1996). ROS scavenging is one of the most common defense responses against abiotic stress (Vranova et al. 2002). Increased ROS-scavenging activity in tolerant genotypes helps to minimize salinity-induced toxicity (Singh MP et al. 2007). The level of the antioxidative response depends on the species, development and metabolic state of the plant, as well as the duration and intensity of stress (Reddy et al. 2004). ROS generation and an increase in the activity of many antioxidant enzymes during salt stress have been reported in rice (Vaidyanathan et al. 2003). To be able to endure oxidative damage under unfavorable conditions such as high/low temperatures, water decit and salinity, plants must possess an ecient antioxidant system (Sairam and Srivastava 2002). Membrane stability has often been used to assess potential drought and salinity tolerance in dierent crops (Blum and Ebrecon 1981). Sairam et al. (2002) used the membrane stability index (MSI) to dierentiate two wheat genotypes growing at dierent salinity levels. Sairam and Srivastava (2002) reported enhanced lipid peroxidation and membrane damage in leaves subjected to levels of salt stress higher than mild stress. MSI is more eective than grain yield reduction in screening large quantities of germplasm at the seedling stage (Farooq and Azam 2006). This study was conceptualized with this in mind and focuses on the response of rice at the early seedling stage to varying levels of salinity as relating to the role of NR, the antioxidant system, including enzymes and ascorbate, the osmoprotectant proline and parameters of membrane reactions in the mitigation of and adaptation to salinity stress.

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Archives of Agronomy and Soil Science Materials and methods Plant materials and method of screening

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Three IRRI genotypes namely IR 74802-3R-7-1-2, IR 73104-B-1-1-3-2-1 and IR 72593-B-19-2-3-1(henceforth IR 74802, IR 73104 and IR 72593) were selected along with FL 478 and IR 29 which served as tolerant and susceptible controls, respectively. FL 478 was selected instead of Pokkali because of its growth duration and plant type because FL 478 is derived from a cross-combination of Pokkali and IR 29. Seeds of each genotype were sown on sterilized sand media. After 5 days, seedlings were transferred to thermocole sheets placed on 12-L plastic trays. Each thermocole sheet had 77 holes (11 6 7 rows). A single seedling was planted per hole, with a total of 11 seedlings per test. The thermocole sheets were placed in a nutrient solution and the salinity treatments were imposed from day 21 onwards using two salinity levels of 60 and 120 mM NaCl. Treatments were replicated three times in a randomized complete block design and two sets were developed to reduce error. The seedlings from the two sets were harvested for enzyme analysis 7 days after initiating salinity stress. Leaves were scored basipetally for salt injury, with leaf number 3 being the oldest and leaf number 7 the youngest. NR activity A known quantity of leaf sample collected from functional leaves was cut into small pieces and placed in a test tube containing 10 mL of ice-cold assay medium (phosphate buer). The enzyme was extracted by suction using a vacuum evaporator for 5 min in the dark. The solution was kept in the dark for 1 h to allow the enzyme reaction. After 1 h, a 2-mL aliquot was taken and 1 mL of zinc acetate (1 M) and 2 mL of 70% ethanol were added. The precipitate was ltered and 1 mL of 1% sulfanilamide in 1.5 N HCl and 1 mL of 0.02% L-naphthyl ethylene diamine dihydrochloride were added. The pink color that developed was read at 540 nm using a UV-spectrophotometer. KNO2 was used to plot a standard graph against which the sample was compared (Nicholas et al. 1976). NR activity was determined by the leaf disc method and expressed as mmol NO2 g71 (fresh weight) h71. Pro content The method of Bates et al. (1973) was used. A known amount of leaf material (0.5 g) was homogenized with 10 mL of 3% aqueous sulfosalicylic acid and centrifuged at 3000 rpm for 10 min. During selective extraction with aqueous sulfosalicylic acid, proteins were precipitated as a complex. Other interfering materials were also removed by absorption to the protein sulfosalicylic acid complex. Two milliliters of the supernatant was taken and 2 mL of glacial acetic acid and 2 mL of an acid ninhydrin mixture were added. The contents were allowed to react at 1008C for 1 h and then kept in an ice bath for 10 min to terminate the reaction. The reaction mixture was mixed vigorously with 4 mL of toluene using a Vortex mixer for 15 20 s. The toluene phase with the reaction product of proline and ninhydrin formed chromospheres (yellow color) and was aspirated from the aqueous phase, warmed to room temperature and the absorbance was read at 520 nm in a spectrophotometer using pure toluene as a blank. The proline content was determined from a standard curve (20100 mg) of pure proline and expressed as mg g71.

4 Antioxidant system AsA content

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Based on the procedure suggested by Sadasivam and Manickam (1992), *1 g of leaf was homogenized with 4% oxalic acid and centrifuged at 5000 rpm for 10 min. The supernatant was made upto 25 mL. To 5 mL of aliquot, 10 mL of 4% oxalic acid was added and titrated against the dye (42 mg of sodium bicarbonate was weighed into a small volume of distilled water and 52 mg of 2,6-dichlorophenol indophenol dye was dissolved and the volume was made up to 200 mL with distilled water). Five milliliters of standard solutions, containing 100 mg mL71 of ascorbic acid was titrated for calibration. The end point was taken to be the disappearance of the pink color, which persisted for a few minutes. The amount of dye consumed was equivalent to the amount of oxalic acid. The amount of ascorbic acid was expressed as mg g71 fresh weight.

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CAT activity CAT activity was assayed from the rate of H2O2 decomposition (extinction coecient of 39.4 mM71 cm71) as measured by the decrease in absorbance at 240 nm, following the procedure of Aebi (1974). Approximately 1 g of rice seedlings was homogenized using phosphate buer and the homogenate was centrifuged; the supernatant was then used as an enzyme extract. The reaction mixture contains 50 mM potassium phosphate buer (pH 7.0) and 40 mL of extract. The reaction was initiated by adding 10 mM H2O2. One unit of CAT is dened as the amount of enzyme that liberates half of the peroxide oxygen from 10 mM H2O2 solution in 100 s at 258C. The CAT activity was expressed as mg H2O2 g71 (fresh weight) min71. POR activity POR activity was determined according to Perur (1962) and Angelini et al. (1990). One gram of leaf was extracted in 0.1 M phosphate buer (pH 7.0), claried by centrifugation and the supernatant was used in the assay. A known volume of the extract was added to a cuvette containing 3 mL of phosphate buer, 3 mL of pyrogallol was then added and the increase in absorbance at 430 nm was recorded. This change in absorbance in minutes was used to calculate the enzyme activity using a spectrophotometer. The POR activity is the change in absorbance at 430 nm in unit g71 min71.

Preparation of leaf extracts for APX and MDA assays APX activity APX activity was determined by modifying the procedure of Nakano and Asada (1981). Fresh leaves (0.5 g) were homogenized in 100 mM potassium phosphate buer (pH 7.8) containing 0.1 mM EDTA, 1% (w/v) polyvinyl pyrrolidone (PVP), 0.5% (V/ V) Triton X-100 at 48C and 5 mM ascorbate. The homogenate was centrifuged at 10 000 rpm for 20 min at 408C. The supernatant was collected to measure APX activity. A known volume of crude extract and reaction solution contained 25 mM potassium phosphate buer, 0.25 mM ascorbate, 0.1 mM EDTA and 0.1 mM

Archives of Agronomy and Soil Science

hydrogen peroxide. The decrease in ascorbate concentration was followed as a decline in the optical density at 290 nm using a UV spectrophotometer, and activity was calculated using the extinction coecient (2.8 mM71 cm71 at 290 nm) for ascorbate. One unit of APX was dened as the amount of enzyme that breaks down 1 mmol ascorbate min71. The amount of APX was expressed as EU mg71 protein.

Membrane stability MDA The amount of MDA derived from unsaturated fatty acid peroxidation of membrane lipids was measured using the method of Dionisio-Sese and Tobita (1998). A crude extract preparation was mixed with the same volume of 0.5% (w/v) thiobarbituric acid solution containing 20% (w/v) tricholoracetic acid. The mixture was heated at 958C for 30 min and the reaction was stopped quickly by placing the sample in an ice bath. The cooled mixture was centrifuged at 10 000 rpm for 10 min and the absorbance of the supernatant was determined at 532 and 600 nm. After subtracting the non-specic absorbance at 600 nm, the MDA concentration was determined using the extinction coecient of 155 mM71 cm71. The amount of MDA was expressed as mM g71. MSI Damage to cell membrane integrity was measured in terms of the percentage of ion leakage by following the method of Deshmukh et al. (1991). Leaf samples (1 cm) were washed in deionized water to remove surface-adhered electrolytes, placed in tubes that included 5 mL of deionized water and shaken gently (100 rpm) for 24 h at room temperature; after incubation, their conductance was measured (T1). Subsequently, the samples were then autoclaved at 1208C for 20 min to kill the tissues completely and shaken for 24 h at room temperature. Final conductance (T2) was then measured. The percentage leakage of electrolytes can be calculated by observing the changes in electrical conductivity before and after treatment at 258C. Statistical analysis The mean data of selected plants for each genotype per replication were subjected to analysis of variance (ANOVA) for all the characters appropriate for randomized complete block design (Panse and Sukhatme 1954) and the analysis was done using CROPSTAT software v. 7.2. Signicant dierences were analyzed based on p 5 0.01, p 5 0.05 and p 5 0.1. Results Mean values for NR were 26.8 (control), 24.5 (60 mM NaCl) and 21.78 mmol NO2 g71 h71 (120 mM NaCl). Genotype IR 73104 showed the highest enzyme activity of 29.5 (control), 26.5 (60 mM NaCl) and 24.0 mmol NO2 g71 h71 (120 mM NaCl) followed by IR 72593 with an enzyme activity of 28.6 (control), 25.6 (60 mM NaCl) and 23.9 mmol NO2 g71 h71 (120 mM NaCl). IR 29, the sensitive control, showed the lowest NR activity at 16.0 mmol NO2 g71 h71 at 120 mM NaCl, in comparison

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with the control (24.6 mmol NO2 g71 h71). In general, NR activity decreased with the increase in salinity (Figure 1a). An eect of salt stress on Pro content was observed and the results were signicant in all interactions (Figure 1b). Genotypes IR 72593 and IR 73104 showed the highest Pro accumulation at 120 mM NaCl (1296 mg g71), followed by FL 478 (1203 mg g71), the lowest accumulation was seen in IR 29 (720 mg g71) at 120 mM NaCl. The average eect of salinity, irrespective of genotype, at 120 and 60 mM were 1135.4 and 552.4 mg g71 (control, 423 mg g71), which indicates stress-induced accumulation of Pro, irrespective of genotype. The AsA content was highest (100.3 mg g71) in genotype IR 72593, followed by IR 773104 (99.56 mg g71), FL 478 (96.61 mg g71), IR 74802 (90.27 mg g71) and IR 29 (83.62 mg g71). The mean values observed for the genotypes were 94.07 mg g71 in control, 85.11 mg g71 in 60 mM NaCl and 78.87 mg g71 in 120 mM NaCl, so the salinity at 120 mM NaCl reduced the AsA content compared with control by 16%; the corresponding value in 60 mM NaCl was 9.5% (Figure 1c). The average mean values for all salinity levels, irrespective of genotype, ranged from 75.92 mg g71 (IR 29) to 79.62 mg g71 (IR 72593). An increase in CAT activity was observed at both levels of salinity stress in all cultivars (Figure 1d). The interaction between genotype and salinity was found to be signicant. In the controls, IR 72593 and IR 29 showed the highest and lowest levels of CAT activity (2.25 and 2.21 mmol H2O2 decomposed per min, respectively), whereas at 60 and 120 mM NaCl, IR 29 gave the lowest level (2.3 and 2.56 mg g71) and IR 72593 (2.89 and 3.43 mg g71) the highest of all the genotypes tested. An increase in POR activity was found in both 60 and 120 mM NaCl. A signicant interaction between the test genotypes and salinity in terms of POR activity was noticed. Mean POR values ranged from 0.280 unit g71 min71 (IR 29) to 1.193 unit g71 min71 (IR 72593). At 120 mM NaCl, genotype IR 72593 gave the highest POR activity of 1.193 unit g71 min71, followed by IR 74802 (1.189 unit g71 min71), FL 478(1.186 unit g71 min71), IR 73104 (1.76 unit g71 min71) and IR 29 (0.72 unit g71 min71) (Figure 2a). APX activity increased signicantly at both 60 and 120 mM NaCl (Figure 2b). Mean APX activity varied from 1201.0 (control) to 1381.0 EU mg71 protein (120 mM NaCl). Among the genotypes, IR 72593 showed the highest values of 1289.0 EU mg71 protein in control, 1320.0 EU mg71 protein in 60 mM NaCl and 1480.0 EU mg71 protein in 120 mM NaCl. IR 29 (1100.0 EU mg71 protein), the sensitive control, showed the lowest values in 120 mM NaCl compared with 60 mM NaCl (1200.0 EU mg71 protein). In this study, APX activity increased with the increase in salinity in all tolerant genotypes, suggesting higher antioxidant enzyme activity at higher salt concentrations; the sensitive genotype showed a negative trend in 120 mM NaCl compared with 60 mM NaCl. The eect of dierent levels of salinity on MDA was statistically signicant (Figure 2c). The average mean MDA activity between salinity levels ranged from 544 mM g71 (IR 29) to 663 mM g71 (IR 74802). Among the genotypes, IR 74802 showed the highest MDA activity at 60 mM NaCl (672 mM g71) and 120 mM NaCl (713 mM g71). IR 29 had the lowest MDA activity, irrespective of stress condition, i.e. 528 mM g71 in controls, 543 mM g71 in 60 mM NaCl and 563 mM g71 in 120 mM NaCl. The MSI was also used as an index of salt tolerance in the ve genotypes. The MSI was similar in the control condition for all ve genotypes. The average mean

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Figure 1. Eect of salinity (120 mM NaCl) on (a) NRase activitya, (b) proline activityc, (c) ascorbic acid contentc and (d) catalase activityb in rice at the seedling stage. Vertical bars indicate the SE of ve replicates for each treatment. aSignicant (LSD0.05) at p 5 0.05. bSignicant (LSD0.05) at p 5 0.01. c Signicant (LSD0.05) at p 5 0.1.

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Figure 2. Eect of salinity (120 mM NaCl) on (a) peroxidase activitya, (b) APX activityc, (c) MDA activitya and (d) MSIa in rice at the seedling stage. Vertical bars indicate the SE of ve replicates for each treatment. aSignicant (LSD0.05) at p 5 0.05. bSignicant (LSD0.05) at p 5 0.01. cSignicant (LSD0.05) at p 5 0.1.

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Figure 3. Comparative response of rice genotypes to dierent salt stresses. FL 478, salttolerant check; IR 29, salt-sensitive check; IR 74802, 72593, 73104, new genotypes.

MSI value was found to decrease with increased salinity compared with the control (Figure 2d). The highest value was found in genotypes IR 73104: 87% in the control, 80% in 60 mM and 64.8% in 120 mM NaCl. The lowest values were recorded in IR 29: 77% in the control, 71.4% in 60 mM NaCl and 41.9%in 120 mM NaCl (Figure 3). Discussion In general, growth is adversely aected when seedlings are subjected to higher levels of salt stress, whereas enzyme activity responds well to the salinity level. Irrespective

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of the salinity level, NR activity was reduced signicantly under salt stress; genotypic variation in NR activity was seen under salt stress, with a lesser reduction seen in IR 73104 than in the other genotypes, similar to the ndings of Flores et al. (2000). The reduced NR activity might be due to a decrease in nitrate content, which in turn may be due to reduced nutrient uptake under salt stress (Yadav et al. 1997). Flores et al. (2000) suggested that the primary cause of a reduction in NR in the leaves is a specic eect associated with the presence of Na salts in the external medium, which might aect the enzyme activity. Proline accumulation is a universal plant response to stress. Our results clearly showed a signicant increase in Pro accumulation in both the tolerant and sensitive genotypes; however, the accumulation was greater in salt-tolerant lines when the salinity increased. An increase in leaf Pro content in the presence of NaCl is in agreement with the ndings of other researchers (Singh AK et al. 1996; Demiral and Turkan 2005). Salt-tolerant genotypes accumulated this organic solute at greater proportions than did salt-sensitive plants, hence, Pro is also considered to act as a compatible osmoticum and, therefore, to be involved in salt resistance (Delauney and Verma 1993). Ascorbic acid is the most abundant, powerful and water-soluble antioxidant, and acts to prevent or minimize the damage caused by ROS in plants (Athar et al. 2008). Increased salinity led to a signicant increase in AsA in the tolerant genotypes (IR 72593, IR 7304), but a slight reduction in the sensitive genotype (IR29). The high level of AsA in tolerant genotypes is known to play a vital role in ROS detoxication in plant cells (Moradi and Ismail 2007). The elevated level of AsA is an indicator of tolerance and is vital in reducing membrane damage during salt stress ((Noctor and Foyer 1998). The tolerant genotype (IR 72593) exhibited maximum POR activity in comparison with the other genotypes and there was a drastic reduction in the sensitive genotype, highlighting the dierences in their salt tolerance. The activities of the antioxidant enzymes CAT, POR and APX increased under salt stress and a strong relationship between these enzymes exists, in accordance with the results of Mittova et al. (2003). CAT, POR and APX activity varied with the intensity of salt stress and were increased at higher salinity levels in tolerant genotypes. Nevertheless, the sensitive genotype (IR 29) showed lower CAT, POR and APX activity, resulting in the premature death of seedlings and a complete cessation of growth. Variable reports documenting either an increase (Rai and Srivastava 2001) or decrease (Zhu 2001) in antioxidant enzyme activities in dierent plant species subjected to salinity stress indicate variability in these activities. POR is highly reactive and linked to degeneration processes leading to disease and ageing (Shigenaga et al. 1994). APX has high substrate specicity for AsA and is the primary hydrogen-peroxide-scavenging enzyme in the chloroplasts and cytosol of plant cells (Dionisio-Sese and Tobita 1998). The activities of antioxidative enzymes such as CAT and APX increased under salt stress in plants and a correlation between levels of these enzyme and salt tolerance exists. These enzymes act as a buer medium when salinity increases, controlling the accumulation of H2O2. The level of MDA was remained static in tolerant genotypes compared with sensitive IR 29 over a prolonged period of 2 weeks, representing the protective role of antioxidant enzymes in the crop growth period and this is in accordance with the results of Moradi and Ismail (2007). Damage to the membrane was monitored by measuring the amount of thiobarbituric acid reactive material (MDA) produced when polyunsaturated fatty acids in the membrane undergo

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peroxidation and this is in accordance with previous reports by Dionisio-Sese and Tobita (1998). Increased H2O2 accumulation (determining CAT activity is the measure of H2O2 decomposition) and lipid peroxidation due to salt stress might have resulted in a decrease in the MSI in the sensitive genotype (IR 29). Lower H2O2 accumulation and lipid peroxidation and higher membrane stability have been reported in salt-tolerant rice genotypes (Islam et al. 2007). In parallel with these observations, we also noticed an increase in lipid peroxidation in 60 and 120 mM NaCl. The occurrence of lipid peroxidation is an indication of the prevalence of free radical reactions and a change in the balance between O2- and H2O2 in leaves. The enhanced activities of CAT, POR and APX in tolerant plants helped in the rapid detoxication of any H2O2 produced, so that a steady level of this ROS is maintained even under high NaCl concentrations. High levels of H2O2 in saltsensitive genotypes resulted in the formation of hydroxyl radicals that may cause lipid peroxidation. This is reected in the greater extent of lipid peroxidation in the sensitive genotype upon exposure to 120 mM NaCl. Salt stress might increase cell damage by increasing the generation of ROS, which leads to the peroxidation of membrane lipids and thus to membrane damage. Resistance to environmental stress may, therefore, depend in part on the inhibition of ROS production or enhancement of antioxidant enzyme activity. The overall superiority of IR 72593 may be due to detoxifying ROS eectively during stress as a consequence of reduced photochemical carbon xation and excess energy (Moradi and Ismail 2007). Peroxidase decomposes H2O2 by oxidation of co-substrates such as phenolic compounds and or antioxidants. Tolerant genotypes (IR 72593 and IR 74802) have higher peroxidase activities than sensitive genotypes (IR 29), showing that peroxidase is involved in reducing the eects of oxidative stress. In plants, an alternative and more eective detoxication mechanism against H2O2 also exists through the operation of the ascorbateglutathione cycle, and APX, which specically uses ascorbate as a physiological reductant, is considered a crucial component in metabolic defense against oxidative stress (Noctor and Foyer 1998). The tolerant genotype suggests that this enzyme has a greater ability to eliminate H2O2. Electrolyte leakage is an indirect measure of membrane stability. The presence of salt induces electrolyte leakage and this is more pronounced when the duration of the stress increases. Loss of internal ions and the cessation of further growth of fresh leaves in the sensitive genotype reduced the leakage, and salt stress even induced greater electrolyte leakage in young leaves than in old, thereby reversing the usual situation in salt-resistant genotypes. By contrast, dierences between the properties of old and young leaves were maintained throughout the experiment (Lutts et al.1996). Dhindsa et al. (1981) previously demonstrated that there are similar changes in the level of lipid peroxidation and in electrolyte leakage during senescence. It is clear that tolerant genotypes have less leakage than sensitive ones, indicating that the compensating mechanism and electrolyte leakage are strongly correlated with other biochemical parameters. Conclusion It can be concluded from this study that, among the genotypes screened for tolerance, IR 72593 exhibited the best performance. The ecient detoxication of H2O2, greater membrane stability and lower ROS production with increased

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antioxidant enzyme activities are characteristics of salt-tolerant genotypes and one of the mechanisms that plays an eective role in combating salinity stress in the early seedling stage of rice. References
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