Influence of Drought Stress on Oxidative Damage and Antioxidant Defense Systems in Tolerant and Susceptible Wheat Genotypes

Drought is one of the major factors limiting crop production in arid and semi-arid regions. Twenty wheat genotypes with wide range of sensitivity to drought, including 18 varieties of bread wheat (Triticum aestivum L.) and two varieties of durum wheat (Triticum turgidum L.) were used in two separate field experiments in 2009-2010 at the Experimental Station of College of Agriculture in Shiraz University. Each experiment was conducted as a randomized completed block design with three replications. The moisture level in one of the experiments was optimum (100% field capacity) while the second experiment was conducted under drought stress (45% field capacity). Several biochemical components including enzymatic (catalase, CAT; peroxidase, POD; superoxide dismutase, SOD and ascorbate peroxidase, APX) and non-enzymatic (proline and carotenoids, Car) antioxidant defense systems and some factors of oxidative damage (hydrogen peroxide, H2O2; lipid peroxidation, LPO and membrane stability index, MSI) were analyzed in the two conditions. Drought stress caused significant increase in enzymatic antioxidant activities, proline content, H2O2 and LPO content at the flowering stage, while Car content and MSI decreased significantly in all genotypes. Drought tolerant genotypes showed the highest enzymatic and non-enzymatic antioxidants, highest MSI and the lowest LPO and H2O2. This trend was reversed in susceptible genotypes. The enzymatic antioxidants had higher correlation than non-enzymatic with oxidative stress factors and yield stability index (YSI). POD showed the highest positive correlation with MSI and the highest negative correlation with LPO. H2O2 and MSI showed the highest correlation with YSI. In present study, Kavir and Alamut varieties were selected respectively as the most tolerant and susceptible genotypes.


Introduction
Drought stress is one of the major factors limiting plant growth and crop productivity in arid and semi-arid regions and with increasing global climate change making the situation more serious.(Golestani and Assad, 1998;Ahmadi et al., 2010) Much of the injury to plants caused by stress exposure is associated with oxidative damage at the cellular level.However in certain tolerant crop plants morpho-physiological and metabolic changes occur in response to drought, which contribute towards adaptation to such unavoidable environmental constraints (Sairam & Sirvastava, 2001).
Wheat is a staple food for more than 35% of the world population and it is also the first grain crop in Iran (Mohammadi et al., 2006).Wheat often experiences drought stress conditions during crop cycle.Thus, improvement of wheat productive for drought tolerance is a major objective in plant breeding programs for arid and semi-arid regions (Shao et al., 2005;Ahmadizadeh et al., 2011).
Drought stress results in stomata closure, which limits CO 2 concentration in leaf mesophyll tissue and reduces NADP + regeneration by the Calvin Cycle.These adverse conditions increase the rate of reactivated oxygen species (ROS) such as hydrogen peroxide (H 2 O 2 ), superoxide (O 2 •− ), singlet oxygen ( 1 O 2 ) and hydroxyl (OH) radicals by enhanced leakage of electrons toward molecular oxygen during photosynthetic and respirator processes (Foyer et al., 1994).These ROS can cause damage to membrane lipids, proteins and DNA leading to cell death (Cadenas, 1989).Plants process very efficient enzymatic (superoxide distumase, SOD; catalase, CAT; ascorbate Peroxidase, APX; Peroxidase, POD and glutathione reductase, GR) and non-enzymatic (carotenoids, ascorbic acid, glutathione and proline) antioxidant defense systems which protect cell and subcellular systems against oxidative damages by scavenging of ROS (Dhindsa et al., 1981;Mittler, 2002).SOD catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide (Alscher et al., 2002).H 2 O 2 can be eliminated by CAT, APX and POD (Asada, 1999;Ramachandra et al., 2004).Carotenoid a lipid soluble antioxidant plays a multitude of functions in plant metabolism including oxidative stress tolerance (Sarvajeet & Narendra, 2010).Accumulation of protective solutes like proline and glycine betaine is a unique plant response to drought stress.Also proline is considered as a potent antioxidant and potential inhibitor of programmed cell death (Bates et al., 1973;Pireivatloum et al., 2010).The objective of the present study was to understand the influence of drought stress on oxidative damage, enzymatic and non-enzymatic antioxidant systems in tolerant, intermediate and susceptible wheat genotypes and also identify the effective biochemical traits in the screening tolerant genotypes to drought.

Plant Material and Experimental Conditions
Eighteen bread wheat genotypes (Triticum aestivum L.) including six drought tolerant genotypes (Azar2, Pishtaz, Toos, Chamran, Kavir and Koohdasht), six intermediate (Roshan, Alvand, Tabasi, Niknejad, cross adl and Darab2) and six susceptible (Shiraz, Shiroudi, Flat, Bahar, Zarin and Alamut) and two durum wheat genotypes (Triticum turgidum L.), Simareh and Yavarus, were also used in two separate field experiments in 2009-2010 at the Experimental Station of College of Agriculture in Shiraz University (52 o 46' E, 29 o 50' N, altitude 1,810 m above sea level).Each experiment was conducted as a randomized completed block design with three replications.Each plot consisted of six 4 m long rows spaced 30 cm apart.The four middle rows were left intact for grain yield determination, and the two outside rows were used for sampling.The moisture level in one of the experiments was optimum (100% field capacity) while the second experiment was conducted under drought stress (45% field capacity), (Table 2).The amount of water needed for irrigation was calculated from the method of Avja and Michael (1987).The characteristics of soil and climates at the experimental station during 2009-2010 are shown in Table 1 and 2 respectively.Flag leaves of flowering stage in two experiments were harvested, weighted and frozen at -70 ° C for later measurement of biochemical traits.

Grain Yield and Yield Stability Index Assay
Grain yield was recorded at physiological maturity stage.The physiological maturity stage was considered when 90% of seed changed color from green to yellowish and stopped photosynthetic activity.Yield stability index (YSI) was calculated using the formula suggested by Bouslama and Schapaugh (1984) as: Where, Ys and Yp represent yield under stress and non-stress conditions, respectively.

Enzymatic Antioxidants Assay
Frozen leaf samples (0.5 g) were used for enzyme extraction.Samples were homogenized with 2 mL of 50 mM phosphate buffer (pH 7.2) using a pre-chilled mortar and pestle.Phosphate buffer contained 1 mM EDTA, 1 mM PMSF, and 1% PVP-40.Then the homogenates were centrifuged at 4 °C and 15,000×g for 15 min.
Superoxide dismutase (SOD, EC 1.15.1.1)activity was assayed by measuring its ability to inhibit the photoreduction of nitroblue tetrazolium (NBT) using the method of Beauchamp and Fridovich (1971).The reaction mixture contained: 50 mM phosphate buffer (pH 7.8), 0.1 mM EDTA, 13 mM methionine, 75 μ M nitroblue tetrazolium (NTB), 2 μM riboflavin and 100 μl of the supernatant.Riboflavin was added as the last component and the reaction was initiated by placing the tubes under two 15 W fluorescent lamps.The reaction was terminated after 15 min by removing the reaction tubes from the light source.Non-illuminated and illuminated reac-tions without supernatant served as calibration standards.Reaction products were measured at 560 nm.One unit of SOD activity was defined as the amount of enzyme that inhibited 50 nitroblue tetrazolium (NBT) photoreduction.
Catalase (CAT, EC 1.11.1.6)activity was measured by following the reduction of H 2 O 2 (ε = 39.4 mM − 1 cm − 1 ) at 240 nm according to the method of Dhindsa et al. (1981).The assay solution contained 50 mM potassium phosphate buffer (pH 7.0) and 15 mM H 2 O 2 .The reaction was started by the addition of 100 µl enzyme extract to the reaction mixture and the change in absorbance was followed 1 min after the start of the reaction.One unit of activity was considered as the amount of enzyme which decomposes 1 mM of H 2 O 2 in one minute.
The tetraguaiacol formed in the reaction has a maximum absorption at 470 nm and thus the reaction can be readily followed spectrophotometrically.The enzyme was assayed in a solution containing 50 mM phosphate buffer (pH 7.0), 5 mM H 2 O 2 and 13 mM guaiacol.The reaction was initiated by adding of 33 µl enzyme extract at 25 °C.One unit of enzyme was calculated on the basis of the formation of guaiacol to tetraguaiacol for 1 min.

Non-enzymatic Antioxidants Assay
The content of proline was extracted and determined by the method of Bates et al. (1973).Leaf tissues (0.5 g) were homogenized in 3 % sulfosalicylic acid and the homogenate was centrifuged at 3,000×g for 10 min.The supernatant was treated with acetic acid and ninhydrin, boiled for 1 h, and then the absorbance was determined at 520 nm.Proline concentration was calculated with a standard curve and expressed as µmolg -1 fresh mass.
The amount of carotenoids (Car) was determined according to Lichtenthaler and Wellburn (1983).Leaf tissues (0.5 g) were homogenized in acetone (80%).Extract was centrifuged at 3,000×g and absorbance was recorded at 646.8 nm and 663.2 nm for chlorophyll assay and 470 nm for Car determine by spectrophotometer.Car and Pigments content were calculated due to the following formulae:

Oxidative Damage Assay
Hydrogen peroxide (H 2 O 2 ) content was determined according to Alexieva et al. (2001).Leaf tissue (0.5 g) was homogenized in ice bath with 5 cm 3 of cold 0.1% (m/v) trichloroacetic acid (TCA).The homogenate was centrifuged (10,000×g, 20 min, 4 °C) and 0.5 cm 3 of the supernatant was added to 0.5 cm 3 of 100 mM potassium phosphate buffer (pH 7.0) and 1 cm 3 of 1 M KI.The absorbance was read at 390 nm.The concentration of H 2 O 2 was determined using a standard curve plotted with a known concentration of H 2 O 2 .
Lipid peroxidation (LPO) rates in plant tissues were determined by measuring the malondialdehyde (MDA) according to the method of Heath and Packer (1968).MDA content was determined with thiobarbituric acid (TBA) reaction.0.5 g tissue sample was homogenized in 5 ml 0.1% trichloroacetic acid (TCA).The homogenate was centrifuged at 10,000×g for 10 min.4 ml of 20% TCA containing 0.5% TBA was added to 1 ml aliquot of the supernatant.The mixture was heated at 95 °C for 30 min and quickly cooled in ice bath.After centrifugation at 10, 000×g for 10 min.The non-specific absorbance of the supernatant at 600 nm was subtracted from the maximum absorbance at 532 nm for MDA measurement.The level of lipid peroxidation was expressed as µmol of MDA formed using an extinction coefficient of 155 mM -1 cm -1 .
Membrane stability index (MSI) estimated according to Sairam (1994).Two sets of leaf tissues (0.1 g) were placed in 10 ml of double-distilled water.One set was kept at 40 °C for 30 min and its conductivity recorded using a conductivity bridge (C 1 ).The second set was kept in a boiling water bath (100 °C) for 10 min and its conductivity also recorded (C 2 ).The membrane stability index was calculated as:

Statistical Analysis of Data
Analysis of variance and Pearson correlations coefficients in all the measurements were conducted by SPSS 16.Means were separated using Tukey's test at P < 0.05.To compare the effects of stress and non-stress, and genotypes by moisture conditions interaction, a combined analysis of variance was used.

Enzymatic Antioxidants Defense Response
The results of the present study showed that considerable variations among genotypes for antioxidant activity were observed when grown under drought stress and non-stress conditions ( Our result clearly indicated efficient role of antioxidant defense machinery in protection of cell systems against oxidative damage.The enzymatic antioxidants had a higher correlation than non enzymatic with all oxidative stress factors (H 2 O 2 , MDA and MSI).It may be reflected more efficient role antioxidant enzymes in compare to non-enzymatic in protects cell systems against oxidative damage.These results are similar to works of Amjad et al. (2011) and Shao et al. (2005).H 2 O 2 and MSI had the highest correlation with YSI in all the traits.Thus, it can be concluded that H 2 O 2 and MSI are more effective indicators for screening drought tolerant genotypes in stress condition.Sairam and Sirvastava (2001) had reported that H 2 O 2 and MSI were good indicators of drought tolerance.

Conclusion
The results showed that genotypes respond differentially to oxidative damage as a result of variations in their antioxidant defense systems.Under water stress condition, activity of CAT, POD, APX and SOD, proline content, H 2 O 2 and LPO significantly (P < 0.01) increased while Car and MSI decreased significantly (P < 0.01).Drought tolerant genotypes which had lowest membrane damage (MDI) and H 2 O 2 content and the highest MSI also showed the highest enzymatic antioxidants activity (CAT, POD, APX and SOD) and non-enzymatic antioxidants (Proline and Car) while drought susceptible genotypes showed the lowest antioxidants defends and MSI, and highest H 2 O 2 and MDA content.Intermediate drought tolerant genotypes showed a moderately response.Also durum wheat indicated similar behavior of tolerant bread wheat under drought stress.We found that enzymatic antioxidants had play more effective role than non-enzymatic antioxidants in protects cell systems against oxidative damage.

Table 1 .
Physical and chemical properties of soil used in the experiments

Table 2 .
Mean temperature, precipitation distribution and total irrigation for each experiment Table 3).Peroxidase (POD) activity increased significantly (P< 0.01) under water stress condition.POX is one of the major enzymes that have a role in the biosynthesis of lignin and defense against water stress by scavenges H 2 O (Mittler, 2002;Sarvajeet & Narendra, 2010)& Narendra, 2010).The highest POD activity were observed in genotypes Toos, Pishtaz, Chamran, Kavir and Koohdasht (drought tolerance, group 1), and the lowest activity in Bahar, Shiraz, Zarin, Alamut and Shiroudi (susceptible, group 3) under water stress condition.The ratio was intermediate in Alvand, Niknejad Cross Adl and Roshan (intermediate tolerance, group 2).From Figure1, we observed that genotypes in group1, group 2 and group 3 had the highest, intermediate and lowest yield stability index (YSI), respectively.Table3.Changes in enzymatic antioxidant (catalase, CAT; superoxide dismutase, SOD; peroxidase, POD and ascorbate peroxidase, APX) activity and non-enzymatic antioxidant (Proline and carotenoids, Car) content in wheat genotypes in response to drought stress.Means of three replicates followed by the same letter in each column and two columns (non-stress and drought stress) related to same indicator are not significantly different according to Tukey's test (probability level of %5).