Abstract: Seedling of two sorghum genotypes (Payam and Sistan) and four wheat genotypes (Bolani, Hirman, Star and Toss) were grown in Hoagland nutrient solution containing 0, 100 and 200 mM NaCl in controlled environment. Antioxidant activities like catalase (CAT), ascobrate peroxidase (APX) and guaiacol peroxidase (GPX) and osmolyte concentrations, proline and soluble carbohydrates were determined in the leaves 20 days after induction of salinity stress. Results showed that the activity of APX, GPX and CAT increased in both sorghum genotypes. Wheat genotypes showed significant differences during the experimental period. By increasing NaCl levels from 0 to 200 mM the activity of APX and GPX decreased, but the activity of CAT increased in all wheat genotypes. At the 100 mM NaCl, the CAT activity in wheat genotypes was higher compared with that in 200 mM NaCl. The increase in salinity stress increased total soluble carbohydrates and proline both in wheat and sorghum genotypes. Results in this study showed sorghum genotype displayed better osmotic adjustment and antioxidant compounds under salt stress and the efficiency of Sistan was better than Payam. Contrarily in wheat, osmotic adjustment (carbohydrate and proline accumulation) was much more effective than antioxidant enzyme activity.
INTRODUCTION
Plant salt tolerance has been generally studied in relation to regulatory mechanisms of ionic and osmotic homeostasis (Ashraf and Harris, 2004). In addition to ionic and osmotic componenrs, salt stress, like other abiotic stress, also leads to oxidative stress through an increase in Reactive Oxygen Species (ROS), such as superoxide (O2−), hydrogen peroxide (H2O2) and hydroxyl radicals (OH·) (Mittler, 2002).
Among the abiotic stress, salt stress causes major problems in cereals like wheat. It has been currently reported that most abiotic stress including NaCl salt stress impose injury in plants by osmotic stress, ionic stress and generating reactive oxygen species (Shalata and Tal, 1998).
During oxidative stress, the excess production of Reactive Oxygen Species (ROS) causes membrane damage that eventually leads to cell death. For protection against ROS, plants contain antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and Glutathione Reductase (GR) or as well as a wide array of non-enzymatic antioxidants (Blokhina et al., 2003). SOD is the major O2− scavenger and its enzymatic action results in H2O2 and O2 formation. The H2O2 produced is then scavenged by CAT and several classes of peroxidases. CAT, which is found in peroxisomes, cytosol and mitochondria, dismutates H2O2 to H2O and O2 (McKersie and Leshem, 1994).
Salt stress imposes osmotic stress, by limiting absorption of water from soil and ionic stress, resulting from high concentrations of potentially toxic salt ions within plant cells. A variety of protective mechanisms have evolved in plants to allow them to acclimatize to these unfavourable environmental conditions for survival and growth. Although, the effects of salinity are studied in variety of glycophytic and halophytic plants, the mechanisms of salt tolerance are not well understood (Hasegawa et al., 2000).
The synthesis and accumulation of low molecular weight metabolites, known as compatible solutes, is a ubiquitous mechanism for osmotic adjustment in plants. Their main role is to increase the ability of cells to retain water without affecting the normal metabolism (Hamilton and Heckarthorn, 2001). Amino acids, sugars, betaines and proline compounds may accumulate, as compatible solutes, in many plant species (Serrano, 1996).
There are large differences in salt tolerance between crop species. One of the main reactions to salt or drought stress is closing of stomata. The C4 plants, in opposite to the C3, are able to utilize very low concentration of carbon dioxide which enables them to assimilate CO2 even during considerable stomatal closure (El Bassam, 1998). This might be one of the probable reason for the difference in resistance to stress between both plant groups. Photosynthesis is a complex process; therefore, it is possible that a number of elements in the C3 and the C4 may differ in resistance to salinity.
The objective of this study was to elucidate the role of some antioxidants and compatible osmolytes. On what basis was this tolerance dependent in four wheat and two sorghum genotypes.
MATERIALS AND METHODS
This study was conducted in a greenhouse at the University of Zabol, Iran during April-June 2007. The experiment was laid-out a completely randomized factorial design with three replicates. Seeds of four wheat genotypes (Bolani, Hirman, Star and Toss) and two sorghum genotypes (Payam and Sistan) were grown in the containers with Hoagland nutrient solution. Surface-sterilized seeds were germinated in the dark on sand, moistened with distrilled water. Seven days old seedling with uniform size were transferred to hydroponic culture in plastic container with 2 L of nutrient solution. Plants were grown under greenhouse conditions with a 12 h photoperiod of natural daylight, maximum and minimum temperatures were 26 and 18°C, respectively and relative humidity was 70% on average.
Three salinity treatments were imposed by adding S0 : 0 (control), S1 : 100 and S2 : 200 mM NaCl to the nutrient solution after 10 day old plants. Twenty days after salt treatment, the plants were harvested. The extracts of mature leaf blades were used to determine soluble carbohydrates (Hendrix, 1993) and proline (Bates et al., 1973) in both crop species.
For free proline content, leaf samples were homogenized in 5 mL of sulphosalycylic acid (3%) using mortar and pestle. A bout 2 mL of extract was taken in test tube and pestle. About 2 mL of extract was taken in test tube and to it 2 mL of glacia acetic acid and 2 mL of ninhydrin reagent were added. The reaction mixture was boiled in water bath at 100°C for 30 min. After cooling the reaction mixture, 6 mL of toluene was added and then transferred to a separating funnel. After thorough mixing, the chromophore containing toluene was separated and absorbace read at 520 nm in spectrophotometer against toluene blank.
Enzyme assays
Ascorbate peroxidase: The enzyme was extracted in 50 mM phosphate buffer
(pH = 7). The activity of ascorbate peroxidase (APX EC 1.11.1.11) was measured
using method of Nakano and Asada (1981).
The APX activity was determined according to Nakano and Asada (1981). The reaction mixture consisted of 50 mM sodium phosphate buffer (pH = 7) containing 0.2 mM EDTA, 0.5 mM ascorbic acid (sigms), 50 mg of BSA (Sigma) and crude enzyme extract. The reaction was started by additin of H2O2 at final concentration of 0.1 mM. Oxidation of ascorbic acid as a decrease in absorbance at 290 nm was followed 2 min after starting the reaction. The difference in absorbance was divided by the ascorbate molar extinction coefficient (2.8 mM-1 cm-1) and the enzyme activity expressed as μmol of H2O2 min-1 mg-1 protein, taking into consideration that 1.0 mol of ascorbate is reqired for the reduction of 1.0 mol of H2O2 (McKersie and Leshem, 1994).
Catalase: Catalase (CAT, EC 1.11.1.6) activity was assayed spectrophotometrically by monitoring the decrease in absrobace of H2O2 at 240 nm. CAT was measured according to the method of Beers et al. (1952). The enzme was extracted in 50 mM phosphate buffer (pH = 7). The assay solution contained 50 mM phosphate buffer and 10 mM H2O2. The reaction was started by addition of enzyme aliquote to the reaction mixture and the change in absorbance was followed 2 min after startingthe reaction. Unite activity was taken as the amont of enzyme, which decomposes 1 M of H2O2 in 1 min.
Guaiacol peroxidase: Total GPX (EC 1.11.1.7) activity was determinde as described by Urbanek et al. (1991) in a reaction mixture (0.2 mL) containing 100 mM phosphate buffer (pH 7.0), 0.1 μM EDTA, 5.0 mM guaiacol, 15 mM H2O2 and 50 μL enzyme extract. The addition of enzyme extract started the reaction and the increase in absorbance was recorded at 470 nm for 1 min. Enzyme activity was quantified by the amount of tetraguaiacol formed using its molar extinction coefficient (26.6 mM-1 cm-1).
Statistical analysis: All data were analyzed with SAS Institute Inc., 6.12. All data were first analyzed by ANOVA to determine significant (p≥0.05) treatment effects. Significant differences between individual means were determined using Fisher’s protected Least Significant Difference (LSD) test. Data points in the figures represent the means±SE of three independent experiments at least three replications per cultivar per treatment combination each.
RESULTS
Enzyme activity: APX activity in leaves for control and salt stressed
plants changed in genotypes of both wheat and sorghum crop species. The activity
of APX increased remarkably in two sorghum genotypes as a result of salt stress.
| Fig. 1: | Changes in the APX content in the leaves of sorghum (S) and wheat (W) genotypes subjected to increasing salinity |
| Fig. 2: | Changes in the GPX contents in the leaves of sorghum (S) and wheat (W) genotypes subjected to increasing salinity |
The activity of APX in Sistan was higher than Payam gentotype. In all of wheat genotypes except Bolani, APX significantly reduced in leaves. The activity of APX was significantly lower in wheat than in the sorghum genotypes (Fig. 1).
The assay for GPX in different salinity levels showed that wheat genotypes
represented significantly lower GPX activity than sorghum genotypes (Fig.
2). In wheat genotypes GPX activity decreased, but in sorghum it increased
when under salt stress. Figure 2 shows that the GPX activity
increased with increasing salinity in sorghum genotypes (Payam and Sistan).
The activity of two enzymes, APX and GPX increased 56.2 and 23.4%, respectively
at 200 mM NaCl compared to control in Siatan genotype. While, the increase of
APX and GPX activity in payam genotype was 51.2 and 54.1%, respectively.
| Fig. 3: | Changes in the CAT contents in the leaves of sorghum (S) and wheat (W) genotypes subjected to increasing salinity |
| Fig. 4: | Changes in the soluble carbohydrate content in the leaves of sorghum (S) and wheat (W) genotypes subjected to increasing salinity |
The activity of CAT was significantly lower in sorghum than wheat genotypes (Fig. 3). Increase in salinity levels significantly increased catalase activity in sorghum genotypes (79.1% in Sistan and 72.6% in Payam). The increasing CAT activity in wheat genotypes were observed only until 100 mM NaCl, after that by increasing salinity level to 200 mM NaCl, its activity decreased (Fig. 3). Wheat genotypes subjected to salinity treatment at the 100 mM NaCl, had the highest CAT activity (Hirman 35.2% and Toss 55.8%, Fig. 3).
Osmolyte concentration: Total soluble carbohydrate increased in all
wheat (Bolani and Toss genotypes, at the 100 mM NaCl salinity treatment, had
the highest carbohydrate) and sorghum genotypes with the increasing salinity
(Fig. 4). Significant differences were observed between the
genotypes of sorghum and wheat for total soluble carbohydrate. Sistan genotype
in sorghum and Bolani and Toss genotypes in wheat had the highest carbohydrate
concentration when under salt stress.
| Fig. 5: | Changes in the proline content in the leaves of sorghum (S) and wheat (W) genotypes subjected to increasing salinity |
In all genotypes of sorghum and wheat, salinity stress stimulated proline accumulation (Fig. 5). Significant differences in proline accumulation were revealed under stress conditions (p<0.01). Payam genotype in sorghum and Toss genotype in wheat had the highest proline content. In this study, we did not find a close relationship between the accumulation of proline and carbohydrate in leaves (R2 = 0.103).
DISCUSSION
In this study, among the antioxidative enzymes, activity of APX, GPX and CAT increased in both sorghum genotypes, but Sistan genotype had the higher enzyme activity than Payam genotype. The activity of these three enzymes increased in tolerant genotype (Sistan), which is similar to the results of Gossett et al. (1993). To tolerant high levels of salts, plants can adopt different strategies. Salinity stress induces Reactive Oxygen Species (ROS) production and lead to oxidative damages. The ROS may react with macromolecules, the proteins and lipid components of membranes causing damage through lipid peroxidation resulting in increased permeability of the membrane. The antioxidant defense system of the plant comprises a variety of antioxidant molecules and enzymes (Arora et al., 2002). Wheat genotypes had different behavior during the experimental period. With increased salinity levels, the activity of APX and GPX decreased but that of CAT increased. At the 100 mM NaCl, the CAT activity in wheat genotypes was higher than that at 200 mM NaCl. Among the wheat genotypes, Toss and Hirman showed the highest CAT activity (Fig. 3). The higher CAT activity indicates that salt tolerant lines had better ability to scavenge H2O2 (Gossett et al., 1994). Willekense et al. (1995) reported that the intracellular levels of H2O2 is regulated by a wide range of enzymes. The CAT functions through an intermediate CAT-H2O2 complex and produces water and dioxygen. Ascorbate (APX) in addition to its role in the scavenging cycle, acts as areductant in the regeneration of α-tocopherol and in the zeaxanthin cycle.
In this study, we found a positive relationship between antioxidant activity and osmolyte concentration, carbohydrates (R2 = 0.33) and praline (R2 = 0.43), in sorghum genotypes. Carbohydrate concentration, beside its role in decreasing water potential, contributes in preventing oxidative damage and maintaining the structure of proteins and membranes under moderate dehydration during drought period (Hoekstra et al., 2001). Carbohydrate also serve as signaling molecules for sugar-responsive genes which leading to different physiological responses like defense responses and turgor-driven cell expansion (Sturm and Tang, 1999).
In this study, the activity antioxidant enzymes and osmolyte concentration increased in both sorghum genotypes. The general comparison of the examined antioxidants in wheat and sorghum genotypes revealed that in the salinity treatments, the salt tolerance is due, at least partially, to the higher constitutive antioxidant enzymes actives and increasing of osmolyte concentrations in the leaves. Between two kinds of plant, sorghum (C4) and wheat (C3), we found which, sorghum for salinity tolerance are use of two mechanisms (antioxidant activity and osmolyte concentration), but in wheat genotypes (C3) the mechanism of osmotic adjustment was acted better than antixodant mechanism. In sorghum genotypes Sistan was better and had the highest antixodant activity and osmolyte concentration.
CONCLUSION
In addition to role of compatible solutes in osmotic adjustment, antioxidant enzymes also play a role in salt tolerance. Sorghum displayed better osmotic adjustment and antioxidant compounds under salt stress and the efficiency of Sistan was better than Payam. Contrarily in wheat, osmotic adjustment (carbohydrate and proline accumulation) was much more effective than antioxidant enzyme activity. Among wheat genotypes, Toss showed greater carbohydrate and proline content and CAT activity.
ACKNOWLEDGMENT
The author thank Dr. Kafi, Faculty of Crop Physiology for his valuable suggestions during the course of investigation.