Salt Pretreatment Enhance Salt Tolerance in Zea mays L. Seedlings

MATERIALS AND METHODS

This experiment has been done in spring of 2006 at the physiological and biochemical laboratories, in the Department of Biology of Faculty of Science of Urmia University. The seeds of Zea mays L. (cv. single cross 704) were cleaned, selected by the size, washed with water and detergent and finally with distilled water and incubated in 25°C to germinate. After 4 days the seedlings with the same size were selected again and 7 seedlings (as a replicate) were pretreated with 50 mM NaCl for 20 h.

Then they were transferred to 200 and 300 mM NaCl for 48 h in an aerated and controlled condition (light density 14000 Lux and day/night temperature 24/20°C and humidity 60%). At the end of treatment, the shoots and roots of seedlings were harvested separately. To show the effect of salt pretreatment and salt stress on the plants, the changes of K⁺-leakage, malondialdehyde content, proline, soluble sugars and the Hill reaction rate were determined.

Measurement of K⁺-leakage: To do this experiment, 4 day old seedlings of Zea mays were pretreated with 50 mM NaCl for 20 h. Then they were transferred to 200 and 300 mM NaCl for 48 h (at above mentioned condition). At the end of treatment the seedlings were harvested and the amount of K⁺-leakage in growth solution of each replicate was determined by flame photometer (model 405 made by Fater Electronic com.) (Chen et al., 2005).

Changes of lipid peroxidation: Lipid peroxidation was determined by measuring the amount of MDA produced by the thiobarbituric acid reaction as described by Heath and Packer (1968). The crude extract was mixed with the same volume of a 0. 5% (w/v) thiobarbituric acid solution containing 20% (w/v) trichloroacetic acid. The mixture was heated at 95°C for 30 min and then quickly cooled in an ice-bath. The mixture was centrifuged at 3000 g for 10 min and the absorbance of the supernatant was monitored at 532 and 600 nm. After subtracting the non- specific absorbance (600 nm), the MDA concentration was determined by its molar extinction coefficient (155 mM^-1 cm^-1) and the results expressed as MDA. g^-1 FW.

Measurement of proline content: To show the changes of proline, the roots and shoots of seedling that were pretreated and treated as above mentioned condition, were dried separately at 60°C for three days. 0.04 g of every sample was grinded in glass powder. The proline content was determined by the method of Bates et al. (1973) using 3% aqueous sulphosalicylic acid for preparing the leaf homogenate. The homogenate was centrifuged at 1000 rpm for 15 min. Two milliliter of the supernatant was mixed with an equal volume of glacial acetic acid and acid ninhydrin and incubated for 1 h at 100°C. Then 4 mL of toluene was added to mixture tube were put in ice and the chromatophore containing fraction was then aspirated from the aqueous phase and its absorbance was determined at 520 nm on a spectrophotometer.

Measurement of soluble sugars: Ten milliliter of 80% ethanol was added to 0.01 g of the powdered root and shoot of each sample to extract the soluble sugars. After one week the amount of soluble sugars was determined by the method of Dubois et al. (1956). Two milliliter of the supernatant of aliquot was mixed with 1 mL of 5% phenol and 5 mL of sulfuric acid. The mixtures incubated for 30 min, after cooling the changes in absorbance were estimated at 485 nm on a spectrophotometer.

Assay of hill reaction rate: To determine the rate of Hill reaction, the shoots of Zea mays seedlings prepared at the same condition were harvested. Leaf spieces were placed in a prechilled blender cup containing 10 mL of ice-cold 0. 5 M sucrose. Then was blended for 15 sec at top speed, the resulting homogenate was squeezed through four layers of prechilled cheesecloth into a prechilled beaker. Equal amounts of this green filtrate were centrifuged at 200 g for 3 min. The supernatant was decanted into additional cold centrifuge tubes and centrifuged again at 1000 g for 7 min. The supernatant was discarded and the chloroplasts precipitates were resuspended immediately in 10 mL of cold 0.5 M sucrose. This process repeated again. At last, it was resuspended in 25 mL of cold 0.1 M phosphate buffer at pH 6.5. 0.5 mL of the chloroplast suspension was placed in a spectrophotometric cuvette and mixed with 0.2 mL of DCPIP (electron acceptor) prepared in extraction buffer; then the absorbance of this solution was determined at 600 nm and repeated every 20 sec after the solution were illuminated with a 100-watt incandescent lamp Choudhury and Biswal (1980).

RESULTS AND DISCUSSION

K⁺-leakage: Analysis of the results has indicated that the K⁺-leakage has been increased significantly by NaCl treatments, its amount in plants treated with 300 mM NaCl was 9.3 times higher than the controls and 2.16 times higher than the plants pretreated with 50 mM NaCl and treated with 300 mM NaCl (Fig. 1). K⁺ is one of the most important and abundant cation in plant cells. There is also anegative correlation between magnitude of K⁺ efflux and membrane stability and salt tolerance of a particular cultivar (Chen et al. 2005; Kronzucker and Szczerba, 2003). Therefore we conclude that the salt stress has caused membrane unstability and this effect has also been confirmed by producing of induced level of MDA in this research.

Lipid peroxidation: The MDA content in the shoots of seedlings was measured as index of the rate of lipid peroxidation. Its amount has been increased significantly in plants treated with 300 mM NaCl (2.9 times higher than the plants pretreated with 50 mM NaCl and treated with 300 mM NaCl and 3.3 times higher than the controls) (Fig. 2). Therefore, these results indicated that salt stress has caused an extensive lipid peroxidation, which has often been used as an indicator of salt induced oxidative damage in membranes (Hernandez and Almansa, 2002). MDA levels have decreased in pretreated plants, reduction of MDA in these plants was probably due to increased antioxidative enzymes activities, which has reduced H₂O₂ levels and membrane damages (Shalata et al., 2001). MDA as a lipid peroxidation product, has been used widely to assess the levels of free radicals in living cells (Hong et al. 2000).

Fig. 1:	The changes of K+-leakage of Zea mays seedlings roots exposed to different concentrations of NaCl (200 and 300 mM) with and with out salt pretreatment (50 mM NaCl) during the period of 48 h. The values represent the mean of three replicates ’ SE

Fig. 2:	The changes of total malondialdehyd of Zea mays seedlings shoots exposed to different concentrations of NaCl (200 and 300 mM) with and with out salt pretreatment (50 mM NaCl) during the period of 48 h. The values represent the mean of three replicates ’ SE

Therefore in this research pretreated plants has been better protected from oxidative damage under salt stress.

Proline content: Proline content in the shoots has been increased in pretreated plants with 50 mM NaCl and treated with 300 mM NaCl (it was 3.3 times higher than control and 2.8 times higher than plants treated only with 300 mM NaCl), also its content in roots pretreated with 50 mM NaCl and treated with 300 mM NaCl was the highest (it was 1.6 times higher than control and 1.4 times higher than plants treated only with 300 mM NaCl). Content of proline in the shoots of seedlings was higher than the roots (Fig. 3). Proline is known to play an important role as an osmoprotectant in plants subjected to hyperosmotic stresses such as drought and soil salinity (Hong et al., 2000; Arshi et al., 2005).

Fig. 3:	The changes of proline content of Zea mays seedlings roots and shoots exposed to different concentration of NaCl (200 and 300 mM) with and with out salt pretreatment (50 mM NaCl) during the period of 48 h. The values represent the mean of three replicates ’ SE

Fig. 4:	The changes of soluble sugars of Zea mays seedlings roots and shoots exposed to different concentrations of NaCl (200 and 300 mM) with and with out salt pretreatment (50 mM NaCl) during the period of 48 h. The values represent the mean of three replicates ’ SE

Stewart et al. (2004) suggested that the capacity to accumulate proline is correlated with salt tolerance. Hong et al. (2000) suggested that increased resistance to oxidative stress is due to some indirect metabolic or physiological consequence of the accumulation of proline and other metabolites. Accumulation of proline in shoots and roots of pretreated plants increased (Fig. 3). Therefore in these plants proline acts as a free radical scavengering and increases salt tolerance of these plants.

Soluble sugars: Soluble sugars content in the shoots of plants have been increased by the salt pretreatment; its amount in shoots of plants pretreated with 50 mM NaCl and treated with 300 mM NaCl was 2 times higher than the shoots of plants only were treated with 300 mM NaCl and 3 times higher than the controls (Fig. 4). The amount of this component in the roots pretreated with 50 mM NaCl and treated with 300 mM NaCl was the highest (it was 2 times higher than control and 1.5 times higher than plants treated only with 300 mM NaCl).

Fig. 5:	Effects of different concentration of NaCl (200 and 300 mM) with and with out salt pretreatment (50 mM NaCl) on Hill reaction rate on Zea may seedlings leaves during the period of 48 h. The values represent the of three replicates ’ SE mean

Content of soluble sugar in the shoots of seedlings was higher than the roots (Fig. 4). Carbohydrate changes has direct relationship with such physiological processes as photosynthesis, translocation and respiration. Adaptation to salt and water stress has been attributed to the stress-induced increase in carbohydrate levels (Ashraf and Garris, 2004; Naeini et al., 2004). Soluble sugars play an important role in osmotic regulation of cells during germination and strees conditions (Gill et al. 2002).

Rate of Hill reaction: Rate of Hill reaction in plants treated with 300 mM NaCl and 200 mM NaCl was, respectively 4.2 and 3.4 times lower than control (Fig. 5). Reduced rate of Hill reaction was because of salt stress damages on photosynthetic photosystems and oxygen evolving complex (Allakhverdiev et al., 2000). Salt stress imposes a water deficit because of osmotic effects on a wide variety of metabolic activities (Parida and Das, 2005). Krieger-Liszkay (2004) proposed that the active oxygen forms destroy the proteins, lipids and important cell components which can secondarily lead to damage in PSII machinery too. Hence, it can expected that Hill reaction is affected by salt stress indirectly. Hill reaction in Zea mays seedlings decreased by 200 and 300 mM NaCl (Fig. 5). This in turn decreases reduction of NADP⁺and phosphorylation of ADP, which will result in a strong inhibition of CO₂ assimilation.

CONCLUSION

We concluded that in pretreated plants, K⁺-leakage and the amount of MDA was decreased in comparison to unpretreated-stressed plants. In contrast amount of proline, soluble sugars and Hill reaction rate was increased. These results indicated the induced tolerance of pretreated plants to salt stress. A better understanding of these mechanisms under salt stress may help us to improve salt tolerance of crop varieties by gradually adaptation of them to saline soils.