Plant exposed to salinity stress exhibit changes in their physiology and metabolism. In general salinity reduces water availability and causes nutritional imbalance in plants. Since there is no enough information about effect of salinity stress on activity of enzymes involved in nitrogen and phosphorous metabolism we studied the effects of different levels of salinity (0, 50, 100, 150 and 200 mM NaCl) on nitrate reductase, acid phosphatase and alkaline phosphatase activity as well as nitrate and phosphate uptake and total nitrogen content in leaves of canola, so a hydroponically experiment was conducted. Canola seeds were sown in an experimental green house under non-saline conditions. After two month, healthy plants were selected for hydroponic culture in Hoagland's nutrient solution supplemented with NaCl. The plants were cultured in plastic pots filled up with perlite and then were put in green house. The plants were watered with fresh nutrient solution made every week. Leaf samples were clipped 0, 7, 14, 21, 30 and 45 days after exposure to salinity stress in order to nitrate reductase, acid phosphatase and alkaline phosphatase assay, measurement of nitrate uptake, phosphate uptake and total nitrogen content. The results showed that nitrate reductase activity was increased in treated plants with 100 mM NaCl, in contrast it was decreased gradually in plants that were exposure to 150 and 200 mM NaCl, in comparison with controls. Decrease of nitrate reductase activity due to salinity stress and nitrate uptake and follow them total nitrogen content were occurred at the same time. Reduction in nitrate reductase activity, nitrate content and total nitrogen content because of high salinity levels may be a physiological response in order to decrease in growth and extra biomass in canola. However, it was observed that salinity stress caused an increase in both acid phosphatase and alkaline phosphatase activity. We monitored phosphate levels in leaves and found that phosphate levels decreased significantly under salinity stress. These results suggest that the induction of acid phosphatase and alkaline phosphatase under salt stress may be due to a phosphorous deficiency.
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Salinity is one of the major abiotic stresses affecting plant productivity. Salinity decreases plant dry matter and leaf area (Amirjani, 2011) and finally decreases crop yield. Also it has reported that salinity changes plant morphological traits (Zadeh and Naeini, 2007). Plants exposed to salt stress undergo changes in their metabolism with the changes taking place in their environment. In addition gene manipulation can improve salt stress tolerance by gene coding of related enzymes (Joseph and Jini, 2011). Nitrate reductase (NR, E.C.220.127.116.11), the first enzyme in the nitrate assimilation pathway, is a limiting factor of plant growth and development (Skriver and Mundy, 1990) also it is influenced by a variety of environmental factors (Solomonson and Barber, 1990) such as heavy metal like lead (Sengar et al., 2008). Many reports on the effects of salinity on nitrate reductase activity in different plants have frequently been contradictory. It has been reported that, NR activity increased with exogenous nitrate concentration (Jha et al., 2007). Bourgeais-Chaillou et al. (1992) reported that in-vitro conditions, NR activity in roots of soybean was higher than control plant as result of salt stress while leaf NR activity remained constant. Acid phosphatase (ACP, E.C 18.104.22.168) and alkaline phosphatase (ALP, E.C 22.214.171.124) are widely distributed in plant (Ourry et al., 1992). Alkaline phosphatase activity increases under conditions of salt, drought and osmotic stress. Hussain and Rai (1991) have been reported the depression of ALP activity under cadmium stress.
Athar et al. (2009) canola (Brassica napus L.) grown mainly for edible oil purpose is a moderately salt tolerant crop. Canola oil is the term given to the oil obtained from the seeds of several species of Brassica genus (Sakr and Arafa, 2009). In view of Bybordi et al. (2010a, b) increasing awareness of the health advantage of canola oil and existing salt tolerance potential, its demand has undoubtedly increased during the last two decades.
This has resulted into increased cultivation of canola on soils where salinity problems already exist. Thus, there is a need for further improvement in the salt tolerance of canola. Bybordi et al. (2010c) during salt stress some plants may have a better protection against reactive oxygen species, at least in part, by increasing the activity of antioxidant enzymes under salt stress.
In this study, we made an attempt to investigate the effect of NaCl stress on nitrate reductase activity and some physiological and other related enzymes in nitrogen and phosphorous metabolism in canola plants.
MATERIALS AND METHODS
In order to study the effects of salt stress on nitrogen and phosphorous metabolism in canola (Brassica napus L. CV SLM046) an experiment was conducted in Baku State University, Faculty of Biology in 2009. Seedlings were raised in an experimental green house under non-saline conditions. After two month, healthy and uniform plants were selected for hydroponic culture in Hoagland's nutrient solution supplemented with NaCl (0, 50, 100, 150 and 200 mM). The plants were cultured in plastic pots filled up with perlite and then were put in green house. The greenhouse was under natural sunlight and temperature was set up to 25°C 3 and 75% relative humidity. The plants were watered with fresh nutrient solution made every week. The pair of leaves from the apex of the growing shoots was harvested 0, 7, 14, 21, 30 and 45 days after exposure to salinity stress in order to nitrate reductase, acid phosphatase and alkaline phosphatase assay, measurement of nitrate uptake, phosphate uptake and total nitrogen content. During each sampling, a new set of plants was used. Nitrate reductase was assayed based on the methods of Sagi et al. (1997). Protein concentration in the enzyme extract was determined following the procedure of Bradford (1976) using Bovine Serum Albumin (BSA) as a standard. Total nitrogen contents in leaves of control and stressed plants were determined following the colorimetric procedure of Baethgen and Alley (1989).
Assay of acid phosphatase: Activity of ACP (EC, 126.96.36.199) was determined as described by Joyce and Grisolia (1960).
Assay of alkaline phosphatase: The activity of ALP (E.C.188.8.131.52) was determined following the procedure of Ihlenfeldt and Gibson (1975).
One unit of ACP or ALP is the number of enzyme required to liberate 1 μ Mole of P-nitro phenol per min. Activity staining of an acid phosphatase native polyaeryl amide gel electrophoresis was performed following the method of Laemmli (1970).
Measurement of phosphate level: Phosphate uptake in control and salinity stressed leaves was measured as method of Ames method (Ames, 1966).
Statistical analysis: Statistical analysis of the results was carried out according to Duncan's Multiple Range Tests. Data were subjected to a two-way ANOVA following the method of Sokal and Rohlf (1995).
RESULTS AND DISCUSSION
Nitrate reductase activity was increased significantly in those plants were subjected to 100 mM NaCl, while it was decreased gradually under 150 and 200 mM treatments compared to control treatment (Table 1). After 45 days of exposure, NR activity decreased in 150 and 200 mM NaCl treated plants, respectively, as compared to control treatment. Decreasing of NR activity due to salinity stress was also accompanied with significant decrease in nitrate uptake and total nitrogen content. According to Table 1, both nitrate and total nitrogen content as well as NR activity were increased because of 100 mM salinity stress in treated plants and decreased in 150 and 200 mM treatments in comparison with control treatment, moreover, total nitrogen content dramatically increased in 100 mM treatment and decreased in 150 and 200 mM, respectively, in comparison with control during the 45 days period of experiment (Table 1). Similarly, in comparison with control treatment, nitrate content increased in treated plants with 50 mM and decreased in treated plants with 150 and 200 mM NaCl, respectively (Table 1). Effects of NaCl on ACP and ALP activity and phosphate uptake in canola leaves through the duration of treatment are shown in Table 2. Forty five days after salinity induction, NaCl caused a significant increase in ACP activity in treated plants with 150 and 200 mM NaCl in comparison with control treatment. Its worth mentioning that, there were no significant changes in ACP activity in treated plants with 50 and 100 mM NaCl. In contrast, ALP activity was increased in treated plant with 50, 100, 150 and 200 mM NaCl in comparison with control (Table 2).
The results in Table 1 show a concentration-dependant decrease in NR activity, nitrate and nitrogen content. Lacuesta et al. (1990) discussed that as NR is a substrate inducible enzyme, its decreased by some researchers to decreased nitrate uptake by plant under salt stress. The decrease of nitrate uptake is accompanied with a high Cl¯ uptake and translocation to the shoot (Flores et al., 2000). Since presence of Cl- into the soil inhibits uptake, NR activity would be affected by flux into the metabolic pools (Abd-El-Baki et al., 2000).
|Table 1:||Effects of different levels of salinity stress on NR activity, nitrate uptake and total nitrogen levels in leaves of canola as a function of days of NaCl treatment|
|Values in columns with the same letter are no significantly different p<0.05|
We observed that there was no significant differences between the two cases on NR activity; these observations suggest that decreasing of NR activity is not due to direct inhibitory of salinity stress and it may be due to a reduction in nitrate uptake in these plants.
Flores et al. (2000) reported that salinity inhibits nitrate uptake and NR activity in leaves of Zea mays. Higher levels of total nitrogen in those plants which were treated with 50 mM may be a natural response for favorable growth of the plants at this concentration, similar these results have been reported earlier. The results appeared in Table 2 show an increase in both ACP and ALP activity due to increase of salinity level also a noticeable change in phosphorus level was observed in stressed plants. Abd-El-Baki et al. (2000) has been reported that activity of ACP and ALP increased in plants were grown under conditions of phosphorus deficiency.
|Table 2:||The Effects of different salinity levels on activity of ACP, ALP, and phosphate levels in leaves of canola measured as a function of days of NaCl treatment|
|Values in columns with the same letter are no significantly different p<0.05|
In conclusion, present results suggest that high level of salinity causes a depression in NR activity, nitrate uptake and total nitrogen content in canola leaves. In addition, salinity leads to significant decrease in phosphate level, thus, salinity induces increase in activity of both ACP and ALP, and it is might be due to inhibition of phosphate uptake. In addition we conclude that uptake is inhibited by high salinity level in canola and also at low availability of nitrate, activity of NR will decrease. The low level of nitrogen in this plant under conditions of high level of salinity may affect on protect synthesis mechanism and then on growth and development (Cramer and Lips, 1995; Cram, 1973; Lefebvre et al., 1990).
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