ION AND MINERAL CONCENTRATIONS IN ROOTS AND LEAVES OF TWO GRAPEVINE CULTIVARS AS AFFECTED BY NITRIC OXIDE FOLIAR APPLICATION UNDER NACL STRESS

Methods and results : The plants were exposed to NaCl at the rate of 0, 25, 50, 75 and 100 mM in nutrient solution and foliar spray of SNP at 0, 0.5, 1 and 1.5 mM under an open hydroponic system. Results indicated that with increasing salinity levels, the Cland Na+ concentrations increased and the K+, Ca2+, Mg2+, NO3-N, Zn2+, Fe2+ concentrations and K+/Na+ ratio decreased in both cultivars. However, application of SNP mitigated the Cland Na+ concentrations and improved the K+, Ca2+, Mg2+ and NO3N concentrations in leaves and roots of both cultivars. The application of SNP did not significantly affect Zn2+ and Fe2+ concentrations under 100 mM NaCl.


INTRODUCTION
The total global area of salt-affected soils has recently been estimated to be approximately 830 million ha (Martinez-Beltran and Manzur, 2005).The salinity of soil and water sources is a serious threat worldwide and in many parts of our country, Iran.Estimated land area affected by salinity varies between 16 and 23 million ha (Siadat et al., 1997).Salinity decreases water potential and causes osmotic effects, specific ion toxicity and/or nutritional disorders (Läuchli and Grattan, 2007).Many reports have shown that salinity reduces the absorption of some nutrients in plants (Garcia and Charbaji, 1993 ;Rogers et al., 2003;Hu and Schmidhalter, 2005).
Grapevine (Vitis vinifera L.) is usually grown in semi-arid areas where drought and salinity are the most prevalent problems (Cramer et al., 2007).Grapevines are considered as moderately sensitive to salinity and the damage is primarily caused by Cl - ions (Fisarakis et al., 2001).It has been shown that the addition of NaCl to nutrient solution increases Na + ions in the vegetative organs of grapevine (Garcia and Charbaji, 1993).Physiological disturbances such as reduction in stomatal conductance and photosynthesis, reduction in both growth and vegetative biomass, and reduction in yield were reported in grapevine cultivars under saline conditions.In severe cases, salt stress symptoms develop into necrotic areas on leaves, starting at leaf margins and progressing inwards (Walker et al., 2004).
It has been documented that some bioregulators ameliorate the deleterious effects of environmental stresses on plants.Under environmental stress conditions (of both abiotic and biotic origins), elevated generation of nitric oxide (NO) occurs in various organs of the plant (Wu et al., 2011).NO is an endogenous signaling molecule in animals and plants, which mediates responses to abiotic and biotic stresses (Zhang et al., 2007).Research findings have indicated that application of exogenous NO increases tolerance of plants to salt, heavy metals, chilling and ultraviolet-B radiation (Tan et al., 2008).The involvement of NO in salt tolerance has drawn much attention in the past few years.The NO function in salt tolerance has been demonstrated in some plant species (Uchida et al., 2002).
Pretreatment with NO donor, SNP (sodium nitroprusside), protected young rice seedlings, resulting in better plant growth and viability (Uchida et al., 2002), promoted seed germination and root growth of yellow lupine seedlings (Kopyra and Gwozdz, 2003), and increased the growth and dry weight of maize seedlings (Zhang et al., 2007) under saline conditions.Zhao et al. (2004) in Phragmites communis and Zhang et al. (2007) in Populus euphratica reported that NO enhances salt tolerance of calluses under salinity through increasing K + /Na + ratio.Strong evidence that NO regulates cytosolic Ca 2+ homeostasis in plant cells was provided by Lamotte et al. (2006).
So, there is a strong potential for NO to reduce ion toxicity and improve mineral nutrient uptake by grapevine roots under salinity.However, there is no report on grapevine in relation to the effect of NO on plant responses in saline conditions.Therefore, the main objective of this research was the evaluation of the effects of SNP, as a NO donor, on mineral composition in two grapevine (Vitis vinifera L.) cultivars which differ in tolerance under salt stress.

Plant materials and growth conditions
Hardwood cuttings (with two nodes) of Vitis vinifera L. cvs.Qarah Shani and Thompson Seedless were collected (winter 2011) and planted in perlite to root.Well rooted cuttings were transplanted to pots filled with perlite and cocopeat (1:1 v/v) in an open hydroponic system.The pots were kept in a greenhouse with a photoperiod of 16:8, a relative humidity of 60±5 % and night and day temperatures of 19±3 and 27±3 °C, respectively.
At the beginning of the experiment, the plants were supplied with 200 mL nutrient solution three times a week.Each time, about 20 % of the nutrient solution was drained from the bottom of the pots.Culture medium was weekly leached by tap water to prevent ion accumulation.The nutrient solution was replaced weekly.All vines were trimmed to a single shoot and axillary buds were removed as they appeared.

NaCl application
Salt stress was implemented to the 100-day-old grapevine plants fertilized with ½ strength Hoagland nutrient solutions.Final concentrations were 25, 50, 75, and 100 mM of NaCl salinity.The control plants only received nutrient solution.The electrical conductivity (EC) of the salt-treated solutions at 25 °C was 1. 4, 3.6, 7.5, and 11.8 ds/m, respectively.To avoid salinity shock, NaCl was gradually added to the nutrient solutions at the rate of 25 mM per day to reach the final salinity level.The trial lasted for seven weeks.

NO foliar spray
SNP (Na 2 [Fe(CN) 5 NO]), as a NO donor, was applied to the foliage at the rate of 0, 0.5, 1 and 1.5 mM.A control group of plants was grown without NaCl and sprayed with deionized water.Tween-20 (0.1 %) was added to all solutions as surfactant.The plants were sprayed with SNP solutions at two-week intervals during the experimental period.

Mineral content in leaves and roots
At the end of the experiment, mature leaves on mid stem and fibrous roots were sampled and rinsed with deionized water, then oven-dried at 70°C for 48 hours.The dried tissues were analyzed for their Na + , Cl -, NO 3 -N, K + , Ca 2+ , Mg 2+ , Zn 2+ , and Fe 2+ contents.
The Na + and K + contents of tissues were analyzed by flame photometry (Fater 405 model, Iran), the Cl - content was analyzed by chloride analyzer (Model 926, Sherwood Scientific Ltd.), and nitrate (NO 3 -N) was colorimetrically analyzed by nitration of salicylic acid (Cataldo et al., 1975).Another group of samples (leaves and roots) was used to determine Ca 2+ , Mg 2+ , Fe 2+ , and Zn 2+ concentrations.Dried samples (0.4 g) were ground and ashed at 550 °C in a porcelain crucible for 5 hours, separately.The white ash was digested in 10 mL HCl (2M), filtered into a 50-mL volumetric flask, and brought to a final volume of 50 mL with distilled water.The concentration of Ca 2+ , Mg 2+ , Fe 2+ , and Zn 2+ was determined by an atomic absorption spectrophotometer (Shimadzu AA-6300, Japan).

Statistical analysis
The experiment was a CRD-based factorial with four replicates.Data were analyzed by SAS 9.1 Statistical Software ( 2002) and means were compared using Duncan's Multiple Range Test at P < 0.01.

Na + and Cl -concentrations
The presence of NaCl, especially at higher level, resulted in an increase in total Na + content in the leaves and roots of both Thompson Seedless and Qarah Shani cultivars ; however, in the leaves of Qarah Shani Na + content was 10.5 % higher than in the leaves of Thompson Seedless.Foliar application of SNP significantly decreased Na + concentration, as compared to salt-exposed plants, in both leaves and roots (Figure 1).
At the end of the salinity stress period, leaf and root Cl -concentration increased with increasing NaCl concentration in nutrient solution.In the presence of 100 mM NaCl in the medium (without SNP), Cl - concentration in the leaves of Thompson Seedless and QarahShani was almost 8.4-and 4.6-fold higher, respectively, compared to the control (without NaCl) and in the roots almost 4-and 6-fold higher, respectively, compared to control plants.The application of SNP mitigated Cl -concentration in roots of both cultivars compared to salt stressexposed plants without SNP (Figure 2).

Ca 2+ and Mg 2+ concentrations
NaCl treatments decreased Ca 2+ concentration in the leaves and roots of both cultivars.In the 100 mM NaCl treatment, Ca 2+ content in the leaves of both cultivars was not significantly different, but was 39 % higher in the roots of Qarah Shani compared to Thompson Seedless.Application of SNP increased Ca 2+ concentration in the roots compared to salinity treatments without SNP application (Table 1).
Salinity significantly decreased Mg 2+ concentration in the leaves of Thompson Seedless but not in leaves of Qarah Shani.The application of SNP did not significantly change Mg 2+ concentration in the leaves of Qarah Shani but increased Mg 2+ concentration in the leaves of Thompson Seedless, compared to NaClexposed plants, when salinity in nutrient solution increased (Table 1).

K + and NO 3 -N concentrations
K + concentration decreased markedly when NaCl concentration increased ; this decrement was more pronounced in the roots than in the leaves.In the 100 mM NaCl treatment, K + content in the leaves of Qarah Shani and Thompson Seedless decreased by 71.4 and 70.1 % when compared to control plants, respectively.The application of SNP resulted in an increase of K + content compared to control in saline conditions (Figure 3).NO 3 -N concentrations in the roots and leaves decreased considerably when the NaCl concentration in the nutrient solution increased.The application of SNP substantially increased the content of NO 3 -N in the salt-treated leaves and roots of both cultivars, when compared to salt-treated plants without SNP (Figure 4).

Fe 2+ and Zn 2+ concentrations
Fe 2+ content in the leaves and roots of NaCl-exposed plants was significantly decreased in both cultivars.In the 100 mM NaCl treatment, Fe 2+ content in the leaves of Thompson Seedless and Qarah Shani was almost 2.3-and 2.6-fold lower compared to control (without NaCl) plants, respectively ; however, this amount was almost 3-fold lower in the roots of both cultivars in comparison with control plants.
concentration of roots was significantly decreased from 172.93 to 56.83 mg/kg DW in Thompson Seedless and from 179.13 to 61.27 mg/kg DW in Qarah Shani with increasing salinity (Table 1).
Salt treatment significantly decreased Zn 2+ content in the leaves and roots when compared to control plants.The decrease in Zn 2+ concentration was alleviated by the application of SNP under low levels of salinity (25 and 50 mM).SNP application did not significantly affect Zn 2+ content in the leaves and roots under the 75 and 100 mM NaCl treatments (Table 1).

DISCUSSION
The present study focused on the possible role of NO in improving tolerance of two grapevine cultivars, Thompson Seedless and Qarah Shani, to salt stress.We assessed the effects of NO on counteracting Na + and Cl -toxicity on mineral nutrition absorption.To date, there are limited reports about NO influences on nutrient absorption under salinity stress.The findings of this research showed that Na + content increased in leaves and roots of both cultivars as NaCl level increased.The Na + concentration in the leaves of Qarah Shani, especially in salinity levels higher than 50 mM, was higher than in Thompson Seedless.
Our results are consistent with previous studies conducted on salt stress in grapevine (Stevens et al., 1996;Singh et al., 2000;Fisarakis et al., 2005) and pistachio (Picchioni et al., 1991).The difference in Na + content in roots showed that the roots have limited ability for Na + accumulation and, thus, Na + is most probably transported to aerial parts.It is reported that Na + is initially retained in the roots of woody plants and then transported to the leaves, causing leaf burn (Tester and Davenport, 2003).
The similarity of the hydrated ionic radii of Na + and K + makes it difficult for cell membrane transport system to discriminate between these two ions and it seems that this is the basis of Na + toxicity under high salinity (Blumwald, 2000).Na + ions can be transported into cells by K + transporters (Parida and Das, 2005).This factor could be the reason for the toxicity of Na + ions, especially under salinity levels higher than 50 mM, in both grapevine cultivars.In grapevine, it has been indicated that Na + and K + ions show strong competition even with addition of small amounts of NaCl in nutrient solution (Troncoso et al., 1999).
-  Our results indicate that SNP application could reduce the absorption of Na + ions in both leaves and roots, especially in salinity levels less than 50 mM.There are few reports about the effect of NO on decreasing Na + ion uptake under salinity stress.Zhang et al. (2004) reported that both NO and NaCl treatments stimulated vacuolar H + -ATPase and H + -PPase activities, resulting in increased H + translocation and Na + /H + exchange.NaCl-induced H + -ATPase and H + -PPase activities were diminished by NO scavenger MB-1 (Zhang et al., 2007).
The higher Cl -concentrations in petioles and laminas reflect the poor capacity of Vitis vinifera L. vines for Cl -exclusion (Downton, 1977).The concentration of Cl -in leaves of salinized grapevines was higher than in roots.The lower Cl -concentrations in leaves of Qarah Shani compared to Thompson Seedless at high salinity indicate a greater ability of Qarah Shani to restrict uptake and/or root-to-shoot transport of Cl - and a possible dilution effect due to distribution of accumulated Cl -throughout plant biomass.However, our study showed that Qarah Shani is a good Cl - excluder and Thompson Seedless a poor Cl -excluder.
In woody perennial species, enhanced Cl -exclusion from leaves/shoots is associated with increased salt tolerance rather than Na + exclusion (Storey et al., 2003).Control of Cl -transport and Cl -exclusion from shoots is correlated with salt tolerance in many species, for example, Citrus and Vitis (Sykes, 1992;Romero-Aranda et al., 1998;Moya et al., 2003) and Pinus banksiana (Franklin and Zwiazek, 2004).
In the present study, NO 3 -N content was significantly reduced in salt-stressed grapevines.It has been reported that an increase in salinity levels in root medium leads to a decrease of nitrogen uptake (Neumann, 1997).Competition between Cl -and NO - 3 uptakes can occur in plants grown under saline stress (Melgar et al., 2008).The increase in uptake and accumulation of Cl -ions in plant tissue generally results in the decrease of NO - 3 accumulation in the plant's aerial parts (Lara et al., 2003).Banuls et al. (1990) indicated that N accumulation in Navel orange scions grafted onto Cleopatra mandarin and Troyer citrange was negatively correlated with Cl - accumulation during salinity stress.
It could be concluded that there is a strong relationship between decreased NO - 3 uptake and increased Cl -content under salt stress in both cultivars.Measurement of NO - 3 under high salinity showed that with increasing Cl -concentration in roots and leaves, NO - 3 content was reduced.Based on our findings, SNP significantly increased NO - had a positive impact on increasing the amount of nitrate.
Increased NaCl salinity caused a significant decrease in the K + content of leaves and roots in both cultivars.
It is now clear that K + ions can enter cells through channels which are often more permeable to Na + under saline conditions (Parida and Das, 2005).
Because of the physicochemical similarity between Na + and K + (i.e., ionic radius and ion hydration energy), the former competes with K + for major binding sites in key metabolic processes in the cytoplasm (Shabala and Cuin, 2008).
The results of the present study showed that the leaf and root K + /Na + ratio of Thompson Seedless and Qarah Shani tended to decrease progressively with increasing salinity.The decrease of K + recorded in roots, which resulted in a decrease in the K + /Na + ratio, may also provide a mechanism by which grapevines achieve ionic balance following uptake of high Na + concentrations in root.As a result, K + /Na + ratio may be a useful criterion for screening salttolerant plants under saline conditions (Munns et al., 2006).In untreated control plants, Qarah Shani had much greater K + /Na + ratio than Thompson Seedless.This implies a competition between Na + and K + in grapevines, resulting in K + /Na + antagonism.The reduction in K + uptake caused by Na + is likely to be the result of the competitive intracellular influx of both ions (Cerda et al., 1995).
The results presented here indicated that application of SNP increased K + /Na + ratio in the roots and leaves of both cultivars under salinity conditions.NO may enhance salt tolerance in plants via increasing the expression of plasma membrane Na + /H + antiporter gene and H + -ATPase gene, which are required for Na + homeostasis and K + acquisition (Qiao and Fan, 2008).In another research, Zhang et al. (2004) reported that NO enhanced salt tolerance in maize seedlings through increasing K + accumulation in roots, leaves and sheathes, while decreasing Na + accumulation.NO induced salt tolerance of Populus euphratica calluses under salt stress via increasing the K + /Na + ratio and was dependent on the increased plasma membrane H + -ATPase activity (Zhang et al., 2007).
Ca 2+ and Mg 2+ concentration in both Thompson Seedless and Qarah Shani cultivars was decreased under the influence of salt stress.Given that Na + readily displaces Ca 2+ from its extracellular binding sites, Ca 2+ availability could be seriously reduced under salinity, especially at low Ca 2+ /Na + ratios.Calcium deficiency, in general, can impair the selectivity and the integrity of cell membrane and permit the passive accumulation of Na + in plant tissue (Hu and Schmidhalter, 2005).
As NaCl concentration in nutrient solution was increased, Mg 2+ content in the roots and leaves of both grape cultivars decreased.These results are in agreement with those of Sivritepe et al. (2010), who reported that NaCl salinity led to decreased Mg 2+ concentration in the leaves and roots of 'Muskule' grafted vines.Mg 2+ concentration of leaves was increased with increasing salinity in own-rooted and 'Ramsey' rootstock-grafted 'Sultana' vines (Walker et al., 1997).Salinity significantly reduced Mg 2+ content in Pistacia vera L. leaves on UCB-1 and P. atlantica (Ferguson et al., 2002) and leaf Mg 2+ concentration in Citrus (Ruiz et al., 1997).It is well known that H + -ATPase in plasma membrane plays an important role in the transport of multiple ions (Shi and Zhu, 2008) and there are investigations indicating that NO could induce H + -ATPase activity (Hayat et al., 2010), which might be responsible for increasing absorption of Ca 2+ and Mg 2+ under salinity stress.Zn 2+ and Fe 2+ concentrations were also gradually decreased in both leaves and roots, depending upon the salinity levels.Changes of Fe 2+ , Zn 2+ , Mg 2+ and Ca 2+ concentration in grapevine indicate that NaCl stress disturbs ionic homeostasis and application of SNP stimulates their maintenance.

CONCLUSION
Salinity disturbs the mineral nutrient contents in plants through its effects on nutrient availability, transport and partitioning.The results of our study indicated that salinity stress reduced the uptake of nutrient elements (NO - 3 , K + , Ca 2+ , Mg 2+ , Fe 2+ and Zn 2+ ), whereas Na + and Cl -uptake increased under NaCl treatments.On the other hand, Cl -inhibited NO - 3 uptake in both cultivars under salinity stress.Also, K + /Na + ratio was decreased progressively with increasing salinity in both cultivars.The reduction in K + uptake caused by Na + is likely to be the result of the competitive feature of both ions.Excessive influx of Na + accompanied by efflux of K + can disturb cellular function in plant.The application of SNP significantly reduced Na + and Cl -concentrations in both roots and leaves, with a stronger effect on leaves.Meanwhile, SNP treatments increased the concentrations of NO - 3 , K + , Mg 2+ and Ca 2+ in roots and leaves of both cultivars in saline conditions.At high levels of NaCl treatments (75 and 100 mM NaCl), application of 1.5 mM SNP did not significantly influence the Fe 2+ and Zn 2+ contents in the leaves and roots of both cultivars.The results of this study highlight the role of SNP in nutrient elements uptake under salinity stress.