Salt overly sensitive 1 (SOS1) gene expression can be regulated via Azospirillum brasilense Sp7 in wheat seedlings under saline condition

Salinity stress reduces plant growth via failure of physiological processes mainly due to the abundance of Na ion. Salt overly sensitive (SOS) signaling pathway is considered as an important component of Na/K homeostasis system in plants, especially under saline condition. Moreover, it is reported that wheat-Azospirillum associated has resulted in an enhanced salinity tolerance. To evaluate involvement of Azospirillum species in regulation of SOS signaling pathway, inoculated and none-inoculated wheat seedlings with Azospirillum brasilense Sp7 were grown for five days. Then uniform seedlings were transferred into saline hydroponic media with and without 200 mM NaCl. The relative expression of TaSOS1 of root, sheath, and blade as well as Na/K ratio was measured after 6, 24 and 48 hours since inoculated and non-inoculated seedling were transferred to NaCl media. Simultaneously Ca, Fe, proline content, root and shoot dry mass and soluble sugars were measured at 72 hour after application of NaCl. Result showed that salinity increased TaSOS1 gene expression, Na, prolin and Na/K ratio but Ca and Fe were decreased in root and shoot of wheat seedlings. Although A. brasilense Sp7 could improve salinity tolerance in wheat via reduction of Na uptake and upregulation of TaSOS1 expression, but do not have any effect in sodium distribution within plant parts. Therefore, salinity could increase TaSOS1 expression in the root, sheath and blade and A. brasilense Sp7 also could reduce the adverse effect of salinity via addition of over expression of TaSOS1.

It has been reported that plant growth promotion rhizobacteria (PGPRs) could induce more mineral uptake (Askary et al., 2009), phytohormones production (Kang et al. 2014) and consequently better tolerance to abiotic stresses (Vargas et al., 2012).El-Dengawy et al. (2011) reported that inoculation of Carob seedlings (Ceratonia siliqua L.) with Azospirillium lipoferum (Beijerinck) Tarrand et al. (ATCC ® 29707 ™ ) under saline condition could improve seedling growth rate, K + /Na + ratio and root characters.In addition, Upadhyay et al. (2012) reported that when wheat co-inoculated with two PGPR strains (Bacillus subtilis (Ehrenberg 1835) Cohn 1872 SU47 and Arthrobacter sp.SU18) were grown under different salinity conditions (2-6 dS m -1 ), an increase in dry biomass, total soluble sugars and proline were observed, meanwhile sodium of wheat leaves was reduced in co-inoculated plants under saline conditions.Creus et al. (1997) also confirmed the reduction of adverse effects of salinity and osmotic stresses on height and dry mass of wheat plants when inoculation with Azospirillium brasilense Tarrand, Krieg & Döbereiner, 1978. Vargas et al. (2012) also showed the upregulation of ethylene receptors genes expression in rice plants upregulated due to the inoculation with A. brasilense Sp245 as well as an increase in transcripts of some genes involved in nutrition uptake in response to A. brasilense. A. brasilense could change the pH of soil via proton efflux and increase availably of plant nutrients in wheat cultivars (Amooaghaie et al., 2002).Meanwhile, change in character of rhizosphere due to PGPRs activities and vis versa change in qurom sensing of bacteria due to the change of rhizosphere is a coincide fenomena which affect plant growth and development in stress condition (Pakdaman et al., 2014).
Among the salt overly sensitive (SOS) genes, SOS1 is a Na + /H + antiporter located in the cell membrane.Its role has already been demonstrated in cytosolic Na + homeostasis by different researchers (Ramezani et al., 2013;Sathee et al., 2015;Shi et al., 2002;Shi & Zhu, 2002;Yadav et al., 2012).It has been reported that TaSOS1 experssion was up-regulated in the root and the shoot of wheat seedlings under saline condition.For example, Yadav et al. (2012) showed that over expression of SOS1 gene in transgenic tobacco caused a higher salt tolerance, elevated seed germination, addition of root and shoot length, less dry mass reduction, higher K + /Na + ratio and more sugars relative to wild-type plants.It has been suggested that the SOS signaling pathway has an important role in ion (Na + and K + ) homeostasis and salt tolerance under saline condition.
For instance, TaSOS1 expression in transgenic Saccharomyces cerevisiae Meyen ex E.C. Hansen (which was already salt sensitive and had high cellular Na + content) caused reduction of Na + and addition of K + of cells (Xu et al., 2008).In addition, Feki et al. (2014) indicated that the Arabidopsis sos1-1 mutant is hypersensitive to both Na + and Li + ions, but its hypersensitivity would have disappeared using TaSOS1 gene.At this condition, better germination and more robust seedling growth, greater water retention capacity, retained low Na + and high K + in their shoots and roots were observed in nutrient solution containing Na + and Li + salts.Their work and complementary studies revealed that TaSOS1 upregulated the ion homeostasis and helped in salinity tolerance (Ramezani et al., 2013).
It has been reported that PGPRs such as Azospirillum species can improve wheat growth and productivity under salinity condition (Upadhyay et al., 2011) and coinoculation of A. brasilense and Rhizobium meliloti (Dangeard 1926) De Lajudie et al. 1994, comb.nov. in different wheat cultivar also showed an enhancement of root colonization and nitrogenize activity (Askary et al., 2008).Help of PGPRs in saline condition in one hand, and increasing 10 % annually saline areas due to the low precipitation, high surface evaporation, weathering of native rocks, irrigation with saline water, and poor cultivation in the other hand, create needs for more research in dual effects of salinity and PGPRs.
It is known that TaSOS1 expression is up-regulated in salt tolerant wheat cultivars (Ramezani et al. 2013) and A. brasilense also increased tolerance to salinity.However, it is not known that Azospirillum species increase salinity tolerance via up or down regulation of TaSOS1 gene in wheat seedlings under salinity condition or this phenomena has been done by different process.Therefore, the aim of this study was to evaluate SOS1 gene expression, Na, K, Ca and Fe uptake, sugar and proline content and biomass production of root, sheath and blade of wheat cultivars inoculated with A. brasilense Sp7 under salinity condition to know more about the effect of A. brasilense Sp7 and TaSOS1 involvment.

Preparation of inoculant and seeding
Standard strain of A. brasilense Sp7 was obtained from NCIMB Company, in Germany and then cultured in an NFb liquid medium supplemented with NH 4 Cl (0.25 g l -

1
) at 30 °C (Baldani & Döbereiner, 1980) in Erlenmeyer flasks for 48 h using a rotary shaker at 200 rpm (logarithmic phase).A high density bacterial culture was obtained by centrifuging at 1000 g for 10 min and was then washed with sterile saline phosphate buffer.Finally, desired concentration (10 7 CFU ml -1 of A. brasilense Sp7) was prepared from the media.
Seeds of a winter semitolerant wheat cultivar named Sardary (Triticum aestivum'Sardari') were obtained from Institute of Agricultural and Research of Isfahan in Iran.Seeds were surface sterilized by dipping in 95 % ethanol for 2 min and then in 1 % sodium hypochlorite (NaOCl) for 1 min followed by six washes in sterile distilled water (Öğüt et al., 2005).Then, sterilized seeds were transferred to autoclaved water agar medium and were kept at 25 ℃ for germination.After 24 hour, uniform germinated seeds were divided into two groups.
The first group was inoculated by submerging the germinated seeds in the solution containing 10 7 CFU ml - 1 of A. brasilense Sp7 and the second group treated without bacteria as control.To verify the inoculation success, the root segments stained with tetrazolium chloride dye and also cross section of inoculated root were prepared.After 3 hour, all seedlings (inoculated and none inoculated) were transferred into pots containing sterile perlite and then irrigated with 1/4 strength of Hoagland's nutrient solution (Hoagland & Arnon, 1950).The pots were kept for 5 days in a glasshouse under photoperiod 16/8 h (light/dark) at 25±2 °C.Then, 200 mM of NaCl (as one dose) was added as salinity treatment into only half of the plants in each group via irrigation water to have inoculated and non-inoculated plants under salinity and optimum saline condition.This experiment was conducted in randomized block design with three replicates.
Samples of roots, sheaths and leaves were collected at 6, 24 and 48 h after salinity applied.Some of the collected plant samples were used for Na and K analysis and some other immediately frozen in liquid nitrogen for Real-time quantitative PCR.Simultaneously, some of the plants was allowed to growth up to 72 hours and then their plant parts were collected for Ca, Fe, dry mass, soluble sugar, proline and root and shoot length analysis.

Real-time quantitative PCR
The total RNA was isolated from frozen roots, sheaths and leaves using Iraizol reagent (RNA biotech, Iran).
After RNA extraction, samples were treated with DNase.Then, the first stranded cDNA was synthesized using the M-MLV reverse transcriptase (Fermatas).Real-time PCR was performed in triplicate using SYBER Green Master Mix (RNA Biotech, Iran).Genespecific primers were designed for an 110 bp fragment of TaSOS1 (Gen Bank Accession No. AY326952).The primer pair was 5'-GGGATGATGAGGAACTTGGG-3' in sense direction and 5'-CTTGTCAGGAACATCGTGGG-3' in anti-sense direction.The primer pair for the housekeeping gene, actin, (Gen Bank Accession No. GI:48927617) was 5'-GTTCCAATCTATGAGGGATACACGC-3' in sense direction and 5'-GAACCTCCACTGAGAACAACATTACC-3' in antisense direction with an amplification length of 422 bp (Xu et al., 2008).The PCR conditions were 94 °C for 4 min followed by 40 cycles of 94 °C for 10 s, 62 °C for 40 s, 72 °C for 60 s, followed by 7 min at 72 °C.Serial dilutions of cDNA were used to obtain optimized standard curve amplification efficiency and the best cDNA concentration for real-time PCR was obtained.The relative expression ratio of target and reference genes were calculated based on its real time efficiencies (E) and crossing point difference (∆Cp) of sample versus control as well as reference versus control, respectively (Pfaffl, 2001).

Dry mass, Na + and K + determination
Each plant part (root, sheath and blade) was weighted separately and then 100 mg of dried mass of each sample was digested with 10 ml 3 % (w/v) aqueous sulfosalicylic acid for 24 hours.Extracted samples were filtered with Whatman No. 1 filter paper.Then, Na + and K + concentrations were measured using flame photometric (Perkin-Elmer Coleman 51-ca), using related standard curves for sodium and potassium.

Determination of soluble sugar content
Soluble sugar content was determined using phenolsulphuric method (Dubois et al., 1956).To do so, 0.01 g of dried plant sample was extracted in distilled water and centrifuged at 3000 rpm for 10 min.The extract (0.5 ml) was treated with 0.5 ml phenol (5 %) and 2.5 ml pure sulphuric acid and then after a mild vortex, their absorbance was measured at 490 nm using Shimadzu double beam UV-visible Spectrophotometer.

Determination of Ca and iron content
Dry mass (100 mg) of each plant sample (roots and shoots separately) was digested in 3 ml of a 1-4 (v/v) mixture of 37 % (v/v) HCl and 65 % (v/v) HNO3 in Teflon cylinders for 7 h at 140 °C.After adjustment of volume to 10 ml with deionized water, Ca and Fe was determined using an atomic absorption spectrophotometer (AAS, Shimadzu model 6200).

Determination of proline content
Roots and shoots proline were determined using Bates et al. (1973) method.100 mg of fresh plant samples was homogenized with 4 ml sulfosalicylic acid (3.0 %) in a mortar.The suspension was centrifuged at room temperature at 3000 rpm for 5 min.The supernatant was mixed well with 4 ml acidic ninhydrin reagent and the reaction mixture was vortexed and the content was placed in a boiling water bath for 60 min.Then, the content was cooled in the ice bath and the mixture was extracted with 4 ml of toluene.The light absorbance of toluene layer was recorded at 520 nm using Shimadzu spectrophotometer (Shimadzu UV-160, Japan) and the concentration of unknown samples was calculated using respected standard curve.

Statistical analysis
The experimental design was completely randomized design with 3 replicates and MSTAT-C software was used for ANOVA.Duncan multiple range test was used (at 5 % level of significance) to compare the mean values of measured indexes.Excel was used to draw the necessary graphs.

Growth, Ca, Fe, and soluble sugars
The results of length, dry mass and soluble sugars of roots and shoots of inoculated and non-inoculated wheat plants under saline and non-saline conditions are presented in Fig. 1.Root length of 8 days old plants was increased significantly in response to inoculation under saline and non-saline conditions.Simultaneously, shoot length was increased significantly under non-saline condition, while no significant difference was seen between inoculated and non-inoculated plant's shoot length under saline condition.
Root dry mass of inoculated plants was increased significantly under saline (25.8 %) and non-saline conditions (30.1 %) in comparison to control plants (non-inoculated and non-saline condition).However, shoot dry mass of inoculated plants did not improve either under saline or non-saline conditions in a short period of time.In contrast, root and shoot soluble sugars were affected by saline condition and shoot soluble sugars in inoculated (16.7 %) and non-inoculated (13.2 %) plants were increased significantly under saline condition.
Calcium concentration in the roots and shoots of inoculated plants not exposed to salinity was increased by 14.1 and 10.63 %, respectively, when compared to control plants (Fig 2).Salinity caused a significant reduction in Ca content of both roots (38.7 %) and shoots (31.26 %) of seedlings.However, inoculation improved Ca concentration in the root and shoot but its amount was still less than the control plants.
In inoculated seedlings, the amount of Fe in the roots and shoots (Fig 2) was the highest (0.51 and 0.21 mg g -1 DM, respectively) meanwhile, a significant reduction was observed in Fe content of the roots (23.68 %) and the shoots (23.68 %) of plants treated NaCl.Inoculation couldn't help the roots to uptake more Fe under salinity condition.Excess of sodium chloride can damage plant growth and development through reducing water and nutrients uptake as well as negative effects on biochemical processes (Akbarimoghaddam et al., 2011).Numerous researchers have reported that growth and morphological indexes such as root length (El-Hendawy et al., 2005), leaf area (Hamdia et al., 2004) as well as soluble sugars, proline (Tavakoli et al., 2016), Ca and Fe content (Askary et al., 2017) are affected significantly by saline condition (Turan, 2008).The result of this study showed that in non-inoculated plant, salinity causes a reduction in root and shoot length.One of the mechanisms that is chosen by plants to cope with environmental stress such as salinity is the accumulation of some organic molecular (such as soluble carbohydrates and proline).It appears that increase in soluble carbohydrates and proline content in the root of wheat seedlings probably cause the better osmotic adjustment and maintained cell turgor for better growth under salinity.However, the soluble sugar of the root and shoot of inoculated and non-inoculated wheat seedlings were increased due to salinity.Meanwhile, proline was increased just in the root of non-inoculated seedlings.This result is similar to the results obtained by Maghsoudi and Arvin (2010).They reported a significant reduction in dry matter of susceptible wheat varieties under saline condition, when compared to the salt tolerate wheat cultivars.Moreover, the results of this study showed that inoculation of wheat plants with A. brasilense Sp7 under saline and non-saline conditions had no significant effect on the amount of root and shoot soluble sugars in short period of time.But, inoculation increased significantly dry mass and length of the roots.Similar results were reported by different researchers.Zarea et al. (2012) showed that Azospirillum had no effect on soluble sugars content of wheat plants.In similar work Hamdia et al. (2004) reported that root dry mass and root length of maize were increased significantly in response to inoculation with A. brasilense.However, growth reduction under saline condition may be either due to lowering the external water potential or ion toxicity on metabolic processes.Other reports indicated that Azospirillum Spp. may produce various plant growth regulators that increase plant growth indexes (dry mass, root and shoot length), nitrogen fixation, and absorption of water and minerals (El-Dengawy et al., 2011, Askary et al., 2017).
In this study, Ca and Fe content of root and shoot seedlings significantly reduced under salt stress while inoculation with A. brasilense shown some improvement in Ca uptake.Upadhyay et al. (2011) reported that some of the native strains of bacteria which separated from the wheat rhizosphere of the soils were able to establish salt tolerance by bacterial secretion such as exopolysaccharides that affect the mineral availability.

Sodium, potassium and Na + /K + ratio
In control condition, sodium content of roots, sheaths and leaf blades showed an increasing trend over time, and so, the sodium content was the highest at 48 hour (at the end of experiment) after salinity applied (Table 1), it means sodium was accumulated during the experiment in all plant parts.The sodium content of roots, sheaths and leaf blades were also increased in same pattern in non-inoculated plants under saline condition as compared to control plants.Under inoculation and non-saline condition, the sodium content of root, sheath and leaf blade were significantly less than control plants.Although salinity raised sodium accumulation in root, sheath and leaf blade of inoculated plants over time, their sodium content were still significantly less than non-inoculated plants that were grown under saline condition.Moreover, the maximum sodium content in root, sheath and leaf blade of non-inoculated plants which exposed to 200 mmol NaCl was 7.94, 4.44 and 2.70 mg g -1 DM, respectively at 48 hour after salinity applied.According to the result (Table 1), we observed that wheat inoculation by A. brasilense could help to prevent the sodium entrance to the roots of plants ( 17.22 % at non-saline and 9.5 % at saline conditions) meanwhile didn't have considerable effect on sodium allocation within plant parts.
Although sodium content was increased in inoculated plants under saline condition, it was significantly less than sodium content of non-inoculated plants which grown under saline condition.In addition, we observed that A. brasilense could help to prevent sodium uptake by root of inoculated wheat plant.This might be due to producing and secretion of bacterial exopolysaccharides to the root environment and reducing the availability of Na + for plant uptake.This result is consistent with the results presented by Upadhyay et al. (2011).They indicated that some of the native strains of bacterial which separated from the wheat rhizosphere in the soils of Varanasi and India were able to establish salt tolerance by bacterial secretion such as exopolysaccharides.
Previous studies revealed that sodium accumulation in germinated seeds was increased gradually under saline condition (Akbarimoghaddam et al., 2011;Hamdia et al., 2004).In addition, El-Dengawy et al. (2011) showed that Carob seedlings (Ceratonia siliqua L.) inoculated with A. lipoferum under saline condition improved the addition of sodium-to-potassium ratio and gave better root characteristics.Al these results show that establishing a cooperation system between wheat and Azospirillum help to facilitate better growth through direct or indirect mechanisms (Hamdia et al., 2004;Nadeem et al., 2006).
Table 1: Mean values of Na + content (mg g -1 D M) in inoculated and non-inoculated wheat plants with Azospirillum brasilense SP7 (10 7 CFU ml -1 ) grown under saline (200 mmol NaCl) and non-saline conditions for 6, 24 and 48 h.The growth condition was light with photon density 650 µmol m -2 s -1 and temperature of 25 ºC.Different letters in each plant parts separately represent a significant difference at 5 % level of significance.) grown under saline (200 mmol NaCl) and non-saline conditions for 6, 24 and 48 h.The growth conditions were light with photon density 650 µmol m -2 s -1 and temperature of 25 ºC.Different letters in each plant parts represent significant differences at 5 % level of significance.
Potassium content of plant's root in treated and nontreated wheat seedlings with A. brasilense Sp7 and Azospirillum plus salinity was higher compared to salt stressed plants alone (Table 2).Also, potassium content of plant's sheath and leaf blade in all treated and nontreated wheat seedlings showed an increasing trend over time (Table 2).Under saline condition, potassium accumulation decreased in root and sheath as compared to control plants while potassium content of leaf blade was preserved.The reduction of potassium content was more severe in root at 48 hour after salinity applied (46.6 %).Potassium accumulation of root, sheath and leaf blade in control plants were 1.94, 3.69 and 3.75 mg g -1 DM, respectively.While potassium content decreased to 1.03, 3.04 and 3.85 mg g -1 DM when salinity applied.However, with inoculation, potassium content increased to 2.16, 4.15 and 5.03 mg g -1 DM in root, sheath and leaf blade, respectively.In dual effect (salinity and inoculation) the potassium content was higher than control condition but less than in inoculated plants.The average potassium content of whole plant was 3.13 (control), 2.64 (salinity), 3.78 (inoculation) and 3.47 mg g -1 DM for dual effects of inoculation and salinity.
In root and sheath of non-treated plants, Na + /K + ratio didn't show variation over time while this ratio in leaf blade was significantly decreased over time (Table 3).Under saline condition, the Na + /K + ratio of noninoculated plant's root, sheath and leaf blade was higher than control plants and also showed an increasing trend over time.Moreover, the maximum Na + /K + ratio in noninoculated plants was seen in root at 48 hour after salinity applied (278 %).Under non-saline condition, Na + /K + ratio of root, sheath and leaf blade in inoculated plants were less than non-inoculated plants.Although the Na + /K + ratio of inoculated plants under saline condition was more than control plants, but it was less than non-inoculated plants treated with salinity.Moreover, the maximum reduction (29.73 %) of Na + /K + ratio was observed in leaf blade of inoculated plants not exposed to NaCl as compared to control plants.
Under saline and non-inoculated condition, potassium content of root and sheath showed a significant reduction compare to control plants.Meanwhile, potassium content of inoculated plants was more than that of non-inoculated plants under saline condition.In addition, under non-saline condition, potassium content of all plant parts was increased due to inoculation.The results of this study show that the wheat plant inoculation with A. brasilense Sp7 has a significant effect on K + accumulation under saline and non-saline conditions.Under saline condition, the competition between uptake of sodium and potassium by noninoculated plants favored sodium ions (Wakeel, 2013), but the wheat seedlings inoculated with Azospirillum increases potassium uptake.Therefore, sodium entry into the cell and potassium leakage out of the cell is decreased; this leads to a reduction of Na + /K + ratio (Fraile-Escanciano et al., 2010;Hamdia et al., 2004;Upadhyay et al., 2011).Ardakani et al. (2011), showed that the potassium content of inoculated wheat plants by A. brasilense was increased and causes better mineral nutrient uptake.The results of this study are also similar to Askary et al. (2009), who showed that A. brasilense Sp7 improves potassium, phosphorus and nitrogen uptake by different wheat cultivars.In addition, the results are the same as Omar et al. (2009), who showed that Na + /K + ratio of wheat plant after inoculation with A. brasilense was decreased due to increasing K + and limiting Na + uptake.In inoculated and non-inoculated plants, Na + /K + ratio of leaf blade significantly was lower than the root.This might be due to less sodium accumulation in leaf blade that have already confirmed by Davenport et al. (2005).

Proline
Inoculated seedlings had lower proline concentration in the root and the shoot when compared to seedlings which were exposed to saline conditions.The lowest amount of proline (0.4 µmol g -1 FM) was measured at the root of inoculated plants not exposed to salinity (P < 0.05, Fig. 2).Salinity didn't have a significant effect on shoot proline, meanwhile, the maximum content of proline was observed in the root of salt-affected that non-inoculated with Azospirillum (P < 0.05, Fig. 3).
Proline production is one of the mechanisms that enable the plant to tolerate adverse effect of environmental stresses.Proline is thought to contribute to osmotic adjustment, detoxification of ROS, and protection of membrane integrity (Heuer, 2010).

Relative TaSOS1 (Triticum aestivum salt overly sensitive 1) gene expression
In control plants, the relative expression ratio of root TaSOS1 didn't show any variation in its transcript level over time (Table 4) while a considerable reduction in TaSOS1 expression was observed in sheath and leaf plants (non-inoculated and non-saline condition) was observed.Although the maximum (224 %) overexpression happened at 24 h after salinity applied, its value declined almost by 50 % afterward (at 48 h).In non-saline condition, the root TaSOS1 relative expression of inoculated plants showed an up-regulation trend during the experiment.While, inoculated roots under salinity condition showed a considerable upregulation (260 %) at 24 h and then down-regulated at 48 h after salinity applied but still the TaSOS1 expression was higher than its control plant.In addition, TaSOS1 over expression was higher in inoculated plants, than non-inoculated under non-saline condition.
Sheath: In non-inoculated plants, the relative expression ratio of TaSOS1 was increased immediately when NaCl was added, then its value was reduced at 24 h, and finally reached to its maximum (120 %) at 48 hour.In non-saline condition, inoculation caused a significant increase in sos1 gene expression as compared to control plants and reached to its maximum at 48 h (188.4 %).In saline and inoculated condition, the relative expression of TaSOS1 was increased in sheath by 119 % when compared to its corresponded control plants.However, TaSOS1 up-regulation due to dual treatments (salinity and inoculation) was significantly higher than inoculation or saline condition.
Leaf blade: There was a significant increase (almost 14 %) in TaSOS1 expression after salinity applied as compared to non-treated plants.In inoculated and nonsaline condition, the relative expression of TaSOS1 showed an increasing trend and reached to 1.66 at 48 h.However, in saline condition, the expression of TaSOS1 in inoculated plants was higher than control plants at 24 (70 %) and 48 h (27 %) after salinity applied.The TaSOS1 mRNA level was higher in inoculated plants treated with NaCl as compared to control plants and also seedlings exposed to salinity.
Table 4: TaSOS1 gene relative expression inoculated and non-inoculated wheat plants with Azospirillum brasilense (10 7 CFU ml -1 ) grown under saline (200 mmol NaCl) and non-saline conditions for 6, 24 and 48 h.The plants were grown under the light density of 650 µmol m -2 s -1 and temperature of 25 ºC.Each value represents the mean of three individual measurements ±SE.Different letters in each plant parts represent significant differences at 5 % level of significance.
The relative expression ratio of TaSOS1 in different plant parts shown that the highest up-regulation of TaSOS1 was observed in root at 24 h whereas the highest in sheath and leaf blade was happend at 48 h after salinity applied (with a delay) as compared to the root.
The result of this study showed that addition of sodium in the root rhizosphere and inoculation of wheat seedlings with A. brasilense Sp7 cause an increase in TaSOS1 expression.This result is similar to that obtained by Ramezani et al. (2013), who showed that under saline condition TaSOS1 and TaSOS4 gene expression would increase in such cases.Moreover, Xu et al. (2008) showed that after 3 h of salt stress implementation, TaSOS1 expression of root was increased immediately and then decreased.But, these changes occurred in the leaf with lower intensity and with a delay (after 9 h of salt stress).
Wheat seedling treated with A. brasilense and salinity showed that TaSOS1 expression was increased in compared to non-inoculated and saline condition.Therefore, it can be concluded that A. brasilense can increase TaSOS1 expression under saline and non-saline condition and can help sodium and potassium ions to be adjusted in plant.Numerous studies have already showed that gene expression changes in host plant after inoculation with plant growth-promoting rhizobacteria (PGPR).For instance, Vargas et al. (2012) rice ethylene receptor gene expression was increased after inoculation with A. brasilense Sp245.In addition, it was reported that some genes involved in nutrient uptake were increased when wheat plant inoculation with A. brasilense (Camilios-Neto et al., 2014).
It seems that the tolerance of wheat plant to high concentration of salt is related to their ability to prevent Na uptake, avoid accumulation of toxic levels of sodium, regulation of osmotic pressure and maintaining adequate amount of potassium especially in the leaf blade.To achieve these goals, some genes related to such indexes should be express differently, e.g.TaSOS1 which is a regulator for Na + uptake and upregulated at salinity condition.Establishment of an associated relationship between wheat plants and PGPR such as A. brasilense may help to regulate expression of such genes (e.g.SOS1) and consequently regulate the balance of cytosolic sodium and potassium.This is coincidence with, reduction of Na + availability for plant uptake via secretion of bacterial exopolysaccharides to the root environment.

CONCLUSION
This study showed that TaSOS1 gene expression, growth, and biochemical indexes increased due to inoculation and saline conditions.Furthermore, the highest up-regulation of TaSOS1 was observed in sheath and leaf blade with a delay as compared to the root.Also, A. brasilense had an important role in preventing the sodium entry into the plant.Further research might explore plasma membrane TaSOS1 antiporter proteins by proteomic techniques in inoculated wheat seedlings under saline condition.

Figure 1 :
Figure 1: Length (A1); dry mass, DM (A2) and soluble sugars (A3) of root and shoot of wheat plants ('Sardari') in non-inoculated and inoculated with A. brasilense Sp7 (10 7 CFU ml -1 ) grown in saline (200 mmol NaCl) and nonsaline conditions.Wheat was grown in 16 h light 8 h dark, photon density 650 µmol m -2 s -1 and temperature of 25 ºC.Plant samples were collected 72 hours after salt application.Each value represents the mean of three measurements ±SE.Different letters represent significant differences at 5 % level of significance

Figure 2 :
Figure 2: Effect of inoculation and salinity on average Ca and Fe content of roots and shoots of wheat seedlings.Differences in small (shoot) and cap (root) letters on the bar graph indicated difference in their mean (n = 3) values based on Duncan's multiple range tests

Figure 3 :
Figure 3: Effect of inoculation and salinity on average (n = 3) proline content of roots and shoots of wheat seedlings.Differences in small and cap letters on the bar graph indicated difference in their mean values based on Duncan's multiple range tests.
blade.When wheat seedlings exposed to 200 mmol NaCl and inoculated with A. brasilense Sp7 (10 7 CFU ml -1 ), TaSOS1 gene expression at mRNA level varied differently in different plant parts.Root: Due to salt stress, TaSOS1 over-expression in non-inoculated plant's root as compared to the control

Table 2 :
Mean (n = 3) values of K + content (mg g -1 D.M) in inoculated and non-inoculated wheat plants with Azospirillum brasilense (10 7 CFU ml -1 ) grown under saline (200 mmol NaCl) and non-saline conditions for 6, 24 and 48 h.The growth conditions were light with photon density 650 µmol m -2 s -1 and temperature of 25 ºC.Different letters in each plant parts represent significant differences at 5 % level of significance.