Changes in Organic and Inorganic Osmolytes of Maize (Zea mays L.) by Sulfur Application Under Salt Stress Conditions

Mineral nutrients have favourable potential in alleviation of salinity problem in plants. Sulfur has specific functions in regulating plant growth, metabolism, enzymatic reactions and osmolyte homeostasis in plants. Hence, an experiment was carried out to explore the role of sulfur in ameliorating salt toxicity in maize by changes in organic and inorganic osmolyte contents. A range of sulfur levels (40, 80 mM) were used to induce salinity tolerance in maize. Various treatments of salinity (25, 75 mM) were applied by using sodium chloride. Results revealed that glycine betaine, proline, total soluble sugars, total soluble proteins and total free amino acids contents were increased by applying salinity while the application of sulfur lowered the proline and increased other studied organic osmolyte contents in all studied maize organs (leaf, shoot, root). The maximum improvement in organic osmolyte contents were found at 40 mM sulfur, however, at 80 mM sulfur proline contents were reduced. Applied salinity increased leaf tissue concentration of Na and decreased that of K, Ca, NO3, PO4, SO4 leading to a severely declined in K/Na and Ca/Na ratio. However, application of sulfur reduced the Na contents and improved K, Ca, NO3, PO4, SO4, K/Na and Ca/Na ratio in the salinity grown plants. Moreover, 40 mM level of sulfur was greatly effective in osmolyte homeostasis at all levels of salinity. This indicated that use of sulfur (40 mM) ameliorated the effect of salinity by changing organic and inorganic osmolyte contents in maize plants.


Introduction
Among various abiotic stresses, salt stress has affected 20% of land used for cultivation and 33% of the irrigated land throughout the world (Machado & Serralheiro, 2017). Overall, 10 million ha of the world land has been degraded due to salinity each year (Pimentel et al., 2004). Salt stress causes disturbances in physiological, biochemical, molecular processes in the plant (Nahar et al., 2016). As a result osmotic stress, imbalance in nutrient transport and accumulation of reactive oxygen species takes place Puniran-Hartley et al., 2014). In such conditions, plants synthesize and accumulate various organic and inorganic osmolytes or osmoprotectants. These include proline, glycine betaine, glucose, isoleucine, mannitol and proteins (Parida & Das, 2005) and various inorganic nutrients (K + , Ca 2+ , NO 3 -, PO 4 3-, SO 4 2-). The functions of these osmolytes are, to balance the ionic transport across the plant cell, scavenge reactive oxygen species, regulate enzyme activity and prevent membrane disintegration (Nahar et al., 2016). However, such strategies are needed that balances the concentrations of various osmolytes for maintaining plant metabolism. As higher concentration of osmolytes become toxic for plant cell.
Sulfur plays a significant role in balancing the osmolyte contents in the plants. Sulfur is a basic constituent of many important compounds that maintain plant growth and development in stress conditions. These compounds include glutathione, vitamins, phytoharmones and various co-enzymes (Spadaro et al., 2010). Sulfur helps in coordination among different physiological and biochemical processes in the plants. Hence, Sulfur improves the cellular function by balancing the organic and inorganic osmolytes that develops salt tolerance in crop plants (Taiz & Zeiger, 2006;Nazar et al., 2014;Riffat & Ahmad, 2016). seriously affected by salinity as maize is moderately sensitive to salinity (Farooq et al., 2015). Therefore, such methods should be devised that increase the salt tolerance of this valuable crop to meet the growing food demand.
Hence, this study focuses on the improvement in salt tolerance potential of maize by sulfur application. To maintain the balance of organic and inorganic osmolytes for development of salt tolerance is another objective of this study.

Plan of Study
A study was conducted to determine the role of sulfur in enhancing salt tolerance by changing the osmolyte contents in maize. The seeds of maize cultivars (Agaitti, 2003;Pak Afgoi, 2003) were acquired from Maize and Millet Institute Sahiwal Pakistan. The seeds were sorted and 10 uniform seeds were sown in plastic pots filled with 10 kg soil.

Treatment Application
Salinity (25, 75 mM) was applied by using sodium chloride. Various levels of sulfur (40, 80 mM) were applied by using potassium sulfate. Both treatments were applied at sowing time. After 15 days of treatment application, sulfur (40, 80 mM) was applied as foliar spray. Then 45 days plants were harvested for the determination of various biochemical attributes.

Determination of Organic Osmolytes
2.3.1 Glycine Betaine Grieve and Grattan (1983) proposed a procedure for the determination of glycine betaine contents. Two reagents 2N H 2 SO 4 and IK-I 2 were prepared. 2N H 2 SO 4 was prepared by mixing 5.6 mL of 36 M H 2 SO 4 and distilled water was used for making final volume100 mL. IK-I 2 was made by mixing 20 g of potassium iodide, 100 mL water and 15.7 g of iodine. Glycine betaine contents were determined by grounding 0.5 g dried plant material in 20 mL of deionized water and shaken for 24 h at 25 o C. The extract was filtered and diluted with 2 N H 2 SO 4 in 1:1 ratio. Then 0.5 mL extract was put in centrifuge tube and kept in ice cooled water for 1 hour followed by addition of 1 mL of IK-I 2 , and vortexed at 0 o C at 10,000 g for 15 min. The supernatant was collected and dissolved in 9 mL of 1-2 dichloroethane. The solution was kept at room temperature for 2-2.5 h. The absorbance of glycine betaine was noted at 365 nm by using spectrophotometer (UV-1100). The values were compared with standard curve.

Proline
Proline contents in plants were determined by the procedure proposed by Bates et al. (1973). Firstly, some reagents were prepared. 6 M phosphoric acid was prepared by diluting 407 mL of 85 % phosphoric acid in 1000 mL distilled water. For the preparation of acid-ninhydrin, 1.25 g of ninhydrin was dissolved in 30 mL glacial acetic acid and 20 mL of 6 M phosphoric acid. 3% sulfuric acid was made by mixing 3 g of sulphosalicylic acid in 100 mL of distilled water. For the determination of proline contents in plant material 0.1 g fresh plant sample was homogenised in 10 mL of 3% sulphosalicylic acid and filtered. Then 2 mL of acid ninhydrin, 2 mL of glacial acetic acid and 1 mL of filterate was heated in water bath at 100 o C for 1 hour and then transferred to ice bath following the addition of 4 mL of toluene. The reaction mixture was vortexed, chromophore having free proline was separated in test tube, kept at room temperature and the proline contents were measured at 520 nm on spectrophotometer (UV-1100). For blank, same procedure was used by using 2 mL of 3% aqueous sulphosalycylic acid. Following formula was used for proline determination. µmoles proline g fresh weight = µg proline/mL × mL of toluene (115.5 µg/mole)/g sample/5 (1)

Soluble Sugars
For the determination of soluble sugars, the procedure given by Yoshida et al. (1976) was followed. Anthrone reagent was made by mixing 1 g anthrone in 1 L conc. H 2 SO 4. For the determination of soluble sugars, 0.1 g fresh plant material was boiled in 5 mL distilled water and the filtrate was diluted to 50 mL with distilled water. To 1 mL of the filtrate, 5 mL of anthrone reagent was added and heated at 90 °C for 20 min. The soluble sugar contents were determined at 620 nm by using spectrophotometer (UV-1100). For standard curve, glucose series (0, 20, 40, 60, 80 and 100 µM) was used.

Total Free Amino Acids
Total free amino acids in plant tissues were measured by the procedure of Hamilton and Van-Slyke (1943). 2% ninhydrine and 10% pyridine solution were prepared in the distilled water. For the determination of total free amino acids, 1 g fresh plant sample was homogenised in 10 mL of phosphate buffer (0.2 M with pH 7.2). To 1 mL of the extract, 1 mL of pyridine (10%) and 1 mL of ninhydrine (2%) were mixed and heated at 100 o C in water bath for 30 min. The volume was maintained 50 mL with distilled water and the absorbance was noted at 570 nm by using spectrophotometer (UV-1100). Following formula was used for calculating total free amino acid.
Total amino acid (mg/g fresh weight) = Graph reading of sample × Volume of sample × Dilution factor Weight of the tissue x 1000 (2)

Total Soluble Proteins
The concentration of total soluble protein was determined by the method given by Bradford (1976). Phosphate buffer saline was prepared by mixing 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.37 mM NaCl and 2 mM KH 2 PO 4 and pH 7.2 was maintained by using HCl. The determination of total soluble protein was done by extracting the 0.5 g fresh plant material in phosphate buffer saline, centrifugation was done and the supernatant was collected. To equal volume of supernatant dye stock was dissolved, vortexed and kept in an incubator for 30 min. The absorbance was noted at 595 nm by using spectrophotometer (UV-1100). The standard curve was drawn by using bovine serum albumin (BSA) of the range (10 to 50 µg mL -1 ).

Determination of Inorganic Osmolytes
2.4.1 Sodium, Potassium, Calcium (Na + , K + , Ca 2+ ) The dried plant sample (0.5 g) was incubated in 5 mL H 2 SO 4 overnight and heated at 350 °C in the digestion block for 30 min. The mixture was cooled; 1 mL of H 2 O 2 was added and again heated for 20 min. These steps were repeated until clear solution was obtained, filtered, and volume was maintained to 50 mL by using distilled water (Wolf, 1982). This extract was used for the determination of Na + , K + , Ca 2+ ions by using flame photometer (Jenway PFP-7). For standard curve a series of standards (10, 20 to 100 ppm of Na + , K + and Ca 2+ ) was prepared.
The actual values were calculated by comparing the values from standard curve and from flame photometer.

Phosphate (PO 4 2-)
The concentration of phosphate ions in plant tissues was determined by following the method of Yoshida (1976). Firstly, two reagents were prepared. For the preparation of molybdate-vanadate solution, 25 g ammonium molybdate was mixed in 500 mL of water, and 1.25 g of ammonium vanadate was mixed in 500 mL of 1N HNO 3 separately, then equal volumes of two solutions were mixed together. For the preparation of nitric acid (2 N), 10 mL of concentrated HNO 3 was mixed in 80 mL of distilled water. The phosphate content was determined by boiling 0.5 g dried plant sample in 5 mL distilled water for 1 h, filtered and 50 mL volume was prepared by using distilled water. 1 mL of extract was mixed with 2 mL of 2 N HNO 3 , volume was maintained to 4 mL with distilled water, 1 mL of molybdate-vanadate reagent was added and the mixture was diluted to 10 mL with distilled water, vortexed, allowed to stand for 20 min and absorbance was noted at 420 nm by using spectrophotometer (UV-1100). For standard curve, stock solution of 25 mg/L PO 4 3was prepared by mixing 0.11 g monobasic phosphate (KH 2 PO 4 ) in 1 L distilled water and standard series was prepared by mixing 1, 2, 3, 4, 5 and 6 mL of 25 mg/L PO 4 3and diluted to 8 mL with distilled water.

Nitrate (NO
For the determination of nitrate contents a procedure proposed by Kowalenko and Lowe (1973) was used. The reagents were prepared. For the preparation of 0.01% TCA, 0.1% CTA stock was prepared. For this purpose, 0.247 g of chromotropic acid disodium salt (CTA) was dissolved in 100 mL of conc. H 2 SO 4 . Then 10 mL of CTA stock was diluted to 100 mL with H 2 SO 4 for the preparation of 0.01% TCA. For the determination of nitrate contents, 0.5 g dried plant sample was boiled in 5 mL of distilled water for 1 h, filtered and diluted to 50 mL by using distilled water. 3 mL extract was mixed with 7 mL of working CTA solution, vortexed and absorbance was noted at 430 nm after 20 min by using spectrophotometer (UV-1100). Water was used for blank. For standard, 0.7216 g of KNO 3 was dissolved in 1 L distilled water for the preparation of 100 mg/L NO 3 stock solution, then a graded series (10,20,30,40,50 and 100 mg/L NO 3 -) was prepared by diluting the stock solution.

Sodiu
Results rev by statistic non-signif Figure 6 The applic organs at b effect (Tab lowered th       Moreover, significant was found contents u root while application cultivars (F plants under stress conditions (Siringam et al., 2012). Moreover, soluble sugars serve as chelating agent that bound Na + with starches and lower the toxic effects of salt on the plants (Xiao et al., 2009). The application of sulfur also increased the soluble sugars contents in both maize cultivars at all levels of treatments. It was supported by the findings of Lunde et al. (2008) who reported the reduction in soluble sugar contents by sulfur deficiency.
In this study, it was found that salinity increased the total soluble protein contents in both studied maize cultivars. It was supported by earlier researches (Chen et al., 2007;Kapoor & Srivastava, 2010). Soluble proteins help to raise the nitrogen level in plants that promotes growth and development under stress conditions. In addition, soluble proteins perform a significant role in osmotic adjustment (Ashraf & Harris, 2004). Sibole et al. (2003) found that by application of salinity (10, 50, 100, 200 mM), the soluble protein contents were increased in the clover plant (Medicago citrna L.). The accumulation of soluble protein contents by salt application has been reported in various plants i.e. barley, maize, sunflower, rice and mung bean (Khosravinejad et al., 2009;Kapoor & Srivastava, 2010). This study showed that the application of sulfur improved the soluble protein contents in maize plants. It may be due to the reason that sulfur is an important part of amino acids the building blocks of proteins (Gardner et al., 1985). Different metabolites of sulfur (i.e. cysteine, thiol) protect the structure of proteins. Hence, sulfur helps in forming the structure and function of proteins in the stress conditions (Malhi & Leach, 2000).
It was found that salt stress enhanced the total free amino acid contents in maize plants. In stress conditions, total free amino acid contents become very high that protects the proteins from degradation (Mansour, 2000). Moreover, this study showed that salt tolerant maize cultivar accumulated high level of total free amino acid in comparison to salt sensitive maize variety. These findings have been supported by previous studies (Ashraf and Tufail, 1995;Ashraf & Fatima, 2004). The application of sulfur improved various amino acid contents in maize plants as sulfur is the constituent of many important amino acids forming various structural and functional proteins in plants (Giovaneli, 1987).
Salt stress causes the disturbance in availability, absorption and transport of nutritional contents in plants (Munns & Tester, 2008). In this study, salinity reduced the beneficial nutrients (K + , Ca 2+ , NO 3 -, PO 4 3-, SO 4 2-, K + /Na + , Ca 2+ /Na + ) in maize plants. It may be due to the reason that salt stress causes the disturbance in external osmotic potential that imbalance the nutrient contents in plants (Murillo-Amador et al., 2002). The imbalance in nutrient contents has been reported in various crops e.g. Lycopersicon esculentum, Spinacia oleracea, Physalis peruviana, as well as in Zea mays (Miranda et al., 2010;Collado et al., 2010).
This study revealed that salt stress increased the sodium (Na + ) contents in the maize plants which are in accordance to the findings of Fortmeier et al. (1995). The rise in sodium (Na + ) contents decreased the plant growth in both studied maize cultivars (Agaitti, 2003;Pak Afgoi, 2003). It may be due to the reason that high sodium (Na + ) contents forms ion-pair and precipitates other ions in plant cell (Hu et al., 2005). The reduction in Ca 2+ , K + , K + /Na + and Ca 2+ /Na + has been reported in this study. The elevated concentration of sodium (Na + ) changes the root permeability and reduces the uptake of calcium (Ca 2+ ) in plants (Greenway & Munns, 1980). This may be due to the competition in uptake of sodium (Na + ) and calcium (Ca 2+ ) contents and due to reduction in soil water potential affecting root pressure (Sonnevelt et al., 1975). Moreover, high concentration of sodium (Na + ) negatively uptake the potassium (K + ) resulting in reduction in carbon fixation, photosynthetic apparatus and ultimately reduces the photosynthesis in plants (Akram et al., 2010). The results of this study revealed that salt tolerant cultivar (Agaitti, 2003) accumulated low sodium (Na + ) and high potassium (K + ) and calcium (Ca 2+ ) contents in comparison to salt sensitive maize variety (Pak Afgoi, 2003). Therefore, Agaitti (2003) showed high K + /Na + and Ca 2+ /Na + ratio. This may be due to the reason that salt tolerant variety compartmentalizes the sodium (Na + ) in the plants thus transport the potassium and calcium (Munns et al., 2006). Thus, salt tolerant cultivar has high K + /Na + ratio. It was supported by previous studies (Song et al., 2009). In salt tolerant variety the restricted uptake of Na + ions maintains plant homeostasis and ultimately overall plant growth. While in salt sensitive variety, plant growth reduced due to disturbance in nutrient homeostasis. These findings are in accordance to previous researches (Eker et al., 2006;Riffat & Ahmad, 2018). Results showed that application of sulfur lowered the Na + ions and improved the Ca 2+ , K + , K + /Na + and Ca 2+ /Na + in the maize plants. Sulfur helps in maintaining nutrient homeostasis in plants and induces salt tolerance (Singh et al., 2011). Sulfur application increases the Ca 2+ and K + ions and decreases the harmful effects of Na + ions in the plants. This results in high K + /Na + and Ca 2+ /Na + ratio that indicate salt tolerance. Thus application of sulfur improves the crop quality and growth and development by maintaining proper nutrient homeostasis in plants under stressful environment (Badr et al., 2002;Prasad et al., 2003).
Results showed that salinity reduced nitrate (NO 3 -) contents in maize plants. It was supported by previous findings of Samra (1985). It may be due to the reason that Na + ions cause slow assimilation of nitrate (NO 3 -) contents. Moreover, salt stress shifts the reduction of nitrate from leaf to root (Frechill et al., 2001;Ullrich, 2002), that disturbs the proper availability of nitrate (NO 3 -) to the other parts of plants. The application of sulfur improved the nitrate (NO 3 -) contents in both studied maize varieties. It was in accordance to the previous studies. Reuveny et al. (1980) reported that the deficiency of sulfur causes the reduction in nitrate reductase activity. However, sulfur application improves the nitrogen metabolism and ultimately improves the nitrate contents in stress conditions (Sexton et al., 1993).
Salt stress also reduced the phosphate (PO 4 3-) contents in maize plants. Champagnol (1979) reported that salt stress reduced the phosphate (PO 4 3-) nutrition in the plants. However, sulfur application at low concentration improved the phosphorous contents in maize plants. These findings are supported by previous researches on various crops i-e. wheat, chickpea and maize, (Islam et al., 2011;Riffat, 2017;Riffat & Ahmad, 2018). Results revealed that salt stress reduced the sulfate (SO 4 2-) contents in both studied maize varieties. Riffat & Ahmad (2018) reported that high concentration of salts reduced the sulfate (SO 4 2-) contents. While, the sulfur application improved the sulfate (SO 4 2-) contents in the maize plants.

Conclusions and Recommendations
Salt stress caused changes in the organic and inorganic osmolytes in the plants. The imbalance in nutrient contents disturbs the normal plant metabolism. To overcome the adverse effects of salinity some natural osmoprotectants get accumulated in the maize plants. Among these organic osmolytes, glycine betaine, proline, total soluble sugars, total soluble proteins and total free amino acids has considerable importance. The application of sulfur (40 mM) not only balanced the organic osmolytes contents by lowering the higher accumulation of proline to avoid toxic effects but also induced salt tolerance in maize plants. Among the inorganic osmolytes, salt stress increased the Na + contents and lowered the beneficial osmolytes in the maize plants. However, sulfur application at 40 mM proved very effective in improving beneficial osmolytes (K + , Ca 2+ , NO 3 -, PO 4 3-, SO 4 2-, K + /Na + and Ca 2+ /Na + ) in the plants. Hence, it is recommended that sulfur at 40 mM is very much effective in balancing organic and inorganic osmolytes for improving salt tolerance potential.