Gibberellic acid and nitrogen efficiently protect early seedlings growth stage from salt stress damage in Sorghum

Salinity one of environmental factor that limits the growth and productivity of crops. This research was done to investigate whether GA3 (0, 144.3, 288.7 and 577.5 μM) and nitrogen fertilizer (0, 90 and 135 kg N ha−1) could mitigate the negative impacts of NaCl (0, 100, and 200 mM NaCl) on emergence percentage, seedling growth and some biochemical parameters. The results showed that high salinity level decreased emergence percentage, seedling growth, relative water content, chlorophyll content (SPAD reading), catalase (CAT) and peroxide (POD), but increased soluble protein content, superoxide dismutase (SOD) activity and malondialdehyde (MDA) content. The SOD activity was decreased by nitrogen. However, the other measurements were increased by nitrogen. The interactive impact between nitrogen and salinity was significant in most parameters except EP, CAT and POD. The seedling length, dry weight, fresh weight, emergence percentage, POD, soluble protein and chlorophyll content were significantly affected by the interaction between GA3 and salinity. The GA3 and nitrogen application was successful mitigating the adverse effects of salinity. The level of 144.3 and 288.7 μm GA3 and the rate of 90 and 135 kg N ha−1 were most effective on many of the attributes studied. Our study suggested that GA3 and nitrogen could efficiently protect early seedlings growth from salinity damage.

Salinity is an essential environmental stress that affects plant growth and causes limitations of crop production in the desert and semi-desert areas in the world 3,4 . Worldwide, there are about 95 million ha from whole world land, 45 million ha of the irrigated area were affected by salinity. Moreover, due to increment of salinity about 1.5 million ha are become out of production 5 .
Salinity stress can significantly inhibit germination and seedling growth, decrease many physiological processes and ultimately reduce crop productivity by causing osmotic stress and/or toxicity of ions as well as by reducing the uptake of important ions such as calcium and potassium 6 . Crop plants can suffer from salinity stress at all growth stages, but germination and early plant stage are known to be more sensitive for most plant species 7,8 . Salinity stress affect all growth stages, but germination and early seedling stage are known to be more sensitive to salinity, causing significantly inhibited germination and seedling growth and ultimately decreased crop productivity through osmotic stress and ion toxicity such as Na + and Cl − , as well as throught reduced absorption of important nutrients such as Ca +2 and K +6,9 . Fertilization is an effective way to supply nutrients for plants, and it is also an important factor to improve the yield and quality of plants 10 . As compared with other nutrients, nitrogen (N) is required most consistently in larger amounts for crop production 11 . N fertilization has a significant impact on plant growth, development, yield components, and quality, and its effective use to can enhance crop yield in agricultural systems 1

. Saline
Seedling length. The nitrogen and GA 3 treatment affected seedling length positively. At the highest salinity level of 200 mM NaCl, seedling length was increased by 15.4% and 13.6% when the seeds were treated with 135 and 90 kg N ha −1 respectively, as compared with 0 mM NaCl (Fig. 1a). However, at the same salinity level, as compared with control of gibberellic acid (0 μM GA 3 ), the levels of 577.5 and 144.3 μM GA 3 increased the seedling length by 53.0% and 32.3% respectively. The seedling length decreased gradually with increased salinity (Fig. 1b). For the interaction between nitrogen and gibberellic acid, the highest seedling length (24.98 cm) value Table 1. Summary of analysis of variance (ANOVA table) for emergence percentage, seedling length, fresh weight, dry weight, Chlorophyll content (SPAD reding), relative water content (RWC), protein content, superoxide dismutase (SOD), catalase (CAT), peroxide (POD), and malondialdehyde (MDA) of sorghum seedlings as effected by salinity, nitrogen and gibberellic acid and their interactions. ns not significant. *Significant at the 0.05 probability level. **Significant at the 0.01 probability level. ***Significant at the 0.001 probability level. www.nature.com/scientificreports/ was determined on 135 kg N ha −1 with 144.3 μM GA 3 , while the lowest seedling length value recorded at the 0 kg N ha −1 with 0 μM GA 3 (Table 3).

Source of variance
Fresh weight and dry weight. Fresh weight and dry weight was increased by GA 3 and application of nitrogen, and decreased with increased salinity level (Table 2). However, at the high salinity level of 200 mM NaCl, as compared with 0 μM GA 3 , 577.5 μM GA 3 level increased fresh weight and dry weight by 50.0% and 41.6%, respectively (Table 2). Moreover, for the interaction between nitrogen and GA 3 , the highest fresh weight (0.343 g plant −1 ) and dry weight (142.6 mg plant −1 ) value were determined on the 135 kg N ha −1 with 288.7 and 135 kg N ha −1 with 144.3 μM GA 3 respectively. While, the lowest fresh weight and dry weight were recorded at the 0 kg N ha −1 with 0 kg N ha −1 ( Table 3). As compared with control of nitrogen, at the high salinity level, the rate of 135 kg N ha −1 increased fresh weight and dry weight by 89.5% and 59.1% respectively (Table 4).
Chlorophyll content (SPAD reading). The chlorophyll content was decreased with increased salinity level. Chlorophyll content was improved by nitrogen application and GA 3 amendment. At the high salinity level of 200 mM NaCl, the rate of 90 kg N ha −1 increased chlorophyll content by 35.0% as compared with 0 kg N ha −1 (Fig. 2a). At the same salinity, the level of 288.7 and 144.3 μM GA 3 had the highest chlorophyll content (17.62 and 17.19 respectively) (Fig. 2b).  Table 3. Effects of different nitrogen rates and different gibberellic acid (GA 3 ) levels on emergence percentage, seedling length, fresh weight, dry weight, relative water content (RWC) protein content (mg g -1 FW), SOD, and POD of sorghum seedlings. Different letters in the same column show significant differences at the 0.05 probability level.   Catalase and peroxidase activities. The catalase (CAT) and peroxidase (POD) activities were enhanced by nitrogen application and exogenous GA 3 , but decreased by increased salinity level. In the interaction between salinity and GA 3 , at the 200 mM NaCl, the highest value of CAT and POD activities values were recorded at the 144.3 μM GA 3 (Fig. 3a) and 577.5 μM GA 3 (Fig. 3b) respectively. in the interaction between nitrogen and GA 3 , the highest value of POD activity (185.7 µg g −1 FW) was observed at 135 kg N ha −1 with 577.5 μM GA 3 , while the lowest POD (25.0 µg g −1 FW) activity value showed at 0 μM GA 3 plus 0 kg N ha −1 (Table 3). At the high salinity level of 200 mM NaCl, the N rates of 135 and 90 kg N ha −1 increased CAT and POD activity by 69.5% and 22.2% respectively as compared with 0 kg N ha −1 (Table 4).
Superoxide dismutase. Superoxide dismutase activity (SOD) was increased by salinity, nitrogen and GA 3 . The highest SOD activity value was recorded at the 200 mM NaCl with 90 kg N ha -1 , however the lowest SOD activity was recorded at 0 mM NaCl plus 0 kg N ha -1 (Fig. 4a). In addition, the highest activity of SOD www.nature.com/scientificreports/ (23.77 µg g -1 FW) was recorded at 288.7 μM + 200 mM NaCl (Fig. 4b). Seeds treated with 90 kg N ha -1 plus 577.5 μM GA 3 increased SOD activity by 147.7% as compared with 0 kg N ha -1 with 0 μM GA 3 (Table 3).
Malondialdehyde and soluble protein content. The malondialdehyde content (MDA) and soluble protein content were increased with salinity, nitrogen rates and GA 3 levels. The highest MDA content (44.71 µg g −1 FW) and soluble protein content (32.3 mg g -1 FW) were recorded at 200 mM NaCl plus 577.5 μM GA 3 and 200 mM NaCl + 288.7 μM GA 3 , respectively (Table 2). Moreover, the highest soluble protein content (36.5 mg g -1 FW) was recorded at 577 μM plus 90 kg N ha −1 ( Table 3). As compared with the 0 mM NaCl + 0 kg N ha −1 , the rate of 90 kg N ha −1 plus 200 mM NaCl increased soluble protein content and MDA content by 20.0% and 43.7%, respectively (Table 4).

Discussion
High salinity stress affects plant growth by changing their physiological parameters 1 . In this study, seedling emergence percentage and seedling growth (root and shoot length, fresh and dry weight) and relative water content were decreased with increased salinity. The reductions in emergence percentage might be due to the decrease in water uptake and enzyme activity caused by salinity 17 . Similar effects were shown in sweet sorghum 17 , wheat 18 , and forage sorghum 19 . In the present study, shoot and root length decreased as salinity level increased. The decline in root and shoot length and development might be due to ions toxicity or reduction in osmotic potential caused by decreased water uptake and nutrient absorption. The impacts of higher salinity on the root growth were more pronounced than shoot growth and caused the reductions in seedlings growth 20,21 . Some studies indicated that reduced growth of the plant due to the proportional increase of sodium 21   www.nature.com/scientificreports/ Khan et al. 22 , Nimir et al. 17 and Ibrahim et al. 3 . The reduced in fresh and dry weight at the salinity stress might be due to the lowest water absorption cause by physiological drought 23,24 . Our findings are in agreement with those of Dheeba et al. 25 , who reported that salinity reduced the fresh and dry weight of plants. Ibrahim et al. 2 noted that the negative correlation between growth characteristics and salinity stress. The result of decreased in RWC under salinity stress similar with the findings of Ibrahim et al. 21 .
In this investigation, nitrogen application increased seedling emergence percentage, seedling growth and RWC, and alleviated the negative effects of salinity. The increase in emergence percentage by introgen is in agreement with Ibrahim et al. 3 who reported that the lowest emergence percentage was achieved at the high salinity with the control of nitrogen. However, these findings are in disagreement with those of Fallahi and Khajeh 26 , who found that nirogen application had a negative effect on seed germination and emergence percentage under saline stress. The increase in seedling growth under salinity stress by nitrogen is in agreement with Ibrahim et al. 3 and Xiong et al. 27 , who reported that nitrogen fertilizer significantly increased root and shoot length, fresh and dry weight under soil salinity conditions.
In the present study, GA 3 treatment enhanced seedling emergence percentage, seedling growth and RWC. GA 3 significantly affected emergence percentage, and alleviated the adverse impact of salinity stress by improving water uptake and increase cellular membrane plasticity 28 , which can stimulate the activity of amylase in cotyledons and the conversion of insoluble starch into soluble sugars for seed germination and promote radical growth 29,30 . Similar results of increased emergence percentage and seedlings length, by GA 3 were reported by Chauhan et al. 24 . However, our results were different from those of Chen et al. 31 and Shaddad et al. 32 , who reported that GA 3 had negative effects on shoot and root length in wheat and soybean (Glycine max) plants.  www.nature.com/scientificreports/ The difference between these two studies probably lies in the differences in crop species and the levels of GA 3 . Shaddad et al. 32 reported that the exogenous gibberellic acid can improve the growth and physiological parameters of the plant, which are responsible for increased fresh and dry weight of the plant. In this study, GA 3 treatment also improved relative water content (RWC). These results were in agreement with those of Ghodrat and Rousta 33 and Chauhan et al. 24 , who reported that the RWC was increased in response to GA 3 application. Soaking seeds with suitable levels of GA 3 plays an important role in the induction of develop salinity tolerance such as; selective accumulation and/or exclusion of ions, ion uptake control by roots and transport into leaves, compartmentalization of ions at the cellular and whole plant levels, synthesis of compatible solutes, change in photosynthetic pathways, alteration in membrane structure, induction of antioxidative enzymes and induction of some plant hormones 34 . Also, overcome limitations created by the environmental stress such as nutritional imbalance, osmotic effects and ion toxicity increases salinity tolerance in the crops 19 . In the study, nitrogen treatment also increased RWC in the leaves of seedlings sorghum under salinity stress. The type of nitrogen sources or nitrogen level may cause the increased in RWC.
Increased in the anti-oxidative enzymes under salt stress could be suggestive of an increased of ROS and improvement of a protective mechanism to decrease oxidative harm triggered by stress in plants. In this study, salinity stress caused a significantly increased in the soluble protein content, malondialdehyde content (MDA), superoxide dismutase activity (SOD). While, catalase (CAT) and peroxidase (POD) activities was decreased with salinity levels increased. This increased in antioxidant enzyme activity might be due to the activation of plant resistance mechanisms 1 . The increased of accumulation protein may be due to rapid accumulation of a specific set of protein in plant 14 . This results are in disagreement with those of Abdoli and Shekafandeh 35 , Bano et al. 36 , Ibrahim et al. 7 , and Hatami et al. 37 . The decreased in protein content under salinity stress may be due to increased sodium content and consequently reduction in potassium concentration in the cell. Increase in SOD activity could increase the ability of the seedlings to scavenge O 2 and remove the accumulation of ROS, which could cause a reduction the membrane damage. An increased of protein content and SOD activity by salinity were also reported by Ali et al. 19 , Zrig et al. 38 , and Nimir et al. 17 . On other hand, Dissimilar result was reported by Qiu et al. 39 in wheat plant, and Sekmen et al. 40 in Gypsophila oblanceolate, who mentioned that SOD, CAT and POD activities were decreased with increased salinity. The difference results between these two studies probably in the difference in crop species. This results were similar with those of Ali et al. 19 , and Ibrahim et al. 7 , who observed that salinity stress increased MDA, CAT and POD activities. In this study, salinity stress inhibited chlorophyll content as SPAD reading. The decreased in chlorophyll content under salinity stress because of the inhibitory effects of ions of several salts on the biosynthesis of different chlorophyll molecules 41 . These results were in accordance with Jamil and Rha 42 .
In this study, we observed that chlorophyll content (SPAD reading) and antioxidant enzymes activity (SOD, and POD) were decreased slightly with increased nitrogen rate. Related findings have been observed by those of Huang et al. 43 and Ibrahim et al. 7 . In this study, CAT was initially increased with increased nitrogen rate. Related increase in the CAT activity have been also observed by those of Ibrahim et al. 7 , and Huang et al. 43 . Moreover, MDA and protein content were increased when the plants were treated with N. These findings are in agreement with Ibrahim et al. 7 .
In this study, the chlorophyll content, soluble protein, MDA content and activity of antioxidant enzymes were increased with increased GA 3 . Our study explained that exogenous GA 3 could increase SOD, CAT and POD activities in sorghum seedlings under salt stress, and improved the seedlings ability to combat oxidative damage. This observation was contrary to the findings of Ali et al. 19 and Zhu et al. 44 , who reported that SOD activity in salt stressed was decreased by exogenous application of hormones. Similar result was reported by Zhu et al. 44 who reported that CAT and POD activities improved by GA 3 amendment was beneficial for okra plants to be more efficient in breaking H 2 O 2 in to O 2 and H 2 O. While, Tuna et al. 45 reported that POD activity were decreased by increased exogenous GA 3 at 144.3 and 288.7 µM in maize plant under. The difference results in POD activity between these two studies probably lies in the difference in crop species and their activity to GA 3 concentration 46 . Related increased in MDA content have been also observed by those of Ali et al. 8 , who reported that MDA content was increased with increased hormone.

Conclusion
Nitrogen was beneficial or even necessary for sorghum growth and development, especially under saline conditions. Our study investigated the effects of external application of GA 3 and nitrogen application on seedlings emergence percentage, seedlings growth and antioxidant enzymes of sorghum seedlings subjected to salinity. The findings from this study showed that seedling emergence percentage, seedling growth, and antioxidant enzymes were inhibited by NaCl salinity stress. Nitrogen and GA 3 had a positive effect on seedlings emergence percentage; seedling growth and antioxidant enzymes by increased these parameters. However, from the present study, it can be concluded that nitrogen management is important when the plant growth in the salinity soil. Further, study to examine the effect of nitrogen and GA 3 is needed to optimize the effectiveness of nitrogen fertilizer and seed treatments with GA 3 on more cultivars of sorghum will help us to see if there is any relationship between nitrogen and GA 3 and salinity tolerance of the seeds during seedling growth stages.

Materials and methods
Experimental site and soil characteristics. A  Institute, Sudan were used in the research. Seeds were selected for color, shape, and symmetric size. Before the study, the sorghum seeds were surface-sterilized with 1% sodium hypochlorite solution for 2 min and then were washed three times with distilled water, and then was dried with air to their original weight (50 g).

Preparation of treatment and experimental design.
This study contented three factors, including three salinity concentrations at 0, 100 and 200 mM NaCl, three rates of nitrogen (0, 90 and 135 kg N ha −1 ), and four gibberellic acid levels were applied: 0, 144.3, 288.7 and 577.5 μM. The study was conducted in an RCBD as a factorial experiment arranged in a split-split-plot with three replications. The main plots included three different salinity levels, the subplots included three different levels of nitrogen fertilizer as urea and the sub-sub-plot included four levels of gibberellic acid. Before seed planting, seeds (50 g) were soaked in 500 mL of one of the gibberellic acid solutions under dark conditions for 12 h, while the control was treated with distilled water. The seeds were dried with forced air for 48 h to their original weight to safe moisture content of seeds. The pots used in this study are 9.5 cm in diameter and 8.5 cm in depth. Each pot was filled with 400 g washed sand. Nitrogen treatment was made by nitrogen solution at the first irrigation with the same amount (10 mL) of 0, 90 and 135 kg N ha −1 . Urea is the source of nitrogen fertilizer. Salinity treatment was made by the NaCl solution in each pot by the first irrigation with the same amount (80 mL) 2 . Ten seeds were sown in each pot at 2 cm in depth. All the pots were placed in the growth chamber (Model PYX-300G-B, Yangzhou Yiwei Automatic Instrument Co. Ltd, Jiangsu, China) for three weeks at 30/25 °C day/night. The relative humidity was maintained at 55-60% and 14/10 h day/ night under a photoactive radiation (PAR) of 500 W m −221,47.

Measurements. Emergence percentage (EP%).
Seedlings were considered emerged when the coleoptiles were visible above the substratum surface. After 10 days, seedling emergence percentage was calculated with the following formula: Seedlings growth attributes. At 21 days after planting, the seedlings of each pot were harvested and washed. Seedling growth parameters were measured including seedling length, fresh weight (FW) and dry weight (DW). DW was recorded after dry the seedlings in the oven at 80 °C for 80 h.
Relative water content. Relative water content (RWC) was measured according to the described by Mäkelä et al. 48 . Leaflet samples were harvested from the three plants. The FW was determined, and the leaves were kept in water for 8 h for saturation weight measurement. The samples were dried in a hot air oven at 80 °C for 72 h to determine DW. The RWC was calculated as the following formula: Chlorophyll content (SPAD reading). Eighteen days after planting, the penultimate leaves of each seedling in each pot were used for SPAD determination with a chlorophyll meter (SPAD-502, chlorophyll meter, Minolta Camera Co., Ltd., Japan). The SPAD reading was recorded for three seedlings. The average of SPAD readings of the seedlings of each pot was calculated.
Determination of biochemical attribute. The soluble protein content was determined according to Bradford 49 . The peroxide (POD) activity was assayed according to the method of Xu and Ye 50 . The activity of superoxide dismutase (SOD) and catalase (CAT) was measured following the method of Janmohammadi et al. 51 . The malondialdehyde (MDA) content was determined following the method of Zhang et al. 52 .
Statistical analysis. The data of each variable were statistically analyzed of variance for RCBD as a factorial design with the statistical package of MSTATC 53 . When F values were significant, means were separated by the least significant difference (LSD) test (P ≤ 0.05 probability) as described by Snedecor  www.nature.com/scientificreports/