Humic Acid and Jasmonic Acid Improves the Growth and Antioxidant Defense System in Salt Stressed - Forage Sorghum Plants

Salinity is one of the primary abiotic stresses that cause several negative physiological and biochemical changes due to the oxidative stress caused by the generation of ROS. The effect of foliar application of jasmonic acid (JA) and humic acid (HA) as a fertilizer on growth and biochemical attributes exposed to salinity stress was investigated. Soil treated with NaCl at levels of 0 (S0), 2 (S1), and 4 g NaCl kg-1 dry soil (S2) and fertilized with 0 (HA0), 3 (HA1), and 6 g HA kg-1 dry soil (HA2). The plant spray with three JA levels (0, 5, and 10mM JA). Under salinity, JA and HA signicantly improved all parameters tested. Salinity stress increased carotenoid, soluble protein content, SOD and MDA. In contrast, salinity stress reduced plant height, leaf area index, relative growth rate, proline content, POD, CAT, and APX. Under S2, HA2 rate increased plant high (9.69%), relative growth rate (70.79%) and CAT (45.47). While, HA1 increased leaf area index (12.45%), chlorophyll content (22.32%), carotenoid contents (38.05%), SOD (20.93), MDA (17.95%), POD (24.64%) and APX (21.67%). At S2, the highest plant height, chlorophyll content, soluble protein content and APX value recorded at 5mMJA, while, the highest value of leaf are index, carotenoid contents, proline, MDA, POD and CAT was achieved at 10mMJA. This study revealed that the level of 10 mM JA and HA1 had a positive effect on forage sorghum plants physiological responses. Furthermore, the results showed that jasmonic acid and humic acid successfully mitigated salinity stress's adverse effects. of antioxidant enzymes combated the adverse effects of stress during plant growth. total chlorophyll content, carotenoid and ant oxidative enzymes parameters of forage sorghum plants subject to salinity. Here, we examine the possibility of mitigating salinity stress by using exogenous JA and HA applications. Therefore, the present study was done to assess the interactive effects of salinity, JA, and HA on chlorophyll a and b, carotenoid, protein, proline, and ant oxidative enzymes parameters of forage sorghum. carotenoid under saline Thus, higher leaf chlorophyll content is one of the additional factors that may have contributed to a higher photosynthetic of plant under saline conditions. The results presented show that foliar application of JA led to a signicant increase in total chlorophyll content and carotenoid content concentration under soil saline stress. 5 , and 43 reported that exogenous JAs signicantly improved the total leaf chlorophyll content exposed to salinity stress. These results suggested that exogenous JA treatment could alleviate salinity stress, allowing plants to increase their tolerance to unfavorable conditions. in our in proline contents with the comparison with the controls was reported by Farahat et al. (2012) during an investigation of exogenous applications of humic acid on seedlings of Khaya senegalensis. The agreement results by increased protein content were showed by 67 and 68 . These results are same with 9 , who reported that the improved accumulation of protein content might be due to the rapid accumulation of a specic protein set. In the present investigation, HA improved the protein content. Similar ndings were reported by 69 and 62 . JA can protect the plant from toxicity ions in the different stages by managing the antioxidant machinery and synthesis of proteins 70 . Enhancement of proline content in the plant under salinity stress is associated with the increment of some enzymes such as Pyrroline-5- carboxylate synthase. In this study, JA improved proline content. An increased in proline contents in comparison with the control by jasmonic acid was reported by Ali. (2020). Our result disagrees with 5 , who reported that jasmonic acid reduced proline content under salt stress in the wheat. study, stress the activity of and MDA content. The increase in with an increase in the of Mn-SOD and Fe-SOD. The reduction in SOD activity could minify the plant's ability to scavenge O 2 radicals favoring an accumulation of ROS, which could cause membrane damage 48 . These results were contrary to 71 , who reported that the SOD activity was decreased under salinity stress in Gypsophila oblanceolate plant. However, agree results were reported for sweet sorghum and sunower by Nimir et al. (2015) and 72 , who noted that salinity stress could increase the same antioxidant enzyme activity. This result similar with the ndings of 73 , and 65 in forage sorghum and sweet sorghum, who suggests that under soil saline, MDA content was substantially increased by an increase in soil salinity as compared with the non-soil salinity. Our study showed that a signicant increase in SOD activity and MDA content was observed in forage sorghum plants treated with JA, suggesting that JA had a good O 2- scavenging ability to protect the plant from oxidative damage. Our study of the increase in SOD activity agree with the results of 57,74 who reported that exogenous JA application of soybean under salinity stress signicantly improved the activities of SOD. However, our study of the an increase in MDA content under salt by jasmonic acid different from the ndings of 75 , and 66 , who reported that JA and MeJA treatment could effectively alleviate NaCl stress-induced by oxidative stress as indicated by the decreased in the MDA, H 2 O 2 and the production rate of O 2- . Similar results were reported by Ali et al. (2019). Jasmonic acid might fulll crucial roles in scavenging radicals, thus inhibiting lipid peroxidation by excess ROS produced under salinity conditions 66 . HA Disagree result results Our study examines the effects of salinity stress on growth parameters, chlorophyll content, carotenoid content and plant defense system of forage sorghum exposed to different humic acid rate as fertilizer and jasmonic acid application as a foliar spry. Soil salinity stress at 4 g NaCl kg -1 soil decreased all study parameters, except protein content, SOD activity, and MDA. In this study, we found that forage sorghum spray with jasmonic acid at 10 mM JA level successfully improved POD and SOD activities. Among different humic acid rate, 3 g HA kg -1 dry soil successfully increased all parameters. These results proposed that 5 mM JA could eciently protect the forage sorghum plant from salty stress damage by enhancing total chlorophyll content, carotenoid content, and antioxidant enzyme. The interaction between 3 g HA kg -1 soil and 5 mM JA was most effective in alleviating salinity stress. Therefore, JA and HA combined applications could enhance the chlorophyll content, carotenoid, and antioxidant enzyme in plants. Therefore, JA and HA application management are required in salt-affected soil to sustain forage growth and increase crop yield and productivity under salinity conditions.


Introduction
Salinity stress is one of the abiotic stress, accumulation of salt stress in plants when passes the different level, resulting in toxicity in plants and led to many changes of physiological and morphological 1,2 . Sodium chloride is essential for structural and functional parts of the vital machinery of the plant cell. The requirement of NaCl for the plant is very low for normal growth and development. Unfortunately, plants nd an ample supply of NaCl through their roots from the soil and accumulated in the system, causing stress and triggering speci c physiological responses 3 . Salinity stress affects all plant growth stages. The root zone is known to be more sensitive to salinity, caused signi cantly inhibit root elongation, and ultimately reduce crop yield by causing osmotic stress and ion toxicity such as Na + and Cl -, as well as by reducing the absorption of essential nutrients such as Ca +2 and K +4,5 .
Reactive oxygen species (ROS) are very harmful to the plant growth and development of most organisms as they affect the structure and function of biomolecules 6,7 . Anti-oxidative enzymes such as catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) remove H 2 O 2 to hydrogen peroxide and dioxygen [6]. The activity of SOD, CAT, and POD increase during biotic and abiotic stresses to protect cells from the potentially hazardous effects of ROS 8 . CAT e ciently scavenges H 2 O 2 and does not require a reducing substrate to perform the task 6,7 . CAT was localized in leaf tissue in peroxisomes to scavenge the H 2 O 2 produced by glycolate oxidase. CAT isozymes differ in biochemical properties and developmental speci city, some related to germination, but their principal role involving probably fatty acid conversion. In contrast, others were related to ligni cation, photorespiration, or the aging process 9 . MDA was produced when polyunsaturated fatty acids in the membrane undergo peroxidation 10 .
Jasmonic acid (JA) is a lipid-derived plant hormone that mediates diverse biological phenomena. It is a member of plant growth regulators named jasmonates, which are important cellular regulators involved in several developmental processes such as seed germination, root growth, fertility, fruit ripening, and senescence. Jasmonate (JAs), including methyl jasmonate (MeJA) and jasmonic acid (JA), stimulates MDA accumulation and inhibition of the Fe-induced release of chelators, intracellular chelators counteract the salt stress, and it suggests that MeJA trigger some other protective mechanisms 11 . 12 observed that high JA levels in wounded leaves caused signi cant changes in the protein pattern of rice plants. Since JAs are the mediator of cellular responses in defense or stress 13 . Ascorbic acid (ASA) is an essential antioxidant, which protects the plants under oxidative stress 6 . Exogenous JA application increases and may regulate ASA metabolism in different plants 13 . 14 reported that ASA increased due to wounding, which led to improved stress resistance. Likewise, 15 also demonstrated enhanced accumulation of AsA Arabidopsis by wounding. MeJA enhanced ASA in Arabidopsis and tobacco suspension cells and in uenced ASA's metabolism 16 . Soares et al. (2010) have demonstrated gradual accumulation of H 2 O 2 in Ricinus communis (L) and a sharp formation of ROS at the initial moment of MeJA and ascribe ditto the decrease in the enzymatic antioxidants activities. In another study with pomegranate (Punicagranatum L.), 18 observed an increase in antioxidant activity in the foliage treated with JAs. 16 found that in the treatment of plants with JAs application, APX activity was upregulated in tobacco plants, and less membrane damage of barley plants. In addition to its role in plant growth and development, jasmonate has been proposed as a critical regulator of plant responses to salinity 19 .
Forage sorghum (Sorghum bicolor (L.) Moench) is an essential crop for fresh fodder, hay, and silage. And is becoming increasingly important in many regions of the world 20 because of its high productivity and high nutritive value, and it can adapt to different environmental conditions, especially in arid and semi-arid areas 21 . Sorghum is considered a moderate tolerance to soil salinity 22 . Due to the progressive salinization of the world arable lands, the exogenous hormone protectants to mitigate salt-induced damages has been more important than ever. However, to our knowledge, there are no reports on the effects of exogenous application of jasmonic acid and humic acid application in forage sorghum on soluble protein, proline content, total chlorophyll content, carotenoid and ant oxidative enzymes parameters of forage sorghum plants subject to salinity. Here, we examine the possibility of mitigating salinity stress by using exogenous JA and HA applications. Therefore, the present study was done to assess the interactive effects of salinity, JA, and HA on chlorophyll a and b, carotenoid, protein, proline, and ant oxidative enzymes parameters of forage sorghum.

Location and climate of the experimental site
A two-year pot experiments were conducted in a greenhouse in two consecutive years (2018 and 2019) at the experimental farm that belongs to the Yangzhou University, Yangzhou, Jiangsu Province, located in west China (lat. 32°39′N ; long. 119°41′E ). Data of monthly, the average temperature was 31°C, the minimum relative humidity was 76%, and cloud cover was 40%, day/night cycle of 16 h/8 h, at 35 1C/26 1C, respectively and a light intensity of 200 mmol photons m 2 s 1 were obtained from the weather station located at the experimental farm.

Soil characteristics
The soil collected from the surface of sandy loam soil (0-20 cm) of the Experimental Farm of Yangzhou University. The soil air-dried and passed through a 10 mm mesh screen. The soil was then separately spread at a thickness of about 70 mm over a piece of polyethylene sheet. The soil suspension was prepared in deionized water at a ratio of 1:2 (w/w) soil: water. The suspension shaken and allowed to stand overnight. After that, the electrical conductivity of the supernatant solution was determined at 0.26 dS/m using a conductivity meter. The soil presented a sandy loam texture with a pH of 7.1, 12.2 g/kg of organic matter, 1.0 g/kg of total N, and 14.1 mg/kg Bray -1 and 77.3 mg/kg of P and K, respectively.

Plant material
Pot performance of the forage sorghum seeds variety Abu sabeein adapted to Sudan conditions, kindly donated from the Agricultural Research Corporation (Madani, Sudan), were evaluated in a randomized complete block design with three replications. Seeds were less than eight months old and had been stored in paper bags under laboratory conditions (RH 40-60% at 15-20℃). Seeds were surface-sterilized with 3% sodium hypochlorite solution for 1 min and then thoroughly rinsed three times with deionized water and air-dried near to their original weight for seeding.
Seeds were germinated on 10 th May in 2018, and on 15 th May in 2019, on seedbed for 15 days at the greenhouse. The strongest and uniform seedlings selected and transferred into pots (32 m × 45 m). The crop was fertilized at the rate of 150 kg ha -1 , as NH 4 NO 3 (nitrogen), 100 kg ha -1 as P 2 O 5 , 100 kg ha -1 , as K 2 O (phosphorus and potassium), and 0.5 kg ha -1 , as ZnSO 4 (zinc) were applied before planting. Another half dose used on the 45 th days of transplanting. Furthermore, the plants were watered regularly to maintain the water level every three day with a tap water. According to local recommendations, conducted the spray of pesticides and weed control. All required approvals were obtained for the study, which complied with all relevant regulations.

Experimental treatments
The study consisted of three experimental factors, three salinity levels, three humic acid rates, and 3 levels of jasmonic acid. Three different humic acid rates supplied as fertilizer, including 0, 373.21, and 746.42 kg HA ha -1 were combined with 3 different NaCl levels including 0. 2 and 4 g NaCl kg -1 dry soil (0.26, 2.3, and 4.7 dS m −1 respectively) making a 9 different treatments solutions was added to the non-saline soil before seedling transferred. The control treatment of soil (without salinity and humic acid) was created by adding tap water (0.26 dSm). The different humic acid rates and salinity levels were chosen based on previous seedlings. Before salinity and humic acid treatment, a 100 g soil sample was collected and oven-dried at 75 o C to constant weight, and the moisture content was calculated.
For the jasmonic acid, on the 20th day after seedling transferred, the plants at each NaCl level and humic acid rates were treated with exogenous jasmonic acid solutions (0, 5, and 10 mM) as a foliar application. The spraying was then repeated every 15th day. During jasmonic acid application, care was taken to avoid any drift of different levels using a plastic shelter to separate each treatment.

Observations and measurements
2.5.1. Plant height (cm) and leaf area index Three plants from every plot will randomly select and tagged, plant height will measure from a point immediately above the soil surface to the top of the plant, and then the mean of height per plant will obtain in cm. Four leaves will randomly select the same plants, two plants selected from each plot to measure leaf area using a leaf area meter.

Relative Growth Rate (RGR):
The RGR was calculated according to 23 : 2.5.3. Preparation of enzyme extracts After 45 days of the seedling transfer, the leaves harvested and immersed in liquid nitrogen for 20 min and stored in a low-temperature freezer to determine enzyme activity, proline, and protein content. Leaf protein extract was made by sodium phosphate dibasic dehydrate and sodium phosphate monobasic dehydrate to makes a phosphate buffer solution. 0.2 g of fresh weight tissue with 2 ml of the phosphate buffer solution crushed, and the slurry centrifuged for 20 min at 4°C. The supernatant, which contained enzyme activity, used as the enzyme source.

Soluble proteins contents
Protein contents were estimated for each extract 24 . A dye stock solution added to the earlier centrifuged samples and incubation at room temperature for 25-30 min. The absorbance of the reaction mixture recorded at 595 nm.

Proline contents
Proline contents were estimated using the protocol of 25 . Fresh samples (0.5g) extracted with sulfosalicylic acid, and the extract ltered to separate the residue. All the ltrates mixed with acidic ninhydrin, orthophosphoric acid, and glacial acetic acid and incubated at 100°C for 30 min. The mixtures cooled, incorporated in toluene, and vortexed. Absorbance recorded at 520nm with a spectrophotometer.

Determination of physiological parameters
The activity of SOD and CAT was determined following the method of 26 . The POD activity was assayed according to the method of 27 . The MDA content was determined following the method of 28 . The APX content was measured according to 29 .

Total chlorophyll content and carotenoid content
The determination of chlorophyll a and b and carotenoid content pigments were created according to the method reported by 30 . Each fresh leaf sample was soaked in acetone solution (80%) in the dark. The extracted samples centrifuged. The absorbance recorded at 453, 645, and 663 nm using a spectrophotometer.

Experimental design and statistical analysis
The study consisted of three experimental factors, three NaCl levels; 0.26 (S0), 2.3 (S1), and 4.7 dS m −1 (S2), three levels of humic acid (0, 373.21, and 746.42 kg HA ha -1 ) designed as HA0, HA1 and HA2 respectively, and 3 levels of jasmonic acid at 0, 5, and 10 mM JA. The experiment designed as a factorial design with three and arranged in a randomized completely block design (RCBD) with three replications for each treatment. There were 81 pots in this study. All pots placed in a greenhouse according to the experimental design.
The data of each variable subjected to analysis of variance (ANOVA) for the factorial RCBD with the statistical package of MSTATC according to this design 31 . When F values were signi cant, means were separated by the least signi cant difference (LSD) test (P ≤ 0.05) of probability as described by 32 .

Results
The results revealed that jasmonic acid, humic acid, salinity, and their interaction signi cantly affected measured parameters on most occasions (Table 1). Table 1 Analysis of variance for effects of jasmonic acid, humic acid, salinity and their combination on growth parameters total chlorophyll content carotenoid contents, protein, proline, and antioxidative enzymes activities of forage sorghum.

Growth parameters as affected by the combination between salinity and humic acid
Growth traits such as plant height (PH), leaf area index (LAI), and relative growth rate (RGR) signi cantly decreased slightly with progression of soil salinity concentration. Without humic acid (HA0), high soil salinity concentration of S2 was reduced the plant height by 9.10% (Fig 1a), leaf area index by 20.01% (Fig. 1b), and relative growth rate by 44.66% (Table 2), in comparison to control (S0) at HA0. The growth parameters was improved by humic acid. At S2, high rate of humic acid of HA2 was increased a plant height by 9.69 (Fig 1a), and relative growth rate by 70.79 (Table 2), in comparison to HA0 (Fig. 1a). At the same salinity level, HA1 rate was increased the leaf area index by 12.45% (Fig. 1b), in comparison to HA0 (Fig. 1b). At the S1, HA1 rate was achieved the highest plant height value ( Fig. 1a), leaf area index value (Fig. 1b), and relative growth rate value (Table 2).

Growth parameters as affected by the combination between salt and jasmonic acid
Regarding the combination between NaCl and JA. Jasmonic acid had the positively effect and improved the PH, LAI and RGR. Both jasmonic acid levels had increased growth parameters. According to the results, at high saline concertation of S2, both jasmonic acid levels of 5 mM and 10 mM, had signi cant increased on plant height by 23.63 and 14.20 % respectively (Fig. 2a), and leaf area index by 10.07 and 14.71% respectively (Fig. 2b), in comparison to salinity control. Under S1, 5mM was increased plant height from 125.55 to 156.16 cm (Fig 2a), and leaf area index from 231.25 to 251.88 cm 2 (Fig 2b).  (Fig. 2b). The total chlorophyll content and carotenoid content was improved by humic acid. At S2, HA1 rate was increased the total chlorophyll content by 22.32 % (Fig. 3a), and carotenoid content by 38.05% (Fig. 3b), in comparison to control (Fig. 1b). At the S1, the highest value of total chlorophyll and carotenoid recorded at the HA2 (Fig2a and b respectively).
3.4. Soluble protein content, proline content chlorophyll content and carotenoid as affected by the combination between jasmonic and salinity Soluble protein content and proline content signi cantly decreased slightly with increased soil salinity concentrations. Without jasmonic acid (0 mM), high salinity concentration of S2 was reduced the proline content by 54.64% (Fig. 4c), in comparison to control (S0) at HA0. Moreover, at the same rate of jasmonic acid (0 mM), S2 increased soluble protein content by 26.18% (Fig. 4d). Regarding the interaction between salinity and jasmonic acid. Jasmonic acid had the positively effect and improved the soluble protein content, proline content, total chlorophyll content and carotenoid content. Both jasmonic acid levels had increased on these parameters. According to the results, at high saline concertation of S2, 5 mM level had highly signi cant increased on chlorophyll content and soluble protein content by 48.21% (Fig. 4a) and 4.52% (Fig. 4c), in comparison to salinity control. At the same salinity level, 10 mM increased carotenoid content by 10.67% (Fig. 4b), and proline content by 58.50% (Fig. 4d).

Jasmonic acid and humic acid improvement the growth parameters and chlorophyll content
Plant height, relative growth rate, chlorophyll content and carotenoid were affected by interaction between jasmonic acid and humic acid rates. According to the results, forage sorghum plants treated with a HA2 and spray with 5 mM JA was achieved the highest value of plant height (263.56 cm) and relative growth rate (5.28). However, treatment of HA1 + 5 mM was recoded the highest an average value of chlorophyll content (3.53 mg g -1 FW) and carotenoid (1.77 mg g -1 FW) ( Table 3). 3.6. Antioxidants enzyme as affected by the salinity and humic acid Salinity stress was signi cantly reduced antioxidants enzyme except SOD and MDA. However, increased salinity stress was signi cantly increased SOD and MDA content. Without treated the plant with humic acid (HA0), high salinity stress of S2 was decreased the POD, CAT, and APX by 33.84% (Fig. 5c), 42.07% (Fig. 5d) and 36.44% (Table 2) respectively, over the control (S0). Moreover, at the same rate of humic acid (HA0), as compared with S0, S2 was increased SOD from 14.34 to 16.96 U g -1 min -1 (Fig. 5a) and MDA from 5.67 to 9.75 U g -1 min -1 (Fig. 5b). The antioxidants enzyme was enhanced and increased by humic acid application. At S2, the medium rate of humic acid of HA1 was increased a SOD by 20.93% (Fig. 5a), MDA by 17.95% (Fig. 5b), POD by 24.64% (Fig. 5c), and APX by 21.67 (Table 2), in comparison to control (HA0). At the same salinity level, HA2 rate was increased the CAT by 45.47% (Fig. 5d) over the control.

Antioxidants enzyme as affected by the combination between JA and salt stress
Regarding the combination between salinity and jasmonic acid. Jasmonic acid had the positively effect and improved the antioxidants enzyme. Both jasmonic acid levels had increased on antioxidants enzyme activities. According to the results, at S2, 5 mM and 10 mM jasmonic acid levels had signi cant increased on MDA by 20.74 and 43.17% respectively (Fig. 6a), POD by 29.67 and 47.54% respectively (Fig, 6b), CAT by 22.61 and 51.78% respectively (Fig. 6c), and APX by 67.84 and 22.57% respectively (Fig. 6d), in comparison to jasmonic acid control (0 mM JA). Under S1, 5mM JA was more effected on the antioxidants enzyme activities except APX. As compared with 0 mM JA, 5 mM JA increased MDA from 6.81 to 8.62 U g -1 min -1 (Fig 6a), POD from 52.91 to 58.75 U g -1 min -1 (Fig 6b), and CAT from 21.23 to 28.23 U g -1 min -1 (Fig. 6c). While 10 mM JA was increased the APX from 6.81 to 8.62 U g -1 min -1 (Fig. 6d). On the other hand, the treatment of JA0 with HA0 recorded the lowest value of soluble protein content and most antioxidants enzyme activities (Table  4).

Discussion
When plants are grown under saline conditions, as soon as the new cell starts its elongation process, the excess of salts modi es the cell wall's metabolic activities, causing the deposition of various materials that limit cell wall elasticity. Cell walls become rigid, and consequently, the turgor pressure e ciency in cell enlargement decreased.

Growth parameters
In this study, NaCl salinity stress signi cantly inhibited plant growth traits of forage sorghum, including plant height, leaf area index, and relative growth rate. Plant height gradually decreased with increased salinity. These results indicated that the inhibition in plant height might be due to ions' toxicity, decreased nutrient absorption and decreased elongation of the internodes. Also reduced internode length and reduced formation of new apical tissues 33,34 . The contrary results were reported by 35 , who noted that the plant height signi cantly increased at the low salinity levels, sill, at the high salinity level, the plant height signi cantly decreased. However, similar ndings were reported by 36 on bean (Vigna aconitifolia L.), 37  The decrease in leaf area index under salinity stress has been attributed to suppressed cell division 39 . The reduction on the leaf area index under salinity stress might be due to shrinkage of the cell contents these lead to reduced development and differentiation of tissues, unbalanced nutrition, and disturbed avoidance mechanism 40 . Our ndings agree with 41 , who noticed a negative correlation between salinity and leaf area index.
Relative growth rate (RGR) depends on canopy photosynthesis per area unit of land. The RGR of plants in the high salinity treatment was lower than that of the other salinity treatments. Our results disagree with 39 they mentioned that the cheatgrass plants in the lowest and medium salt treatments experienced a reduction in RGR by reduced in plant growth and leaf elongation after salinity application. A similar nding with 39 at the nal harvest reported that the RGR decreased in the high salinity level.
Plant hormonal play essential roles in stress responses and adaptation. It is clearly de ned that jasmonic acid (JA) increased in response to salinity 42 . In this study, the foliar spraying of JA alleviated the adverse effects of salinity stress on plant height, leaf area index and relative growth rate.  50 stated that the HA increases plant growth through increased in nutrients uptake to overcome the lack of nutrients, this lead to bene cial effects on growth, production, and quality improvement of agricultural products. In this study, under different salinity levels, the highest plant height, leaf area index, and relative growth rate was recorded at the different humic acid rates at the vegetative growth stage. The increase in the plant height in the HA amended treatments most probably was due to the root zone's improvement 51 . Our results showed that HA could relieve the growth inhibition induced by NaCl in plant height leaf area index, and relative growth rate. Similar impacts were shown by 52 for (Borago o cinalis L.) and 53 for corn plants, who reported that HA increased the plant length. 54 they noted that HA has remarkable effects on the plant's vegetative growth and increases photosynthetic activity and leaf area index. The results were consistent with 55 , who reported that, relative growth rate was enhanced by the treatment with the application of potassium fertilizer and humic acid than control.

Total chlorophyll content and carotenoid content
Salinity stress adversely affected antioxidant enzyme activity 49,56 . The chlorophyll content is widely used as an index to indicate the abiotic tolerance level in plants. Protection of chloroplast and photosynthetic machinery, including the chlorophyll content, is the rst target of defense under stressful conditions 57 . It is well documented that the plants exposed to stressful environments such as salinity resulted in decreased chlorophyll concentration, thereby leading to overall retarded growth 58 . In this study, soil salinity caused a decrease in total chlorophyll content, but increased the carotenoid content on a forage sorghum plant), which is in agreement with some previous studies on different crops e.g., sun ower ( Application of jasmonic acid and humic acid increased the total chlorophyll content and carotenoid content under saline conditions. Thus, higher leaf chlorophyll content is one of the additional factors that may have contributed to a higher photosynthetic of plant under saline conditions. The results presented here show that foliar application of JA led to a signi cant increase in total chlorophyll content and carotenoid content concentration under soil saline stress. 5 , and 43 reported that exogenous JAs signi cantly improved the total leaf chlorophyll content exposed to salinity stress. These results suggested that exogenous JA treatment could alleviate salinity stress, allowing plants to increase their tolerance to unfavorable conditions. Humic acid caused stimulation in photosynthetic pigments may be due to the decrease of pH value and increase in the activity of soil organisms which release more nutrients from the soil such as Fe 59 . As the amount of HA increased in our study, the total chlorophyll content and carotenoid content also increased. These results con rm by 60 and 61 . The HA application can enhance photosynthesis activity like chlorophyll and increase tolerance in stress conditions by increasing the enzyme rubisco 59 .

Soluble protein content and proline content
Improvement of the soluble protein and proline content are an important mechanism that alleviates and protection the plants from the harmful effect of salinity stress 62 1 . In the present investigation, salinity stress increased soluble protein content. However, proline content was decreased with salinity increased. This different result was reported by the results of 63 , who noted that under salinity stress, the soluble protein content was signi cantly reduced. The reduction in soluble protein content when the soil is subjected to salinity may be due to decreased potassium content.
Consequently, increased sodium content, proline synthesis, protease enzyme activity, and hydrolysis of the rubisco enzyme 62 . Moreover, these results were similar to 64 and 65 . Similar results by increased proline content were reported by 65 . These ndings were dissimilar with 66 , who reported that salinity stress signi cantly increased the proline content in wheat and paulownia (Paulownia imperialis L) plants.

Superoxide dismutase and Malondialdehyde contents
Superoxide radicals (O2 -) generated by oxidative metabolisms were detoxi ed by superoxide dismutase (SOD) and converted into H 2 O 2 and O 2 . SOD is one of the enzymes responsible for eliminating O 2 and is considered an essential antioxidant in cells. Improved SOD activity positively correlated with improved protection from damage and adverse effect associated with oxidative stress induced by salinity stress 57 . The malondialdehyde contents (MDA) a product of lipid peroxidation, is generally an indicator of free radical damage to cell membranes causing severe oxidative stress 57,58 . In our study, high salinity stress increased the activity of SOD and MDA content. The increase in SOD activity coincided with an increase in the activities of Mn-SOD and Fe-SOD. The reduction in SOD activity could minify the plant's ability to scavenge O 2 radicals favoring an accumulation of ROS, which could cause membrane damage 48 . These results were contrary to 71 , who reported that the SOD activity was decreased under salinity stress in Gypsophila oblanceolate plant. However, agree results were reported for sweet sorghum and sun ower by Nimir et al. (2015) and 72 , who noted that salinity stress could increase the same antioxidant enzyme activity. This result similar with the ndings of 73 , and 65 in forage sorghum and sweet sorghum, who suggests that under soil saline, MDA content was substantially increased by an increase in soil salinity as compared with the non-soil salinity.
Our study showed that a signi cant increase in SOD activity and showed by 61 reported that the HA fertilizer reduced the activity of SOD in the Maize plant under NaCl salinity stress. Similar results were reported by 76 , who found that HA fertilizer increased the antioxidant enzyme activity, including SOD activity in response to salinity stress. Similar results were reported by 77 , who showed that MDA content under NaCl-stressed plants increased signi cantly by applying a humic acid.

Catalase and peroxidase activity
Enhancement of the anti-oxidative enzymes in plants under saline condition could increment ROS and improve a protecting mechanism to decrease adverse impacts by salt stress. In this study, soil saline stress caused a reduction in catalase (CAT) and peroxidase (POD) activities. Reduced CAT activity under salinity stress might have promoted H 2 O 2 accumulation, which could result in a Haber-Weiss reaction from hydroxyl radicals, which are known to damage biological systems 78 . Our study of decrease in POD and CAT activities also con rmed with the results of 66 and 71 . The discon rmed results has been shown by 79,80 for wheat plant treated with soil saline, who reported that POD activity increase under NaCl stress. Also, discon rmed results were shown by 81 for maize and by 82 for wheat, who noted that salinity stress increased CAT activities.
In this study, foliar application of jasmonic acid could improve CAT and POD activities in the leaves of forage sorghum plants under soil saline condition. Moreover, the highest value of POD activity was shown in the 10mM at high salinity concentration and 5 mM JA under medium salinity concentration and the lowest value of POD have been registered at the control. Our result was similar to the ndings of 57 , who suggested that the wheat and soybean plants exposed to salinity and treatment with JA application signi cantly increase the antioxidant enzyme activities including CAT and POD and play an essential role for ant-oxidative defense required for salt tolerance. Also, 5 noted that the treatment of soybean plants under salt stress by salicylic acid and jasmonic acid promote the antioxidant enzyme activities.
Humic acid (HA) plays an essential role in the plant by improvement the biochemical and physiological processes, synthesize of protein and ability of roots absorption of the nutrients and water 83 , these processes lead to an increase the activation, concentrations and stimulation of the antioxidant's enzymes 84 71 . However, different results by 5,86 who reported that salinity stress reduced APX.
The capability of HA as a scavenger to reactivate oxygen species caused by regulating the direct water ow and solutes between the cytoplasm and vacuolar compartments, the ability to regulate turgor and osmotic pressure, membrane permeability, and cell osmotic balance 87 . In our results, HA was positive effective on APX activity. Increased APX activity under HA application has been reported in maize plants exposed to salinity 61 . 76,88 reported that HA improved APX activity response to salinity stress. Our results showed that exogenous JA was signi cantly increased APX activity. Our work agrees with 89 ndings, who suggested that exogenous JA increased APX activity under salt stress of tobacco (Nicotiana tabacum) plants. Also, 90 report that APX activity increased in plants treated with JA exposed to Pb stress. Moreover, exogenous JA induced the synthesis of antioxidant metabolites that provided additional resistance to neutralize the toxic effects of salt stress generated ROS 66 .

Conclusions
Our study examines the effects of salinity stress on growth parameters, chlorophyll content, carotenoid content and plant defense system of forage sorghum exposed to different humic acid rate as fertilizer and jasmonic acid application as a foliar spry. Soil salinity stress at 4 g NaCl kg -1 soil decreased all study parameters, except protein content, SOD activity, and MDA. In this study, we found that forage sorghum spray with jasmonic acid at 10 mM JA level successfully improved POD and SOD activities. Among different humic acid rate, 3 g HA kg -1 dry soil successfully increased all parameters. These results proposed that 5 mM JA could e ciently protect the forage sorghum plant from salty stress damage by enhancing total chlorophyll content, carotenoid content, and antioxidant enzyme. The interaction between 3 g HA kg -1 soil and 5 mM JA was most effective in alleviating salinity stress. Therefore, JA and HA combined applications could enhance the chlorophyll content, carotenoid, and antioxidant enzyme in plants. Therefore, JA and HA application management are required in salt-affected soil to sustain forage growth and increase crop yield and productivity under salinity conditions.