When plants are grown under saline conditions, as soon as the new cell starts its elongation process, the excess of salts modifies 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 efficiency in cell enlargement decreased.
4.1. Growth parameters
In this study, NaCl salinity stress significantly 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 significantly increased at the low salinity levels, sill, at the high salinity level, the plant height significantly decreased. However, similar findings were reported by 36 on bean (Vigna aconitifolia L.), 37 on cabbage plant (Brassica oleracea capitata L), 38 on cowpea (Vigna unguiculata L.).
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 findings 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 finding with 39 at the final harvest reported that the RGR decreased in the high salinity level.
Plant hormonal play essential roles in stress responses and adaptation. It is clearly defined 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. Alavi et al. (2015) reported that the JA application recorded the highest plant height and leaf area index at the high salinity stress. Similar results were also observed for pepper, wheat, soybean, and rice seedlings 43,44. In contract, Alavi et al. (2015) reported that the JA application not significant effect on measured growth parameters. The increased in leaf area index in this study agrees with reports in wheat 45 and rice plants 46.
Humic acid (HA) can improvement the plant growth by increases the permeability of the cell membrane, this progresses increased water absorption and other nutrients uptake and increased absorbing capacity of roots in the presence of humic acid 47. 48,49 Ali et al. (2019) noticed that the cells might continue their extension growth for more extended periods under the effect of humic acid. 50 stated that the HA increases plant growth through increased in nutrients uptake to overcome the lack of nutrients, this lead to beneficial 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 officinalis 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.
4.2. 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 first 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., sunflower (Ashraf & Sultana, 2000), and wheat (El-Hendawy et al., 2005). Similar results were showed by 59 in common bean and 60 in the pepper plants. The reduction in chlorophyll content under salt stress may be due to the reduction in the carbon use efficiency, synthesis of chlorophyll, uptake of minerals such as Mg and Fe, and increased ethanol and lactate production 59.
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 significant increase in total chlorophyll content and carotenoid content concentration under soil saline stress. 5, and 43 reported that exogenous JAs significantly 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 confirm 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.
4.3. 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 significantly 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 findings were dissimilar with 66, who reported that salinity stress significantly increased the proline content in wheat and paulownia (Paulownia imperialis L) plants.
Jasmonic acid and humic acid application improved the soluble protein content and proline contents of the forage sorghum. A decreased 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 specific protein set. In the present investigation, HA improved the protein content. Similar findings 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.
4.4. Superoxide dismutase and Malondialdehyde contents
Superoxide radicals (O2-) generated by oxidative metabolisms were detoxified by superoxide dismutase (SOD) and converted into H2O2 and O2. SOD is one of the enzymes responsible for eliminating O2- 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 O2 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 sunflower by Nimir et al. (2015) and 72, who noted that salinity stress could increase the same antioxidant enzyme activity. This result similar with the findings 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 significant increase in SOD activity and MDA content was observed in forage sorghum plants treated with JA, suggesting that JA had a good O2- scavenging ability to protect the plant from oxidative damage. Our study of the increase in SOD activity agree with the results of 57,74who reported that exogenous JA application of soybean under salinity stress significantly improved the activities of SOD. However, our study of the an increase in MDA content under salt by jasmonic acid different from the findings 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, H2O2 and the production rate of O2-. Similar results were reported by Ali et al. (2019). Jasmonic acid might fulfill crucial roles in scavenging radicals, thus inhibiting lipid peroxidation by excess ROS produced under salinity conditions 66.
In our results, the application of HA was positively useful under saline conditions to increase the actions of the SOD and MDA content. Disagree result 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 significantly by applying a humic acid.
4.5. 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 H2O2 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 confirmed with the results of 66 and 71. The disconfirmed results has been shown by 79,80 for wheat plant treated with soil saline, who reported that POD activity increase under NaCl stress. Also, disconfirmed 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 findings of 57, who suggested that the wheat and soybean plants exposed to salinity and treatment with JA application significantly 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. In this study, the application of HA improved plant defense systems such as POD and CAT in the forage sorghum plants, and the both rates increased the POD and CAT activities. Many studies have also reported that the increased activities of POD and CAT by application of HA was efficient in improving tolerance of salt in cucumber 85, date plam (Phoenix dactylifera L), and hot pepper 83. However, our result different from the findings of 61 found that the humic acid application decreased CAT and POD activities under salt stress.
4.6 Ascorbate peroxidase activity
The ascorbate peroxidase activity (APX) may help the plant against the H2O2 damage 43. Under salt stress, the APX plays an essential role in protective the plant by reduce H2O2 43,57. Our study showed that increased salinity levels reduced APX activity. A similar result was reported by 66 and 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 flow 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 significantly increased APX activity. Our work agrees with 89 findings, 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.