Comparative Analysis of Defence Response of Soybean by Seed Soaking in Gibberellic Acid to Salinity

Salinity is a major factor limiting crop productivity. Specifically, over seven percentage of the world’s total land and approximately twenty percentage of irrigated land is affected by high salinity (Munns and Gilliham, 2015). In the soil, high salinity deposition leads to osmotic and ion effects, which further caused oxidative stress in plants. The production of reactive oxygen species (ROS) due to salinity denatures the proteins, enzymes, bio-membranes and nucleic acids (Gomez et al., 2004). To cope with oxidative stress, plants have developed an antioxidant defense system, comprising of antioxidant enzymes like superoxide dismutase (SOD), ascorbate peroxidase (APX), peroxidase (POX), catalase (CAT) and glutathione reductase (GR). Salinity regulates these enzymes depending on the plant species, age, degree of stress induced damage and exposure time to stress. Most studies proved that tolerant plant genotypes usually have a better antioxidant defense system under salt stress conditions. Beside this, plants also adapt various biochemical and molecular strategies such as compartmentalization into vacuoles, maintained uptake of ions and accumulating solutes and induced phytohormone levels. 
        
Phytohormones are molecules produced in very low concentrations but able to regulate all cellular processes in plants. Abiotic stress conditions caused to the production of signaling molecules that induce several metabolites including phytohormones like gibberellic acid (GA), abscisic acid (ABA), ethylene (ETH), auxin (IAA), cytokinin (CTK), brassinosteroid (BR) for stress tolerance. Their role in salinity stress is important in modulating physiological responses that lead to the adaptation of plants to stress conditions. Additionally, these hormones have significant effects on plant antioxidant enzyme activities to resist the effects of salinity.
        
Gibberellins (GAs) are a class of phytohormones that control plant growth process including seed germination, leaf expansion, flower initiation, fruit development (Li et al., 2010). However, recent investigations determined that GAs are used to be a regulator to increase stress tolerance in plants. Presoaking treatment or exogenous spraying of GAs in some crops has improved seed performance under salinity (Leite et al., 2003; Saeidi-Sar et al., 2013; Younesi and Moradi, 2014; Sofy et al., 2016; Jiao et al., 2019; Miladinov et al., 2019).
        
Soybean [Glycine max (L.) Merr.] is an important member of the family of Leguminosae, because of its high-quality protein and oil for livestock and human. Although some reports have been determined the positive effects of gibberellic acid on soybean plants there is no data available about the effects of presoaking GA₃ with seeds on the ROS regulation and antioxidant enzymes in cotyledon, radicle and hypocotyl. This is the first report of seed priming of soybean with GA₃ and the change in antioxidant defense system and ROS maintenance in organs.

Table 1 reveals that (FW) of radicle and hypocotyl under salinity was reduced by 62.22% and 64.23% as compared to controls respectively. It has been known that salinity reduced biomass yield seriously in plants and many papers have determined this. In the results, the pretreatment of GA₃ alleviated this value by 29.41% and 2.58 fold compared with salt stress alone. Similar to these results, Maggio et al., (2010) reported that GA₃ induced growth in tomato plants under salinity. This could be explained by the roles of GA₃ in cell division, elongation. However, the FW of cotyledon was not affected by salt stress in this experiment. These results also agree with the reports of Ruffino et al., (2010) who determined the less damage of salt stress on cotyledons of quinoa plants. Considering the DW of soybean organs, the highest reduction was in the radicle by 83.33% while it was 60% in the hypocotyl. In accordance with these findings, Bai et al., (2019) determined that there was a slight reduction in DW values in soybean with salinity. Similar to the fresh weight results, DW of cotyledons also was not changed with stress. This finding indicated that cotyledons have different tolerance capacity comparing with the hypocotyl. Nevertheless, the pretreatment of GA₃ increased this value by 4 fold in radicle and 50% in the hypocotyl. In recent years, Chauhan et al., (2019) determined that GA₃ treated seeds of oat cultivars showed a slight increase in biomass yield. From these results, it could also be said that GA₃ had a positive effect on soybean organs under salinity.
 

        
The pigment content of cotyledons was showed in Table 2. In our results, salinity inhibited photosynthetic pigments in cotyledons significant. This fact had already been proved by a researcher that salt stress had negative effects on photosynthetic mechanisms in soybean (Khan, 2018). This is also an agreement with the results of MDA and H₂O₂ contents in cotyledons (Table 3). However, treatment with GA₃ on soybean seeds improved photosynthetic capacity in cotyledons. It could be suggested that this hormone might play a role in increasing organic matter and maintain water balance in cotyledons. In parallel with this result, Al Mahmud et al., (2019) said that exogenous GA₃ induced chlorophyll content in wheat plants under drought conditions. 
 

 

        
In the results, MDA content was increased with salt stress (150 mM NaCl) by 11.75%, 32.53%, 48.65% in different soybean organs (cotyledon, radicle and hypocotyl) but GA₃ pretreatment reduced this levels under stress condition and the highest reduction (40.36%) was determined in hypocotyl (Table 3). It can be concluded that GA₃ lead to an improvement in hypocotyl rather than cotyledon and radicle of soybean.
        
The present study revealed that salt stress-induced SOD enzyme activity in each part of soybean plants. This result was the agreement with the increased MDA contents with salinity (Table 3). Fig 1 reveals that SOD enzyme activities were increased by 5.95 fold, 42.85% and 3.08 fold with salt treatment as compared to control groups in cotyledon, radicle and hypocotyl but decreased significantly by 64.7%, 50% and 59.25% with GA₃ and salt application according to salt stress alone. In accordance with our results, Amor et al., (2019) reported that SOD enzyme activity was induced with salinity in Cakile maritima. In the present study, GA₃ pretreated plants have decreased SOD activity according to NaCl treated ones. It is suggested that GA₃ may play a role as an antioxidant instead of SOD enzyme. These results also agree with the reports of Chakrabarti and Mukharji (2003) and Ahmad (2010) who reported that there was a decreased activity of SOD enzyme with GA₃ treatment in Vigna radiata and mustard plants respectively. In contrast, Sofy (2016) determined that GA₃ primed soybean seeds had a higher SOD activity according to controls. This paradox can be explained with the differences in concentration of GA₃ and NaCl with different cultivars of soybean. 
 

        
Similar to the results of MDA, salinity also induced H₂O₂ levels by 22.1%, 15.48% and 10.88%, while it was decreased by GA₃ before the application. This amelioration was the highest in radicle by 24.8% (Table 3). Otherwise, there was no change in APX enzyme activity in cotyledon and radicle with salinity, while it was increased remarkably by 3.33 fold in hypocotyl (Fig 2). In contrast, GA₃ treatment induced this activity in cotyledon and radicle but decreased by 33.33% in hypocotyl as compared to salt stress alone (Fig 4). Similarly, Xie et al., (2008) and Verma and Mishra (2005) determined that salt stress did not affect the APX enzyme activity in Brassica juncea and Gossypium hirsutum plants. Nevertheless, there was a significant increase in APX activity as it determined by Weisany et al., (2012) in soybean plants.  However, in the present study, GA₃ treated plants under salinity showed an increased APX activity in cotyledon and radicle according to salt stress alone. This result is also agreement with the reports of Younesi and Moradi (2014) in Medicago sativa. Also, it was determined that the GA₃ application reduced APX enzyme activity in hypocotyl while it was induced with salt stress. This result showed that GA₃ plays antagonist action with salt stress in soybean plants to increase salt tolerance.
 

 

      
Salt stress-induced GR enzyme activity by 86.95% in radicle but did not affect in cotyledon and hypocotyl. However, GA₃ pretreatment under stress-induced this activity in cotyledon (11.76%) and hypocotyl (3.08 fold), while decreased in radicle according to salt stress groups (Fig 3). These results also agree with the reports of the Yassin et al., (2019) and Srivastava et al., (2015) in bread wheat and Sesavium portulacastrum. Otherwise, GA₃ increased GR activity under salinity in cotyledon and hypocotyl, although it was decreased in radicle. Following the evaluation of the all results, the role of GA₃ as an antioxidant (by reducing ROS level) and signal molecule (by inducing antioxidant enzymes activities) in soybean plants under salinity is observed explicitly.

Although GA₃ has been known as a growth regulator for plants they also involved in alleviating salt stress-induced damage. Totally, in the present study, to treat soybean seeds with GA promoted response mechanisms to salinity in soybean plants. It was showed that GA₃ protects soybean plants from salinity and induced biomass yield and photosynthetic capacity significantly as well as maintains the antioxidant enzyme system and lead to reduce oxidative damage. Collectively, this is the report of GA₃ which determined that might behave as a protector in different organs of soybean plants.