Nitric Oxide Enhances Salt Tolerance through Regulating Antioxidant Enzyme Activity and Nutrient Uptake in Pea

Salinity is one of the environmental stress factors that restrict crop production. Salinity shows its direct effect on plants by creating osmotic and ion stress, while its indirect effect is shown by structural degradation and synthesis of toxic compounds. Reduction of the amount of water available due to the osmotic effect causes a decrease in cell expansion and a slowing of shoot development. Nutrient deficiency or nutrient imbalance occurs in plants by increasing Na and Cl ions competing with necessary nutrients such as K⁺, Ca⁺² and NO_-3 (Shams et al., 2019). Much of the cell-level damage caused by salt stress is related to oxidative damage caused by ROS which significantly damages cellular membranes and organelles (Parida and Das 2005). Salinity reduces the leaf number, leaf area, plant fresh weight, dry weight, chlorophyll content and fruit quality (Karlidag et al., 2013).
       
NO is a signal molecule in plants, which has various metabolic functions. It plays an important role in the growth and development of plants. It is evident that NO protects plants from damage caused by oxidative stress conditions in various biological ways (Shams et al., 2019). Excessive ROS accumulation due to salinity conditions can result in oxidative damage and NO is considered a functional molecule to alleviate salt stress in plants via modifying antioxidant metabolism (Lin et al., 2012). Exogenous NO treatment can cause an increase in antioxidant enzyme activity and salt tolerance genes in plants under salinity stress, this leads to increased plant growth and productivity (Arasimowicz and Floryszak- Wieczorek 2007).
       
Pea (Pisum sativum L.) belongs to the family Fabaceae and is a low-cost and nourishing food. It is rich in dietary proteins, complex carbohydrates, essential amino acids, vitamins, fiber and minerals. However, its growth, grain yield, and quality and composition of grains significantly were affected by salinity stress (Nenova, 2008). In this regard, it is beneficial to understand the mechanism of NO on the tolerance of Pisum sativum L to salinity stress. Here, we report a more detailed study of the influence of NO on plant growth, nutrient uptake, MDA and H₂O₂ content, and enzyme activity in pea cultivars under long-term salt stress.

Salinity stress (100 mM of NaCl) decreased shoot fresh and dry weight, and the root fresh and dry weight by 81, 52, 51 and 52%, respectively in Jof cultivar compared to control. However, in Utrillo, the shoots fresh and dry weight and the root fresh and dry weight were reduced by 32, 33, 16 and 26%, respectively, compared to untreated one (Table 1). These results are similar to the findings of Abdel-Latef and Chaoxing (2014) who reported that salinity stress significantly decreased the plant growth in pea. This inhibition of the growth in pea cultivars can be associated with the adverse effect of salinity stress on water availability, transpiration, carbohydrate fixation and ion balance in plants subjected to salinity stress (Shams et al., 2016).
 

       
Based on the results, salinity stresses the biomass values of two pea cultivars, but a different pattern response was found between cultivars. In this respect salinity significantly decreased biomass values in Jof cultivar but it had a slight impact on Utrillo. This might be attributed to differences in salt tolerance across different genotypes. (Sarafi et al., 2018). Based on the data in Table 1. NO treatment (75 μM) was more effective on the growth parameters of Jof than those of Utrillo under salt stress conditions. It increased shoot fresh and dry weight, and root fresh and dry weight by 119, 37, 64 and 31%, respectively in Jof cultivar under 100 mM NaCl. However, it increased shoot fresh and dry weight and root fresh and dry weight in Utrillo cultivar by 15, 26, 13 and 17%, respectively, under 100 NaCl compared to the untreated one. These results pointed out that Jof is more sensitive to salinity than the Utrillo and it is also more responsive to NO treatments (Table 1). These findings are in agreement with those of Hasanuzzaman et al., (2011) who reported that NO supplementation ameliorated plant growth under salinity conditions in wheat. Furthermore, it was reported that NO regulated growth under NaCl stress by promoting antioxidant metabolism and maintaining mineral assimilation in chickpea (Cicer arietinum) (Ahmad et al., 2018).
       
Based on the findings, salinity stress caused a significant reduction in CRV, chlorophyll a, chlorophyll b and chlorophyll a + b (Table 1) and 100 mM NaCl had the highest negative effect. The reduction in chlorophyll content of both pea cultivars under salt stress could be contributed to the adverse effect of NaCl on the chlorophyll pigments and chlorophyll syntheses (Rasoul et al., 2013).  However, the simultaneous application of NO and NaCl reversed the adverse effect of salinity. In this regard,  75 μM NO increased the chlorophyll a, chlorophyll b and chlorophyll a + b content in both cultivars under 100 mM NaCl but it was more effective on Jof cultivar than Utrillo. Similarly, Ahmad et al., (2016) determined that salinity decreased chlorophyll content in chickpea, but combined the application of NO and salinity increased chlorophyll content compared to the control.
       
Cell membrane permeability of pea cultivars used in the study was determined by the measurement of TEC (Table 1). Salinity stress (100 mM NaCl) markedly increased the cell membrane permeability in pea cultivars but it had a lower effect on Utrillo cultivar than the Jof cultivar. The increase in TEC value can be associated with the excessive absorption of Na⁺ and Cl^- and consequently disrupting the cell membrane stability by triggering the over-accumulation of reactive oxygen species (ROS) (Mansour, 2013). On the other hand, 75 μM NO treatment decreased TEC values compared to the control in both cultivars (Table 1). This means that NO treatment mitigated the harmful effect of NaCl on cell membranes. Similarly, it had been shown that oxidative damages in rice seedlings were mitigated by NO applications under salt stress (Wu et al., 2011).
       
The RWC value was decreased in pea cultivars by salinity stress, but the strongest effect of salinity was observed in Jof under 100 mM NaCl. NO treatments had no significant on RWC in all cultivars compared to control. However, in the presence of salinity stress, NO treatment (75 μM) significantly increased RWC in all pea cultivars compared to the plants subjected to NaCl and without NO (Table 1). The findings were similar to those of Shams et al., (2019) who demonstrated that salinity stress decreased RWC and increased TEC, but the application of NO ameliorated the adverse effect of salinity stress. Ahmad et al., (2018) also reported that NaCl negatively affected RWC and chlorophyll content while NO treatments improved RWC and chlorophyll content of tomato.
       
NaCl treatment significantly caused an increase in H₂O₂ and MDA contents in pea cultivars (Table 2). The highest H₂O₂ and MDA content were observed in Jof cultivar under 100 mM NaCl. However, the application of NO reduced the H₂O₂ and MDA content in both cultivars under salinity stress. This increase in H₂O₂ and MDA content can be related to the role of NaCl on the over-accumulation of ROS and stimulation of oxidative stress which causes injury to membranes and other cellular structures (Parida and Das 2005). In harmony with our results, salinity increased the MDA and H₂O₂ content in tomato, but NO treatment decreased MDA and H₂O₂ content (Ahmad et al., 2018).
 

       
APX, POD and SOD enzymes by scavenging ROS perform a crucial role in the antioxidant defense system to remove oxidative damage under environmental stresses in plants. The salinity induced a significant increase in the SOD, APX and POD activities in Utrillo and Jof (Table 2). To mitigate the hazardous effect of NaCl stress, plants can produce higher levels of antioxidant enzyme activities in the cytosol and other organs of the cells. In this experiment, a similar higher activity trend of APX, SOD and POD was detected in pea cultivars under salinity stress (Table 2). The increased enzyme activities in response to salinity stress were reported by Shams et al., (2019) in Capsicum annum.
       
NO is considered a functional molecule in alleviating salt stress in plants through NO modifying antioxidant metabolism (Lin et al., 2012). In the current study, NO (75 µM) increased the POD, SOD and APX activities in both cultivars compared to control in the absence of salinity stress. Also, the application of NO to salinity-stressed pea cultivars stimulated a significant increase in enzyme activities. Similarly, it was reported that NO caused an increase in the activities of SOD, CAT, APX and GR in tomato under salinity stress (Ahmad et al., 2018). This can be attributed to the crucial role of NO on the regulation of genes responsible for the enzyme activities (Ahmad et al., 2016).
       
Salinity stress markedly affected the Na⁺, K⁺ and Ca²⁺ uptake and, consequently, the ratio of K⁺/Na⁺ and Ca²⁺/Na⁺ significantly was decreased in leaves of pea cultivars (Table 2). The decrease in K⁺/Na⁺ and Ca²⁺/Na⁺ ratios can be linked to the over-accumulation of Na⁺ and the decrease in Ca²⁺ and K⁺ concentration in leaves of pea cultivars under NaCl stress. Nonetheless, increase in K⁺/Na⁺ and Ca²⁺/Na⁺ by exogenous NO to salt-stressed plants (50 mM) was reported by Shams et al., (2019), which is similar to our results. Gadelha et al., (2017) reported that NO priming limited the accumulation of toxic ions and ROS by inducing an effective antioxidant system and thus, improving salt tolerance during seedling development.