Effects of Irrigation with Sodium Nitroprusside on Physiological Characteristics of Soybean under Drought Stress

China is the birthplace of soybean [Glycine max (L.) Merr.], which is mostly farmed in the Yangtze River Delta, Jianghan Plain, Huang-Huai Plain and Northeast Plain. It is an important grain and cash crop, playing a significant role in agricultural production and people’s livelihoods (Pagano and Miransari, 2016). Soybeans contain various nutrients such as proteins, fats, dietary fibers and isoflavones (Qin et al., 2022; Tripathi and Misra, 2005). They are often processed into soy flour, soy milk, soy sauce and various soy-based products. The leftover soybean residue is also commonly used in the production of animal feed (Golbitz, 1995; Sun et al., 2021). The growth of soybeans is influenced by various factors, such as drought, pests and diseases and salinity-alkalinity. Drought is a natural phenomenon characterized by a prolonged lack of precipitation (Dai et al., 2022), it has a major impact on agricultural output, environmental stability and local economic growth (Chen et al., 2022). One of the essential processes in plant growth and development is the increase in leaf area, the leaf area of soybean plants is an important factor affecting yield. Studies by Peer et al., (2023); Lestari et al., (2019) have already indicated that drought stress can lead to a reduction in leaf area in plants. Therefore, it is highly necessary to enhance soybean’s drought resistance capabilities.

When free radicalsattack the cell membrane lipid bilayer in plants, membrane lipid peroxidation occurs and MDA is produced (Ribeiro et al., 2023). MDA, H₂O₂ and other chemical levels rise as a result of drought-stressed plants producing reactive oxygen species, or ROS (Begum et al., 2023; Petrović et al., 2023). When there is a significant accumulation of MDA within the plant body, lipid peroxidation is enhanced, which affects normal plant growth (Yasar et al., 2008).

Being a very potent signaling molecule that controls the growth of plants, numerous developmental activities, such as stomatal movement, root formation and seed germination are influenced by NO (Lubyanova et al., 2022; Pandey et al., 2019). NO accumulates in plants when they face biotic and abiotic stress (Zhao et al., 2013) and it reduces a number of stresses via controlling ion homeostasis, metal transport, antioxidant defense systems, oxidative stress and more. Additionally, it has been discovered that NO interacts with a variety of other signaling molecules, including gibberellins (GA), salicylic acid (SA) and abscisic acid (ABA) (Kumar and Ohri, 2023). In the ABA-induced stomatal closure signaling cascade, NO controls potassium and calcium ion channels to maintain ion homeostasis in guard cells (Agurla et al., 2018). Moreover, NO efficiently stimulates the SA signaling pathway’s defense gene expression, strengthening plant resistance to infections (Bellin et al., 2013). In the synthesis of plant gibberellins (GA), NO activates the transcription of key enzymes GA3ox1 and GA3ox2, promoting GA synthesis and breaking seed dormancy (Bethke et al., 2007).

NO can directly scavenge ROS or activate antioxidant defense mechanisms in response to non-biological stress circumstances like drought (Sahay and Gupta, 2017). Research has shown that exogenous NO can regulate the physiological and biochemical processes in fragrant rice to reduce the levels of MDA and H2O2 generated by cadmium stress (Imran et al., 2023). Ahmad et al., (2021) reported that NO in synergy with silicon can reduce the absorption of arsenic in cabbage, thereby alleviating the toxicity of arsenic stress on plants. 

SNP is an exogenous NO donor that releases NO when dissolved. S-nitrosylation is the major pathway for the biologically active transfer of NO, where NO partly forms S-nitrosothiol by covalently binding with cysteine thiol in proteins. This chemical change is quickly becoming recognized as a fundamental aspect of plant life and as a model redox-based post-translational modification (Yu et al., 2014). By boosting the activities of superoxide dismutase and catalase, exogenous SNP can decrease the formation of MDA and H₂O₂ in soybeans during salt stress, increasing the soybeans’ survival rate (Jasid et al., 2009). Given the increasing trend of potential expansion in the range of drought during crop growth seasons, the combined use of exogenous SNP during irrigation can enhance drought resistance in plants. In this experiment, HN44 and HN65 soybeans were irrigated with varying doses of SNP solution during the flowering stage and NO content, MDA content and leaf area per plant were measured, providing theoretical support for the study of soybean’s response to drought stress.

Drought-tolerant Heinong 44 (HN44) and sensitive Heinong 65 (HN65) are the tested soybean cultivars in this study (Wang et al., 2022).

In the experiment, sand culture was employed and the pots were cylindrical plastic containers with a 20 cm diameter and a 40 cm height. After perforating the bottom, it was covered with a mesh and clean river sand was filled into the pots. Six seeds were sown in each pot and distilled water was irrigated once a day, 500 ml each time, until the unifoliate leaves were fully unfolded. Then, thinning was done, leaving three consistently growing soybean seedlings in each pot. Thereafter, modified Hoagland nutrient solution was irrigated once a day, every time 500 ml, until the flowering stage (R2). Drought and exogenous SNP treatments were performed during the flowering stage. PEG-6000 and SNP were dissolved in the modified Hoagland nutrient solution and each treatment was set as shown in Table 1. Irrigating the nutritional solution with 15% PEG-6000 and SNP once a day, 500 ml each time. Samples were taken on day 1, 3, 5 and 7 of the treatment. Three pots were chosen for each treatment, with three soybean plants selected for each pot. The sampling sites were the second and third inverted leaves of the soybean. The collected soybean leaf samples were divided into two parts: one part was immediately measured after obtaining fresh leaves using an icebox for preservation and for further examination, the remaining portion was quickly frozen in liquid nitrogen and kept in an ultra-low temperature freezer. All measurements were conducted in triplicate.



The colorimetric approach was used to determine the MDA content (Hodges et al., 1999). Following the addition of 1 ml of 10% trichloroacetic acid and 0.1 g of the sample to a mortar, the mixture was ground and centrifuged at 12000 g for 10 minutes. After adding 0.2 ml of 0.67% thiobarbituric acid to the homogenate (0.4 ml), the mixture was heated to 100°C for 30 minutes and the sample was boiled. After cooling, the sample was centrifuged twice to obtain the supernatant and the absorbance at 450, 532 and 600 nm was measured.

With a few minor adjustments, the NO content was determined using Ding et al., (1988) methodology as guidance. Using a mortar and pestle, 0.6 g of powdered leaf samples and 3 ml of cold, 50 mM acetic acid buffer (pH 3.6) containing 4% zinc diacetate were combined. The homogenate was centrifuged for 15 minutes at 4°C and 10,000 g to collect the supernatant. The particle was centrifuged after being cleaned with 1 ml of extraction buffer, as previously mentioned. After mixing the two supernatants, 0.1 g of charcoal was added. The filtrate was obtained following vortexing and filtration. 30 minutes were spent incubating a combination of 1 ml of the filtrate and 1 ml of Griess reagent at room temperature. At 540 nm, the absorbance was measured. By comparing the NO content with the NaNO₂- standard curve, it was determined.

The method of measuring the individual leaf area of soybean plants at the R2 stage was conducted using AutoCAD software.

The experimental data were analyzed and plotted using SPSS 22.0 and Excel 2016 for data analysis and visualization.

Variations in soybean leaf NO content with various SNP treatments
 
Following treatment for the drought, soybean leaves’ NO content rose. In HN44 soybean leaves, the NO content increased with increasing SNP concentration and showed an increasing trend with treatment duration. In HN44, compared to the CK, the NO content increased by 35.56%, 23.56%, 56.57% and 126.87% on the 1^st, 3^rd, 5^th and 7^th days after C₁ treatment, respectively, with a significant increase on the 7^th day. After being treated with SNP at concentrations C₃, C₄ and C₅, the NO content in HN44 soybean leaves increased by 0.83%, 11.56% and 25.64%, the C₂ decreases by 1.57%, respectively, on the 3^rd day compared to the 1^st day. The NO content in the soybean leaves treated for 5 days was 16.92%, 16.04%, 11.39% and 6.04% higher than the NO content in the leaves treated for 3 days, respectively. SNP had an effect at different days and the best effect was observed on the 3^rd in HN44 (Table 2).

Although the NO content in C₂ on 5^th and 7^th day was lower than the C₁ treatment, the NO content in the soybean leaves of HN65 increased as the SNP concentration increased. There was an increasing trend with the increase in treatment days (5 days saw the pinnacle of C4’s initial rise and subsequent decline). In HN65, compared to the CK group, the NO content in the soybean leaves increased by 14.40%, 1.46%, 61.49% and 180.85% on the 1^st, 3^rd, 5^th and 7^th day after C₁ treatment, respectively, with the 7^th day showing a significant increase. The NO content in the soybean leaves of HN65 treated with SNP at concentrations C₂, C₃, C₄ and C₅ for 5 days was 54.15%, 39.45%, 72.33% and 30.53% higher than that in the leaves treated for 3^rd, respectively. The NO content in soybean leaves treated with SNP at concentrations C2, C3 and C5 for 7^th days was 3.90%, 7.37% and 15.15% higher than the NO content in the 5^th day treatment, respectively. However, C₄ reduced the NO content by 7.12%. SNP had an effect at different days and the best effect was observed on the 5^th day in HN65 (Table 2).



During drought stress, the NO concentration rose in HN44 and HN65 soybean leaves, with a faster increase rate in the NO content of HN44 soybean leaves compared to HN65, indicating a rapid response to drought stress. After the exogenous application of SNP, the NO content in both soybean leaf types increased, which is in line with findings of Qu et al., (2023). According to Klein et al., (2018), adding exogenous nitric oxide to maize greatly raised the amount of nitric oxide in the plant, which had a beneficial impact on oxidative damage in maize. In HN44, the NO content in the leaves after SNP treatment at concentrations C₃ and C₄ on the 5^th and 7^th day was lower than in the drought group. Following SNP treatment at concentration C₂ on 5^th and 7^th day, the amount of NO in the leaves of HN65 was lower than that of C₁. The decrease in NO content in soybean leaves under drought stress following treatment with lower concentrations of SNP for a while may be related to the instability and easy breakdown of SNP, or it may be the result of the level of NO increase by SNP weakening with the duration of drought stress.

MDA concentration variations in soybean leaves with various SNP treatments

Compared to the CK treatment, under drought stress, the MDA content in HN44 soybean leaves increased by 13.62%, 21.83%, 28.55% and 28.93% at 1^st, 3^rd, 5^th and 7^th, respectively. Significant increases were observed at all time points. The MDA content in HN44 steadily dropped as the SNP concentration rose. The MDA content in the leaves showed a tendency of first reducing and then increasing under the same concentration treatment. Compared to the C₁ treatment, the amount of MDA in the C₅ treatment’s leaves decreased by 12.83%, 36.52%, 28.15% and 22.16% on the 1^st, 3^rd, 5^th and 7^th day, respectively, with significant differences between the treatments. Under concentrations C₂, C₃, C4 and C₅, the MDA content in the leaves decreased by 18.26%, 21.99%, 22.60% and 29.24% on the 3^rd day compared to the 1^st day, respectively. The MDA content in the leaves increased by 12.38%, 7.04%, 6.83% and 16.08% on the 5th compared to the 3^rd, respectively. SNP had an effect at different days and the best effect was observed on the 3^rd in HN44 (Table 3).

In HN65 soybean leaves under drought stress, the amount of MDA rose by 12.09%, 21.83%, 28.55% and 52.08% on the 1^st, 3^rd, 5^th and 7^th day, in that order, showing a significant increase at each time point. With an increase in SNP concentration, the MDA content gradually decreased. Under the same concentration treatment, the MDA content showed an overall trend of initially decreasing and then increasing. On the 1^st, 3^rd, 5^th and 7^th day, the MDA content of the C₅ treatment was significantly lower than that of the C₁ treatment, declining by 14.86%, 20.22%, 30.67% and 30.34%, respectively. The MDA content in the leaves on the 7th day under C₂ concentration significantly increased compared to the 7^th day in the C₁ treatment. Under concentrations C₂, C₃, C₄ and C₅, the MDA content in the leaves decreased by 6.15%, 6.99%, 5.82% and 7.77% on the 5^th compared to the 3^rd, respectively. The MDA content in the leaves increased by 21.62%, 23.92%, 22.58% and 18.51% on the 7^th compared to the 5^th, respectively. SNP had an effect at different days and the best effect was observed on the 5^th in HN65 (Table 3).



Under conditions of drought stress, the MDA content of HN44 and HN65 leaves significantly rose, the MDA level of HN65 leaves was greater, indicating more severe membrane lipid peroxidation. Under different concentrations of SNP treatment, the MDA content of the two soybean leaves decreased to varying degrees. This study’s findings are in line with a study by Sundararajan et al., (2022) that demonstrated sodium nitroprusside’s ability to lower MDA levels in tomato seedlings and increase drought tolerance in tomatoes. When soybeans were treated with varying concentrations of SNP, the MDA level of the two soybean leaves first decreased and subsequently increased as treatment duration increased. The reason for this may be that within a certain period of time, SNP has a mitigating effect on drought stress in HN44 and HN65 soybeans. However, under prolonged drought stress, soybean plants suffer severe damage, cell membranes are compromised and membrane lipid peroxidation becomes more severe. The effect of SNP in alleviating drought stress in soybeans is not significant. Therefore, irrigating soybean plants with SNP can promote an increase in NO content in soybean leaves, effectively reduce the MDA content generated by drought stress, alleviate membrane lipid peroxidation in soybeans and mitigate harmful effects on plant growth.

Variations in soybean plant leaf area per plant following various SNP treatments

Drought treatment reduces the individual leaf area of soybean plants and as the treatment duration increases, the inhibitory effect on leaf area growth becomes stronger. In HN44, compared to CK, the individual leaf area decreased significantly after 1^st, 3^rd, 5^th and 7^th days of treatment in C₁, with reductions of 1.02%, 4.34%, 8.16% and 9.86%, respectively. There were significant differences in the individual leaf area of HN44 soybean plants between C₁ and C₃, C₄, C₅ treatments on the 1^st, 3^rd, 5^th and 7^th day. With the increase in SNP concentration, the individual leaf area of soybean showed a trend of first increasing and then decreasing. Compared with the C1 treatment, the individual leaf area of the C₄ treatment increased by 0.77%, 3.57%, 6.07% and 8.04% on the 1^st, 3^rd, 5^th and 7^th day, respectively. Among the C₂, C₃, C₄ and C₅ treatments, C₄ had the largest leaf area on the 3^rd, 5^th and 7^th days (Table 4).

In HN65, compared to CK, the individual leaf area decreased by 0.99%, 4.37%, 8.12% and 9.38% on the 1^st, 3rd, 5^th and 7^th days, respectively, after the C₁ treatment. There were significant differences in the individual leaf area of HN65 soybean plants between C₁ and C₃, C₄, C₅ treatments on the 5^th and 7^th days. With the increase in SNP concentration, the individual leaf area of soybean showed a trend of first increasing and then decreasing. Compared to the C₁ treatment, the individual leaf area of the C₄ treatment increased by 0.808%, 2.97%, 5.42% and 7.19% on the 1^st, 3^rd, 5^th and 7^th day, respectively. Among the C₂, C₃, C₄ and C₅ treatments, C4 had the largest leaf area on the 1^st, 3^rd, 5^th and 7^th days (Table 4).



Under drought stress, the growth of individual leaf area in both HN44 and HN65 soybeans was suppressed, with a significant decrease in leaf area. As the treatment duration increased, the inhibitory effect became stronger, which is consistent with the findings of the study by Peer et al., (2023). The application of exogenous SNP alleviated the inhibition of individual leaf area growth in both soybean varieties to varying degrees. Among them, the C₄ treatment showed the best alleviation effect, which is consistent with the findings of the study by Chavoushi et al. (2020).

Drought stress significantly increased the contents of NO and MDA and decreased the leaf area per plant of the two cultivars. Treatment with different concentrations of SNP effectively increases leaf NO content, reduces MDA levels, alleviating drought damage in soybean plants and the inhibition of leaf area growth. In practical production, it is recommended to apply a 1000 μmol·g-1 SNP solution when supplementing water during drought disasters, as it effectively alleviates membrane lipid peroxidation damage and enhances drought resistance

This research was funded by the Ministry of Science and Technology of the People's Republic of China, Grant No. 2020YFD1000902. And funded by Natural Science Foundation of Heilongjiang Province of China, Grant No. LH2021C023.

All authors declared that there is no conflict of interest.