Assessment of antioxidant enzyme responses in a biotechnological cotton variety under salt stress conditions

Assessment of antioxidant enzyme responses in a biotechnological cotton variety under salt stress conditions | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Assessment of antioxidant enzyme responses in a biotechnological cotton variety under salt stress conditions Nodira R. Rakhmatova, Azadaxan S. Imamkhodjayeva, Ilxom B. Salahutdinov, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9426942/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In this article, we compared the reaction of two cotton varieties (RNAi Porloq-4 and Coker-312) to different concentrations of NaCl and Na 2 SO 4 and analyzed the activity of superoxide dismutase (SOD), an antioxidant enzyme, and the level of malonic dialdehyde (MDA) in the leaves of seedlings grown in the laboratory. Our goal is to identify changes in specific metabolomic indicators in cotton seedlings of the Porloq-4 variety under the influence of chloride and sulfate salt solutions in a comparative model experiment. We aim to determine the response of the Porloq-4 biotechnological cotton variety (at the level of metabolites) to abiotic stress (salinity). The following tasks were identified to study the effect of low, medium, and high concentrations of NaCl and Na 2 SO 4 solutions on the total chlorophyll content and biochemical parameters (MDA and SOD activity) of Porloq-4 cotton at an early stage of vegetation, compared to the Coker-312 variety. There was an increase in MDA and SOD at an average concentration of sulfate salt, and a slight decrease at a relatively high concentration. When the plants were watered with a relatively low concentration of sodium sulfate salt, MDA and SOD increased by 62%, while at an average concentration (2.0%), SOD increased by 325%. Nevertheless, when high concentrations of Na 2 SO 4 were used, SOD increased by 4.1 times compared to the control experiment. Salt stress reduced the seedling emergence rate, relative biomass, and chlorophyll content; however, MDA content increased. Salt stress markedly increased the superoxide dismutase (SOD) activity. MDA content was markedly higher in salt stress. Abiotic stress Chlorophyll Malonodialdehyde Metabolomics Saline conditions Superoxide dismutase Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Novelty statement In this study, the responses of the biotechnological cotton variety Porloq-4 and the Coker-312 variety to NaCl and Na₂SO₄ salt stress were compared based on antioxidant (SOD) activity and MDA levels. Results showed that Porloq-4 had higher metabolic tolerance to salt stress. At moderate salt concentrations, SOD activity increased by 4.1 times, and MDA content significantly rose. This work contributes to understanding the mechanisms of salt stress tolerance in Porloq-4 through its physiological responses. Introduction Salt resistance is defined as a plant's ability to grow, develop, and reproduce under saline conditions with minimal damage. As you know, any organism is a self-regulating system. To minimize oxidative damage caused by ROS production during salt stress, plants have a complex system of interrelated mechanisms, including antioxidant enzymes, osmolytes, and some non-enzymatic antioxidants (Yu Z et al., 2020 ). To mitigate the adverse effects of salt stress on plants, two complementary approaches are employed: the development of salt-resistant varieties and the enhancement of agrotechnological techniques (Verma V et al., 2016 ; Ku Y et al. , 2018). To obtain more comprehensive information about the biotechnological variety's tolerance to salt stress, we analyzed the activity of certain enzymes in the antioxidant system and the level of malondialdehyde (MDA). Plants respond to salt stress using highly regulated protective enzymes, such as MDA and SOD, to enhance their ability to purify reactive oxygen species. Prolonged exposure to high salinity leads to the accumulation of toxic ions, such as Na+, Cl−, and SO42−, which induce ion toxicity and impair nutrient absorption, thereby exacerbating damage to plant cells and tissues (Isayenkov S et al. , 2019). The undesirable effects of salt stress on plants are manifested in morphology (stunting, chlorosis, impaired seed germination), physiology (inhibition of photosynthesis and nutrient imbalance), and biochemical properties (oxidative stress, electrolyte leakage, membrane disorganization) (Ji X et al., 2022 ; Hannachi S et al., 2022 ). The adverse effects of salinity are particularly severe during the reproductive stage (Rafaliarivony S et al., 2022 ). Over the past 20 years, many studies have been conducted and analyzed on the harm caused by salts and their effects on plants. Increasing plant tolerance to salt stress is a primary focus in the genetic improvement of agricultural crops. The study of salt tolerance is a crucial component of plant biology research, enabling us to understand the complex mechanisms of plant salt tolerance and explore strategies to mitigate the harmful effects of salt stress (Balasubramaniam T et al., 2023 ). In our previous studies, we analyzed the effect of salicylic acid on cotton plants under abiotic stress conditions. Salicylic acid is a phytohormone that is naturally synthesized in plants and plays an important role in the response to both biotic and abiotic stress factors (Rakhmatova N et al., 2023 ). It activates the antioxidant defense system, reduces the level of reactive oxygen species (ROS), increases the activity of antioxidant enzymes (such as SOD, CAT, and APX), and also decreases the content of MDA (malondialdehyde), thereby reducing lipid peroxidation. In addition, salicylic acid enhances plant resistance to salt stress, drought, and infectious diseases. In this study, our objective was to analyze MDA content and SOD activity in cotton plants under salt stress conditions, which is of significant interest to many researchers. Our goal was to identify changes in some metabolic parameters of Porloq-4 cotton seedlings under comparative conditions of a model experiment under the influence of chloride and sulfate salt solutions. For this purpose, the MDA and SOD activity of Porloq-4 cotton at an early stage of vegetation were studied in comparison with Coker-312. Under stress, the level of reactive oxygen species (ROS) increases. These ROS react with lipid peroxide to form MDA. However, since the Porloq-4 variety is a stable variety, our research has shown that it has a much more effective antioxidant system. The enzyme superoxide dismutase (SOD) neutralizes ROS, resulting in a decrease in MDA levels. Materials and Methods The material for this study is the Porloq-4 cotton variety, which was developed by individually selecting lines obtained from the cross of RNAi line Coker-312 with the commercial Namangan-77 variety. The object of this study was the modified Porloq-4 cotton genotype, and the parental Coker-312 genotype was used as a control. To study the salt resistance of Porloq-4, we treated it with NaCl and Na 2 SO 4 solutions at different concentrations (Table 1 ). Table 1 Different concentrations and percentages of chlorine and sodium sulfate in water during different treatments. Cotton varieties NaCl and Na 2 SO 4 Salts Add salt content (mM and %) Porloq-4 Coker-312 Control- non-salinization 0 mM NaCl- mild salinization 100 mM NaCl-moderate salinization 150 mM NaCl-severe salinization 200 mM Control- non-salinization 0% Na 2 SO 4 - mild salinization 1.5% Na 2 SO 4 - moderate salinization 2.0% Na2SO4 - severe salinization 2.5% Table 1 location The work was carried out in a laboratory setting. The germination of seeds in the soil was carried out according to standard technology: the soil was pre-washed and dried. Small pots with drainage are filled with a mixture of soil and sand in a 70:30 ratio. The experiment was performed with four variants of the stress factor (salt) in three repetitions. Five cotton seeds were used in each repetition. Small pots (250 g of soil) were watered daily with 50 ml of water. During sowing, the temperature in the laboratory was maintained at 25–28°C. In all experimental variants, irrigation with a solution of NaCl and Na 2 SO 4 lasted 21 days, starting from the stage of two real leaves. Method for determining the amount of malondialdehyde : The amount of malondialdehyde (MDA) in plant samples (n = 15) was determined according to a modified (Heath R et al. , 1968) protocol: 100 mg of plant material was homogenized in 2 ml of 20% trichloroacetic acid (TCA), making 0.5 ml of additives. The resulting homogenate was centrifuged for 15 min at 10000g, t = 4°C. 1.5 ml of 0.5% thiobarbituric acid (TBA) dissolved in 20% TCA was added to 0.5 ml of the supernatant. Blank reaction: 0.5 ml of distilled water, 1.5 ml of TBA in a 20% TCA solution Control: 0.5 ml of supernatant, 1.5 ml of 20% TСA. Experiment: 0.5 ml of supernatant, 1.5 ml of TBA in 20% TСA. The experimental samples were incubated in a water bath at 95°C for 30 minutes. After incubation, the samples were cooled in an ice bath, and measurements of the control and experimental samples were carried out at wavelengths of 532 nm and 600 nm using a combined reader for Synergy NT microplates, manufactured by BioTek Instruments, USA. The concentration of MDA was calculated using the formula using the molar extinction coefficient: C=(D532 – D600)/(155*0.1)×1000 C is the concentration of MDA, µmol/g of crude weight; 155 – TBA extinction coefficient, mM-1cm-1. 0.1 – weight of plant material, g. Method for determining the activity of superoxide dismutase The activity of the enzyme superoxide dismutase (SOD) (Leonowicz G et al., 2018 ) was determined by the inhibition of superoxide radical in the reaction of autoxidation of adrenaline in an alkaline medium in vitro at a wavelength of 347 nm, with some modifications. To do this, 0.1 ml of distilled water and 0.1 ml of 0.1% (5.46 mM) epinephrine hydrochloride pharmacy solution were added to 2 ml of 0.2 M bicarbonate buffer, pH = 10.65, thoroughly and quickly mixed, placed in a Cary UV 60 spectrophotometer and optical density was determined after 30 seconds for 5 minutes at a wavelength of 347 nm in a quartz cuvette 10 mm thick on a Cary UV 60 (D1) spectrophotometer. Next, 0.1 ml of the enzyme source, in our case, vegetable homogenate, and 0.1 ml of 0.1% epinephrine hydrochloride were added to 2 ml of buffer (pH = 10.65), mixed, and the optical density was measured as described above. The homogenate for determining the activity of SOD was obtained as follows: 100 mg of plant leaves were ground in a porcelain mortar with 1 ml of 10 mM Tris-HCl (pH 7.8). The homogenate was centrifuged at 7000 g at a temperature of + 2, +4°C for 15 minutes. The resulting supernatant was used as an enzyme source. The SOD activity is calculated using the following formulas: А= ((D1 – D0)×54.6×V)/t Where, A is the activity of superoxide dismutase, mM of decomposed adrenaline per minute, D1 is the optical density of the experimental sample, D0 is the control optical density of the control sample, 54.6 mM is the concentration of adrenaline in the cuvette, V is the source of the enzyme in the sample, t is the inhibition time of autoxidation of adrenaline, 5 min. Method for determining the amount of chlorophyll The amount of pigments in the leaves was determined by the generally accepted method (Lichtenthaler H et al. , 1983). To determine the chlorophyll content, discs with a diameter of 4 mm were cut from the sheet plate and incubated in 80% acetone for 24 hours in darkness at room temperature. The optical density of the extract was measured at 645 and 663 NM. Chl a (mg × g − 1 weight of raw material) = (12.72 × A663) - (2.58 × A645). Chl b (mg × g − 1 weight of raw material) = (22.87 × A645) - (4.67 × A663). Also, the total chlorophyll content was measured using the SPAD-502 device. Five indicators for each plant were averaged. Statistical data analysis All data were subjected to analysis of variance (ANOVA) using the OriginPro 2022 software package. The data are presented as an average ± standard error of 3 biological and 3 technical repeats. The significance of the differences between the mean values was determined using the Tukey test. Differences in P < 0.05 were considered significant. Results Agricultural soils irrigated with high-salt water may also contain excessive concentrations of sulfate salts as a result of anthropogenic interference. We have initiated an experiment on the effect of sodium sulfate salt solutions at three concentrations on cotton seedlings at the developmental stage, when four leaves have formed on the plant. Young seedlings grown in pots under laboratory conditions were watered with 1.5%, 2.0% and 2.5% Na 2 SO 4 solutions for 30 days. The leaves were used for the analysis of metabolites according to the appropriate methods. Although the study presents data on the levels of MDA (malondialdehyde) and SOD (superoxide dismutase), their relationship with the overall physiological condition of the plant has not been sufficiently explored. In fact, an increase in MDA content indicates an intensification of lipid peroxidation, which reflects damage to cell membranes. This, in turn, leads to reduced plant growth, a decrease in chlorophyll content in leaf tissues, and visual symptoms of stress, such as leaf yellowing and stunted growth. Moreover, SOD activity indicates the activation of the plant’s antioxidant defense system, which reflects the functioning of mechanisms that counteract stress. However, if this activity is insufficient, reactive oxygen species (ROS) can damage cellular structures and cause irreversible changes in the plant. From this perspective, analyzing changes in MDA and SOD together with plant growth indicators (such as leaf length, root mass), chlorophyll content, and visible stress symptoms allows for a deeper understanding of the physiological effects of salt stress on the plant (Figs. 2 , 3 and 4 ). To assess the biotechnological genotype of Porloq-4 cotton for salinization in the first phases of vegetation, we conducted experiments in which seedlings of the early stage of development (formation of 4 leaves) were exposed to a stressor – NaCl solution at concentrations of 100 mM, 150 mM and 200 mM (plants were watered for 21 days, and then the concentrations of MDA content, as well as the activity of SOD, were determined in the collected leaves. Figure 1 location Agricultural soils that are irrigated with high-salt water may also contain excessive concentrations of sulfate salts due to anthropogenic interference. We conducted an experiment on the effects of sodium sulfate salt solutions at three different concentrations on cotton seedlings at the stage of development when the plant has four leaves. Young seedlings grown in pots in laboratory conditions were watered with 1.5%, 2.0%, and 2.5% Na 2 SO 4 solutions for 21 days. Leaves were used for metabolite analysis according to the relevant methods (Figs. 1 and 2 ). Figure 2 location Abiotic stress causes changes in key physiological components and functions of green plants. An improvement in the monitoring capabilities of such a response is non-destructively represented by determining the relative chlorophyll content using the SPAD-502 device (five readings for each plant were averaged in SPAD units). The assessment of photosynthetic pigments of leaves is an important element of plant stress monitoring. The work of a number of researchers has demonstrated that the SPAD (Soil Plant Analysis Development) readings and the content of plant photosynthetic apparatuses per leaf area are strongly influenced by the salinity and stress of nutrients, but that the general form of their relationship remains largely unaffected by stress. During the analysis of SPAD-502 indicators, it was noted that salt stress at levels of 100 mM, 150 mM, and 200 mM NaCl increased the chlorophyll content in the leaves of the Coker-312 and Porloq-4 genotypes, and increased accordingly with the increase in the salt stress content in the solution used to water the cotton plants. In the control variant, when the plants were watered with regular water, the average chlorophyll content was 1.15 times higher in Porloq-4 than in Coker-312 (Table 2). Table 2. Total chlorophyll in the leaves of cotton seedlings when exposed to NaCl solutions of different concentrations (n = 5; M ± m) N Mean SD SEM Coker-312 0 mM NaCI 5 41.32 5.24995 234785 Coker-312 100 mM NaCI 5 54.96 5.26289 2.35364 Coker-312 150 mM NaCI 5 64.78 4.08864 1.8285 Coker-312 200 mM NaCI 5 55.14 3.82531 1.71073 Porloq-4 0 mM NaCI 5 48.92 5.9061 2.64129 Porloq-4 100 mM NaCI 5 61.1 2.37592 1.06254 Porloq-4 150 mM NaCI 5 65.58 4.34246 1.94201 Porloq-4 200 mM NaCI 5 63.34 3.84877 1.72122 Table 2 location When Porloq-4 plants were treated with a chloride salt solution at a concentration of 100 mM, the SPAD index increased by 1.79 times, while for Coker-312 seedlings it increased by only 1.46 times. At the same time, the total chlorophyll content between the varieties was 23% higher (for Porloq-4). At a NaCl concentration of 150 mM, the chlorophyll content increased by 1.96 times in relation to the control variant. Whereas for Coker-312 plants, this increase was only 1.92 times. And for Coker-312–1.92 times. And between the varieties, the ratio of the increase in the total chlorophyll content with an increase in the concentration of chloride salt was 1.02 times. Already at a concentration of irrigation solution of 200 mM, the SPAD indicator increased to 2.55 times for Porloq in relation to the control variant (Fig. 3 ), and for Coker-312, in 2.30 times. At the same time, the ratio between varieties was also increased in favor of the adaptive mechanisms of cotton plants of the Porloq-4 variety by 1.12 times compared to the Coker-312 variety. Figure 3 location In parallel with this experiment, we carried out analytical work on the effect of Na 2 SO 4 solution on seedlings in order to identify the potential of varieties for salt resistance to this salt. Below are the SPAD-502 indicators for three salt concentration variants (Table 3 ). It should be noted that these concentrations were selected based on a few global studies. It should be noted that we have selected higher osmotic potentials created by the sulfate salt. The osmotic potentials would be the same if concentrations of 100 mM NaCl versus 70 mM Na 2 SO 4 and 150 mM NaCl versus 111 mM Na 2 SO 4 were used. The molecular weight is 2.43 times that of moles. the mass of NaCl. Thus, a 1.5% Na 2 SO 4 solution contains about 0.01 M Na 2 SO 4 Table 3 Total chlorophyll in the leaves of cotton seedlings when exposed to Na 2 SO 4 solutions of different concentrations Coker-312 0% Na2SO4 N Mean SD SEM 5 41.72 4.91345 2.19736 Coker-312 1.5% Na2SO4 5 47.56 3.37757 1.5105 Coker-312 2.0% Na2SO4 5 60.48 4.00961 1.79315 Coker-312 2.5% Na2SO4 5 58.48 3.80421 1.70129 Porloq-4 0% Na2SO4 5 48.92 5.9061 2.64129 Porloq-4 1.5% Na2SO4 5 59.76 3.05418 1.36587 Porloq-4 2.0% Na2SO4 5 68.08 3.86743 1.72957 Porloq-4 2.5% Na2SO4 5 64.08 4.9952 2.23392 Table 3 location As can be seen from the table data, the lower concentrations of sodium sulfate also caused a response and affected the composition of total chlorophyll in the leaves of the Coker-312 seedlings. This resulted in an increase in total chlorophyll, but not to the same extent as in the case of chloride solutions. In the control experiment, the ratio of total chlorophyll (SPAD) was 1.02. However, when exposed to stress, the ratio was 1.06 (between the two varieties). Whereas from the control, the ratio of the increase in chlorophyll increased by 1.26 times for Porloq-4, and by 1.19 times for Coker-312. The dynamics of changes in the total chlorophyll content based on the readings of SPAD-502 when plants are watered with sulfate salt solutions at an early stage of development can be observed in Fig. 4 . Figure 4 location By comparing the total chlorophyll index, we observe increased SPAD values with medium and high levels of chloride salt stress in both Porloq-4 and Coker-312 seedlings, compared with stress from sulfate solution. Nevertheless, it should be noted that in the control variants, the total chlorophyll index in this experiment is higher than in the model of plant treatment with sulfate salt. Thus, the SPAD value increased in the leaves of the Porloq-4 variety compared to the Coker-312 variety. Visually, the Porloq-4 plants also showed better preservation of turgor and green leaf color at all levels of salt exposure compared to Coker-312, which further confirms its greater salt tolerance. Thus, the Porloq-4 variety demonstrated higher salt tolerance, which is evidenced not only by the preservation and even increase in chlorophyll content, but also by the lower variability of SPAD values (low m values), indicating stable physiological responses under stress conditions. Abiotic stress causes changes in key physiological components and functions of green plants. Modulating antioxidant activity can increase plant salt tolerance and serve as a marker for effective selection of salt-resistant varieties. The MDA content makes it possible to evaluate lipid peroxidation. According to our results, salt stress affects lipid peroxidation, as evidenced by both an increase and decrease in the content of MDA under stressful conditions. Figure 5 location Considering the content of MDA and the activity of antioxidant enzymes, it is possible to observe different dynamics in the variants of separate and combined effects of chloride and sulfate salts at varying concentrations in three experimental variants. As mentioned earlier, the concentration of MDA is considered an indicator of oxidative damage to cellular components resulting from the high production of reactive oxygen species (ROS) associated with high concentrations of sodium salts. The average values of MDA recorded by us for six experimental variants in the leaves of Porloq-4 seedlings were as follows: when exposed to a chloride salt solution, there was an increase in MDA by 15%, a decrease by 22.36% (relative to the control group of seedlings), and an increase by 11.58%. In the variant of exposure to sulfate salt, the content of MDA in leaf tissues exceeded that of the control variant by 32%, 5.1%, and 117.9%, respectively, corresponding to increased concentrations of the stress solution. For the Coker-312 variety, the changes in MDA content were as follows: an excess of the control variant by 21.1%, a decrease by 15.53%, and in the third variant, exposure to higher concentrations of NaCl salt by 23.72%. When watering the Na 2 SO 4 seedlings, the increases in MDA were 17.1%, 4.65%, and 22.34% in the three variants, respectively. In this experiment, we observe a relatively reduced indicator response to average salt concentrations compared to exposure to low and higher concentrations of sodium salts (Fig. 5 . A and B). The reaction of the seedlings at an early stage of vegetation to watering with solutions of two combined sodium salts is observed in Fig. 6 . The MDA values increase with increasing salt concentration. Thus, for the first variant of the combination of NaCl and Na 2 SO 4 , indications of an excess of the control variant by 30.6% were noted in Porloq-4 seedlings and 34.4% in the Coker-312 variety in the first variant. For the second variant of the combination of two salts, this indicator increased to 54% and 54.83% for varieties, respectively. Whereas for the third variant, there is an increase in the content of MDA in the leaves of seedlings of both Porloq-4 and Coker-312 varieties by 57% and 64.5%, respectively. It should be noted that in the control variant, the amount of MDA in Porloq-4 exceeded 1.1 times that in the Coker-312 variety. According to the variants, these ratios are 0.97 times for the first variant and 0.99 and 0.95 times. This also suggests that the average salt concentration variant results in a more pronounced response in terms of MDA content. Whereas the effect of medium concentration solutions (150 mM NaCl and 1.5% Na 2 SO 4 ) led to a decrease in the content of MDA relative to the first experimental variant, and even relative to the control variant for chloride salt on the activity of superoxide dismutase (SOD). The greater the generation of ROS, the higher the activity of the SOD enzyme. Similarly, our results showed a greater activity of SOD under salt stress conditions compared to the control. It was found that the activity of SOD increases significantly in the leaves of Porloq-4 seedlings under salt stress. Since the SOD enzyme can catalyze the conversion of superoxide into molecular oxygen and H 2 O 2 , this enzyme is considered the most effective intracellular enzymatic antioxidant. According to the data obtained in this experiment, the activity of SOD increases by 18.9%, 79% and 34%, respectively, with an increase in the concentration of the stressor on Porloq-4 cotton seedlings. Considering the changes that occurred in the leaves of seedlings of the Coker-312 variety, the dynamics of the activity of this enzyme in this case are somewhat different. When watering plants with a solution of 100 mM NaCl, the activity of SOD increased slightly (by 3%), while in the second and third versions of the experiment, the increase in activity was 69% and 11.5%. It should be noted that this gain did not exceed the Porloq-4 grade. And here there is a noticeable decrease in the activity level at the highest concentration of the stressor, compared with the effect of an average concentration of saline solution on cotton seedlings. It has been noted in the scientific literature that the high activity of antioxidant enzymes is associated with both salt resistance and salt sensitivity (Abogadallah, G. 2010). Figure 6 location As a glycophyte, cotton is more resistant to abiotic stresses than other major agricultural crops. And this is despite even the type of salt. However, it was important to consider what the activity of SOD is under the abiotic stress created by the sulfate salt. In the experiment, when the seedlings at the stage of forming 4 leaves began to be watered with a solution of Na 2 SO 4 in three concentrations. This dynamic is noted in our experiment. So, when compared with the control variant, for the first variant, when the concentration of Na 2 SO 4 in the irrigation solution was 1.5%, the activity of SOD increased by 2.31 times and was equal to 231.4%, that is, by 131.4% higher. In the second version of the experiment, when the concentration of Na 2 SO 4 in the irrigation solution was 2.0%, the activity of SOD increased to 260%, although it exceeded that of 12% compared to the first version of the experiment. When a 2.5% solution was applied to cotton seedlings, the activity of SOD was slightly reduced than in the second variant in comparison with the control variant - irrigation with clean water. Discussion Due to the fact that, as a result of a number of reasons, excessive concentrations of various salts, not only chloride but also sulfate, gradually accumulate in soils, there is a need to conduct studies to identify the tolerance of sown crops to these stressors in different combinations. However, most of the world's studies on the quantitative assessment of salt tolerance of plant species were based on experiments in which the predominant salt was NaCl.l. This salt is the most common salt in saline soils, and most salt tolerance studies have been conducted using NaCl alone (Zhang L et al., 2014 ; Reich M et al., 2015 ). Studies of biochemical and physiological parameters of response reactions confirmed the existence of genetic variations in salinity tolerance in cotton (Zhao G et al., 2020 ). At the 150 mM stage, salt stress leads to an increase in reactive oxygen species (ROS) within plant cells. As a result, the plant activates its defense mechanisms, and the activity of antioxidant enzymes, including superoxide dismutase (SOD), is enhanced. This represents an adaptive response aimed at protecting cells from the damaging effects of ROS. However, at a 200 mM NaCl concentration, salt stress becomes excessive and harmful for the plant. Under such conditions, the antioxidant system itself becomes impaired. The synthesis or activity of the SOD enzyme decreases, as the enzymes may be directly damaged by oxidative stress. In addition, the reduced activity may be associated with depletion of energy reserves, suppressed gene expression, and disruption of metabolic processes. In this case, the plant enters a state of damage rather than adaptation (Kohli S et al. , 2019; Abogadallah G, 2010). Therefore, 150 mM NaCl represents a stress response stage, where SOD activity increases and defense mechanisms function effectively. 200 mM NaCl — represents a damage stage, where defensive activity is insufficient and enzyme activity decreases (Fig. 6 . A and B). Although NaCl is one of the most common salts in soils, other salts, such as Na 2 SO 4 , can also be present in high concentrations in some soil types. NaCl and Na 2 SO 4 are even considered to be the main causes of salinization in agricultural lands (Sharif I et al., 2019 ). Agricultural crops have developed several adaptations to salt stress, such as ion homeostasis, osmotic regulation, and various metabolic processes (Pessarakli M et al. , 2010). Salinity has a negative impact on various plant characteristics: the area of leaves exposed to osmotic stress, plant growth, root and shoot growth, and these parameters are the result of the effect of reduced photosynthetic activity, metabolic changes (Volkov V et al. , 2017). However, studies of salt stress involving Na 2 SO 4 are still few and far between, and the mechanism of its toxicity in plants is still poorly understood. Sodium salts have different effects on different crops. For example, for C. demersum L, Na 2 SO 4 was more toxic than NaCl, and high salt concentrations had a significant impact on the morphological and physiological characteristics (Shehzad M et al., 2019 ). Munawar W. (2021) and colleagues noted that the total chlorophyll content in the leaves of some genotypes differed slightly as a result of salt stress. However, a significant increase in total chlorophyll was observed among the group they studied under salt stress (Munawar W et al., 2021 ). Generally, plant species have different tolerance/susceptibility responses to elevated sodium salt concentrations. However, Na 2 SO 4 seems to be more growth-inhibitory in species such as barley, wheat, sugar cane, beet, tomato, wild potato, and others (Al-Nabhan et al. , 2024). In elucidating the plant response to chloride or sulfate salinity stress at the genome level, and performing a combined transcriptome (microarray analysis) and physiological study on rice, it was shown that NaCl was more toxic to seedlings than Na 2 SO 4 . Contrasting genes were expressed under sulfate and chloride salinity, with the difference being most pronounced in the root. Most genes involved in the salt stress response were upregulated in Na 2 SO 4 -treated plants, while more genes were downregulated in NaCl-treated plants. Proline accumulated to a greater extent in NaCl-treated plants (Reginato M et al. , 2021). How can the negative impact of sulfate salt be explained? Perhaps due to the fact that it leads to excessive accumulation of S in agricultural crops (Irakoze W et al., 2022 ). A certain amount of elemental sulfur (S) is essential for higher plants, as it is a constituent of methionine, cysteine, membrane sulfur lipids, cell walls, vitamins, cofactors, and various metabolites with various biological functions (Moreno-Izaguirre et al. , 2015). However, Na 2 SO 4 salinity can also affect S metabolism in plants, resulting in a sharp increase in cysteine content and, in some cases, disruption of carbon metabolism (Davidian J et al. , 2010). Another explanation for how plants can reduce the toxicity of soil salts is the ability of salt-tolerant plants to exclude sodium or compartmentalize it in vacuoles, apoplasm, or trichomes (Aghajanzadeh T et al. , 2018). It has been noted in scientific literature that high activity of antioxidant enzymes is associated with both salt tolerance and salt sensitivity (Flowers T et al. , 2019 In the scientific literature, an increased level of antioxidant enzyme activity can be considered as one of the possible mechanisms of resistance to abiotic stresses (Abbasi H et al., 2016 ). As noted in other studies, sodium sulfate-treated cotton exhibited significant increases in relative conductivity, malondialdehyde content, superoxide dismutase, peroxidase, and leaf catalase activity (Abogadallah G 2010). We have considered the enzyme SOD and MDA since it is noted in the world literature that, due to its function, SOD is considered the main antioxidant enzyme, since it regulates O2 − and H 2 O 2 concentration (Guo J et al., 2024 ). Furthermore, Kohli et al. ( 2019 ), SOD regulates the overproduction of reactive oxygen species (ROS), especially O 2 (Kohli S et al. , 2019). Gene expression and transcriptomics profiling studies in cotton have disclosed that GhCLO (caleosin) genes are responsible for drought and salinity. Higher MDA accumulation and significantly lower SOD activity were found in transformed plants under saline conditions, where GhCLO had a decisive effect on the salt tolerance of cotton (Fu X et al., 2022 ). NaCl treatment and field experiments showed that overexpression of the GaJAZ1 gene significantly enhanced the salt tolerance of cotton, resulting in increased fresh weight, more bolls, and vigorous growth of taller plants (Zhao G et al., 2020 ). It is noted in the world literature that, due to its function, SOD is considered the main antioxidant enzyme, since it regulates O 2 and H 2 O 2 concentrations (Zhao G et al., 2020 ). It was also observed that in cotton tissues, with an increase in the concentration of NaCl, the activity of SOD also increased. This also happened in our experiments. In a comparative aspect, we present a change in the activity of one of the enzymes of the antioxidant system. The activity of SOD increases by 51%, 124% and 71% in the leaves of Porloq-4 plants. Whereas the Coker-312 variety in the same variants increased by 81.1%, 198.6% and 95.9%. This ratio between Porloq-4 and Coker-312 grades is 0.62, 0.63, and 0.74 times for the chloride salt exposure option (Fig. 6 . B). Watering the seedlings with sulfate salt solutions led to a change in the activity of SOD, somewhat lower than in the variant with chloride salt. The activity of SOD in the leaves of Porloq-4 plants in the variant with a relatively low concentration of Na 2 SO 4 (1.5%) increased by 14%, in the second variant – by 9%, and in the third variant – by 6% (Fig. 6 . A). Whereas for the Coker-312 variety, these indicators were as follows: an increase of 1.1%, 10.37% and 39.3%. What is the reaction of seedlings at an early stage of vegetation to stress from two sodium salts at once can be considered. The effect of solutions of combined salts of NaCl and Na 2 SO 4 in one irrigation solution on seedlings caused changes in the activity of SOD of the following order. In the first variant, when relatively low concentrations of NaCl and Na 2 SO 4 were used, the activity of this enzyme of the antioxidant system increased by 44.9% for Porloq-4 and by 27.2% for Coker-312. Whereas in the second variant, activity increased by 99.1% for Porloq-4 and 69.3% for Coker-312. In the third variant, when relatively high concentrations of both salts were used in the solution for watering young plants at the stage of 4 leaves, the activity of SOD increased from the control variant by 83.5% and 65.8% for the varieties, respectively. Thus, it can be noted that the second version of the experiment elicits a greater reaction from one of the plant's antioxidant enzymes, or it can be said that it is more toxic than the other two (Fig. 6 ). Due to the fact that excessive concentrations of various salts, not only chloride, but also sulfate, gradually accumulate in soils for a number of reasons, there is a need to conduct research to identify the tolerance of crops to these stressors. However, most of the world's research on quantifying the salt tolerance of plant species has been based on experiments in which NaCl is the predominant salt. Studies of biochemical and physiological response parameters have confirmed the existence of genetic variations in salinity tolerance in cotton (Saleh B 2012 ). Parameters such as chlorophyll content were used to determine the tolerance of a particular genotype and select the most resistant ones. This selection criterion is related to an increase in yield, the rate of photosynthesis, and the production of dry matter (Harinasut et al., 1996 ). Conclusion It should be noted that, as a result of artificially created salinization stress in laboratory conditions, an increase in the content of malonic dialdehyde (MDA) and the activity of enzymes of the antioxidant system occurs. As the concentration of stress salt increases, an increase in each of the indicators relative to the control variant was mainly revealed. Although in some cases their decrease is also observed, especially in the variant when the strength of the stress salt is relatively high (the third experimental variants). Nevertheless, these levels were higher than in the control variants of the same experiment. Based on this, it can be concluded that the Porloq-4 and Coker-312 genotypes have an adaptive ability, and the response of the Porloq-4 biotechnological variety was more active than that of Coker-312. Experiments to assess the tolerance potential of different genotypes of cotton are also conducted by foreign colleagues. For example, Surian researcher Saleh ( 2012 ) evaluated five cotton varieties ( G.hirsutum L.) for exposure to NaCl concentrations of 0, 50, 100, and 200 mM (Sharif I et al., 2019 ), in order to better understand the reactions of various cotton varieties to salinization stress. Thus, based on the experimental results, we concluded that the analyzed Porloq-4 and Coker-312 cotton varieties react differently to stress from NaCl and Na 2 SO 4 , while Porloq-4 cotton varieties have a more active and responsive antioxidant enzyme system. Declarations Acknowledgements This work was carried out within the framework of the fundamental project FL-9524115083, funded by the Agency for Innovative Development under the Ministry of Higher Education, Science and Innovation of the Republic of Uzbekistan. Ethics Approval Not applicable to this paper. Funding Source This work was supported by the Innovative Development Agency under the Ministry of Higher Education, Science and Innovation Ministry of Uzbekistan [grant numbers FL-9524115083]. Author Contributions NRR, ASI, IBS and VSK carried out the experiments and wrote andrevised the manuscript, performed statistical analysis. SBK, FSR, MZ, RMA, ZZY, RAJ and AAR participated in the experiments, collected the data, and prepared the manuscript. ZTB edited and approved the manuscript. All authors read and approved the final manuscript.. Conflict of interest The authors declare that they have no competing interests. Data Availability Data presented in this study will be available on a fair request to the corresponding author. References Yu Z, X Duan, L Luo, S Dai, Z Ding, G Xia (2020). How Plant Hormones Mediate Salt Stress Responses. Trends Plant Sci 11:1117–1130 Verma V, P Ravindran, Kumar PP (2016). Plant hormone-mediated regulation of stress responses. 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Evolution and Stress Responses of CLO Genes and Potential Function of the GhCLO06 Gene in Salt Resistance of Cotton. Front Plant Sci 1712:801239 Sharif I, S Aleem, J Farooq, M Rizwan, A Younas, G Sarwar, SM Chohan (2019). Salinity stress in cotton: effects, mechanism of tolerance and its management strategies. Physiol Mol Biol Plants 4:807–820 Saleh B (2012). Effect of salt stress on growth and chlorophyll content of some cultivated cotton varieties grown in Syria. Commun Soil Sci Plant Anal 15:1976–83 Harinasut P, K Tsutsui, T Takabe, M Nomura, T Takabe, S Kishitani (1996). Exogenous Glycinebetaine Accumulation and Increased Salt-tolerance in Rice Seedlings. Biosci Biotechnol Biochem 2:366–8 Additional Declarations No competing interests reported. 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The columns represent M ± m (n = 5).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9426942/v1/d284957e8a5f7fb4996758dc.png"},{"id":107705779,"identity":"b3b41df0-fe51-4c3b-b9dc-0fee05062009","added_by":"auto","created_at":"2026-04-24 09:15:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":91487,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSPAD readings of cotton seedling leaves under sulfate stress. The columns represent M ± m (n = 5).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9426942/v1/3fd71c1508c614d2dd25e262.png"},{"id":107597181,"identity":"8b650a4f-83ac-47c0-a880-99cd1a4478cc","added_by":"auto","created_at":"2026-04-23 05:30:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":70738,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMDA in the leaves of cotton seedlings when exposed to different concentrations of salt Na\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eSO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4 \u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e(A) and NaCl (B). On the X-axis - the salt concentration (in % for Na\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eSO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e and in mM for NaCl); the Y-axis is the accumulation of TBA-active products, in μmol MDA/mg wet weight. Data are means ± standard errors (n = 15). p ≤ 0,0001.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9426942/v1/86cc4a22811465ca971f10be.png"},{"id":107705856,"identity":"eb98de58-a93d-4b77-999b-f6bc3d4d085f","added_by":"auto","created_at":"2026-04-24 09:15:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":80138,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe dynamics of SOD in the leaves of cotton seedlings under the influence of different concentrations of salt NaCl and Na\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eSO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e. The Y-axis indicates SOD activity (U / mg of protein). Data are means ± standard errors (n = 15); p ≤ 0,0001\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9426942/v1/b8a72e7aa40e5fcee941cc42.png"},{"id":107709220,"identity":"60f9efd5-11cd-4e38-a6b5-10c0afe1b182","added_by":"auto","created_at":"2026-04-24 09:35:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2897683,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9426942/v1/babfadf8-4a2a-42f7-8776-59522b6dbc47.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Assessment of antioxidant enzyme responses in a biotechnological cotton variety under salt stress conditions","fulltext":[{"header":"Novelty statement","content":"\u003cp\u003eIn this study, the responses of the biotechnological cotton variety Porloq-4 and the Coker-312 variety to NaCl and Na₂SO₄ salt stress were compared based on antioxidant (SOD) activity and MDA levels. Results showed that Porloq-4 had higher metabolic tolerance to salt stress. At moderate salt concentrations, SOD activity increased by 4.1 times, and MDA content significantly rose. This work contributes to understanding the mechanisms of salt stress tolerance in Porloq-4 through its physiological responses.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eSalt resistance is defined as a plant's ability to grow, develop, and reproduce under saline conditions with minimal damage. As you know, any organism is a self-regulating system. To minimize oxidative damage caused by ROS production during salt stress, plants have a complex system of interrelated mechanisms, including antioxidant enzymes, osmolytes, and some non-enzymatic antioxidants (Yu Z et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). To mitigate the adverse effects of salt stress on plants, two complementary approaches are employed: the development of salt-resistant varieties and the enhancement of agrotechnological techniques (Verma V et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Ku Y \u003cem\u003eet al.\u003c/em\u003e, 2018).\u003c/p\u003e \u003cp\u003eTo obtain more comprehensive information about the biotechnological variety's tolerance to salt stress, we analyzed the activity of certain enzymes in the antioxidant system and the level of malondialdehyde (MDA). Plants respond to salt stress using highly regulated protective enzymes, such as MDA and SOD, to enhance their ability to purify reactive oxygen species.\u003c/p\u003e \u003cp\u003eProlonged exposure to high salinity leads to the accumulation of toxic ions, such as Na+, Cl\u0026minus;, and SO42\u0026minus;, which induce ion toxicity and impair nutrient absorption, thereby exacerbating damage to plant cells and tissues (Isayenkov S \u003cem\u003eet al.\u003c/em\u003e, 2019). The undesirable effects of salt stress on plants are manifested in morphology (stunting, chlorosis, impaired seed germination), physiology (inhibition of photosynthesis and nutrient imbalance), and biochemical properties (oxidative stress, electrolyte leakage, membrane disorganization) (Ji X et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Hannachi S et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The adverse effects of salinity are particularly severe during the reproductive stage (Rafaliarivony S et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Over the past 20 years, many studies have been conducted and analyzed on the harm caused by salts and their effects on plants. Increasing plant tolerance to salt stress is a primary focus in the genetic improvement of agricultural crops. The study of salt tolerance is a crucial component of plant biology research, enabling us to understand the complex mechanisms of plant salt tolerance and explore strategies to mitigate the harmful effects of salt stress (Balasubramaniam T et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn our previous studies, we analyzed the effect of salicylic acid on cotton plants under abiotic stress conditions. Salicylic acid is a phytohormone that is naturally synthesized in plants and plays an important role in the response to both biotic and abiotic stress factors (Rakhmatova N et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). It activates the antioxidant defense system, reduces the level of reactive oxygen species (ROS), increases the activity of antioxidant enzymes (such as SOD, CAT, and APX), and also decreases the content of MDA (malondialdehyde), thereby reducing lipid peroxidation. In addition, salicylic acid enhances plant resistance to salt stress, drought, and infectious diseases. In this study, our objective was to analyze MDA content and SOD activity in cotton plants under salt stress conditions, which is of significant interest to many researchers.\u003c/p\u003e \u003cp\u003eOur goal was to identify changes in some metabolic parameters of Porloq-4 cotton seedlings under comparative conditions of a model experiment under the influence of chloride and sulfate salt solutions. For this purpose, the MDA and SOD activity of Porloq-4 cotton at an early stage of vegetation were studied in comparison with Coker-312.\u003c/p\u003e \u003cp\u003eUnder stress, the level of reactive oxygen species (ROS) increases. These ROS react with lipid peroxide to form MDA. However, since the Porloq-4 variety is a stable variety, our research has shown that it has a much more effective antioxidant system. The enzyme superoxide dismutase (SOD) neutralizes ROS, resulting in a decrease in MDA levels.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eThe material for this study is the Porloq-4 cotton variety, which was developed by individually selecting lines obtained from the cross of RNAi line Coker-312 with the commercial Namangan-77 variety. The object of this study was the modified Porloq-4 cotton genotype, and the parental Coker-312 genotype was used as a control. To study the salt resistance of Porloq-4, we treated it with NaCl and Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e solutions at different concentrations (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDifferent concentrations and percentages of chlorine and sodium sulfate in water during different treatments.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCotton varieties\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNaCl and Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e Salts\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAdd salt content (mM and %)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"6\" rowspan=\"7\"\u003e \u003cp\u003ePorloq-4\u003c/p\u003e \u003cp\u003eCoker-312\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl- non-salinization\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 mM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNaCl- mild salinization\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100 mM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNaCl-moderate salinization\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e150 mM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNaCl-severe salinization\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e200 mM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl- non-salinization\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNa\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e- mild salinization\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.5%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNa\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e- moderate salinization\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.0%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNa2SO4 - severe salinization\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.5%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e location\u003c/p\u003e \u003cp\u003eThe work was carried out in a laboratory setting. The germination of seeds in the soil was carried out according to standard technology: the soil was pre-washed and dried. Small pots with drainage are filled with a mixture of soil and sand in a 70:30 ratio. The experiment was performed with four variants of the stress factor (salt) in three repetitions. Five cotton seeds were used in each repetition. Small pots (250 g of soil) were watered daily with 50 ml of water. During sowing, the temperature in the laboratory was maintained at 25\u0026ndash;28\u0026deg;C. In all experimental variants, irrigation with a solution of NaCl and Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e lasted 21 days, starting from the stage of two real leaves.\u003c/p\u003e \u003cp\u003e\u003cb\u003eMethod for determining the amount of malondialdehyde\u003c/b\u003e: The amount of malondialdehyde (MDA) in plant samples (n\u0026thinsp;=\u0026thinsp;15) was determined according to a modified (Heath R \u003cem\u003eet al.\u003c/em\u003e, 1968) protocol: 100 mg of plant material was homogenized in 2 ml of 20% trichloroacetic acid (TCA), making 0.5 ml of additives. The resulting homogenate was centrifuged for 15 min at 10000g, t\u0026thinsp;=\u0026thinsp;4\u0026deg;C. 1.5 ml of 0.5% thiobarbituric acid (TBA) dissolved in 20% TCA was added to 0.5 ml of the supernatant.\u003c/p\u003e \u003cp\u003eBlank reaction: 0.5 ml of distilled water, 1.5 ml of TBA in a 20% TCA solution\u003c/p\u003e \u003cp\u003eControl: 0.5 ml of supernatant, 1.5 ml of 20% TСA.\u003c/p\u003e \u003cp\u003eExperiment: 0.5 ml of supernatant, 1.5 ml of TBA in 20% TСA.\u003c/p\u003e \u003cp\u003eThe experimental samples were incubated in a water bath at 95\u0026deg;C for 30 minutes.\u003c/p\u003e \u003cp\u003eAfter incubation, the samples were cooled in an ice bath, and measurements of the control and experimental samples were carried out at wavelengths of 532 nm and 600 nm using a combined reader for Synergy NT microplates, manufactured by BioTek Instruments, USA.\u003c/p\u003e \u003cp\u003eThe concentration of MDA was calculated using the formula using the molar extinction coefficient:\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eC=(D532 \u0026ndash; D600)/(155*0.1)\u0026times;1000\u003c/h2\u003e \u003cp\u003eC is the concentration of MDA, \u0026micro;mol/g of crude weight;\u003c/p\u003e \u003cp\u003e155 \u0026ndash; TBA extinction coefficient, mM-1cm-1.\u003c/p\u003e \u003cp\u003e0.1 \u0026ndash; weight of plant material, g.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eMethod for determining the activity of superoxide dismutase\u003c/strong\u003e \u003cp\u003eThe activity of the enzyme superoxide dismutase (SOD) (Leonowicz G et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) was determined by the inhibition of superoxide radical in the reaction of autoxidation of adrenaline in an alkaline medium in vitro at a wavelength of 347 nm, with some modifications. To do this, 0.1 ml of distilled water and 0.1 ml of 0.1% (5.46 mM) epinephrine hydrochloride pharmacy solution were added to 2 ml of 0.2 M bicarbonate buffer, pH\u0026thinsp;=\u0026thinsp;10.65, thoroughly and quickly mixed, placed in a Cary UV 60 spectrophotometer and optical density was determined after 30 seconds for 5 minutes at a wavelength of 347 nm in a quartz cuvette 10 mm thick on a Cary UV 60 (D1) spectrophotometer. Next, 0.1 ml of the enzyme source, in our case, vegetable homogenate, and 0.1 ml of 0.1% epinephrine hydrochloride were added to 2 ml of buffer (pH\u0026thinsp;=\u0026thinsp;10.65), mixed, and the optical density was measured as described above.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eThe homogenate for determining the activity of SOD was obtained as follows: 100 mg of plant leaves were ground in a porcelain mortar with 1 ml of 10 mM Tris-HCl (pH 7.8). The homogenate was centrifuged at 7000 g at a temperature of +\u0026thinsp;2, +4\u0026deg;C for 15 minutes. The resulting supernatant was used as an enzyme source.\u003c/p\u003e \u003cp\u003eThe SOD activity is calculated using the following formulas:\u003c/p\u003e \u003cp\u003eА= ((D1 \u0026ndash; D0)\u0026times;54.6\u0026times;V)/t\u003c/p\u003e \u003cp\u003eWhere, A is the activity of superoxide dismutase, mM of decomposed adrenaline per minute, D1 is the optical density of the experimental sample, D0 is the control optical density of the control sample, 54.6 mM is the concentration of adrenaline in the cuvette, V is the source of the enzyme in the sample, t is the inhibition time of autoxidation of adrenaline, 5 min.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eMethod for determining the amount of chlorophyll\u003c/strong\u003e \u003cp\u003eThe amount of pigments in the leaves was determined by the generally accepted method (Lichtenthaler H \u003cem\u003eet al.\u003c/em\u003e, 1983). To determine the chlorophyll content, discs with a diameter of 4 mm were cut from the sheet plate and incubated in 80% acetone for 24 hours in darkness at room temperature. The optical density of the extract was measured at 645 and 663 NM. Chl a (mg \u0026times; g\u0026thinsp;\u0026minus;\u0026thinsp;1 weight of raw material) = (12.72 \u0026times; A663) - (2.58 \u0026times; A645). Chl b (mg \u0026times; g\u0026thinsp;\u0026minus;\u0026thinsp;1 weight of raw material) = (22.87 \u0026times; A645) - (4.67 \u0026times; A663). Also, the total chlorophyll content was measured using the SPAD-502 device. Five indicators for each plant were averaged.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eStatistical data analysis\u003c/strong\u003e \u003cp\u003eAll data were subjected to analysis of variance (ANOVA) using the OriginPro 2022 software package. The data are presented as an average\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of 3 biological and 3 technical repeats. The significance of the differences between the mean values was determined using the Tukey test. Differences in P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered significant.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eAgricultural soils irrigated with high-salt water may also contain excessive concentrations of sulfate salts as a result of anthropogenic interference. We have initiated an experiment on the effect of sodium sulfate salt solutions at three concentrations on cotton seedlings at the developmental stage, when four leaves have formed on the plant. Young seedlings grown in pots under laboratory conditions were watered with 1.5%, 2.0% and 2.5% Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e solutions for 30 days. The leaves were used for the analysis of metabolites according to the appropriate methods.\u003c/p\u003e\n\u003cp\u003eAlthough the study presents data on the levels of MDA (malondialdehyde) and SOD (superoxide dismutase), their relationship with the overall physiological condition of the plant has not been sufficiently explored. In fact, an increase in MDA content indicates an intensification of lipid peroxidation, which reflects damage to cell membranes. This, in turn, leads to reduced plant growth, a decrease in chlorophyll content in leaf tissues, and visual symptoms of stress, such as leaf yellowing and stunted growth. Moreover, SOD activity indicates the activation of the plant\u0026rsquo;s antioxidant defense system, which reflects the functioning of mechanisms that counteract stress. However, if this activity is insufficient, reactive oxygen species (ROS) can damage cellular structures and cause irreversible changes in the plant. From this perspective, analyzing changes in MDA and SOD together with plant growth indicators (such as leaf length, root mass), chlorophyll content, and visible stress symptoms allows for a deeper understanding of the physiological effects of salt stress on the plant (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eTo assess the biotechnological genotype of Porloq-4 cotton for salinization in the first phases of vegetation, we conducted experiments in which seedlings of the early stage of development (formation of 4 leaves) were exposed to a stressor \u0026ndash; NaCl solution at concentrations of 100 mM, 150 mM and 200 mM (plants were watered for 21 days, and then the concentrations of MDA content, as well as the activity of SOD, were determined in the collected leaves.\u003c/p\u003e\n\u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e location\u003c/p\u003e\n\u003cp\u003eAgricultural soils that are irrigated with high-salt water may also contain excessive concentrations of sulfate salts due to anthropogenic interference. We conducted an experiment on the effects of sodium sulfate salt solutions at three different concentrations on cotton seedlings at the stage of development when the plant has four leaves. Young seedlings grown in pots in laboratory conditions were watered with 1.5%, 2.0%, and 2.5% Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e solutions for 21 days. Leaves were used for metabolite analysis according to the relevant methods (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e location\u003c/p\u003e\n\u003cp\u003eAbiotic stress causes changes in key physiological components and functions of green plants. An improvement in the monitoring capabilities of such a response is non-destructively represented by determining the relative chlorophyll content using the SPAD-502 device (five readings for each plant were averaged in SPAD units). The assessment of photosynthetic pigments of leaves is an important element of plant stress monitoring. The work of a number of researchers has demonstrated that the SPAD (Soil Plant Analysis Development) readings and the content of plant photosynthetic apparatuses per leaf area are strongly influenced by the salinity and stress of nutrients, but that the general form of their relationship remains largely unaffected by stress.\u003c/p\u003e\n\u003cp\u003eDuring the analysis of SPAD-502 indicators, it was noted that salt stress at levels of 100 mM, 150 mM, and 200 mM NaCl increased the chlorophyll content in the leaves of the Coker-312 and Porloq-4 genotypes, and increased accordingly with the increase in the salt stress content in the solution used to water the cotton plants. In the control variant, when the plants were watered with regular water, the average chlorophyll content was 1.15 times higher in Porloq-4 than in Coker-312 (Table\u0026nbsp;2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Total chlorophyll in the leaves of cotton seedlings when exposed to NaCl solutions of different concentrations (n = 5; M \u0026plusmn; m)\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"Left\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 173px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;Mean\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003eSEM\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 173px;\"\u003e\n \u003cp\u003eCoker-312 0 mM NaCI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e41.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e5.24995\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e234785\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 173px;\"\u003e\n \u003cp\u003eCoker-312 100 mM NaCI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e54.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e5.26289\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e2.35364\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 173px;\"\u003e\n \u003cp\u003eCoker-312 150 mM NaCI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e64.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e4.08864\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e1.8285\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 173px;\"\u003e\n \u003cp\u003eCoker-312 200 mM NaCI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e55.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e3.82531\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e1.71073\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 173px;\"\u003e\n \u003cp\u003ePorloq-4 0 mM NaCI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e48.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e5.9061\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e2.64129\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 173px;\"\u003e\n \u003cp\u003ePorloq-4 100 mM NaCI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e61.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e2.37592\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e1.06254\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 173px;\"\u003e\n \u003cp\u003ePorloq-4 150 mM NaCI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e65.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e4.34246\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e1.94201\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 173px;\"\u003e\n \u003cp\u003ePorloq-4 200 mM NaCI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e63.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e3.84877\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e1.72122\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eTable\u0026nbsp;2 location\u003c/p\u003e\n\u003cp\u003eWhen Porloq-4 plants were treated with a chloride salt solution at a concentration of 100 mM, the SPAD index increased by 1.79 times, while for Coker-312 seedlings it increased by only 1.46 times. At the same time, the total chlorophyll content between the varieties was 23% higher (for Porloq-4). At a NaCl concentration of 150 mM, the chlorophyll content increased by 1.96 times in relation to the control variant. Whereas for Coker-312 plants, this increase was only 1.92 times. And for Coker-312\u0026ndash;1.92 times. And between the varieties, the ratio of the increase in the total chlorophyll content with an increase in the concentration of chloride salt was 1.02 times.\u003c/p\u003e\n\u003cp\u003eAlready at a concentration of irrigation solution of 200 mM, the SPAD indicator increased to 2.55 times for Porloq in relation to the control variant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), and for Coker-312, in 2.30 times. At the same time, the ratio between varieties was also increased in favor of the adaptive mechanisms of cotton plants of the Porloq-4 variety by 1.12 times compared to the Coker-312 variety.\u003c/p\u003e\n\u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e location\u003c/p\u003e\n\u003cp\u003eIn parallel with this experiment, we carried out analytical work on the effect of Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e solution on seedlings in order to identify the potential of varieties for salt resistance to this salt. Below are the SPAD-502 indicators for three salt concentration variants (Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). It should be noted that these concentrations were selected based on a few global studies. It should be noted that we have selected higher osmotic potentials created by the sulfate salt. The osmotic potentials would be the same if concentrations of 100 mM NaCl versus 70 mM Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and 150 mM NaCl versus 111 mM Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e were used. The molecular weight is 2.43 times that of moles. the mass of NaCl. Thus, a 1.5% Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e solution contains about 0.01 M Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e\u0026nbsp;\u003c/p\u003e\n\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eTotal chlorophyll in the leaves of cotton seedlings when exposed to Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e solutions of different concentrations\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\n \u003cp\u003eCoker-312 0% Na2SO4\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eMean\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003eSEM\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e41.72\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e4.91345\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e2.19736\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eCoker-312 1.5% Na2SO4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e47.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e3.37757\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e1.5105\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eCoker-312 2.0% Na2SO4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e60.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e4.00961\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e1.79315\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eCoker-312 2.5% Na2SO4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e58.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e3.80421\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e1.70129\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003ePorloq-4 0% Na2SO4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e48.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e5.9061\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e2.64129\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003ePorloq-4 1.5% Na2SO4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e59.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e3.05418\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e1.36587\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003ePorloq-4 2.0% Na2SO4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e68.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e3.86743\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e1.72957\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003ePorloq-4 2.5% Na2SO4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e64.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e4.9952\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e2.23392\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003eTable \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\u003e location\u003c/p\u003e\n\u003cp\u003eAs can be seen from the table data, the lower concentrations of sodium sulfate also caused a response and affected the composition of total chlorophyll in the leaves of the Coker-312 seedlings. This resulted in an increase in total chlorophyll, but not to the same extent as in the case of chloride solutions. In the control experiment, the ratio of total chlorophyll (SPAD) was 1.02. However, when exposed to stress, the ratio was 1.06 (between the two varieties). Whereas from the control, the ratio of the increase in chlorophyll increased by 1.26 times for Porloq-4, and by 1.19 times for Coker-312. The dynamics of changes in the total chlorophyll content based on the readings of SPAD-502 when plants are watered with sulfate salt solutions at an early stage of development can be observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e location\u003c/p\u003e\n\u003cp\u003eBy comparing the total chlorophyll index, we observe increased SPAD values with medium and high levels of chloride salt stress in both Porloq-4 and Coker-312 seedlings, compared with stress from sulfate solution. Nevertheless, it should be noted that in the control variants, the total chlorophyll index in this experiment is higher than in the model of plant treatment with sulfate salt.\u003c/p\u003e\n\u003cp\u003eThus, the SPAD value increased in the leaves of the Porloq-4 variety compared to the Coker-312 variety.\u003c/p\u003e\n\u003cp\u003eVisually, the Porloq-4 plants also showed better preservation of turgor and green leaf color at all levels of salt exposure compared to Coker-312, which further confirms its greater salt tolerance.\u003c/p\u003e\n\u003cp\u003eThus, the Porloq-4 variety demonstrated higher salt tolerance, which is evidenced not only by the preservation and even increase in chlorophyll content, but also by the lower variability of SPAD values (low m values), indicating stable physiological responses under stress conditions.\u003c/p\u003e\n\u003cp\u003eAbiotic stress causes changes in key physiological components and functions of green plants. Modulating antioxidant activity can increase plant salt tolerance and serve as a marker for effective selection of salt-resistant varieties. The MDA content makes it possible to evaluate lipid peroxidation. According to our results, salt stress affects lipid peroxidation, as evidenced by both an increase and decrease in the content of MDA under stressful conditions.\u003c/p\u003e\n\u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e location\u003c/p\u003e\n\u003cp\u003eConsidering the content of MDA and the activity of antioxidant enzymes, it is possible to observe different dynamics in the variants of separate and combined effects of chloride and sulfate salts at varying concentrations in three experimental variants. As mentioned earlier, the concentration of MDA is considered an indicator of oxidative damage to cellular components resulting from the high production of reactive oxygen species (ROS) associated with high concentrations of sodium salts. The average values of MDA recorded by us for six experimental variants in the leaves of Porloq-4 seedlings were as follows: when exposed to a chloride salt solution, there was an increase in MDA by 15%, a decrease by 22.36% (relative to the control group of seedlings), and an increase by 11.58%. In the variant of exposure to sulfate salt, the content of MDA in leaf tissues exceeded that of the control variant by 32%, 5.1%, and 117.9%, respectively, corresponding to increased concentrations of the stress solution. For the Coker-312 variety, the changes in MDA content were as follows: an excess of the control variant by 21.1%, a decrease by 15.53%, and in the third variant, exposure to higher concentrations of NaCl salt by 23.72%. When watering the Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e seedlings, the increases in MDA were 17.1%, 4.65%, and 22.34% in the three variants, respectively. In this experiment, we observe a relatively reduced indicator response to average salt concentrations compared to exposure to low and higher concentrations of sodium salts (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. A and B).\u003c/p\u003e\n\u003cp\u003eThe reaction of the seedlings at an early stage of vegetation to watering with solutions of two combined sodium salts is observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003eThe MDA values increase with increasing salt concentration. Thus, for the first variant of the combination of NaCl and Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, indications of an excess of the control variant by 30.6% were noted in Porloq-4 seedlings and 34.4% in the Coker-312 variety in the first variant. For the second variant of the combination of two salts, this indicator increased to 54% and 54.83% for varieties, respectively. Whereas for the third variant, there is an increase in the content of MDA in the leaves of seedlings of both Porloq-4 and Coker-312 varieties by 57% and 64.5%, respectively. It should be noted that in the control variant, the amount of MDA in Porloq-4 exceeded 1.1 times that in the Coker-312 variety. According to the variants, these ratios are 0.97 times for the first variant and 0.99 and 0.95 times. This also suggests that the average salt concentration variant results in a more pronounced response in terms of MDA content.\u003c/p\u003e\n\u003cp\u003eWhereas the effect of medium concentration solutions (150 mM NaCl and 1.5% Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e) led to a decrease in the content of MDA relative to the first experimental variant, and even relative to the control variant for chloride salt on the activity of superoxide dismutase (SOD). The greater the generation of ROS, the higher the activity of the SOD enzyme. Similarly, our results showed a greater activity of SOD under salt stress conditions compared to the control. It was found that the activity of SOD increases significantly in the leaves of Porloq-4 seedlings under salt stress. Since the SOD enzyme can catalyze the conversion of superoxide into molecular oxygen and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, this enzyme is considered the most effective intracellular enzymatic antioxidant.\u003c/p\u003e\n\u003cp\u003eAccording to the data obtained in this experiment, the activity of SOD increases by 18.9%, 79% and 34%, respectively, with an increase in the concentration of the stressor on Porloq-4 cotton seedlings. Considering the changes that occurred in the leaves of seedlings of the Coker-312 variety, the dynamics of the activity of this enzyme in this case are somewhat different. When watering plants with a solution of 100 mM NaCl, the activity of SOD increased slightly (by 3%), while in the second and third versions of the experiment, the increase in activity was 69% and 11.5%. It should be noted that this gain did not exceed the Porloq-4 grade. And here there is a noticeable decrease in the activity level at the highest concentration of the stressor, compared with the effect of an average concentration of saline solution on cotton seedlings. It has been noted in the scientific literature that the high activity of antioxidant enzymes is associated with both salt resistance and salt sensitivity (Abogadallah, G. 2010).\u003c/p\u003e\n\u003cp\u003eFigure \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e location\u003c/p\u003e\n\u003cp\u003eAs a glycophyte, cotton is more resistant to abiotic stresses than other major agricultural crops. And this is despite even the type of salt. However, it was important to consider what the activity of SOD is under the abiotic stress created by the sulfate salt. In the experiment, when the seedlings at the stage of forming 4 leaves began to be watered with a solution of Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e in three concentrations.\u003c/p\u003e\n\u003cp\u003eThis dynamic is noted in our experiment. So, when compared with the control variant, for the first variant, when the concentration of Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e in the irrigation solution was 1.5%, the activity of SOD increased by 2.31 times and was equal to 231.4%, that is, by 131.4% higher. In the second version of the experiment, when the concentration of Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e in the irrigation solution was 2.0%, the activity of SOD increased to 260%, although it exceeded that of 12% compared to the first version of the experiment. When a 2.5% solution was applied to cotton seedlings, the activity of SOD was slightly reduced than in the second variant in comparison with the control variant - irrigation with clean water.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDue to the fact that, as a result of a number of reasons, excessive concentrations of various salts, not only chloride but also sulfate, gradually accumulate in soils, there is a need to conduct studies to identify the tolerance of sown crops to these stressors in different combinations. However, most of the world's studies on the quantitative assessment of salt tolerance of plant species were based on experiments in which the predominant salt was NaCl.l. This salt is the most common salt in saline soils, and most salt tolerance studies have been conducted using NaCl alone (Zhang L et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Reich M et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Studies of biochemical and physiological parameters of response reactions confirmed the existence of genetic variations in salinity tolerance in cotton (Zhao G et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAt the 150 mM stage, salt stress leads to an increase in reactive oxygen species (ROS) within plant cells. As a result, the plant activates its defense mechanisms, and the activity of antioxidant enzymes, including superoxide dismutase (SOD), is enhanced. This represents an adaptive response aimed at protecting cells from the damaging effects of ROS. However, at a 200 mM NaCl concentration, salt stress becomes excessive and harmful for the plant. Under such conditions, the antioxidant system itself becomes impaired. The synthesis or activity of the SOD enzyme decreases, as the enzymes may be directly damaged by oxidative stress. In addition, the reduced activity may be associated with depletion of energy reserves, suppressed gene expression, and disruption of metabolic processes. In this case, the plant enters a state of damage rather than adaptation (Kohli S \u003cem\u003eet al.\u003c/em\u003e, 2019; Abogadallah G, 2010).\u003c/p\u003e \u003cp\u003eTherefore, 150 mM NaCl represents a stress response stage, where SOD activity increases and defense mechanisms function effectively. 200 mM NaCl \u0026mdash; represents a damage stage, where defensive activity is insufficient and enzyme activity decreases (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. A and B).\u003c/p\u003e \u003cp\u003eAlthough NaCl is one of the most common salts in soils, other salts, such as Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, can also be present in high concentrations in some soil types. NaCl and Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e are even considered to be the main causes of salinization in agricultural lands (Sharif I et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Agricultural crops have developed several adaptations to salt stress, such as ion homeostasis, osmotic regulation, and various metabolic processes (Pessarakli M \u003cem\u003eet al.\u003c/em\u003e, 2010). Salinity has a negative impact on various plant characteristics: the area of leaves exposed to osmotic stress, plant growth, root and shoot growth, and these parameters are the result of the effect of reduced photosynthetic activity, metabolic changes (Volkov V \u003cem\u003eet al.\u003c/em\u003e, 2017). However, studies of salt stress involving Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e are still few and far between, and the mechanism of its toxicity in plants is still poorly understood. Sodium salts have different effects on different crops. For example, for \u003cem\u003eC. demersum\u003c/em\u003e L, Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e was more toxic than NaCl, and high salt concentrations had a significant impact on the morphological and physiological characteristics (Shehzad M et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Munawar W. (2021) and colleagues noted that the total chlorophyll content in the leaves of some genotypes differed slightly as a result of salt stress. However, a significant increase in total chlorophyll was observed among the group they studied under salt stress (Munawar W et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Generally, plant species have different tolerance/susceptibility responses to elevated sodium salt concentrations. However, Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e seems to be more growth-inhibitory in species such as barley, wheat, sugar cane, beet, tomato, wild potato, and others (Al-Nabhan \u003cem\u003eet al.\u003c/em\u003e, 2024). In elucidating the plant response to chloride or sulfate salinity stress at the genome level, and performing a combined transcriptome (microarray analysis) and physiological study on rice, it was shown that NaCl was more toxic to seedlings than Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e. Contrasting genes were expressed under sulfate and chloride salinity, with the difference being most pronounced in the root. Most genes involved in the salt stress response were upregulated in Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e-treated plants, while more genes were downregulated in NaCl-treated plants. Proline accumulated to a greater extent in NaCl-treated plants (Reginato M \u003cem\u003eet al.\u003c/em\u003e, 2021). How can the negative impact of sulfate salt be explained? Perhaps due to the fact that it leads to excessive accumulation of S in agricultural crops (Irakoze W et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA certain amount of elemental sulfur (S) is essential for higher plants, as it is a constituent of methionine, cysteine, membrane sulfur lipids, cell walls, vitamins, cofactors, and various metabolites with various biological functions (Moreno-Izaguirre \u003cem\u003eet al.\u003c/em\u003e, 2015). However, Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e salinity can also affect S metabolism in plants, resulting in a sharp increase in cysteine content and, in some cases, disruption of carbon metabolism (Davidian J \u003cem\u003eet al.\u003c/em\u003e, 2010). Another explanation for how plants can reduce the toxicity of soil salts is the ability of salt-tolerant plants to exclude sodium or compartmentalize it in vacuoles, apoplasm, or trichomes (Aghajanzadeh T \u003cem\u003eet al.\u003c/em\u003e, 2018). It has been noted in scientific literature that high activity of antioxidant enzymes is associated with both salt tolerance and salt sensitivity (Flowers T \u003cem\u003eet al.\u003c/em\u003e, 2019 In the scientific literature, an increased level of antioxidant enzyme activity can be considered as one of the possible mechanisms of resistance to abiotic stresses (Abbasi H et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). As noted in other studies, sodium sulfate-treated cotton exhibited significant increases in relative conductivity, malondialdehyde content, superoxide dismutase, peroxidase, and leaf catalase activity (Abogadallah G 2010). We have considered the enzyme SOD and MDA since it is noted in the world literature that, due to its function, SOD is considered the main antioxidant enzyme, since it regulates O2\u0026thinsp;\u0026minus;\u0026thinsp;and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration (Guo J et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Furthermore, Kohli et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), SOD regulates the overproduction of reactive oxygen species (ROS), especially O\u003csub\u003e2\u003c/sub\u003e (Kohli S \u003cem\u003eet al.\u003c/em\u003e, 2019).\u003c/p\u003e \u003cp\u003eGene expression and transcriptomics profiling studies in cotton have disclosed that \u003cem\u003eGhCLO\u003c/em\u003e (caleosin) genes are responsible for drought and salinity. Higher MDA accumulation and significantly lower SOD activity were found in transformed plants under saline conditions, where \u003cem\u003eGhCLO\u003c/em\u003e had a decisive effect on the salt tolerance of cotton (Fu X et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). NaCl treatment and field experiments showed that overexpression of the \u003cem\u003eGaJAZ1\u003c/em\u003e gene significantly enhanced the salt tolerance of cotton, resulting in increased fresh weight, more bolls, and vigorous growth of taller plants (Zhao G et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt is noted in the world literature that, due to its function, SOD is considered the main antioxidant enzyme, since it regulates O\u003csub\u003e2\u003c/sub\u003e and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentrations (Zhao G et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It was also observed that in cotton tissues, with an increase in the concentration of NaCl, the activity of SOD also increased. This also happened in our experiments. In a comparative aspect, we present a change in the activity of one of the enzymes of the antioxidant system.\u003c/p\u003e \u003cp\u003eThe activity of SOD increases by 51%, 124% and 71% in the leaves of Porloq-4 plants. Whereas the Coker-312 variety in the same variants increased by 81.1%, 198.6% and 95.9%. This ratio between Porloq-4 and Coker-312 grades is 0.62, 0.63, and 0.74 times for the chloride salt exposure option (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. B).\u003c/p\u003e \u003cp\u003eWatering the seedlings with sulfate salt solutions led to a change in the activity of SOD, somewhat lower than in the variant with chloride salt. The activity of SOD in the leaves of Porloq-4 plants in the variant with a relatively low concentration of Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e (1.5%) increased by 14%, in the second variant \u0026ndash; by 9%, and in the third variant \u0026ndash; by 6% (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. A). Whereas for the Coker-312 variety, these indicators were as follows: an increase of 1.1%, 10.37% and 39.3%.\u003c/p\u003e \u003cp\u003eWhat is the reaction of seedlings at an early stage of vegetation to stress from two sodium salts at once can be considered.\u003c/p\u003e \u003cp\u003eThe effect of solutions of combined salts of NaCl and Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e in one irrigation solution on seedlings caused changes in the activity of SOD of the following order. In the first variant, when relatively low concentrations of NaCl and Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e were used, the activity of this enzyme of the antioxidant system increased by 44.9% for Porloq-4 and by 27.2% for Coker-312.\u003c/p\u003e \u003cp\u003eWhereas in the second variant, activity increased by 99.1% for Porloq-4 and 69.3% for Coker-312. In the third variant, when relatively high concentrations of both salts were used in the solution for watering young plants at the stage of 4 leaves, the activity of SOD increased from the control variant by 83.5% and 65.8% for the varieties, respectively. Thus, it can be noted that the second version of the experiment elicits a greater reaction from one of the plant's antioxidant enzymes, or it can be said that it is more toxic than the other two (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDue to the fact that excessive concentrations of various salts, not only chloride, but also sulfate, gradually accumulate in soils for a number of reasons, there is a need to conduct research to identify the tolerance of crops to these stressors. However, most of the world's research on quantifying the salt tolerance of plant species has been based on experiments in which NaCl is the predominant salt. Studies of biochemical and physiological response parameters have confirmed the existence of genetic variations in salinity tolerance in cotton (Saleh B \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eParameters such as chlorophyll content were used to determine the tolerance of a particular genotype and select the most resistant ones. This selection criterion is related to an increase in yield, the rate of photosynthesis, and the production of dry matter (Harinasut et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1996\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIt should be noted that, as a result of artificially created salinization stress in laboratory conditions, an increase in the content of malonic dialdehyde (MDA) and the activity of enzymes of the antioxidant system occurs. As the concentration of stress salt increases, an increase in each of the indicators relative to the control variant was mainly revealed. Although in some cases their decrease is also observed, especially in the variant when the strength of the stress salt is relatively high (the third experimental variants). Nevertheless, these levels were higher than in the control variants of the same experiment.\u003c/p\u003e \u003cp\u003eBased on this, it can be concluded that the Porloq-4 and Coker-312 genotypes have an adaptive ability, and the response of the Porloq-4 biotechnological variety was more active than that of Coker-312. Experiments to assess the tolerance potential of different genotypes of cotton are also conducted by foreign colleagues. For example, Surian researcher Saleh (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) evaluated five cotton varieties (\u003cem\u003eG.hirsutum\u003c/em\u003e L.) for exposure to NaCl concentrations of 0, 50, 100, and 200 mM (Sharif I et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), in order to better understand the reactions of various cotton varieties to salinization stress.\u003c/p\u003e \u003cp\u003eThus, based on the experimental results, we concluded that the analyzed Porloq-4 and Coker-312 cotton varieties react differently to stress from NaCl and Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, while Porloq-4 cotton varieties have a more active and responsive antioxidant enzyme system.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was carried out within the framework of the fundamental project FL-9524115083, funded by the Agency for Innovative Development under the Ministry of Higher Education, Science and Innovation of the Republic of Uzbekistan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable to this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Source\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Innovative Development Agency under the Ministry of Higher Education, Science and Innovation Ministry of Uzbekistan [grant numbers FL-9524115083]. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNRR, ASI, IBS and VSK carried out the experiments and wrote andrevised the manuscript, performed statistical analysis. SBK, FSR, MZ, RMA, ZZY, RAJ and AAR participated in the experiments, collected the data, and prepared the manuscript. ZTB edited and approved the manuscript. All authors read and approved the final manuscript..\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData presented in this study will be available on a fair request to the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eYu Z, X Duan, L Luo, S Dai, Z Ding, G Xia (2020). How Plant Hormones Mediate Salt Stress \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Responses. \u003cem\u003eTrends Plant Sci\u003c/em\u003e 11:1117\u0026ndash;1130\u003c/li\u003e\n \u003cli\u003eVerma V, P Ravindran, Kumar PP (2016). 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Exogenous Glycinebetaine Accumulation and Increased Salt-tolerance in Rice Seedlings. \u003cem\u003eBiosci Biotechnol Biochem\u003c/em\u003e 2:366\u0026ndash;8\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Abiotic stress, Chlorophyll, Malonodialdehyde, Metabolomics, Saline conditions, Superoxide dismutase","lastPublishedDoi":"10.21203/rs.3.rs-9426942/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9426942/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn this article, we compared the reaction of two cotton varieties (RNAi Porloq-4 and Coker-312) to different concentrations of NaCl and Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and analyzed the activity of superoxide dismutase (SOD), an antioxidant enzyme, and the level of malonic dialdehyde (MDA) in the leaves of seedlings grown in the laboratory. Our goal is to identify changes in specific metabolomic indicators in cotton seedlings of the Porloq-4 variety under the influence of chloride and sulfate salt solutions in a comparative model experiment. We aim to determine the response of the Porloq-4 biotechnological cotton variety (at the level of metabolites) to abiotic stress (salinity). The following tasks were identified to study the effect of low, medium, and high concentrations of NaCl and Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e solutions on the total chlorophyll content and biochemical parameters (MDA and SOD activity) of Porloq-4 cotton at an early stage of vegetation, compared to the Coker-312 variety. There was an increase in MDA and SOD at an average concentration of sulfate salt, and a slight decrease at a relatively high concentration. When the plants were watered with a relatively low concentration of sodium sulfate salt, MDA and SOD increased by 62%, while at an average concentration (2.0%), SOD increased by 325%. Nevertheless, when high concentrations of Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e were used, SOD increased by 4.1 times compared to the control experiment. Salt stress reduced the seedling emergence rate, relative biomass, and chlorophyll content; however, MDA content increased. Salt stress markedly increased the superoxide dismutase (SOD) activity. MDA content was markedly higher in salt stress.\u003c/p\u003e","manuscriptTitle":"Assessment of antioxidant enzyme responses in a biotechnological cotton variety under salt stress conditions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-23 05:30:01","doi":"10.21203/rs.3.rs-9426942/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2c3c1b6b-beab-499b-a84e-5d0f7fff2ee2","owner":[],"postedDate":"April 23rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-23T05:30:02+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-23 05:30:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9426942","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9426942","identity":"rs-9426942","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}