1. Introduction

The salinization of agricultural lands and the search for efficient usage of these lands is one of the most urgent problems today. The relevance of this issue is determined by the reduced ability to obtain high-quality yields from saline fields, while its complexity is explained by the interaction of biotic (biological traits of plants) and abiotic (type and level of soil salinity, climate conditions) factors. Soil salinity acts as a stress factor on plants, leading to the formation of reactive oxygen species (ROS), which negatively affect plant growth, development, and productivity, even causing plant death [1-5].

Therefore, the selection and purposeful use of salt–tolerant plants is a priority issue. In this context, safflower is important due to its tolerance to drought and saline conditions. Safflower belongs to the Asteraceae family and the Carthamus genus, and cultivated varieties are derived from Carthamus tinctorius L. Mainly, safflower is used for oil extraction. According to scientific sources, safflower oil is rich in linoleic acid, which helps maintain vascular elasticity. Its flowers contain red and yellow pigments and have medicinal properties. The oil is also used for margarine production. Globally, countries such as Kazakhstan, Russia, Mexico, the USA, and India are considered leaders in safflower cultivation [6].

In recent years, evaluating plant tolerance to salinity using antioxidant enzyme activity has gained importance. For example, when bean plants were germinated in 50 and 100 mM NaCl solutions, catalase activity was found to be higher in salt–tolerant genotypes (GS57) than in sensitive ones [7]. Similar studies on sugar beet showed higher ascorbate peroxidase activity in salt–tolerant genotypes [8,9]. An increase in salt concentration in solutions activates ascorbate peroxidase and catalase enzymes [10,11], and this activation helps detoxify hydrogen peroxide, contributing to plant adaptation to salinity [12].

Superoxide dismutase (SOD) plays a vital role in protecting organisms against stress factors. It converts superoxide radicals into hydrogen peroxide and molecular oxygen [13]. In soybean under drought stress, increased lipid peroxidation and elevated levels of SOD, peroxidase, catalase, and proline were observed [14]. Catalase functions as a catalyst in breaking down hydrogen peroxide into water and oxygen [15,16], and it plays a role in photosynthesis, cardiovascular tissues, and seed metabolism [17,18]. Increased catalase activity under low temperatures has been recorded in potato and barley [19]. With 200 mM NaCl salt treatment, accumulation of H₂O₂/O₂ was noted in plant leaves [20]. When two pea (Pisum sativum L.) cultivars differing in salt tolerance were irrigated with 70 mM NaCl solution for 15 days, the salt–tolerant (cv. Granada) showed increased APX, GR, and MDHAR activity, whereas these levels remained unchanged in the sensitive cv. Challis [21]. Thus, antioxidant enzyme activity is a significant indicator in evaluating plant resistance to abiotic factors [22].

Based on the above literature, it is evident that antioxidant activity increases in stress–resistant plants under stress conditions. Hence, antioxidant enzyme activity is a reliable criterion for selecting stress–tolerant genotypes. As mentioned, safflower is drought– and salt–tolerant, thus is usually cultivated on non–irrigated lands. Recent studies identified promising new genotypes for irrigated conditions such as Nari NH–H–5 and Nari NH–H–15. Research is currently ongoing to select autumn types of safflower [23]. However, there is a lack of studies evaluating antioxidant enzyme activity in safflower under saline soil conditions. Therefore, this study aimed to assess the effect of soil salinity on antioxidant enzyme activity.

2. Materials and Methods

As the object of investigation, two accessions of Carthamus tinctorius L. (safflower) were selected: the cultivar “Milyutin–114” and the introduced accession “K–435” from Pakistan. These accessions were studied under field conditions with varying degrees of soil salinity.

Among the antioxidant enzymes, superoxide dismutase (SOD) activity was measured based on the enzyme’s ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT), as described in the method [24]. The reaction mixture consisted of 50 mM potassium–sodium phosphate buffer (pH 7.8), 172 µM NBT, 210 µM methionine, 24 µM riboflavin, and 0.1% Triton X–100. The extracts were incubated under light for 10 minutes, and the absorbance was recorded at 535 nm. SOD activity was expressed in arbitrary units (U) per mg of protein per 10 minutes.

Catalase (CAT) activity was determined according to the method of Sinha [25], with modifications proposed by Mahmoud H.H. [26]. This method is based on the reduction of hydrogen peroxide by dichromate to chromic acetate in the presence of acetic acid. Fresh plant tissue (500 mg) was ground with liquid nitrogen and extracted in 5 mL of 50 mM phosphate buffer (pH 7.8). The homogenate was centrifuged at 12,000×g for 15 minutes. The reaction was initiated by adding 1000 µL of 0.2 M H₂O₂ to 200 µL of the enzyme extract and was stopped after 1 minute by adding a dichromate–acetic acid mixture. The amount of chromic acetate formed was measured spectrophotometrically at 570 nm. Catalase activity was expressed as µmol H₂O₂ decomposed per minute per mg of protein (µmol H₂O₂ min⁻¹ mg⁻¹ protein).

The malondialdehyde (MDA) content, as an indicator of lipid peroxidation, was measured using the thiobarbituric acid (TBA) assay, according to Gur et al. [27]. To 0.5 mL of homogenate, 0.2 mL of 1 mM FeSO₄ and 0.3 mL of 1% Triton X–100 were added, followed by incubation in a water bath at 40°C for 30 minutes with constant shaking. The reaction was stopped by adding 0.2 mL of 3 mM dihydroquercetin. Subsequently, 0.5 mL of 1% Triton X–100, 0.2 mL of 0.6 M HCl, and 0.8 mL of 0.06 M TBA working solution were added. The mixture was heated in a boiling water bath for 10 minutes and then cooled to 15°C for 30 minutes. To stabilize the color, 0.2 mL of 5 mM EDTA and 5–10 mL of ethanol: chloroform mixture (7:3, v/v) were added. The absorbance was measured at 532 nm, with correction for non–specific absorbance at 600 nm. MDA content was expressed as nmol per gram of fresh weight (nmol g⁻¹ FW).

3. Results and Discussions

According to scientific literature, the activity of superoxide dismutase (SOD) increases under unfavorable climatic conditions such as cold, heat, soil salinity, and drought. This observation was previously noted in the literature review. Most importantly, it has been confirmed that SOD activity increases in stress-tolerant genotypes, making it a crucial criterion in selecting plants resistant to stress environments. Superoxide dismutase, as an antioxidant enzyme, works alongside catalase to protect organisms from reactive oxygen species.

For instance, when studying safflower genotypes–salt–tolerant cv. M–CC–190 and salt–sensitive cv. IL–111–in different sodium chloride solutions, the amount of antioxidants increased under saline conditions in the salt–tolerant genotype [26]. Similar findings have been reported by other researchers as well [18-21]. These data indicate that antioxidant enzyme activity is elevated in salt–tolerant genotypes under saline conditions.

The same trend was observed in our research. Under mildly saline soil conditions, the amount of superoxide dismutase in the Milyutin–114 variety was 3.207, while in the K–435 accession, it was 2.803. These indicates that under mildly saline conditions, the SOD content in the K–435 accession was lower than in the control. However, when these genotypes were studied under higher soil salinity levels, the SOD content in Milyutin–114 was 3.203, whereas in K–435 it reached 4.503 (see Figure 1).

Figure 1. Effect of soil salinity level on superoxide dismutase (SOD) enzyme activity

These results show that under mild salinity, SOD content in the control genotype was higher compared to the K–435 accession. With increased soil salinity, however, SOD content in the K–435 accession also increased. The suggests that increased soil salinity acts as a stress factor for safflower genotypes, triggering enhanced SOD activity. In fact, in the K–435 genotype, SOD levels increased by 60% under elevated soil salinity compared to mildly saline conditions, while in the control genotype, this figure was only–0.12%.

Based on these data, the K–435 safflower genotype can be considered salt–tolerant compared to the control, as the activity of the antioxidant enzyme SOD increased under saline conditions in this accession.

Catalase is one of the antioxidant enzymes and is widely present in living organisms. Its primary function is to break down hydrogen peroxide formed under stress conditions. In safflower varieties, catalase content was affected by soil salinity levels in a manner similar to that observed for superoxide dismutase (see Figure 2). In the Milyutin–114 variety, catalase activity under mildly saline soil conditions was measured at 9.560, increasing to 10.747 with higher salinity. A similar pattern was observed in the K–435 accession, with catalase levels rising from 8.115 to 12.482.

Figure 2. Effect of soil salinity level on catalase enzyme activity

These results indicate that catalase activity increased by 12.4% in the Milyutin–114 variety and by 53.8% in the K–435 accession with increased soil salinity. Thus, the higher catalase activity observed in K–435 under saline conditions provides further evidence of its salt tolerance.

Malondialdehyde (MDA) serves as a marker of lipid peroxidation. Scientific sources report that MDA content is generally higher in plant leaves [27]. It has been established that MDA levels fluctuate under the influence of stress factors. When winter and spring wheat varieties were exposed to low temperatures, the MDA content in winter wheat was found to be 1.9 times higher than in spring wheat [28], indicating greater cold tolerance in the winter variety.

According to our results (see Figure 3), under mildly saline soil conditions, the MDA level in the Milyutin–114 variety was 7.179, but decreased to 4.577 with increasing soil salinity. In contrast, the K–435 accession showed the opposite trend: MDA content increased from 6.103 to 7.808. These data reveal that in the K–435 genotype, MDA levels increased by 27.9% with rising soil salinity. This increase in MDA activity under more saline conditions suggests enhanced oxidative response and supports the classification of K–435 as salt–tolerant.

Figure 3. Effect of soil salinity level on malondialdehyde (MDA) activity

4. Conclusions

Based on the obtained results, the following conclusions were drawn:

1. An increase in soil salinity led to enhanced activity of antioxidant enzymes. The amount of superoxide dismutase (SOD) ranged from 2.830 to 3.203 under mildly saline conditions, and increased to 3.203–4.503 with higher salinity levels. In this case, SOD content increased by 0.12% in the Milyutin-114 variety and by 60.0% in the K–435 accession;

2. Catalase content ranged from 8.115 to 9.551 under mildly saline conditions and increased to 10.747–12.481 with rising soil salinity. Catalase activity increased by 12.4% in Milyutin–114 and by 53.8% in K–435;

3. Malondialdehyde (MDA) levels varied by genotype and soil salinity. Under mildly saline conditions, MDA ranged from 6.103 to 7.179, while under more saline conditions, it ranged from 4.517 to 7.808. In the K–435 accession, MDA content increased by 27.9% compared to the control;

4. Increased soil salinity enhanced the activity of antioxidant enzymes (superoxide dismutase, catalase, and malondialdehyde) in the K–435 accession compared to the Milyutin–114 variety, supporting the classification of K–435 as salt–tolerant.