Physiological mechanism of sodium salicylate and folcisteine on alleviating salt stress in wheat seedlings

doi:10.21203/rs.3.rs-3229387/v1

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Physiological mechanism of sodium salicylate and folcisteine on alleviating salt stress in wheat seedlings

https://doi.org/10.21203/rs.3.rs-3229387/v1

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published 18 Dec, 2023

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Soil salinization can significantly affect the normal growth and development of wheat, and can cause wheat death in severe cases, resulting in a decline of grain yield and quality, which poses an important threat to food security in China. In this study, the effects of two plant exogenous growth regulators, sodium salicylate and folcisteine, on the physiology and main characters of wheat under salt stress were studied by hydroponics. The results showed that both plant growth regulators could effectively alleviate the inhibition of wheat growth under salt stress. The morphological and physiological indexes, antioxidant enzyme such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activity and malondialdehyde (MDA) content of wheat after foliar spraying of sodium salicylate and folcisteine under salt stress were determined, which showed that the effect of sodium salicylate was better than that of folcisteine. Meanwhile, the determination results of hydrogen peroxide (H₂O₂) content and superoxide anion (O²⁻) content showed that the effect of folcisteine was better than that of sodium salicylate. Simultaneously, the synergistic effect of the two regulators can be played significantlyin the compound treatment, and the effect is the best. In addition, this study also found that the effect of early regulators was not obvious, but under the continuous action of the regulators, the growth of wheat under stress environment could be improved and the damage of salt stress on wheat growth and development colud be significantly alleviated, which could effectively guarantee the food security and production in China.

Biological sciences/Physiology

Biological sciences/Plant sciences

Soil salinization is one of the main problems threatening the current agricultural production process, and it is also an important factor affecting the global ecological environment. According to statistics, the salinized land area of the world is about 8.0×10⁸ hm², which has suffered different degrees of salinization hazards^1,2. It is estimated that by 2050, more than 50% of the world 's arable land will be affected by salinization, limiting global agricultural production and food supply^3–5.

Soil salinization will cause salt stress on plants, and sodium salt stress has the most significant impact on salt stress, accounting for 45% of the total area of salinized soil. Under the high concentration of Na⁺ environment, it will interfere with the absorption and utilization of potassium, calcium, zinc and other mineral elements in plants, affecting the efficient utilization of mineral nutrients. Moreover, it will also lead to the imbalance of cell osmotic pressure, the destruction of cell membrane structure, and the extravasation of substances in the membrane, which will directly affects the photosynthesis of plants and forces the growth rate of plants to decrease, thus limiting the growth and development process of aboveground and underground parts of plants. Finally, the morphological characteristics are manifested as the decrease of fresh and dry weight of plant tissues. In particularly, the dry and fresh weight of the above ground parts of the leaves decreased significantly^6–7. Meanwhile, salt stress can also cause ion imbalance and high osmotic stress in plant tissues, resulting in increased ion concentration and ion toxicity, which lead to the imbalance of production and removal of reactive oxygen species in the body so as to the excessive accumulation of reactive oxygen species, thereby inducing the antioxidant capacity variation of cells, and ultimately causing oxidative damage. Subsequently the intracellular antioxidant defense system will be stimulated to produce a series of antioxidant enzymes, mainly including SOD, POD and CAT, to slowly remove excessive ROS in the body to alleviate the stress pressure of the outside world, which plays a very important role in plant growth and development^8–10. Excessive salt in plant tissues will directly interfere with plant metabolism, resulting in a large accumulation of osmotic substances in plants. With the extension of time, the harm caused by salt stress will be aggravated, and eventually lead to the whole plant wilt or even death^{7, 11–12}.

Wheat (Triticum aestivum L.), one of the most important food crops worldwide, whose quality and yield directly affect the income of farmers and the sustainable development of agriculture^13–14. In the process of wheat production, it is easy to encounter abiotic stresses such as low temperature, heavy metals and high salt, especially salt stress, which significantly limits the high and stable yield of wheat¹⁵. Therefore, how to improve the salt tolerance of wheat is a research hotspot in agricultural production. Studies have shown that there are two main methods to solve the effect of soil salinization on wheat: one is to clone and analyze the genes related to salt tolerance of wheat, and use transgenic technology to obtain normal expression of the target gene in wheat plants, so as to obtain salt-tolerant wheat varieties¹⁶. Meanwhile, the other is the application of exogenous plant growth regulators, which directly act on wheat, regulate the physiological activity and content of related substances in plants to enhance the salt tolerance of wheat, so as to produce resistance to resist the damage caused by salt stress¹⁷.

Exogenous application of plant hormones is an effective method to cope with salt stress^18–19. Previously studies have interpreted that plant growth regulators can increase plant salt tolerance, and indicated that IAA can effectively regulate the physiological activities of shoots and roots of plants under salt stress and promote their growth and development²⁰. Meanwhile, previously research found that melatonin (MT) can regulate plant growth, photosynthesis and seed germination, and alleviate abiotic or biotic stresses²¹. It can also scavenge free radicals, enhance antioxidant enzyme activity²², and reduce plant damage to cold, heat, salt, drought and other stresses²³. At the same time, brassinolide (BR) can respond to drought, salt stress, low temperature, high temperature and heavy metal stress by regulating a variety of enzymes and exogenous regulators required by the plant itself. Besides, the plant 's own growth potential and advantages are fully utilized to regulate plant growth and development^24–28. In addition, it was found in the study of abscisic acid that it can significantly promote the growth of plant roots and respond to salt stress environment in a timely manner²⁹. Therefore, screening out the growth regulators that can improve the salt tolerance of wheat, which is of great significance to the planting of wheat under stress environment, the improvement of wheat yield and the further guarantee of national food security. Folcisteine, also known as N-acetylthioproline (NATCA), which is originally a pharmaceutical intermediate for the protection of myocardial ischemia, and is also a derivative of organic amino acids. In agriculture, it can be used as a biological stimulant to increase biological activity, cytokines and auxin, regulate the osmotic pressure of crop cells, promote seed germination and cell division and elongation, reduce transplant shock, protect chlorophyll, improve nutrient utilization, maintain water and nutrient transport balance, increase flowering period, increase crop fruit setting rate and fruit yield, delay senescence, enhance stress resistance, and reduce fruit drop. Sodium salicylate, also known as sodium o-hydroxybenzoate (Na-SA), is a derivative of salicylic acid (SA), which has similar biological activity. The common method is to use 0.05% -0.1% concentration for foliar spraying. Since the late 1970s, it has been found that SA has defensive and protective functions for plants invaded by pathogens. SA can induce system acquired resistance (SAR) in plants and plays a critical role in the process of inducing SAR formation. The reason may be that by regulating the level of reactive oxygen species, changing the content of antioxidants and the activity of enzymes among them. Therefore, the plants are induced to produce stress resistance³⁰.

To date, the domestic research on the mitigation mechanism of the combined application of sodium salicylate and folcisteine on wheat under the salt stress environment has not been reported. Therefore, in order to verify whether the two growth regulators can have certain effects on the growth of wheat under salt stress, an indoor hydroponic experiment was carried ou of the current study. By measuring the content of MDA and active oxygen content and the activity of antioxidant enzymes, the mitigation mechanism of sodium salicylate and folcisteine on wheat under stress conditions was preliminarily explored.

Cultivation and collection of experimental materials. Healthy and plump wheat seeds (Bainong 207) were selected, soaked in 10% H₂O₂ solution for 10 min, and then rinsed with distilled water for 3 times to remove residual H₂O₂. Finally, the seeds were placed in a beaker filled with distilled water and treated in dark for 12 h for germination. After germination, the seeds were placed in parallel at a distance of 3 centimeters from the upper edge of absorbent paper soaked in saturated CaSO₄ solution in this way that the pitch is 1 centimeter and the hairy side is on top, and the seeds were covered with another absorbent paper. Soon afterwards, the absorbent paper was rolled up and the seedlings were raised in the greenhouse for 7 days under the condition of avoiding light. After the seedling was completed, the healthy wheat seedlings with consistent growth were selected and transplanted into the moist sponge block in the seedling pot containing 1/2 Hoagland nutrient solution in the greenhouse for growth under hydroponic conditions. After the wheat seedlings grew to the three-leaf stage, they were replaced with full Hoagland nutrient solution culture and 100 mmol/L NaCl was added for salt stress treatment. After 12 h of treatment, folcisteine at a concentration of 25 mg/L and sodium salicylate at a concentration of 0.05 mg/L were used alone by means of foliar spraying, and the regulator was configured with distilled water to the corresponding concentration.

Experimental design. This experiment was conducted in a greenhouse with light-dark ratio of 14/10, light intensity of 400 µmol·m^− 2·s^− 1, the humidity was 60, and the daytime/night temperature was 32/26°C. The growth and development of wheat were carried out by hydroponics. The experiment set up 4 treatment groups and 1 water control group (Table 1), each treatment sets up 3 replicates, a total of 36 wheat seedlings. Samples were collected at 3 d and 6 d after spraying, and 5 wheat plants were collected from each treatment for the determination of morphological indexes and biomass. Otherwise, the leaves of 6 wheat plants from each treatment, which consists of 1 biological replicate for each 2 plants, were temporarily stored in the refrigerator at -80℃ for the determination of harmful substances content and antioxidant enzyme activity in cell tissue after oxidative damage.

Table 1

Experimental treatment. Each experimental treatment was repeated three times in parallel.
Experimental group	Experimental treatment
CK	No salt stress and spraying regulator on leaves
KB	Add salt stress with leaves sprayed with distilled water
ATCA	Add salt stress with leaves sprayed with folcisteine
Na-SA	Add salt stress with leaves sprayed with sodium salicylate
Na-SA + ATCA	Add salt stress with leaves sprayed with folcisteine and sodium salicylate in the meantime

Determination of morphological indexes and biomass accumulation of wheat. At the junction of the rhizomes of wheat, the plant was separated into two parts with an dissecting knife. The leaves and roots were fully spread on the scanning table and scanned using an Epson Expression 12000XL scanner. The morphological indexes, including leaf projection area, total root length, total root area and average root diameter, were analyzed by WinRHIZO Pro 2017 root image analysis software, and the aboveground parts of wheat after shearing were taken for three parallel measurements to obtain the average plant height.

The two divided parts of wheat were dipped the water on the surface with absorbent paper, and the fresh weight was weighed. After then, they was placed in the kraft paper bags, placed in a drying oven 105°C for deactivation at a temperature of 105°C for 30 min, after then dried at 80°C to constant weight, and weighed dry weight in final.

Determination of reactive oxygen species (ROS) content. Determination of hydrogen peroxide (H₂O₂) content. The H₂O₂ level was determined by measuring the absorbance of the purple complex formed by Fe³⁺ and xylenol orange³¹. The detailed method is to weigh the cut 0.1 g leaves and add 600 uL -20°C pre-cooled acetone to grind to form a homogenate, followed by high-speed refrigerated centrifugation with 4°C for 15 min at the speed of 10000 g, and the supernatant is taken for use.

The reaction system was as follows : 200 µL of distilled water, 500 µL of 400 mM sorbitol, 500 µL of 0.8 mM ammonium ferrous sulfate, and 500 µL of 0.4 mM xylenol orange which was dissolved in 25 mM sulfuric acid, were mixed in a 2 mL centrifuge tube to prepare a reaction mixture, followed by adding 300 µL of supernatant. After mixing, the reaction mixture was incubated at 30°C for 30 min, and the OD560 value was measured.

0, 60, 120, 180, 240, 300 µL 100 µM H₂O₂, 750 µL 400 mM sorbitol, 750 µL 0.8 mM ammonium ferrous sulfate and 750 µL 0.4 mM xylenol orange were mixed and incubated at 30°C for 30 min. The standard curve equation was obtained by measuring the OD560 value. The measured OD560 value of the sample is substituted into the standard curve and the actual H₂O₂ content in the sample is calculated.

Determination of superoxide anion (O ²⁻ ) content. O²⁻ levels were measured using the method proposed by Erstner et al.³². The main method is to take fresh weight sample about 0.1 g, add 1 m L 50 m M PBS ( PH = 7.8 ), and subsequently grinding extraction with 4°C for 20 min at the speed of 10000 g, followed by taking the supernatant for use.

The reaction system was as follows : 50 µL 6.25 mM PBS (PH = 7.8), 100 µL 0.25 mM hydroxylamine hydrochloride and 50 µL supernatant were mixed and bathed in 25°C water for 1 h to generate nitrite. The control treatment was equal volume PBS instead of supernatant. Subsequently, 100 µL 4.25 mM sulfanilic acid and 100 µL 1.75 mM α-naphthylamine were added to the above reaction system in turn. After mixing, they were bathed in water at 25°C for 20 min in the dark. The absorbance value of the reaction system solution at 530 nm was determined.

Sodium nitrite (NaNO₂) solutions with concentration gradients of 0, 10, 20, 30, 40 and 50 µM were prepared to make the standard curve equation. The actual O²⁻ content of the sample in the determination was calculated by using the standard curve.

Determination of antioxidant enzyme activity. The crude enzyme extract was prepared according to the method of Zhang et al.³³, that is, the sample was first treated with liquid nitrogen, and then ground by a freezing grinder. After grinding, 0.1 M PBS(pH = 7.5), containing 1 mM EDTA and 1% PVPP, was added to oscillate and mix to form a homogenate, and then centrifuged with the speed of 15000 g at 4°C for 20 min, and the supernatant was taken for later use.

Determination of superoxide dismutase (SOD) activity. The activity of SOD (EC 1.15.1.1) was determined by NBT photoreduction method³⁴. The reaction mixture, containing 20 µL 130 mM methionine solution, 20 µL 30 µM EDTA-Na₂ solution, 135 µL 50 mM PBS(PH = 7.8), 20 µL 0.75 mM NBT solution, was prepared in advance in a 2 mL centrifuge tube, and then 5 µL crude enzyme extract was added. After shaking, it was placed at 25°C and 4000 lx light for 20 min, while the control group replaced the crude enzyme solution with 50 mM PBS. Subsequently placed in the dark. Finally, the absorbance at 560 nm wavelength was measured to measure the SOD enzyme activity.

Determination of peroxidase (POD) activity. POD (EC1.11.1.7) activity was determined by guaiacol method³⁵. 50 mL of 100 mM phosphate buffer (PH = 6.5) was added to 28 µL of guaiacol and then dissolved by heating. After the solution was cooled, 19 µL of 30% H₂O₂ solution was added and mixed evenly to form a reaction mixture. A total of 100 µL reaction mixture and 100 µL crude enzyme extract were added to the centrifuge tube. After mixing evenly, the absorbance change at 470 nm within 3 min was measured once every 30 s, and the absorbance change within 1 min was used to represent the enzyme activity.

Determination of catalase (CAT) activity. The activity of CAT (EC1.11.1.6) was determined according to the method of Chen et al.³⁶. The 20 µL enzyme extract sample was added to 4.98 mL 100 mM phosphate buffer (pH = 7.0), and 50 mM 30% H₂O₂ was added. The absorbance change of H₂O₂ within 1 min was measured at 240 nm to determine the enzyme activity.

Statistical analysis. The data collected were initially analyzed by ANOVA using SPSS software (Ver. 17.0, SPSS Inc.), while statistical differences were determined using Fisher's least significant difference test (α = 0.05), and Graphpad Prism (Ver. 9.0.0) was used to make data graphs.

Ethics declarations. The collection of plant materials is in accordance with relevant institutions, national and international norms and legislation in the study.

Morphological growth index and biomass accumulation. After spraying plant growth regulators on wheat leaves, the plant height and total root length of wheat under salt stress were higher than those of CK group and KB group, indicating that sodium salicylate and folcisteine had obvious alleviating effect on the growth of wheat. The overall trend was basically the same at 3 and 6 days of treatment (Table 2). At 3 days, the plant height of ATCA group, Na-SA group and Na-SA + ATCA group increased by 10%, 24% and 36% respectively compared with KB group. The total root length increased by 16%, 42% and 44%, respectively. The leaf projection area of the Na-SA group and the Na-SA + ATCA group was higher than that of the KB group, and the difference between the treatments was significant. The ATCA group did not have a certain effect on the growth of wheat, but the leaf projection area was slightly lower than that of the KB group. The leaf projection area of the Na-SA group and the Na-SA + ATCA group increased by 11% and 22%, respectively, compared with the KB group. At 6 days, the leaf projection area of each treatment group increased to varying degrees compared with the KB group. The ATCA group, Na-SA group and Na-SA + ATCA group increased by 8%, 21% and 36%, respectively, compared with the KB group, and the difference was very significant.

The root surface area of the three experimental treatment groups increased significantly compared with the KB group (Table 2). At 3 days, the root surface area of the ATCA group, Na-SA group and Na-SA + ATCA group increased by 14%, 41% and 43% respectively compared with the KB group, but the root surface area of the ATCA group was lower than that of the CK group, and the Na-SA + ATCA group had the best effect. At 6 days, the root surface area of ATCA group, Na-SA group and Na-SA + ATCA group increased by 0.3%, 39% and 33% respectively compared with KB group. The root surface area of ATCA group was still lower than that of CK group, and the mitigation effect on wheat growth was non-significant. The overall performance of Na-SA group was the best.

At 3 days, the effect of two growth regulators in ATCA group and Na-SA group on wheat under salt stress was not obvious compared with that in KB group, and there was no difference in the root average diameter between the two treatment groups with ATCA group and Na-SA group increased by 6.25% and 6.27% respectively compared with that of KB group, and was lower than that of CK group (Table 2). The effect of Na-SA + ATCA group was the best, which was significantly different from that of other groups, and increased by 30% and 15% respectively in comparison with KB group and CK group. At 6 days, there exist significant differences among the treatments, and the effect of Na-SA + ATCA group was the best, followed by Na-SA group. In general, the effect of the two regulators on the lateral growth of wheat roots was not significant when they were used alone, while the combined treatment could greattly promote the growth of roots, and whose effect was dramatically higher than that of single regulator treatment.

Table 2

Effects of sodium salicylate and folcisteine on the growth and development of wheat roots and shoots. The lowercase letters in the same column in the table indicate their differences under the condition of P < 0.05.
Time of treatment	Treatment	Plant height (cm)	Total root length (cm)	Leaf projection area (cm²)	Root average diameter (cm)	Root surface (cm²)
3 days	CK	19.37 ± 1.06bc	943.5 ± 6.9ab	14.42 ± 0.88ac	0.340 ± 0.022a	104.4 ± 2.4ab
	KB	16.67 ± 0.69c	839.3 ± 69.7b	12.39 ± 0.90bc	0.304 ± 0.018ab	86.2 ± 11.1b
	ATCA	18.40 ± 0.55bc	969.4 ± 106.4ab	10.42 ± 0.55c	0.323 ± 0.008b	98.3 ± 9.99ab
	Na-SA	20.63 ± 0.61ab	1195.5 ± 128.7a	13.77 ± 0.96ab	0.324 ± 0.002b	121.4 ± 12.2a
	Na-SA + ATCA	22.33 ± 1.07a	1205.4 ± 17.4a	15.08 ± 0.06a	0.392 ± 0.006ab	123.3 ± 2.3a
6 days	KB	20.96 ± 0.64bc	1660.1 ± 153.7bc	23.34 ± 0.95ab	0.325 ± 0.020bc	102.6 ± 4.1a
	CK	19.23 ± 0.26c	1498.6 ± 87.8c	18.82 ± 1.03b	0.309 ± 0.008c	93.3 ± 2.1a
	ATCA	21.90 ± 0.78b	1662.1 ± 50.8bc	20.34 ± 0.51b	0.338 ± 0.003ab	99.6 ± 3.2c
	Na-SA	23.07 ± 1.18b	2011.3 ± 204.7ab	22.87 ± 2.06ab	0.337 ± 0.005ab	129.2 ± 0.2b
	Na-SA + ATCA	26.53 ± 0.78a	2236.9 ± 140.4a	15.08 ± 1.73a	0.428 ± 0.009a	131.7 ± 7.9b

After 3 days of spraying regulators on wheat leaves, the physiological characters under salt stress were significantly alleviated compared with CK group and KB group (Table 3). Among them, the fresh weight of aboveground and dry weight of underground parts showed significant differences among treatments. The fresh weight of aboveground parts in ATCA group, Na-SA group and Na-SA + ATCA group increased by 5%, 26% and 32%, respectively. The dry weight of the underground part increased by 26%, 43% and 47%, respectively. The fresh weight of the underground part and the dry weight of the aboveground part showed no difference. At the same time, the Na-SA group and the Na-SA + ATCA group had a strong mitigation effect on the growth of wheat under stress conditions, while the ATCA group did not significantly improve the wheat stress environment. Among them, the Na-SA + ATCA group had the best growth mitigation effect on each growth index of wheat, which was significantly higher than the effect of single regulator treatment, indicating that the regulator played a synergistic effect between the two. In general, it can be analyzed that the two growth regulators have a regulatory effect on the growth and development of wheat under stress environment.

With the continuous effect of regulators, the physiological characters of wheat increased significantly in general, and each treatment showed significant differences (Table 3). Compared with 3 d, the aboveground fresh weight and underground dry weight of ATCA group increased by 91% and 14% respectively after 6 d. The Na-SA group increased by 55% and 20%, respectively. Na-SA + ATCA group increased by 136% and 38% respectively. It is worth noting that the root fresh weight and shoot dry weight of wheat showed significant differences, and the root fresh weight and shoot dry weight of ATCA group increased by 32%, respectively. The root fresh weight and shoot dry weight of Na-SA group increased by 10% and 13%, respectively. The root fresh weight and shoot dry weight of the Na-SA + ATCA group increased by 5% and 11%, respectively. It was concluded that the mitigation effect of Na-SA on wheat under salt stress was significantly higher than that of ATCA, and the effect of combined treatment of two regulators on each growth index of wheat was significantly higher than that of single regulator treatment, showing a significant synergistic effect.

Table 3

Effects of sodium salicylate and folcisteine on biomass accumulation of wheat. The lowercase letters in the same column in the table indicate their differences under the condition of P < 0.05.
Time of treatment	Treatment	Aboveground fresh weight (g)	Underground fresh weight (g)	Shoot dry weight (g)	Dry weight of the root (g)
3 days	CK	0.701 ± 0.05ab	0.819 ± 0.06a	0.147 ± 0.005a	0.064 ± 0.007ab
	KB	0.597 ± 0.07b	0.743 ± 0.05a	0.124 ± 0.011a	0.058 ± 0.005b
	ATCA	0.628 ± 0.05ab	0.751 ± 0.06a	0.152 ± 0.027a	0.073 ± 0.007ab
	Na-SA	0.750 ± 0.04ab	0.947 ± 0.14a	0.140 ± 0.014a	0.083 ± 0.009ab
	Na-SA + ATCA	0.790 ± 0.02a	1.038 ± 0.10a	0.171 ± 0.020a	0.085 ± 0.003a
6 days	KB	1.091 ± 0.114b	0.91 ± 0.094ab	0.135 ± 0.014ab	0.094 ± 0.003ab
	CK	0.927 ± 0.020b	0.77 ± 0.054a	0.115 ± 0.007c	0.053 ± 0.007c
	ATCA	1.202 ± 0.074b	0.99 ± 0.030a	0.142 ± 0.008bc	0.083 ± 0.009bc
	Na-SA	1.165 ± 0.096b	1.04 ± 0.047a	0.160 ± 0.026ab	0.100 ± 0.018ab
	Na-SA + ATCA	1.867 ± 0.172a	1.09 ± 0.079a	0.189 ± 0.03a	0.117 ± 0.012a

The Concentration of malondialdehyde (MDA). The results showed that after 3 days of treatment, the malondialdehyde concentration in wheat leaves of ATCA group, Na-SA group and Na-SA + ATCA group was significantly lower than that of KB group and CK group. Compared with KB group and CK group, ATCA group decreased by 3% and 7%, respectively. The Na-SA group was reduced by 15% and 18%, respectively, the Na-SA + ATCA group was reduced by 6% and 10%, respectively, and the Na-SA group had the best regulatory effect, which indicated that the regulator had initially begun to play a role in alleviating the salt stress suffered by wheat and reducing the damage of leaf tissue cells, thereby reducing the MDA concentration in the plant. With the continuous effect of the regulator, the effect of single regulator treatment on wheat did not change much after 6 days of treatment. The ATCA group was reduced by 13% and 20% respectively compared with the KB group and the CK group. The Na-SA group decreased by 17% and 25%, respectively, the Na-SA + ATCA group decreased by 25% and 32% respectively, and the MDA concentration was significantly lower than that of the single regulator treatment group, and the Na-SA + ATCA group had the best effect. In general, the MDA concentration of ATCA group, Na-SA group and Na-SA + ATCA group decreased by 17%, 10% and 27% respectively after 6 days compared with that after 3 days (Fig. 1). It can be concluded that Na-SA + ATCA group had the best effect, and there were significant differences among the experimental groups.

The content of reactive oxygen species (ROS). The content of hydrogen peroxide (H₂O₂). The results showed that a large amount of H₂O₂ was produced in the leaf tissue of wheat after salt stress. After 3 days of foliar spraying of regulators, the H₂O₂ content of ATCA group, Na-SA group and Na-SA + ATCA group was significantly lower than that of CK group and KB group, and the effect of Na-SA + ATCA group was the best, which had a significant mitigation effect on the growth of wheat under stress environment, which was 25% and 22% lower than that of CK group and KB group, respectively. After 6 days of foliar spraying, the content of H₂O₂ gradually decreased. The content of H₂O₂ in ATCA group, Na-SA group and Na-SA + ATCA group decreased by 14%, 11% and 15% respectively (Fig. 2). With the continuous action of the two regulators, the effect of ATCA group was better than that of Na-SA group, and the effect of Na-SA + ATCA group was the best, indicating that the two regulators showed obvious synergistic effect on promoting the growth of wheat under salt stress.

The content of superoxide anion (O ²⁻ ). The results showed that the content of superoxide anion (O²⁻) in wheat leaves of KB group was significantly higher than that of the other four treatment groups, indicating that high concentration of salt caused great damage to wheat leaf tissue cells, resulting in a large accumulation of superoxide anion in cell fluid, inhibiting the normal growth and development of wheat (Fig. 3). After the treatment of two regulators, the O²⁻ content in wheat leaves was significantly lower than that of CK group and KB group, and the difference between the experimental groups was very obvious. At 3 d, the superoxide anion content of ATCA group and Na-SA group was basically the same, which was 1% and 4% lower than that of CK group and KB group, respectively. The superoxide anion content of Na-SA + ATCAgroup was the lowest, which was 5% and 3% lower than that of CK group and KB group, respectively. At 6 d, with the continuous regulation, the initial effect of ATCA group and Na-SA group was not obvious, and there was no difference between the treatments. In the later stage, ATCA group gradually played a role in reducing the accumulation of O²⁻ content. There was a significant difference between the two treatment groups. The regulation effect of ATCA group was 1.2 times that of Na-SA group. The effect of Na-SA + ATCA group was still the best, which significantly repaired the damage of wheat tissue cells and reduced the accumulation of intracellular superoxide anion.

The activity of Antioxidant enzyme. The activity of superoxide dismutase (SOD). The results showed that the superoxide dismutase activity of CK group was the lowest at 3 d, while the enzyme activity of other treatment groups was more than 1.7 times that of CK group, and there was no difference between the treatment groups. The enzyme activity of ATCA group, Na-SA group and Na-SA + ATCA group increased by 75%, 76% and 80% respectively compared with CK group, indicating that the regulator had a significant mitigation effect on the growth and development of wheat under salt stress, but there was no difference between the treatment groups. At 6 d, the difference of enzyme activity among treatments was very significant. The superoxide dismutase activity of CK group was still the lowest. The enzyme activity of wheat leaves in Na-SA group was 1.1 times that of ATCA group, and the enzyme activity of Na-SA + ATCA group was the highest, which was 1.2 times that of Na-SA group. The enzyme activity of ATCA group, Na-SA group and Na-SA + ATCA group increased by 121%, 138% and 158% respectively compared with CK group (Fig. 4). In general, the application of the two regulators showed that compared with the single treatment, the composite treatment had a better effect, followed by the Na-SA group.

The activity of peroxidase (POD). At 3 d, the peroxidase activity of Na-SA group was the highest, which was significantly different from other treatment groups, which was 1.1 times and 1.3 times of Na-SA + ATCA group and ATCA group, respectively. Compared with CK group and KB group, the activity increased by 36% and 12%. At the same time, the enzyme activity of other experimental treatment groups was higher than that of CK group. At 6 days, the wheat of Na-SA + ATCA group showed the highest peroxidase activity. The peroxidase activity of Na-SA + ATCA group was 1.2 times that of Na-SA group, which was 101% and 46% higher than that of CK group and KB group (Fig. 5). With the action time of the regulator, the enzyme activity of the Na-SA + ATCA group was the highest, followed by the Na-SA group, but the effect of the Na-SA group could continue to play a significant regulatory role.

The activity of catalase (CAT). At 3 days, from the overall trend, the catalase activity among the treatments was not much different. The enzyme activity of ATCA group, Na-SA group and Na-SA + ATCA group increased by 4%, 5% and 11% respectively compared with CK group, indicating that the regulator had a certain mitigation effect on the growth of wheat. At 6 days, the enzyme activity of ATCA group, Na-SA group, Na-SA + ATCA group was very different from that of CK group and KB group (Fig. 6). The enzyme activity of the three regulator treatment groups was 1.6 times higher than that of CK group and 1.3 times higher than that of KB group. Furthermore, the difference between the treatments was also significant. The enzyme activity of ATCA group, Na-SA group and Na-SA + ATCA group increased by 59%, 71% and 82% respectively compared with CK group. On the whole, it can be analyzed that with the continuous effect of the regulator, the regulation effect is more and more significant, and gradually shows that the Na-SA + ATCA group has the best growth regulation effect on wheat, followed by the Na-SA group.

After crops are subjected to salt stress, high concentration of salt will first have a significant inhibitory effect on the growth and development of seedlings, which directly affects the accumulation of dry matter, resulting in a significant decrease in the growth rate of leaves⁶. It has been reported that with the increase of salt concentration, the biomass of roots, shoots and leaves of crops will be inhibited, resulting in a significant decrease in biomass accumulation⁷. Under the environmental pressure of high concentration salt under salt stress, the reactive oxygen species (ROS) in plants will accumulate in large quantities, thus destroying the cell membrane, leading to a significant decrease in the antioxidant capacity of cells, causing oxidative damage to cells, and also to a certain extent. Destroying the activity of antioxidant enzymes, affecting the metabolism of plants, seriously restricting the normal growth and development of plants^8–9.

During the normal growth and development of plants, once subjected to environmental stress, the growth of plant roots and leaves will be inhibited³⁷. The change of crop morphological characteristics can directly reflect the degree of salt damage ³⁸. Suitable leaf area index and biomass at seedling stage are beneficial to grain filling and yield improvement at later stage. In this study, the effect of ATCA and Na-SA combined treatment was significantly better than that of single regulator in the determination of morphological and physiological indexes and biomass accumulation of wheat under salt stress environment. The effect of Na-SA in a single treatment was better than that of ATCA, which was consistent with the results of Mimouni et al.³⁹. In the regulation of root system, the alleviating effect of regulator on the longitudinal growth of wheat root was higher than that of its transverse growth, and the root morphology was generally slender.

Salt stress can break the dynamic balance of active oxygen scavenging mechanism in plant cells and accumulate a large amount of ROS. It is difficult to remove the accumulated ROS in time for a long time, which will cause oxidative damage to plant cells and eventually lead to the destruction of membrane system, protein and photosynthetic system in cells⁴⁰. In order to resist the damage of external stress, plants regulate their own resistance response. Stress produces a large number of antioxidant enzymes and antioxidants to combine with ROS for redox reaction, mainly including SOD, CAT, POD, etc., which can effectively remove ROS and reduce plant damage⁴¹. In this study, the accumulation of O²⁻and H₂O₂ in wheat leaves was significantly reduced by ATCA and Na-SA foliar spraying, and the oxidative damage of wheat tissue cells was repaired. The regulatory effect of ATCA was slightly better than that of Na-SA. What’s more, the antioxidant enzyme activity in the plant showed that the adjustment of the regulator to the wheat under the stress environment was generally slow in the early stage of leaf spraying treatment, and the difference of antioxidant enzyme activity among the treatments was relatively small. Thus we speculate that it may be due to the early application amount reached the threshold of action, and the effect of plant growth regulator was not obvious on the whole. However, the continuous regulation after spraying the regulator again in the later stage showed significant differences in the enzyme activity among the treatments. The regulation of each enzyme activity in the ATCA and Na-SA combined treatment group was significantly better than that in the single regulator treatment. The effect of Na-SA in single regulator treatment was better than that of ATCA, indicating that the two regulators can significantly improve the activity of antioxidant enzymes in wheat salt stress environment, and can effectively play a role in alleviating salt stress, which is similar to the conclusion of Sihag et al. in the study of sorghum seedlings⁴², to ensure the normal growth and development of wheat.

Salt stress can cause plasma membrane damage, increase plasma membrane permeability, intracellular electrolyte leakage, and increase electrical conductivity⁴³, while plasma membrane decomposition leads to malondialdehyde accumulation^44–45. MDA is an important indicator of plant cell response to stress and cell membrane damage. In this study, the concentration of malondialdehyde (MDA) was significantly reduced after Na-SA leaf spray treatment, and the regulation effect was the best, which greatly alleviated the salt stress pressure of wheat and reduced the damage of leaf tissue cells, thereby reducing the concentration of malondialdehyde. This result is consistent with Chen Hongna et al. in the study of Leymus chinensis seed germination⁴⁶. At the later stage of treatment, the effect of regulators was extremely significant, and the effect of Na-SA treatment was the best, and the regulation of malondialdehyde concentration in the ATCA and Na-SA combined treatment group was significantly better than that in the single regulator treatment group, which could play a synergistic effect, which was similar to the results of Dong et al. in cucumber low temperature stress⁴⁷.

In summary, the normal growth and development of wheat were obviously restricted by the external salt stress environment, and the two plant growth regulators showed different degrees of alleviating effects on the growth of wheat under the salt stress environment. Among the morphological indicators and biomass accumulation, antioxidant enzyme activity and malondialdehyde (MDA) concentration, reactive oxygen species (ROS) content determination indicators, the ATCA and Na-SA combined treatment group was significantly better than the single regulator treatment group, which played a synergistic effect, indicating that the combined treatment of the two growth regulators could significantly alleviate the effects of salt stress on the growth and development of wheat. The above results suggest that it may be due to the two plant growth regulators first reduce the MDA content in plant by increasing the activity of antioxidant enzymes in plants, thereby significantly reducing the accumulation of reactive oxygen species in the plant tissue, and finally alleviating the oxidative damage of reactive oxygen species to the cell membrane and protecting the chlorophyll content in the plant leaves to ensure that the plant is able to photosynthesize enough organic matter for its growth and development. Therefore, the two growth regulators can regulate the normal growth and development of wheat plants under stress environment, and can effectively alleviate the inhibition of wheat growth under salt stress environment.

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Author Contributions

R.-Q. L. and F. Z.: Conceptualization; A.-H. H. and C.-L. W.: methodology; F. Z. and A.-H. H.: software; J.-C. L., L. X. and X.-Y. G.: formal analysis; A.-H. H. and W.-G. L.: investigation; A.-H.H.: resources; C.-L.W.: data curation; A.-H. H.: writing—original draft preparation; R.-Q. L, F.Z. and A.-H. H.: writing—review and editing; J.-C. L: visualization; A.-H. H., R.-Q. L., and F.Z.: supervision; A.-H. H. and W.-G. L.: project administration; R.-Q. L.: funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This study was sponsored by the special fund project for central guiding Henan province local development (No. Z20221343034), the Henan provincial science and technology major project (No. 221100110100), Leading talents of science and technology in the central plain of China (No. 234200510007), Young Backbone Teacher Training Project of Henan Province (2020GGJS166), and Postdoctoral research project in Henan province (HN2022109)

Additional information

Supplementary Information None.

Conflicts of Interest The authors declare no conflict of interest.

Competing interests The authors declare no competing interests.

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Physiological mechanism of sodium salicylate and folcisteine on alleviating salt stress in wheat seedlings

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