Analysis of Effect of Compound Salt Stress on Seed Germination and Salt Tolerance Analysis of Pepper (Capsicum annuum L.)

Summary

The paper below presents a protocol for measuring seed germination, seedling growth, and physiological indexes of two pepper varieties with salinity tolerance differences in response to six mixed salt concentrations. This protocol can be used to evaluate the salt tolerance of pepper varieties.

Abstract

To determine the salt tolerance and physiological mechanism of pepper (Capsicum annuum L.) at the germination stage, the Hongtianhu 101 and Xinxiang 8 varieties, which have large differences in salt tolerance, are employed as the study materials. Six mixed salt concentrations of 0, 3, 5, 10, 15, and 20 g/L derived using equal molar ratios of Na₂CO₃, NaHCO₃, NaCl, CaCl₂, MgCl₂, MgSO_4, and Na₂SO₄ are used. To determine their effects, the related indexes of seed germination, seedling growth, and physiology are measured, and salt tolerance is comprehensively evaluated using membership function analysis. The results show that as the mixed salt concentration increases, the germination potential, germination index, germination rate, seed germination vigor index, root length, and root fresh weight of the two cultivars significantly decrease, whereas the relative salt rate gradually increases. The hypocotyl length and fresh weight aboveground increase first and then decrease, while the malondialdehyde (MDA), proline (Pro) content, catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) activity decrease and then increase. The germination potential, germination index, germination rate, seed germination vigor index, root length, root fresh weight, MDA and Pro content, and CAT activity of the Hongtianhu 101 seeds are higher than those of Xinxiang 8 for all salt concentrations employed here. However, hypocotyl length, fresh weight aboveground, and relative salt rate are lower in Hongtianhu 101 than in Xinxiang 8. The comprehensive evaluation of salt tolerance reveals that the total weighted values of the two membership function indexes increase first and then decrease as the mixed salt concentration increases. Compared with 5 g/L, which has the highest membership function value, the index under salt concentrations of 3 g/L, 10 g/L, and 15 g/L decreases by 4.7%-11.1%, 25.3%-28.3%, and 41.4%-45.1%, respectively. This study provides theoretical guidance for the breeding of salt-tolerant varieties of pepper and an analysis of the physiological mechanisms involved in salt tolerance and salt-tolerant cultivation.

Introduction

Salinity is a major limiting factor for crop productivity worldwide¹. At present, nearly 19.5% of the world's irrigated land and 2.1% of dry land are affected by salinity, and approximately 1% of agricultural land degenerates into saline-alkali land every year. By 2050, 50% of arable land is expected to be affected by salinization^2,3. In addition to natural factors, such as natural rock weathering and salty rainwater near or around the coast, rapid surface evaporation, low rainfall, and unreasonable agricultural management methods have exacerbated the process of soil salinization. Soil salinization inhibits the growth of plant roots and reduces the absorption and transportation of water and nutrients from the plant roots to the leaves. This inhibition results in physiological water shortages, nutritional imbalances, and ion toxicity, which lead to reduced crop productivity and a complete loss of crop yield. The salinization of cultivated land is gradually becoming one of the most critical abiotic stress factors affecting global agricultural food production⁴. Salt stress reduces the arable land available for agriculture, which may result in a significant imbalance between the supply and demand of future agricultural products. Therefore, exploring the effects of soil salinization on crop growth and physiological and biochemical mechanisms is conducive for breeding salt-tolerant varieties, the sustainable utilization of saline soil, and the safety of agricultural products.

Pepper (Capsicum annuum L.) is planted worldwide owing to its high nutritional and medicinal value. For example, capsaicin is an alkaloid responsible for the spicy flavor of pepper. Capsaicin can be used for pain relief, weight loss, improving cardiovascular, gastrointestinal tract, and respiratory systems, and in several other applications⁵. Pepper is also rich in bioactive substances, especially different antioxidant compounds (carotenoids, phenolics, and flavonoids) and vitamin C⁶. Currently, pepper is reported to be the vegetable crop with the largest cultivation area in China, with an annual planting area of more than 1.5 x 10⁶ ha, thereby accounting for 8%-10% of the total vegetable planting area in China. The pepper industry has become one of the largest vegetable industries in China and has the highest output value⁷. However, pepper cultivation is often subjected to a variety of biological (pests and fungi) and abiotic stresses, especially salt stress, which has a direct negative impact on seed germination, growth, and development, resulting in the reduction of pepper fruit yield and quality⁸.

Seed germination is the first stage of interaction between plants and the environment. Seed germination is highly sensitive to fluctuations in the surrounding media, especially soil salt stress, which may exert reversed effects on physiology and metabolism, and eventually disorder the normal growth, development, and morphogenesis of crops⁹. In previous studies, pepper seed germination and seedling growth under salt stress were extensively investigated; however, most studies used NaCl as the only salt for stress induction^10,11,12. However, soil salt damage is mainly due to Na⁺, Ca²⁺, Mg²⁺, Cl^-, CO₃^2-, and SO₄^2- ion toxicity generated by the dissociation of sodium, calcium, and magnesium salts. Owing to the synergy and antagonism between ions, the effects of mixed salt and single salt on crop growth and development may be quite different. However, the corresponding characteristics of pepper seed germination and growth in mixed salt are still unclear. Therefore, two pepper varieties with remarkable differences in salt tolerance are used as materials in this study. Analyzing the effects of different salt concentrations on pepper seed germination, growth, and physiological and biochemical indexes after equimolar mixing of seven salts can reveal the response mechanism of pepper seed germination to salinity stress. It can also provide a theoretical basis for cultivating strong pepper seedlings, as well as high yield and high-quality cultivation in saline cultivated land.

Protocol

NOTE: Here, we present a protocol for assessing the response characteristics and internal mechanisms of pepper seed germination and seedling growth under different mixed salt stresses, which can serve as a reference method for seed salt tolerance evaluation.

1. Experimental preparation

1. Prepare crop seeds for cultivars-Hongtianhu 101 with strong salt tolerance and Xinxiang 8 with low tolerance.
2. Prepare 0.2% KMnO₄ solution as a seed disinfection reagent. First, weigh 4.0 g of KMnO₄, and then add 2,000 mL of distilled water.
   NOTE: Potassium permanganate is usually unstable due to its strong oxidation; accordingly, it is prepared immediately before use.
3. Prepare the mixed salts using seven salts, including sodium carbonate, sodium bicarbonate, sodium chloride, calcium chloride, magnesium chloride, magnesium sulfate, and sodium sulfate¹³. Add the same molar amount of each, which successively account for 14.8%, 11.7%, 8.2%, 15.5%, 13.3%, 16.7%, and 19.8% of the total mass ratio of the mixed salts, respectively.
4. Prepare Petri dishes (single use) and filter paper (medium speed qualitative filter paper), both with a diameter of 9 cm.
   ​NOTE: The material of the Petri dish can be changed; however, the diameter of the Petri dish and filter paper must be the same.

2. Seed soaking and preparation for germination

1. For seed optimization, select pepper seeds with consistent size and full particles from each variety, with an average diameter of 4.2 mm and 3.7 mm for Hongtianhu 101 and Xinxiang 8 seeds, respectively. Calculate the total number of seeds selected according to the test workload.
2. For seed disinfection, soak selected pepper seeds in 0.2% KMnO₄ solution for 15 min and then rinse five times with distilled water.
3. For seed soaking, transfer the sterilized seeds to distilled water and allow them to soak for 24 h. Rinse the seeds several times with distilled water and dry for further use.
   ​NOTE: The soaking time of seeds for different crops may vary.

3. Seed germination and seedling growth

1. Prepare six concentrations of the mixed salts: 0 (control), 3, 5, 10, 15, and 20 g/L. Measure the conductivity of the salt solution using a conductivity meter; the solution conductivity EC values are 0.092, 3.05, 4.73, 8.33, 11.53, and 15.22 ms/cm, respectively.
2. For seed preparation, evenly place 40 pepper seeds in a Petri dish with two layers of filter paper. Prepare the seeds for six experimental treatments and repeat each treatment five times.
3. For seed germination, add a suitable amount of the six mixed salt concentrations to the Petri dish to ensure the filter paper is kept moist. Place the seeds in an air incubator at 28 °C and 80% air humidity for germination in the dark.
4. After seed germination, allow the seedlings to continue to grow in light (light intensity of approximately 450 Lux; light cycle of 12/12h) in the incubator for 14 days after sowing. The temperature and humidity at the seedling growth stage must be the same as those used at the germination stage.
5. Replenish the solution in the culture dish every 12 h to retain a moist filter paper, and completely wash the filter paper every 24 h with the corresponding concentration of the mixed salt solution to keep a constant mixed salt concentration in the Petri dish.
   ​NOTE: The amount of salt solution added to wet seeds can be adjusted according to the seed germination and growth stages. Many methods are available for maintaining a constant concentration of salt solutions in culture dishes. In addition to the methods described in this experiment, the strategy of adding distilled water by weight can be used.

4. Measurement and calculation of indicators

1. Determination of the seed germination indexes
   1. Determine the germination rate daily after sowing, with the radicle breaking seed coat reaching half the seed diameter length as the germination marker.
   2. Calculate the germination rate, germination potential, relative salt rate, germination index, and seed germination vigor index using the following formulas:
      Germination rate (%) = (number of normal germinated seeds on day 7 after sowing/number of tested seeds) × 100
      Germination potential (%) = (number of normal germinated seeds on day 3 after sowing/number of tested seeds) × 100
      Relative salt rate (%) = (control germination rate - treatment germination rate)/control germination rate × 100
      calculated using the seed germination rate on day 7 after sowing
      Germination index (GI) = ∑ [Gt/Dt]
      where Gt refers to the seed germination number at a period of time (t) after sowing and Dt refers to the corresponding germination days
      Seed germination vigor index (VI) = GI x S
      ​where S is the root length
2. Determination of the seedling growth index
   1. On day 14 after sowing, randomly select 10 representative seedlings from each Petri dish and measure the root length and hypocotyl length.
   2. Use a knife to divide the pepper seedlings into two parts: radicle and aboveground parts. Remove the water from the seedlings by wiping, and weigh the seedlings separately to determine the fresh weight.
3. Determine the antioxidant enzyme activity, malondialdehyde (MDA) level, and proline (Pro) content in pepper as follows.
   1. To preserve the pepper seedlings, select representative whole pepper seedlings (approximately 24.0 g) from each treatment on day 14 after sowing. After removing the surface water, immediately freeze the seedlings in liquid nitrogen for 1 min and store them in a refrigerator at an ultra-low temperature (-80 °C).
      NOTE: The sample number of pepper seedlings stored in the ultra-low temperature refrigerator should be sufficient, in case some indicators need to be retested.
   2. Retrieve approximately 1.0 g of seedling sample from each treatment collected in triplicate. Place the seedling sample in a centrifuge tube, add liquid nitrogen, and grind the sample using a grinding rod to determine the physiological indexes of the seedlings. The determined indexes and the measurement scheme are shown below.
   3. Determine the seedling protective enzyme activity (peroxidase [POD], catalase [CAT], superoxide dismutase [SOD]), malondialdehyde (MDA), and proline (Pro) contents using a commercially available kit (spectrophotometry-based) for each factor¹⁴.
      NOTE: Earlier observations revealed no difference in salt stress between the 15 and 20 g/L mixed salt concentrations. As a result, only five salt concentrations (0, 3, 5, 10, and 15 g/L) are measured.
4. Comprehensive evaluation of salt tolerance using the membership function method
   NOTE: The membership function uses a fuzzy mathematics method, which converts qualitative evaluation into quantitative evaluation¹⁵, to evaluate a variety of physiological indexes affected by salt damage.
   1. Calculate the value of the membership function using the following formula by Zhoubin Liu et al.¹⁵:
      Ri = (Xi - Xmin)/(Xmax - Xmin)
      If a trait is negatively correlated with salt tolerance, calculate the inverse membership function using:
      Ri = 1 - (Xi - Xmin)/(Xmax - Xmin)
      ​Accumulate the membership values of each physiological index, where Xi is the measured value of a certain trait, Xmax and Xmin are the maximum and minimum values for Xi, respectively, and Ri is the membership value of that trait.
   2. Include the following relevant indicators: seed germination characteristics (germination potential, germination rate, germination index, and seed germination vigor index), seedling growth characteristics at the germination stage (root length, hypocotyl length, root fresh weight, and fresh weight aboveground), MDA, Pro, and protective enzyme activity (CAT, POD, SOD) for membership function value calculation. The membership function values are obtained from each indicator.
5. Use spreadsheet and SPSS software (version 22.0) to analyze and process the test data and apply the least significant difference (LSD) method for multiple comparisons to identify significant differences. Use Pearson's correlation analysis to investigate the correlation between seed germination and seedling physiological indexes of pepper under compound salt stress.

Representative Results

Seed germination characteristics
As the mixed salt concentration increases, the germination potential and germination index of Hongtianhu 101 and Xinxiang 8 decreases significantly. Both cultivars have a sharp decline in salt concentrations from 0-3 g/L, and a slow and steady decline for salt concentrations from 3-20 g/L (Figure 1A,B). The germination rate of the two varieties gradually decreases as the mixed salt concentrations increase, and the relative salt rate for the varieties gradually increases. There is no significant difference in the germination rate and relative salt rate at salt concentrations of 3-15 g/L. However, the difference is significant at all other salt concentrations (Figure 1C,D). Regarding salt tolerance between the two varieties, the germination potential, germination index, and germination rate of Hongtianhu 101 seeds with increasing mixed salt concentrations are higher than those of Xinxiang 8, whereas the relative salt rate is lower than that of Xinxiang 8.

As the mixed salt concentration increases (0-15 g/L), the seed germination vigor index of the two varieties decreases significantly. When the mixed salt concentration is 15 g/L, the seed germination vigor index of Hongtianhu 101 and Xinxiang 8 decreases by 91.0% and 94.6%, respectively, compared with that of the control. Of note, the decrease is not significant when the mixed salt concentration further increases from 15 to 20 g/L. The seed germination vigor index of Hongtianhu 101 is higher than that of Xinxiang 8 at each mixed salt concentration level (Figure 1E).

Seedling growth characteristics
As the mixed salt concentration increases (0-15 g/L), the root length and root fresh weight of Hongtianhu 101 and Xinxiang 8 decrease significantly. When the mixed salt concentration is 15 g/L, the root length of Hongtianhu 101 and Xinxiang 8 decreases by 89.4% and 91.1%, respectively, and the root fresh weight decreases by 81.7% and 71.2%, respectively, compared to those of the control. However, when the salt concentration is 15-20 g/L, the root length and root fresh weight of the two varieties does not change significantly (Figure 2A,C). The root length and root fresh weight of Hongtianhu 101 are generally higher than those of Xinxiang 8 with increasing mixed salt levels, with obvious differences at concentrations ranging from 0 to 10 g/L.

The hypocotyl length and fresh weight aboveground of the two varieties increase and then decrease with increasing mixed salt concentration. Both indexes reach their highest values at a salt concentration of 5 g/L. Similarly, when the salt concentration is 15-20 g/L, the hypocotyl length and fresh weight aboveground of the two varieties decrease slightly. Based on the varietal differences, the hypocotyl length and fresh weight aboveground of Xinxiang 8 are higher than those of Hongtianhu 101 at each salt concentration (Figure 2B,D).

Membrane lipid peroxidation and osmotic adjustment substance content
As the mixed salt concentration increases, the MDA and Pro contents of the two varieties decrease and then increase. The MDA and Pro contents reach their lowest values at concentrations of 5 g/L and 3 g/L, respectively (Figure 3A,B). The MDA content decreases slightly at salt concentrations of 0-5 g/L and increases rapidly at 10 g/L. Compared with the 5 g/L treatment, the MDA content following the 10 g/L treatment increases by 59.9%-64.8%, and then remains unchanged. The MDA content of Hongtianhu 101 is higher than that of Xinxiang 8 at different salt concentrations (Figure 3A). The decrease in Pro content at 0-3 g/L is not significant and little difference is found between the two varieties. When the salt concentration is 3-15 g/L, the Pro content of Xinxiang 8 slowly increases and remains relatively stable, whereas the Pro content of Hongtianhu 101 increases rapidly. Compared to 3 g/L, the Pro content of Hongtianhu 101 significantly increases by 440.2% at 15 g/L (Figure 3B).

Protective enzyme activity
As the mixed salt concentration increases, the CAT, POD, and SOD activities of the seedlings of Hongtianhu 101 and Xinxiang 8 decrease and then increase, with the lowest values obtained at a concentration of 3 g/L (Figure 4A-C). The CAT and POD activities of the two varieties vary slightly at salt concentrations of 0-5 g/L, and the difference between them is small. Thereafter, the CAT and POD activities of the two varieties increase significantly with increasing salt concentration. Further, the CAT and POD activities of Hongtianhu 101 are higher than those of Xinxiang 8, and the difference between them increases gradually (Figure 4A,B). The SOD activity of the two varieties varies slightly at salt concentrations from 0-10 g/L, and then increases rapidly. The SOD activity of Xinxiang 8 is higher than that of Hongtianhu 101 at salt concentrations from 0-10 g/L; for the remaining concentrations, its activity is lower than that of Hongtianhu 101 (Figure 4C).

Correlation analysis of the seed germination indexes of pepper and comprehensive evaluation of salt stress
Correlation analysis (Table 1) reveals that the root length and root fresh weight for the seedling growth characteristic indexes are significantly positively correlated with the germination indexes (germination potential, germination rate, germination index, seed germination vigor index, etc.); however, no explicit relevance is found between the hypocotyl length, fresh weight aboveground, and germination indexes. A significant negative correlation is found between the indexes of seedling growth characteristics and the activity of protective enzymes (CAT, POD, and SOD). A significant negative correlation is also found between the hypocotyl length and the contents of MDA and Pro, and between the fresh weight of the shoot and the MDA content. With exception to the significant correlation between seed germination vigor index and protective enzyme activity, no significant correlation is found between the germination characteristic indexes and seedling physiological indexes (protective enzyme activity and MDA and Pro content).

The salt tolerance of the two pepper varieties under compound salt stress is evaluated using the membership function method for multiple traits. As the pepper seedlings are not subjected to salt stress when the mixed salt concentration is 0 g/L, their membership function value is not calculated. As a result, only the treatment with salt stress is assessed using membership function analysis. MDA is found to be negatively correlated with the salt tolerance of pepper seedlings and is calculated using the inverse membership function method; other indexes are calculated using the membership function method. Table 2 shows that as the mixed salt concentration increases, the total weighted values of each index function of the two varieties increase and then decrease, ultimately reaching a maximum at a salt concentration of 5 g/L. Compared with the 5 g/L treatment, the values obtained with the 3 g/L, 10 g/L, and 15 g/L salt concentration treatments decrease by 4.7%-11.1%, 25.3%-28.3%, and 41.4%-45.1%, respectively. Therefore, the salt tolerance of pepper subjected to 5 g/L, 3 g/L, 10 g/L, and 15 g/L salt concentration treatments can be categorized as best, second best, bad and worst, respectively.

Figure 1: Effects of increasing mixed salt concentrations on seed germination of pepper. (A), (B), (C), (D), and (E) represent the response characteristics of pepper seed germination potential, germination index, germination rate, relative salt rate, and seed germination vigor index to compound salt stress, respectively. Different lowercase letters in the figure indicate significant differences between treatments, which are analyzed by the Tukey's multiple range test (p < 0.05). Error bars indicate standard deviations (n = 5). Please click here to view a larger version of this figure.

Figure 2: Effects of increasing mixed salt concentrations on seed germination and morphological indexes of pepper. (A), (B), (C), and (D) represent the response characteristics of pepper seedling root length, hypocotyl length, root fresh weight, and fresh weight aboveground to compound salt stress, respectively. Different lowercase letters in the figure indicate significant differences between treatments, which are analyzed by the Tukey's multiple range test (p < 0.05). Error bars indicate standard deviation (n = 5). Please click here to view a larger version of this figure.

Figure 3: Effects of increasing mixed salt concentrations on MDA and Pro content of pepper seedlings. (A) and (B) represent the response characteristics of pepper seedling MDA and Pro content to compound salt stress, respectively. Different lowercase letters in the figure indicate significant differences between treatments, which are analyzed by the Tukey's multiple range test (p < 0.05). Error bars indicate standard deviation (n = 3). Please click here to view a larger version of this figure.

Figure 4: Effects of increasing mixed salt concentrations on CAT, POD, and SOD activities of pepper seedlings. (A), (B), and (C) represent the response characteristics of pepper seedling CAT, POD, and SOD activities to compound salt stress, respectively. Different lowercase letters in the figure indicate significant differences between treatments, which are analyzed by the Tukey's multiple range test (p < 0.05). Error bars indicate standard deviation (n = 3). Please click here to view a larger version of this figure.

Table 1: Correlation analysis between pepper germination and physiological indicators under compound salt stress (n = 30). Pearson's correlation analysis is used to investigate the correlation between seed germination and seedling physiological indexes of pepper under compound salt stress. * p < 0.05; ** p < 0.01. Please click here to download this Table.

Table 2: The weighted membership function value of pepper germination and seedlings' physiological indicators under mixed salt stress. Please click here to download this Table.

Discussion

This research method comprises four key steps that affect the accuracy of the experimental results. First, owing to the poor dissolution of mixed salts caused by the increased solute content in high salt concentration solutions, and the low solubility of reagents such as calcium chloride, which are more difficult to solubilize in water, the weighed reagents must be fully ground in a mortar. Further, the reagents must be dissolved via ultrasonic waves before determining the capacity. Second, the configured salt solution must be completely shaken each time and added to the Petri dish for use. Third, the Petri dishes must retain a suitable water layer after addition of the salt solution, and the water status of each Petri dish must be relatively consistent. Finally, light conditions must be consistent after seed germination.

In this study, the number of test seeds in a single Petri dish can be adjusted by varying the diameter of the selected Petri dish. According to the specific situation of soil salt stress in different planting areas, the proportion of each single salt addition could be adjusted to enable consistency with the actual situation of salt stress in the local soil. While this method is practical, for smaller seeds (such as rape, ice plant, and amaranth) or larger seeds (such as sword beans and broad beans), problems such as operational difficulties in the determination of seedling length and fresh weight, or the availability of very few seeds in a single culture dish to repeat the data, lead to difficulties in studying salt tolerance using this method.

The method described here is used to determine the seed germination characteristics and seedling growth under different mixed salt concentrations and reveal the change mechanism through internal physiological enzyme activity, which is of great significance for objectively evaluating the salt tolerance characteristics of seeds. This technology can provide a technical reference for salt tolerance evaluation of other crops. Seed germination and seedling growth are the stages in which crops are most sensitive to salt stress. Thus, this method can effectively provide a reference for salt-tolerant cultivation and crop breeding.

In most crops, salt stress can inhibit seed germination and seedling growth under biotic stress. Such inhibition may be due to a reduction in seed water uptake by reducing the osmotic potential of the salt solution under saline conditions. Salt ion stress and toxicity may change the activity of protective enzymes (POD, CAT, SOD, etc.) and protein metabolism during seed germination, and destroy endogenous hormone balance^11,16. Zhani et al. reported that the germination process was mainly changed by a reduced germination rate and prolonged germination under salt (NaCl) stress. Further, a significant difference was found in the germination rate among different varieties (10%-50%) at a salt concentration of 8 g/L¹⁷. The present study also reveals that as the mixed salt concentration increases, the germination potential, germination index, germination rate, and seed germination vigor index of Hongtianhu 101 and Xinxiang 8 decrease significantly, and the relative salt rate increases gradually. According to Patanè et al., although salt stress prolonged the germination time of sweet sorghum seeds, increasing salt stress had an adverse effect on the final germination of seeds¹⁸. However, related studies suggest that NaCl treatment can promote pepper seed germination and growth¹⁹, which may be related to differences in salt concentration levels.

Increasing the level of salt stress has a significant effect on the growth of pepper seedlings, and based on this study, the root length and root fresh weight of the two varieties decreased significantly with increasing mixed salt concentrations. This finding is similar to that of Mirosavljević et al., who suggested that root length and root weight decreased as salt stress increased, and the differences among the treatments were significant²⁰. This result indicates that the root system was placed in the soil medium in direct contact with the soil solution, and the root length and root weight were more sensitive to NaCl osmotic stress. Root length and root weight are key indicators of plant response to salt stress. According to this study, as salt concentration increases, the hypocotyl length and fresh weight of the aboveground parts increase and then decrease, with maximum values achieved at 5 g/L. Khan et al. also suggested that as salinity (NaCl) increases (0-9 ms/cm), the shoot length of pepper increases first and then decreases, with the best performance obtained at 3 ms/cm^12,21. The conductivity of salt concentration in this study was 4.73 ms/cm when the value of seedling hypocotyl length and fresh weight aboveground were the highest, which is higher than the value reported by Khan et al.²¹. This result might be due to the high tolerance of pepper aboveground to compound salt stress compared with single salt stress.

Salt stress not only hinders crop growth but also causes significant physiological changes in plants. Salt stress can increase the levels of reactive oxygen species (ROS). If ROS are not cleared in time, membrane lipid peroxidation and oxidative stress, which can cause serious damage to the plant cell membrane, may occur. MDA is the final metabolite of membrane lipid peroxidation, and intracellular MDA concentration is often used as an indicator to assess the degree of damage to plants under stress²². In the present study, as the mixed salt concentration increases, the MDA content of the seedlings of the two varieties decreases first and then increases. Of note, the decrease is not significant at salt concentrations ranging from 0-5 g/L. However, a rapid increase is observed from 5-10 g/L. Thereafter, the values remain unchanged, indicating that the degree of membrane lipid peroxide of pepper seeds changes from general, to rapidly increasing, to stable. Salt stress is speculated to have a serious impact on cell membrane permeability when the mixed salt concentration is greater than 10 g/L. Guzmán-Murillo et al. reported a similar conclusion of a decrease and then an increase in the membrane lipid peroxide level in sweet pepper seedlings as the NaCl concentration increased (0-50 nmol/L). Further, the level of lipid peroxidation was lowest at 25 nmol/L NaCl²³.

Plants have evolved several strategies for coping with salt stress. On the one hand, crops enhance protein stability and membrane integrity by increasing osmotic adjustment substances, such as proline, and reduce the loss of intracellular water, thereby improving salt tolerance²⁴. In the present study, as the mixed salt concentration increases, the Pro content of Xinxiang 8 and Hongtianhu 101 seedlings decreases first and then increases. Notably, the decrease is not significant at concentrations of 0-3 g/L and the difference between the two varieties is not significant, aligning with the experimental results of Muchate et al²⁵. The Pro content of the Hongtianhu 101 pepper variety with good salt tolerance also increases rapidly at salt concentrations of 3-15 g/L. Compared with 3 g/L, the Pro content of 15 g/L significantly increases by 440.2%, whereas the Pro content of Xinxiang 8 with general salt tolerance increases slowly and maintains a relatively stable level at salt concentrations of 3-15 g/L. The scavenging effect of the antioxidant enzymes on ROS induced by salt stress has proven to be the main component of crop defense mechanisms. The activities of CAT, POD, and SOD have been reported to increase under different salt-stress environments, thereby improving its salt tolerance^25,26. Chen et al. showed that as NaCl concentration increases, the activities of SOD, POD, and CAT in tomato seed germination increases gradually, with no significant difference in each index following treatment with 0-50 nmol/L NaCl²⁷. This study also shows that as the mixed salt concentration increases, the activities of CAT, POD, and SOD in the seedlings of Hongtianhu 101 and Xinxiang 8 decrease and then increase. At low salt concentrations (0-3 g/L), the activity of antioxidant enzymes does not change significantly, and a further increase in salt concentration improves salt tolerance.

The adaptability and response characteristics of crops to salt stress are mainly reflected in morphology, structure, physiological ecology, etc. A comprehensive evaluation of compound salt stress is carried out using the membership function value method for multiple traits. This study shows that as the mixed salt concentration increases, the total weighted value of the function value increases first and then decreases. The two varieties reach a maximum at 5 g/L, leading to the best salt tolerance effect. Compared with the 5 g/L treatment, the weighted values following treatment with the salt concentrations of 3 g/L, 10 g/L, and 15 g/L decrease by 4.7%, 25.3%, and 41.4%, respectively, for Hongtianhu 101, and 11.1%, 28.3%, and 45.1%, respectively, for Xinxiang 8. Such findings indicate that the salt tolerance of Hongtianhu 101 is higher than that of Xinxiang 8.

This study provides a comprehensive description of the effects of different concentrations of compound salt stress on pepper seed germination and its physiological enzyme activities, and a technical reference for research on salt tolerance in other crops. Pepper seedlings are less affected by salt stress under simulated mixed salt concentrations (0-5 g/L). Seed germination, radicle elongation, and seedling morphogenesis of pepper are significantly inhibited under high salt stress (>5 g/L), causing serious membrane lipid peroxidation in pepper seedlings. Crops reduce the adverse effects of salt stress by increasing their Pro content and enhancing the activity of protective enzymes (CAT, POD, and SOD). Under salt stress, the proline content and protective enzyme activity of Hongtianhu 101 seedlings are higher, the degree of membrane lipid peroxidation is lower, and seed germination and seedling growth are more obvious than those of Xinxiang 8.

Materials

Name	Company	Catalog Number	Comments
Calcium chloride	Shanghai Experiment Reagent Co., Ltd.,China	Analytical reagent
Centrifugal machine	Shanghai Luxianyi Centrifuge Instrument Co., Ltd., China	TGL-16M
Centrifuge tube	None	None
Conductivity meter	Shanghai Instrument&Electronics Science Instrument Co., Ltd., China	DDSJ-308F
Constant temperature and humidity box	Ningbo Laifu Technology Co., Ltd.,China	PSX-280H
Digital display vernier caliper	Deli Group Co., Ltd.,China	DL90150
Electronic balance	Mettler Toledo Instruments (Shanghai) Co., Ltd.,China	ME802E/02
Filter paper	Hangzhou Fuyang North Wood Pulp and Paper Co., Ltd.,China	GB/T1914-2017
Grinding rod	None	None
Hongtianhu  101	Seminis Seed (Beijing) Co., Ltd.,China	11933955/100147K1-137
Ice machine	Shanghai Kehuai Instrument Co., Ltd., China	IM150G
Liquid nitrogen	None	None
Magnesium chloride	Tianjin Kermel Chemical Reagent Co., Ltd.,China	Analytical reagent
Magnesium sulfate	Tianjin Kermel Chemical Reagent Co., Ltd.,China	Analytical reagent
Petri dish	Jiangsu Yizhe Teaching Instrument Co., Ltd.,China	I-000163
Pocket knife	None	None
Potassium permanganate (KMnO4	Xilong Scientific Co.,Ltd.,China	Analytical reagent
Pure water equipment	Sichuan Youpu Ultrapure Technology Co., Ltd.,China	UPT-I-20T
Sodium bicarbonate	Xilong Scientific Co.,Ltd.,China	Analytical reagent
Sodium carbonate	Xilong Scientific Co.,Ltd.,China	Analytical reagent
Sodium chloride	Xilong Scientific Co.,Ltd.,China	Analytical reagent
Sodium sulfate	Xilong Scientific Co.,Ltd.,China	Analytical reagent
Test kit	Suzhou Keming, Biotechnology Co., Ltd, Suzhou.,China	Spectrophotometer method
Ultra-low temperature freezer	SANYO Techno Solution TottoriCo.,Ltd.	MDF-382
Ultraviolet visible spectrophotometer	Shanghai Precision Scientific Instrument Co., Ltd., China	760CRT
Xinxiang 8	Jiangxi Nongwang High Tech Co., Ltd.,China	GPD Pepper 2017(360013)