Seed priming with salicylic acid enhances salt stress tolerance by boosting antioxidant defense in Phaseolus vulgaris genotypes

Abstract

Salinity stress significantly threatens seed germination, plant growth, and agricultural productivity, necessitating effective mitigation strategies. This study evaluates the potential of salicylic acid (SA) pretreatment to alleviate the detrimental effects of salinity on common bean (Phaseolus vulgaris) genotypes. SA, a phenolic plant hormone, is crucial for regulating growth, stress responses, and essential physiological processes, including seed germination and ion transport. Previous research has established the general benefits of SA in enhancing stress tolerance, but the specific mechanisms and effects on common bean genotypes remain underexplored. This research focuses on the impact of salinity on the germination and seedling growth of various common bean genotypes, the efficacy of SA pretreatment in enhancing these genotypes' tolerance to salinity stress, and the underlying physiological and biochemical mechanisms, particularly involving the antioxidant defense system. The research was conducted in two phases: germination and seedling growth. Ten genotypes and two commercial varieties were exposed to varying salinity levels alongside SA concentrations to assess germination performance. Subsequently, six genotypes and one variety were evaluated for seedling growth under controlled and salt stress conditions (100 mM and 200 mM NaCl), with SA treatments at 0, 0.5, and 1 mM. Results revealed that salinity severely impaired germination traits, which were significantly enhanced by SA pretreatment. During the seedling growth phase, salinity stress resulted in reduced protein, chlorophyll, and carotenoid content, decreased potassium (K⁺) levels, and diminished water content, while increasing electrolyte leakage, malondialdehyde (MDA) levels, sodium (Na⁺) concentrations, enzyme activities, and proline levels. Importantly, SA pretreatment elevated chlorophyll and protein concentrations, improved water retention, and moderated K⁺ and Na⁺ levels, including their ratios under stress conditions. SA pretreatment also significantly enhanced the antioxidant defense system, reducing oxidative damage induced by salinity stress. Principal component analysis (PCA) successfully categorized the genotypes into semi-tolerant, tolerant, semi-sensitive, and sensitive classes based on their stress responses. Notably, the Jules variety exhibited exceptional resilience during both germination and seedling growth stages, indicating its potential as a superior candidate for cultivation in salt-affected regions. This study highlights SA pretreatment as an effective strategy to enhance salinity stress resilience in common bean genotypes. The novelty of this work lies in the detailed elucidation of SA's role in modulating antioxidant defenses and ion homeostasis in different genotypes, providing new insights into breeding programs and agricultural practices aimed at improving crop resilience and productivity in increasingly saline environments.

Introduction

Salinity stress is a significant challenge to agricultural productivity, particularly for leguminous crops like P. vulgaris, which play a crucial role in global food security [1, 2]. This species is not only a major source of protein but also provides essential micronutrients such as iron, zinc, thiamine, and folic acid [3,4,5]. The adverse effects of salinity manifest as osmotic and ionic imbalances, leading to physiological disturbances that impair seed germination, inhibit plant growth, and ultimately reduce crop yields [6, 7].

To overcome these adversities, plants have evolved various physiological and biochemical mechanisms. Osmotic adjustment, ion homeostasis, and antioxidant defense systems play critical roles. The accumulation of osmolytes such as proline and glycine betaine helps maintain cell turgor and osmotic balance under saline conditions [8,9,10]. Ion homeostasis is managed through the selective uptake and compartmentalization of ions, maintaining a higher K⁺/Na⁺ ratio essential for enzyme function and metabolic processes [11, 12]. Under salt stress conditions, the improved growth observed in seedlings derived from seeds primed with the treatments can be attributed to increased membrane stability, elevated chlorophyll content, and a reduced Na⁺/K⁺ ratio [13]. Antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and peroxidase scavenge reactive oxygen species (ROS), protecting cells from oxidative damage [14,15,16].

In recent years, novel approaches such as priming with salicylic acid (SA) have shown promising results in enhancing plant tolerance to abiotic stresses, including salinity [17,18,19,20]. SA modulates various signaling pathways, leading to the activation of stress-responsive genes and the production of protective metabolites [21, 22]. It enhances the antioxidant defense system, increases the activity of ion transporters, and stabilizes cellular structures. SA's role in improving ion homeostasis involves the upregulation of Na⁺/H⁺ antiporters and K⁺ channels, which help maintain ion balance under salt stress [7, 23, 24].

P. vulgaris exhibits a range of salt tolerance levels, making it an interesting model for studying adaptive responses to salinity [25, 26]. Certain landraces and cultivars have shown inherent resilience to salt stress, attributed to their efficient antioxidant systems and ion homeostasis mechanisms. Despite the established role of SA in enhancing salinity tolerance, comprehensive studies on its specific effects and underlying mechanisms in P. vulgaris are limited. Recent studies have highlighted the pivotal role of SA in enhancing salt stress tolerance in P. vulgaris [27, 28]. SA acts as a signaling molecule that modulates various physiological and biochemical pathways, leading to improved plant resilience under saline conditions [22, 29, 30]. It has been shown to regulate ion homeostasis, enhance antioxidant defense mechanisms, and modulate gene expression related to stress responses [31,32,33]. These findings underscore the potential of SA as a key player in developing salt-tolerant crop varieties. Furthermore, SA-mediated priming has been observed to improve the Na⁺/K⁺ balance and maintain membrane integrity, which are crucial for plant survival under salt stress [34].

This study brings a novel perspective to plant stress physiology by focusing on the specific mechanisms through which SA pretreatment enhances salinity stress tolerance in P. vulgaris genotypes. While the role of SA in plant stress responses is generally acknowledged, its precise effects on common bean genotypes under salinity stress remain insufficiently explored. This research addresses this gap by examining the distinct physiological and biochemical pathways activated by SA pretreatment in various common bean genotypes, thereby providing a detailed and genotype-specific understanding of SA's mitigating effects on salinity stress. The dual-phase approach, involving both germination and seedling growth, offers a comprehensive assessment of plant responses at different developmental stages, further adding to the novelty of the study. Understanding these mechanisms can aid in breeding programs and agricultural practices for salt-affected regions, potentially enhancing crop resilience and productivity. In addition to strengthening food security, this research addresses the global challenge of soil salinization, which affects an estimated 20% of irrigated lands worldwide and is projected to worsen with climate change [35].

Additionally, the research stands out due to its methodological rigor and advanced analytical techniques. The use of PCA to categorize the genotypes based on their stress responses provides a sophisticated framework for identifying and classifying tolerance levels. The meticulous examination of key physiological traits, such as chlorophyll content, water retention, electrolyte leakage, and ion homeostasis, coupled with detailed biochemical analyses of antioxidant defense mechanisms, provides a deep insight into the protective role of SA under salinity stress. By incorporating contemporary findings from phytohormonal research and stress physiology, this study seeks to elucidate the fundamental processes through which SA enhances salt tolerance. The identification of the Jules variety as particularly resilient across both developmental stages has significant practical implications for breeding programs and agricultural practices, ultimately contributing to sustainable agriculture in increasingly saline environments. This research not only aims to strengthen food security but also offers practical implications for breeding programs and agricultural practices, thus contributing to sustainable agriculture in increasingly saline environments [36].

Results

Germination and seedling growth

A statistical analysis using analysis of variance (ANOVA) was performed on various seedling traits under conditions of salinity stress and SA pretreatment. The results indicated statistically significant differences across the treatment groups (Table S1).

Salinity stress significantly reduced germination percentage and rate, root and seedling length, and the fresh weight of roots and shoots. Conversely, SA pretreatment mitigated these negative effects, resulting in improved traits. Specifically, genotype 177 exhibited the highest germination percentage across all salinity levels, while genotypes 250, 201, and 266 showed significant declines under higher salinity conditions.

The study also outlines the impact of NaCl and SA treatments on P. vulgaris, detailing the effects on biochemical assays (including CAT, GPX, APX, PPO, and total protein content) and various physiological traits (such as seed germination, seedling growth, and water content) (Fig. 1). The effect of salinity stress and SA treatment on germination percentage is illustrated in Fig. 2, highlighting that SA pretreatment significantly enhances germination under saline conditions.

Fig. 1

Effects of NaCl and SA treatments on P. vulgaris

Fig. 2

The effect of salinity stress and SA treatment on germination percentage. This figure shows the impact of varying salinity levels (0, 100, and 200 mM NaCl) and SA treatments (0, 0.5, and 1 mM) on the germination percentage of different plant cultivars and genotypes. Salinity stress reduced germination, while SA improved it. Genotype 177 had the highest germination percentage across all salinity levels. Mean comparisons are indicated by letters using Duncan’s test

Germination rate analysis

Germination rate, a crucial measure of seedling emergence, was inversely proportional to salinity stress levels. The results indicated a genotype-specific response to increasing salinity, with notable variations in germination rate. Figure 3 demonstrates the impact of salinity stress and SA treatment on the germination rate, showing that SA pretreatment significantly enhances the germination rate, effectively mitigating the detrimental effects of salinity.

Fig. 3

The effect of salinity stress and SA treatment on germination rate. This figure shows the impact of different salinity levels (0, 100, and 200 mM NaCl) and SA treatments (0, 0.5, and 1 mM) on the germination rate of various plant cultivars and genotypes. SA treatment significantly increased germination rate under salinity stress. Genotype 294 had the highest germination rate, while genotypes 201 and 266 had the lowest under higher salinity levels. Mean comparisons are indicated by letters using Duncan’s test

Fresh and dry weight analysis

Salinity stress significantly reduced fresh and dry weights of roots and shoots, whereas SA pretreatment improved these traits. The extent of trait reduction and subsequent recovery following SA application varied significantly among different cultivars (Table S2).

Genotype 284 showed the highest increase in fresh root weight with 1 mM SA pretreatment, while genotypes 167 and 250 showed the lowest. Fresh shoot weight was highest in genotype 269 with 1 mM SA pretreatment and lowest in genotypes 250 and 167 with distilled water pretreatment (Fig. 4).

Fig. 4

The effect of salinity stress and SA pretreatment on fresh root weight. This figure shows the impact of different salinity levels (0, 100, and 200 mM NaCl) and salicylic acid (SA) treatments (0, 0.5, and 1 mM) on the fresh root weight of various plant cultivars and genotypes. Genotype 284 showed the highest increase in fresh root weight with 1 mM SA pretreatment, while genotypes 167 and 250 showed the lowest. Mean comparisons are indicated by letters using Duncan’s test

Principal Component Analysis (PCA)

The PCA revealed that over 83% of the total variance in the data was explained by the first two principal components. The first component, accounting for 60.6% of the variance, was positively correlated with all germination traits, including germination percentage, germination rate, fresh and dry weight of seedlings and hypocotyls, and their lengths, as well as with GPOX, CAT, APX, and PPO enzyme activities, carotenoids, chlorophyll a and b, proline, K⁺, and the K⁺/Na⁺ ratio. Conversely, it was negatively correlated with total protein, MDA, and Na⁺ levels. Genotypes positioned on the right side of the biplot were considered more tolerant to salinity based on these traits. The second component, accounting for 22.7% of the variance, showed positive correlations with root length, fresh and dry weight, shoot dry weight (Shoot DW), total protein, CAT, GPOX, and PPO enzyme activities, and chlorophyll a. However, it was negatively correlated with germination percentage and rate, seedling length, fresh weight of both the shoot and seedling, APX activity, carotenoids, chlorophyll b, total chlorophyll, MDA, proline, Na⁺, K⁺, and the K⁺/Na⁺ ratio. Genotypes below the horizontal line in the biplot were deemed preferable for salinity tolerance.

Consequently, genotypes were classified into different tolerance groups: genotypes 284 and Jules were semi-tolerant, genotypes 217, 177, 294, and 269 were tolerant, genotypes 201 and 167 were sensitive, and genotypes 266 and 250 were semi-sensitive, as depicted in Fig. 5.

Fig. 5

Biplot of PCA based on the first two components. This figure illustrates the PCA of various genotypes, explaining over 83% of the total variance in the data. The genotypes are classified into different tolerance groups based on their positions in the biplot: semi-tolerant (284 and Jules), tolerant (217, 177, 294, and 269), sensitive (201 and 167), and semi-sensitive (266 and 250)

Phase two results, analysis of physiological and biochemical traits under SA pretreatment and salinity stress

Variance analysis indicated a significant impact of salinity stress on physiological traits, Na⁺ and K⁺ content, and the K⁺/Na⁺ ratio. SA pretreatment positively influenced most of these traits.

Relative Water Content (RWC)

RWC decreased with increasing salinity levels across all genotypes. The highest RWC at 0 mM salinity was observed in genotype 201, while the lowest at 200 mM salinity was in genotype 177. The Jules variety and genotypes 167 and 201 exhibited less pronounced reductions in RWC compared to other genotypes, as depicted in Fig. 6.

Fig. 6

RWC of plants under salinity stress. Genotype 201 showed the highest RWC at 0 mM salinity, while genotype 177 had the lowest at 200 mM. Jules, 167, and 201 exhibited smaller reductions in RWC under stress. Stress levels: 0 (control), 100, and 200 mM NaCl. Each treatment was replicated three times (Letters indicate Duncan’s test mean comparisons)

Electrical Conductivity (EC)

Salinity stress increased EC, indicating greater ion leakage and cellular membrane damage. SA pretreatment moderated this effect, reducing the extent of EC increase. The highest EC at 200 mM NaCl was in genotype 269, while the lowest at 0 mM NaCl was in genotype 167. Variance analysis reveals that genotypes 167 and 201, followed by the Jules variety, exhibit a less steep increase in EC with rising salinity stress, indicating lesser damage. In contrast, genotypes 177 and 269 show a more pronounced increase in EC with elevated salinity levels.

Chlorophyll Content (SPAD)

Variance analysis of chlorophyll content data reveals statistically significant primary effects on this trait. The main effects indicates that salinity stress reduces leaf greenness, with this decline intensifying as stress levels increase. Salinity stress reduced chlorophyll content, with the Jules variety showing the highest chlorophyll content and genotypes 177 and 167 the lowest. SA pretreatment helped maintain leaf greenness under salinity stress. Based on results from RWC, electrolyte leakage index (ELI), and chlorophyll content, the cultivar Jules and genotypes 167, 177, and 201 were selected for further analysis of MDA, proline, chlorophyll a, chlorophyll b, total chlorophyll, carotenoids, and other biochemical traits under pretreatment levels of 0 (distilled water) and 1 mM SA.

Malondialdehyde (MDA) content

Under non-stress conditions, the lowest MDA levels were observed in the Jules variety. The highest MDA content at 200 mM salinity was in genotype 177. At a stress level of 100 mM, the Jules variety maintained lower MDA content, indicating greater tolerance compared to other genotypes. Furthermore, the increase in MDA content in genotype 201 and the Jules variety is less than that in genotype 177, indicating that genotype 177 has experienced more damage compared to the other genotypes. SA pretreatment reduced MDA accumulation, indicating reduced lipid peroxidation.

Proline content

The results indicate that proline accumulation increases under salinity stress, with SA pretreatment contributing to this increase as stress levels rise. Specifically, the highest proline content was observed at 200 mM salinity with 1 mM SA pretreatment. Genotype 201 exhibited the greatest increase in proline content with 1 mM SA pretreatment, while genotype 177 showed the least increase with distilled water pretreatment. An ascending trend in proline accumulation with increasing salinity levels was observed across all genotypes. Notably, the Jules variety maintained higher proline levels under severe stress conditions, suggesting a robust osmotic adjustment capability.

Chlorophyll and carotenoid content

Chlorophyll a and b contents decreased with increasing salinity stress, but SA pretreatment mitigated this effect. The highest chlorophyll a content was found in the Jules variety, while the lowest was in genotype 177. SA pretreatment resulted in a less steep decline in chlorophyll a content, particularly in the Jules variety and genotype 167. Regarding chlorophyll b, the highest content was associated with the Jules variety and genotype 201 at 0 mM salinity. However, as salinity levels increased, chlorophyll b content decreased across all genotypes, with the lowest levels observed in genotypes 177 and 201 at 200 mM salinity. The Jules variety and genotype 167 retained more chlorophyll b content under stress conditions.

Carotenoid content also decreased with increasing salinity stress. The highest carotenoid content was found in the Jules variety and genotype 201 at 0 mM salinity, with no significant difference between genotypes at 200 mM salinity. At 100 mM salinity, genotype 167 exhibited lower carotenoid content than others, but the rate of decrease in carotenoid content with increasing salinity levels was less steep in genotype 167, suggesting it preserves more carotenoid content and shows greater tolerance to increasing salinity stress.

Sodium (Na⁺) content

SA pretreatment at 0.5 and 1 mM levels reduced Na⁺ uptake in the leaves across different genotypes, although the extent varied. Genotype 201 showed a significant decrease in Na⁺ absorption with SA pretreatment, while the highest Na⁺ content was observed in genotype 201 at 200 mM salinity. Conversely, the lowest Na⁺ content was found in genotype 177 and the Jules variety at 0 mM salinity. Overall, increased salinity levels led to higher leaf Na⁺ content across all genotypes, but not uniformly. Genotypes 201 and 177 exhibited a steeper increase in Na⁺ content at 200 mM salinity compared to genotype 167 and the Jules variety. At 200 mM salinity, genotypes 201 and 177 had the highest leaf Na⁺ content, with no significant difference between genotype 167 and the Jules variety. However, at 100 mM salinity, genotype 201 had the highest Na⁺ content, with no significant differences among the other genotypes and the Jules variety.

Potassium (K⁺) content

The data demonstrate that leaf K⁺ content decreases as salinity levels rise from 0 to 200 mM. SA pretreatment had varying impacts on K⁺ retention across different genotypes. For instance, the Jules variety exhibited increased K⁺ retention with SA pretreatment, while genotypes 177 and 201 showed a decrease, and genotype 167 did not experience a significant effect. As salinity levels increased, a downward trend in leaf K⁺ retention was observed across all genotypes. The lowest K⁺ content at 200 mM salinity was found in genotypes 177 and 167, while the highest K⁺ content at 0 mM salinity was associated with the Jules variety and genotype 201. Under 200 mM salinity, genotype 201 maintained the highest K⁺ content, followed by the Jules variety. Although genotype 201 retained more K⁺ in its leaves, it is essential to consider the K⁺/Na⁺ ratio to understand which genotype has greater tolerance and has sustained less damage. The balance between these two ions is crucial for assessing stress tolerance, as the content of Na⁺ or K⁺ alone does not indicate plant tolerance to stress.

Potassium to sodium ratio (K⁺/Na⁺)

The K⁺/Na⁺ ratio decreased with increasing salinity levels. SA pretreatment did not significantly alter this ratio under stress conditions. The highest K⁺/Na⁺ ratio was found in the Jules variety at 0 mM salinity, while the lowest ratios at 200 mM salinity were observed in genotypes 167, 177, and 201. The decline in the K⁺/Na⁺ ratio was not consistent across all genotypes; for example, in genotype 167, the ratio initially increased with rising stress levels, although this was not statistically significant, and then decreased as stress intensified. Overall, the Jules variety maintained the highest K⁺/Na⁺ ratio, suggesting better ion homeostasis and less damage by retaining more K⁺ relative to Na⁺ uptake compared to other genotypes. Regarding the impact of SA pretreatment and salinity on the K⁺/Na⁺ ratio, the analysis indicates that at 0 mM salinity, pretreatment led to a reduction in this ratio. However, at 100 and 200 mM salinity levels, there was no significant difference between pretreatment with SA and distilled water.

Biochemical traits

Total protein content

Biochemical changes in plants under stress often involve alterations in total protein concentration due to the breakdown or inhibition of certain proteins and the redirection of cellular energy towards producing stress-specific proteins. Our studies have shown that increasing levels of salinity stress lead to a decrease in total protein content in plant leaves. However, SA pretreatment has somewhat mitigated this effect by increasing total leaf protein content, particularly in genotype 201. The highest protein content was observed in genotypes 177 and 201 at 0 mM salinity stress, while the lowest was at 200 mM salinity stress. This decrease in protein content was not uniform across all genotypes; genotype 167 showed no significant decrease in total leaf protein content when stress levels increased from 100 to 200 mM. In genotype 177, a significant decrease in protein content was observed from 0 to 100 mM salinity, but no further reduction was noted as stress levels rose to 200 mM. A similar pattern was observed for the Jules variety.

Proteins accumulated under salinity stress serve as a nitrogen source for reuse by the plant, aiding in osmotic regulation. Some proteins induced by salinity are cytoplasmic, potentially altering the cytoplasm's viscosity. Plant proteins may increase or decrease in response to salinity stress, and sometimes new proteins are synthesized, which can be crucial for plant growth and development under such conditions. Overall, SA pretreatment played a significant role in maintaining higher total protein content, especially in genotype 201, highlighting its potential in enhancing salinity stress tolerance.

Ascorbate Peroxidase (APX) activity

Salinity stress has been observed to increase the activity of the enzyme APX, while SA pretreatment has led to a decrease in its activity in the studied plants. Specifically, APX activity significantly decreased in the Jules variety and genotype 201 under SA pretreatment, but not in genotypes 167 and 177. As stress levels rose from 0 to 100 mM, APX activity increased, reaching a peak in the Jules variety. However, with more intense stress, the activity level decreased compared to the 100 mM stress level, but remained significantly higher than the control level. The highest APX activity at 200 mM stress was observed in the Jules variety, with no significant difference among other genotypes at this level. At 100 mM stress, genotype 177 showed the highest APX activity after the Jules variety, followed by genotype 201, while genotype 167 exhibited the lowest activity overall.

The decrease in APX activity after the 100 mM level suggests that the enzyme was not able to eliminate ROS produced under salinity stress as effectively, allowing salinity stress at this level to cause damage to plant cells. Another reason for the decrease in APX activity could be the plant's preference to allocate more energy towards growth and development under osmotic stress, thereby preventing the overproduction of this enzyme. This complex response indicates that the plant balances enzyme production against other physiological needs under varying levels of stress. Under both drought and salinity stresses, the plant's strategy for enzyme production varies, highlighting the intricate mechanisms of stress tolerance.

Catalase (CAT) activity.

Salinity stress increases the activity of the CAT enzyme, while SA pretreatment at 200 mM salinity levels leads to a decrease in this activity. The impact of SA pretreatment on CAT activity varies among genotypes. For instance, it caused a considerable decrease in genotypes 167 and 201. The trend of CAT enzyme activity under salinity stress resembles that of APX, initially increasing with rising stress levels and then decreasing. However, overall CAT activity at 200 mM salinity remains higher compared to the control level at 0 mM salinity.

The highest CAT enzyme activity at 100 mM salinity stress was observed in the Jules variety, which also showed the highest enzyme activity among all genotypes at 200 mM salinity. Following the Jules variety, genotype 167 had the highest CAT activity, with no significant difference between genotypes 177 and 201. The decrease in CAT activity after the 100 mM level suggests that the enzyme may not effectively eliminate ROS produced under salinity stress, potentially leading to cellular damage. Another reason for the decrease in CAT activity could be that under osmotic stress, the plant may prioritize growth and development over the production of this enzyme. However, the plant may increase the production of this enzyme at certain stress levels, while at others, it reduces its activity, indicating a complex adaptive response to varying levels of stress.

Glutathione Peroxidase (GPOX) activity

Salinity stress has been observed to increase GPOX enzyme activity at 100 and 200 mM levels. However, SA pretreatment reduces GPOX activity at 200 mM salinity levels. The impact of SA pretreatment on GPOX activity varies among genotypes, causing a decrease in the Jules variety and genotype 177, while leading to a significant increase in genotypes 167 and 201.

The interaction between salinity stress and genotype shows that salinity stress increases GPOX activity across all genotypes, but the rate of this increase is not uniform. In genotype 177, the activity initially rises but does not change significantly as stress levels intensify from 100 to 200 mM. The highest GPOX activity at 200 mM salinity is associated with genotype 167 and the Jules variety, with the lowest observed in genotype 177. Overall, the Jules variety exhibits the highest GPOX activity, suggesting greater tolerance to salinity stress, while genotype 177 shows the least activity.

Polyphenol Oxidase (PPO) activity

Salinity stress significantly increases the activity of the enzyme PPO, while SA pretreatment leads to a decrease in its activity. This reduction is particularly noticeable in genotype 177, whereas SA does not significantly affect other genotypes. Salinity stress increases PPO activity differently across various genotypes. For instance, in the Jules variety, enzyme activity increases with each level of stress, peaking at 200 mM. However, in genotype 177, although enzyme activity initially increases with rising stress levels, it decreases as the stress intensifies. A similar pattern is observed in genotype 167.

Correlation analysis

The correlation analysis, conducted using a heatmap generated by R software, revealed several significant relationships among the measured variables. Specifically, Shoot DW exhibited a strong positive correlation with seedling length, shoot length, root length, total fresh weight (Total FW), and total dry weight (Total DW). Total DW showed a high correlation with root fresh weight (root FW), seedling length, shoot length, root length, and Total FW. Total FW was highly correlated with seedling length, shoot length, and root length. Root length demonstrated a strong correlation with seedling length and shoot length. Shoot length was highly correlated with seedling length (Fig. 7). These findings indicate that the growth parameters are interrelated, with shoot DW, total DW, and total FW being particularly influential in the overall growth dynamics.

Fig. 7

Correlation analysis of growth parameters. This figure shows a heatmap of the correlation analysis among various growth parameters. Notable positive correlations were found, particularly among seedling length, shoot length, root length, Total FW, and Total DW. These correlations suggest significant interrelationships and influence on overall growth dynamics

Discussion

Salinity stress is known to disrupt homeostasis in plants, leading to osmotic stress, ionic toxicity, and oxidative damage [37, 38]. Our findings corroborate previous studies indicating that salinity stress reduces vital physiological parameters such as chlorophyll content, K⁺ levels, and overall protein content, while increasing Na⁺ uptake and ROS production, as evidenced by elevated MDA levels and electrolyte leakage [39, 40]. The application of SA, a phenolic phytohormone, has been shown to induce systemic acquired resistance and enhance antioxidant defense mechanisms in plants [41]. In our study, SA pretreatment effectively improved the stress tolerance of common bean genotypes, as reflected in enhanced germination rates, increased chlorophyll and proline contents, and higher total protein levels under salinity stress. These improvements suggest that SA may modulate the expression of genes involved in stress responses, leading to the activation of physiological pathways that mitigate the detrimental effects of salinity.

SA is synthesized primarily through the isochorismate synthase (ICS) and phenylalanine ammonia-lyase (PAL) pathways and can be modified into various active and inactive forms [42, 43]. SA's crosstalk with ABA and jasmonic acid (JA) can be either synergistic or antagonistic, depending on factors such as species, growth stage, and stress type [22]. It maintains ionic balance, reduces oxidative damage, and improves water-use efficiency through its interactions with other phytohormones. SA's signaling involves NPR1 and redox changes. SA modulates antioxidant defense systems, photosynthesis, osmolyte production, and ion homeostasis under stress, interacting synergistically and antagonistically with other phytohormones like auxin, gibberellin, JA, ABA, and ethylene. Future research should focus on omics techniques, functional genomics, and biotechnological applications to further understand and harness SA's potential in enhancing crop stress tolerance [44].

Interestingly, the study revealed genotypic differences in response to salinity stress and SA pretreatment. The Jules variety, in particular, demonstrated superior stress tolerance, possibly due to its genetic makeup that confers an efficient antioxidant system and ion regulation mechanisms. This variety's ability to maintain higher levels of chlorophyll and proline, along with lower MDA content, suggests a robust defense against ROS and better osmotic adjustment capabilities. The decrease in antioxidant enzyme activities after reaching a certain stress threshold indicates that there might be a limit to the protective capacity of these enzymes under severe stress conditions. This finding aligns with the concept that stress tolerance in plants is a complex trait influenced by multiple physiological and molecular factors, and that the effectiveness of defense mechanisms can be overwhelmed by extreme stress levels.

The study highlights two major insights: (1) seed priming induces persistent physiological changes at the cellular level, leading to sustained stress tolerance, and (2) specific priming agents activate distinct defense pathways, offering broad resistance to abiotic and biotic stressors. The activation of antioxidant systems and the accumulation of secondary metabolites are convergent defense mechanisms shared across different stress types [45]. Mechanistic differences between non-primed and primed plants have been observed in various studies. Primed plants exhibit enhanced germination rates, improved root system development, and increased drought tolerance compared to non-primed plants. Additionally, primed plants show long-term somatic memory and specific epigenomic changes that contribute to their stress tolerance. Comparative transcriptional profiling of primed and non-primed rice seedlings under submergence stress revealed that primed plants had a higher number of differentially expressed genes involved in secondary metabolism, development, and stress response pathways [46].

Submergence stress significantly reduced germination and seedling growth in non-primed rice. However, selenium and SA priming alleviated these negative effects, improving growth parameters. Transcriptome analysis revealed that primed plants exhibited differential expression of numerous transcripts and over-represented genes involved in various metabolic and stress response pathways [46]. Primed plants respond more quickly and cost-effectively to stress by activating defense mechanisms proportional to stress intensity, thereby conserving resources and maintaining productivity without over-allocating resources to stress tolerance. Priming shortens the lag phase during germination, increases DNA repair and enzyme activation rates, and enhances metabolite accumulation necessary for germination.

Primed plants demonstrate improved seedling emergence, growth, vigor, and productivity due to enhanced imbibition capacity and metabolic processes, stabilized membrane potential, and upregulated genes for gibberellic acid biosynthesis and photosynthesis. Priming reprograms antioxidant and polyamine metabolism genes, enhancing salt stress adaptability and improving DNA repair and protein synthesis [47].

The study found that priming lentil seeds with SA improved seedling percentages and growth under salinity stress, particularly in the tolerant variety (IPL-316), due to better photoactivation, antioxidant availability, increased chlorophyll and sugar content, and reduced MDA content. Overall, priming mitigated the negative effects of salinity stress by enhancing various physio-biochemical parameters and improving starch metabolism and membrane stability [48]. Seed priming enhances submergence tolerance by improving starch metabolism, energy production, antioxidant defense, and cell processes, though the specific mechanisms vary by priming agent. This crosstalk between signaling pathways enhances the plant's ability to maintain ionic balance, reduce oxidative damage, and improve water-use efficiency.

The results of this study contribute to the growing body of knowledge on the physiological and biochemical responses of P. vulgaris genotypes to salinity stress and the ameliorative effects of SA pretreatment. Differences between tolerant and susceptible plants have also been documented. Tolerant genotypes possess fewer differentially expressed genes (DEGs) but have higher proportions of upregulated DEGs, which contribute to their stress tolerance. Tolerant plants exhibit significant differences in root system architecture, lignin content, and expression of defense-related genes compared to susceptible plants. For example, tolerant olive cultivars showed increased root lignin content and root membrane permeability after inoculation with Verticillium dahliae, which were not observed in susceptible plants. Additionally, tolerant rice cultivars displayed a higher number of DEGs related to cell surface pattern recognition receptors, Ca²⁺ ion signaling pathways, and the Mitogen-Activated Protein Kinase (MAPK) cascade, which play positive roles in stress response [49, 50].

Crosstalk among plant growth regulators and signaling molecules during biotic and abiotic stresses involves complex networks of regulatory pathways mediated by phytohormones and molecular regulators. The synergistic and antagonistic interactions between stress-responsive hormones and their coordination with regulators fine-tune the defense response with high specificity. For instance, brassinosteroids (BRs) play a crucial role in stress management by modulating gene expression, protein accumulation, and phytohormone content in response to drought. Presenting direct evidence of such crosstalk in the study would strengthen the conclusions and provide a more comprehensive understanding of the mechanisms involved.

Research discusses how SA and its derivatives activate plant defense responses against both abiotic and biotic stresses, enhancing osmotic homeostasis, mineral nutrition uptake, and ROS scavenging capacity. These studies collectively highlight the versatile role of SA in plant stress tolerance, supporting its potential application in sustainable agriculture. Research emphasizes the significance of SA in improving plant abiotic stress tolerance through SA-mediated control of major plant metabolic processes such as photosynthesis, metabolite accumulation, redox homeostasis, and gene regulation. This further supports the potential of SA in enhancing plant resilience under various stress conditions.

This paper highlights SA's role in mitigating cold stress in plants by modulating gene expression and activating physiological pathways that enhance cold tolerance. SA influences various plant processes and interacts with key signaling pathways to increase antioxidant and cold-responsive protein production. However, optimal SA concentration is crucial, as high levels can inhibit seed germination; future research should focus on the detailed molecular mechanisms and cross-talk with other phytohormones. SA plays a crucial role in the transcriptional reprogramming during plant defense responses to biotic and abiotic stress. SA-mediated mechanisms control the transcription of various defense genes in a spatio-temporal manner. SA interacts with ROS and glutathione (GSH) in stressed plants. Redox modifications of regulators and co-regulators involved in SA-mediated transcriptional responses control the temporal patterns of gene expression in response to stress. These redox sensors are coordinated with the dynamics of cellular redox changes during the defense response to biotic and abiotic stress.

Our results underscore the importance of SA in enhancing plant resilience to salinity stress. According to further research, SA plays a crucial role in regulating ion transport processes during salt stress, controlling Na⁺ entry into roots, enhancing H + ATPase activity, preventing stress-induced K⁺ leakage, and increasing K + concentration in shoots. These findings are consistent with our results, which also emphasize the importance of SA in enhancing plant resilience to salinity stress.

Additionally, SA enhances salinity stress tolerance in plants through various physiological and biochemical mechanisms. Research highlights how SA contributes to the regulation of ion homeostasis, antioxidative defense systems, and osmotic balance under saline conditions, emphasizing the complex interplay between SA and other signaling molecules in enhancing plant tolerance to salinity stress. Furthermore, SA induced multiple stress tolerances in bean plants, including heat, chilling, and drought stresses. Seeds imbibed in aqueous solution of SA displayed enhanced tolerance to these stresses. This was attributed to the signaling role of SA, which led to the expression of stress tolerance rather than a direct effect [51]. The induction of multiple stress tolerances by exogenous application of SA and its derivatives has significant practical applications in agriculture, horticulture, and forestry.

Material and methods

Seed germination and priming

A factorial experiment was conducted under a Completely Randomized Design (CRD) with three replications to evaluate the effects of SA pretreatment on the germination and early growth of bean seedlings under salinity stress. The experimental design included an 11-h priming period at a constant temperature of 25 °C, targeting the third stage of root emergence.

Seed preparation and priming

Uniform bean seeds from various genotypes and cultivars were obtained from the Gene Bank of the Department of Agronomy and Plant Breeding at the University of Tehran as indicated in Table 1. These particular genotypes and cultivars were chosen based on their previously reported varying responses to salinity stress, allowing for a comprehensive evaluation of SA pretreatment effects. The seeds were stored at 25 °C for a period of two weeks to ensure uniform viability. Prior to priming, the seeds were disinfected using a 5% sodium hypochlorite solution for 2 min to eliminate potential pathogens [52, 53]. After disinfection, the seeds were thoroughly rinsed with deionized water and air-dried to reach laboratory temperature conditions.

Table 1 Details of the genotypes used for this study

The seeds were then primed in petri dishes under controlled light exposure, which provided a consistent light source to avoid variability. The priming was conducted at a constant temperature of 25 °C. A 0.2% carboxin-thiram solution (1:500 dilution ratio) was applied to the seeds before placing them into the culture dishes to prevent fungal growth [54]. The seed-to-solution ratio used for priming was 1:5 (w/v), ensuring adequate contact between seeds and the priming solution.

Salicylic acid pretreatment

A SA solution was prepared by dissolving 0.0138 g of SA in 2 mL of ethanol, followed by dilution to 1 L with distilled water to achieve a concentration of 0.5 mM [55, 56]. This concentration was selected based on previous studies demonstrating its effectiveness in enhancing salinity tolerance in plants. Ten seeds from each genotype were selected for uniformity in size and shape. The seeds were then subjected to priming treatments: the experimental group was primed in the 0.5 mM SA solution, while the control group received distilled water under identical conditions. After the priming period, the seeds were air-dried back to their original moisture content before germination.

Salinity stress application

Seeds were subjected to salinity stress with NaCl solutions at concentrations of 0, 100, and 200 mM. These specific concentrations were chosen to represent a gradient of stress levels: 0 mM as the control (no stress), 100 mM as moderate stress, and 200 mM as severe stress [57]. The germination process was monitored over a 9-day period in a germinator set at a constant temperature of 25 °C, with daily observations recorded to assess germination rates and seedling growth.

Germination and morphometric analysis

The germination percentage and germination rate were calculated to evaluate the effects of salinity stress and SA pretreatment. Germination percentage was determined by counting the number of germinated seeds out of the total number of seeds placed in the petri dishes, expressed as a percentage [58]. Germination was considered successful when the root extended at least 2 mm from the seed coat. The germination rate was calculated using the formula:

$$\mathrm{Germination}=\frac{\sum\left({\mathrm G}_{\mathrm t}\times{\mathrm T}_{\mathrm t}\right)}{\mathrm T}\\$$

where Gt is the number of seeds germinated at time Tt and T is the total number of germinated seeds.

After the treatments, petri dishes were placed in a germinator set at 25 °C. The number of germinated seeds was counted daily on a cumulative basis for 9 days. After 9 days, five seeds from each genotype were randomly selected to measure seedling length, root length, shoot length, fresh weight, and dry weight of seedlings, roots, and shoots. Seedling, root, and shoot lengths were measured using Digimizer software [59]. For dry weight determination, samples were oven-dried at 60 °C for 24 h.

Selection of cultivars for further study

Six genotypes exhibiting varying degrees of salinity tolerance were identified based on criteria including DW, root length, and shoot length. Biplot analysis, derived from principal component decomposition conducted in preliminary studies, supported this selection process. The selected genotypes were transplanted into pots filled with a soil mixture of clay, sand, and decomposed leaves in a greenhouse. Initially, irrigation was the only treatment. At the fourth leaf stage, different salinity stress levels were applied to assess the impact of SA pretreatment on biochemical responses to salinity. Pre-treated seeds were transplanted, with pots arranged in randomized replicates for factorial experimentation.

Greenhouse conditions and salinity stress application

Greenhouse temperature was maintained at 25 °C with a 16-h photoperiod. Over a 14-day period, irrigation fostered seedling development to the four-leaf stage. Salinity stress was initiated two weeks post-establishment and sustained until the initial phenotypic damage appeared under severe stress. This involved administering 100 mL of saline solution to pots every other day for a fortnight.

Sample collection and preservation.

After salinity stress application, leaf samples were collected for biochemical analysis. Samples were promptly frozen in liquid nitrogen and stored at −80 °C until analysis. Physiological measurements were conducted one day before primary sampling to ensure a comprehensive dataset.

Quantitative assessment of physiological traits

Assessing plant hydration: Relative Water Content (RWC)

RWC, a crucial indicator of plant water status, was measured following the methodology of [60]. Healthy leaves from the central shoot were selected and excised using a punch tool. The fresh weight (FW) of the samples was recorded. To achieve full saturation, the samples were submerged in distilled water for 24 h. After surface drying, the turgid weight (TW) was measured. The samples were then dried in an oven at 72 °C for 72 h to determine the dry weight (DW). RWC was calculated using the formula [61]:

$$\mathrm{RWC}\left(\%\right)=\frac{(\mathrm{FW}-\mathrm{DW})}{(\mathrm{TW}-\mathrm{DW})}\times100$$

Electrolyte Leakage Index (ELI)

The Electrolyte Leakage Index (ELI), which reflects cellular integrity in plant tissues under stress, was assessed using leaf samples from central shoots [62]. Samples were placed in falcon tubes containing 15 cc of distilled water and shaken at 25 °C for 24 h. Initial electrolyte conductivity (EC₁) was measured, followed by boiling the samples at 95 °C for one hour to determine final conductivity (EC₂). ELI was calculated as:

$$ELI\left(\%\right)=\frac{EC1}{EC2}\times100$$

Chlorophyll content estimation

Chlorophyll content, indicative of leaf greenness and overall plant health, was measured using the SPAD 502-MINOLTA chlorophyll meter across different treatment levels. This meter estimates chlorophyll concentration by measuring transmittance of light at specific wavelengths [63]

Photosynthetic pigments quantification

Chlorophyll and carotenoid content were quantified using the acetone extraction method by [64]. Absorbance readings at specific wavelengths were used to calculate the concentrations, using the following equations [65]:

$$\mathrm{Chl}\;\mathrm a\left(\mathrm{mg}/\mathrm{ml}\right)=12.25\times{\mathrm A}_{663.2}-2.79\times{\mathrm A}_{646.8}$$

$$\mathrm{Chl}\;\mathrm b\left(\mathrm{mg}/\mathrm{ml}\right)=21.50\times{\mathrm A}_{646.8}-5.10\times{\mathrm A}_{663.2}$$

$$\mathrm{Total}\;\mathrm{Chl}\left(\mathrm{mg}/\mathrm{ml}\right)=\left(7.15\times{\mathrm A}_{663.2}\right)+\left(18.71\times{\mathrm A}_{646.8}\right)$$

$$\mathrm{Carotenoids}\left(\mathrm{Cx}+\mathrm c\right)\left(\mathrm{mg}/\mathrm{ml}\right)=\frac{\left(1000\times{\mathrm A}_{470}\right)-\left(1.82\times\mathrm{Chl}\;\mathrm a\right)-\left(85.02\times\mathrm{Chl}\;\mathrm b\right)}{198}$$

Ionic balance assessment

The levels of Na⁺ and K⁺, along with their ratio (K⁺/Na⁺), were quantitatively assessed to determine the ionic balance in plant tissues under salinity stress [66]. Plant tissues were first dried at 70 °C for 48 h and then ground to a fine powder. This powder was ashed in a muffle furnace at 550 °C for 6 h. The resulting ash was then treated with 1 M hydrochloric acid (HCl) to extract the elemental contents.

The extracted samples were analyzed for Na⁺ and K⁺ concentrations using flame photometry. For accurate and reproducible measurements, the flame photometer was calibrated using standard solutions of Na⁺ and K⁺. The specific wavelengths used for the determination were 589 nm for Na⁺ and 766.5 nm for K⁺. Calibration curves were prepared by plotting the intensity readings against known concentrations of standard solutions.

These steps ensured precise quantification of Na⁺ and K⁺ levels in the plant tissues, providing essential data for assessing the ionic balance and the effectiveness of salicylic acid pre-treatment in enhancing salt tolerance [67].

Lipid peroxidation quantification

Lipid peroxidation was quantified using the thiobarbituric acid reactive substances (TBARS) method [68]. Trichloroacetic acid and thiobarbituric acid were used for MDA detection, which is a key indicator of oxidative stress. Leaf samples were homogenized in a 0.1% trichloroacetic acid solution and centrifuged. The supernatant was mixed with thiobarbituric acid and incubated at 95 °C for 30 min. After cooling, the absorbance was measured at 532 nm. MDA levels were quantified using a standard curve prepared with known concentrations of MDA.

$$\mathrm{MD}\left(\%\right)=\left[\mathrm{Absorbance}\;\mathrm{at}\;532\;\mathrm{nm}\right]-\left[\mathrm{Absorbance}\;\mathrm{at}\;600\;\mathrm{nm}\right]\times\mathrm{Factor}$$

$$\mathrm{MDA}\left(\%\right)\left(\frac{\mathrm{nmol}}{\mathrm{ml}}\right)=\frac{\mathrm{Absorbance}\;\mathrm{at}\;532\;\mathrm{nm}-\mathrm{Absorbance}\;\mathrm{at}\;600\;\mathrm{nm}}{1.56\ast10^5}$$

Proline accumulation assessment

Proline content in plant tissues was determined using a modified method [69]. Proline was extracted using 3% sulfosalicylic acid and then reacted with acid ninhydrin. The reaction mixture was heated at 100 °C for 1 h, and proline was subsequently separated using toluene. Absorbance was measured at 520 nm. To ensure accurate quantification and avoid variability in the results, a proline standard was used for calibration. Calibration was performed using a series of proline standards to create a standard curve, plotting absorbance against known concentrations of proline. This calibration curve was used to calculate the proline content in the plant tissue samples. The proline content was calculated using the formula:

$$\text{Proline}\left(\upmu\text{mol}\;\text{proline}\cdot\mathrm{g}^{-1}\mathrm{FW}\right)=\left[\frac{\upmu\;\text{proline}\;\cdot\mathrm{ml}\times\mathrm{ml}\;\mathrm{toluene}}{115.5\;\upmu\mathrm{g}\cdot\upmu\mathrm{mol}-1}\right]\times\left[\frac{\mathrm g\;\text{sample}}5\right]$$

Enzymatic activity assays

Biochemical assays were performed to determine total protein content and the activities of specific enzymes. Leaf samples were homogenized in a phosphate buffer and centrifuged to obtain the supernatant, which was stored at −20 °C until analysis for CAT, GPOX, APX, and PPO.

Catalase activity assay

CAT activity was assessed at 25 °C based on the method by [70], measuring the decomposition of H₂O₂. The rate of breakdown provided a quantitative measure of CAT activity:

$$2{\mathrm H}_2{\mathrm O}_2\rightarrow2{\mathrm H}_2\mathrm O+{\mathrm O}_2$$

Standard curves were prepared using known concentrations of H₂O₂ to ensure quantifiable results.

$$\mathrm{Activity}(\upmu\mathrm{mol}\;\mathrm H2\mathrm O2/\min)=\left[\frac{(\mathrm{Abs}.\;\mathrm{initial}-\mathrm{Abs}.\;\mathrm{final})\times\mathrm{Vol}.\;\mathrm{reaction}\;\mathrm{mix}\;(\mathrm{ml})}{\mathrm{Time}\;\mathrm{reaction}\;(\min)\times\lbrack\mathrm{Protein}\;\mathrm{conc}(\mathrm{mg})\rbrack\times\mathrm{Vol}.\;\mathrm{Sample}\;(\mathrm{ml})}\right]$$

Guaiacol peroxidase activity

GPOX activity was measured at 25 °C using a modified method [71]. The assay mixture consisted of phosphate buffer, hydrogen peroxide, guaiacol, and enzyme extract. Absorbance was recorded at 470 nm over time, and activity was calculated based on the absorbance of tetraguaiacol per milligram of protein. Standard curves for guaiacol were used for accurate quantification.

$$4\mathrm{guaiacol}+4{\mathrm H}_2{\mathrm O}_2\rightarrow\mathrm{Tetraguaiacol}+8{\mathrm H}_2\mathrm O\\$$

Activity Calculation:

$$\mathrm{Activity}(\upmu\mathrm{mol}\;\mathrm{Guaiacol}/\min\;\ast\;\mathrm{mg}\;\mathrm{protein})=\left[\frac{(\mathrm{Abs}.\;\mathrm{initial}-\mathrm{Abs}.\;\mathrm{final})\times\mathrm{Vol}.\;\mathrm{reaction}\;\mathrm{mix}\;(\mathrm{ml})}{\mathrm\varepsilon\ast\mathrm{Time}\;\mathrm{reaction}\;(\min)\times\lbrack\mathrm{Protein}\;\mathrm{conc}(\mathrm{mg}/\mathrm{ml})\rbrack\times\mathrm{Vol}.\;\mathrm{Sample}\;(\mathrm{ml})}\right]$$

Ascorbate peroxidase activity

APX activity, crucial for evaluating antioxidative defense, was monitored at 25 °C following [72]. The enzyme catalyzes the conversion of H₂O₂ into water using ascorbate as an electron donor. The decrease in absorbance at 290 nm reflects APX activity. Standard curves for ascorbate were used to ensure precise quantification.

$$\mathrm{Ascorbate}\;({\mathrm C}_6{\mathrm H}_8{\mathrm O}_6)+{\mathrm H}_2{\mathrm O}_2\rightarrow\mathrm{Dehydroascorbate}\;({\mathrm C}_6{\mathrm H}_6{\mathrm O}_6)\;+\;2{\mathrm H}_2\mathrm O$$

Activity Calculation:

$$\mathrm{Activity}(\upmu\mathrm{mol}\;\mathrm{Ascorbate}/\min\;\ast\;\mathrm{mg}\;\mathrm{protein})=\left[\frac{(\mathrm{Abs}.\;\mathrm{initial}-\mathrm{Abs}.\;\mathrm{final})\times\mathrm{Vol}.\;\mathrm{reaction}\;\mathrm{mix}\;(\mathrm{ml})}{\mathrm\varepsilon\ast\mathrm{Time}\;\mathrm{reaction}\;(\min)\times\lbrack\mathrm{Protein}\;\mathrm{conc}(\mathrm{mg}/\mathrm{ml})\rbrack\times\mathrm{Vol}.\;\mathrm{Sample}\;(\mathrm{ml})}\right]$$

Quantification of total protein using bradford assay

Bradford reagent preparation

The Bradford reagent was prepared by dissolving 10 g of Coomassie Brilliant Blue G-250 in 50 mL of 96% ethanol, followed by the addition of 100 mL of 85% orthophosphoric acid [73, 74]. The solution was stirred until the dye dissolved, then diluted to 1 L with distilled water and filtered. The reagent was stored at 4 °C, protected from light.

Stock solution preparation

A stock solution of bovine serum albumin (BSA) was prepared by dissolving 50 mg of BSA in distilled water to achieve a concentration of 1 mg/mL.

Standard curve establishment

BSA standards were prepared at concentrations ranging from 0 to 1000 µg/mL through systematic dilution of the stock solution. Absorbance readings were recorded to establish a calibration curve.

Protein concentration determination

Protein levels in the samples were determined using a plate reader. A tube containing the sample extract was retrieved from −20 °C. In a 96-well plate, 200 µL of Bradford reagent was mixed with 10 µL of the sample extract. After a 20-min incubation at ambient temperature, absorbance was measured at 595 nm [75].

Statistical analysis

Statistical analyses were conducted using R and SAS 9.4 software to ensure rigorous examination of the data. The morphometric analysis, including root and shoot lengths and biomass measurements, was performed in triplicate for each genotype to ensure statistical validity, with three biological replicates considered for each genotype, each consisting of five seedlings. Fresh weights of seedlings were measured immediately after harvesting, and dry weights were determined post-drying. Normality tests were performed on the collected data using Minitab software to confirm appropriate data distribution for analysis.

ANOVA was utilized to compare the effects of SA pretreatment and salinity stress on various measured parameters within a factorial design in a completely randomized framework. When significant differences were detected by ANOVA, post-hoc tests were conducted using Duncan's Multiple Range Test (DMRT) to handle multiple comparisons and identify specific differences between treatment groups. The threshold for statistical significance was set at p < 0.05, with significant differences indicated by letters in the graphs, representing multiple comparison tests at the 5% significance level.

Three-way ANOVA was performed to analyze interactions among salinity stress, SA pretreatment, and genotype. Hypothesis testing was based on the premise that SA pretreatment would significantly enhance salinity tolerance across different genotypes. Correlation analysis was conducted to determine relationships between variables such as shoot DW, seedling length, shoot length, seedling length, total FW, total DW, germination rate, and percentage. Spearman's correlation coefficient was used due to the non-parametric nature of the data. A heatmap was generated using R software to visually represent significant correlations among the measured parameters, providing an intuitive overview of data relationships.

Graphs were plotted using Excel 2016 to visually present the data. This comprehensive statistical analysis allowed for the identification of significant differences between treatments and genotypes, supporting the evaluation of SA pretreatment effectiveness in enhancing salinity tolerance and facilitating the selection of genotypes for further studies.

Conclusion

This study on the effects of salinity stress and SA pretreatment on P. vulgaris genotypes has provided valuable insights into the adaptive mechanisms essential for plant survival and productivity in saline environments. Our research demonstrated that salinity stress negatively impacts various physiological and biochemical parameters, including germination rate, chlorophyll content, electrolyte leakage, and enzyme activities. However, SA pretreatment mitigates these adverse effects, enhancing the plant's ability to cope with salinity stress.

The study revealed that SA pretreatment improved germination traits, increased chlorophyll and proline content, and maintained higher levels of total protein, which are critical indicators of enhanced stress tolerance. Additionally, SA pretreatment modulated ion balance by reducing Na⁺ uptake and preserving a favorable Na⁺/K⁺ ratio, crucial for osmotic adjustment under saline conditions. Significant genotypic variation in response to both salinity stress and SA pretreatment was evident, with certain genotypes like the Jules variety showing remarkable resilience. This genotype maintained higher levels of vital biochemical constituents and exhibited less damage in terms of MDA content and electrolyte leakage, suggesting its potential as a robust candidate for cultivation in salt-affected regions.

Based on the results, the Jules variety demonstrated superior salt tolerance through a multifaceted physiological mechanism. This genotype maintained higher RWC and exhibited increased membrane stability, as evidenced by lower MDA accumulation, indicating reduced lipid peroxidation and cellular damage. The Jules variety also preserved higher chlorophyll and carotenoid content under salinity stress, suggesting enhanced photosynthetic capacity and stress resilience. Additionally, it showed significant proline accumulation, contributing to osmotic adjustment and protection against osmotic stress. Ion homeostasis was effectively maintained in the Jules variety, with a higher K⁺ to Na⁺ ratio, crucial for cellular function and stress mitigation. These combined physiological responses underpin the robust salt tolerance observed in the Jules variety, making it a promising candidate for cultivation in salt-affected regions.

In conclusion, our findings highlight the potential of SA pretreatment as a strategy to enhance salt tolerance in common bean genotypes. This approach could be pivotal for improving legume crop resilience to salinity stress, thereby contributing to agricultural sustainability and food security. The insights gained from this study can be leveraged by future breeding programs to develop new varieties with improved stress tolerance. By integrating SA pretreatment into agricultural practices, it is possible to enhance crop productivity and resilience in increasingly saline environments, ensuring food security in the face of global climate challenges.

Limitations of the study: While our study provides valuable insights, it is important to note that the experiments were conducted under controlled greenhouse and laboratory conditions. Field trials in diverse environmental conditions are necessary to validate the findings. Additionally, the molecular mechanisms underlying the observed physiological responses require further investigation to fully understand the role of SA in enhancing salt tolerance.