Two legume species, namely A. membranaceus and M. sativa, were subjects of experimentation.

Microsoft Excel 2016 and SPSS 24.0 were utilized for statistical data analysis. A correlation model was established, and Origin 2022 was employed for creating figures. Principal component analysis (PCA) was used to obtain the magnitude of each indicator’s contribution and eigenvectors, and the data to compare the magnitude of each indicator’s influence on plant salt tolerance. The salt tolerance of the two legumes was comprehensively evaluated by membership function analysis (MFA).

The relative biomass was calculated using the following formula:

where SB is the biomass of the salt treatment group, and CB is the biomass of the control group.

The salt tolerance coefficient (ω) is the ratio of the average measured value between the salt treatment group and the control group.

The membership function value of each Comprehensive index was calculated using the following formula:

where μ (X_j) represents the membership function value of the comprehensive index of j, X_j represents the comprehensive index value of j, and X_min indicates the minimum value of j. X_max indicates the maximum value of j. The positive correlation between the indexes and salt tolerance was calculated using MFA (Formula 2). The negative correlation between the indexes and salt tolerance was calculated using MFA (Formula 3).

The weight value (W) of each composite indicator was calculated using the following formula:

where: W_j denotes the weight of the composite indicator j among all composite indicators; V_j denotes the contribution percentage of each part of the composite indicator j of the material obtained through PCA.

The salt tolerance index (D) was calculated using the following formula:

The membership function value of each comprehensive index (μ), the weight value (W) and the salt tolerance index (D) of each composite indicator were calculated with reference to the methods of Chen et al. (Chen et al., 2023).

Structural equation modeling (SEM) was employed to analyze the effects of salt stress on plant growth index, physiological index, and protective enzyme system. Before the SEM procedure, variables dimensionality reduction was conducted of by PCA. The parameters of the first principal component were then used in the SEM model. During SEM analyses, the data were fitted to the models using the maximum likelihood estimation method by comparing the model-implied variance–covariance matrix against the observed variance–covariance matrix. All SEM analyses were performed using Amos version 17.0.2 (Amos Development Corporation, Chicago, IL, USA). Model fit was assessed using the chi-square (χ²) test, comparative fit index (CFI), root square mean error of approximation (RSMEA), and goodness of fit index (GFI) (Mi et al., 2015).

With increasement of NaCl concentration, the PBN of both legume species gradually decreased (Figure 1A). The primary branch numbers of A. membranaceus and M. sativa showed no significant difference compared to the control group at NaCl concentrations of 50 mmol/L and 100 mmol/L. However, a considerable difference emerged at 200 mmol/L and 300 mmol/L NaCl concentrations (P<0.05). The variation in PBN among treatments was attributed to NaCl concentration (P<0.001). A. membranaceus exhibited the highest PH at 50 mmol/L and the lowest at 200 mmol/L (Figure 1B). The PH of M. sativa decreased with increasing concentration, showing no significant difference at 50 mmol/L compared to the control group. However, at NaCl concentrations of 100 mmol/L, 200 mmol/L, and 300 mmol/L, there were significant differences from the control group (P<0.05). The differences in PH among treatments were influenced by species (P<0.01) and NaCl concentration (P<0.001).

The LA of both plants initially increased and then decreased with rising salt concentration (Figure 1C), reaching its maximum at 50 mmol/L. At this concentration, the LA of M. sativa significantly differed from the control group (P<0.05). The LA of A. membranaceus decreased by 22.55% compared with the control group at 200 mmol/L and 300 mmol/L NaCl concentrations (P<0.05) and 31.99% (P<0.05). The LA of M. sativa decreased by 12.49% (P<0.05) and 65.96% (P<0.05) at 200 mmol/L and 300 mmol/L salt concentrations, respectively. The differences in LA among treatments were influenced by species (P<0.01), NaCl concentration (P<0.001), and their interactions (P<0.001). The LSD of A. membranaceus in all salt treatment groups was significantly lower than that in the control group (P<0.05; Figure 1D). Under all of salt treatments, M. sativa's LSD was significantly higher than that of A. membranaceus (P<0.05). At 100 mmol/L salt concentration, the LSD of M. sativa was the lowest and significantly lower than that of control group (P<0.05). At 50 mmol/L, 200 mmol/L, and 300 mmol/L salt concentrations, it was significantly higher than the control group (P<0.05). The differences in LSD among treatments were influenced by species (P<0.001), NaCl concentration (P<0.001), and their interactions (P<0.001).

The results indicate varying trends in the overall change of different legumes under different salt concentrations (Figure 2). Specifically, the aboveground biomass (AGB) of A. membranaceus initially increased and then decreased with rising salt concentration, whereas the underground biomass (UGB) showed insignificant changes. In contrast, both AGB and UGB of M. sativa gradually decreased with increasing salt concentration, significantly dropping at 50 mmol/L and 100 mmol/L concentrations (P<0.05), and exhibiting higher levels in the 50 mmol/L and 100 mmol/L treatment groups compared to the stress of 200 mmol/L and 300 mmol/L. Total biomass and AGB trends were similar between the two legume species. The R/S of A. membranaceus and M. sativa in all NaCl-treated groups was significantly lower than in the control (P<0.05), with no significant differences among various salt concentration treatment groups. The variations in AGB, UGB, total biomass, and R/S among treatments were influenced by species (P<0.001), NaCl concentration (P<0.001), and their interactions (P<0.05).

Figure 3 illustrates the different trends in the relative biomass (Formula 1) of the two legume seedlings under NaCl stress. The relative AGB of A. membranaceus was significantly higher than that of the control, except at 300 mmol/L salt concentration, while the UGB was lower than that of the control group. In contrast, the aboveground and UGB of M. sativa was consistently lower than that of the control. The trend in total relative biomass of the two species mirrored that of AGB. A. membranaceus exhibited increased total biomass under 50 mmol/L, 100 mmol/L, and 200 mmol/L salt stress (34.44%, 33.00%, and 51.17% higher than the control, respectively), but under 300 mmol/L salt stress, total biomass decreased by 20.93% compared to the control. M. sativa showed a decrease in total biomass at 50 mmol/L, 100 mmol/L, 200 mmol/L, and 300 mmol/L NaCl concentrations (24.19%, 33.21%, 65.39%, and 74.25% lower than the control, respectively).

Figure 4 reveals significant differences in the MDA content in the aboveground parts of the two legumes. Specifically, at 50 mmol/L and 100 mmol/L salt concentrations, M. sativa had a higher MDA content than A. membranaceus. At 300 mmol/L salt concentration, the MDA content of A. membranaceus was significantly higher than that of M. sativa, while the content of A. membranaceus did not significantly differ at 0–100 mmol/L salt concentration. There were notable differences in the content of MDA in the underground parts of the two plants. The MDA content of A. membranaceus showed no significant difference at 50 mmol/L and 100 mmol/L salt concentrations, but it increased exponentially at 200 mmol/L and 300 mmol/L salt concentrations. The underground MDA content of M. sativa was significantly higher at 200 mmol/L and 300 mmol/L salt concentrations than at 50 mmol/L and 100 mmol/L salt concentrations, and the trend of plant MDA content was similar above and below ground.

With an elevation in NaCl concentration, the SOD activity in the aboveground organs of A. membranaceus gradually diminishes (Figure 5A). At a concentration of 50 mmol/L, the SOD activity in the aboveground parts of plants exhibits no significant deviation from that in the control group and is notably higher than that in other salt treatment groups. M. sativa records the lowest SOD activity at a concentration of 100 mmol/L. Under diverse salt concentrations of A. membranaceus, SOD activity in the underground parts of plants progressively diminishes with the escalating salt concentration (Figure 5B), displaying no significant distinctions at 100 mmol/L, 200 mmol/L, and 300 mmol/L salt concentrations (P>0.05). The SOD activity of M. sativa roots in the treatment group surpasses that in the control group, reaching its peak at a concentration of 200 mmol/L.

POD activity in the aboveground part of A. membranaceus systematically rises with the escalating salt concentration, reaching its zenith at 200 mmol/L (Figure 5C). The POD activity of M. sativa stems and leaves gradually increases but remains significantly lower than that of the control group (P<0.05). POD activity of A. membranaceus roots is significantly lower than that of the control group at concentrations of 50 mmol/L and 100 mmol/L (P<0.05; Figure 5D). The POD activity of M. sativa roots is notably lower than that of the control group at a concentration of 50 mmol/L (P<0.05). The POD activity of M. sativa roots at 100 mmol/L and 200 mmol/L (reaching the highest value) is significantly higher than that of the control group (P<0.05).

The aboveground APX activities in all salt-treated groups of A. membranaceus are significantly higher than those in the control group (P<0.05; Figure 5E). The APX activity of stems and leaves of M. sativa is notably lower than that of the control group only at a 300 mmol/L salt concentration (P<0.05). The root APX activity of A. membranaceus is significantly higher than that of the control group at concentrations of 100 mmol/L, 200 mmol/L, and 300 mmol/L (P<0.05; Figure 5F). The root APX activity of M. sativa at concentrations of 100 mmol/L, 200 mmol/L, and 300 mmol/L is significantly lower than that of the control group (P<0.05).

The CAT activity of stems and leaves of A. membranaceus under all salt treatments was significantly higher than that of the control group (P<0.05; Figure 5G). The CAT activity reached its peak value at a 200 mmol/L salt concentration. Similarly, the CAT activity of the stems and leaves of M. sativa treated with all salt concentrations was significantly higher than that of the control group (P<0.05), with the highest values observed at 200 mmol/L concentration. Generally, after salt treatment, the CAT activity of M. sativa stems and leaves was significantly higher than that of A. membranaceus. The CAT activity of A. membranaceus root was significantly higher than that of the control group at 100 mmol/L and 200 mmol/L salt concentrations (P<0.05; Figure 5H). The maximum value was reached at the 200 mmol/L salt concentration. The root CAT activity of M. sativa under all salt concentrations was also significantly higher than that of the control group (P<0.05), reaching its peak at 200 mmol/L salt concentration.

Correlation analyses were conducted on seedling stage indexes of A. membranaceus (Supplementary Figure 1A), revealing significant associations. The PBN exhibited a noteworthy correlation with UGB, plant SOD activity, and LSD (P<0.05). Additionally, UGB showed a significant correlation with the MDA content of plants (P<0.05). LA demonstrated significant correlations with SOD activity in roots, MDA content in stems and leaves, and PH (P<0.05). The R/S was also found to be significantly correlated with LSD (P<0.05). Furthermore, plant SOD activity exhibited a significant correlation with root APX activity (P<0.05), and plant CAT activity was significantly correlated with stems and leaves APX activity (P<0.05).

In the case of M. sativa seedling stage (Supplementary Figure 1B), each index was subjected to correlation analysis. The PBN was significantly correlated with PH, biomass, MDA content, and APX activity stems and leaves (P<0.05). Biomass displayed significant correlations with plant APX activity, plant CAT activity, and plant MDA content (P<0.05). PH exhibited significant correlations with AGB, MDA content in stems and leaves, and APX activity (P<0.05). The R/S showed a significant correlation with CAT activity of the stems and leaves (P<0.05). Moreover, the MDA content, APX activity, and CAT activity of roots were significantly correlated with each other (P<0.05).

The PCA load chart depicts the correlation coefficients between the original variables and the principal components. For A. membranaceus seedlings, the first three eigenvalues were 47.38%, 26.31%, and 13.78%, respectively, resulting in a cumulative contribution rate of 87.47% (Supplementary Table 5, Supplementary Figure 2). The eigenvector of the load diagram of the PCA and the contribution rate of each principal component revealed that PBN, SOD activity in plant stems, leaves, and roots exhibited higher loads on the first principal component. The second principal component was characterized by APX activity in stems and leaves, AGB, total biomass. The third principal component was associated with CAT activity in roots, POD activity in stems and leaves, and R/S had higher loads (Supplementary Table 6).

According to the load diagram of PCA of M. sativa seedlings, the first four eigenvalues were 46.54%, 17.43%, 13.24%, and 8.80%, leading to a cumulative contribution rate was 85.63% (Supplementary Table 5, Supplementary Figures 3A, B). Therefore, the four principal components were selected as comprehensive indexes to evaluate M. sativa. According to the eigenvector of the load diagram of PCA and the contribution rate of each principal component, the load diagram indicates that root CAT activity, total biomass, UGB, had higher loads on the first principal component. POD activity in stems and leaves, R/S and LA contributed more to the second principal component. SOD activity of stems and leaves, LSD, and PH exhibited higher loads in the third principal component. POD activity in plant stems, leaves and roots and MDA activity of stems and leaves contributed more to the fourth principal component. Extracting these four principal components could effectively represent the information from all indicators, allowing the use of four new variables to replace the original eighteen variables (Supplementary Table 6). However, each load vector represents only the correlation coefficient between the principal component and the corresponding variable, not the coefficient corresponding to each index in each principal component.

The highest salt tolerance index (D) (Formula 5) values were observed at control for both legume plants, indicating that salt treatment reduced the salt tolerance of the plants (Table 1). In the four salt treatment groups, the highest D values of A. membranaceus and M. sativa occurred at concentrations of 50 mmol/L and 200 mmol/L, respectively. The salt tolerance of M. sativa was higher than that of A. membranaceus under each concentration treatment in the comprehensive evaluation.

SEM of A. membranaceus indicated a high fit (χ² = 206.859, df=62, P=0.242; Figure 6A). The results showed that salinity significantly negatively affected PBN, PH, and LA, explaining 88%, 48%, and 56% of their variances, respectively. Salinity had a significant positive effect on MDA, POD, and APX and adversely affected SOD. SOD, POD, APX, CAT, and MDA significantly influenced LSD, with MDA and CAT negatively affecting LSD and SOD, POD, and APX positively affecting LSD. Finally, salinity indirectly affected AGB through MDA, where MDA had an adverse effect on AGB, and Salinity directly negatively affected UGB. Both AGB and UGB significantly influenced R/S, with the negative effect of AGB and the positive effect of UGB together explaining 78% of the total variance of R/S.

SEM of M. sativa also demonstrated a high fit (χ² = 181.459, df=62, P=0.138; Figure 6B). Salinity adversely affected PBN and LA, explaining 68% and 53% of their variances, respectively. Salinity directly affected MDA, APX, and CAT, with significant positive effects on MDA and CAT and an adverse effect on APX. Additionally, salinity significantly influenced LSD only through SOD, CAT, and MDA, which together explained 90% of the total variance of LSD. Salinity directly and adversely affected UGB, explaining 78% of its variance. Salinity indirectly affected AGB through UGB, where UGB positively affected AGB, explaining 77% of its variance. In this model, only UGB had a positive effect on R/S.

The tolerance of plants to saline environments is often evident in their growth characteristics (Munns and Tester, 2008; Verma et al., 2023). Under adverse conditions, plants adapt through changes in morphological characteristics and growth state (Miryeganeh, 2021). In our study, morphological traits such as PBN, PH, and LA exhibited a downward trend to varying degrees under salt stress (Figure 1). High level of salt stress had significant and negative effect on plant growth. Plants readjust their resource allocation patterns to cope with stress under high salinity conditions (Zhang et al., 2020). Leaf fleshy refers to the enlargement of parenchyma tissue in plant organs, leading to the dilution of cell fluid, aiding plants in dealing with salt stress (Ottow et al., 2005). Results indicated that under different salt stress treatments, the LSD varied significantly between the two plants, suggesting that M. sativa had better water absorption and storage capacity than A. membranaceus under salt stress (Figure 1), ensuring water demand for average plant growth (Ogburn and Edwards, 2010).

Plants under salt stress maintain growth by adjusting biomass energy distribution, primarily by reducing carbon assimilation and altering osmotic energy consumption (Zhang et al., 2020). Generally, in saline-alkali environments, plant individual development is shortened, growth is slowed or stopped, and biomass accumulation is reduced. However, for some salt-tolerant plants, low-concentration salt stress can promote growth (Ogburn and Edwards, 2010). Our study indicated that both legumes were affected by salt stress to varying degrees, with the AGB of A. membranaceus slightly increasing under low salt concentrations (Figure 2). Slight salt stress could sometimes stimulate plant growth (Yu et al., 2020). Conversely, M. sativa exhibited a clear downward trend with increasing salt concentration (Figure 2). With increasing stress factors, plants tend to preserve the biomass of the underground part, reflected in the increase in the R/S (Rabhi et al., 2010). The underground parts of plants bear the brunt of environmental stress caused by salt, prompting many plants to adjust root morphology in response to salt stress (Zhang et al., 2023). Some plants inhibit the growth of the underground part in salt environments to avoid excessive exposure to high salt (Zhao et al., 2020). In our experiment, the biomass of the underground portion relative to the aboveground part increased at a salt concentration of 300 mmol/L. More allocation of root biomass and an increase in the R/S may have a greater potential for plant uptake of soil water under salinity stress conditions. (Figures 2, 3).

When the salt content in the plant growing environment increases, it induces changes in the cell membrane function due to salt damage. This results in an elevated rate of electrolyte exosmosis in the cell, leading to a corresponding increase in relative conductivity (Khatri and Rathore, 2022). The imbalance in free radical metabolism in the body causes an increase in the content of certain free radicals (Heyno et al., 2011; Hasanuzzaman et al., 2021). Specifically, superoxide free radicals can trigger lipid peroxidation of unsaturated fatty acids in membrane lipids, causing serious damage to the biofilm system. MDA, a membrane lipid peroxide produced in this process, serves as an indicator reflecting the strength of plant resistance under stress conditions (de Azevedo Neto et al., 2006). Various plants exhibit different levels of antioxidant enzyme activity in relation to their salt stress tolerance (Costa et al., 2010). Our study demonstrated significant differences in MDA content in A. membranaceus, showing an increasing trend with higher salt concentrations (Figure 4). Generally, under stress conditions such as salt, heavy metals, mechanical damage, and high temperature, MDA content in plants tends to increase (Tang et al., 2015). Notably, there was no significant difference in M. sativa for MDA content, possibly due to its high salt tolerance, which mitigates MDA changes through osmotic regulation (Figure 4). With increasing salt concentration, SOD activity, as the first line of defense against reactive oxygen species damage, usually increases rapidly and significantly (Xu et al., 2013). In our study, CAT and POD activities exhibited similar trends to SOD but with varying degrees of enhancement, and the change in SOD activity was not significant (Figure 5). The activities of CAT, POD, and APX all increased to different extents, suggesting the plant’s ability to perform ion regionalization through self-regulation and the mutual influence of osmoregulatory substances, thereby alleviating high salt stress (Malakar and Chattopadhyay, 2021).

The extent of tissue damage in plants responding to salt stress is often reflected in apparent morphological indexes (Xiao and Zhou, 2022). NaCl inhibits plant growth, and this study identified different degrees of damaged morphological characteristics on the fifth day of salt stress treatment. PCA revealed highly significant correlations among the indicators, indicating a certain degree of overlap and crossover in the information reflected by them. A single index was insufficient to gauge salt tolerance (Munns and Tester, 2008). Through PCA, we simplified the data structure, identified which key variables should be retained or excluded to analyze the relationship between each index and salt tolerance, determined reliable salt tolerance evaluation indicators, and adjusted the one-sidedness of a single index. Higher loading values of the indicator for each principal factor indicated a stronger correlation with its principal factors (Chen et al., 2023). In our study, the results of PCA revealed that the morphological parameters (PBN, PH and LA) for A. membranaceu and the biomass parameters (TB, AGB and UGB) for M. sativa was present in the first principal component and had a large load (Supplementary Table 6). In general, the parameters of morphology and biomass were the most apparent changes under salt stress (Kumar et al., 2021).

SEM for two legumes illustrated the response pathway among indices under salt stress. Salinity directly influenced the PBN, PH, and LA of the main stem. Under low-concentration salt stress, the biomass of A. membranaceus increased, maintaining the plant’s normal physiological function through a protective enzyme system (Figure 6). AGB slightly increased by reducing PH, while UGB remained relatively stable. Typically, UGB is less affected by salt stress, allowing more nutrients to be allocated to AGB, providing additional energy for stress resistance (Loudari et al., 2022). However, in our study, the increase in salt concentration did not affect the PH of M. sativa, and the inhibiting effect of salinity on both aboveground and UGB was synchronized (Figure 6). Variations in stress-tolerant growth strategies among species led to different responses to apparent traits, particularly interspecific variation and environmentally controlled biomass allocation processes (Poorter et al., 2012; Tang et al., 2022). Based on the results of comprehensive salt tolerance analysis, the D value decreased with an increase in salt stress concentration, indicating a rise in salt stress and a decrease in plant salt tolerance (Table 1). Simultaneously, the D value of M. sativa exhibited a slight rebound at 100–200 mmol/L. The comprehensive evaluation of salt tolerance results suggested that under the stimulation of a specific threshold salt concentration, plants would gradually establish a tolerance mechanism through a physiological response (Sohrabi et al., 2012). However, our experimental results may have some potential limitations, including the typicality of legume species, the selected parameters, and the control of soil nutrient conditions, etc. We will continue to investigate the salt tolerance mechanism of legumes in future studies.