Effects of Salinity Stress on Growth and Physiological Parameters and Related Gene Expression in Different Ecotypes of Sesuvium portulacastrum on Hainan Island

We conducted a study to examine the growth and physiological changes in 12 different ecotypes of Sesuvium portulacastrum collected from Hainan Island in China. These ecotypes were subjected to different concentrations (0, 200, 400, and 600 mmol/L) of sodium chloride (NaCl) salt stress for 14 days. We also analyzed the expression of metabolic genes related to stress response. Under low salt stress, indicators such as plant height in region K (0 mmol/L: 45% and highest at 200 mmol/L: 80%), internode length (0 mmol/L: 0.38, 200 mmol/L: 0.87, 400 mmol/L: 0.25, and 600 mmol/L: 1.35), as well as leaf area, relative water content, fresh weight, and dry weight exhibited an overall increasing trend with the increase in salt concentration. However, as the salt concentration increased, these indicators showed a decreasing trend. Proline and malondialdehyde contents increased with higher salt concentrations. When the NaCl concentration was 400 mmol/L, MDA content in the leaves was highest in the regions E (196.23%), F (94.28%), J (170.10%), and K (136.08%) as compared to the control group, respectively. Most materials demonstrated a significant decrease in chlorophyll a, chlorophyll b, and total chlorophyll content compared to the control group. Furthermore, the ratio of chlorophyll a to chlorophyll b (Rab) varied among different materials. Using principal component analysis, we identified three ecotypes (L from Xinglong Village, Danzhou City; B from Shuigoupo Village, Lingshui County; and J from Haidongfang Park, Dongfang City) that represented high, medium, and low salt tolerance levels, respectively, based on the above growth and physiological indexes. To further investigate the expression changes of related genes at the transcriptional level, we employed qRT-PCR. The results showed that the relative expression of SpP5CS1, SpLOX1, and SpLOX1 genes increased with higher salt concentrations, which corresponded to the accumulation of proline and malondialdehyde content, respectively. However, the relative expression of SpCHL1a and SpCHL1b did not exhibit a consistent pattern. This study contributes to our understanding of the salt tolerance mechanism in the true halophyte S. portulacastrum, providing a solid theoretical foundation for further research in this field.


Introduction
Sesuvium portulacastrum, also known as Sea-purslane, belongs to the genus Sesuvium in the family Phyllanthaceae and is a succulent herbaceous saline plant that grows adnately in tropical and subtropical near-coastal sands and mudflats on both sides of river inlets, and is highly tolerant to abiotic stresses such as high salt, drought, and heavy metal ions [1]. S. portulacastrum is a perennial succulent herb with multi-branched, prostrate stems, 20-50 cm long, opposite, clasping leaves, flowers solitary in the leaf axils, flowers from April to July, and capsules with ovoid, shiny black seeds with raised tips [2]. It has chlorophyll degradation and one of the earliest studied enzymes in plants and is widely found in higher plants, mosses, and algae. Numerous studies have demonstrated that the chlorophyllase 1 = of plants plays an important role in the chlorophyll degradation pathway [24,[27][28][29][30][31][32][33].
In China, there is only one species of the genus Sesuvium, S. portulacastrum L. This species is mainly distributed on Hainan Island on coastal beaches, with scattered distributions on surrounding beaches and river inlets, but past studies used material from a regional source and did not systematically collect wild resources of S. portulacastrum from different ecotypes in the tropics. We obtained wild S. portulacastrum germplasm material from 16 representative localities through the systematic resource survey of S. portulacastrum in the coastal area of Hainan Island. The tropical ecotype of S. portulacastrum was found to be mainly distributed on sandy beaches at an average altitude of about 7.4 m above sea level, mainly located in the region of 18 • 15 N to 20 • 05 N and 108 • 39 E to 110 • 59 E on Hainan Island. Further identification confirmed that these 16 S. portulacastrum materials could be classified into 12 different ecotypes. Twelve distinct ecotypes of Sesuvium portulacastrum were exposed to salt stress using a monosaline (NaCl) method. This study aimed to examine the morphological and physiological modifications that occur in S. portulacastrum under salt stress. The expression variances of P5CS, LOX, and CHL1 genes in S. portulacastrum were analyzed at various salt concentrations. The objective was to establish a theoretical framework for understanding the physiological and molecular mechanisms involved in salt tolerance in S. portulacastrum.

Plant Material and Growth Conditions
In this experiment, S. portulacastrum from 12 coastal areas of Hainan Island was used as the experimental material. There is a summary of the east longitude and north latitude of the sampling sites, together with physicochemical information for each site, in Table 1. For propagation, 6 cm long stem segments with two nodes and a pair of top leaves were cut from mother plants and cultured in 1/2 Hoagland medium [34]. After 3 weeks, when the adventitious roots start to grow from the nodes, the seedlings from 12 regions were treated with Hoagland nutrient solution with concentrations of 0 mmol/L (blank control), 200 mmol/L, 400 mmol/L, and 600 mmol/L NaCl for 14 days, respectively.

Morphological Measures
The water on the surface of the S. portulacastrum was roughly sucked up with absorbent paper and then the plant was spread on the table. The length from the root to the end of the terminal bud was measured with a ruler, that is, the whole length of the S. portulacastrum, and the height of the S. portulacastrum was recorded as shown in Figure S1. The internode length of the third and fourth nodes from the root of S. portulacastrum was measured with a ruler, which was recorded as the S. portulacastrum internode length. Plant height and internode length had at least three biological replicates. Each section of S. portulacastrum has two opposite leaves. The two opposite leaves in the third section from the root of S. portulacastrum were selected, the front and back sides of the two opposite leaves were measured by a leaf area scanner, and the average value of 8 measurements was taken as the final leaf area.

Determination of Relative Water Content (RWC)
The relative water content (RWC) of leaves for each treatment was calculated according to the formula of Weatherly [35]. RWC = [(fresh weight of leaves − the dry weight of leaves)/(turgid weight of leaves − dry weight of leaves)] ×100.

Determination of Fresh and Dry Weight
All S. portulacastrum material was taken from each treatment group in each area, and the plants were washed with water and wiped to dry their surfaces. Then, the fresh weight of the plants was weighed and recorded. The oven temperature was set to 105 • C, S. portulacastrum was transferred to the oven, then, 15 min later, the plants were taken out and the dry weight was measured.

Determination of Proline Content
Proline content was measured using the method described by [36], with modifications. First, 0.5 g of seedling shoot samples was ground in liquid nitrogen with a mortar and pestle, 10 mL of 3% sulfosalicylic acid was subsequently added, and the solution was then homogenized in a water bath at 99 • C. The supernatant was collected after centrifugation at 12,000× g for 10 min. Subsequently, 2 mL of glacial acetic acid and 3 mL of 2.5% ninhydrin reagent were added to 2 mL of supernatant. The mixture was incubated in boiling water for 60 min, and the reaction mixture was then extracted by adding 4 mL of methylbenzene. The absorbance of the extract liquor was determined at 520 nm, using a spectrophotometer.

Determination of Malondialdehyde (MDA)
MDA levels were determined by reaction with 2-thiobarbituric acid according to [37], with modifications. Briefly, fresh seedling shoot samples (0.3 g each) were homogenized and extracted in 10 mL of 0.25% TBA made in 10% trichloroacetic acid. The mixture was then heated in a water bath shaker at 95 • C for 30 min and rapidly cooled on ice. The absorbance of the supernatants was determined at 532 nm after centrifugation at 5000× g for 10 min. Correction of non-specific absorption was calculated by subtracting the absorbance at 600 nm. The concentration of MDA was expressed as µmol/g FW.

Determination of Chlorophyll Content
Chlorophyll content was determined following the method described by Arnon [38]. Fresh leaves, each containing 0.5 g of plant tissue, were extracted in the dark with 10 mL of 80% acetone. The assay mixture was centrifuged at 10,000× g for 5 min, and the supernatant was removed, mixed with 85% aqueous acetone solution to an appropriate concentration, and the absorbance was measured at 663 and 645 nm. Chlorophyll content was calculated according to the equations given by Arnon.

Total RNA Extraction and Reverse Transcription
An RNAprep Pure Plant kit (Tiangen, China, Beijing) was used to extract total RNA from 3 ecotypes after 14 days of salt stress (3 replicates were used from each treatment). Reverse transcription reactions were completed using 0.5 µg RNA and an iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). The ABI Power SYBR Green PCR Master Mix (Applied Biosystems, Waltham, MA, USA) was used with 5 ng of template. Firststrand cDNA was synthesized from DNaseI-treated total RNA using 1 µL of iScript reverse transcriptase (TaKaRa, Gunma, Japan), 0.8 µg of RNA, and 4 µL of 5× Reaction Mix. Samples were incubated at 25 • C for 5 min, followed by 42 • C for 30 min, 85 • C for 5 min, and finally storage at 4 • C.

Acquisition of Gene-Coding Sequences
Based on the published genetic information of the proline synthase SpP5CS of S. portulacastrum [39], the SpP5CS protein sequence was derived from the results section of this paper. The protein sequences of LOX1 and LOX5 and the CHL1 gene of Arabidopsis thaliana were downloaded from the Tair website. Candidate protein sequences (EVALUE < 1 × 10 −10 ) were screened from S. portulacastrum transcriptome data using local BLAST.
The BioEdit software was used to compare the extracted protein sequences with the previously known sequences. Among them, the sequence of the common region was selected as the target sequence based on the extracted members of SpP5CS and named SpP5CS1. After sequence alignment, the two member sequences closest to LOX1 and LOX5 were selected as the target sequences and named SpLOX1 and SpLOX5, respectively. The two sequences with the least significant difference from the CHL1 sequences of Arabidopsis thaliana were selected as the target sequences and named SpCHL1a and SpCHL1b, respectively.

Quantitative Real-Time PCR (qRT-PCR)
The primer sequence information of the target sequence designed by Primer Premier 5 is shown in Table S1. Meanwhile, GAPDH was used as the reference gene in this study. The SYBR Green qRT-PCR reaction consisted of 5 µL of 2× SYBR Green PCR buffer, 0.5 µL of primers, and 5 ng of templates. The final volume was adjusted to 10 µL with doubledistilled H 2 O. The PCR program was as follows: 50 • C for 2 min; 95 • C for 10 min; 40 cycles at 95 • C for 15 s and 60 • C for 1 min. Data were processed using the 2 −∆∆Ct method [40].

Data Analysis
Microsoft Excel 2010 was used for arranging the raw data and conducting calculations for the mean and standard deviation of both the control group and each treatment trait. To perform principal component analysis (PCA), SPSS 24.0 was employed.

A Comprehensive Evaluation of the Salt Resistance of Different Ecotypes
All indicators were used to determine change rates under salt stress. The salt resistance of different ecotypes of S. portulacastrum was comprehensively evaluated using principal component analysis.

Effects of Salt Stress on Plant Height
After 14 days of salt stress, different ecotypes of S. portulacastrum showed different growth statuses under different salt concentrations. The growth rate of S. portulacastrum in different regions showed significant differences. The growth rate of S. portulacastrum in regions A-J was low, while the growth rate of S. portulacastrum in regions K and L was good, and the growth rate was generally higher than that in other regions. For the same ecotypes of S. portulacastrum, the growth rate of plant height also showed great differences after different salt concentration treatments. After a 200 mmol/L salt treatment, the growth rate of S. portulacastrum height in B, I, K, and L was significantly higher than that in CK (p < 0.05). There was no significant difference in the growth rate of S. portulacastrum height between C and H compared with CK. Compared with the control group, the growth rate of plant height in other S. portulacastrum samples was significantly lower than that in CK. After the 400 mmol/L salt treatment, the growth rate of S. portulacastrum height in areas K and L was significantly lower than that in the 200 mmol/L salt treatment group (p < 0.05), and the growth rate of S. portulacastrum height in other places was significantly higher than that in the 200 mmol/L salt treatment group (p < 0.05). At the same time, under the stress of the 600 mmol/L salt concentration, some S. portulacastrum samples wilted and their growth was inhibited. However, the growth rate of S. portulacastrum height in area K was significantly higher than that in CK (p < 0.05), and higher than that in other regions with the same concentration treatment, and the growth rate of S. portulacastrum height was 44.71% ( Figure 1).

A Comprehensive Evaluation of the Salt Resistance of Different Ecotypes
All indicators were used to determine change rates under salt stress. The salt sistance of different ecotypes of S. portulacastrum was comprehensively evaluated us principal component analysis.

Effects of Salt Stress on Plant Height
After 14 days of salt stress, different ecotypes of S. portulacastrum showed differ growth statuses under different salt concentrations. The growth rate of S. portulacastr in different regions showed significant differences. The growth rate of S. portulacastrum regions A-J was low, while the growth rate of S. portulacastrum in regions K and L w good, and the growth rate was generally higher than that in other regions. For the sa ecotypes of S. portulacastrum, the growth rate of plant height also showed great differen after different salt concentration treatments. After a 200 mmol/L salt treatment, the grow rate of S. portulacastrum height in B, I, K, and L was significantly higher than that in CK < 0.05). There was no significant difference in the growth rate of S. portulacastrum hei between C and H compared with CK. Compared with the control group, the growth r of plant height in other S. portulacastrum samples was significantly lower than that in After the 400 mmol/L salt treatment, the growth rate of S. portulacastrum height in area and L was significantly lower than that in the 200 mmol/L salt treatment group (p < 0. and the growth rate of S. portulacastrum height in other places was significantly hig than that in the 200 mmol/L salt treatment group (p < 0.05). At the same time, under stress of the 600 mmol/L salt concentration, some S. portulacastrum samples wilted their growth was inhibited. However, the growth rate of S. portulacastrum height in a K was significantly higher than that in CK (p < 0.05), and higher than that in other regi with the same concentration treatment, and the growth rate of S. portulacastrum hei was 44.71% ( Figure 1).

Effects of Salt Stress on Internode Length
After 14 days of exposure to salt stress, the internode length of S. portulacastrum displayed significant variations across 12 different regions under varying salt concentrations. Notably, the control group exhibited more noticeable growth in regions F and I, measuring 0.74 cm and 0.61 cm, respectively. At a salt concentration of 200 mmol/L, regions C, D, H, K, and L demonstrated higher internode growth values, with the longest length observed in region K, reaching 0.87 cm. However, as the salt concentration increased to 400 mmol/L and 600 mmol/L, the growth rate of the internodes slowed down, resulting in relatively smaller elongation lengths. Furthermore, in some areas, the elongation process ceased altogether. Overall, the growth rate of internodes slowed down with the increase in salt concentration, which was consistent with the slow growth rate of S. portulacastrum with the increase in salt concentration. Among them, S. portulacastrum in region K were still elongating at the 600 mmol/L concentration, which was significantly different from the control group (p < 0.05), and the maximum length was 1.35 cm (Table 2).

Effects of Salt Stress on Leaf Area
The experimental findings revealed that S. portulacastrum experienced an increase in leaf area in most areas under low salt concentration stress. However, when compared to the control group without stress, the stress impeded leaf growth. Specifically, in region I, the leaf area growth of S. portulacastrum was the highest at 121.44 mm 2 when the salt concentration was 200 mmol/L. When the salt concentration reached 400 mmol/L, apart from regions G and H where the leaf area of S. portulacastrum showed negative growth values, the leaf area in other regions exhibited various degrees of growth. As the salt concentration further increased to 600 mmol/L, the leaf area of Sesuvium portulacastrum in all regions, except for regions J and K, displayed different degrees of reduction. At this time, the symptoms of shrinkage and etiolation of S. portulacastrum leaves appeared, and the growth value of the leaf area appeared negative. It is worth noting that S. portulacastrum in region G showed leaf wilting under 200 mmol/L to 600 mmol/L salt stress, which made leaf area smaller, and the value of the leaf area growth was significantly smaller than that of the control group (p < 0.05) ( Figure 2). On the contrary, the leaf area of the J and K S. portulacastrum increased under all the salt concentration growth conditions ( Figure 2).

Effects of Salt Stress on the Relative Water Content (RWC)
RWC is a relatively sensitive index. The speed of decline in RWC under osmotic stress is closely related to plant stress resistance. Compared with absolute water content, it can better reflect the water status of leaves under osmotic stress ( Figure 3). The RWC of S. portulacastrum in B, D, E, F, H, and L decreased with the increase in salt concentration and the RWC of the control group in area D was the highest, which was 78.86%. The RWC of S. portulacastrum in region J was higher than that of the blank control under all salt concentration treatments, and the maximum value was 76.43% when the salt concentration was 200 mmol/L. When the salt concentration was 0-400 mmol/L, the RWC of S. portulacastrum in region G decreased with the increase in salt concentration. When the salt concentration was 600 mmol/L, the RWC of S. portulacastrum in region G reached the highest value of 90%, which was significantly higher than that in other salt concentration treatment groups (p < 0.05). The RWC of S. portulacastrum in region K at 400 mmol/L salt concentration was significantly higher than that in other salt concentration treatment groups. The RWC of S. portulacastrum in other areas reached the maximum when the salt concentration was 200 mmol/L.

Effects of Salt Stress on the Relative Water Content (RWC)
RWC is a relatively sensitive index. The speed of decline in RWC under osmotic s is closely related to plant stress resistance. Compared with absolute water content, it better reflect the water status of leaves under osmotic stress ( Figure 3). The RWC portulacastrum in B, D, E, F, H, and L decreased with the increase in salt concentration the RWC of the control group in area D was the highest, which was 78.86%. The RW S. portulacastrum in region J was higher than that of the blank control under all salt centration treatments, and the maximum value was 76.43% when the salt concentra was 200 mmol/L. When the salt concentration was 0-400 mmol/L, the RWC of S. port astrum in region G decreased with the increase in salt concentration. When the salt centration was 600 mmol/L, the RWC of S. portulacastrum in region G reached the hig value of 90%, which was significantly higher than that in other salt concentration t ment groups (p < 0.05). The RWC of S. portulacastrum in region K at 400 mmol/L salt centration was significantly higher than that in other salt concentration treatment gro The RWC of S. portulacastrum in other areas reached the maximum when the salt con tration was 200 mmol/L.

Effects of Salt Stress on the Fresh and Dry Weight
The most direct indicator of the effect of salt stress on plant growth is biomass. As shown in Figure 4, when the NaCl concentration was 200 mmol/L, the fresh weight of S. portulacastrum in some areas slightly increased compared with the control group. Among them, E increased by 8.12%, G increased by 16.03%, H increased by 6.02%, and L increased by 14.10% compared with the control. The fresh weight of S. portulacastrum in other areas decreased to different degrees. At the same time, the fresh weight of S. portulacastrum in most areas was greatly affected by the 400 mmol/L NaCl treatment. Except for that of L, which decreased to a lower degree, the fresh weight of S. portulacastrum in the other 11 areas decreased significantly compared with the control (p < 0.05), especially J, which de-

Effects of Salt Stress on the Fresh and Dry Weight
The most direct indicator of the effect of salt stress on plant growth is biomass. As shown in Figure 4, when the NaCl concentration was 200 mmol/L, the fresh weight of S. portulacastrum in some areas slightly increased compared with the control group. Among them, E increased by 8.12%, G increased by 16.03%, H increased by 6.02%, and L increased by 14.10% compared with the control. The fresh weight of S. portulacastrum in other areas decreased to different degrees. At the same time, the fresh weight of S. portulacastrum in most areas was greatly affected by the 400 mmol/L NaCl treatment. Except for that of L, which decreased to a lower degree, the fresh weight of S. portulacastrum in the other 11 areas decreased significantly compared with the control (p < 0.05), especially J, which decreased by 50% compared with the control. Compared with the control, K decreased by 43.12%, A decreased by 42.03%, and E decreased by 26.01%. When the concentration of NaCl reached 600 mmol/L, the S. portulacastrum in most areas was seriously injured, with yellowing, dehydration, and wilting, and the fresh weight was significantly lower than that of the control group. The overall fresh weight of S. portulacastrum decreased gradually with the increase in salt concentration.  As shown in Figure 5, the change in dry weight of S. portulacastrum was similar to the above trend of fresh weight. Under the 200 mmol/L NaCl treatment, the dry weight of S. portulacastrum in some areas was slightly higher than that of the control group treated with 0 mmol/L NaCl. Compared with the control group, E increased by 3.13%, G increased by 15.01%, H increased by 5.02%, L increased by 13.04%. The dry weight of S. portulacastrum in other areas decreased to different degrees. At the same time, under the 400 mmol/L NaCl treatment, the dry weight of S. portulacastrum in the other 11 sites was significantly decreased compared with the control, except the dry weight of L that was increased by 12.5% compared with the control (p < 0.05). G decreased by 46.14%, J decreased by 31.08%, and F decreased by 28.14% compared with the control. When the NaCl concentration reached 600 mmol/L, the dry weight of S. portulacastrum further decreased to different degrees in almost all places. The overall dry weight of S. portulacastrum decreased gradually with the increase in salt concentration. As shown in Figure 5, the change in dry weight of S. portulacastrum was similar to the above trend of fresh weight. Under the 200 mmol/L NaCl treatment, the dry weight of S. portulacastrum in some areas was slightly higher than that of the control group treated with 0 mmol/L NaCl. Compared with the control group, E increased by 3.13%, G increased by 15.01%, H increased by 5.02%, L increased by 13.04%. The dry weight of S. portulacastrum in other areas decreased to different degrees. At the same time, under the 400 mmol/L NaCl treatment, the dry weight of S. portulacastrum in the other 11 sites was significantly decreased compared with the control, except the dry weight of L that was increased by 12.5% compared with the control (p < 0.05). G decreased by 46.14%, J decreased by 31.08%, and F decreased by 28.14% compared with the control. When the NaCl concentration reached 600 mmol/L, the dry weight of S. portulacastrum further decreased to different degrees in almost all places. The overall dry weight of S. portulacastrum decreased gradually with the increase in salt concentration.
The above results indicate that some S. portulacastrum grow faster in environments with a low concentration of NaCl. When the concentration of NaCl exceeds a certain range, the growth rate of S. portulacastrum will slow down, and the S. portulacastrum may become dehydrated and withered with the further increase in NaCl concentration. At the same time, we found that J was relatively more inhibited by NaCl in the S. portulacastrum materials from 12 regions, suggesting that this material was sensitive to salt stress. The dry weight of L was not significantly affected by NaCl and remained at a relatively stable level. Combined with the fact that its fresh weight increased at 200 mmol/L NaCl and showed the smallest decline in 400 mmol/L NaCl, it was speculated that S. portulacastrum samples in region L were not very sensitive to salt stress. The above results indicate that some S. portulacastrum grow faster in environments with a low concentration of NaCl. When the concentration of NaCl exceeds a certain range, the growth rate of S. portulacastrum will slow down, and the S. portulacastrum may become dehydrated and withered with the further increase in NaCl concentration. At the same time, we found that J was relatively more inhibited by NaCl in the S. portulacastrum materials from 12 regions, suggesting that this material was sensitive to salt stress. The dry weight of L was not significantly affected by NaCl and remained at a relatively stable level. Combined with the fact that its fresh weight increased at 200 mmol/L NaCl and showed the smallest decline in 400 mmol/L NaCl, it was speculated that S. portulacastrum samples in region L were not very sensitive to salt stress.

Effects of Salt Stress on Proline Content
As shown in Figure 6, when NaCl concentration was low, the proline content in S. portulacastrum leaves was low, and when NaCl concentration gradually increased, the proline content also showed a gradual increase. When NaCl concentration was 200 mmol/L, the proline content of S. portulacastrum leaves in A-L was significantly increased compared with the control group (p < 0.05). In addition to B and E, the proline content of dentate leaves of S. portulacastrum in other areas was increased by more than 2 times compared with the control. When NaCl concentration was 400 mmol/L, compared with the control group and 200 mmol/L treatment group, the content of proline in S. portulacastrum leaves was significantly increased (p < 0.05), and the increased amplitude was greatly increased. In particular, the proline content of F and J reached 7 times higher than that of the control group. After that, the concentration of NaCl was further increased. Under the treatment of 600 mmol/L NaCl, the proline content in the leaves of S. portulacastrum in some areas was nearly 10 times that of the control group, as shown in J.

Effects of Salt Stress on Proline Content
As shown in Figure 6, when NaCl concentration was low, the proline content in S. portulacastrum leaves was low, and when NaCl concentration gradually increased, the proline content also showed a gradual increase. When NaCl concentration was 200 mmol/L, the proline content of S. portulacastrum leaves in A-L was significantly increased compared with the control group (p < 0.05). In addition to B and E, the proline content of dentate leaves of S. portulacastrum in other areas was increased by more than 2 times compared with the control. When NaCl concentration was 400 mmol/L, compared with the control group and 200 mmol/L treatment group, the content of proline in S. portulacastrum leaves was significantly increased (p < 0.05), and the increased amplitude was greatly increased. In particular, the proline content of F and J reached 7 times higher than that of the control group. After that, the concentration of NaCl was further increased. Under the treatment of 600 mmol/L NaCl, the proline content in the leaves of S. portulacastrum in some areas was nearly 10 times that of the control group, as shown in J.

Effects of Salt Stress on Malondialdehyde (MDA) Content
MDA is the product of lipid metabolism, and its content can directly reflect the degree of stress-induced membrane damage. After 14 days of salt stress, the measurement results showed that MDA content in S. portulacastrum leaves in 12 regions showed an increasing trend with the increase in NaCl concentration (Figure 7). When the treatment condition was 200 mmol/L NaCl concentration, compared with the control group, the MDA content of S. portulacastrum leaves in each region was 13.

Effects of Salt Stress on Malondialdehyde (MDA) Content
MDA is the product of lipid metabolism, and its content can directly reflect the degree of stress-induced membrane damage. After 14 days of salt stress, the measurement results showed that MDA content in S. portulacastrum leaves in 12 regions showed an increasing trend with the increase in NaCl concentration (Figure 7). When the treatment condition was 200 mmol/L NaCl concentration, compared with the control group, the MDA content of S. portulacastrum leaves in each region was 13.   These results indicate that a high concentration of salt may lead to peroxidation of polyunsaturated fatty acids in the cell membrane of S. portulacastrum mesophyll, which may cause cell damage, damage the cell membrane structure, and increase membrane permeability. In conclusion, the accumulation of MDA in S. portulacastrum leaves under salt stress reflects the degree of injury. According to the above results, S. portulacastrum from the 12 regions can be divided into A, B, I, and L ecotypes with high salt tolerance, C, D, F, and K ecotypes with moderate salt tolerance, and E, G, H, and J ecotypes with low salt tolerance. Figure 8A shows the changes in Chla content in 12 different ecotypes of S. portulacastrum leaves. When the concentration of NaCl was 200 mmol/L, the Chla content in S. portulacastrum leaves at E, J, and L had no significant change compared with that in the control group. D, H, and I had values that were significantly higher than that in the control group (p < 0.05), while the content of Chla in other places was significantly lower than that of the control group (p < 0.05). When NaCl concentration was 400 mmol/L, there was no significant difference in the content of Chla in the S. portulacastrum leaves of area B compared with the control group, the content of Chla in the S. portulacastrum leaves of areas H and J was significantly higher than that of the control group, and the content of Chla in other areas was significantly lower than that of the control group (p < 0.05). When salt concentration continued to rise to 600 mmol/L, the Chla content in S. portulacastrum leaves in only region J was significantly higher than that in control group, and the other regions had values that were significantly lower than that in the control group (p < 0.05). In general, the Chla content in S. portulacastrum leaves increased under low concentrations of salt stress in some areas. When the salt concentration continued to increase, the Chla content in S. portulacastrum leaves showed a decreasing trend, generally becoming lower than that in the control group (p < 0.05).

Effects of Salt Stress on Chlorophyll b Content
Valentina et al. demonstrated that the decrease in Chlb content under salt stress can reduce the capture of light energy, the production of reactive oxygen species, and the degradation rate of proteins, making plants more salt resistant. After 14 days of salt stress, the changes in Chlb content in S. portulacastrum of different ecotypes under different salt concentrations were similar to those of Chla. The changes in Chlb content in S. portulacastrum leaves of different regions showed significant differences, and the Chlb content in S. portulacastrum leaves of most regions under different salt concentrations was generally lower than that of the control group ( Figure 8B). The Chlb content of S. portulacastrum leaves in areas A, B, F, and G showed a trend of first decreasing and then increasing, and the Chlb content at different salt concentrations was significantly lower than that in the control group (p < 0.05). The Chlb content of S. portulacastrum in regions K and L decreased continuously with the increase in salt concentration. In addition, the Chlb content of S. portulacastrum in region C did not change significantly before and after treatment with 200 mmol/L and 400 mmol/L salt concentrations, but decreased significantly when the salt concentration increased to 600 mmol/L. The Chlb content of S. portulacastrum in other areas increased first and then decreased.

Effects of Salt Stress on the Ratio of Chlorophyll a to Chlorophyll b (Rab)
The Chla/Chlb (Rab) values of S. portulacastrum leaves treated with different NaCl concentrations were calculated. The results showed that the Rab values of S. portulacastrum leaves of different ecotypes did not show a single change rule under different salt stress levels ( Figure 8C). When NaCl concentration was 200 mmol/L, the Rab values of S. portulacastrum leaves in regions H, I, J, and K were significantly lower than those in the control group (p < 0.05), and the Rab values in region L were significantly higher than those in the control group (p < 0.05). There was no significant difference in the Rab values in other regions compared with the control group. When NaCl concentration was 400 mmol/L, the Rab value of S. portulacastrum leaves in area A was significantly lower than that in the control group (p < 0.05), and Rab values in areas B, E, G, K, and L were significantly higher than that in the control group (p < 0.05). There was no significant difference in Rab value between the other areas and the control group. When the salt concentration increased to 600 mmol/L, the Rab value of S. portulacastrum leaves in most areas was significantly lower than that of the control group (p < 0.05), while the Rab value in area L was not significantly different from that of the control group. Combined with the above analysis, it can be preliminarily speculated that the S. portulacastrum in region L has strong salt tolerance, and the S. portulacastrum in regions B, C, and F also have certain salt tolerance.

Effects of Salt Stress on Total Chlorophyll Content
The effects of different salt concentration treatments on the total chlorophyll content of S. portulacastrum leaves of different ecotypes from 12 regions are shown in Figure 8D. The results showed that when the concentration of NaCl was 200 mmol/L, the chlorophyll content of S. portulacastrum leaves in areas D, H, I, and J was significantly higher than that in the control group (p < 0.05), and there was no significant difference between areas E and L and the control group, while the other areas had values that were significantly lower than that in the control group (p < 0.05). When NaCl concentration was 400 mmol/L, the chlorophyll content of S. portulacastrum leaves in areas H and J was significantly higher than that in the control group (p < 0.05), and the other areas had values that were significantly lower than that in the control group (p < 0.05). When the salt concentration increased to 600 mmol/L, the chlorophyll content of S. portulacastrum leaves in most areas was significantly lower than that of the control group (p < 0.05). It can be seen that with the increase in salt concentration, the chlorophyll content in S. portulacastrum leaves in 12 regions showed various trends.

Comprehensive Evaluation of Salt Resistance Based on Principal Component Analysis
The research conducted at our university demonstrated that the combined contribution of the first two principal components accounted for 71.769% of the total variance (Table S2). Therefore, extracting these two principal components allows for a reduction in the loss of information from the original variables, leading to more satisfactory analysis results. Instead of utilizing the initial six physiological indicators (relative water content, chlorophyll content, malondialdehyde content, proline content, soluble sugar content, and plant height), we employed these two common factors to comprehensively assess the salt tolerance of S. portulacastrum. By employing principal component analysis, we transformed the six physiological indicators into two independent composite indicators, which can be employed for future comprehensive evaluations of S. portulacastrum's salt tolerance.
We calculated and ranked the combined score (Dn values) of salt tolerance principal components for the 12 ecotypes of S. portulacastrum. This calculation was based on the contribution of the principal components and the physiological experimental data of these 12 ecotypes (Table S2). The results indicate that ecotypes L and I exhibit better salt tolerance, while ecotypes K, G, and J display poor salt tolerance, with the remaining ecotypes showing intermediate salt tolerance. These findings are generally consistent with the salinity tolerance ranking of 16 materials presented in previous work at our university. However, individual differences may be attributed to the inclusion of a larger sample size in Table 3.

Analysis of Differential Expression of S. portulacastrum Metabolism-Related Genes under Different Salt Concentrations
Generally, as long as the brightness of 28S rRNA is greater than that of 18S rRNA and the band is clear and the dispersion is slight, the integrity is good and can meet the requirements. As shown in Figure S2 from left to right three salt concentrations are shown: gully slope of Lingshui County (B) (salt-tolerant materials), Haidong Park, Binhai North Road, Dongfang City (J) (poorly salt-resistant materials), Xinglong Village, Danzhou City (L) (highly salt-tolerant materials). Electrophoresis analysis was performed on the extracted RNA samples from three areas and the three different bands of 28S, 18S, and 5S were checked ( Figure S2). The RNA integrity of the 28S/18S bands was found to be between 1.9 and 2.1, indicating that the overall quality of the total RNA meets the requirements of the experiment.

Changes in the Relative mRNA Expression Levels of SpP5CS1, SpCHL1a, and SpCHL1b
Compared with CK, the relative mRNA expression of SpP5CS1 in S. portulacastrum in region B increased after Hoagland nutrient solution treatment with 200 mmol/L NaCl, but the change was not obvious. After treatment with Hoagland nutrient solution with 400 mmol/L NaCl, the relative mRNA expression of SpP5CS1 began to increase significantly (p < 0.01). The relative mRNA expression of SpP5CS1 in S. portulacastrum in region J significantly increased with the increase in salt concentration under Hoagland nutrient solution with three salt concentration gradients (p < 0.01). The relative mRNA expression of SpP5CS1 in S. portulacastrum in region L showed an increasing trend before and after NaCl treatment with three concentration gradients, but the change was not obvious, while when comparing control and after 400 mmol/L NaCl treatment, the relative mRNA expression of SpP5CS1 was significantly increased (p < 0.05) ( Figure 9A).
S. portulacastrum in region B were treated with Hoagland nutrient solution with 200 mmol/L NaCl, and the relative mRNA expression of SpLOX1 was significantly increased compared with CK (p < 0.01). When the salt concentration reached 400 mmol/L, the relative mRNA expression of SpLOX1 had little change compared with the 200 mmol/L treatment group. The relative mRNA expression of SpLOX1 in S. portulacastrum in region J was significantly increased with the increase in salt concentration in Hoagland nutrient solution treated with three salt concentration gradients (p < 0.01). Compared with CK, the relative mRNA expression of SpLOX1 in S. portulacastrum in region L was not significantly different after 200 mmol/L NaCl treatment, while the relative expression of SpLOX1 mRNA was significantly increased after 400 mmol/L NaCl treatment (p < 0.05). ( Figure 9B). In addition, the relative mRNA expression of SpLOX5 in the S. portulacastrum of B, J, and L showed a significant increase with the continuous increase in salt concentration (p < 0.01) ( Figure 9C). S. portulacastrum in region B were treated with Hoagland nutrient solution with 200 mmol/L NaCl, and the relative mRNA expression of SpLOX1 was significantly increased compared with CK (p < 0.01). When the salt concentration reached 400 mmol/L, the relative mRNA expression of SpLOX1 had little change compared with the 200 mmol/L treatment group. The relative mRNA expression of SpLOX1 in S. portulacastrum in region J was significantly increased with the increase in salt concentration in Hoagland nutrient solution treated with three salt concentration gradients (p < 0.01). Compared with CK, the relative mRNA expression of SpLOX1 in S. portulacastrum in region L was not significantly different after 200 mmol/L NaCl treatment, while the relative expression of SpLOX1 mRNA was significantly increased after 400 mmol/L NaCl treatment (p < 0.05). ( Figure  9B). In addition, the relative mRNA expression of SpLOX5 in the S. portulacastrum of B, J, and L showed a significant increase with the continuous increase in salt concentration (p < 0.01) ( Figure 9C).
In the S. portulacastrum of B, J, and L, the relative mRNA expression levels of SpCHL1a and SpCHL1b in the treatment groups were lower than those in the CK group. The relative mRNA expressions of SpCHL1a and SpCHL1b in S. portulacastrum of region J decreased first and then increased with the increase in salt concentration, and the differences were significant (p < 0.01). With the increase in salt concentration, the relative mRNA expression of SpCHL1a in S. portulacastrum of regions B and L decreased gradually. Compared with CK and 200 mmol/L, the relative mRNA expression of SpCHL1a in S. portulacastrum in region B was significantly decreased when the salt concentration was 400 mmol/L (p < In the S. portulacastrum of B, J, and L, the relative mRNA expression levels of SpCHL1a and SpCHL1b in the treatment groups were lower than those in the CK group. The relative mRNA expressions of SpCHL1a and SpCHL1b in S. portulacastrum of region J decreased first and then increased with the increase in salt concentration, and the differences were significant (p < 0.01). With the increase in salt concentration, the relative mRNA expression of SpCHL1a in S. portulacastrum of regions B and L decreased gradually. Compared with CK and 200 mmol/L, the relative mRNA expression of SpCHL1a in S. portulacastrum in region B was significantly decreased when the salt concentration was 400 mmol/L (p < 0.01). However, the relative mRNA expression of SpCHL1a in S. portulacastrum in region L significantly decreased with the increase in salt concentration under Hoagland nutrient solution treated with three salt concentration gradients (p < 0.01). In addition, compared with CK, the relative mRNA expression of SpCHL1b in S. portulacastrum in region B was significantly decreased after salt stress treatment (p < 0.01), while the relative mRNA expression of SpCHL1b in the 400 mmol/L treatment group was significantly increased compared with the 200 mmol/L treatment group (p < 0.05). At the same time, the relative mRNA expression of SpCHL1b in S. portulacastrum in region L was significantly decreased in the treatment group compared with CK (p < 0.05), and there was no significant difference in the relative mRNA expression of SpCHL1b before and after the treatment of 200 mmol/L and 400 mmol/L salt concentrations ( Figure 9D,E).

Discussion
Soil salinity, a significant abiotic stressor, has a notable influence on plant growth and crop yields. The stress caused by salinity can greatly impact global food production [41,42]. To date, around 10% of the Earth's overall land area (equivalent to 950 million hectares), 20% of its arable land (about 300 million hectares), and 50% of the total irrigated land (around 230 million hectares) have been impacted by soil salinization. Moreover, it is projected to affect 50% of the world's cultivated land by 2050, with an alarming rate of expansion [43,44]. Despite making reasonable progress in defining the physiological manifestations, the precise mechanisms underlying the response and tolerance to salt stress have remained elusive [41]. In this study, a total of 12 distinct ecotypes of S. portulacastrum were collected, and the changes in plant physiological indexes and the corresponding gene expression levels in response to varying salt concentrations were investigated. The results showed that various salt tolerance factors showed different changes in S. portulacastrum plants under different salt concentrations. Similarly, the results showed that with the increase in salt concentration, the growth rate of most S. portulacastrum became slow, and the growth rate of plants decreased with the increase in salt concentration. In previous studies, it has been reported that various salt concentrations significantly affected the growth and physiology of plants of different species [15,45]. Furthermore, the plant height of S. portulacastrum increased under low salt concentration in some areas, especially in region L. In addition, the overall internode length showed a slower growth rate with the increased concentration of salt, which corresponded with the slower growth rate of S. portulacastrum with the increase in salt concentration. Our results are consistent with the findings of [46] who reported that salt stress (NaCl) inhibited the growth of Juglans microcarpa L. seedlings. However, when the salt concentration continued to increase, most ecotypes of S. portulacastrum showed leaf wilting due to limited salt tolerance and the leaf area decreased sharply at this time. High salinity causes decreased plant growth primarily due to ion toxicity and nutrient imbalance [47].
Under high salt concentrations, large amounts of Na + and Cl − are transported to the vegetative parts of plants, which antagonistically decrease growth traits, and root and shoot lengths may be due to inappropriate Na + /K + ratios, as higher values of Na + /K + in plant tissue impair the transport of K + and Ca 2+ , disturb plant metabolism, and lead to reduced plant growth [48]. Besides damaging the signal transduction pathway, salinity lowers osmotic potential and reduces water uptake [49]. Salinity also affects leaf initiation, internodal growth, and leaf expansion, thereby reducing shoot growth in plants [48]. Furthermore, under salinity stress, the accumulation of Na + disrupts the uptake of K + , which reduces stomatal conductance and thus creates water-deficit conditions for plants [50]. Reduced stomatal conductance under salt stress may also lead to a reduction in photosynthetic activities [13]. Salinity also results in excessive production of reactive oxygen species (ROS) [14] and a decrease in chlorophyll contents [15]. Moreover, the presence of soluble salts in the soil elevates osmotic pressure and diminishes the overall soil water potential, resulting in reduced water absorption by the roots. Consequently, various plant growth parameters are negatively impacted [51].
The regulation of plant water content is an important part of the salt tolerance of halophytes. During salt stress, salt mainly hinders plant growth and development through osmotic stress. Under osmotic stress, the rate of decrease in relative water content is closely related to plant salt tolerance. Recent reports indicate that salinity reduces the water content in plants by impeding the absorption of water from the soil, resulting in decreased plant growth [52,53]. On the contrary, our findings revealed minimal changes in the relative water content of S. portulacastrum across most regions. However, in select areas experiencing low salt stress, there was an observed increase in relative water content. Typically, under salt stress, the initial physiological reaction is to prevent water loss through transpiration. This is accomplished by reducing the stomatal conductance (Gs) value through the closure of stomata [54,55]. When the salt concentration continued to increase, the relative water content of S. portulacastrum in most regions, except for region G, decreased significantly, which fully demonstrated that S. portulacastrum, as for euhalophytes, was more adapted to low salt stress conditions and even preferred the low-salt environment. Fresh weight and dry weight are the most intuitive performance indexes to determine the salt tolerance level of plants. In the current research, it was observed that a lower concentration of salt can enhance the growth of S. portulacastrum components, resulting in slightly higher fresh weight and dry weight compared to the control group. However, as the salt concentration increased, the fresh weight and dry weight decreased. These results indicated that high salt stress inhibited the growth of S. portulacastrum and significantly reduced their biomass. These findings are consistent with the results from previous studies [56,57]. Plants face difficulties in absorbing water and may even experience dehydration when subjected to NaCl treatment due to the decreased water potential of the surrounding solution. To mitigate or prevent the negative effects of osmotic stress, plants typically employ osmotic adjustment as a mechanism to restore turgor pressure [58]. Numerous experiments have demonstrated a close relationship between the salt tolerance of salt-tolerant plants and organic osmotic regulation [59,60]. Proline serves as the primary osmotic protective agent in plants when they experience salt stress. It possesses several features that contribute to its effectiveness, including its ability to prevent easy penetration of the cell membrane, lack of charge under physiological pH conditions, low molecular weight, and solubility in water [61,62]. Additionally, proline is rapidly generated within the organism and can efficiently accumulate, thereby exerting a strong regulatory effect [63,64]. The significant association between proline accumulation and osmotic stress tolerance has been extensively reported previously [61]. In our study, we observed that as the salt concentration increased, there was a noticeable increase in the proline content. We found that materials with a higher accumulation rate of proline content compared to the control exhibited greater sensitivity to salt stress. Hence, the current findings suggest that the increase in proline levels could serve as a physiological indicator for assessing the impact of NaCl on S. portulacastrum. Similarly, the mRNA expression of SpP5CS1 and SpLOX1 in S. portulacastrum exhibited a regulated trend after NaCl treatment with three different concentrations. In previous studies, it has been reported that delta1-pyrroline-5-carboxylate (P5C) synthetase and lipoxygenase (LOX)-related genes are significantly upregulated under salinity stress [62,[65][66][67][68]. Previously, researchers reported that proline utilization pathway genes are essential for the organization and regulation of the genes involved in L-proline catabolism [69]. Proline dehydrogenase (ProDH) and ∆1-pyrroline-5-carboxylate dehydrogenase (P5CDH) are the two key enzymes in the catabolism of proline [61]. These results suggest that light regulation of salt concentration induces the expression level of SpP5CS1 and SpLOX1 which may maintain the salt tolerance in S. portulacastrum.
In living organisms, the content of MDA, the end product of lipid peroxidation, can represent the damage degree of the lipid membrane, and the change in MDA content is closely related to the permeability of membrane, in which membrane permeability directly reflects the damage degree of membrane lipid, while MDA indirectly reflects the damage degree of the membrane. In this study the vast majority of MDA contents in S. portulacastrum leaves did not change significantly under low salt concentration and, when the salt concentration rose, the MDA content of S. portulacastrum leaves of different ecotypes significantly increased to different extents. Our findings align with a previous study and indicate that salinity stress has a significant impact on the formation of malondialdehyde [70,71]. Enhanced electrolyte leakage (EL) and MDA are considered signs of membrane damage induced by salt stress [72]. Similarly, Sarker and Oba reported a similar rise in MDA in A. tricolor under different salt concentrations [73]. This implies that MDA is highly responsive to elevated salt stress. Chlorophyll content serves as an indicator of photosynthetic capacity and plant growth status. Specifically, chlorophyll a reflects the degree of absorption of long-wave light, while chlorophyll b facilitates the absorption of short-wave light. These functions play a crucial role in the transmission of light energy. Furthermore, this study showed that the contents of chlorophyll a, chlorophyll b, and total chlorophyll and related genes in S. portulacastrum of a few ecotypes in a low-salt environment were increased compared with those in the control group, while the contents of chlorophyll a, chlorophyll b, and total chlorophyll in S. portulacastrum of most ecotypes were decreased compared with those in the control group with the continuous increase in salt concentration. Similar results have been reported in previous studies [74][75][76]. This may be because salt stress damages the chloroplast structure of S. portulacastrum leaves and destroys the dynamic balance between chlorophyll synthesis and degradation, thus reducing the chlorophyll content.

Conclusions
In summary, soil salinity poses a significant threat to plant growth and crop yields, impacting global food production. With a substantial portion of the Earth's land already affected by salinization and projections indicating further expansion, understanding the mechanisms underlying salt stress response and tolerance is crucial. In this study, we investigated the physiological and gene expression changes in S. portulacastrum under varying salt concentrations. Our results demonstrated that salt tolerance factors and growth parameters exhibited distinct responses to salt stress. While low salt concentrations showed positive effects on plant growth, higher concentrations led to reduced growth rates and biomass. S. portulacastrum exhibited different degrees of salt tolerance across ecotypes. Osmotic regulation and proline accumulation were identified as key mechanisms for salt tolerance. The increase in proline levels and the upregulation of SpP5CS1 and SpLOX1 genes further supported their role in salt tolerance. The MDA content reflected the damage to lipid membranes caused by salt stress. Chlorophyll content decreased with increasing salt concentration, indicating chloroplast damage and disrupted photosynthetic capacity. Overall, this study enhances our understanding of the physiological and molecular responses of S. portulacastrum to salt stress, providing insights into salt tolerance mechanisms and potential strategies for crop improvement in salinity-affected areas.
Supplementary Materials: The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/genes14071336/s1, Figure S1: Measurement of internode length; Figure S2: Electrophoresis of total RNA; Table S1: Primers used for quantitative real-time PCR analysis.; Table S2: Loads, characteristic of roots, and contribution rates of variables in principal components.