Transcriptome Analysis and Functional Validation of JAZ Subfamily Genes of Sesuvium portulacastrum Under Salt and Cadmium Stresses

TL;DR

This study explores how Sesuvium portulacastrum responds to salt and cadmium stress by analyzing gene expression and validating the role of JAZ genes in stress tolerance.

Contribution

The study identifies and functionally validates JAZ subfamily genes in S. portulacastrum under salt and cadmium stress for the first time.

Findings

JAZ subfamily genes (SpJAZ1, SpJAZ5, SpJAZ7) are upregulated in S. portulacastrum under salt and cadmium stress.

Overexpression of SpJAZ1, SpJAZ5, and SpJAZ7 in Arabidopsis reduces hydrogen peroxide and chlorophyll loss while increasing proline and root growth under stress.

The JA signaling pathway interacts with stress mitigation pathways in S. portulacastrum under salt and cadmium stress.

Abstract

Plants are frequently exposed to various abiotic stresses during their growth and development. S. portulacastrum possesses inherent tolerance to salinity and heavy metals, yet the underlying molecular mechanisms remain poorly understood. In this study, we performed a comprehensive analysis of S. portulacastrum by integrating full-length transcriptome sequencing and RNA sequencing (RNA-seq) under salt stress conditions. Transcriptome analysis identified 2839 and 1813 DEGs in leaves and 7328 and 754 DEGs in roots at 7 and 14 ds after NaCl treatment, respectively. Pathway enrichment analysis indicated that these DEGs were significantly enriched in pathways associated with Photosynthesis, plant hormone signal transduction, Linoleic acid metabolism, chlorophyll metabolism, and amino acid metabolism. Expression profiling showed that JAZ subfamily genes were significantly upregulated in both leaves and roots under salt and Cd stress. We cloned SpJAZ1, SpJAZ5, and SpJAZ7, and generated their overexpression lines in Arabidopsis. Physiological assays demonstrated that overexpression of SpJAZ1, SpJAZ5, and SpJAZ7 reduced hydrogen peroxide content by 29.07%, 20.62%, and 19.79%, respectively, and lowered the reduction in chlorophyll content (0.12, 0.15, and 0.17 μg/mL vs. 0.22 μg/mL). Meanwhile, proline content was increased in these lines (2.34, 2.08, and 2.05 μg/mL vs. 1.53 μg/mL), alongside enhancements in root length, lateral root number, and water content under salt stress. Importantly, these overexpression lines displayed a similar functional trend under Cd stress. Collectively, our results reveal potential crosstalk between the JA signaling pathway and stress mitigation pathways in S. portulacastrum in response to salt and Cd stresses.

Figures

Full text

1. Introduction

Sesuvium portulacastrum (S. portulacastrum) is a highly reproductive saline plant of the dicotyledonous family Aizoaceae. It is distributed in sandy coastlines and salt marshes in tropical and subtropical regions, and has various ecotypes. S. portulacastrum exhibits its fast reproduction and natural characteristics of drought tolerance, heavy metal tolerance and salinity tolerance in numerous adverse environments, which makes it a potential plant for transforming saline and alkaline soils as well as restoring soils [1], attracting an increasing number of researchers to study its resistance to adversity. S. portulacastrum has a very strong enrichment capacity for cadmium (Cd) [2] and accumulates heavy metals such as chromium, Cd, copper, and zinc, and NaCl with the highest efficiency, in the leaves. The damage caused by Cd to plants is dose-dependent. With the increase in Cd concentration, the biomass of S. portulacastrum leaves and the height of seedlings first increase and then decrease, reaching the highest levels at 10 mg/L and 15 mg/L, respectively [3]. Studies have shown that under low-Cd-concentration conditions, superoxide dismutase (SOD) is the dominant antioxidant enzyme, while under high-Cd toxicity, peroxidase (POD) is the dominant enzyme. Under Cd stress, the cell walls of plant roots, stems and leaves deform and become thinner, and organelles such as chloroplasts and mitochondria deform. The number of nucleoli in the cell nucleus increases to maintain protein synthesis in a stressful environment [4].

Studies showed that more than one billion hectares of land worldwide are affected by salinization [5]. Plants adapt to high-salt environments by regulating ionic balance, activating osmotic stress pathways, mediating plant hormone signals, and adjusting cytoskeleton dynamics and cell wall composition [6,7,8,9]. S. portulacastrum can tolerate salt spray, sand scouring and burial, low soil nutrients and high temperatures. It can accumulate large amounts of sodium (Na). Appropriate Na levels can increase the net photosynthetic rate and promote the growth of S. portulacastrum [10]. S. portulacastrum can maintain normal growth in 400 mM NaCl solution, and when one part of the S. portulacastrum root was immersed in culture without NaCl treatment and the other part was immersed in 800 mM NaCl solution, it was still able to maintain a high growth potential [11]. It was also found that the fresh weight of S.portulacastrum grown in a hydroponic system containing 100–500 mM NaCl was equal to or significantly higher than that of S. portulacastrum grown in the same system, and that the number of roots of plants grown with 100 mM NaCl was similar to that of plants grown without NaCl [1]. Several genes in S. portulacastrum have been shown to promote salt tolerance in plants. For example, the water channel protein gene SpAQP1 from S. portulacastrum increased salt tolerance in transgenic tobacco [12]. Heterologous overexpression of the SpSOS1 and SpAHA1 genes increased salt tolerance in yeast and Arabidopsis [13]. A study has shown that high salinity reduces Cd accumulation in S. portulacastrum, and NaCl attenuates Cd toxicity in S. portulacastrum by maintaining plant water status and redox balance, protecting chloroplast structure and inducing some potential Cd^2+^ chelators such as GSH and proline [14]. Cd also interferes with the salt tolerance mechanism of S. portulacastrum. Cd supply delays the upregulation of salt-induced genes AHA1 and SOS1, and can also induce the activation of the betaine aldehyde dehydrogenase-encoding gene BADH to enhance osmotic stress [11]. However, research on the molecular mechanisms of salt resistance and Cd enrichment in plants is still lacking, which has hindered the development of work related to salt tolerance and heavy metal Cd resistance in S. portulacastrum.

Members of the TIFY gene family are widely distributed in plants, regulating their growth and development and resisting abiotic stress. The TIFY family of transcription factors has been shown to be active in many plant species such as Arabidopsis, kiwifruit, and Gossypium arboreum. The Jasmonate ZIM domain (JAZ) protein family is the subfamily with the highest numerical distribution in the TIFY transcription factor family and plays a significant role in the growth and development, resistance to adverse stress, and hormonal responses of various plants [15,16,17,18]. The expression of BnJAZ52 (BnC08.JAZ1-1) gene in Brassica napus was significantly inhibited under the treatment of abolic acid (ABA), gibberellin (GA), indoleacetic acid (IAA), polyethylene glycol (PEG) and NaCl, while it was induced under the treatment of methyl jasmonate (MeJA), low temperature and waterlogging [19]. Overexpression of FtJAZ12 and FtJAZ12 genes in Tartary Buckwheat (Fagopyrum tataricum) enhances the heat stress tolerance of tobacco [20]. The HaJAZ2/4/5/9 gene in Helianthus annuus is highly expressed under drought- and salt stress-induced conditions [21]. The JAZ proteins were found to directly interact with and inhibit the activities of the transcription factors ROOT HAIR DEFECTIVE 6 (RHD6) and RHD6-LIKE1 (RSL1) to regulate jasmonate-stimulated root hair development [22]. JAZ proteins act as members of an important link in the transduction of jasmonate signals from the SCFCOI1 receptor complex to downstream JA-responsive genes [22]. These studies provide us with a valuable theoretical basis for understanding the functions of JAZ family genes and the molecular mechanisms.

Plants are often exposed to alternating conditions of salt, alkali and heavy metals in the natural environment, making salt, alkali and heavy metal stress a dynamic and complex challenge [23,24]. Although substantial progress has been made in identifying plants’ tolerance to saline–alkali soil and heavy metals, the existing achievements have not fully illustrated the complex molecular mechanisms of plants’ responses to saline–alkali and heavy metal stress [25,26]. Many hub genes have been identified through coexpression analysis, but their functional validation remains insufficient. To address these challenges, this manuscript investigated the transcriptional responses of S. portulacastrum seedlings to salt and Cd stress, identified the key regulatory factors SpJAZ1, SpJAZ5 and SpJAZ7 involved in salt and Cd adaptation, which are involved in JA signaling responses to salt and Cd stress in S. portulacastrum. We cloned the SpJAZ1, SpJAZ5 and SpJAZ7 genes to construct heterologous overexpressed Arabidopsis mutants to verify their function. This in-depth analysis of the full-length transcriptomics of S. portulacastrum under salt and Cd stress provides valuable insights into the transcriptomics of S. portulacastrum. A deeper understanding of the functions and regulatory patterns of the salt and Cd response center genes is crucial for effectively utilizing them to enhance the salt and Cd tolerance of the S. portulacastrum and thereby implement saline–alkali land improvement strategies. It is an important resource for promoting the remediation of contaminated soil by the S. portulacastrum through molecular and genetic engineering strategies.

2. Results and Analysis

2.1. Full-Length Transcriptome Quality and DEGs Analysis

In this study, the PacBio sequencing platform was used to sequence the full-length transcript of S. portulacastrum. The raw sequencing data amounted to 34,282,237,208 bp, with 19,845,789 Subreads generated (average length: 3685 bp; N50 length: 2025 bp). After filtering, 837,519 circular consensus sequence (CCS) reads were obtained (Figure S1A). Statistical results for the length and quality of Reads of Insert (ROI) obtained after off-machine data filtering are shown in Figure S1B,C. The length distribution of full-length non-chimeric (FLNC) transcripts generated via ROI classification is presented in Figure S1D, while the length distribution of consensus transcript sequences after ICE clustering is shown in Figure S1E. Additionally, RNA-seq results revealed the following. After 7 ds of NaCl treatment, the number of reads per sample ranged from 38,187,089 to 41,536,271, with 27,151,803 to 30,581,516 reads aligning to the reference sequence (Table S1). After 14 ds of NaCl treatment, the number of reads per sample ranged from 39,245,175 to 42,796,309, with 29,213,524 to 32,060,343 reads aligning to the reference sequence (Table S1). We sequentially performed functional annotations of the full-length transcripts of S. portulacastrum using six databases: GO, KEGG, KOG, NR, NT, and Swiss-Prot. The number of transcripts with valid functional annotations in each database was as follows (Table S2). The proportion of transcripts simultaneously annotated in multiple databases is presented in Figure S2.

Biological replicates showed strong consistency (Figure 1A), confirming the reliability of the sequencing data for subsequent analyses. Principal component analysis (PCA) revealed distinct differences between treatment groups and the control, as well as between root and leaf samples of S. portulacastrum (Figure 1B). The first and second principal components (PC1 and PC2) accounted for 28.56% and 13.39% of the variance, respectively, with a cumulative variance of 41.95%. As shown in Figure 1C, the number of DEGs in S. portulacastrum under salt stress was as follows: Leaves (7 ds of salt treatment vs. control group): 601 upregulated genes, 2238 downregulated genes, total 2839 DEGs; Roots (7 ds of salt treatment vs. control group): 853 upregulated genes, 960 downregulated genes, total 1813 DEGs; Leaves (14 ds of salt treatment vs. control group): 4642 upregulated genes, 2686 downregulated genes, total 7328 DEGs; Roots (14 ds of salt treatment vs. control group): 429 upregulated genes, 325 downregulated genes, total 754 DEGs. Volcano plots confirmed these DEG trends (Figure S3). These results indicated that leaves of S. portulacastrum exhibited more DEGs than roots, suggesting that leaf tissues may play a more prominent role in acclimation to NaCl stress. Venn diagram analysis further revealed 20 DEGs universally expressed across all four treatment groups. Additionally, 203 DEGs were coexpressed in the NaCl _7Y_vs_CK and NaCl _7G_vs_CK comparisons, while 215 DEGs were coexpressed in the NaCl _14Y_vs_CK and NaCl _14G_vs_CK comparisons (Figure 1D). K-means clustering was used to analyze DEGs in S. portulacastrum roots and leaves under different treatments, classifying them into six subclasses based on similar expression patterns. Subclass 1 had the highest number of DEGs in leaves after 14 d NaCl treatment (7337 DEGs), while subclass 2 had the most in roots after 7-d NaCl treatment (15,183 DEGs), and subclass 4 had the most in leaves after 7-d NaCl treatment (12,751 DEGs). In contrast, subclasses 3 (12, 256 DEGs) and 6 (7722 DEGs) showed the highest DEG counts in the control group (Figure 1E).

2.2. Pathway Enrichment Analysis Under NaCl Stress in S. portulacastrum

GO annotation was performed on the full-length transcriptome to obtain information for each transcript, which was categorized into three main classes: biological process, molecular function and cellular component. Meanwhile, secondary-classification information statistics was conducted, as shown in Figure 2A, which reflects the distribution of the number of genes annotated to each GO term. The secondary classification consists of 50 categories. Among these, for biological process, there were 22 subcategories, with the largest number of genes annotated to cellular process (12,846), metabolic process (12,678), and single-organism process (9304). For cellular component, there were 17 subcategories, with the highest gene counts in cell (11,810), cellular component (11,762), and organelle (8723). For molecular function, there were 11 subcategories, with the majority of genes assigned to catalytic activity (11,239), binding (10,337), and transporter activity (1439). These results enrich the molecular biological data of S. portulacastrum and provide a more robust theoretical basis for further investigating its salt tolerance mechanism. KOG encompasses four major functional categories: Cellular Processes and Signaling, Metabolism, Information Storage and Processing, and Poorly Characterized. To characterize the full-length transcripts of S. portulacastrum, we performed KOG annotation to obtain KOG functional information for these transcripts. The successfully annotated transcripts were classified into 25 KOG subcategories, as shown in Figure 2B. Among these subcategories, the most abundant ones included: General function prediction only (2414 transcripts), Post-translational modification, protein turnover, chaperones (1567 transcripts), and Signal transduction mechanisms (1325 transcripts).

Pathway enrichment analysis was performed based on the KEGG database to investigate specific genes in roots and leaves. KEGG integrates information from multiple sources, including genomes, chemical molecules, and biochemical systems, enabling systematic analysis of metabolic pathways and biological effects associated with gene products and cellular compounds. The annotated transcripts were classified based on their involvement in KEGG metabolic pathways. The 7 d-old aboveground part (leaf) treatment group showed enrichment in multiple metabolic pathways, among which the enrichment pathways of Photosynthesis, Carotenoid biosynthesis, Terpene biosynthesis, Plant hormone signal transduction, and Amino acid metabolism were more significant (Figure 2C). The 14 d aerial (leaf) treatment group emphasized the importance of enrichment pathways such as Photosynthesis, sterol/flavone biosynthesis, Linoleic acid metabolism, Chlorophyll metabolism, Amino acid metabolism and Plant hormone signal transduction (Figure 2D). In the Photosynthesis pathway, we observed the differential expression of two branches of the phosphoenolpyruvate carboxylase (PEPC) gene, PPC1/PPC2, related to the CAM photosynthetic pathway; we tentatively speculate that S. portulacastrum may possess some characteristics of CAM photosynthetic plants (Figure S3). The Plant hormone signal transduction pathway was significantly enriched in the leaves of both the 7 d and 14 d salt treatment groups, indicating that it plays a vital role in S. portulacastrum salt tolerance. Plant hormones play a role in regulating ion balance, osmotic regulation, and antioxidant defense through a complex signaling network during salt stress responses, influencing physiological metabolism and gene expression. In many cases, plant salt tolerance is the result of the synergistic action of multiple hormones. KEGG pathway analysis was conducted on the differentially expressed plant hormone-signaling genes in the leaves that were treated with salt for 14 ds (Figure S4). The results showed that the related genes of IAA, cytokinin, ABA, brassinosteroid, and ethylene signaling pathways showed significant upregulation of the key genes of these hormone pathways. Among them, the JAZ and MYC2 genes on the JA pathway also experienced significant upregulation. Among various Plant hormone signaling pathways, we focused on the JA signaling pathway. JA is synthesized and converted to its active form via the JAR1 (Jasmonic Acid Resistant 1) enzyme: JAR1 catalyzes the conversion of JA to JA-Ile (its active form), initiating downstream signaling. JA-Ile binds to the COI1 (Coronatine-Insensitive 1) receptor, triggering downstream transcriptional regulation to modulate plant physiological responses. Red-marked genes in the JA signaling pathway exhibit significant upregulation, including JAR1, COI1, and MYC2. These genes play crucial roles in enhancing plant disease resistance, stress tolerance, and senescence, among other physiological functions (Figure 2E).

2.3. Identification of the TIFY Gene Family in S. portulacastrum

To evaluate the phylogenetic relationships of the TIFY family, a phylogenetic tree of 152 TIFY proteins from S. portulacastrum, Arabidopsis, Oryza sativa, Salvia miltiorrhiza, Gossypium arboreum, and Triticum aestivum resolved four subfamilies: TIFY, JAZ, PPD, and ZML. Those containing the TIFY and Jas domains belong to the JAZ subfamily. Those with the TIFY domain but lacking the conserved PY structure at the C-terminal end belong to the PPD subfamily. Those with the TIFY, ZnF_GATA and CCT domains belong to the ZML subfamily. Those containing only the TIFY domain belong to the TIFY subfamily. The 15 TIFY family members of S. portulacastrum are scattered among various subfamilies, and the same is true for TIFY family members of other species. SpTIFYs were distributed across all subfamilies, with seven JAZ, two PPD, five ZML, and one TIFY genes. The JAZ subfamily in S. portulacastrum further clustered into six groups, mirroring evolutionary patterns in other species (Figure 3A).

Using sequencing data from our laboratory, we identified a total of 27 candidate SpTIFY proteins via the BLASTP program 2.16.0+. Subsequently, we determined the protein kinase domains and conserved TIFY active center sequences through the Pfam database; fifteen SpTIFY family members were identified, with ORFs ranging from 345 bp (SpTIFY) to 1071 bp (SpPPD2), encoding polypeptides of 114–356aa. The predicted molecular weights spanned 117.86–385.66 kDa, and pI varied between 5.08 (SpZML5) and 9.94 (SpJAZ1). Subcellular localization predictions indicated nuclear localization for all 15 SpTIFY members (Table S3). To assess motif conservation, protein sequences of S. portulacastrum TIFY members were compared with those of Arabidopsis using MEME v5.5.9, revealing 10 conserved motifs (motifs 1–10). All 15 SpTIFYs harbored the highly conserved motif 1 (TIFY domain). SpJAZ1–SpJAZ7 additionally contained motif 2 (Jas domain), while SpZML1–SpZML5 possessed motif 3 (ZnF_GATA) and motif 4 (CCT motif) (Figure 3B). To further analyze the differences in the protein sequences of the TIFY transcription factor in S. portulacastrum, alignment of 15 SpTIFY and 18 AtTIFY proteins using BioEdit v7.7.1.0 highlighted conserved regions, including the N-terminal TIFY domain (Figure 3C). SpJAZ1–SpJAZ7 featured a C-terminal Jas domain with the SLX2FX2KRX2RX5PY signature. Notably, the amino acid sequence of SpJAZ1, SpJAZ5 and SpJAZ7 showed minimal divergence from AtTIFY10a, a known salt tolerance enhancer, suggesting their potential roles in salt tolerance.

2.4. Expression Analysis of TIFY Family Transcripts

To investigate whether JAZ genes are highly expressed under salt stress in S. portulacastrum, we detected the expression levels of JAZ genes as well as all genes belonging to the TIFY family after 400 mM NaCl treatment via quantitative real-time PCR (qRT-PCR). The quantitative experimental results showed that all 15 TIFY genes exhibited high expression levels across different tissues, whereas distinct variations in the expression levels were observed among individual genes of different subfamilies in various tissues. When S. portulacastrum was exposed to salt stress, nearly all members of the JAZ subfamily underwent rapid responses. In stems, four SpJAZ genes (SpJAZ1, SpJAZ5, SpJAZ6, SpJAZ7) and SpPPD1 reached their peak expression levels at 2 h post salt treatment. In leaves, all members of the JAZ subfamily maintained a sustained high expression pattern throughout the salt treatment period compared with the control group, with the exception of SpJAZ2, SpJAZ3 and SpJAZ4, whose expression level first increased and then declined. Members of the PPD and ZML subfamilies showed a trend of an increase followed by a decrease and then a subsequent increase in their expression levels. In addition, SpJAZ1, SpJAZ5, and SpJAZ7 exhibited relatively high expression levels, among which SpJAZ5 performed the best among all the examined genes across all tissues (Figure 4A). The qPCR results showed that under salt stress, the expression level of JAZ1 in leaves at 10 h was 2.33-fold higher than that of the CK group, and it reached a maximum of 4.1-fold higher in roots at 4 h. The expression level of JAZ5 in leaves at 2 h was 4.71-fold higher than that of the CK group, with a maximum of 4.28-fold higher in roots at 2 h. The expression level of JAZ7 in leaves at 2 h was 2.48-fold higher than that of the CK group, and it peaked at 5.68-fold higher in roots at 4 h (Figure 4B). Overall, in contrast to other subfamilies, the JAZ subfamily exhibited relatively high expression levels in various tissues, which indicates that SpJAZs play crucial roles in the response to salt stress.

Through mining and analysis of DEGs’ data, we selected TIFY family genes with high differential expression significance for quantitative analysis. The expression level of the gene was represented by the FPKM value measured by the TIFY family gene transcript. The data results indicate that the TIFY family genes in the S. portulacastrum plants show varying degrees of responses to salt stress in tissues such as roots and leaves, but all are significantly induced by salt stress. Members of the JAZ subfamily were significantly induced by salt stress in both the leaves and roots. This indicates that members of this family may play an important role in the response of seagrass to salt stress. From the transcriptome data obtained from our previous experiments on Cd stress in S. portulacastrum cells, we observed that almost all members of the JAZ subfamily can respond quickly under Cd stress (Figure S5A). Under heavy metal Cd stress conditions, the expression level of SpJAZ1 in the leaves of the S. portulacastrum reached three times that of the control group at 10 h and 2.8 times at 14 ds (Figure S5B). The expression level of SpJAZ7 reached three times that of the control group at 14 ds. The expression level of SpJAZ1 in the root part generally showed immediate downregulation in short-term exposure, with subsequent downregulation persisting through 7–14 d treatments. The expression level of SpJAZ7 remained consistently higher than that of the control group throughout the stress period. The expression level at 14 ds was 1.4 times that of the control group, and the expression patterns at 7 ds and 14 ds matched those of the genome-wide quantitative analysis data. These results suggest that SpJAZ1 and SpJAZ7 may function cooperatively in S. portulacastrum’s Cd stress response, with SpJAZ7 potentially playing a more consistent role in root metal detoxification while SpJAZ1 participates in shoot-specific signaling pathways. Among the JAZ subfamily, SpJAZ1, SpJAZ5 and SpJAZ7 were selected as candidate genes for analysis of their expression patterns under salt stress and Cd stress.

2.5. Cloning and Functional Validation of SpJAZs Genes in S. portulacastrum for Stress Resistance

To investigate the stress resistance conferred by the SpJAZs genes from S. portulacastrum, overexpression vectors SpGFP-JAZ1, SpGFP-JAZ5, and SpGFP-JAZ7 were constructed and introduced into Arabidopsis via Agrobacterium-mediated transformation (Figure S6). Positive transgenic lines were identified (Figure S7), and the T3 generation of stable transformants was selected for subsequent stress resistance assays.

2.5.1. Phenotypic and Physiological Analyses of Transgenic Arabidopsis Under Salt Stress

Notably, SpJAZ1 and SpJAZ7 demonstrated the most pronounced salt tolerance in these assays (Figure 5A). In soil-based experiments under high salt stress, all three transgenic lines exhibited superior survival and growth compared to the control (Figure 5B). Under normal 1/2 MS medium conditions, the root lengths of SpJAZ1 and SpJAZ7 transgenic Arabidopsis were significantly greater than those of the control group (transformed with the empty vector 2300-GFP-HA, CK), while no significant difference was observed for SpJAZ5 (Figure 5C). Additionally, the number of lateral roots in the control group was significantly lower than in all three transgenic lines (Figure 5D). When treated with 150 mM NaCl, the root lengths of SpJAZ1, SpJAZ5, and SpJAZ7 transgenic plants increased by 1.53 cm, 0.53 cm, and 1.37 cm respectively compared to the control group (Figure 5E). Compared to the control group, the number of lateral roots of SpJAZ1, SpJAZ5, and SpJAZ7 transgenic plants increased by 5.66, 2.66, and 4.33 respectively (Figure 5F). These results indicate that overexpression of SpJAZ1, SpJAZ5, and SpJAZ7 significantly enhances salt tolerance in Arabidopsis.

2.5.2. Hydrogen Peroxide Accumulation and Content Determination

Adversity stress can disrupt the balance of reactive oxygen metabolism in plants, leading to excessive accumulation of reactive oxygen species and damage to the membrane system and cell oxidation [6,27]. Hydrogen peroxide, due to its relative stability and ability to cross the membrane for diffusion, is the most important signal-type ROS [28]. The results of this study on the color development and content determination of hydrogen peroxide accumulation sites in plant material after salt stress showed that after salt stress at a certain concentration, the staining of the three different transgenic Arabidopsis materials was lighter than that of the control group, and the stained areas were mostly distributed at the petioles and veins (Figure 6A). In addition, the hydrogen peroxide content of the transgenic materials was significantly lower than that of the control group. OE_JAZ1, OE_JAZ5, and OE_JAZ7 decreased by 29.07%, 20.62%, and 19.79%, respectively (Figure 6B). This indicates that overexpression of SpJAZ significantly reduced the production of hydrogen peroxide. The transgenic Arabidopsis with the SpJAZ1 performed best in this evaluation index of salt tolerance.

2.5.3. Determination of Proline and Chlorophyll Content

Proline, as an important osmotic regulatory substance, accumulates in large quantities when plant cells are subjected to adverse stress. It regulates the plant’s tolerance to adverse conditions through maintaining the osmotic balance inside and outside the protoplast, stabilizing and protecting the cell membrane structure, eliminating free radicals, regulating energy metabolism and activating stress-related signaling pathways [29,30]. The results of this experiment show that the increase in proline before and after salt stress in the three transgenic Arabidopsis lines was significantly higher than that in the control group (the maximum increases were 2.34, 2.08, and 2.05 respectively, vs. 1.53 µg/mL). Among them, the increase in proline in the SpJAZ1 transgenic Arabidopsis was the highest, indicating that transgenic Arabidopsis can accumulate more proline to carry out corresponding metabolic regulation in the salt stress environment, thereby reducing the harm of salt stress to the organism (Figure 7A). Under stress, the amount of active oxygen free radicals in Arabidopsis will continue to increase. When it reaches a certain level, it will damage the chlorophyll in the leaves, which in turn affects the plant’s normal Photosynthesis. The experimental results show that the decrease in chlorophyll in the control group of Arabidopsis under salt stress is significantly higher than that in the three transgenic Arabidopsis (the reductions were 0.22 µg/mL vs. 0.12, 0.15, and 0.17 µg/mL, respectively) (Figure 7B), indicating that the degree of damage to the chloroplast structure in the leaves of the control group of Arabidopsis is higher, while the SpJAZ1 transgenic Arabidopsis suffers the least damage to its chloroplast.

2.5.4. Leaf Moisture Content and Water Loss Rate

Under osmotic stress, the level of relative water content is closely related to plant salt tolerance. The results of this study indicate that the relative water content of the three transgenic Arabidopsis (SpJAZ1, SpJAZ5, and SpJAZ7) was significantly higher than that of the control Arabidopsis (85.7%, 86.81%, 87.66% vs. 77.44%), among which the relative water content of SpJAZ7 transgenic Arabidopsis was the highest (Figure 7C). Moreover, within the 7 h observation period, the in vitro water loss rate of these three transgenic Arabidopsis significantly slowed down, while the in vitro water loss rate of the control Arabidopsis was faster (Figure 7D). This suggests that transgenic Arabidopsis can reduce the rate of water loss while maintaining high water content, thus conferring a certain degree of tolerance under salt stress. In order to verify the function of the JAZ gene in the S. portulacastrum, the common conserved region sequences of three genes, SpJAZ1, SpJAZ5 and SpJAZ7, were cloned and used as target-silencing sequences. SpJAZ-like genes were silenced via virus-induced gene silencing (VIGS) infiltration assay. After one week of salt treatment, compared with the other two control groups, the SpJAZ-like-silenced plants showed poor growth performance, with a large number of leaves exhibiting wilting symptoms. Meanwhile, qRT-PCR analysis revealed that targeting SpJAZ-like genes for silencing led to a significant downregulation of SpJAZ1 expression, whereas the expression levels of SpJAZ5 and SpJAZ7 in the experimental group showed no significant difference from those in the control groups (Figure S8). These results indicated that the constructed silencing vector achieved a satisfactory silencing effect on the SpJAZ1 gene, and also demonstrated that the SpJAZ1 gene positively regulates the salt tolerance of S. portulacastrum.

2.5.5. Effect of Salt Stress on the Expression Level of AtNHX1 in Arabidopsis

Studies have shown that under high salt stress, the plant membrane protein NHX1 can regulate cytoplasmic sodium ion levels via vacuolar ion balance, thereby alleviating salt stress-induced damage to plants. Our experiment, which measured the relative expression levels of AtNHX1 in different Arabidopsis materials following salt stress, revealed that the expression levels of AtNHX1 in the aerial parts of three transgenic Arabidopsis lines were significantly higher than those in the control group (Figure 8A). Notably, the SpJAZ1-overexpressing transgenic line exhibited the highest relative expression level of AtNHX1. In contrast, the relative expression levels of AtNHX1 in the roots of all three transgenic lines were lower than those in the control group (Figure 8B). This root-specific downregulation may be associated with negative feedback regulation of AtNHX1 within the JA signaling pathway in the roots of transgenic plants. These underlying mechanisms require further investigation, such as through protein interaction assays.

2.5.6. Growth and Physiological Indexes of Transgenic Arabidopsis Under Cd Stress

Furthermore, we conducted experiments on these three transgenic Arabidopsis plants under Cd stress conditions. In this experiment, following 14 ds of culturing on 6 µM CdCl_2_-treated 1/2 MS medium, the phenotypes and primary root lengths of SpJAZ1 and SpJAZ5 transgenic Arabidopsis exhibited no discernible difference compared with the control group. The Arabidopsis transfected with the 2300-GFP-HA empty vector (CK) exhibited a slightly longer primary root length than the control group, while the differences in the other phenotypes were less pronounced. Accordingly, subsequent experiments were conducted with an elevated Cd stress concentration, and the SpJAZ7 transgenic Arabidopsis was selected for functional analysis under Cd stress (Figure 9A).

Following a 14 d incubation period on 15 µM CdCl_2_-treated 1/2 MS medium, the seedlings were transferred to nutrient soil for a further two weeks. Subsequently, the treatment was watered with 1 mM CdCl_2_ for three ds. After 14 ds, the transgenic plants showed increased growth, with a higher number of leaves and longer, wider leaves that appeared oblong, in comparison to the control (trans2300-GFP-HA empty vector) Arabidopsis (Figure 9B). In the absence of Cd stress treatment, there was minimal observable difference in phenotype between the control and transgenic Arabidopsis. This suggests that the SpJAZ7 gene, which is located in the S. portulacastrum, can enhance the ability of Arabidopsis to tolerate Cd stress. Following a 7 d and 14 d incubation period in 1/2 MS medium containing 15 μM CdCl_2_, the root lengths of the three transgenic Arabidopsis plants of SpJAZ7 were 0.4 cm, 0.367 cm, and 0.367 cm longer than those of the control plants (Figure 9C,D). Additionally, the lateral root numbers of the transgenic Arabidopsis plants were significantly higher than those of the control plants (8 cm, 8.67 cm, 10.33 cm vs. 7.67 cm) (Figure 9E). This suggests that the SpJAZ7 transgenic Arabidopsis performs better in this evaluation index for tolerance to Cd.

The content of reactive oxygen radicals in plants will increase in adverse environments such as metal Cd stress, and when it reaches a certain level, these free radicals will cause damage to chlorophyll in the leaves, which in turn affects the normal Photosynthesis of the plants, and leads to the death of the plants in severe cases. The results of this study revealed that the amount of changes in chlorophyll content of control Arabidopsis under Cd stress, the changes in chlorophyll content in the control Arabidopsis plants were significantly greater than those in the transgenic Arabidopsis plants (0.16, 0.15, 0.15 vs. 0.20), indicating that the chlorophyll structure in the leaves of control Arabidopsis was damaged to a higher extent, and that the transgenic Arabidopsis suffered from the least degree of damage to chlorophyll (Figure 9F). The regulation of plant water content is an important component of Cd tolerance in plants, and the level of relative water content is closely associated with Cd tolerance under heavy metal Cd stress. The relative water content of the three transgenic Arabidopsis plants of SpJAZ7 was significantly higher than that of the control plants, being 8%, 9.3% and 7% higher, respectively, compared to the control group. This indicates that the transgenic Arabidopsis can maintain a high water content while conferring a certain degree of tolerance under Cd stress (Figure 9G).

3. Discussion

3.1. Vital Role of Transcriptome in the Research of Plant Gene Functions Under Adversity Stress

Affected by factors such as climate warming, increased industrialization and agricultural activities, salt stress and heavy metal pollution have become global environmental problems, seriously threatening and impacting plant growth, soil quality, ecosystem functions and biological health [31,32,33]. Salinity stress triggers the accumulation of H_2_O_2_ in plants, which oxidizes thiol groups of enzymes, leading to their inactivation [34]. Excessive passive uptake of Na^+^ ions under salt stress reduces root osmotic potential, impairs water absorption, and causes cellular dehydration, ultimately damaging membrane integrity, disrupting cell division, and leading to wilting [35]. With the emergence of third-generation sequencing technologies, such as PacBio, transcriptome datasets have become rich and comprehensive. Their research scope has covered nearly all fields, including agriculture and forestry, animal husbandry, biomedicine, and environmental science, playing a crucial role in investigating plant growth and development, stress responses, mutant phenotypes, crop quality, and genetic marker identification [36,37,38]. Full-length transcriptome datasets can serve as valuable resources for studying gene expression, alternative splicing, and evolutionary patterns in plant species [39,40]. To adapt to salt stress, plants have evolved diverse strategies that integrate exogenous salt stress signals with endogenous developmental cues (e.g., plant hormone levels, photosynthetic capacity, energy metabolism, ion homeostasis, and antioxidant enzyme activity) to optimize the balance between growth and stress tolerance. Plant hormones—including ABA and JA, which have received extensive attention in salt stress research—regulate plant growth adaptation by mediating salt signals and help plants establish defense systems via the targeted synthesis, signal transduction, and metabolic regulation of various hormones [41]. Additionally, interactions between plant hormone signals—such as the interaction of JA with other hormone signaling pathways (e.g., ABA [42], ET [43], SA [44], and BR [45]) within complex signaling networks—are central to plants’ responses to biotic and abiotic stresses.

Genes related to photosynthetic antenna proteins and carbon metabolism/carbon fixation pathways in grapevine (Vitis vinifera) are significantly downregulated under salt stress, indicating that salt stress damages the ultrastructure of chloroplasts [46]. The wheat epoxyalkane cyclase gene participates in salt stress responses through the JA biosynthesis pathway and the α-linolenic acid metabolism pathway [47]. The salt tolerance mechanism in sweet sorghum involves multiple processes: increasing Na^+^ exclusion capacity; maintaining root ion homeostasis under salt stress; protecting photosystem structure; enhancing light use efficiency, photosynthetic performance, and sucrose synthase activity; and inhibiting sucrose degradation to sustain high sugar content in shoots under salt stress [48]. In this study, via full-length transcriptome sequencing and RNA-seq analysis, we identified 10,883 salt-responsive DEGs. KEGG functional enrichment analysis revealed that these DEGs were significantly associated with various biological processes and metabolic pathways—including plant hormone signal transduction, Photosynthesis, carbohydrate metabolism, and glycerophospholipid metabolism—and that they significantly activated the JA signaling pathway. This suggests that S. portulacastrum participates in salt stress responses by activating multiple signaling pathways under salt stress conditions.

3.2. Critical Role of JAZ Genes in S. portulacastrum Under NaCl and Cd Stress

Plant hormones exert their functions in plant salt stress tolerance by regulating pathways such as ion homeostasis, osmotic adjustment, antioxidant defense, and growth and development remodeling [49,50]. Auxin (primarily indole-3-acetic acid, IAA) plays a central role in reshaping plant growth patterns through polar transport and signaling pathways, thereby optimizing resource allocation to cope with salt stress. The auxin response factor plays a role in promoting Na^+^ excretion by activating the Na^+^/H^+^ antiporter salt hypersensitive 1 (SOS1) in cellular ion homeostasis [51]. A study has revealed that the amino acid methionine significantly enhances plant salt tolerance. The activation of key genes governing methionine biosynthesis, namely Hcy-S-methyltransferases (HMTs) and methionine synthases (MSs), is controlled by the synergistic interaction between abscisic acid (ABA) and reactive oxygen species (ROS) signaling [52]. This coordinated gene activation subsequently leads to methionine accumulation, which activates ABA signaling and improves plant salt tolerance [52]. In addition to its role in regulating ABA signal transduction, methionine also affects root growth dynamics by inhibiting auxin and cytokinin signaling as well as impeding cell cycle progression. Furthermore, protein post-translational modifications play a crucial role in ABA-mediated abiotic stress responses by regulating core components of signal transduction [53]. ETH activates guard cell Ca^2+^ signaling through the EIN3/EIL1 transcription factors, inducing rapid stomatal closure. Ethylene also enhances jasmonic acid (JA) biosynthesis, and together they co-induce the expression of the PDF1.2 defense gene [54,55].

JA is a key plant hormone that regulates plant growth, development, and stress adaptation to cope with both biotic and abiotic stresses [41,56]. JA has been shown to be associated with salt stress tolerance in plants. In the plant hormone signal transduction pathway, JAZ genes participate in stress responses through the JA signaling cascade [57]. JAZ proteins, which function as critical suppressors in the JA signal transduction pathway, play crucial roles in multiple biological processes—particularly in plant stress responses. Additionally, studies have shown that AtJAZ4, AtJAZ8, and IAA29 in Arabidopsis can interact with WRKY57 to regulate Arabidopsis leaf senescence via a shared JA- and auxin-dependent signaling pathway [58]. Overexpression of the soybean GsJAZ2 gene in transgenic Arabidopsis enhances the plant’s tolerance to saline–alkali stress [59]. In Salvia miltiorrhiza (Danshen), overexpression of SmJAZ3, SmJAZ4, or SmJAZ8 in Arabidopsis significantly enhances salt tolerance by increasing antioxidant enzyme activity, chlorophyll content, proline accumulation, and Na^+^/K^+^ homeostasis; in contrast, SmJAZ1 exerts an antagonistic effect on salt tolerance [60]. Furthermore, a regulatory module comprising SmJAZ proteins, SmbHLH37, SmERF73, and SmSAP4 balances salt tolerance via JA signaling [61]. In Arabidopsis, NaCl stress activates JA signaling by upregulating JA-responsive JAZ genes in roots, in a COI1-dependent manner. JA-Ile sensor assays confirm JA pathway activation in root meristem and elongation zones—an activation that depends on JAR1 function and proteasome activity [57]. In this study, among the pathways enriched in the transcriptome analysis of S. portulacastrum under salt stress, the JA signaling pathway was significantly enriched. JAR1 (jasmonate-resistant 1) catalyzes the conversion of JA to its biologically active form, JA-Ile; JA-Ile then binds to the COI1 (coronatine-insensitive 1) F-box receptor, triggering downstream transcriptional regulation that modulates plant physiological responses. Similarly, a correlation exists between JA signal transduction and Cd detoxification. In potato (Solanum tuberosum), Cd stress results in significant enrichment of JA pathway-related genes—including members of the StOPR and StJAZ families—suggesting conserved mechanisms that link JA signaling to Cd detoxification [62]. Under Cd stress, RNA-Seq analysis revealed differential expression of JAZ genes in white poplar (Populus alba) [63]. In rapeseed, Cd stress significantly induces JAZ subfamilies, with their genes highly expressed across all tissues [64]. This aligns with our findings: Cd stress significantly induces JAZ gene expression in S. portulacastrum, with SpJAZ1 highly expressed in aerial parts and SpJAZ7 showing high expression in both aerial parts and roots. Collectively, these results indicate that JAZ genes play a conserved role in regulating stress responses across diverse plant species—underscoring their conserved function in modulating plant adaptation to abiotic stresses (e.g., Cd and salt).

Within plant cells, TIFY genes regulate the expression levels of JAZ proteins to convey feedback on environmental stresses and participate in plant stress responses—such as drought and salt tolerance. Studies have shown that in Arabidopsis, there are eight AtJAZ genes, corresponding to AtTIFY10a, AtTIFY10b, AtTIFY11a, AtTIFY11b, AtTIFY5a, AtTIFY5b, AtTIFY7, and AtTIFY9; these members exhibit high sequence conservation. All of these AtJAZ genes participate in JA signal responses, interact with AtMYC2 (a key JA signaling regulator) through protein–protein interactions, and employ mechanisms such as alternative splicing to adapt to environmental changes [65]. This study focuses on S. portulacastrum, a true halophyte with high salt tolerance. Through comprehensive bioinformatics analysis, we identified 15 members of the TIFY family in this species; the sequences of these 15 members also exhibit high conservation. Consistent with TIFY family members in other plant species, these 15 TIFY members are classified into distinct subfamilies, including 7 JAZ subfamily genes, 2 PPD subfamily genes, 5 ZML subfamily genes, and 1 gene belonging to the TIFY subfamily. We analyzed and verified the expression levels of TIFY family genes after salt stress in aerial and root parts. Among all genes in the TIFY family, the ones that responded rapidly to salt stress and maintained high expression levels are SpJAZ1, SpJAZ5, and SpJAZ7. Previous studies have shown that AtTIFY10a (from Arabidopsis) is a key gene in the JA signaling pathway: overexpression of AtTIFY10a enhances salt tolerance in Arabidopsis [66]. Notably, three genes in S. portulacastrum—SpJAZ1, SpJAZ5, and SpJAZ7—share the highest sequence homology with AtTIFY10a. Therefore, we can predict the mechanism of action of JAZ genes in S. portulacastrum during JA signaling responses by referencing the characterized functions of JAZ genes in Arabidopsis, thereby clarifying their roles in JA-mediated stress signaling.

Notably, the diversity of JAZ proteins driven by alternative splicing and sequence variation likely underpins functional specialization [67]. In this study, we observed distinct expression patterns of S portulacastrum JAZ subfamily members (SpJAZs) under Cd and salinity stress, suggesting they play stress-specific regulatory roles by interacting with distinct transcription factors or coregulators. Strikingly, SpJAZ7 exhibited pronounced upregulation under Cd stress; its extended gene length relative to other SpJAZs implies potential structural modifications that enable it to bind to unique downstream partners, which diverges from the interactors of canonical JAZ proteins. This functional diversification may reflect adaptive evolution in halophytes for coping with concurrent abiotic stressors in coastal ecosystems.

3.3. Morphological and Physiological Responses of S. portulacastrum to NaCl and Cd

Recent studies have highlighted the association between high salinity tolerance in S. portulacastrum and transcriptional reprogramming [68]. For instance, SpAQP1 (an aquaporin gene in S. portulacastrum) has been shown to mediate salt stress responses by enhancing antioxidant activity, thereby improving salinity tolerance [13]. In this study, we observed that overexpression of SpJAZ1/5/7 in Arabidopsis under salt stress resulted in significant reductions in H_2_O_2_ production and increases in proline content. Exogenous proline application has been demonstrated to enhance plant stress resilience by promoting osmotic adjustment and stimulating growth. For instance, proline supplementation in salt-stressed plants increases antioxidant enzyme activity, thereby mitigating oxidative damage to cell membranes [69]. In this study, we observed that compared with the Arabidopsis control group, three transgenic Arabidopsis lines (OE-SpJAZ1/5/7, overexpressing S. portulacastrum JAZ genes) exhibited significant improvements in multiple physiological, biochemical, and phenotypic traits. These included increased root length, more lateral roots, reduced chlorophyll loss, higher relative water content, and lower water loss rate. In other studies, JAZ genes have also been shown to alleviate the salt stress-induced decline in physiological indicators such as chlorophyll content. For example, heterologous overexpression of maize (Zea mays) ZmJAZ13 in Arabidopsis significantly enhanced the plant’s salt tolerance by increasing chlorophyll content, reducing MDA levels, and boosting antioxidant enzyme activity [70]. Interestingly, studies have shown that salinity can enhance the Cd tolerance of aquatic Chlamydomonas species. For example, under 200 mM NaCl, aquatic Chlamydomonas could survive even with high Cd concentrations in its tissues. This tolerance was attributed to NaCl-induced changes in Cd ion speciation, altered Cd distribution in cells, and enhanced biosynthesis of glutathione (GSH) and proline—all of which alleviated oxidative stress and maintained photosynthetic function [71]. In the present study, under Cd stress, overexpression of SpJAZ7 significantly improved the growth performance of S. portulacastrum, including increases in root length, lateral root number, and relative water content, while also alleviating the reduction in chlorophyll levels.

The NHX gene family plays a crucial role in plants’ response to salt stress by encoding Na^+^/H^+^ antiporters, which help regulate sodium ion transport across cellular membranes [72]. Researchers have successively identified that NHX genes are associated with salt tolerance in Cucurbita L. [73], wheat [74], tomato [75], and Vigna mungo [76]. Stress-inducible expression of AtNHX1 significantly enhanced tolerance to ionic, osmotic, and oxidative stresses under salt stress in transgenic mungbean plants compared to wild-type plants [77]. In this study, our results demonstrate that the relative expression levels of AtNHX1 in the aerial parts of three transgenic Arabidopsis lines were significantly higher than those in the control group. Notably, in the SpJAZ1-overexpressing transgenic line, its expression level was even higher. In contrast, the relative expression levels of AtNHX1 in roots were lower than those in the control group, demonstrating distinct tissue specificity. Previous studies have indicated that SpNHX1 expression is induced by salt stress. Therefore, we hypothesize that the proteins encoded by SpJAZ1, SpJAZ5, and SpJAZ7 may interact with the protein encoded by SpNHX1, with a higher likelihood of interaction between SpJAZ1 and SpNHX1. This suggests that in S. portulacastrum, JAZ proteins may be directly involved in regulating the function of the NHX1 membrane protein.

4. Materials and Methods

4.1. Experimental Material

Material was collected near the waters of Hongsha Pier, Jiyang District, Sanya City, Hainan Province, China, and cuttings of S. portulacastrum with consistent growth environment and physiological status were selected. Stem segments containing four opposite leaves and two nodes were cut from the stock plant, about 5 cm long, and placed into tap water to be cultivated for 14 d under natural conditions.

4.2. Integrated Analysis of Full-Length Transcriptome and RNA-Seq Under Salt Stress

Total RNA was extracted from both salt-stressed (400 mM NaCl) and control samples using the RNAprepPure Plant RNA Kit (Kangwei Biotechnology Co., Ltd., Beijing, China, for polysaccharide- and polyphenol-rich tissues). The salt stress concentration (400 mM NaCl) used for transcriptome sequencing was selected based on the experimental method of Cao et al. [78]. RNA quality was verified prior to library construction (RIN > 8.0, OD260/280 = 1.8–2.2). Full-length cDNA libraries were prepared using the SMARTer PCR cDNA Synthesis Kit (Kangwei Biotechnology Co., Ltd., Beijing, China) and sequenced on the PacBio Sequel platform, yielding 34.28 Gb of raw data. For Illumina RNA-seq, libraries were constructed with the NEBNext Ultra RNA Library Prep Kit (New England Biolabs Co., Ltd., Beijing, China) and sequenced on the NovaSeq 6000 platform, generating 38–42 million reads per sample. Full-length transcriptome data were processed using SMRTlink v8.0 (PacBio) and corrected with Illumina (Novogene, Beijing, China) short reads. Differential expression analysis was performed using DESeq2 (thresholds: |log2FC| > 1, adjusted p-value < 0.05). Functional annotation was conducted using the NR, NT, SwissProt, GO, and KEGG databases, and the genes without functional annotations were analyzed by CDS prediction. Enrichment analysis of salt stress-responsive genes included expression profiling, differential expression analysis, and GO/KEGG pathway enrichment, followed by hierarchical clustering.

4.3. Identification and Expression Analysis of TIFY Gene Family Members in S. portulacastrum

4.3.1. Identification of Members of the TIFY Gene Family in S. portulacastrum

The TIFY candidate protein of S. portulacastrum was obtained by local Blastp [79]. The obtained sequences were further used to identify the TIFY family members of S. portulacastrum using the InterProScan v5.77 [80]. The relative molecular mass and isoelectric point of the protein were predicted using the Calculate tool in the Expasy online database [81]. The subcellular localization was analyzed using the WoLFPSORT internal program.

4.3.2. Analysis of Protein Conserved Motifs

The amino acid sequences were profiled using MEME v5.5.9 to obtain the conserved motifs of the TIFY family members in S. portulacastrum [82].

4.3.3. Multiple Sequence Comparison Analysis

Multiple sequence alignment of S. portulacastrum TIFY proteins was performed using BioEdit tool v7.7.1.0 software [83].

4.3.4. Phylogenetic Tree Construction

Use the Clustal-W program in MEGA 7.0 software to perform multiple sequence alignment of the TIFY protein sequences of S. portulacastrum, Arabidopsis, rice, wheat, Salvia miltiorrhiza and Asiatic cotton. Set the algorithm to neighbor-joining and set the bootstrap to 1000 repeated construction of the phylogenetic tree, and evaluate the constructed phylogenetic tree [84].

4.3.5. Expression Analysis of JAZ Genes

To validate the expression patterns of TIFY family genes identified in transcriptomic analyses under salt and Cd stress, qRT-PCR was performed. For salt stress, S. portulacastrum seedlings were treated with 400 mM NaCl in 1/2 Hoagland nutrient solution for 12 h, with roots, stems, and leaves sampled hourly. For Cd stress, plants were exposed to 25 mg/L CdCl_2_ in 1/2 Hoagland solution for 12 h (roots and leaves sampled every 2 h) or subjected to prolonged treatment for 7 and 14 ds. Tissues from three biological replicates per timepoint were collected. Gene-specific primers for TIFY members were designed using Primer Premier 5.0 (Table S4), with GAPDH serving as the internal reference. Relative expression levels were calculated via the 2^−ΔΔCt^ method, and heatmaps were generated using TBtools-II software v2.225 [85] to visualize tissue- and stress-specific transcriptional dynamics.

4.4. Generation of SpJAZs-Overexpressing Arabidopsis via Agrobacterium-Mediated Transformation

Full-length coding sequences of target genes were amplified from S. portulacastrum cDNA using gene-specific primers (Table S5). A second round of PCR introduced homologous arms to the amplified products. The pCAMBIA2300-GFP-HA vector was digested with BamHI and KpnI, and ligated with the PCR fragments to generate overexpression constructs (Table S5). Recombinant plasmids were transformed into DH5α for amplification, verified by sequencing, and subsequently introduced into GV3101. Wild-type Arabidopsis plants were transformed via floral dip. T0 seeds were surface-sterilized and screened on 1/2 MS medium containing 40 µg/mL kanamycin. Putative transgenic seedlings were transplanted into soil at the two-true-leaf stage. Genomic DNA extracted from one-week-old seedlings was PCR-verified using four independent primer sets (Table S6) to exclude false positives. T1 and subsequent generations (T2, T3) were successively selected under identical antibiotic conditions. All functional assays were conducted using homozygous T3 lines to ensure genetic stability.

4.5. Verification of Transgenic SpJAZs Arabidopsis Stress Resistance Function

4.5.1. Observation of Transgenic Arabidopsis Growth Under Salt Stress and Cd Stress

To assess salt tolerance, 7 d-old seedlings grown on 1/2 MS medium were transferred to either fresh 1/2 MS or 1/2 MS supplemented with 150 mM NaCl for 10 ds, followed by quantification of primary root length and lateral root density. The selection of 150 mM NaCl concentration on the culture medium was based on the experimental method of Cao et al. [78]. For soil-based salt stress assays, seedlings pre-cultured on 1/2 MS medium were transplanted into soil for 14 ds and irrigated with 350 mM NaCl for 5 ds to evaluate growth phenotypes. To screen Cd sensitivity, T3 homozygous seeds of SpJAZ1, SpJAZ5, and SpJAZ7 overexpressing lines were germinated on 1/2 MS medium containing 6 μM CdCl_2_ under controlled light for 14 ds, with root architecture and growth phenotypes analyzed. Based on differential stress tolerance, SpJAZ7-overexpressing lines were selected for further validation. These seedlings were subsequently cultured on 15 μM CdCl_2_-supplemented medium for 14 ds, transplanted into soil, and irrigated with 1 mM CdCl_2_ for 3 ds after acclimatization. Rosette leaf size and root morphology were quantified at 7 and 14 ds post-treatment, with three biological replicates per condition to ensure reproducibility.

4.5.2. Observation of Hydrogen Peroxide Accumulation Sites and Determination of Hydrogen Peroxide Content

In this experiment, the H_2_O_2_ content detection kit was used to determine the H_2_O_2_ content. The samples were cultured on 1/2 MS medium for 7 ds, then transferred to soil for 14 ds, and then irrigated with 350 mM NaCl for 72 h. The hydrogen peroxide content was measured sequentially. By treating each sample and measuring the absorbance of each sample, the hydrogen peroxide content in the tissue can be calculated.

4.5.3. Determination of Proline Content

Arabidopsis seeds were cultured on 1/2 MS medium for 7 d, then transferred to soil for 4 weeks and watered with 350 mM NaCl for 72 h. Arabidopsis seedlings were sequentially sampled before and after watering to determine the Pro content, with three replicates for each treatment. In this experiment, the changes in Pro content were determined using the Pro Content Assay Kit (Shanghai Haling Biotechnology Co., Ltd., Shanghai, China).

4.5.4. Chlorophyll Content and Water Status Analysis Under Cd Stress

For chlorophyll quantification, 7 d-old Arabidopsis seedlings pre-cultured on 1/2 MS medium were transplanted into soil and grown for four weeks before 350 mM NaCl irrigation for 72 h. Chlorophyll levels were measured before and after treatment using acetone extraction, with three biological replicates per condition. To assess Cd effects, T3 SpJAZ7-overexpressing seedlings were germinated on 15 μM CdCl_2_-supplemented 1/2 MS medium for 14 ds, transferred to soil for two weeks, and irrigated with 1 mM CdCl_2_ for 3 ds, followed by chlorophyll analysis.

For water status evaluation, 5-week-old soil-grown plants were subjected to rosette leaf sampling. Leaves from identical nodal positions were rinsed with sterile water, blotted dry, and placed adaxial side up in a climate-controlled chamber. Fresh weight was recorded hourly to calculate leaf water content and detached water loss rate using standardized formulas.

$$[eqn]$$ $$[eqn]$$

4.5.5. Virus-Induced Gene Silencing (VIGS) of JAZ Genes in S. portulacastrum

A TRV silencing vector was constructed, and the corresponding Agrobacterium infection solution was prepared to inoculate S. portulacastrum seedlings. Primers were designed targeting a 405 bp conserved region shared by SpJAZ1, SpJAZ5, and SpJAZ7 as the gene-silencing segment. After obvious leaf yellowing symptoms appeared in the positive control plants, the S. portulacastrum materials were treated with 350 mM NaCl. One week later, the growth status of S. portulacastrum was observed, and the apical leaves of the corresponding materials were harvested and stored in liquid nitrogen. Total RNA was extracted from the leaves to detect the silencing efficiency of the target genes. Both the control and experimental groups were set up with at least three biological replicates. Silent sequence primer information for transgenic Arabidopsis are shown in Table S7.

5. Conclusions

This study provides the first comprehensive analysis of the expression patterns and functional validation of JAZ subfamily genes in S. portulacastrum under salt and Cd stress. By integrating full-length transcriptome and RNA-seq data, we identified 10,883 salt-responsive DEGs. Among these, seven JAZ subfamily members were characterized, and three genes—SpJAZ1, SpJAZ5, and SpJAZ7—were cloned and overexpressed in Arabidopsis. Functional assays demonstrated that overexpression of these genes significantly enhanced the stress tolerance of transgenic plants: SpJAZ1 exerted the most prominent effect on improving salt tolerance, while SpJAZ7 showed excellent performance in Cd detoxification. This establishes a molecular framework for elucidating the mechanisms underlying salt and Cd tolerance in halophytes. Our results indicate a potential interaction between the JA signaling pathway and stress resistance pathways under salt and Cd exposure, providing new insights into the JA-mediated regulatory network. Furthermore, this study deepens our understanding of the role of JAZ proteins in the JA signaling pathway, highlighting their evolutionary conservation and functional plasticity across different plant species. Additionally, this work provides valuable genetic resources and a theoretical basis for screening S. portulacastrum germplasm resources with high Cd tolerance and breeding plants with enhanced adaptability to multiple adverse environments.