Physiological Differences and Transcriptional Regulatory Characteristics of Salt-Tolerant and Salt-Sensitive Grapevine Cultivars Under Salt Stress

Abstract

Salt stress is a major abiotic factor limiting grapevine growth and yield. To elucidate the physiological and molecular regulatory mechanisms underlying salt tolerance in grapevine, this study used ‘Carménère’ (Vitis vinifera) and ‘Pinot Noir’ (Vitis vinifera) as experimental materials. Under 200 mmol/L NaCl stress, the physiological response characteristics of the two cultivars were systematically compared, and transcriptome sequencing combined with qRT-PCR analysis was conducted to explore the molecular basis of their differences in salt tolerance. The results showed that salt stress significantly impaired photosynthetic performance and disrupted cellular homeostasis in grapevine; however, the reductions in relative chlorophyll content (SPAD value), maximum photochemical efficiency of photosystem II (Fv/Fm), and photosynthetic performance were significantly smaller in ‘Carménère’ than in ‘Pinot Noir’, indicating greater stability of the photosynthetic apparatus in ‘Carménère’. Meanwhile, ‘Carménère’ maintained higher activities of antioxidant enzymes and higher levels of non-enzymatic antioxidants, effectively reducing reactive oxygen species accumulation and membrane lipid peroxidation. In addition, under salt stress, ‘Carménère’ accumulated greater amounts of osmotic adjustment substances and maintained lower Na⁺ content and higher K⁺ content, demonstrating a more efficient capacity for osmotic regulation and ion homeostasis. Transcriptomic analysis revealed that the plant hormone signal transduction, MAPK signaling, and glutathione metabolism pathways were significantly enriched in ‘Carménère’, with multiple key genes being coordinately upregulated under salt stress. Taken together, these findings indicate that ‘Carménère’ achieves enhanced salt tolerance through a multilayered signaling regulatory network that coordinates physiological defense responses. This study provides a theoretical basis for elucidating the mechanisms of salt tolerance in grapevine and for the molecular breeding of salt-tolerant cultivars.

1. Introduction

Salt stress is a major abiotic factor affecting agricultural production, with more than one-third of the world’s irrigated land currently impacted by salinization [1]. Soil salinization disrupts soil structure, reduces soil fertility, and imposes ionic toxicity and osmotic stress on crops, thereby leading to significant declines in global crop yields and posing a serious threat to food security and sustainable agricultural development [2]. Grapevine (Vitis vinifera L.), an economically important fruit crop cultivated worldwide, plays a crucial role in agricultural and economic development [3]. However, grapevine growth and development are highly sensitive to environmental stresses [4], among which soil salinization has become a major limiting factor for grape production. This problem is particularly pronounced in northwestern China, a region characterized by arid and semi-arid climates, where long-term water scarcity combined with improper irrigation and fertilization practices has intensified secondary soil salinization. As a result, grape cultivation in this region is exposed to severe saline–alkaline stress, making it a key environmental constraint on high-quality yield formation and sustainable development of the grape industry [5,6]. Salt stress induces osmotic imbalance in plant cells, accumulation of reactive oxygen species (ROS), inhibition of photosynthesis, and disruption of ion homeostasis, ultimately leading to retarded growth, impaired development, and even plant death under severe conditions. These effects greatly restrict grape yield and quality [7,8,9]. Therefore, screening germplasm resources with strong salt tolerance and systematically elucidating their physiological, metabolic, and molecular regulatory mechanisms underlying salt tolerance are of significant theoretical importance and practical value.

At present, substantial progress has been made in research on salt tolerance in grapevine, and related studies have systematically elucidated the core mechanisms by which plants cope with salt stress from both physiological and molecular perspectives. At the physiological level, plants primarily employ the following coordinated strategies to mitigate salt stress. First, ion homeostasis regulation: vacuolar Na⁺/H⁺ antiporters (NHXs) sequester Na⁺ into vacuoles or facilitate its extrusion from the cytoplasm, thereby maintaining low cytosolic Na⁺ concentrations and alleviating ionic toxicity [10]. Second, osmotic adjustment: by accumulating organic osmolytes such as proline (Pro) and soluble sugars (SS), plants reduce cellular osmotic potential and maintain water balance as well as enzyme structural stability [11]. Third, antioxidant defense: salt stress-induced reactive oxygen species (ROS) are scavenged through the activation of antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), in coordination with non-enzymatic antioxidants such as ascorbic acid (AsA) and glutathione (GSH), thereby mitigating oxidative damage [12,13]. At the molecular level, salt stress responses depend on a complex network composed of precise signal transduction and gene expression regulation. Salt stress signals are first perceived by receptors on the cell membrane and are subsequently transmitted via second messengers such as Ca²⁺ and ROS, leading to the activation of the salt overly sensitive (SOS) pathway, mitogen-activated protein kinase (MAPK) cascades, and hormone signaling pathways, particularly those mediated by abscisic acid (ABA) [14]. These pathways do not function independently; rather, they are intricately interconnected and act synergistically to form an integrated regulatory network that finely modulates plant adaptive responses to salt stress.

However, several limitations remain in current studies. Most existing work has focused on the salt tolerance-related physiological responses of a single cultivar or on the isolated analysis of differentially expressed genes, lacking a systematic integration of physiological phenotypes with underlying molecular mechanisms. Moreover, the differences in salt tolerance between ‘Carménère’ and ‘Pinot Noir’ have not been clearly defined in either domestic or international studies. Their coordinated regulation of physiological traits and their molecular response patterns under salt stress remain poorly characterized, which hinders the provision of sufficient theoretical support for salt-tolerant breeding and mechanistic elucidation. Based on the systematic screening of salt tolerance across multiple grapevine cultivars conducted in our research group, and further supported by the independent validation provided by the comprehensive physiological data obtained in the present study, ‘Carménère’ and ‘Pinot Noir’ exhibit markedly contrasting salt tolerance under salt stress. Accordingly, the present study uses these two cultivars as experimental materials and combines physiological measurements with transcriptome sequencing to perform a comparative analysis under salt stress. The aim of this research is to provide a theoretical basis and genetic resources for the development of salt-tolerant grapevine germplasm, cultivation on saline–alkaline soils, and the identification of functional genes associated with salt tolerance.

2. Results

2.1. Physiological and Biochemical Analyses of ‘Carménère’ and ‘Pinot Noir’ Under Salt Stress

In this study, after 7 days of salt stress treatment, distinct phenotypic differences were observed between ‘Carménère’ and ‘Pinot Noir’. The leaves of ‘Pinot Noir’ seedlings exhibited pronounced chlorosis, whereas those of ‘Carménère’ under salt stress showed only slight marginal yellowing and sporadic small brown spots (Figure 1).

Compared with their respective control groups (CK), no significant changes were detected in Fo or Fm in either cultivar under salt stress. However, in ‘Pinot Noir’, Fv/Fm and SPAD values were significantly reduced by 4.71% and 22.66%, respectively, relative to the control, whereas these parameters showed no significant changes in ‘Carménère’ (Figure 2A–D). Overall, salt stress markedly inhibited Fv/Fm and SPAD values in ‘Pinot Noir’ seedlings, indicating that ‘Carménère’ possesses a stronger salt tolerance than ‘Pinot Noir’.

In addition, changes in antioxidant enzymes and non-enzymatic antioxidants were further examined in this study. Compared with their respective control groups (CK), SOD activity was significantly increased in both cultivars under salt stress, whereas POD activity was significantly decreased. Specifically, in ‘Pinot Noir’, SOD activity increased by 37.66% and POD activity decreased by 44.00%; in ‘Carménère’, SOD activity increased by 57.55% and POD activity decreased by 24.10%. CAT activity showed no significant change in ‘Pinot Noir’ but increased significantly by 29.64% in ‘Carménère’. APX activity did not change significantly in either cultivar. The AsA content in ‘Pinot Noir’ decreased significantly by 39.40% under salt stress. GSH content declined significantly in both cultivars, with decreases of 78.0% in ‘Pinot Noir’ and 76.2% in ‘Carménère’ (Figure 3A–F).

Taken together, the two cultivars exhibited marked differences in antioxidant enzyme activities and non-enzymatic antioxidant contents under salt stress. ‘Carménère’ showed a more coordinated and reinforced antioxidant regulatory response, characterized by a greater increase in SOD activity and a significant enhancement of CAT activity. In contrast, the antioxidant response in ‘Pinot Noir’ was relatively weaker, as evidenced by a pronounced reduction in AsA content and more severe depletion of GSH. These results indicate that ‘Carménère’ possesses a stronger tolerance to salt stress.

Under stress conditions, the levels of H₂O₂ and ·O₂⁻ in plants reflect the accumulation of reactive oxygen species and the degree of oxidative stress, whereas malondialdehyde (MDA) content directly indicates the extent of membrane lipid peroxidation damage. Compared with their respective control groups (CK), the H₂O₂ content increased significantly in both cultivars under salt stress, with increases of 94.78% in ‘Pinot Noir’ and 18.4% in ‘Carménère’. The ·O₂⁻ content showed no significant change in ‘Pinot Noir’ but increased significantly by 86.45% in ‘Carménère’. In addition, the MDA content in ‘Pinot Noir’ decreased significantly by 34.44% under salt stress, whereas no significant change was observed in ‘Carménère’ (Figure 4A–C).

Proline and soluble sugars are typical osmotic adjustment substances in plants and can alleviate oxidative damage induced by salt stress by maintaining cellular osmotic balance. Compared with the respective control groups (CK), the Pro content in ‘Pinot Noir’ increased significantly by 106.25% under salt stress, whereas no significant change was observed in ‘Carménère’. The SS content increased significantly in both cultivars, with increases of 42.39% in ‘Pinot Noir’ and 64.59% in ‘Carménère’, respectively (Figure 4D,E).

Sodium (Na⁺) and potassium (K⁺) ions play crucial roles in maintaining intracellular osmotic balance. Compared with their respective control groups (CK), the Na⁺ content in ‘Pinot Noir’ increased significantly by 23.16% under salt stress, whereas no significant change was observed in ‘Carménère’. Meanwhile, the K⁺ content in ‘Pinot Noir’ decreased significantly by 18.07%, whereas that in ‘Carménère’ increased significantly by 24.30% (Figure 4F,G).

Taken together, the two cultivars exhibited pronounced differences in reactive oxygen species metabolism, osmotic adjustment, and ion homeostasis under salt stress. ‘Carménère’ showed a lower degree of ROS accumulation without inducing significant membrane lipid peroxidation, as evidenced by a smaller increase in H₂O₂ content and no significant change in MDA levels, along with significant increases in soluble sugar and K⁺ contents. In contrast, ‘Pinot Noir’ exhibited a greater increase in H₂O₂ accumulation, relied primarily on proline-mediated osmotic adjustment, and was accompanied by marked ion homeostasis imbalance. These results indicate that ‘Carménère’ possesses a more coordinated and integrated physiological regulatory capacity in response to salt stress, further confirming that its salt tolerance is significantly superior to that of ‘Pinot Noir’.

2.2. Correlation Analysis of Physiological Parameters in ‘Carménère’ and ‘Pinot Noir’

Pearson correlation analysis of physiological parameters in ‘Carménère’ and ‘Pinot Noir’ under salt stress revealed marked differences in their physiological regulatory networks (Figure 5A,B). The regulatory network of ‘Pinot Noir’ was characterized by localized coordination but overall disorder, with antagonistic interactions across different modules, whereas ‘Carménère’ exhibited a multi-module synergistic and globally ordered regulatory pattern.

In ‘Pinot Noir’, the following patterns were observed. Photosynthetic system: Fm, Fv/Fm, and SPAD values were significantly positively correlated, indicating coordinated changes between photosynthetic efficiency and chlorophyll content. In contrast, Fo was negatively correlated with Fv/Fm, and Na⁺ content showed significant negative correlations with photosynthetic parameters, suggesting that disruption of ion homeostasis directly inhibited photosynthetic function. Antioxidant system: Although AsA and GSH contents were highly coordinated with POD activity, SOD activity was significantly negatively correlated with POD and CAT activities, indicating an imbalance between H₂O₂ generation and scavenging. MDA content was negatively correlated with SOD activity but showed significant positive correlations with downstream scavenging components (such as POD and AsA), confirming that internal dysregulation of the antioxidant system exacerbated membrane lipid peroxidation. Osmotic adjustment and ion balance: Proline and soluble sugars accumulated coordinately but were significantly negatively correlated with AsA and GSH, reflecting antagonistic interactions between different regulatory systems. Na⁺ accumulation was negatively correlated with K⁺ content and showed significant negative correlations with photosynthetic parameters, further disrupting overall metabolic homeostasis.

In contrast, the regulatory network in ‘Carménère’ was more coordinated: Photosynthetic system: Fo, Fm, Fv/Fm, and SPAD values were significantly positively correlated, and Na⁺ accumulation had a weaker inhibitory effect on photosynthetic parameters, reflecting the system’s higher stability and resistance to stress under the salt treatment. Antioxidant system: SOD and CAT activities were strongly positively correlated with H₂O₂ and ·O₂⁻ levels, while MDA content was negatively correlated with antioxidant enzyme activities, indicating a high level of synchronization between reactive oxygen species (ROS) production and scavenging, effectively mitigating oxidative damage. Osmotic adjustment and ion balance: The accumulation of SS was highly positively correlated with SOD and CAT activities, showing a synergistic relationship between osmotic regulation and the antioxidant system. Na⁺ and K⁺ contents were positively correlated, and Na⁺ accumulation did not induce severe membrane damage (as shown by the negative correlation with MDA), indicating stronger ion homeostasis maintenance ability in ‘Carménère’.

In summary, ‘Pinot Noir’ lacks efficient cross-module coordination among its physiological parameters, with even antagonism present between different systems, leading to an overall disordered regulatory network. In contrast, ‘Carménère’ has formed an orderly network with highly efficient coordination across multiple modules, including photosynthesis, antioxidant defense, osmotic regulation, and ion balance. These results indicate that ‘Carménère’ has significantly better salt tolerance compared to ‘Pinot Noir’.

2.3. Transcriptomic Differential Analysis of VG-17 and VG-20 Under Salt Stress

To investigate the effects of salt stress on gene expression in self-rooted grapevine seedlings of VG-17 (‘Carménère’) and VG-20 (‘Pinot Noir’), transcriptome sequencing was performed using leaf samples collected after 7 days of treatment with 200 mmol/L NaCl, with plants irrigated with an equal volume of water serving as the control. Sequencing data for all samples are summarized in Supplementary Table S2. The results demonstrated high sequencing quality, with Q30 base percentages ranging from 96.17% to 96.89% and GC contents between 44.69% and 45.75%. Alignment to the grape reference genome showed a high mapping rate, ranging from 90.25% to 94.12%. Pearson’s correlation coefficient (R²) was used to evaluate the consistency among biological replicates in the transcriptome datasets. The results showed that the R² values among the three biological replicates for each genotype under both treatments were all greater than 0.9 (Figure 6A), indicating high correlation and good reproducibility among replicates. Furthermore, principal component analysis (PCA) revealed a clear separation of all samples in the multivariate space (Figure 6B), demonstrating significant transcriptomic differences among treatments and genotypes and confirming that the data were suitable for subsequent differential expression analyses.

Using |FC| ≥ 1.5 and FDR < 0.05 as the thresholds, a total of 2866 differentially expressed genes (DEGs; 1564 up-regulated and 1302 down-regulated) and 2788 DEGs (1703 up-regulated and 1085 down-regulated) were identified in the comparisons of VG-17CK vs. VG-17S and VG-20CK vs. VG-20S, respectively (Figure 7A). The Venn diagram (Figure 7B) showed that 764 DEGs were shared between the two comparisons. In addition, 2024 and 2102 specifically expressed DEGs were detected in the VG-17CK vs. VG-17S and VG-20CK vs. VG-20S groups, respectively. These results indicate that the salt-tolerant grape germplasm can effectively induce the expression of its own genes to alleviate the negative effects imposed by salt stress.

2.4. GO Annotation and KEGG Pathway Enrichment Analysis of DEGs

GO enrichment analysis was conducted on the 764 DEGs shared between the two comparisons (VG-17CK vs. VG-17S and VG-20CK vs. VG-20S) identified from the Venn diagram, as well as on the 2102 DEGs derived from the salt-tolerant genotype VG-17. Significant enrichment was observed across all three major Gene Ontology categories: Biological Process (BP), Molecular Function (MF), and Cellular Component (CC) (Figure 8A). Within the BP category, significantly enriched terms included the L-serine catabolic process (GO:0006565), phosphorylation (GO:0016310), and the cytokinin-activated signaling pathway (GO:0009736), all of which are closely associated with plant responses to salt stress. In the MF category, the major enriched functions comprised oxidoreductase activity (GO:0016491), DNA-binding transcription factor activity (GO:0003700), and serine binding (GO:0070905), indicating that these functions are actively involved in regulating the antioxidant defense system in grapevine and are indispensable for alleviating oxidative damage. Regarding the CC category, enriched genes were predominantly localized to the plasma membrane (GO:0005886), nucleus (GO:0005634), and membrane components (GO:0016021), suggesting that these cellular structures play critical roles in salt-stress responses. Overall, the GO enrichment analysis reveals a multilayered and coordinated strategy by which salt-tolerant grapevine genotypes respond to salt stress. The DEGs in the salt-tolerant genotype contribute to enhanced stress resistance and antioxidant defense capacity by participating in key biological processes such as L-serine catabolism, phosphorylation, hormone signal transduction, and oxidoreductase activity, thereby ultimately improving salt tolerance.

KEGG pathway enrichment analysis of the 2866 common DEGs revealed that several pathways were significantly overrepresented, including plant hormone signal transduction (ko04075), the MAPK signaling pathway (ko04016), and glutathione metabolism (ko00480) (Figure 8B). These pathways play pivotal roles in the grapevine response to salt stress. Specifically, the plant hormone signal transduction pathway contributes to enhanced salt tolerance in grapevine by regulating the dynamic balance of endogenous phytohormones. The MAPK signaling pathway further strengthens plant adaptation to stressful environments by activating defense-related responses. In addition, the glutathione metabolism pathway serves a central role in antioxidant defense during plant responses to salt stress, primarily through scavenging reactive oxygen species, maintaining redox homeostasis, repairing oxidative damage, and participating in stress-related signal transduction. Overall, these findings provide important insights into the molecular mechanisms underlying grapevine adaptation to salt stress and identify potential targets for future functional studies.

2.5. Effects of Salt Stress on Plant Hormone Signal Transduction

Salt stress markedly affected the plant hormone signaling system in grapevine. During the response to salt stress, plant hormone signal transduction coordinates growth regulation and stress adaptation through signal perception, downstream transcriptional regulation, and activation of defense-related genes. In this study, a total of 97 differentially expressed genes (DEGs) associated with plant hormone signal transduction were identified, among which 61 genes were upregulated under salt stress conditions (Figure 9A). These genes collectively formed the regulatory networks illustrated in Figure 9B–E.

Among these DEGs, several key regulatory factors exhibited markedly enhanced expression levels in the salt-tolerant genotype VG-17, whereas they showed a down-regulation trend in the salt-sensitive genotype VG-20. Specifically, SCL13 (VIT_00s0463g00020) and SAPK3 (VIT_02s0236g00130), which are involved in hormone signal transduction and stress responses, were significantly upregulated in VG-17, indicating that the salt-tolerant genotype possesses a stronger capacity for stress signal perception and transmission. In addition, TIFY10A (VIT_11s0016g00710), a key regulatory component of the jasmonic acid signaling pathway, was induced by salt stress in VG-17 but suppressed in VG-20, suggesting its potential role in regulating salt-tolerance–related defense responses. Meanwhile, the transcription factors BHLH41 (VIT_11s0016g02070) and BHLH35 (VIT_11s0052g00100) were significantly upregulated in the salt-tolerant VG-17 but showed reduced expression in the salt-sensitive VG-20. These expression patterns indicate that VG-17 can more effectively activate downstream hormone-responsive genes under salt stress, thereby facilitating the coordinated regulation of growth, defense, and adaptive responses.

From a pathway-specific perspective, the elevated expression of AUX1 (VIT_18s0001g03540), GH3 (VIT_03s0091g00310), AUX/IAA (VIT_04s0008g00220, VIT_07s0141g00270, VIT_09s0002g04080, and VIT_14s0081g00010), and SAUR genes (VIT_02s0154g00010, VIT_04s0023g00580, VIT_07s0031g02740, VIT_08s0058g01160, VIT_15s0048g00530, and VIT_16s0098g01150) in VG-17 within the auxin signaling pathway likely contributes to the maintenance of cell elongation and growth regulation under salt stress (Figure 8B). In the gibberellin signaling pathway, the upregulation of DELLA-related genes (VIT_00s0226g00070, VIT_00s0463g00020, VIT_06s0004g04950, VIT_06s0004g04960, and VIT_08s0056g00050) (Figure 9C) may enable the salt-tolerant genotype to achieve a balance between growth restraint and survival under stress conditions. Moreover, the significant upregulation of PYR/PYL receptors and SnRK2 kinases in the abscisic acid (ABA) signaling pathway (Figure 9D), together with the enhanced expression of JAZ (VIT_09s0002g00890 and VIT_11s0016g00710) and MYC2 transcription factors (VIT_02s0025g03450, VIT_11s0016g02070, VIT_11s0052g00100, and VIT_18s0001g08040) in the jasmonic acid signaling pathway (Figure 9E), collectively indicate that VG-17 can integrate multiple hormone signaling pathways under salt stress. This coordinated hormonal regulation facilitates comprehensive control over water status, defense responses, and stress adaptation, thereby contributing to enhanced salt tolerance.

2.6. Effects of Salt Stress on MAPK Signaling

Salt stress markedly activated the MAPK signaling system in grapevine plants. As a key signal amplification module in plant stress responses, the MAPK signaling pathway coordinates antioxidant defense and the expression of stress-responsive genes by integrating reactive oxygen species (ROS) signals, hormone signaling, and downstream transcriptional regulation. In this study, a total of 80 differentially expressed genes (DEGs) associated with MAPK signaling were identified, of which 51 were upregulated under salt stress conditions (Figure 10A).

Among these DEGs, several key regulatory factors exhibited markedly enhanced expression in the salt-tolerant cultivar VG-17, whereas their expression was suppressed in the salt-sensitive cultivar VG-20. Specifically, the transcription factors WRKY11 (VIT_11s0052g00450) and ERF1 (VIT_07s0005g03230) were significantly upregulated in VG-17, indicating that the salt-tolerant cultivar is more capable of efficiently transducing MAPK signaling into downstream transcriptional responses of stress-related genes. Meanwhile, the elevated expression of the ABA receptor PYL4 (VIT_02s0236g00130) and the antioxidant-related gene CAT1 (VIT_18s0122g01320) suggests that MAPK signaling may coordinately regulate salt tolerance through crosstalk with hormone signaling pathways and ROS-scavenging mechanisms.

From the perspective of specific regulatory modules, the MAPK signaling pathway functions through interconnected crosstalk networks, including the H₂O₂-MAPK, ethylene-MAPK, and ABA-MAPK signaling modules (Figure 10B–D). In the H₂O₂-MAPK pathway, the upregulation of WRKY family transcription factors and MPK3/6 enhances the responsiveness to reactive oxygen species signals. In the ethylene-MAPK crosstalk pathway, the pronounced upregulation of ERF1 indicates that MAPK signaling cooperates with ethylene signaling to regulate stress-responsive transcription. In the ABA-MAPK cascade, the increased expression of PYL4 (VIT_02s0236g00130), SnRK2 (VIT_02s0236g00130; VIT_07s0031g03210), and CAT1 (VIT_18s0122g01320) contributes to the enhancement of antioxidant defense capacity under salt stress conditions.

Overall, the coordinated activation of MAPK signaling pathway-related genes in VG-17 indicates that this genotype can more effectively adapt to salt-stress conditions through an integrated regulatory mode encompassing signal integration, transcriptional regulation, and the execution of antioxidant defenses.

2.7. Effects of Salt Stress on the Glutathione Metabolism Pathway

The glutathione metabolism pathway plays a crucial antioxidative regulatory role in plant responses to salt stress, primarily by scavenging reactive oxygen species (ROS) and maintaining cellular redox homeostasis to alleviate oxidative damage. In this study, a total of 11 differentially expressed genes (DEGs) related to glutathione metabolism were identified, of which seven were upregulated under salt stress (Figure 11A), and these genes were involved in the metabolic and regulatory network illustrated in Figure 11B. Further analysis revealed that the glutathione reductase (GR) genes (VIT_00s0317g00050 and VIT_14s0006g00630), as well as several members of the glutathione S-transferase (GST) gene family (VIT_01s0026g01340, VIT_07s0005g00030, VIT_17s0000g02950, VIT_19s0015g02700, and VIT_19s0093g00150), exhibited significantly higher expression levels in the salt-tolerant cultivar VG-17 than in the salt-sensitive cultivar VG-20. The differential expression of these genes suggests that, under salt stress, VG-17 may more effectively eliminate toxic compounds generated by oxidative stress through enhancement of the glutathione cycle and detoxification capacity, thereby mitigating cellular damage. These findings are consistent with the trends observed in antioxidation-related physiological parameters and further support the pivotal role of the glutathione metabolism pathway in the development of salt tolerance in VG-17.

2.8. Differential Expression of Key Salt Tolerance-Related Genes Between VG-17 and VG-20 Under Salt Stress

To further elucidate the molecular response mechanisms of grapevine under salt stress and to identify genes with enhanced expression associated with salt tolerance, this study selected 11 key genes from pathways such as plant hormone signal transduction for validation using quantitative real-time PCR (qRT-PCR). The results are presented as follows:

(1)

Plant hormone signal transduction pathway

To validate genes involved in the plant hormone signaling pathway, SCL13 (VIT_00s0463g00020), SAPK3 (VIT_02s0236g00130), TIFY10A (VIT_11s0016g00710), BHLH41 (VIT_11s0016g02070), and BHLH35 (VIT_11s0052g00100) were selected for analysis. Under salt stress, the expression levels of SCL13, SAPK3, TIFY10A, BHLH41, and BHLH35 in VG-20 were downregulated by 0.73-, 0.80-, 0.88-, 0.56-, and 0.53-fold, respectively, compared with the control. In contrast, in VG-17, the expression levels of SCL13, SAPK3, TIFY10A, BHLH41, and BHLH35 were upregulated by 1.37-, 1.16-, 1.10-, 1.92-, and 1.73-fold, respectively, relative to the control (Figure 12A–E).

(2)

MAPK signal transduction pathway

To validate genes involved in the MAPK signaling pathway, WRKY11 (VIT_11s0052g00450), ERF1 (VIT_07s0005g03230), PYL4 (VIT_02s0236g00130), and CAT1 (VIT_18s0122g01320) were selected for analysis. Under salt stress, the expression levels of WRKY11, ERF1, PYL4, and CAT1 in VG-20 were downregulated by 0.91-, 0.72-, 0.80-, and 1.40-fold, respectively, compared with the control. In contrast, in VG-17, the expression levels of WRKY11, ERF1, PYL4, and CAT1 were upregulated by 1.25-, 1.55-, 1.36-, and 1.56-fold, respectively, relative to the control (Figure 12F–I).

(3)

Glutathione metabolism pathway

To validate genes involved in the glutathione metabolism pathway, DHAR3 (VIT_14s0006g00630) and PARC (VIT_19s0015g02700) were selected for analysis. Under salt stress, the expression levels of DHAR3 and PARC in VG-20 were downregulated by 1.10- and 0.89-fold, respectively, compared with the control. In contrast, in VG-17, the expression levels of DHAR3 and PARC were upregulated by 1.64- and 1.25-fold, respectively, relative to the control (Figure 12J,K).

The qRT-PCR validation results are highly consistent with the gene expression trends observed in the transcriptomic sequencing data, further confirming the reliability and accuracy of the RNA-seq data (Figure 11). A comprehensive analysis of the expression profiles of 11 key genes from three core pathways-plant hormone signaling, MAPK signaling, and glutathione metabolism-revealed significant molecular response differences between the two varieties under salt stress. In the salt-sensitive variety VG-20, the expression of these 11 genes was generally downregulated after salt treatment, indicating a weaker response in salt tolerance-related signaling and antioxidant defense pathways. In contrast, in the salt-tolerant variety VG-17, these genes were significantly upregulated, with the expression levels showing a generally higher fold increase. Notably, genes such as BHLH41, CAT1, and DHAR3 showed upregulation exceeding 1.5-fold.

It is noteworthy that the validated genes are involved in different functional processes, such as stress signal perception, transcriptional regulation, and antioxidant defense execution. Their synergistic upregulation in VG-17 indicates that the salt-tolerant variety is capable of more effectively activating the relevant molecular pathways under salt stress. These results further support the hypothesis that plant hormone signaling, MAPK signaling, and glutathione metabolism pathways, along with their key genes, may collectively regulate VG-17’s adaptive response to salt stress. This provides a reliable basis for further in-depth analysis of the molecular mechanisms underlying salt tolerance and their association with physiological phenotypes.

3. Discussion

Salt stress is a common abiotic stress factor that significantly limits crop growth and agricultural production [15]. According to statistics, approximately 20% of global arable land and 50% of irrigated farmland are affected by varying degrees of salinity stress [16,17], making it a major constraint on sustainable agricultural development. The results of this study show that under 200 mmol/L NaCl treatment, several physiological processes in grapevines were significantly impacted. Salt stress impaired photosynthetic performance, weakened antioxidant system function, and led to a massive accumulation of reactive oxygen species (ROS), while also disrupting ion homeostasis and osmotic balance. These findings are consistent with studies by Wang K [18] on rice and Guo R [19] on maize, which together demonstrate that salt stress disrupts plant physiological processes, thereby hindering their normal growth and development.

Photosynthesis is a fundamental process for plant growth and energy metabolism, and it is also one of the most sensitive physiological processes to salt stress [20]. Salt stress significantly limits photosynthetic capacity by inhibiting chlorophyll synthesis, reducing the efficiency of Photosystem II (PSII) reaction centers, and destabilizing their structural integrity [21,22]. In this study, salt stress caused changes in the photosynthetic indices of both grapevine varieties, but ‘Carménère’ showed significantly smaller declines in SPAD values and Fv/Fm compared to ‘Pinot Noir’, while maintaining higher photosynthetic performance. This indicates that its photosynthetic system is more stable under stress. This result is consistent with the common characteristic of salt-tolerant plants, which alleviate the suppression of photosynthesis by maintaining the integrity of chlorophyll synthesis and photosystem structures. This principle has been validated in salt tolerance studies of various crops [23]. Salt stress induces excessive ROS accumulation in plants, leading to oxidative damage, and an efficient antioxidant system is a key physiological basis for plant salt tolerance [24,25,26]. This study shows that under salt stress, the activities of SOD, POD, CAT, and APX in ‘Carménère’ were significantly higher than those in ‘Pinot Noir’, while its GSH and AsA content decreased less, indicating that ‘Carménère’ can more effectively activate both enzymatic and non-enzymatic antioxidant systems to maintain intracellular redox balance. Correspondingly, the accumulation of H₂O₂, ·O₂⁻, and membrane lipid peroxidation product MDA was significantly suppressed in ‘Carménère’, suggesting stronger ROS scavenging ability and effectively reducing salt stress-induced damage to membrane structures. This is consistent with the conclusions of Arif Y et al. [27] on the impact of salt stress on plants, which found that plants can effectively remove ROS induced by salt stress by significantly upregulating antioxidant enzyme activity and accumulating non-enzymatic antioxidants, thereby alleviating oxidative damage. In addition to oxidative stress, salt stress also disrupts the osmotic balance in plant cells, causing dehydration and metabolic disorder. Plants respond to osmotic pressure imbalance and enhance salt tolerance by accumulating osmotic regulators and maintaining ion homeostasis, which is a key physiological strategy [28]. This study found that ‘Carménère’ accumulated more osmotic regulators (Pro, SS) under salt stress, while maintaining a lower Na⁺ content and higher K⁺ content, indicating more efficient osmotic regulation and ion compartmentalization. This effectively alleviated the physiological damage caused by salt. This is consistent with the findings of Singh P et al. [29] on plant salt stress resilience, which showed that plants can effectively maintain cellular osmotic balance and reduce stress-induced losses by accumulating osmotic regulators and regulating Na⁺/K⁺ absorption and transport. Although the changes in these physiological indices clearly indicate that ‘Carménère’ has stronger tolerance to salt stress, these phenotypic differences are essentially derived from deeper molecular regulation.

Plants need to quickly sense external changes under salt stress and translate these signals into stable molecular responses and physiological adjustments. Previous studies have shown that plant hormone signaling, MAPK cascades, and transcription factors collectively form the core signaling network for plants’ stress responses [30,31]. This study found that the salt-tolerant variety ‘Carménère’ simultaneously activates plant hormone signaling, MAPK signaling, and multiple key transcription factors under salt stress. In contrast, these responses were significantly weaker in the salt-sensitive variety ‘Pinot Noir’. These results suggest that the synergistic activation of multiple pathways may be the molecular basis for ‘Carménère’ salt tolerance.

Abscisic acid (ABA), jasmonic acid (JA), and auxins are hormones that can rapidly respond to osmotic stress and ion toxicity, and regulate the expression of downstream defense genes [32,33,34]. This study found that in ‘Carménère’ (VG-17), SCL13, SAPK3, TIFY10A, BHLH41, and BHLH35 were significantly upregulated under salt stress, while these genes were generally downregulated in ‘Pinot Noir’ (VG-20). These results suggest that ‘Carménère’ can more effectively activate plant hormone signaling pathways, thereby initiating the stress response earlier and laying the foundation for subsequent signal amplification and defense reactions. The MAPK signaling cascade plays a critical role in amplifying signals and integrating multiple pathways in plant stress responses [14]. The MAPK cascade can integrate reactive oxygen species (ROS), hormones, and environmental signals, and transmit the information to downstream transcription factors to induce the expression of defense-related genes [35]. In this study, ‘Carménère’ significantly upregulated WRKY11, ERF1, PYL4, and MAPK cascade-related genes under salt stress, while the expression levels of these genes were significantly reduced in ‘Pinot Noir’. These differences suggest that ‘Carménère’ may achieve sustained amplification and stable transmission of salt stress signals through a stronger MAPK cascade response. Transcription factors play a key role in converting signal perception into physiological responses [36]. Transcription factors such as WRKY, ERF, and BHLH can directly regulate the expression of antioxidant enzymes, osmotic regulation-related genes, and other defense genes under salt stress [37]. This study shows that ‘Carménère’ exhibits synergistic upregulation of multiple transcription factors under salt stress, while ‘Pinot Noir’ shows a weaker transcriptional response under the same treatment. These differences suggest that ‘Carménère’ can not only quickly initiate the stress response but also enhance long-term salt tolerance by maintaining continuous expression of defense genes.

In summary, ‘Carménère’ (VG-17) demonstrates more significant advantages under salt stress in terms of photosynthetic stability, antioxidant defense capacity, and osmotic and ionic regulation. These synergistic physiological responses provide important support for ‘Carménère’ in maintaining strong growth and cellular homeostasis in high-salinity environments. Combined with the transcriptomic analysis results, it can be further observed that these physiological advantages are not isolated, but rather are coordinated by a multi-level signaling regulatory network. Comprehensive analysis reveals that under salt stress, ‘Carménère’ forms a continuous regulatory pattern initiated by hormone signaling, amplified by MAPK cascade responses, and executed through transcription factor mediation. This regulatory pattern converts short-term stress signals into relatively stable molecular responses and continuously induces the expression of genes related to antioxidant defense, osmotic regulation, and ion homeostasis.

Therefore, this multi-level synergistic mechanism of physiological response and transcriptional regulation may constitute an important molecular regulatory basis for the enhanced salt tolerance of ‘Carménère’ compared to ‘Pinot Noir’. Although this study provides an integrated physiological and transcriptomic framework to explain the differential salt tolerance between the two cultivars, several limitations should be acknowledged. In particular, the candidate genes and regulatory pathways identified in this study were inferred based on expression patterns and pathway enrichment analyses, rather than direct functional validation. Therefore, the precise causal roles of specific genes in conferring salt tolerance remain to be experimentally confirmed.

Future studies employing functional approaches, such as gene overexpression, gene knockout, or genome editing, will be essential to verify the contribution of these candidate genes to salt tolerance and to further dissect their regulatory interactions within hormone signaling, MAPK cascades, and downstream transcriptional networks. Such efforts will significantly enhance mechanistic understanding and strengthen the translational potential of these findings for grapevine salt-tolerant breeding.

4. Materials and Methods

4.1. Seedling Cultivation and Treatments

One-year-old self-rooted grapevine seedlings of the cultivars ‘Carménère’ and ‘Pinot Noir’ were used as experimental materials. The seedlings were purchased from Jinguoshu Grape Farm, Qingdao, Shandong Province, China. After rinsing under running water for 12 h, the seedlings were immersed in a rooting agent solution at a concentration of 100 mg/L for 20 min and then transplanted into plastic pots (27.5 cm in diameter and 31 cm in height). Each pot was filled with approximately 0.9 kg of a growth substrate consisting of a 3:1 (v/v) mixture of nutrient soil and vermiculite. After transplanting, the seedlings were thoroughly irrigated and cultivated under natural light conditions (without artificial photoperiod control), with average day/night temperatures maintained at 25 ± 2 °C/18 ± 2 °C. An appropriate amount of water was supplied every 5 days.

After six weeks of growth, grapevine seedlings with 15–20 fully expanded leaves and uniform growth status were selected for salt stress treatment [38]. The experiment included a control group (CK) and a salt treatment group (S), with three biological replicates for each treatment (i.e., six pots per cultivar). For the salt treatment, each pot was irrigated once with 500 mL of 200 mmol/L NaCl solution, whereas the control group received an equal volume of distilled water [39]. After 7 days of treatment, obvious phenotypic changes were observed in the leaves. Fully expanded leaves were collected from each seedling from top to bottom and used for subsequent physiological, biochemical, and transcriptomic analyses.

4.2. Determination of Chlorophyll Fluorescence Parameters and Relative Chlorophyll Content

On day 8 of the salt treatment, the 6th to 8th leaves from the apex of each grapevine plant were selected for the determination of chlorophyll fluorescence parameters and relative chlorophyll content, including minimum fluorescence (Fo), maximum fluorescence (Fm), and the maximum photochemical efficiency of photosystem II (Fv/Fm). Measurements were conducted between 09:00 and 11:00 using a FluorPen FP110/D handheld chlorophyll fluorescence meter equipped with a detachable leaf clip (Photon Systems Instruments; provided by Ekolite Ecological Technology Co., Ltd., Beijing, China). Prior to measurement, all samples were subjected to dark adaptation for 20 min.

Relative chlorophyll content (SPAD value) was measured using a TYS-B handheld chlorophyll meter manufactured by TOP Cloud-Agri Technology Co., Ltd. (Hangzhou, China). At the end of the experiment, fully expanded leaves were harvested from each grapevine seedling, immediately frozen in liquid nitrogen, and stored at −80 °C for subsequent analyses.

4.3. Measurement of Antioxidant Enzyme Activities and Non-Enzymatic Antioxidants

Antioxidant enzyme activities and the contents of non-enzymatic antioxidants were determined using commercial assay kits provided by Suzhou Grace Biotechnology Co., Ltd. (Suzhou, China), with three biological replicates for each parameter. The specific indices measured and the corresponding kit numbers were as follows: superoxide dismutase (SOD, G0102W), peroxidase (POD, G0108W), catalase (CAT, G0106W), ascorbate peroxidase (APX, G0203W), reduced ascorbic acid (AsA, G0201W), and glutathione (GSH, G0206W).

4.4. Determination of Osmotic Adjustment Substances, Reactive Oxygen Species, and Lipid Peroxidation Products

The contents of osmotic adjustment substances, reactive oxygen species, and lipid peroxidation products were determined using commercial assay kits supplied by Suzhou Grace Biotechnology Co., Ltd. (Suzhou, China), with three biological replicates for each index. The specific parameters measured and the corresponding kit numbers were as follows: proline (Pro, G0111W), soluble sugars (SS, G0501W), hydrogen peroxide (H₂O₂, G0112W), superoxide anion (·O₂⁻, G0116W), and malondialdehyde (MDA, G0110W).

4.5. Determination of Na⁺ and K⁺ Contents in Leaves

The contents of sodium (Na⁺) and potassium (K⁺) ions were determined using the flame photometry method [40].

4.6. Transcriptomic Analysis

In this study, to distinguish different experimental components, the transcriptome datasets were designated as VG-17 for ‘Carménère’ and VG-20 for ‘Pinot Noir’. Leaf tissues from VG-17 and VG-20 seedlings were used as materials for transcriptome sequencing. To ensure data accuracy and reliability, three biological replicates were included for each treatment group. Total RNA was extracted from leaf samples of the control (CK) and salt stress (S) groups of VG-17 and VG-20 after 7 days of salt treatment using an RNA extraction kit (Tiangen Biotech Co., Ltd., Beijing, China). RNA purity, concentration, and integrity were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Shanghai, China) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Only RNA samples that met the quality requirements were used for library construction. Library preparation, quality control, and high-throughput sequencing were all performed by Beijing Biomarker Technologies Co., Ltd. (Shunping Road, Nanfaixin Town, Shunyi District, Beijing, China). on an Illumina high-throughput sequencing platform using paired-end 150 bp (PE150) mode. The raw sequencing data were subjected to strict quality control to remove reads containing adapter sequences, reads with more than 10% ambiguous bases (N), and low-quality reads, thereby obtaining high-quality clean data. The Q30 values and GC content were calculated for the clean data. Subsequently, the clean reads were aligned to the grapevine reference genome (Vitis vinifera L., IGGP_12x version, NCBI) using HISAT2 software, and only uniquely mapped reads were retained for subsequent analyses.

Gene expression levels were quantified as fragments per kilobase of transcript per million mapped reads (FPKM). Differentially expressed genes (DEGs) between the control and salt-treated groups were identified separately for VG-17 and VG-20 using the DESeq2 software (version 1.51.3), with the screening criteria set as a false discovery rate (FDR) < 0.05 and a fold change (FC) ≥ 1.5. Functional annotation of the genes was primarily performed based on the following databases: KEGG (Kyoto Encyclopedia of Genes and Genomes), GO (Gene Ontology), COG (Clusters of Orthologous Groups of proteins), and KOG (Eukaryotic Orthologous Groups). In addition, comprehensive annotation was achieved by referencing several authoritative databases, including the NCBI non-redundant protein sequence database (NR), the Protein family database (Pfam), and the manually curated protein sequence database Swiss-Prot.

4.7. Quantitative Real-Time PCR Analysis

To verify the reliability of the transcriptome sequencing results, eleven differentially expressed genes (DEGs) were randomly selected for quantitative real-time PCR (qRT-PCR) analysis to evaluate their consistency with the RNA-seq data. First, total RNA was reverse-transcribed into cDNA using the FastKing RT Kit with gDNase (Tiangen Biotech Co., Ltd., Beijing, China). Subsequently, qRT-PCR was performed using the SYBR Green method (Tiangen Biotech Co., Ltd.) on a LightCycler^® 96 real-time PCR system (Roche Diagnostics). Gene-specific primers were designed based on the full-length coding sequences (CDSs) of the target genes and synthesized by Sangon Biotech (Shanghai, China) Co., Ltd. The gene-specific primers and detailed experimental procedures are listed in Table S1. Relative gene expression levels were calculated using the 2^−ΔΔCt method, with Vitis vinifera glyceraldehyde-3-phosphate dehydrogenase (VvGAPDH) used as the internal reference gene. All experiments were conducted with three biological replicates, and each sample was analyzed with three technical replicates to ensure data reliability.

4.8. Data Analysis

All data were organized using Excel 2024. Statistical analyses were performed in SPSS 26.0, where one-way analysis of variance (ANOVA) was applied for within-cultivar comparisons, with the significance level set at p < 0.05. In addition, SPSS 26.0 was used to conduct correlation analyses between grapevine cultivars and their physiological indices in order to evaluate the relationships among different physiological parameters. Data visualization was carried out using GraphPad Prism 8.0 and TBtools 2.0, and figure editing was performed using Adobe Photoshop 2024 and Adobe Illustrator 2024. All data presented in this study were based on three biological replicates, and each experiment included three technical replicates.

5. Conclusions

This study demonstrates that the salt-tolerant variety ‘Carménère’ exhibits significantly better salt tolerance under 200 mmol/L NaCl stress compared to the salt-sensitive variety ‘Pinot Noir’. This is primarily reflected in less photosynthetic inhibition, stronger antioxidant capacity, and more effective osmotic balance and ion regulation. The physiological indicators and their correlation analysis reveal the systemic advantages of ‘Carménère’ in physiological regulation, indicating that this variety can coordinate photosynthetic protection, redox homeostasis, and ion homeostasis to resist salt stress. Transcriptomic and qRT-PCR validation identified key pathways such as plant hormone signaling, MAPK signaling, and glutathione metabolism, along with several core genes, which may form the molecular basis for the salt tolerance of ‘Carménère’. Overall, this study provides evidence for understanding the salt tolerance mechanisms in grapevines and offers theoretical support for molecular breeding and selection of salt-tolerant varieties.