Multilayered Transcriptional Regulation Underlying Salt Tolerance in Rapeseed (Brassica napus L.) Revealed by Integrated Physiological and Transcriptomic Analyses

Simple Summary

Soil salinity poses a significant threat to global crop production, diminishing plant growth and food security. Rapeseed is an important oil crop, but its productivity is often limited in salty soils. This study investigated the response of rapeseed plants to salt stress by examining alterations in plant growth, stress-induced damage, and gene activity. Increasing salt concentrations resulted in leaf yellowing, reduced growth, and elevated levels of detrimental molecules that damage plant cells. Concurrently, plants produced protective compounds to maintain water balance and mitigate injury. At the molecular level, thousands of genes exhibited altered activity under salt stress, particularly those involved in hormone signaling, antioxidant defense, salt transport within cells, and cell wall reinforcement. We also discovered a group of regulatory genetic molecules that help control these stress responses. Together, our findings show that salt tolerance in rapeseed depends on the coordinated action of multiple protective systems. This knowledge provides useful targets for developing new rapeseed varieties that can better withstand saline soils, helping to maintain crop yields and support sustainable agriculture in areas affected by salinity.

Abstract

Soil salinity represents a significant abiotic constraint limiting the productivity and geographical expansion of rapeseed (Brassica napus L.), yet the coordination among the signaling, hormonal, metabolic, and regulatory layers underlying salt tolerance remains incompletely understood. This study elucidates the physiological, biochemical, and transcriptomic responses of B. napus inbred line 383-5 to moderate salt stress (100 mM NaCl at day 10), identifying key lncRNA–mRNA regulatory networks. Salt stress induced pronounced, dose-dependent growth inhibition, oxidative damage, and osmotic adjustment, accompanied by extensive transcriptional reprogramming. Genome-wide analyses identified 6215 differentially expressed protein-coding genes and 941 salt-responsive long non-coding RNAs (lncRNAs), revealing coordinated regulation of ion transport, redox homeostasis, phytohormone signaling, and secondary metabolism. Functional enrichment analyses highlighted the central involvement of abscisic acid and ethylene signaling pathways, MAPK cascades, membrane transporters, and antioxidant systems. Notably, salt stress strongly activated the phenylpropanoid and lignin biosynthesis pathways, suggesting reinforced cell wall remodeling and enhanced oxidative stress mitigation. Integration of lncRNA–mRNA regulatory networks further indicated that non-coding transcripts act as important modulators linking hormone signaling, redox balance, and metabolic adaptation. Collectively, these results reveal a multilayered and tightly synchronized regulatory framework underlying salinity tolerance in B. napus and provide valuable molecular targets for the genetic improvement of salt-resilient rapeseed cultivars.

1. Introduction

Salinity presents a significant and growing constraint on global crop productivity. It induces osmotic stress, ionic toxicity resulting from Na⁺ and Cl⁻, nutrient imbalances, and secondary oxidative stress, collectively inhibiting growth and accelerating plant senescence [1,2]. Within plants, these stresses are perceived and transmitted through tightly coordinated signaling networks [3]. Key components include Ca²⁺ influx and calcium sensor systems [4], the Salt Overly Sensitive (SOS) pathway, which regulates Na⁺ extrusion and Na⁺/K⁺ homeostasis [5], and reactive oxygen species (ROS) signaling that is coupled to antioxidant defenses [6,7]. These pathways operate alongside MAPK cascades and ABA-centered kinase–phosphatase modules (PYR/PYL–PP2C–SnRK2), which together reprogram gene expression, metabolic processes, and ion transport to restore cellular homeostasis [8,9]. Recent syntheses indicate that SOS core components and their upstream regulators function as multilayered control nodes, integrating developmental and environmental cues. Consequently, this complexity demonstrates why single-pathway models are inadequate for describing salinity tolerance in crops [10,11].

Rapeseed (Brassica napus L.) is a globally significant oilseed crop [12,13]. It is being increasingly cultivated in regions experiencing soil salinization and expansion of saline–alkali lands, posing significant threats to crop establishment and yield stability [14,15]. Genetic and genomic studies have demonstrated substantial natural variation in salt tolerance within B. napus, particularly during germination and early seedling development, offering a valuable foundation for trait dissection and breeding improvement [16,17]. High-throughput screening and association mapping analyses have further identified key loci and phenotypic traits suitable for selection and functional validation [17]. At the molecular level, rapeseed responses to salinity are characterized by pronounced regulation of ion transporters [18], membrane-associated processes [19], phytohormone signaling pathways, and stress-protective metabolic networks [20]. These regulatory patterns have been consistently observed in transcriptomic analyses of roots and seedlings subjected to NaCl stress [21]. Notably, salinity tolerance in rapeseed is not solely determined by ion transport but results from coordinated adjustments across signaling pathways, transcriptional control, and downstream metabolic reinforcement, including redox homeostasis and cell wall remodeling [22,23].

Phytohormones play a central role in coordinating plant responses to salinity stress [24]. Among these, abscisic acid (ABA) functions as a primary integrator of osmotic stress signaling. ABA perception, via PYR/PYL receptors, inhibits PP2C phosphatases and activates SnRK2 kinases, thereby regulating stress-responsive transcription factors, ion channels, and metabolic enzymes [25,26]. ABA signaling also interacts extensively with other phytohormones, including ethylene, jasmonates (JAs), salicylic acid (SA), auxin, gibberellins (GA₃), cytokinins (CKs), and brassinosteroids (BRs), enabling fine-tuning of growth–defense trade-offs under saline conditions [27,28]. In parallel, oxidative signaling acts as both a damage-associated signal and a regulatory information channel during salt stress [29]. Transient bursts of reactive oxygen species (ROS) can amplify hormone-mediated responses, while enzymatic antioxidants and compatible solutes, such as proline and soluble sugars, limit lipid peroxidation and maintain cellular homeostasis [30]. These hormone–redox networks are closely linked to structural and chemical defense mechanisms [31]. Notably, the phenylpropanoid pathway, including lignin and related phenolic compounds, is consistently associated with abiotic stress adaptation through its roles in cell wall reinforcement, antioxidant activity, and stress-induced metabolic reprogramming [32]. In Brassica species, phenylpropanoid metabolism has been further linked to hormone-dependent acclimation responses, suggesting that secondary metabolism can both reflect and stabilize upstream signaling states during stress progression [33].

Beyond protein-coding genes, long non-coding RNAs (lncRNAs) have emerged as important regulatory components of plant stress-response networks. They function through cis- and trans-regulation of neighboring or co-expressed genes, modulation of chromatin and transcriptional activity, and post-transcriptional mechanisms, including target mimicry and competitive endogenous RNA (ceRNA) interactions with microRNAs [34]. Increasing evidence indicates that lncRNAs act as regulatory hubs under high salinity and other abiotic stresses [35,36]. However, their functional roles remain incompletely resolved in most crops due to the strong context dependence of lncRNA-mediated networks, encompassing tissue specificity, developmental stage, genotype, and stress intensity. In Brassica crops, lncRNAs have been identified as key regulators of abiotic stress tolerance and hormone-associated pathways [37]. Nevertheless, integrated and pathway-centered analyses linking phytohormone signaling, redox regulation, transporter activity, and secondary metabolism under salinity stress remain relatively limited.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

Seeds of the Brassica napus inbred line 383-5, developed in our laboratory at the School of Breeding and Multiplication, Sanya Institute of Breeding and Multiplication, School of Tropical Agriculture and Forestry, Hainan University (Sanya, China), were used in this study. The seeds were surface-sterilized by soaking in 75% alcohol for 30 s, followed by 1 min of immersion in 3.5% bleach, and then rinsed three times with sterilized water, each rinse lasting 1 min and occurred in a controlled growth chamber under a 16 h light/8 h dark photoperiod at 23 °C and 80% relative humidity. For the salt stress treatment, uniform seedlings three weeks old, grown in small soil-filled pots, were randomly assigned to four experimental groups, each replicated five times. Three groups were irrigated with 50, 100, or 200 mM NaCl solutions prepared in Hoagland nutrient solution. The fourth group, designated as the control (CK) group, was irrigated with Hoagland’s solution alone. Salt stress was applied for five days, and phenotypic responses were monitored until discernible differences were observed between the treatment and CK groups. Upon the appearance of phenotypic changes, the third to fourth leaves (from apex to base) were harvested, immediately frozen in liquid nitrogen, and stored at −80 °C for RNA-sequencing and subsequent validation analyses.

2.2. Assessment of Physiological Indices

Previously collected samples were used to evaluate various physiological parameters in control (CK) and salt-stressed (100 mM NaCl) plants. To assess antioxidant enzyme activities, 0.5 g of powdered leaf tissue was homogenized in 900 μL of 100 mM phosphate buffer (pH 7.4) following the protocols specified in the respective assay kits. The homogenates were centrifuged at 12,000× g for 15 min at 4 °C, and the resulting supernatants were transferred to new centrifuge tubes for enzymatic analysis. Activities of enzymes, including superoxide dismutase (SOD, Cat. # A001-1), catalase (CAT; Cat. # A007-1), peroxidase (POD, Cat. # A084-3-1), soluble sugars (Cat. # A145-1-1), H₂O₂ (Cat. #: A064-5-1), proline (Cat #: A107-1-1), and malondialdehyde (MDA, Cat. # A003-1-1), were determined using commercial kits supplied by the Nanjing Jiancheng Bioengineering Institute according to the manufacturer’s instructions. Enzyme activities were averaged from technical replicates (n = 5 per assay).

2.3. NBT Staining

Nitroblue tetrazolium (NBT) staining was performed to detect superoxide anion accumulation in Brassica napus leaves under salt stress. Third to fourth fully expanded leaves were excised from control and NaCl-treated plants (50, 100, and 200 mM) and briefly rinsed with distilled water before immersion in a freshly prepared NBT solution (0.5 mg/mL in 50 mM phosphate buffer, pH 7.4). The samples were vacuum-infiltrated for 5–10 min to facilitate reagent penetration, followed by incubation in the dark at room temperature for 2–4 h. After staining, the leaves were boiled in 95% ethanol at 70–80 °C for 10–20 min to remove chlorophyll and enhance the visualization of blue formazan deposits, which indicate superoxide accumulation. Stained leaves were imaged using a digital camera, and the intensity of blue coloration served as a qualitative indicator of oxidative stress in response to salt treatment.

2.4. Total RNA Extraction and Transcriptome Sequencing

Total RNA was extracted from leaf samples of salt-stressed plants (100 mM NaCl) and control plants to construct RNA-seq libraries following the manufacturer’s instructions for the Trizol kit (Invitrogen, Carlsbad, CA, USA). To eliminate genomic DNA (gDNA) contamination, the samples were treated with DNase I (Takara Bio, Dalian, China). The quality and integrity of the RNA were evaluated using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Wilmington, NA, USA) and 2% agarose gel electrophoresis, respectively. High-quality RNA samples were then sent to Shanghai Majorbio Biopharm Technology (Shanghai, China) for library construction and sequencing on the Illumina HiSeq X Ten/NovaSeq 6000 platform. Three biological replicates, derived from independent plants, were grown and treated identically under NaCl stress/control conditions.

2.5. Whole Transcriptome Analysis and Identification

High-quality raw reads were obtained by eliminating contaminants, trimming adaptor sequences, and removing low-quality reads using Trimmomatic (http://www.usadellab.org/cms/?page=trimmomatic, accessed on 24 November 2025). The quality of the filtered reads was assessed using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/download.html#fastqc, accessed on 25 November 2025) before assembly of the sequences. The reference genome assembly of B. napus (ZS11) was retrieved from the BnaIR (B. napus Multi-omics Information Resource; https://yanglab.hzau.edu.cn/BnIR/, accessed on 25 November 2025). The HISAT2 program (https://www.ccb.jhu.edu/software/hisat/index.shtml, accessed on 25 November 2025) was used to map the high-quality filtered reads against the reference genome for each library. StringTie (https://ccb.jhu.edu/software/stringtie/, accessed on 28 November 2025) was used to prepare the assembly from the mapped libraries. Finally, the StringTie merger and gffcompare were used to generate the final assembly and annotation against the reference annotation of B. napus.

To identify long non-coding RNAs (lncRNAs) from the newly assembled transcriptome, the following criteria were established: (i) intronic overlap transcripts (i), intergenic transcripts (u), exonic transcripts (o), and natural antisense transcripts (x); (ii) a length exceeding 200 nucleotides; (iii) more than two exons; and (iv) an expression abundance greater than 0.1 FPKM. To differentiate mRNAs from non-coding RNAs, the coding potential of each transcript was assessed using several programs, including the Coding Potential Calculator (CPC2, score < 0, https://cpc.gao-lab.org/programs/run_cpc.jsp, accessed on 2 December 2025), CNCI (https://github.com/www-bioinfo-org/CNCI, accessed on 3 December 2025), CPAT (https://github.com/liguowang/cpat, accessed on 3 December 2025), PLEK (http://202.200.112.245/plek/, accessed on 5 December 2025), and LGC (https://github.com/GuangyWang/LGC, accessed on 6 December 2025). The results from the aforementioned tools are presented in a Venn diagram. To further distinguish outstanding lncRNAs from protein-coding genes and domains, we conducted BLASTX searches against the Pfam (http://pfam.xfam.org/, accessed on 10 December 2025) and miRBase (https://www.mirbase.org/, accessed on 10 December 2025) databases to differentiate them from small RNAs.

Raw read counts were generated using the featureCounts program (https://rnnh.github.io/bioinfo-notebook/docs/featureCounts, accessed on 15 December 2025) from aligned BAM files. Transcript levels of mRNAs and lncRNAs were quantified as FPKM, and significantly differentially expressed transcripts were predicted using the DESeq R package (version 2.0) [38] with the following parameters: a false discovery rate (FDR) < 0.05 and |log2(fold change)| ≥ 1, as described by Basharat et al. [39].

2.6. Prediction of lncRNAs’ Target Genes

When lncRNAs interact with adjacent target genes, this interaction is characterized as a cis-action. Conversely, candidate genes for trans-acting lncRNAs are identified based on their co-expression patterns. For cis-action analysis, we identified protein-coding genes located within 100 kb upstream and downstream of each lncRNA using bedtools window functions (https://bedtools.readthedocs.io/en/latest/content/tools/window.html, accessed on 21 December 2025). For trans-acting lncRNAs, we employed the Pearson correlation coefficient method to analyze the relationship between lncRNAs and protein-coding genes, as described by Waseem et al. [40], using a threshold of a Pearson correlation |r| > 0.8 and p < 0.01.

2.7. Functional Annotation of Differentially Expressed Genes

Functional annotation of all differentially expressed genes (DEGs) and cis/trans-acting targets of long noncoding RNAs (lncRNAs) was performed using Eggnog-Mapper (in-house standalone version), alongside analyses employing the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) databases. The GO analysis results were categorized into three distinct groups: (i) molecular function (MF), (ii) cellular component (CC), and (iii) biological process (BP). KEGG pathways were identified based on a significance threshold of adjusted p < 0.05. Visual representations, including heatmaps, were generated using ggplot2 in R.

2.8. Statistical Analysis

Physiological and biochemical data were analyzed using GraphPad Prism v8.0 with one-way ANOVA followed by the least significant difference test and Duncan’s tests (p < 0.01, 0.001). Results are presented as the mean ± standard error of the mean (mean ± SEM); graphs were generated in GraphPad Prism v8.0, and figures assembled with CorelDRAW 2022.

3. Results

3.1. Morphological and Physiological Responses

Exposure of Brassica napus inbred line 383-5 seedlings to increasing NaCl concentrations resulted in pronounced, dose-dependent morphological and physiological alterations (Figure 1). Under control (CK) conditions, plants maintained normal growth with healthy green leaves throughout the experimental period (Figure 1a). In contrast, NaCl treatment progressively inhibited seedling growth, with visible reductions in plant vigor apparent after 10 days of exposure. At 50 and 100 mM NaCl, seedlings exhibited moderate growth suppression accompanied by leaf wilting and the onset of chlorosis, whereas severe morphological damage, including extensive leaf yellowing, tissue necrosis, and marked growth retardation, was observed at 200 mM NaCl. Consistent with whole-plant phenotypes, excised leaves collected on day 10 displayed clear salt concentration-dependent deterioration. Leaves from CK plants retained normal coloration and structural integrity, while those from NaCl-treated plants showed progressive chlorosis and lesion development, with the most severe tissue damage occurring under 200 mM NaCl stress (Figure 1a). These observations indicate substantial impairment of leaf physiological status under elevated salinity.

Additionally, histological analyses using NBT staining assessed cell viability and superoxide anion levels in plants subjected to CK and NaCl stresses. We observed that CK leaves exhibited minimal to no blue staining, indicating low basal superoxide production. Under mild stress (50 mM NaCl), faint blue spots appeared on the leaves by day 10, suggesting moderate ROS buildup that antioxidants could largely manage. At 100 mM NaCl, considered moderate stress, faint blue spots indicated limited superoxide buildup. Under severe stress (200 mM NaCl), intense and widespread dark blue staining covered the leaves, confirming a massive oxidative burst that overwhelmed cellular defenses (Figure 1b), as corroborated by parallel elevations in MDA, H₂O₂, and variable enzyme activities (SOD, CAT, POD). This dose-dependent staining pattern demonstrated how escalating salinity disrupted redox homeostasis in B. napus, driving oxidative damage visible both histochemically and biochemically.

To further characterize the physiological responses to salt stress, biochemical parameters were analyzed following 100 mM NaCl treatment (Figure 1c). Activities of antioxidant enzymes were significantly altered; CAT and SOD exhibited significant reductions compared to the control, while POD activity increased slightly but was not statistically significant. Salt stress also resulted in a marked accumulation of oxidative stress markers, as evidenced by significantly elevated levels of MDA and H₂O₂. In parallel, osmoprotective metabolites, including proline and soluble sugars, markedly accumulated under NaCl stress relative to the control. Collectively, these results demonstrate that salt stress induces substantial morphological damage, disrupts antioxidant homeostasis, and promotes osmotic adjustment responses in B. napus seedlings. We further focused on plants with 100 mM NaCl (moderate stress), which induced clear responses without lethality, to perform transcriptomic analysis.

3.2. Genome-Wide Identification and Structural Characterization of lncRNAs

A comprehensive genome-wide analysis was performed on 100 mM NaCl stressed plants versus CK plants to identify long non-coding RNAs (lncRNAs) in B. napus (Figure 2). In total, 100,784 protein-coding mRNAs and 8666 lncRNAs were identified across the genome (Figure 2a). Based on their genomic locations relative to protein-coding genes, lncRNAs were classified into four categories: intergenic lncRNAs (lincRNAs), which constituted the most significant proportion (6205), followed by antisense lncRNAs (1022), sense lncRNAs (1336), and intronic lncRNAs (103), indicating that intergenic transcripts dominate the lncRNA population in B. napus. To improve prediction reliability, lncRNAs were independently identified using five computational tools (CPC2, CNCI, PLEK, LGC, and CPAT). A Venn diagram analysis revealed substantial overlap among these methods, with 3338 lncRNAs commonly predicted by all five tools (Figure 2b), representing a high-confidence lncRNA dataset. Among the individual tools, CPAT identified the highest number of lncRNAs, followed by PLEK, CPC2, LGC, and CNCI (Figure 2c), reflecting differences in coding potential assessment. Chromosomal distribution analysis showed that both mRNAs and lncRNAs were unevenly distributed across the B. napus chromosomes (Figure 2d). Chromosomes with higher gene density also tended to harbor more lncRNAs, although the absolute number of lncRNAs was substantially lower than that of mRNAs on all chromosomes. A subset of transcripts was located on unanchored scaffolds (ChrUn), indicating the presence of lncRNAs in genomic regions not yet assigned to specific chromosomes. Structural feature analysis revealed marked differences between lncRNAs and mRNAs. Length distribution analysis showed that most lncRNAs were relatively short, with the majority falling within the 200–1000 bp range, whereas mRNAs displayed a broader length distribution and were generally longer (Figure 2e). Consistent with this observation, exon number analysis demonstrated that lncRNAs typically contained fewer exons, with most comprising two exons, while mRNAs frequently contained multiple exons, including a substantial proportion with more than ten exons (Figure 2f). These structural characteristics highlight the distinct genomic architecture of lncRNAs compared with protein-coding genes in B. napus.

3.3. Differential Expression Analysis of mRNAs and lncRNAs

To investigate transcriptomic responses to salt stress, differential expression analysis was performed by comparing salt-treated plants with CK plants (Figure 3). A substantial number of both mRNAs and lncRNAs exhibited significant expression changes under salt stress conditions. In total, 6215 mRNAs were identified as differentially expressed, of which, 2662 were upregulated and 3553 were downregulated relative to the control, indicating a predominance of transcriptional repression among protein-coding genes in response to salinity. In parallel, 941 lncRNAs showed significant differential expression, including 516 upregulated and 425 downregulated transcripts, suggesting that lncRNAs also respond dynamically to salt stress, albeit to a lesser extent than mRNAs (Figure 3a). Hierarchical clustering and heatmap visualization further illustrated the distinct expression patterns of differentially expressed transcripts between CK and salt-stressed samples (Figure 3b). Both differentially expressed protein-coding genes and lncRNAs exhibited clear separation between treatment groups across three biological replicates, demonstrating high consistency within replicates and robust transcriptional reprogramming induced by salt stress. In the heatmaps, salt stress led to coordinated upregulation of specific transcript subsets, while others were strongly suppressed, reflecting widespread remodeling of gene expression networks. Notably, the overall expression patterns of lncRNAs mirrored those of mRNA transcripts, indicating that coding and non-coding transcripts are jointly involved in the plant’s transcriptional response to salinity. Collectively, these results demonstrate that salt stress triggers extensive and coordinated changes in both protein-coding and lncRNA transcriptomes in B. napus, highlighting the potential regulatory involvement of lncRNAs in salt stress adaptation.

3.4. Predicted Targets and Functional Enrichment Analysis of Differentially Expressed lncRNAs

It has been found that lncRNAs interact with adjacent and distant protein-encoding genes through cis- and trans-regulatory mechanisms. In the present study, we searched for potential cis-target genes within the regions 100 kb upstream and downstream of the identified DE-lncRNAs. We also analyzed the trans-acting lncRNAs, we employed the Pearson correlation coefficient method. As a result, 247 protein coding transcripts were found to be affected by cis-regulation from 228 DE-lncRNAs (Table S1), and 941 protein coding transcripts were found to be affected by trans-regulation from 490 DE-lncRNAs (Table S2).

To further elucidate the biological functions and pathways associated with salt-stress-responsive genes, Gene Ontology (GO) and KEGG pathway enrichment analyses were performed on DE-lncRNA targets identified in B. napus. GO enrichment analysis revealed that DE-lncRNA targets were significantly overrepresented in multiple biological process categories related to stress perception and adaptation (Figure 4a). Prominent biological processes included responses to abiotic stimuli, toxic substances, chemical stimuli, and water deprivation, indicating that salt stress elicits a broad stress response program. In addition, ion transport-related processes were strongly enriched, reflecting the importance of ionic homeostasis under saline conditions. Within the cellular component category, DE-lncRNA targets were predominantly associated with membrane-related structures, including integral and intrinsic components of the plasma membrane, cell periphery, and plant-type cell wall. These enrichments suggest that membrane-associated proteins play a central role in mediating salt stress responses, likely through regulating transport processes and signal perception. Consistent with this observation, molecular function enrichment highlighted a strong overrepresentation of transmembrane transporter activities, particularly inorganic ion and anion transmembrane transporter activities, as well as oxidoreductase-related functions, indicating enhanced transport and redox regulation under salt stress. KEGG pathway enrichment analysis further supported the involvement of diverse metabolic and signaling pathways in the salt stress response (Figure 4b). Significantly enriched pathways included transporters, signal transduction, and the MAPK signaling pathway, underscoring the activation of intracellular signaling networks in response to salinity. Pathways related to secondary metabolism, such as phenylpropanoid biosynthesis and the biosynthesis of other secondary metabolites, were also enriched, suggesting metabolic reprogramming to enhance stress tolerance. Additionally, enrichment of plant hormone signal transduction pathways indicates hormonal regulation as a key component of the salt stress response. Metabolic pathways associated with lipid, amino acid, carbohydrate, and fatty acid metabolism were likewise significantly represented, reflecting extensive adjustments in primary metabolism to cope with osmotic and ionic stress. Overall, these functional enrichment analyses demonstrate that salt stress induces coordinated transcriptional changes in B. napus involving stress-responsive signaling, membrane transport, metabolic remodeling, and hormone-mediated regulation, collectively contributing to plant adaptation under saline conditions.

3.5. Transcriptomic Modulation of Ethylene and ABA Signaling Pathways

To further elucidate the regulatory mechanisms underlying salt stress responses in Brassica napus, the expression patterns of genes involved in ethylene and abscisic acid (ABA) signaling pathways were examined based on transcriptomic data from predicted targeted genes of lncRNAs (Figure 5). Integration of differentially expressed genes into canonical signaling frameworks revealed substantial transcriptional reprogramming of both hormone-mediated pathways under salt stress. In the ethylene signaling pathway, several key components showed significant changes in expression in response to salinity. Genes encoding upstream regulators, including RAN1 and ethylene receptors (ETR/ERS), displayed altered transcript abundance, suggesting modulation of ethylene perception under stress conditions. Downstream signaling components, such as CTR1, MKK9, and mitogen-activated protein kinases MPK3 and MPK6, also showed differential expression, indicating activation of MAPK cascades associated with ethylene signaling. Furthermore, transcriptional regulators EIN2, EIN3/EIL, and EBF1/2 exhibited distinct expression shifts, reflecting fine-tuned control of ethylene-responsive transcription. Consistent with these changes, the downstream ethylene-responsive transcription factor ERF1 and defense-related genes, including CHI-B and PDE1.2, were differentially expressed, suggesting enhanced activation of ethylene-mediated defense responses under salt stress. Concurrently, significant transcriptional modulation was observed in the ABA signaling pathway. Genes encoding ABA receptors (PYR/PYL) exhibited increased expression under salt stress, accompanied by transcriptional repression of PP2C phosphatases and activation of SnRK2 kinases. This expression pattern is indicative of ABA signal activation and derepression of downstream signaling. Further downstream, MAPK components such as MPK17, MKK3, and MPK10/14 displayed altered expression profiles, supporting the involvement of ABA-dependent MAPK signaling in stress adaptation. In addition, activation of MPK6 was associated with enhanced hydrogen peroxide (H₂O₂) signaling and differential expression of CAT1, linking ABA signaling to redox regulation under salinity stress. Overall, the coordinated transcriptional regulation of ethylene and ABA signaling components highlights extensive hormone-mediated signaling crosstalk in response to salt stress in B. napus. These results indicate that ethylene- and ABA-dependent pathways jointly contribute to stress perception, signal transduction, and downstream defense and adaptation processes, thereby facilitating plant tolerance to saline environments.

3.6. Transcriptomic Landscape of Phytohormone Signaling Pathways

To comprehensively characterize hormone-mediated regulatory responses to salt stress, transcriptomic changes in genes associated with major phytohormone biosynthesis and signaling pathways were systematically analyzed in B. napus (Figure 6). Integration of differentially expressed genes into canonical hormone signaling frameworks revealed extensive and hormone-specific transcriptional reprogramming under saline conditions. Genes involved in auxin biosynthesis and signaling exhibited pronounced expression changes under salt stress. Key components of auxin transport and perception, including AUX1, TIR1, and downstream transcriptional regulators such as ARF, GH3, and SAUR, showed altered transcript abundance, suggesting modulation of auxin-mediated growth regulation. These changes are indicative of suppressed cell elongation and altered developmental processes in response to salt stress. Similarly, genes involved in gibberellin signaling, including GID1 and the growth-repressing DELLA proteins, were differentially expressed, consistent with growth restraint and stress-adaptive growth modulation under saline conditions. The abscisic acid (ABA) signaling pathway showed strong transcriptional activation in response to salt stress. Genes encoding ABA receptors (PYR/PYL), downstream kinases (SnRK2), and transcription factors (ABF) exhibited elevated expression, whereas negative regulators such as PP2C phosphatases showed reduced or contrasting expression patterns. This coordinated regulation reflects the central role of ABA signaling in mediating osmotic stress responses, including stomatal closure and stress-responsive gene activation. In contrast, cytokinin-related genes displayed distinct expression trends, with components involved in cytokinin perception and signaling, such as CRE1, AHP, and B-ARR, showing differential regulation. These changes suggest a rebalancing of cytokinin signaling, potentially contributing to the suppression of cell division and shoot growth under salt stress. Genes associated with jasmonic acid (JA) biosynthesis and signaling, including JAR1, JAZ, and the transcription factor MYC2, were also differentially expressed, indicating activation of JA-mediated stress and defense responses. Concurrently, ethylene biosynthesis and signaling genes, including ETR, CTR1, EIN2, EIN3, and ERF1/2, showed substantial transcriptional modulation, consistent with ethylene’s involvement in stress-induced senescence and defense regulation. Salicylic acid (SA) signaling components, including NPR1, TGA, and PR1, were differentially regulated, suggesting enhanced SA-mediated defense pathways under salt stress. In addition, brassinosteroid (BR) signaling genes, including BRI1, BSK, BZR1/2, and downstream cell cycle regulators, showed altered expression, reflecting adjustments in growth-related signaling. Genes involved in zeatin biosynthesis and signaling pathways also exhibited differential expression, further underscoring the broad hormonal reprogramming induced by salinity. Collectively, these results demonstrate that salt stress triggers extensive and coordinated transcriptional regulation across multiple phytohormone signaling pathways in B. napus. The concurrent modulation of growth-promoting and stress-responsive hormonal networks highlights the complex hormonal crosstalk that underpins adaptive responses to saline conditions.

3.7. Salt Stress Induces Transcriptional Activation of the Phenylpropanoid and Lignin Biosynthesis Pathways

To investigate metabolic reprogramming associated with salt stress, the expression profiles of genes involved in the phenylpropanoid and lignin biosynthesis pathways were examined in B. napus (Figure 7). Transcriptomic mapping of differentially expressed genes onto the canonical phenylpropanoid pathway revealed widespread, coordinated transcriptional activation under salt stress. Genes encoding key upstream enzymes responsible for the entry of carbon flux into the phenylpropanoid pathway, including phenylalanine ammonia-lyase (PAL) and cinnamate-4-hydroxylase (C4H), showed pronounced upregulation under salt stress, indicating enhanced conversion of phenylalanine and tyrosine into cinnamic acid-derived intermediates. Consistently, multiple members of the 4-coumarate-CoA ligase (4CL) family exhibited elevated expression, suggesting increased channeling of hydroxycinnamic acids toward downstream phenylpropanoid metabolism. Further downstream, genes involved in monolignol biosynthesis displayed strong transcriptional induction. Enzymes such as cinnamoyl-CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD), which catalyze the formation of lignin precursors, were markedly upregulated across multiple homologs. In addition, genes encoding caffeic acid O-methyltransferase (COMT) and ferulate-5-hydroxylase (F5H), key determinants of lignin monomer composition, exhibited increased expression, indicating enhanced biosynthesis of guaiacyl (G) and syringyl (S) lignin units under salt stress. Consistent with elevated monolignol production, genes encoding peroxidases (PER), which mediate the oxidative polymerization of lignin monomers into the cell wall matrix, were also transcriptionally activated. This coordinated upregulation suggests an overall enhancement of lignin deposition processes in response to salt stress. In parallel, several UDP-glucosyltransferase (UGT) genes involved in the glycosylation and stabilization of phenolic compounds were upregulated, indicating increased modification and sequestration of phenylpropanoid derivatives. Collectively, these results demonstrate that salt stress triggers robust transcriptional activation of the phenylpropanoid and lignin biosynthesis pathways in B. napus. The enhanced expression of genes associated with monolignol production, lignin polymerization, and phenolic modification suggests reinforced cell wall structure and improved oxidative stress mitigation, highlighting the role of secondary metabolism in plant adaptation to saline environments.

4. Discussion

Exposure of the B. napus inbred line 383-5 to increasing NaCl concentrations elicited clear morphological and physiological changes typical of salt stress injury and acclimation in rapeseed. The dose-dependent inhibition of growth, leaf wilting, chlorosis, and necrosis, especially under 200 mM NaCl, reflects combined osmotic and ionic stress, as widely reported in B. napus and related Brassica cultivars under moderate to severe salinity [41,42]. The leaf deterioration observed at day 10, from healthy green laminae in the CK to chlorotic and necrotic tissue under NaCl treatment, indicates impaired photosynthesis and membrane integrity, consistent with decreased chlorophyll, biomass loss, and elevated membrane injury indices documented in salt-stressed rapeseed seedlings [43,44,45]. Significant declines in CAT and SOD activities under 100 mM NaCl, together with elevated H₂O₂ and MDA levels, indicate disrupted antioxidant capacity and heightened lipid peroxidation [46]. Similar oxidative imbalances have been mitigated in other B. napus genotypes through enhanced antioxidant enzyme activities promoted by treatments such as isosteviol or optimized nitrogen supply [41,46,47]. The minor, non-significant increase in peroxidase (POD) activity suggests a limited compensatory response within the enzymatic ROS-scavenging network that is insufficient to fully counter oxidative damage [41,43,48]. Concurrent accumulation of proline and soluble sugars marks activation of osmotic adjustment mechanisms that maintain turgor and protect macromolecules [48,49]. Plants respond via ion homeostasis, osmolyte accumulation, and antioxidant defenses, but tolerance varies markedly across species, with glycophytes such as rice (Oryza sativa) showing high sensitivity and halophytes like Thellungiella halophila exhibiting robust adaptation [50]. However, the coexistence of high ROS and MDA levels with strong osmolyte accumulation suggests that osmotic regulation alone cannot rescue 383-5 from oxidative injury, underscoring inadequate antioxidant defense as a key driver of salt sensitivity [41,43,48,51,52].

Transcriptome profiling revealed extensive reprogramming of both coding and noncoding genes under NaCl stress, highlighting complex transcriptional control in B. napus adaptation [53]. A total of 6215 differentially expressed mRNAs (DEGs) were detected; more genes were downregulated than upregulated, reflecting broad repression of growth- and photosynthesis-related genes and activation of stress-responsive pathways, as observed in other B. napus studies [54,55,56,57,58,59]. Functional enrichment of DEGs in GO terms related to abiotic stimuli, chemical stress, and water deprivation confirms that salt exposure triggers osmotic, ionic, and oxidative stress pathways, indicating coordinated cellular adjustment [54]. The enrichment of ion transport and membrane components, including Na⁺/H⁺ antiporters, anion channels, and H⁺ pumps, emphasizes the central role of ionic homeostasis maintenance under salinity stress [55]. Likewise, the prominence of oxidoreductase activity highlights redox regulation and ROS detoxification as vital features of salt tolerance [16]. The KEGG pathway analyses identified key signaling and metabolic circuits, MAPK signaling, hormone signal transduction, and phenylpropanoid pathways, demonstrating large-scale reorganization of primary and secondary metabolism that underpins stress resilience [53,56].

Line 383–85 exhibited clear concentration-dependent responses to salinity. At 50 mM NaCl (mild stress), plants showed only slight growth inhibition and leaf wilting, with modest osmolyte accumulation and largely stable antioxidant activities, indicating partial acclimation with limited oxidative damage [60]. At 100 mM NaCl (moderate stress), growth suppression and chlorosis intensified, CAT and SOD activities declined, and H₂O₂, MDA, and osmolyte levels increased, marking a shift from acclimation to evident oxidative injury [61]. Under 200 mM NaCl (severe stress), severe growth arrest, extensive leaf damage, and maximal ROS and MDA levels indicated systemic failure of antioxidant protection [54,62]. In crops such as tomato (Solanum lycopersicum) and rice, salinity stress reduces seed germination, stunts root/shoot growth, and impairs photosynthesis by degrading chlorophyll and closing stomata, leading to up to 50% yield loss at 100–150 mM NaCl. Arabidopsis (Arabidopsis thaliana) mutants like P5CS1 knockouts highlight proline’s role in osmotic adjustment, as its deficiency heightens sensitivity. Wheat (Triticum aestivum) genotypes accumulate glycine betaine and activate NHX antiporters for Na⁺ vacuolar sequestration, yet prolonged exposure causes K⁺/Na⁺ imbalance and ROS buildup [63,64]. Correspondingly, 50 mM mainly affected a smaller set of genes associated with early signaling, ion transport, and osmotic adjustment, whereas 100 mM and 200 mM triggered broad transcriptomic reprogramming, with stronger repression of photosynthesis and growth modules and enhanced activation of MAPK, ABA/ethylene, and phenylpropanoid–lignin pathways [56,62]. Together, these patterns suggest that 50 mM elicits primarily adaptive adjustments, 100 mM represents a critical threshold where acclimation and injury coexist, and 200 mM leads to decompensation of key protective systems [56,65].

Hormone signaling adjustments form a central layer of this regulatory network [55]. Differential expression of ethylene and abscisic acid (ABA) pathway genes (RAN1, ETR/ERS, CTR1, EIN2, EIN3/EIL, PYR/PYL, SnRK2, ABF) indicates strong hormone-mediated coordination of stress signaling. These data support known models where ethylene and ABA integrate growth inhibition, stomatal control, and ROS detoxification under salinity in Brassica and Arabidopsis [56]. Crosstalk among ABA, jasmonic acid (JA), salicylic acid (SA), cytokinin, and brassinosteroid signaling points to a finely balanced hormonal network prioritizing defense and stress endurance over growth under NaCl stress [56,57]. The association of MPK6 activity with H₂O₂ signaling and CAT1 regulation further reinforces the functional link between hormone signaling and redox homeostasis, a theme repeatedly observed in NaCl-tolerant rapeseed genotypes [39,66,67]. Extending beyond ethylene and ABA, the broader phytohormone analysis demonstrates that salt stress reconfigures the entire hormonal landscape [16]. Differential expression of auxin signaling components (AUX1, TIR1, ARF, GH3, SAUR) and gibberellin signaling genes (GID1, DELLA) supports the idea that growth-promoting pathways are actively downregulated to prioritize survival, in agreement with earlier canola and rapeseed transcriptomes showing suppression of cell-elongation and growth modules under salinity stress [56]. The strong activation of ABA signaling components (PYR/PYL, SnRK2, ABF) alongside repression or modulation of the cytokinin (CRE1, AHP, B-ARR), JA (JAR1, JAZ, MYC2), SA (NPR1, TGA, PR1), and brassinosteroid (BRI1, BSK, BZR1/2) pathways reflects a complex hormonal crosstalk network, echoing recent integrative studies where ABA–JA–SA–BR interactions were shown to coordinate defense, ROS management, and growth restraint in B. napus under salt stress [53,54]. The differential regulation of zeatin-related genes further suggests fine-tuning of cytokinin-driven cell division and shoot development, which is typically curtailed under prolonged salinity stress to conserve resources [68,69].

A striking transcriptional activation of phenylpropanoid and lignin biosynthesis genes (PAL, C4H, 4CL, CCR, CAD, COMT, F5H) further indicates cell wall remodeling and enhanced antioxidant defense under salinity stress [32,55,70,71,72]. Strengthened lignification and increased production of phenolic compounds likely improve mechanical stability, reduce ion leakage, and limit oxidative damage [16]. Upregulation of UDP-glycosyltransferases (UGTs) suggests active detoxification and stabilization of phenolics, reinforcing membrane protection and redox balance [73,74,75]. Collectively, these regulatory modules demonstrate that salt tolerance in B. napus depends on tight coordination among ion transport, redox homeostasis, hormone signaling, and secondary metabolism. Our salt-responsive mRNAs included key ROS- and ion-homeostasis genes, consistent with known Brassica/cereal stress modules [76,77]. Notably, altered expression of ascorbate oxidase and MDHAR indicated ascorbate–glutathione cycle remodeling for H₂O₂ detoxification under salinity stress [76,77]. Upregulation of the VOX-like, CPA, CaCA, and Ca²⁺-ATPase genes suggested active Na⁺/H⁺ and Ca²⁺ cycling for ionic balance, aligning with wheat/Brassica stress networks and B. napus SOS/H⁺-pump pathways [65,77,78]. Several DE lncRNAs co-expressed with or targeted these redox/transport genes, resembling salt-responsive lncRNAs in B. juncea/radish that regulate ion transporters and antioxidants via lncRNA–miRNA–mRNA networks [79]. These lncRNA–mRNA pairs likely fine-tune ROS detoxification, Ca²⁺/Na⁺ homeostasis, and hormone signaling in rapeseed salt adaptation.

At the transcriptome architecture level, genome-wide identification of 8666 long noncoding RNAs (lncRNAs) alongside 100,784 protein-coding mRNAs provides a valuable resource for understanding regulatory interactions in B. napus [59,80]. The majority of identified lncRNAs were intergenic and structurally simpler than mRNAs, findings consistent with other plant species [81]. Using five independent coding-potential prediction tools (CPC2, CNCI, PLEK, LGC, and CPAT) and the identification of 3338 lncRNAs in their intersection provides a high-confidence lncRNA set; such multi-tool strategies are recommended by recent benchmarking and pipeline studies (e.g., Plant-LncPipe, PlantLncBoost) to minimize false positives and improve cross-species robustness in plant lncRNA annotation [82]. The larger number of lncRNAs predicted by CPAT relative to other tools is consistent with comparative evaluations showing that different algorithms emphasize distinct sequence features and can vary in sensitivity and specificity, underscoring the importance of integrative prediction frameworks [83]. The uneven chromosomal distribution of lncRNAs [84], with higher counts on gene-rich chromosomes and a presence on unanchored scaffolds (ChrUn) [85], is also consistent with reports that plant lncRNAs tend to cluster in transcriptionally active genomic regions while a proportion remains associated with incompletely assembled or repetitive segments [86]. Structural analyses showing that B. napus lncRNAs are generally shorter (predominantly 200–1000 bp) and contain fewer exons (most with two exons) than mRNAs agree with multiple comparative studies indicating that plant lncRNAs are typically low in exon number, shorter in length, and structurally simpler than protein-coding genes [72]. Together, these features support the emerging consensus that plant lncRNAs represent a distinct class of regulatory transcripts with a unique genomic architecture and distribution, and they provide a robust resource for future functional studies on lncRNA-mediated regulation of development and stress responses in B. napus, complementing earlier stress-focused lncRNA catalogs in this species [39]. In summary, line 383-5 displays clear physiological sensitivity to salinity stress due to weak antioxidant defenses and a limited capacity for redox stabilization. Transcriptomic analysis indicates that effective salt tolerance in B. napus relies on integrated control of antioxidant systems, hormone-signal crosstalk, ion transport, and cell wall fortification, with lncRNAs functioning as secondary regulators within these networks. These insights point to breeding strategies focused on reinforcing redox homeostasis and stress-signaling coordination to enhance B. napus NaCl resilience.

5. Conclusions

In summary, this study provides a comprehensive, systems-level characterization of salinity responses in B. napus, integrating physiological measurements with transcriptomic profiling of both coding and non-coding RNAs. Salt stress elicited marked morphological deterioration, oxidative imbalance, and osmotic adjustment, which were underpinned by extensive transcriptional reprogramming across signaling, transport, and metabolic pathways. The coordinated activation of abscisic acid- and ethylene-mediated signaling networks, MAPK cascades, and redox regulatory systems underscores the central role of hormone–ROS crosstalk in orchestrating stress adaptation. Furthermore, strong induction of phenylpropanoid and lignin biosynthesis genes highlights the importance of secondary metabolism and cell wall reinforcement in enhancing structural stability and stress tolerance under saline conditions.

Importantly, the identification of a large set of salt-responsive lncRNAs and their associated cis- and trans-regulatory targets reveals an additional regulatory layer contributing to the fine-tuning of stress responses. These findings advance current understanding of how multilayered signaling pathways are synchronized to maintain cellular homeostasis under salinity stress. From an applied perspective, the candidate genes and regulatory modules identified here provide a valuable resource for molecular breeding and biotechnological strategies aimed at improving salt tolerance in rapeseed. Future studies integrating functional validation, multi-omics approaches, and field-based evaluations will be essential to translate these molecular insights into resilient cultivars adapted to increasingly saline agroecosystems.