Integrated Physiological and Multi-Omics Analyses Reveal the Coordinated Regulation of Carbon and Nitrogen Metabolism in Rapeseed (Brassica napus L.) Tolerance to Saline-Alkaline Stress

TL;DR

This study explores how different rapeseed varieties respond to saline and alkaline stress, revealing how one variety maintains metabolic balance and defends against stress.

Contribution

The study introduces an integrated multi-omics approach to uncover coordinated carbon and nitrogen metabolism in stress tolerance of Brassica napus.

Findings

H62 rapeseed variety shows enhanced photosynthesis and carbon–nitrogen homeostasis under stress.

Alkaline stress induces more gene expression changes than saline stress in H62.

H62 accumulates more beneficial metabolites like amino acids and phenolic acids compared to X15.

Abstract

Background/Objectives: Soil salinization and alkalization critically limit global agricultural production. This study aimed to investigate the differential response mechanisms of rapeseed (Brassica napus L.) varieties to saline and alkaline stresses at the seedling stage. Methods: Seedlings of a salt-tolerant variety, Huayouza 62 (H62), and a non-salt-tolerant variety, Xiangyou 15 (X15), were exposed to saline (NaCl:Na2SO4 = 1:1) and alkaline (Na2CO3:NaHCO3 = 1:1) stresses. An integrated analysis combining physiology, biochemistry, transcriptomics, and metabolomics was conducted to systematically elucidate their differential stress responses. Results: (1) H62 maintained favorable photosynthetic and carbon–nitrogen homeostasis. Notably, under saline and alkaline stresses, the activity of glutamate dehydrogenase (GDH) in H62 showed a significant increasing trend, whereas it was inhibited in X15. (2) Alkaline stress triggered more differential genes than saline stress, with H62 exhibiting broader transcriptional up-regulation in carbon–nitrogen metabolism. (3) Metabolomic profiling showed that H62 accumulated more beneficial metabolites than X15 under both stresses, such as phenolic acids, amino acids, and their derivatives. (4) In multi-omics analysis, key genes in starch–sucrose and amino acid metabolism in H62 were up-regulated to accumulate osmolytes, enabling an efficient defense network. However, X15’s responses were disordered. Conclusions: H62 leverages robust transcriptional reprogramming to coordinate carbon–nitrogen metabolism, constituting a multidimensional defense network. This study provides potential physiological indicators, candidate genes, and metabolite markers associated with short-term saline–alkaline stress responses, laying a foundation for further exploration of stress response mechanisms.

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1. Introduction

Soil salinization affects nearly 20% of arable land and over 30% of irrigated land worldwide, hindering agricultural production [1,2]. Xinjiang is one of the most severely salinization-affected regions in China, with a salinized land area exceeding 13.36 million hectares [3,4]. Soil salinization leads to two types of stresses, namely saline (mainly containing NaCl and Na_2_SO_4_) and alkaline (mainly containing Na_2_CO_3_ and NaHCO_3_) stresses. Generally, alkaline stress causes more negative impacts on crop enzyme activity and ion balance than saline stress [5,6,7].

Rapeseed (Brassica napus L.) is the third largest oil crop after soybeans and oil palm trees worldwide. It is also a major source of protein feed [8,9]. Currently, its growth and productivity are highly threatened by soil salinization worldwide, particularly during the vulnerable seedling stage. The seedling stage is critical for rapeseed vegetative growth [10]. However, saline and alkaline stresses triggered by soil salinization directly disrupt the cell functions of crop seedlings by reducing cell membrane permeability and triggering oxidative damage. In addition, saline and alkaline stresses interfere with electron transfer in photosynthetic reaction centers and suppress the activity of key enzymes involved in carbon and nitrogen metabolism, obstructing photosynthesis and dry matter accumulation during the seedling stage. This inevitably affects subsequent reproductive growth [11,12,13].

Crops respond to saline and alkaline stresses by co-regulating multiple pathways. Particularly, in the seedling stage, the synergistic effect of responses in the photosynthetic system and carbon–nitrogen metabolism is key for crops’ survival under stress [14]. Carbon metabolism provides energy and osmolytes for seedling growth, and the accumulation of soluble sugars (sucrose, glucose) contributes significantly to its salt tolerance. Starch, as a carbon pool, is regulated by key enzymes, i.e., soluble starch synthase (SSS), sucrose synthase (SS), and sucrose phosphate synthase (SPS) [15]. Nitrogen metabolism supplies nitrogen sources. Its products (soluble proteins, proline, and glutamate) also protect against osmotic stress and oxidative damage, which are crucial for maintaining cellular structural stability [16]. However, nitrogen metabolism depends on key enzymes. Nitrate reductase (NR) is a rate-limiting enzyme for nitrogen assimilation [17,18]. The glutamine synthetase/glutamate synthase (GS/GOGAT) cycle is the main pathway for ammonia assimilation [19]. Glutamate dehydrogenase (GDH) participates in the reuse and detoxification of ammonia under adversities [20].

Metabolomics can systematically reveal the dynamic changes in small-molecule metabolites in crop seedlings under stress. Transcriptomics can reveal the regulatory patterns of differentially expressed genes (DEGs). The integration of the two provides a powerful tool for elucidating the molecular mechanisms of the saline and alkaline stress responses of rapeseed seedlings [21]. However, current multi-omics research on rapeseed mostly focuses on a single stress type (i.e., NaCl stress) or a single variety, rarely analyzing the responses of rapeseed varieties with different salt tolerance to saline and alkaline stresses at the seedling stage. Thus, these studies cannot fully clarify the differential molecular mechanisms underlying the responses to saline and alkaline stresses at the seedling stage, and are unable to establish a complete regulatory network involving photosynthesis, carbon–nitrogen metabolism, metabolites, and genes.

To address these limitations, this study selected two rapeseed varieties with contrasting salt tolerance: the non-salt-tolerant variety Xiangyou 15 and the salt-tolerant genotype Huayouza 62. The differential salt tolerance between these two varieties has been systematically confirmed in our previous study [21], thereby providing a reliable comparative system to dissect the physiological and molecular bases of their differential stress responses. This study simulated saline (NaCl:Na_2_SO_4_ = 1:1 (molar ratio)) and alkaline (Na_2_CO_3_:NaHCO_3_ = 1:1 (molar ratio)) stresses for the two rapeseed varieties at the seedling stage. Then, the leaf photosynthetic parameters, enzymes of carbon–nitrogen metabolism, metabolome, and transcriptome were systematically analyzed. The aims were to clarify (1) the stress response patterns of key metabolites and enzymes of carbon–nitrogen metabolism in different salt-tolerant varieties and the correlations with photosynthetic characteristics; (2) the co-regulation network involving key metabolites and DEGs; and (3) the differences in physiological and molecular mechanisms underlying stress responses between the two varieties. The research will elucidate the early response mechanisms of rapeseed seedlings to saline and alkaline stresses, and identify key physiological and molecular markers that distinguish salt-tolerant and non-salt-tolerant varieties, providing a basis for breeding salt stress-resilient genotypes.

2. Materials and Methods

2.1. Determination of Stress Tolerance Thresholds Based on Growth Parameters

The experiment was conducted at the Experimental Station of the Agricultural College, Shihezi University, Xinjiang, China (44°18′46″ N, 86°03′27″ E). The seeds of two rapeseed varieties with contrasting salt tolerance, the non-salt-tolerant variety Xiangyou 15 (X15) and the salt-tolerant variety Huayouza 62 (H62), were selected based on our previous study [22]. Seeds of uniform size from both varieties were disinfected by soaking in a 0.5% NaClO solution for 10 min and sown in seedling trays. After germination, the seedlings with two leaves were transplanted into pots filled with vermiculite. During this period, the seedlings were irrigated with 1/2 Hoagland nutrient solution every three days.

When the seedlings had three leaves, saline and alkaline stress treatments were carried out. NaCl and Na_2_SO_4_ (1:1) were mixed using 1/2 Hoagland nutrient solution for saline stress treatments, with concentrations of 0, 50, 100, 150, and 200 mmol/L. Na_2_CO_3_ and NaHCO_3_ (1:1) were mixed using 1/2 Hogland nutrient solution for alkaline stress treatments, with concentrations of 0, 25, 50, 75, and 100 mmol/L. The prepared salt solutions were separately added to the pots. The daily water loss was calculated using the weight loss method, followed by water supplementation. After 14-day salt stress, plant samples were collected to determine the stress tolerance thresholds of X15 and H62 by measuring their phenotypic parameters, such as plant height, root length, and fresh weight.

Specifically, one seedling was randomly selected from each pot (three plants per treatment). After cleaning the roots with deionized water, filter paper was used to remove the surface water. A vernier caliper was used to determine the plant height and root length (cm). A balance was used to accurately weigh the leaves and roots to obtain leaf and root fresh weight (g), respectively. Afterwards, the fresh samples were dried in an oven at 105 °C and then dried at 80 °C to a constant weight. The dry weight (g) was determined using a scale. Then, regression equations between salt concentrations and phenotypic parameters were established, and Spearman’s correlation coefficients between the salt concentrations and each phenotypic parameter were calculated. The parameter with the highest correlation was identified and used to determine the saline- and alkaline-stress tolerance thresholds, i.e., the salt concentrations that caused a 50% reduction in the phenotypic parameter compared with the non-stressed control [23].

2.2. Experimental Design

Based on the saline- and alkaline-stress tolerance thresholds determined above, the following treatments were carried out on the seedlings with three leaves: (1) HCK (1/2 Hoagland nutrient solution for H62); (2) XCK (1/2 Hoagland nutrient solution for X15); (3) HSS (saline stress treatment for H62, NaCl:Na_2_SO_4_ = 1:1; concentration: 200 mmol/L); (4) XSS (saline stress treatment for X15, NaCl:Na_2_SO_4_ = 1:1; concentration: 200 mmol/L); (5) HAS (alkaline stress treatment for H62; Na_2_CO_3_:NaHCO_3_ = 1:1; concentration: 50 mmol/L); (6) XAS (alkaline stress treatment for X15; Na_2_CO_3_:NaHCO_3_ = 1:1; concentration: 50 mmol/L). The volume of the salt solutions irrigated was twice that of each pot to ensure that the solution in each pot was completely renewed. Excess solution that drained from the pot bottom was immediately collected and then placed on a tray. After 24 h, the second true leaf was collected from the apex of each plant (biological replicates, n = 3), frozen in liquid nitrogen, and stored in a refrigerator at −80 °C for the analysis of physiological parameters (excluding photosynthesis) and multi-omics.

2.3. Determination of Photosynthetic Parameters

After 24 h stress treatments, the LI-6400XT portable photosynthesis system (LI-COR, Lincoln, NE, USA) was used to determine the photosynthetic parameters of the second true leaf from the apex of each rapeseed seedling on a cloudless morning (9:00–11:00). The instrument was preheated for 30 s. The flow rate, air pressure, and light source were calibrated before the measurement. During the measurement, the photosynthetic photon flux density (PPFD) was maintained at 1000 µmol m^−2^·s^−1^, the reference CO_2_ concentration was set to 400 µmol·mol^−1^, and the leaf temperature and vapor pressure deficit (VPD) were controlled at 25 °C and 1.0–1.5 kPa, respectively. After the environment in the leaf chamber stabilized, the net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular carbon dioxide concentration (Ci), and transpiration rate (Tr) of the rapeseed leaves were determined. Six biological replicates were used for each treatment, and three stable technical readings per leaf were averaged to ensure data reliability.

2.4. Determination of the Products of Carbon Metabolism

Rapeseed leaves were dried at 105 °C in an oven and then dried to a constant weight at 80 °C. The dried samples were ground and sieved through a 100-mesh sieve. Then, 50 mg of the sample and 4 mL of 80% ethanol were added to a 10 mL test tube and placed in a water bath at 80 °C, followed by constant stirring. The samples were centrifuged at 4000 r/min to extract the supernatant. The residues were repeatedly extracted three times with 80% ethanol. The supernatants were mixed and precipitated. After that, 10 mg of activated carbon was added to the supernatant, followed by decolorizing in a water bath at 80 °C for 30 min. After diluting to 10 mL with 80% ethanol and filtering, the filtrate was collected for later use.

The content of soluble sugars was determined using the anthrone-based colorimetry [24]. The sucrose content was determined using the resorcinol-based colorimetry [25]. The glucose content was determined using the glucose oxidase method [26]. The acid hydrolysis was used to decompose starch into glucose, followed by the calculation of the starch content based on the glucose content [27].

2.5. Determination of the Activities of Enzymes of Carbon Metabolism

The activities of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), soluble starch synthase (SSS), sucrose synthase (SS), and sucrose–phosphate synthase (SPS) were determined using the corresponding kits (Solarbio, Beijing, China) according to the manufacturer’s instructions.

2.6. Determination of the Products of Nitrogen Metabolism

The soluble protein content was determined using the Coomassie Brilliant Blue G-250-based colorimetry [28]. The proline content was determined using the ninhydrin-based colorimetry [29].

The glutamic acid (Glu) content was determined using a detection kit (BC1580, 50 tubes/48 samples, Solarbio, Beijing, China) according to the manufacturer’s instructions. The ammonium nitrogen content was determined using the phenol hypochlorite method [30].

2.7. Determination of the Activities of Enzymes Related to Nitrogen Metabolism

The activities of nitrate reductase (NR), glutamine synthetase (GS), glutamate synthetase (GOGAT), and glutamate dehydrogenase (GDH) were determined using the detection kits (Solarbio, Beijing, China) according to the manufacturer’s instructions.

2.8. RNA Extraction and RNA Sequencing

To explore the molecular mechanism of different salt-tolerant rapeseed varieties responding to saline and alkaline stress, 18 frozen leaf samples (2 varieties, 3 treatments, 3 replicates) were used for transcriptome analysis. RNA was extracted using the ethanol precipitation method and CTAB-PBIOZOL. Oligo (dT)-coated magnetic beads were used to enrich the mRNA with polyA tails, followed by fragmentation under the action of divalent cations. With the fragmented mRNA as a template, random primers were used to initiate the synthesis of the first strand of cDNA. After RNaseH degraded the RNA, the second strand was synthesized to obtain double-stranded cDNA. After purification, the ends were repaired, A tails were added, and sequencing adapters were connected. The approximately 200 bp fragment was selected and enriched by polymerase chain reaction (PCR). Finally, Qubit, Agilent 2100 (Agilent Technologies, Santa Clara, CA, USA), and quantitative PCR (qPCR) were used to perform quality control on the library concentration, inserted fragments, and effective concentration. After passing the library quality inspection, the Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA) was used for sequencing.

2.9. Widely Targeted Metabolomic Analysis Based on UPLC-MS/MS (Ultra Performance Liquid Chromatography–Tandem Mass Spectrometry)

A widely targeted metabolomics analysis was conducted by MetWare Biotechnology Co., Ltd. (Wuhan, China). The rapeseed samples frozen at −80 °C were subjected to freeze-drying and ground into powder. Then, 50 mg of powder was mixed with 1.2 mL of 70% methanol extract, followed by extraction with intermittent vortexing. After centrifugation, the supernatant was filtered through a 0.22 μm membrane. The filtrate was used for subsequent UPLC-MS/MS analysis. Extracts were analyzed using the UPLC (ExionLC™ AD, SCIEX, Framingham, MA, USA, https://sciex.com.cn/, accessed on 28 May 2025) and MS/MS (Applied Biosystems 4500 QTRAP, SCIEX, Framingham, MA, USA, https://sciex.com.cn/, accessed on 28 May 2025). The characterization of metabolites was based on the information from the secondary ion mass spectrometry of the self-built database MWDB (Metware database). The interference signals of isotopes and common adduct ions were eliminated during analysis. The multiple reaction monitoring (MRM) of triple quadrupole mass spectrometry was used for metabolite quantification. Chromatographic peaks were integrated, and the integrated areas were used for relative quantification, followed by correction among samples [31].

2.10. Analysis of Differentially Expressed Genes (DEGs) and Differentially Accumulated Metabolites (DAMs)

2.10.1. Transcriptomics Analysis

The raw data were filtered using Fastp (version 0.19.3). The gene annotation was completed using featureCounts (version 1.6.2), and then the Fragments Per Kilobase per Million mapped reads (FPKM) for each gene was calculated based on its length. Differential expression analysis was performed using DESeq2 (version 1.22.1), and DEGs were extracted by |log2Fold Change| ≥ 1 and false discovery rate (FDR) < 0.05. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to further evaluate the biological functions of DEGs in the two rapeseed varieties under saline and alkaline stresses. Enrichment analyses were performed based on the hypergeometric test. For KEGG, a hypergeometric test was performed based on the pathways; for GO, a hypergeometric test was performed based on GO terms.

2.10.2. Metabolomics Analysis

Principal component analysis (PCA) and hierarchical clustering analysis (HCA) were performed on the data using R (www.R-project.org, accessed on 2 July 2025). Pearson’s correlation coefficients between samples were calculated. Differentially accumulated metabolites were extracted by variable importance projection (VIP) > 1 and |Log2FC| ≥ 1.0 using the Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA) model. Finally, KEGG pathway analysis was performed on the DAMs (http://www.kegg.jp/kegg/compound/, accessed on 5 July 2025), and the significantly enriched metabolic pathways were determined through metabolite set enrichment analysis (MSEA).

2.10.3. Integrated Analysis of Transcriptomics and Metabolomics

Co-expression network analysis of DEGs and DAMs was performed (Pearson’s correlation coefficient > 0.8, p-value < 0.05) to study the complex interactions between DEGs and DAMs of rapeseed varieties under saline and alkaline stresses.

2.11. Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR)

To validate RNA-Seq data, 12 genes were selected for RT-qPCR analysis using Tb-Green^®^Premix Ex Taq™II (Takara, Beijing, China) based on the fold changes in expression and significant enrichment in key KEGG pathways. ACTIN7 was used as an internal reference gene [32,33]. Table S1 shows the primers for the 13 genes. RNA extraction followed the method described above. The relative expression of 12 genes was calculated using the 2^−ΔΔCt^ method [34].

2.12. Data Analysis

Data were expressed as mean ± standard error. Statistical analysis was performed using SPSS Statistics 19 (IBM Corp., Armonk, NY, USA). One-way analysis of variance (ANOVA) was used to determine the overall effect of the treatments. Duncan’s test was used for multiple comparisons to determine significant differences between treatments (p < 0.05). Before analysis, the homogeneity of variances and normality were tested using Levene’s test and the Shapiro–Wilk test, respectively. Graphs were drawn using Origin 2021 software (OriginLab Corp., Northampton, MA, USA) and Metware Cloud (https://cloud.metware.cn).

3. Results

3.1. Stress Tolerance Threshold of Rapeseed

The leaf yellowing and growth inhibition of the two rapeseed varieties increased with the increase in salt (saline and alkaline) concentration. This was more prominent under alkaline stress (i.e., under the 100 mol/L alkaline stress treatment, most leaves of the rapeseed varieties turned yellow and fell off). In addition, under each stress treatment, there was a difference in growth inhibition between the two varieties, i.e., the growth inhibition was more severe in X15 than in H62 (Figure 1).

The saline- and alkaline-stress tolerance thresholds of the growth parameters of the two rapeseed varieties are shown in Tables S2 and S3. It was found that the correlation coefficients between leaf fresh weight and salt (saline and alkaline) concentrations were the highest (Figure S1). Based on the stress tolerance thresholds of fresh leaf weight of the two varieties, the salt concentrations for saline and alkaline stress treatments were determined to be 200 and 50 mmol/L, respectively, for subsequent tests.

3.2. Effects of Saline and Alkaline Stresses on the Leaf Photosynthetic Characteristics

Saline and alkaline stresses significantly inhibited the photosynthesis of the two varieties, manifested as significantly lower Pn, Tr, and Gs compared with CK (p < 0.05). The Ci showed differences between varieties and treatments (Figure 2). Under saline stress, the Pn of H62 and X15 was 35.66% and 51.10% lower than that of CK, respectively (p < 0.05). Alkaline stress had a stronger inhibitory effect, with the Pn of H62 and X15 53.87% and 66.96% lower than that of CK (p < 0.05), respectively. Under saline and alkaline stresses, the Pn of X15 was 27.89% and 31.96% lower than that of H62, respectively (p < 0.05). Under saline stress, the Tr of H62 and X15 was 42.40% and 63.35% lower than that of CK (p < 0.05), respectively. Under alkaline stress, the Tr of H62 and X15 was 52.48% and 82.41% lower than that of CK (p < 0.05), respectively. Under saline stress, the Gs of H62 (255.83 ± 12.38 mmol·m^−2^·s^−1^) and X15 (250.53 ± 17.86 mmol·m^−2^·s^−1^) were 34.56% and 68.60% lower than that of CK (p < 0.05), respectively. Under alkaline stress, the Gs of H62 (188.73 ± 7.15 mmol·m^−2^·s^−1^) and X15 (129.80 ± 5.04 mmol·m^−2^·s^−1^) were 51.72% and 68.6% lower than that of CK, respectively (p < 0.05).

3.3. Effects of Saline and Alkaline Stresses on the Products of Carbon Metabolism in Rapeseed Leaves

Saline and alkaline stresses significantly changed the composition of products of carbon metabolism in rapeseed leaves (Figure 3). The contents of soluble sugar, sucrose, and glucose of H62 and X15 leaves under both stresses were significantly higher than those of CK, while the starch content was significantly lower than that of CK. The effect of alkaline stress was stronger than that of saline stress. Among them, the responses in soluble sugar of H62 and X15 were the most prominent under alkaline stress, being 190% and 115.27% higher than that of CK (p < 0.05), respectively. In addition, under this stress, the soluble sugar content of H62 was 28.03% higher than that of X15 (p < 0.05). The response of the sucrose content showed similar variety differences. The sucrose content of H62 and X15 was 76.30% and 28.49% higher than that of CK under alkaline stress (p < 0.05), and the sucrose content of X15 was 31.55% lower than that of H62 (p < 0.05).

3.4. Effect of Saline and Alkaline Stresses on the Enzymes of Carbon Metabolism in Rapeseed Leaves

Saline and alkaline stresses significantly regulated the activity of key enzymes of carbon metabolism in rapeseed leaves (Figure 4). Saline and alkaline stresses inhibited the activities of RuBisCO and SSS compared with CK. The effect of alkaline stress was generally stronger than that of saline stress. Under alkaline stress, the RuBisCO activity of H62 and X15 was 41.23% and 26.08% lower than that of CK (p < 0.05), respectively, and the SSS activity was 50.23% and 31.81% lower than that of CK (p < 0.05), respectively. However, saline and alkaline stresses significantly enhanced the activities of key enzymes involved in sucrose synthesis (SPS and SS) compared with CK. Under alkaline stress, the SPS activity of H62 and X15 was 339.18% and 60.01% higher than that of CK, respectively (p < 0.05). The SS activity of H62 reached its highest under saline stress (i.e., 232.86% higher than that of CK). Under both stresses, the activities of SPS and SS of H62 were higher than those of X15 (p < 0.01).

3.5. Effects of Saline and Alkaline Stresses on the Products of Nitrogen Metabolism in Rapeseed Leaves

Saline and alkaline stresses had significant regulatory effects on key products of nitrogen metabolism in rapeseed leaves (Figure 5). Under both stresses, the contents of osmolytes soluble protein and proline of the two varieties, particularly H62, were significantly higher than those of CK. The responses of nitrogen metabolism intermediates revealed more prominent differences in responses between the two varieties. Glutamate in H62 continued to accumulate under both stresses, while the glutamate content in X15 was significantly lower than that of CK under alkaline stress. Correspondingly, the content of ammonium nitrogen (NH_4_^+^-N) in X15 leaves showed an abnormal surge under alkaline stress (it was 164% higher than that of H62 (p < 0.05)). Notably, the content of ammonium nitrogen in X15 was negatively correlated with GDH activity (r = −0.89, p = 0.003).

3.6. Effect of Saline and Alkaline Stresses on the Enzymes of Nitrogen Metabolism in Rapeseed Leaves

Saline and alkaline stresses significantly and differentially regulated the activity of key enzymes of nitrogen metabolism in rapeseed leaves (Figure 6). Overall, the activities of NR, GS, and GOGAT of the two varieties, particularly X15, under both stresses were all lower than those of CK, and the inhibitory effect of alkaline stress was more significant than that of saline stress. The decreases in enzyme activities of X15 compared to CK were significantly larger than those of H62 (i.e., under alkaline stress, 53.36% vs. 30.23% in GS; 62.63% vs. 23.45% in GOGAT, p < 0.05). On the contrary, there was a difference in the response of GDH activity between the two varieties. The GDH activity of H62 under both stresses, particularly under alkaline stress, was higher than that of CK (p < 0.05). However, the GDH activity of X15 was lower than that of CK (p < 0.05). The GDH activity of H62 under saline and alkaline stresses was 241% and 334% higher than that of X15 (p < 0.05), respectively.

3.7. Transcriptome Analysis

3.7.1. Transcriptome Sequencing and Differentially Expressed Genes

Transcriptome analysis showed that after removing low-quality reads, a total of 867,715,788 clean reads were obtained. The quality score 30 (Q30) and guanine–cytosine (GC) content were 84.12–93.01% and 47.12–47.92%, respectively. In addition, the potential error rate was 0.03%, indicating a high quality of the transcriptome sequencing data (Table S4). The reads and uniquely mapped reads both exceeded 80%, indicating high suitability of the selected reference gene (Table S5). There were 1666, 3593, 1131, and 1952 DEGs identified in HSS vs. HCK, HAS vs. HCK, XSS vs. XCK, and XAS vs. XCK, respectively (Figure 7a). The Venn diagram showed that under saline and alkaline stresses, there were 665 common DEGs in H62 and 389 in X15 (Figure 7c). The heat map showed that saline and alkaline stresses induced transcriptional changes in both varieties (Figure 7b).

3.7.2. GO and KEGG Pathway Analyses

Gene ontology enrichment analysis divided DEGs into three different but interrelated groups: cellular component (CC), biological process (BP), and molecular function (MF). In CC, the terms “cellular anatomical entity” and “protein-containing complex” were significantly enriched by the DEGs. In BP, the terms “cellular process”, “metabolic process”, and “response to stimulus” were significantly enriched. In MF, the terms “binding”, “catalytic activity”, “transcription regulator activity”, and “structural molecule activity” were significantly enriched (Figure 8; Table S6). Therefore, the salt-tolerant rapeseed (H62) mainly responded to saline and alkaline stresses by enhancing photosynthesis, systemic acquired resistance, carboxylic acid binding, and oxidoreductase activity.

Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that a total of 1213 (HSS vs. HCK), 2683 (HAS vs. HCK), 525 (XSS vs. XCK), and 1448 (XAS vs. XCK) DEGs were annotated to 123, 134, 120, and 125 KEGG pathways, respectively. The pathways enriched by the DEGs of H62 were concentrated in secondary metabolite synthesis, lipid metabolism (i.e., glycerophospholipid metabolism), and stress-related signal transduction (i.e., the MAPK pathway). The pathways enriched by the DEGs of X15 were concentrated in maintaining basal metabolism (i.e., ribosome and nucleotide metabolism) and starch and sucrose metabolism (Figure 9; Table S7).

3.7.3. Analysis of Differentially Expressed Genes Related to the Enzymes of Carbon and Nitrogen Metabolism and Gene Functional Verification

This study identified 32 DEGs related to the enzymes of carbon metabolism (RuBisCO (9 DEGs), SS (21), SPS (1), and SSS (1)) and 17 DEGs related to the enzymes of nitrogen metabolism (NR (4), GOGAT (3), GS (1), and GDH (1)) (Figure S2, Table S8). The change trend of related DEGs was similar to that of the enzymes of carbon and nitrogen metabolism. RT-qPCR confirmed the reliability of the transcriptome sequencing data (Figure S3).

3.8. Metabolomics Analysis

3.8.1. Quality Control of Metabolomics Data

A total of 828 metabolites were detected in all samples. The metabolites were divided into phenolic acids (19.44%), lipids (16.18%), amino acids and their derivatives (14.37%), Others (12.80%), alkaloids (8.82%), organic acids (8.45%), flavonoids (7.00%), nucleotides and their derivatives (6.88%), lignans and coumarins (2.90%), terpenes (2.17%), and quinones (0.97%) (Figure S4). According to OPLS-DA analysis, these metabolites were clearly divided into three different groups (Figure 10a). Therefore, there were significant differences in the metabolite profiles of the two rapeseed varieties under saline and alkaline stresses.

For H62, the contents of amino acids and their derivatives, alkaloids, Others, nucleotides and their derivatives, and terpenes under saline stress were significantly higher than those of CK. However, the contents of lipids, organic acids, phenolic acids, flavonoids, lignans, and coumarins were significantly lower than those of CK. Under alkaline stress, the contents of most metabolites (phenolic acids, amino acids and their derivatives, alkaloids, Others, nucleotides and their derivatives) were significantly higher than those of CK.

For X15, the contents of phenolic acids, nucleotides and their derivatives, flavones, lipids, and amino acids and their derivatives under saline stress were significantly higher than those of CK. However, the contents of Others, organic acids, and terpenes were significantly lower than those of CK. Under alkaline stress, the contents of amino acids and their derivatives, nucleotides and their derivatives, and lipid alkaloids were significantly higher than those of CK. The contents of Others, organic acids, and phenolic acids were significantly lower than those of CK (Figure 10b).

The Venn diagram analysis showed that HSS vs. HCK and HAS vs. HCK had 41 identical DAMs. Among these DAMs, the main ones were amino acids and their derivatives (16), alkaloids (11), and organic acids (4). HSS vs. HCK had 33 DAMs up-regulated and 8 down-regulated, while HAS vs. HCK had 36 up-regulated and 5 down-regulated. XSS vs. XCK and XAS vs. XCK had 19 identical DAMs, which were mainly Others (8), organic acids (4), and nucleotides and their derivatives (3). Both comparison groups had 7 DAMs up-regulated and 12 down-regulated (Figure 10c).

A total of 94 (56 up-regulated, 38 down-regulated), 118 (96 up-regulated, 22 down-regulated), 48 (25 up-regulated, 23 down-regulated), and 52 (28 up-regulated, 24 down-regulated) DAMs were identified in HSS vs. HCK, HAS vs. HCK, XSS vs. XCK, and XAS vs. XCK, respectively (Figure 11).

Based on the fold changes in metabolite abundance, the top 20 DAMs in both rapeseed varieties under saline and alkaline stresses were identified. For H62 (Figure 12a,b), in HSS vs. HCK, the up-regulated DAMs included six alkaloids and five amino acids and their derivatives. The down-regulated DAMs included three organic acids, two alkaloids, two Others, one flavonoid, and one phenolic acid. In HAS vs. HCK, the top 20 DAMs included 10 up-regulated DAMs (7 phenolic acids, 2 alkaloids, and 2 amino acids and their derivatives). The down-regulated DAMs included three organic acids, two terpenes, two phenolic acids, two Others, and one flavonoid. For X15 (Figure 12c,d), XAS vs. XCK had seven up-regulated DAMs, including two nucleotides and their derivatives, one phenolic acid, one lipid, one flavonoid, and one amino acid and its derivatives. There were 13 down-regulated DAMs, including 6 Others, 5 phenolic acids, and 2 organic acids.

3.8.2. Kyoto Encyclopedia of Genes and Genomes Enrichment Analysis of Differentially Accumulated Metabolites

A total of 55, 65, 44, and 31 key metabolic pathways were enriched by the DAMs of HSS vs. HCK, HAS vs. HCK, XSS vs. XCK, and XAS vs. XCK, respectively (Table S9). For H62 (Figure 13a,b), among the top 20 enriched KEGG pathways under saline and alkaline stresses, several pathways were consistently enriched by the DAMs of HSS vs. HCK and HAS vs. HCK, including “Aminoacyl-tRNA biosynthesis”, “Phenylalanine, tyrosine, and tryptophan biosynthesis”, “Biosynthesis of various plant secondary metabolites”, “D-Amino acid metabolism”, and “Glucosinolate biosynthesis”. For X15, among the top 20 enriched KEGG pathways under saline and alkaline stresses (Figure 13c,d), “Glycerophospholipid metabolism”, “2-Oxocarbolic acid metabolism”, “carbon fixation by Calvin cycle”, “Pyruvate metabolism”, and “Ether lipid metabolism” were consistently enriched by the DAMs of XSS vs. XCK and XAS vs. XCK.

3.9. Integrated Analysis of Transcriptomics and Metabolomics

Many DEGs and DAMs of HSS vs. HCK, HAS vs. HCK, XSS vs. XCK, and XAS vs. XCK showed highly positive correlations (Figure 14). That is, changes in the accumulation levels of these metabolites may be directly or indirectly affected by the corresponding genes.

In addition, KEGG enrichment analysis of the transcriptomics and metabolomics revealed the top 25 metabolic pathways that were significantly enriched by the DEGs and DAMs in both rapeseed varieties under saline and alkaline stresses (Figure 15). For H62, 12 pathways were enriched by the DEGs and DAMs under both saline and alkaline stresses, including “Metabolic pathways”, “Biosynthesis of secondary metabolites”, “Plant hormone signal transduction”, “Biosynthesis of amino acids”, “Carbon metabolism”, “Arginine and proline metabolism”, “Sulfur metabolism”, “Purine metabolism”, “Valine, leucine, and isoleucine degradation”, “Nucleotide metabolism”, “β-Alanine metabolism”, and “Monobactam biosynthesis”. The KEGG analysis results of the DEGs and DAMs in X15 under saline and alkaline stresses showed that “Metabolic pathways”, “Biosynthesis of secondary metabolites”, “Pentose and glucuronate interconversions”, “Amino sugar and nucleotide sugar metabolism”, “2-Oxocarboxylic acid metabolism”, “Pyruvate metabolism”, “Glucosinolate biosynthesis”, “Ascorbate and aldarate metabolism”, “β-Alanine metabolism”, “Ether lipid metabolism”, “Carbon fixation in photosynthetic organisms”, and “Valine, leucine, and isoleucine biosynthesis” were consistently enriched by the DEGs and DAMs of XSS vs. XCK and XAS vs. XCK. In summary, the DAMs and DEGs of the two rapeseed varieties were mainly enriched in pathways related to carbon and nitrogen metabolism.

3.10. Key Carbon and Nitrogen Metabolism Pathways in Rapeseed Leaves in Response to Stresses

The analysis of pathways related to carbon and nitrogen metabolism in rapeseed in response to saline and alkaline stresses based on the integrated analysis of transcriptomics and metabolomics showed that starch and sucrose metabolism together constituted the main pathway for the distribution, storage, and utilization of carbon fixed by photosynthesis in rapeseed. In the starch and sucrose metabolic pathways, there were significant differences in the molecular responses of the two rapeseed varieties to saline and alkaline stresses (Figure 16). The analysis of DAMs showed that D-glucose was significantly up-regulated (2.34 times) only in HAS vs. HCK, and the changes in other comparison groups were insignificant.

Gene expression analysis showed that the response of H62 to saline stress was relatively conservative, with only a few genes differentially expressed. However, X15 had eight genes up-regulated, five of which were sucrose synthase (SUS) genes. Under alkaline stress, H62 showed significant transcriptional reprogramming. A total of 30 DEGs were significantly up-regulated in the starch and sucrose metabolism pathway, which were widely involved in glycolysis (i.e., AMY, BAM), sugar activation (glgC), sucrose metabolism (SUS, SPS), and trehalose metabolism (TPS, otsB). In contrast, X15 only had 11 genes up-regulated under alkaline stress, and the response level was significantly lower than that of H62.

The integrated metabolomics–transcriptomics analysis of the amino acid biosynthesis pathway revealed significantly different response mechanisms between the varieties under saline and alkaline stresses (Figure 17). A total of 15 DAMs were identified in this pathway. Under saline stress, eight DAMs exhibited significant up-regulation in H62, including seven amino acids (e.g., L-valine and L-proline), while X15 had one up-regulated sugar only. Under alkaline stress, seven amino acids exhibited significant up-regulation in H62; the number was three in X15. Notably, down-regulation of organic acids 2-isopropylmalate and 3-isopropylmalate was detected in both varieties under both stress conditions.

Gene expression analysis showed that under saline stress, the up-regulated DEGs in H62 were primarily involved in proline (P5CS) and citrate (CS) synthesis, while X15’s up-regulated genes were primarily involved in histidine (HIS4) and tryptophan (trpB) synthesis. Under alkaline stress, H62 exhibited a more extensive response, with 20 DEGs up-regulated, primarily involved in tryptophan (trpG), proline (P5CS), and citrate (CS) synthesis pathways. However, X15 had only seven up-regulated DEGs. Meanwhile, H62 had 11 down-regulated DEGs under alkaline stress, involved in tryptophan, isoleucine, and methionine synthesis, while X15 had very few down-regulated genes.

4. Discussion

Saline and alkaline stresses are abiotic stresses that inhibit the growth and development of plants, causing growth retardation and even death [35]. Changes in plant growth parameters can intuitively reflect the degree of stress-induced growth suppression. This study evaluated the tolerance of two rapeseed varieties under saline and alkaline stresses. It was found that, except for root length, the saline and alkaline stress tolerance thresholds of the growth parameters of H62 were significantly higher than those of X15, indicating a higher saline and alkaline stress tolerance of H62.

Photosynthesis is key to carbon metabolism. It synthesizes organic matter such as sugars by fixing CO_2_, and provides a carbon skeleton and energy for processes such as nitrogen metabolism [36,37]. This study found that saline and alkaline stresses inhibited photosynthesis of rapeseed through different physiological mechanisms, and there were significant differences in the response strategies between H62 and X15. The decrease in Pn is usually caused by both stomatal and non-stomatal limitations, and the dominant factors vary depending on varieties and stress types. In this study, under saline stress, the lower Ci of H62 relative to CK was consistent with the lower Gs and Pn. This indicates that its photosynthetic decline is likely predominantly due to stomatal limitation. This reflects the adaptive strategy of H62 by actively regulating stomata, temporarily sacrificing part of carbon assimilation to maintain water balance, and protect the photosynthetic apparatus [13,38]. Under alkaline stress with more severe damage, the Ci of both varieties was significantly higher than that of CK. This pattern suggests that non-stomatal factors likely became the dominant limitation, as reflected by the accumulation of CO_2_ in the intercellular space that was not efficiently assimilated [39]. Notably, the non-salt-tolerant variety X15 already exhibited higher Ci under less severe saline stress. This may indicate an earlier response of its mesophyll cell photosynthetic function to ionic or osmotic disturbance, a response consistent with non-stomatal limitation.

Saline and alkaline stresses severely disrupt the carbon metabolic homeostasis in plants, primarily by carbon reallocation, i.e., inhibiting carbon fixation and enhancing carbon consumption [40]. During the acute 24 h stress phase studied here, when photosynthesis is suppressed, there is a carbon limitation. Plants often reduce starch synthesis and transform starch reserves into soluble sugars and other osmolytes to maintain cellular homeostasis [13,41,42,43]. The results of this study showed that both rapeseed varieties exhibited significantly higher contents of soluble sugars, sucrose, and glucose, as well as lower content of starch in their leaves under both stresses within this short-term window. The significantly inhibited photosynthesis further confirms the aforementioned initial carbon allocation strategy. Additionally, the salt-tolerant variety H62 demonstrated more efficient acute carbon metabolic response than X15, i.e., the contents of soluble sugar, sucrose, and glucose were significantly higher than those of the non-salt-tolerant variety X15, and the decline in starch content was less pronounced. Ribulose-1,5-bisphosphate carboxylase is a key rate-limiting enzyme in photosynthetic carbon assimilation [44]. Soluble starch synthase serves as the core enzyme in starch synthesis, directly catalyzing the polymerization of photosynthetic assimilates into starch chains [45]. This study found that the activities of RuBisCO and SSS in both varieties (particularly X15) under stresses (particularly under alkaline stress) were significantly lower than those of CK. The lower RuBisCO activity leads to lower photosynthetic assimilation capacity, thereby limiting carbon fixation [46]. This aligns closely with the photosynthetic data. The lower SSS activity in both rapeseed varieties under both stresses indicates inhibited starch biosynthesis, explaining the lower starch content. Li et al. (2022) reported that salt-tolerant plants might enhance the adaptability to adversities by activating enzymes related to the sucrose synthesis, accumulating sucrose as an osmolyte [47]. In this study, the activities of SPS and SS in H62 were significantly higher under saline and alkaline stresses than under CK. The SPS activity of H62 under saline stress was 70% higher than that of CK, and the SPS activity of H62 under alkaline stress was 350% higher than that under saline stress. This reflects the specific adaptation mechanism of H62 to high pH under alkaline stress, ensuring sucrose synthesis. Meanwhile, the SS activity under saline and alkaline stresses was nearly 200% higher than that of CK. This is the primary reason why H62 can accumulate more soluble sugar (including sucrose and glucose) to maintain osmotic balance. In contrast, the responses in SPS and SS activities were weaker in X15, resulting in inadequate soluble sugar accumulation and lower osmotic regulation capability. Collectively, the coordinated shifts within 24 h, characterized by reduced starch synthesis, enhanced soluble sugar production, and altered enzyme activities, provide strong evidence for an early, rapid carbon reallocation response aimed at osmoprotection.

The response of key products of nitrogen metabolism to both stresses shows clear variety heterogeneity. Previous studies [48,49] have shown that, as important osmolytes, soluble protein and proline both show a significantly increasing trend under stress. This study found that the increases in the contents of soluble protein and proline in H62 under saline and alkaline stresses relative to CK were greater than those of X15. The accumulation of soluble proteins helps maintain enzyme activity and osmotic balance [50], and the synthesis of proline depends on glutamate precursors. The variety H62 continued to accumulate glutamate under both stresses (especially alkaline stress). In contrast, X15 had a significantly lower glutamate content under alkaline stress compared with CK. The ammonium nitrogen content of both varieties was higher under both stresses (especially alkaline stress) than under CK, but the content was significantly higher in X15 than in H62. The above differences arise from the differential responses of key enzyme activities. Saline and alkaline stresses significantly inhibited the activities of leaf NR, GS, and GOGAT, and the inhibitory effect of alkaline stress was stronger. The decreases in the activities of these three enzymes under both stresses relative to CK were significantly smaller in H62 than in X15. This may be related to stress-induced expression of NiR genes, ensuring the supply of nitrogen. In this study, NiR genes were significantly up-regulated in H62 under saline and alkaline stresses. However, in X15, one was down-regulated, and one was not significantly changed under saline stress; both were down-regulated under alkaline stress. This is consistent with the findings in Arabidopsis of Tang et al. (2022) [51]. The GS/GOGAT cycle is a core pathway for ammonium assimilation [52]. In this study, the activities of GS and GOGAT were higher in H62 than in X15, thus avoiding the excessive accumulation of ammonium in X15 [53]. This is consistent with the difference in ammonium nitrogen content between the two varieties. Notably, in this study, the response of GDH activity showed a significant variety difference. The GDH activities of H62 under both stresses (particularly alkaline stress, 155.15%) were significantly higher than those of CK. Previous studies have shown that GDH plays a reverse catalytic role when the GS/GOGAT cycle is inhibited, providing a compensatory pathway to synthesize glutamate, alleviate ammonium toxicity, and support proline synthesis [20,54]. This explains H62’s higher contents of glutamate, proline, and other osmolytes under alkaline stress. On the contrary, the GDH activity of X15 under both stresses was significantly lower than that of CK, causing the loss of this metabolic pathway. This results in nitrogen metabolism disorders and loss of stress tolerance. This is consistent with the research findings of Cao et al. (2020) in rice [55]. Cao et al. (2020) found that when GDH activity was inhibited under saline stress, glutamate content and stress tolerance showed a decreasing trend [55]. This is consistent with the research findings in rice that when GDH activity was inhibited under saline stress, glutamate content and stress tolerance showed a decreasing trend [55].

Transcriptomics provides a systematic perspective for uncovering the mechanisms of plant stress tolerance and adaptation by analyzing genome-wide gene expression dynamics [56]. This study showed that stress type and variety characteristics jointly shape transcriptional response patterns, with the number of DEGs induced by alkaline stress being consistently higher than that under saline stress. Moreover, H62 had more DEGs than X15 under both stress conditions. These findings suggest that alkaline stress triggers broader transcriptional reprogramming [57]. The variety H62 possesses a more robust stress regulatory network, enabling more systematic defense responses. The GO and KEGG enrichment analyses revealed differences in responses to both stresses between the two varieties, with H62 demonstrating more precise and coordinated regulations. Under saline stress, the DEGs of H62 were mainly enriched in pathways such as photosynthesis-antenna proteins (GO:0009522/23). The photosystem-related genes were up-regulated to protect the photosynthetic apparatus, thus maintaining energy supply [58]. Under alkaline stress, pathways like salicylic acid response (GO:0009751), benzoxazinoid biosynthesis, and organic acid metabolism were synergistically activated to enhance antioxidant capacity, regulate pH, and reinforce cell walls [59,60]. In contrast, X15 showed disordered regulation and metabolic imbalance. Saline stress led to the down-regulation of ribosome biogenesis-related genes (GO:0042274/0042255), indicating a damaged protein synthesis system and delayed defense. Under alkaline stress, the expression of cell wall organization-related genes (GO:0009505) was down-regulated, and the polysaccharide decomposition was enhanced, exacerbating cellular structural damage. Further analysis revealed that saline stress preferentially induced pathways related to energy metabolism and osmotic regulation (i.e., sulfur metabolism, arginine/proline metabolism), while alkaline stress prioritizes pathways associated with pH regulation, reactive oxygen species scavenging, and secondary metabolism.

This study systematically revealed the differential metabolic responses of the two rapeseed varieties to saline and alkaline stresses through metabolomics analysis. A total of 11 types of metabolites were identified, among which phenolic acids (organic acids), lipids, amino acids, and their derivatives were dominant. Previous studies [61,62] have shown that organic acids have a positive impact on the ion and pH homeostasis. In this study, under alkaline stress, the content of phenolic acids (32/38) was significantly up-regulated in the leaves of the salt-tolerant variety H62 but mainly down-regulated in the leaves of the non-salt-tolerant variety X15 (6/8). This indicates that phenolic acid accumulation is a key adaptive strategy of H62 to cope with high pH stress. Amino acids are closely related to abiotic stress tolerance and play an active role in maintaining cell osmotic potential and membrane structure and stability [63,64]. In this study, H62 accumulated more amino acids under stresses (saline stress: 34; alkaline stress: 24). The accumulated amino acids proline, phenylalanine, and serine jointly contribute to osmotic regulation and cell protection [65,66]. However, only two and seven types of amino acids and their derivatives were identified in X15 under saline and alkaline stresses, respectively, and the metabolic response was insignificant. In the adaptation of plants to abiotic stresses such as salt stress, the synthesis and accumulation of secondary metabolites are significantly enhanced, such as alkaloids and flavonoids [67,68,69,70]. In this study, H62 up-regulated the content of alkaloids under both stresses and the content of flavonoids under alkaline stress to cope with oxidative stress. In contrast, the response in the secondary metabolites of X15 was overall disordered and inefficient.

Multi-omics analysis showed that saline and alkaline stresses triggered specific and coordinated regulation of starch and sucrose metabolism and amino acid biosynthesis pathways in rapeseed. In addition, there was a difference in the response pattern between the two varieties. In the starch and sucrose metabolic pathways, the salt-tolerant variety H62 exhibited efficient and synergistic transcriptional reprogramming. Particularly under alkaline stress, its core metabolite D-glucose was specifically and significantly up-regulated (2.34 times). At the same time, 30 DEGs were up-regulated in this pathway, widely covering key links such as starch degradation (i.e., BAM, AMY, bgIX), sucrose synthesis (SUS, SPS), and trehalose metabolism (TPS, otsB). This not only stimulates rapeseed leaves to quickly produce osmolytes, but also provides carbon skeleton and energy for other stress-tolerance reactions [71,72]. In contrast, the genetic response of the non-salt-tolerant variety X15 appeared contradictory and inefficient, with a smaller number of DEGs and a lack of activation of key starch degradation genes, resulting in metabolic network disorder and insufficient metabolite production.

In the amino acid biosynthesis pathway, the salt-tolerant genotype H62 achieved adaptation to saline and alkaline stresses through precise regulation of key amino acid synthesis, whereas the non-salt-tolerant genotype X15 exhibited significant regulatory imbalance. The gene P5CS encodes the rate-limiting enzyme in proline biosynthesis. In rapeseed and other crops, salt stress typically induces the up-regulation of P5CS expression, thereby driving substantial proline synthesis [73]. Consistent with this mechanism, H62 showed up-regulation of five and three P5CS genes under saline and alkaline stresses, respectively, accompanied by a 3.05-fold and 2.1-fold increase in L-proline levels. This demonstrates its excellent transcriptomics–metabolomics synergistic regulation ability. In contrast, although X15 could up-regulate three P5CS genes, it failed to induce the accumulation of the corresponding metabolites, revealing obvious defects in post-transcriptional regulation. At the same time, under saline and alkaline stresses, H62 induced the up-regulation of key genes involved in the synthesis pathways of aromatic (i.e., tryptophan) and sulfur-containing amino acids (i.e., cysteine), such as trpG, trpB, and cysE (particularly under alkaline stress, two trpC genes, two cysE genes, one trpB gene, and one trpG gene were up-regulated). This led to a significant accumulation of tryptophan (up-regulated by 12.12-fold under saline stress and 10.94-fold under alkaline stress). In contrast, in X15, the down-regulation of aroDE under saline stress blocked the shikimic acid pathway, resulting in insignificant accumulation of tryptophan. The synergistic action of these genes and metabolites ensured the supply of multiple protective substances [74]. In summary, the salt-tolerant variety H62 has a comprehensive defense network that integrates rapid mobilization of carbon sources, synthesis of osmolytes, and production of protective amino acids by efficiently and synergistically up-regulating gene expression and metabolite accumulation in multiple metabolic pathways. The regulatory network of the non-salt-tolerant variety X15 exhibits structural defects and insufficient synergy. The key genes (i.e., P5CS, BAM, and SUS) and metabolites (i.e., D-glucose and proline) identified in this study provide important molecular targets for genetic improvement of saline and alkaline stress tolerance of rapeseed.

5. Conclusions

This study systematically revealed the differences in physiological and molecular mechanisms of saline and alkaline stress responses between salt-tolerant (H62) and non-salt-tolerant (X15) rapeseed varieties. The results showed that the divergence in saline and alkaline stress tolerance among rapeseed varieties originated from their differing capabilities to execute coordinated molecular reprogramming during the early stress phase. This difference was manifested across three coordinated levels: (1) Proactive coordination at the metabolic level: H62 rapidly initiated the accumulation of soluble sugars (e.g., sucrose), maintained nitrogen assimilation (GS/GOGAT cycle) homeostasis, and efficiently synthesized osmoprotectants like proline via the GDH pathway, achieving rapid rebalancing of carbon and nitrogen metabolism under external stresses. (2) Precise targeting at the transcriptional level: The transcriptional reprogramming was highly directed in H62, specifically activating key defense pathways such as protection of the photosynthetic apparatus, salicylic acid response, organic acid metabolism, and cell wall reinforcement, providing precise regulatory directives for downstream metabolic adjustments. (3) Systemic integration at the multi-omics level: In core pathways like starch and sucrose metabolism and amino acid biosynthesis, H62 achieved efficient synchronization between gene up-regulation and the corresponding accumulation of metabolites. This “gene–metabolite coordination” formed the foundation for constructing an integrated defense network (encompassing osmoregulation, oxidative stress mitigation, and ion homeostasis), which was precisely the critical component deficient in X15. In summary, saline and alkaline stress tolerance in rapeseed depends not only on the presence or absence of individual genes or metabolites but, more crucially, on the rapid and coordinated mobilization capacity among multiple core pathways during the early stress phase. This study systematically analyzed the mechanisms of saline and alkaline stress response of rapeseed from physiological to molecular levels, providing a scientific basis and key targets for genetic improvement of the stress tolerance of rapeseed.