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Article

Integrated Physiological and Transcriptomic Analyses Reveal the Mechanism of Salt Acclimation-Induced Salinity Tolerance in Tomato Seedlings

by
Nuo Fan
†,
Ruiqing Li
†,
Huixin Liu
,
Ke Zhang
,
Guan Pang
,
Xiaoying Liu
,
Lifei Yang
,
Jin Sun
and
Yu Wang
*
College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2026, 12(2), 159; https://doi.org/10.3390/horticulturae12020159
Submission received: 24 December 2025 / Revised: 24 January 2026 / Accepted: 26 January 2026 / Published: 30 January 2026
(This article belongs to the Special Issue Improvement of Resistance Strategies for Horticultural Plant Cultivation)
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Abstract

Although salt acclimation is a recognized strategy for improving crop salt tolerance, its specific role in tomato (Solanum lycopersicum L.) remains unclear. This study investigated the effects of salt acclimation on enhancing salt tolerance in tomato seedlings through physiological and transcriptomic analyses. Here, we found that T3 acclimation treatment (irrigation with 14 mL of 7.5 g L−1 NaCl solution per plant) effectively conferred enhanced salt tolerance in tomato seedlings, with plant height, stem diameter, leaf area, chlorophyll content, net photosynthetic rate, and soluble protein content increasing by 4.52, 5.13, 3.16, 10.78, 11.85, and 25.96%, respectively, compared with the control. T3 treatment also reduced oxidative damage and ionic stress, as evidenced by reduced electrolyte leakage, lower malondialdehyde content, and a decreased root Na+/K+ ratio, while simultaneously boosting antioxidant enzyme activities. Membership function analysis confirmed T3 as the optimal treatment, with a 9 d duration consistently benefiting multiple cultivars. Transcriptomic analysis revealed that salt acclimation upregulated genes associated with phenylpropanoid biosynthesis, lignin catabolic process, and peroxidase activity, suggesting that these pathways might mediate acclimation-induced salt tolerance through promoting lignin biosynthesis to reduce Na+/K+ ratio and enhancing reactive oxygen species’ scavenging capacity to maintain cellular homeostasis. Our results indicate that tomato seedlings acclimated with 14 mL of 7.5 g L−1 NaCl solution per plant for 9 d significantly improves salt tolerance through coordinated physiological adjustments and transcriptional reprogramming.

1. Introduction

Soil salinization poses a significant threat to global agriculture, degrading ecological resources and limiting crop productivity. In China, saline soils account for approximately 25% of arable land, where they primarily impair plant growth through ion toxicity and oxidative stress caused by reactive oxygen species (ROS), leading to severe reductions in yield and quality [1,2]. Enhancing crop salt tolerance has become an urgent agricultural priority. One promising approach is the use of acclimation techniques, which involve pre-exposing young plants to mild stress to trigger inherent defense mechanisms, thereby enhancing their resilience to subsequent, more severe conditions [3].
Salt acclimation functions by briefly subjecting plants to a non-lethal salt stress, activating a suite of physiological and molecular adaptations. These adaptations can include metabolic adjustments, the synthesis of protective compounds, and altered gene expression, collectively preparing the plant to maintain growth and survive under harsher conditions, resulting in exhibiting stronger salt tolerance, which is the ability of plants to withstand salinity stress [4,5]. The efficacy of salt acclimation has been demonstrated across multiple species. For instance, wheat (Triticum aestivum) plants that were subjected to salt acclimation with 25 mM NaCl for 11 d were able to withstand higher levels of salt stress by maintaining chlorophyll content, net photosynthetic rate (Pn), and antioxidant enzyme activities [6]. Similar benefits have been observed in rice (Oryza sativa), red beet (Beta vulgaris), and barley (Hordeum vulgare) [5,7,8]. Acclimation is also essential for maintaining ion homeostasis in leaves and roots and limiting salt stress-induced increase in Na+/K+ ratio, ultimately enhancing salt tolerance [4,9,10]. It should be noted that the effectiveness of acclimation may vary depending on several specific factors, such as plant genotype, stress intensity, and duration. For instance, the Vandaran rice variety acclimated with 10 mM NaCl significantly enhanced salt tolerance by improving membrane stability, K+ content and K+/Na+ ratio in leaves, but these effects were not observed in the Deejiaohualuo variety [11]. Furthermore, the salt-tolerant olive (Olea europaea) cultivar Frantoio acclimated with 5 mM NaCl for one month was more effective in enhancing salt tolerance than the plants acclimated with 25 mM NaCl [12]. The effects of 15 and 25 mM NaCl solution acclimation on salt tolerance in pea (Pisum sativum) were investigated and it was found that acclimation with 15 mM NaCl solution was optimal for increasing salt tolerance by stimulating K+ sequestration in leaves and roots, as well as for maintaining chlorophyll content, Pn, and relative water content [13]. Therefore, in practical applications, it is essential to optimize the acclimation protocol based on specific crops and stress conditions to achieve the best possible improvement in stress tolerance. Despite its success in other crops, research on salt acclimation in tomato (Solanum lycopersicum L.) remains limited. Investigating tomato’s response to salt acclimation and identifying optimal acclimation conditions are essential for developing effective strategies to enhance its salinity tolerance.
This study uses tomato seedlings as the material and investigates the effects of salt acclimation treatment. We subjected the seedlings to various acclimation concentrations and durations, measuring key physiological indicators (e.g., ion content, antioxidant activity, chlorophyll content), and identified differentially expressed genes (DEGs) under salt stress. Our objective was to determine the optimal acclimation conditions to enhance salt tolerance in tomato seedlings. Our results indicate that tomato seedlings acclimated with 14 mL of 7.5 g L−1 NaCl solution per plant for 9 days is the most effective method for increasing salt tolerance, providing a practical solution to mitigate salt stress in tomato production.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

The tomato cultivars used in this study included Ailsa Craig (AC), Zhongshu No. 4, Zhongza No. 9, Jinpeng No. 1, Hezuo903, Beifan306, and Provence. The seeds of AC were preserved in our lab, while the others were commercially obtained: Zhongshu No. 4 and Zhongza No. 9 from China Vegetable Seed Co., Ltd. (Beijing, China); Jinpeng No. 1 from Shandong Shouhe Seed Industry Co., Ltd. (Shouguang, China); Hezuo903 from Shanghai Hongqiao Tianlong Seed Industry Co., Ltd. (Shanghai, China); Beifan306 from Beijingshi Jingfan Tomato Research Institute (Beijing, China); and Provence from Shouguang Agricultural Development Group Co., Ltd. (Shouguang, China). Tomato seeds were soaked in 55 °C water for 15 min, followed by 6 h at room temperature, and then germinated in the dark at 28 °C. The germinated seeds were sown in a 72-well tray (54 cm × 28 cm × 5.5 cm) filled with commercial seedling substrate (Xingnong Substrate Technology Co., Zhenjiang, China) and placed in a growth chamber. Growth conditions were maintained with a 16/8 h light/dark photoperiod, 300 μmol m−2 s−1 light intensity, 24 °C/18 °C day/night temperatures, and 50–65% relative humidity. When the third leaves were fully expanded, the tomato seedlings were subjected to salt acclimation.

2.2. Experimental Design and Treatments

(1)
Determination of optimal acclimation concentration
To determine the optimal salt acclimation concentration, tomato (AC) seedlings at the three-leaf stage were irrigated with 14 mL of NaCl solution per plant at five different concentrations: (i) CK, distilled water (control); (ii) T1, 2.5 g L−1 NaCl; (iii) T2, 5.0 g L−1 NaCl; (iv) T3, 7.5 g L−1 NaCl; (v) T4, 10.0 g L−1 NaCl. After being treated with different concentrations of salt solution for 2 d, seedlings were transplanted into 250 cm3 pots containing the same substrate. After transplanting for 7 d, each pot was irrigated with 50 mL of 150 mM NaCl every two days for three rounds for the salt stress treatment, and the control plants were irrigated with the same volume of distilled water. Plant phenotype and physiological parameters were measured at 7 d of salt stress treatment.
(2)
Determination of optimal acclimation duration
To determinate the appropriate acclimation duration, tomato (AC) seedlings were watered with 14 mL of 7.5 g L−1 NaCl solution per plant for 0 d, 3 d, 6 d, 9 d, and 12 d (designated TD0 to TD4, respectively; the EC of the substrate was 1.77 mS cm−1 before acclimation but increased to 3.67 mS cm−1 after acclimation). Subsequently, seedlings were transplanted and subjected to the same salt stress regime described above.
(3)
Evaluation acclimation effect across tomato cultivars
To investigate the effect of salt acclimation on salt tolerance in different tomato cultivars, AC and six commonly used commercial cultivars in China, including Zhongshu No. 4, Zhongza No. 9, Jinpeng No. 1, Hezuo903, Beifan306, and Provence, were acclimated with 14 mL of 7.5 g L−1 NaCl solution per plant for 9 d. The non-acclimated seedlings were marked as NA, and the acclimated seedlings were marked as A. The seedlings were transplanted to the same pots as described above after acclimation. After transplanting for 7 d, the seedlings were subjected to the salt stress treatment described above.
(4)
Transcriptomic analysis
For transcriptomic analysis of the DEGs, tomato plants of AC were used to collect root samples after salt stress for 1 d. The root samples contained four treatments with three replicant for each treatment, named NACK, NAS, ACK, and AS. NACK and NAS represent non-acclimated seedlings treated with water and salt, respectively. ACK and AS represent acclimated seedlings treated with water and salt, respectively.

2.3. Measurement of Growth Parameters

Plant height was measured from the stem base to the growth point using a ruler. The stem diameter was measured under 0.5 cm of the cotyledons using vernier calipers. The leaf area of the fourth leaf from the bottom was measured using a digital leaf area meter [YMJ-B (2022), Zhejiang Top Cloud Agriculture Technology Co., Ltd., Hangzhou, China].

2.4. Measurement of Chlorophyll Content and Pn

To measure the content of chlorophyll, tomato leaves (0.1 g) were extracted with 20 mL of 95% ethanol in the dark for 24 h, and absorbance values were measured at 665 nm and 649 nm. The chlorophyll content was calculated as previously described [14].
The first fully expanded leaf from the top was used to measure Pn using a portable photosynthesis system (LI-6400; Li-COR, Lincoln, NE, USA), maintaining the CO2 concentration at 380 μmol mol−1 and photosynthetic photon flux density at 800 μmol m−2 s−1.

2.5. Measurement of Electrolyte Leakage (EL), Malondialdehyde (MDA), Proline, and Soluble Protein Content

The level of EL was measured using the method as previously described [15]. The leaves were washed with distilled water, and ten 1 cm diameter leaf disks were excised, intentionally avoiding the midrib. The disks were placed into test tubes and immersed in 10 mL of distilled water. The tubes were sealed, vacuum-infiltrated to remove intercellular air, and incubated at room temperature for 1 h. The initial electrical conductivity (S1) was measured. Subsequently, the tubes were tightly sealed, heated in a boiling water bath for 10 min, and cooled to room temperature. After thorough mixing, the final electrical conductivity (S2) was recorded. EL (%) was calculated as S1/S2 × 100.
Tomato leaf (0.3 g) was homogenized in 3 mL of ice-cold 50 mM phosphate buffer [PBS, pH 7.8, containing 0.2 mM EDTA and 2% (w/v) polyvinylpyrrolidone K 30], and the homogenate was centrifuged at 12,000× g for 20 min at 4 °C. The supernatant was collected, and MDA content was determined using the thiobarbituric acid method [16]. Proline content was detected as previously described [17]. Soluble protein concentration was determined by the Coomassie brilliant blue dye-binding assay [18], using bovine serum albumin as the standard.

2.6. Measurement of Antioxidant Enzyme Activity

Tomato leaf (0.3 g) was homogenized in 10 volumes (w/v) of ice-cold PBS buffer (pH 7.8), and the homogenate was centrifuged at 3000× g for 10 min at 4 °C. The supernatant was collected and assayed for superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activity using commercial kits (Nanjing Jiancheng Technology Co., Nanjing, China) according to the manufacturer’s protocols.

2.7. Measurement of Na+ and K+ Content

The content of Na+ and K+ in root was determined using an inductively coupled plasma spectrophotometer (ICP; ICP, Optima 8000, PerkinElmer, Waltham, MA, USA) as previously described [19].

2.8. Transcriptome Analysis DEGs

Total RNA was isolated from root samples of tomato plants after salt treatment for 1 d using Trizol® Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacture’s instruction. RNA purity and integrity were assessed using the NanoPhotometer® spectrophotometer (IMPLEN, Calabasas, CA, USA) and the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA), respectively. Sequencing libraries were generated using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, Ipswich, MA, USA) following manufacturer’s recommendations, index codes were added to attribute sequences to each sample, and the library quality was assessed on the Agilent Bioanalyzer 2500 system (Agilent Technologies, Santa Clara, CA, USA). Clustering of the index-coded samples was performed on a cBot Cluster Generation System using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform by Beijing Biomics Biotechnology Co., Ltd. (Beijing, China) and paired-end reads were generated.
Raw data (raw reads) were first processed through in-house Perl scripts. Clean data (clean reads) were obtained by removing reads containing adapters, reads with ploy-N (where the ratio of Ns exceeds 10%), and low-quality reads from the raw data. The tomato reference genome sequence (SL4.0) and annotation files (ITAG4.0) were downloaded from the tomato genome website [http://solgenomics.net/ (accessed on 24 July 2025)]. The index of the tomato reference genome was built using Bowtie v2.2.3, and paired-end clean reads were aligned to the tomato reference genome using TopHat v2.0.12 [20]. Transcript levels were calculated using Fragments Per Kilo-base of exon Per Million Fragments mapped (FPKM). DEGs were analyzed using DESeq2 [21], and the genes with a fold change ≥ 2, a false discovery rates < 0.01, and an adjusted p-value < 0.01 were assigned as differentially expressed. Gene ontology (GO) enrichment analysis of DEGs was implemented by mapping to the GO database [http://www.geneontology.org/ (accessed on 26 July 2025)]. KOBAS 2.0 [https://www.biostars.org/p/200126/ (accessed on 26 July 2025)] was used to test the statistical enrichment of DEGs in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways with a threshold of false discovery rates ≤ 0.05 [22].

2.9. Validation of Gene Expression

To validate the RNA-seq data, the expression level of selected upregulated and downregulated genes was confirmed using quantitative real-time PCR (RT-qPCR). Total RNAs were extracted from tomato roots according to the instructions of the RNAsimple Total RNA Kit (Tiangen, Beijing, China). One μg of total RNAs was used to reverse transcribe to the cDNA template using the HiScript II Q RT SuperMix for qPCR (+gDNA wiper) Kit (Vazyme, Nanjing, China). The RT-qPCR assays were performed using the ChamQ SYBR qPCR Master Mix (Vazyme) in the LightCycler® 480 II Real-Time PCR detection system (Roche, Basel, Switzerland). The PCR conditions consisted of denaturation at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 58 °C for 15 s, and extension at 72 °C for 30 s. The actin gene was used as an internal control. Gene-specific primers were designed according to cDNA sequences as listed in Table S1. Relative gene expression was calculated as previously described [23].

2.10. Statistical Analysis

All data were statistically analyzed using SPSS 20 (SPSS Inc., Chicago, IL, USA), and differences between treatments were analyzed at a p < 0.05 level according to Tukey’s test. All data are expressed as the mean ± SD (standard deviations). A membership function-based approach was applied for integrated assessment of multiple indicators, with weights assigned via the entropy weight method to objectively evaluate overall stress resistance.

3. Results

3.1. Effects of Different Salt Acclimation Concentrations on the Growth and Salt Tolerance of Tomato Seedlings

Salt stress for 7 days significantly inhibited the growth of tomato seedlings, as evidenced by reduced plant height and stem diameter, compared with the non-stress controls. However, T3 treatment mitigated this inhibition, resulting in significantly greater plant height and stem diameter (increases of 4.52% and 5.13%, respectively) compared with CK (Figure 1A,B). Salt stress significantly decreased the leaf area and chlorophyll content of tomatoes, but these parameters were still higher in the T3 treatment than in other treatments (Figure 1C,D). Notably, the Pn of T3 was the highest under salt stress, increasing by 11.85% compared with CK (Figure S1). The EL values in the salt acclimation treatments were higher than those of CK, but the salt stress-induced increase in EL was suppressed in those acclimation treatments compared with CK, with T3 treatment showing the lowest EL value (Figure 1E). As shown in Figure 1F, MDA content in the salt-acclimated group was lower than that in the non-acclimated group, indicating that salt acclimation was beneficial for reducing the MDA content in tomato seedlings under high salt stress. Among them, the MDA content was the lowest in the T3 treatment, indicating that the salt acclimation concentration of T3 was the most effective in reducing MDA content. The results indicated that salt acclimation with an appropriate concentration improved the salt tolerance of tomato seedlings under high salt stress.

3.2. Effects of Different Salt Acclimation Concentrations on Proline and Soluble Protein Content, Na+/K+ Ratio, and Antioxidant Enzyme Activity in Tomato Seedlings

Under normal growth conditions, the proline content in the T4 treatment was significantly higher than that in the other treatments, while no significant difference in proline content was observed between the other treatments (Figure 2A). Under salt stress, the proline content in the T4 treatment was the highest, increasing by 41.77% compared with CK, while the proline content in the T3 treatment was the lowest, decreasing by 35.96% compared with T4 (Figure 2A). The soluble protein content of tomatoes increased compared with CK under salt stress, with the T3 treatment showing a 16.27% increase over CK (Figure 2B).
Salt acclimation increased the Na+/K+ ratio under normal growth conditions but suppressed the increase in the Na+/K+ ratio under high salt stress. The Na+/K+ ratio in the T3 treatment decreased by 19.34% compared with CK (Figure 2C), indicating that salt acclimation effectively reduced the Na+/K+ ratio in tomato seedlings under high salt stress.
Under salt stress, the SOD activity ranked from high to low as follows: T3, CK, T1, T2, and T4. The SOD activity in T3 increased by 5.22%, while the SOD activity in T4 decreased by 73.45% compared with CK (Figure 2D). Under salt stress conditions, the POD activity in the T1 treatment was the highest, which was 5.91 times that of CK (Figure 2E). There was no significant difference in CAT activity between CK, T1, and T2, but the CAT activity in the T3 and T4 treatments decreased by 31.44% and 64.32%, respectively, compared with CK (Figure 2F).

3.3. Analysis of the Membership Function of Salt Tolerance Physiological Parameters Under Different Salt Acclimation Concentrations in Tomato Seedlings

To evaluate the effect of salt acclimation concentrations on salt stress tolerance in tomato seedlings, we conducted a comprehensive analysis using membership functions to integrate multiple physiological indicators. Based on the membership function analysis, the salt tolerance levels across treatments ranked from highest to lowest as follows: T3 > T2 > T1 > CK > T4 (Table S2). Tomato seedlings exhibited the strongest salt tolerance in the T3 treatment, achieving a comprehensive physiological score of 0.418, indicating that the T3 treatment not only effectively promoted seedling growth but also significantly enhanced adaptive capacity under high-salinity stress.

3.4. Effects of Different Salt Acclimation Durations on the Growth and Salt Tolerance of Tomato Seedlings

The plant height and stem diameter in the TD3 treatment were both greater than those in the TD0, increasing by 2.05% and 4.01%, respectively, compared with TD0 (Figure 3A,B). Although salt stress reduced leaf area in all treatments, tomato leaf area in the TD3 treatment was 23.65, 23.27, 16.71, and 17.55% higher than that in the TD0, TD1, TD2, and TD4 treatments, respectively (Figure 3C). Furthermore, chlorophyll content and Pn in the leaves of TD3 also showed the highest level under salt stress (Figure 3D and Figure S2). Under salt stress, the EL value in TD1, TD2, TD3, and TD4 decreased by 5.18, 9.13, 14.64, and 9.16%, respectively, compared with TD0 (Figure 3E). The MDA content in the TD3 treatment was the lowest (Figure 3F).

3.5. Effects of Different Salt Acclimation Durations on the Content of Proline and Soluble Protein, Na+/K+ Ratio, and Antioxidant Enzyme Activity in Tomato Seedlings

After salt stress treatment, the proline content in tomato seedlings significantly increased. Among the treatments, TD0 had the highest proline content, while TD3 had the lowest proline content. Compared with TD0, the proline content in TD1, TD2, TD3, and TD4 decreased by 8.48, 13.12, 20.92, and 11.18%, respectively (Figure 4A). The soluble protein content in TD1, TD2, TD3, and TD4 without salt stress treatment slightly decreased compared with TD0, while the soluble protein content in the acclimated tomato seedlings under salt stress increased compared with the non-acclimated group (Figure 4B). Salt acclimation effectively reduced the Na+/K+ ratio of tomato seedlings under salt stress. The Na+/K+ ratio in TD3 decreased by 26.35% compared with TD0, showing the most significant decrease (Figure 4C).
Salt stress induced a decline in SOD activity, but the TD3 treatment showed the lowest decrease in activity (Figure 4D). POD activity gradually increased with the increase in salt acclimation duration under the normal conditions. In the salt stress group, POD activity initially increased and then decreased as the salt acclimation duration increased, with the TD3 treatment showing the highest POD activity (Figure 4E). The CAT activity displayed a similar trend to POD activity, but the highest level was observed in the TD1 treatment (Figure 4F).

3.6. Analysis of the Membership Function of Salt Tolerance Physiological Parameters Under Different Salt Acclimation Durations in Tomato Seedlings

Based on membership function analysis, salt tolerance across treatments decreased in the following order: TD3 > TD2 > TD4 > TD1 > CK (Table S3). This indicated that tomato plants exhibited maximum salt tolerance under the TD3 treatment, while showing minimal tolerance in CK.

3.7. Effects of Salt Acclimation on Salt Tolerance in Different Tomato Cultivars

To further test the role of salt acclimation in salt tolerance, we selected seven tomato cultivars to subject to salt acclimation followed by 150 mM NaCl stress and analyzed the growth parameters and salt tolerance. Under normal growth conditions, salt-acclimated tomato seedlings showed no significant differences in stem diameter and leaf area compared with non-acclimated controls (Figure 5A,B). However, under salt stress, salt-acclimated seedlings exhibited significantly greater stem diameter and leaf area than non-acclimated seedlings (Figure 5A,B). Under normal conditions, the value of EL marginally increased in salt-acclimated tomato seedlings compared with non-acclimated controls, with significant elevations observed in Zhongshu No. 4 and Zhongza No. 9 (Figure 5C). Conversely, salt-acclimated seedlings exhibited significantly reduced EL values versus non-acclimated seedlings under salt stress, with AC, Zhongshu No. 4, Zhongza No. 9, Jinpeng No. 1, Hezuo903, Beifan306, and Provence decreasing by 15.78, 21.85, 20.89, 20.53, 17.42, 26.07, and 14.65%, respectively (Figure 5C). Furthermore, salt acclimation also attenuated the decline in chlorophyll content and Pn under salt stress (Figure S3). Importantly, under salt stress, salt-acclimated tomato seedlings exhibited significantly reduced Na+/K+ ratios compared with non-acclimated seedlings, decreasing by 9.41, 13.20, 16.03, 7.01, 7.26, 13.24, and 8.79% in AC, Zhongshu No. 4, Zhongza No. 9, Jinpeng No. 1, Hezuo903, Beifan306, and Provence, respectively (Figure 5D). These results suggested that salt acclimation enhanced salt tolerance in tomatoes, with all cultivars exhibiting similar trends in physiological responses to salt stress following acclimation.

3.8. Transcriptome Analysis of DEGs in Salt Stressed Tomato Seedlings

To investigate the potential mechanism of salt acclimation-induced salt tolerance in tomato, a total of 12 cDNA libraries were constructed from NA and A tomato roots at 1 d with or without salt stress. A total of 281,275,017 raw reads was obtained, and 279,069,050 clean reads and 83.35 Gb of sequence data were acquired after removing the adapter and low-quality reads (Table S4). The ratio of clean reads to raw reads was more than 99.12% (Table S4). The average Q30 in the NACK, NAS, ACK, and AS groups was 97.17, 97.32, 97.25, and 97.24%, respectively (Table S4). The average GC content across these groups was 41.82, 42.11, 42.34, and 42.53%, respectively. These results suggested that the sequencing quality and integrity were sufficient for further analysis. All reads were aligned to the tomato genome and the mapping rates of approximately 97.08, 97.86, 98.07, and 98.12% for the NACK, NAS, ACK, and AS groups, respectively (Table S4).
To compare the upregulated and downregulated genes between NA and A plants after salt stress, the DEGs were analyzed based on the results of RNA-seq. A total of 394 (221 upregulated, 173 downregulated), 269 (134 upregulated, 135 downregulated), 78 (53 upregulated, 25 downregulated), and 835 (375 upregulated, 460 downregulated) DEGs were detected in the sample of NACK vs. ACK, NACK vs. NAS, ACK vs. AS, and NAS vs. AS, respectively (Figure 6A; Tables S5–S8). Venn diagram analysis showed that 1283 DEGs were detected and only one DEG was identified across all four groups (Figure 6B). Volcano plots revealed distinct distributions of upregulated and downregulated genes across all four comparisons. Significantly more DEGs were identified in the NACK vs. ACK and NAS vs. AS comparisons than in the NACK vs. NAS and ACK vs. AS comparisons (Figure 6C).
We performed GO functional enrichment analysis for all DEGs across comparison groups and showed that these DEGs were annotated into three fundamental GO classifications, including biological process, cellular component, and molecular function (Figure 7). In the NACK vs. ACK comparison, the significantly enriched GO terms were associated with apoplast (GO:0048046); extracellular region (GO:0005576); hydroquinone: oxygen oxidoreductase activity (GO:0052716), DNA-binding transcription factor activity (GO:0003700); oxidoreductase activity, acting on paired donors, with incorporation or reduction in molecular oxygen (GO:0016705); 1-aminocyclopropane-1-carboxylate oxidase activity (GO:0009815); lignin catabolic process (GO:0046274); plant-type cell wall organization (GO:0009664); and regulation of ethylene biosynthetic process (GO:0010364) (Figure 7A and Table S5). For the NACK vs. NAS comparison, the enriched GO terms with a significant number of DEGs were associated with the extracellular region (GO:0005576); cell wall (GO:0005618); negative regulation of endopeptidase activity (GO:0010951), hydrogen peroxide catabolic process (GO:0042744); cellular oxidant detoxification (GO:0098869); plant-type cell wall organization (GO:0009664); response to oxidative stress (GO:0006979); regulation of ethylene biosynthetic process (GO:0010364); peroxidase activity (GO:0004601); serine-type endopeptidase inhibitor activity (GO:0004867); and heme binding (GO:0020037) (Figure 7B and Table S6). Similarly, in the ACK vs. AS comparison, the GO terms significantly enriched were associated with the extracellular region (GO:0005576); cell (GO:0005623); cell wall (GO:0005618); terpenoid biosynthetic process (GO:0016114); hydrogen peroxide catabolic process (GO:0042744); cell redox homeostasis (GO:0045454); cellular oxidant detoxification (GO:0098869); response to oxidative stress (GO:0006979); magnesium ion binding (GO:0000287); metal ion binding (GO:0046872); and peroxidase activity (GO:0004601) (Figure 7C and Table S7). Furthermore, the highly enriched GO terms in the NAS vs. AS comparison were associated with the extracellular region (GO:0005576); integral component of membrane (GO:0016021); cell wall (GO:0005618); oxidation-reduction process (GO:0055114); hydrogen peroxide catabolic process (GO:0042744); cell wall biogenesis (GO:0042546); heme binding (GO:0020037); oxidoreductase activity, acting on paired donors, with incorporation or reduction in molecular oxygen (GO:0016705); metal ion binding (GO:0046872); and peroxidase activity (GO:0004601) (Figure 7D and Table S8). Importantly, the transcript level of most peroxidase genes in salt-acclimated plants was higher than that in unacclimated plants (Table S9), which was in accord with the highly enriched GO terms associated with the hydrogen peroxide catabolic process, peroxidase activity, and cell redox homeostasis.
To further identify the biological and signal transduction pathways mediated in acclimation-induced salt tolerance, KEGG pathway enrichment analysis was performed to analyze the DEGs across the comparison groups. The results showed that the DEGs were enriched in 54, 41, 27, and 88 typical KEGG pathways in NACK vs. ACK, NACK vs. NAS, ACK vs. AS, and NAS vs. AS comparison, respectively (Figure 8 and Tables S10–S13). In NACK vs. ACK and NACK vs. NAS comparison, the DEGs were mainly enriched in phenylpropanoid biosynthesis, plant hormone signal transduction, starch and sucrose metabolism, plant–pathogen interaction, and MAPK signaling pathway–plant (Figure 8A,B and Tables S10 and S11). The pathways involved in phenylpropanoid biosynthesis, plant hormone signal transduction, ubiquinone and other terpenoid-quinone biosynthesis, and plant–pathogen interaction were highly enriched in ACK vs. AS comparison (Figure 8C and Table S12). Furthermore, phenylpropanoid biosynthesis, protein processing in the endoplasmic reticulum, the MAPK signaling pathway, and zeatin biosynthesis pathways were highly enriched in NAS vs. AS comparison (Figure 8D and Table S13). Across the four comparison groups, commonly highly enriched pathways included phenylpropanoid biosynthesis, in which most of genes’ transcript abundance in this pathway was suppressed by salt stress, but this effect was minimal in acclimated plants (Table S14), suggesting that these pathways might play vital roles in acclimation-mediated salt tolerance.

3.9. Validation of DEGs by RT-qPCR

To verify the reliability of gene transcript results obtained via RNA-seq analysis, eight DEGs were randomly selected for RT-qPCR analysis. As shown in Figure 9, the expression pattern of these eight genes was consistent with the RNA-seq results, confirming the reliability of the RNA-seq data.

4. Discussion

Tomato, as a moderately salt-sensitive crop, is widely cultivated in protected agriculture. However, soil salinization is a global issue that significantly impacts the growth, yield, and quality of tomatoes [24]. Therefore, improving the salt tolerance of tomatoes is crucial for enhancing tomato cultivation in saline soil. Here, we found that the acclimation of tomato seedlings with 14 mL of 7.5 g L−1 NaCl solution per plant for 9 d significantly enhanced salt tolerance. This was evidenced by promoted plant growth, increased antioxidant enzyme activities, and decreased root Na+/K+ ratio under salt stress. Transcriptomic analysis further revealed that the phenylpropanoid biosynthetic pathway might play a critical role in salt acclimation-induced salt tolerance. These findings provide an effective strategy for tomato adaptation to salt stress through appropriate salt acclimation.
This study found that acclimation with 14 mL of 7.5 g L−1 NaCl solution per plant was the optimal concentration for tomato seedlings. At this concentration, tomato seedlings exhibited the best comprehensive score of physiological parameters (Table S2). This might be because moderate salt acclimation concentration stimulated the physiological response of tomatoes, such as increased plant height, stem diameter, Pn, chlorophyll content, POD activity, and CAT activity, while also decreasing membrane damage, as indicated by reduced MDA content and EL value (Figure 1 and Figure 2). In addition, low salt concentration may stimulate processes involved in cell division and expansion, thereby promoting plant growth and development [25]. However, excessive salt acclimation concentration can lead to an imbalance in cell osmotic pressure, leading to physiological dysfunction and severe growth inhibition [12]. Therefore, appropriate acclimation concentrations can help plants adapt to high salt stress by increasing chlorophyll content and photosynthetic rates, enhancing antioxidant capacity, and maintaining ion balance.
The duration of acclimation was equally critical. The results showed that 9 days was the appropriate duration for salt acclimation treatment (Table S3). A shorter salt acclimation treatment period may only allow tomato plants to undergo initial exposure and adaptation to low-salinity environments. In this case, although plants can accumulate some osmoregulatory substances such as prolines and amino acids, their physiological and metabolic processes may not have been fully adjusted, and the improvement in salt tolerance may not be significant [26,27]. In our study, we found that salt acclimation increased proline accumulation (Figure 2A and Figure 4A), indicating that proline acts as a well-known osmolyte contributing to stress protection rather than impaired osmotic adjustment [28,29,30]. As the salt acclimation period extended, plants had more time to adapt to salt stress environments. During this process, plants may enhance their salt tolerance by adjusting physiological and metabolic processes, such as increasing antioxidant enzyme activity to eliminate salt-induced ROS [31]. Indeed, transcriptomic results showed that the DEGs were highly enriched in GO terms related to the hydrogen peroxide catabolic process, cellular oxidant detoxification, and response to oxidative stress (Figure 7). Furthermore, the activity of POD and the transcript abundance of peroxidase genes in acclimated plants were induced under salt stress (Figure 4 and Table S9). Similarly, salt-adapted rice and cucumber (Cucumis sativus L.) increased salt tolerance by enhancing antioxidant enzymatic activity [5,31]. Exogenous application of plant growth regulators, such as melatonin, trehalose, and polyamines, also significantly enhances antioxidant enzyme activity to scavenge salt stress-induced excessive ROS accumulation, ultimately improving plant salt tolerance [32,33,34,35,36]. Antioxidant protection serves as a crucial defense mechanism in plant stress adaptation, with enzyme activity levels acting as a key indicator of plant resistance [37,38]. Therefore, salt acclimation reduces oxidative damage caused by salinity by increasing the activity of antioxidant enzymes in plants. This adaptive mechanism helps plants maintain normal physiological functions and growth under saline stress conditions. Importantly, our findings revealed salt acclimation as a universal enhancer of tomato salt tolerance (Figure 5). While the degree of improvement varied, every cultivar showed consistent qualitative improvements in ion homeostasis and membrane stability following acclimation (Figure 5), indicating the activation of conserved stress-adaptation pathways in tomato.
Salt stress increases root lignification in various plants species, enhancing the mechanical strength of the cell wall to limit the entry of Na+ into the root xylem, resulting in improved salt tolerance [39,40]. Here, we found that the DEGs in the four comparison groups were statistically enriched in the phenylpropanoid biosynthetic pathway, especially in the NACK vs. NAS and NAS vs. AS comparison groups (Figure 8). Salt stress suppressed most of the gene expression associated with lignin biosynthesis, such as lignin-forming anionic peroxidase and cinnamoyl-CoA reductase 1, in tomato plants with or without salt acclimation. However, their abundances in salt-acclimated plants were still higher (Table S14). The phenylpropanoid biosynthetic pathway, a vital secondary metabolic pathway in plants, is responsible for the synthesis of various phenylpropanoid metabolites, such as lignin, flavonoid, terpenoid, anthocyanin, and coumarin [41]. Integrated transcriptomic and metabolomic analyses have revealed that salt stress-induced DEGs and differentially altered metabolites are highly enriched in the phenylpropanoid biosynthetic pathway in several species, including barley (Hordeum vulgare), perennial ryegrass (Lolium perenne), Sophora alopecuroides, and common bean (Phaseolus vulgaris) [42,43,44]. These results indicate that phenylpropanoid biosynthetic pathway-mediated lignin synthesis is essential for adaptation to salt stress. Indeed, salt-acclimated root cells not only upregulate the expression of lignin biosynthesis genes, such as phenylalanine ammonialyase, 4-coumaroyl CoA ligase, and caffeic acid 3-O-methyltransferase (COMT), but also enhance lignin content and cell wall thickness in Arabidopsis [45,46]. Furthermore, knockout of COMT1 results in hypersensitivity to salt stress, indicating that increased lignin content is essential for adaptation to high-salt stress [46]. Importantly, ethylene signal is essential for lignin biosynthesis [47]. In the present study, we found that salt acclimation induced the enrichment of DEGs in 1-aminocyclopropane-1-carboxylate oxidase activity, regulation of the ethylene biosynthetic process, and the lignin catabolic process (Figure 7A). Furthermore, salt acclimation upregulated 1-aminocyclopropane-1-carboxylate oxidase expression (Solyc07g049530), and its expression level in AS was higher than in NAS. Phosphatidylserine synthase 1 mediates salt resistance through activating ethylene signaling to promote lignin biosynthesis in sweet potato (Ipomoea batatas (L.) Lam.) [48]. In this study, we also found that salt acclimation significantly decreased the Na+/K+ ratio in the roots (Figure 4C). Similarly, when Chinese cabbage (Brassica rapa L. ssp. pekinensis) are exposed to salt stress, the salt-tolerant variety induces an increase in lignin content, whereas the salt-sensitive variety does not, leading to significant decrease in the Na+/K+ ratio in both the leaves and roots [44]. The Na+/K+ ratio is an important physiological indicator in plant cells, and cells need a certain range of Na+/K+ ratios to maintain normal physiological responses [49]. When tomatoes are subjected to salt stress, the intracellular Na+ content increases while the K+ content decreases, leading to an increase in the Na+/K+ ratio [50,51]. After salt acclimation, the Na+ content can significantly decrease, and the K+ content can increase in the roots, maintaining Na+/K+ ratio homeostasis suitable for plant growth [4,11]. Therefore, our results suggested that salt acclimation might stimulate ethylene signaling to activate the phenylpropanoid biosynthetic pathway, promoting lignin biosynthesis, reducing the Na+/K+ ratio, and enhancing salt tolerance.

5. Conclusions

In summary, our research provides physiological and molecular insights into salt acclimation as a viable strategy to enhance tomato salt tolerance. We found that tomato seedlings acclimated with 14 mL of 7.5 g L−1 NaCl solution per plant for 9 d significantly increased salt tolerance. Transcriptomic analysis revealed that salt acclimation-induced salt tolerance might depend on ethylene signaling to activate the phenylpropanoid biosynthetic pathway, promoting lignin biosynthesis, resulting in a decrease in the Na+/K+ ratio. These results not only contribute to the sustainable development of protected agriculture but also offer a practical solution to mitigate the global challenge of soil salinization. However, the specific molecular mechanism of ethylene in salt acclimation remains unclear. Future research should investigate whether inhibiting ethylene signaling impairs acclimation-induced salt tolerance. In addition, examining ion localization, specifically the distribution of Na+ in the roots, could provide further insights into how lignification helps limit ion influx, thereby linking structural changes to improved salt tolerance.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/horticulturae12020159/s1, Figure S1: Effects of different salt acclimation concentrations on net photosynthetic rate (Pn) in tomato seedlings; Figure S2: Effects of different salt acclimation durations on net photosynthetic rate (Pn) in tomato seedlings; Figure S3: Effects of salt acclimation on chlorophyll content and net photosynthetic rate (Pn) in different tomato cultivars; Table S1: Primers used for RT-qPCR; Table S2: Analysis of membership functions of salt tolerance physiological parameters under different salt acclimation concentrations in tomato seedlings; Table S3: Analysis of membership functions of salt tolerance physiological parameters under different salt acclimation durations in tomato seedlings; Table S4: Overview of raw and clean reads in acclimated and non-acclimated tomato seedlings exposed to salt stress; Table S5: GO enrichment analysis of differentially expressed genes in NACK vs. ACK; Table S6: GO enrichment analysis of differentially expressed genes in NACK vs. NAS; Table S7: GO enrichment analysis of differentially expressed genes in ACK vs. AS; Table S8: Go enrichment analysis of differentially expressed genes in NAS vs. AS; Table S9: A list of differentially expressed genes in the hydrogen peroxide catabolic process; Table S10: KEGG pathway analysis of differentially expressed genes in NACK vs. ACK; Table S11: KEGG pathway analysis of differentially expressed genes in NACK vs. NAS; Table S12: KEGG pathway analysis of differentially expressed genes in ACK vs. AS; Table S13: KEGG pathway analysis of differentially expressed genes in NAS vs. AS; Table S14: A list of differentially expressed genes in the phenylpropanoid biosynthesis pathway.

Author Contributions

Writing—original draft, N.F., R.L. and Y.W.; methodology, N.F., R.L. and Y.W.; Writing—review and editing, G.P., X.L., L.Y., J.S. and Y.W.; validation, N.F., H.L. and K.Z.; formal analysis, G.P. and X.L.; investigation, R.L.; data curation, N.F., R.L. and K.Z.; visualization, G.P., X.L. and L.Y.; supervision, Y.W.; project administration, Y.W.; funding acquisition, Y.W.; conceptualization, Y.W. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (32472719), the Natural Science Foundation of Jiangsu Province (BK20242064), the Fundamental Research Funds for the Central Universities (KJYQ2025024), the Jiangsu Provincial Association for Science and Technology Youth Science and Technology Talent Support Project (JSTJ-2023-003), and the earmarked fund for China Agriculture Research System (CARS-23).

Data Availability Statement

The original contributions presented in this study are included in the article and Supplementary Materials. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

AAcclimated
ACAilsa craig
CATCatalase
COMTCaffeic acid 3-O-methyltransferase
DEGsDifferentially expressed genes
ELElectrolyte leakage
FKPMFragments Per Kilo-base of exon Per Million Fragments mapped
FWFresh weight
GOGene ontology
KEGGKyoto Encyclopedia of Genes and Genomes
MDAMalondialdehyde
NANon-acclimated
PnNet photosynthetic rate
PODPeroxidase
ROSReactive oxygen species
RT-qPCRQuantitative real-time PCR
SODSuperoxide dismutase

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Figure 1. Effects of different salt acclimation concentrations on the growth and salt tolerance of tomato seedlings. (A) Plant height; (B) Stem diameter; (C) Leaf area; (D) Chlorophyll content; (E) Electrolyte leakage; (F) Malondialdehyde (MDA) content. CK indicates tomato seedlings watered with distilled water. T1, T2, T3, and T4 indicate tomato seedlings watered with 14 mL of 2.5 g L−1, 5.0 g L−1, 7.5 g L−1, and 10.0 g L−1 NaCl solution per plant, respectively. After treatment with different concentrations of salt solution for 2 days, the seedling was transplanted into a 250 cm3 pot. After transplanting for 7 days, each pot was irrigated with 50 mL of distilled water (H2O) or 150 mM NaCl every two days for three rounds, and the growth and salt tolerance parameters were measured at 7 days of salt stress treatment. Results represent the means ± standard deviations (n = 6). Means followed by the same letters do not differ according to Tukey’s test (p < 0.05). FW, fresh weight.
Figure 1. Effects of different salt acclimation concentrations on the growth and salt tolerance of tomato seedlings. (A) Plant height; (B) Stem diameter; (C) Leaf area; (D) Chlorophyll content; (E) Electrolyte leakage; (F) Malondialdehyde (MDA) content. CK indicates tomato seedlings watered with distilled water. T1, T2, T3, and T4 indicate tomato seedlings watered with 14 mL of 2.5 g L−1, 5.0 g L−1, 7.5 g L−1, and 10.0 g L−1 NaCl solution per plant, respectively. After treatment with different concentrations of salt solution for 2 days, the seedling was transplanted into a 250 cm3 pot. After transplanting for 7 days, each pot was irrigated with 50 mL of distilled water (H2O) or 150 mM NaCl every two days for three rounds, and the growth and salt tolerance parameters were measured at 7 days of salt stress treatment. Results represent the means ± standard deviations (n = 6). Means followed by the same letters do not differ according to Tukey’s test (p < 0.05). FW, fresh weight.
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Figure 2. Effects of different salt acclimation concentrations on proline and soluble protein content, Na+/K+ ratio, and antioxidant enzyme activity in tomato seedlings. (A) Proline content; (B) Soluble protein content; (C) Root Na+/K+ ratio; (D) Superoxide dismutase (SOD) activity; (E) Peroxidase (POD) activity; (F) Catalase (CAT) activity. CK indicates tomato seedlings watered with distilled water. T1, T2, T3, and T4 indicate tomato seedlings watered with 14 mL of 2.5 g L−1, 5.0 g L−1, 7.5 g L−1, and 10.0 g L−1 NaCl solution per plant, respectively. After treatment with different concentrations of salt solution for 2 days, the seedling was transplanted into a 250 cm3 pot. After transplanting for 7 days, each pot was irrigated with 50 mL of distilled water (H2O) or 150 mM NaCl every two days for three rounds, and proline and soluble protein content, Na+/K+ ratio, and antioxidant enzyme activity were measured at 7 days of salt stress treatment. Results represent the means ± standard deviations (n = 6). Means followed by the same letters do not differ according to Tukey’s test (p < 0.05). FW, fresh weight.
Figure 2. Effects of different salt acclimation concentrations on proline and soluble protein content, Na+/K+ ratio, and antioxidant enzyme activity in tomato seedlings. (A) Proline content; (B) Soluble protein content; (C) Root Na+/K+ ratio; (D) Superoxide dismutase (SOD) activity; (E) Peroxidase (POD) activity; (F) Catalase (CAT) activity. CK indicates tomato seedlings watered with distilled water. T1, T2, T3, and T4 indicate tomato seedlings watered with 14 mL of 2.5 g L−1, 5.0 g L−1, 7.5 g L−1, and 10.0 g L−1 NaCl solution per plant, respectively. After treatment with different concentrations of salt solution for 2 days, the seedling was transplanted into a 250 cm3 pot. After transplanting for 7 days, each pot was irrigated with 50 mL of distilled water (H2O) or 150 mM NaCl every two days for three rounds, and proline and soluble protein content, Na+/K+ ratio, and antioxidant enzyme activity were measured at 7 days of salt stress treatment. Results represent the means ± standard deviations (n = 6). Means followed by the same letters do not differ according to Tukey’s test (p < 0.05). FW, fresh weight.
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Figure 3. Effects of different salt acclimation durations on the growth and salt tolerance of tomato seedlings. (A) Plant height; (B) Stem diameter; (C) Leaf area; (D) Chlorophyll content; (E) Electrolyte leakage; (F) Malondialdehyde (MDA) content. TD0, TD1, TD2, TD3, and TD4 indicate tomato seedlings watered with 14 mL of 7.5 g L−1 NaCl solution per plant for 0, 3, 6, 9, and 12 d, respectively. After salt acclimation, the seedling was transplanted into a 250 cm3 pot. After transplanting for 7 days, each pot was irrigated with 50 mL of distilled water (H2O) or 150 mM NaCl every two days for three rounds, and the growth and salt tolerance parameters were measured at 7 days of salt stress treatment. Results represent the means ± standard deviations (n = 6). Means followed by the same letters do not differ according to Tukey’s test (p < 0.05). FW, fresh weight.
Figure 3. Effects of different salt acclimation durations on the growth and salt tolerance of tomato seedlings. (A) Plant height; (B) Stem diameter; (C) Leaf area; (D) Chlorophyll content; (E) Electrolyte leakage; (F) Malondialdehyde (MDA) content. TD0, TD1, TD2, TD3, and TD4 indicate tomato seedlings watered with 14 mL of 7.5 g L−1 NaCl solution per plant for 0, 3, 6, 9, and 12 d, respectively. After salt acclimation, the seedling was transplanted into a 250 cm3 pot. After transplanting for 7 days, each pot was irrigated with 50 mL of distilled water (H2O) or 150 mM NaCl every two days for three rounds, and the growth and salt tolerance parameters were measured at 7 days of salt stress treatment. Results represent the means ± standard deviations (n = 6). Means followed by the same letters do not differ according to Tukey’s test (p < 0.05). FW, fresh weight.
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Figure 4. Effects of different salt acclimation durations on the content of proline and soluble protein, Na+/K+ ratio, and antioxidant enzyme activity in tomato seedlings. (A) Proline content; (B) Soluble protein content; (C) Root Na+/K+ ratio; (D) Superoxide dismutase (SOD) activity; (E) Peroxidase (POD) activity; (F) Catalase (CAT) activity. TD0, TD1, TD2, TD3, and TD4 indicate tomato seedlings watered with 14 mL of 7.5 g L−1 NaCl solution per plant for 0, 3, 6, 9, and 12 d, respectively. After salt acclimation, the seedling was transplanted into a 250 cm3 pot. After transplanting for 7 days, each pot was irrigated with 50 mL of distilled water (H2O) or 150 mM NaCl every two days for three rounds, and proline and soluble protein content, Na+/K+ ratio, and antioxidant enzyme activity were measured at 7 days of salt stress treatment. Results represent the means ± standard deviations (n = 6). Means followed by the same letters do not differ according to Tukey’s test (p < 0.05). FW, fresh weight.
Figure 4. Effects of different salt acclimation durations on the content of proline and soluble protein, Na+/K+ ratio, and antioxidant enzyme activity in tomato seedlings. (A) Proline content; (B) Soluble protein content; (C) Root Na+/K+ ratio; (D) Superoxide dismutase (SOD) activity; (E) Peroxidase (POD) activity; (F) Catalase (CAT) activity. TD0, TD1, TD2, TD3, and TD4 indicate tomato seedlings watered with 14 mL of 7.5 g L−1 NaCl solution per plant for 0, 3, 6, 9, and 12 d, respectively. After salt acclimation, the seedling was transplanted into a 250 cm3 pot. After transplanting for 7 days, each pot was irrigated with 50 mL of distilled water (H2O) or 150 mM NaCl every two days for three rounds, and proline and soluble protein content, Na+/K+ ratio, and antioxidant enzyme activity were measured at 7 days of salt stress treatment. Results represent the means ± standard deviations (n = 6). Means followed by the same letters do not differ according to Tukey’s test (p < 0.05). FW, fresh weight.
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Figure 5. Effects of salt acclimation on growth and Na+/K+ ratio in different tomato cultivars. (A) Stem diameter; (B) Leaf area; (C) Electrolyte leakage; (D) Root Na+/K+ ratio. Different tomato cultivar seedlings watered with 14 mL of 7.5 g L−1 NaCl solution per plant for 9 d to achieve salt acclimation. Subsequently, the seedling was transplanted into a 250 cm3 pot. After transplanting for 7 days, each pot was irrigated with 50 mL of distilled water (H2O) or 150 mM NaCl every two days for three rounds, and the growth parameters and Na+/K+ ratio were measured at 7 days of salt stress treatment. The non-acclimated seedlings were marked as NA and the acclimated seedlings were marked as A. Results represent the means ± standard deviations (n = 6). Means followed by the same letters do not differ according to Tukey’s test (p < 0.05).
Figure 5. Effects of salt acclimation on growth and Na+/K+ ratio in different tomato cultivars. (A) Stem diameter; (B) Leaf area; (C) Electrolyte leakage; (D) Root Na+/K+ ratio. Different tomato cultivar seedlings watered with 14 mL of 7.5 g L−1 NaCl solution per plant for 9 d to achieve salt acclimation. Subsequently, the seedling was transplanted into a 250 cm3 pot. After transplanting for 7 days, each pot was irrigated with 50 mL of distilled water (H2O) or 150 mM NaCl every two days for three rounds, and the growth parameters and Na+/K+ ratio were measured at 7 days of salt stress treatment. The non-acclimated seedlings were marked as NA and the acclimated seedlings were marked as A. Results represent the means ± standard deviations (n = 6). Means followed by the same letters do not differ according to Tukey’s test (p < 0.05).
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Figure 6. Overview, Venn diagram, and Volcano plots analysis of differentially expressed genes (DEGs) between treatments. (A) The number of upregulated and downregulated genes in different groups; (B) Venn diagram of DEGs; (C) Volcano plots of DEGs between treatments. Red and blue points represent upregulated and downregulated genes, respectively. Light gray points represent no significant genes. NACK and NAS indicate non-acclimated tomato seedlings treatment with water and salt, respectively. ACK and AS indicate acclimated tomato seedlings treatment with water and salt, respectively.
Figure 6. Overview, Venn diagram, and Volcano plots analysis of differentially expressed genes (DEGs) between treatments. (A) The number of upregulated and downregulated genes in different groups; (B) Venn diagram of DEGs; (C) Volcano plots of DEGs between treatments. Red and blue points represent upregulated and downregulated genes, respectively. Light gray points represent no significant genes. NACK and NAS indicate non-acclimated tomato seedlings treatment with water and salt, respectively. ACK and AS indicate acclimated tomato seedlings treatment with water and salt, respectively.
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Figure 7. Gene ontology (GO) enrichment analysis of differentially expressed genes (DEGs). (A) GO enrichment analysis of DEGs in NACK vs. ACK comparison; (B) GO enrichment analysis of DEGs in NACK vs. NAS comparison; (C) GO enrichment analysis of DEGs in ACK vs. AS comparison; (D) GO enrichment analysis of DEGs in NAS vs. AS comparison. NACK and NAS indicated non-acclimate tomato seedlings treatment with water and salt, respectively. ACK and AS indicate acclimated tomato seedlings treatment with water and salt, respectively.
Figure 7. Gene ontology (GO) enrichment analysis of differentially expressed genes (DEGs). (A) GO enrichment analysis of DEGs in NACK vs. ACK comparison; (B) GO enrichment analysis of DEGs in NACK vs. NAS comparison; (C) GO enrichment analysis of DEGs in ACK vs. AS comparison; (D) GO enrichment analysis of DEGs in NAS vs. AS comparison. NACK and NAS indicated non-acclimate tomato seedlings treatment with water and salt, respectively. ACK and AS indicate acclimated tomato seedlings treatment with water and salt, respectively.
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Figure 8. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differentially expressed genes (DEGs). (A) KEGG enrichment analysis of DEGs in NACK vs. ACK comparison; (B) KEGG enrichment analysis of DEGs in NACK vs. NAS comparison; (C) KEGG enrichment analysis of DEGs in ACK vs. AS comparison; (D) KEGG enrichment analysis of DEGs in NAS vs. AS comparison. NACK and NAS indicate non-acclimated tomato seedlings treatment with water and salt, respectively. ACK and AS indicate acclimated tomato seedlings treatment with water and salt, respectively.
Figure 8. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differentially expressed genes (DEGs). (A) KEGG enrichment analysis of DEGs in NACK vs. ACK comparison; (B) KEGG enrichment analysis of DEGs in NACK vs. NAS comparison; (C) KEGG enrichment analysis of DEGs in ACK vs. AS comparison; (D) KEGG enrichment analysis of DEGs in NAS vs. AS comparison. NACK and NAS indicate non-acclimated tomato seedlings treatment with water and salt, respectively. ACK and AS indicate acclimated tomato seedlings treatment with water and salt, respectively.
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Figure 9. Verification of the differentially expressed selected genes by quantitative real-time PCR (RT-qPCR). (A) Comparison of the NACK vs. ACK; (B) Comparison of the NAS vs. AS. The blue column represents RNA-seq data, and the yellow column represents RT-qPCR data. Results represent the means ± standard deviations (n = 3). NACK and NAS indicate non-acclimated tomato seedlings treatment with water and salt, respectively. ACK and AS indicate acclimated tomato seedlings treatment with water and salt, respectively.
Figure 9. Verification of the differentially expressed selected genes by quantitative real-time PCR (RT-qPCR). (A) Comparison of the NACK vs. ACK; (B) Comparison of the NAS vs. AS. The blue column represents RNA-seq data, and the yellow column represents RT-qPCR data. Results represent the means ± standard deviations (n = 3). NACK and NAS indicate non-acclimated tomato seedlings treatment with water and salt, respectively. ACK and AS indicate acclimated tomato seedlings treatment with water and salt, respectively.
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MDPI and ACS Style

Fan, N.; Li, R.; Liu, H.; Zhang, K.; Pang, G.; Liu, X.; Yang, L.; Sun, J.; Wang, Y. Integrated Physiological and Transcriptomic Analyses Reveal the Mechanism of Salt Acclimation-Induced Salinity Tolerance in Tomato Seedlings. Horticulturae 2026, 12, 159. https://doi.org/10.3390/horticulturae12020159

AMA Style

Fan N, Li R, Liu H, Zhang K, Pang G, Liu X, Yang L, Sun J, Wang Y. Integrated Physiological and Transcriptomic Analyses Reveal the Mechanism of Salt Acclimation-Induced Salinity Tolerance in Tomato Seedlings. Horticulturae. 2026; 12(2):159. https://doi.org/10.3390/horticulturae12020159

Chicago/Turabian Style

Fan, Nuo, Ruiqing Li, Huixin Liu, Ke Zhang, Guan Pang, Xiaoying Liu, Lifei Yang, Jin Sun, and Yu Wang. 2026. "Integrated Physiological and Transcriptomic Analyses Reveal the Mechanism of Salt Acclimation-Induced Salinity Tolerance in Tomato Seedlings" Horticulturae 12, no. 2: 159. https://doi.org/10.3390/horticulturae12020159

APA Style

Fan, N., Li, R., Liu, H., Zhang, K., Pang, G., Liu, X., Yang, L., Sun, J., & Wang, Y. (2026). Integrated Physiological and Transcriptomic Analyses Reveal the Mechanism of Salt Acclimation-Induced Salinity Tolerance in Tomato Seedlings. Horticulturae, 12(2), 159. https://doi.org/10.3390/horticulturae12020159

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