Transcriptomic and Physiological Insights into the Role of Nano-Silicon Dioxide in Alleviating Salt Stress During Soybean Germination

Abstract

Salt stress is a major form of abiotic stress that disrupts soybean germination and early seedling establishment. In this study, physiological, biochemical, and transcriptomic analyses—including germination index, antioxidant enzyme activity, and RNA-seq profiling—were conducted during soybean germination to elucidate early responses to salt stress and biostimulant treatment. A preliminary screening of six biostimulants (nanoparticle zinc oxide (NP-ZnO), nanoparticle silicon dioxide (NP-SiO₂), silicon dioxide (SiO₂), glucose, humic acid, and fulvic acid) revealed NP-SiO₂ as the most effective in promoting germination under salt stress. Under 150 mM NaCl, NP-SiO₂ increased the germination rate and length of the radicle compared with the control, also enhancing peroxidase and ascorbate peroxidase activities while reducing malondialdehyde accumulation, suggesting alleviation of oxidative stress. RNA sequencing revealed extensive transcriptional reprogramming under salt stress, identifying 4579 differentially expressed genes (DEGs) compared with non-stress conditions, while NP-SiO₂ treatment reduced this number to 2734, indicating that NP-SiO₂ mitigated the transcriptional disturbance caused by salt stress and stabilized gene expression networks. Cluster analysis showed that growth- and hormone-related genes suppressed by salt stress were restored under NP-SiO₂ treatment, whereas stress-responsive genes that were induced by salt were attenuated. Hormone-related DEG analysis revealed that NP-SiO₂ down-regulated the overactivation in the abscisic acid, jasmonic acid, and salicylic acid pathways while partially restoring gibberellin, auxin, cytokinin, and brassinosteroid signaling. Overall, NP-SiO₂ at 100 mg/L mitigated salt-induced oxidative stress and promoted early soybean growth by fine-tuning physiological and transcriptional responses, representing a promising nano-based biostimulant for enhancing salt tolerance in plants.

1. Introduction

Salinization poses a major threat to global agriculture, reducing crop productivity, food security, and sustainability. The Food and Agriculture Organization (FAO) estimates that over 1.1 billion hectares of land are affected by salinity worldwide, with arid and semi-arid regions such as the Middle East, Australia, and North Africa being most vulnerable [1,2]. Soybean (Glycine max [L.] Merr.) is valued for its high protein and oil content and is a key global source of food, animal feed, and industrial products, including biofuels [3,4]. Although soybean is only considered moderately salt-sensitive, electrical conductivity levels exceeding 5 dS/m significantly impair germination and growth, leading to substantial yield losses [5]. Given that the germination stage is particularly sensitive to salt stress [6], strategies to improve soybean salt tolerance are required to sustain productivity under salt-stress conditions.

The physiological damage caused by salt stress arises from multiple interconnected processes. For example, excess sodium (Na⁺) and chloride (Cl⁻) ions disrupt cellular structures and interfere with essential metabolic processes [7], while elevated external salt concentrations reduce the soil water potential, limiting water uptake and leading to cellular dehydration and osmotic stress [8]. Sodium ions also displace key cations such as potassium (K⁺), calcium (Ca²⁺), and magnesium (Mg²⁺), resulting in nutrient imbalances and the disruption of ion homeostasis [9]. In addition, salt stress alters plant hormone regulation, particularly affecting abscisic acid (ABA), gibberellins (GAs), and ethylene, which are critical for growth and development [10]. Ion toxicity and osmotic stress consequently trigger excessive production of reactive oxygen species (ROS), which damage cellular integrity by oxidizing membrane lipids, denaturing proteins, and modifying nucleic acids, ultimately impairing vital physiological processes [11,12,13].

Plant biostimulants (PBSs) have been developed to stimulate growth, enhance stress tolerance, and improve overall crop performance [14]. Under salt-stress conditions, PBSs can induce beneficial physiological adjustments in plants, contributing to hormonal regulation, reducing Na⁺ accumulation, enhancing ion homeostasis, and stabilizing cellular membranes. More recently, nano-enabled biostimulants have attracted increasing attention in sustainable agriculture due to their efficiency and multifunctionality. Nanoparticles (NPs), typically ranging from 1 to 100 nm in size, have a high surface-area-to-volume ratio, which improves their absorption and bioactivity in plant tissues [15,16]. Their enhanced reactivity, relatively low ecological toxicity, and minimal input requirements make them promising alternatives to conventional fertilizers and pesticides. As a result, many previous studies have shown that Zn NPs, Ag NPs, SiO₂ NPs, Cu NPs, Fe NPs, and Mn NPs effectively alleviate salt stress in various crops [17]. In addition, PBSs stimulate the activity of antioxidant enzymes such as peroxidase, ascorbate peroxidase, and catalase, thus strengthening the plant defense system against oxidative stress and mitigating ROS-induced damage [18].

The objective of this study was to identify biostimulants capable of mitigating salt stress during soybean germination and to elucidate their underlying transcriptional networks. Soybean seeds were treated with six different biostimulants under salt-stress conditions, and the most effective treatment and dosage were subsequently identified. RNA sequencing of radicle tissues was conducted to characterize transcriptional networks and to identify differentially expressed genes (DEGs) and pathways associated with silicon-mediated salt tolerance.

2. Materials and Methods

2.1. Plant Materials

The Korean soybean variety ‘Haepum’ (Milyang 225), which is widely used for sprout production due to the small size of its seeds (10.4 g per 100 seeds), was selected for the present study. Each replicate consisted of 20 seeds, which were first soaked in 70% ethanol for 5 min and then disinfected in a 0.4% sodium hypochlorite (NaOCl) solution for 20 min. To remove the residual disinfectant, the seeds were rinsed five times with a saline solution. Discolored or damaged seeds were discarded to ensure that only healthy seeds were retained for further use. The sterilized seeds were evenly placed on filter paper inside 12 cm diameter Petri dishes, and each dish received 50 mL of the designated treatment solution to ensure full saturation of both the seeds and the filter medium. To maintain consistent moisture levels, the same treatment solution was reapplied periodically during the germination period. All Petri dishes were incubated in complete darkness at a constant temperature of 24 °C in a growth chamber.

2.2. Preliminary Screening and Biostimulant Selection

Six candidate biostimulants were selected for initial evaluation: nanoparticle zinc oxide and silicon dioxide (NP-SiO₂ and NP-ZnO, respectively), micro-scale silicon dioxide (SiO₂), glucose (Glu), humic acid (HA), and fulvic acid (FA). These candidates and their application rates were chosen according to previous studies demonstrating their effectiveness and non-phytotoxicity in enhancing seed germination and salt-stress tolerance in various crops. Specifically, humic acid, fulvic acid, and glucose were applied at 2.5 mM, whereas nanoparticles were tested at 100 mg L⁻¹ (Table 1).

NP-ZnO and NP-SiO₂ were used in the form of NPs, while the remaining compounds took their standard powdered form. Through previous studies, the particle size distribution and zeta potential of NP-SiO₂ (CAS 7631-86-9, Sigma-Aldrich Inc., St. Louis, MO, USA) and NP-ZnO (Sigma-Aldrich, CAS 1314-13-2) were confirmed. Both nanoparticle compounds exhibited negative surface charges of approximately –20 mV under the experimental conditions, maintaining a stable dispersion state with good colloidal stability [19,20]. All biostimulants were tested at the doses listed in Table 1 under control (0 mM NaCl) and salt-stress (150 mM NaCl) conditions.

To assess stress alleviation, the germination percentage (GP), germination index (GI), and radicle lengths were measured for all of the treatments under both control and salt-stress conditions. Among the six treatments, NP-SiO₂ consistently showed the most favorable results under salt-stress conditions across all measured parameters and was thus selected for subsequent analysis to determine its optimal dosage. Four concentrations (50, 100, 150, and 200 mg/L) were tested under both test conditions, with five replicates per treatment group.

Table 1. Biostimulants used in the preliminary experiment.

Product	Concentration	Company (City, Country)	Particle Size	Reference
Nano-scale zinc oxide (NP-ZnO)	100 mg/L	Sigma-Aldrich Inc. (St. Louis, MO, USA)	<50 nm	[21,22,23]
Nano-scale silicon dioxide (NP-SiO2)	100 mg/L	Sigma-Aldrich Inc. (St. Louis, MO, USA)	10–20 nm	[24,25]
Micro-scale silicon dioxide (SiO2)	100 mg/L	Sigma-Aldrich Inc. (St. Louis, MO, USA)	0.5–10 μm	[26]
Glucose (Glu)	2.5 mM	Sigma-Aldrich Inc. (St. Louis, MO, USA)	-	[27,28]
Humic acid (HA)	2.5 mM	Daejung (Siheung, Republic of Korea)	-	[29,30]
Fulvic acid (FA)	2.5 mM	RNM (Busan, Republic of Korea)	-	[31]

Table 1. Biostimulants used in the preliminary experiment.

Product	Concentration	Company (City, Country)	Particle Size	Reference
Nano-scale zinc oxide (NP-ZnO)	100 mg/L	Sigma-Aldrich Inc. (St. Louis, MO, USA)	<50 nm	[21,22,23]
Nano-scale silicon dioxide (NP-SiO2)	100 mg/L	Sigma-Aldrich Inc. (St. Louis, MO, USA)	10–20 nm	[24,25]
Micro-scale silicon dioxide (SiO2)	100 mg/L	Sigma-Aldrich Inc. (St. Louis, MO, USA)	0.5–10 μm	[26]
Glucose (Glu)	2.5 mM	Sigma-Aldrich Inc. (St. Louis, MO, USA)	-	[27,28]
Humic acid (HA)	2.5 mM	Daejung (Siheung, Republic of Korea)	-	[29,30]
Fulvic acid (FA)	2.5 mM	RNM (Busan, Republic of Korea)	-	[31]

2.3. Germination Parameters

In 2024, germination was assessed over a seven-day period, with each replicate consisting of 20 seeds (n = 3). The number of germinated seeds (defined by a radicle emergence of 2 mm) was recorded daily. The following equations were used to calculate germination-related metrics:

(a)

GP was calculated in accordance with the process outlined by Alsaeedi et al. using Equation (1) [32]:

$$\mathrm{GP} \left(\%\right)={\displaystyle \frac{\mathrm{Number} \mathrm{of} \mathrm{germinated} \mathrm{seeds}}{\mathrm{Number} \mathrm{of} \mathrm{seeds} \mathrm{per} \mathrm{replicate}}}\times 100$$

(1)

(b)

GI was computed to reflect both the speed and uniformity of seed germination based on daily germination data using Equation (2) [33]:

$$\mathrm{GI}=\sum {\displaystyle \frac{{\mathrm{n}}_{\mathrm{i}}}{{\mathrm{d}}_{\mathrm{i}}}}$$

(2)

where n_i indicates the number of seeds that germinated on a given day i and d_i corresponds to the number of days since sowing.

(c)

Radicle length was determined by selecting five germinated seeds with similar growth conditions and calculating the average radicle length.

2.4. Antioxidant Enzyme Activity Analysis

Radicle tissues from 7-day-old seedlings were harvested, immediately frozen in liquid nitrogen, and stored at −80 °C until analysis. Total soluble protein was extracted from 0.2 g of frozen tissue by homogenizing it in 0.2 M potassium phosphate buffer (pH 7.8) supplemented with 0.1 mM EDTA. The homogenate was then centrifuged at 15,294× g for 20 min at 4 °C, and the resulting supernatant was collected to quantify the activity of the antioxidant enzymes peroxidase (POD), ascorbate peroxidase (APX), and catalase (CAT).

POD activity was assessed by monitoring the increase in absorbance at 470 nm, with pyrogallol employed as the substrate. The reaction mixture contained 100 mM potassium phosphate buffer (pH 6.0), 20 mM pyrogallol, and 10 mM hydrogen peroxide. The specific activity was recorded as units per minute per gram fresh weight (U/min/g FW) and calculated using an extinction coefficient (ε) of 12 mM⁻¹·cm⁻¹ [34].

APX activity was evaluated by tracking the decline in absorbance at 290 nm. The assay solution consisted of 50 mM potassium phosphate buffer (pH 7.0), 0.5 mM ascorbic acid, 10 mM hydrogen peroxide, and 0.1 mM EDTA. Enzyme activity was determined at 8 min intervals based on the rate at which the absorbance decreased using a molar extinction coefficient (ε) of 2.8 mM⁻¹·cm⁻¹ [35].

CAT activity was assessed by observing the reduction in absorbance at 240 nm. Each 200 μL reaction mixture contained 140 μL of 50 mM potassium phosphate buffer (pH 7.0) and 50 μL of 10 mM hydrogen peroxide. Enzyme activity was calculated based on the rate of H₂O₂ decomposition using a molar extinction coefficient (ε) of 40 mM⁻¹·cm⁻¹ and expressed as U/min/g FW [36].

2.5. Lipid Peroxidation Assays

Lipid peroxidation was assessed by quantifying the malondialdehyde (MDA) levels. Radicle tissue (approximately 0.2 g) was ground in 1.5 mL of 10% (w/v) trichloroacetic acid (TCA) and centrifuged at 12,000× g for 20 min at 4 °C. Following this, 1 mL of the resulting supernatant was combined with 1 mL of 0.67% (w/v) thiobarbituric acid (TBA) reagent. The mixture was incubated at 95 °C for 30 min to facilitate the reaction. To halt the reaction, the tubes were promptly placed on ice for 5 min. After cooling, a second round of centrifugation was conducted at 2447× g for 5 min. The absorbance of the supernatant was then recorded at 470 nm, 532 nm, and 650 nm using a spectrophotometer (BioTek Instruments Inc., Winooski, VT, USA), and absorbance at 650 nm was used to correct for non-specific background interference. The final MDA concentration was expressed as nanomoles per gram of fresh weight (nmol/g FW) [37,38].

2.6. Transcriptome Sequencing and Analysis

In 2025, total RNA was extracted from 50 to 100 mg of soybean radicle tissue using a MiniBest Plant RNA Extraction Kit (Takara Bio Inc., Kusatsu, Japan) following the manufacturer’s instructions. The RNA concentration and integrity were assessed using a Nano MD spectrophotometer (SCINCO, Seoul, Republic of Korea) and 1% agarose gel electrophoresis. Only samples with RNA integrity values greater than 6.0, as determined by TapeStation analysis (Agilent Technologies, Santa Clara, CA, USA), were used for library construction.

Library preparation and RNA sequencing were outsourced to Seeders Inc. (Daejeon, Republic of Korea). For each sample, 1 μg of total RNA was used to construct strand-specific cDNA libraries using a NEXTFLEX^® Rapid Directional RNA Seq Kit 2.0 (PerkinElmer, Waltham, MA, USA). Poly A mRNA was enriched using oligo(dT) beads, fragmented, and reverse-transcribed into first-strand cDNA, followed by synthesis of the second strand. The resulting double-stranded cDNA was end-repaired, A-tailed, and ligated to sequencing adapters, after which the libraries were PCR amplified and quantified using TapeStation D1000 ScreenTape (Agilent Technologies). Paired-end sequencing (2 × 151 bp) was performed on an Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA).

Raw reads were processed using Trimmomatic (v0.39) to remove adaptors and low-quality bases. The cleaned reads were then aligned to the Glycine max reference genome (Wm82.a4.v1) using HISAT2, and gene-level counts were obtained using HTSeq (v0.11.0). Raw counts were normalized and DEGs were identified using the DESeq2 package in R, with genes meeting the criteria of |log₂ (fold change)| ≥ 1 and an adjusted p-value (FDR) < 0.01 defined as DEGs. Functional enrichment analysis of the DEGs was conducted using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases to determine the transcriptional changes’ biological significance.

2.7. Statistical Analysis

Statistical analyses were conducted using IBM SPSS Statistics software (v27; IBM Corp., Armonk, NY, USA). One-way ANOVA was used to detect differences among treatments, and Tukey’s HSD tests were employed for post hoc multiple comparisons. A significance level of p ≤ 0.05 was used throughout.

3. Results

3.1. Effects of the Biostimulants on Germination Under Control (0 mM NaCl) and Salt-Stress (150 mM NaCl) Conditions

GP is a fundamental physiological indicator used to evaluate seed vigor and environmental adaptability. In a preliminary experiment, six different biostimulants were tested for their potential to enhance salt-stress tolerance during soybean seed germination: NP-ZnO, NP-SiO₂, SiO₂, Glu, HA, and FA. Changes in GP were monitored over a 7-day observation period, and under non-stress conditions, most seeds germinated by the third or fourth day. The HA and Glu treatments promoted germination compared to the untreated control, indicating their potential to enhance germination speed under favorable conditions (Figure 1A). In contrast, exposure to 150 mM NaCl strongly suppressed seed germination in the control, with GP reduced to 30% by day 3, compared to 100% under non-stress conditions (Figure 1B). However, biostimulant application mitigated this inhibitory effect to varying extents. In particular, treatment with 100 mg/L NP-SiO₂ significantly improved germination percentage under salt-stress conditions, restoring it to 88% on day 3.

GI, which accounts for both the speed and uniformity of germination, is also widely used to assess seed vigor and responsiveness to environmental stress. In this study, GI exhibited a trend similar to that of GP: under salt-stress conditions, only NP-SiO₂ treatment led to a statistically significant increase in GI compared to the untreated control (p < 0.05), suggesting that NP-SiO₂ enhanced germination speed under salt stress (Figure 1C,D).

Under non-stress conditions, NP-SiO₂ significantly increased radicle length compared to the control (Figure 1E), and under salt-stress conditions, both NP-SiO₂ and SiO₂ significantly increased radicle length (p < 0.05). In particular, the length of the salt-stress control radicle was 24.2 mm, whereas NP-SiO₂ treatment extended this growth to 102.4 mm, an increase of ~324% (Figure 1F).

Based on these findings, NP-SiO₂ was selected as the most effective biostimulant for improving seed germination and early radicle growth under salt stress, and was selected for further analysis.

3.2. Effects of NP-SiO₂ Dosage on Soybean Germination Under Control (0 mM NaCl) and Salt-Stress (150 mM NaCl) Conditions

To evaluate the influence of NP-SiO₂ dosage on soybean seed germination under both control and salt-stress conditions, a control treatment (0 mg/L) and four NP-SiO₂ concentrations (50, 100, 150, and 200 mg/L) were tested. Under non-stress conditions, most of the seeds germinated by the third day, and the control group had a GP of 98%, while that of the 50 mg/L NP-SiO₂ treatment was slightly lower at 79%, suggesting that NP-SiO₂ did not enhance germination in the absence of stress (Figure 2A). In contrast, under salt-stress conditions, all NP-SiO₂-treated groups exhibited an improved GP relative to the untreated control. The 100 mg/L NP-SiO₂ treatment led to the highest GP, suggesting that ameliorating the salt-induced inhibition of germination was dependent on NP-SiO₂ concentration (Figure 2B).

A similar trend was observed for GI. No significant differences in GI were observed between the treatments under non-stress conditions; however, under salt-stress conditions, GI was significantly higher for the 100 mg/L NP-SiO₂ treatment than the salt-stressed control (p < 0.05), suggesting that NP-SiO₂ concentration influenced not only the overall GP but also the speed and uniformity of germination under salt-stress conditions (Figure 2C,D).

Radicle length also followed a similar trend. Under non-stress conditions, no significant differences were found between treatments (Figure 2E). However, under salt-stress conditions, the 50 and 100 mg/L NP-SiO₂ treatments produced significantly longer radicles than both the control and other concentrations (p < 0.05). In particular, radicle length reached 129 mm in the 100 mg/L NP-SiO₂ treatment, corresponding to an increase of ~362.7% over the salt-stressed control (27.9 mm). Radicle elongation peaked at 100 mg/L and declined at higher concentrations (Figure 2F and Figure 3). Collectively, these results demonstrate that NP-SiO₂ had a concentration-dependent effect on seed germination and early seedling growth under salt-stress conditions, with 100 mg/L identified as the most effective dosage.

3.3. Effects of NP-SiO₂ on POD, APX, and CAT Activity Under Control and Salt-Stress Conditions

To investigate the effect of NP-SiO₂ on the antioxidant defense system, the activities of three major enzymes, POD, APX, and CAT, were examined in soybean seedlings under control and salt-stress conditions. Under non-stress conditions, POD activity remained relatively stable between NP-SiO₂ treatments, except for a slight decrease for the 200 mg/L treatment. In contrast, under salt-stress conditions, POD activity generally increased, with the highest activity observed under 100 mg/L NP-SiO₂ treatment, approximately 2.1 times higher than that of the control (2.14) (Figure 4A,B).

In normal conditions, the control exhibited the highest APX activity, and no significant increases were observed with NP-SiO₂ treatment (Figure 5A). However, under salt-stress conditions, APX activity increased overall, with the 100 mg/L NP-SiO₂ treatment leading to a significant increase from 11.00 (control) to 13.24 (Figure 5B). Similarly, CAT activity remained relatively constant under non-stress conditions (Figure 6A), though under salt-stress conditions a slight increase was observed under 100 mg/L NP-SiO₂ treatment (0.54) compared to the control (0.30) (Figure 6B).

Collectively, the activity of POD, APX, and CAT exhibited a concentration-dependent response, with peak activity in the 100 mg/L NP-SiO₂ treatment followed by a decline at higher concentrations. These results indicate that NP-SiO₂ treatment enhanced the antioxidant machinery in response to salt stress, thus promoting effective ROS scavenging and contributing to improved stress tolerance.

3.4. Effects of NP-SiO₂ on Malondialdehyde (MDA) Levels Under Control and Salt-Stress Conditions

To explore how NP-SiO₂ protects against salt-induced oxidative damage, the levels of MDA, an established marker of membrane lipid peroxidation, were quantified in soybean seedlings. In the absence of salt stress, MDA levels did not differ significantly among the NP-SiO₂ treatments, indicating that they did not notably affect membrane integrity (Figure 7A). In contrast, salt stress triggered a substantial increase in MDA accumulation in the untreated group, indicating greater oxidative damage to cellular membranes (Figure 7B).

However, treatment with NP-SiO₂ at 50 and 100 mg/L effectively suppressed this increase, demonstrating a protective effect against lipid peroxidation. At higher concentrations (150 and 200 mg/L), MDA levels were comparable to those observed in the salt-stressed control, indicating that the protective efficacy of NP-SiO₂ may diminish at excessive dosages.

3.5. Overview of Transcriptome Profiling and Functional Analysis

To investigate transcriptional changes under salt-stress conditions, RNA sequencing (RNA-seq) was conducted on soybean radicle tissues from three treatment groups, each with three biological replicates: control, salt stress alone, and salt stress with NP-SiO₂ treatment. DEGs were identified from two pairwise comparisons: both salt stress and salt stress with NP-SiO₂ treatment compared to the control. Quality control analysis confirmed the reliability of the RNA-seq data, and more than 92% of the clean reads were successfully mapped to the soybean reference genome (Glycine max Wm82.a4.v1), indicating sufficient sequencing quality (Table S1).

In the comparison of the salt-stress group to the control, a total of 4579 DEGs were identified, including 1895 up-regulated and 2684 down-regulated genes (Figure 8A and Table S2). When NP-SiO₂ was applied, the number of DEGs was markedly reduced, decreasing to 1154 and 1766 up- and down-regulated genes, respectively, indicating that NP-SiO₂ alleviates salt-stress-induced differences in gene expression, contributing to the stabilization of the gene expression profiles (Table S3).

GO and KEGG enrichment analysis revealed that, in the comparison of both the salt-stress and the salt-stress with NP-SiO₂ treatment groups to the control, the up-regulated DEGs were significantly enriched in stress-response- and defense-related pathways, including plant−pathogen interactions, MAPK signaling, and glutathione metabolism. In addition, pathways associated with photosynthetic processes, such as porphyrin metabolism, photosynthesis, and photosynthetic antenna proteins, were also up-regulated. Notably, several metabolic pathways, including fatty acid degradation, glucosinolate biosynthesis, glycosphingolipid biosynthesis, ABC transporters, secondary metabolite biosynthesis, and valine, leucine, and isoleucine degradation were specifically enriched under salt stress relative to the control (Figure 8B).

Conversely, the down-regulated DEGs were mainly associated with hormone signal transduction, secondary metabolism, carbohydrate metabolism, and lipid metabolism, with antioxidant- and lipid-related pathways, including alpha linolenic acid metabolism, beta alanine metabolism, and glycerophospholipid metabolism, selectively suppressed under salt stress relative to the control (Figure 8C). Venn diagram analysis illustrated the overlap between the salt stress vs. control and salt stress with NP-SiO₂ vs. control comparisons. Of the up-regulated DEGs, 1078 genes were shared, while 817 and 76 genes were uniquely expressed under salt stress alone and NP-SiO₂ treatment, respectively (Figure 8D and Table S4). For the down-regulated DEGs, 1673 genes were shared, with 1011 specific to salt stress and 93 specific to NP-SiO₂ treatment (Figure 8E and Table S5).

3.6. Hierarchical Clustering of DEGs Under Salt Stress and NP-SiO₂ Treatment

Hierarchical clustering of transcriptome data identified a total of 4748 DEGs, which were grouped into four distinct clusters based on their expression profiles (Figure 9A,B and Table S6). Cluster 1 (1594 genes) consisted of genes that were up-regulated under salt stress and remained at comparable levels following NP-SiO₂ treatment, and Cluster 3 (970 genes) included genes that were down-regulated under salt stress and showed little change with the application of NP-SiO₂, indicating that these gene sets were minimally affected by NP-SiO₂ treatment.

In contrast, Cluster 2 (1807 genes) contained genes that were down-regulated under salt stress but recovered toward control levels following NP-SiO₂ treatment. KEGG enrichment analysis revealed that these genes were predominantly associated with the plant hormone signal transduction pathway, which exhibited the strongest enrichment with 137 annotated genes (Figure 9C), as well as with phenylpropanoid biosynthesis, amino sugar and nucleotide sugar metabolism, ascorbate and aldarate metabolism, and multiple secondary metabolite biosynthesis pathways, including flavonoid, isoflavonoid, and carotenoid biosynthesis. Several metabolic processes, such as alpha linolenic acid metabolism, beta alanine metabolism, and sphingolipid metabolism, were also represented. These results suggest that Cluster 2 plays a central role in hormone-mediated signaling, antioxidant defense, and secondary metabolism, highlighting its importance in NP-SiO₂-mediated stress adaptation.

Similarly, Cluster 4 (377 genes) consisted of genes up-regulated under salt stress but with weaker up-regulation in response to NP-SiO₂ treatment, suggesting the modulation of salt-induced transcriptional activation under NP-SiO₂ treatment. KEGG enrichment analysis showed that Cluster 4 genes were most commonly involved in pathways related to central carbon metabolism and energy processes, including pyruvate metabolism, glyoxylate and dicarboxylate metabolism, and fatty acid degradation, as well as in phenylpropanoid and carotenoid biosynthesis (Figure 9D). These functional categories indicate that Cluster 4 genes are associated with central metabolic and energy-related processes, and that NP-SiO₂ treatment attenuates their salt-induced up-regulation, thereby contributing to the fine-tuning of metabolic adjustment and energy balance under stress conditions.

3.7. Differential Expression of Hormone-Related Genes and Their Roles in Salt Tolerance Under NP-SiO₂ Treatment

Transcriptome profiling revealed that soybean hormone signaling pathways were extensively affected by salt stress and modulated by NP-SiO₂ treatment (

Table 2. NP-SiO₂-responsive genes involved in hormone pathways and their likely roles in salt-stress tolerance in soybean.

Hormone	Gene ID	Symbol	Category	Cluster	Log2 Fold Change		Likely Role in Salt-Stress Tolerance
					S_vs_C	SSI_vs_C
ABA	Glyma.01G225100.1	HAI2	ABA_Signaling	C4	7.69	6.36	Reduces ABA hypersignaling, contributing to stomatal regulation
	Glyma.01G018800.1	PYL12|RCAR6	ABA_Signaling	C2	−3.10	−1.86	Restores ABA receptor activity, maintaining appropriate signaling
	Glyma.07G017300.1	ABI4	ABA_Signaling	C2	−3.74	−2.21	Recovery of ABA signaling promotes balanced stress response
	Glyma.11G095100.1	BG2|PR2	ABA_Biosynthesis	C4	5.6	4.31	Suppresses ABA-induced PR proteins, preventing unnecessary defense
	Glyma.19G194500.1	ABF4	ABA_Signaling	C4	3.14	1.69	Attenuates ABA overactivation, preventing growth arrest
	Glyma.19G224400.1	AIT1|NRT1.2	ABA_Transport	C2	−3.08	−1.60	Restores ABA transport, supporting hormone homeostasis
GA	Glyma.09G183500.1	PIF1|PIL5	GA_Signaling	C2	−3.40	−2.24	GA–light crosstalk recovery supports photosynthesis and ROS defense
	Glyma.10G190200.1	GAI	GA_Signaling	C4	2.2	1.91	Reduces DELLA overactivation, restoring cell elongation
	Glyma.13G218200.1	GA2ox1	GA_Biosynthesis	C4	2.71	2.06	Moderates GA catabolism induction, balancing GA levels
	Glyma.16G089000.1	DXR	GA_Biosynthesis	C2	−3.64	−2.26	Recovers GA biosynthesis, supporting elongation
	Glyma.16G200800.3	GA20ox1|GA5	GA_Biosynthesis	C2	−3.19	−1.94	Promotes GA biosynthesis, enhancing growth recovery
SA	Glyma.02G023400.1	TRX5	SA_Signaling	C2	−5.03	−3.05	Restores redox balance, enhancing ROS detoxification
	Glyma.02G063500.1	MES7	SA_Synthesis	C2	−2.67	−1.33	Recovery of SA homeostasis → maintenance of ABA/ROS crosstalk
	Glyma.18G238900.1	BSMT1	SA_Synthesis	C4	2.86	1.75	Attenuates SA overactivation, stabilizing signaling crosstalk
JA	Glyma.02G142200.1	DAD1	JA_Biosynthesis	C2	−2.62	−1.40	Enhances JA-mediated lipid signaling, contributing to ROS defense
	Glyma.03G159000.1	DGL	JA_Biosynthesis	C4	3.51	2.3	Reduces JA overactivation, saving energy under salt stress
	Glyma.13G030300.1	LOX2	JA_Biosynthesis	C4	2.68	1.64	Suppresses excessive JA/ROS induction, preventing cell damage
CK	Glyma.13G324700.1	LOG4	CK_Biosynthesis	C4	5.67	4.48	Suppresses CK overactivation, maintaining growth–defense balance
	Glyma.14G175100.1	UGT85A1	CK_Biosynthesis	C2	−4.60	−2.19	Restores cytokinin biosynthesis, enhancing growth
	Glyma.20G057500.1	UGT85A1	CK_Biosynthesis	C2	−1.64	−0.60	Restores cytokinin biosynthesis, enhancing growth
BR	Glyma.02G256800.1	CPD|CBB3|DWF3	BR_Biosynthesis	C2	−2.55	−1.08	Recovers BR biosynthesis, supporting elongation
	Glyma.03G002900.1	CDG1	BR_Signaling	C2	−2.16	−0.95	Restores BR signaling, supporting development
	Glyma.11G204700.1	BRI1|CBB2|DWF2	BR_Signaling	C2	−4.31	−3.15	Restores BR receptor function, maintaining developmental control
	Glyma.13G352800.1	CDG1	BR_Signaling	C2	−3.98	−2.67	Recovers BR kinase signaling, regulating growth
AUX	Glyma.03G063900.1	AUX1	AUX_Transport	C2	−3.14	−1.67	Restores auxin transport, supporting directional radicle growth
	Glyma.07G164600.4	PIN4	AUX_Transport	C2	−2.58	−1.34	Recovers auxin efflux, enhancing radicle development
	Glyma.17G139400.1	NRT1.1	AUX_Transport	C2	−2.74	−1.22	Restores nitrate/auxin transport, maintaining growth
ETH	Glyma.07G017300.1	EREBP|ERF13	ETH_Signaling	C2	−3.74	−2.21	Recovery of ERF13 supports ROS detoxification and ion homeostasis
	Glyma.09G008400.1	ACO1	ETH_Biosynthesis	C2	−2.29	−1.64	Recovery of ACC oxidase maintains ethylene biosynthesis under salt stress
	Glyma.18G059700.1	ETO1	ETH_Biosynthesis	C2	−2.50	−1.75	Moderation of ethylene overproduction helps maintain the hormonal balance

S_vs_C: Salt stress (S) relative to control (C). SSI_vs_C: Salt stress with NP-SiO₂ treatment (SSI) relative to control (C). Box color indicates gene expression level, from green (down-regulated) to red (up-regulated).

). Eight major hormonal pathways, including ABA, GA, cytokinin (CK), auxin, brassinosteroid (BR), ethylene, jasmonic acid (JA), and salicylic acid (SA), were represented among the DEGs.

In the ABA pathway, stress-induced up-regulation of ABF4, BG2, and HAI2 was observed, though their expression levels were reduced under NP-SiO₂ treatment compared to salt stress alone. Conversely, ABI4, AIT1, and PYL12, which were strongly down-regulated under salt stress, partially recovered following NP-SiO₂ treatment. GA-related genes, including DXR, GA20ox1, and PIF1, were strongly repressed under salt stress but up-regulated with NP-SiO₂ treatment, while in contrast, GAI and GA2ox1 expression, which was induced by salt stress, was reduced under NP-SiO₂ treatment.

CK biosynthesis and metabolism were also influenced by salt stress and NP-SiO₂ treatment. LOG4 showed strong induction under salt stress, whereas NP-SiO₂ treatment attenuated this increase. In contrast, UGT85A1, which was suppressed by salt stress, was partially restored under NP-SiO₂ treatment. In the auxin pathway, auxin transporters (AUX1, NRT1.1, and PIN4) were down-regulated in the presence of salt, but their expression levels recovered with NP-SiO₂ treatment.

Brassinosteroid-related genes, such as BRI1, CDG1, and CPD, were significantly repressed under salt stress, and their expression was partially recovered by NP-SiO₂ treatment, with the same pattern observed for ethylene-related genes (ACO1, ERF13, and ETO1).

In the JA pathway, DGL and LOX2 were strongly induced under salt stress, but this induction was weaker under NP-SiO₂ treatment, while DAD1, which was suppressed by salt stress, was moderately restored. In terms of SA-associated genes, MES7 and TRX5 were suppressed under salt stress but recovered after NP-SiO₂ treatment, whereas BSMT1 was strongly induced by salt stress, with its expression attenuated under NP-SiO₂ treatment.

Overall, these results indicate that NP-SiO₂ treatment altered the expression of key hormone-related genes, suppressing stress-induced overactivation in ABA, JA, and SA signaling while partially restoring growth-associated pathways, including GA, CK, auxin, and BR, under salt-stress conditions.

4. Discussion

Plant biostimulants (PBSs) are substances or microorganisms that, when applied to seeds, plant tissues, or the rhizosphere, stimulate natural physiological processes to enhance nutrient uptake, improve tolerance to abiotic stress, and ultimately increase crop quality and yield [39,40]. In this study, the effects of different biostimulants were evaluated during the most salt-sensitive developmental stage of soybean germination, and the most effective treatment was identified. Furthermore, transcriptomic analysis was conducted to elucidate the molecular mechanisms underlying the enhanced salt tolerance conferred by the selected biostimulants.

Among the six previously reported PBSs with proven efficacy, treatment with NP-SiO₂ at 100 mg L⁻¹ significantly improved germination percentage (GP) and germination index (GI) under salt-stress conditions, indicating a marked enhancement in seed vigor and salt tolerance [24]. Mechanistically, this treatment activated major antioxidant enzymes (POD, APX, and CAT) to scavenge excessive reactive oxygen species (ROS) produced under salt stress, while concurrently reducing the level of malondialdehyde (MDA), a key marker of oxidative membrane damage. These findings suggest that 100 mg/L NP-SiO₂ represents the optimal concentration for mitigating salt-induced oxidative stress during soybean germination. In contrast, higher concentrations (150 mg/L and 200 mg/L) decreased antioxidant activity and increased MDA content to levels comparable to salt stress alone, indicating that excessive NP-SiO₂ may negatively affect redox homeostasis.

Previous studies support these observations. In pepper seeds, treatment with 300 mg/L NP-SiO₂ significantly recovered germination percentage, germination index, and radicle length under salt stress, accompanied by reduced H₂O₂ (−24.8%) and O₂⁻ (−8.4%) accumulation [41]. Similarly, in tomato seeds, 100 mg/L NP-SiO₂ enhanced germination performance, activated antioxidant enzymes, and modulated hormonal conditions by decreasing ABA while increasing auxin and GA levels. Notably, the nano-scale form of SiO₂ exhibited higher Si uptake and accumulation than its bulk counterpart, demonstrating its superior bioavailability [24]. Collectively, NP-SiO₂ enhances salt tolerance during germination through cell wall reinforcement, osmotic regulation, and ROS scavenging, benefiting from its intrinsic high surface-area-to-volume ratio and reactive physicochemical properties.

RNA-seq analysis provided molecular evidence for NP-SiO₂-mediated transcriptional stabilization under salt stress. Salt treatment induced 4579 differentially expressed genes (DEGs), indicating a substantial transcriptional reprogramming. However, NP-SiO₂ treatment reduced this number to 2920 DEGs, suggesting that the biostimulant not only activates certain stress-responsive genes but also restores transcriptional networks toward non-stress conditions.

Hierarchical clustering further revealed distinct regulatory modules. Cluster 2 genes, enriched in pathways related to hormone signaling (ABA, GA), phenylpropanoid and flavonoid biosynthesis, and ROS detoxification, were markedly down-regulated by salt stress but recovered to control levels following NP-SiO₂ treatment. Specifically, the recovery of PYL12/RCAR6 and ABI4 (ABA signaling), PIF1 and GA20ox1 (GA signaling), and TRX5 and ERF13 (ROS regulation) indicates that NP-SiO₂ maintains a balanced interplay between hormonal signaling and the antioxidant defense system under stress. This transcriptional pattern strongly parallels previous findings in other plant species, where antioxidant enzymes such as SOD, CAT, APX, and POD, which were down-regulated under salt stress, were restored to control levels following NP-SiO₂ treatment [42]. Similarly, NP-SiO₂ has been shown to recover the expression of stress-responsive transcription factors (e.g., AP2/ERF family) and ion-homeostasis-related genes (e.g., NHX transporters), reinforcing ROS detoxification and hormonal balance [41]. Together, these observations indicate that NP-SiO₂ reactivates the ROS-scavenging network at the transcriptional level, maintaining redox balance and ion homeostasis across diverse plant species.

Conversely, Cluster 4 genes, including those involved in energy and carbon metabolism, hormone signaling, and ROS detoxification, such as HAI2, ABF4, CAT, TRX5, GA2ox1, and DXR, were strongly induced under salt stress but attenuated following NP-SiO₂ treatment. The suppression of these overactivated genes suggests that NP-SiO₂ mitigates excessive ABA-dependent signaling and ROS-mediated responses, thereby reducing unnecessary metabolic energy consumption and restoring homeostatic balance. Similar trends were reported in previous studies, where NP-SiO₂ treatment enhanced photosynthetic performance and PSII efficiency in pepper, alleviating metabolic overload under salt stress [41], and normalized hyperactivated antioxidant enzyme levels in pea, reducing ROS-dependent energy expenditure [42]. These findings indicate that NP-SiO₂ performs a dual regulatory role, restoring suppressed antioxidant and hormonal pathways (Cluster 2) while simultaneously moderating overactivated stress metabolism (Cluster 4), ultimately optimizing the trade-off between growth and defense.

Finally, the ecological implication of NP-SiO₂ application should be considered for practical agricultural use. Recent studies have shown that NP-SiO₂ is environmentally benign and nontoxic at agricultural concentrations [43], therefore being a safe and sustainable nanomaterial for modern agriculture, although further research is needed to clarify its long-term behavior in soil ecosystems. Overall, current evidence supports NP-SiO₂ as an environmentally safe and sustainable biostimulant that enhances crop resilience under salt stress [44].

5. Conclusions

NP-SiO₂ demonstrated strong potential to mitigate the adverse effects of salt stress during soybean seed germination. Under salt stress induced by 150 mM NaCl, NP-SiO₂ treatment significantly enhanced germination performance, particularly at a concentration of 100 mg/L, where the most notable improvements in GP, radicle elongation, and physiological recovery were observed. Biochemical analysis revealed that NP-SiO₂ application reduced lipid peroxidation, as evidenced by decreased MDA levels, and enhanced the antioxidant defense system by increasing the activity of the key enzymes POD, APX, and CAT. Transcriptomic analysis further provided molecular evidence supporting the biostimulant’s protective role under salt stress. Although NP-SiO₂ is recognized as a safe and sustainable nanomaterial, its long-term ecological effects in soil remain to be fully elucidated. Overall, this study demonstrates that NP-SiO₂ significantly enhances soybean tolerance to salt stress through physiological, biochemical, and molecular mechanisms. These findings provide mechanistic insights into its protective action and position silicon-based nanomaterials as a promising strategy for improving the abiotic stress resilience of crops.