Next Article in Journal
Efficacy of Biological Products in Managing Root Pathogens in Melons
Previous Article in Journal
Desert Locust Management Is Plagued by Human-Based Impediments
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Agronomy
Volume 15
Issue 9
10.3390/agronomy15092103
agronomy-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

A Plant Growth-Promoting Bacterial Isolate, Bacillus velezensis 41S2, Enhances Seed Protein, Isoflavone Accumulation, and Stress Resilience in Soybean Under Salt–Alkaline Soil Conditions

by
Han Zheng
1,2,
Shutian Hua
1,2,
Zhe Li
2,
Ziyan Wang
2,
Donglin Zhao
2,
Changliang Jing
2,
Yiqiang Li
2,
Chengsheng Zhang
2,
Yanfen Zheng
2,
Youqiang Wang
2,* and
Mingguo Jiang
1,*
1
Guangxi Key Laboratory of Polysaccharide Materials and Modification, School of Marine Sciences and Biotechnology, Guangxi Minzu University, Nanning 530008, China
2
Marine Agriculture Research Center, Tobacco Research Institute of Chinese Academy of Agricultural Sciences, Qingdao 266101, China
*
Authors to whom correspondence should be addressed.
Agronomy 2025, 15(9), 2103; https://doi.org/10.3390/agronomy15092103
Submission received: 6 August 2025 / Revised: 24 August 2025 / Accepted: 28 August 2025 / Published: 31 August 2025
(This article belongs to the Section Soil and Plant Nutrition)
Browse Figures
Versions Notes

Abstract

Salt–alkaline soil poses a significant challenge to soybean productivity. While plant growth-promoting rhizobacteria (PGPR) offer a sustainable strategy for stress mitigation, their field-level application remains underexplored. Here, a field experiment was conducted in the Yellow River Delta of Shandong, China, a typical salt–alkaline region. In this study, we evaluated the effectiveness of Bacillus velezensis 41S2 in enhancing soybean performance under salt–alkaline soil through integrated field trials and transcriptomic analysis. Inoculation with strain 41S2 significantly improved plant biomass, yield components, and seed yield under salt–alkaline soil, and notably increased seed protein and isoflavone contents. Physiological analyses revealed that strain 41S2 markedly reduced hydrogen peroxide (H2O2) accumulation, indicating alleviation of oxidative stress. Moreover, strain 41S2 modulated the levels of soluble sugars and amino acids, contributing to osmotic regulation and carbon–nitrogen (C-N) metabolic balance. Transcriptome profiling further indicated that strain 41S2 upregulated genes involved in antioxidant response, C–N metabolism, and phenylpropanoid biosynthesis, highlighting its role in coordinating multilayered stress response pathways. Overall, these findings highlight the potential of B. velezensis 41S2 as a multifunctional bioinoculant for improving salt tolerance and presents a promising tool for sustainable crop production and ecological restoration in salt–alkaline soil.

1. Introduction

Soil salinization is a significant environmental challenge facing global agriculture, severely limiting crop yields and threatening sustainable agricultural development. According to the Food and Agriculture Organization [1], approximately 1.38 billion hectares are affected by salinization. The formation of salt–alkaline soils is closely linked to improper irrigation practices, rapid soil water evaporation, and rising groundwater levels [2,3]. Over time, salt accumulation degrades soil structure and diminishes its capacity to retain water and nutrients, thereby impairing seed germination and crop productivity [4]. Under salt–alkaline conditions, excessive sodium ions (Na+) disrupt water and nutrient uptake in plants [5]. Salt stress impairs plant function primarily through three interconnected mechanisms: ion toxicity, osmotic stress, and oxidative stress [6,7]. These mechanisms collectively disturb cellular metabolism, increase the production of reactive oxygen species (ROS), and lead to lipid peroxidation and photosynthetic inhibition [8,9,10]. In addition, compared with salt stress, alkaline salts (e.g., Na2CO3 and NaHCO3) not only trigger similar stresses but also impose high-pH toxicity and nutrient deficiencies. To cope with alkaline stress, plants are required to regulate their intracellular pH to sustain ion balance [11]. In short, salt–alkaline stress has a profound impact on the normal growth and development of crops, leading to reductions in both yield and quality.
To cope with salt stress, plants have evolved a range of complex adaptive mechanisms, including the activation of salt-responsive signaling pathways, such as the salt overly sensitive pathway, the biosynthesis of osmoprotectants like proline and betaine to maintain cellular osmotic balance, and the enhancement of antioxidant enzyme activities [12,13]. In addition, plants alleviate the detrimental effects of salt stress by maintaining Na+/K+ homeostasis and regulating the expression of stress-responsive genes [14,15]. Although traditional breeding and genetic engineering hold great promise for improving plant salt tolerance—particularly through enhanced performance of certain transgenic lines under controlled conditions—their effectiveness in field environments remains poorly validated [16,17]. This is largely because the inherent physiological responses of plants are insufficient to sustain stable yield and quality under prolonged salt–alkaline stress. Hence, complementary approaches beyond genetic improvement are required. Although genetic engineering holds great promise for improving plant salt tolerance—particularly through enhanced performance of certain transgenic lines under controlled conditions—its effectiveness in field environments remains poorly validated. This is largely due to the inherent limitations of plant physiological responses, which are often insufficient to maintain stable yield and quality under prolonged or severe salt stress [18]. Consequently, in recent years, the application of plant growth-promoting rhizobacteria (PGPR) has emerged as a promising and sustainable biological strategy to enhance crop growth and productivity in salt–alkaline soil [19].
A growing body of research has demonstrated that PGPR enhances plant salt tolerance through a variety of physiological and molecular mechanisms. These include modulation of the antioxidant defense system, accumulation of osmolytes, regulation of phytohormone levels, improvement of nutrient uptake, and the induction of systemic resistance [20,21]. For instance, many PGPR strains produce 1-aminocyclopropane-1-carboxylate deaminase, which reduces ethylene levels and alleviates salt-induced growth inhibition [22], or secrete indole-3-acetic acid to stimulate root growth and maintain K+/Na+ homeostasis [23]. In addition, PGPR inoculation also commonly enhances the activities of antioxidant enzymes, such as superoxide dismutase and catalase, helping plants fight oxidative damage. Among various PGPR genera, Bacillus species have received particular attention due to their stress resilience and high biosynthetic capacity. In rice, Bacillus pumilus JPVS11 improved salt tolerance by modulating antioxidant activity and soil enzyme functions [24]. Likewise, Bacillus safensis TS3 significantly promoted the growth and yield of radish and oat under salt stress [25]. Therefore, understanding the physiological and molecular mechanisms by which PGPR improve salt tolerance will support their broader application. Nevertheless, the majority of such findings are restricted to controlled environments, and little is known about whether these mechanisms operate effectively in salt–alkaline soils. Given the combined stressors of high salinity, alkalinity, and nutrient imbalance in these soils, it is uncertain whether PGPR—especially Bacillus strains—can confer comparable benefits under field conditions.
Soybean (Glycine max), one of the most economically important crops worldwide, is a major source of plant-based protein and oil, and is widely utilized across the food, feed, and industrial sectors [26,27]. Beyond its nutritional value, soybean seeds are rich in bioactive compounds such as isoflavones and saponins, which not only exhibit notable antioxidant, anticancer, and antidiabetic properties but also play crucial roles in plant responses to environmental stress [28,29]. While PGPRs are widely recognized for their role in promoting plant growth, the central challenge under salt stress is to understand the physiological and molecular mechanisms by which they enhance stress tolerance [30]. Addressing this knowledge gap is critical for extending their application in sustainable agriculture. At the same time, the impact of PGPR on crop quality traits, an aspect closely linked to nutritional value and market competitiveness, remains insufficiently studied. Therefore, investigating how PGPR regulates both stress resilience and quality formation in soybean under salt stress is of great scientific and practical relevance. In our previous study, we isolated a Bacillus velezensis strain, 41S2, with typical PGPR characteristics from the rapeseed rhizosphere and demonstrated its ability to significantly enhance host nitrogen uptake and utilization efficiency [31]. More recently, we discovered that this strain also demonstrates strong salt tolerance, a trait that not only supports its long-term survival in salt–alkaline soils but also further emphasizes its potential applications. Given the advantages of Bacillus in commercial development, 41S2 was ultimately identified as an ideal candidate bioinoculant for evaluating its role in enhancing soybean adaptation to salt–alkaline soils.
In this study, we conducted field trials using the previously isolated PGPR strain Bacillus velezensis 41S2 to systematically evaluate its effects on soybean growth, yield, and quality under salt–alkaline soil. Coupled with transcriptomic analyses, this investigation aimed to elucidate the molecular mechanisms underlying the observed phenotypic responses. Specifically, the objectives of this study were to (i) uncover the biological mechanisms by which B. velezensis 41S2 enhances salt tolerance in soybean and (ii) assess its regulatory effects on soybean yield and quality under natural salt–alkaline soil. This research contributes to a deeper understanding of PGPR-mediated salt stress tolerance and expands current knowledge regarding the influence of PGPR on crop quality, ultimately providing a novel, sustainable approach for managing salinized agricultural systems.

2. Materials and Methods

2.1. Experimental Design

To investigate the effects of B. velezensis 41S2 on soybean performance under natural salt–alkaline soil, a field experiment was conducted from June to October 2024 in Dongying, Shandong Province, China (37°17′37.55″ N, 118 °38′1.33″ E). The soil type is coastal salt–alkali soil. The basic physicochemical properties of the soil are as follows: organic matter content 12.38 g/kg, total nitrogen 1.15 g/kg, alkali-hydrolyzable nitrogen 48.07 mg/kg, available phosphorus 6.25 mg/kg, available potassium 104.93 mg/kg, pH value 8.90, and a salt content of 0.3%. The soybean cultivar Zhonghuang 13 was used as the experimental material. Two treatments were established: uninoculated control and inoculation with strain 41S2. Each treatment was replicated three times in a randomized complete block design. Each plot measured 6 m × 14 m, with approximately 1100 soybean plants per plot.
To evaluate the salt tolerance of strain 41S2, five NaCl concentrations (0.0%, 1.0%, 2.0%, 3.0%, and 4.0%) were prepared by supplementing the nutrient broth (NB) medium with the corresponding NaCl levels, followed by sterilization at 121 °C for 20 min. Strain 41S2 was first cultured in NB medium for 24 h, after which 196 µL of NB medium containing different NaCl concentrations was dispensed into 96-well plates under aseptic conditions. Each NaCl concentration was tested in triplicate. Subsequently, 4 µL of bacterial suspension was inoculated into the wells, and cultures were incubated at 28 °C for 2 days. The optical density at 600 nm (OD600) was measured using a microplate reader to determine bacterial growth under salt stress [32].
Following the salt tolerance assessment, strain 41S2 was cultured overnight in NB, harvested by centrifugation at 5000 r/min for 5 min, washed twice with sterile 0.9% NaCl solution, and resuspended to an optical density of OD600 = 0.2. A two-step method was employed for inoculation with strain 41S2. Initially, soybean seeds were coated with a bacterial suspension and mixed with a starch–peat carrier before sowing to ensure uniform bacterial coverage of the seeds. Subsequently, as previously described methods [33], root irrigation with a bacterial suspension was conducted during the soybean flowering stage. Specifically, each plant in the inoculated group received 20 mL of bacterial suspension (OD600 = 0.2), whereas the control group was treated with the same volume of sterile water. Ten days after bacterial root drenching, at the pod-setting stage, agronomic traits of plants inoculated with strain 41S2 were evaluated, including plant height, nodule number and weight, and root and shoot biomass. Simultaneously, RNA-seq samples were collected.

2.2. Carbon and Nitrogen Content Analysis

At the maturity stage, soybean seeds from both uninoculated and B. velezensis 41S2-inoculated plants were harvested, and seed yield was recorded. The dried samples were ground and sieved, and the total carbon (C) and total nitrogen (N) contents were measured using a FlashSmart Elemental Analyzer (Thermo Fisher, Waltham, MA, USA), based on the dry combustion method [34]. Each treatment consisted of six biological replicates.

2.3. Determination of Protein Content

The protein content in the seeds was determined using the Coomassie Brilliant Blue G-250 assay. Briefly, 0.100 g of finely ground soybean sample was placed into a 50 mL centrifuge tube, with three independent replicates for each treatment. Protein extraction was performed at a material-to-liquid ratio of 1:400 by shaking at 30 °C and 220 r/min for 90 min, followed by centrifugation at 3500 r/min for 5 min. The supernatant was diluted 10-fold, and 1 mL of the diluted extract was mixed with 1 mL of CBG reagent. The absorbance was measured at 595 nm [35].

2.4. Determination of Isoflavone Content

The isoflavone content was determined using a UV–visible spectrophotometric method. Briefly, 0.100 g of ground soybean sample was placed into a 15 mL centrifuge tube, with each treatment processed in triplicate. Then, 5 mL of 80% methanol was added to each tube. The samples were subjected to ultrasonic extraction at 50 °C for 1 h, with an ultrasonic frequency of 40 kHz and a power of 300 W. After extraction, the samples were centrifuged at 12,000 r/min for 10 min. The supernatant was diluted 5-fold, and 2 mL of the diluted extract was used to measure absorbance at 260 nm using a UV–visible spectrophotometer [36].

2.5. Quantitative Analysis of Hydrogen Peroxide (H2O2)

Soybean root samples were collected on the 60th day after sowing under salt stress conditions to determine H2O2 content. The measurement was performed using a commercial H2O2 assay kit (Solarbio Science & Technology, Beijing, China), following the manufacturer’s instructions. This colorimetric assay relies on the reaction of H2O2 with titanium sulfate to form a yellow titanium-peroxide complex, which exhibits maximum absorbance at 415 nm.
Approximately 0.1 g of fresh root tissue was homogenized in 1 mL of acetone solution in an ice bath. The homogenate was centrifuged at 9200 r/min for 10 min at 4 °C, and the supernatant was collected for subsequent reaction. According to the manufacturer’s instructions, the sample was mixed with the appropriate reagents and incubated at room temperature. After centrifugation, the precipitate was retained and resuspended with a chromogenic reagent. Following a 5 min incubation at room temperature, 200 μL of the reaction mixture was transferred to a 96-well microplate, and the absorbance was measured at 415 nm. The H2O2 content was calculated accordingly (Solarbio Science & Technology, Beijing, China).

2.6. RNA Extraction, Library Construction, and Transcriptome Sequencing

To minimize border effects, two soybean plants were randomly selected from the center of each plot and pooled as a composite sample. Roots from control and B. velezensis 41S2-inoculated plants were collected for RNA-seq analysis, with three biological replicates per treatment. Total RNA was extracted using TRIzol® Reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. RNA concentration and purity were measured using a NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific, USA), and RNA integrity was assessed using the 5300 Bioanalyzer System (Agilent Technologies, Santa Clara, CA, USA). Only RNA samples meeting stringent quality control criteria (OD260/280 = 1.8–2.2, OD260/230 ≥ 2.0, RIN or RQN ≥ 6.5, 28S:18S ≥ 1.0, and total RNA yield > 1 μg) were selected for library construction.
Library preparation was carried out by Shanghai Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). For each sample, 1 μg of total RNA was used to prepare the library using the Illumina® Stranded mRNA Prep, Ligation protocol. Poly (A)+ mRNA was enriched with Oligo (dT) magnetic beads and fragmented. Double-stranded cDNA was synthesized using random hexamer primers, followed by end-repair, A-tailing, and adapter ligation. The libraries were PCR-amplified (10–15 cycles), size-selected for fragments of 300–400 bp, and quantified using a Qubit 4.0 fluorometer. Paired-end sequencing (PE150) was performed by the service provider.

2.7. RNA Data Processing

Raw reads were quality-filtered and trimmed using fastp [37]. Clean reads were mapped to the soybean reference genome (Glycine max, version Gmax_275; Wm82.a2.v1) obtained from Phytozome using HISAT2 [38]. Transcript assembly was conducted using StringTie [39]. Gene expression levels were quantified as transcripts per million using RSEM [40]. Differentially expressed genes (DEGs) were identified using DESeq2 with thresholds of |log2 FC| ≥ 1 and FDR < 0.05 [41]. Functional enrichment analyses of DEGs were performed using GOatools and SciPy for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, respectively [42,43]. Fisher’s exact test was used for enrichment analysis, and the Benjamini–Hochberg method was applied to control the false discovery rate. p-values < 0.05 were considered statistically significant.

2.8. Statistical Analysis

In this study, the results are presented as the mean ± standard deviation. The differences between the inoculated and uninoculated 41S2 treatments were assessed using a Student’s t-test. Statistical significance is indicated by asterisks: * p < 0.05, ** p < 0.01, and *** p < 0.001. Data visualization was performed using Prism 10.1.2 (GraphPad Software, USA) and the Majorbio Cloud Platform (https://cloud.majorbio.com/).

3. Results

3.1. Effects of B. velezensis 41S2 on Soybean Growth Performance Under Salt–Alkaline Soil

To evaluate the effects of the PGPR strain B. velezensis 41S2 on soybean salt–alkaline tolerance, growth performance was compared between inoculated and uninoculated plants under salt–alkaline soil. Prior to inoculation, we evaluated the salt tolerance of strain 41S2, and the results demonstrated a strong capacity for salt tolerance, suggesting considerable potential for adaptation in salt–alkaline soils (Figure S1). Field inoculation trials demonstrated that strain 41S2 inoculation significantly improved soybean growth (Figure 1a). Compared with the control, plants treated with strain 41S2 exhibited a 15%increase in plant height, and shoot and root biomass were increased by 61%and 55%, respectively (Figure 1b–d). In addition, strain 41S2 markedly promoted root nodulation (Figure 1e). The total nodule weight was 59% higher than that of the control (Figure 1f), indicating strain 41S2 enhanced symbiotic interaction between soybean roots and rhizobia. These findings demonstrate that strain 41S2 significantly enhances soybean adaptability to salt stress, highlighting its potential for application in soybean cultivation under salt–alkaline soil.

3.2. B. velezensis 41S2 Maintains ROS Homeostasis Induced by Salt–Alkaline Soil

To elucidate the molecular mechanisms by which B. velezensis 41S2 enhances salt tolerance in soybean, RNA-seq analysis was performed on root tissues from both inoculated and uninoculated plants grown in salt–alkaline soil. A total of 853 differentially expressed genes (DEGs) were identified, including 340 upregulated and 513 downregulated genes (Figure 2a). GO annotation analysis revealed that DEGs were significantly associated with stress response, oxidoreductase activity, and ion binding, indicating potential involvement in antioxidant defense and maintenance of ion homeostasis under salt stress conditions (Figure 2b). Further GO enrichment analysis indicated that DEGs induced by strain 41S2 were predominantly involved in oxidative stress responses, particularly redox metabolism and hydrogen peroxide (H2O2) catabolism (Figure 2c). Notably, strain 41S2 inoculation strongly upregulated peroxidase (POX) genes, which are crucial for H2O2 scavenging and the maintenance of ROS homeostasis (Figure 2d). For example, compared with the non-inoculated treatment, the expression level of the POX gene Glyma.03G038100 in 41S2-treated plants was upregulated 6.3-fold (p < 0.01). Similarly, the expression level of the CYP gene Glyma.09G142200 was upregulated 9.9-fold (p < 0.01). To validate this regulatory pathway, H2O2 levels were measured in the roots of both inoculated and uninoculated plants. The results showed that strain 41S2 inoculation effectively reduced H2O2 accumulation compared to the control (Figure 2e), indicating enhanced antioxidant capacity. Collectively, these findings suggest that B. velezensis 41S2 mitigates salt-induced oxidative stress in soybean by activating antioxidant defense, thereby improving plant adaptability to salt–alkaline soil.

3.3. B. velezensis 41S2 Enhances Plant N and C Metabolism Under Salt–Alkaline Soil

KEGG enrichment analysis revealed that inoculation with B. velezensis 41S2 significantly affected several key metabolic pathways in soybean roots under salt–alkaline soil (Figure 3). DEGs were significantly enriched in phenylpropanoid biosynthesis, N metabolism, and starch/sucrose metabolism in 41S2-inoculated plants (Figure 3a). In the N metabolism pathway, the expression of the genes Glyma.16G153400 and Glyma.02G07220, which encode the enzyme glutamate dehydrogenase (GDH), was significantly upregulated in response to induction by strain 41S2 (Figure 3b). GDH plays a central role in ammonium assimilation and N flux regulation, suggesting that 41S2 enhances N assimilation by facilitating the direct conversion of ammonium to L-glutamate (Figure 3b). For C metabolism, inoculation with strain 41S2 significantly activated the starch and sucrose metabolism pathway, particularly promoting the synthesis of D-glucose. Under salt stress, genes encoding β-glucosidase (BGL) and invertase (INV) were markedly upregulated, leading to increased D-glucose accumulation (Figure 3c). These carbohydrates serve as crucial energy sources, feeding into glycolysis and the tricarboxylic acid (TCA) cycle to generate ATP and reducing equivalents (NADH/NADPH), which are essential for meeting the elevated energy demands under saline stress. These results suggest that B. velezensis 41S2 enhances plant salt tolerance by coordinately modulating C and N metabolic pathways, thereby supporting metabolic homeostasis under salt–alkaline conditions.

3.4. B. velezensis 41S2 Enhances Soybean Seed Yield Under Salt–Alkaline Soil

At the maturity stage, the effect of B. velezensis 41S2 inoculation on soybean seed yield was evaluated (Figure 4). Under salt–alkaline soil, 41S2-inoculated plants exhibited a significant yield advantage compared to the control (Figure 4a). Specifically, the number of pods per plant and seed yield per plant increased by 45% and 75%, respectively, relative to the control (Figure 4b,c). These findings demonstrate the substantial yield-enhancing potential of strain 41S2 in soybean cultivation under salt–alkaline soil. This provides a practical and effective microbial strategy for improving soybean productivity in saline-affected soils, with significant implications for broader field application.

3.5. B. velezensis 41S2 Improves Soybean Seed Quality Under Salt–Alkaline Soil

To investigate the potential impact of B. velezensis 41S2 on soybean seed quality, we measured key quality-related parameters in seeds from both 41S2-inoculated and uninoculated plants (Figure 5). The results showed that inoculation with strain 41S2 significantly increased the C and N accumulation in soybean seeds, with levels 80% and 83% higher, respectively, compared to the control (Figure 5a,b). This suggests that strain 41S2 promotes the efficient allocation of C and N nutrients into the seeds. Notably, the protein and isoflavone contents of 41S2-inoculated seeds were also significantly enhanced by 88% and 77%, respectively (Figure 5c,d), indicating a marked improvement in the nutritional quality of soybean seeds. Overall, these findings demonstrate that B. velezensis 41S2 not only enhances yield but also improves the quality of soybean seeds under salt–alkaline soil, reinforcing its potential as a beneficial microbial agent for sustainable agriculture in salt-affected areas.

4. Discussion

PGPR offers a sustainable solution for enhancing crop growth in saline soils. Understanding how PGPR helps plants cope with salt stress is key to developing effective microbial inoculants. In this study, we identified B. velezensis 41S2 as a beneficial strain that significantly enhanced soybean adaptability under salt–alkaline soil. However, the specific biological mechanisms underlying the salt tolerance conferred by strain 41S2 remained unclear. To address this, we conducted integrated physiological and transcriptomic analyses to elucidate the molecular basis of 41S2-mediated salt tolerance, providing mechanistic insights for the application of PGPR in saline agriculture.

4.1. B. velezensis 41S2 Enhances the Antioxidant Capacity of Soybean Under Salt–Alkaline Soil

B. velezensis 41S2 improved soybean growth and reduced oxidative damage under salt–alkaline soil (Figure 1). Transcriptome analysis indicated that strain 41S2 reprogrammed genes associated with ROS metabolism and osmotic regulation (Figure 2 and Figure 3). Salt-induced ROS accumulation disrupts cellular homeostasis, leading to oxidative injury across organelles such as chloroplasts, mitochondria, and peroxisomes [44,45]. Several studies have demonstrated PGPR-mediated ROS detoxification under salt stress. For instance, Bacillus subtilis CNBG-PGPR-1 enhances salt tolerance in tomato by activating ROS-scavenging pathways linked to methionine-mediated ethylene signaling [46], while Ensifer sp. GMS14 suppresses H2O2 accumulation in soybean [33]. In our study, strain 41S2 inoculation substantially increased the expression of antioxidant-related genes, with POX and CYP450 families showing the strongest responses (Figure 2c,d). The CYP superfamily contributes not only to hormone biosynthesis and secondary metabolism but also to ROS detoxification [47]. CYP81D5 in wheat, for example, enhances ROS removal and confers salt tolerance. Similarly, POXs, as efficient H2O2-scavenging enzymes, play a central role in maintaining ROS homeostasis [48,49]. Notably, TaPRX-2A regulates ROS levels and improves osmotic stability through ABA-dependent antioxidant mechanisms [50]. These findings suggest that 41S2 alleviates oxidative stress by selectively enhancing the transcription of POX (e.g., Glyma.03G038100) and CYP450 (e.g., Glyma.09G142200) genes, thereby establishing a mechanistic link between microbial inoculation and improved redox homeostasis under salt–alkaline conditions.

4.2. Regulation of C and N Metabolic Homeostasis by B. velezensis 41S2 Under Salt–Alkaline Soil

Salt stress disrupts cellular homeostasis primarily through osmotic imbalance and ion toxicity [51], impairing the coordination of central metabolic pathways [52,53]. In this study, we found that B. velezensis 41S2 reprogrammed soybean root metabolism, particularly enhancing C and N pathways under salt–alkaline soil (Figure 3). These pathways are vital for distributing energy, producing stress-related compounds, and maintaining overall homeostasis. Salt stress frequently leads to C/N ratio disruption, which limits plant growth and development [54,55]. While many PGPRs enhance salt tolerance by facilitating N uptake [56,57], strain 41S2 specifically upregulated GDH genes, suggesting activation of an ammonium assimilation (Figure 3b). This pathway supports N flux toward glutamate, a precursor for stress-responsive amino acids, such as proline and arginine, which contribute to osmotic protection, membrane stabilization, and ROS detoxification [58]. For instance, exogenous proline has been shown to improve salt tolerance in soybean by enhancing osmotic balance and antioxidant defense [59].
Salt stress also inhibits carbon metabolism by impairing photosynthesis [60,61]. Interestingly, strain 41S2 upregulated the expression of genes encoding BGL and INV, enzymes that break down complex sugars into glucose (Figure 3c). This likely promotes downstream energy pathways like glycolysis and the TCA cycle, providing ATP and NAD(P)H to counter salt stress. Similarly, the salt-tolerant PGPR strain KR-17 has been reported to enhance carbohydrate accumulation in radish roots, thereby alleviating the detrimental effects of salt stress [62]. In addition to their energy-providing role, soluble sugars also function as osmoprotectants. Exogenous sucrose has been reported to alleviate salt-induced damage in sunflower and canola by modulating osmolyte levels, antioxidant enzyme activities, and gene expression [63]. Thus, the sugar-mediated response observed in 41S2-inoculated plants likely complements proline accumulation, representing a coordinated metabolic adaptation to osmotic stress. Taken together, B. velezensis 41S2 confers salt tolerance by fine-tuning C and N metabolic networks to support energy balance, osmotic regulation, and cellular resilience.

4.3. B. velezensis 41S2 Enhances Soybean Yield and Quality Under Salt–Alkaline Soil

PGPR have been widely reported to enhance plant performance and yield under abiotic stress by optimizing rhizosphere dynamics and nutrient availability [64,65]. Consistent with these reports, 41S2 significantly increased pod number and seed yield under salt–alkaline soil (Figure 4). These benefits likely result from the strain’s capacity to mitigate stress-induced damage and restore productivity. As soybean inherently engages in symbiotic N fixation through rhizobia [66,67], the observed enhancement in nodule formation in 41S2-inoculated plants (Figure 1e,f) suggests a synergistic effect that boosts N acquisition and contributes to improved yield outcomes. However, such effects may result from potential interactions between 41S2 and native rhizobia, as well as broader rhizosphere microbial communities, which together enhance nitrogen fixation efficiency and overall plant resilience under salt stress. Future research should integrate metagenomic or colonization studies to further elucidate these complex relationships.
Importantly, B. velezensis 41S2 also positively influenced soybean seed quality. Previous studies have demonstrated that certain PGPR, such as Massilia spp., can significantly increase seed oil content in soybean, highlighting a close link between beneficial microbes and seed quality traits [68]. Similarly, our results showed that 41S2 significantly increased protein and isoflavone contents in soybean seeds. Protein synthesis depends on sufficient C skeletons and N supply, both of which are commonly disrupted under salinity [69,70]. Our findings suggest that, under salt stress, 41S2 inoculation helps maintain C–N homeostasis by enhancing L-glutamate biosynthesis and energy metabolism via sucrose degradation, thereby increasing both C and N contents in seeds (Figure 3 and Figure 5a–c). Moreover, KEGG analysis revealed activation of the phenylpropanoid biosynthesis pathway (Figure 3a), suggesting that 41S2 may promote flavonoid production. These compounds serve dual roles: enhancing stress tolerance and acting as important nutritional markers in soybean [71,72]. Notably, we observed a significant increase in seed isoflavone levels (Figure 5d), potentially linked to PGPR-induced activation of flavonoid biosynthetic genes. These improvements in seed quality may also reflect indirect effects of 41S2 colonization on the rhizosphere microbial community, which could modulate nutrient availability and secondary metabolism. Although research in this area remains limited, our findings underscore the multifaceted role of 41S2 in promoting both yield and nutritional quality. Overall, B. velezensis 41S2 represents a promising microbial inoculant for enhancing soybean resilience and productivity in salt–alkaline soil. In addition, this study was limited to salt–alkaline soil conditions. Future research should introduce additional control treatments under controlled conditions to more precisely elucidate the growth-promoting effects of strain 41S2.

5. Conclusions

This study presents a comprehensive evaluation of the PGPR (B. velezensis 41S2) under field conditions, demonstrating its capacity to confer salt tolerance in soybean through a multifaceted regulatory framework (Figure 6). Field trials and molecular analyses revealed that strain 41S2 enhances plant performance under salinity via promoting biological N fixation, activating antioxidant defense systems, and stabilizing C and N metabolic homeostasis. Additionally, 41S2 inoculation significantly increased both seed yield and nutritional quality, particularly in terms of protein and isoflavone accumulation. Collectively, these findings advance our understanding of PGPR-mediated stress mitigation and offer a strong foundation for the development of targeted microbial bioproducts to improve crop resilience in saline environments.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy15092103/s1, Figure S1: Growth of strain 41S2 under different NaCl concentrations.

Author Contributions

H.Z.: Investigation, visualization, formal analysis, writing—original draft; S.H.: investigation, methodology; Z.L.: investigation, methodology; Z.W.: investigation, formal analysis; D.Z.: methodology, formal analysis; C.J.: investigation, formal analysis; Y.L.: conceptualization, writing—review and editing; C.Z.: conceptualization, writing—review and editing, funding acquisition; Y.Z.: investigation, supervision; Y.W.: project administration, formal analysis, writing—review and editing; M.J.: conceptualization, supervision, funding acquisition, project administration, writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Science and Technology Major Project of Guangxi (AA18242026), the National Natural Science Foundation of China (32402669), the Youth Innovation Program of the Chinese Academy of Agricultural Sciences (Y2025QC35), and the Young Talent of Lifting Engineering for Science and Technology in Shandong, China (SDAST2025QTA075).

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. FAO. Global Status of Salt-Affected Soils; FAO: Rome, Italy, 2024; ISBN 978-92-5-139307-9. [Google Scholar]
  2. Mehrabi, S.S.; Sabokdast, M.; Bihamta, M.R.; Dedičová, B. The Coupling Effects of PGPR Inoculation and Foliar Spraying of Strigolactone in Mitigating the Negative Effect of Salt Stress in Wheat Plants: Insights from Phytochemical, Growth, and Yield Attributes. Agriculture 2024, 14, 732. [Google Scholar] [CrossRef]
  3. Xiao, F.; Zhou, H. Plant Salt Response: Perception, Signaling, and Tolerance. Front. Plant Sci. 2023, 13, 1053699. [Google Scholar] [CrossRef]
  4. van Zelm, E.; Zhang, Y.; Testerink, C. Salt Tolerance Mechanisms of Plants. Annu. Rev. Plant Biol. 2020, 71, 403–433. [Google Scholar] [CrossRef] [PubMed]
  5. Zhao, S.; Zhang, Q.; Liu, M.; Zhou, H.; Ma, C.; Wang, P. Regulation of Plant Responses to Salt Stress. Int. J. Mol. Sci. 2021, 22, 4609. [Google Scholar] [CrossRef] [PubMed]
  6. Fang, S.; Hou, X.; Liang, X. Response Mechanisms of Plants Under Saline-Alkali Stress. Front. Plant Sci. 2021, 12, 667458. [Google Scholar] [CrossRef]
  7. Fu, H.; Yang, Y. How Plants Tolerate Salt Stress. Curr. Issues Mol. Biol. 2023, 45, 5914–5934. [Google Scholar] [CrossRef]
  8. Arif, Y.; Singh, P.; Siddiqui, H.; Bajguz, A.; Hayat, S. Salinity Induced Physiological and Biochemical Changes in Plants: An Omic Approach towards Salt Stress Tolerance. Plant Physiol. Biochem. 2020, 156, 64–77. [Google Scholar] [CrossRef] [PubMed]
  9. Munns, R.; Passioura, J.B.; Colmer, T.D.; Byrt, C.S. Osmotic Adjustment and Energy Limitations to Plant Growth in Saline Soil. New Phytol. 2020, 225, 1091–1096. [Google Scholar] [CrossRef]
  10. Zhou, H.; Shi, H.; Yang, Y.; Feng, X.; Chen, X.; Xiao, F.; Lin, H.; Guo, Y. Insights into Plant Salt Stress Signaling and Tolerance. J. Genet. Genom. 2024, 51, 16–34. [Google Scholar] [CrossRef]
  11. Yang, S.; Xu, Y.; Tang, Z.; Jin, S.; Yang, S. The Impact of Alkaline Stress on Plant Growth and Its Alkaline Resistance Mechanisms. Int. J. Mol. Sci. 2024, 25, 13719. [Google Scholar] [CrossRef]
  12. Ali, A.; Petrov, V.; Yun, D.-J.; Gechev, T. Revisiting Plant Salt Tolerance: Novel Components of the SOS Pathway. Trends Plant Sci. 2023, 28, 1060–1069. [Google Scholar] [CrossRef]
  13. Wang, X.; Chen, Z.; Sui, N. Sensitivity and Responses of Chloroplasts to Salt Stress in Plants. Front. Plant Sci. 2024, 15, 1374086. [Google Scholar] [CrossRef]
  14. Wu, T.-Y.; Goh, H.; Azodi, C.B.; Krishnamoorthi, S.; Liu, M.-J.; Urano, D. Evolutionarily Conserved Hierarchical Gene Regulatory Networks for Plant Salt Stress Response. Nat. Plants 2021, 7, 787–799. [Google Scholar] [CrossRef] [PubMed]
  15. Zhao, C.; Zhang, H.; Song, C.; Zhu, J.-K.; Shabala, S. Mechanisms of Plant Responses and Adaptation to Soil Salinity. Innovation 2020, 1, 100017. [Google Scholar] [CrossRef] [PubMed]
  16. Joshi, R.; Mangu, V.R.; Bedre, R.; Sanchez, L.; Pilcher, W.; Zandkarimi, H.; Baisakh, N. Salt Adaptation Mechanisms of Halophytes: Improvement of Salt Tolerance in Crop Plants. In Elucidation of Abiotic Stress Signaling in Plants: Functional Genomics Perspectives; Pandey, G.K., Ed.; Springer: New York, NY, USA, 2015; Volume 2, pp. 243–279. ISBN 978-1-4939-2540-7. [Google Scholar]
  17. Kotula, L.; Caparros, P.G.; Zörb, C.; Colmer, T.D.; Flowers, T.J. Improving Crop Salt Tolerance Using Transgenic Approaches: An Update and Physiological Analysis. Plant Cell Environ. 2020, 43, 2932–2956. [Google Scholar] [CrossRef]
  18. Qin, H.; Li, Y.; Huang, R. Advances and Challenges in the Breeding of Salt-Tolerant Rice. Int. J. Mol. Sci. 2020, 21, 8385. [Google Scholar] [CrossRef]
  19. Egamberdieva, D.; Wirth, S.; Bellingrath-Kimura, S.D.; Mishra, J.; Arora, N.K. Salt-Tolerant Plant Growth Promoting Rhizobacteria for Enhancing Crop Productivity of Saline Soils. Front. Microbiol. 2019, 10, 2791. [Google Scholar] [CrossRef] [PubMed]
  20. Giannelli, G.; Potestio, S.; Visioli, G. The Contribution of PGPR in Salt Stress Tolerance in Crops: Unravelling the Molecular Mechanisms of Cross-Talk between Plant and Bacteria. Plants 2023, 12, 2197. [Google Scholar] [CrossRef]
  21. Gao, Y.; Zou, H.; Wang, B.; Yuan, F. Progress and Applications of Plant Growth-Promoting Bacteria in Salt Tolerance of Crops. Int. J. Mol. Sci. 2022, 23, 7036. [Google Scholar] [CrossRef]
  22. Ha-Tran, D.M.; Nguyen, T.T.M.; Hung, S.-H.; Huang, E.; Huang, C.-C. Roles of Plant Growth-Promoting Rhizobacteria (PGPR) in Stimulating Salinity Stress Defense in Plants: A Review. Int. J. Mol. Sci. 2021, 22, 3154. [Google Scholar] [CrossRef]
  23. Chinachanta, K.; Shutsrirung, A.; Santasup, C.; Pathom-Aree, W.; Luu, D.T.; Herrmann, L.; Lesueur, D.; Prom-u-thai, C. Rhizoactinobacteria Enhance Growth and Antioxidant Activity in Thai Jasmine Rice (Oryza sativa) KDML105 Seedlings under Salt Stress. Plants 2023, 12, 3441. [Google Scholar] [CrossRef] [PubMed]
  24. Kumar, A.; Singh, S.; Mukherjee, A.; Rastogi, R.P.; Verma, J.P. Salt-Tolerant Plant Growth-Promoting Bacillus pumilus Strain JPVS11 to Enhance Plant Growth Attributes of Rice and Improve Soil Health under Salinity Stress. Microbiol. Res. 2021, 242, 126616. [Google Scholar] [CrossRef]
  25. Chebotar, V.K.; Zaplatkin, A.N.; Chizhevskaya, E.P.; Gancheva, M.S.; Voshol, G.P.; Malfanova, N.V.; Baganova, M.E.; Khomyakov, Y.V.; Pishchik, V.N. Phytohormone Production by the Endophyte Bacillus safensis TS3 Increases Plant Yield and Alleviates Salt Stress. Plants 2023, 13, 75. [Google Scholar] [CrossRef]
  26. Cheng, Y.; Cheng, X.; Wei, K.; Wang, Y. Comparative Transcriptome Analysis of Salt-Tolerant and -Sensitive Soybean Cultivars under Salt Stress. Int. J. Mol. Sci. 2024, 25, 9818. [Google Scholar] [CrossRef]
  27. Lu, P.; Dai, S.-Y.; Yong, L.-T.; Zhou, B.-H.; Wang, N.; Dong, Y.-Y.; Liu, W.-C.; Wang, F.-W.; Yang, H.-Y.; Li, X.-W. A Soybean Sucrose Non-Fermenting Protein Kinase 1 Gene, GmSNF1, Positively Regulates Plant Response to Salt and Salt–Alkali Stress in Transgenic Plants. Int. J. Mol. Sci. 2023, 24, 12482. [Google Scholar] [CrossRef]
  28. Kim, I.-S. Current Perspectives on the Beneficial Effects of Soybean Isoflavones and Their Metabolites on Plants. Food Sci. Biotechnol. 2022, 31, 515–526. [Google Scholar] [CrossRef]
  29. Soliman, M.H.; Abdulmajeed, A.M.; Alhaithloul, H.; Alharbi, B.M.; El-Esawi, M.A.; Hasanuzzaman, M.; Elkelish, A. Saponin Biopriming Positively Stimulates Antioxidants Defense, Osmolytes Metabolism and Ionic Status to Confer Salt Stress Tolerance in Soybean. Acta Physiol. Plant. 2020, 42, 114. [Google Scholar] [CrossRef]
  30. Nagrale, D.T.; Chaurasia, A.; Kumar, S.; Gawande, S.P.; Hiremani, N.S.; Shankar, R.; Gokte-Narkhedkar, N.; Renu; Prasad, Y.G. PGPR: The Treasure of Multifarious Beneficial Microorganisms for Nutrient Mobilization, Pest Biocontrol and Plant Growth Promotion in Field Crops. World J. Microbiol. Biotechnol. 2023, 39, 100. [Google Scholar] [CrossRef] [PubMed]
  31. Wang, Y.; Zhao, D.; Li, Z.; Yang, Y.; Peng, Z.; Meng, C.; Ma, S.; Xu, K.; Li, Y.; Ding, G.; et al. Kaempferol Drives Genotype-Specific Microbiota Bacillaceae to Enhance Nitrogen Acquisition in Rapeseed. J. Adv. Res. 2025, in press. [CrossRef]
  32. Sharma, A.; Dev, K.; Sourirajan, A.; Choudhary, M. Isolation and Characterization of Salt-Tolerant Bacteria with Plant Growth-Promoting Activities from Saline Agricultural Fields of Haryana, India. J. Genet. Eng. Biotechnol. 2021, 19, 99. [Google Scholar] [CrossRef]
  33. Wang, Y.; Yang, Y.; Zhao, D.; Li, Z.; Sui, X.; Zhang, H.; Liu, J.; Li, Y.; Zhang, C.-S.; Zheng, Y. Ensifer Sp. GMS14 Enhances Soybean Salt Tolerance for Potential Application in Saline Soil Reclamation. J. Environ. Manag. 2024, 349, 119488. [Google Scholar] [CrossRef]
  34. Taliercio, E.; Gillenwater, J.; Woodruff, L.; Fallen, B. Glycine Soja, PI424025, Is a Valuable Genetic Resource to Improve Soybean Seed-Protein Content and Composition. PLoS ONE 2024, 19, e0310544. [Google Scholar] [CrossRef]
  35. Jin, W.; Zhou, H.; Zhao, H.; Pei, Y.; Su, F.; Li, Y.; Luo, T. Isolation, In Vitro Antioxidant Capacity, Hypoglycemic Activity and Immunoactivity Evaluation of Polysaccharides from Coriandrum sativum L. Antioxidants 2025, 14, 149. [Google Scholar] [CrossRef]
  36. Hsia, S.-Y.; Hsiao, Y.-H.; Li, W.-T.; Hsieh, J.-F. Aggregation of Soy Protein-Isoflavone Complexes and Gel Formation Induced by Glucono-δ-Lactone in Soymilk. Sci. Rep. 2016, 6, 35718. [Google Scholar] [CrossRef] [PubMed]
  37. Chen, S.; Zhou, Y.; Chen, Y.; Gu, J. Fastp: An Ultra-Fast All-in-One FASTQ Preprocessor. Bioinformatics 2018, 34, i884–i890. [Google Scholar] [CrossRef] [PubMed]
  38. Kim, D.; Paggi, J.M.; Park, C.; Bennett, C.; Salzberg, S.L. Graph-Based Genome Alignment and Genotyping with HISAT2 and HISAT-Genotype. Nat. Biotechnol. 2019, 37, 907–915. [Google Scholar] [CrossRef]
  39. Pertea, M.; Pertea, G.M.; Antonescu, C.M.; Chang, T.-C.; Mendell, J.T.; Salzberg, S.L. StringTie Enables Improved Reconstruction of a Transcriptome from RNA-Seq Reads. Nat. Biotechnol. 2015, 33, 290–295. [Google Scholar] [CrossRef]
  40. Li, B.; Dewey, C.N. RSEM: Accurate Transcript Quantification from RNA-Seq Data with or without a Reference Genome. BMC Bioinform. 2011, 12, 323. [Google Scholar] [CrossRef]
  41. Love, M.I.; Huber, W.; Anders, S. Moderated Estimation of Fold Change and Dispersion for RNA-Seq Data with DESeq2. Genome Biol. 2014, 15, 550. [Google Scholar] [CrossRef] [PubMed]
  42. Klopfenstein, D.V.; Zhang, L.; Pedersen, B.S.; Ramírez, F.; Warwick Vesztrocy, A.; Naldi, A.; Mungall, C.J.; Yunes, J.M.; Botvinnik, O.; Weigel, M.; et al. GOATOOLS: A Python Library for Gene Ontology Analyses. Sci. Rep. 2018, 8, 10872. [Google Scholar] [CrossRef]
  43. Virtanen, P.; Gommers, R.; Oliphant, T.E.; Haberland, M.; Reddy, T.; Cournapeau, D.; Burovski, E.; Peterson, P.; Weckesser, W.; Bright, J.; et al. SciPy 1.0: Fundamental Algorithms for Scientific Computing in Python. Nat. Methods 2020, 17, 261–272. [Google Scholar] [CrossRef] [PubMed]
  44. Hasanuzzaman, M.; Bhuyan, M.H.M.B.; Zulfiqar, F.; Raza, A.; Mohsin, S.M.; Mahmud, J.A.; Fujita, M.; Fotopoulos, V. Reactive Oxygen Species and Antioxidant Defense in Plants under Abiotic Stress: Revisiting the Crucial Role of a Universal Defense Regulator. Antioxidants 2020, 9, 681. [Google Scholar] [CrossRef]
  45. Mittler, R.; Zandalinas, S.I.; Fichman, Y.; Van Breusegem, F. Reactive Oxygen Species Signalling in Plant Stress Responses. Nat. Rev. Mol. Cell Biol. 2022, 23, 663–679. [Google Scholar] [CrossRef]
  46. Feng, L.; Li, Q.; Zhou, D.; Jia, M.; Liu, Z.; Hou, Z.; Ren, Q.; Ji, S.; Sang, S.; Lu, S.; et al. B. subtilis CNBG-PGPR-1 Induces Methionine to Regulate Ethylene Pathway and ROS Scavenging for Improving Salt Tolerance of Tomato. Plant J. 2023, 117, 193–211. [Google Scholar] [CrossRef]
  47. Singh, A.; Panwar, R.; Mittal, P.; Hassan, M.I.; Singh, I.K. Plant Cytochrome P450s: Role in Stress Tolerance and Potential Applications for Human Welfare. Int. J. Biol. Macromol. 2021, 184, 874–886. [Google Scholar] [CrossRef]
  48. Kidwai, M.; Ahmad, I.Z.; Chakrabarty, D. Class III Peroxidase: An Indispensable Enzyme for Biotic/Abiotic Stress Tolerance and a Potent Candidate for Crop Improvement. Plant Cell Rep. 2020, 39, 1381–1393. [Google Scholar] [CrossRef]
  49. Wang, P.; Liu, W.-C.; Han, C.; Wang, S.; Bai, M.-Y.; Song, C.-P. Reactive Oxygen Species: Multidimensional Regulators of Plant Adaptation to Abiotic Stress and Development. J. Integr. Plant Biol. 2024, 66, 330–367. [Google Scholar] [CrossRef] [PubMed]
  50. Su, P.; Yan, J.; Li, W.; Wang, L.; Zhao, J.; Ma, X.; Li, A.; Wang, H.; Kong, L. A Member of Wheat Class III Peroxidase Gene Family, TaPRX-2A, Enhanced the Tolerance of Salt Stress. BMC Plant Biol. 2020, 20, 392. [Google Scholar] [CrossRef] [PubMed]
  51. Hasanuzzaman, M.; Fujita, M. Plant Responses and Tolerance to Salt Stress: Physiological and Molecular Interventions. Int. J. Mol. Sci. 2022, 23, 4810. [Google Scholar] [CrossRef]
  52. Liang, M.; Hu, F.; Xie, D.; Chen, Z.; Zheng, Q.; Xie, Q.; Zheng, F.; Liu, D.; Jian, S.; Chen, H.; et al. Physiological Measurements and Transcriptome Survey Reveal How Semi-Mangrove Clerodendrum Inerme Tolerates Saline Adversity. Front Plant Sci. 2022, 13, 882884. [Google Scholar] [CrossRef]
  53. Patel, M.K.; Kumar, M.; Li, W.; Luo, Y.; Burritt, D.J.; Alkan, N.; Tran, L.-S.P. Enhancing Salt Tolerance of Plants: From Metabolic Reprogramming to Exogenous Chemical Treatments and Molecular Approaches. Cells 2020, 9, 2492. [Google Scholar] [CrossRef]
  54. Cui, J.; Zhang, E.; Zhang, X.; Wang, Q. Silicon Alleviates Salinity Stress in Licorice (Glycyrrhiza uralensis) by Regulating Carbon and Nitrogen Metabolism. Sci. Rep. 2021, 11, 1115. [Google Scholar] [CrossRef]
  55. Zhang, X.; He, P.; Guo, R.; Huang, K.; Huang, X. Effects of Salt Stress on Root Morphology, Carbon and Nitrogen Metabolism, and Yield of Tartary Buckwheat. Sci. Rep. 2023, 13, 12483. [Google Scholar] [CrossRef]
  56. AbuQamar, S.F.; El-Saadony, M.T.; Saad, A.M.; Desoky, E.-S.M.; Elrys, A.S.; El-Mageed, T.A.A.; Semida, W.M.; Abdelkhalik, A.; Mosa, W.F.A.; Al Kafaas, S.S.; et al. Halotolerant Plant Growth-Promoting Rhizobacteria Improve Soil Fertility and Plant Salinity Tolerance for Sustainable Agriculture—A Review. Plant Stress 2024, 12, 100482. [Google Scholar] [CrossRef]
  57. Alishahi, F.; Alikhani, H.A.; Khoshkholgh-Sima, N.A.; Etesami, H. Mining the Roots of Various Species of the Halophyte Suaeda for Halotolerant Nitrogen-Fixing Endophytic Bacteria with the Potential for Promoting Plant Growth. Int. Microbiol. 2020, 23, 415–427. [Google Scholar] [CrossRef] [PubMed]
  58. El Moukhtari, A.; Cabassa-Hourton, C.; Farissi, M.; Savouré, A. How Does Proline Treatment Promote Salt Stress Tolerance During Crop Plant Development? Front. Plant Sci. 2020, 11, 1127. [Google Scholar] [CrossRef] [PubMed]
  59. Yan, L.; Lu, M.; Riaz, M.; Tong, K.; Yu, H.; Gao, G.; Niu, Y. Exogenous Proline Enhances Salt Acclimation in Soybean Seedlings: Modifying Physicochemical Properties and Controlling Proline Metabolism through the Ornithine-Glutamate Dual Pathway. Ecotoxicol. Environ. Saf. 2025, 294, 118012. [Google Scholar] [CrossRef] [PubMed]
  60. Lu, C.; Li, L.; Liu, X.; Chen, M.; Wan, S.; Li, G. Salt Stress Inhibits Photosynthesis and Destroys Chloroplast Structure by Downregulating Chloroplast Development–Related Genes in Robinia Pseudoacacia Seedlings. Plants 2023, 12, 1283. [Google Scholar] [CrossRef]
  61. Zahra, N.; Al Hinai, M.S.; Hafeez, M.B.; Rehman, A.; Wahid, A.; Siddique, K.H.M.; Farooq, M. Regulation of Photosynthesis under Salt Stress and Associated Tolerance Mechanisms. Plant Physiol. Biochem. 2022, 178, 55–69. [Google Scholar] [CrossRef]
  62. Shahid, M.; Al-Khattaf, F.S.; Danish, M.; Zeyad, M.T.; Atef Hatamleh, A.; Mohamed, A.; Ali, S. PGPR Kosakonia Radicincitans KR-17 Increases the Salt Tolerance of Radish by Regulating Ion-Homeostasis, Photosynthetic Molecules, Redox Potential, and Stressor Metabolites. Front Plant Sci. 2022, 13, 919696. [Google Scholar] [CrossRef]
  63. Sevgi, B.; Leblebici, S. Exogenous Sucrose Alleviates Salt Stress in Sunflower (Helianthus annuus L.) and Canola (Brassica napus L.) by Modulating Osmotic Adjustment and Antioxidant Defense System. Physiol. Mol. Biol. Plants 2025, 31, 405–418. [Google Scholar] [CrossRef]
  64. El-Saadony, M.T.; Saad, A.M.; Mohammed, D.M.; Fahmy, M.A.; Elesawi, I.E.; Ahmed, A.E.; Algopishi, U.B.; Elrys, A.S.; Desoky, E.-S.M.; Mosa, W.F.A.; et al. Drought-Tolerant Plant Growth-Promoting Rhizobacteria Alleviate Drought Stress and Enhance Soil Health for Sustainable Agriculture: A Comprehensive Review. Plant Stress 2024, 14, 100632. [Google Scholar] [CrossRef]
  65. Iqbal, M.Z.; Singh, K.; Chandra, R. Recent Advances of Plant Growth Promoting Rhizobacteria (PGPR) for Eco-Restoration of Polluted Soil. Clean. Eng. Technol. 2024, 23, 100845. [Google Scholar] [CrossRef]
  66. Chen, Y.; Zhou, X. Symbiotic Nitrogen Fixation: The Role of Rhizobia in Enhancing Legume Growth and Soil Fertility. Mol. Microbiol. Res. 2024, 14, 109–118. [Google Scholar] [CrossRef]
  67. Ni, H.; Hou, X.; Tian, S.; Liu, C.; Zhang, G.; Peng, Y.; Chen, L.; Wang, J.; Chen, Q.; Xin, D. Insights into the Early Steps of the Symbiotic Interaction between Soybean (Glycine max) and Sinorhizobium fredii Symbiosis Using Transcriptome, Small RNA, and Degradome Sequencing. J. Agric. Food Chem. 2024, 72, 17084–17098. [Google Scholar] [CrossRef]
  68. Han, Q.; Zhu, G.; Qiu, H.; Li, M.; Zhang, J.; Wu, X.; Xiao, R.; Zhang, Y.; Yang, W.; Tian, B.; et al. Quality Traits Drive the Enrichment of Massilia in the Rhizosphere to Improve Soybean Oil Content. Microbiome 2024, 12, 224. [Google Scholar] [CrossRef]
  69. Akhtar, K.; ul Ain, N.; Prasad, P.V.V.; Naz, M.; Aslam, M.M.; Djalovic, I.; Riaz, M.; Ahmad, S.; Varshney, R.K.; He, B.; et al. Physiological, Molecular, and Environmental Insights into Plant Nitrogen Uptake, and Metabolism under Abiotic Stresses. Plant Genome 2024, 17, e20461. [Google Scholar] [CrossRef] [PubMed]
  70. Meng, F.; Feng, N.; Zheng, D.; Liu, M.; Zhang, R.; Huang, X.; Huang, A.; Chen, Z. Exogenous Hemin Alleviates NaCl Stress by Promoting Photosynthesis and Carbon Metabolism in Rice Seedlings. Sci. Rep. 2023, 13, 3497. [Google Scholar] [CrossRef]
  71. Trush, K.; Pal’ove-Balang, P. Biosynthesis and Role of Isoflavonoids in Legumes under Different Environmental Conditions. Plant Stress 2023, 8, 100153. [Google Scholar] [CrossRef]
  72. Wang, Y.; Liu, W.; Li, W.; Wang, C.; Dai, H.; Xu, R.; Zhang, Y.; Zhang, L. Integrative Analysis of Metabolome and Transcriptome Reveals Regulatory Mechanisms of Flavonoid Biosynthesis in Soybean under Salt Stress. Front. Plant Sci. 2024, 15, 1415867. [Google Scholar] [CrossRef] [PubMed]
Agronomy 15 02103 g001
Figure 1. Effects of B. velezensis 41S2 on soybean growth phenotype under salt–alkaline soil. (a) Growth phenotypes. (b) Plant height. (c) Shoot dry weight. (d) Root dry weight. (e) Total number of nodules per plant. (f) Root nodule weight per plant. Soybean seedlings are grown in salt–alkaline soil without (CK) or with strain 41S2 (41S2). Values are means ± SD, n ≥ 12 independent biological replicates. Asterisks indicate significant differences between CK and 41S2 treatments: * p < 0.05 and ** p < 0.01 by two-tailed Student’s t-test.
Figure 1. Effects of B. velezensis 41S2 on soybean growth phenotype under salt–alkaline soil. (a) Growth phenotypes. (b) Plant height. (c) Shoot dry weight. (d) Root dry weight. (e) Total number of nodules per plant. (f) Root nodule weight per plant. Soybean seedlings are grown in salt–alkaline soil without (CK) or with strain 41S2 (41S2). Values are means ± SD, n ≥ 12 independent biological replicates. Asterisks indicate significant differences between CK and 41S2 treatments: * p < 0.05 and ** p < 0.01 by two-tailed Student’s t-test.
Agronomy 15 02103 g001
Agronomy 15 02103 g002
Figure 2. B. velezensis 41S2 maintains ROS homeostasis under salt–alkaline soil. (a) Volcano plot showing the DEGs under salt–alkaline soil (41S2 vs. CK). Red: up-regulated genes; blue: down-regulated genes. (b) GO annotation analysis; CC: cellular component, MF: Molecular function, BP: biological process. (c) GO enrichment analysis. (d) Expression patterns of POX and CYP genes. (e) H2O2 concentration. Soybean seedlings are grown in salt–alkaline soil without (CK) or with 41S2 (41S2). Values are means ± SD, with three independent biological replicates. Asterisks indicate significant differences between CK and 41S2 treatments: * p < 0.05 by two-tailed Student’s t-test.
Figure 2. B. velezensis 41S2 maintains ROS homeostasis under salt–alkaline soil. (a) Volcano plot showing the DEGs under salt–alkaline soil (41S2 vs. CK). Red: up-regulated genes; blue: down-regulated genes. (b) GO annotation analysis; CC: cellular component, MF: Molecular function, BP: biological process. (c) GO enrichment analysis. (d) Expression patterns of POX and CYP genes. (e) H2O2 concentration. Soybean seedlings are grown in salt–alkaline soil without (CK) or with 41S2 (41S2). Values are means ± SD, with three independent biological replicates. Asterisks indicate significant differences between CK and 41S2 treatments: * p < 0.05 by two-tailed Student’s t-test.
Agronomy 15 02103 g002
Agronomy 15 02103 g003
Figure 3. B. velezensis 41S2 enhances plant N and C metabolism. (a) KEGG enrichment analysis. (b) N metabolism. GDH: Glutamate dehydrogenase. GS: Glutamine synthetase. (c) Starch and sucrose metabolism. INV: Invertase. BGL: β-glucosidase.
Figure 3. B. velezensis 41S2 enhances plant N and C metabolism. (a) KEGG enrichment analysis. (b) N metabolism. GDH: Glutamate dehydrogenase. GS: Glutamine synthetase. (c) Starch and sucrose metabolism. INV: Invertase. BGL: β-glucosidase.
Agronomy 15 02103 g003
Agronomy 15 02103 g004
Figure 4. B. velezensis 41S2 improves soybean seed yield. (a) Comparison of soybean yield per plant in salt–alkaline soil. (b) Total number of soybean pods per plant. (c) Seed yield. Values are means ± SD with six independent biological replicates. Asterisks indicate significant differences between CK and 41S2 treatments: *** p < 0.001 by two-tailed Student’s t-test.
Figure 4. B. velezensis 41S2 improves soybean seed yield. (a) Comparison of soybean yield per plant in salt–alkaline soil. (b) Total number of soybean pods per plant. (c) Seed yield. Values are means ± SD with six independent biological replicates. Asterisks indicate significant differences between CK and 41S2 treatments: *** p < 0.001 by two-tailed Student’s t-test.
Agronomy 15 02103 g004
Agronomy 15 02103 g005
Figure 5. B. velezensis 41S2 improves seed quality under salt–alkaline soil. (a,b) C and N accumulation per plant. (c) Soluble protein content per plant. (d) Isoflavone content per plant. Values are means ± SD, with six independent biological replicates. Asterisks indicate significant differences between CK and 41S2 treatments: ** p < 0.01 and *** p < 0.001 by two-tailed Student’s t-test.
Figure 5. B. velezensis 41S2 improves seed quality under salt–alkaline soil. (a,b) C and N accumulation per plant. (c) Soluble protein content per plant. (d) Isoflavone content per plant. Values are means ± SD, with six independent biological replicates. Asterisks indicate significant differences between CK and 41S2 treatments: ** p < 0.01 and *** p < 0.001 by two-tailed Student’s t-test.
Agronomy 15 02103 g005
Agronomy 15 02103 g006
Figure 6. Schematic illustration depicting the role of B. velezensis 41S2 in promoting soybean production in salt–alkaline soil. The schematic diagram was created in BioRender. Wang Y.Q. (2025) https://app.biorender.com/illustrations/68b12ff5000b247d39abbe7f?slideId=c4abcc64-7681-4fbb-afda-db6586140d0e, accessed on 24 August 2025.
Figure 6. Schematic illustration depicting the role of B. velezensis 41S2 in promoting soybean production in salt–alkaline soil. The schematic diagram was created in BioRender. Wang Y.Q. (2025) https://app.biorender.com/illustrations/68b12ff5000b247d39abbe7f?slideId=c4abcc64-7681-4fbb-afda-db6586140d0e, accessed on 24 August 2025.
Agronomy 15 02103 g006
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Zheng, H.; Hua, S.; Li, Z.; Wang, Z.; Zhao, D.; Jing, C.; Li, Y.; Zhang, C.; Zheng, Y.; Wang, Y.; et al. A Plant Growth-Promoting Bacterial Isolate, Bacillus velezensis 41S2, Enhances Seed Protein, Isoflavone Accumulation, and Stress Resilience in Soybean Under Salt–Alkaline Soil Conditions. Agronomy 2025, 15, 2103. https://doi.org/10.3390/agronomy15092103

AMA Style

Zheng H, Hua S, Li Z, Wang Z, Zhao D, Jing C, Li Y, Zhang C, Zheng Y, Wang Y, et al. A Plant Growth-Promoting Bacterial Isolate, Bacillus velezensis 41S2, Enhances Seed Protein, Isoflavone Accumulation, and Stress Resilience in Soybean Under Salt–Alkaline Soil Conditions. Agronomy. 2025; 15(9):2103. https://doi.org/10.3390/agronomy15092103

Chicago/Turabian Style

Zheng, Han, Shutian Hua, Zhe Li, Ziyan Wang, Donglin Zhao, Changliang Jing, Yiqiang Li, Chengsheng Zhang, Yanfen Zheng, Youqiang Wang, and et al. 2025. "A Plant Growth-Promoting Bacterial Isolate, Bacillus velezensis 41S2, Enhances Seed Protein, Isoflavone Accumulation, and Stress Resilience in Soybean Under Salt–Alkaline Soil Conditions" Agronomy 15, no. 9: 2103. https://doi.org/10.3390/agronomy15092103

APA Style

Zheng, H., Hua, S., Li, Z., Wang, Z., Zhao, D., Jing, C., Li, Y., Zhang, C., Zheng, Y., Wang, Y., & Jiang, M. (2025). A Plant Growth-Promoting Bacterial Isolate, Bacillus velezensis 41S2, Enhances Seed Protein, Isoflavone Accumulation, and Stress Resilience in Soybean Under Salt–Alkaline Soil Conditions. Agronomy, 15(9), 2103. https://doi.org/10.3390/agronomy15092103

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop