Ion Homeostasis, Osmotic Adjustment, and ROS Detoxification Underlie Pea Salinity Tolerance Induced by Pseudomonas putida RT12

Abstract

In saline soil, legumes are restricted in their growth potential by osmotic stress, ion toxicity, and oxidative damage. We evaluated five halotolerant plant growth-promoting bacteria and selected Pseudomonas putida RT12 for its exceptional EPS production, tolerance to 600 mM NaCl, strong biofilm development, and plant growth-promoting traits (ACC-deaminase 2.86 µM·mg⁻¹; IAA 144 µM·mL⁻¹). RT12 was evaluated on two varieties of peas (peas2009 and 9800-10) with and without inoculation at 0, 75, and 150 mM NaCl concentrations. RT12 markedly protected growth under severe salinity: at 150 mM, shoot length rose to 23.13 cm (peas2009) and 17.44 cm (9800-10), in contrast to 11.18 cm and 12.32 cm in uninoculated specimens; root length and dry weight demonstrated comparable recovery (root length increased from 11.00 to 22.25 cm; dry weight of peas2009 from 0.15 to 0.17 and 0.41 to 0.71 g). RT12 sustained photosynthesis (total chlorophyll increased from 43.5 to 54.5), enhanced relative water content (to 94.1% and 97.2%), elevated osmolytes (total soluble proteins rose from 7.34 to 18.12 µg·g⁻¹ FW; total soluble sugars increased from 19.1 to 41.3 mg·g⁻¹ FW), and augmented antioxidant activities (catalase increased from 2.11 to 4.70; superoxide dismutase rose from 1.20 to 4.83; peroxidase increased from 0.08 to 0.18), while reducing malondialdehyde/hydrogen peroxide levels. RT12 was significant as it inhibited the accumulation of Na⁺ (from 23.95 to 16.32 mg·g⁻¹ DW), elevated K⁺ levels (from 17.76 to 29.12 mg·g⁻¹ DW), and restored the K⁺/Na⁺ ratio to normal (from 0.74 to 1.59) in inoculated plants compared to non-inoculated ones. A multivariate analysis linked growth protection to ionic homeostasis, osmotic control, and the detoxification of reactive oxygen species (ROS). RT12 is a promising bioinoculant for cultivating peas in saline-affected soils.

1. Introduction

Peas (Pisum sativum L.) are an important staple crop because of their high nutritional content and consumption globally [1]. It is an important legume vegetable that contains dietary fiber, carbs, proteins, and vital micronutrients [2]. Increased attention is being directed towards pea production for several reasons. This encompasses a growing global population, heightened awareness of the health advantages of peas, and a rise in farmers adopting environmentally sustainable agricultural practices [3]. Peas are globally farmed as a cool-season leguminous grain. The primary producers are China, Canada, France, Australia, India, and the United States [4]. Legumes, such as peas, hold significance beyond their nutritional benefits. They also contribute to sustainable agriculture by fixing atmospheric nitrogen [5]. Minimum tillage practices and the rotation of legumes such as peas can mitigate erosion, enhance organic matter, rebuild soil structure, and provide subsequent crops with increased nitrogen [6,7].

However, abiotic stresses provide a significant danger to the horticultural yield of plants like peas. These encompass factors such as salinity, drought, and extreme temperatures [8]. Moreover, climate change and global warming are intensifying the occurrence, frequency, and severity of abiotic stresses [9]. This endangers the viability and productivity of peas. Salinity is a crucial abiotic factor that might negatively impact pea cultivation. They reduce agricultural yield, especially in regions that receive rainfall and irrigation. Among abiotic stresses salinity, for instance, arises from the increasing utilization of poor-quality water for irrigation and soil salinization, adversely affecting crops globally [10]. Excessive salt in the soil induces osmotic stress, hindering plants’ ability to absorb water. It disrupts ion homeostasis, rendering plants toxic and inhibiting their growth [11,12]. Salinity stress is a significant environmental challenge for plants, disrupting numerous physiological, biochemical, and structural processes, hence impeding growth and productivity. Excessive soil salinity significantly alters the morphology, functionality, and chemical composition of peas [13,14]. Elevated salt concentrations induce osmotic stress, limiting water absorption in plants and diminishing turgor pressure. This impedes the development of roots and shoots, the accumulation of biomass, and the proliferation of leaves [15,16]. Reduced leaf area results in diminished photosynthesis, exacerbated by stomatal closure and inadequate aquaporin regulation. This results in decreased transpiration and carbon assimilation [17,18].

Salinity also alters the equilibrium of nutrients and water, the metabolism of proteins and lipids, and the accessibility of micronutrients, including essential ones such as zinc [19,20]. Higher salt concentrations also compromises the cell wall, altering the rigidity and pliability of the tissue [21]. Elevated salt concentrations in germinating seeds inhibit water absorption, retard germination, and impede seedling development [22]. Elevated salt concentrations induce oxidative stress, leading to an accumulation of reactive oxygen species (ROS) that can damage cellular membranes, proteins, and DNA [1]. Plants react by enhancing the activity of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR). They synthesize osmolytes such as proline and glycine betaine to maintain osmotic equilibrium and safeguard cellular structures [23,24]. Salinity alters hormonal signaling, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) activity, and the expression of genes and transcription factors that regulate ion transport and stress responses [25]. Due to ion toxicity (the substitution of Na⁺ for K⁺ and the conformational changes in proteins caused by Na⁺ and Cl⁻), osmotic stress (the excessive buildup of sodium in cell walls resulting in osmotic stress and cell death), and nutrient deficiencies (N, Ca, K, P, Fe, Zn), salt-sensitive plants cannot survive or grow poorly in saline environments in addition to reduced soil water absorption [26].

Finding affordable methods to reduce salinity and maintain crop productivity is crucial in developing nations like Pakistan [27]. Through the formation of biofilms, halophilic enzymes, exopolysaccharide (EPS), and appropriate solutes, plant growth-promoting bacteria (PGPB) that can thrive in saltier environments aid plants in becoming more salinity-resistant [28]. In addition to fixing nitrogen, these bacteria also dissolve nutrients and produce indole-3-acetic acid (IAA), 1-aminocyclopropane-1-carboxylate (ACC) deaminase, and siderophores, among other plant growth-promoting (PGP) characteristics. When combined, these characteristics aid in the rhizosphere growth of plants [29,30]. Biofilms, predominantly composed of EPS, protect bacterial cells against salinity, desiccation, pH fluctuations, and many stressors [31,32]. The EPS matrix, comprising polysaccharides, lipids, and functional groups such as hydroxyl, amino, and carboxyl, is crucial for binding Na⁺ and maintaining root hydration, hence enhancing the plant’s salt tolerance [33,34,35,36]. Plant growth promoting rhizobacteria (PGPR) that establish biofilms facilitate nutrient cycling, mitigate plant diseases, and enhance soil health and agricultural productivity [37,38]. Because of their adaptability and competitive advantage in local soils, indigenous biofilm-forming PGPR perform better in field conditions than non-biofilm-forming strains [39,40]. As a result, these strains with many functions are thought to be viable inoculants for sustainable agricultural production. However, only a small number of biofilm-forming PGPR have been shown to improve the growth of crops in saline conditions, including maize, rice, wheat, cucumbers, legumes, and rice [40,41]. Alcaligenes sp. AF7, Azotobacter chroococcum C5, Bacillus pumilus JPVS11, Burkholderia cepacia, Enterobacter cloacae, Jeotgalicoccus huakuii, Klebsiella variicola SURYA6, Kocuria rhizophila 14asp, and Pseudomonas putida are among the PGPR that can produce EPS and survive in salty conditions. These organisms have demonstrated potential in promoting plant growth in various crops under salt stress [1,42,43,44,45].

We propose that a halotolerant PGPR strain exhibiting multifunctional plant growth-promoting characteristics, especially its capacity to produce resilient and dense biofilms, can deliver consistent and dependable advantages in saline environments. As the strain showed previously promising results in Brassica juncea [46], we believe that it will improve plant growth attributes and alleviate the negative effects of salt in pea plants. To assess this hypothesis, we employed Pseudomonas putida as an inoculum in pea plants grown in a controlled pot-soil setting. This study assesses its function in alleviating growth suppression due to salinity and investigates the regulation of physiological processes, antioxidant defence mechanisms, and overall plant performance resulting from inoculation. Our research offers new insights into the potential of P. putida as a bioinoculant to improve pea resilience against salt stress through evaluations of growth, metabolism, and stress responses.

2. Materials and Methods

2.1. Preliminary Screening of Acquired Strains for EPS Production

We received five halotolerant bacterial strains (RT1, RT2, RT12, RT13, and RT14) previously isolated from the rhizosphere and rhizosphere of cotton plants [47]. Among these, RT12, identified as Pseudomonas putida, exhibited the highest EPS production and salinity tolerance. The isolates originated from the rhizosphere of cotton plants [47]. LB broth supplemented with NaCl concentrations ranging from 0 to 600 mM was used to culture the bacteria, which were then incubated at 32 ± 2 °C with 180 rpm agitation to assess their ability to synthesize exopolysaccharides (EPS) under salt stress. The cultures were spun for 20 min at 7000 rpm after incubation (Biobase-BJPX-H50IV), and cold ethanol (1:3 v/v) was added to create EPS, which was then kept overnight at 4 °C. Following a 24 h drying process at 58 °C, the EPS pellets were weighed. For subsequent research, we selected strains with mucoid colony morphologies and higher EPS production in saline circumstances.

2.2. Quantification of EPS Production Under Salinity

The selected strains were cultured in nutrient broth supplemented with NaCl (0–600 mM) and incubated at 30 °C for 48 h at 160 rpm for precise measurement. Chilled ethanol (3:1 v/v) was employed to precipitate EPS and was maintained at 4 °C for 24 h. The cultures were centrifuged at 15,000 rpm for 20 min, and the EPS pellets were desiccated at 58 °C for 24 h prior to weighing [35,48].

2.3. Assessment of Salt Tolerance, Flocculation, and Sodium Absorption in Halotolerant Strains

We further evaluated flocculation, growth, and Na⁺ absorption to examine the effects of EPS on bacterial adaptation to salinity. The strains were cultivated in tryptic soy broth containing 0, 200, 400, and 600 mM NaCl. After 24 h of incubation, the optical density at 600 nm (Agilent 8453 UV-visible Spectroscopy System, Guangzhou China), was assessed to monitor growth and salt tolerance [49]. Flocculation was assessed by filtering cultures through Whatman No. 1 paper, thereafter drying at 60 °C, and recording the dry weight [50]. Bacterial pellets were rinsed with sterile distilled water, digested overnight in 0.1 N HCl, and subsequently analysed for salt uptake using a flame photometer [51].

2.4. Screening of the Strains for Additional PGP Traits

We conducted standard assays for indole-3-acetic acid (IAA) production, ACC deaminase activity, phosphate solubilization, siderophore production, and enzyme activities (including protease, cellulase, amylase, and catalase) to ascertain that the selected strains possess the potential for broader plant growth-promoting applications. We employed conventional methodologies of [52,53,54,55,56,57,58].

2.5. Morphological and Molecular Profiling of the Strain

The morphological and biochemical characterization of the identified strains was performed using the Qts-24 microbial identification kit from DEStO laboratory in Karachi.

We isolated bacterial genomic DNA for molecular characterization by adhering to the technique detailed by Ahmed et al. [59]. The 16S rRNA gene was amplified using the universal primers 27F (5′-AACTGAAGAGTTTGATCCTGGCTC-3′) and 1492R (5′-TACGGTTACCTTGTTACGACTT-3′) from the isolated DNA. The initial phase of thermal cycling involved denaturation at 96 °C for 5 min. This was succeeded by 30 cycles at 96 °C for 1 min each, 56 °C for 1 min for primer annealing, and 72 °C for 1 min for extension. The final stage of the extension was conducted at 72 °C for 10 min, after which the reaction was cooled to 4 °C. We employed the Genome-to-Genome Distance Calculator (GGDC) 2.1 to analyze the obtained sequence.

2.6. Soil Processing, Seed Disinfection, and Microbial Inoculation

We obtained topsoil from Swat University in Charbagh, Pakistan (34.85° N, 72.45° E), sieved it, allowed it to air dry, and subsequently autoclaved it at 121 °C for 20 min. The ICARDA handbook and other established methodologies were employed to examine the soil’s physicochemical characteristics, including organic matter, pH, electrical conductivity, and nutrient composition [60,61,62], with results presented in Table S1.

The National Agriculture Research Centre (NARC) in Islamabad, Pakistan, supplied two varieties of peas: “peas2009” and “9800-10”. The specimens were surface sterilized using 0.1% HgCl₂ and 10% ethanol, followed by rinsing with distilled water [1]. We cultured P. putida RT12 in LB medium, subsequently centrifuged it, and resuspended it in double-distilled water to achieve an OD₆₀₀ of 1.0. Uniform seeds were immersed in the bacterial suspension for four hours, whereas control seeds were subjected to pure water treatment [1]. Five treatments were administered in triplicate, both with and without the inclusion of P. putida RT12 (Figure 1). Eight uniform seeds were sown in 1 kg of autoclaved soil contained within pots measuring 14 × 18 cm. The seeds were sown at a depth of 2 cm [1]. Following a period of two weeks, the seedlings were reduced to five per pot and irrigated daily with 50 mL of water. Administering 50 mM NaCl every three days induced salinity stress until the ultimate concentrations of 75 mM and 150 mM were attained.

2.7. Crop Experiments

Following 40 days of salt exposure, plants were taken out for physiological and morphological analysis. Distilled water was used to wash the roots in order to remove rhizosphere dirt. Plant samples for biochemical and physiological assays were either frozen at −80 °C or dried in an oven. Relative water content (RWC), fresh weight (FW), dry weight (DW), root length (RL), and shoot length (SL) were measured using established protocols as previously described in our experiments [1,27]. The DW was determined after oven-drying, while the FW was noted right after harvest. The RL and SL were measured in centimeters using a laboratory scale.

2.8. P. putida Enhances Morphophysiological Attributes of Pea Under Salinity

2.8.1. Assessing Photosynthetic Pigment Analysis and Relative Water Contents

Prior to the plants being harvested down, the priorly described protocol was used to measure the amount of chlorophyll in the leaves [63]. Following an 8-week period of salt stress, following equations were employed to determine the concentration of photosynthetic pigments in DMSO at various wavelengths: 480, 649, and 665 nm.

Where

Ch-a = 12.47A665.1 − 3.62A649.1

Ch-b = 25.06A649.1 − 6.5A665.1

Total Chlorophyll = (8.2 × A665) + (20.2 × A665)

Carotenoid = (1000A480 − 1.29Ca − 53.78Cb)/220

The relative water content (RWC) was calculated using the methodology outlined by El Nahhas et al. [64].

2.8.2. Nutrient Analysis of Peas Under Salinity and RT12 Inoculation

We employed atomic absorption spectroscopy (AAS) to quantify the mineral content, including sodium (Na⁺), calcium (Ca²⁺), magnesium (Mg²⁺), and potassium (K⁺). We accomplished this by combining nitric acid with perchloric acid in a 3:1 ratio as previously described [1].

2.8.3. Analysis of Plant Osmoprotectants

The quantification of osmoprotectants in the shoot tissues of wheat plants exposed to salt stress was performed. The proline concentration was evaluated utilizing the method of [65]. The total soluble sugars (TSS) in the plants subjected to salt stress were also quantified by following the previously described protocol [66]. Five milliliters of 80% ethanol were employed to homogenize 0.1 grammes of fresh leaf tissue, which was thereafter centrifuged at 10,000 rpm for 10 min. We combined the supernatant with 3 mL of anthrone reagent, subjected it to boiling for 12 min, allowed it to cool to ambient temperature, and subsequently incubated it at 25 °C for 20 min. We quantified absorbance at 625 nm and utilized a glucose standard curve to determine the total soluble solids (TSS) concentration, thereafter expressed as g g⁻¹ fresh weight (FW).

The total soluble protein (TSP) assay method used bovine serum albumin (BSA) as the standard and adhered to the Mendez and Kwon [67] protocol. utilizing Bovine Serum Albumin (BSA) as the standard. Fresh leaves (0.1 g) were homogenized in 1 mL of phosphate buffer (pH 7.5) and centrifuged at 3000 rpm for 10 min. Protein concentrations were determined by assessing absorbance at 650 nm.

2.8.4. Plant Antioxidants Analysis

The method of Beauchamp and Fridovich [68] was followed to determine SOD assay. Catalase (CAT) activity was estimated by following the protocol of Luck [69]. For POD analysis with some modifications, the protocol previously described by Reddy et al. [70], was followed.

2.8.5. Evaluation of Oxidative Burst

The MDA (malondialdehyde) level was evaluated to determine the extent of lipid peroxidation using the previously published thiobarbituric technique [27]. The extinction coefficient of MDA, which is 155 mM cm, was used to compute the concentration. To ascertain endogenous H₂O₂ content, the protocol from Li et al. [71] was followed.

2.9. Statistical Analysis

The data was statistically analyzed utilizing STATIX version 8.1. A two-way ANOVA was conducted with Fisher’s LSD method to assess the significance level (p < 0.05) among the means. For comparison of the morphological attributes with biochemical and physiological parameters through PCA, Mantel test and RDA we used R software (version 4.1.1, R Core Team, 2013).

3. Results

3.1. Initial Screening of Strains for Halotolerance and EPS Production

The five acquired strains were evaluated under saline conditions. When evaluated in saline conditions, RT12 generated the highest number of EPS (Figure 2). With increasing NaCl concentrations, RT12’s EPS production grew gradually; the control had the lowest amount (0.022 mg·mL⁻¹), while 10 mg·mL⁻¹ NaCl had the maximum (0.049 mg·mL⁻¹). Growth curve analysis revealed that all strains suffered from increased NaCl levels in comparison to the control (Figure S1). Despite the decrease in this figure, RT12 consistently improved with saline treatments, indicating superior salt tolerance (Figure 2). These data indicate that RT12 not only produces greater quantities of EPS but also continues to proliferate despite the presence of salt stress. This renders it the most promising strain among those examined.

3.2. Bacterial Survival, Flocculation and Sodium Uptake Under Saline Regime

The RT12 strain can thrive in salty water with NaCl levels as high as 600 mM (Figure 3A). As salinity goes up, the flocculation of bacteria in RT12 goes up too, reaching a high of 91.7% at 600 mM NaCl compared to the control (Figure 3B). When the amount of NaCl increases, the amount of Na⁺ that is absorbed rises up as well. Bacterial absorption of Na⁺ increases to 19% at 600 mM relative to the control (Figure 3C). The most biofilm is generated when the NaCl concentration is 400 mM. When the concentration goes up to 600 mM, the biofilm declines quickly. RT12 can build strong biofilms in salty environments, which means that it exploits this protective strategy to help it cope with increased levels of salinity (Figure 3D).

3.3. PGP Potential of Strain RT12

Our results reveal that multi plant growth-promoting (PGP) traits are shown in Strain RT12 qualitatively, such as the production of indole-3-acetic acid (IAA), siderophores, ammonia, hydrogen cyanide (HCN), ACC-deaminase, exopolysaccharides (EPS), and many enzymes, as shown in Tables S2 and S3. Quantitative investigation (Table S2, Figure 2) demonstrated that under saline circumstances, RT12 exhibited elevated levels of ACC-deaminase (2.86 µM mg⁻¹), EPS (3.78 mg mL⁻¹), and IAA (144 µM mL⁻¹), underscoring its enhanced capacity to promote plant development and stress resilience relative to other strains.

3.4. Molecular Profiling of the Selected Strains

By using 16S rRNA for molecular profiling, the bacteria that were chosen were identified as Pseudomonas putida RT12, respectively (Figure S2).

3.5. RT12 Enhances Pea Varieties’ Growth in Saline Conditions

The growth parameters of two pea varieties (peas2009 and 9800-10) were significantly affected by salt stress and bacterial inoculation (Figure 4). Under controlled conditions, peas2009 had a greater shoot length (18.67 cm) than 9800-10 (14.51 cm). Salinity stress significantly diminished shoot length in both cultivars, with the minimum measurements recorded at 150 mM NaCl (T2; 11.18 cm in peas2009 and 12.32 cm in 9800-10). In contrast, inoculation with Pseudomonas putida RT12 (T3) significantly enhanced shoot length, achieving 23.13 cm in peas2009 and 17.44 cm in 9800-10, above the control group. Combined treatments (T4 and T5) exhibited partial recovery, with T4 (75 mM NaCl + RT12) outperforming T5 (150 mM NaCl + RT12) as given in Figure 4A. The root length exhibited a comparable pattern. Control plants exhibited root lengths of 17.5 cm (peas2009) and 23.33 cm (9800-10). Salt stress markedly diminished root length, with the most pronounced reduction observed in T2 (11 cm in peas2009 and 15.65 cm in 9800-10). RT12 inoculation (T3) significantly improved root growth in both varieties (22.25 cm and 26.44 cm, respectively). Among the combined treatments, T4 showed significant enhancement relative to salinity alone; however, T5 proved to be less beneficial, especially in variety 9800-10 (13.53 cm) (Figure 4B). Fresh weight (FW) in peas was significantly diminished by salt in 2009, decreasing from 3.29 g (control) to 1.49 g in T2. In contrast, during 9800-10, salinity did not significantly reduce FW, with values ranging from 3.41 to 3.58 g under T1 and T2. RT12 inoculation (T3) resulted in the largest fresh weight in both cultivars, measuring 3.71 g in peas2009 and 5.11 g in 9800-10. Combined treatments enhanced fresh weight relative to salinity alone, with T4 demonstrating superior recovery compared to T5 (Figure 4C). Dry weight (DW) also decreased under salinity stress, with the lowest measurements recorded in T1 and T2 for peas2009 (0.15 g and 0.17 g, respectively). Inoculation with RT12 (T3) elevated dry weight to 0.41 g in peas2009 and 0.41 g in 9800-10. T4 exhibited the highest dry weight across all treatments, measuring 0.71 g and 0.44 g, respectively, as shown in Figure 4D.

3.6. RT12 Mitigates the Reduction of Pigments and Water in Peas Induced by Salt

Salinity and bacterial inoculation significantly influenced the chlorophyll, carotenoids, and relative water content (RWC) of various pea types (Figure 5). Salinity stress decreased chlorophyll a concentration in both varieties, with the lowest levels recorded at 150 mM NaCl (T2). In peas2009, chlorophyll a diminished from the control (C) to T2; however, inoculation with Pseudomonas putida RT12 (T3) enhanced chlorophyll a compared to salinity alone. The integrated treatments performed significantly better, with T5 (150 mM NaCl + RT12) exhibiting the highest chlorophyll a level in both peas2009 and 9800-10 (Figure 5A). Chlorophyll b exhibited a similar trend. Salinity stress (T1 and T2) resulted in a significant reduction in chlorophyll b, with T2 exhibiting the most pronounced decrease. Inoculation with RT12 (T3) significantly increased chlorophyll b levels, whereas T4 and T5 further enhanced these levels, with T5 exhibiting the highest concentrations of both types (Figure 5B). Salinity significantly reduced total chlorophyll concentration, particularly in T2. Conversely, inoculation alone (T3) and in conjunction (T4, T5) increased levels beyond control values. T5 (150 mM NaCl + RT12) exhibited the highest total chlorophyll, indicating the efficacy of the bacterial treatment (Figure 5C). Carotenoids exhibited a comparable trend, with salinity (T1, T2) diminishing pigment concentrations, particularly in peas 2009. Inoculation with RT12 (T3) significantly enhanced carotenoid levels, and the combination of the two treatments further improved accumulation. T5 contains the highest concentration of carotenoids, succeeded by T4 for both categories. Under salinity stress, relative water content (RWC) significantly decreased, with T2 exhibiting the lowest values for both types (Figure 5D). Inoculation with RT12 (T3) improved relative water content (RWC) compared to salinity alone, and the combination of both treatments significantly enhanced water quality. T4 and T5 exhibited the highest relative water content, with no significant difference observed between the two (Figure 5E). This suggests that the salt-induced dehydration was successfully prevented by RT12 inoculation.

3.7. Proline and Antioxidants Elevated in Inoculated Treatments

Salinity stress markedly increased proline accumulation in both pea varieties, with maximum levels observed at 150 mM NaCl (T2), particularly in 9800-10 (99.51 µmol g⁻¹ FW). Inoculation with Pseudomonas putida RT12 (T3) elevated proline levels, which peaked at 134.21 µmol g⁻¹ FW in treatment T5 (9800-10) when combined with other treatments (T4 and T5) as shown in Figure 6A. The catalase (CAT) activity exhibited a comparable trend, with the minimal values observed in the controls (2.11 and 1.90 µmol g⁻¹ FW min⁻¹ in peas-2009 and 9800-10, respectively). Salinity alone (T1, T2) caused no changes, whereas RT12 inoculation (T3) significantly elevated CAT activity. The highest activity was seen in T5, with values of 4.70 and 4.89 µmol g⁻¹ FW min⁻¹ for peas-2009 and 9800-10, respectively (Figure 6B). Both inoculation and combination therapies significantly boosted the activity of superoxide dismutase (SOD). The controls exhibited the lowest values (1.19 and 2.92 µmol g⁻¹ FW min⁻¹), while T5 demonstrated the highest values (4.83 and 4.64 µmol g⁻¹ FW min⁻¹) as shown in Figure 6C. The activity of peroxidase (POD) was minimal in both the control and salinity-only treatments; however, it significantly increased upon the addition of RT12, whether administered alone or in conjunction with salt stress. The peak POD activity occurred in T5, measuring 0.18 and 0.23 µmol g⁻¹ FW min⁻¹ in peas-2009 and 9800-10, respectively (Figure 6D).

3.8. Regulation of TSS and TSP Under RT12 Inoculation in Pea

The accumulation of total soluble proteins and total soluble sugars in both pea varieties, Peas-2009 and 9800-10, was significantly influenced by salt stress and inoculation with Pseudomonas putida RT12 as shown in Figure 7. The control plants of both varieties exhibited the lowest concentrations of total soluble proteins, with Peas-2009 measuring 7.34 µg/g FW and 9800-10 measuring 6.91 µg/g FW. The stress induced by salinity resulted in a gradual increase, attaining 8.44 and 9.12 µg/g FW at 75 mM NaCl (T1) and 10.12 and 11.22 µg/g FW at 150 mM NaCl (T2), respectively. The incorporation of P. putida RT12 (T3) significantly enhanced protein accumulation, yielding 15.11 and 17.12 µg/g FW in Peas-2009 and 9800-10, respectively (Figure 7A). The amalgamated treatments (T4 and T5) significantly augmented protein content, with T5 exhibiting the highest values (18.12 and 21.23 µg/g FW). A similar occurrence transpired with total soluble sugar. The control plants exhibited the lowest sugar concentrations, measuring 19.1 mg/g FW in Peas-2009 and 32.11 mg/g FW in 9800-10, respectively. Salt stress alone elevated sugar concentrations to 25.08 and 34.11 mg/g FW at 75 mM NaCl (T1) and 28.22 and 37.22 mg/g FW at 150 mM NaCl (T2). Inoculation with P. putida (T3) further enhanced sugar accumulation to 28.89 and 37.12 mg/g FW. The amalgamated treatments exhibited the most significant development, with T5 (150 mM NaCl + RT12) yielding the highest sugar content (41.32 and 45.06 mg/g FW in Peas-2009 and 9800-10, respectively) as described in Figure 7B.

3.9. Modulation of Lipid Peroxidation and ROS Accumulation Under Salinity Stress

Salinity stress markedly increased oxidative damage in pea plants, as indicated by elevated levels of malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) as described in Figure 8. In the control condition, MDA levels were modest, measuring 39.1 and 44.05 µg⁻¹ FW in Peas-2009 and 9800-10, respectively. The H₂O₂ concentrations were 52.07 and 67.13 µmol g⁻¹ FW. Exposure to 75 mM NaCl (T1) markedly increased lipid peroxidation and reactive oxygen species (ROS) accumulation, with malondialdehyde (MDA) levels rising to 77.15 and 82.07 U g⁻¹ FW and hydrogen peroxide (H₂O₂) concentrations escalating to 65.17 and 79.33 µmol g⁻¹ FW in the two variants, respectively. The effect was more severe at 150 mM NaCl (T2), with MDA levels of 89.23 and 95.12 U g⁻¹ FW and H₂O₂ values of 79.22 and 91.94 µmol g⁻¹ FW, indicating substantial oxidative stress. Conversely, inoculation with Pseudomonas putida RT12 (T3) effectively mitigated oxidative damage by maintaining MDA and H₂O₂ levels comparable to the control, with only minor elevations above baseline values. The amalgamated treatments (T4 and T5) offered additional corroboration of RT12’s protective efficacy, as both MDA and H₂O₂ concentrations were significantly reduced compared to treatments including only salinity. Modest increases were observed under T5; nevertheless, the values (53.27 and 61.20 µg g⁻¹ FW for MDA; 58.23 and 78.29 µmol g⁻¹ FW for H₂O₂) remained significantly inferior to those of T2 (Figure 8B). This evidence indicates that RT12 can diminish lipid peroxidation and the accumulation of reactive oxygen species (ROS). Overall, the data indicate that salt exposure markedly elevated oxidative stress markers in pea plants. Inoculating the plants with P. putida RT12 significantly mitigated this effect, safeguarding them from membrane damage and injuries induced by reactive oxygen species (ROS).

3.10. Modulation of Ionic Equilibrium Under Saline Regime in Pea

Salinity stress significantly altered the ionic composition of pea plants, with Na⁺ being the most affected osmolyte (

Table 1. Modulation of sodium (Na⁺), potassium (K⁺), calcium (Ca²⁺), magnesium (Mg²⁺), and potassium-to-sodium ratio (K⁺/Na⁺) in Pisum sativum cultivars (peas2009 and 9800-10) inoculated with Pseudomonas putida RT12 under salt stress. Values represent mean ± standard error (SE). Different letters (a–f) within a column indicate statistically significant differences among treatments according to Tukey’s HSD test at p < 0.05.

	Na+ (mg·g−1 DW)		K+ (mg·g−1 DW)		Ca+ (mg·g−1 DW)		Mg+ (mg·g−1 DW)		K/Na (mg·g−1 DW)
	peas2009	9800-10	peas2009	9800-10	peas2009	9800-10	peas2009	9800-10	peas2009	9800-10
C	15.85 ± 0.06 f	16.98 ± 0.03 f	17.76 ± 0.16 e	20.69 ± 0.02 e	18.59 ± 0.09 f	18.98 ± 0.01 d	18.47 ± 0.02 d	28.92 ± 0.01 f	1.12 ± 0.04 d	1.21 ± 0.04 c
T1	18.54 ± 0.01 c	19.68 ± 0.03 c	17.18 ± 0.15 f	20.78 ± 0.08 d	18.9 ± 0.5 e	17.32 ± 0.1 f	10.98 ± 0.12 e	37.11 ± 0.02 c	0.92 ± 0.03 e	1.05 ± 0.04 d
T2	23.95 ± 0.01 a	24.3 ± 0.06 a	17.87 ± 0.13 d	19.37 ± 0.06 f	19 ± 0.9 d	17.85 ± 0.08 b	10.09 ± 0.16 f	40 ± 0.02 b	0.74 ± 0.01 f	0.79 ± 0.03 e
T3	16.32 ± 0.02 e	17.32 ± 0.05 e	23.45 ± 0.06 c	25.88 ± 0.03 b	20.45 ± 0.3 c	24.96 ± 0.04 b	24.73 ± 0.3 a	48.28 ± 0.1 a	1.43 ± 0.03 b	1.49 ± 0.03 a
T4	16.46 ± 0.03 d	17.45 ± 0.07 d	26.32 ± 0.04 b	24.22 ± 0.04 c	21.67 ± 0.05 b	25.4 ± 0.1 a	21.43 ± 0.04 b	33.15 ± 0.2 e	1.59 ± 0.1 a	1.38 ± 0.04 b
T5	21.99 ± 0.05 b	20.73 ± 0.04 b	29.12 ± 0.07 a	27.96 ± 0.05 a	24.74 ± 0.2 a	23.23 ± 0.2 c	19.73 ± 0.05 c	34.71 ± 0.3 d	1.32 ± 0.2 c	1.38 ± 0.05 b

). In the control plants, the Na⁺ concentration was 15.85 mg g⁻¹ DW in Peas-2009 and 16.98 mg g⁻¹ DW in 9800-10. Upon increasing the salinity level to 150 mM NaCl (T2), the concentration of Na⁺ in the water significantly escalated, attaining values of 23.95 and 24.30 mg g⁻¹ DW. This indicates that the ions were significantly perilous under elevated stress levels. Conversely, inoculation with Pseudomonas putida RT12 suppressed Na⁺ buildup, as demonstrated by T3, where values remained close to control levels, and in the combination treatments, where T4 (75 mM NaCl + RT12) significantly reduced Na⁺ to 16.46 and 17.45 mg g⁻¹ DW in comparison to salt treatments alone. Despite elevated salt levels (T5), plants infected with RT12 absorbed less Na⁺ than their non-inoculated counterparts. This finding indicates that RT12 may diminish Na⁺ absorption. Besides controlling Na⁺, RT12 significantly enhanced the absorption of other cations, particularly K⁺. Under salt stress, K⁺ levels decreased; however, in inoculated plants, they significantly increased, peaking in T5 at 29.12 and 27.96 mg g⁻¹ DW. Identical positive outcomes occurred for Ca²⁺ and Mg²⁺. Their levels decreased under salt stress but increased with the addition of bacteria, particularly in T3 and T4. The inoculation restored the K⁺/Na⁺ ratio, which significantly declined under salinity (0.74 in Peas-2009 and 0.79 in 9800-10 at T2), to elevated levels. Peas-2009 attained a value of 1.59 under T4, indicating improved ionic homeostasis. Overall, our findings indicate that salinity led to excessive accumulation of Na⁺ and an ionic imbalance. Nonetheless, P. putida RT12 mitigated stress by restricting Na⁺ absorption and augmenting the acquisition of K⁺, Ca²⁺, and Mg²⁺. This maintained a more balanced K⁺/Na⁺ ratio in both pea varieties (Table 1).

3.11. Correlation of Morphological Parameters with Biochemical Attributes

Principal component analysis (PCA) indicated that both pea varieties distinctly grouped according to treatment (Figure 9). The initial two principal components in peas2009 accounted for 51.94% and 31.84% of the overall variance, respectively, distinguishing the control group from the salt-stressed plants (T1 and T2). Inoculation solely with Pseudomonas putida RT12 (T3) positioned the samples nearer to the control, but the combination treatments (T4, T5) placed them in an intermediate position, indicating that the effects of salinity were only partially mitigated (Figure 9A). In the 9800-10 experiment, control and infected plants (T3) clustered together, while salt treatments (T1 and T2) were distinctly separated. The infected salt-stressed groups (T4 and T5) were positioned between the control and non-inoculated salt groups, further demonstrating that RT12 provides protection (Figure 9B). Redundancy analysis (RDA) clarified a substantial portion of the variation in physiological indicators (Figure 9C,D). In peas2009, salt stress (T1, T2) was significantly associated with oxidative stress indicators (MDA, H₂O₂) and Na⁺ buildup. Conversely, characteristics such as chlorophyll pigments, carotenoids, relative water content (RWC), antioxidant enzymes (SOD, CAT, POD), and K⁺/Na⁺ ratio exhibited a favorable correlation with RT12 inoculation (T3–T5) as shown in Figure 9C. Analogous associations were observed in 9800-10, where salinity correlated with oxidative and ionic stress, and RT12 enhanced the accumulation of osmolytes (proline, soluble proteins), antioxidant activity, and photosynthetic efficiency. The Mantel test further corroborated these patterns (Figure 9D). Robust positive associations were identified between Na⁺ and oxidative stress indicators (MDA, H₂O₂). Conversely, growth-related measures (shoot length, root length, fresh and dry biomass) exhibited a positive correlation with K⁺, Ca²⁺, Mg²⁺, and the K⁺/Na⁺ ratio. The associations were significantly more evident in the inoculation treatments (T3–T5), suggesting that RT12 improved ionic equilibrium and strengthened physiological integration under salt stress conditions. The multivariate investigations demonstrate that P. putida RT12 markedly alleviated salinity-induced stress in both pea varieties by diminishing oxidative damage, enhancing antioxidant defenses, and maintaining ionic homeostasis.

4. Discussion

4.1. Halotolerance and PGP Traits of P. putida RT12

When exposed to salt, P. putida RT12 produced the highest amounts of ACC-deaminase, IAA, and EPS (Tables S2 and S3; Figure 2). Both bacterial adaptation and the improvement of plant development under stress conditions depend on these multifunctional characteristics [72]. While EPS synthesis and biofilm formation facilitate bacterial adhesion to surfaces and buffer ions in the rhizosphere, which aids plants in retaining moisture and obtaining nutrients, ACC-deaminase activity reduces stress-induced ethylene levels [73,74]. The improved halotolerance of RT12 (Figure 3) illustrates its metabolic flexibility and capacity to withstand osmotic stress, consistent with prior research indicating that Pseudomonas spp. mitigates salinity effects through similar mechanisms [75,76,77,78,79,80,81].

4.2. Alleviation of NaCl-Induced Growth Inhibition in Pea Cultivars by RT12 Inoculation

The RT12 inoculation markedly improved pea growth under saline circumstances (Figure 4), demonstrating the bacterium’s role in alleviating osmotic and ionic constraints. The augmented biomass of shoots and roots is likely attributable to the bacteria producing IAA and ACC-deaminase, which modulate ethylene levels [82,83]. Biofilms composed of extracellular polymeric substances facilitate root contact with soil, thereby enhancing water and nutrient absorption [35,81]. Cereals and legumes infected with EPS- and ACC-deaminase-producing plant growth-promoting bacteria (PGPB) have shown comparable improvements in growth traits [1,84,85,86]. Our findings confirm the role of PGPB, particularly Pseudomonas putida RT12, in mitigating salt stress and promoting sustainable crop growth in saline conditions.

4.3. RT12 Sustains Photosynthetic Performance and Water Retention in Salinity-Stressed Pea

While RT12 retained chlorophyll, carotenoids, and the amount of water in the cells, salinity typically disrupts pigment synthesis and the interaction between water and other substances (Figure 5). The improved pigment stability indicates reduced oxidative bleaching and extended photosynthetic efficiency [87,88]. This activity might be the consequence of osmolyte accumulation and bacterial IAA-induced stomatal control, which preserve chloroplast integrity. The findings align with those observed in rice and soybeans, where inoculation with Pseudomonas bacteria producing ACC-deaminase maintained effective photosynthesis under saline circumstances [89,90,91].

Inoculation with Pseudomonas putida RT12 significantly increased pigment levels in both cultivars in the current experiment. Under T3, peas2009 exhibited a 25% increase in chlorophyll a (31.12) and complete restoration of chlorophyll b (18.15). 9800-10 exhibited an increase of 32.12 (+9%) in chlorophyll a and a restoration of 24.96 in chlorophyll b compared to the control (Figure 5A–C). In T5, the total chlorophyll content surpassed that of the controls, with peas2009 measuring 49.27 and 9800-10 measuring 72.25. These enhancements correspond to the outcomes observed when Enterobacter PR14, a producer of ACC-deaminase, was introduced to rice [92]. Similarly, in another study the inoculation of PGPR restored photosynthetic pigments under saline circumstances in soybean [93]. Carotenoids exhibited increases upon inoculation in our current experiment, indicating enhanced protection against light damage. In peas2009, carotenoid levels increased from 9.09 (control) to 13.15 in T4 (+45%), whereas in 9800-10, they increased from 7.46 to 15.11 in T5 (+102%) as given in Figure 5D. Comparable improvements have been recorded in Capsicum annuum infected with Burkholderia cepacia under salt stress [94].

4.4. RT12 Enhances Antioxidants to Alleviate Oxidative Stress

RT12 enhanced the antioxidant system of pea plants by elevating the activities of CAT, SOD, and POD (Figure 6) and promoting the accumulation of proline and soluble metabolites (Figure 7). Proline is recognized for its ability to safeguard cells against osmotic stress and eliminate reactive oxygen species (ROS) [8,95]. The increase in proline is seen under bacterial inoculation indicates superior osmotic adjustment, consistent with findings in rice and tomato subjected to PGPB inoculation [96,97,98]. These reactions collaborate to reduce oxidative damage by eliminating reactive oxygen species and protecting cell membranes [99], in different plants inoculated with Pseudomonas putida [100,101], and in pea plants with ACC deaminase-producing Pseudomonas aeruginosa [102]. Our results support the conclusions of Hassan et al. [103], and Liu et al. [104] who showed reduced oxidative damage in maize inoculated with Bacillus and Plancoccus species, similarly in P. sativum plants inoculation with EPS producing PGPR showed increased antioxidant enzyme activity in under saline stress. The synthesis of EPS may assist in preventing the ingress of Na⁺ and maintaining redox equilibrium stability. Reports indicate that bacteria enhance enzymatic defences in legumes and cereals inoculated with halotolerant plant growth promoter bacteria (PGPB) [105]. The elevated accumulation of proline and the overexpression of CAT, SOD, and POD in inoculated plants suggest that P. putida RT12 aids pea plants in managing oxidative stress, hence facilitating their growth in saline environments.

4.5. RT12 Role in TSP and TSS Production in Salt-Stressed Conditions

Plants subjected to high salt concentrations typically endure osmotic stress, impeding their ability to absorb water and disrupting cellular equilibrium. Plants accumulate appropriate solutes, such as soluble carbohydrates and proteins, to maintain osmotic balance and safeguard their cells [106]. In our study, both pea cultivars exhibited significant increases in total soluble proteins (TSP) and total soluble sugars (TSS) during salt stress (Figure 7). The gains were significantly more substantial following inoculation with Pseudomonas putida RT12. The results demonstrate that RT12 facilitates osmolyte accumulation, hence improving cellular osmoprotection, stress tolerance, and resilience in pea plants. Comparable augmentations in Total Soluble Protein (TSP) and Total Soluble Sugars (TSS) resulting from bacterial inoculation have been recorded in Pisum sativum [107], Triticum aestivum [108], Triticum monococcum [109], and Oryza sativa [110]. The strong reaction demonstrated by both pea cultivars in this study highlights the critical role of PGPB in mitigating salt-induced osmotic stress.

4.6. RT12 Revive Nutrients Uptake and Protect Pea from Salt-Induced Ionic Imbalance

Salt stress substantially impacted nutrient absorption patterns in pea plants, as indicated by increased Na⁺ accumulation and reduced K⁺, Ca²⁺, and Mg²⁺ uptake in uninoculated controls as given in Table 1. In uninoculated plants decrease in the nutrient’s uptake was observed which suggests that the ions were out of balance because of the salinity stress. However, inoculation with Pseudomonas putida RT12 reduced Na⁺ toxicity by reducing the uptake of Na⁺ and restored the balance of other minerals. For example, Na⁺ levels declined to 16.32 and 17.32 mg g⁻¹ DW at T3 in peas2009 and 9800-10, respectively, when compared to the uninoculated stressed plants (T2). The K⁺/Na⁺ ratio improved significantly, increasing from 1.43 in peas2009 to 1.49 in 9800-10. Similarly, RT12 inoculation enhanced K⁺ accumulation, with values rising from 17.87 mg g⁻¹ DW at T2 to 29.12 and 27.96 mg g⁻¹ DW at T5 in peas2009 and 9800-10, respectively. Comparable enhancements were observed for Ca²⁺ and Mg²⁺. These findings are supported by other studies in plants exposed to saline regime [111,112]. Our results are also in agreement with a study where plants exposed to saline conditions and inoculated with ACCD producing bacterial strains showed altered Na⁺ and K⁺ selectivity as well as decreased absorption and transportation into the entire plant body [113,114]. Prior studies also has shown that bacterial inoculation in plants improves root growth, nutrient absorption, organic acid generation, decrease in pH, and siderophores in the rhizosphere which are in agreement with our findings [115,116]. More Na⁺ is taken in and stored by plants under salinity, causing osmotic and ionic stress, oxidative damage, and reduced K⁺ uptake by salt-stressed plants Table 1. This nutritional absorption modification affects K⁺/Na⁺ ratio. Similar results were founded in K. rhizophila-inoculated pea plants, in which improved their K⁺/Na⁺ ratio, shielding transport proteins against Na⁺ accumulation [1]. Low K⁺/Na⁺ concentrations improve plant sensitivity and osmotic potential adjustments [117,118]. Similarly, it regulate stomatal movement, enzyme activation, and stress tolerance depend on K⁺ concentrations [119].

In both varieties, a PCA, RDA and Mantel analysis of all plant parameters was carried out (Figure 9 and Figure 10). Through the inoculation of strain RT12, all plant growth parameters that show better response of these qualities under both normal and stressful conditions were obtained. The multivariate analysis calculates the variance and correlation between different response variables and input variables [119]. Most recently, Mantel multivariate analysis has been used to find patterns and connections between different data sets [120,121]. Salt stress and inoculation of RT12 overall effects on plant responses in the current study’s Mantel and Pearson’s correlation biplot were found to differ from each other and the control in terms of how they affected plant responses.

5. Conclusions

This study demonstrates that inoculating pea plants (cultivars peas2009 and 9800-10) with ACC deaminase (ACCD) and exopolysaccharide (EPS)-producing Pseudomonas putida RT12 significantly enhanced their salinity tolerance through alterations in morphology, physiology, and biochemistry. Under salt stress, uninoculated plants demonstrated stunted growth, diminished shoot and root biomass, and restricted leaf development, mostly attributable to high Na⁺ accumulation (up to 23.9 mg g⁻¹ DW) and a marked decrease in the K⁺/Na⁺ ratio (0.74). Inoculated plants exhibited elongated shoots and roots, increased fresh and dry biomass, and expanded leaf area. This improvement occurred due to a reduced uptake of Na⁺ (16.3 mg g⁻¹ DW in T3) and an increased uptake of K⁺ (29.1 mg g⁻¹ DW), Ca²⁺ (24.7 mg g⁻¹ DW), and Mg²⁺ (24.7 mg g⁻¹ DW), resulting in a K⁺/Na⁺ ratio of 1.59. These alterations in ion homeostasis directly facilitated cellular turgidity and growth. In physiological terms, inoculated plants exhibited approximately 45% higher chlorophyll levels and nearly 20% increased relative water content. These changes enhanced their efficiency in photosynthesis and improved their water utilization. Osmoprotectant accumulation was markedly increased biochemically, with soluble sugars escalating from 19.1 to 41.3 mg g⁻¹ FW and proteins from 7.3 to 18.1 μg g⁻¹ FW, thereby reinforcing osmotic control and the detoxification of reactive oxygen species. However, this study is limited to controlled greenhouse conditions and two pea cultivars, and further field trials across diverse cultivars and environments are needed to validate the broader applicability of these findings.