, Abiotic stresses are significant barriers to overcome for increasing crop productivity, critically impacting both the amount and quality of yields globally. The stresses disrupt cellular homeostasis through osmotic imbalance, ion toxicity, and excessive reactive oxygen species (ROS) generation. These disturbances lead to a wide range of morphological changes (e.g., reduced leaf area, altered root architecture), physiological disruptions (e.g., decreased photosynthetic efficiency, impaired water relations), and metabolic reprogramming (enhanced stress metabolite synthesis) which in turn adversely affect plant growth, development, and productivity (Kohler et al., 2008; Liu et al., 2013; Ma et al., 2020). It is concerning that a large portion of arable land is currently under substantial stress due to soil drought and high salinity (Kopecká et al., 2023; Tarolli et al., 2024). Furthermore, climate change and continuous fertilization are exacerbating these environmental challenges (Kaushal and Wani, 2016; Kopecká et al., 2023).
Diverse approaches, such as classical and biotechnological plant breeding (Ashraf and Akram, 2009) and agronomic practices (Kumar et al., 2021), have been adopted to enhance crop performance and mitigate the effects of abiotic stresses. However, these methods often face challenges, including high costs, time-consuming processes, potential negative environmental impacts, and social concerns (Hassan et al., 2021; Kotula et al., 2020).
Recently, plant growth-promoting rhizobacteria (PGPR) have gained prominence as environmentally friendly and cost-effective biostimulants for improving plant health and yield under challenging conditions (Etesami et al., 2023; Mellidou and Karamanoli, 2022). PGPR confer beneficial effects through specific mechanisms, including the modulation of key phytohormones (auxin, cytokinin, gibberellin), induction of critical antioxidative enzymes such as catalase (CAT) and superoxide dismutase (SOD), facilitation of nutrient uptake through siderophore production and phosphate solubilization, and upregulation of stress-responsive gene networks (Barghi et al., 2021; Etesami and Maheshwari, 2018).
Members of the Bacillus genus are among the most prevalent naturally occurring PGPR in soil (Tiwari et al., 2017). Bacillus spp., known as free-living nitrogen fixers, can induce the expression of nitrate and ammonium transporters in Arabidopsis, which enhances the plant’s ability to uptake nitrate and ammonium from the soil, which is crucial for coping with nutrient deficiency stress (Calvo et al., 2019). Inoculating wheat seeds with Bacillus sp. strains can confer seedlings tolerance to cold stress by modulating abscisic acid-related gene expression and stimulating proline accumulation (Zubair et al., 2019). B. cereus Pb25 strain has also been found to improve salt tolerance in mung beans by upregulating antioxidant enzyme activities (Islam et al., 2016). Moreover, exogenous treatments of certain indole-3-acetic acid-producing Bacillus species can boost plant growth and crop yield in high saline environments caused by heavy agrochemical use or under water scarcity conditions (Mellidou and Karamanoli, 2022).
Among vegetable crops, lettuce (Lactuca sativa L.) stands out for its global economic importance and susceptibility to abiotic stresses. Its shallow root system, high transpiration rate, and rapid growth make it physiologically vulnerable to water and ionic imbalances, underscoring the need for sustainable mitigation strategies (Abdelkader et al., 2024). To alleviate these challenges in lettuce, PGPRs were also employed (Hong and Lee, 2017). Treating lettuce plants with the PGPR strain Pseudomonas mendocina enhanced CAT activity, which helped safeguard the plants against oxidative stress induced by drought conditions (Kohler et al., 2008). Moreover, inoculation of lettuce with a consortium comprising Klebsiella sp. improved growth and heat tolerance by augmenting nutrient uptake and water-use efficiency under heat stress (Chan et al., 2024).
However, given the strain-specific and context-dependent nature of PGPR-plant interactions across different species, developmental stages, and environmental conditions, systematic characterization of beneficial strains and their functional effects remains crucial for agricultural optimization. To our knowledge, Bacillus subtilis W1 strains have received limited attention as PGPR in lettuce. Moreover, the B. subtilis W1-like strain (hereafter, BsW1L), a geographically distinct isolate, has not been investigated for its role in promoting stress tolerance. Therefore, in this study, we explored the effects of the Bacillus subtilis W1-like strain (hereafter, BsW1L) on the tolerance responses of lettuce plants under drought and high salinity stress conditions. Soil drenching with the BsW1L effectively enhanced the plant’s tolerance to drought and high salinity. Additionally, we analyzed various physiological and biochemical changes in BsW1L-treated plants compared to mock-treated lettuce plants. These results indicate that the BsW1L strain could boost the resilience of lettuce plants to drought and salt stress, presenting practical strategies for sustainable agriculture in the face of environmental changes.
Materials and Methods
Plant material, inoculation, and stress treatments
Lettuce seeds (cv. New Red Fire) were sterilized by immersing them in 70% ethanol for 30 s, then in a 2% NaOCl solution for 5 min, and finally rinsed several times with sterile distilled water. The sterilized seeds were sown on Murashige and Skoog medium (pH 5.8) (Duchefa, Haarlem, Netherlands) containing 1.5% sucrose and 0.8% plant agar. The seedlings were maintained at 21°C and approximately 60% relative humidity under a 16/8 h light/dark cycle for 16 days in a controlled growth chamber. Light was provided using six cool white fluorescent lamps positioned approximately 20 cm from the plants. Conditions supported normal lettuce growth, and light was uniform across all treatments to avoid confounding effects on chlorophyll ratios. The BsW1L was originally isolated from the rhizosphere soil of field-grown corn in Isfahan, Iran, through serial dilution and plating on tryptic soy agar. For taxonomic identification, genomic DNA was extracted with the Gspin Total DNA Extraction Kit (iNtRON, Seongnam, Korea), and the 16S rRNA gene was amplified with universal primers 785F and 907R (Lane, 1991). Sequence analysis of the resulting 1,514 bp amplicon revealed 99.72% identity with B. subtilis strain W1 (GenBank accession no. KC441816.1). Based on this high sequence similarity, the isolate was designated as B. subtilis W1-like (BsW1L).
For experimental use, the strain was cultured overnight in tryptic soy broth (Merck, Germany) at 32°C and diluted to approximately 1 × 108 colony-forming unit (CFU)/mL. Three uniform-sized 4-day-old lettuce seedlings were transplanted into plastic pots containing 150 g of loamy soil, and 30 mL of the freshly prepared BsW1L suspension was drenched onto the soil to facilitate rhizosphere colonization, while distilled water was used as a control (Supplementary Fig. 1).
Drought stress was initiated 5 days after transplanting by applying 20.25 mL of distilled water per pot every two days, corresponding to 50% of the irrigation volume required to maintain field capacity (FC). Control plants received 40.5 mL per pot at the same interval, representing 100% FC. At this level, the soil moisture content was approximately 27% (w/w) based on gravimetric measurements (Supplementary Figs. 1B and 2A). To induce high salt stress, 30 mL of a 200 mM NaCl solution was applied to the pots every two days (Supplementary Fig. 1B and 2B). The experiment included six treatment groups: C (control) - well-watered, mock (no BsW1L), no stress; B (BsW1L only) - well-watered, BsW1L-inoculated, no stress; D (Drought stress only) - drought-stressed, mock; S (Salt stress only) - salt-stressed (treated with 200 mM NaCl), mock; BD (BsW1L + drought) - BsW1L-inoculated, drought-stressed; BS (BsW1L + salt) - BsW1L-inoculated, salt-stressed. Each treatment included three biological replicates, and each biological replicate comprised three technical replicates (i.e., three pots), with each pot containing three plants. Consistency among biological replicates was assessed by comparing both the direction and magnitude of treatment effects across independent experimental replicates.
Measurement of plant growth parameters
Eleven days after stress treatment, the effects of BsW1L on the plant tolerance against stresses were assessed by evaluating growth parameters for each treatment, total fresh weight (FW, g), and root and shoot lengths (cm). Leaf area (cm2) was also measured to determine growth promotion traits. This time point was selected based on preliminary trials, as it captured both early stress responses and emerging adaptive mechanisms. At this stage, lettuce plants were in the early vegetative growth, which our initial observations showed to be most responsive to microbial treatment and stress-mitigation effects.
For chlorophyll measurement, leaf discs (0.2 g) from 3 plants were collected, placed in a Falcon tube with 3 mL of 80% acetone, and stored at −10°C overnight. Chlorophyll a and b absorbances were determined at 663 nm and 645 nm, respectively, using a UV-visible spectrophotometer (EPOCH2TSC, BioTek, Winooski, VT, USA). The concentrations of chlorophyll a, chlorophyll b, and total chlorophyll were calculated using the following equations (Arnon, 1949): Chlorophyll a = (0.0127D663 − 0.00269D645), Chlorophyll b = (0.0229D645 − 0.0468D663), and Total chlorophyll = Chlorophyll a + Chlorophyll b (where D663 and D645 are optical densities at 663 nm and 645 nm, respectively).
RT-qPCR analysis of stress-responsive genes
RNA was isolated using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the supplier’s instructions. To remove any DNA contaminants, 3 μL of 10× buffer, 2 μL of DNase I (200 U/mL, Roche, Monza, Italy), and 25 μL of extracted RNA were mixed and incubated at 37°C for 30 min. Next, 270 μL of DEPC-treated distilled water and PCI (phenol:chloroform:isoamyl alcohol at 25:24:1) were added, and the solution was centrifuged at 16,000 ×g for 20 min at 4°C. A 200 μL supernatant was retained for ethanol precipitation, and resulting RNA pellet was resuspended in 50 μL of DEPC-treated RNase-free water. For cDNA synthesis, 10.5 μL of RNA (~1 μg) was mixed with 10 μM oligo dT and 2 μL of 10 mM dNTP, then heated at 65°C for 5 min. Following this, 5× first-strand buffer, 0.5 μL of reverse transcriptase (Invitrogen, Carlsbad, CA, USA), and 2 μL of 0.1 mM DTT were added, and the mixture was incubated for 1 h at 42°C, then at 70°C for 15 min. The cDNA was diluted 10 times and stored at −20°C. For RT-qPCR, cDNA, specific primers, and SYBR Green PCR Master Mix (Bio-Rad, Hercules, CA, USA) were combined in a 20 μL reaction. The PCR protocol involved an initial 10 min at 95°C, followed by 45 cycles of 95°C for 15 s and 60°C for 30 s. Melting curve analysis was performed between 60-95°C with 0.2°C increments for 60 s. Gene expression related to detoxification and antioxidant response was quantified using specific primers (Supplementary Table 1). Data from three biological replicates with three technical replicates each were normalized to LsACTIN expression, and relative mRNA levels were calculated by the 2−ΔΔCT method (Livak and Schmittgen, 2001).
Measurement of stress-responsive enzymatic activity and concentration of key metabolite
To examine how drought and increased salt conditions affect stress-responsive elements, we measured the enzymatic activities of CAT, ascorbate peroxidase (APX), and SOD and the levels of hydrogen peroxide (H2O2), malondialdehyde (MDA), proline, and total soluble sugar (TSS) in the first true leaves of lettuce seedlings.
For CAT activity (EC 1.11.1.6), a 0.1 mL aliquot of crude protein extracts of lettuce leaf tissues was mixed with a reaction solution containing 100 mM phosphate buffer (pH 7.8), 0.1 μM EDTA, and 0.1% hydrogen peroxide (H2O2) (Chen and Zhang, 2016). The breakdown of hydrogen peroxide was tracked immediately and at 15-s intervals over 1 min using a spectrophotometer set to 240 nm. The CAT activity was measured using the molar extinction coefficient ɛ = 39.4 mM/cm for the breakdown of hydrogen peroxide.
SOD activity (EC 1.15.1.1) was assessed using the approach outlined by Chen and Zhang (2016). For the assay, the reaction solution contained 50 mM sodium phosphate buffer with 0.1 mM EDTA, 12 mM methionine, 75 μM NBT, and 50 mM Na2CO3. Enzyme extract and buffer (100 μL) were added as the sample and blank, respectively. Following this, 300 μL of 0.1 mM riboflavin was introduced, bringing the total volume to 2 mL. The solution was agitated and then exposed to 15 W fluorescent light for 15 min. The absorbance was measured at 560 nm with a spectrophotometer. One unit of SOD activity was defined as the enzyme amount needed to achieve 50% inhibition of NBT reduction.
APX activity (EC 1.11.1.1) was evaluated according to Nakano and Asada (1981). The assay involved adding 0.1 mM hydrogen peroxide to a reaction mixture containing 50 mM phosphate buffer (pH 7.8), 0.1 mM EDTA, 0.5 mM ascorbate, and 0.1 mL of plant crude extract. Ascorbate oxidation was monitored at 290 nm over 3 min. The activity of APX was calculated using ascorbate’s molar extinction coefficient of 2.8/mM/cm.
The hydrogen peroxide levels in leaf tissues were determined using a spectrophotometric method based on its reaction with potassium iodide (KI), as outlined by Alexieva et al. (2001). The supernatant (0.5 mL) from a 0.1% trichloroacetic acid (TCA) leaf extract was mixed with 0.5 mL of 100 mM potassium phosphate buffer and 2 mL of a 1 M KI solution. This mixture was incubated in darkness for one hour, and the absorbance was subsequently recorded at 390 nm. The concentration of hydrogen peroxide was calculated by comparing the absorbance values to a standard curve generated from known hydrogen peroxide concentrations.
We adapted a standard protocol to assess MDA levels (Heath and Packer, 1968). Lettuce leaves (0.2 g) were homogenized in 1.5 mL of 5% TCA and then centrifuged at 10,000 ×g for 10 min at 4°C. The supernatant (0.5 mL) was combined with 1 mL of 5% thiobarbituric acid in 20% TCA and heated at 95°C for 25 min. After cooling on ice, the mixture was centrifuged again at 10,000 ×g at 4°C. Absorbance was measured at 532 nm and 600 nm using a spectrophotometer. MDA concentration was calculated using an extinction coefficient of 155 mM/cm.
Proline was extracted from 0.5 g of leaf samples using a 3% (w/v) aqueous sulfosalicylic acid solution. The concentration of proline was then determined with ninhydrin reagent based on the method described by Bates et al. (1973). Absorbance readings were taken at 520 nm, and proline concentrations were calculated using a calibration curve and expressed as μg proline per gram of FW.
To determine the sugar content in lettuce seedlings, we followed a modified approach inspired by DuBois et al. (1956). Approximately 100 mg of tissue was homogenized in 3 mL of 80% (v/v) methanol and subsequently heated at 70°C for 30 min. After this step, 500 μL of the extract was mixed with an equal amount of 5% phenol solution, followed by 1.5 mL of concentrated sulfuric acid (H2SO4). The solution was kept in the dark for 15-20 min, and absorbance was read at 490 nm using a spectrophotometer (EPOCH2TSC, BioTek, Winooski, VT, USA).
Statistical analysis
Statistical analysis was conducted on one representative biological replicate (comprising three technical replicates, each with three plants; n = 3), selected based on its consistency with other biological replicates, which exhibited similar trends in direction and magnitude of response. Assumptions of normality and homogeneity of variance were evaluated using the Shapiro-Wilk and Levene’s tests, supplemented by visual inspection of residual plots. As the data appeared symmetric with comparable variances, one-way ANOVA followed by Tukey’s post-hoc test was performed using Minitab version 21.3.1. Statistical significance was set at P ≤ 0.05. The number of replicates used in each analysis is provided in the corresponding figure legends.
Results
Tolerance to drought and salinity is improved in lettuce plants treated with BsW1L strain
Considering that beneficial rhizobacteria often exhibit broad host ranges across different plant species, we tested the ability of the BsW1L strain, isolated from the corn rhizosphere, to enhance stress tolerance in lettuce by exposing 9-day-old seedlings to drought and salt stress. In the absence of stress treatment, applying the BsW1L strain did not notably enhance the FW of lettuce seedlings (Fig. 1A). Shoot and root length and leaf area in 20-day-old lettuce seedlings treated with the BsW1L strain were comparable to those of the control group when plants grew under non-stress conditions, although a modest increase in leaf area was observed (Fig. 1). While BsW1L treatment led to a significant increase in chlorophyll a content compared with those in control plants, it failed to increase total chlorophyll or chlorophyll b levels (Fig. 1E, Supplementary Fig. 3).
Drought and salt stress reduced the plant’s total FW by 19.90% and 20.39%, respectively (P < 0.05), along with notable declines in other growth parameters such as shoot and root height and leaf area (Fig. 1, Supplementary Fig. 4). In contrast, application of the BsW1L strain helped plants mitigate the adverse effects of these stresses on total FW, shoot length, leaf area, and chlorophyll content (Fig. 1). Compared to the lettuce seedlings only subjected to drought and salt stresses, lettuce seedlings treated with the BsW1L displayed enhanced FW of seedlings, showing a significant increase of 12.99% and 10.24%, respectively (P < 0.05) (Fig. 1A). Direct application of the BsW1L strain to the soil also resulted in significantly enhanced growth of shoots and roots by 25% and 13.5%, respectively, under drought stress and an increase of 21.7% in shoot growth under salt stress (P < 0.05), outperforming the mock treatments (Fig. 1B and C). However, we did not see any increment in the root length of lettuce plants treated with the BsW1L strain compared with those in mock-treated plants under salt stress conditions (Fig. 1C). Furthermore, plants co-incubated with the BsW1L exhibited larger leaf areas, with 5.73% and 6.83% increases compared to those receiving the stress treatments (P < 0.05) (Fig. 1D, Supplementary Fig. 4). Furthermore, plants treated with the BsW1L strain showed a remarkable increase in chlorophyll a and total chlorophyll levels compared to those receiving the drought and salt treatments. Compared to non-inoculated stressed plants, BsW1L treatment under drought stress increased chlorophyll a and total chlorophyll levels by 9.3% and 11.69%, respectively. Similarly, under salt stress, chlorophyll a increased by 6.9% and total chlorophyll by 10.53% (P < 0.05). However, the increase in chlorophyll b was not statistically significant (Fig. 1E, Supplementary Fig. 3). Note that the proliferation of the BsW1L strain was not impacted by the drought conditions or the salt concentration added to the soil (Supplementary Fig. 5A and B). These results highlight the capacity of the BsW1L strain to mitigate the adverse effects of drought and salt stresses on plant growth and chlorophyll content.
The Bacillus strain modulates gene expression and antioxidant enzyme activity in lettuce grown under drought and salt stresses
Environmental stresses typically lead to the overproduction of ROS in plants (Huang et al., 2019). To investigate whether exogenous treatments with the BsW1L strain regulated antioxidant responses in plants, mRNA levels and enzymatic activities of CAT, APX, and SOD were evaluated in the leaves of lettuce plants grown under drought or salt stresses, with or without treatment by the BsW1L strain. Except for LsCAT, which showed a moderate increase, BsW1L treatment did not significantly alter mRNA levels or enzyme activities compared to control plants when plants grew under normal growth conditions (Fig. 2). Exposure to drought and salinity stresses led to an increase in mRNA levels of LsCAT by 1.40- and 1.52-fold (Fig. 2A), and LsAPX by 1.191- and 1.158-fold (Fig. 2B), respectively, in lettuce leaves compared with those in plants grown under non-stress conditions. Compared to the drought treatment without the bacterium, application of the BsW1L strain increased LsCAT expression by 1.58-fold and LsAPX by 1.17-fold in plants under drought stress (Fig. 2A and B). At the same time, LsSOD transcription remained unaffected (Fig. 2C). Following salt treatment, the BsW1L application also elevated LsCAT mRNA expression in lettuce plants by 1.28-fold (Fig. 2A). In contrast, the mRNA levels of LsAPX and LsSOD remained unchanged relative to the salt treatment alone (Fig. 2B and C).
In line with these transcriptional changes, lettuce plants inoculated with the BsW1L strain exhibited a significant increase in CAT and APX activities in response to drought stress with no observed changes in SOD activity (Fig. 2D-F). Moreover, after salt exposure, the BsW1L treatment boosted CAT activity, although it did not produce any noticeable effects on APX and SOD activity (Fig. 2D-F). Overall, these findings suggest that the BsW1L strain may enhance the transcription and enzymatic activities of antioxidant enzymes, facilitating the detoxification of ROS produced during oxidative stress and thereby promoting drought and salt tolerance in plants.
BsW1L strain mitigates oxidative stress in lettuce under drought and salt stress
Considering the increased enzymatic activities by application of the BsW1L strain, we presumed that levels of hydrogen peroxide would be reduced in plants treated with the BsW1L strain. Lettuce plants exposed to drought and salt stresses displayed a notable increase in hydrogen peroxide levels. As with no change of enzymatic activity, BsW1L treatment could not affect hydrogen peroxide levels in non-stressed lettuce plants. Expectedly, treatment of BsW1L decreased the level of hydrogen peroxide, compared with untreated samples, in leaves grown under stress conditions (Fig. 3A).
MDA is an indicator for assessing oxidative stress damage in plants (Bharti et al., 2014; Morales and Munné-Bosch, 2019). Under drought stress conditions, MDA levels in the leaves were lower in plants treated with the BsW1L strain than in those not subjected to the drought. Similarly, under salt stress, BsW1L treatment led to a modest reduction in MDA accumulation relative to mock-treated plants (Fig. 3B). These results suggest that the BsW1L strain can enhance the antioxidant activity of plants, thereby reducing oxidative damage by drought and high salt stress.
Osmolyte accumulation in lettuce is enhanced by the BsW1L treatment under drought and salinity conditions
Plants cultivated with the BsW1L strain exhibited enhanced resilience and stronger antioxidant responses than mock-treated plants when exposed to drought and high salt stresses. To assess whether BsW1L treatments could influence osmoprotectant levels, we analyzed endogenous proline, a key metabolite (Verslues and Sharma, 2010), and TSS levels in lettuce leaves grown under various conditions. Treatment of the BsW1L strain did not significantly alter proline levels in lettuce plants under non-stressed conditions (Fig. 4A). However, plants subjected to drought and high salt stresses demonstrated elevated proline levels compared to non-stressed plants. Interestingly, lettuce plants cultivated in the BsW1L-treated soils showed a significant increase in proline levels under drought stress by 19.01% (P < 0.05) compared to those treated with a drought without BsW1L. On the other hand, BsW1L treatment did not increase proline levels under salt stress; instead, it caused a slight reduction of 13.2% (Fig. 4A).
TSS are recognized as compatible osmolytes that assist plants in coping with environmental stresses by increasing osmolytes synthesis (Tiwari et al., 2017). Stressed plants exhibited a modest increase in TSS accumulation compared to their non-stressed counterparts (Fig. 4B). BsW1L inoculation further enhanced TSS levels in seedlings subjected to drought and high salinity, resulting in a significant increase compared to uninoculated plants. Specifically, TSS content in inoculated seedlings increased from 0.58 ± 0.03 to 0.75 ± 0.04 μg/g FW under drought stress and from 0.60 ± 0.02 to 0.76 ± 0.05 μg/g FW under salt stress (P < 0.05). In contrast, inoculated seedlings under non-stressed conditions exhibited TSS levels comparable to those of the control group, indicating that the observed increase in TSS is primarily stress-induced (Fig. 4B). Collectively, these findings suggest that the application of the BsW1L strain mitigates oxidative stress in plants by regulating osmolyte contents, as well as antioxidant enzymatic activities, in plants under both drought and high salt conditions.
Discussion
PGPR, such as Bacillus spp., Rhizobium spp., and Pseudomonas spp., enhances tolerance to abiotic stress and induces resistance against pathogen infection through both direct and indirect interactions with plants (Ayuso-Calles et al., 2023; Barghi and Jung, 2024; Zboralski and Filion, 2023). In this study, we investigated the potential of the BsW1L strain to enhance lettuce tolerance to drought and salt stress by focusing on physiological and biochemical responses. While no substantial changes in growth parameters were observed under non-stress conditions, BsW1L improved tolerance responses in stress-exposed lettuce plants by preserving chlorophyll content, enhancing antioxidant defense systems, and modulating osmolyte accumulation. The conditional effect of PGPR has already been documented. For instance, treatments of Bacillus strains, such as B. subtilis HAS31 and B. zanthoxyli HS1 strains, did not promote growth under favorable conditions but significantly enhanced stress tolerance in potato, cabbage, and cucumber under drought and heat stress (Barghi and Jung, 2024; Batool et al., 2020). Thus, these results support the role of PGPR that provide vital protection and resilience to plants exposed to environmental stresses (Barghi and Jung, 2024; Poulaki and Tjamos, 2023).
BsW1L treatment improved FW, shoot length, and leaf area in plants under both drought and salt stress compared to mock plants. However, root length increased only under drought conditions. This drought-specific root elongation aligns with previous reports showing that PGPR-induced root growth provides advantages under water-limited conditions but may not be beneficial under other stress types (Sandhya et al., 2010; Sharifi et al., 2022).
Maintaining chlorophyll content is a critical component of stress tolerance, as it is often compromised by abiotic stresses such as drought and salinity (Chauhan et al., 2023). The application of PGPR can mitigate these stress-induced chlorophyll losses (Barnawal et al., 2012; Curá et al., 2017), although their effects on chlorophyll a and b accumulation vary depending on the bacterial strain and plant species (Dube et al., 2024; Li et al., 2024). For example, Prittesh et al. (2020) observed significant variation in chlorophyll content in rice plants treated with different PGPR strains under saline conditions, emphasizing the context-dependent nature of these interactions. In our study, BsW1L treatment under non-stress conditions selectively increased chlorophyll a without significantly affecting chlorophyll b or total chlorophyll content. However, under drought and salt stress, BsW1L markedly enhanced both chlorophyll a and total chlorophyll, while chlorophyll b levels remained unchanged across all treatments. This differential response indicates that BsW1L’s influence on total chlorophyll is stress-dependent, likely reflecting a protective role during periods of elevated oxidative stress. Given that chlorophyll a is predominantly located within the photosystem II reaction centers—key sites for photochemical energy conversion—its selective preservation suggests that BsW1L reinforces core photosystem components rather than augmenting peripheral light-harvesting capacity. In contrast, chlorophyll b, localized mainly in the light-harvesting complex II (LHCII) at the photosystem periphery, is more vulnerable to oxidative degradation. The observed stabilization of chlorophyll a appears to be mediated by enhanced CAT activity, which facilitates H2O2 detoxification and maintains chloroplast integrity. Despite uniform and non-stressful light conditions, the increased ROS burden under drought and salinity, together with limited activation of APX and SOD, may have exacerbated LHCII photooxidation. As LHCII-bound chlorophyll b depends on localized antioxidant defenses for stability, the lack of sufficient enzymatic protection likely impaired its preservation, recovery, or accumulation. The observed reduction in MDA further supports the role of BsW1L in maintaining membrane stability and protecting photosynthetic core structures (Tanaka and Tanaka, 2011). These findings indicate that BsW1L preferentially stabilizes chlorophyll a and preserves photosynthetic efficiency under oxidative stress conditions.
High levels of ROS caused by salinity and drought stress, disrupt plant growth through oxidative damage (Wang et al., 2024). PGPR regulates the antioxidant enzymes’ mRNA expression and their activity in plants to enhance resilience against oxidative damage caused by abiotic stresses (Desoky et al., 2020; Vardharajula et al., 2011). In line with Bharti et al. (2016), our results show that the BsW1L modulates the antioxidant defense system in lettuce under drought and salt stress. BsW1L treatment significantly upregulated CAT gene expression and enzymatic activity under drought and salt stresses, whereas APX expression and activity were enhanced primarily under drought. These findings align with studies in wheat, where PGPR improved antioxidant enzyme activity (Khan et al., 2020; Liu et al., 2020). The lack of increased LsSOD expression and activity under salt stress suggests prioritization of CAT and APX for efficient ROS detoxification, consistent with Sharma et al. (2012). Taken together, despite the lack of significant changes in SOD activity, the enhancement of CAT and APX under stress conditions indicates a selective modulation of the antioxidant defense system by BsW1L.
The differential activation of CAT and APX reflects the distinct oxidative stress signatures imposed by drought and salt stress. The consistent upregulation of CAT expression and its enzyme activity under both conditions-particularly under salt stress-highlights its broad-spectrum ROS-scavenging capacity and essential role in managing cellular H2O2 accumulation (Sharma et al., 2012). Salt stress induces ROS primarily through Na+ toxicity and membrane destabilization, leading to widespread oxidative damage (Miller et al., 2010). The high catalytic efficiency of CAT and its independence from reducing equivalents make it critical for detoxifying excess H2O2 and preventing spillover into the cytoplasm under sustained oxidative stress. In contrast, drought stress leads to stomatal closure and reduced CO2 availability, which disrupts photosynthetic electron transport and enhances ROS generation-particularly singlet oxygen and H2O2-in chloroplasts (Miller et al., 2010). The increased APX expression and activity observed under drought reflects its integration into the ascorbate-glutathione cycle, enabling efficient and localized scavenging of chloroplast-derived H2O2 at the major site of ROS production (Sharma et al., 2012).
Furthermore, MDA was lower in BsW1L-treated seedlings compared to control plants under stress conditions. This reduction aligns with earlier studies showing that decreased lipid peroxidation is a hallmark of enhanced stress tolerance in plants (Bharti et al., 2014; Chiappero et al., 2019). Together, these findings highlight the targeted modulation of antioxidant pathways in BsW1L-treated lettuce, which facilitates ROS detoxification and enhances stress tolerance.
Proline and TSS are pivotal biochemical markers in plant stress responses, contributing to osmotic regulation and stress tolerance (Chiappero et al., 2019; Tiwari et al., 2017). Studies have shown that the application of certain types of PGPR enhances the accumulation of proline and TSS in plants under abiotic stresses like osmotic stress (Liu et al., 2020; Tiwari et al., 2017). For instance, Azotobacter sp. increased plant resilience in response to salt stress by elevating proline levels (Yasin et al., 2018). Similarly, Pseudomonas spp. inoculation in maize under drought stress boosted proline and soluble sugar content, improving stress tolerance (Sandhya et al., 2010). In this study, BsW1L-treated plants under drought stress exhibited elevated proline and TSS levels compared to controls. Under salt stress, however, BsW1L-treated plants showed reduced proline levels but a significant increase in TSS. These findings align with reports that PGPRs can reduce proline levels in stressed plants (Chiappero et al., 2019; Shukla et al., 2012). For example, B. zanthoxyli HS1 reduced proline levels in cabbage under heat stress (Barghi and Jung, 2024), and B. amyloliquefaciens SQR9 decreased proline in maize under salt stress (Chen et al., 2016). The underlying mechanism for reduced proline accumulation under salt stress with BsW1L treatment likely involves enhanced proline catabolism via upregulation of proline dehydrogenase and possible suppression of P5CS, thereby limiting proline biosynthesis. Moreover, improved ion homeostasis, achieved through better Na+ exclusion and K+ retention, further alleviates osmotic stress, diminishing the cellular requirement for proline as an osmoprotectant (Munns and Tester, 2008; Szabados and Savouré, 2010).
The consistent enhancement of TSS by BsW1L under both drought and salt stress suggests that soluble sugars may also serve as central regulators of stress adaptation. Beyond osmoprotection, sugars function as signaling molecules that influence stress-responsive gene expression, modulate hormone signaling pathways (e.g., abscisic acid [ABA] and ethylene), and support antioxidant defenses (Couée et al., 2006). BsW1L may promote carbon allocation toward sugar biosynthesis by regulating key enzymes such as sucrose phosphate synthase and invertase, which could modulate transcription factors and stimulate ROS detoxification pathways, thereby strengthening plant resilience under adverse conditions.
The contrasting patterns of proline and total TSS accumulation under drought versus salt stress reflect fundamentally different cellular strategies for osmotic adjustment and stress mitigation. Under drought stress, the simultaneous increase in proline and TSS represents a classical osmotic adjustment response, where both function as compatible solutes contributing to osmotic regulation, cellular structure stabilization, and ROS scavenging (Hayat et al., 2012; Tiwari et al., 2017). Although proline synthesis from glutamate is metabolically costly, its multifunctional protective properties justify this investment under drought conditions. In contrast, under salt stress, the reduction in proline levels alongside increased TSS suggests a more energy-efficient osmotic strategy. Soluble sugars effectively maintain osmotic balance at a lower metabolic cost compared to amino acid synthesis (Hare et al., 1999). This shift may reflect the PGPR’s enhancement of the plant’s intrinsic salt tolerance mechanisms, reducing the need for proline as an emergency osmolyte. This differential response suggests that the BsW1L strain appears to finely modulate compatible solute production to optimize the metabolic cost-benefit balance of stress tolerance.
Impacts of PGPR on plants in response to abiotic stresses vary depending on the bacterial strain, plant species, and stress type (Mishra et al., 2011; Rodríguez-Vázquez and Mesa-Marín, 2023). For instance, Bacillus strains enhance antioxidant activity in potatoes under drought stress (Gururani et al., 2013) but reduce it in wheat (Kasim et al., 2013). Similarly, Pseudomonas spp. exhibit variable effects on wheat depending on the type of stress encountered (Ali et al., 2011; Bakaeva et al., 2022). This variability is evident in our study, where BsW1L differentially modulated antioxidant responses and proline levels under drought and salt stress, suggesting a targeted activation of biochemical pathways to optimize plant survival under specific conditions.
Our research suggests that the rhizospheric BsW1L strain may enhance induced systemic tolerance (IST) in lettuce seedlings under drought and salt stress by modulating oxidative stress responses and osmolyte accumulation. While these responses align with features of induced IST, we acknowledge the absence of direct evidence, leaving IST as a plausible hypothesis that requires further molecular and spatial validation. Notably, BsW1L may conditionally modulate tolerance depending on the type and presence of stress, potentially minimizing fitness trade-offs. These findings advance our understanding of plant-microbe interactions under abiotic stress and highlight the strain’s potential for developing sustainable and stress-resilient cropping systems. Further validation under field conditions is required to support its practical application.
The Na+/K+ ratio plays a critical role in mitigating salt-induced osmotic and ionic stress by preserving cellular integrity and metabolic functions. Salt stress typically leads to Na+ accumulation and K+ depletion, disrupting ion homeostasis and impairing physiological processes. Several PGPRs have been documented to enhance plant salt tolerance through mechanisms involving Na+ exclusion and K+ retention, which regulate ion transporter expression and maintain favorable Na+/K+ ratios (Sharifi and Ryu, 2017). Although ion concentrations were not directly assessed in this study, the enhanced stress tolerance observed in BsW1L-treated lettuce suggests a potential role in maintaining ionic balance similar to these established PGPR mechanisms. Future studies should investigate whether BsW1L contributes to Na+ exclusion or K+ retention, possibly via the regulation of ion transporter genes. To further clarify the molecular basis of BsW1L-induced stress tolerance, future studies should include expression profiling of key ABA biosynthesis and signaling genes, along with proline biosynthesis genes such as P5CS, to elucidate the proposed stress-specific regulatory mechanisms. Additionally, identifying bioactive compounds released by BsW1L and their molecular targets will be crucial for developing innovative and sustainable agricultural strategies to address environmental challenges.
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