Harnessing Rhodopseudomonas palustris strains for salt stress mitigation in Arabidopsis thaliana

Abstract

Background

Soil salinity severely limits plant growth and agricultural productivity. 5-Aminolevulinic acid (ALA), a precursor in tetrapyrrole biosynthesis, has been reported to alleviate salinity stress and is frequently proposed as a key component by which purple non-sulfur bacteria enhance plant stress tolerance. This study compared the treatments of three Rhodopseudomonas palustris strains (PS3, TPN1, and YSC3) with ALA under salinity stress to understand the importance of ALA production for their potential ability to alleviate salinity stress, using Arabidopsis thaliana, a salt-sensitive model plant in which NaCl concentrations above 30 mM induce growth inhibition and physiological stress responses, making it well suited for assessing salinity tolerance mechanisms.

Results

Both bacterial and ALA treatments increased the photosynthetic efficiency, root growth, relative water content, and oxidative balance of salt-stressed plants. These treatments maintained chlorophyll biosynthetic capacity and modulation of ion transport-related responses, consistent with improved ionic homeostasis under salinity stress. Among the strains, TPN1 performed the best, exhibiting altered expression of antioxidant genes, reduced lipid peroxidation, and decreased electrolyte leakage, which indicates improved membrane integrity. The outcomes were associated with the ability of TPN1 to maintain halotolerance and key plant growth-promoting traits, including the production of extracellular polysaccharides and indole-3-acetic acid, under high salinity. Notably, strains with comparatively lower extracellular ALA outputs conferred benefits comparable to those observed with ALA treatment, suggesting that plant stress mitigation is not solely dependent on ALA concentration.

Conclusions

We identified ALA-producing R. palustris strains, particularly TPN1, that help enhance plant tolerance to salinity through coordinated changes in ion regulation, antioxidant balance, and photosynthetic performance, and demonstrate that ALA production may not be the primary factor contributing to R. palustris' enhancement of plant salt tolerance.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-026-08255-w.

Introduction

Soil salinization, exacerbated by climate change and poor irrigation practices, poses a critical threat to global food security. More than 20% of cultivated land is affected by salinity, a figure projected to reach 50% by 2050 [1–3]. Saline soils, typically defined by an electrical conductivity equivalent to or above 4 dS m-1 (≈ 40 mM NaCl), can reduce crop yields by 50–80%, depending on the plant species [1, 4]. Excess sodium chloride (NaCl) causes osmotic and oxidative stress, disrupts ion homeostasis, and ultimately suppresses plant growth and productivity [5, 6]. These adverse effects highlight the urgent need to develop effective strategies to increase crop salt resilience [7, 8].

Biological strategies, such as salt-tolerant cultivars and beneficial soil microorganisms, help maintain ion homeostasis, osmotic balance, and antioxidant capacity under saline conditions [9–11]. Among these, plant growth-promoting rhizobacteria (PGPR) are widely recognized for promoting root development, regulating phytohormone levels, and modulating stress-responsive metabolic pathways [12–14].

Additionally, the exogenous application of 5-aminolevulinic acid (ALA), a universal metabolic precursor in tetrapyrrole biosynthesis [15, 16], has been widely reported to enhance photosynthesis, antioxidant defenses, and osmotic adjustment under salt stress [17–19]. Consequently, ALA-producing PGPR, such as those of purple non-sulfur bacteria (PNSB), are frequently proposed to confer salt stress tolerance primarily through microbial ALA production [20–23]. For example, the culture supernatants of R. palustris TN114, an ALA-producing PNSB, have been reported to promote rice growth under salinity stress by increasing the chlorophyll content and antioxidant enzyme activity [22, 24, 25]. However, it remains unclear whether ALA production alone is sufficient to explain these beneficial effects. Most studies have relied on ALA applications or comparisons of ALA-producing strains [18, 20, 26] to assume that microbial ALA supply is the dominant driver of plant stress mitigation [21, 24, 25]. Direct comparisons among multiple ALA-producing strains with ALA applications are scarce. Moreover, PGPR exhibit a range of beneficial traits, including phytohormone modulation, exopolysaccharide production, and ACC deaminase activity, that have been independently linked to enhanced plant tolerance to salinity [27, 28]. The relative contributions of these traits compared with ALA production therefore remain unresolved.

To address this gap, we examined three R. palustris strains- PS3, TPN1, and YSC3-previously isolated from paddy fields by our group [28]. Although all three strains harbor key ALA biosynthetic genes (hemA and hemO) [29], preliminary analyses revealed substantial variation in their ALA production capacities and salt tolerance-related traits. This diversity enables direct assessment of whether microbial ALA output correlates with, or sufficiently explains, PNSB-mediated salt tolerance.

Arabidopsis thaliana Col-0 was selected as a model system for mechanistic evaluation of salt stress due to its well-characterized salt sensitivity [30, 31]. By integrating physiological, biochemical, and molecular analyses, this study compares the effects of different ALA-producing R. palustris strains with ALA applications under salinity stress to examine whether strain-specific microbial functions contribute independently of ALA production.

Materials and methods

Evaluation of the salt tolerance of different R. palustris strains

The R. palustris strains, PS3 (BCRC910564/DSM 29314), TPN1, and YSC3, were isolated from farmlands in northern Taiwan. Salt tolerance was assessed in PNSB media, as previously described [32], supplemented with NaCl at concentrations ranging from 10 to 20 g L⁻¹ (≈ 171 to 342 mM). The original PNSB medium contained 0.5 g L⁻¹ NaCl (≈ 8.55 mM NaCl). The strains were grown in liquid PNSB at 37 °C with shaking at 220 rpm for 21 h to stationary phase. Each bacterial culture was prepared in triplicate. 10 μL of each culture (1 × 10⁹ CFU mL⁻¹) was seeded onto PNSB agar plates containing the corresponding NaCl concentration and incubated at 37 °C for 72 h. Salt tolerance was classified according to the Larsen scale [33].

Determination of the bacterial growth curve of different R. palustris strains

For growth curve analysis, single colonies were pre-cultured in liquid PNSB media at 37 °C with shaking at 220 rpm for 21 h. Subsequently, 5 μL of each culture was inoculated into 50 mL fresh PNSB media supplemented with 10, 15, or 20 g L⁻¹ NaCl. Cultures were incubated at 37 °C with shaking at 220 rpm. Bacterial growth was monitored every hour for over 30 h by determining colony-forming units (CFU mL⁻¹). Growth curves were plotted as log CFU mL⁻¹ versus time. All experiments were conducted in triplicate.

Quantitative analysis of ALA production

For preliminary quantification, ALA production in bacterial cultures was determined after 48 h of culture, following the method described by Burnham [34], which employs modified Ehrlich's reagent. For a more precise quantitative analysis of ALA, liquid chromatography‒mass spectrometry (LC‒MS) was performed as described previously [35], with some modifications. A total of 20 μL of either the ALA standard or bacterial sample was added to 80 μL of acetonitrile, followed by centrifugation at 13,000 × g for 5 min to precipitate proteins and derivatize the samples. The supernatant was dried under nitrogen gas, redissolved in 100 μL of 95% acetonitrile with 0.1 M HCl, and then filtered through a nylon membrane filter (0.22 μm) (Sartorius, Germany). Chromatographic separation was carried out on an Atlantis Premier BEH Z-HILIC column (2.1 × 100 mm, 1.7 μm particle size; Waters, USA) via an Ultimate 3000 UHPLC system (Thermo Scientific, USA) with the column oven maintained at 40 °C. The mobile phases consisted of the following: solvent A, 0.1% formic acid in acetonitrile; solvent B, 0.1% formic acid in water; and solvent C, 100 mM ammonium formate (HCOO-NH₄) in water. A 2 μL injection volume was used. Separation was achieved via gradient elution at a constant flow rate of 0.4 mL min⁻¹ (Table S3). Detection was performed on a Q Exactive mass spectrometer (Thermo Scientific, USA) equipped with an electrospray ionization (ESI) source operating in positive ionization mode. The MS parameters are provided in Table S4.

Determination of extracellular polysaccharides produced by different R. palustris strains

Extracellular polysaccharide (EPS) production by R. palustris strains was assessed following the methods of Aoudi et al. [36], with modifications. Strains were cultured on nPNSB agar (± 3% glucose) and incubated at 37 °C for 72–96 h, where mucoid colony morphology was taken as indicative of EPS production. For the salt stress assays, the plates were supplemented with 10, 15, or 20 g L⁻¹ NaCl. For EPS extraction, strains were grown in 50 mL of nPNSB broth at 37 °C with shaking (220 rpm) until the OD₆₀₀ = 1.0. Cultures were heat-treated (100 °C, 15 min) and centrifuged (12,000 × g, 10 min, 25 °C), and the supernatant was filtered through a 0.20 µm membrane. EPSs were precipitated with 2 volumes of cold 95% ethanol, incubated overnight at 4 °C, and pelleted by centrifugation. The crude EPS was redissolved in distilled and deionized water (DDW), freeze-dried (72 h), and quantified.

Plant material and growth conditions

Arabidopsis thaliana Columbia-0 (Col-0) seeds were surface sterilized and sown on half-strength Murashige and Skoog (1/2 MS) media supplemented with 0.8% (w/v) Bacto-agar (Focus Bio, Australia) [37]. The plates were sealed with 3 mm Micropore tape to prevent dehydration and contamination and placed horizontally in a growth chamber under the following conditions: 12 h light/12 h dark photoperiod, 74 - 93 μmol m⁻² s⁻¹ (LED, Philips GreenPower, Netherlands) light intensity, constant temperature of 23 °C and relative humidity of 60 - 70%.

Determination of plant growth-promoting traits under salt stress

Distinct NaCl concentrations were employed for bacterial and plant assays to account for differences in osmotic sensitivity and ion regulation. Bacterial cultures required higher NaCl levels (g L^-1 range) to induce physiological stress comparable to that observed in plants (mM range) [10, 38].

Col-0 seedlings were initially grown on half-strength MS media for 7 days, then transferred to treatment plates and monitored for an additional 21 days to assess their physiological responses under normal and salt-stress conditions. Two experimental conditions were established: (a) nonstress control and (b) salt stress control. For the nonstress experiments, the seedlings were transferred to fresh 1/2 MS media and grown at 23 °C. For the salt stress treatments, the seedlings were transferred to 1/2 MS media supplemented with 50 mM NaCl. Under both conditions, the roots of each seedling were inoculated individually with one of the following treatments: 20 μL of bacterial suspension (final density = 1 × 10⁷ CFU mL⁻¹; prepared by diluting stationary-phase cultures (≈ 1 × 10⁹ CFU mL⁻¹) to the desired concentration), 20 μL of 10 mg L⁻¹ commercial ALA (Sigma-Aldrich, USA), or 20 μL of distilled and deionized water (mock treatment). Plants subjected to different treatments were cultivated under the conditions mentioned above. Each treatment consisted of 15 biological replicates (n = 15). A concentration of 50 mM NaCl was used for agar plate assays because it consistently induced measurable growth inhibition in Arabidopsis without causing severe developmental arrest under in vitro conditions [32, 33].

Root length, number of lateral roots, number of leaves, and fresh weight (biomass) were determined to assess morphophysiological responses. The plants were carefully removed from the Petri dishes, and their roots were gently cleaned of adhering agar using tweezers. To further evaluate the potential of R. palustris strains to promote plant growth and enhance salt stress tolerance in Arabidopsis thaliana (Col-0), preliminary pot trials were conducted under 100 mM NaCl stress, as described in the supplementary information. A higher NaCl concentration was used to account for soil buffering capacity, ion adsorption, and spatial heterogeneity, which reduce the effective salt stress experienced by plant roots compared to agar-based systems [39]. For both plate and pot experiments, bacterial inoculation and ALA supplementation were applied once at the start of the experiment, prior to the imposition of salt stress (50 or 100 mM NaCl).

Photosynthetic efficiency and pigment analysis

The maximum efficiency of photosystem II (PSII) activity (Fv/Fm) was determined via a fluorometer (Hansatech Instruments Ltd., United Kingdom) as an indicator of PSII functionality and potential stress-induced damage. The total chlorophyll (Chl; Chl a + Chl b) content and chlorophyll a/b ratio was determined spectrophotometrically (Ultrospec 2100 Pro, United Kingdom) following the methods of Cohen et al. [40].

Determination of hydrogen peroxide (H₂O₂) content

The H₂O₂ content was determined to evaluate the effectiveness of the R. palustris strains in scavenging H₂O₂, following a method described previously [41]. Briefly, 100 mg of leaf tissue from each treatment group was homogenized in an ice bath with 5 mL of 0.1% (w/v) trichloroacetic acid (TCA) (Bioman, China). The homogenate was centrifuged at 12,000 × g for 15 min, and 0.5 mL of the supernatant was mixed with 0.5 mL of 10 mM potassium phosphate buffer (pH 7.0) (J.T. Baker, USA) and 1 mL of 1 M potassium iodide (KI) (Sigma Aldrich, USA). The absorbance was measured at 390 nm via an Ultrospec 2100 Pro spectrophotometer (Amersham Biosciences, UK), and the H₂O₂ concentration was determined via a standard calibration curve.

Determination of nonenzymatic antioxidant activity

The total phenolic content (TPC) was determined via the Folin‒Ciocalteu method as described previously [42]. One mL of extract (200 μg mL⁻¹) was mixed with 2 mL of 10% (w/v) Folin-Ciocalteu reagent (Sharlab, Spain). After 5 min, 2 mL of 7.5% Na₂CO₃ (Bio Basic, Canada) was added, and the mixture was incubated at 50 °C for 10 min with intermittent agitation. The reaction mixture was then cooled, and the absorbance was measured at 765 nm via a UV spectrophotometer (Ultrospec 2100 Pro, UK). A reagent blank without extract was used as a reference. The outcome data were expressed as mg of gallic acid equivalents per gram of dry extract (mg GAE⁻¹ g⁻¹). Flavonoid and anthocyanin contents were determined spectrophotometrically as described previously [43].

Determination of antioxidant enzyme activity

For the antioxidant enzyme assays, 0.1 g of excised leaves from each treatment group was ground and homogenized in 1 mL of 50 mM sodium phosphate buffer (pH 6.8) (Bio Basic, Canada). The homogenate was centrifuged at 12,000 × g for 20 min at 4 °C, and the supernatant was filtered through a Millex® membrane (Merck Millipore, Cork, Ireland) prior to use in triplicate enzyme activity assays.

Catalase (CAT) (EC 1.11.1.6) activity was determined via a previously described method [44]. The assay was conducted at 30 °C in a 1 mL reaction mixture containing 0.2 mL of enzyme extract and 0.7 mL of 100 mM sodium phosphate buffer (pH 7.0) (Bio Basic, Canada) supplemented with 1 M freshly prepared H₂O₂ (Sigma Aldrich, USA). CAT activity was determined spectrophotometrically by measuring the decomposition of H₂O₂ at 240 nm. A standard curve was prepared using known H₂O₂ concentrations, and the CAT activity in the samples was calculated accordingly.

Ascorbate peroxidase (APX) (EC 1.11.1.11) activity was determined according to previous work [45] by measuring the decrease in absorbance at 290 nm resulting from ascorbate oxidation. The 3 mL reaction mixture contained 150 mM potassium phosphate buffer (pH 7.0) (J.T. Baker, USA), 0.75 mM EDTA (J.T. Baker, USA), 1.5 mM freshly prepared ascorbic acid (Merck, Germany), and 6 mM freshly prepared H₂O₂ (Sigma Aldrich, USA).

Determination of water status, proline content, lipid peroxidation, and electrolyte leakage

The relative water content (RWC) of the leaves was determined via the following equation:

where FW, FTW and DW denote the leaf fresh weight, fully turgid weight (after 48 h of immersion in distilled and deionized water), and leaf dry weight (after oven drying at 60 °C to constant mass), respectively.

Proline content was determined following a previously described method (Bates et al., 1973), with modifications [46]. Briefly, 0.5 g of each leaf sample was ground and homogenized in 2.5 mL of 3% (w/v) sulfosalicylic acid (Sigma Aldrich, USA). Insoluble polyvinylpolypyrrolidone (PVPP; 250 mg) (Sigma Aldrich, USA) was added, and the mixture was vortexed for 30 s and then centrifuged at 9,300 × g for 10 min (TOMY MX-305 high-speed refrigerated microcentrifuge, Japan). Two millilitres of the supernatant were mixed with 2 mL of glacial acetic acid (99.8% ACS grade) (Fisher Scientific, USA) and 2 mL of 2.5% (w/v) acid ninhydrin (Thermo Scientific, USA) at 100 °C for 1 h. The mixture was cooled in an ice bath, and the reaction mixture was then extracted with 4 mL of toluene (vortex-mixed vigorously for 1 min). The absorbance of the toluene phase was measured at 520 nm via a 10 mm optical path cell (Ultrospec 2100 Pro, UK). The proline content was determined from a standard curve and expressed on a fresh weight basis.

The content of malondialdehyde (MDA), an indicator of lipid peroxidation, was determined as described previously [47]. Approximately 0.1 g of each leaf sample was homogenized in 1% (w/v) trichloroacetic acid (TCA) and centrifuged at 10,000 × g for 5 min at 20 °C. The supernatant (0.5 mL) was mixed with 1.5 mL of a solution containing 20% TCA and 0.5% thiobarbituric acid (Sigma Aldrich, USA), vortexed for 15 s, and incubated in a water bath at 95 °C for 60 min. The mixture was chilled in an ice bath and centrifuged again at 9,300 × g for 10 min. Absorbance was assessed at 532 nm and corrected for nonspecific absorbance at 600 nm (Ultrospec 2100 Pro, UK). The MDA content was calculated via an extinction coefficient of 155 mM⁻¹ cm⁻¹.

Electrolyte leakage was determined according to a previous report [48]. Fifteen leaf discs (1 cm in diameter) from the third node (from the base) were rinsed with deionized water and placed in 30 mL of DDW in 50 mL Falcon tubes. After 48 h at room temperature in the dark, the initial conductivity (EC₁) was determined via a conductivity meter (Ohaus, ST3100M-F, China). The samples were then incubated at 95 °C for 20 min and cooled to room temperature, after which the final EC (EC₂) was determined. Electrolyte leakage was determined according to the equation: (EC₁/EC₂) × 100.

Expression analysis of genes related to ALA metabolism, the antioxidant response, and salt tolerance

Total RNA was extracted from Col-0 seedlings via TRIzol reagent (Invitrogen, USA) and the Direct-zol™ RNA MiniPrep Kit (Zymo Research, USA) following the instructions provided by the manufacturer. RNA samples (2.2 μg) were treated with the TURBO DNA-free™ Kit (Invitrogen, USA) to remove genomic DNA contamination. First-strand cDNA was subsequently synthesized via SuperScript™ IV Reverse Transcriptase (Invitrogen, USA) with oligo(dT)₂₀ primers. The quantity and quality of the RNA were measured via a NanoDrop ND-1000 spectrometer (NanoDrop Technologies, Inc., DE, USA).

Quantitative reverse transcription‒PCR (RT‒qPCR) was conducted using LightCycler 480 SYBR Green I Master Mix (Roche Diagnostics, Germany). Each 10 μL reaction contained 4.5 μL of cDNA template (50-fold diluted), 5 μL of SYBR Green I Master Mix, 0.5 μL of each primer (10 mM), and nuclease-free water. The thermocycling conditions were as follows: preincubation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 1 s.

Gene expression was normalized to that of the ACTIN2 (AT3G18780) housekeeping gene as described previously [49]. Relative expression levels were calculated as log₂-fold changes via the 2^−ΔΔCT method [50]. The expression profiles of genes involved in ALA metabolism (HEMA1 (AT1G58290) and CHLH (AT5G13630)), the ROS pathway (CAT2 (AT4G35090), APX1 (AT1G07890), and APX2 (AT3G09640)), and salt tolerance (NHX1 (AT5G27150) and HKT1 (AT4G10310)) were analyzed via RT‒qPCR. The primer sequences are listed in Table S7. Before conducting gene expression analysis, primer sets were examined for their amplification specificity by analyzing the melting curve and sequence of the amplicons.

Statistical analysis

All the statistical analyses were performed via R (version 4.3.3, 2024–02–29). One-way and two-way ANOVA were performed to evaluate treatment effects. For two-way ANOVA, the main effects of salt stress, treatment, and their interaction were evaluated. Post hoc comparisons were conducted via Tukey’s HSD test at a significance level of P < 0.05.

Results

Quantification of extracellular ALA production in three R. palustris strains

Extracellular 5-aminolevulinic acid (ALA) production by R. palustris strains PS3, TPN1, and YSC3 under general growth conditions (0.5 g L⁻¹ NaCl) was preliminarily quantified via colorimetric analysis (Table S1). After 48 h of incubation, PS3 produced the highest ALA concentration (48.98 ± 9.80 μM), followed by TPN1 (47.28 ± 14.72 μM) and YSC3 (21.35 ± 5.81 μM). Although colorimetric assays provide a simple method to quantify 5-ALA, they generally show bias due to low specificity, limited sensitivity, and matrix interference. To ensure accurate quantification of extracellular ALA, LC‒MS analysis was employed to reveal strain- and salinity-dependent variations among the three R. palustris strains (Table 1). Although lower yields were detected with this method, at 0.5 g L⁻¹ NaCl, PS3 consistently produced the highest ALA concentration (2.50 ± 0.30 μM), followed by TPN1 (1.48 ± 0.31 μM) and YSC3 (0.95 ± 0.13 μM). However, at 10 g L⁻¹ NaCl, only TPN1 presented increased ALA production and the highest production (2.03 ± 0.21 μM), exceeding that of PS3 (1.35 ± 0.17 μM) and YSC3 (0.86 ± 0.15 μM). At 15 g L⁻¹ NaCl, the extracellular ALA levels decreased markedly across all the strains; the PS3 and TPN1 concentrations fell below the detection limit (< 0.5 μM), whereas YSC3 maintained a comparatively stable level (0.74 ± 0.11 μM). The extracellular ALA concentrations detected via LC–MS (0.86 - 2.50 μM at 0.5 - 10 g L^-1 NaCl) were substantially lower than the synthetic ALA dose of 10 mg L^-1 (~ 75 μM) applied in the plant assays [51]. This discrepancy suggests that the physiological effects of bacterial inoculation may not be attributed solely to extracellular ALA levels, instead reflect localized delivery, cumulative production over time, or synergistic bacterial traits. Moreover, the intracellular ALA produced by all R. palustris strains was undetectable by LC–MS, except for TPN1 at 15 g L^-1 NaCl, which was detectable but unquantified as it fell below the quantification limit (< 0.5 μM) (Table S2). The extremely low intracellular ALA represents a limitation of the present study when directly comparing bacterial ALA production with exogenous ALA application.

Table 1.

Effect of salinity on extracellular 5-ALA production by R. palustris strains, as quantified by LC‒MS analysis

R. palustris strains	NaCl concentrations (g L−1)	5-ALA production (μM)
PS3	0.5	2.50 ± 0.30a
	10	1.35 ± 0.17bcd
	15	< 0.50e
TPN1	0.5	1.48 ± 0.31bc
	10	2.03 ± 0.21ab
	15	< 0.5e
YSC3	0.5	0.95 ± 0.13 cd
	10	0.86 ± 0.15cde
	15	0.74 ± 0.11de

Data represent the means ± SDs (n = 3 biological replicates, each from independently cultured flasks). Different letters indicate statistically significant differences according to Tukey’s HSD test (p < 0.05)

Salt tolerance, growth response and EPS formation of R. palustris strains under varying NaCl concentrations

We evaluated the growth of three R. palustris strains cultivated with different concentrations of NaCl. Bacterial growth was monitored over 30 h by measuring colony-forming units (CFU mL⁻¹) on a logarithmic scale. Under general growth conditions (0.5 g L⁻¹ NaCl, i.e., ≈ 8.56 mM NaCl in nPNSB medium), all strains presented similar growth rates (Fig. 1A). NaCl supplementation inhibited bacterial growth in a concentration-dependent manner, with strain-specific differences in tolerance. TPN1 could tolerate up to 20 g L⁻¹ (≈ 342.23 mM) NaCl, whereas PS3 and YSC3 could tolerate up to 15 g L⁻¹ NaCl (≈ 256.80 mM) (Fig. 1C). According to Larsen’s classification [33], all three strains can be classified as slightly resistant (Table S5).

Fig. 1.

Effect of salinity on the growth of R. palustris strains PS3, TPN1 and YSC3 under (A) control (0.5 g L⁻¹), (B) 10 g L⁻¹ NaCl, (C) 15 g L⁻¹ NaCl, and (D) 20 g L⁻¹ NaCl in nPNSB medium

To assess the effect of salinity on EPS, the EPS yields were quantified under the same conditions. All strains exhibited a salt-inducible but salt-sensitive EPS production profile, with maximum secretion observed at 10 g L⁻¹ NaCl, followed by a marked decrease at higher concentrations (Table 2). Strains TPN1 and YSC3 exhibited the highest EPS dry weights (14.09 g L⁻¹ and 14.02 g L⁻¹, respectively) at 10 g L⁻¹ NaCl. Although increased EPS production under moderate salinity may be associated with bacterial stress responses, its composition and the mechanisms underlying its ion-binding capacity and potential roles in Na⁺ sequestration, rhizosphere modification, or plant stress mitigation remain hypothetical and warrant further study [52].

Table 2.

Effect of salinity on the EPS dry weight of R. palustris strains

R. palustris strains	NaCl concentrations (g L−1)	EPS dry weight (g L−1)
PS3	0.5	10.49 ± 1.36ab
	10	10.15 ± 1.44ab
	15	4.91 ± 0.52 cd
	20	0.86 ± 0.45d
TPN1	0.5	10.46 ± 1.17ab
	10	14.09 ± 0.71a
	15	7.20 ± 1.06bc
	20	1.18 ± 0.81d
YSC3	0.5	10.51 ± 0.48ab
	10	14.02 ± 1.13a
	15	6.04 ± 0.90bc
	20	1.08 ± 0.47d

Data represent the means ± SDs (n = 3 biological replicates, each from independently cultured flasks). Different letters indicate statistically significant differences according to Tukey’s HSD test (p < 0.05)

Preliminary screening for the optimal exogenous ALA concentration

Transgenic Arabidopsis carrying a yeast ALA synthase gene (YHem1) can increase endogenous ALA levels and improve plant tolerance to salt stress [53]. However, a suitable exogenous ALA concentration for Arabidopsis has not been determined. To identify the optimal concentration for growth promotion in A. thaliana Col-0, we treated 7-day-old seedlings with 5, 10, 15, or 20 mg L⁻¹ ALA for 7 days under non-stress conditions. As shown in Figure S1, treatment with 10 mg L⁻¹ ALA significantly increased the biomass (94.48%), root length (65.97%), and lateral root number (239.24%) relative to those of the untreated controls. Moreover, the 10 mg L⁻¹ ALA treatment improved the photosynthetic efficiency, increasing the F_v/F_m ratio by 2.90% and the total chlorophyll content by 35.10% (Figures S2A and S2B, respectively). The Chl a/b ratio was markedly reduced due to the high Chl a content in the plants under the 10 mg L⁻¹ ALA treatment (Figure S2B). On the basis of these findings, 10 mg L⁻¹ ALA was identified as the most effective treatment and was selected for subsequent experiments.

R. palustris inoculation enhances vegetative growth under saline stress

Compared with the mock treatment, both the ALA (10 mg L⁻¹) treatment and R. palustris inoculation (2.0 × 10⁷ CFU per plant) significantly improved A. thaliana growth, as indicated by increased biomass, root length, leaf number, and lateral root growth under both non-stressed and saline-stressed conditions, although salinity markedly retarded plant growth (Fig. 2). Notably, inoculation with strains PS3 and TPN1 enhanced vegetative growth more effectively than did ALA treatment, with TPN1 showing the most pronounced effects across all parameters. Compared with the saline-stressed control, TPN1 increased the biomass by 41.38% (Fig. 2B), root length by 51.64% (Fig. 2C), lateral root number by 48.17% (Fig. 2D), and leaf number by 28.20% (Fig. 2E).

Fig. 2.

Effect of ALA treatment and R. palustris inoculation on the growth performance of A. thaliana Col-0 plants under non-stress and saline (50 mM NaCl) conditions. A Representative photographs of plants under non-stress and saline conditions, comparing uninoculated controls with plants treated with ALA (10 mg L⁻¹) or plants inoculated with R. palustris strains PS3, TPN1, and YSC3 (2.0 × 10⁷ CFU per plant, respectively). B-E Quantitative analysis of vegetative growth traits under the same conditions: (B) biomass (mg), (C) root length (cm), (D) number of lateral roots, and (E) number of leaves. Data represent means ± SD (n = 15 individual plants). Different letters indicate statistically significant differences according to Tukey’s HSD test (p < 0.05)

The preliminary pot trials revealed trends consistent with those of the plate assays. Both ALA and R. palustris strains, especially PS3 and TPN1, significantly increased vegetative growth parameters (Figure S3A). Among the treatments, TPN1 performed slightly better than ALA did, increasing the biomass by 29.96% (Figure S3B), root length by 183.58% (Figure S3C), leaf number by 41.38% (Figure S3D), and lateral root number by 66.67% (Figure S3E) compared with those of the nontreated control plants.

Under saline conditions, these effects were even more pronounced. The TPN1 and PS3 treatments both outperformed ALA, with the TPN1-inoculated plants exhibiting the most vigorous morphology under 100 mM NaCl stress. Specifically, compared with saline-stressed, non-inoculated control plants, TPN1-treated plants presented substantial increases in biomass (358.48%) (Figure S3B), root length (160%) (Figure S3C), leaf number (87.5%) (Figure S3D), and lateral root number (150%) (Figure S3E).

R. palustris inoculation improves A. thaliana photosynthetic efficiency under saline stress

Under non-stressed conditions, the maximum quantum yield of photosystem II (PSII) (Fv/Fm) remained consistent (0.80 - 0.83) across all the treatments (Fig. 3A). Under saline stress, the lowest Fv/Fm value (0.75) was recorded in the mock treatment control. Both ALA treatment and R. palustris inoculation significantly increased the Fv/Fm ratio to 0.78, reflecting enhanced photosynthetic performance. However, no significant differences were observed among the experimental treatments under saline stress (Fig. 3A). The pot trials displayed a similar pattern (Figure S4A). Under saline stress, only R. palustris-inoculated plants presented a statistically significant recovery of Fv/Fm from 0.77 in the saline-stressed control to 0.80 in the PS3- and YSC3-inoculated plants and 0.81 in the TPN1-inoculated plants. In contrast, ALA-treated plants presented a modest increase to 0.79.

Fig. 3.

Effect of ALA treatment and R. palustris inoculation on photosynthetic efficiency and chlorophyll content in A. thaliana Col-0 under non-stress and saline (50 mM NaCl) conditions. A Maximum quantum efficiency of PSII (Fv/Fm), (B) total chlorophyll content (μg mg⁻¹ FW), (C) chlorophyll a (Chl a) and chlorophyll b (Chl b) levels and (D) ratio of chlorophyll a (Chl a) to chlorophyll b (Chl b) contents. Plants were either uninoculated (control), treated with exogenous ALA (10 mg L⁻¹), or inoculated with R. palustris strains PS3, TPN1, and YSC3 (2.0 × 10⁷ CFU per plant, respectively). Data represent means ± SD (n = 15 individual plants). Different letters indicate statistically significant differences according to Tukey’s HSD test (p < 0.05)

Compared with the mock treatment, inoculation with R. palustris strains also increased the total chlorophyll content under both non-stressed and saline-stressed conditions (Fig. 3B). Under non-stressed conditions, TPN1 presented the greatest increase (19.41%), followed by YSC3 (16.78%), ALA (15.30%), and PS3 (11.68%), although these differences were not statistically significant (Fig. 3B). Under saline stress, the chlorophyll content increased by 20.30% with TPN1, followed by 15.53% with YSC3 and 2.4% with PS3, whereas ALA treatment resulted in an 18.95% increase compared with the mock treatment. Pot experiments further confirmed the superior effect of TPN1, which increased the chlorophyll content by 49.87% under non-stressed conditions and by 124.40% under saline stress compared with the respective controls (Figure S4B).

In non-stressed plants, the chlorophyll a/b ratios ranged from 3.22 ± 0.07 (ALA) to 3.54 ± 0.60 (PS3) and 3.11 ± 0.10 (YSC3), with TPN1-treated seedlings having a value of 3.69 ± 0.19, whereas the value was 3.03 ± 0.70 in the control (Fig. 3C). Under saline stress, the ratio decreased to 1.36 ± 0.37 in the control plants. ALA treatment partially mitigated this decrease (2.01 ± 0.02), whereas PS3 and TPN1 restored the ratio to 2.36 ± 0.9 and 2.83 ± 0.28, respectively. YSC3 maintained an intermediate value of 2.09 ± 0.28. The change in the chlorophyll a/b ratio was due to a reduction in the chlorophyll a content; however, the chlorophyll b content did not significantly change under salinity stress (Fig. 3B and C). These results suggest that both TPN1 and PS3 are more effective at stabilizing chlorophyll under salt stress, thereby indicating improved stability of the light-harvesting complex under these conditions.

R. palustris inoculation enhances the physiological resilience of A. thaliana under saline stress

H₂O₂ accumulation, a hallmark of oxidative stress, was significantly reduced under saline conditions by all the treatments (Fig. 4A). Compared with the mock treatment, each treatment effectively mitigated oxidative stress under salt stress. Although PS3- and TPN1-inoculated plants presented the greatest numerical reductions in H₂O₂ levels (31.06% and 30.31%, respectively), these values were not significantly different from the reduction observed with ALA treatment (25.86%).

Fig. 4.

Effect of ALA treatment and R. palustris inoculation on oxidative stress markers, secondary metabolite levels and antioxidant enzyme activities in A. thaliana under non-stress and saline (50 mM NaCl) conditions. A Hydrogen peroxide (H₂O₂) content, (B) anthocyanins levels, (C) Flavonoid content, (D) total phenolic compounds (TPC), E) Catalase (CAT) activity, and (F) Ascorbate peroxidase (APX) activity was measured in A. thaliana Col-0 plants grown under non-stress and saline (50 mM NaCl) conditions. Treatments included uninoculated controls, ALA application (10 mg L⁻¹), and inoculation with PS3, TPN1, and YSC3 (2.0 × 10⁷ CFU per plant, respectively). Data represent means ± SD (n = 15 individual plants). Different letters indicate statistically significant differences according to Tukey’s HSD test (p < 0.05)

Compared with the untreated saline-stressed control, TPN1 significantly increased the anthocyanin content by 233.75% (Fig. 4B) and the flavonoid content by 155.30% (Fig. 4C). In contrast, ALA-treated plants presented a 52.50% increase in anthocyanin content and a 60.52% increase in flavonoid content. Under non-stressed conditions, the total phenolic content (TPC) did not differ significantly between untreated and treated plants (Fig. 4D). However, under saline stress, only the TPC of the TPN1-inoculated plants significantly increased, with a 56.06% increase relative to that of the untreated saline-stressed control plants.

Antioxidant enzyme activities were also significantly affected by salinity. Both catalase (CAT) and ascorbate peroxidase (APX) activities were induced by salinity in mock treatment controls. Similarly, the enzyme activity was greater in the ALA-treated plants under saline stress than in the non-stressed plants. CAT activity, however, did not significantly change, and APX1 activity was significantly lower in all R. palustris-inoculated plants under saline stress than under non-stressed conditions (Fig. 4E, F). Under non-saline conditions, the CAT activity did not increase significantly with either ALA treatment or bacterial inoculation compared with that of the untreated control. However, under saline stress, these strains reduced the CAT activity by 77.99%, 79.18% and 78.69%, respectively. In contrast, ALA treatment increased the CAT activity by 41.17% relative to that of the salt-stressed control. A similar pattern was observed for APX activity (Fig. 4F). Under non-stressed conditions, ALA treatment increased APX activity by 26.37%, whereas the APX activity in the PS3, TPN1, and YSC3 treatments significantly increased by 62.51%, 62.31% and 47.15%, respectively. Upon salt stress, bacterial treatments markedly reduced APX activity, with reductions of 47.25% (PS3), 48.19% (TPN1), and 44.47% (YSC3), whereas ALA treatment increased APX activity by 10.84% under saline-stressed conditions. This trend is consistent with the observed reduction in H₂O₂ content, suggesting alleviated oxidative stress and altered antioxidant regulation. However, reduced CAT and APX activities may just reflect changes in antioxidant demand or regulatory adjustments rather than a direct reduction in overall ROS production.

Compared with that of the untreated control plants, the leaf relative water content (RWC) significantly improved in all treated plants under saline stress (Fig. 5A). The greatest increase was observed with TPN1 (28.03%), followed by ALA (20.66%), YSC3 (18.52%), and PS3 (17.14%). The accumulation of proline, an important osmoprotectant and stress marker, was elevated in all treated plants under both non-stressed and saline-stressed conditions (Fig. 5B). Compared with the untreated control plants, the TPN1-treated plants presented the greatest increase in proline content (46.74%), whereas the ALA-treated plants presented a 37.69% increase in proline content. Under saline stress, proline levels increased by 25.44% (PS3), 38.90% (TPN1), and 37.06% (YSC3), whereas ALA improved the proline content by 30.88% (Fig. 5B). Malondialdehyde (MDA), a marker of membrane lipid peroxidation and oxidative membrane damage, was significantly reduced in all treated plants under saline stress (Fig. 5C). Notably, the MDA content decreased by 55.01%, 50.88%, and 52.63% in plants inoculated with PS3, TPN1, and YSC3, respectively, whereas ALA treatment led to a 41.34% reduction compared with the saline-stressed control. Electrolyte leakage, which increased significantly under saline stress, was also remarkably mitigated by TPN1 treatment, reducing membrane leakage to approximately half that of the untreated salt-stressed plants (Fig. 5D).

Fig. 5.

Effect of ALA treatment and R. palustris inoculation on water status, osmolyte accumulation, and membrane integrity in A. thaliana Col-0 under non-stress and saline (50 mM NaCl) conditions. A Relative water content (RWC), B proline content, C malondialdehyde (MDA), and D electrolyte leakage. Treatments included uninoculated controls, exogenous ALA application (10 mg L⁻¹), and inoculation with PS3, TPN1, and YSC3 (2.0 × 10⁷ CFU per plant, respectively). Data represent means ± SD (n = 15 individual plants). Different letters indicate statistically significant differences according to Tukey’s HSD test (p < 0.05)

R. palustris inoculation modulates stress-related gene expression under salinity

To elucidate the transcriptional responses associated with salt stress tolerance, we analyzed the expression of genes involved in chlorophyll biosynthesis (HEMA1, CHLH), reactive oxygen species (ROS) detoxification (CAT2, APX1, APX2), and sodium transport (NHX1, HKT1) in A. thaliana following inoculation with three R. palustris strains (PS3, TPN1, YSC3), as well as exogenous ALA treatment. The expression of these genes was evaluated under nonstress and salt stress conditions (50 mM NaCl for 21 days) via RT‒qPCR and normalized to that of ACTIN2.

Under nonstress conditions, HEMA1 and CHLH were upregulated in TPN1- and PS3-inoculated plants, whereas YSC3 inoculation and ALA treatment resulted in negligible changes (Fig. 6A). Salt stress drastically suppressed HEMA1 expression in the control plants; however, the HEMA1 transcript levels were maintained at levels comparable to those in the non-stressed control plants under all the treatments. CHLH displayed a similar trend, with elevated expression across treatments under both conditions, with the highest expression observed in TPN1 (Fig. 6B).

Fig. 6.

Relative expression of genes involved in ALA biosynthesis, antioxidant defense, and salt stress responses in A. thaliana Col-0 under non-stress and saline (50 mM NaCl) conditions. A-B qRT-PCR analysis of genes involved in ALA biosynthesis: A HEMA1 and B CHLH. C-E qRT-PCR analysis of antioxidant-related genes: C CAT2 D APX1 and E APX2. F-G qRT-PCR analysis of salt-stress-responsive genes: F NHX1 and G HKT1.Treatments included uninoculated controls, exogenous ALA application (10 mg L⁻¹), and inoculation with PS3, TPN1, and YSC3 (2.0 × 10⁷ CFU per plant, respectively). Data represent means of means ± SD of three biological replicates (n = 3). The effects of "stress" and "treatment," and their interaction, were evaluated using two-way ANOVA. Different letters indicate statistically significant differences according to Tukey’s HSD test (p ≤ 0.05)

For the ROS-scavenging genes, the treatments did not alter the expression of CAT2, APX1, or APX2 under nonstress conditions (Fig. 6C and D). Under salt stress, similar to the results of the control group, ALA treatments led to a moderate increase in both CAT2 and APX1 expression, whereas all R. palustris inoculations generally resulted in a significant reduction in their transcript levels. In contrast, APX2 was downregulated in the control group but was consistently upregulated in all the experimental treatment groups under salt stress (Fig. 6E). The TPN1- and YSC3-inoculated plants presented the strongest responses, followed by the PS3- and ALA-inoculated plants, suggesting a compensatory role for APX2 when CAT2 and APX1 were suppressed. However, transcript abundance does not always directly correlate with enzyme activity; therefore, the observed transcriptional changes were interpreted as indicative of regulatory trends rather than definitive measures of functional antioxidant capacity. The expression of ion transporter genes (NHX1 and HKT1) was not significantly affected by treatment under nonstress conditions (Fig. 6F and G). Under salt stress, both genes were strongly upregulated in all the experimental treatments. For NHX1, transcript abundance was highest in ALA-treated plants (4.56-fold greater than in the non-stressed control), followed by plants inoculated with TPN1 (4.44-fold), PS3 (4.33-fold), and YSC3 (3.59-fold). For HKT1, the strongest induction was observed in plants treated with TPN1 (3.53-fold), followed by those treated with ALA (3.42-fold), YSC3 (3.00-fold), and PS3 (2.55-fold).

Discussion

Model validation and salinity stress in A. thaliana

Soil salinity is a major abiotic constraint to global agriculture, disrupting water and ion homeostasis, impairing nutrient uptake, and causing oxidative stress [54–56]. In this study, salt-sensitive A. thaliana Col-0 plants were exposed to 50 mM NaCl (~ 5 dS m⁻¹) for 21 days, resulting in marked reductions in growth and physiological performance, consistent with established salt sensitivity thresholds for this accession [30, 31, 57]. These responses support the suitability of this system for evaluating salinity-associated physiological changes and stress mitigation strategies.

ALA and ALA-producing bacteria as potent stress alleviators

5-Aminolevulinic acid (ALA) has been associated with increased salinity tolerance by improving photosynthesis, osmolyte accumulation (e.g., proline), ion transporters (e.g., SOS1, NHX1, HKT1), and antioxidant defenses [58–60]. However, the high cost of ALA limits its agronomic application [52], prompting interest in microbial ALA producers, such as Rhodopseudomonas spp., as sustainable alternatives [61]. R. palustris is a plant growth-promoting rhizobacterium (PGPR), reported to secrete ALA and exhibit additional plant-beneficial traits linked to stress-associated plant responses, including IAA production and EPS synthesis [62–64]. In rice, several R. palustris strains have been associated with improved plant growth under saline conditions, effects frequently attributed to increased ALA biosynthesis and stress tolerance [65, 66].

In the present study, we compared three R. palustris strains (PS3, TPN1, and YSC3) with ALA treatment to assess strain-dependent associations with salt stress responses in A. thaliana Col-0 plants.

Our findings revealed that all three R. palustris strains exhibit slight salt resistance according to Larsen’s classification [33] (Table S5), consistent with their mesophilic nature, but varied in salinity thresholds and ALA production profiles, reflecting strain-specific osmoadaptive responses (Fig. 1A-D) (Table 1) [67–69]. Among the strains, TPN1 exhibited enhanced ALA production at 10 g L⁻¹ NaCl and demonstrated a higher salinity threshold compared to PS3 and YSC3 [70–72].This strain-specific, salt-responsive adjustment of ALA biosynthesis, aligns with reports of metabolically flexible Rhodopseudomonas strains under saline stress [22, 73, 74]. Although the regulatory mechanisms underlying this response remain to be elucidated, this supports the potential of TPN1 for further evaluation under saline conditions.

Both exogenous ALA and R. palustris inoculation partially recovered salt-induced growth inhibition, most notably through changes in root architecture (Fig. 2; Figure S3). TPN1 exhibited the best performance, characterized by increased lateral root formation and biomass accumulation under salinity (Fig. 2; Figure S3). It was associated with its high extracellular IAA production at 0.5 and 10 g L⁻¹ NaCl (Table S6) [75, 76]. Consistently, PGPRs with high IAA production have been shown to improve water and nutrient acquisition through root adaptations under saline conditions [77, 78]. While IAA production represents a plausible contributing factor, the present data do not resolve the relative contribution of bacterial IAA to the observed plant responses.

Physiological adjustment and photosynthetic performance under salinity

Under saline stress, plants physiologically adjust to osmotic responses, including the accumulation of proline, changes in sodium transport, and the activation of ROS-scavenging systems, to maintain water content and reduce oxidative stress [79, 80]. Both exogenous ALA treatment and R. palustris inoculation effectively maintained photosynthetic ability and relative water content, especially in TPN1-inoculated plants (Figs. 3 and 5). Salt stress suppressed the expression of two key genes in the chlorophyll biosynthetic pathway, HEMA1 and CHLH, while TPN1-inoculated plants maintained the highest transcript abundance under salinity (Fig. 6), consistent with TPN1’s ability to maintain Arabidopsis chlorophyll content and photosynthesis efficiency (Figs. 3 and 5). The higher proline accumulation in TPN1-inoculated plants is also associated with enhanced relative water content. The ability of a PGPR to enhance plant salt tolerance through tetrapyrrole regulation and proline accumulation under salinity stress has been reported in other plant systems [53, 81], suggesting the potential of TPN1 as a promising salt stress alleviator. However, exogenous ALA did not alter HEMA1 or CHLH expression under either non-stressed conditions or saline conditions (Figs. 6A and B), indicating different regulatory mechanisms triggered by ALA.

Salt stress is known to disrupt membrane integrity through ionic toxicity and oxidative damage, often reflected by increased lipid peroxidation and electrolyte leakage [82, 83]. In this study, electrolyte leakage (EL) and malondialdehyde (MDA), the two markers of membrane destabilization, were elevated under salinity (Fig. 5C and D). Both exogenous ALA and R. palustris inoculation reduced EL and MDA levels under salinity, indicating reduced membrane damage [84]. PGPRs have been reported to potentially enhance ion regulation and mitigate oxidative damage, thereby reducing damage under salt stress [85–87]. Despite the effective mitigation from both treatments, it is possible that R. palustris provides broader protection than exogenous ALA treatment, given that PGPRs contain many beneficial traits to their hosts [88, 89].

ROS-related responses further differentiated the ALA supplementation from R. palustris inoculation treatments, though both treatments successfully attenuated H₂O₂ accumulation (Fig. 4A), consistent with markedly reduced membrane destabilization [60, 90, 91]. Enzymatic and nonenzymatic antioxidants are widely implicated in scavenging ROS and protecting membranes under salinity [92–94]. In nonenzymatic systems, both ALA and R. palustris inoculation, especially TPN1, protected plants with elevated antioxidant compounds under salinity [95, 96] (Figs. 4B, C, and D), consistent with reported PGPR-mediated enhancement of stress tolerance [79].

In contrast, enzymatic antioxidant responses differed among treatments. While exogenous ALA was associated with increased CAT and APX activities, R. palustris inoculation correlated with lower activities of both enzymes under salinity (Fig. 4E and F) [97–99]. These results indicate that ALA and R. palustris modulate plant oxidative stress through diverse pathways. To understand the transcriptional regulations in the CAT and APX genes, their transcript abundance was measured. CAT2 and APX1 were upregulated, while APX2 was downregulated under salinity stress (Fig. 6). Consistent with enzyme activities, ALA treatment induced all three genes, while R. palustris suppressed CAT2 and APX1, which were upregulated in control plants, but induced APX2, which was downregulated under salinity (Fig. 6). The reduction in both ROS content and enzymatic antioxidant responses led us to consider the potential of ion trapping [100, 101].

EPS secretion and ion homeostasis

Several studies have reported that under salinity stress, PGPR secrete extracellular polymeric substances (EPSs) that can bind metal ions and enhance bacterial attachment to root surfaces via biofilm formation [102, 103]. All three R. palustris strains, especially TPN1, exhibited salinity-responsive EPS production profiles, with maximal secretion observed at 10 g L⁻¹ NaCl (Table 2). Given that EPSs produced by purple non-sulfur bacteria are rich in anionic residues such as uronic acids with Na⁺- binding capacity, the EPS secreted by TPN1 may hypothetically contribute to localized Na⁺ buffering in the rhizosphere, potentially alleviating ionic stress at the root-soil interface [104].

Salt-stressed A. thaliana plants inoculated with TPN1 displayed increased transcript levels of the ion homeostasis genes NHX1 and HKT1 (Figs. 6F and G). NHX1 and HKT1, which mediate vacuolar Na⁺ sequestration and Na⁺ unloading from the xylem into parenchyma cells, respectively, are widely recognized as key contributors to salt tolerance [101, 105]. Their coordinated induction is associated with improved ionic compartmentalization rather than direct stress avoidance [106–108]. Similar transcriptional responses have been reported in plants inoculated with EPS-producing PGPR, where improved Na⁺/K⁺ balance and enhanced ion transporter activity were observed under salinity stress [105, 109, 110]. Conversely, exogenous ALA treatment also led to elevated NHX1 and HKT1 expression under saline stress, in agreement with previous studies reporting that ALA application supports ionic homeostasis by promoting Na⁺ sequestration and maintaining metabolic function in stressed tissues [75, 91, 111].

Proposed dual-layer model for salinity tolerance:

The mitigation of salt stress by R. palustris TPN1 is best interpreted as a synergistic, two-tiered regulatory framework. The ALA-dependent pathway is associated with plant-intrinsic metabolic shifts, where ALA correlates with stabilized chlorophyll biosynthesis and heme-based antioxidant machinery. [58, 93]. In parallel, the ALA-independent pathway is linked to microbially mediated traits, such as EPS-facilitated Na⁺ buffering [97] and IAA-related root remodeling [63], which likely modify the immediate stress environment. Together, these pathways suggest a coordinated, multi-level response that appears more robust than the effects associated with exogenous ALA application alone.

Conclusion

This study demonstrated that inoculation with the R. palustris strain TPN1 was associated with enhanced salt stress tolerance in A. thaliana, exhibiting stronger overall physiological responses than exogenous 5-aminolevulinic acid (ALA) under the tested conditions. By modulating ROS-related processes, osmotic adjustment, ion homeostasis, and chlorophyll biosynthesis, TPN1 elicited broader stress-associated physiological and molecular responses than ALA treatment (summarized in Fig. 7). These responses were accompanied by increased expression of chlorophyll biosynthesis genes (HEMA1 and CHLH) and key ion transporters (HKT1 and NHX1), coinciding with improved Na⁺/K⁺ balance, water status, and photosynthetic performance under salinity stress. The elevated EPS production observed in TPN1 may further contribute to these responses by influencing rhizospheric Na⁺ dynamics, though direct Na⁺ binding was not quantified in this study. Consistent physiological trends were observed in preliminary pot trials conducted under 100 mM NaCl stress, supporting the robustness of TPN1-associated salt stress mitigation across experimental systems. Collectively, these findings suggest that R. palustris-mediated enhancement of salt tolerance extends beyond ALA-related mechanisms and involves additional microbial traits that enhance plant stress responses.

Fig. 7.

Schematic diagram of the proposed mechanisms by which R. palustris enhances salt-stress tolerance in A. thaliana. Salt stress negatively affects plants through multiple processes, including (1) reactive oxygen species (ROS) accumulation, ion leakage, lipid peroxidation, and impaired photosynthetic activity. Inoculation with R. palustris counteracts these constraints through several mechanisms: (2) secretion of phytohormones such as indole-3-acetic acid (IAA) and microbial 5-aminolevulinic acid (ALA), which enhance root development and activate plastidial chlorophyll biosynthesis pathways; (3) production of extracellular polymeric substances (EPS), contributing to rhizospheric Na⁺ buffering and improved root protection; (4) osmotic adjustment through proline accumulation and increased relative water content; (5) accumulation of non-enzymatic antioxidants, such as total phenolics, flavonoids, and anthocyanins; (6) modulation of enzymatic antioxidants (CAT, APX) and selective induction of stress-responsive isoforms (e.g., APX2); and (7) transcriptional activation of ion homeostasis genes NHX1 and HKT1, which promote Na⁺ sequestration and retrieval. Collectively, these processes (8) restore photosynthetic efficiency, chlorophyll content, membrane stability, and biomass accumulation, resulting in enhanced tolerance of A. thaliana to salinity stress

Abbreviations

ALA

5-Aminolevulinic acid

AMF

Arbuscular mycorrhizal fungi

APX

Ascorbate peroxidase

CAT

Catalase

CFU

Colony-forming units

Chl

Chlorophyll

Chl a/b

Chlorophyll a/b ratio

CHLH

Magnesium chelatase H subunit (gene, chlorophyll biosynthesis)

DDW

Distilled and deionized water

EL

Electrolyte leakage

EPS

Extracellular polysaccharides

ESI

Electrospray ionization

FTW

Fully turgid weight

Fv/Fm

Maximum quantum efficiency of photosystem II

HEMA1

Glutamyl-tRNA reductase (gene, chlorophyll biosynthesis)

HKT1

High-affinity K⁺ transporter 1

IAA

Indole-3-acetic acid

LC–MS

Liquid chromatography–mass spectrometry

LED

Light-emitting diode

MDA

Malondialdehyde

Mg-branch

Magnesium branch (of chlorophyll biosynthesis)

MS

Murashige and Skoog medium

NaCl

Sodium chloride

NHX1

Na⁺/H⁺ exchanger 1

Na⁺/K⁺

Sodium/potassium ions

nPNSB

Nutrient medium for phototrophic non-sulfur bacteria

PNSB

Purple non-sulfur bacteria

PGPR

Plant growth-promoting rhizobacteria

PVPP

Polyvinylpolypyrrolidone

qPCR/RT-qPCR

Quantitative (reverse transcription) polymerase chain reaction

ROS

Reactive oxygen species

R. palustris

Rhodopseudomonas palustris

RWC

Relative water content

SD

Standard deviation

SOS1

Salt overly sensitive 1 (gene, Na⁺/H⁺ antiporter)

TCA

Trichloroacetic acid

TPC

Total phenolic content

Authors’ contributions

Swarnali Roy: Writing – original draft, visualization, methodology, investigation, data curation, conceptualization. Pei-Yin Lin: Methodology, Investigation. Ting-Jang Lu: Resources. Jen-Chih Chen: Methodology, Supervision, Writing – review & editing; Chi-Te Liu: Writing – review & editing, Supervision, Resources, Conceptualization, Project administration, Funding acquisition.

Funding

This study was supported by grants from the National Science and Technology Council (NSTC) of Taiwan (NSTC 113–2321-B-002–037, 114–2321-B-002–014, and 114–2218-E-002–021).

Data availability

All the data are available upon request to the corresponding author.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.