Comparative Immunomodulatory Efficacy of Chemogenic and Biogenic Manganese Nanoparticles for Inducing Arsenic Stress Resilience in Rapeseed

Abstract

Arsenic (As) contamination poses a critical threat to agricultural productivity, affecting rapeseed (Brassica napus L.), an agronomically important crop. A comparative assessment was performed to evaluate the efficacy of chemogenic and biogenic manganese nanoparticles (C-MnNPs and B-MnNPs) for mitigating As toxicity. B-MnNPs were biosynthesized using cell-free filtrate of Bacillus pumilus MAY4, while C-MnNPs were obtained from Cwnano Co., Ltd. (Shanghai, China). Greenhouse assays demonstrated that both C-MnNPs and B-MnNPs alleviated detrimental effects of As; however, B-MnNPs exhibited superior performance compared to their chemical counterparts. Compared to As-stressed plants, B-MnNPs enhanced leaf and root biomass (26.4% and 56.15%, respectively), net photosynthetic rate (64.8%), and stomatal conductance (50%). B-MnNPs more effectively reduced oxidative stress markers by activating antioxidant defense systems in both leaf and root tissues. Furthermore, B-MnNPs reduced in planta As accumulation while significantly improving uptake of essential nutrients, including potassium, phosphorous, magnesium, and manganese, etc., in rapeseed plants. Expression studies revealed that B-MnNPs upregulated antioxidant defense and redox homeostasis related stress-responsive genes under induced As stress. Biochemical assays further confirmed the enrichment of stress-responsive phytohormones, including salicylic acid, jasmonic acid, and abscisic acid, in B-MnNP-treated As-stressed rapeseed plants, indicating activation of multi-tier defense response by B-MnNPs to cope with As stress. These findings establish B-MnNPs as a highly effective nano-enabled strategy for managing As toxicity in the rapeseed cultivation system. This research provides critical insights into the molecular and physiological mechanisms underlying MnNP-mediated stress tolerance and offers a promising green nanotechnology approach for heavy metal-resilient crops.

1. Introduction

The global issue of environmental pollution, particularly heavy metal (HM) contamination, has become a significant threat to agricultural productivity and food security [1]. HMs such as lead (Pb), cadmium (Cd), and arsenic (As) contaminate agricultural soil and water, posing toxic risks to crops and human health [2,3]. Among these, the persistence of As contamination, often sourcing from industrial discharge, mining, and agricultural runoff, has intensified its impact on agroecosystems worldwide [4,5]. Arsenic is particularly damaging, leading to reduced photosynthesis, growth, and overall yield in sensitive plants [6]. For example, it has been reported that As stress reduced the growth of rice, maize, wheat, and tomato by inducing oxidative damage and negatively impacting the normal physiological or biochemical functions such as nutrient acquisition and photosynthesis [7,8,9,10,11,12,13]. Since oxidative stress is a primary mechanism of As toxicity, developing strategies to counteract these effects is essential for improving crop tolerance and ensuring food security [14]. Arsenic exposure at sufficiently high levels has been shown to severely reduce growth, disrupt photosynthesis, and induce oxidative injury in rapeseed cultivars [15,16]. Furthermore, studies have demonstrated that heavy metal accumulation in rapeseed tissues can pose potential risks of metal transfer into extracted oil fractions, raising concerns for food safety in contaminated regions [17]. Given its economic importance and extensive cultivation, enhancing As tolerance in rapeseed represents a vital step toward sustainable agricultural management in contaminated regions.

To overcome the detrimental effects of HM stress, numerous strategies have been investigated to enhance plant resilience, among which nanotechnology has emerged as a promising approach [18]. Manganese nanoparticles (MnNPs) have demonstrated considerable potential to alleviate oxidative stress, facilitate nutrient absorption, and boost photosynthetic efficiency in plants [19]. Although chemically synthesized NPs (C-NPs) have been widely employed for mitigating abiotic stress, numerous concerns persist regarding their environmental compatibility, persistence, and potential bio-toxicity [20,21,22]. In contrast, biologically synthesized NPs (B-NPs), produced through eco-friendly microbial or plant-based processes, offer a sustainable and non-toxic alternative. These B-NPs demonstrated improved effectiveness in mitigating HM stress compared to their chemical counterparts, with minimum environmental risks [23]. Given these, B-NPs represent a promising innovation for devising modern sustainable agricultural practices.

Previous studies have demonstrated that B-NPs can enhance nutrient uptake, photosynthetic performance, and tolerance to HM toxicity [24,25]. Among these, B-MnNPs gained significant attention for their ability to enhance Mn bioavailability while mitigating As stress in plants. Mn, a vital micronutrient involved in antioxidant defense and stress regulation, is efficiently delivered through B-MnNPs, minimizing the risks associated with excess Mn accumulation [26,27]. For example, Anas, et al. [28] revealed that MnO₂ NPs effectively mitigated Cd stress in wheat (Triticum aestivum L.) by reducing Cd uptake, restoring essential minerals and enhancing antioxidant defenses and anatomical recovery. In another study, nano-priming with B-MnO NPs synthesized using Bacillus subtilis effectively mitigated Pb toxicity in tomato (Solanum lycopersicum L.), enhancing growth parameters and antioxidant defenses while reducing Pb accumulation [29]. Given these, MnNP-based HM mitigation strategies could be instrumental in shaping the more resilient crop cultivation system.

Although the HM mitigation potential of MnNPs has been reported previously [19,28,30], comparative efficacy of C-MnNPs and B-MnNPs as well as precise metal mitigating mechanisms are still elusive. In this study, the functional capability of C-MnNPs and B-MnNPs in mitigating As stress in rapeseed was compared. The results demonstrated that B-MnNPs more effectively alleviated As-induced damage and enhanced rapeseed resilience to metal stress by strengthening key physiological and biochemical processes. Overall, B-MnNPs could simultaneously provide essential nutrients to regulate key processes and enhance protection to crops under challenging environments.

2. Materials and Methods

2.1. Isolation, Screening, and Identification of Mn-Resistant Strain

The Mn-resistant bacterial strain Bacillus pumilus MAY4 was isolated from the rhizosphere soil of rapeseed cultivated at the experimental field of Zhejiang University, Hangzhou, China (29°56′42″ N, 119°35′54″ E), using the serial dilution method [31]. Briefly, 1 g of rhizosphere soil was suspended in sterile saline solution and serially diluted up to 10⁻⁵. A 100 μL aliquot from each dilution was spread on freshly prepared nutrient agar medium and incubated at 28 ± 2 °C for 48 h. Distinct colonies were selected based on morphological variation and purified through repeated streaking [27]. The isolates were screened for Mn resistance using the minimum inhibitory concentration (MIC) method against MnCl₂·4H₂O following established procedures (Table S2). The most Mn-tolerant isolate was selected for molecular identification. Genomic DNA was extracted using a commercial bacterial DNA extraction kit (Sangon Biotech, Shanghai, China), and the 16S rRNA and rpoB genes were amplified using universal primers (fD1/rD1 and rpoB-F/rpoB-R, respectively). The purified PCR products were sequenced commercially (Zhejiang Youkang Biotech, Hangzhou, China), and sequence similarity was analyzed using BLASTn (v5.0). Multiple sequence alignment was performed in ClustalW, and a phylogenetic tree was constructed in MEGA 7.0 using the Maximum-Likelihood method with 1000 bootstrap replicates [27].

2.2. Biosynthesis of B-MnNPs

Extracellular biosynthesis of MnNPs was performed using the cell-free filtrate of B. pumilus MAY4 as a bioreductant and stabilizing medium, following previously described protocols with minor modifications [31]. The bacterial strain was cultured in nutrient broth at 28 ± 2 °C for 24 h under shaking conditions (150 rpm). After incubation, the culture was centrifuged at 6000× g for 10 min, and the cell-free supernatant was collected through filtration. For nanoparticle synthesis, 100 mL of 10 mM MnCl₂·4H₂O solution was added to an equal volume of bacterial filtrate under constant agitation (200 rpm) at 28 ± 2 °C for 72 h. The formation of biogenic MnNPs was indicated by a color change from pale yellow to dark brown. The reaction mixture was centrifuged at 10,000× g for 15 min to collect the MnNPs, which were then washed several times with deionized water and ethanol to remove residual impurities. The purified nanoparticles were freeze-dried for 6 h, ground into a fine powder, and stored in airtight containers for further characterization.

2.3. Characterization of Nanomaterials

Commercial C-MnNPs (99% purity, MnO form, 30–45 nm) were obtained from Cwnano Co., Ltd. (Shanghai, China). As these NPs were supplied with certified physicochemical specifications (size, purity, composition), no further characterization was performed. B-MnNPs were characterized using a suite of analytical techniques. Fourier-transform infrared spectroscopy (FTIR; NICOLET iS50, Thermo Fisher Scientific, Waltham, MA, USA) was conducted in the 4000–400 cm⁻¹ range to identify functional groups associated with biomolecular reduction and stabilization of nanoparticles, following the procedure of [32]. X-ray diffraction (XRD; D8 Advance, Bruker, Germany) with Cu Kα radiation (λ = 1.5406 Å) was used to determine crystalline structure over a 2θ range of 10–80°, and crystallite size was estimated using the Scherrer equation as described previously [33]. Morphological characteristics were examined using scanning electron microscopy (SEM; ZEISS GEMINI 300, Oberkochen, Germany) operated at 10 kV to assess surface features and aggregation patterns. High-resolution structural visualization was performed using transmission electron microscopy (TEM; Hitachi HT-7820, Tokyo, Japan) operated at 200 kV to determine nanoparticle shape, size distribution, and dispersion. Elemental composition and purity of B-MnNPs were confirmed via energy-dispersive X-ray spectroscopy (EDS) attached to the SEM instrument.

2.4. Plant Growth Materials and Experimental Setup

Healthy seeds of rapeseed (cv. ZD-635) were collected from the College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China. The seeds were surface sterilized with 1% sodium hypochlorite (NaOCl) for 10 min, followed by several rinses with sterile distilled water to remove any residual disinfectant. Sterilized seeds were then germinated on moist filter paper in Petri dishes and incubated in the dark at 25 °C for three days. C-MnNPs (99% purity, MnO spherical form, 30–45 nm size) were purchased from Cwnano Co., Ltd. (Shanghai, China), while sodium arsenate (Na₂HAsO₂·7H₂O) was procured from Sigma-Aldrich Co. (St. Louis, MO, USA). Uniformly germinated seedlings were transferred to 25% Hoagland nutrient solution for seven days, followed by 50% Hoagland solution for an additional eight days to ensure acclimatization. After 15 days of pre-culture, seedlings were transferred to 100% Hoagland nutrient solution and maintained under controlled growth chamber conditions, 24/16 °C (day/night) temperature, 65% relative humidity, 16 h light/8 h dark photoperiod, and a light intensity of 400 µmol m⁻² s⁻¹. The nutrient solution was continuously aerated using an air pump and renewed twice weekly to ensure adequate nutrient supply and prevent microbial contamination. The pH was adjusted daily to 6.0 ± 0.1 using 0.1 M NaOH or HCl, and evaporative losses were compensated with deionized water. Following acclimatization, plants were exposed to six treatment conditions based on preliminary screening studies (unpublished): (1) Control (CK)—Hoagland solution only; (2) C-MnNPs—Hoagland + 100 µM C-MnNPs; (3) B-MnNPs—Hoagland + 100 µM B-MnNPs; (4) As stress—Hoagland + 200 µM As; (5) As + C-MnNPs—Hoagland + 200 µM As + 100 µM C-MnNPs; and (6) As + B-MnNPs—Hoagland + 200 µM As + 100 µM B-MnNPs, as described previously [34,35]. The experiment followed a completely randomized design (CRD). Each treatment consisted of three biological replicates, with four plants per replicate (total n = 12 plants per treatment). For physiological, biochemical, and gene-expression analyses, composite samples were prepared by pooling tissues from all four plants within each replicate to minimize individual variability. All measurements were conducted at the replicate level unless otherwise specified.

2.5. Determination of Morphological Parameters

At the end of the treatment period, the morphological attributes of rapeseed were evaluated. Plants were gently removed from the nutrient solution and rinsed with deionized water to remove any adhering residues. The shoot length (SL) and root length (RL) were measured using a measuring scale, ensuring proper alignment for accuracy. For biomass determination, plants were separated into shoots and roots, and their fresh weights (LFW and RFW) were immediately recorded using an analytical balance (AUW220D, Shimadzu, Japan). The samples were then oven-dried at 70 °C until a constant weight was achieved to obtain the dry weights (LDW and RDW). All measurements were taken from three biological replicates per treatment, and mean values were used for statistical analysis [36].

2.6. Determination of Photosynthesis and Gas Exchange Parameters

The gas exchange parameters, including net photosynthetic rate (Pn), stomatal conductance (Gs), and transpiration rate (Tr), were determined on fully expanded upper leaves using a portable photosynthesis system (LI-6400XT, LI-COR Biosciences, Lincoln, NE, USA), as described by [37]. Measurements were performed between 09:00 and 11:00 h on cloudless days to minimize diurnal variation. During each measurement, the chamber conditions were maintained at a photosynthetic photon flux density (PPFD) of 1000 µmol m⁻² s⁻¹, CO₂ concentration of 400 µmol mol⁻¹, leaf temperature of 25 ± 1 °C, and relative humidity of 60–70%, with slight modification of the reported methodology [38]. The maximum quantum yield of photosystem II (Fv/Fm) was recorded using a pulse-amplitude modulated chlorophyll fluorometer (PAM-2500, Heinz Walz GmbH, Effeltrich, Germany). Leaves were dark-adapted for 30 min prior to measurement, and data were calculated automatically using the fluorometer integrated software (https://www.walz.com/, accessed on 28 February 2025) [39]. For pigment determination, chlorophyll a (Chl a) and chlorophyll b (Chl b) contents were quantified according to the Arnon method, as reported by [40]. Fresh leaf tissue (0.1 g) was homogenized in 80% (v/v) acetone and centrifuged at 10,000× g for 10 min. Absorbance of the supernatant was recorded at 663 nm and 645 nm using a UV-visible spectrophotometer (UV-2600, Shimadzu, Kyoto, Japan). Chlorophyll concentrations (mg g⁻¹ FW) were calculated using standard Arnon equations following [41], as follows:

Chl a = 12.7 × A₆₆₃ − 2.69 × A₆₄₅

Chl b = 22.9 × A₆₄₅ − 4.68 × A₆₆₃

Chlorophyll values were expressed on a fresh-weight basis.

2.7. Determination of Reactive Oxygen Species Activity

The contents of malondialdehyde (MDA), hydrogen peroxide (H₂O₂), and superoxide anion (O₂•⁻) in leaf and root tissues were determined following standard spectrophotometric protocols. Fresh plant tissue (0.5 g) was homogenized in 5 mL of ice-cold 50 mM phosphate buffer (pH 7.0) and centrifuged at 12,000× g for 15 min at 4 °C. The resulting supernatant was used for all biochemical assays. Lipid peroxidation was estimated by quantifying MDA formation using the thiobarbituric acid (TBA) reaction, as described by [42]. H₂O₂ content was measured following the potassium iodide (KI) method, where the reaction mixture was incubated in the dark, and absorbance was recorded at 390 nm, according to the procedure outlined in [43]. The O₂^•− generation rate was determined by monitoring the reduction of nitroblue tetrazolium (NBT) at 530 nm, as reported by [44]. For in vivo visualization of ROS accumulation, fresh root segments were stained using 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) for H₂O₂ detection and dihydroethidium (DHE) for O₂^•− localization, following the protocol of [45]. After staining, roots were gently rinsed in phosphate buffer and immediately examined under a confocal laser scanning microscope (Olympus FV3000, Tokyo, Japan). Fluorescence imaging was performed using excitation/emission settings of 488/530 nm for H₂DCFDA and 518/606 nm for DHE, and representative images were captured from three biological replicates.

2.8. Determination of Antioxidant Enzyme Activity

Activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) were assayed in leaf and root tissues following standard spectrophotometric methods. Fresh tissue (0.5 g) was homogenized in 50 mM phosphate buffer (pH 7.0) containing 1% polyvinylpyrrolidone (PVP) and centrifuged at 12,000× g for 20 min at 4 °C. The resulting supernatant served as the enzyme extract. SOD activity was quantified by measuring the inhibition of nitroblue tetrazolium (NBT) photoreduction, following the method described by [46]. CAT activity was determined by monitoring the decomposition of H₂O₂ at 240 nm, as per the procedure reported in [47]. APX activity was measured by recording the decline in absorbance at 290 nm due to ascorbate oxidation, as outlined in [48], and GR activity was evaluated by following the oxidation rate of NADPH at 340 nm, as described by [44]. Enzyme activities were expressed on a fresh-weight basis, and all assays were performed using three biological replicates.

2.9. Determination of Element Contents

Leaf and root samples were oven-dried at 65 °C and finely ground to a uniform powder. Approximately 0.20 g of dried tissue was digested using a HNO₃–HClO₄ acid mixture (3:1, v/v) in a microwave digestion system. The digested samples were evaporated to near dryness, diluted with ultrapure water, and filtered through 0.22 µm polytetrafluoroethylene (PTFE) membranes. Elemental concentrations of As, Mn, Fe, K, Mg, and P were determined using inductively coupled plasma–optical emission spectrometry (ICP–OES; NexION^TM 300X, PerkinElmer, Shelton, CT, USA). Standard calibration curves and certified reference materials were used for quality control, and all measurements were conducted using three biological replicates [49].

2.10. Quantification of Phytohormones

The levels of salicylic acid (SA), jasmonic acid (JA), and abscisic acid (ABA) in rapeseed leaves were quantified using enzyme-linked immunosorbent assay (ELISA) kits (Shanghai Enzyme-Linked Biotech Co., Ltd., Shanghai, China), following the manufacturer’s instructions, as described by [50]. Fresh leaf tissue (0.1 g) was homogenized in cold extraction buffer (9× volume), incubated on ice, and centrifuged at 8000× g for 30 min at 4 °C. The resulting supernatant was used for hormone determination. Absorbance was measured at 450 nm using a microplate reader (Thermo Scientific, Waltham, MA, USA), and concentrations were calculated using standard curves supplied with the kit. Each sample included three biological and three technical replicates.

2.11. Gene Expression Analysis

Reverse transcriptase-quantitative PCR (RT-qPCR) was performed to evaluate the expression of stress-related genes in rapeseed. Primers for target genes were selected based on previous laboratory study to ensure specificity and amplification efficiency [51], as shown in Table S1. First-strand cDNA was synthesized from total RNA using the PrimeScript RT reagent kit (Takara Bio, Kusatsu, Japan). RT-qPCR was conducted on a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) using SYBR Premix Ex Taq II (Takara Bio, Kusatsu, Japan) in a 20 µL reaction volume. The amplification program consisted of an initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Gene expression levels were calculated using the 2⁻ΔΔCt method, with normalization to the internal reference gene and comparison to the control group. Three biological and three technical replicates were included for each gene, with BnaActin used as the internal reference gene, as per reported methodology by [52].

2.12. Observation of Leaf Ultrastructure

Leaf stomatal structure was examined using SEM. Fresh, fully expanded leaves were rinsed with distilled water to remove surface impurities and immediately fixed in 2.5% glutaraldehyde (v/v) prepared in 0.1 M phosphate buffer (pH 7.2) at 4 °C for 24 h. The samples were then washed three times with phosphate buffer and dehydrated through a graded ethanol series (30%, 50%, 70%, 90%, and 100%; 15 min each). Dehydrated samples were dried using a critical point dryer (Leica EM CPD300, Leica Microsystems, Wetzlar, Germany) and mounted on aluminum stubs with double-sided carbon adhesive tape. Specimens were sputter-coated with gold using a sputter coater (Leica EM ACE200, Wetzlar, Germany) to ensure electrical conductivity. Observations of stomatal morphology and aperture were carried out under a SEM (ZEISS GEMINI 300, Oberkochen, Germany) operated at an accelerating voltage of 10 kV, and representative micrographs were recorded for analysis [51].

2.13. Statistical Analysis

All experiments were conducted with three biological replicates. The data were normalized wherever necessary and analyzed using one-way analysis of variance (ANOVA) in Statistix software (version 8.0). Results are presented as mean ± standard error (SE). Statistical significance was determined using Fisher’s least significant difference (LSD) test, with lowercase letters indicating significant differences at p < 0.05. Graphs were generated using Prism software (version 8.0).

3. Results

3.1. Taxonomic Characterization of the Isolated Strain

The taxonomic identity of the isolated bacterial strain was determined using phylogenetic analysis and sequence similarity searches based on both 16S rRNA and rpoB gene sequences. BLASTn analysis of the 16S rRNA gene sequence revealed a 100% sequence identity with B. pumilus NJM4 (accession no. EU234500), indicating that the isolate belongs to the B. pumilus species. Similarly, analysis of the rpoB gene sequence showed 100% sequence identity with B. pumilus strain Bp30 (accession no. JX183185), further supporting this taxonomic classification. The phylogenetic trees constructed using the maximum likelihood method based on the 16S rRNA (PQ489455) and rpoB (PQ498405) gene sequences consistently clustered with the B. pumilus strains. Taken together, both sequence similarity searching and phylogenetic tree conclusively identified the isolate MAY4 as belonging to the species B. pumilus (Figure 1A,B).

3.2. Characterization of Nanomaterials

The morphological and structural characteristics of B-MnNPs were comprehensively investigated through various spectroscopic and microscopic techniques. FTIR spectroscopy revealed multiple absorption bands at 3303, 2925, 1657, 1546 and 1408 cm⁻¹, which correspond to various bioactive molecules adsorbed on the nanoparticle surface (Figure 2A). XRD analysis exhibited broad diffraction peaks confirming the semi-crystalline nature of the synthesized B-MnNPs (Figure 2B).

SEM micrographs demonstrated densely packed, granular nanoparticles with rough surfaces and minimal agglomeration, suggesting uniform distribution of B-MnNPs (Figure 2C). TEM images further revealed nearly spherical particles with nanoscale dimensions and homogeneous dispersion, corroborating the morphological observations obtained through SEM analysis (Figure 2D). EDX analysis displayed prominent signals corresponding to Mn and O as principal constituents with minor peaks attributed to C, thereby confirming the elemental composition and successful synthesis of B-MnNPs (Figure 2E). Collectively, these comprehensive characterization results substantiate the successful biogenic synthesis of MnNPs exhibiting favorable morphological features, crystallinity, and surface functionality.

Collectively, the FTIR, SEM, TEM, and EDS results demonstrate that B-MnNPs possessed multiple surface functional groups, including O–H/N–H, aliphatic C–H, amide I and II, and COO⁻-related vibrations, confirming the presence of extracellular biomolecules from Bacillus pumilus MAY4 acting as natural reducing and capping agents. These organic moieties likely contribute to particle stabilization and may influence aggregation behavior, as indicated by the slight clustering observed in SEM images. In contrast, the C-MnNPs used in this study were supplied as a standardized MnO formulation (99% purity) without organic coatings, suggesting inherent physicochemical differences between biogenic and chemogenic nanoparticles that may partly explain their differential behavior in plant systems.

3.3. Effect of MnNPs on Plant Biomass

Exposure substantially suppressed overall plant growth (Figure S1), with pronounced declines in biomass and growth development of rapeseed. Compared with untreated controls, As stress reduced LFW by 28.4%, LDW by 47%, RFW by 47%, and RDW by 50%, while SL and RL declined by 46.2% and 41.7%, respectively. Application of MnNPs mitigated these losses, with B-MnNPs conferring a markedly stronger recovery than C-MnNPs. Relative to As-stressed plants, C-MnNPs increased LFW, LDW, RFW, and RDW by 13.2%, 60%, 54%, and 44.5%, whereas B-MnNPs improved these parameters by 26.4%, 74%, 50%, and 78%, respectively. Similarly, B-MnNPs improved SL and RL by 66.7% and 56%, while C-MnNPs restored them by 47.4% and 38%, compared with As-stressed plants. These morphological improvements were evident through enhanced leaf expansion, vigorous shoot elongation, and well-developed root systems, highlighting the capacity of B-MnNPs to nearly restore growth to control levels (Figure 3A–F).

3.4. Effect of MnNPs on Photosynthesis and Gas Exchange Parameters

Arsenic stress significantly impaired the photosynthetic apparatus of rapeseed, reflected in marked declines in chlorophyll pigments, photosynthetic rates, and photochemical efficiency. Under As exposure, chlorophyll a and b contents decreased by approximately 98% and 43%, respectively (Figure 4A,B), relative to untreated controls, indicating pigment degradation and chloroplast dysfunction. The reductions in pigment concentration were accompanied by a decline in net photosynthetic rate by 45%, stomatal conductance by 39.5%, and transpiration rate by 39.7%, highlighting stomatal limitation and disrupted gas exchange (Figure 4C–F).

Similarly, the maximum quantum efficiency of PSII (Fv/Fm) declined by 40.8%, revealing photo-inhibition and impaired electron transport (Figure 4F). Treatment with MnNPs alleviated these inhibitory effects, with B-MnNPs showing a substantially greater restorative impact than C-MnNPs. Relative to As-stressed plants, C-MnNPs enhanced chlorophyll a and b contents by 49.38% and 43.48% and improved Pn, Gs, and Tr by 38.90%, 32.6%, and 29.3%, respectively. In contrast, B-MnNPs markedly elevated chlorophyll pigments and gas-exchange traits, increasing Pn, Gs, and Tr by 64.8%, 50%, and 47.6%, respectively, and restoring Fv/Fm values close to control levels (Figure 4A–F). The improved pigment retention and PSII efficiency under B-MnNPs were reflected in greener foliage and stronger photochemical stability, suggesting that biogenic MnNPs preserved the integrity of photosynthetic machinery and mitigated oxidative photodamage induced by As stress (Figure 4F).

3.5. Effect of MnNPs on ROS Activity

Arsenic treatment resulted in substantial accumulation of these oxidative stress markers in both leaf and root compared to control (Figure 5A–F). Specifically, MDA content, an indicator of lipid peroxidation, increased significantly by 60.88% in leaves and 76.14% in roots under As stress alone. Similarly, H₂O₂ levels were elevated by 77.18% in leaves and 79.57% in roots, while O₂^•− generation showed marked increments of 74.55% in leaves and 79.27% in roots under As exposure. The application of C-MnNPs and/or B-MnNPs significantly mitigated the As-induced oxidative damage. The treatment of B-MnNPs demonstrated more pronounced reductions in oxidative stress markers compared to C-MnNPs applications. The chemically synthesized C-MnNPs reduced MDA levels by 22.72% and 25.31% in leaves and roots, H₂O₂ by 25.92% and 25.06%, and O₂^•− by 24.14% and 24.80%, respectively, compared to As treatment alone.

B-MnNPs exhibited superior efficacy in reducing oxidative stress parameters when compared to their chemically synthesized counterparts. The maximum reductions observed with B-MnNPs were 30.74% and 33.94% for MDA, 33.37% and 32.78% for H₂O₂, and 35.22% and 36.17% for O₂^•− in leaves and roots, respectively, compared to As treatment.

Confocal microscopic analysis of root tissues further corroborated these biochemical findings. Fluorescent staining for H₂O₂ (red fluorescence) and O₂^•− (green fluorescence) revealed intense accumulation of both ROS in root cells under As stress alone, manifested as maximum red and green fluorescence signals throughout the root architecture (Figure 5G,H). Roots treated with C-MnNPs and/or B-MnNPs displayed reduced red and green fluorescence intensity compared to As stress, indicating partial alleviation of ROS accumulation. The B-MnNP treatment showed the most effective suppression of both H₂O₂ and O₂^•− generation in root tissues, as evidenced by substantially diminished red and green fluorescence signals, approaching levels comparable to control conditions. These confocal observations demonstrate that C-MnNPs and/or B-MnNPs, especially the B-MnNPs, effectively alleviate oxidative stress in rapeseed under As toxicity by reducing the accumulation of lipid peroxidation and ROS.

3.6. Effect of MnNPs on Antioxidant Defense Systems

The activities of SOD, CAT, APX and GR were quantified in leaf and root tissues of rapeseed to understand the antioxidant enzyme responses under As stress and following C-MnNP and/or B-MnNP applications. Arsenic treatment significantly modulated antioxidant enzyme activities compared to control (Figure 6A–H). Specifically, SOD activity increased by 51% in leaves and 76% in roots under As stress alone. CAT activity increased by 96.8% in leaves and 142% in roots, while APX activity increased by 131.4% in leaves and 208.8% in roots under As exposure. GR activity showed increases of 51.4% in leaves and 72.5% in roots under As stress. Exogenous application of C-MnNPs and B-MnNPs effectively restored antioxidant enzyme homeostasis. The chemically synthesized C-MnNPs increased SOD activity by 8.77% and 11.18% in leaves and roots, increased CAT activity by 17% and 20.21%, and enhanced APX activity by 17.04% and 20.2%, respectively, compared to As treatment alone. GR activity was elevated by 11.5% in leaves and 16.29% in roots with this treatment. B-MnNPs exhibited superior efficacy in modulating antioxidant enzyme activities. The application of B-MnNPs increased SOD activity by 30.46% and 30.38% in leaves and roots, inclined CAT activity by 45% and 53.7%, and elevated APX activity by 50.6% and 60.2%, respectively, compared to As stress alone. GR activity showed an enhancement of 35.8% in leaves and 39.4% in roots with B-MnNP treatment. These findings demonstrate that C-MnNPs and B-MnNPs, particularly the B-MnNPs, effectively restore antioxidant enzyme balance in rapeseed under As toxicity by normalizing enzyme activities in both leaf and root tissues.

3.7. Effect of MnNPs on Element Concentration and Arsenic Accumulation

Arsenic exposure markedly disrupted nutrient homeostasis in rapeseed, reducing essential mineral uptake while increasing As accumulation in plant tissues (Figure 7A,B). In comparison with the control, As-stressed plants exhibited a sharp rise in As concentration in both leaves and roots coupled with a corresponding decline in Mn, Fe, K, Mg, and P contents (Figure 7C,D and Figure 8A–H). The depletion was the most pronounced for Mn and Fe, which decreased by 89% and 57%, respectively, in leaves, and by 84% and 48% in roots, indicating interference with metal transport and assimilation pathways. Application of MnNPs mitigated these imbalances, with B-MnNPs showing superior efficacy to their chemically synthesized counterparts. Relative to As-stressed plants, C-MnNPs reduced As accumulation in leaves and roots by 27.60% and 24.50%, respectively, while increasing leaf Fe, K, Mg, and P contents by 46.67%, 57.7%, 63%, and 53.8%, respectively. In terms of roots, the corresponding increases were 32%, 85.5%, 24%, and 50%. The biogenic B-MnNPs, however, further decreased As accumulation by 41.4% and 40.68% in leaves and roots, respectively. In addition, they significantly increased Mn, Fe, K, Mg, and P concentration in leaves and in roots, exhibiting the trend B-MnNPs > C-MnNPs > Ck > B-MnNPs + As > C-MnNPs + As > As. The enhanced nutrient status under B-MnNP treatment corresponded with improved photosynthetic efficiency and growth attributes, suggesting that biogenic nanoparticles not only facilitated Mn bioavailability but also modulated overall nutrient uptake and translocation under As stress (Figure 7A–D and Figure 8A–H).

3.8. Effect of MnNPs on Phytohormone Profile

The levels of SA, ABA, and JA were quantified to elucidate the phytohormone-mediated stress signaling responses under As stress and following C-MnNP and/or B-MnNP treatments (Figure 9A,B). Arsenic stress alone significantly elevated all three phytohormones, increasing SA by 17.19%, JA by 35.85%, and ABA by 73.61% compared to control conditions. Exogenous application of C-MnNPs further enhanced phytohormone accumulation by 5.21% for SA, 22.87% for JA, and 14.13% for ABA over control. B-MnNPs demonstrated maximum phytohormone accumulation under As stress, with increases of 10.92% for SA, 55.42% for ABA, and 28.26% for JA over control conditions. These results indicate that B-MnNPs exhibited superior capacity to modulate phytohormone biosynthesis and accumulation compared to chemically synthesized counterparts, particularly under As stress. The elevated levels of SA, ABA, and JA in plants treated with As + B-MnNPs suggest enhanced activation of multiple defense signaling pathways, contributing to improved stress tolerance and adaptive responses in rapeseed (Figure 9A–C). Interestingly, despite the higher accumulation of ABA, which is typically associated with stomatal closure, the corresponding stomatal aperture measurements did not show a proportional reduction. This apparent mismatch likely reflects a temporal and functional disconnect between hormone accumulation and stomatal responses under C-MnNP and/or B-MnNP treatments. Moreover, the simultaneous elevation of SA and JA may modulate or counteract ABA-induced stomatal closure, resulting in a physiological state where hormone levels and stomatal behavior do not follow a direct linear relationship.

3.9. Effect of MnNPs on Gene Expression

Arsenic exposure induced significant transcriptional reprogramming in rapeseed, as evidenced by distinct gene expression profiles in both leaves and roots (Figure 10A,B). In leaves, As stress significantly upregulated genes associated with phenolic metabolism and oxidative responses, including BnaCAD and BnaPPO, as well as antioxidant and detoxification-related genes such as BnaDHAR, BnaMDHAR, BnaCHLG, BnaGSH, BnaCAO, BnaSOD, BnaCAT, BnaAPX, and BnaGR. However, the highest expression occurred in As + B-MnNP-treated plants, followed by As + C-MnNPs, indicating that MnNPs, particularly biogenic MnNPs (B-MnNPs), enhanced the transcriptional activation of these genes beyond the As stress response. This pattern suggests that B-MnNPs more effectively bolstered antioxidant defenses and the ascorbate–glutathione cycle, mitigating the redox imbalance caused by As toxicity. The overall transcriptional pattern in leaves followed the order As + B-MnNPs > As + C-MnNPs > As > B-MnNPs > C-MnNPs > Control, reflecting a progressive enhancement of gene expression with MnNP treatments under As stress (Figure 10A).

In roots, a similar transcriptional trend was observed, with As stress markedly inducing BnaCAD, BnaSOD, BnaDHAR, BnaMDHAR, BnaAPX, BnaCAT, BnaGSH, BnaCHLG, BnaCAO, and BnaGR (Figure 10B). The highest expression levels were observed in As + B-MnNP-treated plants, followed by As + C-MnNPs, surpassing the upregulation seen under As stress alone. This indicates a robust compensatory antioxidant response amplified by MnNPs, with B-MnNPs being more effective than C-MnNPs in reactivating these genes. The enhanced expression of BnaSOD, BnaCAT, and BnaGSH in MnNP-treated plants underscores strengthened enzymatic and non-enzymatic antioxidant defenses, consistent with improved cellular resilience against As toxicity. The overall transcriptional pattern in roots followed the order As + B-MnNPs > As + C-MnNPs > As > B-MnNPs > C-MnNPs > Control, confirming that biogenic MnNPs most effectively restored redox-related gene activity, aligning with reduced ROS levels and increased biomass observed under these treatments.

3.10. Leaf Ultrastructure Observation

To elucidate the influence of both C-MnNPs and B-MnNPs on stomatal aperture under As stress, microscopic examination of stomata on the epidermis of rapeseed was undertaken. Under As stress conditions, considerable structural impairment to the stomatal apparatus of rapeseed was observed (Figure 11). Conversely, supplementation of combined treatment of C-MnNPs and B-MnNPs substantially ameliorated the As-induced structural damage in stomata opening. Furthermore, quantitative assessment of the stomatal aperture revealed that As stress induced pronounced stomatal closure in rapeseed leaves. However, plants treated with C-MnNPs and B-MnNPs exhibited enhanced stomatal opening compared to As stress alone (Figure 11). Notably, the B-MnNPs demonstrated superior ameliorative effects compared to their chemically synthesized counterparts. These observations demonstrate that B-MnNPs effectively mitigate the deleterious effects of As on stomatal functioning in rapeseed.

4. Discussion

Arsenic is among the most toxic metalloids affecting global food crops, impairing plant growth, photosynthesis, and nutrient homeostasis through severe oxidative stress and metabolic disruption [53,54]. The present study demonstrated that B-MnNPs significantly alleviated As-induced toxicity in rapeseed, outperforming C-MnNPs in restoring physiological, biochemical, and molecular functions. The enhanced mitigation capacity of B-MnNPs reflects their improved bioavailability, surface functionalization by microbial biomolecules, and environmentally compatible interaction with plant tissue [31,55]. It is important to note that the enhanced performance of B-MnNPs may also be influenced by their surface chemistry. FTIR and EDS analyses revealed that B-MnNPs retained biomolecular residues originating from the microbial filtrate, which likely act as capping and stabilizing agents. Such organic coatings are absent in C-MnNPs, potentially contributing to differences in dispersion, aggregation, and biological interaction. Under As exposure, rapeseed plants exhibited pronounced growth inhibition, reduced leaf expansion, and suppressed biomass accumulation, which aligns with previous findings that As disrupts cell division and nutrient assimilation [56,57,58,59]. Treatments with MnNPs, particularly B-MnNPs, markedly recovered shoot and root growth, restoring biomass close to control levels. These improvements are likely linked to MnNP-mediated activation of photosynthetic and enzymatic systems that regulate redox homeostasis, as reported in previous studies [27,55]. The superior performance of B-MnNPs can be attributed to their better biocompatibility and surface attachment with plant tissues. Further, the organic capping layer provided by Bacillus pumilus MAY4 metabolites enhanced NP dispersibility, Mn ion release, and biocompatibility with plant tissues [23,24,25,60,61,62].

The restoration of photosynthetic machinery under B-MnNP treatment reflects improved chlorophyll retention, higher gas-exchange efficiency, and enhanced PSII stability; these findings fall in line with the recent studies on B-MnNP-mediated HM stress alleviation [19,63]. As-induced inhibition of Pn, gs, and Fv/Fm in rapeseed indicates both stomatal and non-stomatal limitations on photosynthesis, a well-documented response to As toxicity in rice, mustard, wheat, and rapeseed [56,57,58,64,65,66]. The reactivation of these parameters by B-MnNPs suggests effective protection of chloroplast membranes and enhanced activity of Mn-dependent enzymes such as the oxygen-evolving complex of PSII. This agrees with reports showing that Mn supplementation stabilizes the photosynthetic apparatus that mitigates HM-induced photo-inhibition [67]. Moreover, SEM analyses confirmed that B-MnNPs preserved stomatal aperture and guard-cell integrity, facilitating optimal CO₂ diffusion and transpiration even under As stress, a response previously observed in nanoprimed crops under metal toxicity [51,68].

Arsenic toxicity is primarily mediated by oxidative stress through overproduction of ROS, leading to lipid peroxidation and membrane instability [69,70]. In this study, As stress sharply increased MDA, H₂O₂, and O₂^•− accumulation in both leaves and roots, consistent with elevated ROS generation reported in As-exposed crops. However, B-MnNP treatment substantially suppressed ROS levels and restored redox balance, supported by confocal visualization showing weaker fluorescence intensity in B-MnNP-treated roots compared to As-stressed ones. This reduction in oxidative stress corresponds to the enhanced activity and expression of antioxidant enzymes, including SOD, CAT, APX, and GR [71,72,73]. Such Mn-dependent antioxidant activation has been similarly observed in nanoprimed wheat and carrot plants under Cd and Pb stress [28,74]. The stronger induction of antioxidant enzymes under B-MnNP treatment compared to C-MnNPs likely reflects the biological surface coating of the biogenic nanoparticles. FTIR and EDS results confirmed the presence of microbial biomolecules on B-MnNPs, which can act as natural capping and stabilizing agents. Such organic residues enhance nanoparticle compatibility with plant tissue, facilitate Mn delivery to redox-active centers, and may participate in signaling pathways that upregulate antioxidant machinery. These properties are absent in chemically synthesized MnNPs, which lack functionalized surfaces, providing a mechanistic explanation for the superior activation of SOD, CAT, APX, and GR in B-MnNP-treated plants.

In addition to redox regulation, MnNPs markedly influenced nutrient dynamics under As stress. The substantial decline in Fe, K, Mg, and P contents in As-treated plants indicates impaired ion transport and competitive interference between As and essential mineral nutrients, a phenomenon previously documented in rice and spinach [75,76]. B-MnNPs effectively restored nutrient homeostasis by improving Mn availability and membrane permeability, thereby enhancing Fe and P translocation and reducing As accumulation in both roots and shoots. This result aligns with the findings of [31], who demonstrated that biogenic metal NPs facilitate nutrient uptake through rhizospheric modulation and improved root architecture. The lower As translocation factor observed in B-MnNP-treated plants suggests that Mn facilitates nutrient uptake, particularly P uptake through phosphate transporters, rather than As, thereby restricting As through metal ion chelation and reducing mobility within the plant, as recently reported [19]. Moreover, the marked reduction in As concentrations in both roots and shoots under B-MnNP treatment suggests that their biologically functionalized surface may provide additional reactive groups capable of binding or adsorbing As species. Such surface-mediated interactions can lower the pool of freely available As for root uptake and translocation. In contrast, C-MnNPs lack these bio-derived functional sites, which likely limits their capacity for As complexation.

Phytohormones serve as central mediators of plant stress responses, orchestrating complex signaling networks that regulate physiological and biochemical adaptations to environmental challenges [77,78,79,80]. Under HM stress, plants typically exhibit dysregulated hormonal homeostasis, with elevated ABA levels promoting stomatal closure and osmotic adjustment, while SA and JA activate defense gene expression and antioxidant metabolism, responses well-documented in As-exposed crops including rice, soybean, and rapeseed [37,81,82,83]. In this study, the superior performance of B-MnNPs in modulating phytohormone biosynthesis suggests a more refined regulatory mechanism compared to C-MnNP treatment under As stress. This enhanced efficacy can be attributed to the biocompatibility of B-MnNPs, which possess bacterial-produced metabolites as surface coatings that facilitate cellular recognition and uptake while minimizing cytotoxicity, a characteristic advantage of B-NPs previously demonstrated in alleviating salt, Cd, and Cr toxicity in various crops such as rice, mung-bean, and maize [84,85,86]. The pronounced ABA accumulation under B-MnNP treatment likely optimized stomatal regulation without causing complete closure, thereby maintaining a balance between reduced As uptake via transpiration and adequate CO₂ assimilation for photosynthesis. Simultaneously, the elevated SA and JA levels indicate activation of complementary defense pathways under As stress. This coordinated hormonal modulation suggests that B-MnNPs function as immunomodulators that recalibrate the plant stress responsive machinery; similar results were reported for bio-CuNPs in rice [55]. Although increased ABA levels typically promote stomatal closure under stress [87], the higher accumulation under As + B-MnNPs did not correspond to a proportional reduction in stomatal aperture. This mismatch likely reflects a delay between hormone buildup and guard-cell signaling, together with the concurrent rise in SA and JA, which can modulate or attenuate ABA sensitivity. Such cross-talk can decouple ABA levels from stomatal behavior, explaining the non-linear response observed in our study. Collectively, the B-MnNP-mediated enhancement of phytohormone signaling reflects a sophisticated reprogramming of defense-responsive phytohormones, conferring As tolerance to rapeseed.

At the molecular level, gene expression analysis corroborated the physiological and biochemical data. Stress upregulated most antioxidant-related genes (BnaAPX, BnaCAT, BnaSOD, and BnaGR) and detoxification-associated transcripts (BnaPCs, BnaMDHAR, and BnaDHAR), along with stress markers such as BnaPPO and BnaCAD. Treatment with B-MnNPs further enhanced these trends, strongly upregulating ROS-scavenging and redox-regenerating genes, consistent with improved antioxidant enzyme activities. The upregulation of BnaGSH and BnaPAL further suggests that B-MnNPs activate both enzymatic and non-enzymatic defense pathways to counteract As-induced oxidative damage. Similar transcriptional enhancement patterns under biogenic nanoparticles have been reported in diverse crops. In Citrullus lanatus, bio-functionalized MnNPs suppressed Fusarium wilt through infection disruption and host defense response potentiation, upregulating ROS-scavenging and phenylpropanoid-pathway genes [31]. Likewise, biosynthetic selenium (Se) NPs mitigated antimony toxicity in rice by improving antioxidant defense and modulating stress-responsive gene expression [88]. In Cucumis sativus, Mn₃O₄ nanozymes enhanced resistance to salinity stress by boosting endogenous antioxidant metabolites and activating redox regulatory networks [89]. Moreover, MnNPs in Capsicum annuum controlled salinity-modulated molecular responses through priming of MnSOD and associated ROS-detoxifying genes [90].

Furthermore, B-SiO₂ NPs modulated phenylpropanoid pathway genes (BnaPAL, BnaCAD, and BnaPPO) in rapeseed under Cd stress, demonstrating nanoparticle-induced enhancement of lignin biosynthesis and phenolic metabolism [51]. Similarly, biogenic metallic nanoparticles (AgNPs and FeNPs) altered lignification-related gene expression in Glycine max, particularly upregulating BnaPAL and BnaCAD, signifying activation of the phenylpropanoid pathway [61]. Together, these reports support our findings that B-MnNPs act as efficient elicitors of molecular defense networks in B. napus, coordinating antioxidant, phenylpropanoid, and glutathione-related gene expression to maintain ROS homeostasis and mitigate HM toxicity.

Stomatal structure and function represent critical determinants of plant stress tolerance, as guard-cell integrity and aperture regulation directly influence gas exchange, transpirational cooling, and the uptake of both essential nutrients and toxic elements. Arsenic induces severe structural damage to the stomatal apparatus through oxidative injury to guard-cell membranes and disruption of ion homeostasis. The resulting stomatal closure, although initially serving to restrict further metal entry, ultimately impairs photosynthetic efficiency by reducing CO₂ influx and disturbing the leaf energy balance under As stress. Similar reductions in stomatal conductance and photosynthetic performance under metalloid exposure have been reported in wheat, sugar beet, and glossy privet plants [91,92,93,94]. The superior ability of B-MnNPs to maintain stomatal structure and aperture likely stems from their bioactive coatings, which protect guard-cell membranes from ROS damage and lipid peroxidation, preserving normal turgor function. Furthermore, the improved hormonal balance, particularly regulated ABA levels, appears to facilitate a controlled stomatal response that conserves water without inducing full closure, unlike the severe constriction observed under As stress alone. The preservation of stomatal function by B-MnNPs represents a pivotal stress adaptation mechanism in rapeseed under As stress.

Collectively, these findings provide a mechanistic framework wherein B-MnNPs alleviate As toxicity by improving Mn bioavailability, enhancing antioxidant defense, restoring nutrient equilibrium, and stabilizing photosynthetic function (Figure 12). The microbial synthesis route confers unique biochemical interfaces on NPs, enabling controlled Mn release and minimal environmental toxicity compared with conventional C-NPs. Thus, B-MnNPs not only mitigate As-induced physiological and molecular disturbances but also offer a sustainable, biologically compatible strategy for improving crop performance in metal-contaminated environments.

5. Conclusions

This study demonstrated the superior efficacy of B-MnNPs over C-MnNPs in mitigating As toxicity in B. napus. Biosynthesis using Bacillus pumilus MAY4 represents an environmentally sustainable approach with enhanced biocompatibility. B-MnNPs significantly ameliorated As-induced phytotoxicity through multiple mechanisms, substantially improving plant biomass, photosynthetic efficiency, and chlorophyll content. The nanoparticles effectively mitigated oxidative stress by reducing ROS and lipid peroxidation while enhancing antioxidant enzyme activities. Notably, B-MnNPs reduced As accumulation in both leaves and roots while simultaneously enhancing uptake of essential minerals. Gene expression revealed upregulation of stress-responsive genes, and elevated phytohormone levels indicated activation of defense signaling pathways. Ultrastructural observations confirmed restoration of stomatal functionality. These findings establish B-MnNPs as a promising eco-friendly nanotechnology-based solution for managing metal stress in agricultural systems. Future research should focus on field-scale validation to facilitate practical implementation in As-contaminated regions.