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Article

Synthesis, Antibacterial Properties, and Physiological Responses of Nano-Selenium in Barley (Hordeum vulgare L.) Seedlings Under Cadmium Stress

by
Hongyan Sun
1,*,
Xin Lian
1,
Runge Yao
1,
Bingjie Shang
1,
Siyu Yi
1,
Jia Yu
1,
Bo Zhang
1 and
Xiaoyun Wang
2,*
1
School of Environment and Resources, Taiyuan University of Science and Technology, Taiyuan 030024, China
2
Institute of Soil and Water Conservation, Shanxi Agricultural University, Taiyuan 030045, China
*
Authors to whom correspondence should be addressed.
Agronomy 2025, 15(12), 2750; https://doi.org/10.3390/agronomy15122750
Submission received: 25 October 2025 / Revised: 19 November 2025 / Accepted: 27 November 2025 / Published: 28 November 2025
(This article belongs to the Special Issue Regulatory Network of Crop to Environmental Stress: Genetic and Biochemical Characterization)
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Abstract

Selenium (Se) nanoparticles have emerged as a vital tool in enhancing plant resilience to multiple stress factors. So, the present study was designed to synthesize nano-Se, evaluate its antibacterial properties, and to investigate the effects of nano-Se at 2, 5, 10, and 15 μM on the growth and physiological responses of barley seedlings under Cd stress. The results showed that nano-Se with an average size of 24.71 nm exhibited strong antibacterial activity against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Notably, 5 μM nano-Se reduced Cd concentrations in leaves and roots by 19.46% and 31.07%, respectively, while enhancing root length, shoot/root fresh weight (FW), and dry weight (DW) compared to Cd-stressed plants. Furthermore, exogenous nano-Se significantly increased chlorophyll, protein, amino acid content, and enhanced photosynthetic performance compared to Cd treatment alone. Nano-Se further boosted the activity of antioxidant enzymes and concurrently reduced malondialdehyde (MDA), hydrogen peroxide (H2O2), proline, total flavonoids, and total phenols levels. Moreover, nano-Se supplementation under Cd stress promoted the uptake of essential nutrient elements and increased sugar content. Our results collectively suggest that nano-Se application during Cd stress may enhance photosynthesis, promote carbohydrate metabolism, and mitigate oxidative damage, thereby improving barley growth under Cd toxicity.

Graphical Abstract

1. Introduction

Soil heavy metal pollution has been identified as a global environmental problem, which has a significant impact on agriculture and food safety [1]. Among them, Cadmium (Cd) was identified as the most prevalent pollutant. In China, the exceedance of Cd was 7.0% among soil pollutants [2]. Cd pollutants can enter crops through contaminated soil, posing a threat to food security and causing adverse effects on human health. The harm of Cd to plants was mainly manifested as leaf chlorosis, decrease in rhizomes, and excessive accumulation of reactive oxygen species (ROS) [3]. Cd can also accumulate in plant cells and interfere with normal cellular physiological activities, including photosynthesis, nutrient absorption, and the functioning of antioxidant enzymes [4]. According to a soil survey performed in China, at least 13,330 ha of farmland in 11 provinces has been contaminated by varying degrees of Cd [5]. Therefore, it is crucial to develop eco-friendly strategies to prevent the accumulation of Cd in plant tissues and mitigate its detrimental effects on plant growth. In this regard, nanobiotechnology offers a promising approach to enhance Cd tolerance in plants.
In recent decades, significant progress has been made in the rational use of nanomaterials to enhance plant tolerance under various stress conditions, including nano-selenium (Se) [6]. Se is regarded as an essential micronutrient element for plants, animals, and humans; its deficiency can lead to specific diseases [7]. Moreover, Se can enhance plant resilience against heavy metals and other stressors [8,9]. However, it plays indispensable roles within only a tiny and narrow concentration range in plants [1,10]. That is to say, low concentrations of Se can promote plant growth and development, enhance quality, and increase the yield of plants grown in contaminated soil, while high concentrations may induce oxidative stress, thereby reducing crop yield and causing metabolic disorders [11]. Therefore, in agricultural production, the effective dosage range of sodium selenite (Na2SeO3) is quite narrow, which poses risks of micronutrient accumulation and food safety concerns.
In contrast, nano-Se demonstrates a wider range of optimal concentration for application and toxic concentration than selenite and selenate. It also offers higher bioavailability, biosafety, and bioactivity, making it an increasingly attractive option for agricultural use. For instance, Zsiros et al. described that at the same concentration, nano-Se had a more significant promoting effect on tobacco organogenesis and root growth compared to Se [12]. Ghanbari et al. reported that the application of nano-Se significantly improved the growth and phytochemical compounds of lemon verbena [13]. Furthermore, nano-Se was more effective than traditional Se forms at equivalent concentrations in alleviating drought stress in wheat [14]. Additionally, appropriate concentrations of nano-Se have demonstrated positive effects on the growth of rice, wheat, barley, tomato, cabbage, tobacco, and mustard, both in environments with and without Cd contamination [15,16,17,18]. Additionally, nano-Se exhibits significant protective effects under abiotic stresses and can mitigate stress impacts on plants through various mechanisms. Some studies have indicated that exogenous nano-Se enhances Cd tolerance in plants primarily by increasing the activities of antioxidant enzymes [19], promoting the fixation of heavy metals in plant cell walls [20], improving photosynthetic efficiency [16], and altering the transcript levels of Se-related genes [17,21].
Barley (Hordeum vulgare L.) is commonly recognized as a staple food in North Africa and certain regions of Asia. However, barley is relatively sensitive to Cd toxicity compared to other cereal crops. In barley, 1 μM of Cd exposure significantly inhibited root elongation, plant height, and root and shoot fresh and dry weight [22,23]. Therefore, it is significant to understand the role of nano-Se in promoting adaptive responses of barley seedlings to cope with Cd stress. Accordingly, this research was carried out to synthesize nano-Se, evaluate its antibacterial activity, and investigate its potential to enhance Cd stress tolerance in barley plants. We hypothesized that nano-Se could mitigate the adverse effects of Cd stress on barley seedlings. Accordingly, the effects of four concentrations of nano-Se on barley seedlings under Cd stress were examined by analyzing growth parameters, pigment content, photosynthetic performance, Cd and other related element concentrations, oxidative stress levels, and the levels of secondary metabolites. The objectives of this study were to determine the optimal concentrations of nano-Se for alleviating Cd toxicity and to elucidate the physiological and biochemical mechanisms by which nano-Se enhances barley’s resistance to Cd stress. This research provides a theoretical foundation for developing safe and sustainable barley production technologies.

2. Materials and Methods

2.1. Synthesis and Characterization of Nano-Se

Chitosan (Cs, ≥75 deacetylation), sodium selenite (Se), and ascorbic acid (AsA) were obtained from Sigma-Aldrich (Shanghai, China). All other chemicals used in this study were of analytical grade and obtained from Sangon Biotech (Shanghai, China). Nano-Se was prepared using Chen et al.’s method [24]. In total, 250 mL of sodium selenite (Na2SeO3, 0.1 M) was mixed with 300 mL of chitosan solution (Cs, 12 g/L) at 30 °C with stirring at 100 r/min for 30 min. Subsequently, 250 mL of ascorbic acid (AsA, 0.5 M) was added dropwise to the mixture. Deionized water was then added to adjust the total volume to 1 L. The reaction mixture was maintained at 30 °C for an additional 1 h to complete the synthesis [18]. The morphology and composition of nano-Se were analyzed using TEM (JEM-2100, JEOL Ltd., Tokyo, Japan). Additionally, the solution was characterized by UV-Vis spectrophotometry within the wavelength range of 300 nm to 800 nm.

2.2. Antibacterial Activity of Nano-Se

The antibacterial activity of nano-Se was tested against the Gram-negative E. coli (ATCC 25322) and Gram-positive S. aureus (ATCC25922). Firstly, E. coli and S. aureus were inoculated in LB liquid medium and cultured by shaking at 37 °C at 100 r/min for 6 h until the bacterial suspension reached the desired concentration (~106 CFU/mL). The cultured bacterial solution was diluted and inoculated into an LB liquid medium containing different concentrations of nano-Se (0, 0.005, 0.025, 5, 15, 25 mM). Subsequently, the bacteria were incubated at 37 °C with continuous shaking at a speed of 100 r/min for 8 h. The effects of various treatments were observed by measuring absorbance at 600 nm; the ultimate concentration, antibacterial rate (%) (1), and bacterial viability (%) (2) were calculated according to Tuyen et al. [25].
Antibacterial rate (%) = (Absorbance of control − Absorbance of the test)/Absorbance of control × 100
Bacterial viability (%) = Absorbance of the test/Absorbance of control × 100
Finally, 50 μL of each bacterial suspension was spread onto the surface of plate count agar (PCA) medium. The plates were then incubated at 37 °C for 8 h. The effects of different concentrations of nano-Se on the growth of the strains were evaluated by observing the growth of the colonies.

2.3. Plant Material and Experimental Designs

A hydroponic experiment was conducted at Taiyuan University of Science and Technology. Commonly cultivated barley cultivar “Xiyin 2” was selected for the experiment. When barley seedlings reached the two-leaf stage, healthy and uniform plants were transplanted into 3 L hydroponic containers filled with 2.8 L of basal nutrient solution (BNS) (mg/L): (NH4)2SO4, 48.2; MgSO4·7H2O, 154.88; K2SO4, 15.9; KNO3, 18.5; Ca(NO3)2·4H2O, 86.17; KH2PO4, 24.8; Fe-citrate· 5H2O, 7; MnCl2·4H2O, 0.9; ZnSO4·7H2O, 0.11; CuSO4·5H2O, 0.04; HBO3, 2.9; H2MoO4, 0.01. The pH of the BNS was regulated to 5.9 ± 0.1 by adding 1 mol/L of hydrochloric acid (HCl) or sodium hydroxide (NaOH) [3]. The bucket was covered with a lid with five evenly spaced holes (three plants per hole), and placed in a greenhouse with a 12 h photoperiod (300 μmol/m2/s) at 22/20 °C (day/night), the relative humidity was 60–65%, and the nutrient solution was continuously aerated with pumps and renewed every 5 days. After 5 days of hydroponic cultivation, nano-Se and Cd were added to the corresponding nutrient solution, with concentrations selected based on our previous studies [3,18] and preliminary experimental results. Six experimental treatments with three replicates each were established based on preliminary experiments: Control (BNS), Cd (50 μM CdCl2), four nSe treatments (2, 5, 10, and 15 μM nano-Se) in combination with Cd treatment (50 μM CdCl2). Following a 15-day treatment period, all seedlings at the five-leaf stage were harvested, stored at −80 °C for further analysis, or immediately used for various biochemical assays.

2.4. Plant Growth, Biomass, and Element Determination

After 15 days of treatment, all plants were uprooted and separated into shoots and roots. The roots were immersed in 20 mM EDTA for 20 min to eliminate metals adhered to the root surface, followed by a rinse with purified water. The height of the plants and the length of the roots were measured simultaneously. The fresh weights (FWs) of both shoots and roots were recorded before determining their dry weights (DWs) after drying in an oven at 80 °C for 72 h. Dried plant materials were ashed at 550 °C, followed by digestion with 30% HNO3. The levels of Cd, Cu, Zn, Ca, and Mg were quantified using flame atomic absorption spectroscopy [3].

2.5. Photosynthetic Pigment Content and Photosynthetic Parameters

The first fully expanded leaves were chosen for assessing pigment levels and photosynthetic parameters. Pigments were extracted in a 1:1 v/v mixture of acetone and ethanol, and the absorbance of the extraction solution was measured using a spectrophotometer at wavelengths of 645 nm, 663 nm, and 470 nm, respectively [26]. The net photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance (Gs), and intercellular carbon dioxide concentration (Ci) were measured using a fully automatic portable photosynthesis system (LC Pro-SD, ADC BioScientific Ltd., Hoddesdon, UK).

2.6. Lipid Peroxidation, Hydrogen Peroxide, and Antioxidant Enzyme Activities

After a 15-day treatment, 0.3 g of fresh plant samples were homogenized in 8 mL 0.05 M phosphate-buffered solution (pH 7.8), and then subjected to centrifugation at 12,000× g for 10 min. The supernatant was collected to evaluate lipid peroxidation, hydrogen peroxide, and all enzyme activities.
Hydrogen peroxide (H2O2) content was determined following the procedure described by Zhou et al. [27]. The H2O2 content was assayed by monitoring the A410 of the titaniumperoxide complex. Lipid peroxidation levels were assessed by quantifying the content of malondialdehyde (MDA). MDA content was determined by the thiobarbituric acid reaction [28]. The activities of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), and peroxidase (POD, EC 1.11.1.7) were determined following the protocols reported by Zhang et al. [29]. The NBT method was used to determine the activity of the SOD enzyme, and the degree of inhibition of the SOD enzyme on the reduction in nitrotetrazolium chloride blue was evaluated using a UV spectrophotometer (Model UV-1800, Shimadzu corp., Kyoto, Japan) at 560 nm. The reaction mixture for POD consisted of 100 μL of enzyme extract, 100 μL of guaiacol (1.5%, v/v), 100 μL of H2O2 (300 mM), and 2.7 mL of 25 mM PBK with 2 mM EDTA (pH 7.0). Increases in the absorbance were measured spectrophotometrically at 470 nm (an extinction coefficient of 26.6 mM/cm). The assay mixture for CAT contained 100 μL of enzyme extract, 100 μL of H2O2 (300 mM), and 2.8 mL of 50 mM phosphate buffer with 2 mM EDTA (pH 7.0). CAT activity was assayed by monitoring the decrease in the absorbance at 240 nm as a consequence of H2O2 consumption (an extinction coefficient of 39.4 mM/cm). Ascorbate peroxidase (APX, EC 1.11.1.11) activity was determined according to Nakano and Asada [30]. The reaction mixture consisted of 100 μL of enzyme extract, 100 μL of ascorbate (7.5 mM), 100 μL of H2O2 (300 mM), and 2.7 mL of 25 mM potassium phosphate buffer with 2 mM EDTA (pH 7.0). The oxidation of ascorbate was determined by the decrease in absorbance at 290 nm (an extinction coefficient of 2.8 mM/cm).

2.7. Carbohydrate Measurement

Fresh plant tissues were digested in 80% ethanol and heated in an 85 °C water bath for 30 min. Then, the sample was centrifuged at 1000× g for 5 min, and the precipitate was extracted with 80% ethanol again. The extraction process was repeated three times, and the extracts were combined for sugar quantification. The residue was then extracted with perchloric acid and used for starch measurement. The determination of soluble sugar and starch was conducted using the anthrone-concentrated sulfuric acid method at a wavelength of 630 nm. Sucrose was quantified using the anthrone–potassium nitrate method at 620 nm. The reducing sugar content was measured with 3,5-dinitrosalicylic acid reagent at a wavelength of 520 nm [31].

2.8. Total Flavonoids, Phenols, and Proline Measurement

The total flavonoid content can be measured via spectrophotometry [32] and a rutin standard curve and expressed as mg rutin equivalents per g fresh weight. The total phenol content was measured using the Folin–Ciocalteu reagent method, according to Terpinc et al. [33], and expressed as mg gallic acid equivalents per g fresh weight. The content of proline was evaluated using the technique provided by Bates et al. [34].

2.9. Amino Acid and Soluble Protein Evaluation

Amino acid content was measured as recommended by Huang et al. [35]. Following the method detailed by Bradford, the soluble protein content was measured [36].

2.10. Statistics

The results were the average of at least three independent replicates. Statistical analyses were performed with a data-processing system (DPS) statistical software package (version v21.05) using ANOVA followed by Duncan’s multiple range tests to evaluate significant differences among different treatments, with different letter means at a significance level of p ≤ 0.05.

3. Results

3.1. Characterization of Nano-Se

TEM characterization revealed that the synthesized samples possessed rod-shaped nanostructures, with an average particle size of approximately 24.71 nm (Figure 1A). In the UV spectrum of the synthesized nano-Se, a distinct absorption peak was observed within the range of 300 to 350 nm, indicating its nanoscale characteristics. The absorption spectrum remained consistent over time, demonstrating the stability of the particles (Figure 1B).

3.2. Analysis of the Antibacterial Activity of Nano-Se

The growth of E. coli and S. aureus in the control group remained robust; however, the addition of nano-Se inhibited the growth and reproduction of both bacteria to varying degrees (Figure 2A,B). For E. coli, no significant inhibition was observed at a concentration of 5 μM of nano-Se; however, at concentrations above 5 mM, the E. coli was significantly inhibited, with the antibacterial rate ranging from 85.4% to 92.4% as the concentration increased from 5 mM to 25 mM. The antibacterial effects against S. aureus were comparable to those observed for E. coli. Figure 2D,E demonstrate that the antibacterial effect increased with the increase in nano-Se concentration, and the antibacterial activity to both bacteria reached the maximum at 25 mM. The corresponding antibacterial rate for E. coli and S. aureus was 92.4% and 72.5%, respectively. Conversely, bacterial viability exhibited an opposite trend, reaching its lowest point at 25 mM nano-Se, with E. coli and S. aureus accounting for 7.6% and 27.5%, respectively.

3.3. Plant Growth Parameters

Barley plants subjected to Cd stress exhibited significant growth inhibition. Compared to untreated plants, Cd stress led to a reduction in root length and plant height by 16.63% and 29.08%, respectively. Furthermore, Cd treatment decreased the root FW and DW by 38.40% and 18.15%, while the shoot FW and DW were reduced by 37.14% and 21.41%, respectively, in comparison to the control group (Figure 3). Nevertheless, exogenous nano-Se (2, 5, 10, and 15 μM) effectively mitigated the growth inhibition caused by Cd stress. The mitigating effects of the four nSe treatments (Cd + nSe) on barley seedlings initially increased, but then diminished after reaching a specific threshold concentration of nano-Se. The lowest growth traits were observed with the application of 15 μM nano-Se. The highest growth parameters were observed when 5 μM of nSe (Cd + nSe5) was applied, resulting in a 16.52% increase in root length compared to the Cd treatment alone, nearly reaching the levels of the control group (Figure 3A). Additionally, the application of 5 μM nSe (Cd + nSe5) increased root and shoot FW by 58.34% and 31.45%, respectively, while the root and shoot DWs increased by 30.43% and 18.63%, compared to the plants subjected to Cd stress alone (Figure 3B,C).

3.4. Photosynthetic Pigment Contents and Parameters

The changes in plant pigments in barley under Cd treatment and nSe application were illustrated in Figure 4. In contrast with the control plants, the contents of chlorophyll a (Chl a), chlorophyll b (Chl b), and chlorophyll a + b (Chl a + b) were markedly reduced by 58.46%, 78.14%, and 60.46%, respectively, under 50 μM Cd stress. Barley plants treated with nSe (Cd + nSe) at all levels exhibited an increase in photosynthetic pigment content compared to those treated with Cd alone. Exogenous nSe (Cd + nSe) treatments caused an increase of 27.26–108.69% in chlorophyll a, 65.84–459.40% in chlorophyll b, 29.43–128.47% in chlorophyll a + b, and 16.09–141.39% in carotenoids in barley leaves compared to the Cd-alone treatments. Moreover, the highest growth percentage was observed in plants supplied with 5 μM nSe.
Treatment with Cd resulted in reductions of 11.50%, 11.54%, and 43.09% in photosynthetic rate (Pn), transpiration rate (Tr), and stomatal conductance (Gs), respectively, compared to the control group. However, there was a significant increase in intercellular carbon dioxide concentration (Ci). The application of 5 μM of nano-Se effectively mitigated the changes induced by Cd. Specifically, compared to the Cd-only treatment, Pn, Tr, and Gs increased by 39.75%, 37.39%, and 50.00%, respectively, while Ci decreased by 48.54% (Figure 5).

3.5. Cd and Nutrient Element Concentration

In the absence of Cd and/or nano-Se application, leaf and root Cd levels were below the minimum detectable concentration, and thus, they were not included in Table 1. Compared to the Cd-only treatment, applying nano-Se lowered Cd levels in both leaf and root tissues; 5 μM nSe (Cd + nSe5) resulted in the most considerable reductions of 19.46% and 31.07% in leaves and roots, respectively.
In addition, Cd treatment markedly increased Cu concentration in the roots while decreasing Zn and Mn concentrations in both the leaves and roots. Conversely, the addition of nano-Se resulted in a notable restoration of Zn, Mn, and Cu levels, with the exception of root Zn concentration under Cd + nSe10 and Cd + nSe15, as well as Mn concentrations under Cd + nSe15. For example, Zn concentrations in the Cd + nSe5 treatments were 95.84% higher in leaves and 26.10% higher in roots, compared to plants subjected to Cd stress alone. Furthermore, Cd stress significantly reduced Ca and Mg concentrations in both roots and leaves. The Cd-induced reduction in Ca and Mg levels was notably alleviated by the application of nano-Se, with the Cd + nSe5 group exhibiting the highest concentrations of both minerals. In the Cd + nSe5 treatment, Ca concentrations were 53.23% higher in leaves and 37.20% higher in roots, compared to the Cd-only treatment.

3.6. Accumulation of Lipid Peroxidation, Hydrogen Peroxide, and Antioxidant Enzyme Activities

Cd stress significantly induced lipid peroxidation and H2O2 generation in barley seedlings (Figure 6). The increase in MDA and H2O2 levels in the leaves and roots caused by Cd was notably reduced by the application of 2, 5, and 10 μM of nano-Se under Cd stress. Both MDA and H2O2 levels initially decreased and then began to rise with increasing concentrations of nano-Se. Among the Cd + nSe treatments, the application of 5 μM of nano-Se under Cd stress resulted in the most significant reductions: MDA and H2O2 contents in leaves/roots decreased by 16.76%/49.96% and 49.58%/41.53%, respectively, compared with the Cd-alone treatment. However, the levels of MDA and H2O2 were significantly elevated following the Cd + nSe15 treatment compared to the Cd-only treatment.
The effects of Cd and varying concentrations of nano-Se on the activities of SOD, POD, CAT, and APX in barley seedlings are presented in Table 2. Cd stress induced a considerable increase in root SOD, leaf/root POD, root CAT, and leaf/root APX activities, with increases of 7.04%, 116.06%/19.13%, 73.65%, and 27.02%/18.83%, respectively, compared to the control. When nano-Se was added, the antioxidant enzyme response of barley seedlings exhibited varying trends. Compared to the Cd-alone treatment plants, the activities of root SOD, leaf/root CAT, and leaf/root APX under the Cd + nSe treatment exhibited an initial increase, followed by a decrease as the concentration of nano-Se rose. The application of Cd + nSe5 resulted in maximum increases of 8.81% for root SOD, 53.67%/71.62% for leaf/root CAT, and 18.17%/41.56% for leaf/root APX compared to Cd treatment alone. However, the activity of POD under Cd + nSe5 treatment was markedly reduced compared to treatment with Cd alone, showing reductions of 10.46% in leaves and 13.24% in roots, respectively.

3.7. Carbohydrate Content

Compared with the control group, Cd treatment alone significantly increased the soluble sugar content by 26.89% in leaves and 35.95% in roots. Additionally, the sucrose content in roots and the starch content in leaves also increased markedly. Furthermore, the reducing sugar level in roots decreased by 7.62%, while other carbohydrates showed no substantial distinctions compared with the untreated group (Table 3).
On the other hand, the data indicated that Cd + nSe5 treatment improved soluble sugar levels in both leaves and roots compared to the Cd-only treated plants. Additionally, both the Cd + nSe2 and Cd + nSe5 treatments noticeably enhanced the levels of reducing sugars, with the 5 µM nano-Se treatment showing a more pronounced effect, resulting in an increase of 86.73% in leaves and 14.59% in roots, compared with the Cd-only treatment. Furthermore, exogenous application of 5 µM nSe (Cd + nSe) markedly increased sucrose content in roots, and starch content in both leaves and roots, compared to the Cd-alone treatment (Table 3).

3.8. Total Flavonoid, Total Phenol, and Proline Content

Compared to the untreated plants, Cd treatment markedly raised the levels of total flavonoids and total phenols (Figure 7). However, the application of all nano-Se levels (Cd + nSe) decreased leaf total flavonoid content, with the percentage reduction ranging from 7.40% to 23.21% compared to Cd stress alone. The percentage exhibited a trend of first decreasing and then increasing as the concentration of nano-Se rose, and the nSe5 treatment showed the most significant reduction, with total flavonoid content in leaves decreasing by 23.21% compared to the Cd treatment alone. Total phenol content in roots displayed a variation pattern similar to that of the total flavonoid content in leaves, with the lowest levels observed under the Cd + nSe5 treatment. However, the root total phenols content under high levels of nano-Se treatment (nSe10 and 15) was higher than that observed with Cd treatment alone. Nevertheless, the nano-Se treatment had no obvious effect on the total flavonoid content in roots or the total phenol content in leaves.
Proline contents in leaves and roots under Cd-alone treatment were 11.99% and 32.73%, respectively, higher than those in the control group. Compared to cadmium-treated plants, 5 µM and 10 µM of nano-Se (Cd + nSe) significantly reduced the proline content in leaves and roots. Notably, 5 µM of nano-Se showed a stronger inhibitory effect, decreasing leaf and root proline levels by 35.25% and 31.25%, respectively. Furthermore, the application of 15 µM of nano-Se significantly increased proline content in both leaves and roots (Figure 7C).

3.9. Analysis of Amino Acid and Soluble Protein Content

Compared with untreated control plants, Cd treatment significantly reduced amino acid content in both leaves and roots. The decrease in amino acid content induced by Cd was notably enhanced by exogenous nano-Se (2, 5, 10, 15 μM) addition. With the increase in nano-Se concentration, the growth percentage increased first and then decreased. Cd + nSe5 treatment had the most beneficial effect on amino acid content, which increased 34.85% and 47.32% in leaves and roots, respectively, compared with Cd alone treatment (Figure 8A). Additionally, a similar trend was observed in soluble protein content; Cd treatment led to a decrease, while the addition of nano-Se (Cd + nSe) resulted in an increase in soluble protein content in both leaves and roots. The most significant increase was recorded with the Cd + nSe5 treatment (Figure 8B).

4. Discussion

Heavy metal pollution in agricultural soil has become a critical issue. The bioaccumulation of heavy metals in plants not only disrupts essential metabolic processes but also hinders normal plant development, ultimately limiting crop production [1]. Therefore, there is an urgent need for strategies to mitigate heavy metal contamination. The production and development of metal nanoparticles have garnered significant attention in the fields of agriculture. Many researchers are interested in various methods to synthesize Se nanoparticles because of their low toxicity and high bioactivity [12]. Consequently, the potential of Se nanoparticles has gained popularity in several fields of agriculture [15,16], like using Se nanoparticles to alleviate Cd toxicity effects on rice [16]. The formation of Se nanoparticles typically involves a redox system consisting of inorganic selenium and reducing agents like ascorbic acid and cysteine [21,37]. The previous studies have shown that low doses of Se nanoparticles, synthesized using sodium selenite and ascorbic acid, promoted barley growth under Cd stress [21]. However, the Se nanoparticles are unstable without stabilizers due to their high surface energy, which makes them difficult to transport and poorly absorbed by plants [38]. To obtain stable Se nanoparticles, stabilizing agents, such as polysaccharides and proteins, are commonly added during the preparation process to prevent particle aggregation and enhance their biological activity. Chitosan, a biocompatible and biodegradable polysaccharide, has been widely demonstrated as a safe and effective encapsulation matrix for bioactive compounds [39]. Chitosan also has several favorable biological properties, such as a penetration-enhancing effect, antibacterial, biocompatibility, and low toxicity. Thus, chitosan serves as an ideal stabilizer for the synthesis of Se nanoparticles, gaining significant attention in modern nanotechnology research [40]. In the present study, nano-Se was synthesized using ascorbic acid and chitosan, and its dose-dependent effects on Cd-stressed barley seedling growth, photosynthesis, physiology, and antioxidative capacity were assessed after 15 days of exposure.

4.1. Characterization and Antibacterial Activity of Nano-Se

TEM study revealed a rod-shaped structure of the nano-Se, with an average particle size of approximately 24.71 nm, consistent with the findings reported by Peng et al. [38]. Particle size distribution analysis revealed that the synthesized nano-Se was at the nanoscale. The absorption spectrum demonstrated the stability of the particles (Figure 1). Research on nano-Se has demonstrated its antimicrobial potential; however, the antibacterial efficacy of Se nanoparticles varies considerably with synthesis methods. Song et al. suggested that KGM-SeNPs had a significant inhibitory effect on S. aureus in a dose-dependent manner when the Se concentration was no more than 2.67 mmol/L, but the inhibitory effect of KGM-SeNPs against E. coli was not observed [41]. In the present study, the addition of nano-Se inhibited the growth and reproduction of E. coli and S. aureus (Figure 2), and the antibacterial effect increased with the increase in nano-Se concentration. These results demonstrated that the stabilizer chitosan and nano-Se may have a synergistic effect on the antibacterial activity. The antibacterial mechanisms of nano-Se are attributed to Se nanoparticle-induced osmotic imbalance, which destabilizes essential biochemical bonds in the bacterial membrane. This compromises bacterial cell integrity, generates intracellular ROS, and damages DNA upon cellular entry, ultimately leading to cell death [42]. Moreover, these results indicate that plant-absorbed nano-Se may disturb endogenous microbiota homeostasis and modulate ROS levels in plants.

4.2. Exogenous Nano-Se Mitigates Cd-Induced Inhibition in Plant Growth, Reduces Cd Accumulation, and Counteracts Nutrient Element Changes

Cd treatment significantly decreased the root length, FW, and DW of barley seedlings (Figure 3), which was consistent with findings from our previous study [3]. Notably, Cd-induced growth suppression in barley was markedly alleviated by the application of optimal concentrations of nano-Se, particularly in root length and biomass under Cd + nSe5 treatment. Furthermore, the application of nano-Se showed potential beneficial effects in promoting wheat plant growth under Cd stress, while simultaneously reducing Cd accumulation, as reported in recent studies [17,23]. Our results demonstrated that exogenous nano-Se application (Cd + nSe) effectively reduced Cd concentration in both leaves and roots of barley seedlings. Similarly, Wang et al. reported that nano-Se supplementation significantly decreased Cd uptake in wheat under Cd stress [17]. Furthermore, nanomaterials like nano-Se not only mitigate heavy metal toxicity but also enhance nutrient availability and promote plant growth, as evidenced by previous studies [15].
In this study, Cd stress disrupted the normal absorption and distribution of essential minerals (Cu, Zn, Mn, Ca, Mg) in barley, leading to obvious imbalances in plant mineral composition (Table 1). Notably, exogenous nano-Se at optimal concentrations reversed the Cd-induced reductions in Cu, Zn, Mn, Ca, and Mg levels, as well as the abnormal elevation of the root Cu concentration observed under Cd stress alone. These findings suggested that nano-Se could alleviate Cd toxicity by restoring mineral homeostasis and counteracting the detrimental effects of Cd on plant nutrient metabolism.

4.3. Exogenous Nano-Se Improves Cd-Induced Inhibition in Pigments and Photosynthesis

Cd stress severely disrupted the photosynthetic apparatus of barley seedlings, suppressing the synthesis and accumulation of photosynthetic pigments [3]. In this research, application of 2–10 μM nano-Se (Cd + nSe) reversed the decrease in chlorophyll and carotenoid levels caused by Cd stress, with a concentration of 5 μM having the largest growth percentage (Figure 4). Meanwhile, the application of 5 μM of nano-Se under Cd stress significantly improved photosynthetic parameters (Figure 5). Se might accelerate Chl biosynthesis by enhancing electron transport in the respiratory chain and promoting respiration [43]. Additionally, Padmaja et al. [44] demonstrated that Se can interact with key enzymes in the Chl synthesis pathway—specifically, porphobilinogen deaminase and 5-aminolevulinic acid dehydratase—and thereby facilitates chlorophyll biosynthesis, subsequently improving photosynthesis, plant growth, and development. Moreover, Qi et al. reported that relative to Cd-only treatment, provision of Se nanoparticles at any tested concentration (5, 10, and 20 mg/L) resulted in significantly higher chlorophyll content on day 12 of Cd exposure [45].
In addition, many researchers have reported that nanoparticles improve Pn and photoprotection, which results in higher crop yield under various abiotic stress conditions [46]. In the present study, 5 μM nano-Se-treated barley plants showed higher Pn, Tr, and Gs than those of Cd treatment alone (Figure 5). Previous studies have reported that foliar application of nanoparticles significantly increases the chlorophyll content in plants, which reveals that nanoparticles support plants in synthesizing additional light-harvesting complexes to capture large amounts of light energy, leading to increased Pn [47]. Moreover, compared to those under Cd-stressed conditions, coriander plants treated with nano-Se exhibited improved gas exchange characteristics [15]. Additionally, nano-Se enhanced gas exchange characteristics in Solanum melongena L. seedlings under Cd stress, as reported by Ahmed et al. [48]. Meanwhile, under Cd treatment, chlorophyll and carotenoid contents were significantly reduced, while application of nano-Se (Cd + nSe) reversed the decrease caused by Cd stress, which helped to improve the photoprotection and thereby maintain the Pn in barley seedlings. Consistent with our findings, previous studies have demonstrated that nano-Se plays multifaceted roles in plants, including regulating photosynthetic and respiratory processes, enhancing stress tolerance and antioxidant capacity, and mitigating heavy metal toxicity [49].

4.4. Exogenous Nano-Se Offsets Cd-Induced Alterations in the Antioxidant System

Cadmium (Cd) stress triggered excessive ROS production and accumulation in plants, resulting in cellular metabolic disorders and disrupting normal plant growth and metabolism [50]. MDA, an end-product of membrane lipid peroxidation, serves as a key biomarker for evaluating oxidative damage to cell membranes [16]. Among ROS, H2O2 is a primary mediator of Cd-induced oxidative stress, as its overaccumulation exacerbates membrane lipid peroxidation and cellular toxicity [50]. In this study, Cd stress significantly elevated MDA and H2O2 levels in both leaves and roots of barley (Figure 6), consistent with oxidative stress mechanisms reported previously [3,22]. However, supplementation with exogenous nano-Se (2, 5, and 10 μM) markedly reduced H2O2 and MDA concentrations compared to Cd stress alone, demonstrating that appropriate concentrations of nano-Se could effectively mitigate Cd-induced oxidative damage.
On the other hand, plants under oxidative stress can activate antioxidant defense systems to counteract elevated ROS levels [3]. SOD is mainly responsible for converting superoxide anions (O2•−) into H2O2, while POD and APX are critical antioxidant enzymes responsible for scavenging H2O2 and other ROS. Notably, the relationship between ROS production and antioxidant enzyme activity in plants is dose-dependent, particularly in response to nanoparticle exposure [15]. In this study, Cd stress increased leaf/root POD, root SOD/CAT, and leaf/root APX activities. However, supplementation with suitable levels of nano-Se (Cd + nSe) further enhanced root SOD, leaf/root CAT, and leaf/root APX activities compared to Cd stress alone, with 5 μM of nano-Se showing the most pronounced effects (Table 2). These results aligned with recent findings demonstrating that nano-Se reduced Cd concentration in Cd-stressed wheat by alleviating oxidative stress (lower MDA and H2O2 levels) through enhanced antioxidant enzyme activity [17].

4.5. Exogenous Nano-Se Counteracts Cd-Induced Changes in Carbohydrate Content, Total Flavonoids, Total Phenols, and Proline Content

Carbohydrates are essential for the normal plant function, as biomass accumulation is primarily derived from carbohydrates produced by photosynthesis [51]. In the present study, Cd stress significantly increased leaf/root soluble sugar levels, as well as root sucrose content in barley plants (Table 3). This enhancement of soluble sugar represents an effort by barley to enhance Cd stress tolerance, which may represent an adaptive mechanism for plants to cope with adverse stress environments. Furthermore, sucrose acts not only as a signaling molecule regulating gene expression but also serves as an osmoprotectant, safeguarding biomolecules and membranes. Similarly, starch serves as a critical energy reserve supporting plant growth and development [52]. Recent studies indicated that nanomaterial additives can significantly modulate starch and sucrose metabolism [53]. Consistent with these findings, exogenous 5 μM of nano-Se (Cd + nSe5) increased the levels of leaf/root soluble sugar, reducing sugar, root sucrose, and leaf/root starch, compared to Cd stress alone (Table 3). These results demonstrated that 5 μM of nano-Se can enhance carbohydrate metabolism and antioxidant capacity, thereby mitigating Cd toxicity and improving barley growth.
Total flavonoids and total phenols, as key antioxidant substances, play a critical role in mitigating oxidative stress by directly scavenging ROS under adverse conditions [54]. Furthermore, proline acts as a stress-responsive osmolyte that accumulates under stress conditions. In this study, Cd stress significantly enhanced the contents of total flavonoids, total phenols, and proline in barley plants (Figure 7), indicating that plants activate antioxidant accumulation as a defense mechanism against Cd-induced oxidative stress. This aligns with previous findings demonstrating that elevated flavonoid and phenolic antioxidants contribute to heavy metal detoxification [55]. Additionally, nano-Se supplementation (Cd + nSe) reduced leaf total flavonoids and root total phenols in barley seedlings, with 5 μM of nano-Se having the most significant effect (Figure 7A,B), potentially due to differential regulation of secondary metabolite biosynthesis under the combined treatments [56]. A similar phenomenon was reported by Huang et al., where foliar nano-Se application decreased total flavonoids and phenols in summer tea while enhancing product quality [57]. On the other hand, the application of nano-Se (5 and 10 μM) significantly decreased leaf/root proline content compared with the Cd treatment alone (Figure 7C). This reduction in proline may be attributed to enhanced Cd tolerance induced by exogenous nano-Se, as it reduced the need for osmotic pressure protection mechanisms. Consistent with this observation, Ikram et al. reported a positive impact of nano-Se in modulating proline levels to improve Huanglongbing tolerance in Kinnow mandarin plants [58]. Specifically, they demonstrated that nano-Se supplementation significantly reduced proline content compared to disease plants, which aligns with the outcomes of the present study.

4.6. Exogenous Nano-Se Abates Cd-Induced Changes in Amino Acid and Soluble Protein

The fundamental growth and metabolic processes of plants require amino acids for protein synthesis, energy production, and nitrogen utilization. These organic compounds play a pivotal role in maintaining plant health and immune responses by facilitating nutrient transport and exchange within plant systems, thereby ensuring proper growth and stress resistance [59]. In the present study, Cd treatment induced a significant reduction in amino acid and protein content in barley seedlings, while nano-Se supplementation notably alleviated this adverse effect, with the application of 5 μM of nano-Se (Cd + nSe5) group exhibiting the most pronounced ameliorative effect (Figure 8). These findings align with previous research demonstrating that nano-Se enhances the biosynthesis of primary metabolites (including free amino acids and soluble proteins), concurrently promoting the growth and development of chili plants under Cd stress [21]. Furthermore, nano-Se has been shown to enhance sugarcane tolerance to Xanthomonas albilineans infection through increased amino acid content and elevated antioxidant enzyme activities [60].

5. Conclusions

In the present study, the synthesis of nano-Se was successfully achieved using ascorbic acid and chitosan as natural reducing and stabilizing agents. The nano-Se exhibited significant antibacterial activity against E. coli and S. aureus. Additionally, application of nano-Se at specific concentrations enhanced barley’s resistance to Cd stress. Within a certain range, exogenous nano-Se substantially reduced Cd concentration in both leaves and roots of barley under Cd stress. As the nano-Se application increased, the Cd reduction percentage increased gradually at first and then began to decrease after reaching the critical nano-Se concentration of 5 μM. Furthermore, Cd + nSe treatment significantly promoted barley growth by enhancing root length, plant biomass, chlorophyll content, and improving photosynthesis under Cd stress. However, nano-Se application exceeding a certain threshold diminished its growth-promoting effects. Moreover, nano-Se alleviated Cd toxicity by balancing mineral nutrient uptake, activating antioxidant defense systems, and reducing oxidative damage in barley seedlings. Additionally, the mitigating effects were correlated with balanced carbohydrate metabolism, elevated total flavonoid and phenolic contents, and increased proteins and amino acids compared to Cd-alone treatment. Overall, 5 μM nano-Se may serve as an effective exogenous application strategy to enhance barley plants’ resistance to Cd toxicity and improve growth performance. However, further research is needed to elucidate the interaction mechanisms between nano-Se and Cd in plants through more detailed physiological and genetic perspectives.

Author Contributions

Formal analysis, supervision, resources, validation, writing—review and editing, H.S.; conceptualization, writing—original draft, project administration, writing—review and editing, X.W.; investigation, methodology, data curation, formal analysis, visualization, writing—original draft, X.L.; methodology, data curation, formal analysis, writing—original draft, R.Y.; methodology, data curation, formal analysis, B.S.; methodology, data curation, formal analysis, S.Y.; methodology, formal analysis, validation, J.Y.; methodology, formal analysis, resources, B.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Key Research and Development Project of Shanxi Province (No. 201903D221066), and the National Natural Science Foundation of China (No. 31401319).

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
CdCadmium
SeSelenium
μMμmol/L
E. coliEscherichia coli
S. aureusStaphylococcus aureus
FWFresh weight
MDAMalondialdehyde
H2O2Hydrogen peroxide
ROSReactive oxygen species
Na2SeO3Sodium selenite
PCAPlate count agar
DWDry weights
PnPhotosynthetic rate
TrTranspiration rate
GsStomatal conductance
CiIntercellular carbon dioxide concentration
SODSuperoxide dismutase
CATCatalase
APXAscorbate peroxidase
PODPeroxidase

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Agronomy 15 02750 g001
Figure 1. TEM image ((A), inset: particle size distribution histogram) and absorption spectrum curve (B) of synthesized nano-Se.
Figure 1. TEM image ((A), inset: particle size distribution histogram) and absorption spectrum curve (B) of synthesized nano-Se.
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Figure 2. Antibacterial effects of different concentrations of nano-Se on E. coli (A) and S. aureus (B), determination of absorbance values (C), as well as antibacterial rate and survival rate (D,E). Data are presented as means, with error bars representing the standard deviation (SD) (n = 3). Different letters above the columns indicate significant differences among different nano-Se concentrations (p ≤ 0.05). mM, mmol/L.
Figure 2. Antibacterial effects of different concentrations of nano-Se on E. coli (A) and S. aureus (B), determination of absorbance values (C), as well as antibacterial rate and survival rate (D,E). Data are presented as means, with error bars representing the standard deviation (SD) (n = 3). Different letters above the columns indicate significant differences among different nano-Se concentrations (p ≤ 0.05). mM, mmol/L.
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Figure 3. Effect of nano-Se on the growth parameters (A–C) and morphological changes (D) of barley seedlings under Cd stress. pl, plant. FW, fresh weight. DW, dry weight. Cd, Cd + nSe2, Cd + nSe5, Cd + nSe10, and Cd + nSe15 correspond to 50 μM Cd, 50 μM Cd + 2 μM nSe, 50 μM Cd + 5 μM nSe, 50 μM Cd + 10 μM nSe, and 50 μM Cd + 15 μM nSe, respectively. Data are presented as means, with error bars representing the standard deviation (SD) (n = 3). Different letters above the columns indicate significant differences among treatments (p ≤ 0.05).
Figure 3. Effect of nano-Se on the growth parameters (A–C) and morphological changes (D) of barley seedlings under Cd stress. pl, plant. FW, fresh weight. DW, dry weight. Cd, Cd + nSe2, Cd + nSe5, Cd + nSe10, and Cd + nSe15 correspond to 50 μM Cd, 50 μM Cd + 2 μM nSe, 50 μM Cd + 5 μM nSe, 50 μM Cd + 10 μM nSe, and 50 μM Cd + 15 μM nSe, respectively. Data are presented as means, with error bars representing the standard deviation (SD) (n = 3). Different letters above the columns indicate significant differences among treatments (p ≤ 0.05).
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Figure 4. Effect of nano-Se on photosynthetic pigments (mg/g FW) in barley leaves under Cd stress. FW, Fresh weight. Cd, Cd + nSe2, Cd + nSe5, Cd + nSe10, and Cd + nSe15 correspond to 50 μM Cd, 50 μM Cd + 2 μM nSe, 50 μM Cd + 5 μM nSe, 50 μM Cd + 10 μM nSe, and 50 μM Cd + 15 μM nSe, respectively. Data are presented as means, with error bars representing the standard deviation (SD) (n = 3). Different letters above the columns indicate significant differences among treatments (p ≤ 0.05).
Figure 4. Effect of nano-Se on photosynthetic pigments (mg/g FW) in barley leaves under Cd stress. FW, Fresh weight. Cd, Cd + nSe2, Cd + nSe5, Cd + nSe10, and Cd + nSe15 correspond to 50 μM Cd, 50 μM Cd + 2 μM nSe, 50 μM Cd + 5 μM nSe, 50 μM Cd + 10 μM nSe, and 50 μM Cd + 15 μM nSe, respectively. Data are presented as means, with error bars representing the standard deviation (SD) (n = 3). Different letters above the columns indicate significant differences among treatments (p ≤ 0.05).
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Figure 5. Effect of nano-Se on photosynthetic parameters in barley leaves under Cd stress. Pn, net photosynthetic rate; Tr, transpiration rate; Gs, stomatal conductance; Ci, intercellular CO2 concentration. Cd, Cd + nSe2, Cd + nSe5, Cd + nSe10, and Cd + nSe15 correspond to 50 μM Cd, 50 μM Cd + 2 μM nSe, 50 μM Cd + 5 μM nSe, 50 μM Cd + 10 μM nSe, and 50 μM Cd + 15 μM nSe, respectively. Data are presented as means, with error bars representing the standard deviation (SD) (n = 3). Different letters above the columns indicate significant differences among treatments (p ≤ 0.05).
Figure 5. Effect of nano-Se on photosynthetic parameters in barley leaves under Cd stress. Pn, net photosynthetic rate; Tr, transpiration rate; Gs, stomatal conductance; Ci, intercellular CO2 concentration. Cd, Cd + nSe2, Cd + nSe5, Cd + nSe10, and Cd + nSe15 correspond to 50 μM Cd, 50 μM Cd + 2 μM nSe, 50 μM Cd + 5 μM nSe, 50 μM Cd + 10 μM nSe, and 50 μM Cd + 15 μM nSe, respectively. Data are presented as means, with error bars representing the standard deviation (SD) (n = 3). Different letters above the columns indicate significant differences among treatments (p ≤ 0.05).
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Figure 6. Effect of nano-Se on lipid peroxidation (A), hydrogen peroxide content (B) in barley seedlings under Cd stress. Cd, Cd + nSe2, Cd + nSe5, Cd + nSe10, and Cd + nSe15 correspond to 50 μM Cd, 50 μM Cd + 2 μM nSe, 50 μM Cd + 5 μM nSe, 50 μM Cd + 10 μM nSe, and 50 μM Cd + 15 μM nSe, respectively. Data are presented as means, with error bars representing the standard deviation (SD) (n = 3). Different letters above the columns indicate significant differences among treatments (p ≤ 0.05).
Figure 6. Effect of nano-Se on lipid peroxidation (A), hydrogen peroxide content (B) in barley seedlings under Cd stress. Cd, Cd + nSe2, Cd + nSe5, Cd + nSe10, and Cd + nSe15 correspond to 50 μM Cd, 50 μM Cd + 2 μM nSe, 50 μM Cd + 5 μM nSe, 50 μM Cd + 10 μM nSe, and 50 μM Cd + 15 μM nSe, respectively. Data are presented as means, with error bars representing the standard deviation (SD) (n = 3). Different letters above the columns indicate significant differences among treatments (p ≤ 0.05).
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Figure 7. Effect of nano-Se on TFC (A), TPC (B), and proline content (C) in barley seedlings under Cd stress. TFC, total flavonoids content; TPC, total phenols content. Cd, Cd + nSe2, Cd + nSe5, Cd + nSe10, and Cd + nSe15 correspond to 50 μM Cd, 50 μM Cd + 2 μM nSe, 50 μM Cd + 5 μM nSe, 50 μM Cd + 10 μM nSe, and 50 μM Cd + 15 μM nSe, respectively. Data are presented as means, with error bars representing the standard deviation (SD) (n = 3). Different letters above the columns indicate significant differences among treatments (p ≤ 0.05).
Figure 7. Effect of nano-Se on TFC (A), TPC (B), and proline content (C) in barley seedlings under Cd stress. TFC, total flavonoids content; TPC, total phenols content. Cd, Cd + nSe2, Cd + nSe5, Cd + nSe10, and Cd + nSe15 correspond to 50 μM Cd, 50 μM Cd + 2 μM nSe, 50 μM Cd + 5 μM nSe, 50 μM Cd + 10 μM nSe, and 50 μM Cd + 15 μM nSe, respectively. Data are presented as means, with error bars representing the standard deviation (SD) (n = 3). Different letters above the columns indicate significant differences among treatments (p ≤ 0.05).
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Figure 8. The effect of nano-Se on amino acid (A) and soluble protein content (B) in barley seedlings under Cd stress. Cd, Cd + nSe2, Cd + nSe5, Cd + nSe10, and Cd + nSe15 correspond to 50 μM Cd, 50 μM Cd + 2 μM nSe, 50 μM Cd + 5 μM nSe, 50 μM Cd + 10 μM nSe, and 50 μM Cd + 15 μM nSe, respectively. Data are presented as means, with error bars representing the standard deviation (SD) (n = 3). Different letters above the columns indicate significant differences among treatments (p ≤ 0.05).
Figure 8. The effect of nano-Se on amino acid (A) and soluble protein content (B) in barley seedlings under Cd stress. Cd, Cd + nSe2, Cd + nSe5, Cd + nSe10, and Cd + nSe15 correspond to 50 μM Cd, 50 μM Cd + 2 μM nSe, 50 μM Cd + 5 μM nSe, 50 μM Cd + 10 μM nSe, and 50 μM Cd + 15 μM nSe, respectively. Data are presented as means, with error bars representing the standard deviation (SD) (n = 3). Different letters above the columns indicate significant differences among treatments (p ≤ 0.05).
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Table 1. Effect of nano-Se on the element content of barley seedlings under Cd stress.
Table 1. Effect of nano-Se on the element content of barley seedlings under Cd stress.
TissuesTreatmentElement Concentration (mg/kg DW) *
CdCuZnMnCaMg
LeafControlnd25.77 ± 3.5 b4.56 ± 0.2 c6.65 ± 0.6 a6378 ± 64.1 a436 ± 7.2 b
Cd16.37 ± 4.6 a35.26 ± 1.8 a3.54 ± 0.3 d2.53 ± 0.3 d3976 ± 25.4 c321 ± 9.3 c
Cd + nSe215.27 ± 3.8 ab19.74 ± 2.4 c5.81 ± 0.4 b4.34 ± 0.5 b5525 ± 46.3 ab471 ± 3.2 b
Cd + nSe513.19 ± 2.8 c17.01 ± 2.0 d6.93 ± 0.7 a6.96 ± 0.4 a6092 ± 60.2 a551 ± 5.1 a
Cd + nSe1014.70 ± 3.5 b22.01 ± 2.9 bc6.63 ± 0.8 a3.43 ± 0.2 c6039 ± 61.3 a541 ± 6.2 a
Cd + nSe1516.02 ± 4.0 a25.00 ± 3.2 b3.40 ± 0.5 d2.99 ± 0.6 d46.75 ± 38.0 b477 ± 4.1 b
RootControlnd65.36 ± 3.6 b19.08 ± 4.7 a15.00 ± 5.7 a1531 ± 89.1 a2038 ± 64.1 a
Cd192.63 ± 4.8 a69.09 ± 3.8 a13.65 ± 3.6 c8.57 ± 2.8 c885 ± 42.4 c1167 ± 36.2 d
Cd + nSe2171.34 ± 4.7 b33.72 ± 2.3 d16.10 ± 4.0 b9.12 ± 3.5 bc1067 ± 51.2 b1318 ± 45.1 c
Cd + nSe5132.78 ± 3.6 c33.67 ± 2.4 d17.22 ± 4.3 b13.28 ± 5.4 b1214 ± 67.1 b1894 ± 59.0 b
Cd + nSe10182.73 ± 4.6 ab34.28 ± 2.9 d11.80 ± 3.4 d12.11 ± 3.8 b1113 ± 54.3 b1419 ± 51.4 c
Cd + nSe15190.23 ± 4.7 a39.65 ± 3.2 c11.50 ± 3.1 d7.77 ± 1.7 d712 ± 34.1 c1068 ± 26.3 d
* DW, dry weight. nd = not detected. Cd, Cd + nSe2, Cd + nSe5, Cd + nSe10, and Cd + nSe15 correspond to 50 μM Cd, 50 μM Cd + 2 μM nSe, 50 μM Cd + 5 μM nSe, 50 μM Cd + 10 μM nSe, and 50 μM Cd + 15 μM nSe, respectively. Data are presented as means ± standard deviation (SD). Different lowercase letters indicate significant differences (p < 0.05) among the six treatments.
Table 2. The effect of nano-Se on antioxidant enzyme activity in barley seedlings under Cd stress.
Table 2. The effect of nano-Se on antioxidant enzyme activity in barley seedlings under Cd stress.
TissuesTreatmentSOD
(U/g)		POD
(μmol/min/g FW *)		CAT
(nmol/min/g FW)		APX
(nmol/min/g FW)
LeafControl1170.2 ± 15.0 a20.9 ± 2.0 c1555.3 ± 10.3 e24.3 ± 2.3 c
Cd1142.3 ± 20.4 a45.2 ± 2.7 a1532.3 ± 3.4 e30.9 ± 4.0 b
Cd + nSe21090.4 ± 38.5 a45.2 ± 7.2 a1850.3 ± 8.8 d34.0 ± 4.4 a
Cd + nSe51147.5 ± 14.4 a40.5 ± 2.6 b2354.6 ± 3.4 a36.5 ± 1.6 a
Cd + nSe101066.0 ± 7.6 a47.2 ± 5.5 a2061.0 ± 17.8 b35.1 ± 2.4 a
Cd + nSe151091.7 ± 4.1 a48.0 ± 1.5 a1985.2 ± 13.0 bc35.2 ± 1.4 a
RootControl587.5 ± 14.0 d36.0 ± 2.4 c94.2 ± 5.7 e19.8 ± 3.2 a
Cd628.9 ± 12.4 c42.9 ± 2.1 a163.7 ± 9.5 d23.5 ± 1.5 e
Cd + nSe2641.9.1 ± 8.9 b42.3 ± 1.7 a186.6 ± 4.1 c30.9 ± 2.3 c
Cd + nSe5684.3 ± 19.8 a37.2 ± 3.7 c280.9 ± 14.0 a33.3 ± 1.5 b
Cd + nSe10628.4 ± 10.9 c40.1 ± 3.4 b212.2 ± 13.1 b28.3 ± 4.4 c
Cd + nSe15581.4 ± 12.0 d40.9 ± 3.1 b208.2 ± 2.5 b26.6 ± 1.6 d
* FW, fresh weight. Cd, Cd + nSe2, Cd + nSe5, Cd + nSe10, and Cd + nSe15 correspond to 50 μM Cd, 50 μM Cd + 2 μM nSe, 50 μM Cd + 5 μM nSe, 50 μM Cd + 10 μM nSe, and 50 μM Cd + 15 μM nSe, respectively. Data are presented as means ± standard deviation (SD). Different lowercase letters indicate significant differences (p < 0.05) among the six treatments.
Table 3. Effect of nano-Se carbohydrate content in barley seedlings under Cd stress.
Table 3. Effect of nano-Se carbohydrate content in barley seedlings under Cd stress.
TissuesTreatmentCarbohydrate Content (mg/g FW *)
Souble SugarReducing SugarSucroseStarch
LeafControl1.00 ± 0.06 c0.96 ± 0.04 b12.38 ± 0.5 a35.10 ± 2.2 d
Cd1.28 ± 0.06 b0.90 ± 0.05 b13.20 ± 0.6 a40.52 ± 1.2 cd
Cd + nSe21.22 ± 0.05 b1.55 ± 0.04 a13.98 ± 0.5 a41.00 ± 1.3 cd
Cd + nSe51.65 ± 0.04 a1.68 ± 0.06 a14.00 ± 0.4 a48.10 ± 1.5 c
Cd + nSe101.19 ± 0.05 b0.98 ± 0.05 b12.08 ± 0.4 a51.44 ± 1.4 b
Cd + nSe151.29 ± 0.07 b1.04 ± 0.06 b13.36 ± 0.3 a60.81 ± 1.2 a
RootControl0.74 ± 0.01 c1.03 ± 0.01 a5.60 ± 0.2 c43.12 ± 2.3 b
Cd1.01 ± 0.03 b0.95 ± 0.02 ab6.99 ± 0.3 b45.12 ± 1.2 b
Cd + nSe20.92 ± 0.02 b1.08 ± 0.03 a6.60 ± 0.2 b46.69 ± 1.4 b
Cd + nSe51.31 ± 0.01 a1.09 ± 0.01 a8.10 ± 0.3 a55.90± 1.6 a
Cd + nSe100.97 ± 0.01 b0.95 ± 0.02 ab7.11 ± 0.2 b47.57 ± 1.8 b
Cd + nSe150.93 ± 0.01 b0.93 ± 0.01 ab6.74 ± 0.3 b48.99 ± 1.1 b
* FW, fresh weight. Cd, Cd + nSe2, Cd + nSe5, Cd + nSe10, and Cd + nSe15 correspond to 50 μM Cd, 50 μM Cd + 2 μM nSe, 50 μM Cd + 5 μM nSe, 50 μM Cd + 10 μM nSe, and 50 μM Cd + 15 μM nSe, respectively. Data are presented as means ± standard deviation (SD). Different lowercase letters indicate significant differences (p < 0.05) among the 6 treatments.
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Sun, H.; Lian, X.; Yao, R.; Shang, B.; Yi, S.; Yu, J.; Zhang, B.; Wang, X. Synthesis, Antibacterial Properties, and Physiological Responses of Nano-Selenium in Barley (Hordeum vulgare L.) Seedlings Under Cadmium Stress. Agronomy 2025, 15, 2750. https://doi.org/10.3390/agronomy15122750

AMA Style

Sun H, Lian X, Yao R, Shang B, Yi S, Yu J, Zhang B, Wang X. Synthesis, Antibacterial Properties, and Physiological Responses of Nano-Selenium in Barley (Hordeum vulgare L.) Seedlings Under Cadmium Stress. Agronomy. 2025; 15(12):2750. https://doi.org/10.3390/agronomy15122750

Chicago/Turabian Style

Sun, Hongyan, Xin Lian, Runge Yao, Bingjie Shang, Siyu Yi, Jia Yu, Bo Zhang, and Xiaoyun Wang. 2025. "Synthesis, Antibacterial Properties, and Physiological Responses of Nano-Selenium in Barley (Hordeum vulgare L.) Seedlings Under Cadmium Stress" Agronomy 15, no. 12: 2750. https://doi.org/10.3390/agronomy15122750

APA Style

Sun, H., Lian, X., Yao, R., Shang, B., Yi, S., Yu, J., Zhang, B., & Wang, X. (2025). Synthesis, Antibacterial Properties, and Physiological Responses of Nano-Selenium in Barley (Hordeum vulgare L.) Seedlings Under Cadmium Stress. Agronomy, 15(12), 2750. https://doi.org/10.3390/agronomy15122750

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