Selenium nanoparticles from Dermacoccus abyssi MT1.1ᵀ improve water status, biomass, and antioxidant defense in tomato under salt stress

Summary

Salt stress severely limits plant growth and agricultural productivity. Nanoparticles have emerged as a promising strategy to alleviate salinity stress. This study investigated the biogenic synthesis of selenium nanoparticles (SeNPs) using the culture supernatant of Dermacoccus abyssi MT1.1^T and evaluated their effects on tomato germination and seedling growth under salt stress. Successfully synthesized SeNPs were spherical, nanoscale, negatively charged, and moderately stable. Application of SeNPs at 5 mg mL⁻¹ showed no phytotoxic effects on tomato germination under 50, 100, and 150 mM NaCl. Under 150 mM NaCl, SeNP-treated seedlings exhibited increased fresh and dry weights and enhanced accumulation of proline and total soluble sugars, leading to improved relative water content. Furthermore, SeNPs alleviated salt-induced oxidative stress by reducing hydrogen peroxide levels and enhancing antioxidant enzymes, including catalase and guaiacol peroxidase. These findings highlight biogenic SeNPs from D. abyssi MT1.1^T as a potential eco-friendly nanobiostimulant for improving tomato salt tolerance.

Graphical abstract

Highlights

• •Deep-sea actinobacteria successfully biosynthesize selenium nanoparticles
• •Biogenic SeNPs show no toxicity to tomato germination and seedlings growth
• •SeNPs alleviate salt stress in tomato by enhancing osmolytes and antioxidant defenses

Microbiology; Applied microbiology; Plant biology; Interaction of plants with organisms; Plant physiology

Introduction

Soil salinity is a critical abiotic stressor that significantly threatens agricultural productivity by adversely affecting plant physiology, growth, and yield. High concentrations of soluble salts in the soil interfere plant water uptake by inducing osmotic stress, which leads to nutrient imbalances. This excessive accumulation of sodium ions (Na⁺) results in ion toxicity and oxidative stress, further compromising cellular homeostasis and metabolic processes in plants.^1,2,3,4 Therefore, the development of innovative, efficient, and environmentally sustainable strategies to enhance crop resilience and productivity has become a critical priority in agriculture.

Nanobiotechnology offers a promising solution in agriculture by enabling the development of nanomaterials with unique physicochemical properties, such as high surface area, enhanced bioactivity, and improved bioavailability.⁵ Selenium nanoparticles (SeNPs) have gained considerable attention due to their multifunctional roles in plant growth promotion and stress tolerance. Selenium (Se) is a trace element that, at low concentrations, plays a beneficial role in enhancing plant growth and resilience to various abiotic and biotic stresses, including salinity, drought, heavy metal toxicity, and phytopathogen attack.^5,6 However, excessive Se levels can promote the generation of reactive oxygen species (ROS), induce oxidative stress, and cause phytotoxic effects such as reduced biomass, leaf area, and impaired seed formation.⁵ To overcome the challenges associated with selenium toxicity while harnessing its beneficial properties, the synthesized SeNPs exhibit enhanced bioactivity, stability, and bioavailability, making them promising candidates for agricultural applications as compared to bulk selenium.⁵ Biosynthesized SeNPs produced from either microbial or plant-mediated processes are particularly attractive due to their environmental compatibility and capacity to promote seed germination and seedling growth in crops, such as tomato (Solanum lycopersicum) and barley (Hordeum vulgare).^7,8 In addition, SeNPs have demonstrated efficiency in alleviating various abiotic stresses, including drought, salinity, heat, and heavy metal accumulation in plants.^9,10

SeNPs can be synthesized using biological, chemical, or physical methods. However, many conventional chemical and physical approaches are expensive and often require high energy input and toxic reagents, raising concerns regarding environmental safety and human health. Particularly, chemical synthesis of SeNPs typically uses inorganic selenium salts (e.g., Na₂SeO₃), reduces by chemical agents (e.g., ascorbic acid) and often requires synthetic stabilizers/surfactants to prevent aggregation. Chemical synthesis offers fast reactions and easy parameter control but generates toxic residues and requires post-synthesis purification steps.¹¹ In contrast, green synthesis using biological systems represents a cost-effective and environmentally friendly alternative. Biosynthetic approaches employ various organisms, including plants, fungi, yeasts, and bacteria to reduce selenite into nanoscale elemental selenium.^12,13 Among these, microbial-mediated synthesis is particularly promising due to the scalability, adaptability, and metabolic versatility of microbes. Microbial biomolecules not only facilitate nanoparticle synthesis but also act as reducing and stabilizing agents, to ensure integrity and functionality of the obtained nanoparticles under diverse conditions. Bacteria, in particular offers several advantages for nanoparticle synthesis such as rapid growth rates and easy to culture under controlled conditions. This approach reduces the need for hazardous chemical reagents, lowers production costs, and aligns well with environmentally sustainable practices.

Actinobacteria, a diverse group of Gram-positive bacteria belonging to the phylum Actinomycetota. They are well known for their secondary metabolite production, which includes antimicrobial compounds, enzymes, metal-chelating agents, and plant growth promoting compounds.^14,15,16 These properties make actinobacteria ideal candidates for sustainable agricultural biotechnology. Actinobacteria have demonstrated significant potential in environmentally friendly nanoparticle biosynthesis with applications in agriculture. Nanoparticles synthesized from actinobacteria have been used to reduce the reliance on agrochemicals for antimicrobial agents against phytopathogens. For example, Streptomyces griseoplanus-derived silver nanoparticles effectively inhibited Macrophomina phaseolina, a phytopathogenic fungus,¹⁷ while copper nanoparticles from S. capillispiralis exhibited antimicrobial activity against human pathogens and showed potential in plant disease management.¹⁸ Selenium nanoparticles synthesized by actinobacteria have also shown diverse bioactivities, including antiviral activity (e.g., SeNPs from Streptomyces minutiscleroticus against dengue virus), cytotoxicity against HT-29 human colon cancer cell line,¹⁹ and antibacterial against pathogenic bacteria.²⁰

Despite these advances, research on SeNPs synthesized by actinobacteria for alleviating salt stress in plants remains limited. Given the urgent need for sustainable solutions to salinity-induced agricultural decline, exploration of novel actinobacterial strains from underexplored environments is of particular interest. Therefore, the objective of this study was to synthesize selenium nanoparticles using the culture filtrate of Dermacoccus abyssi MT1.1ᵀ, an actinobacterium isolated from deep-sea sediment, and to evaluate their potential in enhancing seed germination and early seedling growth of tomato under normal and salt stress conditions. Furthermore, this study aimed to assess the biochemical responses of tomato seedlings to SeNP treatment under salinity stress, thereby elucidating the potential of SeNPs as a biogenic strategy for salinity stress mitigation.

Results and discussion

Successful biosynthesis of selenium nanoparticles using the culture supernatant of Dermacoccus abyssi MT1.1^T

In this study, selenium nanoparticles (SeNPs) were biosynthesized using the culture supernatant of Dermacoccus abyssi MT1.1ᵀ and 8 mM sodium selenite (Na₂SeO₃) under shaking conditions at 37 °C. A visible color change from yellow to red was observed after 24 h, with increased intensity after 72 h, indicating the reduction of Se⁴⁺ ions to elemental selenium (Se⁰) in nanoparticle form (Figures 1A and 1B). This colorimetric shift is a typical indicator of SeNP formation and has been reported in similar biosynthesis processes using various microorganisms, including Streptomyces spp.^19,20 The use of a cell-free culture supernatant in this study confirms that SeNP synthesis occurred via an extracellular biogenic process mediated by the reduction of selenite (SeO₃²⁻).

Figure 1.

Biosynthesis of SeNPs from D. abyssi MT1.1^T supernatant

(A) The supernatant of D. abyssi MT1.1^T; (B) the formation of a characteristic red color of SeNPs; and (C) UV-visible spectrum of the synthesized SeNPs after 72 h incubation.

The described process yielded 32.7 ± 0.005 mg of SeNPs per 100 mL of reaction mixture. A relatively higher yield of 50 mg per 100 mL was previously reported using Streptomyces sp. M10A65 and 1 mM Na₂SeO₃,²⁰ suggesting that both strain specificity and precursor concentration influence synthesis efficiency.

The UV-visible absorption spectrum of the biosynthesized SeNPs exhibited a characteristic peak at 288 nm (Figure 1C), indicating the successful formation of SeNPs. Previous studies have shown that SeNPs display broad maximum absorption peaks typically ranging from 270 to 575 nm, depending on biological source and the synthesis methods. For example, SeNPs synthesized using Ralstonia eutropha, Salinicoccus iranensis, and Lactobacillus paracasei HM1 exhibited absorption maxima at 270, 294, and 300 nm, respectively.^21,22,23 In contrast, SeNPs synthesized from the culture supernatant of Lactobacillus acidophilus exhibited a peak at 385,²⁴ and those derived from Streptomyces griseoruber showed a maximum absorbance at 575 nm.¹⁹ Bacterial synthesized SeNPs generally show different UV-vis spectra which reflects the difference in their atomic structure, particle size, shape, and surface chemistry influenced by the synthesis methods.^25,26 The absorption peak at 288 nm observed in the present study is consistent with the presence of nanoscale elemental selenium.

Transmission electron microscopy (TEM) revealed that the SeNPs were spherical with polydisperse distribution (Figures 2A and 2B). The size distribution of 185 nanoparticles based on TEM analysis is presented in Figure 2C, with a calculated average particle diameter of 195.5 ± 50.2 nm. Scanning electron microscopy (SEM) confirmed the spherical morphology and highlighted the rough external surface texture of the particles (Figures 2D and 2E). These findings are in agreement with previous studies that reported the size of SeNPs typically between 20 and 250 nm.^19,20,22

Figure 2.

Selenium nanoparticles imaged by TEM and SEM

(A and B) TEM images.

(C) Particles size distribution histogram.

(D and E) SEM images.

Elemental composition analysis by energy-dispersive X-ray spectroscopy (EDS) confirmed the presence of selenium (Se), carbon (C), and oxygen (O) (Figure 3). While Se originates from the reduced selenite, the carbon and oxygen are likely associated with organic biomolecules from the bacterial supernatant acting as capping and stabilizing agents. Fourier transform infrared spectroscopy (FTIR) further confirmed the involvement of functional groups in the biosynthesis and stabilization of SeNPs (Figure 4A). As shown in Table 1, the band at 3280 cm⁻¹ corresponds to O–H and N–H stretching vibrations, indicative of proteins or polysaccharides involved in the reduction process.¹⁹ Additional bands corresponding to amide A, I, II, and III regions suggest protein binding on the nanoparticle surface, enhancing both stability and biological activity of the synthesized nanoparticle.^19,20

Figure 3.

EDS spectrum with the corresponding elemental composition of SeNPs-synthesized from D. abyssi MT1.1^T supernatant

Figure 4.

SeNP characterization

(A) FTIR spectrum.

(B) XRD pattern.

(C) Hydrodynamic size distribution curves, and (D) Zeta potential curve of SeNPs-synthesized from D. abyssi MT1.1^T supernatant.

Table 1.

Relevant functional groups in the synthesized SeNPs using FTIR technique

Band no.	Wavenumber (cm−1)	Relevant functional group
1	3280	amide A in proteins (O–H and N–H stretching)
2	2962	–CH3 (C–H stretching)
3	2927	–CH2 (C–H stretching)
4	1636	amide I in proteins
5	1532	amide II in proteins
6	1447	–CH2/–CH3 in protein, lipids, polyesters, etc.
7	1386	carboxyl group (–COO– stretching)
8	1234	amide III in proteins
9	1067	polysaccharides/polyesters (C–O/C–C/C–O–H/C– O–C)
10	532	Se–O

Data taken from Kamnev et al.^27,28; Tendenedzai et al.²⁹; Tugarova et al.³⁰

X-ray diffraction (XRD) analysis revealed a broad diffraction peak at 2θ ranging from 15° to 35°, as shown in Figure 4B. This result suggests that the synthesized SeNPs are predominantly in an amorphous phase. This result aligns with previous studies showing that SeNPs produced through biological routes often possess a low degree of crystallinity.^31,32,33

The hydrodynamic size distribution (i.e., the size of particles surrounded by water molecules as the dispersant) of the obtained SeNPs is illustrated in Figure 4C. Dynamic light scattering (DLS) analysis showed an average hydrodynamic diameter of 162 nm for the synthesized SeNPs, confirming their nanoscale range even in a hydrated state. The slight difference between TEM and DLS sizes can be attributed to the hydration shell surrounding the particles during DLS measurements.

Zeta potential analysis revealed an average surface charge of −27.0 mV of the synthesized SeNPs, as presented in Figure 4D, suggesting moderate colloidal stability. The presence of carboxyl groups (–COO^-) on the nanoparticle surface, as indicated by the FTIR spectral peak at 1386 cm⁻¹, likely contributes to this negative surface charge and overall stability (Table 1). The absolute zeta potential value of greater than ±30 mV generally indicates high colloidal stability.³⁴ Although the colloidal stability of the synthesized SeNPs in this study fall slightly below that threshold, it remains adequate for short-term biological applications, including plant treatments, where immediate uptake and interaction with plant tissues are expected.

Selenium nanoparticles mitigate salt stress during tomato seed germination

In this study, the impacts of 5 mg mL⁻¹ SeNPs on tomato seed germination were evaluated under varying NaCl concentrations. Under non-stress condition, seeds treated with sodium selenite (5 mg mL⁻¹) exhibited significantly reduced germination, with only 49% germination (Figure 5A) and a germination index of 46% (Figure 5B), indicating a clear phytotoxic effect. This observation aligns with previous reports that high concentrations of selenium can induce oxidative stress through excessive ROS generation, thereby inhibiting germination and early seedling development.⁵ In contrast, seeds treated with SeNPs under the same conditions showed markedly improved performance. Both germination percentage and germination index values were approximately 50% higher than those observed with sodium selenite treatment and were comparable to those of the control treatment. These findings suggest that SeNPs, unlike their ionic counterparts, exhibit reduced phytotoxicity and may possess beneficial properties that support seed development under optimal conditions.^35,36

Figure 5.

Effect of the synthesized selenium nanoparticles (5 mg mL⁻¹) on tomato seed germination under non-stress (0 mM NaCl) and salt stress at 50, 100, and 150 mM NaCl

(A) seed germination percentage; (B) seed germination index.

Data are presented as mean ± SD. Different letters indicate statistically significant differences among treatments within each salt concentration (p < 0.05) based on one-way ANOVA followed by Duncan’s multiple range test. n = 3 biological replicates (15 seeds per replicate).

Under salt stress, a dose-dependent inhibitory effect of NaCl on seed germination was observed. Germination percentage and germination index declined with increasing salinity, with the most pronounced inhibition observed at 150 mM NaCl. Notably, no germination occurred in the sodium selenite-treated seeds under this high-salinity condition, further highlighting its toxicity. At low NaCl concentration (50 mM NaCl), SeNPs effectively mitigated salt stress, maintaining germination percentages above 80% (Figure 5A). However, the protective effect of SeNPs diminished at higher salinity levels, indicating that the efficacy of SeNPs may be limited under severe salt stress. Previous study showed that 25 mg L⁻¹ SeNPs supplementation improved germination in mustard under 200 mM NaCl stress when applied via Hoagland’s solution.¹⁰ Furthermore, the application of 150 μM SeNPs synthesized by Comamonas testosterone improved seed germination and germination rate of rapeseeds under 150 mM NaCl stress.³⁷ This improvement was attributed to enhanced water uptake and the upregulation of genes involved in triacylglycerol (TAG) catabolism, which play a critical role in mobilizing energy reserves during seed germination under stress conditions. This discrepancy suggests the effectiveness of SeNPs in mitigating salt stress may be attributed to differences in plant species, SeNPs concentration, synthesis method, or mode of application, which merits further investigation.

Selenium nanoparticles enhance growth and physiological-biochemical responses of tomato seedlings under salt stress

Selenium nanoparticles (SeNPs) have attracted considerable interest in agricultural research due to their potential to mitigate various abiotic stresses in plants, particularly drought³⁸ and salinity.³⁹ Salt stress disrupts numerous biochemical and physiological processes in plants, primarily by promoting excessive accumulation of ROS, such as hydrogen peroxide (H₂O₂), superoxide anion (O₂⁻), and hydroxyl radicals (·OH). These ROS induce oxidative stress which disrupt cellular homeostasis and cause oxidative damage through lipid peroxidation, protein oxidation, DNA degradation, and initiation of programmed cell death.^40,41 To mitigate ROS toxicity, plants have evolved complex antioxidant defense systems comprising of both enzymatic components—such as catalase (CAT), superoxide dismutase (SOD), peroxidases (e.g., APX, and GPX)—and non-enzymatic antioxidants, including ascorbate, glutathione, phenolic compounds, and proline. The balance between ROS production and antioxidant capacity determines the extent of oxidative damage under stress conditions.

In the present study, H₂O₂ accumulation was used as a marker of oxidative stress in tomato seedlings. Under non-stress conditions, similar H₂O₂ levels were detected in seedlings treated with SeNPs and those in untreated controls (Figure 6A), indicating no oxidative burden associated with SeNP exposure. However, under salt stress, the control seedlings exhibited a dramatic 212% increase in H₂O₂ content relative to the non-stressed control, consistent with expected ROS accumulation in response to salt-induced oxidative stress. Various plant species, including mungbean,⁴² strawberry,⁴³ tomato,^44,45 and wheat⁴⁶ showed similar findings. Notably, SeNP treatment significantly reduced H₂O₂ levels by 38% in salt-stressed tomato seedlings, suggesting an active role in oxidative stress mitigation (Figure 6A). This finding aligns with previous studies showing that SeNP application effectively reduces ROS levels under salinity. For example, SeNPs at 10 μM and 100 μM concentrations lowered H₂O₂ accumulation in salt-stressed strawberry,⁴³ while 0.05%–0.1% SeNPs significantly reduced H₂O₂ content in wheat subjected to 12 dS m⁻¹ NaCl stress.⁴⁶ These results support the hypothesis that SeNPs contribute to ROS detoxification, possibly by enhancing antioxidant enzyme activities or stabilizing membrane structures against salt-induced oxidative damage.

Figure 6.

Effect the synthesized selenium nanoparticles (5 mg mL⁻¹) on hydrogen peroxide content, antioxidant activity, and antioxidative enzyme activity of tomato seedling grown under non-stress (0 mM NaCl) and salt stress (150 mM NaCl)

(A) hydrogen peroxide content; (B) total antioxidant activity; (C) catalase activity; (D) ascorbate peroxidase (APX) activity and (E) guaiacol peroxidase (GPX) and (F) superoxide dismutase (SOD) activity.

Data are presented as mean ± SD. Different letters indicate statistically significant differences among treatments (p < 0.05) based on two-way ANOVA followed by Duncan’s multiple range test. n = 3 biological replicates.

Previous studies have demonstrated that SeNPs can enhance the activity of antioxidant enzymes, thereby improving plant tolerance to various abiotic stresses, including salinity.^43,46 Catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APX), and guaiacol peroxidase (GPX) play crucial roles in scavenging ROS and maintaining cellular redox homeostasis. In the present study, the effects of SeNPs synthesized from the culture supernatant of Dermacoccus abyssi MT1.1ᵀ on the activities of antioxidant enzymes in tomato seedlings subjected to salt stress were investigated (Figures 6B–6F). Under non-stress condition, GPX activity was slightly increased in tomato seedlings treated with SeNPs compared to the untreated control (Figure 6E), suggesting a priming effect of SeNPs on the antioxidative enzymatic machinery. Under salt stress conditions, SeNP treatment significantly enhanced the activities of CAT and GPX in tomato seedlings (Figures 6C and 6E). CAT is a key enzyme responsible for the breakdown of H₂O₂ into water and oxygen, thus playing a central role in mitigating oxidative damage during stress.⁴⁷ Similarly, GPX contributes to H₂O₂ detoxification by using, a phenolic compound, guaiacol as an electron donor in redox reactions.⁴⁸ These increases in CAT and GPX activities in response to SeNPs treatment suggest a targeted enhancement of H₂O₂-scavenging pathways under salt stress. Our findings are in agreement with earlier reports where commercial or plant-extract-mediated SeNPs increased antioxidant enzyme activities under salinity. For instance, Soleymanzadeh et al.⁴³ reported that 10 μM commercial SeNPs enhanced CAT activity in strawberry under salt stress. Similarly, SeNPs (0.01%–0.1%) synthesized using lemon leaf extract increased SOD, CAT, and peroxidase (POD) activities in wheat under saline conditions.⁴⁶ Additionally, chemically synthesized SeNPs (0.5–1 mg L⁻¹) were found to enhance the activities of glutathione peroxidase (GSH-PX), glutathione reductase (GR), CAT, and SOD in salt-stressed soybean.⁴⁹

In this study, APX (Figure 6D) and SOD (Figure 6F) activities of SeNP-treated seedlings showed no significant difference with untreated plants under salt stress. APX is the enzyme responsible for detoxifying H₂O₂ into water using ascorbate as electron donor. It plays a key role in the ascorbate-glutathione cycle, by maintaining cellular redox balance under stress conditions.⁵⁰ However, in this study, APX activity of SeNP-treated seedlings was lowered than untreated plants under salt stress (Figure 6D). Similarly, SOD activity also did not change significantly following SeNP application under salt stress (Figure 6F).

SOD and APX are not selenoprotein type of enzymes. SOD does not contain selenium—its catalytic center requires metal cofactors like Cu/Zn, Mn, or Fe, not selenium.⁵¹ APX also does not require selenium—it is a heme-containing enzyme that uses iron and ascorbate as substrates and is part of the ascorbate-glutathione cycle.⁵² Since there is no direct biochemical requirement of Se from SeNPs for both enzymes, therefore they are not “preferentially induced” by selenium availability. Both enzymes are induced directly by the oxidative stress. SOD responds to levels of superoxide anion (O₂⁻) while APX responds to H₂O₂ and requires ascorbate.⁵³

In response to salt-induced oxidative stress, SeNPs synthesized from the supernatant of D. abyssi MT1.1^T also elevated total antioxidant activity, expressed as mg gallic acid equivalents (GAE) g⁻¹ FW, of tomato seedlings as shown in Figure 6B. SeNP-treated seedlings under salt stress exhibited an additional 12% increase in total antioxidant activity compared to untreated stressed plants (Figure 6B). This increase together with an observed elevation in CAT and GPX activities suggested that SeNPs synthesized from D. abyssi MT1.1ᵀ enhance both enzymatic and non-enzymatic antioxidant defenses, which represents a key mechanism to mitigate salt-induced oxidative stress in tomato seedlings.

Salt stress induces osmotic stress due to the accumulation of soluble salts in the soil surrounding plant roots, thereby lowering the soil water potential. This reduction in water potential impairs the plant’s ability to absorb water and essential nutrients through the roots, ultimately disrupting cellular homeostasis and physiological functions.^40,47,48 In response to osmotic stress, plants activate various adaptive mechanisms, including the synthesis and accumulation of compatible solutes, also known as osmolytes to facilitate osmotic adjustment.⁴⁷ These small, non-toxic organic molecules contribute to osmotic adjustment by balancing the cellular osmotic pressure, thereby maintaining cell turgor and minimizing water loss. Common osmolytes include proline, soluble sugars (e.g., sucrose, glucose, and fructose), glycine betaine, and polyols such as mannitol and sorbitol.⁴⁷ In the present study, we focus on the change in proline and soluble sugar concentrations as indicator of plant responses to induced salt stress at 150 mM.

Proline and soluble sugars are widely recognized as key biochemical indicators of salinity tolerance in plants.^54,55 Proline plays several protective functions under stress conditions, including scavenging ROS, stabilizing proteins and cellular structures, and maintaining redox balance.⁵⁴ Soluble sugars, on the other hand, contribute to osmotic adjustment, protect macromolecules by stabilizing proteins and membranes, and act as an energy source to support metabolic processes during stress.^56,57

In the present study, treatment with SeNPs led to significant accumulation of both proline (Figure 7A) and total soluble sugars (Figure 7B) in tomato seedlings under both non-stressed and salt-stressed conditions compared to untreated controls. Under salt stress (150 mM NaCl), tomato seedlings treated with SeNPs exhibited a 166% increase in proline content and a 144% increase in soluble sugar levels relative to the salt-stressed control. This high proline concentration suggests that SeNPs play a role in enhancing osmotic stress tolerance by promoting the accumulation of compatible solutes. Similar trends have been observed in other plant species. For example, SeNP seed priming at 1 mg L⁻¹ significantly enhanced proline and sugar levels in quinoa grown under 200 and 400 mM NaCl stress.⁵⁸ Similarly, foliar application of SeNPs at concentrations of 0.5–1.5 mg L⁻¹ in soybean exposed to 4000 mg L⁻¹ sea salt stress increased total soluble sugar accumulation.⁴⁹

Figure 7.

Effect the synthesized selenium nanoparticles (5 mg mL⁻¹) on biochemical parameters of tomato seedling growth under non-stress (0 mM NaCl) and salt stress (150 mM NaCl)

(A) proline content; (B) total soluble sugar content; (C) Relative water content (RWC).

Data are presented as mean ± SD. Different letters indicate statistically significant differences among treatments (p < 0.05) based on two-way ANOVA followed by Duncan’s multiple range test. n = 3 biological replicates.

Relative water content (RWC) is a key physiological parameter used to assess plant water status and tolerance to dehydration, particularly under salt stress conditions.^54,59 Salt stress induces an excessive accumulation of sodium (Na⁺) and chloride (Cl⁻) ions in plant tissues, which reduces water uptake, resulting in a lower RWC value. Therefore, RWC is widely used as a reliable indicator of plant water status and the ability to maintain cellular turgor pressure under stress. In the present study, tomato seedlings treated with SeNPs under non-stress conditions exhibited a high RWC value of 99%, similar to the untreated control, indicating no adverse effects of SeNPs on plant water balance (Figure 7C). However, a 34% reduction in RWC was observed after exposure to 150 mM NaCl, reflecting the plant’s inability to retain water under high salinity conditions. This decrease is consistent with previous findings in which high salinity negatively impacted cellular water balance and membrane stability.^60,61 In this study, the application of SeNPs improved a RWC by approximately 11% compared to the salt-stressed control. The observed RWC of 72% indicated an improved water status, which are essential for normal physiological functions (Figure 7C). The improved RWC in tomato seedlings reflects an enhanced water status, suggesting an increased tolerance to salt stress. This was accompanied by a better growth in SeNP-treated seedlings compared with the salt-stressed control, as indicated by an increased in fresh and dry weight (Figures 8C and 8D). Similarly, a foliar spray of 20 mg L⁻¹ SeNPs improved the RWC of strawberry plants under salt stress by 5%–6%.³⁹ This increase suggests that SeNPs contribute to maintaining water homeostasis in tomato seedlings under salt stress. One possible mechanism is the SeNP-induced accumulation of compatible solutes such as proline and soluble sugars, which aid in osmotic adjustment and help preserve cellular pressure.

Figure 8.

Effect the synthesized selenium nanoparticles (5 mg mL⁻¹) on tomato seedling growth under non-stress (0 mM NaCl) and salt stress (150 mM NaCl)

(A) Shoot length.

(B) Root length.

(C) Fresh weight.

(D) Dry weight.

(E) Total chlorophyll content of tomato seedlings.

Data are presented as mean ± SD. Different letters indicate statistically significant differences among treatments (p < 0.05) based on two-way ANOVA followed by Duncan’s multiple range test. n = 3 biological replicates.

In the present study, tomato seedlings treated with 5 mg mL⁻¹ SeNPs exhibited normal growth, as indicated by shoot length, root length, fresh weight, dry weight, and photosynthetic pigments which were comparable to those of untreated controls (Figure 8). These results suggest that SeNPs at this concentration do not exhibit phytotoxic effects and are safe for use in tomato cultivation under optimal growth conditions. In contrast, exposure to 150 mM NaCl significantly impaired tomato seedling growth, as evidenced by reductions in all measured morphological parameters and total chlorophyll when compared to the non-stressed control group (Figure 8). These findings clearly demonstrate the detrimental effects of salt stress on tomato seedling development, which is consistent with previous studies on growth inhibition of tomato due to salinity-induced osmotic and oxidative stress.⁴⁴ The morphology of tomato seedlings grown under non-stressed and salt-stress conditions is shown in Figure S4.

In this study, the application of SeNPs significantly enhanced biomass production in tomato seedlings subjected to 150 mM NaCl-induced salt stress, as indicated by increased fresh and dry weights compared to untreated controls (Figures 8C and 8D). This finding demonstrates the potential of SeNPs to mitigate the detrimental effects of salinity on plant growth and development. Consistent with previous results, Zahedi et al.³⁹ reported that foliar application of 10–20 mg L⁻¹ commercial SeNPs improved biomass and fruit yield in strawberry plants under varying levels of salt stress (25, 50, and 75 mM NaCl). Similarly, Desouky et al.⁴⁹ showed that foliar spraying with 1 mg mL⁻¹ chemically synthesized SeNPs significantly increased pod dry weight, and overall seed yield in soybean under sea salt stress ranging from 1000 to 4000 mg L⁻¹. Furthermore, Zafar et al.⁴⁶ found that SeNPs derived from lemon leaf extract enhanced biomass and yield per plant, and 1000-grain weight in wheat cultivated under 1% (w/v) NaCl stress. These findings collectively support the hypothesis that SeNPs can enhance plant growth and productivity under salinity stress, likely through mechanisms involving improved water status (RWC value), enhanced antioxidant defense, and modulation of stress-related metabolic pathways. The observed increase in biomass may also reflect SeNP-mediated improvements in nutrient uptake and photosynthetic efficiency, as suggested in prior studies,^39,43,46,58 although these parameters were not assessed in the current work.

Therefore, our result showed the potential of biogenic SeNPs from a deep-sea actinobacterium, D. abyssi MT1.1^T supernatant in promoting plant growth under salt stress and further application in the agricultural sector. Regarding safety concern, the released SeNPs in plant production areas can be accumulated in soil. However, based on previous study, SeNPs exhibit a slow-release behavior in soil, promote beneficial soil microorganisms, and show lower toxicity compared with selenite (SeO₃²⁻), which can negatively affect microbial diversity.⁶² Moreover, SeNPs do not readily enter microbial cells, indicating a higher level of environmental safety. Reported SeNP concentrations that exhibited toxicity in animals⁶³ were generally above 50 mg/kg. For example, the median lethal dose (LD₅₀) of SeNPs in mice was 113.0 mg Se/kg,⁶⁴ while Wang et al.⁶⁵ and Zhang et al.⁶⁶ reported an LD₅₀ of 92.1 mg Se/kg. Additionally, Zhang et al.⁶⁷ reported LD₅₀ values of 61.6 mg Se/kg for female mice and 72 mg Se/kg for male mice. These literatures and low concentration (5 mg mL⁻¹) of used in this study support the safe use of SeNPs for agricultural applications with minimal ecological risk.

Biogenic selenium nanoparticles (SeNPs) were successfully synthesized using the culture supernatant of the deep-sea actinobacterium Dermacoccus abyssi MT1.1ᵀ and sodium selenite (Na₂SeO₃) as the precursor. This biosynthesis approach is environmentally sustainable and avoids the use of toxic solvents or hazardous chemical agents. The use of a cell-free supernatant simplifies downstream processing and enhances biosafety, supporting a green nanotechnology platform. In addition, the synthesized SeNPs showed no phytotoxicity in tomato during seed germination and seedling stages as compared to selenium salt precursor. The functional characteristics—nanoscale size, surface chemistry, and stability—support the potential of these SeNPs for biological applications in particular plant treatments. This study also provides evidence that biogenic SeNPs from Dermacoccus abyssi MT1.1ᵀ significantly alleviate salt-induced oxidative stress in tomato seedlings through multiple mechanisms including reduction of H₂O₂ accumulation, upregulation of key antioxidant enzymes (CAT and GPX), and increased total antioxidant capacity. SeNPs also enhanced osmotic stress tolerance by promoting the accumulation of compatible solutes, proline and total soluble sugar. The physiological and biochemical improvements of tomato seedlings reflected in a better biomass and water status. In summary, these findings highlight the potential of biogenic SeNPs derived from D. abyssi MT1.1ᵀ as a promising tool for mitigating salt-induced osmotic and oxidative damages and promoting crop resilience especially in salt affected areas.

Limitations of the study

This study evaluated a single concentration of SeNPs (5 mg mL⁻¹) at the tomato seedling stage. Although selected based on germination results, the optimal concentration may vary across developmental stages and environmental conditions. As the study focused only on early growth, long-term effects on plant development, yield, and fruit quality were not assessed. Therefore, the findings may not fully reflect field conditions, where soil heterogeneity and microbial interactions could influence SeNP effectiveness. Moreover, the persistence, transformation, and accumulation of SeNPs in soil-plant systems, as well as their potential environmental and ecological risks, were not investigated.

Resource availability

Lead contact

Requests for further information and resources should be directed to and will be fulfilled by the lead contact, Wasu Pathom-aree (wasu.p@cmu.ac.th).

Materials availability

This study did not generate new unique reagents.

Data and code availability

• •All data reported in this paper will be shared by the lead contact upon request.
• •This paper does not report original code.
• •Information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Acknowledgments

This research was supported by CMU Junior Research Fellowship Program for budget year 2023, Chiang Mai University, Thailand (Contract No. JRCMU2566R_042).

Author contributions

Conceptualization, research design, methodology, investigation, data analysis, discussion and conclusion, writing – original draft, writing-review and editing, funding acquisition, P.R.; writing-review and editing, S.S.; data analysis, writing – original draft, writing-review and editing, S.H.; investigation, M.M.; methodology, investigation, data analysis, S.I.; conceptualization, research design, data analysis, discussion and conclusion, supervision, writing-review and editing, W.P.-a. All authors have read and agreed to the published version of the manuscript.

Declaration of interests

The authors declare no competing interests.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work, the authors used “ChatGPT3.5 and ChatGPT4.0” to improve the readability, and language of the manuscript. After using these tools, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Bacterial strain
Dermacoccus abyssi MT1.1T	Pathom-aree et al.68	DSM17573T and NCIMB 14084T
Chemicals, peptides, and recombinant proteins
Sodium selenite	Sigma-Aldrich	Cat#214485-100G
Sodium hypochlorite (6% w/w available chlorine)	HAITER, Industrial (Thailand)	N/A
Sodium chloride	Loba Chemie	Cat#05820
Acetone	RCL Labscan	Cat#AR1003-G2.5L
Absolute ethanol	RCL Labscan	Cat#AR1380-P2.5L
Ninhydrin	Loba Chemie	Cat#04912
Sulfuric acid 98%	ACL Labscan	Cat#AR11930G2.5L
Phenol	Ambion	Cat#AM9712
Trichloroacetic acid	RCL Labscan	Cat#AR1317-G500G
Potassium iodide	RCL Labscan	Cat#AR1245-P500G
2,2-diphenyl-1-picrylhydrazyl (DPPH)	Sigma-Aldrich	Cat#D9132-1G
Methanol	RCL Labscan	Cat#LC1115-G2.5L
Di-potassium hydrogen phosphate (K2HPO4)	VWR chemicals	Cat#26931.263
Potassium dihydrogen orthophosphate anhydrous (KH2PO4)	RCL Labscan	Cat#AR1153-P1KG
Polyvinylpyrrolidone (PVPP)	Sigma-Aldrich	Cat#PVP360-100G
Hydrogen peroxide	Sigma-Aldrich	Cat#8.22287.1000
Nitroblue tetrazolium chloride (NBT)	Sigma-Aldrich	Cat#N6876-50 MG
L-Methionine	Sigma-Aldrich	Cat#M9625-25G
Riboflavin	Sigma-Aldrich	Cat#R4500-25G
EDTA	Vivantis	Cat#PC0706-1 KG
L-ascorbic acid	Kemaus	Cat#KA79
Guaiacol	Sigma-Aldrich	Cat#G5502-100G
Gallic acid	Sigma-Aldrich	Cat#398225-100G
Tryptic soy broth	Difco, BBL	Cat#211825
Experimental models: Organisms/strains
Tomato (Solanum lycopersicum)	Chia Tai, Thailand	N/A
Software and algorithms
IBM SPSS Statistics 22	International Business Machines (IBM)Corporation, Armonk, New York.	https://www.ibm.com/support/pages/downloading-ibm-spss-statistics-22
Other
Peatmoss	Potground H, Klasmann, Germany	N/A

Experimental model and study participant details

Actinobacterial strain and culture condition

Dermacoccus abyssi MT1.1^T, previously isolated from sediment collected in the Mariana Trench (Pathom-aree et al.⁶⁹), was utilized for nanoparticle biosynthesis. The strain was routinely cultivated on tryptic soy agar (TSA) for 7 days at 37 ±2°C. For selenium nanoparticle synthesis, 100 mL of tryptic soy broth (TSB) in 250 mL Erlenmeyer flasks was inoculated with 1 mL of cell suspension (adjusted to OD₆₀₀ =1, approximately 10⁸ CFU/mL) and incubated at 37±2°C on a rotary shaker at 140 rpm for 7 days. The cell-free culture supernatant was harvested for biosynthesis process.

Plant materials

Commercial plum tomato (Solanum lycopersicum) seeds were purchased from Chia Tai, Thailand, and used as the experimental model. Experiments were conducted during the seed germination (Day 0–7) and early seedling growth stages (up to 60 days post-germination). For seedling cultivation, seeds were sown in sterilized peat moss (Potground H, Klasmann, Germany) characterized by a 70:30 white peat to black peat ratio. The plants were maintained on a plant growth rack at a controlled room temperature of 30± 2°C under a 12 h light/12 h dark photoperiod provided by full-spectrum LED grow lights (AC220V T20). All plant experiments were conducted under controlled laboratory conditions in accordance with laboratory biosafety. No specific institutional permission was required for the use of this commercial tomato cultivar.

Method details

Actinobacterial strain and growth conditions

Dermacoccus abyssi MT1.1^T was cultivated on tryptic soy agar (TSA) for 7 days. Subsequently, the cells were harvested and suspended in tryptic soy broth (TSB). The number of cells was adjusted to approximately 10⁸ CFU/mL, corresponding to the optical density of 1 at 600 nm. One mL of the standardized suspension was then inoculated into 100 mL of TSB in a 250 mL Erlenmeyer flask. The culture was incubated at 37±2°C on a rotary shaker at 140 rpm for 7 days. After incubation, the culture was centrifuged at 7,000 rpm for 15 minutes to collect the cell-free supernatant, which was used for selenium nanoparticle synthesis.

Biosynthesis of selenium nanoparticles

The SeNPs were synthesized using a modified method based on Ranjitha and Ravishankar Ranjitha & Ravishankar, (2018). A schematic representation of the synthesis and purification of selenium nanoparticles was shown in Figure S1. Preliminary experiments were conducted to determine the optimal concentration of sodium selenite (Na₂SeO₃) for SeNPs synthesis. The supernatant of D. abyssi MT1.1^T was mixed with different concentrations of Na₂SeO₃ solutions (8, 10, 12, 14, and 16 mM) in a 1:1 ratio (Figure S2). Among these, the 8 mM Na₂SeO₃ solution yielded the highest amount of SeNPs. Therefore, in this study, the supernatant of D. abyssi MT1.1^T was mixed with 8 mM Na₂SeO₃ solution in a 1:1 ratio and incubated at 37 ±2°C on a shaker at 140 rpm for 72 hours. The SeNPs were collected by centrifugation at 7,000 rpm for 20 minutes at 4°C, then washed twice with deionized water, and the resulting pellets were resuspended in deionized water. The SeNPs suspension was characterized using ultraviolet (UV)–visible spectroscopy (SpectraMax iD3) at wavelengths 200-700 nm. In addition, SeNP pellets were lyophilized for determination of the yield and expressed as mg dry weight of the lyophilized SeNPs per 100 mL of D. abyssi MT1.1^T supernatant-Na₂SeO₃ mixtures. Dried SeNPs powder was kept at -20°C for future use.

Characterization of selenium nanoparticles

The size, shape and surface morphology of the synthesized SeNPs were examined using transmission electron microscopy (TEM; JEM-2010, JEOL) and scanning electron microscopy (SEM; JSM-IT800, JEOL) equipped with energy dispersive spectroscopy (EDS). Fourier-transform infrared spectroscopy (FTIR; Bruker Tensor 27, Kaller, Germany) was performed to identify functional groups and potential bioactive compounds associated with the synthesized SeNPs. The crystalline structure and phase purity of SeNPs were examined using X-ray diffractometer (XRD; Rigaku SmartLab). Dynamic light scattering (DLS) was performed using a particle size analyzer (Nanosizer, Horiba SZ-100 series) to determine the hydrodynamic size distribution of the SeNPs. Zeta potential measurements were also conducted using the same instrument to evaluate the surface charge of the nanoparticles.

Effect of selenium nanoparticles on tomato seed germination under salt stress

A preliminary dose–response assay (1–10 mg mL^-1) was conducted to determine the optimal SeNP concentration for tomato seed germination (Figure S3). Although germination percentage was not significantly affected, 5 mg mL^-1 resulted in the highest germination index (GI), indicating enhanced early seedling development, particularly root growth. Higher concentrations (6–10 mg mL^-1) reduced GI; therefore, 5 mg mL^-1 was selected for subsequent experiments. To evaluate the effect of the synthesized SeNPs on tomato (Solanum lycopersicum) seed germination under both non-stress and salt stress conditions, tomato seeds (Chia Tai, Thailand) were first surface sterilized by soaking in 70% (v/v) ethanol for 3 mins, followed by treatment with 5% (v/v) sodium hypochlorite for 5 mins, then rinsed six times with sterile deionized water to remove residual sterilizing agents.⁴⁵ The experiment was arranged in a factorial completely randomized design (CRD) with two factors: selenium treatment (three levels) and salinity (four levels: 0, 50, 100, and 150 mM NaCl). Sterilized seeds were soaked for 10 h in one of three treatments: (1) deionized water (control), (2) 5 mg mL^-1 SeNPs, or (3) 5 mg mL^-1 sodium selenite (Na₂SeO₃). For each treatment combination, 15 seeds were randomly sown in plastic trays (15 × 10 × 5.5 cm) containing 150 g of sterile peat moss (white peat: black peat, 70:30; Potground H, Klasmann, Germany), with three replicate trays per treatment. Seeds were incubated in darkness for 24 h, subsequently subjected to salt stress by daily irrigation with sodium chloride (NaCl) solutions at concentrations of 0, 50, 100, or 150 mM. Distilled water was used for the non-saline control (0 mM NaCl). All trays were maintained under a 12 h light/12 h dark photoperiod for 7 days. At the end of the incubation period, germinated seeds were counted, and germination percentage was calculated (Equation 1). In addition, root length of seedlings was measured and used to calculate the germination index (Equation 2).

$$\text{Seed}\text{germination}(\%)=\frac{\text{No}.\text{of}\text{germinated}\text{seeds}}{\text{No}.\text{of}\text{total}\text{seeds}}\times 100$$ (Equation 1)

$$\text{Germination}\text{index}(\%)=\frac{{\text{seed}\text{germination}}_{\text{treatment}}(\%)\times {\text{average}\text{root}\text{length}}_{\text{treatment}}}{{\text{seed}\text{germination}}_{\text{control}}(\%)\times {\text{average}\text{root}\text{length}}_{\text{control}}}\times 100$$ (Equation 2)

Effects of selenium nanoparticles on tomato seedlings under salt stress

Tomato seeds were sown into plastic pot (6 cm diameter × 6 cm height) containing 60 g of sterilized planting substrate (white peat: black peat, 70:30; Potground H, Klasmann, Germany), with three seeds per pot. After one week, two seedlings were removed from each pot, leaving one uniform seedling per pot for further analysis. At 30 days after germination, seedlings were arranged in a factorial completely randomized design (CRD) with two factors: selenium nanoparticle (SeNP) treatment (control and 5 mg mL^-1 SeNPs) and salinity condition (0 and 150 mM NaCl). Seedlings received one of two treatments: (1) control (deionized water) or (2) 5 mg mL^-1 SeNPs. For SeNP treatment, 200 μL of SeNP solution was applied weekly to each seedling until harvest. The salinity conditions included non-stressed (0 mM NaCl), in which seedlings were irrigated daily with distilled water, and salt-stressed (150 mM NaCl), in which seedlings were irrigated daily with sodium chloride solution. The same volume of solution was applied across all treatments. The first day of salt treatment was designated as day 1 after transplanting (DAT1), and irrigation was continued daily for 30 days (DAT30). At the end of the experimental period (DAT30), tomato seedlings were harvested, and evaluated for key morphological parameters, including shoot length, root length, fresh weight (FW), and dry weight (DW) with three plants used as biological replicates for each treatment (n=3). Turgid weight (TW) was also determied after soaking the plant root in distilled water for 48 h in the dark at room temperature. Relative water content (RWC) was calculated according to the formula described by Oukarroum et al.⁷⁰

{RWC (%) = [(FW –[ DW)/ (TW − DW) × 100]}

Evaluation of biochemical parameters

Biochemical parameters were determined using fresh leaf samples collected from three tomato seedlings per treatment (n=3) at 30 days after transplanting (DAT30). Chlorophyll and carotenoid contents were determined following the method described by Arnon.⁷¹ Briefly, leaf tissues were homogenized in 80% (v/v) acetone and centrifuged at 5,000 rpm for 10 minutes to extract pigments. The absorbance of the resulting supernatant was measured at 480 nm, 645 nm, and 663 nm using a spectrophotometer. Total chlorophyll, and carotenoid concentrations were calculated using the following equations:

Total chlorophyll (mg g^-1) = 20.2 ×OD₆₄₅ +18.2×OD₆₆₃ × V/(1000× W)

Carotenoid (mg g^-1) = 4.695× OD₄₈₀ - 0.268 ×V/(1000× W)

V = volume of acetone (mL); W = weight of leaves (g).

Proline accumulation in tomato leaves was quantified using a colorimetric method described by Bates et al.⁷² with slight modifications.⁴⁴ Fresh leaf was homogenized in 95% (v/v) ethanol, and the homogenate was centrifuged at 1,500 rpm for 5 minutes to obtain the supernatant. The supernatant was then mixed with ninhydrin reagent and incubated in a boiling water bath at 100 °C for 1 hour. The reaction was stopped by placing the tubes in an ice bath. An equal volume of toluene was then added to each tube, and the mixture was vortexed for 20 seconds. The upper organic phase was carefully collected, and absorbance was measured at 520 nm using a UV-Vis spectrophotometer (SpectraMax iD3, Molecular Devices, USA). Proline concentration was determined from a standard curve of known concentrations of L-proline.

Total soluble sugar content in tomato leaves was estimated using a modified phenol sulfuric acid method, based on Shukla et al.⁷³ and adapted by Rangseekaew et al.⁴⁴ Fresh leaf samples (10 mg) were homogenized in 1 mL of 80% (v/v) ethanol using a mortar and pestle and heated in a water bath at 75 °C for 15 minutes, followed by centrifugation at 12,000 rpm for 15 minutes to collect the supernatant. For quantitative analysis of sugar, 250 μL of each supernatant was mixed with 250 μL of 80% (v/v) ethanol, 2.5 mL of concentrated sulfuric acid, and 0.5 mL of 5% (w/v) phenol. The reaction mixtures were incubated at room temperature for 20 minutes and the optical density was measured at 520 nm using a UV–Vis spectrophotometer (SpectraMax iD3, Molecular Devices, USA). Total soluble sugar concentrations were determined from a glucose standard curve.

Evaluation of antioxidative parameters

The concentration of hydrogen peroxide (H₂O₂) in fresh tomato leaves collected from three plants per treatment (n=3) was determined following the method of Velikova et al.⁷⁴ with modifications as detailed in Rangseekaew et al.⁴⁴ Leaf tissue was homogenized in ice bath using 0.1% (w/v) trichloroacetic acid and centrifuged at 10,000 rpm for 15 minutes. The obtained supernatant was mixed with 10 mM potassium phosphate buffer (pH 7.0) and 1 M potassium iodide (KI). UV–Vis spectrophotometer (SpectraMax iD3, Molecular Devices, USA) was used to measure the optical density of the mixture at 390 nm. Hydrogen peroxide concentration was estimated using a standard curve prepared from known concentrations of H₂O₂.

Antioxidant activity was evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay, following the method of Bhadoriya et al.⁷⁵ Fresh leaf samples collected from three plants per treatment (n = 3) were extracted with methanol. Leaf samples were extracted with methanol, and 50 μL of the extract was mixed with 150 μL of 0.1 mM DPPH solution. The mixture was subjected to a dark incubation for 20 minutes. UV–Vis spectrophotometer (SpectraMax iD3, Molecular Devices, USA) was used to measure the optical density of the mixture at 517 nm. The DPPH radical scavenging activity was determined as the percentage reduction in absorbance relative to the control using the following formula:

DPPH scavenging activity (%) = [(Ac-(As-Asc))/Ac] × 100

Ac = absorbance of the DPPH control; As = absorbance of the sample with DPPH; Asc = absorbance of the sample without DPPH.

Total antioxidant content was estimated using a standard calibration curve of gallic acid and expressed as milligrams of gallic acid equivalents (mg GAE) per gram of fresh weight.

Analysis of enzymatic antioxidant activities

Enzymatic antioxidant activities in tomato leaves were analyzed using fresh leaf tissue collected from three plants per treatment (n = 3). The tissue was extracted in 50 mM potassium phosphate buffer (pH 7.0) supplemented with 1% (w/v) polyvinylpyrrolidone (PVPP), using a homogenizer, as described by Singh and Jha.⁷⁶ The extract was centrifuged at 7,500 rpm for 15 minutes at 4 °C, and the obtained supernatant was used for the determination of catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APX), and guaiacol peroxidase (GPX).

Catalase (CAT) activity

CAT activity was determined by measuring the decomposition of hydrogen peroxide (H₂O₂) at 240 nm, according to the method of Aebi⁷⁷ and Islam et al.⁴² The 2 mL reaction mixture consisted of 0.1 mL of crude enzyme extract, 1.7 mL of 50 mM phosphate buffer (pH 7.0), and 0.2 mL of 25 mM H₂O₂. The decrease in absorbance at 240 nm was measured using a UV–Vis spectrophotometer (SpectraMax iD3, Molecular Devices, USA). One unit of CAT activity was defined as a 0.1 unit min^-1 change in an optical density, and results were expressed as units mg^-1 protein.

Superoxide Dismutase (SOD) activity

SOD activity was determined based on its ability to inhibit the photochemical reduction of nitroblue tetrazolium chloride (NBT), as described by Islam et al.⁴² and Singh and Jha.⁷⁶ The reaction mixture contained 50 mM phosphate buffer (pH 7.8), 13 mM methionine, 75 μM NBT, 2 μM riboflavin, 0.1 mM EDTA, and the crude enzyme extract. After incubation under fluorescent light (15 W, 4,000 lux) for 10 minutes, the absorbance was measured at 560 nm using a UV–Vis spectrophotometer (SpectraMax iD3, Molecular Devices, USA). SOD activity was expressed as units mg^-1 protein where one unit was the amount of enzyme required to inhibit 50% of NBT reduction.

Ascorbate peroxidase (APX) activity

APX activity was evaluated following the modified method of Morales-Espinoza et al.⁷⁸ The reaction mixture consisted of 100 μL of crude enzyme extract, 500 μL of 10 mM ascorbic acid, 400 μL of 5% (v/v) sulfuric acid (H₂SO₄), and 1 mL of 100 mM H₂O₂. The absorbance of the reaction mixture was measured immediately after mixing at 266 nm (T₀) and after 1 minute (T₁) using a UV–Vis spectrophotometer (SpectraMax iD3, Molecular Devices, USA). The reaction was stopped by adding 400 μL of 5% (v/v) H₂SO₄. APX activity (units mg^-1 protein) was calculated based on the difference in absorbance (T₀ – T₁) with 0.01 unit min^-1 change in absorbance was equivalent to one unit.

Guaiacol peroxidase (GPX) activity

GPX activity was measured following the method of Zhang et al.⁷⁹ with modifications. The 2.5 mL reaction mixture contained 2.3 mL of 50 mM phosphate buffer (pH 7.0), 0.05 mL of 0.1% (v/v) guaiacol, 0.1 mL of 0.1% (v/v) H₂O₂, and 0.05 mL of crude enzyme extract. The mixture was incubated at room temperature for 5 minutes, and the absorbance at 470 nm was recorded using UV–Vis spectrophotometer (SpectraMax iD3, Molecular Devices, USA). A 0.001 unit min^-1 change in absorbance was defined as one unit of GPX activity and expressed as units mg^-1 protein.

Quantification and statistical analysis

Statistical analysis of the tomato seed germination experiment was performed using one-way analysis of variance (ANOVA) under a completely randomized design (CRD). Data were compared among treatments (control, 5 mg mL^-1 SeNPs, and 5 mg mL^-1 Na₂SeO₃) within each salinity level (0, 50, 100, and150 mM NaCl). Results are presented as mean values of three replicates (15 seeds per replicate). Different letters indicate significant differences among treatments within each salinity condition according to Duncan’s multiple range test (p < 0.05). For the tomato seedling experiment, data were analyzed using two-way ANOVA based on a factorial completely randomized design (CRD) with two factors: selenium nanoparticle (SeNP) treatment and salinity (NaCl concentration). Duncan’s multiple range test was applied for post hoc comparisons. All results are presented as mean ± standard deviation (SD). Data were tested for normality prior to analysis and confirmed to follow a normal distribution. Statistical analyses were performed using SPSS software (version 22). Mean values that were significantly different are indicated by different letters at p < 0.05.