The biosynthesis and characterization of AgNPs synthesized from F. solani IOR 825 were performed as described previously by Trzcińska-Wencel et al. (2023b). Briefly, AgNPs were synthesized using fungal autolysate in water. For this purpose, the fungal strain was grown in Potato Dextrose Broth (PDB, A&A Biotechnology, Gdańsk, Poland) at 26°C for 7 days, followed by centrifugation (6500 × g, 10 min), biomass washing with sterile distilled water, and resuspending in water (100 mL of water for 10 g of biomass) for 3 days for autolysis. Autolysate was centrifuged (4000 × g, 5 min), filtered by sterile filter paper, and used for challenging with 100 mM silver nitrate (AgNO₃; final concentration of 1 mM). The reaction mixture was sunlight-treated and incubated for 7 days in darkness. After this incubation period, AgNPs were centrifuged (13000 × g, 1 hour) and dried at 37°C. The mass of AgNPs was determined in mg, and the powder was used to prepare the stock solution of 2048 µg mL⁻¹ for further analyses. For maize grain treatment, the AgNPs stock solution was used to prepare final (32, 128, and 512 µg mL⁻¹) concentrations (Trzcińska-Wencel et al., 2023b).

The biosynthesis of AgNPs was confirmed using UV–Vis spectroscopy (NanoDrop One, Thermo Fisher Scientific, Waltham, MA, USA) at the wavelength range 200–700 nm with a resolution of 1 nm. The size and shape of AgNPs were determined using transmission electron microscopy (TEM) (FEI, Tecnai 12 Netherland) after applying AgNPs solution on a carbon-coated copper grid with 400-μm mesh size and drying at room temperature for 24 hours. The size of AgNPs was measured based on TEM micrographs using the ImageJ software. X-ray diffraction (XRD) (X’ Pert PRO Analytical X6 diffractometer, PANalytical, Almelo, Netherlands) with Cu Kα (λ = 1.54056 Å) radiation source and Ni were used with a filter in the 2θ range 5°–120°. The functional groups on the surface of AgNPs were determined by Fourier transform infrared (FTIR) spectroscopy (Spectrum 2000, Perkin-Elmer, Waltham, MA, USA). Before analysis, AgNPs powder was combined with KBr (1:100 ratio, w/w) and analyzed in the range 400–4000 cm⁻¹ at a resolution of 4 cm⁻¹. The size distribution and surface potential of AgNPs in Milli-Q water solution were evaluated by nanoparticle tracking analysis (NTA LM20, NanoSight Limited, Amesbury, UK), dynamic light scattering (DLS), and Zeta potential measurement (Zetasizer Nano-ZS 90, Malvern, UK). The software provided by the equipment manufacturer was used to analyze the obtained results, namely, NTA, version 2.3 Build 0033, and Zetasizer Software, version 6.32.

Maize (Z. mays) grains (Torseed S.A, Toruń, Poland) were sterilized in previously selected concentrations (32 and 128 µg mL⁻¹) of AgNPs as described by Trzcińska-Wencel et al. (2023b), while a concentration of 512 µg mL⁻¹ was added to this study to expand significantly the concentration range of AgNPs used for the treatment of grains. Briefly, for each variant, 25 grains were selected and sterilized at room temperature for 30 min with 25 mL of 30% hydrogen peroxide (H₂O₂) and 70% ethanol (1:1, v:v) or with 25 mL of AgNPs solutions at the concentrations of 32, 128, and 512 µg mL⁻¹ and washed five times with sterile distilled water. Grains were placed on ½ Murashige and Skoog (MS) agar and germinated at 22°C ± 2°C for 14 days. The 14-day-old plantlets at the V2 growth stage were harvested, their length and fresh and dry weight were measured, and the plant material for biochemical analysis was frozen in liquid nitrogen immediately after harvesting and stored at −80°C. All these parameters were estimated for roots, leaves, stems, and caryopses separately ( Supplementary Figure S1 ).

The parameters of grain germination were calculated as follows:

where G% is the germination percentage, Ʃn is the total number of grains germinated after 14 days, and N is the total number of grains sown (Scott et al., 1984).

where MGT is the mean germination time, f is the number of germinated grains at day x, x is the number of days from sowing, and Ʃn is the total number of germinated grains (Orchard, 1977).

where GRI is the germination rate index and G1, G2, …, Gx is the germination percentage in the subsequent days after sowing (Esechie, 1994).

where G% is the germination percentage and PL is the length of plantlets.

where G% is the germination percentage and PDW is the dry weight of plantlets (Abdul-Baki and Anderson, 1973).

The length of the shoots and roots was measured using a ruler and expressed in cm; the fresh and dry weight of the leaves, stems, roots, and caryopses were determined in mg.

The plant material was washed and dried at 50°C for 48 hours, then finely powdered, and used for energy dispersive spectroscopy (EDS) analysis to assess elemental composition using a scanning electron microscope (LEO Electron Microscopy model 1430 VP Ltd., UK) coupled with an energy dispersive X-ray spectrometer (Quantax 200 with XFlash 4010 detector, Bruker AXS, Karlsruhe, Germany) (Kumari et al., 2024).

The total chlorophyll content in maize leaves was assayed according to the method described by Witham et al. (1971). Shortly, the powdered leaf tissue (0.5 g) was extracted with 1.5 mL of 80% cooled acetone and then centrifuged at 5000 × g for 5 min (Thermo Fisher Scientific, USA). The supernatant was drained into a 15-mL test tube, and the extraction of the remaining pellet was repeated five times (until the green color disappeared). Supernatants were combined, and the absorbance of the samples was read at λ_645nm and λ_663nm using a U-1800 spectrophotometer (Hitachi, Tokyo, Japan). The amount of total chlorophyll was calculated based on the following formula:

where V is the final volume of the extract (mL) and FW is the fresh weight of the leaf (g).

The data analysis was performed using GraphPad Prism version 10.0.0 (GraphPad Software, Boston, MA, USA). The results were shown as a mean ± standard error (SE). The means were then compared to determine statistical significance (if p < 0.05) by one-way ANOVA and post-hoc Tukey’s test. The growth, oxidative stress, and antioxidant parameters quantified in the organs of plantlets developed from AgNPs-treated grains were used to perform principal component analysis (PCA) and hierarchical cluster analysis (HCA) with R 4.4.2 (R Foundation for Statistical Computing, Vienna, Austria) using the factoextra, ggplot2, and dendextend R packages (Galili, 2015; Kassambara and Mundt, 2020; Wickham, 2016).

TEM analysis confirmed the formation of spherical AgNPs with sizes ranging from 8.9 to 47.9 nm and an average diameter of 22.97 ± 9.4 nm ( Figure 1A ). UV–visible absorption spectroscopic analysis of the mycosynthesized AgNPs showed a characteristic peak at 419 nm ( Figure 1B ). The results of nanoparticle tracking analysis (NTA) confirmed that the AgNPs exhibited an average size of 43 ± 36 nm and most frequently a diameter of 23 nm ( Figure 1C ). The results of DLS indicated that average hydrodynamic diameters of AgNPs were found to be 27.44 (14%) and 108.4 nm (86%), as shown in Figure 1D , while Zeta potential values were found to be −19.5 (61.3%) and −2.93 mV (38.6%) ( Figure 1E ). The diffractogram of AgNPs showed peaks at 38.63, 46.41, 65.10, and 77.09 corresponding to (1 1 1), (2 0 0), (2 2 0), and (3 1 1) planes of the face-centered cubic (fcc) silver crystal, respectively ( Figure 1F ). FTIR spectra showed absorption bands at 3429.21 cm⁻¹ (N–H stretching, amines), 2924.03 cm⁻¹ (C–H stretching, alkane), 2852.77 cm⁻¹ (C–H stretching, alkane), 1743.8 cm⁻¹ (C–H bending, aromatic compound), 1631.89 cm⁻¹ (C=C stretching, alkene), and 1384.44 cm⁻¹ (C–H bending, alkane) ( Figure 1G ).

The surface sterilization of maize grains with AgNPs from F. solani IOR 825 at the concentration range tested did not affect the germination percentage (%G), mean germination time (MGT), and germination rate index (GRI), as shown in Table 1 . The gradual improvement in plant growth was observed after treatment with increasing AgNPs concentrations, when compared to control, and reflected in the increase in shoot length, fresh weight of leaves, and stem of plants developed from AgNPs-treated grains ( Figure 2 ; Supplementary Table S1 ). The strongest effect was observed for AgNPs treatment at a concentration of 512 µg mL⁻¹, with improvements in shoot (13.3%) and root (11%) lengths, as well as fresh weight of leaves (22.3%) and stems (39.2%) and dry weight of leaves (37.8%) and stems (43.1%). This was also demonstrated in vigor indexes I and II, which increased to 4334.3 (I) and 8096.1 (II), compared to the controls, which were 3980.6 and 6259.7, respectively ( Table 1 ). However, approximately 20% reduction in caryopsis weight was observed. Nevertheless, the stimulatory effect of the AgNPs treatment on root development was observed, and the difference with the untreated control did not pass the significance test.

The analysis of elemental composition of plants developed from AgNPs-treated grains showed no significant difference in Ag content between tested and control samples, as shown in Supplementary Table S2 .

AgNPs treatments at concentrations of 32 and 128 µg mL⁻¹ showed negligible effect on total chlorophyll content in the leaves of plantlets. The application of AgNPs at a concentration of 512 µg mL⁻¹ resulted in a decrease in chlorophyll content by 11.8% (0.05 mg g FW⁻¹) ( Figure 3 ; Supplementary Table S1 ).

Changes in the activity of individual components of the antioxidant system for the tested plants, concerning non-treated plantlets, given as percentages (%), are shown in Supplementary Table S1 .

The results of PCA and HCA showing general alterations and correlations of growth and individual biochemical parameters among organs of plantlets developed from grains treated with AgNPs are demonstrated in Figure 7 . The first two components, PC1 and PC2, allow us to represent high values of the initial variability of the data (44.8% and 27.8%, respectively). The results showed that APX activity, MDA, and CAT activity showed the highest contribution to PC1 (14.0%, 12.6%, and 12.3%, respectively), while the contribution of variables to PC2 was explained by SOD activity (16.8%), FW (12.9%), and GSH content (12.2%) ( Supplementary Table S3 ). The highest positive correlation was found for ASC and total ASC, along with POX and APX ( Figures 7A, B ). The effect of the individual concentrations of AgNPs (32, 128, and 512 µg mL⁻¹) among plantlet organs (leaves, caryopses, stems, and roots) based on biochemical and growth parameters were assayed by HCA ( Figure 7C ). All organs were classified as separate groups, including untreated samples. In both leaves and stems, changes in biochemical and growth parameters under AgNPs treatments at concentrations of 32 and 128 µg mL⁻¹ were nearest to each other, followed by concentrations of 512 µg mL⁻¹ and controls. The caryopses showed dose-dependent alteration, where the two highest concentrations (128 and 512 µg mL⁻¹) caused a similar response pattern, followed by a concentration of 32 µg mL⁻¹ and controls. In roots, the effects of AgNPs treatment at the concentration of 32 µg mL⁻¹ were found to be similar to those of controls, followed by concentrations of 128 and 512 µg mL⁻¹.

Biological synthesis of NPs addresses the need to develop environmentally friendly, efficient, and safe methods of producing NPs. Many studies have shown that fungi display potential for the biosynthesis of NPs, as they efficiently produce the biomass along with a variety of chemical compounds responsible for the reduction of silver ions to silver NPs and possess a high tolerance to metals (Borehalli Mayegowda et al., 2023). It was reported that fungal enzymes are involved in the synthesis of AgNPs (Ahmad et al., 2003; Hamedi et al., 2017). In a study conducted by El-Sayed and El-Sayed (2020), the protein-rich cell-free filtrate of F. solani KJ 623702 was successfully used for the synthesis of AgNPs, CuNPs, and ZnONPs.

The physicochemical properties of biosynthesized AgNPs and consequently their biological interactions or reactivity are highly dependent on synthesis conditions including the biological source of reducing and stabilizing agents, type, and concentration of precursor salt, as well as the temperature and pH of the reaction mixture (Sidhu et al., 2022; Trzcińska-Wencel et al., 2023a). The results of our study confirmed that the fungal strain F. solani IOR 825 is a system for the efficient synthesis of AgNPs with small size (10–50 nm) and the presence of natural origin capping biomolecules on their surface. This capping can be involved in the reduction of silver ions (Ag⁺) and the formation of AgNPs and affects their stability and antimicrobial activity. Moreover, the coating modulates the interaction between nanoparticles and biological surfaces, which affects the potential uptake of nanoparticles (Huang et al., 2022; Wypij et al., 2022). In accordance with our results, El-Sayed and El-Sayed (2020) found that FTIR analysis of AgNPs from F. solani KJ 623702 showed peaks attributed to N–H bending, C–H stretching vibrations of protein methylene groups, and O–H stretching of carboxylic acids. The authors, based on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and FTIR results, suggested that the process of biogenic synthesis of NPs is related to the presence of various fungal-origin compounds (containing sulfur, nitrogen, and phosphorus), proteins with β-sheet and a carbonyl group of amino acid residues, and glycoprotein-containing polysaccharides with α-glycosidic bond (El-Sayed and El-Sayed, 2020). The nanoparticle surface properties such as surface charge and hydrophobicity/hydrophilicity are responsible for electrostatic repulsion between individual nanoparticles that prevent aggregation. This makes nanoparticles more mobile and more available for plants than bare nanoparticles. In contrast to chemically synthesized NPs, which are most frequently coated with polyvinylpyrrolidone (PVP), Arabic gum (AG), citrate, and cetyltrimethylammonium bromide (CTAB), the biological NPs are coated with molecules of natural origin (plants or microbial extracts). Consequently, the biological coating is considered less toxic than the chemical one (Sidhu et al., 2022; Wypij et al., 2022). Similar findings were described in our previous studies by Trzcińska-Wencel et al. (2023b, 2023c), who showed the potential of using the fungi of the genus Fusarium for the efficient, low-cost, simple, and environmentally friendly synthesis of metal nanoparticles with desirable physical and chemical properties to provide biocompatibility and biological activity as well as their potential for use in multiple applications, including agriculture.

Although silver is not an essential element for plant growth as compared to other micronutrients such as copper, zinc, iron, or magnesium, its high antimicrobial activity is an advantage in many applications (Duffy et al., 2018; Kakian et al., 2024). The nanoscale form of silver can provide their slow and controlled release over time, thus prolonging antimicrobial protection for plants (Bernardo-Mazariegos et al., 2019; Cao et al., 2025). Moreover, as the biogenic AgNPs exhibit higher antimicrobial activity than biogenic CuNPs, ZnNPs, and FeNPs, their use at a lower effective dose can reduce the toxicity of nanoproducts released into the environment (Asghar et al., 2018; Trzcińska-Wencel et al., 2023b). Therefore, biosynthesized AgNPs are excellent alternatives to conventional fungicides, which are based on copper compounds (Ding et al., 2019; Yen et al., 2019; Khan et al., 2021; Trzcińska-Wencel et al., 2024).

The germination of seeds and early growth of seedlings are identified as critical stages in the development and establishment of plants (Rajjou et al., 2012). A high threat of microbial contamination of seeds used for sowing provides a risk of fungal pathogen growth and infection of germinating seeds or developing seedlings (Magan et al., 2004). Seed priming is an effective method to prepare seeds for sowing to increase germination efficiency and achieve improved seedling growth (Abbasi Khalaki et al., 2021). As previously reported by Trzcińska-Wencel et al. (2023b), the use of AgNPs for grain pretreatment eliminated grain-borne pathogens at the lowest effective AgNPs concentration of 32 µg mL⁻¹ with no impact on grain germination efficiency. In the present paper, we decided to broaden the study on the effect of AgNPs on grain germination and plantlet growth using the effective concentration (32 µg mL⁻¹), its fourfold higher concentrations (128 and 512 µg mL⁻¹), and prolonged (14 days) plant growth period. Simultaneously, the disinfecting effect of the used AgNPs concentrations on grains was confirmed, as no microbial growth was visually detected on the surface of the germinating grains or plant growth medium ( Supplementary Figure S2 ). The results showed no impact of all tested AgNPs concentrations on grain germination when compared with control. Nonetheless, the treatment accelerated plantlet development, as manifested by the improved growth of shoot and reduction of grain weight in comparison with the control, indicating a stimulatory effect of the AgNPs on maize development. Similarly, seed pre-sowing treatments with bio-AgNPs improved the growth of licorice (Glycyrrhiza glabra) (Kim et al., 2023), maize (Z. mays) (Karim et al., 2023), onion (Allium cepa L.) (Acharya et al., 2019), watermelons (Citrullus lanatus) (Acharya et al., 2020), winged bean (Psophocarpus tetragonolobus) (Kamal Kumar et al., 2020), wheat (Triticum aestivum) (Mondéjar-López et al., 2023), and black gram [Vigna mungo (L.) Hepper] (Krishnasamy et al., 2024). In the study reported by Soliman et al. (2020), seed priming with increasing AgNPs concentrations gradually improved the germination of seeds and growth of maize (Z. mays L.), fenugreek (Trigonella foenum-graecum L.), and onion (A. cepa L.). Moreover, stimulatory effects on the growth and development of seedlings after seed nanopriming with the AgNPs synthesized using chemical and physical methods were reported for common beans (Phaseolus vulgaris) (Savassa et al., 2021) and beans (Prażak et al., 2020). However, in the other experimental system, namely, medium supplementation with chemically synthesized nanoparticles at a concentration of 100 ppm, the phytotoxic effects were observed on Bacopa monnieri (Krishnaraj et al., 2012). Higher phytotoxicity was observed under foliar application of AgNPs than root exposure in soybean and rice (Li et al., 2017), while the application of PVP-coated AgNPs resulted in limited germination and a decrease in the growth of T. aestivum L. (Vannini et al., 2014).

As numerous studies have suggested the adverse effects of NPs (especially at high concentrations), there is a need for extensive research on the interactions that occur between NPs and plants (Ding et al., 2019; Khan et al., 2021; Trzcińska-Wencel et al., 2024). The bioaccumulation and translocation of AgNPs in the treated plants depend on the plant (e.g., species and stage of growth), method of application, time of exposure, or dose used. The biological activity of AgNPs is determined by their physicochemical features, mainly by size, chemical composition, and surface properties. These parameters influence the uptake and transport of NPs within plant organs, their interactions with cellular components (e.g., cell wall penetration), and the release of silver ions, subsequently contributing to their effect on plant growth (Nandini et al., 2023; Trzcińska-Wencel et al., 2024). AgNPs in the environment undergo various biotransformations such as aggregation, ion release, sulfidation, or complexation with organic matter that determine their bioavailability to plants or toxicity. It has been noticed that AgNPs transport throughout the plant tissues via both apoplastic and symplastic pathways (Ali et al., 2021; Cao et al., 2025). However, our study showed that the Ag was undetected in any of the plantlet organs after 14 days of pre-sowing treatment of grains. In contrast, in a study conducted by Savassa et al. (2021), the seed treatments with 1, 10, and 100 mg L⁻¹ of chemically synthesized AgNPs, Ag₂SNPs, and AgNO₃ (control) resulted in silver accumulation and biotransformation in P. vulgaris seeds. Ag was detected in the seed coat depending on the Ag source and concentration used. After 5 days from seed treatment, the Ag from AgNO₃ was detected in the epidermis, while the Ag from AgNPs and Ag₂SNPs was mainly located in the internal layer (parenchyma cells) of seeds. The study of Koley et al. (2023) implies that seed treatment with AgNPs led to their accumulation at a low dose and then the gradual release of Ag⁺ ions, which stimulated the antioxidant system with accelerated germination and growth of legume plants. In turn, Yan et al. (2023) reported that AgNPs stimulated the germination process of rice seeds due to the AgNPs uptake during the imbibition process. AgNPs were accumulated in decreasing concentrations in the seed coat, embryo, and endosperm. In contrast, the metabolomic and transcriptomic analyses revealed that AgNPs increased in stress signaling molecule synthesis, showing their potential as nanobiostimulators to ensure long-term stress memory (Yan et al., 2023).

AgNPs may induce ROS generation and strengthen antioxidant system response by the enzymatic and/or non-enzymatic pathways involving both a wide range of enzymes (e.g., superoxide dismutase, catalase, peroxidases) and low-molecular-weight compounds (e.g., ascorbic acid, glutathione, proline, and tocopherols) (Sharma et al., 2019). In the present study, the treatment of maize grains with AgNPs specifically affected cellular redox agents in plant organs subjected to analysis. Symptomatically, as revealed by the lipid peroxidation assay, the treatments did not cause severe oxidative stress in any organ. This finding shows that optimal AgNPs treatments do not increase the risk of oxidative damage. However, the slight but significant increase in the rate of MDA formation and a decrease in total chlorophyll content occurred in leaves, but solely in plants challenged with the highest AgNPs concentration. Similarly, the decrease in photosynthetic pigments was reported in Brassica sp. seedlings exposed to AgNPs synthesized from Aloe vera extract (Vishwakarma et al., 2017) and in Lupinus termis after exposure to AgNPs synthesized from the leaf extract of Coriandrum sativum (Al-Huqail et al., 2018). In our study, the photosynthetic rate and the maximum efficiency of photosystem II were not analyzed in AgNPs-treated maize plants. Therefore, it remains to be determined to what extent the decrease in chlorophyll content affects photosynthetic efficiency. However, this issue will be addressed in our forthcoming study. Other authors have shown that a decrease in chlorophyll content adversely affects photosynthesis. Under decreased chlorophyll levels, excess electron flow may result in an imbalance between the donor and acceptor sites of photosystem II. This results in molecular oxygen reduction generating harmful ROS and photosystem damage (photoinhibition) (Bhattacharjee, 2019). The increase in lipid peroxidation and decrease in chlorophyll content in leaves of plants exposed to the highest AgNPs concentration may be related to increased photoinhibition occurring at excessive nanoparticle concentration. Although further experiments are required to assess if maize leaves become more susceptible to photooxidation under non-optimal AgNPs concentrations, these findings stress the necessity of optimizing the treatment procedure thoroughly since treating seeds with excessive AgNPs concentrations may bring about some detrimental consequences.

Although no significant oxidative damage was detected in AgNPs-treated plants, several components of the cellular redox systems were affected by treatments. Usually, the parameters were altered in an organ-specific manner. In leaves, the H₂O₂ levels were moderately reduced in the AgNPs-treated plants. The decrease in the oxidant content may be partly due to a substantial reduction in the activity of SOD, which is a H₂O₂-producing enzyme. The stable total glutathione levels and a small increase in the ASC content may also contribute to reducing the H₂O₂ level in leaves. Therefore, the ascorbate is supposed to scavenge H₂O₂ in a non-enzymatic manner since no increase in the APX activity can be observed (Kunert and Foyer, 2023). Gupta et al. (2018) evaluated the stimulatory effects of AgNPs on rice seedlings and suggested that growth promotion was related to efficient ROS scavenging mechanisms, including changes in glutathione–ascorbate cycle and activities of involved enzymes (ascorbate peroxidase and glutathione reductase) in leaves. Contrary to our results, authors have suggested low alterations of SOD activity in response to AgNPs treatment. Soliman et al. (2020) reported an increase in ascorbate and glutathione contents in leaves of Z. mays, A. cepa, and T. foenum-graecum, as well as an increase in expression levels and activities of antioxidant enzymes in AgNPs-treated seedlings. In turn, our results indicate that the H₂O₂-scavenging enzymes, namely, POX and CAT, differentially reacted to AgNPs treatment. A negligible increase in POX activity and a substantial decrease in CAT activity were observed. When compared to APX and other H₂O₂-scavenging enzymes, CAT has a low affinity to its substrate. Therefore, the enzyme is involved in H₂O₂ scavenging when oxidants are accumulated to high levels (Heck et al., 2010; Černý et al., 2018). Since the H₂O₂ content decreased under the AgNPs treatment, the amount of the enzyme may be adjusted accordingly by decreasing its abundance. Earlier studies demonstrated that CAT activity is dependent on the high availability of H₂O₂ (Scandalios et al., 1983; Rodríguez-Ruiz et al., 2019).

The growth of the AgNPs-treated plant roots was maintained at the control level. The H₂O₂ content was decreased in roots. This may be linked to the rise in the level of the H₂O₂-scavenging agent, namely, ascorbate. Furthermore, increased ascorbate content was accompanied by high activities of the H₂O₂-consuming enzymes, namely, POX and APX. At this stage of plant development, significant root growth is observed that is correlated with a wide range of physiological processes, such as auxin metabolism, cross-linking of cell wall components, or cell elongation, in which peroxidases are involved (Passardi et al., 2005; Majda and Robert, 2018). POX is a prevalent apoplast-targeted enzyme. However, the majority of ascorbate is located in the cytosol, where APX isoforms are present. Therefore, we believe that the increasing ASC levels in the roots of the AgNPs-treated plants may contribute to maintaining high APX activity, which in turn prevents excessive H₂O₂ accumulation. These mechanisms may be responsible for the reduction in H₂O₂ content in the roots of the AgNPs-treated plants. In contrast, decreased CAT activity and increased SOD activity may favor H₂O₂ accumulation (Kumari et al., 2021). Acting simultaneously, the aforementioned factors may contribute to H₂O₂ homeostasis in roots under the AgNPs treatment. In turn, Kim et al. (2023) observed improvements in shoot and root elongation of mung bean (Vigna radiata) after 12-hour seed treatment with biosynthesized AgNPs (at concentrations of 12.5, 25, and 50 ppm). In contrast to our findings, other authors suggested inhibition in root development after direct exposure of roots to AgNPs in soil or medium (Cvjetko et al., 2017). In the study reported by Guilger-Casagrande et al. (2022), the effect of soil exposure of soybean seedlings to AgNPs resulted in a reduction in dry weight. In addition, the increase in H₂O₂ and lipid peroxidation (higher MDA content) in seedling roots was observed. Considering the inconclusive results, further research is still required to understand the effects of AgNPs on root development in plants in view of different species and the conditions of NPs application.

The stimulatory effect of AgNPs treatments on maize was observed as the reduction in biomass of the caryopses. This was accompanied by a consistent decrease in H₂O₂ content and APX, CAT, and SOD activities. Simultaneously, the redox balance of ascorbate turned out to be further reduced, and the content of total glutathione substantially increased. The latter effect was more pronounced under treatments with higher AgNPs concentrations. The increase in total glutathione concentration significantly distinguished the caryopses from other organs. Glutathione is a versatile molecule, with many functions that go beyond common antioxidant roles. Glutathione is a source of sulfur for protein biosynthesis, plays a crucial role as an antioxidant, and represents the potential as a highly reducing chemical barrier to prevent over-oxidation of cellular components through its direct interaction with peroxides or as a substrate for ROS-neutralizing enzymes (Noctor et al., 2012). Since ASC levels were relatively low in caryopses, GSH is supposed to be a major low-molecular-weight antioxidant in this organ. Consequently, the reduction in H₂O₂ level in caryopses may be a consequence of AgNPs-dependent accumulation of GSH in this organ. It is suggested that the detoxification of Ag in plant cells is related to the direct bonding of Ag with GSH through the −SH group or utilization of GSH for increased production of phytochelatins involved in metal detoxification (Kaur et al., 2021; Larue et al., 2014). Enhanced antioxidant protection in the caryopses, due to AgNPs-stimulated GSH accumulation, may be important for mitigating the relatively high oxidative damage in this organ, as exemplified by high lipid peroxidation. However, the latter is not related to AgNPs treatment. It should be kept in mind that at this developmental stage, plant growth is still important and supported by seed reserves (Kennedy et al., 2004). Therefore, a complete analysis of the metabolic status of caryopses in a 2-week-old maize plant is required to interpret the behavior of redox agents analyzed in this study.

Our study shows that the activities of certain enzymes decreased at AgNPs treatment. Studies on plants overexpressing CAT, APX, or SOD have shown that these enzymes act synergistically to keep the homeostasis of cellular redox state, being increased or decreased in an orchestrated manner (Scandalios et al., 1983; Faize et al., 2011; Wang et al., 2017; Che et al., 2020). We suspect that the decrease in the enzyme activities, observed in all organs except the stem, may reflect their synchronized response to a decrease in H₂O₂ resulting from the AgNPs treatment. In stems, the increase in the H₂O₂ content was in line with the enhanced stem biomass gain and stem lengthening resulting from AgNPs administration. Therefore, we assume that it may be a growth-related process, involving rather an apoplast H₂O₂, whereas CAT is a cytosolic enzyme and may not directly respond to changing apoplast H₂O₂ levels (Majda and Robert, 2018; Marzol et al., 2022). In this organ, H₂O₂ homeostasis may be regulated by the ascorbate pool, which turned out to be more reduced when challenged with AgNPs.

To sum up, the possible mechanisms of AgNPs action in plantlets that are responsible for alterations in cellular redox metabolism during the AgNPs-dependent maize growth stimulation are presented in Figure 8 . The overall decrease in H₂O₂ accumulation in all organs (except stem) suggests that the efficiency of redox reactions has increased after grain treatments with AgNPs. The results indicate that specific plantlet organs showed a varying response and that the induced effect depended on the applied dose. The alterations of the determined oxidative stress parameters were stronger with the application of higher AgNPs concentrations to grains. However, our results show no evidence of the induction of severe oxidative stress by AgNPs in maize plantlets, as overproduction of ROS and its consequences due to the incapability of an antioxidative defense system for efficient ROS scavenging were not detected.