The three wheat varieties selected in this study were the high-gluten wheat variety Huamai1168, the medium-gluten wheat variety Huamai2152, and the low-gluten wheat variety WanXimai0638. These varieties are representative selenium-enriched wheat cultivars, screened through the application of a bio-nano-selenium nutrient solution in the wheat-growing areas of the middle and lower reaches of the Yangtze River in China (Huang S. et al., 2020). The bio-nano-selenium nutrient solution we used in the experiment was a patented product developed by our research team (Patent No. ZL201610338121.6), and the pure selenium content of this solution was ≥5000 mg L^-1, and the average particle size of nano-selenium was 126 ± 0.5 nm. The experimental site was located at the Nanhu Experimental Farm of Hubei Academy of Agricultural Sciences in Wuhan City, Hubei Province (114°18’ E, 30°29’ N, elevation 30 m), with a soil pH of 6.29.The average rainfall in 2022 was 1,042.3 mm, which belongs to the year of partial dryness, and the growing season of wheat is from November to May of the following year.

The experiment was conducted using a completely randomised block design, with selenium treatment and control groups for each variety, each replicated three times, for a total of 18 plots. The flowering period of each plot was determined when more than 50% of the wheat plants were in flower, and wheat spikes within the plots were tagged on the day of flowering, with 120 spikes tagged per plot. The selenium treatment group was foliar sprayed on the 4th day of wheat flowering, with 29 mL of bio-nano-selenium nutrient solution applied per plot, equivalent to 145 mg of pure selenium. This application was converted to 4,500 mL of bio-nano-selenium nutrient solution per hectare, providing 23,000 mg of pure selenium, at a concentration of 30 mg L^-1. The control group was sprayed with an equal amount of aqueous solution at the same time. Each plot had an area of 63 m² (25 m × 2.5 m), with a planting row spacing of 0.25 m. Other field management practices were consistent with the local wheat field management protocol.

On the 10th, 15th, 20th, 25th and 30th days after foliar spraying of the bio-nano-selenium nutrient solution, the saber leaves from five hanging spikes in each plot were collected. The samples were placed in ice packs immediately after sampling and stored at 4°C. The following parameters were measured: catalase (CAT) activity, peroxidase (POD) activity, superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, reduced glutathione (GSH) content, and proline (Pro) content. The assay methods are described in Supplementary Table S1 .

After full maturation of each material, 6 plants were randomly selected for harvesting, threshing and drying until the grain moisture content fell below 14%. Grain with pests and diseases were excluded. The morphological traits of the grain from each material were measured using the Wanshen SC-G Automatic Grain Analysis System. Measured traits included grain area (Ga, mm²), grain perimeter (Gp, mm), grain width (Gw, mm), grain length (Gl, mm), grain length-width ratio (Lwr), grain roundness (Gr) and grain diameter (Gd, mm), thousand grain weight (Tgw, g), and the factor from density (Ffd). The assay method is described in Supplementary Table S1 .

After the test materials fully matured, 10 plants were randomly selected from each experimental plot to examine their agronomic traits. Eight trait parameters were measured for each plant: plant height (PH, cm), number of effective spike (ES), Length of main spike (LMS, cm), panicle neck length of main spike (LFPMS, cm), rachis internode length of main spike (RLMS, cm), spikelets on main spike (SMS), number of spikelets per plant (NSPP), and number of grains per plant (GNP). The average of the 10 measured values was taken as the phenotypic value of each trait in the plot. The assay methods are described in Supplementary Table S1 .

Approximately 250 g of soil was collected at a depth of 20 cm below the surface in each plot planted with Huamai1168 using the 5-point sampling method. The samples were mixed well, dried, and then 500 g of soil was taken to examine the basic physical and chemical properties and total selenium content in the soil of both the selenium-treated and control groups. The basic physical and chemical properties of the soil were tested according to the method of Zhou et al. (2022). The total and organic selenium contents of wheat grains harvested from each plot were determined after wheat maturity. The total and organic selenium contents of spikes, leaves, stems and roots of Huamai1168 were also examined. The methods for determining total and organic selenium content are provided in Supplementary Table S1 . The biotransformation efficiency of selenium by wheat can be calculated by the following equation:

We used Origin version 9 to draw bar graphs and SPSS version 22.0 for LSD (Least-Significant Difference) test to evaluate the differences in the target traits among the varieties. Correlation analysis (Pearson correlation) and Mantel_test (a statistical method used to assess the correlation between two distance or similarity matrices) were performed by cor and mantel_test functions in R, and results were visualized by the ggplot2, linkET, and igraph packages in R 3.6.0.

As shown in Figure 3A , the CAT activity of all three wheat varieties gradually declined as wheat entered the filling stage. In each time period, the CAT activity in the selenium-treated group showed varying degrees of decline compared to the control group. Huamai1168 exhibited the largest decline, with an average of 22.55% over the five periods; Huamai2152 showed the second-largest decline, with an average decrease of 15.51%; and Wanximai0638 had the smallest decline, with an average decrease of 14.12%. The results of POD activity for the three wheat varieties are shown in Figure 3B , which demonstrated an overall trend of increase followed by a decrease. After exogenous selenium supplementation, POD activity increased by an average of 38.42% in Huamai1168, increased by 6.09% in Huamai2152, and decreased by5.26% in Wanximai0638 compared to the control. Figure 3C shows the results for SOD activity, where all three wheat varieties exhibited an increasing followed by a decreasing trend over the five periods. The SOD activities in the selenium-treated groups were higher than that in the control, in the order: Huamai1168 > Huamai2152 > Wanximai0638. MDA content gradually increased as wheat filling ( Figure 3D ), and was enhanced to varying degrees after selenium treatment. The pattern of change in MDA content was more consistent with SOD activity. Figure 3E presents the GSH content, which first increased and then decreased over the filling period. GSH content was enhanced after foliar selenium supplementation, and the enhancement effect was proportional to the gluten content of the wheat varieties. The results for proline content are shown in Figure 3F . The proline content of all three wheat varieties increased over the grouting period, with an overall improvement in proline content following bio-nano-selenium spraying. Among the varieties, Huamai2152 showed the most improvement, while Huamai1168 showed the least.

The results of agronomic traits examined in three wheat varieties after foliar spraying of the bio-nano-selenium nutrient solution are shown in Table 1 . After comparison, we found that bio-nano-selenium had positive effects on Plant height (PH), number of effective spike (ES), panicle neck length of the main spike (LFPMS), rachis internode length of main spike (RLMS) and number of spikelets per plant (NSPP). The greatest effects were observed on RLMS, ES and NSPP, which increased by 21.95%, 19.03% and 17.82%, respectively, while the effects on PH and LFPMS were smaller, with increased of 8.13% and 3.82%, respectively. Meanwhile, we found that bio-nano-selenium decreased three traits: length of main spike (LMS), spikelets on main spike (SMS), and grain number per plant (GNP), by 2.16%, 1.16% and 1.13%, respectively.

All three selenium content indicators of wheat grain were significantly and positively correlated with 10dCAT and 15dSOD. There were also negative correlations between morphological traits of the grain and antioxidant indicators in the leaves ( Figure 5 ). CAT activity was negatively correlated with POD activity, Pro content, SOD activity, MDA content, GSH content, and Lwr, while it was positively correlated with Gr. POD activity was positively correlated with SOD activity, MDA content, and GSH content, but negatively correlated with Tgw, Ga, Gw, Gd and Ffd. Pro content was generally negatively correlated with grain morphology. SOD activity was positively correlated with MDA content and GSH content, but negatively correlated with Tgw, Ga, Gw, Gd and Ffd. MDA content and GSH content were negatively correlated with traits related to seed morphology. Tgw was negatively correlated with Ga, but positively correlated with Ga, Gp, Gl, Gw, Gd and Ffd. Ga was positively correlated with Gp, Gl, Gw and Gd. Gp and Gl were positively correlated with Gd and Lwr, but negatively correlated with Gr. Gw was positively correlated with Ffd and Gd. lwr was significantly negatively correlated with Gr.

In our study, the selenium content of wheat was significantly and positively correlated with antioxidant enzyme activities (e.g., CAT, SOD) after exogenous selenium supplementation, suggesting that bio-nano-selenium application not only increased the selenium content of wheat but also enhanced its antioxidant defense system. However, it was also found that there may be a trade-off between selenium application and grain yield. It has been shown that selenium, as a cofactor of antioxidant enzymes, significantly enhances the antioxidant capacity of wheat and its tolerance to environmental stresses through multiple mechanisms, such as modulation of the antioxidant defense system (Alsamadany et al., 2023), enhancement of sulfur metabolism (Boldrin et al., 2016), lowering of peroxide levels (Elkelish et al., 2019), and modulation of relevant gene expression (Huang et al., 2023). These physiological mechanisms provide protection for wheat growth under adverse environmental conditions. However, during the reproductive growth stage of wheat, although selenium application enhances antioxidant enzyme activities, it may also alter the plant’s amino acid metabolism (Xiao et al., 2023), protein synthesis (Xia et al., 2020), and carbon and nitrogen metabolism (Zahedi et al., 2019), which in turn affects grain yield. Therefore, in the production of selenium-enriched wheat, rational control of the timing of selenium application is particularly important. Only by ensuring the enhancement of selenium content while optimizing both yield and quality can the best growth benefits be achieved (Wang et al., 2020).

Bio-nanomaterials, especially nano-selenium, significantly contribute to overall plant growth and development by promoting root growth, enhancing water and nutrient uptake, and boosting antioxidant capacity (Samynathan et al., 2023). In this study, we examined the selenium content in various parts of wheat after maturation and found that wheat absorbed selenium primarily through the roots. Selenium was mainly accumulated in the spikes, while the selenium content in the seeds was relatively low, with the selenium content in the spikes being 1.905 times higher than that in the seeds. After foliar exogenous supplementation with bio-nano-selenium, selenium was primarily accumulated in the leaves, and the selenium accumulation capacity in the seeds increased compared to the control. Specifically, the selenium content in the spikes was 2.048 times higher than that in the seeds, and the efficiency increased by 7.5%. We hypothesized that foliar spraying of selenium enables direct uptake by wheat leaves, with subsequent translocation to the seed kernels through the phloem (Xia et al., 2020), thus shortening the process of selenium uptake from the roots. In addition, bioactive molecules attached to the surface of nanoparticles, such as polysaccharides, peptides, and phytohormones, promoted root development by enhancing interactions with plant cells (Yuan et al., 2022). These bioactive molecules also helped the plant efficiently maintain the flow of water and nutrients by regulating ionic balance and osmotic adjustments within the cells, which in turn improved photosynthetic efficiency and the plant’s resilience (Rasheed et al., 2024). Nodes in wheat play an important role in the transport and distribution of mineral elements (Xiao et al., 2024), and foliar spraying of nano-selenium may further accelerate the transport of selenium from leaves to seeds by promoting the expression or activity of transporter proteins in the nodes. However, the specific mechanism of action still needs to be verified by further experiments.

In this study, we also preliminarily investigated the effects of foliar spraying of bio-nano-selenium on soil physicochemical indicators in wheat. Some studies have shown that the application of nano-selenium can improve soil quality (Samynathan et al., 2023), which is consistent with our findings. Bio-nano-selenium resulted in a significant increase in ammonium nitrogen (308.40%) and nitrate nitrogen (92.87%), which may be attributed to the fact that bio-nano-selenium enhances the nitrification capacity of the soil by promoting the growth and reproduction of microorganisms such as nitrifying bacteria, thus accelerating the conversion of ammonium nitrogen to nitrate nitrogen (Liu J. et al., 2021). Meanwhile, the pH of the selenium-treated group increased by 1.91%, which may be due to changes in microbial activities or the interaction of selenium with other soil constituents, altered the soil’s chemical composition (Zhou et al., 2020). Soil organic matter increased by 21.84%, which we speculate may be due to bio-nano-selenium promoting plant growth, positively influencing microbial activity and decomposition processes, and thus increasing the amount of organic matter in the soil (Liu et al., 2021). In addition, calcium and magnesium in the soil increased by 8.09% and 46.20%, respectively. We speculate that bio-nano-selenium increased the biomass and activity of wheat roots, which caused the root system to release more organic acids, dissolving more calcium and magnesium from the soil particles and increasing their availability in the soil (Samynathan et al., 2023). However, the exact mechanism needs further investigation. On the other hand, selenium application also led to decreases in some soil physicochemical indices. In our study, hydrolyzed nitrogen in the soil of selenium-treated group decreased by 4.35%, effective phosphorus decreased by 31.05%, and quick-acting potassium decreased by 6.48%. We hypothesize that this is because bio-nano-selenium promotes plant growth, increasing the plant’s demand for nitrogen, phosphorus, and potassium, which in turn reduces their content in the soil (Samynathan et al., 2023). SO₄> ^2- was reduced by 53.03%, which may be due to vigorous plant growth after selenium application, leading to increased uptake of SO₄> ^2-, thus reducing sulphate content in the soil (Samynathan et al., 2023). Alternatively,selenium application may stimulate the activity of soil microorganisms, particularly those involved in sulfur cycling, accelerating the conversion and metabolism of sulfur and consuming sulfate in the soil (Zhuang et al., 2024). The specific mechanism still requires further experimental verification.

Although foliar spraying of bio-nano selenium shortened the process of selenium uptake by wheat roots and enhanced seed uptake efficiency in this study, the translocation and uptake of bio-nano-selenium in wheat plants also affected soil physiological indices through the root system. In addition to promoting selenium uptake and transformation in wheat, bio-nano-selenium also improved the uptake and utilization of other nutrients in the soil. This not only enhanced the biotransformation efficiency of selenium in wheat and its effective accumulation in the plant, but also had a positive overall effect on the healthy growth of wheat.