Nanosilica increases tolerance to Cu toxicity applied to soybean seeds by modulating pigment increase and N nutritional efficiency and inhibiting Cu absorption

Nanosilica increases tolerance to Cu toxicity applied to soybean seeds by modulating pigment increase and N nutritional efficiency and inhibiting Cu absorption | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Nanosilica increases tolerance to Cu toxicity applied to soybean seeds by modulating pigment increase and N nutritional efficiency and inhibiting Cu absorption Patrícia Messais Ferreira, Luan Mori, Renato de Mello Prado, Gelza Carliane Marques Teixeira, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8843881/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 10 You are reading this latest preprint version Abstract The treatment of soybean seeds with copper (Cu) can induce toxicity in seedlings depending on the applied dose and seed storage time after treatment. Silicon (Si) has the potential to mitigate Cu toxicity; however, the underlying mechanisms remain poorly understood. This study provides the first evaluation of soybean responses to toxic Cu doses in seed treatment and investigates whether nanosilica application via fertigation after sowing can alleviate this toxicity. Two experiments were conducted under controlled conditions using soybean grown in Oxisol. The first experiment assessed the nutritional responses of seedlings subjected to seed treatment with increasing Cu doses (0, 1, 4, 7, 15, and 29 g kg⁻¹ of seed as CuO) combined with two storage periods (48 and 96 h). The second experiment evaluated the same Cu doses in combination with the absence or presence of Si (2 mmol L⁻¹) applied via fertigation. Seed treatment with Cu was feasible up to 4 g kg⁻¹ when seed storage did not exceed 48 h. Higher Cu doses induced oxidative stress and reduced nitrogen use efficiency. Nanosilica application mitigated Cu toxicity at doses up to 7.4 g kg⁻¹ by reducing Cu accumulation in shoots and oxidative stress. This study establishes reference limits for Cu seed treatment and strategies to prevent toxicity in soybean. Biological sciences/Biotechnology Physical sciences/Nanoscience and technology Biological sciences/Plant sciences micronutrient oxidative stress heavy metal abiotic stress Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction In recent years, seed treatment with micronutrients has significantly advanced in soybean cultivation, showing greater potential for elements with low plant demand, such as copper [ 1 ]. This application method offers advantages, including better application uniformity, improved nutrient uptake by plants, and lower application costs [ 2 ], ultimately benefiting plant growth [ 3 ]. However, research on seed treatment with Cu in soybean remains limited. While seed treatment before sowing is now common, the maximum storage duration of Cu-treated seeds without posing toxicity risks to soybean seedlings and the optimal Cu dose remain unknown. This is particularly relevant because annual crops at the seedling stage are highly sensitive to micronutrient toxicity, including copper [ 4 ]. One commonly used Cu source is copper oxide, which dissolves in acidic environments [ 5 ], offering the advantage of slow micronutrient release. Despite Cu’s importance for plant protein synthesis, which enhances photosynthesis [ 6 ] and biological nitrogen fixation (BNF) in legumes [ 7 – 9 ]. Cu is a highly toxic element [ 10 ] that induces lipid peroxidation and cellular electrolyte leakage [ 11 ], increasing membrane permeability [ 12 ]. It can also reduce the concentration of antioxidant compounds, such as phenols [ 13 ], thereby exacerbating oxidative stress. This stress is further aggravated by structural damage to chloroplasts, particularly at the thylakoid level [ 14 ], reducing chlorophyll synthesis [ 15 , 16 ], electron flow, and photosystem II (PSII) quantum efficiency [ 17 ]. Additionally, excess Cu in plants can reduce nitrogen content in maize leaves and roots [ 18 ] by inhibiting NO₃ uptake and upward translocation, modulating NO₃ transporter gene expression levels [ 19 ], nitrate reductase activity, and protein synthesis [ 20 ]. A promising strategy to mitigate the harmful effects of Cu toxicity in soybean plants is the use of silicon (Si). Si functions as a beneficial element in the development of many plant species [ 21 ] and is absorbed by dicotyledons through aquaporins [ 22 ]. Si plays a crucial role in metabolism, physiological activity, and structural integrity, particularly due to its antioxidant effects, which enhance plant tolerance to various stresses [ 23 ], including heavy metal toxicity [ 21 , 24 – 27 ]. In cucumber plants, Si has been shown to mitigate Cu toxicity [ 28 ]. One key mechanism is the co-deposition of Si with Cu in the cell wall, where Cu binds to phenolic compounds [ 29 ], reducing its intracellular bioavailability [ 30 – 32 ]. Additionally, Cu compartmentalization in root vacuoles may prevent its translocation to more sensitive tissues, such as young leaves [ 33 ]. Another crucial benefit of Si is its ability to enhance root nodulation in legumes [ 34 – 36 ]. One mechanism that plants use to tolerate Cu is the complexation with nitrogen compounds, such as glutathione/cysteine, in the cytosol of root and leaf cells [ 37 ] and in the cell wall [ 38 ]. In this context, it is crucial that Si acts quickly to counteract potential Cu toxicity damage in plants that received micronutrients through seed treatment. This effect can be achieved with innovative sources such as nano-silica, which may enhance its rapid absorption by plants. This occurs because the particles of this source are extremely small, within the nanometric scale (1 to 100 nm) [ 39 ], and can provide superior agronomic performance compared to conventional Si sources [ 40 ]. The effects of Si in mitigating heavy metal toxicity are well known [ 41 ] unlike in soybean plants that received Cu via seed treatment. It is believed that the most promising strategy for quickly mitigating this stress, especially in the seedling stage of soybeans, which is sensitive to any stress, would be the use of Si. This potential of Si is relevant because it has been reported that in just 72 hours after the application of Si, there are clear effects on the mitigation of stress (water deficit) seen in young sugarcane plants [ 42 ]. The challenge of research on Cu seed treatment must progress to ensure that this technology can meet the nutritional demands of soybean cultivation in the field without posing toxicity risks. In this context, we propose the following hypotheses: i) The risk of toxicity to soybean seeds with Cu depends on the dose of Cu mixed into the seed and the storage time after application of the micronutrient. ii) Soybean at the early growth stage is highly sensitive to Cu toxicity, as it induces oxidative stress that degrades photosynthetic pigments and is also expected to impair N uptake and its utilization in plant metabolism. iii) The application of Si via fertigation after soybean sowing, in the form of nano-silica, has an immediate effect in enhancing Cu toxicity tolerance by increasing the Cu dose threshold for safe seed treatment. This study aimed to evaluate Cu doses in seed treatment, different seed storage durations, and whether nano-silica application via fertigation after sowing mitigates Cu toxicity in soybean plants, as well as the mechanisms involved. If confirmed, these hypotheses will pave the way for optimizing Cu seed treatment in terms of storage time and ideal Cu dose while also expanding the use of nano-silica-based nanotechnology via fertigation in soybean cultivation. This approach would mitigate potential Cu toxicity risks after sowing without harming the environment and consequently prevent reductions in plant population in the field. This has practical relevance, as uneven soybean plant distribution in the field can reduce soybean yields (Matsuo et al., 2018). 2. Results Cu doses and storage time affected Cu accumulation in soybean seedlings (p < 0.01). Cu accumulation in shoots and roots increased according to a quadratic polynomial fit at both times (48 and 96 h after seed treatment) (Fig. 1 a, c, e). The maximum Cu accumulation was 1,461.7, 717, and 2,039.5 mg per plant in shoots, roots and the whole plant, respectively, corresponding to the doses of Cu 17.6, 32.4, and 20.2 g kg -1 seed for soybean plants whose seeds were treated 48 h before planting (Fig. 1 a, c, e). For seeds treated 98 h before planting, the maximum Cu accumulation was 1,417, 1,212, and 2,679 mg per plant at Cu doses 18.5, 19.1, and 18.8 g kg -1 seeds in shoots, roots and the whole plant, respectively. Only the single factor Cu dose (D) was significant (p < 0.01) for shoot and root dry mass (Fig. 1 b, d,). There was a quadratic polynomial fit at both treatment duration times, except for shoot dry mass at 96 h, which followed a decreasing linear fit (Fig. 1 d). The maximum dry matter accumulation was 3 and 0.8 g at Cu 4.15 and 13.9 g kg -1 seed doses in shoots and roots, respectively, that received seed treatment 48 h before planting. In the treatment with 96 h before planting, the highest mass was 0.6 g in roots, corresponding to the dose of Cu 10.12 g kg -1 seeds (Fig. 1 d) The dry mass accumulation of the whole plant depended on Cu doses and storage time (Fig. 1 f). Dry mass accumulation increased in plants that received seed treatment 48 h before planting and followed a quadratic polynomial fit, showing a maximum accumulation of 3.4 g at the Cu dose 1.96 g kg -1 seeds. Plants that received seed treatment 96 h before planting showed a decrease in dry mass, following a decreasing linear fit. The accumulation of Cu in the plant depends on the doses of Cu and Si ( p < 0.01). The maximum accumulation of Cu in shoots was 61.4 and 25.5 mg per plant, corresponding to the doses of Cu 24.8 and 15.2 g kg -1 seeds, in soybean plants grown in the absence and presence of Si, respectively (Fig. 2 a). In roots, the maximum Cu accumulation corresponds to 64 and 44.6 mg per plant at the doses of Cu 29.1 and 17.9 g kg -1 seed in the absence and presence of Si, respectively (Fig. 2 c). In the whole plant, the maximum Cu accumulation was 119.5 and 68.8, corresponding to the doses of Cu 23.1 and 17.6 g kg -1 seed, in plants grown without and with Si fertigation, respectively. The interaction between the factors (Si × D) was significant for Si accumulation in shoots and the whole plant (p < 0.01); for roots, only Si was significant (p < 0.01). Si accumulation increased in shoots, roots, and the whole plant following a quadratic polynomial fit when plants were fertigated with Si. In the Si-free condition, no mathematical model was significant for shoots (y = 7.1), roots (y = 7.2), and the whole plant (y = 14.2). The maximum Si accumulation in shoots, roots, and the whole plant was 13.2, 10.5, and 23.8 mg per plant, corresponding to the doses of Cu 13, 14.6, and 13.5 g kg -1 seed, respectively (Fig. 2 b, d, f). The rate of electrolyte leakage in the plant depends on the dose of Cu and Si (p < 0.01). There was an increasing linear fit in the presence of Si, while for the absence of Si there was a decreasing linear fit (Fig. 3 a). For phenolic compounds, only the isolated factors Si and Cu dose were significant (both p < 0.01). There was a quadratic polynomial fit for the absence of Si, while in the presence of Si there was no mathematical fit (y = 27.7). The supply of Si via fertigation favored a greater increase of phenolic compounds in soy plants independent of the dose studied. On the other hand, in the absence of Si, there was a decrease in the production of these compounds. The maximum phenolic compounds produced in the absence of Si was 24 g EAG 100 g -1 at the Cu dose equivalent to 7 g kg -1 seeds (Fig. 3 b). Only the isolated effect of Si was significant for nodule number (p < 0.01) (Fig. 3 c). Both Si conditions showed no mathematical fit for nodule number (y = 41 and y = 60) in the absence and presence of Si, respectively. Si supply promoted an increase in nodule number by 46% compared to the absence of Si (Fig. 4 a). The isolated factors of Si (p < 0.01) and Cu doses (p < 0.01) were significant for N accumulation in shoots, roots, and in crude protein content. There was a quadratic polynomial fit for Si conditions, except for N accumulation in shoots in the -Si condition, which followed a decreasing linear fit (Fig. 3 d-f). The maximum N accumulation in soybean plants grown under Si fertigation was 38.4, 13.8, and 52.4 mg per plant in shoots and roots, corresponding to the doses of Cu 11.3, 14.2, and 12.4 g kg -1 seeds, respectively. In the absence of Si, the maximum N accumulation was 11 and 43.3 at the doses of Cu 11.5 and 12.4 g kg -1 seeds in roots and the whole plant, respectively (Fig. 3 d,e). The protein content in the whole plant had a quadratic fit with Cu doses. In the presence of Si, the maximum content was 79.8 g kg -1 and in the absence it was 68.1 g kg -1 at the Cu doses 16 and 12.4 g kg -1 seeds, respectively (Fig. 3 f). Cu uptake and translocation efficiency depended on Cu and Si doses (p < 0.01). Cu uptake efficiency showed a quadratic polynomial fit in the absence and presence of Si (Fig. 4 a, c). In the presence of Si, the maximum Cu absorption efficiency was 0.05 g g -1 , and the translocation efficiency was 36% observed at the Cu concentrations 18.8 and 14.2 g kg -1 seed, respectively. In the absence of Si, the maximum absorption efficiency was lower: 0.1 g g -1 , obtained at a higher dose of Cu of 32.25 g kg -1 of seed. The maximum use efficiency, on the other hand, was higher (64.3%), but still at a higher Cu dose of 18 g kg -1 seed compared to the + Si condition. Only the isolated effects of Si and Cu doses showed significant effects for N uptake efficiency (both p < 0.01) (Fig. 4 b) and N translocation efficiency – Si (p < 0.01) and Cu dose (p < 0.05) (Fig. 4 d). For N use efficiency, only the single factor Cu dose (p < 0.01) showed a significant effect (Fig. 4 f). There was a quadratic polynomial fit for N uptake efficiency in + Si and -Si conditions for translocation efficiency in -Si condition and for N use in + Si condition. Also for N translocation efficiency, no mathematical model fitted the + Si condition (y = 74). The N use efficiency in the absence of Si followed a decreasing linear fit. The maximum N uptake efficiency was 0.03 and 0.04 g g -1 at the Cu dose 12.5 and 12.8 g kg -1 seeds dose in both Si conditions (-Si and + Si, respectively). The maximum N translocation efficiency was 71.2%, corresponding to a Cu concentration of 9.1 g kg -1 seed in -Si. The maximum N use efficiency in the presence of Si was 383 g g -2 , corresponding to Cu dose of 10.7 g kg -1 seeds. The shoot dry mass (p < 0.01) depended on Cu doses and Si supply (Fig. 4 e). There was a quadratic polynomial fit in the presence of Si and a decreasing linear fit in the absence of Si. The maximum shoot dry matter production, 2.7 g, corresponded to the doses of Cu of 7.4 g kg -1 seed, respectively. This section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn. 3. Discussion 3.1 Unraveling the Risk of Cu Toxicity in Soybean Seedlings as a Function of Cu Doses via Seed Treatment and Seed Storage Duration The application of Cu via seeds resulted in an increase in the accumulation of Cu both in the seedling phase and in the initial growth of soybean plants. This shows that the strategy of treating the seeds before sowing the soybeans is efficient to supply them in the initial growth phase of the crop, thus avoiding the deficiency of this micronutrient. Therefore, copper oxide applied to soybean seeds can quickly release Cu, allowing the plant to absorb it even at the seedling stage probably because of the small particle size (3 µm) of the source. However, the use of high doses of Cu in seed treatment, even in the form of oxide, can more easily induce losses by toxicity due to the increased accumulation and efficiency of Cu absorption by the plant. This Cu absorption occurs even if there are outer layers of seed teguments composed of highly lignified and impermeable cells and cuticles [ 43 ] [ 44 ] [ 45 ], which hinders nutrient absorption. Translocation efficiency, which represents the amount of nutrient transported to the shoot, decreased as higher amounts of Cu were applied. This effect can be explained by the limited capacity of plants to remobilize Cu from roots to shoots [ 1 ]. However, the use efficiency of these elements decreased with the increase in the doses supplied via seeds because, as there is an increase in the availability of the element, the capacity of use by plants tends to decrease [ 46 ]. This study shows for the first time the sensitivity of soybean crops to excess Cu via seeds, especially at doses above 4 g kg -1 seed. Furthermore, increasing the storage time of Cu-treated seeds from 48 h to 96 h increased Cu toxicity in soybean seedlings, as it enhanced Cu uptake by the seedlings. This raises a still unknown warning: to avoid storing seeds treated with CuO for periods longer than 48 h, requiring that this procedure of seed fertilization be done only at the moment of sowing. We clearly observe that excess Cu in soybean without a Si supply greatly impaired N in the plant, decreasing N accumulation and the number of nodules in relation to the efficiency of uptake and transport of Cu in the plant at all Cu doses. However, the greatest damage occurred in the use efficiency of N in the plant. There are even reports that excess Cu in the plant can decrease the N content in the leaf and root of corn [ 18 ] by inhibiting NO 3 uptake and upward translocation, modulating the expression level of NO 3 transporter genes [ 19 ] and impairing N metabolism by decreasing the expression of genes encoding nitrate reductase [ 20 ]. We reveal here that Cu toxicity in plants from seeds treated with Cu impairs the growth of soybean plants from different mechanisms that occur simultaneously with excess Cu in leaves and with impaired N uptake and metabolism. These effects combined potentiate strong plant stress. These effects of excess Cu in seeds were accompanied by an increase in oxidative stress, which was indicated by high rates of electrolyte leakage index due to a decrease in antioxidant compounds such as phenolic compounds, reflecting in the decrease of plant dry mass. In general, the hypothesis can be accepted:(i) Soybean seed treatment with copper oxide may pose a toxicity risk to seedlings, which can be minimized with an appropriate Cu dose and seed storage duration. We found that Cu seed treatment is viable as long as the storage period does not exceed 48 hours and Cu doses remain up to 4 g kg⁻¹. Beyond these limits, seedling growth is compromised, particularly in terms of shoot biomass accumulation. 3.2 Mechanisms of Si in Mitigating Cu Toxicity Induced by High Cu Doses in Seed Treatment The objective of this research is to propose a new strategy using Si to mitigate the harmful effects of Cu toxicity in soybean plants that have undergone Cu seed treatment. This is important because the first study revealed that soybean is sensitive to Cu toxicity caused by seed treatment with this micronutrient (> 4 g kg⁻¹ Cu) when Si is not supplied. However, with Si application, plants may tolerate higher Cu doses, though further research is needed to confirm this potential benefit. This beneficial element is known to mitigate different biotic [ 47 ] [ 48 ] or abiotic stresses [ 49 ] [ 50 ]. However, there is no information on the immediate effects of Si in mitigating seed-applied Cu toxicity in any species. The challenge for Si to mitigate this type of stress is having to exert an immediate effect from seedling emergence until the first weeks of initial crop growth. For this, with the emergence of innovative sources using nanotechnology, such as nanosilica, and due to small particle diameter of less than 10 nm and high specific surface area (300 m 2 g -1 ), it can result in rapid absorption, favoring its immediate benefits in plants. There are even reports that Si can mitigate stress in short periods of 72 h as seen in sugarcane plants under water deficit [ 51 ] using conventional sources; however, if using nanosilica, the expectation of relatively rapid effects of Si on plants increases. Such expectations of beneficial effects of Si mitigating Cu toxicity were confirmed in soybean plants when high doses of Cu were supplied via seeds from different mechanisms. Si provided a decrease in Cu uptake and translocation from root to shoots in soybean. This can be justified by the effects of Si in decreasing apoplasmic metal transport [ 52 ]. Yet, the deposition of Si in the proximity of the endodermis may decrease the porosity of the cell wall of inner root tissues, forming a physical barrier that decreases the concentration of Cu in the xylem, [ 27 ]. In addition, Si can decrease the uptake of metals such as Cu in plants and decrease the transfer of Cu in their tissues [ 52 ] [ 53 ] [ 54 ] by stimulating the production of root exudates, which can chelate metals [ 55 ] or even cause complexation and polymerization of Si with metals such as Cu [ 56 ] [ 57 ] in a form that may be less toxic [ 58 ] [ 59 ]. Another plant strategy for complexing Cu in the plant aiming to increase tolerance to Cu is its complexation with nitrogen compounds, such as glutathione and cysteine, both in roots and leaves, in the cellular cytosol [ 60 ] [ 37 ] and in the cell wall [ 38 ] This possible effect occurred because Si favored increased N accumulation in root and shoot parts of the plant due to improved N uptake efficiency and consequently increased translocation of this nutrient to plant shoots. N was metabolized, increasing crude protein in the plant. This effect of Si on N uptake and translocation may be associated with its effect on increasing the expression of efficient nutrient transporters in the plant [ 32 ]. Another explanation for the increase in plant N, although of lesser magnitude, is the possible effect of Si on the improvement of BNF, which was observed through an increase in the number of nodules mediated by Si in relation to its absence at most doses of Cu in the seed treatment. This effects of Si in increasing the number of nodules were previously evidenced in cowpea plants [ 61 ] alfalfa [ 35 ] and soybean [ 62 ] This effect is attributed to increased flavonoid production in the nodule, favoring root nodulation [ 36 ], but further research is important for a better understanding of these effects. Therefore, the effects of Si in complexing Cu in the plant aiming to increase tolerance to Cu toxicity by the different mechanisms commented above decreases the accumulation of free Cu at toxic levels in shoots of soy plants, a fact also verified in wheat plants [ 27 ]. It will therefore reflect in the reduction of oxidative stress [ 63 ]. This is because excess Cu in leaves induces lipid peroxidation [ 11 ] and intense proton extrusion into the cells with a markedly increased membrane permeability [ 12 ]. This causes structural damage to thylakoids [ 14 ] and impairs plant physiological processes. Therefore, Si decreasing excessive Cu accumulation in leaves has an important contribution to indirectly prevent oxidative stress. There are other direct effects of Si in decreasing oxidative stress by an antioxidant action, promoting increased synthesis of antioxidant compounds such as phenols [ 64 ] or even due to an increased activity of antioxidant enzymes decreasing the production of reactive oxygen species (ROS) [ 53 ][ 65 ], inhibiting lipid peroxidation [ 66 ], and avoiding degradation of cell compounds. This direct antioxidant effect of Si in decreasing the damage by Cu toxicity occurred because there was an increase in phenolic compounds at all Cu doses and at the same time there was a decrease in cell electrolyte leakage. The study found that the soybean crop is sensitive to Cu toxicity when supplied via seed treatment, providing evidence that hypothesis (ii) can be accepted. That is, soybean at the early growth stage is sensitive to Cu toxicity because it induces oxidative stress, leading to the degradation of photosynthetic pigments. Additionally, Cu toxicity is expected to impair N uptake and its utilization in the plant's metabolism. However, in irrigated areas, fertigation with Si (2 mmol L -1 ) can be used after crop emergence to mitigate metal toxicity at Cu doses up to 7,4 g kg -1 seeds because Si induces different mechanisms for decreasing shoot Cu content and oxidative stress. It is observed that hypothesis (iii) can be accepted: "The use of Si via fertigation after soybean sowing in the form of nano-silica has an important immediate effect in enhancing tolerance to Cu toxicity by increasing the Cu dose threshold for seed treatment". In addition, it was indicated for the first time that the mechanism of action of Si to relieve the Cu toxicity by a mechanism that starts by in-creasing the uptake of N and its use in the metabolism and at the same time decreases the uptake and root-shoot transport of Cu, avoiding reaching the leaves, which are a more sensitive organ to stress; consequently, tissues with low Cu content will reflect in the reduction of oxidative stress. Future perspectives are that further studies on nutritional metabolomics understand the effects of Si on genes expressing Cu and N transporters in the root in Cu toxic soybean plants. 4. Material and Methods 4.1 Plant Material and Growth Conditions Two experiments were conducted at the State University of São Paulo (Unesp), Campus of Jaboticabal, São Paulo, Brazil, from January to February 2022. Soybean seeds not treated with fungicides and insecticides of the cultivar AS 3590 IPRO developed by Brasmax and widely cultivated in Brazil, were used. In soybean seeds, the mass of 1.000 seeds (300 g) and germination rate (95%) were determined following the procedures described by the Rules for Seed Analysis [ 67 ]. The first experiment was conducted under laboratory conditions. The average temperature was 25°C and the relative humidity was 45%. In the second experiment, the plants were kept in a controlled environment (greenhouse) for five days after emergence and then transferred to a full sun environment to increase the incidence of light. During the experimental period, meteorological data were recorded daily using a digital thermo-hygrometer (U23-001, Sigma Sensors, Brazil): air temperature (average maximum of 31 ± 5ºC and minimum of 20 ± 2°C) and air humidity (average maximum of 88 ± 3% and minimum of 39 ± 6%). 4.2 Experimental design and treatments 4.2.1 Experiment I The experimental design was entirely randomized in a 6 x 2 factorial design of six doses of Cu (0, 1, 4, 7, 15, and 29 g kg -1 of seed, equivalent to 0, 65, 260, 455, 975, and 1,885 g ha -1 of Cu considering 65 kg of seeds per ha) combined with two storage periods of seeds (48 h and 96 h). After treatment, the seeds were placed in paper bags and stored in ambient conditions (air temperature: 25°C; relative humidity: 45%). Cu doses were applied via seeds using cupric oxide (Cu: 342.5 g L -1 and particle size = 3 µm (D90)). At the dose 0 g kg -1 of Cu, the seeds were treated only with 1.5 ml deionized water (same volume as the suspensions with copper oxide). Polypropylene containers with a capacity of 1 dm 3 were filled with textured sand medium previously washed under running water, HCl solution (0.3%, v:v), and deionized water. In each container, 50 seeds of each treatment were sown. This constituted an experimental plot with three replications. Plots were irrigated daily with deionized water until the sand saturation point. 4.2.2 Experiment II The treatments consisted of a 6 x 2 factorial design of with six doses (D) of Cu (0, 1, 4, 7, 15, and 29 g kg -1 of seed) combined with the presence (+ Si) and absence (-Si) of Si, with five replications arranged in completely randomized blocks. Seeds treated with Cu were stored for 24 h in paper bags in environmental conditions (25°C, 45% RH) and then sown. The Si supply was given using as source nanosilica (Bindzil 830, particle diameter 8.5–9.7 nm, 168.3 g L -1 of Si, specific surface area: 300 m 2 g -1 , pH: 10.5, density: 1.2 g cm -3 , Na 2 O: 0.5%, viscosity: 7Cp) from the company AkzoNobel ® . In the + Si condition, a concentration of 2 mmol L -1 was applied via fertigation simulating a 2.5-mm blade on the pot surface. The application of Si was performed daily, starting five days after full emergence until the end of the experiment. The pH value of the solution was adjusted to 5.5 ± 0.2 with hydrochloric acid or sodium hydroxide 1.0 mol L -1 as required. Eight soybean seeds were sown in each pot. Five days after emergence, the seeds were thinned, removing all shoots of the seedling along with cotyledons, leaving only two plants per pot, which constituted one experimental unit. At sowing, an inoculant containing Bradyrhizobium elkanii strains (Biomax ® premium) at a concentration of 3.6 mL L -1 was applied in sowing furrows with a volume corresponding to 3 ml of solution per pot. The plants were irrigated daily, maintaining 70% of the water retention capacity of the soil by the method of weighing the pots, as [ 68 ] proposed. Polypropylene containers with a capacity of 2 dm 3 were used. They were filled with 1.5 dm 3 of soil samples classified as Entisol [ 69 ]. Soil chemical analysis was performed for fertility purposes according to the method [ 70 ] described. The results were as follows: pH CaCl 2 = 5; organic matter = 11 g dm -3 ; P resin = 11 mg dm -3 ; S = 18 mg dm -3 ; B < 0.12 mg dm -3 ; Cu = 0.2 mg dm -3 ; Fe = 9 mg dm -3 ; Mn = 1.7 mg dm -3 ; Ca = 11 mmolc dm -3 ; Mg = 6 mmolc dm -3 ; K = 1.9 mmolc dm -3 ; Al = 0 mmolc dm -3 ; H + Al= 20 mmolc dm -3 ; BS (base sum) (Ca + Mg+K) = 18.5 mmolc dm -3 ; CEC (cation exchange capacity) (SB + H+Al) = 38.1 mmolc dm -3 ; and V (base saturation) (V=SBx100/CTC) = 49%. The Si content was 3 mg dm -3 , as determined by the method described by [ 71 ]. Soil particle size was 540 g kg -1 sand, 380 g kg -1 clay, and 90 g kg -1 silt [ 72 ]. Calcined limestone (Relative Total Neutralizing Power = 125%; CaO = 58.5%; MgO = 9%) was incorporated to raise the base saturation to 50% and provide calcium. Phosphorus fertilization was performed at a dose of 100 mg dm -3 of P using simple superphosphate as source. The remaining nutrients were supplied via fertigation. Potassium (K), boron (B), zinc (Zn), and manganese (Mn) were applied at doses of 150, 1, 4, and 6 mg dm -3 of soil, respectively. The sources of these nutrients were potassium chloride, boric acid, zinc sulfate, and manganese sulfate, respectively. Fe was supplied via leaf application using iron chelate as source at a concentration of 0.15 g L -1 15 days after sowing. 4.3 Variables analyzed 4.3.1 Electrolyte leakage index The electrolyte leakage index was determined using five leaf discs (129 mm² each) taken from the middle third of the first fully developed trefoil. The discs were placed in a beaker containing 20 mL of deionized water at room temperature for 2 h. After this period, the initial electrical conductivity (EC1) was measured using a conductivity meter (AK51, Akso, Brazil). Subsequently, the samples were placed in an autoclave at 121°C for 20 min and, after cooling, a new reading of electrical conductivity was taken to determine the final electrical conductivity (EC2). Extravasation was estimated using the equation proposed by [ 73 ]: EC1/EC2 × 100. 4.3.2 Determination of total phenol levels The levels of total phenols were determined according to the method proposed by [ 74 ]. For extraction, 0.1 g of fresh leaves (leaf discs) were collected and transferred to 15-mL falcon tubes wrapped with aluminum foil. 2 mL of concentrated methanol were added and left to stand for three hours in the dark. Afterwards, the samples were filtered and 3 mL of methanol solution were added. For the colorimetric reaction, 1 mL of the filtered extract was collected and transferred to a 15-mL falcon tube wrapped in aluminum foil. The volume was completed by adding 10 mL of deionized water and 0.5 mL of Folin-Ciocauteau 2 N. The solution was kept at rest for three minutes, and 1.5 mL of 20% sodium carbonate was added and kept at rest for two hours. After this time, the absorbance of samples was read with a spectrophotometer (765 nm). 4.3.3 Dry mass production The plants were collected and partitioned into shoots and root, washed in tap water, detergent solution (0.1% v/v), hydrochloric acid solution (0.3% v/v), and deionized water. Afterwards, they were placed in paper bags and dried in an oven with forced air circulation (65 ± 5°C) until reaching a constant mass. After drying, the material was weighed on an analytical scale to obtain dry mass. 4.3.4 Number of nodules The number of nodules was determined after the collection of plants by removing the nodules present on the roots and counting them manually. 4.3.5 Analysis of silicon (Si) and copper (Cu) Si contents in shoots and roots were determined according to the method described by [ 75 ], by wet digestion and by the addition of hydrogen peroxide (H 2 O 2 ) and sodium hydroxide (NaOH). The reaction was induced in an oven at 120°C for four hours. The determination of Si content was performed in a spectrophotometer at 410 nm according to the colorimetric method described by [ 71 ]. The Cu content was determined from dry samples previously ground in a Wiley mill following the methodology of [ 76 ]. Based on the contents and dry mass, the accumulation of Cu in shoots and roots of plants was calculated. 4.3.6 Determination of total nitrogen (N) and crude protein content The N content was determined by adding concentrated sulfuric acid to samples of previously dried and ground plant material, followed by distillation and titration with sulfuric acid [ 76 ], and then the crude protein (CP) contents [ 77 ]. 4.3.7 Cu and N uptake, transport efficiency, and N utilization Cu and N nutrient efficiencies of plants were obtained using Cu and N accumulation data and plant dry mass, following the indications of [ 78 ]. For this, formulas were used to calculate the uptake efficiency (whole plant accumulation/root mass), transport ((aboveground accumulation/whole plant accumulation) x 100), and use ((whole plant dry mass) 2 /whole plant accumulation). 4.4 Statistical Analysis Data were submitted to analysis of variance by F test. Qualitative data were compared by Student t test ( p ≤ 0.05). Quantitative data were analyzed using linear or polynomial regression, choosing the significant model with the highest coefficient of determination and the AgroEstat ® statistical program [ 79 ]. 5. Conclusions Seed treatment with Cu oxide causes toxicity in soybean plants in the seedling stage and during initial growth at doses greater than 4 g kg -1 , especially if the seed is stored for longer than 48 hours. It was found that the use of Si via fertigation, applied five days after soybean plant emergence, can mitigate Cu toxicity in soybean plants that underwent seed treatment with doses of up to 7.4 g kg⁻¹ Cu. Si-nanosilica mitigated Cu toxicity in soybean plants by decreasing the efficiency of absorption and transport of Cu to the shoot and at the same time favored the increase in flux and efficiency of N absorption and protein content in the plant. plant and consequently attenuated the oxidative stress given the decrease in the extravasation of cellular electrolytes. This research established, for the first time, standards for copper seed treatment in soybean plants and strategies to prevent toxicity risks. Declarations Conflicts of Interest The authors declare that they have no conflict of interest. Funding This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001 (Author benefited PMF). The founders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. There was no additional external funding received for this study. Author Contribution Conceptualization, P.M.F. and G.C.M.T.; methodology, P.M.F., L.M., G.C.M.T. and R.M.P.; validation, P.M.F. and G.C.M.T; formal analysis, P.M.F., L.M; investigation, P.M.F., L.M; resources, R.M.P.; data curation, P.M.F., L.M and G.C.M.T.; writing—original draft preparation, P.M.F. R.M.P and G.C.M.T; writing—review and editing; visualization, P.M.F. and G.C.M.T.; supervision, R.M.P.; project administration, R.M.P.; funding acquisition, R.M.P and M.A.N.D. All authors have read and agreed to the published version of the manuscript. Data Availability All relevant data are within the paper and its Supporting Information files. References de Prado, M. R. Mineral nutrition of tropical plants. Mineral nutrition of tropical plants (2021). https://doi.org/10.1007/978-3-030-71262-4 doi:10.1007/978-3-030-71262-4. Conceição, G. M. et al. Mineral supplementation of soybean seeds with different initial nutrient levels. Acta Sci. Agron. 42 , e42484 (2020). Galrão, E. Z. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8843881","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":614394334,"identity":"2afcf52c-8b83-45a7-a8af-5e343ea0a8be","order_by":0,"name":"Patrícia Messais Ferreira","email":"data:image/png;base64,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","orcid":"","institution":"São Paulo State University","correspondingAuthor":true,"prefix":"","firstName":"Patrícia","middleName":"Messais","lastName":"Ferreira","suffix":""},{"id":614394335,"identity":"b2199a1c-236c-4ca5-8097-18b901a3eb9b","order_by":1,"name":"Luan Mori","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Luan","middleName":"","lastName":"Mori","suffix":""},{"id":614394336,"identity":"6466c9f4-1b7b-4f34-b704-cc4d500e54ba","order_by":2,"name":"Renato de Mello Prado","email":"","orcid":"","institution":"São Paulo State University","correspondingAuthor":false,"prefix":"","firstName":"Renato","middleName":"de Mello","lastName":"Prado","suffix":""},{"id":614394337,"identity":"6831f504-f907-4221-8505-fdf69efc1d26","order_by":3,"name":"Gelza Carliane Marques Teixeira","email":"","orcid":"","institution":"Federal University of Lavras","correspondingAuthor":false,"prefix":"","firstName":"Gelza","middleName":"Carliane Marques","lastName":"Teixeira","suffix":""},{"id":614394338,"identity":"a56d6060-f240-4754-8241-16fd363cab9a","order_by":4,"name":"Marcos Altomani Neves Dias","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Marcos","middleName":"Altomani Neves","lastName":"Dias","suffix":""}],"badges":[],"createdAt":"2026-02-10 17:27:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8843881/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8843881/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105827716,"identity":"f0f3b6c9-5410-4282-8e1d-088d348e27f6","added_by":"auto","created_at":"2026-03-31 14:14:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":222316,"visible":true,"origin":"","legend":"\u003cp\u003eCu accumulation in shoots (a), roots (c), and the whole plant (e), dry mass in shoots (b), root (d), and whole plant (f) of soybean plants grown under doses of Cu: 0, 1, 4, 7, 15, and 29 g kg\u003csup\u003e-1\u003c/sup\u003e of seed. ** and *: Significant at p\u0026lt;0.01. and p\u0026lt;0.05, respectively; ns: not significant by F test. Same letters do not differ between seeds treated 48 and 96 h before planting by Student t test at p\u0026lt;0.05 probability.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8843881/v1/7d0fab353e9151c5b10547a6.png"},{"id":105827709,"identity":"75d4b6cd-b8e9-4bf9-80a9-50f1eb4f95b2","added_by":"auto","created_at":"2026-03-31 14:14:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":217040,"visible":true,"origin":"","legend":"\u003cp\u003eCu accumulation in shoots (a), roots (c), and whole plant (e), Si accumulation in shoots (b), roots (d), and the whole plant (f) in soybean plants grown under Cu doses: 0, 1, 4, 7, 15, and 29 (g kg\u003csup\u003e-1\u003c/sup\u003e seed) in the presence (+Si) and absence (-Si) of Si. ** and *: Significant at p\u0026lt;0.01 and p\u0026lt;0.05, respectively; ns: not significant by F test. Same letters do not differ between the presence and absence of Si by Student t test at \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 probability.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8843881/v1/2d4b5005d08c4086e65ded21.png"},{"id":105827700,"identity":"7c527bba-2aec-47df-aa84-9ec0a00868b2","added_by":"auto","created_at":"2026-03-31 14:13:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":41873,"visible":true,"origin":"","legend":"\u003cp\u003eElectrolyte extravasation index (a), phenolic compounds (b), number of nodules (c), N accumulation in shoots (d), roots (e) and protein content in the whole plant (f) in soybean plants grown with Cu doses 0, 1, 4, 7, 15, and 29 g kg\u003csup\u003e-1\u003c/sup\u003e seed in the presence (+Si) and absence (-Si) of Si. ** and *: Significant at p\u0026lt;0.01 and p\u0026lt;0.05 probability, respectively; ns: not significant by F test. Same letters do not differ between the presence and absence of Si by Student t test at p\u0026lt;0.05 probability.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8843881/v1/0c25268eea5f8cf11622dce2.png"},{"id":105827707,"identity":"04ab4548-b06a-47f7-8b4c-3a139b02c2b5","added_by":"auto","created_at":"2026-03-31 14:13:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":210727,"visible":true,"origin":"","legend":"\u003cp\u003eCu uptake and translocation efficiency (a, c), N uptake, translocation and use efficiency (b, d, f), and shoot dry mass (e) of soybean plants grown under Cu doses 0, 1, 4, 7, 15, and 29 g kg\u003csup\u003e-1\u003c/sup\u003e seed in the presence (+Si) and absence (-Si) of Si. ** and *: Significant at p\u0026lt;0.01 and p\u0026lt;0.05 probability, respectively; ns: not significant by F test. Sam letters do not differentiate between the presence and absence of Si by Student t test at p\u0026lt;0.05 probability.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8843881/v1/4228af93f9569dbae5d96a43.png"},{"id":105904503,"identity":"5461afbc-b763-4ce2-bf0b-f2ddbafdc81b","added_by":"auto","created_at":"2026-04-01 10:09:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2437928,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8843881/v1/3099501d-e0f5-49b4-b7d8-d8855760753b.pdf"},{"id":105827697,"identity":"f9a94a1b-f845-4413-9f41-90b8e7146ed6","added_by":"auto","created_at":"2026-03-31 14:13:55","extension":"rar","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":25592,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Materials: \u003c/strong\u003eS1 File. Supporting information data. This file contains all data of the experiment.\u003c/p\u003e","description":"","filename":"datascience.rar","url":"https://assets-eu.researchsquare.com/files/rs-8843881/v1/60227189bcd4124d8ac1e005.rar"}],"financialInterests":"No competing interests reported.","formattedTitle":"Nanosilica increases tolerance to Cu toxicity applied to soybean seeds by modulating pigment increase and N nutritional efficiency and inhibiting Cu absorption","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIn recent years, seed treatment with micronutrients has significantly advanced in soybean cultivation, showing greater potential for elements with low plant demand, such as copper [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This application method offers advantages, including better application uniformity, improved nutrient uptake by plants, and lower application costs [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], ultimately benefiting plant growth [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, research on seed treatment with Cu in soybean remains limited. While seed treatment before sowing is now common, the maximum storage duration of Cu-treated seeds without posing toxicity risks to soybean seedlings and the optimal Cu dose remain unknown. This is particularly relevant because annual crops at the seedling stage are highly sensitive to micronutrient toxicity, including copper [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOne commonly used Cu source is copper oxide, which dissolves in acidic environments [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], offering the advantage of slow micronutrient release. Despite Cu\u0026rsquo;s importance for plant protein synthesis, which enhances photosynthesis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and biological nitrogen fixation (BNF) in legumes [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCu is a highly toxic element [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] that induces lipid peroxidation and cellular electrolyte leakage [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], increasing membrane permeability [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. It can also reduce the concentration of antioxidant compounds, such as phenols [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], thereby exacerbating oxidative stress. This stress is further aggravated by structural damage to chloroplasts, particularly at the thylakoid level [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], reducing chlorophyll synthesis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], electron flow, and photosystem II (PSII) quantum efficiency [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Additionally, excess Cu in plants can reduce nitrogen content in maize leaves and roots [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] by inhibiting NO₃ uptake and upward translocation, modulating NO₃ transporter gene expression levels [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], nitrate reductase activity, and protein synthesis [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA promising strategy to mitigate the harmful effects of Cu toxicity in soybean plants is the use of silicon (Si). Si functions as a beneficial element in the development of many plant species [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and is absorbed by dicotyledons through aquaporins [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Si plays a crucial role in metabolism, physiological activity, and structural integrity, particularly due to its antioxidant effects, which enhance plant tolerance to various stresses [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], including heavy metal toxicity [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In cucumber plants, Si has been shown to mitigate Cu toxicity [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. One key mechanism is the co-deposition of Si with Cu in the cell wall, where Cu binds to phenolic compounds [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], reducing its intracellular bioavailability [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Additionally, Cu compartmentalization in root vacuoles may prevent its translocation to more sensitive tissues, such as young leaves [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Another crucial benefit of Si is its ability to enhance root nodulation in legumes [\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. One mechanism that plants use to tolerate Cu is the complexation with nitrogen compounds, such as glutathione/cysteine, in the cytosol of root and leaf cells [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] and in the cell wall [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this context, it is crucial that Si acts quickly to counteract potential Cu toxicity damage in plants that received micronutrients through seed treatment. This effect can be achieved with innovative sources such as nano-silica, which may enhance its rapid absorption by plants. This occurs because the particles of this source are extremely small, within the nanometric scale (1 to 100 nm) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], and can provide superior agronomic performance compared to conventional Si sources [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe effects of Si in mitigating heavy metal toxicity are well known [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] unlike in soybean plants that received Cu via seed treatment. It is believed that the most promising strategy for quickly mitigating this stress, especially in the seedling stage of soybeans, which is sensitive to any stress, would be the use of Si. This potential of Si is relevant because it has been reported that in just 72 hours after the application of Si, there are clear effects on the mitigation of stress (water deficit) seen in young sugarcane plants [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe challenge of research on Cu seed treatment must progress to ensure that this technology can meet the nutritional demands of soybean cultivation in the field without posing toxicity risks. In this context, we propose the following hypotheses: i) The risk of toxicity to soybean seeds with Cu depends on the dose of Cu mixed into the seed and the storage time after application of the micronutrient. ii) Soybean at the early growth stage is highly sensitive to Cu toxicity, as it induces oxidative stress that degrades photosynthetic pigments and is also expected to impair N uptake and its utilization in plant metabolism. iii) The application of Si via fertigation after soybean sowing, in the form of nano-silica, has an immediate effect in enhancing Cu toxicity tolerance by increasing the Cu dose threshold for safe seed treatment. This study aimed to evaluate Cu doses in seed treatment, different seed storage durations, and whether nano-silica application via fertigation after sowing mitigates Cu toxicity in soybean plants, as well as the mechanisms involved. If confirmed, these hypotheses will pave the way for optimizing Cu seed treatment in terms of storage time and ideal Cu dose while also expanding the use of nano-silica-based nanotechnology via fertigation in soybean cultivation. This approach would mitigate potential Cu toxicity risks after sowing without harming the environment and consequently prevent reductions in plant population in the field. This has practical relevance, as uneven soybean plant distribution in the field can reduce soybean yields (Matsuo et al., 2018).\u003c/p\u003e"},{"header":"2. Results","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eCu doses and storage time affected Cu accumulation in soybean seedlings (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Cu accumulation in shoots and roots increased according to a quadratic polynomial fit at both times (48 and 96 h after seed treatment) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, c, e).\u003c/p\u003e\u003cp\u003eThe maximum Cu accumulation was 1,461.7, 717, and 2,039.5 mg per plant in shoots, roots and the whole plant, respectively, corresponding to the doses of Cu 17.6, 32.4, and 20.2 g kg\u003csup\u003e-1\u003c/sup\u003e seed for soybean plants whose seeds were treated 48 h before planting (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, c, e). For seeds treated 98 h before planting, the maximum Cu accumulation was 1,417, 1,212, and 2,679 mg per plant at Cu doses 18.5, 19.1, and 18.8 g kg\u003csup\u003e-1\u003c/sup\u003e seeds in shoots, roots and the whole plant, respectively.\u003c/p\u003e\u003cp\u003eOnly the single factor Cu dose (D) was significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) for shoot and root dry mass (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, d,). There was a quadratic polynomial fit at both treatment duration times, except for shoot dry mass at 96 h, which followed a decreasing linear fit (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed).\u003c/p\u003e\u003cp\u003eThe maximum dry matter accumulation was 3 and 0.8 g at Cu 4.15 and 13.9 g kg\u003csup\u003e-1\u003c/sup\u003e seed doses in shoots and roots, respectively, that received seed treatment 48 h before planting. In the treatment with 96 h before planting, the highest mass was 0.6 g in roots, corresponding to the dose of Cu 10.12 g kg\u003csup\u003e-1\u003c/sup\u003e seeds (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed)\u003c/p\u003e\u003cp\u003eThe dry mass accumulation of the whole plant depended on Cu doses and storage time (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). Dry mass accumulation increased in plants that received seed treatment 48 h before planting and followed a quadratic polynomial fit, showing a maximum accumulation of 3.4 g at the Cu dose 1.96 g kg\u003csup\u003e-1\u003c/sup\u003e seeds. Plants that received seed treatment 96 h before planting showed a decrease in dry mass, following a decreasing linear fit.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe accumulation of Cu in the plant depends on the doses of Cu and Si (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The maximum accumulation of Cu in shoots was 61.4 and 25.5 mg per plant, corresponding to the doses of Cu 24.8 and 15.2 g kg\u003csup\u003e-1\u003c/sup\u003e seeds, in soybean plants grown in the absence and presence of Si, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). In roots, the maximum Cu accumulation corresponds to 64 and 44.6 mg per plant at the doses of Cu 29.1 and 17.9 g kg\u003csup\u003e-1\u003c/sup\u003e seed in the absence and presence of Si, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). In the whole plant, the maximum Cu accumulation was 119.5 and 68.8, corresponding to the doses of Cu 23.1 and 17.6 g kg\u003csup\u003e-1\u003c/sup\u003e seed, in plants grown without and with Si fertigation, respectively.\u003c/p\u003e \u003cp\u003eThe interaction between the factors (Si \u0026times; D) was significant for Si accumulation in shoots and the whole plant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01); for roots, only Si was significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Si accumulation increased in shoots, roots, and the whole plant following a quadratic polynomial fit when plants were fertigated with Si. In the Si-free condition, no mathematical model was significant for shoots (y\u0026thinsp;=\u0026thinsp;7.1), roots (y\u0026thinsp;=\u0026thinsp;7.2), and the whole plant (y\u0026thinsp;=\u0026thinsp;14.2). The maximum Si accumulation in shoots, roots, and the whole plant was 13.2, 10.5, and 23.8 mg per plant, corresponding to the doses of Cu 13, 14.6, and 13.5 g kg\u003csup\u003e-1\u003c/sup\u003e seed, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, d, f).\u003c/p\u003e \u003cp\u003eThe rate of electrolyte leakage in the plant depends on the dose of Cu and Si (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). There was an increasing linear fit in the presence of Si, while for the absence of Si there was a decreasing linear fit (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFor phenolic compounds, only the isolated factors Si and Cu dose were significant (both p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). There was a quadratic polynomial fit for the absence of Si, while in the presence of Si there was no mathematical fit (y\u0026thinsp;=\u0026thinsp;27.7). The supply of Si via fertigation favored a greater increase of phenolic compounds in soy plants independent of the dose studied. On the other hand, in the absence of Si, there was a decrease in the production of these compounds. The maximum phenolic compounds produced in the absence of Si was 24 g EAG 100 g\u003csup\u003e-1\u003c/sup\u003e at the Cu dose equivalent to 7 g kg\u003csup\u003e-1\u003c/sup\u003e seeds (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eOnly the isolated effect of Si was significant for nodule number (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Both Si conditions showed no mathematical fit for nodule number (y\u0026thinsp;=\u0026thinsp;41 and y\u0026thinsp;=\u0026thinsp;60) in the absence and presence of Si, respectively. Si supply promoted an increase in nodule number by 46% compared to the absence of Si (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eThe isolated factors of Si (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and Cu doses (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) were significant for N accumulation in shoots, roots, and in crude protein content. There was a quadratic polynomial fit for Si conditions, except for N accumulation in shoots in the -Si condition, which followed a decreasing linear fit (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed-f).\u003c/p\u003e \u003cp\u003eThe maximum N accumulation in soybean plants grown under Si fertigation was 38.4, 13.8, and 52.4 mg per plant in shoots and roots, corresponding to the doses of Cu 11.3, 14.2, and 12.4 g kg\u003csup\u003e-1\u003c/sup\u003e seeds, respectively. In the absence of Si, the maximum N accumulation was 11 and 43.3 at the doses of Cu 11.5 and 12.4 g kg\u003csup\u003e-1\u003c/sup\u003e seeds in roots and the whole plant, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed,e).\u003c/p\u003e \u003cp\u003eThe protein content in the whole plant had a quadratic fit with Cu doses. In the presence of Si, the maximum content was 79.8 g kg\u003csup\u003e-1\u003c/sup\u003e and in the absence it was 68.1 g kg\u003csup\u003e-1\u003c/sup\u003e at the Cu doses 16 and 12.4 g kg\u003csup\u003e-1\u003c/sup\u003e seeds, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eCu uptake and translocation efficiency depended on Cu and Si doses (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Cu uptake efficiency showed a quadratic polynomial fit in the absence and presence of Si (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, c). In the presence of Si, the maximum Cu absorption efficiency was 0.05 g g\u003csup\u003e-1\u003c/sup\u003e, and the translocation efficiency was 36% observed at the Cu concentrations 18.8 and 14.2 g kg\u003csup\u003e-1\u003c/sup\u003e seed, respectively. In the absence of Si, the maximum absorption efficiency was lower: 0.1 g g\u003csup\u003e-1\u003c/sup\u003e, obtained at a higher dose of Cu of 32.25 g kg\u003csup\u003e-1\u003c/sup\u003e of seed. The maximum use efficiency, on the other hand, was higher (64.3%), but still at a higher Cu dose of 18 g kg\u003csup\u003e-1\u003c/sup\u003e seed compared to the +\u0026thinsp;Si condition.\u003c/p\u003e \u003cp\u003eOnly the isolated effects of Si and Cu doses showed significant effects for N uptake efficiency (both p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb) and N translocation efficiency \u0026ndash; Si (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and Cu dose (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). For N use efficiency, only the single factor Cu dose (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) showed a significant effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). There was a quadratic polynomial fit for N uptake efficiency in +\u0026thinsp;Si and -Si conditions for translocation efficiency in -Si condition and for N use in +\u0026thinsp;Si condition.\u003c/p\u003e \u003cp\u003eAlso for N translocation efficiency, no mathematical model fitted the +\u0026thinsp;Si condition (y\u0026thinsp;=\u0026thinsp;74). The N use efficiency in the absence of Si followed a decreasing linear fit. The maximum N uptake efficiency was 0.03 and 0.04 g g\u003csup\u003e-1\u003c/sup\u003e at the Cu dose 12.5 and 12.8 g kg\u003csup\u003e-1\u003c/sup\u003e seeds dose in both Si conditions (-Si and +\u0026thinsp;Si, respectively). The maximum N translocation efficiency was 71.2%, corresponding to a Cu concentration of 9.1 g kg\u003csup\u003e-1\u003c/sup\u003e seed in -Si. The maximum N use efficiency in the presence of Si was 383 g g\u003csup\u003e-2\u003c/sup\u003e, corresponding to Cu dose of 10.7 g kg\u003csup\u003e-1\u003c/sup\u003e seeds.\u003c/p\u003e \u003cp\u003eThe shoot dry mass (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) depended on Cu doses and Si supply (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). There was a quadratic polynomial fit in the presence of Si and a decreasing linear fit in the absence of Si. The maximum shoot dry matter production, 2.7 g, corresponded to the doses of Cu of 7.4 g kg\u003csup\u003e-1\u003c/sup\u003e seed, respectively.\u003c/p\u003e \u003cp\u003eThis section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"3. Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e \u003cb\u003e3.1 Unraveling the Risk of Cu Toxicity in Soybean Seedlings as a Function of Cu Doses via Seed Treatment and Seed Storage Duration\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe application of Cu via seeds resulted in an increase in the accumulation of Cu both in the seedling phase and in the initial growth of soybean plants. This shows that the strategy of treating the seeds before sowing the soybeans is efficient to supply them in the initial growth phase of the crop, thus avoiding the deficiency of this micronutrient. Therefore, copper oxide applied to soybean seeds can quickly release Cu, allowing the plant to absorb it even at the seedling stage probably because of the small particle size (3 \u0026micro;m) of the source.\u003c/p\u003e \u003cp\u003eHowever, the use of high doses of Cu in seed treatment, even in the form of oxide, can more easily induce losses by toxicity due to the increased accumulation and efficiency of Cu absorption by the plant. This Cu absorption occurs even if there are outer layers of seed teguments composed of highly lignified and impermeable cells and cuticles [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], which hinders nutrient absorption.\u003c/p\u003e \u003cp\u003eTranslocation efficiency, which represents the amount of nutrient transported to the shoot, decreased as higher amounts of Cu were applied. This effect can be explained by the limited capacity of plants to remobilize Cu from roots to shoots [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. However, the use efficiency of these elements decreased with the increase in the doses supplied via seeds because, as there is an increase in the availability of the element, the capacity of use by plants tends to decrease [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study shows for the first time the sensitivity of soybean crops to excess Cu via seeds, especially at doses above 4 g kg\u003csup\u003e-1\u003c/sup\u003e seed. Furthermore, increasing the storage time of Cu-treated seeds from 48 h to 96 h increased Cu toxicity in soybean seedlings, as it enhanced Cu uptake by the seedlings.\u003c/p\u003e \u003cp\u003eThis raises a still unknown warning: to avoid storing seeds treated with CuO for periods longer than 48 h, requiring that this procedure of seed fertilization be done only at the moment of sowing.\u003c/p\u003e \u003cp\u003eWe clearly observe that excess Cu in soybean without a Si supply greatly impaired N in the plant, decreasing N accumulation and the number of nodules in relation to the efficiency of uptake and transport of Cu in the plant at all Cu doses. However, the greatest damage occurred in the use efficiency of N in the plant. There are even reports that excess Cu in the plant can decrease the N content in the leaf and root of corn [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] by inhibiting NO\u003csub\u003e3\u003c/sub\u003e uptake and upward translocation, modulating the expression level of NO\u003csub\u003e3\u003c/sub\u003e transporter genes [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and impairing N metabolism by decreasing the expression of genes encoding nitrate reductase [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe reveal here that Cu toxicity in plants from seeds treated with Cu impairs the growth of soybean plants from different mechanisms that occur simultaneously with excess Cu in leaves and with impaired N uptake and metabolism. These effects combined potentiate strong plant stress.\u003c/p\u003e \u003cp\u003eThese effects of excess Cu in seeds were accompanied by an increase in oxidative stress, which was indicated by high rates of electrolyte leakage index due to a decrease in antioxidant compounds such as phenolic compounds, reflecting in the decrease of plant dry mass.\u003c/p\u003e \u003cp\u003eIn general, the hypothesis can be accepted:(i) Soybean seed treatment with copper oxide may pose a toxicity risk to seedlings, which can be minimized with an appropriate Cu dose and seed storage duration. We found that Cu seed treatment is viable as long as the storage period does not exceed 48 hours and Cu doses remain up to 4 g kg⁻\u0026sup1;. Beyond these limits, seedling growth is compromised, particularly in terms of shoot biomass accumulation.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Mechanisms of Si in Mitigating Cu Toxicity Induced by High Cu Doses in Seed Treatment\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe objective of this research is to propose a new strategy using Si to mitigate the harmful effects of Cu toxicity in soybean plants that have undergone Cu seed treatment. This is important because the first study revealed that soybean is sensitive to Cu toxicity caused by seed treatment with this micronutrient (\u0026gt;\u0026thinsp;4 g kg⁻\u0026sup1; Cu) when Si is not supplied. However, with Si application, plants may tolerate higher Cu doses, though further research is needed to confirm this potential benefit.\u003c/p\u003e \u003cp\u003eThis beneficial element is known to mitigate different biotic [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] or abiotic stresses [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. However, there is no information on the immediate effects of Si in mitigating seed-applied Cu toxicity in any species.\u003c/p\u003e \u003cp\u003eThe challenge for Si to mitigate this type of stress is having to exert an immediate effect from seedling emergence until the first weeks of initial crop growth. For this, with the emergence of innovative sources using nanotechnology, such as nanosilica, and due to small particle diameter of less than 10 nm and high specific surface area (300 m\u003csup\u003e2\u003c/sup\u003e g\u003csup\u003e-1\u003c/sup\u003e), it can result in rapid absorption, favoring its immediate benefits in plants. There are even reports that Si can mitigate stress in short periods of 72 h as seen in sugarcane plants under water deficit [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] using conventional sources; however, if using nanosilica, the expectation of relatively rapid effects of Si on plants increases.\u003c/p\u003e \u003cp\u003eSuch expectations of beneficial effects of Si mitigating Cu toxicity were confirmed in soybean plants when high doses of Cu were supplied via seeds from different mechanisms. Si provided a decrease in Cu uptake and translocation from root to shoots in soybean. This can be justified by the effects of Si in decreasing apoplasmic metal transport [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Yet, the deposition of Si in the proximity of the endodermis may decrease the porosity of the cell wall of inner root tissues, forming a physical barrier that decreases the concentration of Cu in the xylem, [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition, Si can decrease the uptake of metals such as Cu in plants and decrease the transfer of Cu in their tissues [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] by stimulating the production of root exudates, which can chelate metals [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] or even cause complexation and polymerization of Si with metals such as Cu [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] in a form that may be less toxic [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e] [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnother plant strategy for complexing Cu in the plant aiming to increase tolerance to Cu is its complexation with nitrogen compounds, such as glutathione and cysteine, both in roots and leaves, in the cellular cytosol [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e] [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] and in the cell wall [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] This possible effect occurred because Si favored increased N accumulation in root and shoot parts of the plant due to improved N uptake efficiency and consequently increased translocation of this nutrient to plant shoots. N was metabolized, increasing crude protein in the plant. This effect of Si on N uptake and translocation may be associated with its effect on increasing the expression of efficient nutrient transporters in the plant [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnother explanation for the increase in plant N, although of lesser magnitude, is the possible effect of Si on the improvement of BNF, which was observed through an increase in the number of nodules mediated by Si in relation to its absence at most doses of Cu in the seed treatment. This effects of Si in increasing the number of nodules were previously evidenced in cowpea plants [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e] alfalfa [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] and soybean [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e] This effect is attributed to increased flavonoid production in the nodule, favoring root nodulation [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], but further research is important for a better understanding of these effects.\u003c/p\u003e \u003cp\u003eTherefore, the effects of Si in complexing Cu in the plant aiming to increase tolerance to Cu toxicity by the different mechanisms commented above decreases the accumulation of free Cu at toxic levels in shoots of soy plants, a fact also verified in wheat plants [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. It will therefore reflect in the reduction of oxidative stress [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. This is because excess Cu in leaves induces lipid peroxidation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and intense proton extrusion into the cells with a markedly increased membrane permeability [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This causes structural damage to thylakoids [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and impairs plant physiological processes. Therefore, Si decreasing excessive Cu accumulation in leaves has an important contribution to indirectly prevent oxidative stress.\u003c/p\u003e \u003cp\u003eThere are other direct effects of Si in decreasing oxidative stress by an antioxidant action, promoting increased synthesis of antioxidant compounds such as phenols [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e] or even due to an increased activity of antioxidant enzymes decreasing the production of reactive oxygen species (ROS) [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e][\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e], inhibiting lipid peroxidation [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e], and avoiding degradation of cell compounds. This direct antioxidant effect of Si in decreasing the damage by Cu toxicity occurred because there was an increase in phenolic compounds at all Cu doses and at the same time there was a decrease in cell electrolyte leakage.\u003c/p\u003e \u003cp\u003eThe study found that the soybean crop is sensitive to Cu toxicity when supplied via seed treatment, providing evidence that hypothesis (ii) can be accepted. That is, soybean at the early growth stage is sensitive to Cu toxicity because it induces oxidative stress, leading to the degradation of photosynthetic pigments. Additionally, Cu toxicity is expected to impair N uptake and its utilization in the plant's metabolism.\u003c/p\u003e \u003cp\u003eHowever, in irrigated areas, fertigation with Si (2 mmol L\u003csup\u003e-1\u003c/sup\u003e) can be used after crop emergence to mitigate metal toxicity at Cu doses up to 7,4 g kg\u003csup\u003e-1\u003c/sup\u003e seeds because Si induces different mechanisms for decreasing shoot Cu content and oxidative stress. It is observed that hypothesis (iii) can be accepted: \"The use of Si via fertigation after soybean sowing in the form of nano-silica has an important immediate effect in enhancing tolerance to Cu toxicity by increasing the Cu dose threshold for seed treatment\".\u003c/p\u003e \u003cp\u003eIn addition, it was indicated for the first time that the mechanism of action of Si to relieve the Cu toxicity by a mechanism that starts by in-creasing the uptake of N and its use in the metabolism and at the same time decreases the uptake and root-shoot transport of Cu, avoiding reaching the leaves, which are a more sensitive organ to stress; consequently, tissues with low Cu content will reflect in the reduction of oxidative stress.\u003c/p\u003e \u003cp\u003eFuture perspectives are that further studies on nutritional metabolomics understand the effects of Si on genes expressing Cu and N transporters in the root in Cu toxic soybean plants.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Material and Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Plant Material and Growth Conditions\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTwo experiments were conducted at the State University of S\u0026atilde;o Paulo (Unesp), Campus of Jaboticabal, S\u0026atilde;o Paulo, Brazil, from January to February 2022. Soybean seeds not treated with fungicides and insecticides of the cultivar AS 3590 IPRO developed by Brasmax and widely cultivated in Brazil, were used. In soybean seeds, the mass of 1.000 seeds (300 g) and germination rate (95%) were determined following the procedures described by the Rules for Seed Analysis [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe first experiment was conducted under laboratory conditions. The average temperature was 25\u0026deg;C and the relative humidity was 45%. In the second experiment, the plants were kept in a controlled environment (greenhouse) for five days after emergence and then transferred to a full sun environment to increase the incidence of light.\u003c/p\u003e \u003cp\u003eDuring the experimental period, meteorological data were recorded daily using a digital thermo-hygrometer (U23-001, Sigma Sensors, Brazil): air temperature (average maximum of 31\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u0026ordm;C and minimum of 20\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) and air humidity (average maximum of 88\u0026thinsp;\u0026plusmn;\u0026thinsp;3% and minimum of 39\u0026thinsp;\u0026plusmn;\u0026thinsp;6%).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Experimental design and treatments\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e4.2.1 Experiment I\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe experimental design was entirely randomized in a 6 x 2 factorial design of six doses of Cu (0, 1, 4, 7, 15, and 29 g kg\u003csup\u003e-1\u003c/sup\u003e of seed, equivalent to 0, 65, 260, 455, 975, and 1,885 g ha\u003csup\u003e-1\u003c/sup\u003e of Cu considering 65 kg of seeds per ha) combined with two storage periods of seeds (48 h and 96 h). After treatment, the seeds were placed in paper bags and stored in ambient conditions (air temperature: 25\u0026deg;C; relative humidity: 45%). Cu doses were applied via seeds using cupric oxide (Cu: 342.5 g L\u003csup\u003e-1\u003c/sup\u003e and particle size\u0026thinsp;=\u0026thinsp;3 \u0026micro;m (D90)). At the dose 0 g kg\u003csup\u003e-1\u003c/sup\u003e of Cu, the seeds were treated only with 1.5 ml deionized water (same volume as the suspensions with copper oxide).\u003c/p\u003e \u003cp\u003ePolypropylene containers with a capacity of 1 dm\u003csup\u003e3\u003c/sup\u003e were filled with textured sand medium previously washed under running water, HCl solution (0.3%, v:v), and deionized water. In each container, 50 seeds of each treatment were sown. This constituted an experimental plot with three replications. Plots were irrigated daily with deionized water until the sand saturation point.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e4.2.2 Experiment II\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe treatments consisted of a 6 x 2 factorial design of with six doses (D) of Cu (0, 1, 4, 7, 15, and 29 g kg\u003csup\u003e-1\u003c/sup\u003e of seed) combined with the presence (+\u0026thinsp;Si) and absence (-Si) of Si, with five replications arranged in completely randomized blocks. Seeds treated with Cu were stored for 24 h in paper bags in environmental conditions (25\u0026deg;C, 45% RH) and then sown.\u003c/p\u003e \u003cp\u003eThe Si supply was given using as source nanosilica (Bindzil 830, particle diameter 8.5\u0026ndash;9.7 nm, 168.3 g L\u003csup\u003e-1\u003c/sup\u003e of Si, specific surface area: 300 m\u003csup\u003e2\u003c/sup\u003e g\u003csup\u003e-1\u003c/sup\u003e, pH: 10.5, density: 1.2 g cm\u003csup\u003e-3\u003c/sup\u003e, Na\u003csub\u003e2\u003c/sub\u003eO: 0.5%, viscosity: 7Cp) from the company AkzoNobel\u003csup\u003e\u0026reg;\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn the +\u0026thinsp;Si condition, a concentration of 2 mmol L\u003csup\u003e-1\u003c/sup\u003e was applied via fertigation simulating a 2.5-mm blade on the pot surface. The application of Si was performed daily, starting five days after full emergence until the end of the experiment. The pH value of the solution was adjusted to 5.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 with hydrochloric acid or sodium hydroxide 1.0 mol L\u003csup\u003e-1\u003c/sup\u003e as required.\u003c/p\u003e \u003cp\u003eEight soybean seeds were sown in each pot. Five days after emergence, the seeds were thinned, removing all shoots of the seedling along with cotyledons, leaving only two plants per pot, which constituted one experimental unit.\u003c/p\u003e \u003cp\u003eAt sowing, an inoculant containing \u003cem\u003eBradyrhizobium elkanii\u003c/em\u003e strains (Biomax\u003csup\u003e\u0026reg;\u003c/sup\u003e premium) at a concentration of 3.6 mL L\u003csup\u003e-1\u003c/sup\u003e was applied in sowing furrows with a volume corresponding to 3 ml of solution per pot. The plants were irrigated daily, maintaining 70% of the water retention capacity of the soil by the method of weighing the pots, as [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e] proposed.\u003c/p\u003e \u003cp\u003ePolypropylene containers with a capacity of 2 dm\u003csup\u003e3\u003c/sup\u003e were used. They were filled with 1.5 dm\u003csup\u003e3\u003c/sup\u003e of soil samples classified as Entisol [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. Soil chemical analysis was performed for fertility purposes according to the method [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e] described. The results were as follows: pH CaCl\u003csub\u003e2\u003c/sub\u003e= 5; organic matter\u0026thinsp;=\u0026thinsp;11 g dm\u003csup\u003e-3\u003c/sup\u003e; P resin\u0026thinsp;=\u0026thinsp;11 mg dm\u003csup\u003e-3\u003c/sup\u003e; S\u0026thinsp;=\u0026thinsp;18 mg dm\u003csup\u003e-3\u003c/sup\u003e; B\u0026thinsp;\u0026lt;\u0026thinsp;0.12 mg dm\u003csup\u003e-3\u003c/sup\u003e ; Cu\u0026thinsp;=\u0026thinsp;0.2 mg dm\u003csup\u003e-3\u003c/sup\u003e; Fe\u0026thinsp;=\u0026thinsp;9 mg dm\u003csup\u003e-3\u003c/sup\u003e; Mn\u0026thinsp;=\u0026thinsp;1.7 mg dm\u003csup\u003e-3\u003c/sup\u003e ; Ca\u0026thinsp;=\u0026thinsp;11 mmolc dm\u003csup\u003e-3\u003c/sup\u003e; Mg\u0026thinsp;=\u0026thinsp;6 mmolc dm\u003csup\u003e-3\u003c/sup\u003e; K\u0026thinsp;=\u0026thinsp;1.9 mmolc dm\u003csup\u003e-3\u003c/sup\u003e; Al\u0026thinsp;=\u0026thinsp;0 mmolc dm\u003csup\u003e-3\u003c/sup\u003e; H\u0026thinsp;+\u0026thinsp;Al= 20 mmolc dm\u003csup\u003e-3\u003c/sup\u003e; BS (base sum) (Ca\u0026thinsp;+\u0026thinsp;Mg+K)\u0026thinsp;=\u0026thinsp;18.5 mmolc dm\u003csup\u003e-3\u003c/sup\u003e; CEC (cation exchange capacity) (SB\u0026thinsp;+\u0026thinsp;H+Al)\u0026thinsp;=\u0026thinsp;38.1 mmolc dm\u003csup\u003e-3\u003c/sup\u003e; and V (base saturation) (V=SBx100/CTC)\u0026thinsp;=\u0026thinsp;49%. The Si content was 3 mg dm\u003csup\u003e-3\u003c/sup\u003e, as determined by the method described by [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. Soil particle size was 540 g kg\u003csup\u003e-1\u003c/sup\u003e sand, 380 g kg\u003csup\u003e-1\u003c/sup\u003e clay, and 90 g kg\u003csup\u003e-1\u003c/sup\u003e silt [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCalcined limestone (Relative Total Neutralizing Power\u0026thinsp;=\u0026thinsp;125%; CaO\u0026thinsp;=\u0026thinsp;58.5%; MgO\u0026thinsp;=\u0026thinsp;9%) was incorporated to raise the base saturation to 50% and provide calcium. Phosphorus fertilization was performed at a dose of 100 mg dm\u003csup\u003e-3\u003c/sup\u003e of P using simple superphosphate as source.\u003c/p\u003e \u003cp\u003eThe remaining nutrients were supplied via fertigation. Potassium (K), boron (B), zinc (Zn), and manganese (Mn) were applied at doses of 150, 1, 4, and 6 mg dm\u003csup\u003e-3\u003c/sup\u003e of soil, respectively. The sources of these nutrients were potassium chloride, boric acid, zinc sulfate, and manganese sulfate, respectively. Fe was supplied via leaf application using iron chelate as source at a concentration of 0.15 g L\u003csup\u003e-1\u003c/sup\u003e 15 days after sowing.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Variables analyzed\u003c/h2\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e4.3.1 Electrolyte leakage index\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe electrolyte leakage index was determined using five leaf discs (129 mm\u0026sup2; each) taken from the middle third of the first fully developed trefoil. The discs were placed in a beaker containing 20 mL of deionized water at room temperature for 2 h. After this period, the initial electrical conductivity (EC1) was measured using a conductivity meter (AK51, Akso, Brazil). Subsequently, the samples were placed in an autoclave at 121\u0026deg;C for 20 min and, after cooling, a new reading of electrical conductivity was taken to determine the final electrical conductivity (EC2). Extravasation was estimated using the equation proposed by [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]: EC1/EC2 \u0026times; 100.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e4.3.2 Determination of total phenol levels\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe levels of total phenols were determined according to the method proposed by [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. For extraction, 0.1 g of fresh leaves (leaf discs) were collected and transferred to 15-mL falcon tubes wrapped with aluminum foil. 2 mL of concentrated methanol were added and left to stand for three hours in the dark. Afterwards, the samples were filtered and 3 mL of methanol solution were added. For the colorimetric reaction, 1 mL of the filtered extract was collected and transferred to a 15-mL falcon tube wrapped in aluminum foil. The volume was completed by adding 10 mL of deionized water and 0.5 mL of Folin-Ciocauteau 2 N. The solution was kept at rest for three minutes, and 1.5 mL of 20% sodium carbonate was added and kept at rest for two hours. After this time, the absorbance of samples was read with a spectrophotometer (765 nm).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e4.3.3 Dry mass production\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe plants were collected and partitioned into shoots and root, washed in tap water, detergent solution (0.1% v/v), hydrochloric acid solution (0.3% v/v), and deionized water. Afterwards, they were placed in paper bags and dried in an oven with forced air circulation (65\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u0026deg;C) until reaching a constant mass. After drying, the material was weighed on an analytical scale to obtain dry mass.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e4.3.4 Number of nodules\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe number of nodules was determined after the collection of plants by removing the nodules present on the roots and counting them manually.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e4.3.5 Analysis of silicon (Si) and copper (Cu)\u003c/h2\u003e \u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eSi contents in shoots and roots were determined according to the method described by [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e], by wet digestion and by the addition of hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) and sodium hydroxide (NaOH). The reaction was induced in an oven at 120\u0026deg;C for four hours. The determination of Si content was performed in a spectrophotometer at 410 nm according to the colorimetric method described by [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe Cu content was determined from dry samples previously ground in a Wiley mill following the methodology of [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. Based on the contents and dry mass, the accumulation of Cu in shoots and roots of plants was calculated.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e4.3.6 Determination of total nitrogen (N) and crude protein content\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe N content was determined by adding concentrated sulfuric acid to samples of previously dried and ground plant material, followed by distillation and titration with sulfuric acid [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e], and then the crude protein (CP) contents [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e4.3.7 Cu and N uptake, transport efficiency, and N utilization\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eCu and N nutrient efficiencies of plants were obtained using Cu and N accumulation data and plant dry mass, following the indications of [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. For this, formulas were used to calculate the uptake efficiency (whole plant accumulation/root mass), transport ((aboveground accumulation/whole plant accumulation) x 100), and use ((whole plant dry mass)\u003csup\u003e2\u003c/sup\u003e/whole plant accumulation).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e4.4 Statistical Analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eData were submitted to analysis of variance by F test. Qualitative data were compared by Student t test (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05). Quantitative data were analyzed using linear or polynomial regression, choosing the significant model with the highest coefficient of determination and the AgroEstat\u003csup\u003e\u0026reg;\u003c/sup\u003e statistical program [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eSeed treatment with Cu oxide causes toxicity in soybean plants in the seedling stage and during initial growth at doses greater than 4 g kg\u003csup\u003e-1\u003c/sup\u003e, especially if the seed is stored for longer than 48 hours.\u003c/p\u003e \u003cp\u003eIt was found that the use of Si via fertigation, applied five days after soybean plant emergence, can mitigate Cu toxicity in soybean plants that underwent seed treatment with doses of up to 7.4 g kg⁻\u0026sup1; Cu.\u003c/p\u003e \u003cp\u003eSi-nanosilica mitigated Cu toxicity in soybean plants by decreasing the efficiency of absorption and transport of Cu to the shoot and at the same time favored the increase in flux and efficiency of N absorption and protein content in the plant. plant and consequently attenuated the oxidative stress given the decrease in the extravasation of cellular electrolytes.\u003c/p\u003e \u003cp\u003eThis research established, for the first time, standards for copper seed treatment in soybean plants and strategies to prevent toxicity risks.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicts of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study was financed in part by the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior - Brasil (CAPES) - Finance Code 001 (Author benefited PMF). The founders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. There was no additional external funding received for this study.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization, P.M.F. and G.C.M.T.; methodology, P.M.F., L.M., G.C.M.T. and R.M.P.; validation, P.M.F. and G.C.M.T; formal analysis, P.M.F., L.M; investigation, P.M.F., L.M; resources, R.M.P.; data curation, P.M.F., L.M and G.C.M.T.; writing\u0026mdash;original draft preparation, P.M.F. R.M.P and G.C.M.T; writing\u0026mdash;review and editing; visualization, P.M.F. and G.C.M.T.; supervision, R.M.P.; project administration, R.M.P.; funding acquisition, R.M.P and M.A.N.D. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll relevant data are within the paper and its Supporting Information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ede Prado, M. R. Mineral nutrition of tropical plants. \u003cem\u003eMineral nutrition of tropical plants\u003c/em\u003e (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/978-3-030-71262-4\u003c/span\u003e\u003cspan address=\"10.1007/978-3-030-71262-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e doi:10.1007/978-3-030-71262-4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eConcei\u0026ccedil;\u0026atilde;o, G. M. et al. Mineral supplementation of soybean seeds with different initial nutrient levels. \u003cem\u003eActa Sci. 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Preprint at (2020).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"micronutrient, oxidative stress, heavy metal, abiotic stress","lastPublishedDoi":"10.21203/rs.3.rs-8843881/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8843881/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe treatment of soybean seeds with copper (Cu) can induce toxicity in seedlings depending on the applied dose and seed storage time after treatment. Silicon (Si) has the potential to mitigate Cu toxicity; however, the underlying mechanisms remain poorly understood. This study provides the first evaluation of soybean responses to toxic Cu doses in seed treatment and investigates whether nanosilica application via fertigation after sowing can alleviate this toxicity. Two experiments were conducted under controlled conditions using soybean grown in Oxisol. The first experiment assessed the nutritional responses of seedlings subjected to seed treatment with increasing Cu doses (0, 1, 4, 7, 15, and 29 g kg⁻\u0026sup1; of seed as CuO) combined with two storage periods (48 and 96 h). The second experiment evaluated the same Cu doses in combination with the absence or presence of Si (2 mmol L⁻\u0026sup1;) applied via fertigation. Seed treatment with Cu was feasible up to 4 g kg⁻\u0026sup1; when seed storage did not exceed 48 h. Higher Cu doses induced oxidative stress and reduced nitrogen use efficiency. Nanosilica application mitigated Cu toxicity at doses up to 7.4 g kg⁻\u0026sup1; by reducing Cu accumulation in shoots and oxidative stress. This study establishes reference limits for Cu seed treatment and strategies to prevent toxicity in soybean.\u003c/p\u003e","manuscriptTitle":"Nanosilica increases tolerance to Cu toxicity applied to soybean seeds by modulating pigment increase and N nutritional efficiency and inhibiting Cu absorption","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-31 14:13:34","doi":"10.21203/rs.3.rs-8843881/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-07T05:32:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-24T13:24:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"304916174067429410011994548074981459874","date":"2026-04-24T13:08:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-10T00:22:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"181785604079088324786486363716305144971","date":"2026-04-03T01:03:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-30T08:30:28+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-02-13T22:01:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-11T10:41:19+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-11T10:37:42+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-02-10T16:36:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ddad4370-b4ec-4767-a1a4-017f8d35895d","owner":[],"postedDate":"March 31st, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-07T05:32:44+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":65375833,"name":"Biological sciences/Biotechnology"},{"id":65375834,"name":"Physical sciences/Nanoscience and technology"},{"id":65375835,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2026-05-07T05:41:02+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-31 14:13:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8843881","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8843881","identity":"rs-8843881","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}