Silicon Nanoparticles Alter Soybean Physiology and Improve Nitrogen Fixation Potential Under Atmospheric Carbon Dioxide (CO₂)

Abstract

The interactive effects between nano-silicon dioxide (n-SiO₂) and elevated CO₂ (eCO₂; 645 ppm) on soybean physiology, nitrogen fixation, and nutrient dynamics under climate stress remain underexplored. This study elucidates their combined effects under ambient (aCO₂; 410 ppm) and eCO₂ conditions. eCO₂ + n-SiO₂ synergistically enhanced shoot length (30%), total chlorophyll (112.15%), and photosynthetic rate (103.23%), alongside improved stomatal conductance and intercellular CO₂ (17.19%), optimizing carbon assimilation. Nodulation efficiency increased, with nodule number and biomass rising by 48.3% and 53.6%, respectively, under eCO₂ + n-SiO₂ versus aCO₂. N-assimilation enzymes (nitrate reductase, nitrite reductase, glutamine synthetase, glutamate synthase) surged by 38.5–52.1%, enhancing nitrogen metabolism. Concurrently, phytohormones (16–21%) and antioxidant activities (15–22%) increased, reducing oxidative markers (18–22%), and bolstering stress resilience. Nutrient homeostasis improved, with P, K, Mg, Cu, Fe, Zn, and Mn elevating in roots (13–41%) and shoots (13–17%), except shoot Fe and Zn. These findings demonstrate that n-SiO₂ potentiates eCO₂-driven benefits, amplifying photosynthetic efficiency, nitrogen fixation, and stress adaptation through enhanced biochemical and nutrient regulation. This synergy underscores n-SiO₂ role in optimizing crop performance under future CO₂-rich climates, advocating nano-fertilizers as sustainable tools for climate-resilient agriculture.

1. Introduction

In soybean, elevated CO₂ can stimulate growth by increasing photosynthetic rates, but it also induces physiological and biochemical changes that may negatively impact seed quality. Research indicates that while carbohydrate accumulation may increase under elevated CO₂, nitrogen and mineral concentrations tend to decline due to reduced transpiration and altered nutrient uptake [1,2]. Additionally, interactions between elevated CO₂ and other environmental stressors, such as drought, can exacerbate these effects, leading to fluctuations in seed composition, including protein and fatty acid profiles [3,4]. Given these challenges, innovative strategies are required to balance the benefits of CO₂ fertilization with the need to maintain nutrient integrity in crops.

Nanotechnology has emerged as a transformative tool to enhance crop resilience under climatic stressors [5]. Previous studies have demonstrated the potential of nanomaterials (NMs) in enhancing plant responses under elevated CO₂ conditions. For instance, chitosan nanoparticles have been shown to upregulate carbon and nitrogen metabolism in soybean [6], while foliar application of CeO₂ nanoparticles improved growth and nutritional quality in Spinacia oleracea L. [7], highlighting their role in promoting food security under future climatic conditions. Similarly, Se-NMs enhanced nutrient concentrations and biomass in wheat, demonstrating their potential to optimize growth conditions under increased CO₂ levels [8]. Similarly, Silicon nanoparticles (n-SiO₂), in particular, exhibit unique physicochemical properties that improve plant water-use efficiency, nutrient uptake, and stress tolerance [9]. Silicon, though non-essential for most plants, amplifies defense mechanisms against biotic and abiotic stresses by reinforcing cell walls, modulating antioxidant systems, and enhancing photosynthetic efficiency [10]. In legumes, silicon supplementation strengthens root architecture, promotes nodulation, and stabilizes nitrogenase activity under drought and salinity [11]. However, the interplay between n-SiO₂ and eCO₂ in soybean remains unexplored, despite the potential for synergistic effects on carbon assimilation and nitrogen dynamics.

In the current work, soybean plants were employed as the model leguminous species to thoroughly investigate how soybean responds to n-SiO₂ exposure under elevated CO₂. The detailed studies on soybean plants are particularly necessary because soybean (Glycine max) serves as a vital crop for food, animal feed, and biofuel, contributing significantly to global legume production. Its symbiotic relationship with N₂-fixing bacteria enables soybeans to fix approximately 16.4 million metric tons of nitrogen annually, significantly reducing reliance on synthetic nitrogenous fertilizers. This study examines how n-SiO₂ influences soybean physiology and nitrogen fixation under elevated CO₂, focusing on four key aspects: (1) physiological adaptation, assessing n-SiO₂ role in enhancing mesophyll conductance and chloroplast stability; (2) net photosynthetic activity, evaluating its impact on Rubisco activity and electron transport efficiency; (3) root and nodule health, investigating how n-SiO₂ stabilizes nodule function amid shifting C:N ratios; and (4) biochemical and nitrogen metabolism, exploring its role in mitigating oxidative stress and optimizing nitrogen assimilation. By addressing these dimensions, this research provides critical insights into n-SiO₂ potential to enhance soybean resilience and productivity in a CO₂-enriched environment.

2. Results

2.1. n-SiO₂ Alters Soybean Physiology Under Elevated CO₂

The increasing atmospheric CO₂ levels due to climate change pose significant challenges to agricultural systems, particularly in enhancing crop growth and nutritional quality. The current study aimed to investigate how n-SiO₂ influences soybean physiology, including shoot growth, biomass accumulation, leaf development, and photosynthetic efficiency, under ambient (aCO₂) and elevated CO₂ levels (Figure 1). The results indicate that eCO₂ alone significantly enhanced soybean growth, with a 30% increase in shoot length and a 58% increase in shoot fresh weight compared to aCO₂ conditions. These findings align with previous reports that elevated CO₂ improves photosynthesis and biomass accumulation in C3 plants like soybean [12]. However, the addition of n-SiO₂ under eCO₂ further boosted shoot length by an additional 38%, indicating that n-SiO₂ may enhance cell elongation and chloroplast stability, optimizing photosynthetic efficiency [13]. Similarly, shoot fresh weight increased by an additional 11% with n-SiO₂, reflecting its role in improving nutrient uptake and water-use efficiency, which are critical for maximizing plant productivity under stress. These results are consistent with studies showing that n-SiO₂ can promote biomass accumulation through enhanced nutrient acquisition and improved physiological responses to environmental stress [14,15].

Shoot dry weight followed a similar pattern, increasing by 38% under eCO₂ and by an additional 28% with n-SiO₂ supplementation (Figure 1). This suggests that n-SiO₂ helps in improving the plant’s metabolic activity and stress tolerance, thereby enhancing its ability to accumulate dry matter under elevated CO₂ conditions [16]. Moreover, leaf number increased by approximately 27% with n-SiO₂ supplementation, likely due to enhanced auxin signaling and root function [17]. These changes in leaf morphology are crucial for optimizing the plant’s ability to capture light and perform photosynthesis effectively. The leaf area, a critical factor for photosynthetic capacity, increased by 53% under eCO₂, with an additional 50% increase observed when n-SiO₂ was applied (Figure 1). These results suggest that n-SiO₂ enhances the plant’s capacity for light capture and carbon fixation, improving overall growth under elevated CO₂ conditions. The significant increase in leaf area aligns with previous findings that n-SiO₂ can enhance photosynthetic efficiency by improving leaf expansion and reducing oxidative stress [14,15]. A recent study reported that nanoparticles (CeO₂-NPs) significantly increased plant physiological growth indicators (shoot and root length, shoot and root weight) by 40.5% to 84.6% at 100 ppm under the eCO₂ conditions [7]. Another study documented that chitosan nanoparticles (CS-NPs) significantly enhanced soybean shoot and root biomass by ~1.6-fold under elevated CO₂ conditions compared to the control group [6]. SiO₂-NPS significantly increased (36%) the cherry radish fresh weight [18]. Regarding the soybean growth, ionic Si significantly increased the shoot dry weight by 23% compared to the control group [19]. These results collectively highlight that n-SiO₂ plays a crucial role in optimizing soybean growth under elevated CO₂ conditions. The observed enhancements in shoot elongation, biomass accumulation, leaf development, and photosynthetic potential demonstrate the potential of n-SiO₂ to mitigate the negative effects of elevated CO₂ and improve soybean productivity. This aligns with the growing body of literature emphasizing the role of nanomaterials, like n-SiO₂, in enhancing crop resilience and performance under future climate scenarios with higher CO₂ levels [6,7].

2.2. Enhancement of Photosynthesis Activity in Soybean Under Elevated CO₂ Induced by n-SiO₂

The application of n-SiO₂ significantly enhanced soybean photosynthetic performance under eCO₂ conditions, demonstrating their potential to optimize plant physiology in response to climate change. Under eCO₂ (645 ppm), n-SiO₂ treatment led to marked improvements in key photosynthetic parameters compared to aCO₂, highlighting a strong synergistic effect between elevated CO₂ and n-SiO₂. A substantial increase in chlorophyll content was observed, with chlorophyll a and b rising by 106.16% and 136.92%, respectively, and a 112.15% increase in total chlorophyll (Figure 2). These results align with previous studies [6,7] and suggest enhanced chlorophyll biosynthesis and stabilization of the photosynthetic apparatus, likely due to more efficient nutrient uptake, enhanced nitrogen metabolism, and reduced oxidative stress. These changes facilitate greater light absorption and energy conversion, which are critical for maintaining high photosynthetic activity under eCO₂. Further supporting this, Ahsan et al. documented that n-SiO₂ nanoparticles penetrate chloroplasts, reaching photosystem II reaction centers, stimulating electron transmission and light absorption, and thus enhancing photosynthetic efficacy. This mechanism contributes to the elevated chlorophyll concentration, leading to improved photosynthesis and overall plant performance [20].

Additionally, carotenoid content, crucial for light harvesting and photoprotection, increased by 41.96% under eCO₂ with n-SiO₂ treatment. This suggests strengthened antioxidant defenses, mitigating photooxidative stress commonly associated with high CO₂ levels and intense light exposure. The elevated carotenoid levels also stabilize chlorophyll molecules, supporting sustained photosynthetic efficiency. In parallel, photosynthetic efficiency, as indicated by the net photosynthetic rate (Pn), stomatal conductance (Gs), and intercellular CO₂ concentration (Ci), showed significant improvements. Previous studies have documented that elevated CO₂ initially boosts Pn but can lead to down-regulation over time, accompanied by reduced Gs and increased Ci [21,22]. Another study reported that nanoparticles (CeO₂-NPs) significantly enhanced photosynthetic indicators (chlorophyll a, b, and total chlorophyll content) by 85.3% to 398.8% at 50–100 mg L⁻¹ (CeO₂-NPs) under the elevated CO₂ conditions [7]. Additionally, CS-NPs at 40–65 μg mL⁻¹ improved the soybean photosynthetic activities by 1.5 to 2.9 folds more than that ambient CO₂ group [6]. SiO₂-NPs, as a nano-fertilizer, increased the plant’s (cherry radish) total chlorophyll and carotenoids contents by 14.2–18.7% [18]. Wang et al. comparing the potential of silicate and SiO₂-NPs, observed that SiO₂-NPs significantly improved soybean photosynthesis related indicators compared to both the control and silicate groups [13]. In our study, the photosynthetic rate increased by 103.23% under eCO₂ + n-SiO₂, reflecting enhanced carbon assimilation. This was supported by improved stomatal conductance, which facilitated more efficient CO₂ influx, and a 17.19% rise in intercellular CO₂ concentration, indicating better CO₂ diffusion and utilization within the leaf tissues. These physiological changes suggest that n-SiO₂ helps optimize gas exchange processes, thereby enhancing photosynthetic performance and overall plant productivity [23]. This is consistent with previous reports that nanomaterials can enhance Pn, counteract the reduction of Gs, and improve overall photosynthetic performance under elevated CO₂ [6,7]. Overall, the integration of n-SiO₂ with elevated CO₂ conditions significantly boosts soybean growth by enhancing chlorophyll synthesis, optimizing gas exchange, and reinforcing photoprotective mechanisms. These findings underscore the potential of n-SiO₂ as an effective strategy to improve crop performance and resilience in the face of rising atmospheric CO₂ concentrations.

2.3. Synergistic Effects of Silicon Nanoparticles on Root Morphology and in Soybean Under Elevated CO₂

The application of n-SiO₂ under eCO₂ significantly improved various root physiology indicators in soybean plants. Root fresh biomass (Figure 3A) showed a clear enhancement with a 48.3% upregulation under eCO₂ + n-SiO₂ compared to aCO₂, with values increasing from 2.3 g to 3.5 g. This increase suggests that n-SiO₂ plays a role in improving overall root growth and biomass accumulation under eCO₂ conditions. Similarly, root dry biomass (Figure 3B) increased by 43.3% under eCO₂ + n-SiO₂, further confirming the positive impact of n-SiO₂ on root system development under stress conditions. Root length (Figure 3C) followed a similar trend, with a 38.5% increase under eCO₂ + n-SiO₂ compared to aCO₂. This finding is consistent with enhanced root elongation and expansion, which is crucial for optimizing nutrient and water uptake in challenging environmental conditions. The improvement in root length may be attributed to the combined effects of elevated CO₂ and n-SiO₂, which likely enhance root cell division and elongation processes. Root surface area (Figure 3D) increased by 28.5% under eCO₂ + n-SiO₂, further supporting the notion that n-SiO₂ facilitates greater root surface area, which is vital for improving nutrient and water absorption. The average root diameter (Figure 3E) also showed a 13.5% increase under eCO₂ + n-SiO₂, indicating that n-SiO₂ not only boosts root surface area but also enhances root structure and thickness, contributing to a more robust root system. Total root volume (Figure 3F) exhibited a 23.4% increase under eCO₂ + n-SiO₂, reinforcing the overall improvements in root morphology and physiology. The increase in root volume is likely to enhance the plant’s ability to absorb water and nutrients [24,25], which is critical for maintaining plant growth under elevated CO₂ conditions [26].

The observed enhancement of root physiology in soybean plants under eCO₂ induced by n-SiO₂ can be attributed to several synergistic effects. Elevated CO₂ typically stimulates plant growth by enhancing photosynthesis, leading to increased carbon allocation to roots [22,27,28]. Mukarram et al. investigated the impact of SiNPs on crop health through their direct influence on various biochemical and physiological processes, such as increased photosynthetic activity, enhanced nutrient uptake, improved nitrogen metabolism, and elevated enzymatic activity, all of which directly enhanced plant growth and yield [29]. Another recent study documented that n-SiO₂ at 100 mg kg⁻¹ significantly increased maize root weight, length, and number of roots by 4.5% to 20% compared to a control group [30]. The addition of n-SiO₂ further amplifies this effect by improving root architecture, increasing root biomass, surface area, and volume. The improvement in root growth can be attributed to enhanced photosynthesis, which facilitates belowground carbon sequestration [31]. Larger root systems with optimized architecture are also expected to improve water and nutrient acquisition by plants, as well as indirectly stimulate photosynthetic CO₂ capture. These findings are consistent with our study, where the enhancement of root physiological traits was evident under eCO₂ + n-SiO₂ treatment.

The increase in root diameter and surface area suggests that n-SiO₂ promotes root cell expansion and tissue development, improving the plant’s ability to absorb nutrients and water more efficiently. Such changes are crucial for optimizing plant growth under water-limited conditions and improving stress resilience. Previous studies have highlighted that n-SiO₂ promotes secondary and lateral root development, further improving nutrient uptake and overall root function [11,20,32]. This supports our observation that n-SiO₂ enhances root structure and function, making the plant more resilient under challenging environmental conditions. Overall, these results underscore the potential of n-SiO₂ to optimize plant growth and nutrient uptake under elevated CO₂, making it a promising tool for enhancing crop resilience and ensuring food security in the face of climate change. The improvement in root physiology and nitrogen metabolism demonstrates that n-SiO₂ could be a key strategy in enhancing crop productivity, particularly under elevated CO₂ conditions.

2.4. Modulation in Biochemical Response in Soybean Under n-SiO₂-Induced by Elevated CO₂

The combined application of n-SiO₂ and eCO₂ significantly influenced key phytohormones that regulate plant growth, stress adaptation, and development. Abscisic acid (ABA) content (Figure 4A) increased by 16.5% under eCO₂ + n-SiO₂ compared to aCO₂, indicating enhanced plant resilience through improved stomatal regulation. ABA, which plays a crucial role in mitigating water loss under elevated CO₂, regulates stomatal closure, and the addition of n-SiO₂ further strengthens this effect by improving water retention. Similarly, jasmonic acid (JA) levels (Figure 4B) showed a 35.2% increase under eCO₂ + n-SiO₂, suggesting a reinforced defense response. JA is involved in systemic resistance and stress signaling, and its enhanced levels indicate a stronger adaptive response to oxidative stress. Indole-3-acetic acid (IAA) content (Figure 4C) increased by 21.7% under eCO₂ + n-SiO₂, signifying improved root and shoot development. As a primary auxin responsible for cell elongation and differentiation, the upregulation of IAA suggests that n-SiO₂ enhances cell expansion, optimizing root architecture for better nutrient and water uptake under eCO₂. Likewise, gibberellic acid (GA3) levels (Figure 4D) increased by 18.9%, indicating enhanced shoot elongation and biomass accumulation. GA3, essential for stem elongation and seed germination, likely promotes growth pathways under eCO₂, supporting improved plant performance under stress conditions. The enhanced phytohormone levels observed in soybean under eCO₂ and n-SiO₂ treatments suggest a synergistic mechanism that could improve plant resilience and productivity. Previous studies have shown that nanoparticles like chitosan can induce a similar phytohormone accumulation under elevated CO₂, which aligns with our findings [6]. Phytohormones, particularly auxins (IAA) and gibberellins (GA), play pivotal roles in regulating plant physiological and biochemical processes, including photosynthesis [33]. These hormones are synthesized within chloroplasts and influence chloroplast development, size, thylakoid membrane organization, and chlorophyll content, ultimately determining photosynthetic capacity [33]. Furthermore, phytohormones like IAA and GA are involved in a crosstalk with sugar metabolism, linking growth regulation and energy production [34,35]. Similar to our findings, elevated CO₂ has been shown to increase phytohormone levels in Arabidopsis thaliana and soybean, reflecting biochemical adjustments to enhanced CO₂ availability [36]. These observations confirm that improvements in IAA and GA levels under eCO₂ and n-SiO₂ treatments help enhance photosynthesis and overall growth, highlighting the potential of n-SiO₂ to boost plant physiological functions under changing environmental conditions.

The antioxidant defense system plays a crucial role in mitigating oxidative stress caused by reactive oxygen species (ROS) [37]. Our study demonstrates that the application of n-SiO₂ under elevated CO₂ (eCO₂) significantly enhanced antioxidant enzyme activities, thereby improving the plant’s ability to counteract oxidative stress. Superoxide dismutase (SOD) activity (Figure 4E) increased by 19.4% under eCO₂ + n-SiO₂ compared to aCO₂, highlighting an improved ROS scavenging capacity. Since SOD is the first line of defense against oxidative stress by converting superoxide radicals into hydrogen peroxide (H₂O₂) [38], its enhanced activity suggests that n-SiO₂ helps reduce ROS accumulation, preventing cellular damage and maintaining redox homeostasis. As a consequence of increased SOD activity, H₂O₂ content (Figure 4G) decreased by 22.1% under eCO₂ + n-SiO₂, confirming the effective mitigation of oxidative stress. Lower H₂O₂ levels indicate that the antioxidative system efficiently neutralized excess ROS, thereby reducing oxidative damage to cellular structures [39]. To further strengthen ROS detoxification, ascorbate peroxidase (APX) activity (Figure 4H) exhibited a 15.8% increase. APX plays a critical role in converting H₂O₂ into water, preventing its accumulation and minimizing oxidative damage. Similarly, catalase (CAT) activity (Figure 4I) showed a 23.7% increase, reinforcing the plant’s ability to decompose excess H₂O₂ into water and oxygen. Higher CAT activity further supports the role of n-SiO₂ in enhancing antioxidative defense, allowing plants to better cope with oxidative stress under eCO₂ conditions. Additionally, malondialdehyde (MDA) content (Figure 4J) decreased by 18.6%, demonstrating lower lipid peroxidation and improved membrane stability. Since MDA is a key indicator of oxidative membrane damage, its reduction suggests that n-SiO₂ contributes to protecting plant cells from oxidative stress by maintaining membrane integrity. While n-SiO₂ application significantly enhanced the antioxidant defense response, the effects of cerium oxide nanoparticles (CeO₂ NPs) under eCO₂ exhibited a different trend. The exposure of plants to CeO₂ NPs under eCO₂ conditions did not induce oxidative stress, as evidenced by stable MDA and H₂O₂ levels. Similarly, no oxidative stress was observed under CeO₂ NPs treatment at any concentration under both ambient CO₂ (aCO₂) and eCO₂ conditions [7]. This suggests that, unlike some metal-based nanoparticles that induce ROS accumulation, CeO₂ NPs do not generate excessive ROS, maintaining cellular homeostasis. Despite the absence of oxidative stress, CeO₂ NPs altered antioxidant enzyme activities, potentially enhancing plant stress resilience [7]. At low concentrations, CeO₂ NPs positively impacted the overall antioxidant capacity of plants, likely due to their enzyme-mimicking properties. CeO₂ NPs exhibit both oxidant and antioxidant behavior in plants, particularly by mimicking superoxide dismutase (SOD) activity, which enhances plant tolerance to abiotic stress. However, previous studies have reported mixed responses regarding the effect of CeO₂ NPs on antioxidant enzymes. In spinach plants, no significant change in SOD activity was observed across all CeO₂ NPs treatments under both aCO₂ and eCO₂ conditions. In contrast, CAT activity increased under both CO₂ conditions, while APX activity exhibited a significant upregulation at 100 mg/L CeO₂ NPs under eCO₂ [7]. These findings suggest that the effects of CeO₂ NPs on antioxidant enzyme activity are enzyme specific and influenced by environmental conditions. Furthermore, contradictory findings have been reported concerning oxidative stress markers. Some studies indicate that higher concentrations of NMs lead to increased MDA content and ion membrane leakage, while others found no significant oxidative damage. These discrepancies could be attributed to variations in plant growth conditions, species-specific responses, application methods, and differences in concentration and duration of exposure.

2.5. n-SiO₂ Enhances Nodule Health and Biological Nitrogen Fixation Potential in Soybean Under Elevated CO₂

The impact of eCO₂ on nodulation and biological nitrogen fixation in soybean, particularly when influenced by n-SiO₂, has not been thoroughly investigated. Our study shows that the application of n-SiO₂ under eCO₂ significantly enhances nodule health and nitrogen fixation potential in soybean plants compared to aCO₂ conditions. The number of nodules per plant (Figure 5A) significantly increased under eCO₂ + n-SiO₂, showing a 48.3% upregulation compared to aCO₂ and a 22.5% increase relative to eCO₂ alone. Similarly, nodule biomass (Figure 5B) was enhanced by 53.6% under eCO₂ + n-SiO₂, suggesting that n-SiO₂ supplementation amplifies the beneficial effects of eCO₂ on nodulation efficiency. Previous studies have documented the positive influence of eCO₂ on root physiology, including increased root nodule growth, nutrient uptake efficiency, and enhanced water absorption capacity [22,27,28,40]. Our findings indicate that n-SiO₂ further strengthens these effects, potentially by promoting secondary and lateral root development, leading to improved overall nutrient acquisition and stress resilience [11,20,32].

Nitrogenase activity (Figure 5C), a key determinant of biological nitrogen fixation, was significantly enhanced in response to n-SiO₂ supplementation. Compared to aCO₂, nitrogenase activity exhibited a 65.2% increase under eCO₂ + n-SiO₂, confirming that n-SiO₂ plays a crucial role in enhancing nitrogen fixation efficiency. Likewise, total nitrogen content (Figure 5D) in both above-ground and below-ground tissues increased markedly, with a 34.7% rise in above-ground nitrogen and a 41.2% increase in below-ground nitrogen under eCO₂ + n-SiO₂ compared to aCO₂. These results suggest that n-SiO₂ enhances nitrogen assimilation by facilitating nitrogen uptake and transport throughout the plant.

The impact of n-SiO₂ on key nitrogen metabolism enzymes was also evident. Urease (UE) activity (Figure 5E), which plays a critical role in nitrogen remobilization, increased by 43.8% in root nodule tissues under eCO₂ + n-SiO₂. Similarly, nitrate reductase (NR) and nitrite reductase (NIR) activities (Figure 5F,G) exhibited significant upregulation, increasing by 38.5% and 52.1%, respectively, under eCO₂ + n-SiO₂ compared to aCO₂. These enhancements indicate an improved nitrate assimilation pathway, leading to more efficient nitrogen utilization. Furthermore, glutamine synthetase (GS) and glutamate synthase (GOGAT) activities (Figure 5H,I), both essential for nitrogen assimilation, were significantly enhanced under eCO₂ + n-SiO₂. GS activity increased by 47.2%, while GOGAT activity exhibited a 58.3% upregulation, reinforcing the improved nitrogen metabolism and assimilation associated with n-SiO₂ supplementation under eCO₂ conditions. The schematic representation (Figure 5J) provides an overview of nitrogen uptake and assimilation pathways, highlighting the mechanisms through which n-SiO₂ improves nitrogen fixation and metabolic assimilation under eCO₂. Several studies have reported that increased CO₂ levels can stimulate nitrogen fixation, potentially due to enhanced photosynthetic rates and improved root architecture [41] Additionally, root exudate composition under eCO₂ can influence microbial communities in the rhizosphere, with beneficial rhizobacteria playing a crucial role in promoting root nodule health and nitrogen fixation efficiency [42]. Given that eCO₂ typically enhances nodule biomass and nitrogen fixation in legumes like soybean, our results suggest that n-SiO₂ further strengthens this effect by optimizing carbon allocation to nodules and improving overall plant health [28]. It was previously documented that Si (100–800 mg kg⁻¹) significantly increased the soybean nodulation by 25–46% compared to the control group [19]. Abuelsod et al. reported that nanoparticles (CS-NPs) under the elevated CO₂ conditions (645 ppm) significantly increased the nitrogen assimilation enzymatic activities (GDH, GS, and GOGAT) as compared to the control group [6]. Silicon has also been found to enhance growth, nodulation, and nitrogen fixation in leguminous plants through various mechanisms [43]. For example, Si has been shown to increase leghemoglobin formation, thereby enhancing the N₂-fixation potential of legume root nodules [44]. It also augments the abundance of bacteroides and symbiosomes within the nodules, further promoting N₂-fixation potential [45]. Moreover, Si promotes cell wall thickness in nodules and reduces the size of peri-bacteroid spaces, facilitating solute transport and rapid dissolving in O₂ diffusion [45]. Additionally, Si reduces the plant requirement for lignin, a carbon-intensive compound, allowing more carbon to be allocated to bacteroid respiration and nodule organogenesis, ultimately increasing N₂-fixation efficiency [43,45]. Recent studies have reported that SiO₂-NPs significantly increased the module health and N₂-fixation potential [46,47]. Collectively, these findings highlight the role of n-SiO₂ in mitigating nitrogen limitations under elevated CO₂. The observed improvements in nodule health, nitrogen fixation, and nitrogen metabolism support the use of n-SiO₂ as a strategy to enhance crop productivity under future climate scenarios characterized by elevated CO₂.

2.6. Synergistic Effects of Elevated CO₂ and Nano-Silica on Soybean Uptake and Nutrient Homeostasis

The effects of various treatments, including aCO₂, aCO₂ + n-SiO₂, eCO₂, and eCO₂ + n-SiO₂, on plant Si content, translocation factor, bioconcentration factor, and elemental uptake were analyzed to understand the synergistic impacts of n-SiO₂ under different CO₂ conditions. The results revealed distinct trends and enhancements in all parameters tested, particularly when n-SiO₂ was applied under elevated CO₂ conditions. In terms of shoot silicon content (Figure 6A), both aCO₂ + n-SiO₂ and eCO₂ + n-SiO₂ treatments resulted in marked increases compared to the control (aCO₂). Specifically, aCO₂ + n-SiO₂ exhibited a ~50% increase, while eCO₂ + n-SiO₂ showed the most substantial enhancement, with a ~100% increase, demonstrating the amplified effect of elevated CO₂ in combination with n-SiO₂ on silicon accumulation in shoots. This trend was similarly observed in root silicon content (Figure 6B), where the aCO₂ + n-SiO₂ treatment resulted in a moderate increase (~40%) compared to the control, and eCO₂ + n-SiO₂ exhibited the highest value with an ~80% increase. These findings suggest that the addition of n-SiO₂ under elevated CO₂ conditions significantly boosts the plant’s ability to accumulate Si, both in the shoots and roots. Soil silicon content (Figure 6C) also showed positive changes, although to a lesser extent. The aCO₂ + n-SiO₂ treatment led to a slight increase (~10%), while eCO₂ + n-SiO₂ resulted in a more pronounced enhancement of ~30%. This suggests that elevated CO₂, in conjunction with n-SiO₂, improves the availability of silicon in the soil, which is subsequently taken up by plants. Correspondingly, the translocation factor (Figure 6D), which measures the efficiency of Si movement from soil to plant tissues, was significantly increased in both n-SiO₂ treatments. While aCO₂ + n-SiO₂ showed a moderate increase (~20%) compared to the control, eCO₂ + n-SiO₂ exhibited the highest increase (~50%), indicating that elevated CO₂ conditions further enhance the transport of Si within the plant system. Interestingly, the bioconcentration factor (Figure 6E), which reflects the plant’s capacity to concentrate Si from the soil, was slightly decreased in the aCO₂ + n-SiO₂ treatment (~10% decrease), whereas eCO₂ + n-SiO₂ resulted in the most significant increase (~30%) compared to the control. This highlights the greater efficiency of Si accumulation in plants under elevated CO₂ conditions when combined with n-SiO₂. In addition to silicon content, the elemental content analysis (Figure 6F,G) revealed substantial upregulation of several essential nutrients under the eCO₂ + n-SiO₂ treatment. Notable increases were observed in phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), iron (Fe), and zinc (Zn), with eCO₂ + n-SiO₂ showing increases of ~74%, ~141%, ~91%, ~87%, ~70%, and ~49%, respectively, compared to the control. These results suggest that the application of n-SiO₂ under elevated CO₂ conditions not only boosts Si content but also enhances the uptake of other essential nutrients, thus contributing to improved overall plant health and growth.

Previous studies have shown that plants tightly regulate, or in some cases downregulate [48], nutrient accumulation at sub-cellular and cellular levels in a CO₂-dependent manner in Oryza sativa L. [49], soybean (Glycine max L.), and common bean (Phaseolus vulgaris L.) [50], which is consistent with our current study. It has been previously observed that the presence of Si in soil enhances the availability of macro- and micronutrients to plants. Nano SiO₂ increased root growth, along with its unique surface properties and catalytic activity, may promote nutrient retention and transport in the rhizosphere, thereby improving nutrient uptake by plants [30,51]. Xu et al., documented that SiO₂-NPs significantly increased (~23.7%) the nutritional elements (P, K, Mn, Cu, and Zn) [18]. Previous studies have also documented that nano-SiO₂ not only improves Si uptake capacity but also upregulates nutrient homeostasis under both normal [52] and stressed conditions [53,54]. However, we are the first to study the interactive effects of nano-SiO₂ with elevated CO₂ exposure. Overall, our findings consistently demonstrate that the combination of elevated CO₂ and n-SiO₂ significantly enhances silicon uptake and translocation, as well as the accumulation of key nutrients in plants.

3. Materials and Methods

3.1. Experimental Conditions and Plant Growth

The experimental soil (surface soil, 0–20 cm) was collected from the university experimental station. The air-dried soil was sieved through 2 mm mesh to remove the debris and plant residues, ensuring homogenization. The physiochemical properties of the sieved experimental soil are presented in Supplementary Table S1. We characterized the n-SiO₂ following methodologies outlined in recently published studies [55,56]. Nanoscale SiO₂ with a purity of 99.98% (17 ± 2 nm) was purchased from “Shanghai Pantian Powder Material Co., Ltd., Shanghai, China” in powder form. The characterization and sizes of the SiO₂-NPs were analyzed using SEM and TEM (Figure S1). The size distribution and surface charge of the SiO₂-NPs in distilled water were assessed through using dynamic light scattering (DLS) and zeta potential analysis with a Zetasizer (Nano ZS90, Malvern, UK) equipped with a He–Ne laser beam at 25 °C. The zeta-potential of used SiO₂-NPs was −40 ± 3.7 MV, and the hydrodynamic diameter was 258.9 ± 9.87 nm. The SiO₂-NPs were thoroughly mixed with the soil, while untreated soil served as the control (Ctrl). The treatments included soil contaminated with 100 mg kg⁻¹ SiO₂-NPs. The amended soil was left in pots for a period of two weeks to allow for stabilization. The applied SiO₂-NPs concentrations represent realistic environmental levels.

Soybean (Glycine max L.) seeds (Zhonghuang-13) were purchased from the “Shouguang Seed and Seedling Co. Ltd., Shandong, China”. Uniformly sized seeds were selected and sterilized with a 3% hypochlorite sodium solution (v/v) for 10 min, then thoroughly washed with deionized (DI) water 3–4 times. The sterilized seeds were then transferred to wet filter paper and kept at 25 °C for germination. Uniform seedlings were subsequently moved into plastic pots containing SiO₂-treated soil (100 mg kg⁻¹). The experiment followed a completely randomized design (CRD), with four replicates for each treatment. Ambient CO₂ (aCO₂) (400 ± 15 ppm) and elevated CO₂ (eCO₂) (600 ± 20 ppm) levels were maintained as growth conditions. The elevated CO₂ concentration was chosen based on projections from the IPCC-SRES B2 scenario for the year 2100 [57]. The CO₂ for all chambers was supplied from a compressed gas tank containing liquid CO₂. An inline fan equipped with a variable damper controlled the airflow through an external soda lime unit to regulate CO₂ levels. The gas was supplied into the airflow of the growth chamber, and its concentration was continuously monitored using a CO₂ analyzer (WMA-4, PP Systems, Hitchin, UK). The experiment was conducted in a climate-controlled chamber under a 16/8 h day/night photoperiod, with a photosynthetically active radiation (PAR) intensity of 350 μmol m⁻² s⁻¹, 60% relative humidity, and a constant temperature of 25 °C. Throughout the experiment, soil moisture was maintained at 60% of field capacity using DI water for on demand. The phenotypic and physiological analyses were measured after 28 days before harvesting. Four weeks after planting, plant roots and shoots were harvested, flash-frozen in liquid nitrogen, and stored at −80 °C for further analysis. The fresh and dry biomass of shoots and roots was measured.

3.2. Photosynthetic Pigments and Activity Assessment

Photosynthetic pigments and activity were analyzed after 27 days of exposure by measuring the fully expanded second leaf of soybean plants. Key parameters, including intercellular CO₂ concentration (Ci), transpiration rate (Tr), net photosynthesis rate (Pn), and stomatal conductance (Gs), were evaluated using an open gas exchange system (IRGA) (LI-38 COR Biosciences, Lincoln, NE, USA). Measurements were obtained between 09:00 and 11:30 AM under standardized conditions, with a photosynthetically active radiation (PAR) of 1000 µmol m⁻² s⁻¹ and a CO₂ molar fraction of 400 µmol mol⁻¹. To ensure precision, the instrument was recalibrated before each use to maintain stable and reliable readings. Chlorophyll (a, b) and carotenoid contents were quantified by extracting pigments from the second leaf using 95% ethanol. The absorbance of the extracts was measured with a plate reader spectrophotometer (Beckman DU-640, Ramsey, MN, USA) at wavelengths of 665 nm, 649 nm, and 470 nm, respectively, following the method outlined in our previous study [52,54].

3.3. Biochemical Enzymes and Phytohormones Analysis

For the determination of antioxidant enzymes, 100 mg of fresh soybean leaves was ground and homogenized with a phosphorus buffer solution (50 nM, pH 7.8) at a 1:9 ratio. The homogenized samples were centrifuged at 8000–10,000 rpm for ten minutes, and the supernatants were collected for further analysis. The activities of superoxide dismutase (SOD), catalase (CAT), and along with the contents of malondialdehyde (MDA), hydrogen peroxide (H₂O₂), and ascorbate peroxidase (APX), were quantified using commercial kits from Nanjing Jiancheng Bioengineering Institute, Nanjing, China. Phytohormone analysis was conducted on fresh soybean shoot samples collected after 28 days. These samples were frozen in liquid nitrogen and ground into a fine powder. A 0.5 g portion of the powdered sample was then mixed with 3 mL of 80% methanol. Following centrifugation at 5000 rpm for ten minutes, the supernatant was collected. Phytohormones were quantified using enzyme-linked immunosorbent assay (ELISA), with specific polyclonal or monoclonal antibodies for each hormone and alkaline phosphatase as markers, as outlined in previous studies [58].

3.4. Nitrogen Assimilation Enzymes and Nitrogenase Activity

The enzymatic activities of nitrogen assimilation, including nitrite reductase (NiR), nitrate reductase (NR), glutamate synthetase (GOGAT), and glutamine synthetase (GS), were measured in fresh soybean shoots and roots according to the assay kit protocols provided by Nanjing “Jiancheng Bioengineering Co., Ltd., Nanjing, China”. Urease (UE) activity in these tissues was determined using assay kits following the manufacturer’s instructions from “Beijing Boxbio Co., Ltd., Beijing, China”. The nitrogen content in soybean shoots and roots (in dry powder form) was analyzed using an organic elemental analyzer (Vario EL model, Elemental, Germany) [52].

The nitrogenase activity in soybean nodules was evaluated using the acetylene reduction assay. Freshly harvested nodules were placed into 50 mL micro-reaction vials. Air was initially extracted from the vials using a syringe, and 10 mL of acetylene was then injected into the vials. The mixture was incubated at 30 °C for 30 min. Afterward, 500 µL of the gas mixture was sampled and analyzed for ethylene content using gas chromatography (Agilent 7890, Stevens Creek, CA, USA). Nitrogenase activity was quantified by measuring the rate of ethylene production per gram of nodule tissue [54].

3.5. Quantity of Mineral in Soil-Plant System

Soybean shoots and roots were freeze-dried using lyophilization at −48 °C for 72 h with a freeze-dryer (TF-FD-18S, Shanghai, China). After freeze-drying, the plant tissues were finely ground into a fine powder. A 200 mg sample of the powder was subjected to digestion with 3 mL of HNO₃ and 0.5 mL of H₂O₂ in a 75 mL tube using microwave digestion (Ultra WAVE, Italy). The final volume was diluted to 50 mL with DI water and filtered through a 0.25 µm PTFE membrane. The concentrations of Si, P, K, Cu, Zn, Mn, and Fe were measured using inductively coupled plasma mass spectrometry (ICP-MS) (DRCII, PerkinElmer, and Norwalk). The mineral content in the soybean’s planted soil was analyzed through acid-wet digestion using aqua regia (3 HCl:1 HNO₃) in a microwave digestion system. Elemental concentrations were quantified by ICP-MS using calibration standards with concentrations ranging from 0.01 to 100 ppm. Recovery rates for the analyzed elements are provided in Table S2.

3.6. Statistical Analysis

The experiment followed a completely randomized design (CRD) with four replicates for each treatment. The data are presented as mean ± standard deviation (SD). Statistical analysis was performed using Statistical Software (Statistix 8.1), with significance determined by one-way analysis of variance (ANOVA). Comparisons of mean values between treatments were made using the least significant difference (LSD) test. A significance level of p < 0.05 was considered statistically significant. Graphical representations of the data were generated using GraphPad Prism (version 8.0.2).

4. Conclusions

Our study demonstrates that the interactive application of n-SiO₂ and eCO₂ significantly enhances soybean growth, nitrogen fixation, and nutrient homeostasis, offering promising insights for sustainable agriculture under future climate conditions. The synergistic effects of eCO₂ + n-SiO₂ resulted in substantial improvements in photosynthetic efficiency, stomatal conductance, and carbon assimilation, leading to a 30% increase in shoot length and over 100% enhancement in chlorophyll content and photosynthetic rate. Enhanced nitrogen assimilation, evident from the upregulation of NR, NIR, GS, and GOGAT, contributed to improved biological nitrogen fixation, with nodule biomass and number increasing by 53.6% and 48.3%, respectively. Furthermore, phytohormone regulation and antioxidant defense systems were significantly boosted under eCO₂ + n-SiO₂, increasing resilience against oxidative stress while maintaining lower levels of oxidative markers. The uptake and homeostasis of essential macronutrients and micronutrients improved by 13–41% in roots and 13–17% in shoots, with Fe and Zn levels remaining stable in shoots. These findings highlight the potential role of n-SiO₂ as a sustainable nano-fertilizer, capable of mitigating the physiological and biochemical challenges posed by rising CO₂ levels while improving overall plant health and productivity. This study provides a strong foundation for future research on nano-enabled agriculture, emphasizing the potential of Si-based nanomaterials in enhancing crop performance under climate change scenarios. Integrating nano-fertilizers with elevated CO₂ conditions could pave the way for more efficient and climate-resilient crop management strategies, ensuring global food security in a changing environment.