Genotype-Dependent Effects of Silicon on Cell Wall Composition and Antioxidant Responses in Oats Under Nitrogen Deficiency

TL;DR

This study explores how silicon affects oat plants under nitrogen deficiency, showing that silicon improves plant resilience by altering cell wall composition and antioxidant activity.

Contribution

The study reveals genotype-dependent effects of silicon on cell wall composition and antioxidant responses in oats under nitrogen deficiency.

Findings

Silicon reduced shoot nitrogen content but increased silicon accumulation in both oat genotypes under nitrogen deficiency.

Silicon decreased lipid peroxidation in both genotypes under nitrogen-deficient conditions.

Silicon increased cellulose and antioxidant activity in the N-sensitive genotype but reduced lignin and TAL activity in the N-tolerant genotype.

Abstract

Nitrogen (N) availability strongly regulates plant growth and metabolism, and its deficiency constrains plant development and yield. Silicon (Si) has been reported to enhance plant tolerance to multiple stresses; however, its influence on N metabolism in oats remains poorly understood. This study aimed to investigate the effects of Si on cell wall composition and antioxidant responses in oat genotypes grown under N limitation. Two oat genotypes with contrasting N tolerance were hydroponically cultivated under N-deficient (0.5 mM) or N-sufficient (5 mM) conditions in combination with 0 or 2 mM Si. Growth parameters, N and Si uptake, cell wall structural components, phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) activities, antioxidant responses, and oxidative damage were evaluated. In both genotypes grown under N deficiency, Si supply reduced shoot N content while enhancing Si accumulation. Moreover, Si application decreased lipid peroxidation in both genotypes under N-deficient conditions. In the N-sensitive genotype, Si increased cellulose deposition and antioxidant activity, whereas in the N-tolerant genotype, Si reduced lignin content and TAL activity. We conclude that Si supplementation improves the metabolic performance of oat genotypes under N-deficient conditions by modulating nutrient uptake, antioxidant responses, and cell wall composition.

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1. Introduction

Oat (Avena sativa L.) is a widely cultivated cereal crop of considerable nutritional, agronomic, and economic importance worldwide. However, global oat production is increasingly constrained by multiple biotic and abiotic stressors that negatively affect plant growth, physiological performance, and yield [1]. Among mineral nutrients, nitrogen (N) is a primary determinant of photosynthetic capacity, biomass accumulation, and overall crop productivity [2,3]. Plants predominantly acquire N in the form of nitrate (NO_3_^−^) or ammonium (NH_4_^+^), and both the chemical form and external availability of N strongly influence plant physiological responses [4,5]. Nitrogen deficiency limits chlorophyll biosynthesis and protein synthesis, resulting in reduced growth and yield, whereas excessive N supply disrupts cellular homeostasis, impairs root function, and compromises structural integrity [6,7].

Oat cultivars exhibit marked genotypic variation in N uptake capacity, assimilation efficiency, and tolerance to N imbalance, which are closely associated with differences in root architecture, transporter activity, and metabolic regulation [3,8]. Under N-deficient conditions, N-sensitive oat genotypes typically display chlorosis, reduced tillering, and impaired grain filling, whereas N-tolerant genotypes are able to sustain photosynthetic activity and root development [9,10]. Structural traits, including plant height, culm thickness, and cell wall composition (cellulose, hemicellulose, and lignin), are strongly regulated by soil N availability [11,12,13]. In this sense, N limitation disrupts carbon (C) and N metabolism, promoting C redistribution into cell wall structural components such as cellulose, hemicellulose, and lignin. Beyond their structural role, these compounds contribute to stress adaptation by reinforcing plant structure and modulating oxidative responses [14,15,16,17]. Therefore, changes in cell wall composition as a consequence of N deficiency could serve as key markers of physiological adjustment and potential tolerance to N deficiency in cereals.

Silicon (Si), although not classified as an essential nutrient, is widely recognized as a beneficial element for many plant species, particularly grasses, such as oat. Silicon enhances plant growth and resilience to a wide range of biotic and abiotic stresses [18,19,20,21] and plays an important role in cell wall architecture by modulating lignin biosynthesis and forming covalent and hydrogen bonds with cellulose and hemicellulose, thereby increasing mechanical strength and structural rigidity [22]. Environmental stress conditions commonly induce the overproduction of reactive oxygen species (ROS), leading to oxidative damage, and Si has been shown to mitigate this damage by regulating both enzymatic (superoxide dismutase, catalase, peroxidase) and non-enzymatic (ascorbate, glutathione) antioxidant defense systems [19,23,24,25].

Growing evidence indicates that Si interacts closely with N nutrition, influencing N uptake, transport, assimilation, and internal remobilization, particularly under N-deficient conditions [26,27,28]. Silicon supplementation has been reported to alleviate the detrimental effects of N deficiency by sustaining photosynthetic performance, improving plant water status, and enhancing antioxidant capacity, responses that are often accompanied by increased accumulation of phenolic compounds and flavonoids [27,28,29,30]. In addition, Si-mediated improvements in carbon metabolism suggest a key role for Si in maintaining carbon–nitrogen balance under nutritional stress [31,32,33]. While these beneficial effects have been documented in several crop species, they remain highly species- and genotype-dependent, and are strongly influenced by N form and availability [34]. Although Si has been proposed as a potential mitigator of the negative effects associated with N deficiency through the modulation of growth and physiological homeostasis, this aspect remains under-researched. Moreover, the molecular and metabolic mechanisms underlying Si–N interactions, particularly those linking N nutrition with secondary metabolism, are still poorly understood. Taken together, these observations highlight a critical knowledge gap regarding the combined effects of Si and N imbalance on the physiological, biochemical, and structural responses of oat genotypes. Addressing this gap is essential for developing sustainable fertilization strategies that maximize yield, minimize environmental impact, and account for genotypic variability in nutrient responses. Therefore, this study aimed to study the effects of Si on cell wall composition and antioxidant responses in oat genotypes grown under N limitation.

2. Results

2.1. Plant Parameters and N-Si Content

In all treatments, shoot and root dry weight were consistently higher in the N-tolerant genotype than in the N-sensitive genotype (Figure 1A,B). Under N deficiency, the N-tolerant genotype showed a significant increase in root dry weight, whereas shoot dry weight remained unchanged. In contrast, the N-sensitive genotype did not exhibit significant changes in shoot or root dry weight in response to either N or Si application. Overall, Si supply did not significantly affect total plant dry weight in either genotype, except for a slight reduction in shoot dry weight under N-deficient conditions.

Shoot length was greater in the N-tolerant genotype across all N and Si treatments (Figure 1C). In the N-sensitive genotype, shoot length decreased under N deficiency in the absence of Si, whereas in the N-tolerant genotype shoot length increased under the same condition. Root length did not significantly vary among treatments in the N-tolerant genotype. In the N-sensitive genotype, root length increased under N deficiency; however, this increase was attenuated by Si supply (Figure 1D).

As expected, shoot N content decreased in both genotypes under N deficiency, and this reduction was further enhanced by Si application (Figure 2A). Under N-deficient conditions, root N content was not significantly affected by either N or Si treatments. Under N sufficiency, Si supplementation increased shoot N content by at least 28% in the N-sensitive genotype, whereas no significant changes were observed in the N-tolerant genotype (Figure 2B).

Silicon content increased markedly in both genotypes following the addition of 2 mM Si, regardless of N level (Figure 2C,D). In shoots, Si accumulation was higher under N-sufficient conditions in both genotypes (Figure 2C). Conversely, in roots, Si content decreased at higher N supply (Figure 2D).

2.2. Cell Wall Compounds in Oat Plants

Cellulose concentration was higher in shoots of the N-tolerant genotype than in the N-sensitive genotype, regardless of N or Si doses, except in plants treated with 5 mM N and 2 mM Si (Figure 3A). No differences in cellulose concentration were observed between the application of 0.5 or 5 mM N in the N-sensitive genotype. Nevertheless, increases of 65% and 40% were observed following Si application at 0.5 and 5 mM N, respectively. In contrast, in the N-tolerant genotype, a reduction of 37% in cellulose was recorded under N sufficiency and Si supply compared to plants without Si.

Overall, the N-sensitive genotype showed higher hemicellulose content than the N-tolerant genotype (Figure 3B). In addition, a reduction in hemicellulose was observed in the N-sensitive genotype under N-deficient conditions, either with or without Si. Conversely, in the N-tolerant genotype, Si application increased hemicellulose concentration at 0.5 mM N and 5 mM N.

Overall, lignin concentration was higher in the N-tolerant than in the N-sensitive genotype, irrespective of N or Si treatments (Figure 3C). In the N-sensitive genotype, no differences in lignin concentration were observed in response to Si application under either N-deficient or N-sufficient conditions. However, lignin concentration increased under N deficiency, irrespective of Si supply, compared with plants grown under N-sufficient conditions. In contrast, in the N-tolerant genotype, Si application reduced lignin concentration under both N treatments, with a more noticeable effect under N limitation.

Modifications in the cell wall components of culm vascular bundles were also observed in both oat cultivars in response to N and Si addition (Figure 3D). In the N-tolerant cultivar, an increase in cellulose deposition was observed in plants subjected to N deficiency, with a further increase recorded under Si supply. In the N-sensitive genotype, cellulose was reduced by N limitation, but Si augmented it. Although Si also increased cellulose in culms of N-sensitive genotype supplied with 5 mM N, it decreased cellulose in N-tolerant genotype. The N-tolerant genotype exhibited a decrease in lignin accumulation under N deficiency. Such reduction in lignin was additionally diminished by Si application in plants grown under N deficiency or N sufficiency. Nitrogen deficiency also decreased lignin in the N-sensitive cultivar, but Si enhanced it. In this genotype, a decrease in lignin accumulation was induced by Si under N sufficiency.

2.3. Pal and Tal Activities Assay

A higher phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) activities were found in the N-sensitive genotype than the N-tolerant genotype (Figure 4A,B). In the N-sensitive genotype, no significant differences in PAL activity were observed across the N and Si treatments. However, TAL activity was highest in plants exposed to N deficiency, regardless of Si application. In contrast, Si reduced PAL and TAL activities in the N-tolerant genotype exposed to sufficient N supply by about 35% and 28%, respectively. Similarly, the application of 2 mM Si decreased TAL activity by about 47% under N-deficient conditions (Figure 4B).

2.4. Antioxidant Responses

No changes in shoot total phenols were observed in both oat genotypes across the different N doses (Figure 5A). However, Si application decreased phenolic concentration in the N-sensitive genotype under both N treatments. In contrast, in the N-tolerant genotype, Si increased phenols concentration in plants grown under N-deficient conditions. In roots, total phenols were not significantly affected by N treatments in the N-tolerant genotype (Figure 5B), whereas Si application decreased the phenolic concentration at both N rates. A reduction in root phenol concentration was also induced by N deficiency in the N-sensitive genotype. Nevertheless, Si increased the root phenols under N limitation, but a reduction of about 48% was observed when Si was applied to 5N-treated plants.

In shoots and roots, antioxidant activity decreased in both genotypes under N deficiency (Figure 5C). In the N-tolerant genotype, Si application did not significantly affect shoot free radical scavenging activity. However, the N-sensitive genotype exhibited a slight reduction in shoot antioxidant activity following Si application under N sufficiency. In roots, the N-tolerant genotype showed higher antioxidant activity than the N-sensitive genotype (Figure 5D). Moreover, Si application reduced root antioxidant capacity in both genotypes grown under N-sufficient conditions. However, Si enhanced free radical scavenging activity in the N-sensitive genotype grown under N-deficient conditions.

2.5. Oxidative Damage

Membrane lipid peroxidation (measured as TBARS levels) was assessed as an indicator of oxidative damage in oat plants. An increase of about 24% in lipid peroxidation was observed only in shoots of the N-sensitive genotype cultivated at deficient N levels (Figure 5E). In contrast, Si decreased the TBARS levels in shoot and roots of both oat genotypes irrespective of N level. Accordingly, Si significantly decreased shoot lipid peroxidation in the N-sensitive genotype by about 28% and 11% for 0.5 and 5 N levels, respectively. Similarly, a reduction of about 42% and 32% was induced by Si in the N-tolerant genotype grown at deficient and sufficient N levels, respectively. In roots, both oat genotypes also showed a decrease in TBARS levels of at least 42% as a result of Si application when plants were grown under 0.5 or 5 mM N (Figure 5F).

2.6. Principal Component Analysis

Principal component analysis (PCA) of the pooled dataset revealed a clear multivariate separation between the N-sensitive and N-tolerant oat genotypes (Figure 6A). The first two principal components explained 55.2% of the total variance (PC1 = 32.4%, PC2 = 22.8%). PC1 was primarily driven by biomass production, shoot N uptake, lignin content, root phenolics, and antioxidant capacity (DPPH), which were positively associated with the N-tolerant genotype. In contrast, PAL and TAL activities, shoot phenolic concentration, root length, and TBARS levels showed higher loadings toward the N-sensitive genotype. These variables represent the main physiological drivers distinguishing genotype performance under contrasting N–Si conditions. PC2 was mainly influenced by Si accumulation, cellulose content, and shoot elongation on the positive axis, whereas lipid peroxidation (TBARS) contributed negatively. This axis therefore captured the balance between structural reinforcement and oxidative damage associated with Si supply.

In addition to the global PCA including both genotypes, separate PCAs were conducted for each genotype (Figure 6B,C) to specifically evaluate how the treatments (0.5N–0Si, 5N–0Si, 0.5N–2Si, and 5N–2Si) were distributed within each genetic background. In the N-sensitive genotype, the first two principal components explained 67.0% of the total variance (Figure 6B). The 0.5N–0Si treatment was closely associated with shoot and root TBARS and PAL activity. In contrast, the 5N–0Si treatment was positioned on the opposite side of PC1 and was mainly associated with higher antioxidant capacity, phenolic compounds, and hemicellulose content, reflecting a shift toward improved redox balance and structural stabilization under sufficient N supply. Under N deficiency, Si addition (0.5N–2Si) induced a marked displacement along PC1 toward cellulose and lignin accumulation, together with increased Si uptake. Conversely, the 5N–2Si treatment separated primarily along PC2 and was associated with higher shoot and root Si accumulation and increased shoot length.

For the N-tolerant genotype, PC1 and PC2 together explained 75.6% of the total variance (Figure 6C), showing a more consolidated response pattern. The 0.5N–0Si treatment clustered with biomass reduction, oxidative stress markers, and PAL activity, reflecting metabolic adjustment under N limitation. The 5N–0Si treatment occupied a distinct region associated with enhanced antioxidant capacity and nutrient uptake, consistent with its intrinsic N efficiency. Silicon addition under N deficiency (0.5N–2Si) grouped with cellulose content, root dry weight, and shoot length, exhibiting coordinated structural and growth reinforcement. Finally, the 5N–2Si treatment showed the strongest separation and was closely associated with increased Si accumulation and enhanced antioxidant activity.

3. Discussion

The mechanisms involved in the interactions between N and Si in plants remain largely unknown. However, several studies suggest that this interaction may play a crucial role in modulating plant growth, nutrient balance, and stress responses [27,28,29,30,35]. In this study, clear genotypic differences were observed between N-tolerant and N-sensitive oat genotypes in their morphological, physiological, and biochemical responses to different levels of N and Si applied. Overall, our findings demonstrate that Si supply positively influenced the structural and metabolic adjustments of both genotypes, alleviating oxidative stress and modifying cell wall composition according to N availability.

The N-tolerant genotype exhibited greater shoot and root biomass compared to the N-sensitive genotype across all N–Si treatments. This superior growth performance could be attributed to a more efficient system for N acquisition, as demonstrated by the greater N accumulation found in the N-tolerant genotype, in agreement with previous reports [8,9,35]. Silicon application resulted in a reduction in shoot N content, an effect also reported by Gou et al. [36], who suggested that Si application may alter N uptake and internal allocation, potentially through interference with transport and assimilation processes. In some species, reductions in tissue N content following Si supplementation have been attributed to a dilution effect associated with enhanced biomass production [32,37]. However, because no substantial biomass stimulation was observed in the present study, an alternative explanation may involve Si-mediated modulation of nitrate or ammonium transporter expression, altered root membrane permeability, or changes in N remobilization efficiency, as suggested for cereals under stress conditions [38,39]. Such adjustments may reflect an optimization of N use induced by Si under variable N levels. Further studies addressing gene expression and nutrient fluxes are needed to clarify these underlying mechanisms.

On the other hand, both genotypes accumulated more Si in the shoots under N-sufficient treatment, whereas Si content in roots decreased with increasing N. This inverse trend supports the hypothesis that high N availability promotes Si translocation to the aerial parts, possibly to reinforce cell wall rigidity and optimize photosynthetic efficiency [40,41,42,43]. Enhanced Si deposition in aerial tissues has been associated with improved mechanical support and reduced transpiration rates in grasses [44].

Cell wall composition was strongly influenced by both N and Si supply. The N-tolerant genotype displayed consistently higher cellulose and lignin concentrations than the N-sensitive genotype, consistent with cellulose and lignin accumulation patterns observed in culm tissues. Nitrogen deficiency is known to restrict protein synthesis while promoting carbon reallocation toward structural carbohydrates and phenylpropanoid derivatives, as part of a compensatory C-N balance mechanism [45,46,47]. In the N-sensitive genotype, the Si-induced increase in cellulose deposition under N deficiency suggests enhanced carbon partitioning toward structural reinforcement. In contrast, in the N-tolerant genotype, Si supply reduced lignification and TAL activity, indicating a coordinated downregulation of phenylpropanoid metabolism. Because lignin biosynthesis is energetically demanding and tightly linked to PAL and TAL activities, reduced lignification under Si supplementation may reflect a shift in metabolic energy allocation away from secondary wall thickening toward growth maintenance or stress mitigation [48,49].

This reduction in lignin content in the N-tolerant genotype may also imply a potential trade-off between structural rigidity and resource use efficiency. Lignin plays a central role in stem strength and lodging resistance in cereals [50,51,52,53,54,55]. Therefore, decreased lignin deposition could theoretically increase susceptibility to lodging under field conditions. Nevertheless, Si deposition in the cell wall can partially compensate for reduced lignification by forming Si–cellulose complexes that enhance mechanical strength [16,22,56]. In this context, Si may contribute to maintaining stem stability while reducing the metabolic cost associated with lignin biosynthesis, potentially improving N use efficiency. However, because this study was conducted under hydroponic conditions, the implications of reduced lignin for lodging risk under agronomic environments remain speculative and warrant field validation.

The antioxidant capacity of both oat genotypes was also affected by N and Si application. Overall, the N-tolerant genotype exhibited higher free radical scavenging activity across all treatments, supporting its greater physiological resilience. Conversely, a decrease in phenols occurred in both genotypes following Si application under N-deficient conditions. This finding supports the notion that Si modulates the phenylpropanoid pathway under nutrient stress, as previously reported in barley under aluminum toxicity [51,52]. The reduction in MDA accumulation following Si application highlights its protective role against oxidative damage. Both genotypes benefited from Si supply, with reductions in lipid peroxidation of up to 40% under N deficiency and 23% under N sufficiency. These results are consistent with previous reports demonstrating that Si enhances membrane stability and mitigates ROS-induced oxidative damage by regulating both enzymatic and non-enzymatic antioxidant systems in different species under plants stressors [19,23,24,25,57,58].

Finally, the PCA provides a comprehensive framework linking these individual responses (Figure 6). Separation along PC1 was primarily driven by biomass production, N accumulation, lignin content, and antioxidant capacity, which were clustered with the N-tolerant genotype. In contrast, PAL and TAL activities, root elongation, shoot phenolics, and TBARSs were associated with the N-sensitive genotype. This indicates that N tolerance in oats is characterized by an integrated strategy combining efficient nutrient acquisition, structural reinforcement, and controlled oxidative status, whereas sensitivity is associated with heightened stress-responsive metabolism and oxidative imbalance. PC2 was largely influenced by Si accumulation and cellulose content, with an inverse relationship to lipid peroxidation. This axis highlights the structural–oxidative balance modulated by Si supply. Within each genotype, Si shifted treatment clusters toward traits associated with cell wall reinforcement and reduced oxidative damage under N deficiency. Thus, Si acts as a metabolic modulator that redirects carbon allocation and stabilizes cellular redox status, but its magnitude and direction depend strongly on the underlying genetic architecture.

Taken together, these results indicate that Si modulates the response of oat genotypes to N deficiency through an integrated network involving nutrient uptake, cell wall biosynthesis, and antioxidant defenses. Although the underlying mechanisms by which Si enhances plant performance under N-limited conditions remain to be fully elucidated, this study provides the first evidence of genotype-dependent Si effects in oats grown under N deficiency. Further research should be therefore focused on elucidating the metabolic and molecular mechanisms by which Si influences N uptake and assimilation, as well as primary and secondary metabolic pathways in oat plants.

4. Materials and Methods

4.1. Plant Growth Conditions and Yield Determination

Two oat (Avena sativa L.) commercial genotypes exhibiting contrasting nitrogen (N) tolerance were selected based on a preliminary hydroponic screening of ten commercial oat genotypes grown under different N levels (0.5, 5, and 10 mM N). The screening evaluated root dry weight, root length, and lipid peroxidation after 21 days of growth (Supplementary Figure S1). Based on their contrasting responses to N deficiency, cultivar AV4 was classified as N-sensitive, whereas cultivar AV6 was identified as N-tolerant.

Seeds were surface-sterilized in 2% (v/v) sodium hypochlorite for 15 min and germinated on moist filter paper for 10 days. Uniform seedlings were then transferred to 4.5 L pots (48 plants per pot) containing continuously aerated basal nutrient solution as described by Hoagland [59]. Plants were grown under hydroponic conditions for 14 days to allow for acclimation. After the acclimation period, N and Si treatments were applied. The experimental treatments consisted of N deficiency (0.5 mM N) and N sufficiency (4 mM N), supplied as NH_4_Cl (Merck, Darmstadt, Germany), combined with 0 or 2 mM Si supplied as Na_2_SiO_3_ (Merck, Darmstadt, Germany). The control treatment corresponded to N sufficiency without Si application (4 mM N + 0 mM Si). Plants were maintained under these N–Si treatments for 21 days. The total experimental period from germination to harvest was 45 days.

The experiment followed a completely randomized factorial design with three replicates per treatment. At harvest, shoots and roots were collected for growth and biochemical analyses. Subsamples intended for biochemical analyses were stored at −20 °C and −80 °C until use. For yield determination, shoots and roots were dried at 65 °C for 48 h to obtain dry weight (DW). Plant growth was further assessed by measuring the length of the longest shoot and root from 10 randomly selected plants per experimental replicate.

4.2. Nitrogen and Si Content

Total N content (g) in plant samples was determined using the Kjeldahl method described by Goyal et al. [60]. Briefly, samples were digested with concentrated sulfuric acid (H_2_SO_4_, from Sigma–Aldrich, Darmstadt, Germany) with a catalyst (SeCu, from Merck, Darmstadt, Germany). Following digestion, the mixture was alkalinized with sodium hydroxide (NaOH), liberating ammonia (NH_3_), which was distilled and collected in a boric acid (H_3_BO_3_) solution. The amount of ammonia was quantified by titration with standardized hydrochloric acid (HCl, from Sigma–Aldrich, Darmstadt, Germany). Nitrogen content was calculated based on the volume of acid consumed during titration. For Si (mg) determination, dried shoot and root tissues were digested with nitric acid (HNO_3_) at 70 °C for 5 h. After digestion, hydrofluoric acid (HF, 40%) and deionized water were added, and the mixture was left to stand overnight. The following day, boric acid (H_3_BO_3_, 2% w/v) was added, and the volume was adjusted to 25 mL with distilled water. Silicon was measured using flame atomic absorption spectrometry (FAAS, PinAAcle 500 Perkin Elmer, Waltham, MA, USA) at 251.6 nm, following the method described by Pavlovic et al. [61].

4.3. Structural C-Compounds Analyses

Lignin content (%) in dried shoot samples was quantified following the Brinkmann method [62]. Samples were sequentially washed with 80% methanol, 1 M sodium chloride (NaCl, from Winkler, Santiago, Chile), 0.5% Triton X-100, distilled water, and acetone, then dried at 80 °C for 48 h. The resulting pellet was incubated with 25% acetyl bromide (Merck, Darmstadt, Germany) at 70 °C for 30 min, and absorbance was measured in a spectrophotometer at 280 nm. Alkaline lignin was used as the calibration standard. Cellulose concentration (%) was determined by the methodology described by Foster et al. [63]. Cell wall material was isolated through sequential treatments with ultrapure water, ethanol, chloroform, and acetone, followed by drying at 65 °C. Cellulose was extracted from 5 mg of isolated cell wall material using concentrated sulfuric acid (H_2_SO_4_) at 90 °C for 1 h, and quantified via the phenol–sulfuric acid method by measuring absorbance at 490 and 493 nm. Hemicellulose (%) was estimated as the difference between total lignin and cellulose concentrations. To visualize lignin and cellulose distribution within plant tissues, fresh culm sections were stained with Safranin O (0.1%) and rojo Congo (0.5%), respectively. Laser Scanning Confocal Microscopy (CLSM; Olympus FV1000, Arquimed, Tokyo, Japan) was used for analyzing lignin and cellulose at excitation/emission wavelengths of 543/590 nm and 497/600 nm, respectively [64]. Image analysis was performed using Image Processing Software (FV10-ASW v0.200c; Olympus, Tokyo, Japan). Safranin and rojo Congo fluorescence intensity was expressed as Relative Fluorescence Units (RFUs), and average RFU values were calculated from multiple regions of interest within each image.

4.4. Plant Biochemical Analyses

For enzyme analyses (PAL and TAL), fresh leaf material was homogenized in extraction buffer (50 mM Tris-HCl, pH 8.5, 14.4 mM 2-mercaptoethanol, 5% w/v PVPP) and centrifuged (Eppendorf, Hamburgo, Germany). Total soluble protein was quantified using the Bradford assay [65]. PAL and TAL enzyme activities were determined following the method of Beaudoin-Eagan and Thorpe [66], using L-phenylalanine or L-tyrosine as substrates. After incubation at 40 °C for 60 min, reactions were stopped with 5 M hydrochloric acid (HCl), and product formation was measured spectrophotometrically at 290 nm (trans-cinnamic acid) and 333 nm (p-coumaric acid). Enzyme activities were expressed as nmol product mg^−1^ protein min^−1^.

Total soluble phenols in the shoot and root of oat plants were quantified spectrophotometrically at 765 nm using the Folin–Ciocalteu reagent, according to the Slinkard and Singleton method [67]. The concentration of total phenols (µg CAE g^−1^ FW) was determined using chlorogenic acid as a standard.

The antioxidant capacity (Trolox eq. mg g^−1^ FW) in oat genotypes was assessed using the method outlined by Chinnici et al. [68], using the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical and Trolox as a standard. The absorbance of samples was recorded at 515 nm with a spectrophotometer (Perkin Elmer, Waltham, MA, USA).

In fresh shoot and root samples, oxidative damage was assessed by measuring thiobarbituric acid reactive substances (TBARSs) and expressed as nmol malondialdehyde (MDA) g^−1^ FW according to the modified method of Du and Bramlage [69]. Absorbance was recorded at 532, 600, and 440 nm to correct for interference from TBARS–sugar complexes.

4.5. Data Analysis

All variables were checked for data quality, including outlier detection and assessment of distributional assumptions. When required, Box–Cox transformations were applied. Two-way ANOVA was used to evaluate the effects of genotype (N-tolerant and N-sensitive), treatment (N or Si levels), and their interaction, using Type II sums of squares. Significant effects were explored using Tukey-adjusted post hoc comparisons. Statistical significance was set at p ≤ 0.05. All analyses were performed in R. Principal component analysis (PCA) was conducted on centered and scaled variables related to growth, nutrient uptake, oxidative stress, antioxidant responses, and cell wall composition. Components explaining at least 80% of cumulative variance were retained, and the most informative variables were identified based on weighted squared loadings. PCA biplots were generated separately for each genotype, grouping samples by fertilization treatment or Si level and including 95% confidence ellipses. PCA was used as an exploratory tool, whereas ANOVA assessed inferential effects.

5. Conclusions

This study demonstrates that Si contributes to mitigating the responses of oat plants to N deficiency in a genotype-dependent manner, influencing oxidative stress, antioxidant response, and cell wall composition. The N-tolerant genotype showed superior growth, higher antioxidant capacity, and lower oxidative damage across treatments, reflecting its intrinsic ability to cope with limited N availability. Silicon application significantly reduced lipid peroxidation in shoots and roots of both genotypes, particularly under N deficiency, highlighting its protective role in maintaining membrane stability. In the N-sensitive genotype, Si promoted structural adjustments by increasing cellulose deposition and enhancing root antioxidant activity under N limitation, thereby partially compensating for its lower stress tolerance. Conversely, in the N-tolerant genotype, Si reduced lignin accumulation and downregulated TAL activity. These findings provide new insights into the interaction between N and Si in cereals, particularly in oats, and highlight the importance of considering genotypic variation when developing sustainable fertilization strategies. From an agronomic perspective, Si incorporation may represent a complementary strategy to mitigate the adverse effects of N limitation in cereal production systems, particularly by enhancing structural stability and oxidative stress tolerance under reduced N inputs. Therefore, integrating Si fertilization into nutrient management programs may improve oat resilience under limited N supply, contributing to more sustainable and resource-efficient production systems.