Silicon Enhances Rice Tolerance to Drought and Blast Disease Through Modulating ROS Accumulation and Stress-Related Genes

TL;DR

Silicon helps rice plants better handle drought and blast disease by balancing harmful oxygen molecules and activating protective genes.

Contribution

The study reveals how silicon improves rice stress tolerance through specific gene regulation and ROS management.

Findings

Silicon at 2–4 mM optimally improves rice tolerance to drought and blast disease.

Silicon activates genes like OsNCED3, OsDREB2A, and OsCatB to enhance drought resistance via ABA signaling.

Silicon boosts blast resistance by activating defense genes and improving ROS-scavenging capacity.

Abstract

Silicon (Si) serves as a beneficial element that enhances plant resistance to both abiotic and biotic stresses. Although its positive effects have been widely investigated, the molecular mechanisms by which silicon improves stress tolerance in rice (Oryza sativa L.) remain unclear. Here, we show that Si displayed an optimal improved effect at concentrations of 2–4 mM in hydroponic system, and Si enhanced rice tolerance to drought and blast disease by maintaining reactive oxygen species (ROS) homeostasis and reducing root cell damage. In addition, Si at 4 mM upregulated the ABA biosynthesis gene OsNCED3, stress- and ABA-responsive genes OsDREB2A and OsLEA5, as well as the catalase gene OsCatB, while suppressing the drought-responsive negative regulator OsWRKY5, thereby enhancing drought tolerance through an ABA-dependent signaling pathway. Si at 4 mM enhanced resistance to rice blast by activating defense-related genes OsPBZ1, OsPR10a, OsPR5 and OsWRKY45 while simultaneously boosting ROS-scavenging capacity. Collectively, our results demonstrate that Si enhances rice tolerance to drought and blast disease through the coordinated modulation of ABA signaling, ROS homeostasis, and stress-related gene expression.

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Full text

1. Introduction

Silicon (Si) is the second most abundant element in soil, primarily existing in the form of aluminosilicates and quartz (silicon dioxide), which account for approximately 75% to 95% of the inorganic matter in soil [1]. As a non-essential yet beneficial element that significantly promotes plant tolerance to multiple environmental stresses, Si is primarily absorbed by plant roots in the form of an uncharged, non-reactive monomeric species, Si (OH)4, under physiological pH conditions [2,3]. The Si content varies considerably among different plant species, ranging from 0.1% to 10% of shoot dry weight. Rice is a typical Si-accumulating crop, exhibiting remarkable capacity for Si uptake and accumulation [4,5]. Studies have shown that Si application during the adult stage of rice growth leads to high Si accumulation in various tissues, which significantly increases the tillers number, the primary branch number per panicle, the seed setting rate, and the straw yield; moreover, it further enhances grain yield by improving lodging resistance, stem strength, and leaf erectness [6,7]. Particularly during the panicle reproductive growth stage, Si application reduces water loss from the husk, resulting in higher hydrostatic pressure that promotes cell expansion. It also significantly increases the grain number per panicle and the 1000-grain weight, while enhancing photosynthetic efficiency without altering leaf area or whole-plant biomass [8]. Moreover, exogenous Si alleviates oxidative stress in wheat, maize, and tomato bud seedling by improving their defense mechanisms and antioxidant system, thereby accelerating seed germination under drought stress [9,10].

Si plays a crucial role in plant responses to both biotic and abiotic stresses. Under drought conditions, Si application significantly reduces the permeability of the cytoplasmic membrane and inhibits the production of highly toxic oxygen free radicals while activating H^+^-ATPase in the root plasma membrane, helping to maintain specific cellular functions by preserving cell wall osmotic pressure [7,11,12,13]. In addition, Si regulates the root-to-shoot ratio and enhances root water uptake capacity by modulating levels of polyamines (PAs) and 1-aminocyclopropane-1-carboxylic acid (ACC), thereby significantly improving drought tolerance in species such as wheat, barley, rice, maize, tomato, cotton, and oilseed rape [14,15,16]. It is worth noting that the role of Si in enhancing drought tolerance is independent of whether or not the crop is classified as a high Si accumulator [11].

Si regulates the biosynthesis of compatible organic solutes in plants under salt stress by increasing the content of proline and betaine, which help stabilize proteins and membrane structure, thereby alleviating the osmotic stress induced by salt stress [17,18]. Moreover, it helps maintain ion homeostasis by reducing sodium ion uptake and translocation, while promoting Na^+^ efflux in plants subjected to salt stress [19,20]. In rice, Si application leads to Si deposition in the root endodermis, forming a physical barrier that enhances cell wall lignification and suberization [21]. Si also promotes K^+^ uptake, improves the K^+^/Na^+^ selectivity in plant cells, and maintains cellular K^+^ homeostasis by upregulating the K^+^ transporter gene HAK1, thus mitigating ionic toxicity caused by salt stress [22,23]. Si supplement further supports plant growth under stress: on the one hand, it stimulates root development and enhances water and nutrient uptake under drought stress; on the other hand, it increases the biomass of aboveground parts of plants under salt stress and reduces the damage caused by salt stress on plants [24,25,26].

Furthermore, Si plays a pivotal role in mitigating nutrient imbalance stresses in plants. It enhances nitrogen (N) use efficiency by promoting amino acid remobilization [27], increases phosphorus (P) availability through chemical competition for soil sorption sites [28,29], and facilitates calcium (Ca) uptake by stimulating plasma membrane H^+^-ATPase activity [14]. Si also improves root function, thereby alleviating membrane lipid peroxidation and oxidative stress associated with potassium (K) deficiency [30,31]. Through these coordinated mechanisms, Si synergistically promotes the absorption and utilization of macronutrients such as N, P, Ca, and K. In the case of micronutrients, Si regulates their homeostasis by modulating root oxidation capacity, altering their distribution within plant tissues, and facilitating metal sequestration via coprecipitation or increased cell wall binding sites. Specifically, Si promotes iron (Fe) translocation to shoots to mitigate deficiency-induced chlorosis [32], ensures uniform manganese (Mn) distribution in the leaves to reduce localized toxicity [33], delays zinc (Zn) transport to the shoots [34], and enhances copper (Cu) binding sites, thereby decreasing Cu phytotoxicity [35].

Si enhances plant resistance to biotic stresses through several mechanisms. On the one side, Si promotes the deposition of silicates on the surface of plant cell walls and root systems, forming a strong silica layer that acts as a physical barrier, making plant surfaces more difficult to be invaded by fungal pathogens, including rice blast [24,36], sheath blight [37], and brown spot [21,38]. Si deposition also renders plant tissues more resistant to chewing and digestion by insects [39], including white-backed planthopper (WBPH) [40], brown planthopper (BPH) [41], and rice leaf folder (RLF) [42]. On the other side, Si induces biochemical defense responses in plants, which play a crucial role in resistance to disease stress [37]. Si application enhances the activity of disease resistance related enzymes such as chitinase, β-1,3-glucanase, peroxidase, and phenylalanine ammonia lyase (PAL), creating a chemical barrier that decomposes pathogen cell wall and inhibits their growth and spread [19,43,44,45]. Additionally, Si enhances direct defense against phloem feeders by promoting callose deposition, thereby impeding insect feeding. Beyond this direct effect, Si also initiates indirect defense by modulating herbivore induced plant volatiles (HIPVs) to enhance the attraction of natural enemies [46,47]. Moreover, Si modulates the synthesis and signaling of hormones such as salicylic acid (SA), jasmonic acid (JA), and ethylene (ETH), thereby activating the plant’s immune response network [48]. Interestingly, silicate anion also acts as a competitive anion in the soil, the availability of beneficial ions like sulfate and phosphate. This improves nutrient uptake and metabolic homeostasis, ultimately enhancing plant tolerance to biotic stresses [49,50].

Proper accumulation of Si is indispensable for normal plant growth, and Si uptake in rice is mediated by various types of Si transporters. The Si influx transporter, Lsi1, localized on the distal side of the exodermis and endodermis in roots, facilitates the passive absorption of silicic acid (H_4_SiO_4_) from the soil into the root epidermis and across cell membranes [4,51]. Subsequently, Lsi2, located on the proximal side of the exodermis, consumes energy to actively transport silicic acid out of root cells into the intercellular space, enabling Si to enter the xylem vessels [52,53,54]. Together, Lsi1 and Lsi2 enable the unidirectional transport of Si from the soil into the plant. The Casparian strip forms a dual-layer barrier that effectively prevents Si backflow from the stele, and Si transporters function optimally in cells equipped with this structure. It is noteworthy that the expression patterns and subcellular localization of Si transporters vary considerably across plant species [55,56]. Lsi6 is expressed in xylem parenchyma cells of leaf blades and leaf sheath. It is responsible for unloading Si from the xylem into the panicles and leaves, thereby mediating Si distribution in the shoot and promoting grain development. This process contributes to increased grain number per panicle and higher thousand-grain weight in rice [8,57,58]. GEN‘s research showed that the double-layered Casparian strips prevent Si from leaking back out of the xylem, helping maintain the Si concentration gradient. Furthermore, Lsi1 and Lsi2 synergize with the Casparian strips through their polar localization, forming an efficient Si uptake system in rice [55].

The absence of Si in standard Yoshida nutrient solutions maintains hydroponic rice plants in an unnatural, Si-deficient state [59]. Therefore, in this study, we investigated the physiological changes and ROS accumulation in rice under drought and rice blast disease, subject to different Si concentrations in the hydroponic condition. By understanding the responses of rice seedlings to varying levels of exogenous Si at both physiological and molecular levels, the findings reveal that Si application enhances ABA synthesis and acts dependently on the ABA signaling pathway. Concurrently, Si significantly enhances ROS-scavenging capacity, maintaining ROS homeostasis and thereby mitigating stress-induced oxidative damage. These results will help to accurately screen the optimal Si application rate for enhancing rice resistance to drought and rice blast.

2. Results

2.1. Main Text

2.1.1. Optimal Exogenous Silicon Concentration for Alleviating Drought Stress in Rice Seedlings

Exogenous Si at specific concentrations effectively mitigates the inhibitory effects of drought stress on rice seedling growth. To investigate the optimal Si concentration for enhancing drought tolerance, two-week-old seedlings pretreated with various Si concentrations were subjected to a 5-day drought treatment. Under drought stress, seedlings in the control group exhibited leaf wilting, curling, and yellowing (Figure 1A). Exogenous Si at concentrations ranging from 1 to 4 mM alleviated drought-induced damage in rice seedlings and promoted the recovery of leaf expansion under drought stress. Application of 4 mM Si significantly increased the survival rate of seedlings and reduced leaf water loss (Figure 1B,C). Under drought stress, chlorophyll content in leaves was significantly reduced compared to control plants, Si treatment within the 1–6 mM range significantly alleviated this reduction (Figure 1D). Similarly, shoot length and biomass were markedly inhibited by drought stress, but these traits were significantly improved by 1–4 mM Si under drought conditions (Figure 2A,B). Root growth inhibition caused by drought stress was partially alleviated by Si concentrations of 2–6 mM. Regarding root biomass, drought stress led to a significant decrease, and exogenous Si at 4–6 mM partially restored the reduction caused by drought when compared to the 0–2 mM treatments (Figure 2C,D). Furthermore, drought stress significantly suppressed photosynthesis and transpiration rates. Application of Si in the 1–6 mM range effectively alleviated the decline in photosynthesis under drought stress and promoted transpiration in leaves (Figure 2E,F). Collectively, these results indicate that exogenous Si at concentrations of 2–4 mM effectively alleviates growth inhibition in rice seedlings under drought stress.

2.1.2. Silicon Application Reduces ROS Accumulation to Enhance Drought Resistance in Rice Seedlings

Reactive oxygen species (ROSs) are produced and accumulate rapidly in plants exposed to drought stress, where they act as signaling molecules that activate downstream stress responses [60]. To evaluate the influence of Si on ROS accumulation under drought conditions, we employed 3,3-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining to visualize ROSs in the root tips. Under drought stress, a marked accumulation of ROS was observed in rice roots. Compared to drought-stressed plants without Si supplementation, application of 1 mM Si reduced ROS accumulation. Furthermore, treatments with 2 mM and 4 mM Si markedly decreased the accumulation of both H_2_O_2_ and O_2_^−^ in seedling roots compared to the untreated control. In contrast, Si at 6 mM had minimal effect on mitigating ROS accumulation (Figure 3A,B). Quantitative analysis confirmed that drought stress significantly elevated H_2_O_2_ and O_2_^−^ contents, while both 2 mM and 4 mM Si significantly lowered these levels, with the 4 mM treatment showing the most pronounced reduction (Figure 3C,D). Drought stress also substantially suppressed the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). However, these activities were significantly enhanced in plants treated with 2 mM and 4 mM Si compared to untreated control (Figure 3E–G). Similarly, the transcript level of ROS-scavenging genes, including OsCatB, OsAPX1, OsPOX1, were significantly upregulated under drought stress following silicon application, particularly at 2 mM and 4 mM Si (Figure 4A–C). As an osmotic regulator, proline protects protein and membrane structures under stress. Si treatment significantly increased the expression of the proline synthesis gene OsP5CR in rice seedlings, supporting more efficient ROS scavenging, which correlated with enhanced proline content and supported more efficient ROS scavenging (Figure 4D). To investigate Si-mediated protection against cellular damage under drought, Evan’s blue staining and absorbance quantification analysis were performed on seedling root tips. The results demonstrated that Si at 4 mM considerably reduced root cell damage compared to both the control and treatments with 1 mM or 6 mM Si (Figure 4E,F). Drought stress induces lipid peroxidation in cell membranes, leading to increased MDA content. Si applications from 1 to 6 mM consistently reduced MDA content in seedlings, indicating a significant decrease in oxidative damage (Figure 4G). These findings indicate that Si at 2–4 mM mitigates drought stress in rice seedlings by maintaining ROS homeostasis and reducing oxidative damage in root cells.

2.1.3. Silicon Treatment Upregulates the Expression of Stress Response Genes

To assess the effect of Si on stress-responsive gene expression under drought, the transcript levels of several key genes were analyzed by RT-qPCR. Abscisic acid (ABA) is a well-established hormone in plant stress responses [40]. Under drought stress, Si treatment markedly upregulated the expression of three stress- and ABA-responsive genes, including OsNCED3, OsDREB2A, and OsLEA3, which contribute to drought tolerance in rice (Figure 5A–C). In contrast, the expression of the negative drought regulator OsWRKY5 was elevated under drought stress relative to the control. However, application of Si at 2, 4, and 6 mM downregulated OsWRKY5 expression, with the lowest expression level observed at 4 mM under drought conditions (Figure 5D). These findings indicate that Si application at 2–4 mM enhances drought tolerance at the seedling stage by modulating the expression of stress-responsive genes through ABA-dependent mechanisms.

2.1.4. Silicon Application Improves Resistance to Rice Blast Disease in Seedlings

In addition to enhancing abiotic stress tolerance, Si supplementation has been shown to improve resistance against biotic stresses in rice. To identify the optimal Si concentration for blast resistance, two-week-old seedlings grown under varying Si regimes were spray-inoculated with M. oryzae isolate Guy11 (Figure 6A). Seven days after spray inoculation, the phenotypic identification of leaf blast was evaluated based on a standardized scoring system where grades 0–2 represent resistant phenotypes and grades 3–5 indicate susceptibility [61]. Phenotypic analysis revealed that, without the application of silicon, seedlings exhibited typical susceptible responses, with a high proportion of leaves displaying grades 3–5. However, Si application significantly increased the number of leaves rated as grade 0 or 1, while substantially reducing lesion counts compared to the control. At 4 mM Si, fewer infected leaves developed grade 5 and more were classified as grade 0 relative to the 2 mM treatment. However, 6 mM Si resulted in a higher lesion counts and more grade 5 infections than the 2 mM and 4 mM treatments, indicating greater susceptibility to rice blast (Figure 6A,B). To further investigate the effect of Si on rice blast susceptibility, punch inoculation was performed on seedling leaves. Seven days after inoculation, seedlings without Si application exhibited large lesions developed around the inoculation sites, while Si treatment reduced lesion area, with the smallest lesions observed at 4 mM Si compared to the control (Figure 6C,D). To quantify fungal biomass in inoculated leaves following different Si treatments, total DNA was extracted from spray-inoculated seedling leaves and analyzed by quantitative PCR. The results showed that fungal DNA accumulation was lowest in seedlings treated with 4 mM Si (Figure 6E). Several pathogenesis related proteins, OsPBZ1, OsPR10a, and OsPR5, were used as immune markers to evaluate rice disease resistance, and OsWRKY45 was also proven to positively regulate blast resistance [62,63]. RT-qPCR analysis showed that under blast infection, the expression levels of these defense-related genes were induced in seedlings without Si supplement. Si application markedly upregulated the expression of OsWRKY45, OsPBZ1, OsPR10a, and OsPR5 (Figure 6F–I). Our findings demonstrate that 4 mM Si effectively enhances blast resistance in rice seedlings.

2.1.5. Silicon Application Reduces ROS Accumulation in Seedling Leaves After Blast Infection

Cellular ROS plays a key role in both Pattern-Triggered Immunity (PTI) and effector-triggered immunity (ETI) in plant immune responses, and the activation of plant immune responses is usually accompanied by a rapid ROS burst [64]. We first detected H_2_O_2_ levels in the leaves inoculated with isolates Guy11 using DAB staining. The results showed that, upon blast infection, H_2_O_2_ accumulation was markedly induced in leaves of seedlings without Si treatment, as revealed by DAB staining. Si application significantly reduced H_2_O_2_ content after infection, with the most pronounced effects observed at 2 mM and 4 mM Si. However, the plants treated with 6 mM Si exhibited noticeably stronger staining intensity than those treated with 4 mM Si (Figure 7A). We further quantified H_2_O_2_ and O_2_^−^ contents in infected leaves. The results showed that the accumulation of H_2_O_2_ and O_2_^−^ in Si-treated seedlings was lower than that in the untreated control group. Specifically, 2 mM Si markedly reduced O_2_^−^ content in infected leaves (Figure 7B,C). Additionally, we evaluated the activities of antioxidant enzymes SOD and POD and CAT in the blast-infected leaves under different Si treatments. In the control group, infection suppressed the activities of these antioxidant enzymes. Compared to the control, Si application led to a substantial increase in activities of SOD, POD and CAT, enhancing ROS-scavenging capacity in infected tissues (Figure 7D–F). Consistent with these findings, the expression levels of ROS-scavenging enzymes OsCatB, OsAPX1, and OsPOX1, along with the proline synthase OsP5CR, were upregulated under blast infection with Si treatment (Figure 7G–J). Collectively, these results indicate that Si applied at 2–4 mM reduces blast-induced ROS accumulation in rice seedlings.

3. Discussion

Rice, as a typical Si-accumulator crop, has been shown in previous studies to benefit from Si application during the seedling stage, which significantly promotes root development stress resistance [4,16,52]. However, the specific concentration range and underlying physiological mechanisms through which Si application improves drought and blast resistance in rice seedlings remain unclear. To address this knowledge gap, we established a wide gradient of Si treatments to evaluate its protective effects under both drought stress and rice blast infection. Our results provide new insights into the role of Si in enhancing drought tolerance and blast resistance at the seedling stage.

ROS regulates diverse physiological processes in plants, including growth and development, gene expression, programmed cell death, and responses to multiple abiotic and biotic stresses. Under abiotic stresses, ROS such as hydrogen peroxide, singlet oxygen, superoxide anion, and hydroxyl radicals are rapidly generated, initiating critical signaling cascades and oxidative responses [65]. While H_2_O_2_ acts as an important signaling molecule, its over-accumulation causes oxidative damage to cellular structures and inhibits root hydraulic conductance. To counteract ROS, plants employ an integrated antioxidant system involving enzymes such as SOD, POD, CAT, and APX. Among these, APX shows high affinity for H_2_O_2_ and enables fine-tuned regulation, whereas CAT helps maintain H_2_O_2_ homeostasis under stress [41]. Si application under drought stress has been shown to effectively mitigate oxidative damage, suppress the formation of suberin lamellae structures in roots induced by H_2_O_2_, and disrupts the hydrophobic barrier of the root system, thereby enhancing water permeability [11,12]. Si effectively mitigates oxidative damage in plants under salt and drought stress by regulating the enzymatic antioxidant system [66]. This process helps prevent membrane lipid peroxidation triggered by MDA accumulation, protects the structural and functional integrity of cell membranes, reduces membrane permeability, and enhances root water uptake capacity [67,68]. Furthermore, recent studies reveal that plasma membrane intrinsic proteins (PIPs) regulate immune responses in various plants by mediating H_2_O_2_ transport. In Arabidopsis, flg22-induced phosphorylation of AtPIP2;1 activates H_2_O_2_ influx into guard cells to trigger the stomatal defense [69]. Similarly, OsPIP2;2 in rice and TaPIP2;10 in wheat facilitate the transport of apoplastic H_2_O_2_ into the cytoplasm, thereby conferring resistance to fungal and bacterial pathogens [70,71].

Our results revealed that supplementation with 2–4 mM Si in hydroponic cultures systems significantly enhanced the activities of ROS-scavenging enzymes SOD, POD and CAT in seedling leaves under both drought stress and rice blast infection (Figure 3 and Figure 7). DAB and NBT staining experiments further confirmed that Si reduced the accumulation of H_2_O_2_ and O_2_^−^ in roots under drought stress, as well as the H_2_O_2_ levels in leaves with rice blast infection. Notably, this protective effect was observed only under stress conditions, which is consistent with previous studies indicating that Si supplementation does not markedly alter plant metabolic activity or physiological function in the absence of stress [65]. Additionally, this study found that under drought and rice blast infection conditions, Si application under stress significantly upregulated the expression of ROS-scavenging genes OsAPX1, OsCatB, and OsPOX1, together with the proline synthesis gene OsP5CR, consistent with elevated SOD, CAT, and POD activities (Figure 4A–D and Figure 7G–J). These findings further supported that Si application enhances plant tolerance under both abiotic and biotic stress by boosting ROS-scavenging capacity. In addition to enhancing enzymatic antioxidants, Si also strengthens the non-enzymatic defense system by modulating key components such as glutathione (GSH) and ascorbic acid (ASC) [16]. Under drought stress, Si application elevates glutathione reductase (GR) activity and ASC content in wheat leaves. On the one hand, Si upregulates major enzymes involved in the ascorbate–glutathione cycle, including glutathione reductase (GR), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR), and glutathione synthetase (GS), which improves the efficiency of the antioxidant recycling system. On the other hand, Si enhances the transcription of key enzyme genes in the flavonoid biosynthesis pathway, such as PAL, CHS, F3H, DFR, and ANS, thereby strengthening the non-enzymatic antioxidant capacity and ultimately alleviating drought-associated oxidative damage [66]. Collectively, by enhancing antioxidant capacity, Si application helps maintain ROS homeostasis in plants under stress conditions.

Plants activate complex signaling pathways in response to biotic and abiotic stresses, coordinating a wide range of metabolic and developmental processes. Among these, ABA signaling plays a central role in stress adaptation. Drought stress triggers ABA-dependent signaling plays a central role, leading to the activation of transcription factors that enhance drought resistance mechanisms. Specifically, drought stress markedly upregulates the expression of ABA biosynthesis metabolic genes OsNCED3 and catalase OsCATB [72]. ABA treatment also suppresses the expression of OsWRKY5, and loss-of-function mutations in OsWRKY5 and the R2R3-type MYB transcription factor OsMYB2 enhance plant sensitivity to ABA, leading to of multiple stress- and ABA-responsive genes such as OsLEA3, OsDREB2A, OsRAB16A, collectively improving drought tolerance [73]. Notably, dehydration-responsive element binding protein (DREB2A) participates in regulating the synthesis of ROS-scavenging enzymes and unfolded protein degradation enzymes [74]. The NCED gene family regulates water stress sensitivity in plants and shows induced expression under osmotic stress. In rice, members such as OsNCED3, OsNCED4, and OsNCED5 participate in ABA biosynthesis and contribute to enhanced abiotic stress tolerance [75]. Research by Liu et al. demonstrated that Si application in tobacco enhances ABA levels by modulating the expression of genes involved in ABA biosynthesis and metabolism, thereby improving plant adaptation to abiotic stress [76]. Specifically, Si upregulates key ABA biosynthesis-related genes, leading to increased endogenous ABA content. This elevated ABA level helps alleviate hydrogen-peroxide-induced impairment of aquaporin function. Concurrently, Si enhances the expression of aquaporin genes, which improves root hydraulic conductivity (Lpr) and helps maintain leaf water balance. Collectively, this regulatory pathway enhances plant tolerance to salt and drought stress [77,78]. In rice, Si application has been shown to induce the upregulation of genes such as DREB2A, OsNAC5, OsRDCP1, and OsRAB16b. Among these, elevated OsDREB2A expression contributes to improved drought tolerance [79,80]. Furthermore, Si regulates NAC transcription factors, including OsNAC5, which function through ABA-dependent signaling pathways to enhance stress tolerance [81,82]. Under drought conditions, loss-of-function of OsWRKY5 promotes ABA-dependent stomatal closure, thereby improving drought resistance in rice [73]. Our study found that Si application during the seedling stage significantly increased the expression of OsNCED3, OsDREB2A, OsLEA3, and OsCatB under drought conditions, while downregulating the drought stress negative regulator OsWRKY5 (Figure 5). This indicates that Si promotes ABA synthesis in rice under drought stress and regulates osmotic stress tolerance through ABA-dependent mechanisms. Our findings confirm that Si enhances osmotic stress tolerance in rice through ABA-mediated pathways. Despite these findings, the interactions between plant hormones and Si under stress remain poorly understood. Further omics analyses are required to elucidate how Si initiates these stress responses and coordinates metabolic balance between stress adaptation and growth-related pathways.

The thylakoid membrane is a fundamental structure for photosynthesis in plants, exhibits high sensitivity to environmental changes. The thylakoid membrane dynamically adjusts its structure and function in response to stress to maintain efficient light energy conversion. Si supplementation preserves chloroplast structural integrity and increases the number of thylakoid membrane protein complexes, thereby enhancing chlorophyll content. Consequently, Si improves light capture and energy transfer efficiency in crops under salt and drought stress [83]. Si serves as a key structural component of cell walls, significantly enhancing stem mechanical strength, promoting chlorophyll synthesis, and maintaining leaf erection under non-stress conditions [59]. Our findings confirm that Si application alleviates chlorosis in hydroponic rice seedlings. Specifically, rice seedlings grown in a Si-deficient nutrient solution exhibited chlorotic symptoms and reduced stem strength when subjected to drought stress (Figure 1A). In contrast, treatments with Si ranging from 2 to 4 mM markedly increased chlorophyll content and survival rate in rice seedlings (Figure 1B,D). Furthermore, under drought stress, Si at concentrations of 1 to 4 mM substantially mitigated stress-induced declines in photosynthetic and transpiration rates (Figure 2E,F), consistent with earlier reports [16], which suggested that Si plays a crucial role in the response of plants to abiotic stress. These improvements are attributed to Si’s role in promoting leaf water status, photosynthetic efficiency, and mineral nutrient uptake. Nevertheless, the molecular mechanisms through which Si mediates enhanced photosynthetic performance under abiotic stress require further clarification.

In summary, our study demonstrates that Si enhances drought resistance in rice seedlings by maintaining ROS homeostasis, leading to mitigation of oxidative damage in root cells, and regulating stress-responsive genes to enhance drought tolerance in rice seedlings. Furthermore, Si application enhances ROS-scavenging capacity and reduces ROS accumulation in the leaves infected with blast fungus M oryzae, thereby improving blast resistance in hydroponically grown seedlings. These findings provide critical concentration guidelines for future field production. Our study advances the understanding of physiological mechanisms through which Si confers resilience to both drought and disease in rice seedlings, and offers a theoretical basis for Si fertilizer application in rice cultivation. Further studies are required to elucidate the molecular mechanisms by which Si regulates the response of rice seedlings to biotic and abiotic stresses, particularly its concentration-dependent regulatory effects. Such insights will facilitate precision management of Si fertilization to effectively promote rice growth and development under diverse environmental conditions.

4. Materials and Methods

4.1. Plant Materials and Growth Conditions

The rice subspecies japonica cultivar Zhonghua11 (ZH11) was used in this experiment. Selection of filled ZH11 rice seeds for sterilization with sodium hypochlorite, the plants were hydroponically cultured in normal Yoshida nutrient solution [84]. The plants were hydroponically cultured in standard rice culture solutions without silicate and grown in the incubator at 28 °C for 16 h (day) and 25 °C for 8 h (night) until three-leaf and one-tiller stage. The nutrient solution was replaced every three days. Each treatment group consisted of 48 seedlings.

4.2. Silicon and Drought Treatment

Rice plants at the three-leaf and one-tiller stage were treated with different concentrations of exogenous Si (Na_2_SiO_3_·9H_2_O, 0, 1, 2, 4 and 6 mM) for one week. Plants were transferred to hydroponic solutions containing both varying concentrations of exogenous silicon and 15% polyethylene glycol (PEG-6000) for five days to simulate drought stress, and then the samples were collected for phenotypic analysis, physiological assays, and statistical analysis. Seedlings that resume growth, with leaves regreening and unfurling, are counted as surviving plants. The survival rate is calculated as the number of surviving plants divided by the total number of treated plants. To determine the rate of water loss, the fully expanded penultimate leaves of seedlings treated with different concentrations of Si were placed on filter paper under laboratory conditions (22 °C and 25–35% humidity). Fresh leaf weight was recorded at 30, 60, 120, and 240 min after cutting. Water loss rate was calculated as a percentage of the initial fresh weight.

4.3. Silicon and Rice Blast Fungus Resistance Evaluation

The M. oryzae isolate Guy11 was used to evaluate the blast disease resistance and the method of spray-inoculation applied was described as previously with some modifications [85]. Rice seedlings were treated with different concentrations of Si until the three-leaf and one-tiller stage for one week. For spraying inoculation assays, the surface of rice leaves was equably sprayed with a conidial suspension containing 5 × 10^5^ spores/mL and 0.02% Tween 20. The inoculated seedlings were then placed in a transparent plastic box under high temperature and high humidity conditions for 24 h to facilitate infection, followed by a cycle of 8 h of darkness and 16 h of light for an additional week. For spray-inoculated leaves, samples were collected to calculate relative fungal biomass. DNA was extracted from infected leaves, and fungal biomass was quantified via qRT-PCR using the rice gene OsUBQ as an internal reference. Punch Inoculation was performed on four-week-old seedlings using a 10 μg/mL 6-benzylaminopurine (6-BA) solution with pH 7.0. The solution was dispensed into sterile Petri dishes. Each punch spot was inoculated with 5 μL of a spore suspension (5 × 10^5^ spores/mL). Following inoculation, the leaves were placed in the 6-BA solution and incubated in darkness at 25 °C. After 24 h, the treatment was switched to a 16 h light/8 h dark cycle. Lesion area was quantified using ImageJ 7 days post-inoculation.

4.4. RNA Extraction and Quantitative Real-Time PCR (qRT-PCR) Analysis

Tissue fragments of rice seedlings treated with drought and rice blast were used for RNA extraction and gene expression analysis. Total RNA was isolated using Trizol reagent (15596026CN, Invitrogen, Carlsbad, CA, USA). RNA samples were reverse-transcribed using HiScript III All-in-one RT SuperMix Perfect (R333, vazyme, Nanjing, China). qRT-PCR was performed on a QuantStudio 1 Plus (Thermo Fisher, Shanghai, China) using ChamQ Blue Universal SYBR qPCR Master Mix (Q312, vazyme, Nanjing, China). Each treatment included three biological replicates. Relative gene expression was calculated using the 2^−∆∆Ct^ method, with the rice OsActin gene acting as an internal reference for normalization. The primers for qPCR are listed in Supplementary Table S1.

4.5. Determination of MDA Content and SOD, POD, CAT Activity

Leaf samples were used to measure malondialdehyde (MDA) content and superoxide dismutase (SOD) activity. MDA content was quantified using an MDA assay kit (Solarbio, BC0020, Beijing, China) according to the manufacture instructions. The seedling leaves (0.1 g) were grinded with 100 μL extraction solution, and the supernatant was removed by centrifugation at 4 °C for 10 min. Reagents were added in steps and a blank control group was included. The reaction solution was incubated in a 100 °C water bath for 60 min, and then centrifuged at 10,000× g for 10 min. The absorbance of the supernatant was measured at 532 nm and 600 nm. MDA content was calculated according to the formula provided in the instruction manual. Each sample was analyzed with three technical replicates.

SOD activity was measured using the SOD assay kit (Biosharp, BL903A, Hefei, China), based on the colorimetric measurement of WST-8 product formation. The leaves (0.1 g) were ground with 1 mL of pre-cooled PBS. The homogenate was centrifuged at 4 °C, and the supernatant was incubated with reaction reagent at 37 °C for 30 min. Absorbance was measured at 450 nm, and SOD enzyme activity was calculated according to the provided with the assay kit. Each sample was analyzed with three technical replicates.

POD activity was measured using the POD assay kit (KTB1150, Abbkine, Wuhan, China). The leaves (0.1 g) were ground with pre-cooled Extraction Buffer, and the supernatant was taken after centrifugation. Absorbance was measured at 460 nm. POD enzyme activity was calculated according to the provided with the assay kit.

CAT activity was measured using the CAT assay kit (KTB1040, Abbkine, Wuhan, China). The leaves (0.1 g) were ground with 1× Lysis buffer, the supernatant was taken after centrifugation. Absorbance was measured at 540 nm. CAT enzyme activity was calculated according to the provided with the assay kit.

4.6. Determination of Photosynthetic Rate, Transpiration Rate, and Chlorophyll Content

The photosynthetic rate and transpiration rate were measured in the morning under strong indoor lighting conditions using a Li-6400 Portable Photosynthesis System (Licor, Lincoln, NE, USA). After calibrating the instrument, the measurements were taken on fully expanded leaves that were undamaged and of a uniform length, avoiding the position of the leaf vein.

Chlorophyll content was determined using a spectrophotometric method with 95% ethanol extraction. The leaves (0.1 g) were cut into small pieces and grinded with 95% ethanol. The homogenate was transferred to a 10 mL centrifuge tube and incubated in the dark for 3 h. The mixture was shaken periodically to ensure complete decolorization of the tissue. The supernatant was diluted to 25 mL with 95% ethanol, and absorbance was measured at 665 nm and 649 nm. The chlorophyll content was calculated according to the following formula: Chlorophyll a (mg/L) = 13.95 × A_665_ − 6.88 × A_649_, Chlorophyll b (mg/L) = 13.95 × A_665_ − 6.88 × A_649_. Total Chlorophyll content = Chlorophyll a + Chlorophyll b.

4.7. DAB, NBT and Evan’s Blue Staining

DAB staining was used to localize H_2_O_2_ in tissues. Leaves or root tips were immersed in 1% DAB staining and incubated in the dark for 12 h. After incubation, samples were rinsed five times with distilled water, treated with tissue decolorizing solution, and heated in an 80 °C water bath for 40 min. The samples were then transferred to tissue preservation solution for 30 min. For NBT staining, used to detect O_2_^−^ localization, root tips were excised and immersed in 0.1% NBT solution (pH 7.8). The samples were evacuated for 30 min and then incubated at room temperature for another 30 min. Decolorization was performed using 95% ethanol in an 80 °C water bath. For Evan’s blue staining, rice seedling roots were washed and then immersed in 0.25% Evans Blue solution (SL7200, Coolaber, Beijing, China) diluted with PBS for 24 h. After staining, the roots were rinsed with water to remove excess dye and then boiled in anhydrous ethanol: glycerol (9:1) for 30 min before being photographed. For quantification of cell damage, the stained roots were excised and immersed in 1% SDS solution for 1 d. The absorbance of the extracted solution was measured at 600 nm, using the most intensely stained sample as a control. Images of DAB-, NBT- and Evan’s blue-stained tissues were captured using a stereoscopic microscope (Nikon DS-Ri2).

4.8. Statistical Analysis

Data are presented as mean ± standard deviation (SD). Statistical analysis was determined by one-way ANOVA with Tukey’s multiple comparison test. The physiological activity measurements were based on at least three independent biological replicates for each experiment and biomass was calculated.

5. Conclusions

Our study demonstrates that Si improves drought and blast resistance in rice seedlings by modulating ROS homeostasis- and stress-related gene expression. These findings provide direct evidence that Si application at optimal concentrations alleviates root cell damage and enhances drought tolerance through the regulation of ABA biosynthesis and stress-responsive genes. Simultaneously, it upregulates defense-related genes and strengthens ROS-scavenging capacity, leading to increased resistance to rice blast.