Potassium Silicate Supplementation Accelerates Recovery from Combined Salinity–Waterlogging Stress in Maize

Abstract

In reclaimed and poorly drained soils, combined salinity–waterlogging stress markedly inhibits the early vegetative growth of maize. In this study, maize seedlings at 12 days after sowing (DAS) were subjected to combined stress by immersing the entire root system in 200 mM NaCl for 7 d (stress; ST), then transferred to recovery conditions and supplied potassium at equivalent activity (5 mM K⁺; soil drench) as KH₂PO₄ (ST + K + P), K₂SO₄ (ST + K + S), and potassium silicate (ST + K + Si) at 0 and 5 days after treatment (DAT). Morphological traits, chlorophyll fluorescence, and gas-exchange parameters were measured at PreTR (immediately after stress termination), 5 DAT, and 10 DAT. Phytohormone, mineral nutrient profiles, oxidative stress markers and redox status, osmotic and metabolic parameters, and the expression patterns of key ion transport and stress-responsive genes were quantified at 0 and 10 DAT. The effects of K supplementation were evident across the growth- and photosynthesis-related indicators. Treatment groups (ST + K + Si, ST + K + S, and ST + K + P) exhibited significantly higher carbon fixation capacity than ST at 10 DAT. The Na/K ratio was also notably reduced in all K-supplemented groups, indicating that ionic homeostasis was restored with K supplementation through improvements in various stress response indicators such as phytohormones, osmotic adjustment, and antioxidant responses. The potassium- and silicon-treated group showed the greatest recovery effect, which may reflect the physiological characteristics of cereal species. Overall, these findings provide foundational data for the development of cultivation technology to expand the cultivation area of maize.

1. Introduction

Maize (Zea mays L.) is a major global crop, serving as a key source of food and livestock feed [1]. Although maize is well-adapted to various environments due to its C₄ photosynthetic traits [2], it is susceptible to abiotic stress such as drought, waterlogging, high temperatures and salt during its early growth stage [3]. This is because the root system and photosynthetic apparatus are not fully developed. In poorly drained soils like reclaimed lands, salinity and waterlogging stress frequently occur simultaneously [4]. Under these combined stresses, waterlogging impairs the exclusion of toxic Na⁺ from plants [5], and the accumulated salt further weakens the osmotic regulation ability of the roots already stressed by oxygen deficiency [6]. Consequently, the synergistic effect of these two combined stresses severely inhibits seedling growth, which directly affects final productivity and quality [7].

Under anerobic conditions caused by waterlogging, mitochondrial respiration is restricted [8], leading to a severe shortage of ATP [9]. This energy deficit compromises the maintenance of the plasma membrane potential, impairing the selective uptake of ions [10]. As a result, Na⁺ enters the cells more readily through non-selective cation channels [11], while K⁺ retention becomes increasingly difficult [12]. This leads to a rapid collapse of the cytosolic Na⁺/K⁺ ratio [13]. The ionic imbalance disrupts K⁺-dependent enzyme activities and stomatal regulation [14], while also aggravating cell membrane damage via lipid peroxidation [15]. These metabolic and ionic disruptions significantly undermine the seedling’s physiological integrity [16].

Supplying K⁺ during the recovery phase after combined salinity–waterlogging stress represents a viable strategy to restore ionic homeostasis. Plants maintain cytosolic ion balance through electrochemical gradients driven by plasma membrane H⁺-ATPase and a complex network of transporters [17]. Sufficient K⁺ stabilizes this transport system and the membrane potential, thereby mitigating stress-induced depolarization [18]. This stabilization effectively restricts Na⁺ influx and minimizes cytosolic K⁺ leakage [19]. Furthermore, K⁺ is also essential for maintaining turgor pressure and enzyme activities for nitrogen assimilation [20]. Collectively, proper K⁺ supplementation is helpful for restoring metabolic functions and alleviating physiological damage [20]. K⁺ is typically applied as a salt combined with various anions, such as phosphate, sulfate, silicate, nitrate or chloride in practical agricultural fields. For instance, phosphates like KH₂PO₄ replenish the inorganic phosphate (Pi) pool essential for ATP synthesis and membrane biosynthesis [21]. Sulfates like K₂SO₄ promote the synthesis of cysteine and glutathione via the sulfur assimilation pathway and strengthen the plant’s antioxidant capacity [22]. In contrast, although silicon is not classified as an essential element, silicates provide distinct structural advantages [23]. Silicon is deposited as silica (SiO₂·nH₂O) in cell walls [24]. It physically reinforces the root barriers and effectively reduces the apoplastic bypass flow of Na⁺ to the shoots [25]. Cereal crops like maize are particularly proficient at utilizing these transport mechanisms to maximize stress tolerance [26]. In addition, Si is also known to stabilize photosynthetic machinery [27] and modulate stress signaling by regulating the ethylene biosynthesis pathway [28].

However, most studies have predominantly focused on restoring Na⁺/K⁺ homeostasis [29], with limited attention paid to the specific physiological roles of the accompanying anions. Based on this background, the present study aimed to extend the analysis beyond simple assessments of Na⁺/K⁺ ratios. We investigated how different anions (phosphate, sulfate and silicate) accompanying K⁺ influence the physiological recovery following combined salinity–waterlogging stress. We hypothesized that each formulation would provide specific advantages in addition to the basic effect of K⁺ supply. To validate this hypothesis, we evaluated growth, ion balance, phytohormonal and metabolic adjustments, and antioxidant responses to determine the comparative efficacy of each treatment and identify the optimal strategy for stress recovery from combined salinity–waterlogging stress.

2. Materials and Methods

2.1. Plant Materials and Experimental Design

Maize (Zea mays L.) seeds were sown in plastic pots containing a commercial horticultural substrate and cultivated within a controlled growth chamber. The environmental conditions were strictly regulated at a day/night temperature of 28 °C/24 °C, a 16 h photoperiod, and 60% relative humidity. A total of 12 days after sowing (DAS), seedlings with fully expanded second leaves were screened for uniformity and selected for the experiment. This timing targets the fully autotrophic stage, which is highly susceptible to stress [30]. To prevent substrate displacement and ensure root stability during the waterlogging treatment, the soil surface was mulched with a 1 cm layer of weathered granite soil.

The experiment was designed as Scheme 1 to assess recovery from combined stress. The seedling root systems were fully immersed in a 200 mM NaCl solution [31] for a duration of 7 days. Preliminary tests confirmed this duration induces severe yet reversible damage, avoiding the lethality of longer exposures. Upon completion of this stress period (PreTR), the soil of the pots was thoroughly washed with distilled water to eliminate residual salinity. Subsequently, to evaluate the efficacy of different potassium sources for recovery, the plants were treated via soil drenching with one of the following solutions: potassium silicate (ST + K + Si; K₂O +SiO₂), potassium sulfate (ST + K + S; K₂SO₄), or potassium phosphate (ST + K + P; KH₂PO₄). All potassium salt treatments were normalized to provide an equivalent K+ activity of 5 mM. To prevent potential nitrogen deficiency during the rapid recovery phase, all treatment solutions were supplemented with 1 mM Ca(NO₃)₂·4H₂O. These treatments were applied at two time points: immediately upon stress termination (0 DAT) and 5 days post-treatment (5 DAT). A 10-day recovery period was selected to sufficiently evaluate physiological and morphological restoration.

2.1.1. Assessment of Morphological Recovery

Phenotypic recovery was quantified by measuring key morphological traits: plant height, stem diameter, and leaf width at PreTR, 5 DAR, and 10 DAT. Plant height was determined by measuring the distance from the soil line to the tip of the longest extended leaf. Stem diameter was recorded at the widest section of the stem base using digital vernier calipers. To assess lateral expansion, leaf width was measured at the broadest part of the second leaf.

2.1.2. Photosynthetic Gas Exchange and Chlorophyll Fluorescence

To evaluate the restoration of photosynthetic apparatus and function, physiological measurements were conducted on the second leaf at PreTR, 5 DAR, and 10 DAT. Gas exchange parameters, including net photosynthetic rate (A), stomatal conductance (g_s), intercellular CO₂ concentration (c_i), and transpiration rate (E), were quantified using a portable photosynthesis system [32] (LC-ProT, ADC BioScientific Ltd., Hoddesdon, UK). Simultaneously, chlorophyll fluorescence kinetics were assessed to determine the effective quantum yield of PSII (Φ _PSII), the fraction of open reaction centers (qL), and the maximum quantum yield of PSII (F_v/F_m) using a handheld fluorometer [33] (MultispeQ V2.0, PhotosynQ Inc., East Lansing, MI, USA). All measurements were performed under standardized environmental conditions to ensure data consistency.

2.1.3. Elemental Profiling via ICP-OES

For the analysis of ion content, 0.1 g of freeze-dried seedling tissue was subjected to acid digestion in 3 mL of concentrated nitric acid (HNO₃) at 90 °C for 3 h. The digested extracts were diluted to a final volume of 30 mL with deionized water and filtered through a 0.45 μm cellulose acetate syringe filter. The concentrations of key mineral elements (Na, K, P, Si, Mg, Fe, and Ca) were determined using an Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES, Thermo Fisher Scientific, Waltham, MA, USA). This quantification followed a standard acid digestion protocol validated for plant biological samples to ensure accuracy and reproducibility. Analyses were conducted at PreTR and 10 DAT.

2.1.4. Quantification of Endogenous Abscisic Acid (ABA)

At PreTR and 10 DAT, the quantification of endogenous ABA was conducted. Briefly, 0.5 g of freeze-dried tissue was extracted using a solvent mixture consisting of 95% isopropanol and 5% glacial acetic acid [34]. To validate quantification accuracy, 100 ng of ABA was added as an internal standard.

Prior to analysis, the extracts were reacted via methylation with diazomethane for derivatization. The samples were then analyzed using an Agilent 6890N Gas Chromatograph equipped with a 5973 Network Mass Selective Detector (Agilent Technologies, Palo Alto, CA, USA). The system operated in Selected Ion Monitoring (SIM) mode, detecting ions at m/z 190 and 162 for methyl-ABA (Me-ABA) and m/z 194 and 166 for the deuterated internal standard (Me-d₆-ABA). Data acquisition and processing were conducted using Lab-Base software (ThermoQuest, Manchester, UK).

2.1.5. Determination of Osmolytes: Total Soluble Sugars (TSSs) and Proline

Osmotic adjustment capabilities were assessed by monitoring total soluble sugars (TSSs) and proline accumulation at PreTR and 10 DAT. For TSS analysis, 80% ethanolic extracts were reacted with anthrone reagent (0.2% anthrone in 98% H₂SO₄) at 100 °C for 10 min, and absorbance was measured at 620 nm. TSS contents were calculated based on a standard curve ranging from 0.0 to 100.0 μg·mL⁻¹ D-glucose.

Free proline content was determined using a modified ninhydrin assay. An amount of 200 mg of dried plant tissue was extracted in 10 mL of 70% (v/v) ethanol at 95 °C for 20 min. After cooling to room temperature, the extract was centrifuged at 10,000× g and 4 °C for 10 min. A 0.5 mL aliquot of the supernatant was mixed with 1 mL of a reaction solution containing 1% (w/v) ninhydrin, 60% (v/v) acetic acid, and 20% (v/v) ethanol. The mixture was incubated at 95 °C for 20 min, cooled to room temperature, and vortexed. The absorbance was measured at 520 nm using a microplate reader, and proline concentrations were calculated based on a standard curve ranging from 0.04 to 1.0 mM L-proline.

2.1.6. Oxidative Stress Markers and Histochemical Staining

To evaluate oxidative damage and antioxidant capacity, several biochemical markers were analyzed at PreTR and 10 DAT. Hydrogen peroxide (H₂O₂) content was determined according to the method of Loreto and Velikova [35]. An amount of 50 mg of dried leaf tissue was homogenized in 0.1% (w/v) trichloroacetic acid (TCA), centrifuged, and the supernatant was mixed with 10 mM potassium phosphate buffer (pH 7.0) and 1 M KI. Absorbance was recorded at 390 nm. Superoxide dismutase (SOD) activity was determined by measuring the inhibition of the photochemical reduction in nitroblue tetrazolium (NBT) at 560 nm under 4000 lux illumination. The reaction mixture comprised 50 mM potassium phosphate buffer (pH 7.8), 13 mM methionine, 75 μM NBT, 100 μM EDTA, and 2 μM riboflavin. Peroxidase (POD) activity was assayed based on the rate of pyrogallol oxidation in the presence of H₂O₂, with the increase in absorbance monitored at 420 nm. The crude enzyme was reacted with 1% pyrogallol and 50 μM H₂O₂ in a 100 mM phosphate buffer (pH 6.8). Protein quantification for all enzyme activities was performed using the Bradford assay with bovine serum albumin (BSA) as a standard [36]. Non-enzymatic antioxidant capacity was assessed by measuring total phenolic content (TPC) using the Folin–Ciocalteu assay [37]. Ethanolic extracts were incubated with Folin–Ciocalteu reagent and Na₂CO₃, and absorbance was read at 750 nm. Lipid peroxidation was estimated by quantifying malondialdehyde (MDA) via the thiobarbituric acid (TBA) assay [38]. Extracts were heated with TBA/TCA solution at 95 °C, and absorbance was measured at 532 nm, with non-specific turbidity corrected at 600 nm.

Visual detection of reactive oxygen species (ROSs) was performed at 10 DAT. Superoxide anion (${\mathrm{O}}_2^{-}$) accumulation was visualized using Nitroblue tetrazolium (NBT) [39] and 3,3′-Diaminobenzidine (DAB) staining [40], respectively. Leaf segments were immersed in staining solutions containing 10 mM NaN₃ and incubated in the dark for 6 h. Chlorophyll was subsequently removed by boiling in 90% ethanol to enhance visualization. The cleared leaves were preserved in a 50% glycerin solution for imaging and analysis.

2.1.7. Gene Expression Analysis (qRT-PCR)

To validate physiological recovery at the molecular level, the expression patterns of key stress-responsive and ion transport-related genes (e.g., ZmADH1, ZmACS6, ZmHAK5, ZmSOS1, and ZmAHA2) were analyzed. Total RNA was isolated from leaf tissues pulverized in liquid nitrogen using the Biofact™ Total RNA Prep Kit (Biofact, Daejeon, Republic of Korea) according to the manufacturer’s instructions. The integrity and concentration of the extracted RNA were verified before proceeding to first-strand cDNA synthesis, which was performed using the Solg™ RT Kit (SolgBio, Seoul, Republic of Korea). The resulting cDNA served as a template for quantitative PCR, conducted on a CFX384 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The reaction mixture consisted of Solg™ 2× Multiplex Real-Time PCR Smart Mix containing SYBR Green to facilitate the detection of specific amplicons. The thermal cycling conditions were initiated with enzyme activation at 50 °C for 2 min and 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 30 s and a combined annealing/extension step at 60 °C for 1 min. To ensure the absence of non-specific amplification or primer-dimer formation, a melting curve analysis was performed following the final cycle.

Relative transcript abundance was quantified using the 2^−ΔΔCT method [41]. The target gene expression levels were normalized against the internal reference gene, ZmActin, to account for variations in template concentration. All assays were executed with three biological replicates, each comprising three technical replicates to ensure statistical robustness. The primer sequences utilized in this study are detailed in Supplementary Table S1.

2.1.8. Statistical Analysis

All experimental data were obtained from at least three independent biological replicates. Each biological replicate consisted of six individual pots, and technical replicates were incorporated during the biochemical assays. Data are presented as the mean ± standard error (SE). To evaluate statistical significance among treatment groups, a one-way analysis of variance (ANOVA) was performed, followed by Tukey’s Honestly Significant Difference (HSD) test to determine significant differences at p < 0.05. To comprehensively assess the multifaceted recovery patterns of maize seedlings, principal component analysis (PCA) and Euclidean distance calculations were conducted based on the integrated physiological, biochemical, and transcriptional datasets. All statistical analyses and data visualizations were performed using R Studio (version 4.5.1).

3. Results

3.1. Effects of Potassium Source Supplementation on the Recovery of Phenotypic Growth

Vegetative growth status is a fundamental indicator for evaluating plant stress adaptability [42] and potential productivity. To assess the efficiency of different potassium sources, the morphological traits of the maize seedlings, including plant height, stem diameter, and leaf width, were measured (Figure 1).

Initial measurements at PreTR revealed growth inhibition by combined stress. Compared to the NT, the ST seedlings showed a reduction of approximately 32.1% in plant height (Figure 1a), 27.5% in stem diameter (Figure 1b), and 31.1% in leaf width (Figure 1c). At 5 DAT, there were no statistically significant growth recovery effects observed by potassium treatment. Although the stem diameter growth rate of the ST + K + Si group was approximately 28.7% higher than that of the ST group, no statistically significant growth recovery was observed at 5 DAT. By 10 DAT, significant growth recovery effects were observed in volumetric parameters such as stem diameter and leaf width due to potassium salt supplementation. Stem diameters of all potassium-treated groups exceeded the ST baseline (10.28 mm). The ST + K + P, ST + K + S, and ST + K + Si groups showed increases of 11.3%, 9.4%, and 13.2%, respectively. In the case of leaf width, the recovery effect was most pronounced in the silicate-treated group. Compared to the ST group (3.38 cm), the ST + K + Si and ST + K + S groups exhibited increases of 9.2% and 3.6%, while the ST + K + P group showed no substantial improvement. While plant height showed no significant variation among the stress-exposed groups at 10 DAT, the silicate treatment with potassium resulted in statistically higher values for both stem diameter and leaf width compared to the ST.

3.2. Potassium Silicate Enhances Photosynthetic Recovery and Photochemical Efficiency During Stress Alleviation

Photosynthetic capacity serves as a primary indicator of plant health, as carbon assimilation provides the energy substrate for vegetative recovery [43]. Therefore, gas exchange parameters, including net photosynthetic rate (A), intercellular CO₂ concentration (c_i), and stomatal conductance (g_s), were quantified. Concurrently, chlorophyll fluorescence kinetics were analyzed to provide an immediate and sensitive diagnosis of the photosynthetic apparatus affected by various stresses [44]. The maximum quantum yield of PS II (F_v/F_m), effective quantum yield (Φ_PSII), and the fraction of open reaction centers (qL) were monitored to assess the structural integrity and electron transport efficiency of PS II under combined stress and recovery conditions (Figure 2) [45]. Combined stress severely impaired the photosynthetic apparatus. At PreTR, the net photosynthetic rate of ST was 0.95 μmol·m⁻²·s⁻¹, indicating a severe suppression of metabolic activity relative to NT (21.82 μmol·m⁻²·s⁻¹) (Figure 2a). This decline was accompanied by a drastic reduction in gs to 0.01mol·m⁻²·s⁻¹ (Figure 2b) and E to 0.18mmol·m⁻²·s⁻¹ (Figure 2c), alongside a decrease in photochemical efficiency as Φ_PSII dropped to 0.56 (Figure 2g). These results suggest that photosynthesis was primarily limited by both stomatal closure and structural damage to PS II.

Potassium salt supplementation reactivated the photosynthetic apparatus. By 5 DAT, all potassium salt-treated groups exhibited significantly higher photosynthetic rates (Figure 2a) than the ST group, at 9.72 μmol·m⁻²·s⁻¹. The recovery was initially most rapid in the ST + K + S group, at 15.07 μmol·m⁻²·s⁻¹, followed closely by the ST + K + Si group (14.48 μmol·m⁻²·s⁻¹). Consistently, the photochemical indicators in these groups showed an improvement at 5 DAT. Φ_PSII value was 0.731 in ST + K + S and 0.735 in ST + K + Si (Figure 2g). By 10 DAT, the recovery trends diverged, and the silicate-treated groups showed the highest efficiency. The net photosynthetic rate of the ST + K + Si group reached 10.48 μmol·m⁻²·s⁻¹, exceeding the NT control (10.12 μmol·m⁻²·s⁻¹), while the ST + K + P and ST + K + S groups were 9.69 μmol·m⁻²·s⁻¹ and 9.53 μmol·m⁻²·s⁻¹, respectively (Figure 2a). The silicate-treated group also maintained high photochemical efficiency, recording the highest Φ_PSII (0.649) and qL (0.736) among all treatment groups. Stomatal conductance and transpiration rate of ST + K + Si were 0.07 mol·m⁻²·s⁻¹ and 1.45 mmol·m⁻²·s⁻¹, respectively.

3.3. Potassium Supply Restores Na⁺/K⁺ Homeostasis Disrupted by Salt Stress

To evaluate ionic disruption induced by salt stress and the effectiveness of potassium salt treatments during recovery, the concentrations of major mineral elements (Na, K, P, Si, S, Ca, Mg, and Fe) were quantified at PreTR and 10 DAT, together with the Na⁺/K⁺ ratio as an integrative indicator of ion homeostasis (Figure 3). ICP-OES analysis verified the elemental uptake, notably showing significant Si accumulation in the ST + K + Si group compared to the ST control (Figure 3a). At PreTR, the ion homeostasis of the ST group was profoundly disrupted. The cytosolic Na⁺/K⁺ ratio in the ST group surged to 1.16, representing a marked imbalance compared to 0.01 in the NT control (Figure 3b). The spike was driven by a substantial accumulation of Na⁺, which reached 86.37 ppm in the ST group, whereas the NT control remained at 1.54 ppm. Conversely, the K⁺ concentration of the ST group decreased to 74.21 ppm, which was significantly lower than the 118.99 ppm observed in the NT control. By 10 DAT, all potassium salt treatments effectively lowered the Na⁺/K⁺ ratio compared to the ST group (0.48). The ST + K + S group recorded the lowest value of 0.16, mainly due to the highest K⁺ accumulation (135.19 ppm). The ST + K + Si and ST + K + P groups also maintained stable ion ratios of 0.24 and 0.25, respectively. While the phosphate-treated group showed the lowest Na⁺ concentration (19.05 ppm), the higher K⁺ uptake efficiency of the ST + K + S and ST + K + Si groups resulted in a more optimal ion homeostasis.

3.4. Potassium Silicate Modulates ABA Accumulation and Osmolyte Dynamics During Stress Recovery

To evaluate the biochemical responses during stress and recovery, the concentrations of ABA and key osmolytes, including proline and total soluble sugar (TSS), were quantified (Figure 4) [46]. At PreTR, the ST group exhibited a significant increase in all measured parameters compared to the NT control. The ABA concentration of the ST group surged to 149.81 ng·g⁻¹ DW, while the NT control remained at 139.93 ng·g⁻¹ DW (Figure 4a). This was accompanied by a proline content of 1.59 mM·g⁻¹ DW in the ST group, which was nearly three times higher than the 0.56 mM·g⁻¹ DW recorded in the NT control (Figure 4c). Similarly, the TSS content showed an increase in the ST group (57.53 mg·g⁻¹ DW) relative to the NT control (54.70 mg·g⁻¹ DW) (Figure 3b).

By 10 DAT, these biochemical indicators markedly decreased in all stressed groups. The ABA levels of the ST (28.16 ng·g⁻¹ DW) and ST + K + Si (27.19 ng·g⁻¹ DW) groups dropped significantly (Figure 4a), reaching levels substantially lower than the 115.38 ng·g⁻¹ DW of the NT control. Proline concentration also returned to near-basal levels (Figure 4c), with the ST + K + Si group (0.26 mM·g⁻¹ DW). The TSS content of the NT control maintained the highest level at 63.14 mg·g⁻¹ DW (Figure 4b). Among the potassium salt-treated groups, distinct differences were observed, with the ST + K + Si group maintaining the highest TSS content (49.57 mg·g⁻¹ DW), whereas the ST + K + S and ST + K + P groups recorded lower values of 44.36 mg·g⁻¹ DW and 38.24 mg·g⁻¹ DW, respectively.

3.5. Potassium Salts Mitigate Oxidative Damage and ROS Accumulation Under Stress

To evaluate the oxidative damage and antioxidant responses, the concentrations of malondialdehyde (MDA), hydrogen peroxide (H₂O₂), and total phenol content (TPC) were measured [47], alongside histochemical staining with NBT and DAB [48]. In addition, to link oxidative damage with enzymatic antioxidant capacity as suggested, the activities of SOD and POD were analyzed [49,50].

At PreTR, the ST group showed higher levels of MDA (137.44 μmol·g⁻¹ DW) and H₂O₂ (34.57 μmol·g⁻¹ DW) compared to the NT control, which recorded 107.63 and 31.52 μmol·g⁻¹ DW, respectively (Figure 5g,e). By 10 DAT, the H₂O₂ content of the NT control recorded the highest level at 45.20 μmol·g⁻¹ DW, while the ST group was at 42.87 μmol·g⁻¹ DW. Conversely, the potassium salt-treated groups maintained lower H₂O₂ levels, with the ST + K + P group showing the lowest concentration (33.79 μmol·g⁻¹ DW), whereas the ST + K + S and ST + K + Si groups recorded 35.64 and 36.70 μmol·g⁻¹ DW, respectively. Consistent with these ROS levels, the ST + K + P group showed the highest activities of SOD and POD among all treatments (Figure 5c,d). These enhanced activities led to MDA content of 85.74 μmol·g⁻¹ DW (Figure 5g), which was lower than the 108.58 μmol·g⁻¹ DW observed in the NT control. In contrast, the ST + K + Si group exhibited moderate enzyme activities and maintained MDA levels similar to the NT control.

These quantitative results were consistent with the histochemical observations, where the ST group exhibited more intense blue (NBT) and dark brown (DAB) precipitates in the leaf tissues (Figure 5a,b), indicating significant accumulation of ${\mathrm{O}}_2^{-}$ and H₂O₂. The intensity of staining was visibly reduced in potassium salt-treated groups compared to the ST group.

3.6. Transcriptional Regulation of Stress-Responsive and Ion Transport Genes

To validate the observed physiological recovery at the molecular level, the transcriptional changes in key marker genes associated with ion transport, anerobic metabolism, and stress signaling were analyzed (detailed with their functional integration in Supplementary Table S3) (Figure 6). ZmADH1 (alcohol dehydrogenase 1), a crucial enzyme in the fermentative pathway, serves as a primary indicator of waterlogging stress [51]. At 10 DAT, its expression showed a significant decline in all potassium salt-treated groups compared to the ST group. The ST + K + Si group exhibited the most pronounced reduction, with a decrease of 29% relative to the ST group. The ST + K + P and ST + K + S groups also showed reductions of approximately 28% and 17.5%, respectively. This decline indicates that the metabolic burden induced by hypoxic stress was effectively relieved following the combined stress termination and potassium supplementation.

The Na⁺ efflux gene ZmSOS1 (salt overly sensitive 1) encodes a plasma membrane Na⁺/H⁺ antiporter mediating active Na⁺ exclusion [52], while ZmAHA2 (plasma membrane H⁺-ATPase) provides the necessary proton motive force for this active ion transport. While the ST group maintained high transcriptional levels of ZmSOS1, the ST + K + Si group showed a marked suppression, achieving a reduction of 62.0% compared to the ST group. This down-regulation suggests that the rapid restoration of internal ion homeostasis by silicate treatment reduced the physiological necessity for activating the energy-intensive ZmSOS1 detoxification pathway. Furthermore, the expression of ZmAHA2 was increased by 17.6% in the ST + K + Si group compared to the ST control.

The transcriptional responses of the high-affinity K⁺ transporter ZmHAK5 and the ethylene biosynthesis gene ZmACS6 were also evaluated to assess nutritional status and senescence signaling. ZmHAK5 gene expression was significantly downregulated in all potassium salt-treated groups. The ST + K + Si group showed a reduction of 65.3% relative to the ST group. ZmACS6 gene expression remained stable in the silicate-treated group, showing a slight decrease of 3.5% compared to the ST group. This stability provides molecular evidence for the role of silicon in mitigating stress-induced senescence signals, thereby preventing premature leaf death during the recovery phase.

4. Discussion

4.1. Silicate Confers Coordinated Morphological and Metabolic Advantages During Recovery

The restoration of morphological traits serves as a primary indicator of recovery from the combined salinity–waterlogging stress [5]. While longitudinal growth recorded as plant height showed a gradual recovery, notable improvements were recorded in volume growth indicators such as stem diameter and leaf width, particularly in the ST + K + Si group. These results indicated that the plant prioritized structural thickening and lateral expansion in the short-term resumption. This morphological improvement is considered to provide the physiological foundation for the subsequent resumption of vertical growth. This morphological resilience is likely attributable to the superior photosynthetic performance observed in the silicate-treated plants.

In this study, differential responses to the various potassium salt treatments revealed distinct restoration patterns. While the ST + K + S group exhibited the lowest Na+/K+ ratio, this ionic advantage did not lead to vigorous growth. In contrast, the ST + K + Si group maintained higher photosynthetic efficiency, as measured by A and F_v/F_m, even surpassing the NT control by 10 DAT. This indicates that while sulfate is effective in literal ionic titration, silicate provides a more comprehensive defense through structural reinforcement [53]. The high efficiency in the ST + K + Si group is considered to have directly influenced the metabolic status, which is supported by TSS content [54]. This metabolic availability is thought to have served as a critical energy source for structural recovery and overall biomass production.

4.2. Potassium Silicate Resets ABA–Proline Signaling to Enable Growth Resumption

The physiological recovery effects are further supported by the synergistic regulation of ABA and proline [55]. While ABA levels typically increase in proportion to stress intensity [56], a contrasting trend was observed in this study. At 10 DAT, the ST + K + Si group exhibited a distinct hormonal readjustment, with the ABA levels declining significantly below the NT baseline. This sharp decline in the ABA is considered to indicate the release of the physiological brake imposed during the stress phase. This change is thought to have allowed the plants to transition effectively from defense to growth [57].

This physiological shift is considered to have minimized the metabolic burden typically associated with sustained stress defense. Consequently, this recalibration is thought to offer an opportunity for more efficient allocation of resources toward the resumption of growth. This coordinated response is interpreted as a primary reason why the silicate-treated plants exhibited a faster recovery in leaf and stem thickness compared to the phosphate or sulfate treatments. In addition, the stabilization of proline [58] is considered to confirm the restoration of water and ion status. Its reduction in the ST + K + Si group, coupled with low ABA levels, is likely to reflect that ionic and osmotic homeostasis was sufficiently restored, especially when compared to the higher proline levels maintained in the other potassium salt-treated groups.

The transcriptional responses provide molecular evidence for the distinct advantages of the silicate treatment. As evidenced by the lowest MDA and H₂O₂ levels, the phosphate treatment seems to be the most effective in directly scavenging reactive oxygen species (ROSs) [59]. The higher activities of SOD and POD in the ST + K + P group suggest a strong enzymatic defense that led to an excessive reduction in oxidative markers. While this strategy minimizes oxidative damage, it requires significant metabolic energy [60].

In contrast, the silicate treatment promotes a recovery mechanism that is more integrated and efficient in energy use [61]. Considering the overall physiological indicators, including growth, silicate can potentially alleviate stress by integrating biochemical antioxidant capacity with structural reinforcement [62]. Instead of simply maximizing enzyme activities, silicate treatment maintained a physiological balance closest to the NT control. This restoration of homeostasis supports superior systemic recovery and biomass accumulation, providing a more sustainable strategy for stress adaptation by optimizing the use of internal resources [63].

Specifically, the Si-mediated deposition in cell walls serves as a physical barrier that reduces Na+ influx [25], fundamentally limiting the initial induction of oxidative stress. This structural integrity contributes to the maintenance of a favorable Na⁺/K⁺ ratio, a key determinant of salinity–waterlogging recovery [7]. The silicate-treated group achieved this ionic balance through the coordinated regulation of ZmSOS1 and ZmHAK5, effectively optimizing K⁺ uptake while minimizing Na⁺ accumulation. The 62.0% reduction in ZmSOS1 expression in the ST + K + Si group suggests that internal ion homeostasis was achieved with high energy efficiency, as the physical barrier reduced the necessity for the energetically costly active exclusion of toxic Na⁺. Furthermore, the lowest expression of ZmADH1 in the ST + K + Si group reflects a rapid metabolic transition from anerobic fermentation back to aerobic respiration [64]. This implies that the silicate treatment allowed the plants to escape from the waterlogging stress more quickly than the other groups. Moreover, the stabilization of ZmACS6 suggests that the silicate treatment effectively suppressed signals related to leaf senescence, which is typically promoted by ethylene under stress conditions [65]. While the phosphate treatment was primarily effective in mitigating the oxidative burden, the silicate treatment synergistically integrated these molecular responses, leading to a more comprehensive restoration of the entire plant. In conclusion, the synergy between K+ and silicate is considered to provide the most efficient pathway for maize seedlings to overcome combined stress by balancing biochemical protection and structural resilience.

4.3. Integrated Physiological and Molecular Recovery Revealed by PCA

The PCA biplot (PC1: 43.57%, PC2: 29.63%) shown in Figure 7 provides a visual summary of the physiological and molecular shifts across the treatment groups at 10 DAT. Distinct clustering was observed, with the NT and ST groups positioned at opposite ends of the PC1 axis, highlighting the profound impact of the combined salinity–waterlogging stress. Notably, among the potassium treatments, the ST + K + Si group clustered closest to the NT group, indicating that the silicate treatment resulted in the most effective restoration of the overall physiological status. In contrast, the ST + K + S and ST + K + P groups showed a larger separation from the NT baseline, suggesting that while sulfate and phosphate provided partial mitigation, they were less effective in achieving a systemic recovery.

Vector analysis further corroborates the distinct advantages of the silicate treatment. Stress and damage indicators, including the Na⁺/K⁺ ratio, MDA, proline, and stress-responsive genes such as ZmSOS1 and ZmADH1, were strongly oriented toward the ST group. Conversely, vectors for growth parameters (stem diameter, leaf width) and photosynthetic efficiency (A, F_v/F_m, Φ_PSII) aligned with the NT and ST + K + Si quadrants. Collectively, these PCA results confirm that the integration of biochemical protection and structural reinforcement by silicate offers the most efficient pathway for the comprehensive restoration of maize seedlings under combined stress.

5. Conclusions

In conclusion, this study confirms that potassium (K⁺) supplementation is an effective strategy to accelerate the recovery of maize seedlings from the combined salinity–waterlogging stress. While all potassium salt treatments helped physiological restoration, the efficiency varied significantly depending on the accompanying anion (phosphate, sulfate, and silicate). Notably, potassium silicate (ST + K + Si) demonstrated the most comprehensive recovery effect, as shown by its closest proximity to the NT control in the PCA. While the ST + K + S group was effective in lowering the Na⁺/K⁺ ratio, the silicate treatment outperformed others in regaining photosynthetic capacity and growth vitality. This superiority is attributed to the synergetic contribution by K⁺ and silicate, which silicate reinforces the physical barrier to block entry, while activating metabolism. This accompanying function minimized energy loss and optimized the physiological balance. At the molecular level, this recovery was supported by the rapid down-regulation of stress-related genes (ZmADH1, ZmACS6) and the stabilization of ion transporters. Furthermore, the strategic shift in hormonal signals allowed the plants to efficiently redirect metabolic energy from defense back to growth. Collectively, our findings suggest that potassium silicate is a superior practical solution for enhancing crop resilience against complex climate stresses, particularly for silicon-accumulating crops like maize. Future targeted metabolic studies will be essential to clarify the specific mechanistic roles of silicate, phosphate, and sulfate in driving the metabolic pathways during this recovery process.