Exogenous silicon induces aluminum tolerance in white clover (Trifolium repens) by reducing aluminum uptake and enhancing organic acid secretion

White clover culture and Al stress treatments

We used ‘Haifa’ seeds from Barenbrug China Company as our experimental material in this study. Prior to being used, the seeds were sterilized by rinsing them three times with distilled water after immersing them in a 0.1% mercuric chloride solution for 5 min. A meticulously regulated growth chamber (GHP-500; Jiangnan Instrument Factory, Ningbo, China) with a 12-h photoperiod, 75% relative humidity, and a day/night temperature of 23 °C was used throughout the entire experiment. There was 750 μmol m⁻² s⁻¹ of light. After 7 days of germination, the seedlings were carefully transferred into plastic trays and cultivated in Hoagland’s solution at a 1/4 strength for 3 days. They then spent an additional 23 days growing in the solution at half strength. Before being exposed to aluminum stress, the white clover plants were first subjected to a 4-day pretreatment with either 0 or 1 mM K₂SiO₃. Using 4 mM AlCl₃ dissolved in 1/2 strength Hoagland’s solution, aluminum stress was induced. Four distinct treatment groups were formed, namely: (1) CK (control): plants were kept in 1/2 concentration of Hoagland’s solution for 19 days without receiving any additional Al or Si; (2) Si (exogenous Si treatment): plants were pretreated with Si for 4 days before being exposed to 1/2 concentration of Hoagland’s solution for 15 days; (3) Al (Al stress): plants were exposed to 1/2 concentration of Hoagland’s solution for 4 days, followed by 15 days of Al stress, with pH kept at 4.5; and (4) Al+Si (exogenous Si treatment followed by Al stress): plants were pretreated with Si for 4 days before being exposed to 15 days of Al stress, with pH kept at 4.5. Every therapy was administered in growth chambers at random.

Measurement of growth index

Ten independent biological replicates were assigned to each treatment group after dividing the plants from the four treatment groups into shoot and root sections for length measurement. The shoot and root tissues were then placed in an oven (DHG-2200B; Sheng Yuan Instrument Co., Ltd., Zhengzhou, China) at 65 °C until they reached a constant dry weight.

The physiological parameters

Four independent biological replicates were assigned to each treatment, and meticulous measurements of physiological indices were conducted on the second leaves of multiple stems and the entire root system of each plant. 0.1 g of leaves and roots were carefully placed in centrifuge tubes with 35 ml of distilled water to test for electrolyte leakage (EL). The tubes were then incubated for a full day. Then, a conductivity meter (DDS-307; Lei-Ci, China) was used to measure the initial conductance (Ci). The tubes were then autoclaved for 20 min at 140 °C, and the maximum conductance (Cmax) was determined. Finally, the formula (%) = Ci/Cmax × 100 was used to calculate EL (Blum & Ebercon, 1981). The formula used to compute the relative water content (RWC) of leaves is RWC (%) = ((FW − DW)/(SW − DW)) × 100%. Here, FW stands for the weight of the leaves when they were fresh, SW for their weight after they were saturated after soaking in ultrapure water for 24 h, and DW for their weight when they were dried in an oven for 24 h at 80 °C to a consistent weight. 0.15 g of fresh tissue was cleaned before being submerged in 15 ml of dimethyl sulfoxide solution. It was then left in the dark at room temperature for 8 h, or until the leaves turned translucent, to extract chlorophyll (Chl). The extract’s absorbance was then measured using a spectrophotometer (Spectronic Instruments, Rochester, NY, USA) at 663 and 645 nm (Barnes et al., 1992). Using the Chlorophyll Fluorescence System (Pocket PEA, Hansatech, UK), white clover leaf samples were dark-adapted for 20 min. PIABS and PSII Fv/Fm measurements were taken. PIABS serves as a comprehensive photosynthetic index and a plant health status indicator. In order to extract malondialdehyde (MDA), 0.1 g of leaves and roots were first crushed separately using liquid nitrogen. Following this, the mixture was centrifuged for 30 min at 12,000 rpm and 4 °C using 2 mL of phosphate buffer (50 mM, pH 7.8). One ml of reaction solution containing 0.5% thiobarbituric acid and 20% trichloroacetic acid was mixed with the supernatant. After centrifugation, 1 mL of the reaction solution containing 20% trichloroacetic acid and 0.5% thiobarbituric acid was mixed with 0.5 mL of the supernatant. Following centrifugation, 1 mL of reaction solution containing 20% trichloroacetic acid and 0.5% thiobarbituric acid is mixed with 0.5 mL of supernatant, and the mixture is heated for 15 min at 95 °C. The mixture was quickly cooled in an ice water bath before being centrifuged for 10 min at 8,000 rpm. The OD600 and OD532 values were subtracted to determine the absorbance of the supernatant (Dhindsa, Pamela & Thorpe, 1981).

Measurement of Al and Si content

After 15 days of aluminum stress, the leaves and root tissues of white clover were carefully placed in an oven at 65 °C. Samples with a dry weight of 1 g were digested with 5 mL of hydrofluoric acid (HF) and 2 mL of hydrogen peroxide (H₂O₂). Each treatment comprised four independent biological replicates, and the aluminum and silicon content was determined using inductively coupled plasma mass spectrometry (ICP-MS).

Root staining

The plant roots were carefully submerged in deionized water for 5 min, irrespective of whether they had been exposed to aluminum stress for 15 days. The samples were subsequently rinsed in deionized water for an additional 15 min after being stained with hematoxylin staining solution for 15 min. The staining solution consisted of 0.1% hematoxylin, 0.01% KIO₃, and 0.2 mM NaOH (Delhaize, Ryan & Randall, 1993). The LEICA MC 190 HD stereomicroscope was used to examine the stained roots’ hematoxylin staining patterns after they had been placed on a level surface.

Measurements of internal and external OAs

Regardless of whether they had received Si pretreatment or not, white clover plants were transferred to trays filled with 1/2 strength Hoagland’s solution containing either 0 or 4 mM AlCl₃ and left for a full day. Then, 0.2 g of root tips were collected to measure the concentration of citrate and malate, and the solution was collected to measure the exudation of these two compounds. A spectrophotometer was used to measure the exudation and concentration of citrate and malate. G0864W and G0862W kits (Suzhou Geruisi Bio-Technology Co. Ltd., Suzhou, China) were used to measure the OD470 and OD450 readings, respectively.

Micro-distribution and compartmentation of ions in root

The elemental distribution and mineral ion compartmentation in the root samples were carefully investigated using an energy dispersive X-ray (EDX) analyzer attached to a scanning electron microscope (SEM). The plant root tips were carefully cut into 1 cm pieces with a sharp knife, rapidly submerged in liquid nitrogen, and then freeze-dried for 24 h at −80 °C. For each treatment, three distinct samples were meticulously collected for X-ray microanalysis. After deducting background X-ray counts, the net K-shell X-ray peak counts were used to automatically quantify the relative weights of the mineral ions in the root’s epidermal, cortical, and stelar layers (Javaid et al., 2019).

Statistical analyses

In this study, all data were presented as the mean ± standard error (SE) of at least three replicates. Using SPSS version 25, the Levene’s test for homogeneity of variance was conducted, followed by Tukey’s post hoc multiple comparison correction technique. In particular, the process included selecting “Data Analysis,” conducting a one-way ANOVA test, and then performing multiple comparisons afterward. Both the Tukey and LSD approaches were selected, with a significance threshold of 0.05. Principal component analysis (PCA) was conducted using Origin 2021 software. Principal component analysis was chosen as the method, and a 2D component plot was selected. All other settings were left at their default values.

Growth and physiological response of white clover plants under treatments

Figure 1A illustrates the growth phenotype of white clover under various treatments. It is clear that exogenous silicon had a mitigating impact on aluminum stress, but aluminum clearly hindered the growth of white clover. The measurements of length and dry weight under various treatment conditions further confirmed this observation. Both the length and dry weight significantly decreased under Al stress (54% and 41%, respectively, compared to the control in shoots and roots, and 62% and 68%, respectively, compared to the shoots and roots). On the other hand, silicon treatments substantially reduced the inhibition of dry weight (45% increase in shoots and 20% in roots) and length (37% increase in shoots and 46% in roots) compared to aluminum treatments. Furthermore, under normal conditions, additional silicon had a favorable effect on white clover growth, as evidenced by a significant increase in the dry weight of both shoots and roots (Figs. 1D, 1E).

We carefully examined the physiological characteristics of the plants to confirm the impact of silicon on aluminum stress. There were no appreciable differences in the phenotypic or physiological indices between the silicon-treated plants and the untreated control group (Figs. 2A–2H). Nevertheless, exposure to aluminum stress resulted in a significant drop in the leaves’ relative leaf water content (RGH) and chlorophyll levels, as well as a decrease in photosynthetic efficiency and overall health index, irrespective of silicon treatment. However, by the nineteenth day of aluminum stress, plants treated with silicon showed significant increases of 1.15 times for relative leaf water content (Fig. 2A), 1.4 times for chlorophyll content (Fig. 2B), and 3.88 times and 1.14 times for photosynthetic efficiency and health index, respectively (Figs. 2C, 2D). Furthermore, aluminum stress induced a significant increase in conductivity and MDA content in both leaves and roots of the plants. Conversely, under silicon treatment conditions, conductivity decreased by 15.27% in leaves and 18.32% in roots (Figs. 2E, 2F), while MDA content decreased by 42.21% in leaves and 67.23% in roots (Figs. 2G, 2H).

Exogenous Si pretreatment increased the endogenous Si concentration and decreases Al accumulation under Al stress

Endogenous silicon (Si) content in shoots and roots increased by 60% and 74%, respectively, in Si-treated shoots and roots compared to the control. However, there were no significant differences in endogenous silicon content between the control and aluminum treatments, as well as between the silicon and Al+Si treatments (Figs. 3A, 3B). Aluminum (Al) stress was found to cause a substantial increase in Al content in shoots and roots (80- and 48-fold increases, respectively, compared to the control), with Al predominantly accumulating in the roots (over 24 g·kg⁻¹). Additionally, compared to aluminum-stressed plants, the aluminum concentration in the roots and shoots of the Al+Si treatments was significantly lower by 65% and 29%, respectively (Figs. 3C, 3D). Hematoxylin staining, an indicator of aluminum absorption, was used to confirm the uptake of Al in the roots under various treatments (deeper staining indicating a higher concentration of physiologically active Al). Compared to the roots in Al treatments, the roots in Al+Si treatments exhibited a lighter hematoxylin stain. In contrast, only the lateral root development sites in Al+Si treatments were darkly colored, while the entire root surface in Al treatments appeared dark (Figs. 3E, 3F). SEM analysis of root cross-sections revealed that aluminum damaged the cortical, stellar, and epidermal tissues of the roots, leading to a reduction in the xylem area and eventual collapse. In contrast to aluminum treatments, silicon priming resulted in a larger xylem area and reduced collapse (Fig. 3G).

Exogenous Si improves the exudation and concentration of OAs under Al toxicity

In our studies, aluminum dramatically increased the exudation of malate and citrate. When compared to normal plants, the exudation of citrate and malate increased by approximately 12 and 2 times, respectively, in Al-stressed plants. Furthermore, there was no discernible difference in citrate exudation between the two treatments, but malate exudation was 71% higher in the Al+Si treatment than in the Al treatment (Figs. 4A, 4B). In the Al and Al+Si treatments, compared to the control, the concentrations of citrate and malate increased by 35% and 41%, and 77% and 172%, respectively. These increases indicate that malate was more sensitive to silicon stimulation than citrate in response to aluminum stress in white clover (Figs. 4C, 4D).

Micro-distribution and compartmentation of ions in white clover root tips

In order to understand how Si affects the distribution of mineral elements (Si²⁺, Al³⁺, Cl⁻, Mg²⁺, K⁺, and Zn²⁺) in roots across four treatments, energy-dispersive X-ray (EDX)-based mineral element microanalysis was conducted by scanning the cross-section of root tips and analyzing energy spectra. The cross-sectional image of the root revealed that the cortical and epidermal regions were the primary areas where newly added silicon accumulated, with the smallest xylem area observed in the aluminum treatments (Fig. 5A). When white clover plants were treated with exogenous silicon, the concentration of Si peaked, and the relative contents of Cl⁻, Mg²⁺, K⁺, and Zn²⁺ increased by 97%, 8%, 28%, and 9%, respectively, compared with the control. The relative weights of Mg²⁺, K⁺, and Zn²⁺ were all lower under Al stress (62%, 41%, and 95% lower than under control conditions, respectively). On the other hand, Al+Si treatments protected the xylem from Al stress and decreased the relative concentration of Al³⁺ by 44%. Additionally, in comparison to aluminum stress, they increased the relative weight of Mg²⁺, K⁺, and Zn²⁺ by 96%, 53%, and 356%, respectively (Fig. 5). Using EDX-based methods, mineral element microanalysis was conducted in the epidermal, cortical, and stelar layers of the root, as further reported (Fig. 6). The relative weight of silicon in the epidermal, cortical, and stelar layers increased dramatically in the silicon treatments compared to the control, reaching 18.5-fold, 17.5-fold, and 10.0-fold, respectively. Significant silicon buildup was observed in the epidermal and cortical layers, which was found to have a positive effect on the accumulation of Cl⁻, Mg²⁺, K⁺, and Zn²⁺. In contrast, aluminum stress significantly decreased the relative weight of Mg²⁺, K⁺, and Zn²⁺ in the cortical layers by 10%, 44%, and 64%, in the stelar layers by 139%, 40%, and 60%, and in the epidermal layers by 23%, 40%, and 55% compared to the control. The relative weight of Al³⁺ in the cortical, stelar, and epidermal layers increased by 1.72%, 1.26%, and 0.72%, respectively, compared to the control, as predicted. The epidermal layers acquired the highest amount of Al³⁺. In the meantime, K⁺ levels in the root epidermal, cortical, and stelar layers significantly increased in the Al+Si treatments, and the relative weight of Zn²⁺ in the root epidermal and cortical layers similarly increased. In contrast to aluminum treatments, the administration of silicon did not significantly alter the relative weight of Mg²⁺ in either the cortical or epidermal layers (Figs. 6D–6F).

Principal component analysis of the impact of different treatments on the growth of white clover

The morphological and physiological traits of seedlings under various treatments were thoroughly examined using principal component analysis (PCA) to evaluate the mitigating effect of exogenous silicon under aluminum stress. PC1 and PC2, which clearly separated the various treatments, accounted for 75.2% and 16.6% of the overall variance, respectively, as shown in Fig. 7. Notably, the treatments with aluminum stress alone and in combination with exogenous silicon were precisely distributed in the first and fourth quadrants, respectively, while the control (CK) treatment was clearly located in the third quadrant and the exogenous silicon treatment in the second quadrant. Additionally, in PC1, there were noteworthy positive relationships between the following parameters: electrolyte conductivity of the shoots and roots; MDA content in the shoots; Al content in the shoots; and concentrations of citrate, malate, citrate exudate, and malate exudate. Significantly and favorably correlated with citrate concentration, malate concentration, citrate exudation, and malate exudation, respectively, was the silicon content in shoots and roots in the analyses that included PC2. This indicates that exogenous silicon treatments were successful in promoting the secretion of organic acids, thereby significantly reducing the toxicity of aluminum. The silicon content of shoots and roots was also significantly negatively correlated with the MDA content of shoots, the Al content of roots, the Al content of shoots, and the electrolyte conductivity of both. This demonstrates how the administration of exogenous silicon greatly reduces oxidative damage, enhances plant resilience to aluminum stress, and significantly decreases the uptake and translocation of aluminum in white clover (Fig. 7).

We present a well-developed conceptual model that clearly illustrates the essential role of silicon in reducing the toxicity of aluminum, drawing on insights obtained from comprehensive investigations. When white clover is exposed to aluminum stress, exogenous silicon pretreatment plays a critical role in reducing the negative effects. It accomplishes this by greatly boosting the production of organic acids and carefully controlling the absorption of vital mineral elements (Fig. 8).