Application of nano silicon dioxide in relieving stress of polystyrene microplastics on hibiscus mutabilis

Abstract

The invention relates to the technical field of plant conditioners, in particular to application of nano silicon dioxide in relieving stress of polystyrene microplastics on hibiscus mutabilis. The invention finds that the influence of polystyrene micro-plastic on the stress of hibiscus mutabilis can be relieved to a certain extent during treatment by nano-silica, the intervention of the nano-silica causes the increase of secretion of melatonin, and the combined action of the nano-silica and melatonin makes the effect more remarkable and shows a synergistic effect. The nano silicon dioxide directly acts on the surfaces of cells to form a protective layer, so that adsorption and damage of PS-MPs to the cells are reduced, synthesis of melatonin is promoted, and a melatonin signal channel is activated. The activated melatonin signaling pathway further regulates the activity of carbon and nitrogen metabolism key enzymes, promotes carbon and nitrogen metabolism, improves synthesis and accumulation of carbohydrates and proteins, enhances the activity of an antioxidant system, removes active oxygen and reduces oxidative damage.

Description

Technical Field

[0001] This invention relates to the field of plant conditioner technology, and in particular to the application of nano-silica in alleviating the stress of polystyrene microplastics on water hyacinth. Background Technology

[0002] Carbon and nitrogen metabolism are indispensable components of plant physiology, and they are interdependent and mutually restrictive. Carbon metabolism provides energy, reducing agents, and the carbon skeleton for nitrogen metabolism, while nitrogen metabolism provides enzymes and photosynthetic pigments for carbon metabolism. The coordination of carbon and nitrogen metabolism directly affects plant growth, development, yield formation, and quality. When plants are subjected to external stresses, maintaining stable carbon and nitrogen metabolism plays a crucial role in their survival and growth.

[0003] Water hyacinth (Pistia stratiotes L.) plays a crucial role in aquatic ecosystems, significantly contributing to the maintenance of aquatic ecological balance. However, with increasingly severe microplastic pollution in the environment, the growth and development of water hyacinth, its physiological characteristics, and its internal homeostasis are facing enormous challenges. Polystyrene microplastics (PS-MPs) inhibit the growth and metabolism of water hyacinth. Currently, there is a lack of substances that can alleviate the inhibitory effects of PS-MPs on the growth and metabolism of water hyacinth. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides the application of nano-silica in alleviating the stress of polystyrene microplastics on water hyacinth. This invention discovers that nano-silica can alleviate the stress of polystyrene microplastics on water hyacinth, promoting its growth and metabolism.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides the application of nano-silica or nano-silica combined with melatonin in alleviating the stress of polystyrene microplastics on water lilies, wherein the water lily stress includes one or more of the following: 1) Inhibits the growth of water hyacinth; 2) Inhibits carbon and nitrogen metabolism in water hyacinth; 3) Inhibits the antioxidant activity of water hyacinth; 4) Reduces the melatonin content in water hyacinth and / or inhibits the melatonin signaling pathway in water hyacinth.

[0006] Preferably, the inhibition of water hyacinth growth includes reducing one or more of the following: fresh weight, root length, leaf area, and plant height of water hyacinth.

[0007] Preferably, the nano-silica promotes the synthesis of melatonin from water lilies and / or activates the melatonin signaling pathway.

[0008] Preferably, the nano-silica promotes carbon and nitrogen metabolism in water lilies, and increases the synthesis and accumulation of carbohydrates and proteins.

[0009] Preferably, the nano-silica enhances the activity of the antioxidant system of water lily, scavenges reactive oxygen species, and reduces oxidative damage.

[0010] This invention provides a method for alleviating the stress of polystyrene microplastics on water lilies, comprising: applying a nano-silica suspension to the water lilies; wherein the nano-silica particles have a diameter of 10-30 nm.

[0011] Preferably, the method of application includes spraying.

[0012] Preferably, the spraying amount per water lily plant is 4.8 mg based on the mass of nano silica.

[0013] Preferably, the preparation method of the nano-silica suspension includes: mixing nano-silica and water, sequentially performing ultrasonic treatment, volume adjustment and stirring to obtain the nano-silica suspension.

[0014] Preferably, the ultrasonic treatment has a power of 150 W, a temperature of 25°C, and a time of 30 min; the stirring time is 5 min.

[0015] Beneficial effects: PS-MPs have a significant negative impact on the growth, carbon and nitrogen metabolism, antioxidant system, and melatonin signaling pathway of *Hylocereus undatus*. This invention discovers that treatment with nano-silica can alleviate these negative effects to some extent. The intervention of nano-silica leads to an increase in melatonin secretion, and the combined effect of nano-silica and melatonin is more significant, exhibiting a synergistic effect. Nano-silica forms a protective layer on the cell surface by directly acting on it, reducing the adsorption and damage of PS-MPs to cells, while simultaneously promoting melatonin synthesis and activating the melatonin signaling pathway. The activated melatonin signaling pathway further regulates the activity of key enzymes in carbon and nitrogen metabolism, promotes carbon and nitrogen metabolism, increases the synthesis and accumulation of carbohydrates and proteins, enhances the activity of the antioxidant system, scavenges reactive oxygen species, and reduces oxidative damage. Melatonin (MT), through regulating the plant's antioxidant system, hormone balance, and gene expression, works synergistically with nano-silica to alleviate the stress of polystyrene microplastics on *Hylocereus undatus*, promoting its growth and recovery. This invention provides technical support for mitigating the harm of microplastic pollution to aquatic plants. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 The results of cultivating water lily in different groups; Figure 2Transmission electron microscope image of polystyrene nanoparticles, scale bar 30 μm; Figure 3 The effect of nano-silica on PS concentration and growth characteristics in *Hibiscus syriacus* under polystyrene nanoparticle stress; where different letters represent statistically significant differences (n = 4, P < 0.05). Figure 4 Scanning electron microscope images of cross-sections of leaves of *Hydrocotyle vulgaris* under different treatments; Figure 5 Laser confocal scanning images of the root system of *Hydrocotyle vulgaris* under different treatments; Figure 6 The distribution of differentially expressed genes (DEGs) is shown; where A is a Venn diagram of differentially expressed gene expression in plants under different stresses; B is the number of upregulated and downregulated genes in plants under different mediating and stress conditions; and C is a volcano diagram of upregulated and downregulated differentially expressed genes in plants under different stresses. Figure 7 Analysis of PCA under different stress groups; Figure 8 The results of the identification and correlation analysis of the weighted gene co-expression network (WGCNA) modules are shown. Among them, A is a clustering tree constructed based on different genes with topological overlap, and each module is assigned a color; B is a heatmap of the relationship between modules and samples, with green and red indicating positive and negative correlations with stress, respectively; C is the correlation between the identified modules and different groups under different stresses. Figure 9 The results of gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses for hub differentially expressed genes (DEGs) are shown. In A, the significance of upregulated and downregulated differentially expressed genes in the top 20 pathways on KEGG is presented in microplastic (NPs), silicon, and silicon-microplastic (Si-NPs) treatments. The x-axis represents enrichment factors, the y-axis represents pathway names, bubble size represents the number of differentially expressed genes involved, and bubble color represents the degree of pathway enrichment. In B, the results of GO functional pathway enrichment analysis for differentially expressed genes in silicon-microplastic (Si-NPs) treatment are shown. Differentially expressed genes can be divided into three ontology categories (biological processes (BPs), molecular functions (MFs), and cellular components (CCs)). Figure 10This section lists the top 20 common pathways with significant KEGG upregulation and downregulation of differentially expressed metabolites compared to CK and NPs-SiO2, NPs and CK, and SiO2 and NPs-SiO2. In section A, the X-axis represents enrichment factors, and the Y-axis represents pathway names. Bubble size indicates the number of differentially expressed metabolites involved, and bubble color indicates the degree of pathway enrichment. The section also includes changes in the expression of differentially expressed metabolites (DEMs) and KEGG pathway enrichment analysis from metabolomics data. Section B is an enrichment chord plot: representing significantly enriched GO terms for differentially expressed genes / transcripts. The left side shows genes / transcripts, arranged in descending order of log2FC. A larger log2FC indicates a greater fold change in upregulated gene / transcript expression, a smaller log2FC indicates a greater fold change in downregulated gene / transcript expression, and a log2FC closer to 0 indicates a smaller fold change in gene / transcript expression. The right side shows GO term information for significantly enriched differentially expressed genes / transcripts. (KEGG data is also included.) Pathway enrichment chord plot: Represents the significantly enriched pathways corresponding to differentially expressed genes / transcripts. The left side shows genes / transcripts, arranged in descending order of log2FC. The larger the log2FC, the greater the fold change in the expression of upregulated genes / transcripts; the smaller the log2FC, the greater the fold change in the expression of downregulated genes / transcripts; the closer the log2FC is to 0, the smaller the fold change in the expression of genes / transcripts. The right side shows the KEGG pathway information on the significantly enriched differentially expressed genes / transcripts. Detailed Implementation

[0018] This invention provides the application of nano-silica or nano-silica combined with melatonin in alleviating the stress of polystyrene microplastics on water lilies, wherein the water lily stress includes one or more of the following: 1) Inhibits the growth of water hyacinth; 2) Inhibits carbon and nitrogen metabolism in water hyacinth; 3) Inhibits the antioxidant activity of water hyacinth; 4) Reduces the melatonin content in water hyacinth and / or inhibits the melatonin signaling pathway in water hyacinth.

[0019] In one embodiment, inhibiting the growth of *Hylocereus undatus* includes reducing one or more of the following: fresh weight, root length, leaf area, and plant height. In another embodiment, the nano-silica promotes the synthesis of melatonin in *Hylocereus undatus* and / or activates the melatonin signaling pathway. In yet another embodiment, the nano-silica promotes carbon and nitrogen metabolism in *Hylocereus undatus*, increasing the synthesis and accumulation of carbohydrates and proteins. In yet another embodiment, the nano-silica enhances the activity of the antioxidant system in *Hylocereus undatus*, scavenging reactive oxygen species and reducing oxidative damage.

[0020] This invention discovers that treatment with nano-silica can alleviate the negative effects of polystyrene microplastics (PS-MPs) on water hyacinth to a certain extent. The intervention of nano-silica leads to an increase in melatonin secretion, and the combined effect of nano-silica and melatonin is more significant, exhibiting a synergistic effect. Nano-silica forms a protective layer on the cell surface by directly acting on it, reducing the adsorption and damage of PS-MPs to cells, while simultaneously promoting melatonin synthesis and activating the melatonin signaling pathway. The activated melatonin signaling pathway further regulates the activity of key enzymes in carbon and nitrogen metabolism, promotes carbon and nitrogen metabolism, increases the synthesis and accumulation of carbohydrates and proteins, enhances the activity of the antioxidant system, scavenges reactive oxygen species, and reduces oxidative damage. Melatonin (MT), through regulating the plant's antioxidant system, hormone balance, and gene expression, works synergistically with nano-silica to alleviate the stress of polystyrene microplastics on water hyacinth, promoting its growth and recovery. This invention provides technical support for mitigating the harm of microplastic pollution to aquatic plants and for the protection and restoration of aquatic ecosystems.

[0021] Based on the above advantages, this invention provides a method for alleviating the stress of polystyrene microplastics on water lilies, comprising: applying a nano-silica suspension to the water lilies; wherein the nano-silica particles have a diameter of 10-30 nm. As one embodiment, the nano-silica particles have a diameter of 20 nm.

[0022] As one implementation method, the application method includes spraying.

[0023] As one implementation method, the spraying amount per water lily plant is 4.8 mg based on the mass of nano-silica.

[0024] As one embodiment, the preparation method of the nano-silica suspension includes: mixing nano-silica and water, sequentially performing ultrasonic treatment, volume adjustment and stirring to obtain the nano-silica suspension.

[0025] In one embodiment, the ultrasonic treatment power is 150 W, the temperature is 25°C, and the time is 30 min; the stirring time is 5 min.

[0026] To further illustrate the present invention, the application of the nano-silica provided by the present invention in alleviating the stress of polystyrene microplastics on water lilies is described in detail below with reference to embodiments and accompanying drawings, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0027] Example 1 1) Plants and growing conditions The *Hydrocotyle vulgaris* samples used in this invention were purchased from the Ye Jinxuan Botanical Breeding Garden in Songnan Village, Luzhi Town, Wuzhong District, Suzhou City. Healthy, uniformly sized, and disease-free mother plants were selected and brought back to the laboratory of Guizhou Minzu University. They were placed in a 1 / 4-strength Hoagland nutrient solution (15×16×20cm cylindrical container), and 1.5 L of NP-free artificial freshwater (NaHCO3 13.0 μg / mL, KCl 1.2 μg / mL, MgSO4·7H2O 24.7 μg / mL, and CaCl2·2H2O 58.5 μg / mL) was added for 7 days of pre-culturing. The nutrient solution was changed every 5 days. During the culturing period, stable environmental conditions were maintained: relative humidity 60%, daytime temperature 26℃, nighttime temperature 20℃, and light intensity 350 μmol·m⁻¹. −2 ·s −1 Provide 14 hours of light per day. After the water lilies have adapted to the laboratory environment, select water lilies with similar growth at the single-leaf stage and reproduce asexually in the dark using ionized water to produce new seedlings from the parent plant (4-5 days). After incubation, select uniform seedlings and transfer them to the same culture conditions as the parent plant for the following experiments.

[0028] 2) Measurement and sampling of growth parameters Seedlings were divided into a control group (CK), a nano-silica treatment group (NPsSiO2, NPs-SiO2, or SiO2), a nano-plastic treatment group (NPs or Ps), and a nano-plastic and nano-silica composite treatment group (NPsSiO2-NPs, Ps+Si, or NPs-SiO2), with three replicates in each group. Polystyrene nanoplastic particles with a diameter of 50 nm and a purity ≥99% were selected and purchased from Jiangsu Zhichuan Technology (Suzhou) Co., Ltd. Before use, the nano-plastic particles underwent ultrasonic dispersion treatment at a power of 150 W for 20 min to ensure uniform dispersion in the solution. Solutions for different treatment groups were prepared according to the experimental design concentration, with the nano-plastic treatment group concentration set at 1 mg / L. Artificial freshwater free of NPs and Cd was selected as the control group (CK). Nano-silica (100361) was provided by Jiangsu Xianfeng Nanotechnology Co., Ltd., with a particle diameter of 20 nm and a purity ≥99%. The concentration of the nano-silica treatment group was 20 mg / L, and the solvent was artificial fresh water that did not contain NPs and Cd.

[0029] Specific methods for preparing nano-silica solution: Weigh 0.02 g of nano-silica and add it to an appropriate amount of artificial fresh water (800 mL). Stir magnetically for 10 min until initially dispersed. Transfer the solution to a 500 mL beaker and place it in an ultrasonic instrument (power 150 W, temperature set 25℃) for ultrasonic treatment for 30 min (to ensure uniform dispersion of particles and avoid agglomeration). After ultrasonication, add artificial fresh water to the target volume (1 L) and stir magnetically again for 5 min to obtain a 20 mg / L nano-silica solution. Quality control: Dynamic light scattering (DLS) was used to detect the particle size distribution and zeta potential of nano-silica in the solution to ensure uniform dispersion (target particle size 20±5 nm, absolute value of zeta potential > 30 mV). Seedling treatment operation steps Treatment method: During the seedling stage and early growth stages, a constant spraying distance and pressure were used to minimize variability introduced by differences in leaf absorption efficiency (6 applications during the seedling stage and 6 applications during the early vegetative growth stage, 20 mL each time, totaling 240 mL per pot). Replication and duration: Each group had 3 replicates, with each replicate containing 10 seedlings. The experimental period was set at 28 days (adjusted according to the seedling growth stage). The application rate of nano-silica was 4.8 mg / plant. Seedling growth status (plant height, number of leaves, fresh weight, etc.) was recorded daily.

[0030] Experimental environment control Greenhouse conditions: Temperature 25±2℃ (daytime) / 18±2℃ (nighttime); Light duration 16h light / 8h darkness, light intensity 300 μmol·m −2 ·s −1 Humidity 60%±5%. Dispersion control of nano-silica: During ultrasonic treatment, the power (150 W) and time (30 min) must be strictly controlled to avoid particle denaturation caused by high temperature; it should be used immediately after dispersion, and if stored, it should be refrigerated (4℃) and used within 24 hours. Concentration accuracy verification: The silicon content in the solution was determined by inductively coupled plasma optical emission spectrometry (ICP-OES), confirming that the deviation between the actual concentration and the theoretical concentration was ≤5%. Operational repeatability: Micropipettes (accuracy ±1%) must be used for irrigation to ensure that each seedling receives the same amount of solution. After acclimatizing seedlings of *Hylocereus undatus* in a greenhouse for 30 days, healthy plants with a height of 15 cm and abundant foliage were selected for the experiment. The plants were collected, rinsed, and separated into leaves and roots. Water samples were collected from glass containers. After recording the fresh weight (FW), the plant tissue was divided into three parts: these were rapidly frozen in liquid nitrogen and then transferred to a freezer at -80°C for biochemical assays and transcriptomic analysis. The plant roots were preserved by immersing them in pre-cooled 2.5% glutaraldehyde culture dishes, and the root cell structure was observed and photographed using a scanning electron microscope (SEM, SMZ25, Nikon, Japan).

[0031] 3) Cd content analysis and Cd content analysis (cadmium content analysis) Both root and leaf samples were digested using a 1:3 volume ratio of HCl and HNO3. After digestion, the products were transferred to 50 ml volumetric flasks and diluted with deionized water. Inductively coupled plasma mass spectrometry (ICP-MS) was used to determine the cadmium content in the samples.

[0032] The enrichment factor is calculated as follows: Cd-CCF = total Cd content in the roots and leaves of water hyacinth / total Cd content in the water. The translocation factor is calculated as follows: Cd - TCF = Cd content in water hyacinth leaves / Cd content in water hyacinth roots.

[0033] 4) Physiological and bioinformatics measurements Floating aquatic plants, with roots growing below the water surface, are more sensitive to changes in water pollutants than their leaves. This experiment used standard reagent kits from Shanghai Enzyme-Linked Biotechnology Co., Ltd. to determine relevant indicators. Antioxidant enzyme activity assays can reflect the plant's antioxidant defense capabilities. 0.5g of *Hydrocotyle vulgaris* leaves were weighed, added to pre-cooled 50 mmol / L phosphate buffer (pH 7.8, containing 1% polyvinylpyrrolidone), homogenized in an ice bath, and centrifuged at 12000g for 20 min at 4℃. The supernatant was used as the enzyme extraction solution. Superoxide dismutase (SOD) activity was determined using the nitroblue tetrazolium (NBT) photoreduction method; peroxidase (POD) activity was determined using the guaiacol method; and catalase (CAT) activity was determined using ultraviolet spectrophotometry. These antioxidant enzymes can scavenge reactive oxygen species and alleviate oxidative damage; changes in their activity can reflect the stress response of *Hydrocotyle vulgaris* to nanoplastics and cadmium stress.

[0034] Malondialdehyde (MDA) content is a key indicator for assessing the degree of lipid peroxidation in plant cell membranes. The experiment used the thiobarbituric acid (TBA) colorimetric method. 0.5 g of *Hydrocotyle vulgaris* leaves were mixed with 5 mL of 10% trichloroacetic acid (TCA) solution, homogenized in an ice bath, and centrifuged at 4℃ and 10000g for 15 min. 2 mL of the supernatant was collected, and 2 mL of 0.6% TBA solution was added. The mixture was then heated in a boiling water bath for 15 min, cooled, and centrifuged again. The MDA content was calculated by measuring the absorbance at a specific wavelength using a spectrophotometer. Increased MDA content indicates exacerbated oxidative damage to the cell membrane, which helps to understand the effects of nanoplastics and cadmium stress on the cell membrane integrity of *Hydrocotyle vulgaris*.

[0035] The determination of osmotic regulation substance content is of great significance for studying the osmotic regulation mechanism of plants under stress. The contents of soluble sugars, soluble proteins, and proline were determined using the anthrone colorimetric method, Coomassie Brilliant Blue G-250 staining method, and acidic ninhydrin method, respectively. When plants are under stress, they accumulate these substances to reduce cell osmotic potential, maintain water balance, and enhance stress resistance. Measuring their contents can reveal the osmotic regulation strategies of *Hydrocotyle vulgaris* under nanoplastic and cadmium stress.

[0036] 5) Transcriptomics analysis RNA extraction and sequencing Total RNA was extracted from the tissue of *Hydrocotyle vulgaris* using the Trizol reagent method.

[0037] RNA concentration and purity were determined using a NanoDrop 2000 spectrophotometer, ensuring an A260 / A280 ratio between 1.8 and 2.2 and an A260 / A230 ratio greater than 2.0. RNA integrity was assessed by agarose gel electrophoresis, observing the brightness and clarity of the 28S and 18S rRNA bands to ensure no significant RNA degradation.

[0038] Total RNA extracted was sent to a sequencing company for transcriptome sequencing. The sequencing platform used was an Illumina HiSeq2500, employing the PE150 sequencing strategy. Prior to sequencing, RNA samples underwent library construction. After passing quality checks, the constructed libraries were subjected to high-throughput sequencing.

[0039] Plant non-targeted metabolomics detection and targeted metabolism validation.

[0040] In the determination of carbohydrate and nitrogen metabolite content, for sugars, 0.1g of frozen sample was pulverized and mixed with 1ml of 80% ethanol, centrifuged at 12000 rpm for 15 minutes, and the resulting sample was dissolved in 80% acetonitrile. Sucrose, fructose, and glucose were quantitatively analyzed using an Agilent 1260 high-performance liquid chromatography (HPLC) kit. Starch content was determined using a Sigma-Aldrich STA20 kit. For nitrogen metabolite determination, nitrate was quantified, ammonia content was determined, and free amino acid content was assessed using an Agilent 1260 HPLC kit. For soluble protein content determination, 0.1g of frozen sample was pulverized in liquid nitrogen, added to a phosphate buffer containing specific components, homogenized, and centrifuged at 4℃ and 11000×g for 15 minutes. The absorbance was measured at 595nm using a Shimadzu UV-2550 spectrophotometer, and the absorbance was determined using a Sigma-Aldrich bovine serum albumin standard curve.

[0041] The endogenous melatonin in plants was quantitatively determined using an Agilent 1260 HPLC system. 0.5 g of fresh plant sample was pulverized into a fine powder under liquid nitrogen at low temperature, and 5 mL of methanol was added. The sample was centrifuged at 10000×g for 30 minutes at 4°C, and the methanol was removed by nitrogen purging. The residue was dissolved in 0.2 mL of 0.1 M Na₂HPO₄-acetonitrile (65:35) solution and filtered through a 0.22 μm filter membrane. 5 µL of the filtrate was injected into a C18 column at 30°C and eluted at a flow rate of 0.5 mL / min. The concentration was monitored at 220 nm, and the concentration was determined using the Sigma-Aldrich melatonin standard curve.

[0042] Chlorophyll content and photosynthetic parameters were measured. Approximately 0.2 g of leaf sample stored at -80℃ was mixed with 5 mL of 80% acetone and ground into a homogenate. The homogenate was then centrifuged at 4℃ and 5000 g for 10 minutes. The absorbance was measured at wavelengths of 645 nm, 652 nm, and 663 nm using a Shimadzu UV-2550 spectrophotometer. After 7 days of stress treatment, a LI-6800 portable photosynthesis analysis system (LI-COR, USA) was used with a 2×3 cm red-blue light source leaf chamber and a light intensity of 600 µmol·m⁻¹. −2 ·s −1The photosynthetic rate of maize plants was evaluated at a flow rate of 500 µmol / s. A 0.1 g frozen sample was weighed and homogenized in 100 mM bicine-NaOH buffer (pH = 7.8) containing 1 mM EDTA, 5 mM DTT, 5 mM MgCl2, and 1 mM MPMSF. The homogenate was centrifuged at 15000 × g for 5 minutes at 4 °C. The enzyme solution was then added to a mixture containing 200 mM Tris-HCl (pH = 8.5), 1 mM RuBP, 10 mM NaHCO3, 5 mM MgCl2, 0.1 mM DTT, 1 mM ATP, 5 units of phosphoglycerate kinase, 5 units of glyceraldehyde-3-phosphate dehydrogenase, and 0.2 mM NADH. The absorbance at 340 nm was recorded using a Shimadzu UV-2550 spectrophotometer. AGPase and AMY activities were measured. SPS activity assay, SuSy and INV activity assay, CS and PEPC activity analysis, and NR activity assay were performed. 0.1 g of frozen sample was ground into powder in liquid nitrogen and added to an extraction buffer containing 50 mM KH₂PO₄-KOH (pH = 7.5), 2 mM EDTA, 2 mM dithiolitol, and 1% polyvinylpyrrolidone. After homogenization, the mixture was centrifuged at 4°C and 20000×g for 20 minutes. At the start of the reaction, 700 µL of reaction buffer (50 mM KH₂PO₄-KOH [pH = 7.5], 10 mM KNO₃) was added. All measurements were performed in the morning, with the relative humidity maintained at 50%. After a 30-minute dark adaptation period, chlorophyll fluorescence parameters were measured using a Walz PAM2500 instrument (Germany).

[0043] Measurement of carbon and nitrogen metabolism parameters In related experimental studies, enzyme activity and metabolite content were precisely measured. For enzyme activity analysis, for AGPase, 0.1g of frozen sample was weighed and homogenized in 100mM bicine-NaOH buffer (pH = 7.8) containing 1mM EDTA, 5mM DTT, 5mM MgCl2, and 1mM PMSF. The homogenate was then centrifuged at 4℃ and 15000×g for 5 minutes. The enzyme solution was then added to a specific component mixture, and the absorbance at 340nm was recorded using a Shimadzu UV-2550 spectrophotometer. AMY activity, SPS, SuSy, and INV activities, and CS and PEPC activities were also measured. For NR activity measurement, 0.1g of frozen sample was first ground in liquid nitrogen, then added to an extract containing specific components. After homogenization, the mixture was centrifuged at 4℃ and 20000×g for 20 minutes, and then a specific reaction buffer was added. Rubisco activity assays, as outlined by Bota et al., assessed nitrite concentration after incubating specific reaction systems at 28°C for 15 minutes. GS and GOGAT enzyme activities, GDH enzyme activity, and protease activity were also measured. Each treatment was performed in four biological replicates.

[0044] In the data processing stage, one-way ANOVA combined with IBM SPSS 26.0 (Chicago, USA) was used for statistical analysis. If the data did not meet the assumptions of normality and homogeneity of variance, a nonparametric Kruskal-Wallis (NET T-2) test was performed. Charts and graphs were created using Origin 2021 and Adobe Illustrator software. Visualization of metagenomic and metabolomics data was achieved using an online platform (www.majorbio.com).

[0045] result Impact on the growth of water lily Polystyrene microplastic treatment significantly inhibited the growth of *Hymenochloa chinensis*. Compared with the control group, its fresh weight, root length, leaf area, and plant height were all significantly reduced (P<0.05). This is consistent with the negative effects of microplastics on plant growth in previous studies, indicating that polystyrene microplastics have a significant stress effect on the growth of *Hymenochloa chinensis*. Figures 1-3 The growth of *Hylocereus undatus* improved with nano-silica treatment compared to the polystyrene microplastic treatment group, with increased growth indicators, indicating that nano-silica treatment can alleviate the inhibitory effect of polystyrene microplastics on *Hylocereus undatus* growth to some extent. After a period of nano-silica treatment, melatonin (MT) levels increased, and the recovery of *Hylocereus undatus* growth indicators was more significant, suggesting a synergistic effect between nano-silica and melatonin in alleviating the inhibitory effect of polystyrene microplastics on *Hylocereus undatus* growth. Figure 5This is because nano-silica can enhance the stability of plant cell walls, reduce the damage to cells caused by polystyrene microplastics, and regulate the physiological metabolic processes of plants; MT, as a signaling molecule and antioxidant, regulates the plant's hormone balance and antioxidant system, and promotes cell division and elongation. The synergistic effect of the two creates more favorable conditions for the growth of water hyacinth. Figure 4 ).

[0046] Effects of water lily on the expression of genes related to the melatonin signaling pathway Under polystyrene microplastic treatment, the expression levels of key genes for melatonin synthesis in *Hymenochloa crus-galli*, such as tryptophan decarboxylase (TDC) and tryptophan 5-hydroxylase (T5H), decreased significantly, while the expression levels of key genes for melatonin degradation, such as cytochrome P450 monooxygenase (CYP71A12), increased significantly. Figures 6-9 This indicates that polystyrene microplastic stress inhibits melatonin synthesis and promotes its decomposition, leading to a decrease in melatonin levels in plants. Figure 9 After treatment with nano-silica, the expression levels of TDC and T5H were upregulated by 21.2% and 34.5%, respectively, while the expression level of CYP71A12 was downregulated by 12.7%. This indicates that nano-silica can regulate the expression of genes related to the melatonin signaling pathway, promote melatonin synthesis, and inhibit its degradation, thereby increasing the melatonin content in plants. This may be because nano-silica alters the microenvironment within plant cells, affecting the transcription and translation processes of related genes. MT further upregulated the expression levels of TDC and T5H, increasing them by 5.7% and 8.9% respectively compared to the addition of nano-silica alone, while further downregulating the expression level of CYP71A12, decreasing it by 2.3% compared to the addition of nano-silica alone. This indicates that MT can enhance the regulatory effect of nano-silica on the expression of genes related to the melatonin signaling pathway, further increasing the melatonin content in plants. Figure 9 This may be because MT, as a signaling molecule, synergistically activates the signal transduction pathway related to melatonin synthesis with nano-silica, promoting efficient gene expression. The synergistic effect of these two molecules, by regulating the expression of genes related to the melatonin signaling pathway, maintains the dynamic balance of melatonin in the plant, thereby enhancing the resistance of *Hibiscus syriacus* to PS-MPs stress. This allows the plant to better regulate its physiological processes under stress, maintaining relative stability in growth and metabolism. Figure 10 ).

[0047] Under PS-MPs stress, SiO2-mediated increase in melatonin content in *Hylocereus undatus* was observed. In the process of investigating the effect of SiO2 nanoparticles on the melatonin content in water lilies under microplastic (NPs) stress, a series of complex and ingenious physiological regulatory mechanisms were discovered.

[0048] When water hyacinth is subjected to nanoplastic stress, the physiological balance within cells is disrupted, leading to a massive accumulation of reactive oxygen species (ROS) that severely damage cell structure and function, including the inhibition of melatonin synthesis. Studies have shown that under PS-MPs stress, the expression levels of key genes for melatonin synthesis in water hyacinth, such as tryptophan decarboxylase (TDC) and tryptophan 5-hydroxylase (T5H), significantly decrease, while the expression levels of key genes for melatonin degradation, such as cytochrome P450 monooxygenase (CYP71A12), significantly increase, resulting in a decrease in melatonin content within the plant. The intervention of nano-silica alters this situation. First, nano-silica exerts its effect by altering the microenvironment within plant cells. Nano-silica possesses unique physicochemical properties, enabling it to interact with the cell surface and influence intracellular ion balance and signal transduction. When nano-silica enters plant cells, it may regulate some key intracellular signaling molecules, thereby affecting gene transcription and translation processes. In the melatonin signaling pathway, nano-silica upregulated the expression levels of TDC and T5H, and downregulated the expression level of CYP71A12, respectively. This indicates that nano-silica can promote the synthesis of melatonin, inhibit its decomposition, and thus increase the melatonin content in plants.

[0049] From a cellular perspective, nano-silica stabilizes cell membrane structure and function, reducing PS-MP damage and thus providing a relatively stable internal environment for melatonin synthesis. The cell membrane is a crucial barrier for material exchange and signal transduction between the cell and its external environment. PS-MP stress can increase cell membrane permeability and exacerbate lipid peroxidation, thereby affecting various intracellular physiological processes. Nano-silica enhances cell membrane stability, reduces the accumulation of lipid peroxidation products such as malondialdehyde (MDA), and maintains intracellular redox balance. This stable cellular environment is conducive to maintaining the activity of melatonin synthesis-related enzymes, promoting melatonin synthesis.

[0050] Furthermore, nano-silica indirectly affects melatonin synthesis by regulating hormone balance within plants. Plant hormones play crucial roles in plant growth, development, and stress responses, coordinating with each other to regulate physiological processes. Nano-silica may influence the synthesis, transport, and signal transduction of plant hormones such as auxin and cytokinin, which are closely related to melatonin (MT) synthesis. For example, auxin can promote MT synthesis by regulating the expression of related genes. By regulating plant hormone balance, nano-silica creates a favorable hormonal environment for melatonin synthesis, thereby promoting a recovery in MT levels.

[0051] Once the melatonin content in plants recovers, melatonin, as a multifunctional signaling molecule and antioxidant, further plays its important role in plant stress resistance. Melatonin can regulate the plant's antioxidant system, activating the activity of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), enhancing the plant's ability to scavenge ROS and reducing oxidative damage. Simultaneously, melatonin can also regulate the plant's carbon and nitrogen metabolism, promoting photosynthesis and nitrogen assimilation, providing sufficient energy and material basis for plant growth. Under NP stress, melatonin, through these effects, synergistically works with nano-silica to alleviate the stress of PS-MPs on water hyacinth, promoting its growth and recovery.

[0052] Effects on carbon metabolism of water lily Regarding carbon metabolism, NPs treatment significantly reduced the content of soluble sugars and starches in *Hylocereus undatus* leaves. This is because polystyrene microplastics interfere with the photosynthetic process, affecting carbon assimilation and reducing carbohydrate synthesis. Simultaneously, the activities of key carbon metabolism enzymes such as sucrose phosphate synthase (SPS), sucrose synthase (SS), and amylase (AMY) were also inhibited, further hindering carbon metabolism pathways. This is because nano-silica may promote the expression of photosynthesis-related genes, increase the synthesis of photosynthetic pigments, thereby enhancing photosynthetic efficiency and promoting carbon assimilation and carbohydrate accumulation. When nano-silica treatment was applied for a period of time, the resulting increase in melatonin also had a positive impact on carbon metabolism, increasing the accumulation of soluble sugars and starches and enhancing the activity of related enzymes. This is because melatonin can regulate the plant's antioxidant system, reduce damage to chloroplasts from reactive oxygen species, maintain the stability of photosynthetic enzymes, and thus promote carbon metabolism. When nano-silica and melatonin act together, the soluble sugar and starch contents in the leaves of *Hylocereus undatus* are significantly higher than those in the single-treatment groups, and the related enzyme activities are also at higher levels. This indicates that the synergistic effect of the two can more effectively promote chlorophyll synthesis or inhibit its decomposition, optimize the photosynthetic process, improve the light energy utilization efficiency of *Hylocereus undatus*, further enhance carbon assimilation capacity, and provide more energy and material basis for plant growth.

[0053] Effects on nitrogen metabolism of water lily Treatment with polystyrene microplastics (PS-MPs) reduced the content of free amino acids and proteins in the leaves of *Hylocereus undatus*, and inhibited the activity of key nitrogen metabolism enzymes such as nitrate reductase (NR), glutamine synthase (GS), and glutamate synthase (GOGAT). This indicates that polystyrene microplastics interfere with the absorption, conversion, and assimilation of nitrogen, affecting protein synthesis. Treatment with nano-silica increased the content of free amino acids and proteins, and also enhanced the activities of NR, GS, and GOGAT, indicating that silica nanoparticles can promote nitrogen metabolism. This may be because nano-silica improves the growth environment of plant roots, enhances the root system's ability to absorb nitrogen, and simultaneously regulates the hormonal balance within the plant, promoting the synthesis and expression of nitrogen metabolism-related enzymes. Increased MT levels within the plant also have a positive regulatory effect on nitrogen metabolism, increasing the content of free amino acids and proteins and enhancing the activity of related enzymes. This is because MT in plants can regulate nitrogen metabolism pathways, promoting nitrogen absorption and assimilation by activating related signaling pathways. In the PS-MPs + nano-silica treatment group, the contents of free amino acids and proteins were significantly higher than those in the PS-MPs treatment group, and the activities of NR, GS, and GOGAT were also at a high level. This indicates that the synergistic effect of nano-silica and melatonin (MT) effectively alleviates the inhibition of nitrogen metabolism in water hyacinth by polystyrene microplastics, promotes nitrogen absorption and assimilation, is beneficial to protein synthesis, and provides sufficient nitrogen source for plant growth and development.

[0054] Effects on the antioxidant system of water lily PS-MPs treatment significantly increased MDA content in *Hymenochloa crus-galli* leaves, indicating intensified membrane lipid peroxidation and oxidative damage to cells. Simultaneously, while the activities of SOD, POD, and CAT increased initially, they gradually decreased with prolonged treatment, suggesting that the plant's antioxidant defense system initially attempted to cope with stress, but its antioxidant capacity gradually became insufficient as stress intensified. Nano-silica treatment significantly reduced MDA content, while the activities of SOD, POD, and CAT remained at high levels, indicating that silica nanoparticles can enhance the antioxidant capacity of *Hymenochloa crus-galli* and mitigate oxidative damage. This may be because nano-silica induces upregulation of the expression of plant antioxidant enzyme genes, promoting the synthesis of antioxidant enzymes and thus enhancing the scavenging capacity of reactive oxygen species. MT also reduced MDA content and increased antioxidant enzyme activity because melatonin can regulate plant antioxidant signaling pathways, activate antioxidant enzyme activity, and enhance the plant's antioxidant defense system. In the PS-MPs + nano-silica treatment group, the MDA content was significantly lower than that in the PS-MPs treatment group, while the activities of SOD, POD, and CAT were significantly higher than those in the polystyrene microplastic treatment group. This indicates that the synergistic effect of nano-silica and MT effectively activated the antioxidant system of *Hylocereus undatus*, improved the plant's ability to scavenge reactive oxygen species, alleviated oxidative stress caused by PS-MPs, protected the integrity of plant cells, and maintained normal physiological functions of cells.

[0055] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of nano-silica or nano-silica combined with melatonin in alleviating the stress of polystyrene microplastics on water lilies, wherein the water lily stress includes one or more of the following: 1) Inhibits the growth of water hyacinth; 2) Inhibits carbon and nitrogen metabolism in water hyacinth; 3) Inhibits the antioxidant activity of water hyacinth; 4) Reduces the melatonin content in water hyacinth and / or inhibits the melatonin signaling pathway in water hyacinth.

2. The application according to claim 1, characterized in that, The inhibition of water hyacinth growth includes reducing one or more of the following: fresh weight, root length, leaf area, and plant height.

3. The application according to claim 1, characterized in that, The nano-silica promotes the synthesis of melatonin from water lilies and / or activates the melatonin signaling pathway.

4. The application according to claim 1, characterized in that, The nano-silica promotes carbon and nitrogen metabolism in water lilies, and increases the synthesis and accumulation of carbohydrates and proteins.

5. The application according to claim 1, characterized in that, The nano-silica enhances the activity of the antioxidant system of water lily, scavenges reactive oxygen species, and reduces oxidative damage.

6. A method for alleviating the stress of polystyrene microplastics on water lilies, characterized in that, include: A nano-silica suspension was applied to water lily; the nano-silica particles had a diameter of 10~30nm.

7. The method according to claim 6, characterized in that, The application method includes spraying.

8. The method according to claim 7, characterized in that, Based on the mass of nano-silica, the spraying amount per water lily plant is 4.8 mg.

9. The method according to claim 6, characterized in that, The preparation method of the nano-silica suspension includes: mixing nano-silica and water, sequentially performing ultrasonic treatment, volume adjustment and stirring to obtain the nano-silica suspension.

10. The method according to claim 9, characterized in that, The ultrasonic treatment was performed at a power of 150 W, a temperature of 25°C, and a time of 30 min; the stirring time was 5 min.

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