Method for improving accumulation and transport capacity of cadmium by sedum plumbizincicola

Abstract

This invention discloses a method for enhancing the cadmium accumulation and translocation capacity of *Sedum aizoon*, relating to the field of phytoremediation of heavy metal pollution. The invention includes the following steps: adding a 5 μM selenium ion concentration solution to a cadmium-polluted water environment; planting *Sedum aizoon* seedlings in the cadmium-polluted water environment for cultivation management; plant pretreatment: after culturing *Sedum aizoon* seedlings in unpolluted soil for at least three generations, selecting uniformly sized and healthy *Sedum aizoon* plants for pre-culturing in Hoaglands nutrient solution for two weeks to induce rooting; hydroponic treatment: transferring healthy plants to Hoaglands nutrient solution containing selenium for cultivation, and harvesting the plants after 28 days. This invention, by simulating a high-concentration cadmium-polluted water environment and applying selenium during the growth period of *Sedum aizoon*, promotes the absorption and accumulation of cadmium by *Sedum aizoon*, increases plant biomass, alleviates the toxic effects of cadmium on plants, and achieves efficient remediation of cadmium-polluted environments by *Sedum aizoon*.

Description

Technical Field

[0001] This invention relates to the field of phytoremediation of heavy metal pollution, specifically to a method for improving the cadmium accumulation and translocation capacity of the hyperaccumulating plant, Sedum spectabile. Background Technology

[0002] Heavy metal pollution caused by human activities such as industrial and agricultural production has become a global environmental problem. Cadmium, in particular, is a heavy metal element that is highly harmful to plant growth and development. Cadmium is highly mobile and easily absorbed by plant roots. When excessive cadmium is ingested by plants, it inhibits plant growth and development and can even lead to cadmium poisoning. Traditional physical and chemical remediation techniques are not suitable for large-scale cadmium pollution remediation due to their high costs. Phytoremediation is considered an eco-friendly cadmium pollution remediation technology, and its remediation effect largely depends on the ability of hyperaccumulating plants to absorb and accumulate heavy metals. However, under heavy metal stress, plants exhibit slow growth and low biomass, which affects phytoremediation efficiency and hinders its large-scale application. To alleviate cadmium toxicity to plants and improve phytoremediation efficiency, it is necessary to address the negative impacts of cadmium on plant physiological growth by improving plant antioxidant levels, protecting photosynthetic organs, and promoting plant growth and development.

[0003] Selenium is a beneficial micronutrient for plant growth and development, playing a crucial role in promoting plant growth and development under heavy metal stress. After absorbing adequate selenium, plants can reduce cadmium toxicity through various metabolic mechanisms. Appropriate selenium levels can promote plant growth and development, increase photosynthetic pigment content, improve antioxidant defense systems, enhance carbohydrate and nitrogen assimilation, and improve the absorption of other essential elements, thus effectively alleviating cadmium-induced plant stress. Under low to medium concentrations of cadmium pollution, selenium mitigates the adverse effects on crop growth and development; however, low to medium concentrations of cadmium pollution do not significantly affect the growth and development of hyperaccumulating plants, while high concentrations do. Research on the effects of selenium on mitigating cadmium toxicity in hyperaccumulating plants and on cadmium absorption and translocation under high concentrations of cadmium pollution is limited. Studies have found that selenium can activate plant protection mechanisms, reduce oxidative stress, and increase the absorption of arsenic by *Sedum aizoon*. To date, no research has been reported on the effects of selenium on the growth, development, and cadmium accumulation of the hyperaccumulating plant *Sedum sarmentosum* under cadmium pollution. Therefore, it is essential to explore the effects of selenium on the accumulation and translocation of cadmium in associated mineral Sedum. Summary of the Invention

[0004] This invention addresses the limitations of hyperaccumulating plants in phytoremediation technologies, such as slow growth and low biomass, by proposing a method to enhance the cadmium accumulation and translocation capacity of *Sedum aizoon*. By simulating a high-concentration cadmium-polluted aquatic environment, selenium is applied during the growth period of *Sedum aizoon*, thereby promoting the absorption and accumulation of cadmium, increasing plant biomass, mitigating the toxic effects of cadmium on plants, and achieving efficient remediation of cadmium-polluted environments using *Sedum aizoon*.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] A method for improving the ability of Sedum sarmentosum to accumulate and translocate cadmium includes the following steps: preparing a selenium solution with a selenium ion concentration of 5 μM and adding it to a cadmium-polluted water environment; planting Sedum sarmentosum seedlings in the cadmium-polluted water environment and carrying out cultivation management; wherein the selenium ion concentration in the selenium solution is added in the form of Na2SeO3 and calculated as Se.

[0007] (1) Plant pretreatment: After the seedlings of Sedum sarmentosum were cultured in non-polluted soil for at least three generations, the Sedum sarmentosum plants of uniform size and healthy growth were selected and pre-cultured in Hoaglands nutrient solution for two weeks to root.

[0008] (2) Hydroponic treatment: Select healthy plants with the same growth after two weeks of pre-culture and transfer them to Hoagland nutrient solution containing 0-100 μM selenium solution for culture. Harvest the plants after 28 days.

[0009] The healthy Sedum companion plant is defined as a plant with 4-8 true leaves and a plant length of 6-7 cm.

[0010] The plant pretreatment and hydroponic treatment were conducted under the following conditions: a 16-hour light cycle, day and night temperatures of 26°C and 20°C respectively, a relative humidity of 70%, and a light intensity of 300 µmol / m². 2 Maintain continuous ventilation for 24 hours, change the Hoagland nutrient solution every 3 days, and adjust the pH of the Hoagland nutrient solution to 5.8 with 0.1 mol / L NaOH solution or 0.1 mol / L HCl solution.

[0011] Compared with existing technologies, the beneficial effects are as follows: This invention provides a method to improve the ability of Sedum sarmentosum to accumulate and translocate cadmium. By utilizing the low-promoting and high-inhibiting effect of selenium on plant growth and cadmium absorption, the cadmium content and biomass of Sedum sarmentosum are increased, thereby improving the ability of Sedum sarmentosum to accumulate and translocate cadmium and effectively promoting the efficient remediation of cadmium-polluted areas by Sedum sarmentosum. Attached Figure Description

[0012] Figure 1 The effects of different cadmium and selenium treatments on the biomass of associated mineral Sedum;

[0013] Figure 2 The effects of different cadmium and selenium treatments on the cadmium concentration in associated mineral Sedum aizoon;

[0014] Figure 3 Cadmium translocation coefficient of Sedum sarcodactylon under different cadmium and selenium treatments;

[0015] Figure 4 The effects of different cadmium and selenium treatments on the photosynthetic pigment content of associated mineral Sedum aizoon;

[0016] Figure 5 Effects of different cadmium and selenium treatments on the activities of catalase (A), superoxide dismutase (B) and peroxidase (C) in Sedum aizoon;

[0017] Figure 6 The effects of different cadmium and selenium treatments on the malondialdehyde content of associated mineral Sedum sarcodactylum;

[0018] Figure 7 Transmission electron micrographs (2.0 μm) of root tip cells of *Sedum aizoon* under control (A), 100 μM cadmium (B), and 100 μM cadmium plus 5 μM selenium (C); where: CW represents the cell wall; N represents the nucleus; M represents the mitochondria; GB represents the Golgi apparatus.

[0019] Figure 8 Statistics on the number of differentially expressed genes in different comparison groups;

[0020] Figure 9 KEGG classification analysis of CK vs Cd comparison groups;

[0021] Figure 10 KEGG enrichment analysis of CK vs Cd comparison group;

[0022] Figure 11 KEGG classification analysis of Cd vs Cd / Se5 comparison groups;

[0023] Figure 12 KEGG enrichment analysis of Cd vs Cd / Se5 comparison groups.

[0024] Figure 1-6 In the figures, data are the mean plus standard error of three replicates (n=3). Data were analyzed using Duncan multiple comparisons. Different lowercase letters in each figure represent significant differences (p<0.05) between treatments with different concentrations of cadmium or selenium solutions. Se0, Se5, Se25, Se50, and Se100 represent selenium concentrations of 0, 5, 25, 50, and 100 μM, respectively. Cd0, Cd50, Cd100, and Cd150 represent cadmium concentrations of 0, 50, 100, and 150 μM, respectively. Detailed Implementation

[0025] The specific embodiments are described in detail below with reference to the accompanying drawings, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Unless otherwise specified, the raw materials and reagents used in the embodiments are commercially available.

[0026] In this embodiment, hydroponic cultivation was carried out using a polyethylene plastic box with a diameter of 17.5 cm and a height of 8.5 cm.

[0027] In the examples, sodium selenite (Na2SeO3) and cadmium chloride (CdCl22.5H2O) were both purchased from Sinopharm Chemical Reagent Co., Ltd.

[0028] The basic composition of the Hoagland nutrient solution used in the examples is as follows: Ca(NO3)2·4H2O 2.00 mM, CuSO4·5H2O 0.20 μM, KH2PO4 0.10 mM, MgSO4·7H2O 0.50 mM, KCl 0.10 mM, K2SO4 0.70 mM, H3BO3 10.00 μM, MnSO4·H2O 0.50 μM, ZnSO4·7H2O 0.50 μM, (NH4)6Mo7O 24 0.01 μM.

[0029] Example 1

[0030] To simulate a cadmium-polluted aquatic environment, a hydroponic experiment was conducted, with each culture box containing different concentrations of selenium and cadmium solutions and 1L of Hoagland nutrient solution.

[0031] *Sedum sarmentosum* was collected from an abandoned lead-zinc mine in Tianli Village, Qujiang District, Quzhou City, Zhejiang Province. Seedlings were cultured in uncontaminated soil for at least three generations. Uniformly sized and healthy plants were then pre-cultured in Hoaglands nutrient solution for two weeks to induce rooting. Six healthy plants of similar growth were selected as experimental material, with six plants per pot. Cadmium solutions of 0 (control group), 50, 100, and 150 μM were prepared by dissolving CdCl22.5H2O in deionized water to simulate cadmium pollution in the aquatic environment. The experiment was conducted in four groups, with three replicates per group, for a total of 12 pots. Samples were harvested and processed after 28 days of hydroponics.

[0032] The laboratory culture environment (i.e., plant pretreatment and hydroponic treatment) was as follows: the pH of the Hoagland nutrient solution was adjusted to 5.8 using 0.1 mol / L NaOH or 0.1 mol / L HCl solution, and changed every 3 days, with continuous aeration for 24 hours. The hydroponically grown Sedum sarmentosum was placed in an artificial climate chamber with day and night temperatures of 26°C and 20°C, respectively, with a 16-hour photoperiod and a light intensity of 300 µmol / m².2 / s, humidity around 70%. To maintain a constant nutrient solution volume, replenish the volume lost due to transpiration daily with Hoagland nutrient solution.

[0033] Example 2

[0034] Based on the operation in Example 1, a selenium solution with a concentration of 5 μM was prepared by dissolving Na2SeO3 in deionized water. Similarly, cadmium solutions with concentrations of 0, 50, 100 and 150 μM were prepared and mixed with it. The experiment was carried out in 4 groups, with 3 replicates in each group, and a total of 12 pots were cultured.

[0035] Example 3

[0036] Based on the operation in Example 1, a selenium solution with a concentration of 25 μM was prepared by dissolving Na2SeO3 in deionized water. Similarly, cadmium solutions with concentrations of 0, 50, 100 and 150 μM were prepared and mixed with it. The experiment was carried out in 4 groups, with 3 replicates in each group, and a total of 12 pots were cultured.

[0037] Example 4

[0038] Based on the operation in Example 1, a selenium solution with a concentration of 50 μM was prepared by dissolving Na2SeO3 in deionized water. Similarly, cadmium solutions with concentrations of 0, 50, 100 and 150 μM were prepared and mixed with it. The experiment was carried out in 4 groups, with 3 replicates in each group, and a total of 12 pots were cultured.

[0039] Example 5

[0040] Based on the operation in Example 1, a selenium solution with a concentration of 100 μM was prepared by dissolving Na2SeO3 in deionized water. Similarly, cadmium solutions with concentrations of 0, 50, 100 and 150 μM were prepared and mixed with it. The experiment was carried out in 4 groups, with 3 replicates in each group, and a total of 12 pots were cultured.

[0041] The treatment concentrations of Examples 1-5 above are shown in Table 1:

[0042] Table 1 Cadmium and selenium concentrations in different embodiments

[0043]

[0044] Examples 1-5: Samples were harvested and processed after 28 days of cultivation. Before collection and processing, plant roots were soaked in 20 mM EDTA-Na2 solution for 15 min to remove adsorbed metal ions from the root surface. The plants were then rinsed several times with deionized water and dried. Samples were separated into roots, stems, and leaves. One portion of the samples was flash-frozen in liquid nitrogen and stored in a -80°C cryogenic freezer for physiological index analysis. Another portion of the samples was blanched at 105°C for 30 min, then dried at 60°C to constant weight for later use. The dry weight (DW) was recorded, i.e., biomass determination (results are shown below). Figure 1 (As shown). After drying and grinding, the samples were used to determine the cadmium content in plant roots, stems, and leaves.

[0045] Under abiotic stress, plant biomass is one of the important indicators reflecting plant health. Figure 1 It was found that the addition of 5, 25, 50, and 150 μM selenium increased the biomass of *Sedum aizoon* at different cadmium concentrations, while the biomass decreased by 12.93% only at the 100 μM selenium and 150 μM cadmium treatments. This indicates that within the above concentration range, the addition of 5 μM selenium had the best promoting effect on the growth of *Sedum aizoon*.

[0046] Cadmium content determination:

[0047] The cadmium content in *Sedum aizoon* was determined according to the national food safety standard "Determination of Cadmium in Food" (GB 5009.15-2014). Plant samples were weighed and digested with a nitric acid-perchloric acid mixture (v:v=4:1). After the digest was brought to a final volume, the absorbance of cadmium in the solution was determined using a flame atomic absorption spectrometer (TAS-990, Beijing Purkinje General Instrument Co., Ltd., China). The cadmium content was quantitatively analyzed and the cadmium translocation coefficient was calculated using the standard curve method. The results are shown below. Figure 2 and Figure 3 As shown.

[0048] Depend on Figure 2 It was found that the addition of 5 μM selenium significantly increased the cadmium content in the leaves of *Sedum aizoon* under different cadmium concentrations, with differences in the magnitude of cadmium content changes between stems and roots. The increase in cadmium content in the leaves was most significant under the 5 μM selenium and 100 μM cadmium treatments, increasing by 27.62%. The addition of 25, 50, and 100 μM selenium decreased the cadmium content in the leaves of *Sedum aizoon* under different cadmium concentrations, but significantly increased the cadmium content in the roots. The changes in the cadmium translocation coefficient of *Sedum aizoon* were calculated. Figure 3It was found that adding 5 μM selenium increased the stem / root translocation coefficient by 1.26% under 100 μM cadmium treatment; adding 5 μM selenium increased the leaf / stem translocation coefficient by 2.06% under 50 μM cadmium treatment. Adding high concentrations of selenium (50 or 100 μM) reduced the cadmium translocation coefficient of *Sedum aizoon* under different cadmium concentrations. Therefore, within the above concentration range, adding 5 μM selenium increased the cadmium content in the roots, stems, and leaves of *Sedum aizoon* under different cadmium concentrations, thus improving its cadmium translocation capacity.

[0049] Determination of photosynthetic pigment content:

[0050] The content of photosynthetic pigments was determined using the 80% acetone extraction method. Fresh plant leaves were washed and dried, avoiding the veins. 0.20 g of leaves from the same location were weighed and placed in a porcelain mortar. 2 mL of 80% acetone was added, and the mixture was ground into a homogenate. The homogenate was filtered into a 25 mL brown volumetric flask. The residual pigment on the filter paper was washed into the flask with 80% acetone, and the mixture was brought to the mark and mixed thoroughly. The absorbance was measured at 663 nm, 646 nm, and 470 nm using a microplate reader. The contents of chlorophyll a, chlorophyll b, chlorophyll, and carotenoids were calculated using the following formulas. The results are shown below. Figure 4 As shown.

[0051]

[0052]

[0053]

[0054]

[0055] Depend on Figure 4 It was found that the addition of 5, 25, and 50 μM selenium significantly increased the chlorophyll a and chlorophyll b contents of *Sedum aizoon* at 50 and 100 μM cadmium concentrations. Among these, the addition of 5 μM selenium resulted in the highest chlorophyll content (1.17 mg / g) at the 50 μM cadmium concentration. The addition of different selenium concentrations decreased the carotene content of *Sedum aizoon* at both 100 and 150 μM cadmium treatments. However, the addition of only 5 and 50 μM selenium increased the carotene content by 7.50% and 2.50%, respectively, at the 50 μM cadmium treatment. This indicates that within the above concentration range, the addition of 5 μM selenium significantly promoted the photosynthesis of *Sedum aizoon*.

[0056] Antioxidant enzyme activity assay

[0057] Weigh 0.30 g of fresh plant leaves and place them in a pre-cooled porcelain mortar at 4°C. Add 3 mL of potassium phosphate buffer (pH 7.8) and grind into a homogenate. Transfer the homogenate to a clean 10 mL centrifuge tube. Centrifuge the homogenate at 4°C and 8000 g for 10 min. Take the supernatant as the crude extract for enzyme activity determination.

[0058] (1) Assay of catalase (CAT) activity

[0059] Take 100 μL of crude extract, add 2.8 mL of EDTA-containing phosphate buffer (pH 7.0) and 100 μL of hydrogen peroxide solution, respectively, and use a UV-Vis spectrophotometer to measure the initial absorbance value A1 at a wavelength of 240 nm. After the reaction is completed by 1 min, record the final absorbance value A2. Calculate the CAT activity based on the change in absorbance value before and after the reaction.

[0060] Depend on Figure 5 As shown in Figure A, without the addition of exogenous selenium, the CAT activity of *Sedum aizoon* significantly decreased by 36.19%–71.52% under different cadmium concentrations. Under the interaction of selenium and cadmium, the CAT activity of *Sedum aizoon* showed a trend of first increasing and then decreasing. Specifically, the treatments of 5 μM selenium + 100 μM cadmium and 50 μM selenium + 50 μM cadmium significantly increased the plant's CAT activity, by 190.95% and 161.29%, respectively. The addition of 50 or 100 μM selenium reduced CAT activity.

[0061] (2) Assay of superoxide dismutase (SOD) activity

[0062] SOD activity was determined using the nitrotetrazole blue (NBT) colorimetric method. 2.725 mL of a mixed solution consisting of phosphate buffer (pH 7.8), EDTA-Na2 solution, methionine solution, nitrotetrazole blue solution, and riboflavin solution was added to a sample tube, followed by 25 mL of crude extract and 25 mL of hydrogen peroxide solution. An equal volume of the mixed solution and hydrogen peroxide solution was added to a blank tube (without the crude extract). Both sample and blank tubes were placed under sunlight at 4000 Lx for 20 min. A control tube consisting of 2.725 mL of the mixed solution, 25 mL of deionized water, and 25 mL of hydrogen peroxide solution was prepared and placed in the dark. The absorbance of the control tube was measured at 560 nm using a microplate reader.

[0063] Depend on Figure 5As shown in Figure B, without the addition of exogenous selenium, the SOD activity of the plants decreased by 31.24% and 27.27% under 100 and 150 μM cadmium treatments, respectively. The addition of 5 and 25 μM selenium, at different cadmium concentrations, both increased the SOD activity of *Sedum aizoon*. The treatment with 5 μM selenium and 150 μM cadmium showed the most significant increase in SOD activity, increasing by 133.31%. The addition of high concentrations of selenium (50 or 100 μM) significantly reduced the SOD activity of *Sedum aizoon*.

[0064] (3) Peroxidase (POD) activity assay

[0065] POD activity was determined using the guaiacol colorimetric method. 5 μL of crude extract was added to a sample tube, followed by 120 μL of guaiacol solution, 30 μL of hydrogen peroxide solution (300 mM), 30 μL of EDTA-containing phosphate buffer (pH 7.0), and 60 μL of deionized water. The mixture was rapidly mixed and transferred to a micro-volume quartz cuvette. The absorbance (A1) was measured at 470 nm for 30 seconds using a UV-Vis spectrophotometer. After 1 min of reaction, the final absorbance (A2) was recorded. POD activity was calculated based on the changes in absorbance before and after the reaction.

[0066] Depend on Figure 5 As shown in Figure C, without the addition of exogenous selenium, the POD activity of *Sedum aizoon* decreased under different cadmium concentrations. Under the interaction of selenium and cadmium, the addition of 5-50 μM selenium increased the POD activity of the plants to varying degrees. The treatment with 25 μM selenium and 150 μM cadmium showed the most significant increase in POD activity, increasing by 344.40%. The addition of 100 μM selenium decreased the POD activity of *Sedum aizoon*.

[0067] Therefore, within the above concentration range, the addition of 5 μM selenium to different concentrations of cadmium treatments increased the activity of antioxidant enzymes in the plants and alleviated the toxicity of high concentrations of cadmium to Sedum sarmentosum.

[0068] Malondialdehyde (MDA) content determination:

[0069] The malondialdehyde (MDA) content was determined using the thiobarbituric acid (TBA) colorimetric method. 0.10 g of fresh plant leaves were weighed and placed in a pre-cooled porcelain mortar at 4°C. A 10% trichloroacetic acid solution was added, and the mixture was ground into a homogenate, which was then transferred to a 10 mL centrifuge tube. The homogenate was centrifuged at 12000 g for 10 min at 4°C. 2 mL of the supernatant was collected in a 10 mL test tube, and 2 mL of a 0.67% thiobarbituric acid solution was added. The mixture was reacted in a boiling water bath for 10 min, rapidly cooled in an ice bath, and then centrifuged again. A suitable amount of the supernatant was then transferred to a 96-well microplate, and the absorbance was measured at 450 nm, 532 nm, and 600 nm using a microplate reader. The MDA content was calculated, and the results are shown below. Figure 5 As shown.

[0070] As Figure 6 It was found that, without the addition of exogenous selenium, the malondialdehyde (MDA) content in the leaves of *Sedum aizoon* increased under different cadmium concentrations. With the addition of 5 μM selenium, the MDA content decreased by 31.49-49.21% under different cadmium concentrations. With the addition of high concentrations of selenium (50 or 100 μM), the MDA content increased by 4.53%-240.26% under different cadmium concentrations. This indicates that within the above concentration range, the addition of 5 μM selenium reduced the degree of membrane lipid peroxidation in plant cells and alleviated cadmium-induced oxidative damage.

[0071] Root tip cell ultrastructure:

[0072] Figure 7 The study revealed changes in the ultrastructure of *Sedum aizoon* root tip cells. In the control group, the root tip cells had smooth, continuous cell walls, clear nuclei, round nucleoli, and clearly visible oval mitochondria and Golgi bodies. Under 100 μM cadmium treatment, *Sedum aizoon* root tip cells exhibited a series of morphological changes, such as cell deformation and rupture, cell wall thickening, and loss of some organelles. The addition of 5 μM selenium improved the structure of the cell membrane and vacuoles in root tip cells, maintaining cell integrity and revealing clear cell walls and well-differentiated organelles. This indicates that under high cadmium stress, the addition of 5 μM selenium improved the ultrastructure of root tip cells and alleviated cadmium-induced membrane damage.

[0073] Root transcriptome:

[0074] To determine the regulatory mechanism of selenium treatment on *Sedum aizoon* seedlings under cadmium stress, this invention prepared root samples of *Sedum aizoon* seedlings under different treatments for RNA-seq experiments, analyzed transcriptome data, and screened differentially expressed genes (DEGs) between the control (CK) and other treatments. This invention collected samples from three treatments: CK (blank), Cd (100 μM cadmium), and Cd / Se5 (100 μM cadmium + 5 μM selenium). Three replicates were taken from each treatment, for a total of nine samples, for RNA-seq transcriptome analysis. The RNA-seq was performed by Shanghai Meiji Biotechnology Co., Ltd. After library construction, sequencing was performed using the Illumina platform. After quality control and assembly of the sequencing data, differential gene expression analysis was conducted.

[0075] Differential expression analysis between sample groups was performed using DESeq2 software, with fold change (FC) ≥ 2 and false detection rate (FDR) < 0.05 as screening criteria. Figure 8 As shown, a total of 12,626 differentially expressed genes were identified in the two comparison groups, including 9,288 upregulated genes and 3,338 downregulated genes. In the CK vs Cd comparison group, there were 2,264 differentially expressed genes, including 1,623 upregulated genes and 641 downregulated genes; in the Cd vs Cd / Se5 comparison group, there were 10,362 differentially expressed genes, including 7,665 upregulated genes and 2,697 downregulated genes.

[0076] To further understand the metabolic pathways involved in the differentially expressed genes of *Sedum aizoon* in response to selenium-cadmium interaction, the KEGG database was used to classify and enrich differentially expressed genes in different comparison groups. In the CK vs Cd comparison group, the differentially expressed genes were... Figure 9 KEGG classification analysis of the CK vs Cd comparative group showed that 100 μM cadmium treatment upregulated the number of genes in pathways such as environmental adaptation, translation, and carbohydrate metabolism, and increased the number of genes in replication and repair pathways compared to downregulated pathways; Figure 10 KEGG enrichment analysis of the CK vs Cd comparison group showed that differentially expressed genes downregulated by 100 μM cadmium treatment were mainly enriched in pathways such as isoflavone synthesis, taurine and low-taurine metabolism, and butyrate metabolism, while differentially expressed genes upregulated by cadmium treatment were mainly enriched in pathways such as photosynthesis, ribosomes, and carbon fixation by photosynthetic organisms. In the Cd vs Cd / Se5 comparison group, [the following data were observed]. Figure 11 KEGG classification analysis of the Cd vs Cd / Se5 comparative group showed that treatment with 5 μM selenium and 100 μM cadmium upregulated the number of genes involved in pathways such as translation, carbohydrate metabolism, folding, sorting and degradation, transport and catabolism, and energy metabolism; Figure 12KEGG enrichment analysis of the Cd vs Cd / Se5 comparative group revealed that the differentially expressed genes upregulated by 5 μM selenium and 100 μM cadmium treatment were mainly enriched in pathways such as photosynthesis, photosynthetic antennal proteins, cutin, primary alkaloid and wax biosynthesis, and glycerol lipid metabolism. The differentially expressed genes downregulated by cadmium treatment were mainly enriched in pathways such as zeatin biosynthesis, extracellular polysaccharide biosynthesis, plant hormone signal transduction, and sesquiterpene and triterpene biosynthesis. Therefore, the addition of 5 μM selenium can alleviate the toxicity of cadmium to plants by inhibiting the accumulation of reactive oxygen species, protecting photosynthetic organs, and improving plant metabolic activities, thereby promoting plant growth.

[0077] The method of this invention improves the biomass, cadmium content, and cadmium translocation capacity of *Sedum aizoon* by adding 5 μM selenium, while adding high concentrations of selenium (50 or 100 μM) significantly inhibits the cadmium translocation capacity of *Sedum aizoon*. In phytoremediation of cadmium-contaminated environments, applying appropriate concentrations of selenium can improve the remediation efficiency of *Sedum aizoon* in cadmium-contaminated environments.

Claims

1. A method for improving the ability of associated mineral Sedum to accumulate and translocate cadmium, characterized in that, The process includes the following steps: preparing a selenium solution with a selenium ion concentration of 5 μM and adding it to a cadmium-polluted water environment; planting Sedum sarmentosum seedlings in the cadmium-polluted water environment and carrying out cultivation management; the selenium ion concentration in the selenium solution is added in the form of Na2SeO3 and calculated as Se. (1) Plant pretreatment: After the seedlings of Sedum sarmentosum were cultured in non-polluted soil for at least three generations, the Sedum sarmentosum plants of uniform size and healthy growth were selected and pre-cultured in Hoaglands nutrient solution for two weeks to root. (2) Hydroponic treatment: Select healthy plants with the same growth after two weeks of pre-culture and transfer them to the selenium-containing cadmium-polluted water environment prepared above for cultivation. Harvest the plants after 28 days. The healthy Sedum companion plant is defined as a plant with 4-8 true leaves and a plant length of 6-7 cm. The plant pretreatment and hydroponic treatment were conducted under the following conditions: a 16-hour light cycle, day and night temperatures of 26°C and 20°C respectively, a relative humidity of 70%, and a light intensity of 300 µmol / m². 2 Maintain continuous ventilation for 24 hours, change the Hoagland nutrient solution every 3 days, and adjust the pH of the Hoagland nutrient solution to 5.8 with 0.1 mol / L NaOH solution or 0.1 mol / L HCl solution.

2. The method for improving the cadmium accumulation and translocation capacity of associated mineral Sedum as described in claim 1, characterized in that, To maintain a constant volume of nutrient solution, the volume lost due to transpiration is replenished daily with Hoagland nutrient solution.

3. The method for improving the cadmium accumulation and translocation capacity of associated mineral Sedum as described in claim 1, characterized in that, The basic composition of Hoagland nutrient solution is: Ca(NO3)2·4H2O 2.00 mM, CuSO4·5H2O 0.20 μM, KH2PO4 0.10 mM, MgSO4·7H2O 0.50 mM, KCl 0.10 mM, K2SO4 0.70 mM, H3BO3 10.00 μM, MnSO4·H2O 0.50 μM, ZnSO4·7H2O 0.50 μM, (NH4)6Mo7O 24 0.01 μM.

4. The method for improving the cadmium accumulation and translocation capacity of associated mineral Sedum as described in claim 1, characterized in that, A 5 μM selenium solution was prepared by dissolving Na₂SeO₃ in deionized water. Similarly, four cadmium solutions (0, 50, 100, and 150 μM) were mixed with it. The cadmium solution was added as CdCl₂ and calculated as Cd. After 28 days of cultivation, samples were harvested and processed. Before collection and processing, plant roots were immersed in a 20 mM EDTA-Na₂ solution for 15 min to remove adsorbed metal ions from the root surface. The plants were then rinsed several times with deionized water and dried. Samples were separated into roots, stems, and leaves. One portion of the samples was flash-frozen in liquid nitrogen and stored in a -80°C cryogenic freezer for physiological index analysis. Another portion of the samples was blanched at 105°C for 30 min and then dried at 60°C to constant weight for later use. The dry weight (DW) was recorded, representing biomass determination. After drying and grinding, the cadmium content in the roots, stems, and leaves was determined. The addition of 5 μM selenium showed the best growth-promoting effect on *Sedum aizoon* under different cadmium concentrations.

5. The method for improving the cadmium accumulation and translocation capacity of associated mineral Sedum as described in claim 1, characterized in that, To determine the cadmium content in *Sedum aizoon*, plant samples were weighed and digested with a nitric acid-perchloric acid mixed solution (v:v=4:1). After the digest was brought to a final volume, the absorbance of cadmium in the solution was measured using a flame atomic absorption spectrometer. The cadmium content was quantitatively analyzed and the cadmium translocation coefficient was calculated using the standard curve method. The addition of 5 μM selenium to different concentrations of cadmium treatments increased the cadmium content in the roots, stems, and leaves of *Sedum aizoon*, thus improving the plant's ability to translocate cadmium.

6. The method for improving the cadmium accumulation and translocation capacity of associated mineral Sedum according to claim 1, characterized in that, Under high cadmium stress, the addition of 5 μM selenium increased the activity of antioxidant enzymes in the plants and alleviated the toxicity of high cadmium to Sedum sarmentosum.

7. The method for improving the cadmium accumulation and translocation capacity of associated mineral Sedum as described in claim 1, characterized in that, The photosynthetic pigment content was determined by 80% acetone extraction. Under high cadmium stress, the addition of 5 μM selenium significantly increased the photosynthetic pigment content of Sedum sarmentosum.

8. The method for improving the cadmium accumulation and translocation capacity of associated mineral Sedum according to claim 1, characterized in that, The malondialdehyde (MDA) content was determined using the thiobarbituric acid (TBA) colorimetric method. Under high cadmium stress, the addition of 5 μM selenium reduced the MDA content in plant cells and decreased the degree of membrane lipid peroxidation.

9. The method for improving the cadmium accumulation and translocation capacity of associated mineral Sedum according to claim 1, characterized in that, Under high cadmium stress, the addition of 5 μM selenium improved the ultrastructure of root tip cells and alleviated cadmium-induced membrane damage.

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