Method for reducing phthalate accumulation in plants using 2,4-epibrassinolid and applications

Abstract

This invention discloses a method and application of 2,4-epibrassinolide to reduce phthalate accumulation in plants. The method involves intervening in phthalate accumulation in plants with exogenous 2,4-epibrassinolide, specifically by spraying an aqueous solution of 2,4-epibrassinolide onto plant seedlings grown in phthalate-contaminated soil. This invention reduces PAE residues in plants through exogenous application of the plant hormone EBR. It has been found to promote not only the metabolism of short-chain PAEs but also the metabolism of some low-bioavailability long-chain PAEs. The method is simple to operate, highly efficient, and has a wide range of effects. The treatment process regulates plant growth, does not cause secondary pollution, and is environmentally friendly.

Description

Technical Field

[0001] This invention belongs to the field of agricultural risk management technology, specifically relating to a method and application of using 2,4-epibrassinolone to reduce the accumulation of phthalates in plants. Background Technology

[0002] Phthalic acid esters (PAEs) are persistent organic pollutants in the environment. Widely used as plasticizers in daily life to make plastic products more flexible and durable, plasticizers have recently received increasing negative attention due to the fact that PAEs do not irreversibly bind to the polymer matrix, making them easily released into the environment and detected in various environmental matrices such as air, water, soil, and sediment. PAEs are widely and frequently detected in various environmental zones globally, and studies have shown that PAEs can cause significant harm to the nervous, cardiovascular, and reproductive systems through inhalation or skin absorption / exposure. The USEPA has listed diethyl phthalate (DEP), dibutyl phthalate (DnBP), dimethyl phthalate (DMP), di(2-ethylhexyl) phthalate (DEHP), di-n-octyl phthalate (DnOP), and butyl benzyl phthalate (BBP) as key environmental pollutants. my country has also identified DMP, DEP, and DnOP as priority environmental pollutants for control.

[0003] In recent years, due to the widespread use of plastic film and greenhouses, as well as the input of wastewater irrigation and atmospheric deposition, large amounts of plant-based airborne emulsions (PAEs) have been found in farmland soil samples around the world. Once in the environment, PAEs are absorbed and accumulated by plants through various means. PAEs are then ingested by humans through the food chain, ultimately posing a threat to human health.

[0004] 2,4-epibrassinolide (EBR), also known as brassinolide or brassinosteroid, is a novel plant endogenous hormone that can regulate plant growth and development, enhance plant detoxification capabilities, and has been shown to inhibit the growth of human cancer cell lines without affecting the growth of normal cells. Current research demonstrates that EBR can promote the metabolism of pollutants in plants and reduce pollutant residues by upregulating the gene expression of detoxification enzymes, including P450 monooxygenase and carboxylesterase (CXE). Currently, there are no reports of exogenous EBR intervention in PAE accumulation in plants; therefore, this invention proposes a method for reducing PAE accumulation in plants using EBR. Summary of the Invention

[0005] To address the problems existing in the prior art, one objective of this invention is to provide a method for reducing the accumulation of phthalic acid esters (PAEs) in plants using 2,4-epibrassinolide. This method is simple to operate, low in cost, does not cause secondary pollution to plants and soil, is fast-acting, and is of great significance in reducing the accumulation of PAEs in plants. Furthermore, when the plant is ingested, it will not cause harm to human health.

[0006] Specifically, this is achieved through the following technical solution: a method for reducing the accumulation of phthalates in plants using 2,4-epibrassinolide. This method uses exogenous 2,4-epibrassinolide to intervene in the accumulation of phthalates in plants. Specifically, an aqueous solution of 2,4-epibrassinolide is sprayed onto plant seedlings grown in soil contaminated with phthalates.

[0007] Furthermore, the aqueous solution concentration of the 2,4-epibrassinolide is 0.05-0.1 μM.

[0008] Further, the phthalate ester includes at least one of dimethyl phthalate, diethyl phthalate, diisobutyl phthalate, di-n-butyl phthalate, di(2-ethylhexyl) phthalate, and di-n-octyl phthalate.

[0009] Furthermore, the spraying method is foliar spraying, spraying until water drips from the leaves.

[0010] Furthermore, the concentrations of various phthalic acid pollutants in phthalate-contaminated soils range from 1 to 10 mg / kg.

[0011] Furthermore, the plants are crops, including but not limited to lettuce, carrots, tomatoes, and wheat during their growth period.

[0012] Another object of the present invention is to provide the application of 2,4-epibrassinolone in reducing the accumulation of phthalates in plants.

[0013] This invention reduces PAE residues in plants by exogenously spraying the plant hormone EBR. It has been found that this method not only promotes the metabolism of short-chain PAEs but also significantly improves the metabolism of some long-chain PAEs with low bioavailability. Furthermore, the method is simple to operate, highly efficient, and has a wide range of effects. The treatment process regulates plant growth, does not cause secondary pollution, and is environmentally friendly. Attached Figure Description

[0014] Figure 1 The effect of EBR on the accumulation of PAEs in lettuce;

[0015] Figure 2 The effect of EBR on the accumulation of MPAEs in lettuce;

[0016] Figure 3 The effect of EBR on the expression of P450 enzyme genes in lettuce;

[0017] Figure 4 The effect of EBR on CXE gene expression in lettuce. Detailed Implementation

[0018] The present invention will be further described in detail below with reference to specific embodiments in order to better understand the technical solution.

[0019] In this embodiment of the invention, EBR (CAS No. 78821-43-9) was purchased from Sinopharm Chemical Co., Ltd. (Nanjing, Jiangsu, China). The solid powder was first dissolved in anhydrous ethanol, and then diluted with water to a concentration of 0.05-0.1 μM.

[0020] The room temperature described in this invention is 20-25℃.

[0021] In this embodiment of the invention, the concentration of PAEs in the soil is 5 mg / kg, that is, the concentrations of DMP, DEP, DiBP, DnBP, DEHP, and DnOP are all 5 mg / kg.

[0022] Example 1

[0023] Sample cultivation: Growing lettuce was transplanted into ceramic pots (outer diameter × height, 14 × 12 cm) containing 700 g of PAE-contaminated soil. The soil moisture content was 60% of the maximum field capacity, and the plants were exposed to 12 h of light per day at room temperature. The experiment consisted of three treatments: a control group, a PAE group, and a PAE+EBR group. For the EBR treatment group, 0.1 μM EBR solution was sprayed onto the leaves until dripping water was observed 24 h after transplanting. For the other experimental groups, distilled water was used instead, and spraying was performed every five days. 24 h after transplanting was recorded as day 0. Samples were collected at days 0, 4, 8, 12, 16, and 20 of the EBR treatment. The plant samples after cultivation were frozen at -80℃ to terminate the experiment. Subsequently, the plant samples were placed in a freeze dryer and dried at -55℃ for 48 h to remove moisture.

[0024] Extraction and purification of PAEs in samples: 0.2 g of each plant sample was placed in a glass centrifuge tube, and 10 mL of acetonitrile was added for PAE extraction. The mixture was vortexed for 10 min, sonicated for 30 min, and then centrifuged at 3000 rpm for 10 min. The supernatant was transferred to a glass centrifuge tube containing 40 mg of graphitized carbon black, vortexed for 1 min, and then filtered through a 0.45 μm filter membrane. The sample was dissolved in 1 mL of n-hexane by rotary evaporation and nitrogen blowing, and stored in a 2 mL brown vial at -20℃ for GC-MS analysis. The peak areas of each PAE in the plant samples were determined by gas chromatography-mass spectrometry (GC-MS). Based on the standard curves of each PAE (Table 1), the concentrations of each PAE during cultivation were calculated. The results are shown below. Figure 1 As shown in the figure. Under the above conditions, during the cultivation process, the concentrations of various PAEs in lettuce treated with EBR were always lower than those in the PAE group. After 20 days of EBR treatment, the residual amounts of DMP, DEP, DiBP, DnBP, DEHP, and DnOP in the EBR-treated lettuce were 20.8%, 30.9%, 3.2%, 5.5%, 34.3%, and 8.3% lower, respectively, than those in the PAE-treated lettuce. This indicates that EBR treatment can reduce the accumulation of PAEs in plants.

[0025] Table 1. PAEs Marking Equations

[0026] PAEs Standard curve equation <![CDATA[Correlation coefficient (R 2 )]]> DMP y = 90.986x - 378.94 <![CDATA[R 2 =0.9997]]> DEP y = 1159.4x - 19611 <![CDATA[R 2 =0.9993]]> DiBP y = 51.098x - 777.88 <![CDATA[R 2 =0.9994]]> DnBP y = 11.855x - 268.93 <![CDATA[R 2 =0.9974]]> DEHP y = 11.855x - 268.93 <![CDATA[R 2 =0.9986]]> DnOP y = 134.67x - 1737.7 <![CDATA[R 2 =0.9994]]>

[0027] Example 2

[0028] According to the cultivation method in Example 1, this invention scientifically verifies the effect of EBR on PAEs accumulation in plants by detecting the contents of monobutyl phthalate (MnBP) and monoethylhexyl phthalate (MEHP), the metabolites corresponding to DnBP and DEHP, as well as the gene expression of metabolic enzymes (P450 enzymes and CXE) in plants. The concentration changes of MPAEs in lettuce are shown below. Figure 2 As shown, in the early stage of cultivation, compared with the PAE group, the concentration of MPAEs in lettuce treated with EBR increased faster, while the accumulation of PAEs was slower. Furthermore, during the cultivation process, the concentration of metabolites in lettuce treated with EBR was higher, indicating that EBR can accelerate the metabolism of PAEs in plants, thereby reducing the residue of PAEs in plants.

[0029] The relative expression levels of metabolic enzyme genes in lettuce during cultivation are as follows: Figure 3 (P450 enzyme) and Figure 4(CXE) is shown. Under the above conditions, after PAEs entered the plant, the relative expression of P450 enzyme genes gradually increased with the increase of culture time, reaching its maximum at 8 days of treatment, playing a major role in the early stage of PAE metabolism. EBR treatment can increase P450 gene expression under PAE stress at all time points (16%-35% higher than the PAE group), indicating that EBR can increase the water solubility of PAEs by increasing P450 enzyme gene expression. Compared with the control group, PAEs interfered with CXE production in the early stage of culture, resulting in a decrease in CXE gene expression in lettuce. After 4 days of culture, the CXE gene expression gradually increased with the culture time. However, the plant's own ability to resist stress is limited. EBR application increased CXE gene expression under PAE stress at all time points (33%-42% higher than the PAE group), indicating that EBR can accelerate the hydrolysis of PAEs in plants by increasing carboxylesterase gene expression. The expression levels of metabolic enzyme genes in lettuce changed during 4-8 days of cultivation, and the accumulation of PAEs in lettuce decreased at this time, indicating that P450 enzymes and CXE have a positive effect on the metabolism of PAEs in plants. The application of EBR increased the gene expression of P450 enzymes and CXE, thereby promoting the metabolism of PAEs in plants and reducing the accumulation of PAEs in plants.

Claims

1. A method for reducing the accumulation of phthalate esters in plants by 2,4-epibrassinolid, characterized in that This method uses exogenous 2,4-epibrassinolactone to intervene in the accumulation of phthalates in plants. Specifically, an aqueous solution of 2,4-epibrassinolactone is sprayed onto plant seedlings cultured in phthalate-contaminated soil. The concentration of the aqueous solution of 2,4-epibrassinolactone is 0.05-0.1 μM.

2. The method for 2,4-epibrassinolide to reduce accumulation of phthalate esters in plants according to claim 1, characterized in that, The phthalate esters include at least one of dimethyl phthalate, diethyl phthalate, diisobutyl phthalate, di-n-butyl phthalate, di(2-ethylhexyl) phthalate, and di-n-octyl phthalate.

3. The method for 2,4-epibrassinolid reducing phthalate ester accumulation in plants according to claim 1, characterized in that The spraying method is foliar spraying, spraying until water drips from the leaves.

4. The method for 2,4-epibrassinolid reducing phthalate ester accumulation in plants according to claim 1, wherein The concentrations of various phthalic acid pollutants in phthalate-contaminated soil range from 1 to 10 mg / kg.

5. The method for reducing phthalate accumulation in plants using 2,4-epibrassinolide as described in claim 1, characterized in that... The plants mentioned are crops, including but not limited to lettuce, carrots, tomatoes, and wheat during their growth period. 6.2,4-Epibrassinolactone in reducing phthalate accumulation in plants, characterized in that, Specifically, the application involves spraying an aqueous solution of 2,4-epibrassinolide onto plant seedlings grown in phthalic acid-contaminated soil, wherein the concentration of the aqueous solution of 2,4-epibrassinolide is 0.05-0.1 μM.

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