Method for relieving tin stress of plants through calcium folate and application of method

Abstract

The invention discloses a method for relieving plant tin (Sn < 4 + >) stress through calcium folate and application, and belongs to the technical field of plant stress resistance and heavy metal pollution remediation. Aiming at the problems of high cost, single target spot, insufficient metabolic regulation and the like caused by dependence on a chelating agent or an antioxidant in the prior art, multiple-effect synergistic alleviation is realized by exogenously applying calcium folate with a specific concentration: plant leaf shedding and root length shortening induced by Sn < 4 + > are inhibited, active oxygen (H2O2 and O2-) is effectively removed, membrane lipid peroxidation damage is reduced, and the yield of the plant is increased. The antioxidant enzyme activity and chlorophyll synthesis capability are recovered, and the photosynthetic function is improved. In a molecular mechanism, a folic acid synthesis pathway is activated and related gene expression is regulated and controlled. The method can synchronously improve plant stress resistance and heavy metal restoration efficiency, can reduce Sn < 4 + > bio-availability when applied to aquatic plants, can block heavy metal food chain transmission when applied to terrestrial crops, and provides an innovative technical scheme for ecological management and agricultural safety production.

Description

Technical Field

[0001] This invention relates to the field of plant stress resistance and heavy metal pollution control technology, specifically a method for alleviating tin stress in plants using calcium folic acid and its application. Background Technology

[0002] Tin (Sn) 4+ Sn, a widespread industrial heavy metal pollutant, primarily originates from human activities such as electronics manufacturing, electroplating processes, tin mining, and metallurgical wastewater discharge. Its accumulation in the environment poses a serious threat to ecosystems and agricultural production. At the plant physiological level, Sn... 4+ Stress can cause irreversible damage through multiple pathways: on the one hand, Sn 4+ It can directly target the ultrastructure of chloroplasts, causing thylakoid membrane rupture and grana stacking disorder, thereby disrupting the integrity of the photosynthetic electron transport chain, inactivating photosystem II (PSII), and resulting in chlorophyll degradation and a significant decrease in photosynthetic efficiency; on the other hand, Sn 4+ It can induce reactive oxygen species (ROS) in plant cells, such as hydrogen peroxide (H2O2) and superoxide anion (O2). - The explosive accumulation of these substances triggers a chain reaction of membrane lipid peroxidation, leading to the massive production of toxic products such as malondialdehyde (MDA), ultimately resulting in increased cell membrane permeability, extravasation of cell contents, and programmed cell death. Furthermore, Sn... 4+ It also inhibits plant root growth, leading to shortened root length and obstructed lateral root development, further weakening the plant's ability to absorb water and nutrients and exacerbating the growth inhibition effect.

[0003] Currently, regarding plant Sn 4+ Stress relief techniques mainly rely on two types of methods: one is chemical chelating agents (such as citric acid, ethylenediaminetetraacetic acid (EDTA), etc.), which work by reacting with Sn... 4+ The formation of stable chelates reduces their bioavailability, but excessive application of such substances can easily disrupt the pH balance of soil or water, leading to soil acidification, nutrient leaching, and imbalance of the microecological community structure, causing secondary environmental risks. Secondly, exogenous antioxidants (such as glutathione, vitamin C, etc.) alleviate oxidative damage by directly scavenging ROS, but these substances have short action cycles and single targets, and can only achieve instantaneous antioxidant protection. They cannot synergistically regulate the activity integration of the plant's endogenous antioxidant enzyme system (such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT)), nor can they intervene in the dynamic balance of the plant's metabolic network under stress.

[0004] Folic acid (vitamin B9), as a core coenzyme in plant one-carbon metabolism, plays a crucial role in nucleic acid synthesis, amino acid metabolism, and the maintenance of redox balance. Its metabolic homeostasis is closely related to plant stress resistance. However, existing research largely focuses on the indirect regulatory role of key folic acid synthesis genes (such as methylenetetrahydrofolate reductase (MTHFR)) under abiotic stress, while studies on the function of folic acid derivative calcium folic acid in heavy metal stress responses have not yet been conducted. Crucially, the question remains whether exogenous calcium folic acid can directly interfere with Sn. 4+ There are currently no research reports on the molecular mechanisms by which plants maintain ROS metabolic balance and repair photosynthetic damage under stress, and whether they achieve systemic stress resistance enhancement by activating key genes in the folate synthesis pathway (such as aminodeoxycarylate synthase (ADCS) and folate polyglutamate synthase (FPGS)) and metabolic regulatory genes (such as γ-glutamyl hydrolase 1 (GGH1)).

[0005] Therefore, it is necessary to develop a multi-pathway synergistic method based on calcium folic acid to alleviate plant Sn. 4+ The coercive approach is of great significance for overcoming the limitations of existing technologies and improving the effectiveness of heavy metal pollution control. Summary of the Invention

[0006] The technical problem to be solved by this invention is the existing method for mitigating plant tin (Sn) 4+ The techniques for addressing stress have significant limitations. Specifically, reliance on chemical chelating agents easily leads to soil acidification and microecological imbalance; reliance on exogenous antioxidants can only temporarily scavenge reactive oxygen species (ROS) and cannot synergistically regulate the functional integration of endogenous antioxidant enzyme systems and the dynamic balance of folic acid metabolism. Furthermore, research on the function of calcium folic acid in the response to heavy metal stress is still lacking. Whether it mediates Sn by activating key genes in the folic acid synthesis pathway (such as ADCS and FPGS) and metabolic regulatory genes (such as GGH1) remains unclear. 4+ The molecular mechanism of stress relief is still unclear, and there is an urgent need to provide a multi-effect synergistic method for the application of calcium folic acid to overcome the above-mentioned technical bottlenecks.

[0007] The technical solution adopted in this invention is: a method for alleviating tin stress in plants using calcium folic acid, comprising the following steps:

[0008] (a) Prepare a folic acid calcium treatment solution with a concentration of 100-1600 μmol / L;

[0009] (b) Apply the treatment solution to the Sn-treated area. 4+ In plant culture systems under stress;

[0010] (c) Stress relief is achieved by regulating the endogenous folate metabolic pathway and antioxidant system function in plants.

[0011] As a further aspect of the present invention: the plant is duckweed (Lemna minor) or Arabidopsis thaliana.

[0012] When the object being treated is duckweed, the concentration of the calcium folic acid treatment solution is 800 μmol / L;

[0013] When the target organism is Arabidopsis thaliana, the concentration of the folic acid calcium treatment solution is 80 μmol / L.

[0014] As a further aspect of the present invention, the regulation in step (c) includes the following synergistic effects:

[0015] (a) Repair the function of the plant photosynthetic system and restore chlorophyll synthesis capacity and photosynthetic parameter levels;

[0016] (b) Regulate the activity of antioxidant enzyme systems and reduce reactive oxygen species (H2O2, O2). - Accumulation and membrane lipid peroxidation damage;

[0017] (c) Activate the plant folic acid biosynthesis pathway and promote the expression of ADCS, FPGS and GGH1 genes.

[0018] A calcium folic acid composition comprising:

[0019] (a) The active ingredient of calcium folic acid and a plant-acceptable carrier, wherein the carrier is a buffer solution or deionized water with a pH of 5.5-6.5;

[0020] (b) The concentration of the calcium folic acid is 100-1600 μmol / L.

[0021] Applications of a calcium folic acid composition in the following areas:

[0022] (a) Repair Sn 4+ Polluted environment;

[0023] (b) Reduce Sn 4+ Ecotoxicity risks.

[0024] As a further aspect of the present invention: when applied to aquatic plants, it reduces the Sn content in the water. 4+ Bioavailability; when applied to terrestrial crops, it blocks Sn 4+ Through the food chain.

[0025] The beneficial effects of this invention are:

[0026] 1. This invention achieves the effect of exogenous application of calcium folic acid on the saturation of plants (Sn). 4+The multi-effect synergistic relief of stress significantly improved the growth status and physiological functions of plants under stress conditions. Regarding growth indicators, experiments on duckweed showed that treatment with 800 μmol / L calcium folic acid increased leaf proliferation by 101.5%, and increased fresh weight and dry weight to 3.2 times and 5.8 times that of the stress group, respectively, completely reversing the effects of Sn. 4+ Induced leaf drop; in Arabidopsis, treatment with 80 μmol / L calcium folate restored root length to 96% of the control group, effectively alleviating Sn-induced leaf drop; 4+ This leads to growth inhibition.

[0027] 2. This invention can effectively regulate the oxidative stress balance in plants and reduce oxidative damage caused by tin stress. Calcium folic acid treatment can significantly reduce reactive oxygen species (H2O2, O2) in plants. - The explosive accumulation of malondialdehyde (MDA) in duckweed decreased by 34%, and in Arabidopsis thaliana by 41.4%. At the same time, by precisely regulating the activities of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), the activities of SOD, POD, and CAT in duckweed decreased by 36.8%, 50.9%, and 24.1%, respectively, thus restoring them from the abnormally high state induced by stress to the normal range and avoiding the metabolic burden caused by the overactivation of the oxidative system.

[0028] 3. This invention can effectively repair the function of the plant photosynthetic system and maintain efficient photosynthesis. In duckweed, folic acid calcium treatment restored the contents of chlorophyll a, b, and total chlorophyll to normal levels, increased the actual photochemical efficiency of photosystem II (Y(II)) by approximately 76.9%, increased the maximum photochemical efficiency (Fv / Fm) by approximately 28.8%, and increased the electron transport rate (ETR) by approximately 81.8%, significantly alleviating Sn 4+ The disruption of chloroplast structure and the photosynthetic electron transport chain ensures the plant's core needs for energy synthesis and metabolism.

[0029] 4. This invention achieves a systematic enhancement of plant stress resistance by activating endogenous metabolic pathways. Calcium folic acid can promote the expression of key genes for folic acid synthesis such as ADCS and FPG, as well as the GGH1 metabolic regulatory gene, restoring the balance of endogenous folic acid metabolism in plants. It constructs a multi-pathway synergistic response mechanism of "ROS scavenging-photosynthetic repair-metabolic regulation" at the molecular level. Compared with traditional chelating agents or antioxidants, it avoids secondary environmental risks and the limitations of single-target action, significantly improving the plant's resistance to Sn. 4+ Sustained tolerance to stress.

[0030] 5. This invention has broad application value, taking into account both ecological governance and agricultural safety production needs. Applying it to aquatic plants (such as duckweed) can reduce Sn levels in water. 4+Its bioavailability helps to restore tin-polluted water environments; when applied to terrestrial crops (such as Arabidopsis thaliana), it can block the transfer of tin through the food chain, reduce the risk of heavy metal accumulation, and provide an innovative technical solution for ecological restoration and sustainable agricultural production in areas polluted by heavy metals. Attached Figure Description

[0031] Figure 1 This invention relates to a method for alleviating tin stress in plants using calcium folic acid and the phenotypic analysis of duckweed under tin stress using calcium folic acid.

[0032] Figure 2 This invention relates to a method for alleviating tin stress in plants using calcium folic acid and its application in regulating ROS and antioxidant systems to alleviate Sn stress. 4+ Oxidative damage to duckweed.

[0033] Figure 3 This invention relates to a method for alleviating tin stress in plants using calcium folic acid and the application of calcium folic acid to Sn. 4+ The effects of stress on chlorophyll content and photosynthetic function of duckweed.

[0034] Figure 4 This invention relates to a method for alleviating tin stress in plants using calcium folic acid and its application. 4+ The effect of stress on folic acid content in duckweed.

[0035] Figure 5 This invention relates to a method for alleviating tin stress in plants using calcium folic acid, and the application of calcium folic acid in alleviating tin stress by regulating ROS metabolism. 4+ Oxidative damage in Arabidopsis thaliana caused by stress Figure 1 .

[0036] Figure 6 This invention relates to a method for alleviating tin stress in plants using calcium folic acid, and the application of calcium folic acid in alleviating tin stress by regulating ROS metabolism. 4+ Oxidative damage in Arabidopsis thaliana caused by stress Figure 2 .

[0037] Figure 7 This invention relates to a method for alleviating tin stress in plants using calcium folic acid and its application in calcium folic acid-dependent folic acid synthesis and metabolic pathways to alleviate Sn stress. 4+ Coercion. Detailed Implementation

[0038] The present invention will be specifically described below through embodiments. It should be noted that the following embodiments are only for further explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above-described invention are still within the scope of protection of the present invention.

[0039] 1. Phenotypic analysis of duckweed under tin stress using calcium folic acid.

[0040] After sterilization with 10% sodium hypochlorite solution, Lemna minor was rinsed three times with sterile deionized water and transferred to Hoagland nutrient solution for pre-acclimatization culture. The culture was placed in an artificial climate chamber (constant temperature 24±1℃, photoperiod set to 16 hours light / 8 hours dark alternation, light intensity 120 μmol·m⁻¹). -2 ·s -1 After the plants reached a growth equilibrium, folic acid calcium concentration gradient treatment groups were set up (0, 100, 200, 400, 800, 1600 μmol / L). Each group was supplemented with 100 μmol / L SnCl4 to construct Sn. 4+ Stressful environment. Treatment lasted for 7 days. Constant temperature and light conditions were maintained during the experiment, and plant phenotypic changes were recorded daily. After the treatment, samples were taken for physiological and biochemical index testing.

[0041] The results showed that 800 μmol / L folic acid calcium significantly inhibited Sn. 4+ Stress-induced leaf drop and chlorophyll degradation in duckweed Figure 1 A), the number of leaves increased by 101.5% ( Figure 1 B), fresh weight and dry weight increased to 3.2 times and 5.8 times that of the stress group, respectively. Figure 1 CD). When the concentration exceeds 800 μmol / L, growth indicators decrease with increasing concentration. Therefore, 800 μmol / L is the optimal concentration for alleviating Sn. 4+ The optimal concentration for stress.

[0042] Folic acid calcium regulates ROS and the antioxidant system to alleviate Sn 4+ Oxidative damage to duckweed

[0043] After the duckweed's growth stabilized, three treatment groups were set up: (1) control group, (2) stress group (Sn 4+ ): Add 100 μmol / L SnCl4 solution, (3) treatment group (Sn 4+ +Calcium Folic Acid): 100 μmol / L SnCl4 and 800 μmol / L calcium folic acid solution were added simultaneously. All treatment groups had three biological replicates, maintained under identical culture conditions, and samples were taken 7 days after treatment for analysis: root tip and leaf samples were collected, and H2O2 and O2 were detected using DAB and NBT staining methods, respectively. - The distribution of ROS was observed under a microscope. An equal amount of sample was taken, and the activities of SOD, POD, CAT, and MDA content were measured using a spectrophotometer according to the kit instructions to assess oxidative stress and antioxidant response.

[0044] Experimental results show that Sn 4+Stress caused a significant reduction in the root length of duckweed compared to the control group, while treatment with 800 μmol / L calcium folic acid restored the length of newly formed roots to normal levels. Figure 2 AB). ROS histochemical analysis showed that Sn 4+ Stress significantly promotes the exchange of H2O2 and O2 in root tips and leaves. - The accumulation of folic acid calcium, while folic acid calcium treatment effectively reduces its formation ( Figure 2 CD). Under stress conditions, the activities of SOD, POD, and CAT in duckweed increased to several times that of the control group; after folic acid calcium intervention, the activities of antioxidant enzymes returned to the normal range, and the MDA level decreased by 34%. Figure 2 EH).

[0045] Folic acid calcium for Sn 4+ The effects of stress on chlorophyll content and photosynthetic function of duckweed

[0046] Approximately 0.2 g of fresh, treated duckweed sample was weighed and added to pre-cooled 80% acetone solution. Chloroplast pigment extraction was performed in a dark environment for 3 hours until complete dissolution. Absorbance was measured at specific wavelengths of 663 nm and 645 nm using a UV-Vis spectrophotometer, and the contents of chlorophyll a, chlorophyll b, and total chlorophyll were calculated. The photosynthetic performance of the duckweed was evaluated using the Walz (Germany) IMAGING-PAM chlorophyll fluorescence dynamic analysis system: after 30 minutes of dark adaptation, the sample was treated with 281 μmol·m⁻¹ chlorophyll. -2 ·s -1 Activation light with high quantum flux density was used to determine key parameters of photosystem II—actual quantum yield Y(II), maximum photochemical efficiency Fv / Fm, and linear electron transport rate ETR—to comprehensively analyze the photosynthetic mechanism's influence on Sn. 4+ Characteristics of response to stress.

[0047] The results show that Sn 4+ Stress significantly reduced the chlorophyll a, b, and total chlorophyll content of duckweed. After adding 800 μmol / L calcium folic acid, the chlorophyll content significantly recovered (Figures 3A–C), indicating that it helps maintain chloroplast structure and photosynthetic pigment stability. Fluorescence parameter analysis showed that calcium folic acid significantly improved photosynthetic function: the actual photochemical efficiency Y(II) increased from 0.130 to 0.230 (Figure 3D); the maximum photochemical efficiency Fv / Fm increased from 0.510 to 0.657 (Figure 3E); and the electron transport rate ETR increased from 15.843 to 28.793 (Figure 3F). This indicates that calcium folic acid can effectively alleviate Sn. 4+ Stress damages the photosynthetic system of duckweed, hindering its efficient operation.

[0048] Sn 4+Effects of stress on folic acid content in duckweed

[0049] Different treatment groups (control, Sn) 4+ (Stressed) duckweed samples were rapidly frozen in liquid nitrogen, freeze-dried, ground into powder, and 0.5 g was accurately weighed and added to a 10 mL centrifuge tube. 2 mL of pre-cooled 0.01 mol / L hydrochloric acid solution was injected, and the samples were extracted by sonication in an ice bath for 30 min. The supernatant was collected by centrifugation at 12,000 × g for 5 min at 4 °C. The extraction was repeated once, and the supernatants were combined and diluted to 5 mL with the extract. After vortexing and mixing, the samples were filtered through a 0.22 μm aqueous filter membrane and stored at -20 °C for analysis. An ultra-high performance liquid chromatography (UHPLC) system (column: XBridge BEH C18, 4.6 × 150 mm, 2.5 μm; column temperature 35℃) was used. The mobile phase consisted of ammonium formate aqueous solution (phase A) and methanol (phase B). A gradient elution program was used: the proportion of phase B increased from 5% to 95% within 0-10 min, the flow rate was 1 mL / min, the injection volume was 10 μL, and the detection wavelength was 270 nm. Mass spectrometry was performed in ESI positive ion mode (scanning range m / z 100-800), using folic acid characteristic ions ([M+H)). + Qualitative and quantitative analysis was performed using m / z 442.1.

[0050] The results showed that LC-MS detection indicated that Sn 4+ Stress significantly reduced the endogenous folic acid content in duckweed ( Figure 4 AB), which interferes with its synthesis or metabolic balance.

[0051] Implementation 2: Folic acid calcium alleviates Sn by regulating ROS metabolism. 4+ Oxidative damage in Arabidopsis thaliana caused by stress

[0052] Sterilized Arabidopsis thaliana (Col-0) seeds were sown in culture containers containing 1 / 2 MS medium. Three treatment groups were set up: a blank control group, a Sn control group, and a Sn-based control group. 4+ After 7 days of treatment, the root length recovery rate of Arabidopsis thaliana was analyzed using ImageJ software in the stress group (100 μmol / L SnCl4) and the folic acid calcium relief group (80 μmol / L folic acid calcium + SnCl4). Simultaneously, the activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), as well as the content of malondialdehyde (MDA) in the leaves, were measured to evaluate the mechanism of action of folic acid calcium in regulating ROS metabolism.

[0053] The results showed that at 80 μmol / L, calcium folic acid significantly restored Arabidopsis thaliana's resistance to Sn. 4+ The stress tolerance recovery rate reached 96%. Figure 5 A-5C). Further analysis shows that Sn4+ Stress significantly activated the activities of SOD, POD, and CAT and increased MDA levels; while folic acid calcium intervention significantly decreased the activities of these antioxidant enzymes and MDA levels. Figure 6 (A–D).

[0054] Folic acid calcium depends on folic acid synthesis and metabolic pathways to alleviate Sn 4+ coercion

[0055] The ADCS / FPGS and GGH1 genes play a role in Sn synthesis and metabolism. 4+ Regulatory mechanisms of stress

[0056] Wild-type Arabidopsis thaliana (Col-0) and folate metabolism-related mutants of ADCS / FPGS and GGH1 were selected as experimental materials. They were sown in 1 / 2 MS medium containing 100 μmol / L SnCl4 and cultured for 7 days. Root length of seedlings in each group was measured and differences were compared. Simultaneously, total RNA was extracted from plant tissues using the TRIzol method, and cDNA was obtained by reverse transcription. Using the ACTIN2 gene as an internal control, qRT-PCR amplification was performed using SYBR Green fluorescent dye to detect the expression levels of ADCS / FPGS and GGH1 genes, analyzing their role in folate metabolism response to SnCl4. 4+ Regulatory mechanisms under stress.

[0057] The results show that Sn 4+ Under stress, the root lengths of ADCS / FPGS and GGH1 gene deletion mutants (adcs / fpgs and ggh1) were shorter than those of the wild-type Col-0 (Fig. 7A), indicating that gene deletion enhances the plant's resistance to Sn. 4+ Sensitivity. qRT-PCR showed that Sn 4+ Stress significantly induced the expression of ADCS / FPGS and GGH1 genes. Figure 7 B).

[0058] Physiological and biochemical tests showed that exogenous calcium folic acid treatment significantly enhanced the resistance of duckweed and Arabidopsis thaliana to Sn. 4+ Resistance to stress. Specifically, folic acid calcium effectively alleviates Sn by regulating the balance of reactive oxygen species (ROS) metabolism, inhibiting membrane lipid peroxidation, increasing the activity of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), and restoring photosynthetic function. 4+ Stress-induced oxidative damage and growth inhibition.

[0059] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for alleviating tin stress in plants using calcium folic acid, characterized in that, Includes the following steps: (a) Prepare a folic acid calcium treatment solution with a concentration of 100-1600 μmol / L; (b) Apply the treatment solution to the Sn-treated area. 4+ In plant culture systems under stress; (c) Stress relief is achieved by regulating the endogenous folate metabolic pathway and antioxidant system function in plants.

2. The method for alleviating tin stress in plants using calcium folic acid according to claim 1, characterized in that, The plant in question is duckweed or Arabidopsis thaliana; When the object being treated is duckweed, the concentration of the calcium folic acid treatment solution is 800 μmol / L; When the target organism is Arabidopsis thaliana, the concentration of the folic acid calcium treatment solution is 80 μmol / L.

3. The method for alleviating tin stress in plants using calcium folic acid according to claim 2, characterized in that, The regulation in step (c) includes the following synergistic effects: (a) Repair the function of the plant photosynthetic system and restore chlorophyll synthesis capacity and photosynthetic parameter levels; (b) Regulate the activity of antioxidant enzyme systems to reduce the accumulation of reactive oxygen species and membrane lipid peroxidation damage; (c) Activate the plant folic acid biosynthesis pathway and promote the expression of ADCS, FPGS and GGH1 genes.

4. A folic acid calcium composition according to claim 3, characterized in that, Include: (a) The active ingredient of calcium folic acid and a plant-acceptable carrier, wherein the carrier is a buffer solution or deionized water with a pH of 5.5-6.5; (b) The concentration of the calcium folic acid is 100-1600 μmol / L.

5. The application of the folic acid calcium composition according to claim 4, characterized in that, Applications in the following areas: (a) Repair Sn 4+ Polluted environment; (b) Reduce Sn 4+ Ecotoxicity risks.

6. The application of the folic acid calcium composition according to claim 5, characterized in that: When applied to aquatic plants, it reduces Sn in the water. 4+ Bioavailability; when applied to terrestrial crops, it blocks Sn 4+ Through the food chain.

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