Application of polyacrylic acid modified Mn3O4 nanoparticles in improvement of crop photosynthesis and / or cadmium resistance

Abstract

The invention belongs to the technical field of crop stress resistance, and particularly relates to application of polyacrylic acid modified Mn3O4 nanoparticles in improvement of crop photosynthesis and / or cadmium resistance. The polyacrylic acid modified Mn3O4 nano-particles can remarkably improve leaf pigment synthesis, Rubisco enzyme activity and antioxidant enzyme activity, so that the photosynthesis and cadmium stress resistance of rice are improved. The invention also provides a preparation method of the polyacrylic acid modified Mn3O4 nanoparticles, the preparation process is simple, and the action effect is obvious. The polyacrylic acid modified Mn3O4 nanoparticles not only can improve the photosynthesis of rice leaves under cadmium stress and promote the growth and development of rice, but also can relieve the toxic action of cadmium on the rice and improve the cadmium tolerance of the rice.

Description

Technical Field

[0001] The invention belongs to the technical field of crop stress resistance, and particularly relates to application of polyacrylic acid-modified Mn3O4 nanoparticles in improving crop cadmium resistance. Background Art

[0002] Heavy metal pollution in soil, especially excessive cadmium in soil, has become one of the important factors threatening the stability of rice production and food security in my country. Cadmium is a toxic heavy metal element. Its accumulation in the soil not only affects soil quality, but also enters the plant body through rice roots and accumulates in rice grains, posing a potential risk to human health.

[0003] Photosynthesis is a vital physiological process in the growth of crops such as rice. Photosynthesis is the basis for the formation of crop dry matter and yield. Under normal circumstances, leaves convert light energy into chemical energy and synthesize organic matter through photosynthesis, providing the necessary energy and material basis for the growth and development of rice. However, the reduction of leaf photosynthetic rate under cadmium stress is one of the main reasons for the reduction of rice yield. This is mainly because cadmium can interfere with the structure and function of chloroplasts, affect the synthesis and stability of photosynthetic pigments, reduce the efficiency of light energy capture, and inhibit the key enzyme activity of photosynthetic electron transport chain and carbon fixation process, thereby hindering the smooth progress of photosynthesis. The reduction of photosynthetic rate is directly related to the growth rate and biomass accumulation of rice, which in turn affects the final yield. Therefore, how to improve the ability of rice photosynthesis to resist cadmium stress is of great significance to ensure rice production and food security.

[0004] Nanoparticles are a general term for nanomaterials that have activities similar to cellular antioxidant enzymes. In recent years, researchers have discovered a variety of nanomaterials that can improve the ability of crops to resist stress, such as cerium oxide nanomaterials, silver nanoparticles, and gold nanoparticles. However, all of the above nanomaterials contain heavy metal elements, which poses certain biosafety concerns. Manganese, as a trace nutrient necessary for plants, can participate in a variety of metabolic reactions during plant growth and development.

[0005] Although Mn3O4 nanoparticles have been used in the field of biomedicine to alleviate acute liver damage associated with iron poisoning in mice, they have not yet been put into practical use in the field of agricultural science, especially in improving the photosynthesis efficiency of crops and enhancing their resistance to cadmium stress. Therefore, how to effectively apply Mn3O4 nanoparticles in agricultural science to improve the photosynthesis performance of crops and enhance their tolerance to cadmium stress is an urgent problem to be solved in this field. Summary of the invention

[0006] In view of this, the present invention provides a polyacrylic acid modified Mn3O4 nanoparticle and applies it to the growth process of rice to solve the defects and shortcomings of cadmium stress inhibiting rice photosynthesis and environmental pollution.

[0007] The above-mentioned object of the present invention is achieved by the following technical solutions:

[0008] Application of polyacrylic acid modified Mn3O4 nanoparticles in improving crop photosynthesis and / or cadmium resistance.

[0009] Photosynthesis involves gas exchange and fluorescence parameters. Gas exchange parameters include: net photosynthetic rate A, stomatal conductance g s , leaf conductivity g m , Maximum carboxylation rate of Rubisco enzyme in leaves V cmax , maximum electron transfer rate of leaves J max and triose phosphate transport rate TPU; fluorescence parameters include: maximum photochemical quantum yield F of photosystem II v / F m , leaf electron transfer rate ETR, photosystem II reaction center open ratio q p 、 Actual quantum efficiency of photosystem II φ PSII 、The proportion of light energy used for heat dissipation φ NPQ 、The proportion of light energy dissipated in other formsφ NO .

[0010] Preferably, the method for preparing the polyacrylic acid modified Mn3O4 nanoparticles comprises the following steps:

[0011] S1: dissolving manganese acetate tetrahydrate in ethanol to obtain a solution with a concentration of 0.04-0.2 g / ml, dispersing polyacrylic acid in ethanol to obtain a solution with a concentration of 0.2-1.6 g / ml, and then mixing the two solutions to obtain a mixed solution, wherein the mixing ratio is calculated based on the mass ratio of the solute manganese acetate tetrahydrate to the solute polyacrylic acid in the solution to be 1:7.3;

[0012] S2: placing the mixed solution obtained in S1 into a reactor for heating treatment, cooling and centrifuging to obtain a supernatant;

[0013] S3: Purify the supernatant obtained in S2 to obtain polyacrylic acid modified Mn3O4 nanoparticles.

[0014] The present invention also provides the use of polyacrylic acid modified Mn3O4 nanoparticles in improving the pigment content, Rubisco enzyme activity or antioxidant enzyme activity in crop leaves.

[0015] The pigments include total chlorophyll, carotenoids, chlorophyll a, chlorophyll b, and the ratio of chlorophyll a to b;

[0016] Antioxidant enzymes include superoxide dismutase, peroxidase, and catalase.

[0017] Preferably, the reactor in step S2 is a polytetrafluoroethylene-lined reactor, and the heating treatment temperature is 100° C.-180° C., and the time is 12-26 hours.

[0018] Preferably, the centrifugal speed of the centrifugal treatment in step S2 is 3000-8000 r / min, and the time is 30 min-90 min.

[0019] Preferably, the purification in step S3 is to transfer the supernatant to a dialysis bag for dialysis treatment, the molecular weight cutoff of the dialysis bag is 8KD-14KD, and the dialysis time is 12h-26h.

[0020] Preferably, the application method is: mixing the polyacrylic acid modified Mn3O4 nanoparticles with a surfactant to prepare a dispersion, and spraying the dispersion evenly on the leaves of the crop.

[0021] Preferably, the surfactant Silwet L-77 in the dispersion accounts for 0.05% of the polyacrylic acid modified Mn3O4 nanoparticle dispersion.

[0022] Preferably, the concentration of the polyacrylic acid modified Mn3O4 nanoparticles in the dispersion is 100-300 mg / L.

[0023] Preferably, the crop is rice.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The polyacrylic acid-coated Mn3O4 nanoparticles (PAA@Mn3O4) of the present invention increase the photosynthetic rate of leaves by increasing the content of total chlorophyll, carotenoids, chlorophyll a and chlorophyll b, and the ratio of chlorophyll a to b, thereby enabling crop leaves to grow healthily.

[0026] (2) The polyacrylic acid-coated Mn3O4 nanoparticles (PAA@Mn3O4) of the present invention promote the rice leaves to capture and utilize light energy more effectively by increasing the activity of Rubisco enzyme, accelerate the fixation of carbon dioxide and the generation of organic matter, thereby improving the efficiency of photosynthesis and providing energy and substances for crops.

[0027] (3) The polyacrylic acid-coated Mn3O4 nanoparticles (PAA@Mn3O4) of the present invention also improve the photosynthesis of rice by increasing the activity of the antioxidant enzyme system in rice leaves, thereby enhancing the ability of rice leaves to resist cadmium stress.

[0028] (4) The polyacrylic acid-coated Mn3O4 nanoparticles (PAA@Mn3O4) of the present invention have low preparation cost, readily available raw materials, and a simple preparation process. At the same time, they have good water solubility, biocompatibility, and no toxic side effects.

[0029] (5) The dispersion prepared by mixing the polyacrylic acid-coated Mn3O4 nanoparticles (PAA@Mn3O4) of the present invention with a surfactant is simple to use and has an obvious effect.

[0030] (6) The polyacrylic acid-coated Mn3O4 nanoparticles (PAA@Mn3O4) of the present invention can provide the essential trace element Mn for plant growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0032] Figure 1 TEM image, hydrodynamic diameter and potential schematic diagram of PAA@Mn3O4, where Size represents the hydrodynamic diameter and Charge represents the potential change;

[0033] Figure 2 This is an analysis diagram of leaf photosynthetic parameters of the control (CK), cadmium stress (Cd) and PAA@Mn3O4 (Cd+PMO) under cadmium stress treatment in Example 5;

[0034] Figure 3 This is a fluorescence parameter analysis diagram of photosystem II of rice leaves in Example 5;

[0035] Figure 4 This is an analysis chart of soluble protein and pigment content in rice leaves in Example 6;

[0036] Figure 5 This is the analysis diagram of Rubisco enzyme and antioxidant enzyme activity in rice leaves in Example 7;

[0037] in Figure 2-Figure 5CK group: the first pot of seedlings was cultivated normally throughout the experimental period; Cd group: the second pot was treated with cadmium stress 7 days after transplantation; Cd+PMO group: the third pot of seedlings was sprayed with the dispersion 5 days after cadmium stress treatment. DETAILED DESCRIPTION

[0038] Embodiments of the present invention are described in detail below, and the examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals represent the same or similar elements or elements with the same or similar functions from beginning to end. The embodiments described below by reference to the accompanying drawings are exemplary, are only used to explain the present invention, and are not to be construed as limitations of the present invention. In the embodiments, if specific techniques or conditions are not indicated, the techniques or conditions described in the literature in this area or the product specifications are carried out. The reagents used or the instruments that are not indicated by the manufacturer are all conventional products that can be obtained commercially.

[0039] In one aspect of the present invention, the present invention provides a use of polyacrylic acid modified Mn3O4 nanoparticles in improving crop photosynthesis and / or cadmium resistance. Preferably, the crop is rice.

[0040] In some embodiments of the present invention, the application method is as follows: dissolving the polyacrylic acid modified Mn3O4 nanoparticles to a concentration of 100-300 mg / L and then spraying the solution on the leaves, preferably 200 mg / L.

[0041] In another aspect of the present invention, the present invention provides a method for preparing polyacrylic acid modified Mn3O4 nanoparticles, comprising the following steps:

[0042] In step S1, manganese acetate tetrahydrate is dissolved in ethanol to obtain a solution with a concentration of 0.04-0.2 g / ml, polyacrylic acid is dispersed in ethanol to obtain a solution with a concentration of 0.2-1.6 g / ml, and the two solutions are mixed to obtain a mixed solution, wherein the mixing ratio is calculated as 1:7.3 according to the mass ratio of the solute manganese acetate tetrahydrate and the solute polyacrylic acid in the solution. Preferably, the concentration of manganese acetate tetrahydrate is 0.04 g / ml, and the concentration of polyacrylic acid is 0.29 g / ml.

[0043] Further, in S2, the dispersion obtained in S1 is placed in a reactor for heating treatment, and then cooled and centrifuged to obtain a supernatant; in this step, the reactor is a polytetrafluoroethylene-lined reactor, the heating temperature is 100°C-180°C, preferably 120°C, the time is 12-26h, preferably 24h. The centrifugal speed is 3000-8000r / min, preferably 6000r / min, and the centrifugal time is 30min-90min, preferably 60min.

[0044] In step S3, the supernatant obtained in S2 is purified to obtain polyacrylic acid modified Mn3O4 nanoparticles. In this step, the purification is to transfer the supernatant to a dialysis bag for dialysis treatment, the molecular weight cutoff of the dialysis bag is 8KD-14KD, preferably 10KD, and the dialysis time is 12-26h, preferably 24h.

[0045] Furthermore, the present invention can also calculate the concentration of Mn3O4 nanoparticles modified by polyacrylic acid, and the specific operation is as follows: dry the freeze-dried bottle to constant weight and record its mass (m1); take an appropriate amount of dialyzed solution and put it into the freeze-dried bottle dried to constant weight, ensure that the solution is evenly distributed in the container, and use an electronic balance to weigh the total mass of the freeze-dried bottle after adding the solution (m2). Put the freeze-dried bottle containing the solution into a freeze dryer until the weight is constant, accurately weigh the weight of the dried product (m3), and calculate the concentration of the Mn3O4 nanoparticles modified by polyacrylic acid based on the original weight of the sample and the weight lost after drying.

[0046] Another aspect of the present invention also provides polyacrylic acid modified Mn3O4 nanoparticles prepared by the above preparation method.

[0047] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0048] The production processes, experimental methods or detection methods involved in the embodiments of the present invention, unless otherwise specified, are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, and are very clear and unambiguous in the relevant application fields. Technical personnel in the field can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0049] The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in terms of their sources, and are all conventional products that can be purchased through regular commercial channels, or can be prepared according to conventional methods well known to those skilled in the art.

[0050] Example 1

[0051] Preparation and characterization of polyacrylic acid modified Mn3O4 nanoparticles

[0052] Weigh 0.2g of tetrahydrated manganese acetate and 1.46g of PAA (MW 1800), dissolve / disperse in 5mL of ethanol respectively, vortex the above two solutions at 2500r / min for 15min to mix them evenly, add them dropwise into 15mL of 30wt% ammonia water to obtain the final mixed solution, then put the final mixed solution into a 25mL polytetrafluoroethylene-lined reactor, hydrothermally react at 120℃ for 24h, cool, divide into 2ml centrifuge tubes, centrifuge at 6000r / min for 60min to remove large agglomerates, then collect the supernatant and dialyze with a dialysis bag (MW 10000) for 24h, change water every 8h, and obtain polyacrylic acid modified Mn3O4 nanoparticles. Then put the dialyzed solution into a freeze-dried bottle dried to constant weight, and use an electronic balance to weigh the total mass (m2) of the freeze-dried bottle after adding the solution. The freeze-dried bottle containing the solution was placed in a freeze dryer until the weight was constant, and the weight of the dried product (m3) was accurately weighed. The concentration of the polyacrylic acid-modified Mn3O4 nanoparticles was calculated based on the original weight of the sample and the weight lost after drying.

[0053] Transmission electron microscopy (HRTEM) technology is used to observe the morphology of nanomaterials. When preparing samples, ordinary carbon support film (300 mesh) copper mesh is used as a carrier, and 30μL-50μL of sample is dripped onto each copper mesh and vacuum dried. Subsequently, the sample copper mesh is mounted on the HRTEM, the acceleration voltage is set to 200kV, and the image is optimized by adjusting the focal length and sample position to achieve the best clarity, so as to record the morphology and structural characteristics of the sample, and perform data analysis to obtain the size, shape, and other parameters of the nanomaterial.

[0054] Example 2

[0055] Weigh 0.4g of tetrahydrated manganese acetate and 3.07g of PAA (MW 1800), dissolve / disperse in 5mL of ethanol respectively, vortex the above two solutions at 2500r / min for 15min to mix them evenly, add them dropwise into 15mL of 30wt% ammonia water to obtain the final mixed solution, then put the final mixed solution into a 25mL polytetrafluoroethylene-lined reactor, hydrothermally react at 170℃ for 26h, cool, divide into 2ml centrifuge tubes, centrifuge at 8000r / min for 90min to remove large agglomerates, then collect the supernatant and dialyze with a dialysis bag (MW 10000) for 26h, change water every 8h, and obtain polyacrylic acid modified Mn3O4 nanoparticles. Then put the dialyzed solution into a freeze-dried bottle dried to constant weight, and use an electronic balance to weigh the total mass (m2) of the freeze-dried bottle after adding the solution. The freeze-dried bottle containing the solution was placed in a freeze dryer until the weight was constant, and the weight of the dried product (m3) was accurately weighed. The concentration of the polyacrylic acid-modified Mn3O4 nanoparticles was calculated based on the original weight of the sample and the weight lost after drying.

[0056] Example 3

[0057] Weigh 0.1g of tetrahydrated manganese acetate and 0.76g of PAA (MW 1800), dissolve / disperse in 5mL of ethanol respectively, vortex the above two solutions at 2500r / min for 15min to mix them evenly, add them dropwise into 15mL of 30wt% ammonia water to obtain the final mixed solution, then put the final mixed solution into a 25mL polytetrafluoroethylene-lined reactor, hydrothermally react at 100℃ for 12h, cool, divide into 2ml centrifuge tubes, centrifuge at 3000r / min for 30min to remove large agglomerates, then collect the supernatant and dialyze with a dialysis bag (MW 10000) for 12h, change water every 8h, and obtain polyacrylic acid modified Mn3O4 nanoparticles. Then put the dialyzed solution into a freeze-dried bottle dried to constant weight, and use an electronic balance to weigh the total mass (m2) of the freeze-dried bottle after adding the solution. The freeze-dried bottle containing the solution was placed in a freeze dryer until the weight was constant, and the weight of the dried product (m3) was accurately weighed. The concentration of the polyacrylic acid-modified Mn3O4 nanoparticles was calculated based on the original weight of the sample and the weight lost after drying.

[0058] like Figure 1 The figure is a schematic diagram of the characterization of the polyacrylic acid modified Mn3O4 nanoparticles prepared in Examples 1-3 of the present invention, wherein Figure 1This is a TEM schematic diagram of PAA@Mn3O4, which shows that PAA@Mn3O4 appears to be spherical, has good dispersion, and uniform particle size; its potential diagram shows that its hydrodynamic diameter is 9.2±1.1nm and the potential is -17.2±1.6mV. The negatively charged surface potential and smaller hydrodynamic diameter indicate that PAA@Mn3O4 has good water solubility.

[0059] The following Examples 4, 5, 6, and 7 are analyzed by spraying 200 mg / L of PAA@Mn3O4 prepared in Example 1 as an example.

[0060] Example 4

[0061] Rice seed disinfection, sowing, transplanting and foliar spraying

[0062] This experiment uses healthy rice seeds of the Zhongjia Zao 17 variety. First, select some rice seeds with full grains and uniform size, disinfect them with 0.3% hydrogen peroxide solution, soak them at room temperature for 24 hours, and then transfer them to a culture dish for germination. When they grow to the two-leaf and one-heart stage, they are transplanted to a hydroponic box. Each culture pot has 12 holes, and two plants are transplanted in each hole. The nutrient solution is prepared according to the formula of the International Rice Nutrition Institute.

[0063] Treatment of rice:

[0064] CK group: The first pot of seedlings was cultivated normally throughout the experimental period

[0065] Cd group: Cadmium stress (1.68 mg L -1 Cd) treatment

[0066] Cd+PMO group: The third pot of seedlings was sprayed with dispersion 5 days after cadmium stress treatment.

[0067] Prepare dispersion solution: 200mg / LPAA@Mn3O4 mixed solution containing 0.05% SilwetL-77 by volume

[0068] Subsequently, the foliar spraying method was adopted. 10 mL was sprayed into each pot and then placed in a weak light place to adapt for 3 hours. On the third day after spraying the nanoparticles, various indicators of the newly fully expanded leaves were measured.

[0069] Example 5

[0070] Determination of photosynthetic and fluorescence parameters in rice leaves

[0071] Detection using LiCor-6800 portable photosynthetic instrument

[0072] Subsequently, the gas exchange parameters of fully expanded leaves of the three treated rice plants were measured in an artificial climate chamber from 9:00 to 12:00 every day. The six parameters, including net photosynthetic rate, stomatal conductance, mesophyll conductance, maximum carboxylation rate of Rubisco enzyme in leaves, maximum electron transfer rate in leaves and triose phosphate transport rate, were measured using a LiCor-6800 portable photosynthetic meter.

[0073] The environmental conditions in the photosynthetic instrument leaf chamber were set as follows: light intensity was 1500 μmol m -2 , leaf temperature was 25°C, relative humidity was 60%, and CO2 concentration on the leaf surface was 400 μmolmol -1 .

[0074] The results are as follows Figure 2 As shown: Rice leaves treated with cadmium stress significantly reduced the net photosynthetic rate, stomatal conductance, mesophyll conductance, maximum carboxylation rate of leaf Rubisco enzyme, maximum electron transfer rate and triose phosphate utilization rate.

[0075] However, compared with rice leaves treated with cadmium stress, the net photosynthetic rate, mesophyll conductance, maximum carboxylation rate of Rubisco enzyme, maximum electron transfer rate and triose phosphate utilization rate of rice leaves increased significantly after application of PAA@Mn3O4.

[0076] This result shows that spraying PAA@Mn3O4 on leaves can improve the ability of rice photosynthesis to resist cadmium stress.

[0077] After the photosynthetic rate and stomatal conductance stabilized, the data were recorded and the fluorescence parameters were recorded for calculating the mesophyll conductance. Then the CO2 response curve was measured to calculate the maximum carboxylation rate of Rubisco enzyme in leaves, the maximum electron transfer rate in leaves and the triose phosphate transport rate. The CO2 response curve was set to a gradient of 400, 300, 200, 100, 50, 400, 400, 600, 800, 1000, 1500 μmolmol -1 .

[0078] After the daytime experiment, the light in the artificial climate chamber was turned off. After the rice plants were adapted to the dark for at least 2 h, the maximum quantum efficiency of the photosystem II of the newly fully expanded leaves was measured using a LiCor-6800 portable photosynthetic instrument. The light intensity in the leaf chamber was set to 0 μmol / m -2 s -1 , other environmental settings are the same as daytime.

[0079] Then, combined with the fluorescence data obtained during the day, the leaf electron transfer rate, the open proportion of the reaction center of photosystem II, the actual quantum efficiency of photosystem II, the proportion of light energy lost through heat dissipation, and the proportion of light energy dissipated through other forms can be calculated.

[0080] The results are as follows Figure 3 As shown: Rice leaves treated with cadmium stress significantly reduced the maximum quantum efficiency of photosystem II, electron transfer rate, open ratio of photosystem II reaction center, and actual quantum efficiency of photosystem II, but significantly increased the proportion of light energy used for heat dissipation;

[0081] However, compared with rice leaves treated with cadmium stress, the electron transfer rate, the open ratio of photosystem II reaction centers, and the actual quantum efficiency of photosystem II in rice leaves increased significantly after application of PAA@Mn3O4, and the proportion of light energy used for heat dissipation decreased significantly.

[0082] This result shows that spraying PAA@Mn3O4 on leaves can improve the ability of photosystem II in rice leaves to resist cadmium stress.

[0083] Example 6

[0084] Determination of soluble protein and pigment content in rice leaves

[0085] The soluble protein and pigment content in leaves jointly affect the ability of photosynthesis. Changes in the soluble protein content in leaves can affect the photosynthetic rate by regulating the activity of photosynthesis-related enzymes, and the pigment content in leaves directly affects the efficiency of photosynthesis. When the pigment content increases, the leaves can capture more light energy, thereby increasing the photosynthetic rate.

[0086] In order to determine the soluble protein content of the leaves of the three treated rice plants, the following steps were performed: First, about 0.1 g of fresh leaf samples were weighed from each treated rice plant, and then 1 mL of distilled water was added to each sample and homogenized in an ice bath. Then, the resulting mixture was centrifuged at 12,000 r / min for 10 minutes at 4°C, after which the supernatant was taken out and placed on ice for detection. During the detection process, the supernatant was placed in an acidic solution and Coomassie Brilliant Blue G-250 was added, which combined with the protein to form a blue complex; the complex had a maximum absorption peak at 620 nm, and the depth of its color was proportional to the concentration of the protein.

[0087] To determine the chlorophyll content of the leaves of the three treated rice plants, first weigh about 0.1g of fresh leaf samples from each treated rice plant, then add 1mL of acetone to each sample and add a small amount of about 50mg of extract. In the dark or weak light, use a mortar to fully grind the mixture of leaves and acetone, then transfer the ground mixture to a 10mL test tube, then rinse the mortar with 80% acetone to ensure that all chlorophyll is transferred to the test tube. Then, continue to add 80% acetone to the test tube until the total volume reaches 10 ml. Place the test tube in the dark and let it stand for 3h so that the chlorophyll is fully dissolved in acetone. Subsequently, observe that the tissue residue at the bottom of the test tube has completely turned white, and carefully aspirate the supernatant in the test tube with a pipette. This part of the supernatant will be used for subsequent chlorophyll content determination.

[0088] Chlorophyll a and chlorophyll b have maximum absorption at 645nm and 663nm. Chlorophyll a, chlorophyll b and total chlorophyll content can be calculated according to empirical formula.

[0089] The method for determining carotenoid content is the same as that for chlorophyll.

[0090] Carotenoids have a maximum absorption peak at 470nm, and the carotenoid content is obtained through an empirical formula.

[0091] The results are as follows Figure 4 As shown: the rice leaves treated with cadmium stress significantly reduced the contents of soluble protein, total chlorophyll, carotenoids, chlorophyll a, and chlorophyll b, but significantly increased the ratio of chlorophyll a to chlorophyll b;

[0092] However, compared with rice leaves treated with cadmium stress, the contents of soluble protein, total chlorophyll, carotenoids, chlorophyll a, chlorophyll b, and the ratio of chlorophyll a to chlorophyll b in rice leaves increased significantly after application of PAA@Mn3O4.

[0093] The results showed that spraying PAA@Mn3O4 on leaves could improve photosynthesis by increasing the content of osmotic regulating substances and pigments.

[0094] Example 7

[0095] Determination of Rubisco and antioxidant enzyme activities in rice leaves

[0096] The activity of Rubisco and antioxidant enzymes in leaves jointly affect photosynthesis and plant stress resistance. Increasing the activity of Rubisco can reduce the consumption of photorespiration and increase the carbon assimilation rate of photosynthesis; while increasing the activity of antioxidant enzymes builds a strong antioxidant defense line for rice leaves, effectively resisting the negative effects of cadmium stress. Antioxidant enzymes include SOD, POD, and CAT. SOD can catalyze the dismutation reaction of superoxide anion radicals, converting them into oxygen and hydrogen peroxide, thereby reducing the oxidative stress caused by cadmium stress; POD can decompose hydrogen peroxide and other peroxides to prevent them from damaging cell structures; CAT further catalyzes the decomposition of hydrogen peroxide into water and oxygen, completely eliminating its potential harm.

[0097] Rubisco enzyme activity assay

[0098] In order to determine the Rubisco enzyme activity of the leaves of the three treated rice plants, about 0.1 g of fresh leaf samples were first weighed from each treated rice plant, 1 mL of extract was added to each sample, and then the sample and extract mixture was placed in an ice bath and preliminarily homogenized using a homogenizer. Then ultrasonic disruption was performed, and the parameters were set as follows: 200 W, disruption for 3 s, rest for 7 s, and total time for 1 min under ice bath conditions. The disrupted mixture was then centrifuged at 8000 g for 10 min at 4 ° C, and the supernatant was taken for subsequent enzyme activity determination.

[0099] Under the catalysis of Rubisco, one molecule of ribulose-1,5-bisphosphate (RuBP) combines with one molecule of CO2 to produce two molecules of 3-phosphoglyceric acid (PGA). Under the action of 3-phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase, PGA produces glyceraldehyde-3-phosphate and oxidizes reduced coenzyme I (NADH).

[0100] The absorbance change of the reaction system was measured at a wavelength of 340 nm using a spectrophotometer. The change in absorbance is proportional to the oxidation rate of NADH, which can reflect the activity of the Rubisco enzyme. The oxidation rate of NADH was calculated based on the time curve of the absorbance change. The oxidation rate can be further converted into the activity unit of the Rubisco enzyme.

[0101] Determination of superoxide dismutase (SOD) activity

[0102] The treatment of rice plant leaves after the three treatments was the same as that of Rubisco enzyme activity determination, but the difference was that the centrifugation was performed at 12000r / min for 10 minutes at 4°C. Then, superoxide anions (O 2- ), while O2- It can react with WST-8 to produce a water-soluble dye, formazan, which absorbs at 450nm. The role of SOD is to remove O 2- , thereby inhibiting the formation of formazan. Therefore, the activity of SOD is judged by observing the depth of yellow color of the reaction solution. The darker the yellow color of the reaction solution, the lower the SOD activity, and vice versa.

[0103] Determination of peroxidase (POD) activity

[0104] The treatment operations for the leaves of rice plants after the three treatments were the same as the determination of superoxide dismutase (SOD) activity.

[0105] POD catalyzes the oxidation of specific substrates by H2O2 and has characteristic light absorption at 470nm.

[0106] Determination of catalase (CAT) activity

[0107] The treatment operations for the leaves of rice plants after the three treatments were the same as the determination of superoxide dismutase (SOD) activity.

[0108] H2O2 has a characteristic absorption peak at 240nm. CAT can decompose H2O2, causing the absorbance of the reaction solution at 240nm to decrease with reaction time. The CAT activity can be calculated based on the rate of change of absorbance.

[0109] The results are as follows Figure 5 As shown: The activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) in rice leaves treated with cadmium stress were significantly increased.

[0110] However, compared with rice leaves treated with cadmium stress, the activities of rice Rubisco, superoxide dismutase, peroxidase and catalase were significantly increased after application of PAA@Mn3O4.

[0111] The results showed that spraying PAA@Mn3O4 on leaves can improve the photosynthesis of rice leaves by further increasing the activity of antioxidant enzymes and activating Rubisco enzyme.

[0112] From the above examples, it can be seen that the gas exchange parameters, fluorescence parameters, soluble proteins, Rubisco enzymes and antioxidant enzyme activities are all increased compared with the rice leaves treated with cadmium stress. It can be seen that the polyacrylic acid modified Mn3O4 nanoparticles provided by the present invention not only improve photosynthesis but also improve the cadmium resistance of rice leaves.

[0113] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of polyacrylic acid modified Mn3O4 nanoparticles in improving crop photosynthesis and / or cadmium resistance.

2. The application according to claim 1, characterized in that: The method for preparing the polyacrylic acid modified Mn3O4 nanoparticles comprises the following steps: S1: dissolving manganese acetate tetrahydrate in ethanol to obtain a solution with a concentration of 0.04-0.2 g / ml, dispersing polyacrylic acid in ethanol to obtain a solution with a concentration of 0.2-1.6 g / ml, and then mixing the two solutions to obtain a mixed solution, wherein the mixing ratio is calculated based on the mass ratio of the solute manganese acetate tetrahydrate to the solute polyacrylic acid in the solution to be 1:7.3; S2: placing the mixed solution obtained in S1 into a reactor for heating treatment, and then cooling and centrifuging to obtain a supernatant; S3: Purify the supernatant obtained in S2 to obtain polyacrylic acid modified Mn3O4 nanoparticles.

3. Application of polyacrylic acid modified Mn3O4 nanoparticles in increasing the pigment content, Rubisco enzyme activity or antioxidant enzyme activity of crop leaves.

4. The use according to claim 2, characterized in that: The reactor in step S2 is a reactor lined with polytetrafluoroethylene, and the heating treatment temperature is 100° C.-180° C., and the time is 12 h-26 h.

5. The use according to claim 2, characterized in that: The centrifugal speed of the centrifugal treatment in step S2 is 3000-8000 r / min, and the time is 30 min-90 min.

6. The use according to claim 2, characterized in that: The purification in step S3 is to transfer the supernatant to a dialysis bag for dialysis treatment, the molecular weight cutoff of the dialysis bag is 8KD-14KD, and the dialysis time is 12-26h.

7. The use according to claim 1, characterized in that: The application method is: mixing the polyacrylic acid modified Mn3O4 nanoparticles with a surfactant to prepare a dispersion, and spraying the dispersion evenly on the leaves of the crops. In the dispersion, the surfactant Silwet L-77 accounts for 0.05% of the polyacrylic acid modified Mn3O4 nanoparticle dispersion.

8. The use according to claim 9, characterized in that: The concentration of the polyacrylic acid modified Mn3O4 nanoparticles in the dispersion is 100-300 mg / L.

9. The use according to claim 1, characterized in that: The crop is rice.

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