Application of coumarin-like compounds or their salts in preparing products for improving plant resistance by closing stomata

Abstract

The present invention provides an application of a coumarin-like compound or a salt thereof in the preparation of a product for improving plant resistance by closing stomata. The structure of the coumarin-like compound or its salt is shown in Formula I: #imgabs0# wherein R1, R2, R3, R4, R5, and R6 are independently selected from H, halogen atoms, methyl, hydroxyl, carboxyl, nitro, benzene ring, methoxy, trifluoromethyl, amino, or an oligosaccharide composed of 1-4 monosaccharides; or any site combination of R1 and R2, R2 and R3, or R3 and R4 and the C atoms of the benzene rings to which they are bonded form a ring of #imgabs1# or R5 and R6 and the C atoms of the double bonds to which they are bonded form a ring of #imgabs2#. This application first discovered that coumarin-like compounds can induce plant stomatal closure, and further experiments verified that such substances improve plant resistance by regulating plant stomata, providing new ideas for plant disease prevention and control and drought resistance research.

Description

Technical Field

[0001] The present invention relates to the field of agricultural technology, in particular to application of a coumarin-like compound or a salt thereof in preparing a product for improving plant resistance by closing stomata. Background Art

[0002] Plant diseases have always been an important challenge in agricultural production. Currently, chemical control is one of the main means, and more than 300 active ingredients of fungicides are known to be used to control various plant diseases. However, the long-term and large-scale use of chemical fungicides has led to an increase in the resistance of plant pathogens, and the effectiveness of traditional fungicides has gradually weakened. Therefore, it is imperative to find new disease control strategies to replace traditional fungicides.

[0003] Stomata are small holes surrounded by a pair of guard cells in the epidermis of the aboveground part of plants. Their opening and closing movement is the main means for plants to regulate gas exchange with the environment. A large number of studies have shown that guard cells can respond to a variety of signals from the plant's internal and environmental sources to cause stomatal movement, including light, CO2, water stress, plant hormones, and plant secondary metabolites, etc., to regulate gas exchange, transpiration water loss, and defend against microbial invasion. Studies have shown that the activity of a variety of transport proteins located on the plasma membrane is crucial for the opening and closing movement of stomata. In the process of stomatal closure, the activation of anion channels is the driving force. Its activation will induce depolarization of the plasma membrane, which will induce the activation of outward potassium ion channels and inhibit inward potassium ion channels. The loss of major osmotic pressure regulating ions such as potassium ions and anions will lead to an increase in intracellular water potential, causing guard cells to lose water and shrink, and ultimately causing stomatal closure. Reactive oxygen species (ROS) and nitric oxide (NO) as second messenger molecules play an important role in regulating the activity of guard cell transport proteins and the process of stomatal closure. Cytoplasmic calcium ions are important second messengers in stomatal movement. BAPTA is a selective calcium ion chelator with high selectivity for calcium ions. Therefore, BAPTA is widely used as an intracellular buffer to study the movement of Ca through the plasma membrane. 2+ Permeability channels transfer Ca from intracellular storage or influx 2+ The impact of release.

[0004] Drought, as a multidimensional stress, can cause a series of changes in plants from the phenotypic, physiological, biochemical, to the molecular level. Severe drought can lead to the termination of photosynthesis and metabolic disorders, and ultimately lead to plant death. About 33% of the world's arable land is in a state of insufficient water supply. Drought has become one of the most important adverse factors in agricultural production and the top natural disaster in my country. In order to reduce the impact of drought on the growth of crops and plants, screening and breeding drought-resistant crops and plants is currently recognized as an effective method. At the same time, breeding and planting drought-resistant and water-saving plants can also play a positive role in the restoration of arid environments, but it takes a long time to breed drought-resistant varieties and they are not broad-spectrum. Therefore, broad-spectrum drought-resistant agents have practical application value in solving the problem of crop drought resistance and water conservation.

[0005] The use of chemicals to induce stomatal closure as a strategy for crop drought resistance and water conservation has become an important area of ​​concern for researchers. In addition to ABA, there are other compounds that have shown the potential to regulate the opening and closing of stomata, such as methyl jasmonate. These compounds can regulate the opening and closing of stomata by affecting ion channels, hormone signaling pathways or growth regulation, thereby reducing water evaporation and improving the drought resistance of plants. In practical applications, the use of chemicals to induce stomatal closure can help crops better retain water under drought conditions and maintain normal physiological and biochemical metabolism, thereby alleviating the adverse effects of drought on crops. This strategy provides new possibilities for agricultural production and is expected to provide more effective solutions for drought-resistant breeding and crop production.

[0006] In addition, studies have found that plants are not helpless in the face of pathogen invasion. Plants can recognize signal molecules from pathogens called plant immune elicitors and induce stomatal closure to prevent pathogen invasion. This plant immune response is called stomatal immunity. Studies have shown that pathogens can secrete toxins and proteins called effectors to overcome stomatal immunity and even induce stomatal opening to successfully invade the host, such as toxins secreted by bacteria, such as coronatine and syringolin A and effectors, such as AvrB and HopM1; oxalic acid and toxins secreted by fungi, such as fusicoccin (FC), etc. In summary, the interaction between stomata and pathogens is an important factor in determining the occurrence of diseases, and the study of its mechanism is currently a cutting-edge topic in the field of plant disease resistance and immunity research. Many important crop diseases, such as wheat leaf rust, grape downy mildew, cucumber downy mildew, etc., are caused by pathogens that specifically invade stomata.

[0007] In recent years, researchers have discovered some potential compounds that can enhance plant resistance by affecting stomatal movement. Some compounds can act on ion channels to regulate intracellular ion concentrations, thereby affecting the opening and closing of stomata. For example, some potassium channel regulators can change intracellular potassium ion levels, thereby affecting stomatal movement; some compounds can affect plant signal transduction pathways, such as hormone signaling pathways or the production of secondary messengers, thereby regulating the opening and closing of stomata; some jasmonic acid analogs can affect stomatal movement by activating gibberellin signaling pathways. Based on the theory of stomatal immunity, inducing stomatal closure through chemical substances is a new strategy to inhibit plant diseases. The discovery and application of these compounds will provide new ideas and strategies for plant protection, and are expected to promote the development of plant resistance research and provide more solutions and possibilities for agricultural production. Summary of the invention

[0008] The main purpose of the present invention is to provide an application of a coumarin-like compound or a salt thereof in the preparation of a product for improving plant resistance by closing stomata, so as to provide a new compound for improving plant resistance by utilizing the stomatal immunity theory.

[0009] In order to achieve the above object, according to a first aspect of the present invention, there is provided a use of a coumarin-like compound or a salt thereof in preparing a product for improving plant resistance by closing stomata, wherein the structure of the coumarin-like compound or the salt thereof is shown in Formula I: Wherein, R1, R2, R3, R4, R5, and R6 are each independently selected from H, a halogen atom, a methyl group, a hydroxyl group, a carboxyl group, a nitro group, a benzene ring, a methoxy group, a trifluoromethyl group, an amino group, or an oligosaccharide composed of 1 to 4 monosaccharides; or any combination of R1 and R2, R2 and R3, or R3 and R4, and the C atoms of the benzene rings to which they are bonded form a ring of Or R5 and R6 and the C atoms of the double bond to which they are bonded form a ring

[0010] Further, R1 is selected from H, a chlorine atom, a nitro group, a methoxy group or a hydroxyl group; R2 is selected from H, a chlorine atom, an amino group, a methoxy group or a hydroxyl group; R3 is selected from H, glucose, a hydroxyl group, a methoxy group or an amino group; R4 is selected from H, glucose or a hydroxyl group; R5 is selected from H, an amino group, a hydroxyl group, a benzene ring, a methyl group or a trifluoromethyl group; R6 is selected from H, a chlorine atom, a bromine atom, a methyl group, a trifluoromethyl group or a carboxyl group.

[0011] Furthermore, the coumarin-like compounds include any one or more of the following:

[0012] Further, the salt of the coumarin-like compound includes any one or more of the following:

[0013] Further, the plant includes angiosperms, gymnosperms and / or ferns; preferably, angiosperms include monocots and dicots; preferably, dicots include tobacco, grapes and cucumbers; preferably, monocots include wheat; preferably, plant resistance includes disease resistance and / or drought resistance; preferably, disease resistance includes resistance to any of the following diseases: wheat leaf rust, cucumber downy mildew and grape downy mildew.

[0014] In order to achieve the above object, according to the second aspect of the present invention, a coumarin-like compound or a salt thereof is provided, and the structure of the coumarin-like compound or the salt thereof is shown in the following formula I: Wherein, R1, R2, R3, R4, R5, and R6 are each independently selected from H, a halogen atom, a methyl group, a hydroxyl group, a carboxyl group, a nitro group, a benzene ring, a methoxy group, a trifluoromethyl group, an amino group, or an oligosaccharide composed of 1 to 4 monosaccharides; or any combination of R1 and R2, R2 and R3, or R3 and R4, and the C atoms of the benzene rings to which they are bonded form a ring of Or R5 and R6 and the C atoms of the double bond to which they are bonded form a ring

[0015] Further, R1 is selected from H, a chlorine atom, a nitro group, a methoxy group or a hydroxyl group; R2 is selected from H, a chlorine atom, an amino group, a methoxy group or a hydroxyl group; R3 is selected from H, glucose, a hydroxyl group, a methoxy group or an amino group; R4 is selected from H, glucose or a hydroxyl group; R5 is selected from H, an amino group, a hydroxyl group, a benzene ring, a methyl group or a trifluoromethyl group; R6 is selected from H, a chlorine atom, a bromine atom, a methyl group, a trifluoromethyl group or a carboxyl group.

[0016] Furthermore, the coumarin-like compounds include any one or more of the following:

[0017] Further, the salt of the coumarin-like compound includes any one or more of the following:

[0018] In order to achieve the above object, according to the third aspect of the present invention, a product for improving plant resistance by closing stomata is provided, the product comprising the above-mentioned coumarin-like compound or its salt in the application or the above-mentioned coumarin-like compound or its salt.

[0019] In order to achieve the above-mentioned purpose, according to the fourth aspect of the present invention, a method for improving plant resistance is provided, which comprises dissolving or directly spraying the above-mentioned product for improving plant resistance by closing stomata on the surface of plant leaves; preferably, plant resistance includes disease resistance and / or drought resistance.

[0020] In order to achieve the above-mentioned purpose, according to the fifth aspect of the present invention, there is provided a coumarin-like compound or its salt in the above-mentioned application or the application of the above-mentioned coumarin-like compound or its salt as a plant stomatal closure inducer.

[0021] By applying the technical solution of the present invention, the present application discovered for the first time that coumarin-like compounds can induce the closure of plant stomata. Further experiments verified that such substances improve the resistance of plants by regulating plant stomata, and are especially able to effectively inhibit plant diseases caused by pathogens that use plant stomata as invasion channels, providing new ideas for plant disease prevention and control and drought resistance research. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0023] Figure 1 A schematic diagram showing the statistical results of the measurement of stomatal aperture changes induced by coumarin compounds in Example 2 of the present application;

[0024] Figure 2 A schematic diagram showing the results of coumarin compounds inducing ROS accumulation in tobacco guard cells in Example 3 of the present application is shown;

[0025] Figure 3 A schematic diagram showing the results of the coumarin compounds inducing NO accumulation in tobacco guard cells in Example 3 of the present application is shown;

[0026] Figure 4 A schematic diagram showing the regulating effect of calcium ions in the process of plant stomatal closure induced by test sample 1 in Example 4 of the present application is shown;

[0027] Figure 5 A schematic diagram showing the regulating effect of calcium ions in the process of inducing plant stomatal closure by test sample 2 in Example 4 of the present application;

[0028] Figure 6 A schematic diagram showing the regulating effect of calcium ions in the process of inducing plant stomatal closure by test sample 3 in Example 4 of the present application is shown;

[0029] Figure 7 A comparison chart of the water-retaining and drought-resistant activities of the test sample 1 and the control sample on wheat in Example 5 of the present application is shown;

[0030] Figure 8 A comparison chart of the water-retaining and drought-resistant activities of the test sample 2 and the control sample on wheat in Example 5 of the present application is shown;

[0031] Fig. 9A comparison chart of the water-retaining and drought-resistant activities of the test sample 3 and the control sample on wheat in Example 5 of the present application is shown;

[0032] Fig.10 A comparison chart of the water-retaining and drought-resistant activities of test samples 1, 2, 3 and the control sample on cucumber in Example 6 of the present application is shown;

[0033] Fig.11 A comparison chart of the grape downy mildew inhibition activities of the test sample 1 and the control sample in Example 7 of the present application is shown;

[0034] Fig.12 A comparison chart of the grape downy mildew inhibition activity of the test sample 2 and the control sample in Example 7 of the present application is shown;

[0035] Fig.13 A comparison chart of the grape downy mildew inhibition activities of the test sample 3 and the control sample in Example 7 of the present application is shown;

[0036] Fig.14 A comparison chart of wheat leaf rust inhibition activity of test sample 1 and control sample in Example 8 of the present application is shown;

[0037] Fig.15 A comparison chart of wheat leaf rust inhibition activity of the test sample 2 and the control sample in Example 8 of the present application is shown;

[0038] Fig.16 A comparison chart of the wheat leaf rust inhibition activity of the test sample 3 and the control sample in Example 8 of the present application is shown. DETAILED DESCRIPTION

[0039] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0040] As mentioned in the background technology, during the growth of plants, the opening and closing of stomata is the main means for plants to regulate gas exchange with the environment, and is also an important path for various pathogens to invade plants. It is currently found that plant proteins that can control the opening and closing of stomata are mostly evolved by pathogens to produce related factors that can destroy stomatal immunity. Therefore, whether there are chemical substances other than plant proteins that can control the opening and closing of stomata is of certain significance to improving plant resistance. Through research and experimental common sense, the present application finally discovered a class of coumarin compounds that can close stomata, and found that this compound can improve the disease resistance and drought resistance of a variety of plants, especially the resistance to diseases caused by stomata as invasion channels. On this basis, the applicant proposed a series of schemes of the present application.

[0041] In a first typical embodiment of the present invention, there is provided a use of a coumarin-like compound or a salt thereof in the preparation of a product for improving plant resistance by closing stomata, wherein the structure of the coumarin-like compound is shown in Formula I:

[0042]

[0043] Wherein, R1, R2, R3, R4, R5, and R6 are each independently selected from H, a halogen atom, a methyl group, a hydroxyl group, a carboxyl group, a nitro group, a benzene ring, a methoxy group, a trifluoromethyl group, an amino group, or an oligosaccharide composed of 1 to 4 monosaccharides; or any combination of R1 and R2, R2 and R3, or R3 and R4, and the C atoms of the benzene rings to which they are bonded form a ring of R5 and R6 and the C atoms of the double bond to which they are bonded form a ring:

[0044] When the above compounds are applied to plant leaves, the chemical substances or salts act on the plants themselves, which can promote the closure of plant stomata, close the channels through which the plants themselves exchange substances with the outside world through the stomata, avoid the harm to the plants caused by the opening of stomata, and thus improve the resistance of the plants. This is completely different from the working principle of the chemical substances in the prior art that act on the pests infecting the plants and kill the invading organisms to improve the insect resistance of the plants.

[0045] In a preferred embodiment, R1 is selected from H, a chlorine atom, a nitro group, a methoxy group or a hydroxyl group; R2 is selected from H, a chlorine atom, an amino group, a methoxy group or a hydroxyl group; R3 is selected from H, glucose, a hydroxyl group, a methoxy group or an amino group; R4 is selected from H, glucose or a hydroxyl group; R5 is selected from H, an amino group, a hydroxyl group, a benzene ring, a methyl group or a trifluoromethyl group; R6 is selected from H, a chlorine atom, a bromine atom, a methyl group, a trifluoromethyl group or a carboxyl group.

[0046] In a preferred embodiment, the coumarin-like compound includes any one or more of the following:

[0047]

[0048] Any salt of a coumarin-like compound that can embody the function of the coumarin-like compound in closing plant stomata is applicable to the present application. In a preferred embodiment, the salt of the coumarin-like compound includes any one or more of the following:

[0049]

[0050] The above preferred compounds or their salts have a better effect on controlling the closure of plant stomata than other compounds conforming to the general structure.

[0051] Any plant that can produce stomatal closure response to the compounds of the present application is applicable to the present application. In a preferred embodiment, the plants include angiosperms, gymnosperms and / or ferns; preferably, angiosperms include monocots and dicots. Since the compounds of the present application can improve plant resistance, they have certain practical application value and can better ensure the normal growth of experimental model plants or crop plants. Any plant that needs to improve its resistance is applicable to the present application. In a preferred embodiment, dicots include tobacco, grapes and cucumbers; preferably, monocots include wheat.

[0052] Since the compounds of the present application can adjust the state of the plant stomata to a closed state, the effect of closing the channel for external invasion of plants or water loss of the plants themselves can be achieved. The use of the compounds of the present application can avoid any harm to plants caused by stomata. In a preferred embodiment, plant resistance includes disease resistance and / or drought resistance, that is, the closure of stomata can avoid excessive loss of self-water or invasion of external pathogens. Any disease that enters the plant through the stomata to complete the infection can be defended by stomatal closure. In a preferred embodiment, disease resistance includes resistance to any of the following diseases: wheat leaf rust, cucumber downy mildew and grape downy mildew.

[0053] In a second typical embodiment of the present invention, a coumarin-like compound or a salt thereof is provided, and the structure of the coumarin-like compound or the salt thereof is shown in Formula I:

[0054]

[0055] Wherein, R1, R2, R3, R4, R5, and R6 are each independently selected from H, a halogen atom, a methyl group, a hydroxyl group, a carboxyl group, a nitro group, a benzene ring, a methoxy group, a trifluoromethyl group, an amino group, or an oligosaccharide composed of 1 to 4 monosaccharides; or any combination of R1 and R2, R2 and R3, or R3 and R4, and the C atoms of the benzene rings to which they are bonded form a ring of R5 and R6 and the C atoms of the double bond to which they are bonded form a ring:

[0056] When the above compounds are applied to plant leaves, the chemical substances or salts act on the plants themselves, which can promote the closure of plant stomata, close the channels through which the plants themselves exchange substances with the outside world through the stomata, avoid the harm to the plants caused by the opening of stomata, and thus improve the resistance of the plants. This is completely different from the working principle of the chemical substances in the prior art that act on the pests infecting the plants and kill the invading organisms to improve the insect resistance of the plants.

[0057] In a preferred embodiment, R1 is selected from H, a chlorine atom, a nitro group, a methoxy group or a hydroxyl group; R2 is selected from H, a chlorine atom, an amino group, a methoxy group or a hydroxyl group; R3 is selected from H, glucose, a hydroxyl group, a methoxy group or an amino group; R4 is selected from H, glucose or a hydroxyl group; R5 is selected from H, an amino group, a hydroxyl group, a benzene ring, a methyl group or a trifluoromethyl group; R6 is selected from H, a chlorine atom, a bromine atom, a methyl group, a trifluoromethyl group or a carboxyl group.

[0058] In a preferred embodiment, the coumarin-like compound includes any one or more of the following:

[0059]

[0060]

[0061] Any salt of a coumarin-like compound that can embody the function of the coumarin-like compound in closing plant stomata is applicable to the present application. In a preferred embodiment, the salt of the coumarin-like compound includes any one or more of the following:

[0062]

[0063] The above preferred compounds or their salts have a better effect on controlling the closure of plant stomata than other compounds conforming to the general structure.

[0064] In a third typical embodiment of the present invention, a product for improving plant resistance by closing stomata is provided, the product comprising the above-mentioned coumarin-like compound or its salt in the application or the above-mentioned coumarin-like compound or its salt, and a basic buffer for dissolving the coumarin-like compound or its salt; wherein the basic buffer comprises: 10mM MES, pH=5.6, 5mM potassium chloride and 50μM calcium chloride. In a preferred embodiment, the concentration of the coumarin-like compound or its salt in use of the product is 10-1000μM, preferably 50-100μM. The coumarin-like compound or its salt dissolved in the basic buffer can be stored and used more stably, and is configured to a suitable concentration for more convenient subsequent direct use.

[0065] In a fourth typical embodiment of the present invention, a method for improving plant resistance is provided, the method comprising spraying the above-mentioned product for improving plant resistance by closing stomata on the surface of plant leaves; preferably, plant resistance includes disease resistance and / or drought resistance. Spraying a suitable amount of a coumarin-like compound or a salt thereof on the surface of leaves can effectively control the closure of plant stomata to achieve the purpose of providing plant resistance. Among them, resistance includes disease resistance and / or drought resistance. Disease resistance includes resistance to any of the following diseases: wheat leaf rust, cucumber downy mildew and grape downy mildew.

[0066] In a fifth typical embodiment of the present invention, there is provided a coumarin-like compound or a salt thereof in the above application or a use of the coumarin-like compound or a salt thereof as a plant stomatal closure inducer.

[0067] The present application is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in the present application.

[0068] The plant used in the examples of the present application is Nicotiana benthamiana, which comes from the Crop Stomatal Biology and Regulation Technology Laboratory of Modern Agricultural Research Institute of Peking University. The basic buffer solution is composed of 10mM MES (Tris, pH=5.6), 5mM potassium chloride and 50μM calcium chloride. The coumarin-like compound solution is prepared by the basic buffer solution.

[0069] Stomatal aperture refers to the degree of stomatal opening, and its unit is length unit, usually μm;

[0070] Stomatal conductance refers to the amount of water passing through a unit leaf area per unit time, and its unit is mmol. -1 ·m -2 ·s -1 .

[0071] Example 1: Detection of the effect of coumarin-like compounds on plant stomatal aperture

[0072] The specific method is as follows:

[0073] (1) Take a 4 mm tobacco leaf disc and place it in a basic buffer at 150 μmol -1 ·m -2 ·s -1 Incubate for 2 h under light of 80°C;

[0074] (2) placing the above tobacco leaf disc in a 50 μM solution of different coumarin-like compounds and incubating for 2 h under the same conditions as step (1) to serve as a test sample; in addition, placing a control sample in a 1000-fold dilution of DMSO and incubating for another 2 h under the same conditions as step (1);

[0075] (3) Use ImajeJ software to measure the stomatal aperture of each test sample and the control sample and calculate the mean value and standard deviation.

[0076] The calculation formula of the relative opening of the pores is as follows:

[0077]

[0078] The results are shown in the table below, and it can be seen that the above-tested coumarin-like compounds can induce stomatal closure.

[0079]

[0080]

[0081]

[0082]

[0083] Among them, * indicates 0.05>P>0.01; ** indicates P<0.01.

[0084] Example 2: Detection of the effect of coumarin-like compounds on plant stomatal aperture

[0085] Taking the coumarin compounds 1, 2, and 3 in Example 1 as an example, the specific method is as follows:

[0086] (1) Take a 4 mm tobacco leaf disc and place it in a basic buffer at 150 μmol -1 ·m -2 ·s -1 Incubate for 2 h under light of high intensity, using the same basic buffer composition as in Example 1;

[0087] (2) placing the above tobacco leaf discs in a 1000-fold dilution of DMSO and a 100 μM solution of coumarin compounds 1, 2, and 3, respectively, and incubating for 2 h under the same conditions as step (1), and recording them as control samples, test samples 1, 2, and 3, respectively;

[0088] (3) Observe the opening and closing of the stomata of the tobacco leaf disc after incubation under a microscope and take photos (e.g. Figure 1 The stomatal aperture of tobacco leaf discs under different treatments was measured and the mean value and standard deviation were calculated (as shown in Figure 1 shown).

[0089] from Figure 1 The statistical results of the stomatal opening measurements show that the stomatal openings of test samples 1, 2, and 3 are significantly more closed than those of the control sample. Figure 1 The stomatal opening of tobacco leaf discs shows that the stomata of tobacco leaf discs incubated with coumarin compounds 1, 2, and 3 are completely closed, indicating that coumarin compounds show good activity in inducing stomatal closure.

[0090] Example 3: Detection of physiological changes in plants during the process of coumarin-like compounds inducing stomatal closure

[0091] Taking the coumarin compounds 1, 2, and 3 in Example 1 as an example, the experiment was carried out, and the specific method is as follows:

[0092] (1) Thirty-day-old tobacco leaves were taken and crushed. Equal amounts of crushed leaf tissues were placed in a basic buffer solution at 150 μmol -1 ·m -2 ·s -1 Incubate for 2 h under light conditions;

[0093] (2) adding 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA, TargetMol) at a concentration of 50 μM to the mixture in step (1) as a reactive oxygen species fluorescence probe, and the solvent was pure DMSO;

[0094] (3) The mixture obtained in step (2) was divided into four parts, and 1000-fold diluted DMSO and 50 μM concentration of coumarin compounds 1, 2, and 3 were added respectively, and incubated for 40 min, and recorded as control sample, test sample 1, 2, and 3, respectively;

[0095] (4) Fluorescence changes were observed using a confocal microscope (Nikon ECLIPSE Ti2) (excitation wavelength 480 / 30 nm; absorption wavelength 510 nm; dichroic wavelength 505 nm), and the fluorescence intensity was measured using ImajeJ software.

[0096] The experimental results are as follows Figure 2 As shown in the figure, the fluorescence intensity of plant guard cells of test samples 1, 2, and 3 was significantly higher than that of DMSO, indicating that the content of reactive oxygen species (ROS) in plant guard cells increased significantly after treatment with coumarin-like compounds 1, 2, and 3, indicating that the reactive oxygen species (ROS) in guard cells are involved in mediating the process of stomatal closure induced by coumarin-like compounds. Figure 3 As shown, the fluorescence intensity of plant guard cells of test samples 1, 2, and 3 was significantly higher than that of DMSO, indicating that the NO content in plant guard cells increased significantly after treatment with coumarin-like compounds 1, 2, and 3, indicating that guard cell NO is involved in mediating the process of coumarin-like compounds-induced stomatal closure.

[0097] Example 4: Detection of the regulatory role of calcium ions in the process of plant stomatal closure induced by coumarin-like compounds

[0098] Taking the coumarin compounds 1, 2, and 3 in Example 1 as an example, the experiment was carried out, and the specific method is as follows:

[0099] (1) Take a 4 mm tobacco leaf disc and place it in a basic buffer at 150 μmol -1 ·m -2 ·s -1 Incubate for 2 h under strong light, using the same basic buffer composition as in Example 1;

[0100] (2) treating the leaf discs in (1) with 100uM test sample 1, test sample 2, test sample 3 and 100uM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) respectively, and incubating for 2h under the same conditions as step (1), and recording them as control sample, test sample 1, 2, 3 respectively;

[0101] (3) Observe the opening and closing of the stomata of the tobacco leaf discs after incubation under a microscope and take photos. Also measure the stomatal opening of the tobacco leaf discs under different treatments (e.g. Figure 4 , Figure 5 , Figure 6 shown).

[0102] The experimental results are as follows Figure 4 , Figure 5 , Figure 6 As shown, extracellular Ca 2+ The chelator BAPTA significantly inhibited the stomatal closure induced by coumarin-like compounds, indicating that guard cell Ca 2+ Involved in mediating coumarin-induced stomatal closure.

[0103] Example 5: Detection of the effects of coumarin-like compounds on water retention and drought resistance in wheat

[0104] Taking the coumarin compounds 1, 2, and 3 in Example 1 as an example, the experiment was carried out, and the specific method is as follows:

[0105] (1) Three groups of wheat leaves with basically the same growth conditions were selected, one part of the wheat leaves in each group was used as a control sample, and the other part was used as a test sample. The test samples in the three groups were immersed in a test solution of coumarin-like compounds 1, 2, and 3 with a concentration of 100 μM for 10 min, and were recorded as test samples 1, 2, 3 and control samples, respectively. The control samples were immersed in a 1000-fold dilution of DMSO;

[0106] (2) The control sample and test samples 1, 2, and 3 were placed in a room at a temperature of 23°C, a relative humidity of 60%, and a light intensity of 35 μmol -1 ·m -2 ·s -1 Keep it still under the conditions and continue taking pictures;

[0107] (3) Record the initial and final weights of the control sample and test samples 1, 2, and 3.

[0108] Figure 7 , Figure 8 , Fig. 9The growth status comparison diagrams of test samples 1, 2, 3 and control samples are shown respectively, where the upper diagram shows the initial state of each sample, and the lower diagram shows the state of each sample after standing for 30 minutes. It can be seen that the wheat leaves in the control sample wilt after 30 minutes of light exposure, indicating that the water loss is serious and insufficient to support the leaves to stand upright, while the wheat leaves in the test sample remain upright; at the same time, the water loss of test samples 1, 2, and 3 is less than that of the control samples. It can be seen that the above-mentioned coumarin-like compounds have the activity of promoting wheat water retention and drought resistance at a concentration of 100μM.

[0109] Example 6: Detection of the effect of coumarin compounds on drought resistance of cucumber

[0110] Taking the coumarin compounds 1, 2, and 3 in Example 1 as an example, the experiment was carried out, and the specific method is as follows:

[0111] (1) Six cucumber plants with basically the same growth conditions were selected, three cucumbers were used as control samples, and the other three were used as test samples. The three test samples were sprayed with test solutions of coumarin-like compounds 1, 2, and 3 with a concentration of 100 μM, and the control samples were recorded as test samples 1, 2, and 3, respectively. The control samples were sprayed with a 1000-fold dilution of DMSO on the cucumber plants;

[0112] (2) The control sample and test samples 1, 2, and 3 were placed in a room at a temperature of 20°C, a relative humidity of 60%, and a light intensity of 120 μmol -1 ·m -2 ·s -1 Keep it still under the conditions and continue taking pictures;

[0113] Fig.10 The figure is a comparison of the drought resistance of test samples 1, 2, 3 and the control sample. It can be seen from the figure that the leaves of the cucumber plants in the control sample wilted after 12 days of drought treatment, indicating that the water in them was severely lost and insufficient to support the leaves to stand upright, while the cucumber plants in the test sample remained upright. It can be seen that the above-mentioned coumarin-like compounds have the activity of promoting cucumber drought resistance at a concentration of 100 μM.

[0114] Example 7: Detection of the inhibitory activity of coumarin-like compounds against grape downy mildew

[0115] Taking the coumarin compounds 1, 2, and 3 in Example 1 as an example, the experiment was carried out, and the specific method is as follows:

[0116] (1) Three groups of grape leaves with basically the same growth conditions were selected. One part of the grape leaves in each group was used as a control sample, and the other part was used as a test sample. 12 mm leaf disks were taken from the three groups of grape leaves and placed in a basic buffer solution at a strength of 130 μmol -1·m -2 ·s -1 The mixture was floated for 2 h under illumination conditions of , wherein the composition of the basic buffer was the same as that of Example 1;

[0117] (2) The grape leaves of the test samples were placed in a test solution of coumarin compounds 1, 2, and 3 at a concentration of 100 μM, and the control samples were placed in a 1000-fold dilution of DMSO, and floated for 2 h under the same conditions as step (1), and recorded as test samples 1, 2, and 3;

[0118] (3) Add equal volumes of Downy Mildew sporangium suspension (2×10 5 / mL), and continue floating for 6h;

[0119] (4) The leaf disks of the control sample and test samples 1, 2, and 3 were placed in a culture dish with the back side facing upward and transferred to an incubator for 6 days. The incubator parameters were 19° C., 80% humidity, and 3500 Lx light (Light:Dark=12:12).

[0120] Method for detecting the number of sporangia: Use 1 mL of water to rinse a single leaf disk to obtain a sporangium suspension, and then use a hemacytometer to count the number of sporangia in the sporangium suspension under a microscope.

[0121] Test results such as Fig.11 , Fig.12 and Fig.13 As shown, the leaf disc infection status of test samples 1, 2, 3 and control samples 6 days after inoculation with downy mildew and the number of downy mildew sporangia detected in grapes 6 days after infection are shown. It can be seen that no colonies were produced in the grape leaf discs treated with coumarin-like compounds 1, 2, and 3, and no downy mildew sporangia were observed. It can be seen that the coumarin-like compounds showed inhibitory activity against grape downy mildew at a concentration of 100 μM.

[0122] Example 8: Study on the inhibitory activity of coumarin-like compounds against wheat leaf rust

[0123] Take coumarin compounds 1, 2, and 3 as examples. The specific test method is as follows:

[0124] (1) Three groups of wheat at the one-leaf-one-heart stage with basically the same growth conditions were selected, and one part of the wheat in each group was used as a control sample, and the other part was used as a test sample. The test samples were sprayed with 100 μM concentrations of coumarin-like compounds 1, 2, and 3, respectively, and the control samples were sprayed with a 1000-fold dilution of DMSO;

[0125] (2) 2 hours later, the cells were inoculated with wheat leaf rust and cultured in the dark at 20°C and 80% relative humidity for 24 hours;

[0126] (3) The cells were cultured in an environment with a temperature of 23°C, a relative humidity of 80%, and a light intensity of 5000 Lx (Light:Dark=12:12), and the antibacterial effect was measured on the 14th day.

[0127] Fig.14 , Fig.15 and Fig.16 The growth status comparison diagrams of the control sample and the test sample show that the control sample leaves have large rust spots on their surface, indicating that they are all infected with wheat leaf rust, while the test sample leaves have only a few spots on their surface, and their growth status is significantly better than the control sample. It can be seen that coumarin compounds show inhibitory activity against wheat leaf rust at a concentration of 100 μM.

[0128] From the above description, it can be seen that the above-mentioned embodiments of the present invention achieve the following technical effects: a new use of the coumarin-like compounds in the present application is found to induce the closure of plant stomata. By regulating the plant stomata with coumarin-like compounds, the water retention and drought resistance and disease control properties of the plants can be well promoted. In particular, plant diseases caused by pathogens that use plant stomata as invasion channels can be effectively inhibited, providing a new idea for the prevention and control of plant diseases.

[0129] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of a coumarin-like compound or a salt thereof in the preparation of a product for improving plant resistance by closing stomata, characterized in that: The coumarin-like compound is one or more of the following: The plant resistance is disease resistance.

2. The use according to claim 1, characterized in that: The plant is an angiosperm.

3. The use according to claim 2, characterized in that: The angiosperms include monocots and dicots.

4. The use according to claim 3, characterized in that: The dicotyledonous plants include tobacco, grapes and cucumbers.

5. The use according to claim 3, characterized in that: The monocotyledonous plants include wheat.

6. The use according to claim 1, characterized in that: The disease resistance includes resistance to any one of the following diseases: wheat leaf rust, cucumber downy mildew and grape downy mildew.

7. A coumarin-like compound or a salt thereof, characterized in that: The coumarin-like compound is:

8. A product for improving plant resistance by closing stomata, characterized in that: The product comprises the coumarin-like compound or its salt in the use of any one of claims 1 to 6 or the coumarin-like compound or its salt in claim 7; The plant resistance is disease resistance.

9. A method for improving plant resistance, characterized in that: The method comprises spraying the product for improving plant resistance by closing stomata as claimed in claim 8 directly on the surface of plant leaves or spraying it on the surface of plant leaves after dissolving; The plant resistance is disease resistance.

10. Use of the coumarin-like compound or its salt in any one of claims 1 to 6 or the coumarin-like compound or its salt in claim 7 as a plant stomatal closure inducer.

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