Application of Sinapic Acid in Regulation of Stomatal Opening and Closing in Plants

Abstract

The present application belongs to the field of plant physiology technology, and specifically relates to the application of sinapinic acid in regulating the opening and closing of plant stomata. Sinapic acid SA is involved in regulating the opening of UV-B-induced stomata. During the UV-B-induced stomatal activity, UV-B radiation treatment can induce the second messenger H2O2, NO and cytoplasmic Ca in leaves. 2+ The level of sinapic acid SA is increased by converting to sinapoylmalate SM and reducing the second messenger H2O2, NO and cytosolic Ca through the accumulation of SM. 2+ The level of sinapic acid synthesis metabolism related genes is regulated. BRT1 and SNG1 The inventors conducted a preliminary study on the physiological performance of Arabidopsis leaves under UV-B radiation, especially the opening and closing of stomata. Based on the relevant conclusions, it has very important theoretical value and application prospects for improving and enhancing the ability of crops in high-latitude areas or in summer to resist ultraviolet damage.

Description

Technical Field

[0001] The present application belongs to the field of plant physiology technology, and specifically relates to the application of sinapinic acid in regulating the opening and closing of plant stomata. Background Art

[0002] In plants, sinapic acid esters synthesized with sinapic acid SA as a precursor usually refer to: sinapoylcholine (SC), sinapoylglucose (SG), and sinapoylmalate (SM). Since sinapic acid esters are directly related to the synthesis of flavonoids, anthocyanins, lignin and other substances, the study of sinapic acid ester metabolic pathways is of great significance for the study of plant physiological metabolic regulation.

[0003] In the model plant Arabidopsis, due to its rich content of sinapic acid esters, the research on its sinapic acid ester metabolic pathway has been relatively clear. Studies have shown that the main key enzymes involved in sinapic acid ester metabolism include: sinapic acid-glucosyltransferase, sinapic acyl glucose malate transferase, and sinapic acyl glucose choline transferase. Among them, sinapic acid-glucosyltransferase is used to catalyze the synthesis of SG from sinapic acid, and SG is the precursor for the synthesis of SM and SC. Sinapic acyl glucose malate transferase (SMT) was originally discovered in carrot extracts and is responsible for catalyzing the formation of SM from SG. The catalytic effect of sinapic acyl glucose choline transferase (SCT) is to transfer the sinapic acid ester part of SG to the hydroxylated acceptor molecule to synthesize SC.

[0004] As sessile organisms on land that need sunlight to grow and develop, plants are inevitably exposed to ultraviolet wavelengths (Ultraviolet Radiation, UV-R, 200-400 nm), which account for 7% of the sun's electromagnetic radiation. Although UV-C (Ultraviolet Radiation-C, 200-280 nm) and most of the UV-B (Ultraviolet Radiation-B, 280-315 nm) radiation are absorbed by ozone in the stratosphere, some UV-B radiation is still transmitted to the earth's surface. As a signal stimulus, low-dose UV-B has a direct impact on plant morphogenesis and metabolism, and also participates in inducing plant photomorphogenesis and UV-B acclimation response, thereby enabling plants to tolerate UV-B, such as: de-etiolation of seedlings, adaptation and tolerance to UV-B, day and night alternation, inhibition of thermal morphogenesis, phototropism, inhibition of shading response, influence on plant leaf development, induction of stomatal movement, etc.; high-dose UV-B may cause DNA damage, induce the production of reactive oxygen species (ROS), affect cell vitality and integrity, and inhibit plant growth.

[0005] Normally, when plants are subjected to UV-B stress, they will quickly initiate a series of defense mechanisms, including the removal of ROS, the biosynthesis of flavonoids and their derivatives, the biosynthesis of sinapinic acid esters, the synthesis of pathogenicity-related defense proteins, and DNA repair mechanisms. Therefore, it is very important to understand the transduction mechanism of UV-B signals in cells.

[0006] Stomata are a microporous tissue structure on the plant epidermis, composed of a pair of guard cells. The appearance of stomata is an important sign of the evolution of plants from aquatic to terrestrial organisms. During the physiological and metabolic activities of plants, stomatal movement is regulated by adjusting the turgor pressure of guard cells, which in turn has a direct impact on physiological activities such as respiration, photosynthesis, gas exchange during transpiration, and water evaporation. Studies have shown that stomatal movement is related to changes in the volume of vacuoles in guard cells, and changes in the volume of guard cell vacuoles are affected by the accumulation of osmolytes in the cells to form a reversible regulation. Therefore, the dynamic changes in the volume and structure of guard cell vacuoles are crucial to achieving stomatal movement.

[0007] Studies have shown that the chemical properties of osmotic regulators involved in stomatal movement depend on the type and growth conditions of the plant. Most studies have shown that inorganic potassium ions (K + ), chloride ion (Cl - ), and organic solutes malate and sucrose are the main influencing factors; among them, K + , Cl - It can be obtained from outside the plastid, while malic acid and sucrose can be obtained through starch degradation or by fixing CO2. The accumulation of these osmotic substances can reduce the osmotic potential of guard cells, promote guard cells to absorb water and swell, and thus lead to stomatal opening. There are many other signals that respond to stomatal movement, such as soil moisture, temperature, humidity, light intensity, UV-B, CO2, air pollutants and other factors. Therefore, studying the regulatory mechanism of stomatal movement is crucial for the study of plant physiological activities.

[0008] Although UV-B radiation accounts for a relatively small proportion of the entire spectrum, it has a great impact on higher plants. In order to reduce the impact of UV-B radiation on plants, plants themselves metabolize and synthesize "sunscreens" such as phenolic substances, sinapate, flavonoids, etc. These synthetic substances will be transported to the upper epidermis of the leaves to prevent UV-B radiation from penetrating the upper epidermis, thereby reducing the impact of ultraviolet radiation.

[0009] During the screening and research of Arabidopsis plant-related mutants, it was found that the phenylpropanoid metabolic pathway can affect the sensitivity and tolerance of plants to UV-B by regulating the changes in the content of sinapate esters. Sinapic acid and its derivatives are intermediates in the phenylpropanoid secondary metabolic pathway and are identified as a class of hydroxycinnamic acid derivatives with ultraviolet absorption ability that are abundant in Arabidopsis and other cruciferous plants. Although sinapate esters have been reported in many aspects of plant growth and development, as well as resistance to external environmental stress, it is still unclear whether the metabolism of sinapate esters is directly related to UV-B and its specific regulatory mechanism. Therefore, studying the relationship between sinapate ester metabolism and the UV-B signal transduction pathway has important biological significance for revealing the molecular mechanism of sinapate esters regulating plant growth and development and response to adverse stress. Summary of the invention

[0010] The purpose of the present application is to provide the application of sinapinic acid in regulating the opening and closing of plant stomata, especially regulating the closure of plant stomata induced by ultraviolet B (UV-B), thereby laying a certain foundation for the improvement of related plant varieties.

[0011] The technical solution adopted in this application is described in detail as follows.

[0012] The application of sinapinic acid in regulating the opening and closing of plant stomata. Sinapic acid is involved in regulating the opening of stomata induced by UV-B; specifically:

[0013] For Arabidopsis leaves, a certain intensity of UV-B (0.5 Wm -2 ), under a certain radiation duration (4h), the leaf temperature increased, the stomatal opening decreased, and the water loss rate decreased;

[0014] In terms of the specific degree of leaf stomatal opening, a certain intensity of UV-B (0.5 Wm -2 ), under certain radiation duration (4h), sinapic ester-related mutants sng1 and brt1 The stomatal opening of the leaves of the strain was smaller than that of the wild type (WT);

[0015] sng1 The mutant is a mutant whose leaves lose the ability to convert sinapoylglucose (SG) into sinapoyl ester;

[0016] brt1 The mutant is a mutant whose leaves lack the ability to convert sinapinic acid into SG;

[0017] Furthermore, UV-B radiation treatment can induce the second messengers H2O2, NO and cytosolic Ca 2+The level of UV-B increased, and it also promoted the accumulation of sinapinic acid malic acid SM; when exogenous sinapinic acid SA (0.5 mM) or malic acid (6 mM) was applied, the content of endogenous sinapinic acid SM was increased, which in turn promoted stomatal closure, thereby regulating the effect of UV-B on plant leaves.

[0018] Based on the above results, when plant leaves are facing possible damage caused by UV-B radiation, plant adaptability can be improved by exogenously applying sinapinic acid SA or malic acid; or by utilizing genetic engineering technology to regulate the expression of genes related to sinapinic acid metabolism (especially SM expression) to improve the physiological activities of plants under UV radiation.

[0019] In this application, the inventors conducted a preliminary study on the physiological performance of Arabidopsis leaves under UV-B radiation, especially on the opening and closing of stomata, with the aim of studying the physiological performance of plants under UV-B radiation. The results showed that sinapic acid esters can participate in and inhibit the stomatal closure process induced by UV-B radiation, and this process is related to the second messengers H2O2, NO and cytoplasmic Ca in plants. 2+ Based on this theory, it has very important theoretical value and application prospects for improving and enhancing the ability of crops in high latitudes or in summer to resist ultraviolet damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 To screen the irradiation intensity and irradiation time of UV-B; among them:

[0021] A and B represent the stomatal aperture of WT under different radiation intensities and times, respectively;

[0022] C is 0, 0.5, 0.7 and 0.9 w / m 2 After the guard cells were treated with UV-B for 2 h, they were then treated with 10 μg / mL FDA and 0.9 w / m 2 Fluorescence images collected after UV-B treatment of guard cells followed by treatment with 5 mg / mL PI alone for 10 min;

[0023] In each experiment, three epidermal strips were measured, and each experiment was repeated three times; the selected confocal laser images represent the same results of approximately nine measurements, and the scale bar of all fluorescent images is 50 μm;

[0024] Figure 2 For WT, sng1 , brt1Analysis of water loss rate, stomatal aperture and leaf surface temperature under normal light and UV-B conditions;

[0025] A is WT under normal growth conditions, sng1 and brt1 Detached rosette leaf water loss analysis;

[0026] B is 0.5 W / m 2 UV-B conditions for WT, sng1 and brt1 Analysis of water loss in detached rosette leaves after treatment for different durations; both sets of data were obtained from three independent repeated experiments, *P < 0.05, **P < 0.01;

[0027] C: The epidermal strips with open stomata were placed in MES buffer and exposed to normal light and 0.5 W / m 2 Treated under UV-B conditions for 2 h, the scale bars of the images in C are all 10 μm;

[0028] D is the statistical analysis of the stomatal pore size of epidermal strips, the data are from three independent repeated experiments (n = 150), *P < 0.05, **P < 0.01;

[0029] E is WT, sng1 and brt1 Infrared imaging false color images of in vitro leaves; the scale bar of all images in Figure E is 2 cm; n = 50 in vitro plants tested for each genotype;

[0030] F is the quantitative statistical analysis of leaf temperature of the images shown in Figure E using the infrared software FLIR Tools (version 6.4), **P < 0.01;

[0031] Figure 3 Treatment with sinapic acid and malic acid increased the accumulation of endogenous SM in Arabidopsis leaves; wherein:

[0032] A is the peak time and position of SM, SG and sinapinic acid standard separated by HPLC;

[0033] B Quantitative statistics of isolated SM, data are from three independent repeated experiments, *P < 0.05;

[0034] Figure 4 Malic acid is involved in regulating UV-B-induced stomatal movement; among them:

[0035] A and C show the effects of different concentrations of sinapinic acid on WT and sng1 , brt1 Under normal light, dark conditions and 0.5 W / m 2The treatment was performed under UV-B conditions for 2 h, and the scale bars in all images are 10 μm;

[0036] B and D are statistical analyses of the stomatal pore diameters of the epidermal strips in Figures A and C. The data are from three independent repeated experiments (n = 150), **P < 0.01;

[0037] Figure 5 The lack of SM content in Arabidopsis leaves indirectly induced an increase in H2O2 content and the scavenging effect of sinapinic acid and malic acid on H2O2; among them:

[0038] A is a fluorescence image showing the effect of exogenous application of CAT, sinapinic acid and malic acid on the H2O2 content in guard cells under normal light and UV-B conditions. The scale bars of all images are 5 μm.

[0039] B is the quantitative statistics of the fluorescence intensity of 50 μM H2DCFDA treated in the dark for 10 min in Figure A. The data are from three independent repeated experiments (n = 150), **P < 0.01;

[0040] Figure 6 The lack of SM content in Arabidopsis leaves indirectly induced an increase in NO content and the scavenging effect of sinapinic acid and malic acid on NO; among them:

[0041] A is the fluorescence image showing the effect of exogenous c-PTIO, sinapinic acid and malic acid on NO content in guard cells under normal light and UV-B conditions. The scale bars of all images are 5 μm.

[0042] B is the quantitative statistics of the fluorescence intensity of (A) treated with 10 μM DAF-2DA in the dark for 30 min. The data are from three independent repeated experiments (n = 150), **P < 0.01;

[0043] Figure 7 The effect of UV-B radiation on Ca in guard cells 2+ The influence of concentration; among which:

[0044] A is to convert YC3.6, YC3.6 sng1 and YC3.6 brt1 The epidermal strips with open stomata were placed in MES buffer and heated at 0.5 W / m 2 UV-B treatment for 40 min. The scale bar of all images under low magnification is 50 μm, and the scale bar of all images under high magnification is 5 μm;

[0045] B is to convert YC3.6, YC3.6 sng1 and YC3.6 brt1The epidermal strips with open stomata were placed in MES buffer and heated at 0.5 W / m 2 Effect of UV-B on Ca2+ in guard cells after 20 min, 40 min and 60 min treatment 2+ The concentrations were statistically analyzed;

[0046] C is the Ca in Figure A 2+ Statistical analysis of concentrations, statistical data were obtained from three independent repeated experiments (n = 150), **P < 0.01. DETAILED DESCRIPTION

[0047] The present application is further explained below in conjunction with the embodiments. Before introducing the specific embodiments, some experimental backgrounds in the following embodiments are briefly introduced as follows.

[0048] Biomaterials

[0049] Arabidopsis wild mutant brt1 (AT3G21560), sng1 (AT2G22990) (The results show that brt1 The content of sinapoylmalic acid in the material is 60-70% lower than that in normal plants; sng1 Materials (plants that accumulate more sinapinic acid glucose than normal plants) were provided by Professor Clint Chapple of Purdue University, USA;

[0050] Experimental reagents:

[0051] Sinapic acid, malic acid and H2DCFDA (chemically reduced fluorescein dichlorofluorescein diacetate), products of Sigma, USA;

[0052] CAT (catalase), FDA (fluorescein diacetate), PI (inorganic phosphate), DMSO (dimethyl sulfoxide), and MES (2-morpholineethanesulfonic acid) were purchased from Solarbio;

[0053] Acetonitrile and methanol, products of MREDA;

[0054] Experimental equipment:

[0055] High performance liquid chromatograph Agilent 1260, a product of Agilent Technologies, USA.

[0056] Example 1

[0057] Since the main purpose of this application is to explore the specific functions and effects of sinapate esters when plants respond to UV-B irradiation, it is first necessary to clarify the UV-B irradiation conditions that plants can adapt to. To this end, taking the model plant Arabidopsis thaliana as an example, this example preliminarily clarifies the UV-B irradiation intensity and irradiation time. The specific experimental situation is briefly described as follows.

[0058] The leaf epidermal strips of WT Arabidopsis thaliana (Col-0, Columbia 0) grown for three weeks were used as experimental samples, and the stomatal aperture of the leaf epidermal strips was used as the evaluation index. The leaves were irradiated with UV-B of different intensities and durations. Figure 1 Specifically:

[0059] From the perspective of radiation intensity, Figure 1 As shown in A, the intensity of the -2 , 0.5 Wm -2 , 0.7W m -2 After 4 h of UV-B irradiation on WT epidermis, 0.5 Wm -2 The degree of stomatal closure in the epidermal strips of the experimental group was higher than that of 0.3 W m -2 The experimental groups showed significant statistical differences, and considering that guard cells were -2 Physiological status under irradiation conditions is better than 0.7 Wm -2 Therefore, the optimal UV-B irradiation intensity was 0.5 Wm -2 .

[0060] From the perspective of irradiation duration, the same irradiation intensity (0.5 Wm -2 ) were used to treat WT epidermal strips for 0 h, 1 h, 2 h, 3 h, and 4 h, respectively. The results are shown in Figure 1 As shown in B, it can be seen that the stomatal opening is more obviously closed at 4 h (or 2 h) than in the previous time periods, so 4 h (or 2 h) is selected as the appropriate treatment time.

[0061] In order to further clarify that stomatal closure in the above experimental process is not caused by guard cell damage caused by UV-B irradiation, the inventors further conducted experimental verification. The specific experimental results are as follows.

[0062] Normal light (light intensity of 150 μmol m 2 s 1 )、0.5 W / m 2 , 0.7 W / m 2 and 0.9 W / m 2Arabidopsis WT epidermal strips treated with UV-B for 2 h were placed in Tris-KCl buffer containing 10 μg / mL FDA (which can pass through the cell membrane, be hydrolyzed by intracellular esterases, produce polar compounds, accumulate in the cell, and show green fluorescence when excited by blue light) and 5 mg / mL PI (which can pass through the damaged cell membrane, bind to DNA and RNA, and form a bright red fluorescent complex visible in the nucleus of dead cells) and incubated at room temperature in the dark for 10 min. Excess fluorescent dye on the epidermal strips was washed off with fresh Tris-KCl buffer and slides were prepared for observation immediately.

[0063] The results are as follows Figure 1 C. It can be seen that: 0.5 W / m 2 , 0.7 W / m 2 The physiological state of guard cells in the experimental group under UV-B conditions was almost no different from that under normal conditions; however, guard cells under 0.9 W / m2 UV-B were almost all labeled with PI dye, and the activity of guard cells was significantly reduced. These results show that: 2 , 0.7 W / m 2 UV-B intensities of 0.9 W / m 2 UV-B intensity irradiation can cause serious damage to guard cells, which also explains the above 0.5 W / m 2 Stomatal closure induced by UV-B is not caused by damage to guard cells, but is caused by the influence of other metabolites.

[0064] Example 2

[0065] Sinapoylglucose malate transferase (SMT) was originally discovered in carrot extracts and is encoded by the SNG1 gene (AT2G22990). It is responsible for catalyzing SG to form SM and is one of the key synthases of sinapoyl glucose metabolites. The conversion of SA (sinapoylglucose) to SG (sinapoylglucose) in the metabolic pathway is the responsibility of the BRT1 (AT3G21560) gene.

[0066] In order to clarify whether stomatal closure under UV-B irradiation is regulated by sinapic acid ester metabolites, and to clarify whether the content of sinapic acid ester metabolites is associated with other physiological phenotypes of plants under UV-B irradiation, the inventors used the SM deletion mutant ( sng1 ), SG deletion mutant ( brt1 , which can be partially synthesized SM) was used as the research material, and relevant experiments were carried out. The specific experimental process is briefly introduced as follows.

[0067] Select the ones with good growth status and about three weeks old (without bolting) sng1Mutants, brt1 The mutant plants were treated under normal light for 2 h to adapt to the environment, and then 0.5 W / m 2 The plants were irradiated under UV-B conditions (normal light treatment was used as a control), and then the roots were removed for a water loss experiment.

[0068] The specific operation of the water loss experiment is as follows:

[0069] First, the air conditioning temperature of the dehydration platform room was set to about 22°C 1 h in advance; before the experiment began, the materials were placed in the room 30 min in advance to adapt to the environment;

[0070] Then, put the weighing paper into the electronic balance (note the "peeling"), and then remove the roots and soil of the whole Arabidopsis thaliana plant (not bolted) that is about three weeks old and has not yet grown, and put it on the weighing paper (each material should be repeated at least three times, and the weight of the material should be kept as consistent as possible);

[0071] Finally, set the time interval for collecting weight to 5 min, start weighing, and record the relevant measurement data.

[0072] The specific experimental results are shown in Figure 2. From the analysis, it can be seen that normal light treatment ( Figure 2 A), the water loss rate of mutant plants was significantly reduced, and the SM deletion mutant ( sng1 ) has the slowest water loss rate. Under different UV-B radiation durations ( Figure 2 B), the water loss rate of mutant plants was significantly reduced, and the SM deletion mutant ( sng1 ) also had the slowest water loss rate. The water loss rate is directly related to the degree of stomatal opening (i.e., the slower the water loss rate, the lower the degree of stomatal opening). Therefore, it can be considered that sinapinic acid ester metabolism plants are directly involved in the regulation of stomatal opening under UV-B treatment conditions, and the importance of SM sinapinic acid malate is higher than that of SG sinapinic acid glucose.

[0073] Furthermore, in order to specifically clarify the effect of UV-B irradiation on stomatal aperture, three-week-old WT, sng1 , brt1 The fifth rosette leaf was removed, the epidermis was torn off, and the remaining mesophyll cells on the epidermis were brushed off with a brush, and then the epidermis was placed in fresh stomatal buffer; after being placed under normal light for 2 h, one part continued to be treated under normal light as a control, and the experimental group was treated with 0.5 W / m 2 The samples were irradiated with UV-B for 2 hours and then observed. Figure 2 C. Figure 2 As shown in D.

[0074] It can be seen that under UV-B radiation treatment, compared with the control, sng1 The stomatal opening of the mutant was significantly changed. brt1 The stomatal opening of the mutant was not significantly changed. This result preliminarily indicated that SM was directly involved in the regulation of stomatal opening and closing under UV-B irradiation.

[0075] In order to further evaluate the physiological changes of plants under UV-B irradiation, the inventors further used the ThermaCAMSC1000 (FILR system) infrared camera to perform infrared imaging analysis on the leaf surface temperature of wild type and mutant plants (the plants were about three weeks old, without bolting, and the treatment method was the same as above). The results are as follows Figure 2 E. Figure 2 As shown in F.

[0076] Analysis shows that at 0.5 W / m 2 After 2 h of UV-B treatment, the leaf temperature of each treatment group increased compared with that of normal light. sng1 The leaf temperature of the group was significantly different. This result is consistent with the previous experiment, that is, compared with normal light, under UV-B treatment, sng1 The leaf temperature of the group increased significantly, the stomatal aperture decreased significantly, and the water loss rate decreased significantly. In other words, sinapinic acid SM directly participated in a series of physiological activities of leaves under UV-B conditions.

[0077] Example 3

[0078] On the basis of Example 2, in order to further clarify the relationship between the change of SM content and stomatal aperture under UV-B induction, the inventors further conducted relevant experiments with the help of HPLC detection technology. The specific experimental results are briefly described as follows.

[0079] It should be noted that during the experiment, when using HPLC technology to detect SM, the specific operation can be referred to as follows:

[0080] The fifth rosette leaf of each treatment group was selected, 0.1 g was weighed for each replicate and quickly placed in liquid nitrogen (at least three replicates for each genotype), quickly ground into powder, and then transferred to a 1.5 mL centrifuge tube, 1 mL of 50% methanol (v / v) extract was added, and then quickly shaken to mix;

[0081] The mixed sample was heated and extracted at 65°C for 30 min. After the extraction, it was centrifuged at 11,000 rpm for 10 min at room temperature.

[0082] The supernatant was aspirated with a 1 mL syringe and filtered through a 0.22 μm organic filter into the inner tube of a brown liquid phase vial to detect the content of related substances.

[0083] During the HPLC detection process, the HPLC elution procedure and related detection parameters are as follows:

[0084] XBridge C18 (4.6 × 150 mm) column; column temperature 25 °C;

[0085] Table 1. Elution program

[0086]

[0087] Phase A is the aqueous phase, phase B is the organic phase, and the flow rate is 1 mL / min;

[0088] The number of injections was 1, and the injection volume was 10 μL;

[0089] The UV detection wavelength range was 200-550 nm, and the extraction peak wavelength was 330 nm.

[0090] When determining the content, first prepare a standard solution of sinapic acid ester with different concentration gradients, adopt the above-mentioned determination procedure, use the different concentrations of sinapic acid ester content as the X-axis, and use the peak area corresponding to each concentration as the Y-axis to prepare a standard curve of sinapic acid ester content, and then quantify the determination results of the sample to be tested by comparing them with the standard curve.

[0091] In the specific experiment, WT, sng1 Mutants, brt1 The fifth rosette leaf of the mutant plant was floated in stomatal buffer (MES-KOH, 10 mM MES, 50 mM KCl, 0.1 mM CaCl2, pH adjusted to 6.15 with NaOH) and placed under normal light for 2 h to promote stomatal opening. Then, 0.5 mM sinapinic acid and 6 mM malic acid were exogenously applied, and then placed under normal light and UV-B conditions for 2 h. After the treatment, the changes in the content of sinapinic acid esters were determined by referring to the above-mentioned HPLC detection method (peak time reference Figure 3 A).

[0092] The results are as follows Figure 3 As shown ( Figure 3 B). The analysis shows that:

[0093] In normal wild-type WT plants, sinapic acid, malic acid, and UV-B treatment can promote the increase of SM content in leaves. Under normal light conditions, exogenous application of malic acid has the best effect on increasing endogenous SM; under ultraviolet UV-B treatment conditions, although exogenous application of malic acid or sinapic acid can also increase endogenous SM content, the difference in the increase effect is not obvious; at the same time, the effect of ultraviolet UV-B treatment on increasing endogenous SM content is also significantly better than the effect of exogenous application of malic acid or sinapic acid. In other words, under UV-B induction, the endogenous sinapoylmalic acid SM content of leaves can be significantly increased; at the same time, exogenous application of malic acid or sinapic acid can also promote the increase of endogenous sinapoylmalic acid SM content.

[0094] The mutant brt1 In the treatment of wild-type plants, the changes in SM content were similar to those in normal wild-type plants, but it was obvious that the effect of exogenous malic acid was greater than that of exogenous sinapic acid. In other words, the changes in malic acid content played a dominant role in the regulation of endogenous SM content.

[0095] In summary, it can be preliminarily concluded that sinapinic acid SA further synthesizes SM to resist stomatal opening induced by UV-B radiation; and in this process, malic acid can also be converted into SM through a certain metabolic mechanism to participate in regulating the degree of stomatal opening; and even under UV-B treatment conditions, exogenously applied sinapinic acid and malic acid can also participate in UV-B-induced stomatal movement by converting into SM.

[0096] Example 4

[0097] In the above-mentioned Example 3, the inventors have preliminarily proved that sinapinic acid is involved in stomatal opening activity under UV-B irradiation conditions. In order to further clarify whether the change in sinapinic acid content has an effect on the regulation of related gas control activities, the inventors further conducted relevant experiments. The specific experimental results are briefly described as follows.

[0098] Three-week-old Arabidopsis WT, sng1 , brt1 After the stomata were opened, different concentrations of sinapinic acid (0.1 mM, 0.3 mM, 0.5 mM, 0.7 mM, 0.9 mM, and 1.1 mM) were added to the stomatal buffer (reference example 3). One group was placed under normal light as a control, and one group was placed under UV-B conditions. After 2 h, the samples were examined under a microscope and photographed.

[0099] The results are as follows Figure 4 As shown. The analysis shows that:

[0100] Under normal lighting conditions (such as Figure 4 A. Figure 4As shown in Figure 2, even for different mutant strains, appropriately low concentrations of sinapinic acid treatment (not exceeding 0.3 mM) can increase the degree of stomatal openness, and reach the maximum degree of openness under the condition of 0.3 mM treatment concentration; but when the sinapinic acid concentration exceeds 0.3 mM, stomatal closure activity begins to be induced, and shows obvious dose-dependent characteristics (i.e., the higher the sinapinic acid concentration, the higher the degree of stomatal closure); and the stomatal openness of different mutant strains is reduced to varying degrees compared with the wild type.

[0101] However, under UV-B treatment conditions ( Figure 4 C. Figure 4 D) Even for different mutant strains, the stomatal openness of the UV-B treatment group was lower than that of the wild type, that is, UV-B treatment induced stomatal closure activity; in this case, appropriately low concentrations of sinapinic acid treatment (not exceeding 0.5mM) could increase the stomatal openness and reach the maximum openness under the condition of 0.5mM treatment concentration; but when the sinapinic acid concentration exceeded 0.5mM, stomatal closure activity began to be induced and showed obvious dose-dependent characteristics (that is, the higher the sinapinic acid concentration, the higher the stomatal closure).

[0102] By comparing the stomatal opening under normal light treatment conditions and UV-B treatment conditions, it can be seen that increasing the content of sinapinic acid can supplement and offset the stomatal closing effect brought about by UV-B, but compared with the treatment under normal light conditions, the concentration of sinapinic acid treatment needs to be appropriately increased to achieve the same degree of stomatal opening.

[0103] Example 5

[0104] In order to further clarify the regulation pathway of stomatal activity under UV-B induction by sinapinic acid, the inventors investigated the H2O2, NO, Ca 2+ The content changes were measured and the specific experimental process is briefly described as follows.

[0105] 1. Changes in H2O2 content

[0106] The epidermal strips of leaves of three-week-old plants were floated in stomatal buffer and cultured under normal light for 2 h. Then, 100 units / mL catalase (CAT), 0.5 mM sinapinic acid, and 6 mM malic acid were added to the stomatal buffer, and then treated under normal light and UV-B conditions for 2 h. After the treatment, the epidermal strips were transferred to Tris-KCl buffer containing 50 μM H2DCFDA. After incubation at room temperature for 10 min under dark conditions, they were immediately rinsed with Tris-KCl buffer, and then the fluorescence image of H2O2 was observed and recorded using a fluorescence microscope. The results are shown in Figure 2. Figure 5 As shown. The analysis shows that:

[0107] Compared with normal light conditions, UV-B irradiation treatment significantly increased the H2O2 content, while the application of exogenous CAT, sinapinic acid or malic acid could significantly reduce the H2O2 content, that is, it can be considered that the absence of SM indirectly induced the increase of H2O2 content;

[0108] Under UV-B irradiation conditions, the H2O2 content in the wild type and mutant after treatment with sinapinic acid and malic acid had no significant difference compared with that after treatment with CAT, which also preliminarily confirmed that SM generated by the conversion of sinapinic acid and malic acid indirectly inhibited the generation of H2O2.

[0109] 2. Changes in NO Content

[0110] Referring to the above operation, the epidermal strips with open stomata of leaves of three-week-old plants were placed in MES-KCl containing 200 μM NO scavenger (c-PTIO), 0.5 mM sinapinic acid, and 6 mM malic acid, respectively, and were treated under normal light and UV-B radiation for 2 h. After the treatment, the epidermal strips were placed in Tris-KCl containing 10 μM DAF-2DA in the dark at room temperature for 30 min. The excess DAF-2DA on the sample was washed off with fresh Tris-KCl buffer, and the sample was quickly placed under a confocal microscope to observe and record NO fluorescence images. The results are shown in Figure 6 As shown. The analysis shows that:

[0111] Similar to the aforementioned changes in H2O2, UV-B irradiation treatment significantly increased NO accumulation compared with normal light conditions, while the application of exogenous c-PTIO, sinapinic acid, or malic acid could significantly reduce NO content, that is, it can be considered that the absence of SM indirectly induced an increase in NO content;

[0112] Under UV-B irradiation conditions, there was no significant difference in the NO content in the wild type and mutant after treatment with sinapinic acid and malic acid compared with that after treatment with c-PTIO. This also preliminarily confirmed that SM generated by the conversion of sinapinic acid and malic acid indirectly inhibited the accumulation and clearance of NO.

[0113] (III) Ca 2+ Content changes

[0114] It should be noted that YC3.6 (a Ca 2+ Fluorescent protein probes) to measure Ca 2+ concentration, and external stimulation will cause the cytoplasmic Ca 2+ oscillations, so before a specific experiment, to ensure the best measurement signal (i.e., the need for Ca 2+ The most suitable UV-B treatment duration must be determined first.

[0115] In the specific experiment, refer to the above operation, the epidermal strips were exposed to UV-B (0.5 W / m 2 ) were irradiated for 20 min, 40 min, and 60 min, and then Ca 2+ The results are as follows Figure 7 The results showed that when the treatment time reached 40 min, compared with the normal growth state, different strains (WT, sng1 , brt1 ) 2+ The concentrations all reached the relative highest value, so in subsequent experiments, the UV-B irradiation duration of 40 min was selected as the experimental condition.

[0116] Specific Ca 2+ When measuring the content change, refer to the above operation, put the lower epidermis of the torn mature rosette leaves into the stomatal buffer, first place it under normal light for 2 hours to promote stomatal opening, then transfer it to UV-B radiation for 40 minutes, and use laser confocal microscope to quickly capture images after pressing, and use ImageJ software to perform statistical analysis on the images. Figure 7 As shown. The analysis shows that:

[0117] Under normal light conditions, SM deletion mutants sng1 Intracellular Ca 2+ The concentration was significantly higher than that of wild type and brt1 Mutants, at the same time brt1 Mutant intracellular Ca 2+ The concentration was also higher than that of the wild type;

[0118] After UV-B treatment, intracellular Ca 2+ concentrations were significantly increased, and the SM deletion mutant sng1 Intracellular Ca 2+ The concentration increased significantly.

[0119] Based on the above results, it can be seen that under UV-B treatment conditions, Arabidopsis leaves will respond by closing stomata and reducing water loss rate, and in this process, plant leaves will regulate stomatal opening by changing the content of sinapinic acid metabolism; further, through Ca 2+ In other words, based on this regulatory pathway, the effects of UV-B irradiation on leaves can be regulated by exogenously applying sinapinic acid, malic acid, etc.

Claims

1. A method for regulating stomatal opening and closing of Arabidopsis thaliana, characterized in that: Under normal light conditions, 6 mM malic acid was used to regulate the opening and closing of stomatal pores in Arabidopsis leaves.

Images