CN113969289A - Application of sinapic acid in plant stomatal opening and closing regulation - Google Patents
Application of sinapic acid in plant stomatal opening and closing regulation Download PDFInfo
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- CN113969289A CN113969289A CN202111490900.5A CN202111490900A CN113969289A CN 113969289 A CN113969289 A CN 113969289A CN 202111490900 A CN202111490900 A CN 202111490900A CN 113969289 A CN113969289 A CN 113969289A
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- stomatal
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- sinapic acid
- plant
- regulation
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- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
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- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The application belongs to the technical field of plant physiology, and particularly relates to application of sinapic acid in plant stomata opening and closing regulation. Sinapic acid SA participates in regulating and controlling UV-B induced stomatal aperture regulation. UV-B radiation treatment induces second messenger H in leaves during UV-B induced stomatal activity2O2NO and cytoplasmic Ca2+(ii) an increased level of; whereas sinapinic acid SA reduces the second messenger H by conversion to sinapoyl malate SM and by accumulation of SM2O2NO and cytoplasmic Ca2+And thus regulate the stomatal activity. The present application is based on sinapinic acid anabolism related genesBRT1AndSNG1the inventor carries out preliminary research on the physiological performance of arabidopsis related leaves under the condition of UV-B radiation, particularly the open and close conditions of air holes by deleting mutant materials. Based on relevant conclusions, the method has very important theoretical value and application prospect for improving and enhancing the capability of resisting ultraviolet injury of crops in high latitude areas or summer.
Description
Technical Field
The application belongs to the technical field of plant physiology, and particularly relates to application of sinapic acid in plant stomata opening and closing regulation.
Background
In plants, sinapinate synthesized from sinapinic acid SA as a precursor generally refers to: sinapoylcholine (SC), Sinapoylglucose (SG), Sinapoylmalate (SM). Because sinaponate is directly related to synthesis of flavonoids, anthocyanidins, lignins and the like, research on the metabolic pathway of sinaponate has important significance on the research on regulation and control of plant physiological metabolism.
In the model plant Arabidopsis thaliana, because the sinapinate content is rich, the research on the sinapinate metabolic pathway is clear. It has been shown that the major key enzymes involved in sinapinate metabolism include: sinapic acid-glucosyltransferase, sinapoyl glucose malate transferase, and sinapoyl glucose choline transferase. Wherein sinapinic acid-glucosyltransferase catalyzes the synthesis of SG from sinapinic acid, which is a precursor for SM and SC synthesis. While sinapoyl glucose malate transferase (SMT) was originally found in carrot extracts and is responsible for catalyzing the formation of SM by SG. Sinapoyl glucosylcholinesterase (SCT) catalyzes the transfer of the sinapoate moiety of SG to a hydroxylated acceptor molecule to synthesize SCs.
Plants, which are sessile organisms on land requiring sunlight for growth and development, are inevitably exposed to Ultraviolet wavelengths (UV-R, 200-. Low dose UV-B as a signal stimulus, which has a direct effect on the morphogenesis and metabolism of the plant, also participates in inducing the photomorphogenic and UV-B acclimatizing responses of the plant, thereby making the plant tolerant to UV-B, such as: seedling de-yellowing, adaptation and tolerance of UV-B, day and night alternation, inhibition of occurrence of heat morphology, phototaxis, inhibition of shading reaction, influence on development of plant leaves, induction of stomatal movement, etc.; high doses of UV-B may cause DNA damage, induce the production of Reactive Oxygen Species (ROS), affect cell viability and integrity, and inhibit plant growth.
Generally, when plants are subjected to UV-B stress, a series of defense mechanisms can be rapidly started by the plants, including: ROS elimination, biosynthesis of flavonoids and derivatives thereof, biosynthesis of sinapinate, synthesis of pathogenic-related defense proteins, DNA repair mechanisms, and the like. It is therefore very important to understand the transduction mechanism of UV-B signals in cells.
Stomata is a microporous tissue structure on the plant epidermis and is composed of a pair of guard cells, and the appearance of stomata is an important mark for the evolution of plants from aquatic life to terrestrial life. During the physiological metabolism of the plant, the stomatal movement is regulated and controlled by regulating the turgor pressure of guard cells, and further the physiological activities such as respiration, photosynthesis, gas exchange and water evaporation in the transpiration process are directly influenced. It is thought that stomatal movement is associated with changes in vacuolar volume within guard cells, which in turn are affected by the accumulation of permeants in the cells, resulting in a reversible regulation. Therefore, it is important to ensure the dynamic changes of the vacuole of the cell in volume and structure to realize the movement of the stomata.
It is considered that the chemical properties of osmoregulators involved in stomatal movement depend on the species of plants and growth conditions, and most of the studies considered inorganic potassium ion (K)+) Chloride ion (Cl)-) And the organic solutes malic acid (Malate), sucrose (sucrose) are major influencing factors; wherein K+、Cl-Can be obtained from outside of plastid, and malic acid and sucrose can be degraded by starch or fixed with CO2The accumulation of these osmotic agents is obtained to reduce the osmotic potential of the guard cells and promote the swelling of the guard cells by water absorption, which leads to the opening of the stomata. Other signals responsive to pore movement are also numerous, such as soil moisture content, temperature, humidity, light intensity, UV-B, CO2And air pollutants and the like, so that the research on the regulation and control mechanism of stomatal movement is very important for the research on the physiological activities of plants.
UV-B radiation, although relatively small over the entire spectral range, has a large effect on higher plants. To mitigate the effects of UV-B radiation on plants, the plants themselves are metabolized to synthesize "sunscreens" such as the phenolics erucate, flavonoids, etc., which are transported to the upper cortex of the leaves for accumulation to prevent UV-B radiation from penetrating the upper cortex and thus mitigating the effects of UV radiation.
In the screening and research process of related mutants of Arabidopsis plants, it is found that the phenylpropanoid metabolic pathway can influence the sensitivity and tolerance of plants to UV-B by regulating and controlling the content change of sinapinate. Sinapic acid and derivatives belong to the intermediate products of phenylpropanoid secondary metabolic pathways and are identified as ultraviolet-absorbing hydroxycinnamic acid derivatives abundant in arabidopsis thaliana and other crucifers. Although sinapinate has been reported in the aspects of plant growth and development and resistance to external environmental stress, whether sinapinate metabolism is directly related to UV-B and the specific control mechanism thereof are still unknown. Therefore, the research on the relation between the sinaponate metabolism and the UV-B signal transduction pathway has important biological significance for disclosing the molecular mechanism of sinaponate for regulating and controlling the plant growth and development and responding to the adversity stress.
Disclosure of Invention
The application aims to provide the application of sinapic acid in regulation and control of plant stomata opening and closing, especially regulation and control of ultraviolet B (UV-B) induced plant stomata closing, so that a certain foundation is laid for improvement of related plant varieties.
The technical solution adopted in the present application is detailed as follows.
The sinapic acid is applied to the regulation of the opening and closing of plant stomata, and the sinapic acid participates in regulating and controlling the UV-B induced stomata opening regulation; specifically, the method comprises the following steps:
for Arabidopsis thaliana leaves, certain light intensity UV-B (0.5 Wm)-2) Under the condition of treatment for a certain radiation time (4 h), the temperature of the blades is increased, the opening degree of air holes is reduced, and the water loss rate is reduced;
for a specific degree of blade air hole opening, a certain light intensity UV-B (0.5 Wm)-2) And the sinapinate related mutant is treated for a certain radiation time (4 h)sng1Andbrt1the opening degree of the leaf stomata of the strain is smaller than that of a Wild Type (WT);
sng1the mutant is a mutant with the capability of losing the Sinapoyl Glucose (SG) of the leaves to be converted into sinapoyl gallate;
brt1the mutant is a mutant with the leaf blade lacking the capacity of converting sinapinic acid into SG;
further, the method comprises the following steps: UV-B radiation treatment induces second messenger H in leaves2O2NO and cytoplasmic Ca2+While also promoting the accumulation of sinapoyl malate, SM; in the case of exogenous administration of sinapinic acid SA (0.5 mM), or malic acid (6 mM), the content of endogenous sinapoyl malic acid SM can be increased, and stomatal closure can be promoted, so that the influence of UV-B on plant leaves can be adjusted.
Based on the above results, plant adaptability can be improved by applying erucic acid SA or malic acid exogenously when the plant leaves are possibly damaged by UV-B radiation; or regulating the expression level of sinapinic acid metabolism related genes (especially SM expression level) by using genetic engineering technology to improve the physiological activities of plants under the condition of UV radiation.
In the application, the physiological research of plant leaves under the UV-B radiation condition is taken as a research aim, and based on arabidopsis thaliana sinapinic acid anabolism related genes BRT1 and SNG1 deletion mutants as research materials, the inventor conducts research on the physiological performance of arabidopsis thaliana related leaves under the UV-B radiation condition, particularly the physiological performance of arabidopsis thaliana related leaves under the condition of stomatal pore opening and closingA preliminary study was conducted. The result shows that the sinapinate can participate and inhibit the stomatal closure process induced by UV-B irradiation, and the process is related to the second messenger H in plants2O2NO and cytoplasmic Ca2+The level content is relevant. Based on the theory, the method has very important theoretical value and application prospect for improving and enhancing the capability of resisting ultraviolet injury of crops in high latitude areas or summer.
Drawings
FIG. 1 shows the intensity and duration of UV-B screening; wherein:
a and B represent the air hole opening of the WT at different radiation intensities and times, respectively;
c is 0, 0.5, 0.7 and 0.9 w/m respectively2After UV-B treatment of guard cells for 2h, the cells were treated with 10. mu.g/mL FDA and 0.9 w/m of FDA in the dark2After the guard cells are treated by UV-B, the fluorescence images collected for 10min are treated by 5 mg/mL PI alone;
in each experiment, 3 skin strips were measured, and each experiment was repeated three times; the selected confocal laser images represent the same results for approximately 9 measurements, all fluorescence images being at a scale of 50 μm;
FIG. 2 shows the components WT,sng1、brt1Analyzing the water loss rate, the air hole opening degree and the blade surface temperature under normal light and UV-B conditions; wherein:
a is WT under normal growth conditions,sng1Andbrt1analyzing water loss of the in-vitro lotus throne leaves;
b is 0.5W/m2For WT under the UV-B condition,sng1Andbrt1analyzing the water loss of the in-vitro rosette leaves after different time durations of treatment; two sets of data were three independent replicates of P< 0.05，**P < 0.01；
C, placing the skin strip with open pores in MES buffer solution under normal light and 0.5W/m2Processing for 2h under the UV-B condition, wherein the scale of the picture in the C is 10 mu m;
d is the pore size of the statistical analysis skin bars, data are three independent replicates (n = 150), P < 0.05, P < 0.01;
e is WT,sng1Andbrt1the non-detached leaf of (1) is subjected to infrared imaging to obtain a false color image; wherein the scale bar for all images in Panel E is 2 cm; n = 50 non-isolated plants tested for each genotype;
f is a quantitative statistical analysis of the leaf temperature of the images shown in panel E, respectively, by means of the infrared software of FLIR Tools (version 6.4) × P < 0.01;
FIG. 3 is a graph showing that sinapic acid and malic acid treatment increased the accumulation of endogenous SM in Arabidopsis leaves; wherein:
a is the peak-appearing time and position of SM, SG and sinapic acid standard substance separated by HPLC;
b, quantitative statistics of isolated SM, data from three independent replicates,. P < 0.05;
FIG. 4 shows malic acid participating in regulating UV-B induced stomatal movement; wherein:
a and C are exogenous substances applied to WT with different concentrations of sinapinic acid,sng1、brt1Under normal light and dark conditions and 0.5W/m2Treating for 2h under the condition of UV-B, and the scale bar in all images is 10 mu m;
b and D are statistical analyses of the pore size of the epidermal strips in panels a and C, with data from three independent replicates (n = 150); P < 0.01;
FIG. 5 is an indirect induction of H by the absence of SM content in Arabidopsis thaliana leaves2O2Increased content and sinapic acid, malic acid to H2O2The cleaning effect of (1); wherein:
a is H in guard cells under normal light and UV-B conditions by applying CAT, sinapic acid and malic acid exogenously2O2Content-affected fluorescence images, all images having a scale bar of 5 μm;
b is the fluorescence intensity in panel a treated by 50 μ M H2DCFDA in the dark for 10min, data are three independent replicates (n = 150); P < 0.01;
FIG. 6 shows that the absence of SM content in Arabidopsis thaliana leaves indirectly induces the increase of NO content and the scavenging effect of sinapic acid and malic acid on NO; wherein:
a is a fluorescence image of influence of exogenous application of c-PTIO, sinapic acid and malic acid on NO content in guard cells under normal light and UV-B conditions, and the scale bar of all images is 5 mu m;
b is the fluorescence intensity from 10 μ M DAF-2DA in (a) treated in the dark for 30 min, data are three independent replicates (n = 150); P < 0.01;
FIG. 7 shows UV-B radiation vs. Ca in guard cells2+The effect of concentration; wherein:
a is selected from YC3.6, YC3.6sng1And YC3.6brt1The stomatal skin strip of (2) was placed in MES buffer at 0.5W/m2And treating for 40min under the UV-B condition. The scale of all images under the low power lens is 50 μm, and the scale of all images under the high power lens is 5 μm;
b is selected from YC3.6, YC3.6sng1And YC3.6brt1The stomatal skin strip of (2) was placed in MES buffer at 0.5W/m2Treating under UV-B condition for 20 min, 40min, and 60 min, and then treating Ca in guard cells2+Carrying out statistical analysis on the concentration;
c is Ca in the graph A2+Statistical analysis of concentrations, statistical data were three independent replicates (n = 150). times.P< 0.01。
Detailed Description
The present application is further illustrated by the following examples. Before describing the specific embodiments, a brief description will be given of some experimental background cases in the following embodiments.
Biological material
Arabidopsis thaliana wild mutantbrt1（AT3G21560）、sng1(AT 2G 22990) (the measurement results showed,brt1the content of sinapoyl malic acid in the material is reduced by 60-70% compared with that in normal plants;sng1materials accumulate an excess of sinapoyl glucose over normal plants), provided by professor Clint chappel, university of przege, usa;
experimental reagent:
sinapic acid, malic acid, and H2DCFDA (chemical reduction fluorescein dichlorofluorescein diacetate), sigma usa;
CAT (catalase), FDA (fluorescein diacetate), PI (inorganic phosphate), DMSO (dimethyl sulfoxide), MES (2-morpholinoethanesulfonic acid), and the like, available from Solarbio corporation;
acetonitrile and methanol, products of MREDA corporation;
experimental equipment:
high performance liquid chromatography Agilent 1260, a product of Agilent, usa.
Example 1
Since the main objective of the present application is to investigate the specific functions and actions of sinapinate in plants responding to UV-B irradiation, it is necessary to specifically define the UV-B irradiation conditions that the plants can adapt to. For this reason, this example was preliminarily clarified with respect to the UV-B irradiation intensity and irradiation time, taking the model plant Arabidopsis thaliana as an example. The specific experimental conditions are briefly described below.
The leaf surface skin strips of WT arabidopsis thaliana (Col-0, Columbia 0 type) which grows for three weeks are taken as experimental samples, the pore opening degree of the leaf surface skin strips is taken as an evaluation index, and the leaves are irradiated by UV-B with different intensities and different time lengths. The results are shown in FIG. 1. Specifically, the method comprises the following steps:
from the irradiation intensity viewpoint, as shown in FIG. 1A, the intensity of 0, 0.3 Wm is used-2、0.5 Wm-2、0.7 W m-2UV-B of (1) 0.5Wm after 4h of irradiation of the WT skin strips-2The degree of air hole closure of the skin strip of the test group is already more than 0.3W m-2The experimental groups showed significant statistical differences, taking into account that guard cells were at 0.5Wm-2The physiological state under irradiation is better than 0.7 Wm-2Test groups, therefore, the optimum irradiation intensity of UV-B was selected to be 0.5Wm-2。
From the irradiation time period perspective, the same irradiation intensity (0.5 Wm)-2) In the case of the WT skin strips, 0 h, 1 h, 2h, 3 h, and 4h irradiation treatment was performed, and as a result, as shown in fig. 1B, it can be seen that the gas hole opening was closed more significantly at 4h (or 2 h) than at the first several time periods, and therefore 4h (or 2 h) was selected as the appropriate treatment time.
To further clarify that the stomatal closure during the above experiment is not caused by the damage of guard cells due to UV-B irradiation, the inventors further performed experimental verification, and the specific experimental conditions are as follows.
Respectively collecting normal light (illumination intensity is 150 μmol m)2 s1）、0.5 W/m2、0.7 W/m2And 0.9W/m22h, respectively placing the Arabidopsis WT epidermis strips into Tris-KCl buffer solutions containing 10 ug/mL FDA (capable of penetrating cell membranes, hydrolyzing by intracellular esterase to generate polar compounds, gathering in cells, and showing green fluorescence when excited by blue light), 5 mg/mL PI (capable of penetrating damaged cell membranes, combining with DNA and RNA to form bright red fluorescence complexes visible in dead cell nuclei) and incubating at room temperature for 10min under dark conditions. Excess fluorochrome on the skin strips was washed with fresh Tris-KCl buffer and immediately visualized by sectioning.
The results are shown in FIG. 1C. It can be seen that: 0.5W/m2、0.7 W/m2The physiological state of the guard cells in the experimental group under the UV-B condition is almost not obviously different from that under the normal state; while guard cells under 0.9W/m 2 UV-B can be almost all marked by PI dye, and the activity of the guard cells is obviously reduced. These results show that: 0.5W/m2、0.7 W/m2The UV-B intensity of (1) does not cause extensive, non-specific damage to guard cells, but 0.9W/m2UV-B intensity radiation causes severe damage to guard cells and also illustrates the aforementioned 0.5W/m2 UV-B induced stomatal closure is not caused by damage to guard cells, but by other metabolic effects.
Example 2
Sinapoyl glucose malate transferase (SMT), originally found in carrot extracts, is encoded by the SNG1 gene (AT 2G 22990), and is responsible for catalyzing the formation of SM by SG, and belongs to one of the key synthetases of sinapoyl ester metabolites; while the conversion of SA (sinapic acid) to SG (sinapoyl glucose) in the metabolic pathway is responsible for the BRT1 (AT 3G 21560) gene.
To determine whether the pore closure is regulated by sinapinate metabolites under UV-B irradiation and whether the sinapinate metabolites content has the same physiological phenotype as other plant under UV-B irradiationIn connection with, the inventors deleted the SM mutant: (sng1) SG deletion mutant (A)brt1SM can be partially synthesized) as a research material and relevant experiments were performed, and the specific experimental procedures are summarized as follows.
Selecting those with good growth state and about three weeks (not bolting)sng1Mutant,brt1Treating the mutant plant under normal light for 2h to adapt to the environment, and then 0.5W/m2Irradiation treatment under UV-B conditions (with normal light treatment as a control), followed by removal of the root system, and water loss experiments were performed.
The specific operation of the water loss experiment is as follows:
firstly, setting the air-conditioning temperature of a water-loss platform room to be about 22 ℃ in advance for 1 hour; before the experiment begins, the materials are put into a room 30 min in advance to adapt to the environment;
then, putting the weighing paper into an electronic balance (note that the weighing paper is peeled off), then removing roots and soil of the whole arabidopsis thaliana which grows for about three weeks (the arabidopsis thaliana is not bolting), and putting the arabidopsis thaliana on the weighing paper (at least three times of each material are repeated, and the weight of the material is kept consistent as much as possible);
and finally, setting the time interval for collecting the weight to be 5 min, starting weighing, and recording related measurement data.
The specific experimental results are shown in fig. 2. Analysis shows that the water loss rate of mutant plants is obviously reduced in normal light treatment (figure 2A), and SM deletion mutant (A)sng1) The rate of water loss is slowest. Under different UV-B radiation duration conditions (figure 2B), the water loss rate of mutant plants is obviously reduced, and SM deletion mutants (A and B)sng1) The rate of water loss is also slowest. The rate of water loss is directly related to the degree of stomatal opening (i.e., the slower the rate of water loss, reflecting the lower the degree of stomatal opening), and thus, it is believed that sinapoyl metabolising plants are directly involved in stomatal opening regulation under UV-B treatment conditions, and that the importance of SM sinapoyl malate is higher than that of sinapoyl glucose SG.
Further, in order to specifically clarify the influence of UV-B irradiation on the stomatal aperture, WT of three weeks old and good growth state was selected,sng1、brt1Tearing off the epidermis of the fifth rosette leaf, brushing off mesophyll cells remained on the epidermis strip by using a writing brush, and then putting the epidermis strip into a fresh air hole buffer solution; after being placed under normal light for 2h, part of the test group was treated under normal light as a control, and the test group was subjected to a treatment of 0.5W/m2Carrying out UV-B irradiation treatment, and flaking and observing after 2 hours. The results are shown in FIGS. 2C and 2D.
It can be seen that under the UV-B radiation treatment conditions, compared to the control,sng1the mutant has a significant change in the degree of stomatal opening, andbrt1the change in stomatal aperture of the mutant was not significant enough. This result preliminarily indicates that SM is directly involved in stomatal opening and closing regulation in the case of UV-B irradiation induction.
In order to further evaluate the physiological change of plants under the condition of UV-B irradiation, the inventor further uses a Therma CAMSC1000 (FILR system) infrared camera to carry out infrared imaging analysis on the leaf surface temperature conditions of the wild type and the mutant (the plant sizes are all about three weeks old, the bolting is not carried out, and the processing method is the same as the method described above). The results are shown in FIGS. 2E and 2F.
Analysis can see that the concentration is 0.5W/m2 After 2h of treatment under the UV-B condition, the temperature of each treated leaf is increased compared with that of normal illumination, but the temperature of each treated leaf is increasedsng1The temperature of the leaves is obviously different. This result is consistent with the previous experiments, namely: compared with normal illumination, under the UV-B treatment condition,sng1the temperature of the blades is obviously increased, the opening degree of the air holes is obviously reduced, and the water loss rate is obviously reduced. In other words, sinapoyl malate SM directly participates in the series of physiological activities of the leaves under UV-B conditions.
Example 3
Based on example 2, in order to further clarify the relationship between the variation of SM content and the opening degree of pores under the UV-B induction, the inventors further performed related experiments by means of HPLC detection technology, and the specific experimental conditions are briefly described as follows.
In the experimental process, when detecting SM by using HPLC technology, the specific operations may refer to the following:
selecting the fifth rosette leaves of each treatment group, weighing 0.1 g of the fifth rosette leaves repeatedly, quickly placing the fifth rosette leaves in liquid nitrogen (at least three repeats of each genotype), quickly grinding the fifth rosette leaves into powder, then transferring the powder into a 1.5 mL centrifuge tube, adding 1 mL of extracting solution 50% methanol (v/v), and quickly oscillating and shaking the mixture uniformly;
heating and extracting the uniformly mixed sample at 65 ℃ for 30 min; after extraction, centrifuging at 11000 rpm for 10min at room temperature;
the supernatant was aspirated with a 1 mL syringe and filtered through a 0.22 μm organic filter into the cannula of a brown liquid phase vial to determine the content of the relevant substance.
In the HPLC detection process, the high performance liquid elution program and related detection parameters are referred to as follows:
XBridge C18 (4.6 × 150 mm) chromatography column; the column temperature is 25 ℃;
TABLE 1 elution procedure
The phase A is a water phase, the phase B is an organic phase, and the flow rate is 1 mL/min;
sampling for 1 time, wherein the sampling amount is 10 mu L;
the ultraviolet detection wavelength range is 200-550 nm, and the extraction peak wavelength is 330 nm.
When the content is measured, firstly, preparing standard sinapinate solutions with different concentration gradients, adopting the measuring program, taking the sinapinate content with different concentrations as an X axis and taking peak areas corresponding to the concentrations as a Y axis, making a standard curve of the sinapinate content, and then, quantifying the measuring result of a sample to be measured by comparing the standard curve with the measuring result of the sample to be measured.
In a specific experiment, three weeks old WT, and,sng1Mutant,brt1The fifth rosette leaf of the mutant plant floats in a stomatal buffer (MES-KOH, 10 mM MES, 50 mM KCl, 0.1 mM CaCl)2pH is adjusted to 6.15 by NaOH), and the mixture is placed under normal light for 2h to promote stomata to open, 0.5mM sinapinic acid and 6 mM malic acid are respectively and externally applied, and then the mixture is respectively placed under normal light and UV-B conditions for 2 h. After the treatment is finished, the HPLC detection method is referred toChanges in sinapinate content were measured (peak time refer to FIG. 3A).
The results are shown in FIG. 3 (FIG. 3B). Analysis can see that:
in normal wild type WT plants, sinapic acid, malic acid, and UV-B treatments all promoted an increase in the SM content inside the leaves. Under normal illumination, the best effect of increasing endogenous SM is achieved by applying malic acid from an external source; under the ultraviolet UV-B treatment condition, although the exogenous application of malic acid or sinapic acid can also increase the content of endogenous SM, the increase effect difference is not obvious; meanwhile, the effect of increasing the content of endogenous SM by ultraviolet UV-B treatment is also obviously better than the effect of applying malic acid or sinapic acid from an external source. In other words, under the condition of UV-B induction, the content of endogenous mustard acyl malic acid SM in the leaves can be obviously increased; meanwhile, the increase of the content of endogenous sinapoyl malic acid SM can be promoted under the condition of exogenously applying malic acid or sinapic acid.
And the mutantbrt1In this case, the SM content change was similar to that of normal wild-type plants, but it was clearly seen that the effect of exogenous malic acid was greater than that of exogenous sinapic acid. In other words, malic acid content variation dominates endogenous SM content regulation.
In conclusion, it can be preliminarily considered that: sinapinic acid SA is protected against UV-B radiation induced stomatal opening by further synthesis of SM; in the process, malic acid can also be converted into SM through a certain metabolic mechanism so as to participate in regulating and controlling the opening degree of stomata; furthermore, exogenously applied sinapic acid, malic acid, can also participate in UV-B induced stomatal movement by conversion to SM, even under UV-B treatment conditions.
Example 4
In the foregoing example 3, the inventors have preliminarily demonstrated that sinapic acid participates in stomatal opening activity under UV-B irradiation conditions, and further conducted related experiments in order to further clarify whether changes in sinapic acid content have an influence on regulation of related pneumatic activity. The specific experimental conditions are briefly described below.
Selecting three-week-old arabidopsis WT with consistent growth state,sng1、brt1After the air hole is opened, the air is blown inDifferent concentrations of sinapinic acid (0.1 mM, 0.3mM, 0.5mM, 0.7 mM, 0.9 mM, 1.1 mM) were added to the well buffer (reference example 3), one group was placed under normal light as a control, and one group was placed under UV-B conditions and photographed 2h later by microscopic examination.
The results are shown in FIG. 4. Analysis can see that:
under normal light conditions (as shown in FIGS. 4A and 4B), even for different mutant strains, the appropriate low sinapic acid treatment concentration (not more than 0.3 mM) can increase the stomatal opening degree, and the maximum opening degree is reached under the condition of the treatment concentration of 0.3 mM; but when the sinapinic acid concentration exceeds 0.3mM, the induction of stomatal closure activity is initiated and a significant dose-dependent profile is exhibited (i.e., the higher the sinapinic acid concentration, the higher the degree of stomatal closure); the stomata opening degree of different mutant strains is reduced in different degrees compared with the wild type.
Whereas under UV-B treatment conditions (FIG. 4C, FIG. 4D), even for different mutant lines, the stomata opening degree of the UV-B treated group was lower than that of the wild type, i.e., UV-B treatment induced stomata closing activity; in this case, a suitably low sinapic acid treatment concentration (no more than 0.5 mM) increases stomatal opening and reaches maximum patency at the treatment concentration of 0.5 mM; but when sinapinic acid concentration exceeded 0.5mM, it began to induce stomatal closure activity and exhibited a significant dose-dependent profile (i.e., higher sinapinic acid concentration, higher stomatal closure).
Comparing the pore opening under the normal illumination condition with that under the UV-B treatment condition, it can be seen that the pore closing effect brought by the UV-B can be supplemented and counteracted by increasing the sinapic acid content, but compared with the treatment under the normal illumination condition, the same pore opening degree can be reached by properly increasing the sinapic acid treatment concentration.
Example 5
To further clarify the regulation pathway of sinapinic acid for stomatal activity in the context of UV-B induction, the inventors have shown that H is commonly involved in the regulation pathway2O2、NO、Ca2+The content change condition is measured, and the specific experimental process is simpleThe following is introduced.
(1) H2O2Content change condition
Floating epidermal strips of three-week-old plant leaves in a pore buffer solution, culturing for 2H under normal light, adding 100 units/mL Catalase (CAT), 0.5mM sinapic acid and 6 mM malic acid into the pore buffer solution, treating for 2H under normal light and UV-B conditions, transferring the epidermal strips into Tris-KCl buffer solution containing 50 mu M H2DCFDA after treatment, incubating for 10min under dark condition at room temperature, immediately washing with Tris-KCl buffer solution, and observing and recording H by using a fluorescence microscope2O2The fluorescence image of (1). The results are shown in FIG. 5. Analysis can see that:
UV-B irradiation treatment significantly increased H compared to normal light conditions2O2The content of the active ingredient can be obviously reduced by using exogenous CAT, sinapic acid or malic acid2O2Content, that is, it can be said that deletion of SM indirectly induces H2O2An increase in the content;
while in wild type and mutant H after erucic acid and malic acid treatment under UV-B irradiation2O2The content of the compound (A) is not significantly different from that of the compound (A) after CAT treatment, and the fact that SM generated by converting sinapic acid and malic acid indirectly inhibits H can be preliminarily confirmed2O2And (4) generating.
(II) variation of NO content
Referring to the above procedure, the leaf open-stomached epidermal strips of three-week-old plants were placed in MES-KCl containing 200. mu.M NO scavenger (c-PTIO), 0.5mM sinapic acid, 6 mM malic acid, respectively, and treated under normal light and UV-B radiation for 2 hours, respectively, after which the epidermal strips were dark-treated in Tris-KCl containing 10. mu.M DAF-2DA at room temperature for 30 min. And (4) flushing redundant DAF-2DA on the sample by using fresh Tris-KCl buffer solution, and quickly placing the sample under a confocal microscope to observe and record an NO fluorescence image. The results are shown in FIG. 6. Analysis can see that:
and the foregoing H2O2The variation is similar, compared with the normal illumination condition, at UV-B irradiationThe accumulation of NO is obviously increased, and the content of NO can be obviously reduced by virtue of the application of exogenous c-PTIO, sinapic acid or malic acid, namely, the deletion of SM can be considered to indirectly induce the increase of the content of NO;
under the condition of UV-B irradiation, the content of NO in the wild type and the mutant after the sinapic acid and the malic acid are treated has NO significant difference compared with that after the c-PTIO treatment, and the fact that the SM generated by the conversion of the sinapic acid and the malic acid indirectly inhibits the accumulation and the elimination of NO can be preliminarily confirmed.
(III) Ca2+Content change condition
It should be noted that YC3.6 (a Ca) was used in the experiment2+Fluorescent protein probe) to determine Ca2+Concentration of cytosolic Ca in guard cells due to external stimulus2+Shaking, and therefore before a particular experiment, to ensure an optimal assay signal (i.e., Ca is required)2+Highest concentration) and therefore it is first necessary to determine the optimum UV-B treatment duration.
In a specific experiment, the skin strips were treated at UV-B (0.5W/m) with reference to the above procedure2) Respectively irradiating for 20 min, 40min and 60 min under the condition, and measuring Ca2+And (4) concentration. The results are shown in FIG. 7. The results showed that when the treatment time reached 40min, the different strains (WT, Cb) were grown as compared to the normal growth state,sng1、brt1) Ca in (1)2+The concentration reaches the relative highest value, so that the UV-B irradiation time is selected to be 40min as the experimental condition in the subsequent experiment.
Specifically Ca2+When the content change condition is measured, referring to the operation, the lower surface skin of the torn mature lotus throne leaves is placed in the stomatal buffer solution, the stomatal buffer solution is firstly placed under normal light for 2h to promote stomatal opening, then the mature lotus throne leaves are transferred to UV-B to be radiated for 40min, images are rapidly captured by using a laser confocal microscope after tabletting, and the images are subjected to statistical analysis by means of ImageJ software. The results are shown in FIG. 7. Analysis can see that:
under normal light conditions, SM deletion mutantssng1Intracellular Ca2+The concentration is obviously higher than that of the wild type andbrt1mutant, simultaneouslybrt1Mutant intracellular Ca2+The concentration is also higher than that of wild herbsShaping;
whereas after UV-B treatment, intracellular Ca, whether wild-type or mutant2+The concentration is obviously increased, and SM deletion mutantsng1Intracellular Ca2+The concentration is increased very significantly.
From the above results, it can be seen that under the UV-B treatment conditions, the arabidopsis leaves respond by closing the stomata and reducing the rate of water loss, and in the process, the plant leaves regulate the stomata opening by the metabolic content change of sinapic acid; further, by Ca2+、H2O2NO-mediated associated signaling pathways to reduce damage to the leaf. In other words, based on this regulatory pathway, the effect of UV-B irradiation on the leaves can be regulated by exogenous application of sinapic acid, malic acid, and the like.
Claims (4)
1. The application of sinapic acid in plant stomata opening and closing regulation is characterized in that sinapic acid SA participates in regulation of UV-B induced stomata opening regulation.
2. Use of sinapic acid for stomatal modulation in plants according to claim 1, characterized in that during UV-B induced stomatal activity, UV-B radiation treatment induces second messengers H in leaves2O2NO and cytoplasmic Ca2+(ii) an increased level of; while sinapinic acid SA reduces second messenger H by conversion to SM and by accumulation of SM2O2NO and cytoplasmic Ca2+And thus regulate the stomatal activity.
3. The use of sinapic acid in plant stomatal opening and closing regulation according to claim 1, wherein the effect of UV-B on plant leaves is regulated by promoting an increase in the content of endogenous sinapoyl malate SM under UV-B irradiation treatment conditions with exogenous application of sinapic acid SA 0.5mM or malate 6 mM.
4. The use of sinapic acid for the regulation of stomatal opening and closing in plants according to claim 1, wherein the physiological activities of plants under UV radiation are regulated by regulating the expression level of SNG1 or BRT1 gene by using genetic engineering techniques.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008150165A1 (en) * | 2007-06-05 | 2008-12-11 | Expressive Research B.V. | Resistance to abiotic stress in plants |
| WO2012167023A2 (en) * | 2011-06-02 | 2012-12-06 | Board Of Regents, The University Of Texas System | Regulation of stomatal apertures by apyrases and extracellular nucleotides |
| CN107148971A (en) * | 2017-04-28 | 2017-09-12 | 河南大学 | Application of the sinapic acid in terms of seed sprouting, root growth and seedling development |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008150165A1 (en) * | 2007-06-05 | 2008-12-11 | Expressive Research B.V. | Resistance to abiotic stress in plants |
| WO2012167023A2 (en) * | 2011-06-02 | 2012-12-06 | Board Of Regents, The University Of Texas System | Regulation of stomatal apertures by apyrases and extracellular nucleotides |
| CN107148971A (en) * | 2017-04-28 | 2017-09-12 | 河南大学 | Application of the sinapic acid in terms of seed sprouting, root growth and seedling development |
Non-Patent Citations (3)
| Title |
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| WEIQIANG LI ET AL.: "Sinapate Esters Mediate UV-B-Induced Stomatal Closure by Regulating Nitric Oxide, Hydrogen Peroxide, and Malate Accumulation in Arabidopsis thaliana", 《PLANT CELL PHYSIOL》, vol. 63, no. 12, pages 1890 * |
| 毕宝弟: "芥子酸代谢调控拟南芥种子萌发和气孔运动的机制", 《中国知网硕士电子期刊》, no. 6, pages 41 * |
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