CN104170823A - Small molecule compound for enhancing plant stress resistance - Google Patents

Abstract

The invention relates to a small molecular compound for enhancing plant stress resistance. It is revealed for the first time that the small molecule compound AM1 has similar physiological activities to ABA, which can significantly enhance the tolerance of plants to abiotic stresses widely present in the environment (common ones include drought, cold, salinity, osmotic pressure, heat, etc.), with High application value.

Description

A small molecular compound that enhances plant stress resistance

technical field

The invention belongs to the fields of biotechnology and botany; more specifically, the invention relates to a small molecular compound for enhancing plant stress resistance.

Background technique

Abscisic acid (ABA) is a key factor to balance the metabolism of plant endogenous hormones and related growth active substances. It has the ability to promote plants to absorb water and fertilizer in balance and coordinate internal metabolism. It can effectively regulate plant root/shoot and vegetative growth It plays an important role in improving the quality and yield of crops. By applying ABA, the application amount of chemical pesticides can be reduced, and it has important physiological activity and application value in many aspects such as improving the quality of agricultural products. In addition, exogenous ABA can cause the rapid closure of leaf stomata and inhibit transpiration, which can be used to preserve flowers or prevent wilting during the transportation of crop seedlings; ABA can also control flower bud differentiation and regulate flowering. It has great application value in flower gardening. ABA is a pure natural plant growth regulator, which can improve the growth quality, seed setting rate and quality of crops in adverse growth environments such as low temperature, drought, cold spring, salinity, pests and diseases, increase the yield per unit area of low- and medium-yielding fields, and reduce chemical pesticides Dosage. ABA can also be widely used in greening construction such as urban lawns and gardens, as well as in water-saving agriculture and facility agriculture in the western region, as well as restoration and reconstruction of ecological vegetation, which is of great significance to the development of China's agricultural industrialization. However, the current bottleneck lies in the fact that pure (+)-ABA with natural activity is unstable and difficult to synthesize artificially, and the production cost is extremely high. Due to the high price and the difference in activity, ABA has not been widely used in agricultural production. Scientists from all over the world are looking for ways to produce natural ABA cheaply.

After decades of research on ABA signaling, protein phosphatases 2C (Protein Phosphatases2C, PP2Cs) and SNF1-related kinase subfamily 2 (Subfamily2 of the SNF1-related kinases, SnRK2kinases) have been identified as downstream signaling hubs of ABA receptors. Recently, a family of START proteins comprising 14 members was identified as ABA receptors, named PYR1 (Pyrabactin Resistance 1)/PYR1-like (PYL) receptors (for simplicity, hereinafter referred to as PYL receptors). ABA binding to the PYL receptor enhances the binding ability of the latter to certain specific PP2C protein phosphatases (including ABI1, ABI2 and HAB1, etc.), and at the same time inhibits the activity of these PP2C protein phosphatases, and then activates the downstream SnRK2 protein kinase , thereby activating the subsequent stress response. Recent structural studies have shown that the PYL receptor and the PP2C protein phosphatase form a co-receptor for ABA and identified a conserved mechanism associated with ABA sensing and signaling.

Park, S.Y.et al.<Abscisic acid inhibits type2C protein phosphatases via thePYR/PYL family of START proteins>Science324, 1068-71 (2009) mentioned that the small molecule compound pyrabactin has the function similar to ABA, but its physiological activity is lower than that of ABA An order of magnitude, and can only bind to a few PYL receptors, it mainly acts as an ABA analogue in the seed germination stage, and cannot enhance the drought tolerance of plants in seedling and mature stages, and has low application value in agricultural production.

Therefore, it is necessary to further develop ABA substitute products in this field. Once such a small molecular compound is found, the economic, social and environmental benefits produced by it will be very significant.

Contents of the invention

The object of the present invention is to provide cheap natural abscisic acid substitutes with abscisic acid (Abscisic Acid, ABA) activity.

In the first aspect of the present invention, there is provided a use of a compound of formula (I) or a salt thereof for enhancing plant stress resistance or for preparing an agricultural preparation for enhancing plant stress resistance,

In another preferred example, the stress resistance is abiotic stress resistance involving ABA.

In another preferred example, the abiotic stress resistance involved in ABA includes, but is not limited to: drought tolerance, cold tolerance, salt-alkali tolerance or osmotic pressure tolerance, etc.

In another preferred example, the plants include: plants containing ABA receptors of the PYR/PYL family.

In another preferred example, the plants include: economic crops and food crops.

In another preferred example, the plants include but are not limited to land plants.

In another preferred example, the plants include but are not limited to: Brassicaceae plants (including Arabidopsis or Brassica, such as Arabidopsis thaliana, Chinese cabbage, Chinese cabbage), Solanaceae plants (including Nicotiana , such as tobacco), grasses (including Oryza, such as rice).

In another preferred example, the plants include, but are not limited to: Arabidopsis thaliana, cabbage, rapeseed, tobacco, tomato, pepper, wheat, rice, barley, corn, sorghum, oats, rye, sugarcane, cotton, soybean , beets, sunflowers, etc.

In another preferred embodiment, the compound of formula (I) or its salt is also used for: promoting the interaction between ABA receptor PYL protein and multiple PP2C protein phosphatases; or weakening the transpiration of leaves.

In another aspect of the present invention, there is provided a method for enhancing plant stress resistance, applying a compound of formula (I) or a salt thereof to plants:

In a preferred embodiment, an effective amount of the compound of formula (I) or a salt thereof is applied to the plants.

In another preferred example, the effective amount is 1-500 μM; preferably 5-200 μM; more preferably 20-100 μM;

In another preferred example, the stress resistance is abiotic stress resistance involving ABA.

In another preferred example, the abiotic stress resistance involved in ABA is at least one of drought tolerance, cold tolerance, salt-alkali tolerance, osmotic pressure tolerance or heat tolerance.

In another preferred embodiment, the plant is a plant containing ABA (abscisic acid) receptors of the PYR/PYL family.

In another preferred example, the plants include, but are not limited to: Brassicaceae plants, Solanaceae plants, Gramineae plants.

In another preferred example, the plants include, but are not limited to: Arabidopsis thaliana, tobacco, cabbage, rape, tomato, pepper, wheat, rice, barley, corn, sorghum, oats, rye, sugar cane, cotton, soybean , Beets, Sunflowers.

In another aspect of the present invention, there is provided an agricultural formulation comprising: an effective amount of a compound of formula (I) or a salt thereof; and an agriculturally acceptable carrier;

In a preferred example, in the agricultural formulation, the content of the compound of formula (I) or its salt in the agricultural formulation is 1-500 μM; preferably 5-200 μM; more preferably 20-100 μM; and/ or

In another preferred example, the agricultural preparation is a concentrated preparation, wherein the content of the compound of formula (I) is 1-1000 mM; preferably 10-500 mM, such as 50 mmM, 100 mM, 200 mM, 400 mM.

The agriculturally acceptable carrier includes (but not limited to): surfactants (such as Tween-20, Silwet-77, preferably with a content of 0.02% (v/v)).

Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein.

Description of drawings

Figure 1. AM1 is a broad-spectrum and highly effective PYL receptor agonist.

a, Two-dimensional structures of AM1 and ABA.

B. Activity experiment of protein phosphatase HAB1. AM1 at the working concentration of ABA can significantly inhibit the latter's phosphatase activity by promoting the binding of multiple PYL receptors (PYR1, PYL1, PYL2, PYL3, PYL5, PYL7) to protein phosphatase HAB1.

c-e, AlphaScreen experiments verify the interaction between PYL receptors and protein phosphatase HAB1. The values in the table below the figure are EC50 values.

Figure 2. Yeast two-hybrid experiments confirm that AM1 can promote the interaction between the PYL receptor family and multiple PP2C protein phosphatases.

a. Yeast growth in medium without isoleucine and tryptophan (-Leu/-Trp).

b. Growth of yeast on DMSO control medium without isoleucine, tryptophan and histidine (-Leu/-Trp/-His).

c. Growth of yeast on a medium containing 2 μM ABA without isoleucine, tryptophan and histidine (-Leu/-Trp/-His).

d, Yeast growth on the medium containing 2 μM AM1 without isoleucine, tryptophan and histidine (-Leu/-Trp/-His).

Figure 3. Crystal structure of PYL2-AM1-HAB1 complex.

a, Schematic diagram of the three-dimensional structure of the complex.

b, Local schematic diagram of the two-dimensional structure of the complex.

Figure 4. AM1 treatment can induce the expression of stress-related genes in plants.

a, Stress-related genes in wild-type Arabidopsis seedling plants were both up-regulated after 50 μM ABA and AM1 treatment, while the up-regulated levels were significantly reduced in PYL receptor triple mutants.

b. The results of RNA-seq showed that compared with the DMSO-treated control group, there were 4529 and 4454 genes in the ABA and AM1-treated sample groups that were expressed differently, and 3644 genes were expressed in both ABA- and AM1-treated sample groups. There are differences in expression, accounting for 80.5% and 81.8% of the two, respectively.

c. Among the above 3644 genes, the most significant differences are the genes related to plant response to drought, cold, osmotic pressure and salt stress.

Figure 5. AM1 can significantly inhibit seed germination.

a) Photographs of PYL receptor triple mutant strains (pyr1; pyl1; pyl4) 4 days after germination. All the three PYL receptor mutants opened cotyledons, and the wild-type plants in the DMSO control group all opened the cotyledons, while most of the wild-type plants in the experimental group containing 1 μM ABA and AM1 only grew radicles, and the germination was significantly inhibited.

b) The germination rate statistics of PYL receptor triple mutant strains (pyr1; pyl1; pyl4) 4 days after germination, with the cotyledon opening as the germination standard.

Figure 6. AM1 enhances drought tolerance of plants by reducing transpiration.

a, Soil drought experiment, two-week-old wild-type Arabidopsis thaliana stopped water supply and sprayed AM1 or ABA solution on the leaves every other day. After 2 weeks, most of the plants sprayed with the DMSO control solution were withered and could not recover after water supply was restored, while the plants sprayed with AM1 or ABA remained green and quickly recovered after one day of water supply.

b. The survival rate of wild-type Arabidopsis thaliana after 2 weeks of drought is calculated according to the survival rate after 1 day of water supply recovery.

c, the water loss rate of leaves of wild-type plants in four weeks.

Detailed ways

The present invention discloses a natural abscisic acid substitute with abscisic acid (Abscisic Acid, ABA) activity - abscisic acid analog 1 (ABA mimic1, AM1), the small molecule compound AM1 of the present invention has a similar physiological activity to ABA , can bind to most of the PYL receptors, play the role of ABA analogs in the germination and growth stages, and can significantly enhance the stress resistance of plants (such as drought tolerance, cold resistance, etc.), due to the vast majority of food crops and economic All crops contain PYR/PYL family ABA receptors, and the compound has high application value. The invention solves the defects that the natural ABA has low chemical stability, high production cost and is difficult to apply to agricultural production.

Compounds and their uses

The present invention at first provides a kind of compound as shown in structural formula (I):

The present invention also includes isomers, racemates, salts, hydrates or precursors of the above-mentioned compounds, as long as they also have the effect of enhancing plant stress resistance (preferably abiotic stress resistance, such as drought tolerance) effect. The "salt" refers to the salt formed by the reaction of the compound with inorganic acid, organic acid, alkali metal or alkaline earth metal. These salts include (but are not limited to): (1) salts formed with inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid; (2) salts formed with organic acids such as acetic acid, oxalic acid, succinic acid, tartaric acid , methanesulfonic acid, maleic acid, or arginine. Other salts include those formed with alkali or alkaline earth metals such as sodium, potassium, calcium or magnesium, in the form of esters, carbamates, or other conventional "precursors". Compounds possess one or more asymmetric centers. These compounds may thus exist as racemic mixtures, individual enantiomers, individual diastereoisomers, diastereomeric mixtures, cis or trans isomers.

The "biological stress" refers to the stress exerted by some organisms (such as pests, pathogens). The "abiotic stress" is the environmental impact caused by abiotic, such as: drought, cold, salinity, osmotic pressure, heat, flood, mineral deficiency and unfavorable pH, etc.

The "precursor of the compound" means that after being applied to the plant, the precursor of the compound undergoes metabolism or chemical reaction in the plant to be converted into a compound of the structural formula (I), or a compound of the chemical structural formula (I). composition of salts or solutions.

Those skilled in the art should understand that after knowing the structure of the compound of the present invention, the compound of the present invention can be obtained by various methods well known in the art and using known raw materials, such as chemical synthesis or from organisms (such as animals or plants) ), these methods are included in the present invention.

Synthetic chemical modification, protected functional group methodology (protection or deprotection) is very helpful for the synthesis of application compounds, and is a well-known technique in the prior art, such as R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); TWGreene and PGM Wuts, Protective Groups in Organic Synthesis, ^3rd Ed., John Wiley and Sons (1999); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed. ., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) are disclosed.

A kind of method of synthetic formula (I) compound is as follows:

Based on the inventor's new discovery, the present invention provides the purposes of the compound shown in formula (I) or its isomer, racemate, salt, hydrate or precursor, they should act on ABA receptor, should It has similar purposes with ABA, preferably, it can be used to enhance plant stress resistance (such as drought tolerance, cold resistance, salt-alkali tolerance, osmotic pressure resistance or/and heat resistance); or for preparing drought-resistant, cold-resistant , plants with enhanced salt-alkali tolerance, osmotic pressure tolerance or/and heat tolerance.

In the present invention, there is no particular limitation on the plants (or crops) applicable to the present invention, as long as they have PYR/PYL family ABA receptors and their downstream signaling pathways, such as various crops, floral plants, or forestry plants. Said plant can be, for example (not limited to): dicotyledonous plant, monocotyledonous plant or gymnosperm plant. More specifically, said plants include (but are not limited to): wheat, barley, rye, rice, corn, sorghum, sugar beet, apple, pear, plum, peach, apricot, cherry, strawberry, raspberry, blackberry, bean , lentils, peas, soybeans, canola, mustard, poppies, olean, sunflowers, coconuts, castor oil plants, cocoa beans, peanuts, gourds, cucumbers, watermelons, cotton, flax, hemp, jute, citrus, lemons, grapes Grapefruit, spinach, licorice, asparagus, cabbage, Chinese cabbage, bok choy, carrot, onion, potato, tomato, green pepper, avocado, cinnamon, camphor, tobacco, nuts, coffee, eggplant, sugar cane, tea, pepper, grapevine , oyster grass, bananas, natural rubber trees and ornamental plants.

As a preferred mode, the "plants" include but are not limited to: Brassicaceae plants (including Arabidopsis or Brassica, such as Arabidopsis thaliana, Chinese cabbage, Chinese cabbage), Solanaceae plants (including Nicotiana, such as Nicotiana), Poaceae plants (including Oryza, such as rice).

Agricultural formulations

As used herein, the term "agricultural formulation of the present invention" is generally an agricultural plant growth regulator, which contains a compound of formula (I) or its isomer, racemate, salt, hydrate or precursor as an agent for enhancing plant stress resistance. properties (such as drought tolerance) active ingredient; and agriculturally acceptable carrier or excipient.

In the present invention, the term "comprising" means that various components can be used together in the mixture or composition of the present invention. Accordingly, the terms "consisting essentially of" and "consisting of" are included in the term "comprising".

In the context of the present invention, an "agriculturally acceptable" ingredient is a substance with a reasonable benefit/risk ratio that is suitable for use on plants without undue adverse side effects such as toxicity, irritation and allergic reactions.

In the present invention, "agriculturally acceptable carrier" is an agrochemically acceptable carrier for delivering the compound of formula (I) or its isomer, racemate, salt, hydrate or precursor of the present invention to plants. Accepted solvents, suspending agents or excipients. The carrier can be liquid or solid. Agriculturally acceptable carriers suitable for use in the present invention include, but are not limited to: water, buffers, DMSO, surfactants such as Tween-20, and combinations thereof. Any agriculturally suitable carrier known to those skilled in the art is acceptable and may be used in the present invention. Optionally, the formulation may also include at least one surfactant, herbicide, fungicide, insecticide or fertilizer.

The present invention also provides a method for preparing the agricultural formulation, including using the compound represented by formula (I). An effective amount of the compound of formula (I) can be mixed with an agriculturally acceptable carrier to obtain the agricultural preparation of the present invention, and the content of the active ingredient in the composition can be, for example, 1-500 μM; preferably 5-200 μM; more preferably The ground is 20-100 μM. The agricultural preparation can also be a concentrated preparation, wherein the content of the compound of formula (I) is 1-1000mM; preferably 10-500mM, such as 50mM, 100mM, 200mM, 400mM.

The dosage form of the agricultural preparation of the present invention can be various, as long as it can make the active ingredient reach the plant effectively. From the standpoint of ease of preparation and application, the preferred agricultural formulation is a spray or solution formulation.

The present invention also provides a method for enhancing plant stress resistance (such as drought tolerance), comprising the steps of: applying a compound of formula (I) or its isomer, racemate, salt, hydrate or precursor to the plant . The active ingredient is administered in an effective amount.

Application can be by various known methods, for example, by spraying, spraying, dusting or broadcasting the composition on plant foliage, propagating material, or brushing or splashing or otherwise bringing the plant into contact with the composition, in the case of seeds , by coating, wrapping or otherwise treating the seeds. As an alternative to treating plants or seeds directly before planting, the formulations according to the invention can also be introduced into the soil or other medium in which the seeds are to be sown. In some embodiments, a carrier may also be used. The carrier can be solid or liquid as mentioned above.

The main advantages of the present invention are:

The small molecular compound contained in the invention can accurately simulate the action of the plant hormone abscisic acid (ABA) in plants, and significantly enhance the stress resistance (such as drought resistance, cold resistance, etc.) of plants. The advantages are as follows:

1) Use less. The present invention only needs to be used when plants encounter drought, and does not need to be used throughout the entire growth cycle;

2) Easy to use. It is soluble in water at the working concentration and can be directly used with existing agricultural sprinklers;

3) Strong specificity and small side effects. After spraying, it mainly enhances the stress resistance of plants (such as drought tolerance, cold resistance), and has no effect on other physiological functions of plants;

4) Compared with abscisic acid, the chemical synthesis of the present invention is simple and convenient, and the cost is low; and

5) Does not involve any transgenic technology.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Experimental methods not indicating specific conditions in the following examples are usually according to conventional conditions such as edited by J. Sambrook et al., Molecular Cloning Experiment Guide, Third Edition, Science Press, 2002, or according to the conditions described in the manufacturer suggested conditions.

I. Materials and methods

plant growth

The model plant Arabidopsis thaliana used in the experiment includes the Columbia (Col-0) ecotype and the PYL triple mutant pyr;pyl1;pyl4 based on the Col-0 ecotype. The practical tobacco used in the experiment was Nicotiana benthamiana.

The growth temperature of Arabidopsis was 22°C, the growth temperature of tobacco was 24°C, and the light intensity was 75 μmol m ^–2 s ^–1 . The latter is 8 hours light/16 hours dark.

Unless otherwise specified, the plant growth medium used in the experiment was 1/2MS (Murashige and Skoog) solid medium containing 1% (w/v) sucrose and 0.6% (w/v) agar.

The compounds used in the experiment were purchased from Life Chemical Inc, dissolved in DMSO, stored at a concentration of 100 mM, and diluted with water to the corresponding concentration when used.

protein expression and purification

Arabidopsis gene PYL1 (amino acid sequence 36-211) with 6×His and SUMO double tag sequence, PYL2 (amino acid sequence 14-188) and Arabidopsis gene HAB1 (amino acid sequence 172-511) with Biotin tag sequence For the construction method of the recombinant plasmid, please refer to "A gate-latch-lock mechanism for hormonesignalling by abscisic acid receptors" (<Nature>Vol462, 3December2009), other Arabidopsis PYL genes with 6×His and SUMO double tag sequences, including PYR1 , PYL3, PYL4, PYL5, PYL6, PYL7, PYL8, PYL9 and PYL10 (the above 9 genes are all gene coding sequences), the construction method of the recombinant plasmid is the same as that of PYL1/PYL2.

Transfer the above-mentioned recombinant plasmid into competent cells BL21(DE3), inoculate into 200ml LB liquid medium containing Amp resistance, and culture at 200rpm at 37°C overnight; inoculate into 2L LB containing Amp resistance at 1:50-1:100 Expand culture in liquid medium, culture at 200 rpm at 37°C for 3-4 hours, and culture at low temperature at 16°C until about OD ₆₀₀ =0.8-1.0. The recombinant plasmid of PYR1 or PYL1 or PYL2 with 6×His and SUMO double tag sequence was induced overnight with 100 μM IPTG, while the HAB1 recombinant plasmid with Biotin tag sequence was induced simultaneously with 100 μM IPTG and 40 μM biotin.

The bacterial liquid after induction for 16 hours was collected by centrifugation in a low-speed large-capacity centrifuge, and centrifuged at 4000 rpm for 20 min at 4°C. Resuspend the bacteria with 50ml of extraction buffer (containing 20mM Tris, pH8.0, 200mM NaCl and 10% (v/v) glycerol) per 2L of bacterial liquid, and then perform 1000Pa pressure disruption at 4°C for 3-5 times. The broken cells were subjected to ultracentrifugation at 16,000 rpm for 30 min, repeated twice, and the supernatant was collected and passed through an affinity chromatography column.

For the PYR/PYL protein with 6×His and SUMO double tag sequences, select a 50ml affinity chromatography Ni column (50ml Ni-NTA column, purchased from GE); first use 10% buffer B (containing 20mM Tris, pH8 .0, 200mM NaCl, 500mM imidazole and 10% glycerol) equilibrated with 600ml, then eluted with 200ml50% buffer B, and finally eluted with 100ml100% buffer B; the protein used for crystal analysis and ulp1 enzyme were 1000:1 The molar ratio was mixed for enzyme digestion and dialyzed overnight; the digested protein was passed through an affinity chromatography Ni column again; the collected solution was passed through HiLoad26 with elution solution (containing 25mM Tris, pH 8.0, 200mM Mammonium acetate, 1mM dithiotreitol and 1mM EDTA) /60Superdex200 gel filtration column (purchased from GE Company) for further separation and purification of protein.

For HAB1 protein with Biotin tag sequence, pass through 50ml MBP affinity chromatography column (purchased from GE); first use 10% buffer C (containing 20mM Tris, pH8.0, 200mM NaCl, 10mM Maltose and 10% glycerol) Equilibrate 600ml, then elute with 200ml50% buffer C, and finally elute with 100ml100% buffer C; collect the solution (containing 20mM Tris, pH8.0, 200mM NaCl and 10% glycerol) through HiLoad26/60Superdex200 Gel filtration column for further separation and purification of protein.

Protein Crystal Analysis

Before crystallization, the digested PYL2 and HAB1 proteins were mixed with the compound at a molar ratio of 1:1:5, concentrated to 6 mg/ml for crystallization. Use the hanging drop method to point the crystals, mix 1 μl complex protein and 1 μl wellbuffer to incubate at room temperature (20°C); the well buffer of PYL2-AM1-HAB1 complex crystals contains 0.1M Succinic acid and 15% PEG3350, PYL2-AM2/AM3- The well buffer of HAB1 (unless otherwise stated, "/" means "or") complex crystals contains 0.2M ammonium sulfate, 0.1M BIS-Tris, pH6.0, 25% PEG3350. Crystals can be seen after one day and grow to 100-120μm in about 3-4 days. The crystals were subjected to X-ray diffraction at the Shanghai Synchrotron Radiation Light Source and the diffraction data were collected. The collected data were processed by HKL2000, and the structure of the complex was analyzed according to the published structural model of the PYR/PYL receptor.

AlphaScreen experiment

AlphaScreen kit was purchased from Perkin Elmer, 100μl experimental system contained 10×buffer (50mM MOPS, pH7.4, 50mM NaF, 50mM CHAPS, 0.1mg/ml bovine serummalbumin), each 100nM HAB1 with Biotin tag sequence and 6× PYR1/PYL1/PYL2 protein of His and SUMO double tag sequence, 100μM (+)-ABA/pyrabactin/AM1/AM2, 5ug/ml donor beads and acceptor beads (Perkin Elmer), after incubation at room temperature for 1.5 hours in the dark, place in The Envision plate reader (purchased from Perkin Elmer) was read according to the set AlphaScreen program.

HAB1 Phosphatase Activity Assay

The reaction system contained 50mM imidazole, pH 7.2, 5mM MgCl ₂ , 0.1% β-mercaptoethanol, 0.5μgml ^-1 BSA, 100nM HAB1 protein with Biotin tag sequence, 500nM PYL receptor with 6×His-SUMO double tag sequence The body protein and the corresponding concentration of the compound were incubated at room temperature for 30 minutes, and the phosphorylated polypeptide containing 11 amino acids was added as the substrate to continue the reaction for 20 minutes. The phosphorylated polypeptide is the 170-180 amino acid of the SnRK2. Phosphorylated serine (HSQPK _p STVGTP, purchased from GenScript) is a known HAB1 target site. After 20 minutes, a chromogenic reagent (purchased from BioVision) was added, and the absorbance at a wavelength of 650 nm was read with a microplate reader.

Yeast two-hybrid experiment

14 PYL receptor proteins (PYR1, PYL1-13) of Arabidopsis thaliana were amplified by PCR method, respectively cloned into the multiple cloning site of pGADT7 (purchased from Clontech) with corresponding restriction endonucleases, and amplified The coding regions (CDS) of four PP2C phosphatases (HAB1, HAB2, ABI1, PP2CA) were cut with corresponding restriction enzymes and cloned into the multiple cloning site of pBD-GAL4Cam vector (purchased from Stratagene). After the vector was identified by sequencing, it was co-transformed into the yeast strain AH109 (purchased from Clontech) according to the corresponding PYL-PP2C combination (refer to Figure 2), and placed on SD medium (-L/-T) without Leu and Trp, 30 Cultivate for 2 days. Take yeast plaques in good growth state and dilute to OD ₆₀₀ =0.3, then dilute 5 times and 25 times with water respectively, take 1 μl each in 1) SD medium without Leu and Trp(-L/-T) (as a positive Control), 2) SD culture without Leu, Trp and His(-L/-T/-H) and containing 1mM3-AT and 2μM(+)-ABA/AM1/AM2 or 0.05%DMSO (as a negative control) cultured at 30°C for another 2 days and then photographed.

Table 1, PCR primers (the underlined part is the restriction endonuclease site)

Gene Expression Analysis

(1) RNA extraction

Col-0 Arabidopsis seedlings grown for 10 days on 1/2 MS solid medium were treated with 50 μM (+)-ABA/AM1/AM2 or 0.05% DMSO (control) for 6 hours, using TRIzol reagent (purchased from Invitrogen) according to the instructions Total RNA was extracted, DNase (purchased from Qiagen) without RNase was added during the extraction process to remove residual genomic DNA, and the concentration of the obtained total RNA was determined by NanoDrop spectrophotometer (purchased from Thermo Scientific) for reverse transcription or RNA sequencing.

(2) Reverse transcription and fluorescent quantitative PCR

The reverse transcription reaction was carried out using the TransScript RT kit (purchased from Invitrogen) according to its experimental procedure. 2 μg of total RNA was taken from each sample for reverse transcription, and the obtained cDNA was used as a template for fluorescent quantitative PCR. The fluorescent quantitative PCR system used the SYBR Premix Ex Taq ^TM II kit (purchased from TaKaRa) and configured according to its instructions, and used the CFX96 fluorescent quantitative PCR instrument (purchased from BIO-RAD) to perform the reaction. Three biological replicates were taken for each treatment and two experimental replicates were performed, and the ACT7 gene was used as an internal reference.

Table 2. Fluorescent quantitative PCR primers

(3) Transcriptome sequencing and data analysis

Transcriptome sequencing was completed by Meiji Company using Illumina's HiSeq2000 high-throughput sequencer. Two biological replicates were sent for each treatment, and each sample exceeded 10M effective data (clean reads, average length >49bp). The differentially expressed genes of 50 μM (+)-ABA/AM1/AM2 treatment relative to DMSO treatment were obtained using SAM, and the expression correlation between different samples was represented by Spearman correlation coefficient.

Seed germination, soil drought and leaf dehydration experiments in vitro

(1) Germination of seeds

Arabidopsis Col-0 ecotype and PYL receptor triple deletion mutant (pyr1; pyl1; pyl4) (see "AbscisicAcid Inhibits Type2C Protein Phosphatases via the PYR/PYL Family of STARTProteins", "Science" Vol324, 22May2009) After being sterilized with NaClO, they were placed in a refrigerator at 4°C for vernalization for 3 days, and then seeded on 1/2 MS solid medium containing 1 μM (+)-ABA/AM1/AM2 or 0.05% DMSO. Two strains were sown simultaneously on each medium with a diameter of 6 cm, 15 seeds were sown for each strain, and 4 replicates were set for each compound. The medium was cultured under long-day light at 22°C, and the opening of the cotyledons was used as a sign of germination, and the germination rate was counted every day.

(2) Soil drought experiment

Arabidopsis Col-0 ecotype seeds were sterilized with NaClO and placed in a 4°C refrigerator for vernalization for 3 days, then sowed on 1/2MS solid medium, and after 4 days of growth, select seedlings with good growth and uniform size and move them into 8× 7 x 6cm ³ flower pots. Each flower pot is equipped with the same weight of soil and moved into the same number of plants (six plants) to reduce experimental errors. All flower pots are placed in 22 ° C for short-day cultivation, and watering is stopped after ten days for drought treatment. A solution containing 0.02% tween-20 and 50 μM (+)-ABA/AM1/AM2 or 0.02% tween-20 and 0.05% DMSO (control) was sprayed to the leaves one day, and the position of the flower pot was changed to reduce the experimental error. Resume watering after about 12-14 days. Photographs were taken before and after rehydration. The soil drought experiment of tobacco is similar to that of Arabidopsis thaliana. Each pot contains only one plant. All plants are cultivated in long-day sunlight at 24°C. After one month, watering is stopped for drought treatment. During this period, 0.05 A solution of % tween-20 and 50 μM (+)-ABA/AM1/AM2 or 0.05% tween-20 and 0.05% DMSO (control), while changing the position of the flowerpot. Resume watering after about 14 days.

(3) Leaf dehydration experiment in vitro

Arabidopsis thaliana Col-0 ecotype plants grown for 1 month under short-day conditions at 22°C were sprayed with 0.02% tween-20 and 50 μM(+)-ABA/AM1/AM2 or 0.02% tween-20 and 0.05% A solution of DMSO (control) was left standing for another 3 hours. The lotus leaves of the same size were cut and weighed, then exposed to the air, and the fresh weight was weighed regularly.

(4) Cold experiment

The process of seed vernalization and Arabidopsis growth was the same as the soil drought experiment. Three-week-old plants were placed in a 4°C growth chamber for 5 days, and sprayed with 0.02% tween-20 and 50 μM (+)-ABA on the leaves every day. /AM1/AM2 or a solution of 0.02% tween-20 and 0.05% DMSO (control). After five days, they were removed from the incubator and placed in a normal growth environment.

II. Example

Embodiment 1, small molecule compound AM1

1. Structure

The small molecule compound AM1 (ABA mimic1) contained in the present invention has a different chemical structure from ABA, consisting of a xylene linked to a sulfonyl group and a quinoline derivative linked to an amino group, and has no optical activity, as shown in Figure 1a.

2. In vitro biochemical experiments, PP2C protein phosphatase activity experiments

In vitro biochemical experiments show that AM1, as a broad-spectrum and highly efficient PYL receptor agonist, can bind to multiple PYL receptors, promote the latter's binding and inhibit the PP2C protein phosphatase activity.

The PP2C protein phosphatase activity experiment using SnRK2.6 phosphorylated polypeptide as the substrate showed that in the presence of AM1, multiple PYL receptors can bind to PP2C protein phosphatase (HAB1), thereby inhibiting the phosphorylation of SnRK2.6 by HAB1. Dephosphorylation of the polypeptide (Figure 1b), the above results show that AM1 is a broad-spectrum PYL receptor agonist similar to ABA.

3. Alpha Screen

AlphaScreen results showed that AM1 has similar PYL receptor affinity and binding ability to ABA (Fig. 1c-e). The binding ability of AM1 to PYL receptor and PP2C protein phosphatase (HAB1) was detected by AlphaScreen technology. _EC50 value (half effect concentration, which is the concentration at which 50% of the maximum effect is produced. The lower the _EC50 value, the higher the affinity of the compound for the receptor) and peak signal (represents the reading at which the compound binds to the receptor at its maximum , the higher the signal peak, the stronger the binding ability of the compound to the receptor) indicated that in the presence of AM1, the three PYL receptors, including PYR1 (Fig. 1c), PYL1 (Fig. 1d) and PYL2 (Fig. 1e), interacted with HAB1 has a similar affinity and binding ability to ABA, which is significantly better than Pyrabactin, and the binding ability of three PYL receptors (PYR1, PYL1 and PYL2) to HAB1 has a dose-dependent AM1 effect. The above results prove that AM1 is a highly effective PYL receptor agonist similar to ABA.

AM1 can promote the binding of three PYL receptors, PYR1(c), PYL1(d) and PYL2(e), to protein phosphatase HAB1, and the interaction has a dose-dependent effect. The affinity (EC50 value) and binding strength (peak Photon counts) of AM1 for the three PYL receptors are superior to those of Pyrabactin.

4. Yeast two-hybrid experiment

The present inventors conducted a yeast two-hybrid experiment, and the PYL receptor protein fused with the binding domain (binding-domain, BD) and the PP2C protein phosphatase protein combined with the activation domain (activation-domain, AD) were jointly transfected according to the corresponding combination. into the yeast.

As a result, as shown in Fig. 2a, the yeast can grow normally on the medium without isoleucine and tryptophan (-Leu/-Trp), indicating that the yeast transformation is successful. As shown in Figure 2b, the yeast grows on the DMSO control medium without isoleucine, tryptophan and histidine (-Leu/-Trp/-His), five PYL receptors such as PYR1 and PYL1-4 are associated with Co-transformed strains with PP2C protein phosphatase failed to grow normally. As shown in Figure 2c, yeast grows on a medium containing 2 μMABA and does not contain isoleucine, tryptophan and histidine (-Leu/-Trp/-His), five PYL receptors such as PYR1 and PYL1-4 The co-transformation strain with PP2C protein phosphatase can grow normally. It was shown that the interaction of these PYL receptors with PP2C protein phosphatase was dependent on the presence of ABA. As shown in Figure 2d, the yeast grew on a medium containing 2 μM AM1 without isoleucine, tryptophan and histidine (-Leu/-Trp/-His), four PYLs including PYR1 and PYL1-3 were affected The co-transformation strains of body and PP2C protein phosphatase can grow normally, indicating that AM1 at a low concentration can promote the interaction between these ABA-dependent PYL receptors and PP2C protein phosphatase.

The results showed that a low concentration (2μM) of AM1 can promote the interaction between most PYL receptors and multiple PP2C protein phosphatases, and this concentration is only 1/5 of the working concentration of Pyrabactin reported in the literature. Unlike ABA, AM1 at this concentration cannot promote the interaction between PYL4 and PP2C protein phosphatase, which is consistent with the results of protein phosphatase activity detection experiments. The above results further prove that AM1 is a broad-spectrum and efficient PYL receptor agonist.

Example 2, the structure of small molecule compound AM1, PYL receptor protein and HAB

The inventors have detected the crystal structure of the PYL2-AM1-HAB1 complex. From the schematic diagram of the three-dimensional structure of the complex, the oxygen atom on the quinoline derivative of AM1 can pass through the free water molecule and the tryptophan at the 385th position of HAB1 (W385 ) to form a hydrogen bond, thereby further bringing HAB1 and PYL2 closer (Fig. 3a); from the partial schematic diagram of the two-dimensional structure of the complex, AM1 exists in the pocket structure of PYL2, and through the oxygen atom on the sulfonyl group, it interacts with multiple active sites of PYL2 The dots are hydrogen bonded (Fig. 3b). Therefore, AM1 binds in the pocket structure of PYL2, and binds to the key amino acid active sites of PYL2 and HAB1 through a mechanism similar to ABA, thereby further promoting and stabilizing the binding of PYL2 and HAB1. This result explains why AM1 is a broad-spectrum and highly effective PYL receptor agonist.

Example 3, AM1 can cause gene expression similar to ABA

The present inventors analyzed the effect of adding AM1 on gene expression in plants. Gene expression analysis showed that the addition of AM1 caused gene expression similar to that of ABA (Fig. 4). After 50 μM AM1 treatment, the expression levels of six known abscisic acid-induced genes related to environmental stress in 10-day-old wild-type Arabidopsis (Col-0) seedling plants increased significantly, which was close to that after 50 μM ABA treatment. The expression level of the corresponding gene in the PYL receptor triple mutant strain (pyr1; pyl1; pyl4) has a limited increase. This result indicates that the induction of environmental stress-related genes by AM1 is through the ABA signal mediated by the PYL receptor. pathway (Fig. 4a).

The results of RNA sequencing (RNA-seq) showed that compared with the DMSO control group, the expression levels of about 4500 genes were significantly changed after 50 μM AM1 and ABA treatment, and more than 80% of the genes (3644) were simultaneously Induced by AM1 and ABA (Fig. 4b), and the most significant differences in these genes relative to the control group were genes related to plant resistance to drought, cold, osmotic pressure and salt stress (Fig. 4c). This result indicates that AM1 induces physiological changes highly similar to ABA, and mainly focuses on environmental stress responses.

Example 4, AM1 can cause physiological responses similar to ABA

(1) Arabidopsis drought resistance (or drought tolerance)

Physiological experiments showed that AM1 treatment elicited similar physiological responses to ABA. The exogenous addition of 1 μM AM1 to the medium can significantly inhibit the seed germination of wild-type Arabidopsis plants (Col-0), but the PYL receptor triple mutants (pyr1; pyl1; pyl4) are not affected, which indicates that AM1 The inhibitory effect on seed germination is carried out through the ABA signaling pathway mediated by the PYL receptor, as shown in FIG. 5 .

In order to further explore the effect of AM1 on the drought resistance of plants, wild-type Arabidopsis plants (Col-0) grown in soil for two weeks were stopped from water supply, and plants of the same size were selected for soil drought experiments, during which they were sprayed with The aqueous solution of 50 μ M AM1 or ABA, added the surfactant Tween-20 of 0.02% (v/v) simultaneously in the solution to enhance the penetration of the spray to the leaf epidermis. After two weeks of drought treatment, all the plants sprayed with ABA survived after the water supply was restored, and more than 95% of the wild-type plants sprayed with DMSO control solution were withered and could not recover after water supply was restored, while 80% of the plants sprayed with AM1 Still can continue to grow, as shown in Figure 6a-b.

Leaves of four-week-old Arabidopsis plants were sprayed with the corresponding solution for 3 hours, and the leaves of the same size were removed, and the fresh weight of the leaves was recorded regularly. As shown in Figure 6c, the water loss rate of detached leaves showed that the increased drought resistance of AM1 was due to the weakened transpiration and enhanced water retention of leaves. This result indicated that plants could reduce the transpiration of leaves by spraying AM1 when encountering drought environment, thus enhancing the drought tolerance of plants.

(2) Tobacco drought resistance (or drought tolerance)

Nicotiana benthamiana growing for 1 month in a long-day environment stopped watering, and selected plants of the same size for a soil drought experiment. During this period, an aqueous solution containing 50 μM AM1 or ABA was sprayed once every other day, and 0.05% ABA was added to the solution at the same time (v/v) surfactant Tween-20 to enhance the penetration of the spray on the leaf epidermis. After two weeks of drought treatment, all the plants sprayed with ABA survived after water supply was restored, and most of the wild-type plants sprayed with DMSO control solution were withered and could not recover after water supply was restored, while most of the plants sprayed with AM1 could continue grow.

(3) Arabidopsis cold resistance (or called cold resistance)

In order to further explore the effect of AM1 on plant cold resistance, wild-type Arabidopsis plants (Col-0) of the same size grown in soil for three weeks were placed in a growth chamber at 4°C for 5 days, during which they were sprayed with 50 μM AM1 or In the aqueous solution of ABA, 0.02% (v/v) surfactant Tween-20 was added to the solution to enhance the penetration of the spray on the leaf epidermis.

After 5 days of cold treatment, the plants sprayed with ABA all survived, while the wild-type plants sprayed with DMSO control solution were mostly withered (more than 70%), and they could not recover after being moved back to the normal growth environment, while the plants sprayed with AM1 were mostly Some can still grow.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (14)

1. A use of a compound of formula (I) or a salt thereof, for enhancing plant stress resistance or for preparing an agricultural preparation for enhancing plant stress resistance; 2. The use according to claim 1, characterized in that said stress resistance is abiotic stress resistance involving ABA. 3. The use according to claim 2, characterized in that, the abiotic stress resistance in which ABA participates is at least one of drought tolerance, cold tolerance, salt-alkali tolerance, osmotic pressure tolerance or heat tolerance. 4. The use according to claim 1, wherein the plant is a plant containing ABA receptors of the PYR/PYL family. 5. The use according to claim 4, wherein said plants include but are not limited to: Brassicaceae plants, Solanaceae plants, Gramineae plants. 6. The use according to claim 1, wherein said plants include but are not limited to: Arabidopsis, tobacco, cabbage, rape, tomato, pepper, wheat, rice, barley, corn, sorghum, oat, Rye, sugar cane, cotton, soybeans, sugar beet, sunflower. 7. A method for enhancing plant stress resistance, characterized in that, the compound of formula (I) or its salt is applied to plants: 8. The method according to claim 7, characterized in that said stress resistance is abiotic stress resistance involving ABA. 9. The method according to claim 8, characterized in that, the abiotic stress resistance in which ABA participates is at least one of drought tolerance, cold tolerance, salt-alkali tolerance, osmotic pressure tolerance or heat tolerance. 10. The method of claim 7, wherein the plant is a plant containing ABA receptors of the PYR/PYL family. 11. The method according to claim 10, wherein said plants include but not limited to: Brassicaceae plants, Solanaceae plants, Poaceae plants. 12. The use according to claim 7, wherein said plants include but are not limited to: Arabidopsis, tobacco, cabbage, rape, tomato, pepper, wheat, rice, barley, corn, sorghum, oat, Rye, sugar cane, cotton, soybeans, sugar beet, sunflower. 13. An agricultural formulation, characterized in that the agricultural formulation comprises: a compound of formula (I) or a salt thereof; and an agriculturally acceptable carrier; 14. The agricultural formulation according to claim 13, characterized in that, The content of the compound of formula (I) or its salt in the agricultural preparation is 1-500 μM; preferably 5-200 μM; more preferably 20-100 μM; and/or The agriculturally acceptable carrier includes: surfactant.