JP7197888B2 - plant stomatal opening inhibitor - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act (Part 1) Website publication date April 8, 2018 Website address https://academic. oup. com/pcp/article-abstract/59/8/1581/5053956? redirectedFrom=fulltext (Part 2) Website publication date April 9, 2018 Website address (Nagoya University Institute of Transformative Bio-Molecules website) http://www. it bm. nagoya-u. ac. jp/ja/research/2018/04/SCL-Kinoshita. php (Nagoya University press release) http://www. it bm. nagoya-u. ac. jp/ja/research/20180409_PCP_Kinoshita_ITbM. pdf (Part 3) Posting date of website June 1, 2018 Website address http://www. bio. nagoya-u. ac. jp/paper/2018-31/11. html

TECHNICAL FIELD The present invention relates to a plant stomatal opening regulator, a plant stomatal opening inhibitor, and the like.

Higher terrestrial plants regulate the amount of carbon dioxide uptake and transpiration necessary for photosynthesis through stomata opening regulation in the epidermis of leaves. For example, when exposed to water shortages, it is known to close stomata and inhibit transpiration in order to maintain water within the plant body. It is also known that stomata are opened in response to light, promoting the uptake of carbon dioxide necessary for photosynthesis. Therefore, by artificially adjusting the stomata opening, effects such as promotion of photosynthesis, promotion of growth, and improvement of drought resistance can be expected.

As an example of artificial regulation of stomatal opening, a method of overexpressing plasma membrane H ⁺ -ATPase (AHA2) in guard cells has been reported (Patent Document 1, Non-Patent Document 1). According to this method, stomatal opening can be promoted, along with photosynthetic rate and plant growth. In addition, a method for improving drought tolerance by overexpressing magnesium chelatase H subunit in guard cells and promoting stomatal closure has also been reported (Non-Patent Document 2).

Since these methods are based on genetic recombination technology,
1. For organisms for which gene recombination technology has not been established, establishment and optimization of recombination technology are necessary each time.
2. It takes time to produce genetically modified crops (GMO) (for example, it takes about half a year to produce the current generation of transgenic poplar, and more than half a year to obtain next-generation GMO seeds for rice).
3. Approval is required for each GMO created.

On the other hand, if it is a substance that can regulate stomatal opening by applying it to plants that are already growing, there is no need to take the procedures of producing and obtaining approval for GMOs for various plants. Useful.

WO2014/142334

PNAS, January 7, 2014, vol.111, no.1, pp.533-538 Front Plant Sci, October 30, 2013, vol.4, Article 440

An object of the present invention is to provide a plant stomatal opening regulator.

As a result of intensive studies in view of the above problems, the present inventors have found that each compound represented by the general formula (1) has a pore opening regulating action. The present invention was completed as a result of further studies based on this finding.

The present invention includes the following inventions.

Section 1.
General formula (1):

[In the formula: R ¹ and R ³ are the same or different, =O or -OR ¹¹ (R ¹¹ represents a hydrogen atom, an optionally substituted alkyl group, or an optionally substituted alkanoyl group.) indicates R ² and R ⁴ are the same or different and represent an optionally substituted alkyl group, an optionally substituted cycloalkyl group, an optionally substituted aryl group, or a halogen atom. n represents 0 or an integer of 1 to 3; A double line consisting of a solid line and a dotted line, which may be the same or different, indicates a single bond or a double bond (provided that the ring in the formula is an unsaturated carbocyclic ring). ]
A plant stomatal opening regulator containing at least one selected from the group consisting of the compound represented by the following, salts thereof, and solvates thereof.

Section 2.
The compound has the general formula (1A):

[In the formula: R ¹ , R ² , R ³ , R ⁴ , n, and the double line of solid and dotted lines are the same as above. ]
Item 2. The plant stomatal opening regulator according to Item 1, which is a compound represented by:

Item 3.
Item 3. The plant stomatal opening regulator according to Item 1 or 2, wherein said R ¹ and R ³ are hydroxy groups.

Section 4.
Item 4. The plant stomatal opening regulator according to any one of items 1 to 3, wherein said R ² and R ⁴ are alkyl groups.

Item 5.
Item 5. The plant stomatal opening regulator according to any one of Items 1 to 4, wherein n is 0 or 1.

Item 6.
The compound has the general formula (1AA):

[In the formula: R ¹ , R ² , R ³ and the double line of the solid line and the dotted line are the same as above. ^R41 represents a hydrogen atom or an alkyl group. ]
Item 6. The plant stomatal opening regulator according to any one of items 1 to 5, which is a compound represented by:

Item 7.
Item 7. The plant stomatal opening regulator according to any one of Items 1 to 6, which is a plant stomatal opening inhibitor.

Item 8.
Item 8. An agent for improving drought resistance, comprising the plant stomatal opening regulator according to any one of items 1 to 7.

Item 9.
Item 8. A method for improving drought tolerance, comprising applying the plant stomatal opening regulator according to any one of Items 1 to 7 to a plant.

Item 10.
Item 9. The method for improving drought tolerance according to Item 9, which comprises bringing the plant stomatal opening regulator according to any one of Items 1 to 7 into contact with the stomata of the plant.

Item 11.
General formula (1):

[In the formula: R ¹ and R ³ are the same or different, =O or -OR ¹¹ (R ¹¹ represents a hydrogen atom, an optionally substituted alkyl group, or an optionally substituted alkanoyl group.) indicates R ² and R ⁴ are the same or different and represent an optionally substituted alkyl group, an optionally substituted cycloalkyl group, an optionally substituted aryl group, or a halogen atom. n represents 0 or an integer of 1 to 3; A double line consisting of a solid line and a dotted line, which may be the same or different, indicates a single bond or a double bond (provided that the ring in the formula is an unsaturated carbocyclic ring). ]
A cell membrane proton pump phosphorylation inhibitor containing at least one selected from the group consisting of the compound represented by the formula, salts thereof, and solvates thereof.

According to the present invention, a plant stomatal opening regulator can be provided. It is also possible to provide drought tolerance improvers such as plant growth promoters.

Schematic diagram of the signaling mechanism of stomatal opening. Fig. 1 shows the results of measurement of stomata opening-regulating action of Compound 1 (Test Example 1). The vertical axis indicates the pore opening. In the horizontal axis, "DMSO" indicates the case where the stomatal opening measurement solution was used as the test liquid (negative control), and "ABA" used a solution in which abscisic acid was dissolved in the stomatal opening measurement solution as the test liquid. "Compound 1" indicates the case in which a solution prepared by dissolving compound 1 in the stomatal opening measurement solution was used as the test liquid. * indicates a p-value of less than 0.05 relative to the negative control. Fig. 1 shows the results of measurement of stomata opening-regulating action of Compound 1 (Test Example 1). The vertical axis indicates the pore opening. In the horizontal axis, "DMSO" indicates the case where the stomatal opening measurement solution is used as the test liquid, and "Compound 1" indicates the case where compound 1 is dissolved in the stomatal opening measurement solution as the test liquid. show. "FC" indicates the case where Fusicoccin was further dissolved. * indicates p-value for DMSO + FC less than 0.05. Fig. 2 shows the measurement results of phototroropine phosphorylation (Test Example 2-1). On the left side of the photograph, "Phots" indicates phosphorylated phototropin, and "14-3-3" indicates 14-3-3 protein. In the upper part of the photograph, "DMSO" indicates the case in which DMSO was used as the test solution, "Compound 1" indicates the case in which the DMSO solution of Compound 1 was used as the test solution, and R is 80 μmol m ^-2 s ^-1 Red. RB indicates the case of irradiation with light for 30 minutes, and RB indicates the case of irradiation with 20 μmol m ⁻² s ⁻¹ blue light for 1 minute in addition to the conditions of R. Fig. 2 shows the measurement results of potassium channel (KAT1) activity (Test Example 2-2). "Compound 1" indicates the case where compound 1 is allowed to act. The vertical axis indicates the relative value of KAT1 activity to the case where Compound 1 is not acted (1). Fig. 2 shows the measurement results of phosphorylation of cell membrane proton pumps (Test Example 2-3). "DMSO B" indicates the case of using DMSO as a test solution and light treatment, "Compound 1" indicates the case of using a DMSO solution of compound 1 as a test solution and light treatment, "DMSO D" It shows the case where DMSO was used as a test solution and dark treatment was performed without light treatment. The vertical axis indicates the signal intensity of the phosphorylated cell membrane proton pump relative to DMSO B (100). * indicates p-value for DMSO B less than 0.02. Fig. 2 shows the measurement results of phosphorylation of cell membrane proton pumps (Test Example 2-3). "DMSO" indicates the case where DMSO was used as the test solution and was treated in the dark, and "Compound 1" indicates the case where a DMSO solution of Compound 1 was used as the test solution and was treated in the dark. "FC" indicates that Fusicoccin was added to the test solution. The vertical axis indicates the signal intensity of the phosphorylated cell membrane proton pump relative to DMSO + FC (100). * indicates p-value for DMSO + FC less than 0.02. 3 shows the results of a germination test (Test Example 3-1). "DMSO" indicates the case where DMSO was used as the test solution, "Compound 1" indicates the case where the DMSO solution of compound 1 was used as the test solution, and "ABA" used the DMSO solution of abscisic acid as the test solution. indicate the case. The vertical axis indicates the germination rate. Fig. 3 shows measurement results of expression levels of abscisic acid-responsive genes (Test Example 3-2). "DMSO" indicates the case where DMSO was used as the test solution, "Compound 1" indicates the case where the DMSO solution of compound 1 was used as the test solution, and "ABA" used the DMSO solution of abscisic acid as the test solution. indicate the case. The vertical axis indicates the expression level. * indicates p-value for DMSO less than 0.05. 6 shows the measurement results of phosphorylation of 61 kDa protein (Test Example 3-3). "DMSO" indicates the case where DMSO was used as the test solution, "Compound 1" indicates the case where the DMSO solution of compound 1 was used as the test solution, and "ABA" used the DMSO solution of abscisic acid as the test solution. indicate the case. The top picture shows the phosphorylated 61 kDa protein and the bottom picture shows the 14-3-3 protein. The results (photographs) of Test Example 4 (rose) are shown. The upper row shows the photograph before treatment, and the lower row shows the photograph 6 hours after treatment. The results (photographs) of Test Example 4 (oats) are shown. The left side shows the picture before treatment and the right side shows the picture 20 minutes after treatment.

As used herein, the expressions "contain" and "include" include the concepts "contain", "include", "consist essentially of" and "consist only of".

In one aspect of the present invention, the general formula (1):

A plant stomatal opening regulator (herein referred to as "the plant stomata of the present invention It may be indicated as "opening regulator".). This will be explained below.

1. active ingredient
R ¹ and R ³ are the same or different and represent ═O or —OR ¹¹ (R ¹¹ represents a hydrogen atom, an optionally substituted alkyl group, or an optionally substituted alkanoyl group). R ¹ and R ³ are the same or different, preferably -OR ¹¹ . Particularly preferably both R ¹ and R ³ are hydroxy groups.

The alkyl group (R ¹¹ ) shown in R ¹ or R ³ includes both linear and branched groups. The number of carbon atoms in the alkyl group is not particularly limited, and is, for example, 1-8, preferably 1-6. The number of carbon atoms is particularly preferably 1, 4 or 6. On the other hand, in some embodiments of the present invention, the lower limit of carbon number of the alkyl group is, for example, 2, 3, 4, 5, 6, 7. Specific examples of the alkyl group include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group, sec-butyl group, n-pentyl group, neopentyl group, n -hexyl group, 3-methylpentyl group, n-heptyl group, n-octyl group and the like. The alkyl group may be substituted with halogen atoms such as fluorine, chlorine, bromine and iodine atoms.

The alkanoyl group (R ¹¹ ) represented by R ¹ or R ³ includes a group represented by -C(=O)-R ¹² . ^R12 is an optionally substituted alkyl group. The alkyl group includes both straight-chain and branched-chain ones. The number of carbon atoms in the alkyl group is not particularly limited, and is, for example, 1-8, preferably 1-6. The number of carbon atoms is particularly preferably 1, 4 or 6. On the other hand, in some embodiments of the present invention, the lower limit of carbon number of the alkyl group is, for example, 2, 3, 4, 5, 6, 7. Specific examples of the alkyl group include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group, sec-butyl group, n-pentyl group, neopentyl group, n -hexyl group, 3-methylpentyl group, n-heptyl group, n-octyl group and the like. The alkyl group may be substituted with halogen atoms such as fluorine, chlorine, bromine and iodine atoms.

The position of R3 is para ^, meta or ortho to ^R1 , preferably para.

R ² and R ⁴ are the same or different and represent an optionally substituted alkyl group, an optionally substituted cycloalkyl group, an optionally substituted aryl group, or a halogen atom. R ² and R ⁴ are the same or different, preferably an optionally substituted alkyl group, more preferably an alkyl group.

The alkyl group represented by R ² or R ⁴ includes both linear and branched (preferably branched). The number of carbon atoms in the alkyl group is not particularly limited, and is, for example, 1-12, preferably 2-8, more preferably 3-6, still more preferably 4-5, still more preferably 4. Specific examples of the alkyl group include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group, sec-butyl group, n-pentyl group, neopentyl group, n -hexyl group, 3-methylpentyl group, n-heptyl group, n-octyl group, n-nonyl group, n-decyl group, n-undecyl group, n-dodecyl group and the like. Among these, a tert-butyl group is particularly preferred. The alkyl group may be substituted with halogen atoms such as fluorine, chlorine, bromine and iodine atoms.

The number of carbon atoms in the cycloalkyl group represented by R ² or R ⁴ is not particularly limited, and is, for example, 3-10, preferably 4-8, more preferably 5-7, still more preferably 6. Specific examples of the cycloalkyl group include cyclopropyl group, cyclobutyl group, cyclopentyl group, cyclohexyl group, cycloheptyl group, cyclooctyl group and the like. The cycloalkyl group may be substituted with halogen atoms such as fluorine, chlorine, bromine and iodine atoms.

The aryl group represented by R ² or R ⁴ is not particularly limited, but preferably has 6 to 12 carbon atoms, more preferably 6 to 12 carbon atoms, and even more preferably 6 to 8 carbon atoms. The aryl group can be either monocyclic or polycyclic (eg, bicyclic, tricyclic, etc.), but is preferably monocyclic. Specific examples of the aryl group include phenyl, naphthyl, biphenyl, pentalenyl, indenyl, anthranyl, tetracenyl, pentacenyl, pyrenyl, perylenyl, fluorenyl, and phenanthryl groups. and preferably a phenyl group. The aryl group may be substituted with halogen atoms such as fluorine, chlorine, bromine and iodine atoms.

The halogen atom represented by R ² or R ⁴ is not particularly limited, and examples thereof include fluorine atom, chlorine atom, bromine atom and iodine atom.

n represents 0 or an integer of 1 to 3; n is preferably 0 or 1, more preferably 0.

When n is 1 or more, the position of R ³ is para, meta, or ortho to R ² , preferably R ³ (one of a plurality of R ³ when n is 2 or more) The position is Para.

A double line consisting of a solid line and a dotted line, which may be the same or different, indicates a single bond or a double bond (provided that the ring in the formula is an unsaturated carbocyclic ring). When R ¹ and R ³ are —OR ¹¹ , general formula (1) is preferably general formula (1a):

is mentioned.

In one aspect of the present invention, among general formula (1), preferably general formula (1A):

[In the formula: R ¹ , R ² , R ³ , R ⁴ , n, and the double line of solid and dotted lines are the same as above. ]
and more preferably general formula (1AA):

[In the formula: R ¹ , R ² , R ³ and the double line of the solid line and the dotted line are the same as above. ^R41 represents a hydrogen atom or an alkyl group. ]
can be mentioned.

R ⁴¹ is preferably a hydrogen atom. The alkyl group represented by ^R41 is the same as the alkyl group represented by ^R2 or ^R4 .

When R ¹ and R ³ are —OR ¹¹ , general formula (1A) is preferably general formula (1Aa):

is mentioned.

When R ¹ and R ³ are —OR ¹¹ , general formula (1AA) is preferably general formula (1AAa):

is mentioned.

The compound represented by formula (1) may include stereoisomers and optical isomers, but these are not particularly limited.

The salt of the compound represented by formula (1) is not particularly limited as long as it is an agriculturally acceptable salt. As the salt, both acid salts and basic salts can be employed. Examples of acid salts include mineral salts such as hydrochloride, hydrobromide, sulfate, nitrate, phosphate; acetate, propionate, tartrate, fumarate, maleate, malic acid. Salts, organic acid salts such as citrate, methanesulfonate, p-toluenesulfonate, etc. Examples of basic salts include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as salts; salts with ammonia; morpholine, piperidine, pyrrolidine, monoalkylamine, dialkylamine, trialkylamine, mono(hydroxyalkyl)amine, di(hydroxyalkyl)amine, tri(hydroxyalkyl) Examples thereof include salts with organic amines such as amines.

The compound represented by general formula (1) can also be a hydrate or solvate. Solvents include, for example, agriculturally acceptable organic solvents (eg, ethanol, glycerol, acetic acid, etc.).

As the compound represented by the general formula (1), for example, a known compound can be obtained (for example, a commercially available product) and used. Examples of known compounds include the following compounds.

Moreover, as the compound represented by the general formula (1), one synthesized according to or according to a known synthesis method may be used. The compound represented by the general formula (1) can be synthesized, for example, with reference to previous reports (Gageaa, B. C., Parvulescua, A. N., Ponceletb, G., Parvulescu, V. I. Catalysis Lett. 2005, 105, 219.). . Examples of compounds that can be synthesized are shown below.

2. Use The plant stomatal opening regulator of the present invention contains at least one selected from the group consisting of compounds represented by formula (1), salts thereof, and solvates thereof. In addition, regulation of stomatal opening can be used to regulate photosynthesis and, furthermore, to regulate plant growth. Therefore, at least one selected from the group consisting of the compound represented by the general formula (1), salts thereof, and solvates thereof can be used as an active ingredient of photosynthesis regulators, plant growth regulators, and the like. can.

In particular, the compound represented by the general formula (1) has the effect of suppressing stomatal opening of plants (especially stomatal opening to light or chemicals (such as fushicoccin)). In addition, it is known that transpiration is suppressed and water content in the plant is maintained by suppressing stomatal opening of the plant. Therefore, at least one selected from the group consisting of the compound represented by the general formula (1), salts thereof, and solvates thereof is a plant stomatal opening inhibitor, a drought tolerance improver, a freshness-keeping agent, etc. It can be used as an active ingredient. It is also known that pathogenic microorganisms enter plants through stomata openings. Therefore, at least one selected from the group consisting of the compound represented by formula (1), salts thereof, and solvates thereof can be used as an active ingredient such as a disease resistance improver.

In addition, suppression of the phosphorylation of the cell membrane proton pump is considered to be one part of the stomatal opening regulating action mechanism of the compound represented by general formula (1). Therefore, at least one selected from the group consisting of compounds represented by general formula (1), salts thereof, and solvates thereof can be used as an active ingredient of a cell membrane proton pump phosphorylation inhibitor. . The target cell membrane proton pump is not particularly limited as long as it is expressed in guard cells. For example, for Arabidopsis, AHA1 (AT2G18960), AHA2 (AT4G30190), AHA3 (AT5G57350), AHA4 (AT3G47950), AHA5 (AT2G24520), AHA6 (AT2G07560), AHA7 (AT3G60330), AHA8 (AT3G42640), AHA9 (AT601) , AHA10 (AT1G17260), AHA11 (AT5G62670), etc.また、イネの場合であれば、OSA1（LOC_Os03g48310）、OSA2（LOC_Os07g09340）、OSA3（LOC_Os12g44150）、OSA4（LOC_Os05g25550）、OSA5（LOC_Os08g14360）、OSA6（LOC_Os02g55400）、OSA7（LOC_Os04g56160）、OSA8（LOC_Os03g01120）、OSA9 (LOC_Os03g08560), OSA10 (LOC_Os06g08310), etc.

The target plant for the plant stomatal opening regulator of the present invention is not particularly limited as long as it is a plant having stomata. For example, it can be widely applied to plants such as angiosperms (dicotyledonous plants, monocotyledonous plants, etc.), gymnosperms, and pteridophytes. Specific examples include eggplants such as tomatoes, green peppers, peppers, and eggplants; melons such as cucumbers, pumpkins, melons, and watermelons; vegetables such as cabbage, broccoli, and Chinese cabbage; green onions, green onions, garlic and other green onions, soybeans, peanuts, green beans, peas, adzuki beans and other beans, strawberries and other fruit vegetables, radish, turnips, carrots, burdock and other tap roots, taro, cassava, potatoes , sweet potatoes, potatoes such as Chinese yam, soft vegetables such as asparagus, spinach, mitsuba, etc., flowers such as lisianthus, stock, carnations, and chrysanthemums, grains such as rice, wheat, barley, oats, and corn, bentgrass, and kouraishiba. oil crops such as rapeseed and groundnut, sugar crops such as sugarcane and sugar beet, fiber crops such as cotton and rush, fodder crops such as clover, sorghum and dent corn, apples, pears, grapes , deciduous fruit trees such as peaches, citrus fruits such as mandarin oranges, lemons and grapefruits, and woody plants such as azaleas, azaleas and cedars.

The plant stomatal opening regulator of the present invention may consist only of the above-described agents, but may contain various additives in addition to the above-described agents depending on the dosage form, mode of application, etc. described later. The content of the drug in the plant stomatal opening regulator can be appropriately determined according to the dosage form, mode of application, etc., which will be described later. As a more specific example, when the plant stomatal opening regulator of the present invention, which is a liquid agent, is brought into contact with the stomata, the content of the above-mentioned agent is 1 to 500 μM, preferably 10 to 200 μM, more preferably 50 to 50 μM. About 150 μM is exemplified.

The dosage form of the plant stomatal opening regulator of the present invention is not particularly limited as long as it is an agriculturally acceptable dosage form. Examples include liquids, solids, powders, granules, granules, wettable powders, flowables, emulsions, pastes, dispersants and the like.

Additives are not particularly limited as long as they are agriculturally acceptable additives. Examples thereof include carriers, surfactants, spreading agents, spray adjuvants, thickeners, bulking agents, binders, vitamins, antioxidants, pH adjusters, volatilization inhibitors, pigments and the like.

Among these, spray adjuvants containing surfactants such as polyoxyethylenehexitane fatty acid esters and plant water repellency inhibitors such as silicone surfactants are particularly preferred. In one aspect of the present invention, it is desirable to use these two components in combination. The content of the spray adjuvant may vary depending on the dosage form. For example, in the case of a liquid formulation, it is, for example, 0.01 to 0.3% by mass, preferably 0.03 to 0.1% by mass, based on 100% by mass of the plant stomatal opening regulator of the present invention. is. In addition, the content of the water repellency inhibitor may vary depending on the dosage form. 0.05% by mass.

The mode of application of the plant stomatal opening regulator of the present invention is not particularly limited as long as it is a known mode (or a mode to be developed in the future) as a mode of use of agricultural chemicals. Examples thereof include spraying, dripping, coating, mixing or dissolving in a plant growing environment (in soil, water, solid medium, liquid medium, etc.). Since the target of action of the plant stomatal opening regulator of the present invention is the stomata, it is preferable to apply the plant stomatal opening regulator of the present invention by bringing it into contact with the stomata of the plant.

EXAMPLES The present invention will be described in detail below based on examples, but the present invention is not limited by these examples.

Test example 1. Measurement test of stomatal opening regulation effect 1
The stomatal opening regulating action of Compound 1 below was measured.

<Test Example 1-1. Preparation of small pieces of epidermis>
A part of the stem of Commelina benghalensis was placed in a planter containing a mixed soil of vermiculite and peat moss, and grown for 2 to 4 weeks in a room with natural light by the window (room temperature 25 to 28°C). The planters were placed in a darkroom and darkened overnight. After dark treatment, under weak red light, the epidermis on the underside of fully developed leaves was peeled off with tweezers and cut with scissors to produce 2.0-3.0 mm square pieces.

<Test example 1-2. Preparation of test solution>
As a test solution, stomatal opening measurement solution (5 mM MES-BTP [pH 6.5], 50 mM KCl, 0.1 mM CaCl ₂ , 0.5% DMSO), test compound (compound 1: 50 μM or abscisic acid : 20 μM) and fucicoccin dissolved in these solutions (10 μM) were prepared.

<Test example 1-3. Measurement test>
50 μl/well of each test solution was dispensed into 5 wells of a 96-well plate, and leaf discs were immersed therein. Chemical treatment was performed for 4 hours under fluorescent lamp irradiation. After drug treatment, stomatal closure was evaluated under observation images (600x) with a stereoscopic fluorescence microscope (Leica M205FA). The short diameter of 30 pores (hereinafter referred to as "pore opening") was measured for each leaf disk. The above measurement test was performed twice in total, and the average value and standard deviation (SD) were obtained based on the measured values obtained two times in total for each test solution.

<Test Example 1-4. Results>
The results are shown in FIGS. 2 and 3. FIG. Compound 1 was found to have stomatal regulating activity.

Test example 2. Analysis of mechanism of regulating stomatal opening Signal transduction mechanism of stomatal opening is shown in FIG. Blue light is accepted by phototropin, activating the cell membrane proton pump and inducing potassium ion uptake. This increases the osmotic pressure, entraps water, and increases the volume of guard cells, thereby opening the stomata. In this test example, it was analyzed where compound 1 acts in the above mechanism.

<Test Example 2-1. Phosphorylation of Phototropin>
Blue light- and phosphorylation-dependent binding of a 14-3-3 protein to phototropins in guard cell protoplasts from broad bean (Vicia faba) was previously reported (Kinoshita et al. (2003)). Plant Physiol. 133: 1453-1463.) by protein blot analysis using the 14-3-3 protein as a probe. DMSO or Compound 1 in DMSO was used as the test solution. The working concentration of Compound 1 was 50 μM. 14-3-3 protein in guard cells from broad bean (Vicia faba) has been previously reported (Kinoshita, T. and Shimazaki, K. (1999) Blue light activates the plasma membrane H ⁺ -ATPase by phosphorylation of the C- terminus in stomatal guard cells. EMBO J. 18: 5548-5558.) were detected by Western blotting.

<Test example 2-2. Potassium channel (KAT1) activity>
The mMESSAGE mMACHINE T7 kit (Ambion , Austin, Tex.) were used to synthesize capped complementary RNA encoding KAT1 and injected into Xenopus oocytes. A two-electrode voltage-clamp technique was performed using an AxoClamp 2B voltage-clamp amplifier (Axon Instruments, Foster City, Calif.) according to the following protocol: holding potential of −40 mV. +10mV to -170 mV in -20mV increments for 500ms. The external solution was 120 mM KCl, 1 mM MgCl2, ₁ mM CaCl2, 10 mM 4-( ₂ -hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.3, adjusted with NaOH) and 0.2% (w/v) containing Compound 1, in DMSO. The working concentration of Compound 1 was 50 μM.

<Test Example 2-3. Phosphorylation of Cell Membrane Proton Pump>
Hayashi et al. (2011) Immunohistochemical detection of blue light-induced phosphorylation of the plasma membrane H ⁺ - ATPase in stomatal guard cells. Plant Cell Physiol. 52: 1238-1248.). DMSO, a DMSO solution of compound 1, or a solution obtained by adding fucicoccin to these solutions was used as the test solution. The working concentration of Compound 1 was 50 μM, and the working concentration of Fusicoccin was 10 μM.

<Test Example 2-4. Results>
The results of Test Example 2-1 are shown in FIG. 4, the results of Test Example 2-2 are shown in FIG. 5, and the results of Test Example 2-3 are shown in FIGS. From Figures 4 to 7, it was found that suppression of phosphorylation of the cell membrane proton pump is one aspect of the stomatal opening regulating action mechanism of Compound 1.

Test example 3. Analysis of Effect on Abscisic Acid Response Abscisic acid is known to induce various side effects (abscisic acid response) although it has a stomatal opening control effect. Therefore, the effect of compound 1 on the abscisic acid response was analyzed.

<Test Example 3-1. Seed Germination>
Arabidopsis thaliana seeds were used according to a previous report (Tsuzuki et al. (2011) Mg-chelatase H subunit affects ABA signaling in stomatal guard cells, but is not an ABA receptor in Arabidopsis thaliana. J. Plant Res. 124: 527-538). A seed germination test was performed. DMSO or a DMSO solution of compound 1 or abscisic acid was used as the test solution. The working concentrations of compound 1 and abscisic acid were 50 μM.

<Test Example 3-2. Expression of Abscisic Acid-Responsive Gene>
Expression levels of abscisic acid-responsive genes (RAB18 (At5g66400) and RD29B (At5g52300)) in Arabidopsis seedlings were measured by quantitative reverse transcription polymerase chain reaction. Relative quantification was performed using the comparative cycle threshold method and the relative amounts of amplified RAB18 or RD29B products were normalized to the relative amounts of the internal control (TUB2). Specifically, 2-week-old seedlings were incubated in liquid MS medium (pH 5.8) containing 50 μM abscisic acid or compound 1 at 24° C. for 3 hours. Total RNA was extracted and first strand cDNA was prepared from abscisic acid or compound 1 treated plants. Quantitative RT-PCR was performed using Power SYBR Green PCR Master Mix and the StepOne® Real-Time PCR System (Applied Biosystems, Carlsbad, Calif.). RAB18, RD29B and TUB2 cDNAs were amplified by PCR using specific primers.

<Test Example 3-3. Phosphorylation of 61 kDa protein>
Takahashi et al. (2007) Protein phosphorylation and binding of a 14-3-3 protein in Vicia guard cells in response to ABA. Cell Physiol. 48: 1182-1191.) were evaluated by protein blot analysis using the 14-3-3 protein as a probe. Guard cell protoplasts were treated with 20 μM ABA or 50 μM Compound 1 for 20 minutes at 24°C.

<Test Example 3-4. Results>
The results of Test Example 3-1 are shown in FIG. 8, the results of Test Example 3-2 are shown in FIG. 9, and the results of Test Example 3-3 are shown in FIG. From Figures 8 to 10, it was found that Compound 1 had no effect on the abscisic acid response.

Test example 4. Drought tolerance test Rose leaves and 7-day-old oat seedlings were added with test solutions (0.02% Silwet L77 (Biomedical Science, Tokyo, Japan), 0.05% Approach BI (Maruwa Biochemical, Tokyo, Japan), 0.5% DMSO or 100 μM compound 1) was sprayed and left at 24° C. for 3 hours under a white fluorescent lamp of 50 μmol m ⁻² s ⁻¹ and 70% relative humidity. Leaves were then removed and incubated for 24 hours under 50 μmol m ⁻² s ⁻¹ white fluorescent light and 35-50% relative humidity for 6 hours (rose) or 20 minutes (oats).

The results (photographs) for roses are shown in FIG. 11, and the results (photographs) for oats are shown in FIG. From Figures 11 and 12, it was found that compound 1 improved the drought tolerance.

Claims (5)

General formula (1):

[In the formula, R ² represents a branched alkyl group having 3 to 6 carbon atoms . ]
A plant stomatal opening inhibitor containing at least one selected from the group consisting of a compound represented by the following, salts thereof, and solvates thereof. A drought tolerance improver containing the plant stomatal opening inhibitor according to claim 1 . A method for improving drought tolerance, comprising applying the plant stomatal opening inhibitor according to claim 1 to a plant. 4. The method for improving drought tolerance according to claim 3 , which comprises bringing the plant stomatal opening inhibitor according to claim 1 into contact with the stomata of the plant. General formula (1):

[In the formula, R ² represents a branched alkyl group having 3 to 6 carbon atoms . ]
A cell membrane proton pump phosphorylation inhibitor containing at least one selected from the group consisting of the compound represented by the formula, salts thereof, and solvates thereof.

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