Plant growth regulator, plant growth regulation method, plant growth regulation device, plant growth regulation system, and plant growth regulation kit

Abstract

To provide a novel method for promoting plant growth by controlling opening and closing of plant stomata.SOLUTION: A plant growth regulator comprises an abscisic acid (ABA) agonist that induces closure of stomata and does not inhibit opening of stomata.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a plant growth regulator, a plant growth regulation method, a plant growth regulation device, a plant growth regulation system, and a plant growth regulation kit.

Background Art

[0002] Conventionally, as plant growth regulators, selective herbicides for harmful plants (Patent Document 1), agents for regulating the growth of specific plant species of interest (Patent Document 2), and agents having an action of enhancing pest and disease resistance (Patent Document 3) have been proposed.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0004] An object of the present invention is to provide a new method for promoting plant growth by controlling the opening and closing of plant stomata.

Means for Solving the Problems

[0005] The present inventors have first clarified that although pyrabactin induces stomatal closure like ABA, unlike ABA, it does not inhibit stomatal opening, and further have found that by utilizing this property of pyrabactin, the opening and closing of plant stomata can be controlled. The present invention has been completed by further examination based on such findings and includes the following aspects.

[0006] Item 1. A plant growth regulator comprising an abscisic acid (ABA) agonist that induces stomatal closure and does not inhibit stomatal opening. Item 2. The plant growth regulator according to Item 1, which is used in combination with a stomatal opening promoting factor. Item 3. The plant growth regulator according to Item 1 or 2, wherein the ABA agonist is a non-ABA analog type. Item 4. The plant growth regulator according to Item 1 or 2, wherein the ABA agonist binds to a dimeric ABA receptor. Item 5. The plant growth regulator according to Item 1 or 2, wherein the ABA agonist is pyrabactin. Item 6. (A) A step of supplying a plant growth regulator containing an ABA agonist that induces stomatal closure and does not inhibit stomatal opening to a plant A plant growth regulation method comprising the above. Item 7. (B) A step of supplying a stomatal opening promoting factor to a plant The plant growth regulation method according to Item 6, further comprising the above. Item 8. A plant growth regulation device comprising means for supplying an ABA agonist that induces stomatal closure and does not inhibit stomatal opening to a plant. Item 9. The plant growth regulation device according to Item 8, further comprising means for supplying a stomatal opening promoting factor to a plant. Item 10. (A) A measurement unit for measuring the degree of stomatal opening of a plant; (B) A first supply unit for supplying an ABA agonist that induces stomatal closure and does not inhibit stomatal opening to a plant; and (C) A second supply unit for supplying a stomatal opening promoting factor to a plant A plant growth regulation system comprising the above. Item 11. A first agent containing an ABA agonist that induces stomatal closure and does not inhibit stomatal opening; and A second agent containing a stomatal opening promoting factor A plant growth regulation kit comprising the above.

Advantages of the Invention

[0007] According to the present invention, a new method for promoting plant growth by controlling the opening and closing of plant stomata can be provided.

Brief Description of the Drawings

[0008]

Figure 1

Figure 2

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Figure 8

Mode for Carrying Out the Invention

[0009] The terms used in this specification have the meanings commonly used in the relevant technical field. These terms, their definitions, and meanings can be easily understood by referring to various public information such as various academic documents, educational texts, technical documents, web pages, etc. that are available or accessible at the time of filing this application. In the case where there is a discrepancy between the description in this specification and the description in the public information, the description in this specification shall be preferentially applied.

[0010] 1. Plant growth regulator of the present invention The plant growth regulator of the present invention contains an abscisic acid (ABA) agonist that induces stomatal closure and does not inhibit stomatal opening.

[0011] "Plant growth regulator" means an agent used for growing plants, and is distinguished from agents used for imparting resistance to external stresses such as drying resistance agents and low-temperature adaptation agents, and agents used for promoting fruit coloring. More specifically, "plant growth regulator" includes agents used for increasing the weight of the above-ground part and / or underground part of plants, agents used for promoting seed ripening, and the like.

[0012] "Stomata" are small pores on the surface of the plant body. Stomata are present in the epidermis of almost all plants having vascular bundles and are composed of a small pore and two guard cells surrounding it. When the stomata open, gas exchange between the plant body and the atmosphere is carried out, thereby enabling the absorption of CO2 by photosynthesis, transpiration, etc. Also, transpiration cools the temperature of the leaves, and the stomata present in the petals also help in the uptake of oxygen as a respiratory substrate. On the other hand, when the stomata close, these functions are stopped. That is, by controlling the opening and closing of the stomata, the plant body can regulate these biological functions.

[0013] Stomata are abundant in the epidermis of green tissues, especially on the leaf surface and stem, but also in petals, sepals, stamens, the pericarp of bananas, grapes, apples, etc., and even in roots and rhizomes. In plants with stomata on both sides of the leaf (amphistomatous leaves), the number of stomata on the lower surface is usually greater. There are also those that exist only on the back of the leaf (hypostomatous leaves), such as many woody plants, and those that exist only on the upper surface (epistomatous leaves), such as water lilies. The density of stomata often varies at the tip, center, base, and periphery of the leaf and also changes depending on environmental conditions. In many monocotyledonous plants and gymnosperms, such as duckweeds, onions, corn, and sugarcane, the stomata are arranged parallel to the leaf veins, while in dicotyledonous plants, such as broad beans and tobacco, there is no specific orientation. In saxifrage, the stomata form clusters and are present in patches on the leaf surface, sharing a stomatal cavity. The shape of the guard cells is roughly divided into two major types: kidney-shaped and dumbbell-shaped. In gymnosperms and dicotyledonous plants, they are kidney-shaped, and the stomata are elliptical. Dumbbell-shaped stomata are limited to some monocotyledonous plants, and in the Poaceae family, such as corn and sugarcane, all stomata are dumbbell-shaped and the shape of the stomata is slit-like.

[0014] The opening and closing of stomata are induced by morphological changes controlled by the uneven thickness of the cell wall and the orientation of cellulose microfibrils as the volume of the guard cells increases and decreases. When K + , Cl - , and malate ions accumulate in the guard cells, the water potential of the guard cells decreases, water flows in, the turgor pressure of the guard cells increases, and the stomata open. At this time, the driving force for the uptake of K + against the concentration gradient is formed by the plasma membrane proton-ATPase. This enzyme is activated by the blue light absorbed by phototropin, transports H + outside the guard cells, and forms a negative membrane potential inside the cell. In response to this membrane potential, the voltage-dependent inward rectifying K + channels on the same membrane open, and K + is electrophoretically taken up and accumulated inside the guard cells. In the afternoon, it has been reported that sucrose replaces K + and Cl - and acts as an osmotic substance to lower the water potential and maintain the afternoon stomatal aperture, but the molecular mechanism is unclear.

[0015] Stomatal closure is induced by the plant hormone abscisic acid and high concentrations of CO2. For example, when the concentration of abscisic acid increases, the cell membrane anion channel is activated, and malic acid and Cl - accumulated in the guard cells flow out according to the concentration gradient. As a result, the membrane potential of the guard cells shifts to the positive side (depolarization), and the depolarization-dependent outward rectifying K + channel on the same membrane is activated, and now the accumulated K + flows out. At this time, the cell membrane proton-ATPase that drives stomatal opening is inhibited by abscisic acid, which helps to keep the membrane potential on the depolarized side. In this way, anions and K + flow out continuously, and the stomata close. The inward rectifying K + channel and the outward rectifying K + channel involved in stomatal opening are different proteins.

[0016] The ABA agonist used in the present invention (hereinafter sometimes referred to as "the ABA agonist of the present invention") induces stomatal closure and does not inhibit stomatal opening. Examples of such ABA agonists include pyrabactin (the compound of the following formula (Ia)). Other such ABA agonists can be obtained by selecting from among ABA agonists those having the action of inducing stomatal closure and not inhibiting stomatal opening. Since the fact that pyrabactin has the above action was first discovered by the present inventors, such knowledge can also be used as an index in the above selection to indicate the commonality in terms of the structure and / or properties with pyrabactin.

[0017]

Chemical formula

[0018] For example, a compound of the following formula (Ia) having similarity with pyrabactin in terms of structure can be used as an ABA agonist in the present invention.

Chemical formula

[0019] In the above, R 1 ~R 3 are preferably H.

[0020] In the above, R 4 is preferably 1 to 4 R 4aIt may be a substituted aryl group or heteroaryl group, more preferably an unsubstituted aryl group or heteroaryl group.

[0021] In the above, X is preferably Br.

[0022] Since pyrabactin is a non-ABA analog type, as the ABA agonist used in the present invention, a non-ABA analog type is preferred. Examples of non-ABA analog type ABA agonists include, in addition to pyrabactin, quinabactin (compound of the following formula (II)), sinabactin (compound of the following formula (III)), and mandipropamid (compound of the following formula (IV)).

[0023]

Chemical formula

[0024]

Chemical formula

[0025]

Chemical formula

[0026] For example, a compound of the following formula (IIa) having similarity in structure to quinabactin can be used as an ABA agonist in the present invention.

Chemical formula

[0027] Examples of the compound of formula (IIa) include AMF4 (compound of the following formula (V)) and the like.

[0028]

Chemical formula

[0029] For example, a compound of the following formula (IIIa), which has a similarity in structure to sinabactin, can be used as an ABA agonist in the present invention.

Chemical formula

[0030] Since pyrabactin binds to the dimeric ABA receptor, as the ABA agonist used in the present invention, those that bind to the dimeric ABA receptor are preferred.

[0031] The ABA agonists used in the present invention include modified forms of pyrabactin. Such modified forms can be prepared by known methods. For example, they can be prepared by chemically modifying pyrabactin, or alternatively, by modifying the steps in the known production method of pyrabactin.

[0032] The ABA agonists used in the present invention induce stomatal closure and do not inhibit stomatal opening, and the fact that they have such a function can be confirmed by a closure induction assay and an opening inhibition assay. These assays are specifically carried out as follows.

[0033] <Closure Induction Assay> Excised rosette leaves are floated on an opening buffer containing 5 mM KCl, 50 μM CaCl2, and 10 mM MES-Tris (pH 6.15) for 2 hours under light (80 μmol m -2 s -1 ) to pre-open the stomata. Next, pyrabactin (product number B-3538, Sigma-Aldrich Inc., St. Louis, MO, USA) or ABA (Sigma-Aldrich Inc.) is added to the opening buffer, and the mixture is further incubated for 2 hours under light. The opening width is measured under a microscope after releasing the epidermal specimens by blender treatment with a commercially available Waring blender (BB-700, Waring Products, Torrington, MO, USA) for 30 seconds after incubation.

[0034] <Opening Inhibition Assay> Rosette leaves cut from dark-adapted plants (overnight in the dark for 8 hours) are treated with a blender under dim green light (without blue and red light) before the start of the light period. The excised epidermal specimens are suspended in the opening buffer in the dark. Subsequently, after incubating in the dark for 2 hours, they are incubated in the light for 2.5 hours in the presence or absence of pyrabactin or ABA. The opening width is measured under a microscope.

[0035] The plant growth regulator of the present invention contains the ABA agonist of the present invention as an active ingredient. The plant growth regulator of the present invention preferably contains the ABA agonist of the present invention at 0.4 to 40 weight ppm based on the whole plant growth regulator. Alternatively, the plant growth regulator of the present invention contains the ABA agonist of the present invention at 0.4 to 30 ppm, 0.4 to 20 ppm, 0.4 to 10 ppm, 0.4 to 5 ppm, 0.7 to 40 ppm, 0.7 to 30 ppm, 0.7 to 20 ppm, 0.7 to 10 ppm, 0.7 to 5 ppm, 1 to 40 ppm, 1 to 30 ppm, 1 to 20 ppm, 1 to 10 ppm, 1 to 5 ppm, 2 to 40 ppm, 2 to 30 ppm, 2 to 20 ppm, 2 to 10 ppm, or 2 to 5 ppm based on the whole plant growth regulator.

[0036] The plant growth regulator of the present invention may further contain other components. Examples of other components generally include those components formulated in plant growth regulators. For example, as other active ingredients, components having a plant growth regulating effect may be included.

[0037] The plant growth regulator of the present invention may further contain at least one additive selected from the group consisting of carriers such as solid carriers or liquid carriers, surfactants, binders, tackifiers, thickeners, colorants, antifreezing agents, anti-caking agents, disintegrants, anti-decomposition agents, preservatives, etc., as necessary. The content ratio of each additive can be appropriately adjusted within a range that does not impair the effects of the present invention.

[0038] Examples of liquid carriers include monohydric alcohols, polyhydric alcohols, ketones, ethers, aliphatic hydrocarbons, aromatic hydrocarbons, halogenated hydrocarbons, esters, lactones, amides, nitriles, sulfur compounds, vegetable oils, water, etc. These may be used alone or in combination of two or more.

[0039] Although not particularly limited, examples of monohydric alcohols include methanol, ethanol, propanol, isopropanol, butanol, etc.

[0040] Although not particularly limited, examples of the polyhydric alcohol include ethylene glycol, diethylene glycol, propylene glycol, hexylene glycol, polyethylene glycol, polypropylene glycol, glycerin and the like.

[0041] Although not particularly limited, examples of the polyhydric alcohol derivative include propylene glycol ether and the like.

[0042] Although not particularly limited, examples of the ketone include acetone, methyl ethyl ketone, methyl isobutyl ketone, diisobutyl ketone, cyclohexanone, isophorone and the like.

[0043] Although not particularly limited, examples of the ether include ethyl ether, dioxane, cellosolve, dipropyl ether, tetrahydrofuran and the like.

[0044] Although not particularly limited, examples of the aliphatic hydrocarbon include normal paraffin, naphthene, isoparaffin, kerosene, mineral oil and the like.

[0045] Although not particularly limited, examples of the aromatic hydrocarbon include benzene, toluene, xylene, solvent naphtha, alkyl naphthalene and the like.

[0046] Although not particularly limited, examples of the halogenated hydrocarbon include dichloroethane, chloroform, carbon tetrachloride and the like.

[0047] Although not particularly limited, examples of the ester include ethyl acetate, diisopropyl phthalate, dibutyl phthalate, dioctyl phthalate, dimethyl adipate and the like.

[0048] Although not particularly limited, examples of the lactone include γ-butyrolactone and the like.

[0049] Although not particularly limited, examples of the amide include dimethylformamide, diethylformamide, dimethylacetamide, N-alkylpyrrolidinone, and the like.

[0050] Although not particularly limited, examples of the nitrile include acetonitrile and the like.

[0051] Although not particularly limited, examples of the sulfur compound include dimethyl sulfoxide and the like.

[0052] Although not particularly limited, examples of the vegetable oil include soybean oil, rapeseed oil, cottonseed oil, castor oil, and the like.

[0053] The plant growth regulator of the present invention can be used for plant cultivation. Since the plant growth regulator of the present invention contains an abscisic acid (ABA) agonist that induces stomatal closure and does not inhibit stomatal opening, it can be effectively used in plants with a large number of stomata or plants in which stomata are actively functioning. As such plants, herbaceous plants rich in leaves are preferred, but the use is not limited to woody plants and the like. The plant growth regulator of the present invention is limited only by the fact that it contains an abscisic acid (ABA) agonist that induces stomatal closure and does not inhibit stomatal opening, and can regulate plant growth in various ways under various conditions including the number, state, temperature, humidity, light conditions, and atmospheric pressure of other stomata, and its action is not limited to that under a specific environment. Specific plants that can be exemplified include a wide range of plants existing in nature, such as monocotyledonous plants such as rice, lily, tulip, miscanthus, and corn, dicotyledonous plants with combined petals such as morning glory, dandelion, azalea, camellia, and eggplant, dicotyledonous plants with separate petals such as rape, pea, cherry, and petunia, gymnosperms such as pine, cedar, ginkgo, cycad, and cypress, and fern plants such as bracken, horsetail, polypodium, and pteris, but are not limited thereto.

[0054] The plants targeted by the plant growth regulator of the present invention are not particularly limited and can be widely selected. The plant growth regulator of the present invention can be applied to any plant that opens and closes stomata, but applying it to plants of industrial value can bring great benefits to producers. Such plants include edible plants, forage plants, experimental plants, ornamental plants, industrial plants (industrial crops), and the like.

[0055] Examples of edible plants include cereals, vegetables, and fruits.

[0056] Examples of cereals include seeds derived from Gramineae crops such as rice (paddy rice), wheat, barley, rye, buckwheat, oats, millet, foxtail millet, Japanese millet, sorghum, corn, and seeds (beans) of leguminous crops such as broad bean, chickpea, lentil, quinoa, soybean, adzuki bean, or plants of other families, for example, buckwheat (Polygonaceae), amaranth (Amaranthaceae), quinoa (Chenopodiaceae), etc.

[0057] Examples of vegetables include cruciferous plants such as cabbage, komatsuna, Chinese cabbage, tatsoi, chingensai, broccoli, cauliflower, daikon radish, turnip, mizuna, and cauliflower; umbelliferous plants such as carrot, celery, parsley, mitsuba, ashitaba, and Florence fennel; cucurbitaceous plants such as cucumber, pumpkin, watermelon, bitter gourd, zucchini, winter melon, and bottle gourd; alliaceous plants of the subfamily Allium such as onion, leek, Japanese leek, garlic chives, garlic, rakkyo, and leek; solanaceous plants such as eggplant, tomato, pepper, paprika, chili pepper, ornamental pepper, and potato; leguminous plants such as kidney bean, pea, edamame (soybean), and peanut; malvaceous plants such as okra and moringa; asteraceous plants such as lettuce, shungiku, burdock, edible chrysanthemum, artichoke, chicory, trevis, endive, goldenrod, and yacon; zingiberaceous plants such as ginger and myoga; convolvulaceous plants such as sweet potato and kudzu; labiatae plants such as perilla and sesame; gramineous plants such as corn and bamboo shoots; asparagaceous plants such as asparagus; araceous plants such as taro and elephant yam; chenopodiaceous plants such as spinach, okahijiki, beet, and Swiss chard; and pedaliaceae plants such as sesame, but are not limited thereto.

[0058] Examples of fruits include lardizabalaceae such as akebia; acerola; avocado; apricot; strawberry; fig; Japanese apricot; citrus fruits such as mandarin orange, orange, persimmon, loquat, citrus fruits, kiwifruit, horned melon, kumquat, chestnut, guava, grapefruit, cherry, pomegranate, watermelon, star fruit, plum, pear, cherry plum, Chinese pear, dragon fruit, durian, pear, nectarine, banana, pineapple, passion fruit, papaya, loquat, grape, blueberry, prune, berries (such as blackcurrant, gooseberry, cranberry, huckleberry, blackberry, raspberry, etc.), quince, mangosteen, mango, melon, peach, yuzu, lychee, lime, apple, lemon, etc., but are not limited thereto.

[0059] Forage plants are synonymous with forage crops and are plants that are cultivated for use as livestock feed, directly or after processing, and are used as livestock feed. Typical examples include corn, rye grass, timothy grass, soybeans, etc.

[0060] Experimental plants refer to plants used for purposes such as academic research. Examples include Arabidopsis thaliana, tobacco, tomato, Japanese millet, etc.

[0061] Ornamental plants are plants in which the flowers, leaves, or the entire plant body are used for ornamental purposes. For example, they are used as cut flowers, potted plants (flowering potted plants, tree potted plants, foliage plants, etc.), flower bed seedlings, trees (potted trees), ground cover plants, lawns, and others (such as potted plants and succulents). Examples of plants in which the flowers are admired include, but are not limited to, morning glory, hydrangea, carnation, gerbera, baby's breath, cosmos, cyclamen, lily of the valley, violet, dandelion, tulip, pink, rose, pansy, viola, sunflower, lily, lavender, etc. Examples of plants in which the leaves are admired include, but are not limited to, olive, cactus, dracaena, pachira, poinsettia, pothos, monstera, etc.

[0062] Industrial plants (industrial crops) include fiber plants such as cotton, hemp, kenaf, etc.; tatami plants such as rush; Japanese paper plants such as mulberry, mitsumata, and butterbur; oil plants such as rapeseed, safflower, sunflower, sesame, perilla, olive, etc.; sweet sugar plants such as sugarcane, sugar beet, licorice (stevia), etc.; starch and paste plants such as sweet potato, potato, corn, konjac, etc.; preference plants such as tea, tobacco leaves, etc.; spice plants such as lavender, jasmine, etc.; resin plants such as lacquer, hazelnut, etc.; dye plants such as indigo, safflower, red cabbage, etc.; medicinal plants such as pyrethrum, peppermint, angelica, young barley leaves, turmeric, kale, etc., but are not limited to these.

[0063] The plant growth regulator of the present invention is characterized by containing an abscisic acid (ABA) agonist that induces stomatal closure and does not inhibit stomatal opening. Although it seemingly causes stomatal closure and appears to inhibit plant growth, in another stage that brings about stomatal opening, the mechanism of stomatal opening proceeds as normal, thus enabling the creation of a process that promotes plant growth. In this regard, it is different from conventional abscisic acid (ABA) agonists that merely induce stomatal closure. Therefore, after inducing stomatal closure using the plant growth regulator of the present invention, it is possible to perform operations such as irradiating blue light to open the stomata again. For example, in situations where it is desired to freely control stomatal closure and opening in response to changes in temperature stress or drought stress, the plant growth regulator of the present invention demonstrates its true value. More specifically, in situations where the water supply is insufficient, it is difficult to supply water regularly, or in such environments, the growth of plants can be optimally controlled by freely manipulating stomatal closure and opening. For example, the case of growing plants under high-temperature and dry conditions will be described as an example. Normally, as long as the plant to be grown is regarded as a plant under drought stress and the plant growth regulator of the present invention is administered without light stimulation, the stomata of this plant will remain closed. On the other hand, when it is time to water the plant, at that time, blue light can be irradiated to open the stomata. When the stomata open, the water absorption of the plant will be promoted. If it seems to be exposed to excessive drought stress again, the stomata can be closed again by performing light interruption again and administering the plant growth regulator of the present invention.

[0064] The plant growth regulator of the present invention can be applied to plants in open field cultivation, but the true value of the present invention is more effectively demonstrated under more controlled plant cultivation conditions. For example, although agricultural greenhouses that control temperature, humidity, and water supply in greenhouses have already been put into practical use, the reproducibility of plant yields and the like varies greatly, and the current situation is that the results are unpredictable. The causes may include differences in the daily handling of producers, subtle changes in the soil, variations in the seedlings themselves, etc., but it is still difficult to obtain completely consistent results at present.

[0065] Therefore, the plant growth regulator of the present invention can exhibit its performance better when plants are placed under more controlled growth conditions. For example, in a plant factory where the growth conditions of plants can be quantitatively controlled, when the plant growth regulator of the present invention is used, it is possible to more quantitatively evaluate the effects of the plant growth regulator of the present invention.

[0066] Generally, a plant factory refers to "among the protected horticulture facilities that control the growth environment (light, temperature, humidity, carbon dioxide concentration, nutrients, water, etc.) of plants within the facility for cultivation, a cultivation facility that can perform annual and planned production of plants such as vegetables by performing advanced environmental control and growth prediction based on the monitoring of the environment and growth. That is, it means a system that artificially creates an environment suitable for cultivation and produces plants systematically. "Plant factory" can be divided into a "fully artificial light type" that produces in a closed environment without using sunlight and controls the environment, and a "sunlight utilization type" that produces mainly using sunlight in a semi-closed environment such as a greenhouse and uses supplementary lighting on rainy and cloudy days and high-temperature suppression technology in summer. As of February 2022 in Japan, approximately 400 "plant factories" (190 fully artificial light type, 176 sunlight utilization type, 38 combined type) are in operation, and the operating entities include food manufacturers, the food service industry, venture companies, construction companies, electrical appliance manufacturers, etc.

[0067] By using the plant growth regulator of the present invention in the process of production management in a plant factory, it is possible to brake the growth of plants by closing the stomata to block water absorption and gas exchange through the stomata, and then restart the plant growth process again by irradiating blue light.

[0068] 2. Plant growth regulation method of the present invention The plant growth regulation method of the present invention (A) A step of supplying a plant growth regulator containing the ABA agonist of the present invention to a plant is included.

[0069] The plant growth regulation method of the present invention (B) A step of supplying a stomatal opening promoting factor to a plant may further be included. Thereby, the opening and closing of stomata in plants can be adjusted, and their growth can be regulated.

[0070] The stomatal opening promoting factor is not particularly limited, and known ones can be widely selected. For example, red light, blue light, artificial auxin, natural auxin, artificial cytokinin, natural cytokinin, and cytokinin degrading enzyme inhibitor (forchlorfenuron), etc. can be mentioned.

[0071] 3. Device for plant growth regulation of the present invention The plant growth regulation device of the present invention includes means for supplying the ABA agonist of the present invention to a plant.

[0072] The means for supplying the ABA agonist of the present invention to a plant is not particularly limited, but for example, a sprayer, manual application, mechanical application, injection, transdermal patch, supply through an aqueous solution absorbed from the roots (including soil or hydroponics), absorption from the cut surface of the stem or leaf, and absorption of water vapor, etc. can be mentioned.

[0073] The plant growth regulating device of the present invention may further comprise means for supplying a stomatal opening promoting factor to the plant. Thereby, the opening and closing of stomata in the plant can be adjusted, and its growth can be regulated. The means for supplying a stomatal opening promoting factor to the plant is not particularly limited. For example, when the stomatal opening promoting factor is light, natural light, means for generating natural light (combustion means such as candles and gunpowder), and artificial light sources such as electric bulbs and LEDs can be mentioned. In the case of other than light, a sprayer, manual application, mechanical application, injection, transdermal patch, supply through an aqueous solution absorbed from the roots (including soil or hydroponic cultivation), absorption from the cut surface of the stem or leaf, and absorption of water vapor can be mentioned.

[0074] 4. System for plant growth regulation of the present invention The plant growth regulating system of the present invention (A) a measuring unit for measuring the stomatal aperture of a plant; (B) a first supply unit for supplying the ABA agonist of the present invention to the plant; and (C) a second supply unit for supplying a stomatal opening promoting factor to the plant comprises.

[0075] The measuring unit is not particularly limited. For example, a microscope, an infrared gas analyzer, a thermography camera and a thermocouple, a CCD camera, a CMOS camera, a MOS camera, a single-lens reflex camera, and a mirrorless camera can be mentioned.

[0076] The first supply unit and the second supply unit are not particularly limited. For example, a sprayer, manual application, mechanical application, injection, transdermal patch, supply through an aqueous solution absorbed from the roots (including soil or hydroponic cultivation), absorption from the cut surface of the stem or leaf, and absorption of water vapor can be mentioned.

[0077] 5. Kit for plant growth regulation of the present invention The plant growth regulating kit of the present invention a first agent containing the ABA agonist of the present invention; and a second agent containing a stomatal opening promoting factor comprises.

[0078] For the stomatal opening promoting factor, those described above can be used.

Example

[0079] 1. Materials and Methods 1-1. Plant Materials Arabidopsis thaliana (L.) Heynh. wild type (Col-0), pyr1 pyl1 pyl2 pyl4 pyl5 pyl8 hexuple mutant (Gonzalez-Guzman et al., 2012), pyr1 pyl1 pyl2 pyl4 quadruple mutant (Park et al., 2009), and atrbohD / F double mutant (Kwak et al., 2003) were grown in a soil mixture consisting of 70% (v / v) vermiculite (Asahi Industry) and 30% (v / v) Kureha soil (Kureha Chemical). The temperature and relative humidity in the growth chamber were controlled at 22°C ± 2°C and 60% ± 10%, respectively. The photoperiod was a 16-hour light / 8-hour dark regime, and the photon flux with white fluorescent tubes was 80 μmol m -2 s -1 . The alleles of each mutant were as follows: pyr1, Q169stop; pyl1, SALK_054640; pyl2, GT2864; pyl4, SAIL_517_C08; pyl5, SM3_3493; pyl8, SAIL_1269_A02; atrbohD, SALK_070610, and atrbohF, SALK_059888 (Park et al., 2009; Gonzalez-Guzman et al., 2012).

[0080] 1-2. Measurement of Stomatal Aperture Induction of stomatal closure and inhibition of opening were measured as previously described (Munemasa et al., 2019). In the closure induction assay, excised rosette leaves were placed on an opening buffer containing 5 mM KCl, 50 μM CaCl2, and 10 mM MES-Tris (pH 6.15) for 2 hours under light (80 μmol m -2 s -1Float it under () to pre-open the stomata. Next, add pyrabactin (product number B-3538, Sigma-Aldrich Inc., St. Louis, MO, USA) or ABA (Sigma-Aldrich Inc.) to the opening buffer and incubate for an additional 2 hours under light. The opening width was measured under a microscope after incubation following the release of the epidermal specimen by blender treatment for 30 seconds with a commercially available Waring blender (BB-700, Waring Products, Torrington, MO, USA) unless otherwise specified.

[0081] In the aperture inhibition assay, rosette leaves excised from dark-adapted plants (overnight in the dark for 8 hours) were treated with a blender under dim green light (without blue and red light) before the start of the light period. The excised epidermal specimens were suspended in the opening buffer in the dark. Subsequently, after incubation in the dark for 2 hours, they were incubated in the light for 2.5 hours in the presence or absence of pyrabactin or ABA. The opening width was measured under a microscope.

[0082] 1-3.H 2 O 2 and Measurement of NO Generation The generation of H2O2 and NO in guard cells was examined using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA, Sigma-Aldrich Inc.) and diaminofluorescein-2-diacetate (DAF-2DA, Sigma-Aldrich Inc.), respectively, as described elsewhere (Ying et al., 2016; Munemasa et al., 2019). Briefly, the epidermis was collected from leaves of 4- to 6-week-old plants by blender treatment, incubated in the light in the opening buffer for 2 h, and then incubated with 50 μM H2DCF-DA or 5 μM DAF-2DA for 30 min at room temperature to load the dyes into guard cells. Subsequently, it was rinsed with distilled water on a nylon mesh to remove excess dye. The epidermis loaded with the dyes was continuously treated with 10 μM pyrabactin or ABA (or solvent control) for 20 min. Fluorescence of guard cells was imaged using a fluorescence microscope (BZ-8000, Keyence Corporation, Osaka, Japan). The fluorescence intensity of guard cells was semi-quantified using ImageJ software (http: / / imagej.net).

[0083] 1-4. Measurement of Cytosolic pH Change According to Ying et al. (2016), changes in the cytosolic pH (pHcyt) of guard cells were examined using 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester (BCECF-AM, Dojindo, Kumamoto, Japan). Epidermal tissues isolated from 4- to 6-week-old plants by blender were incubated in the light in the opening buffer at 80 μmol m -2 s -1It was incubated for 3 hours under

[0084] 1-5. Patch-Clamp Measurement inward-rectified K + current (I kin ) was measured by whole-cell patch clamp of isolated guard cell protoplasts according to previous reports (Ying et al., 2016).

[0085] 1-6. Cell Membrane H in Guard Cells + -ATPase Phosphorylation According to previous reports (Hayashi et al., 2011), blue light-induced phosphorylation of plasma membrane H + -ATPase in guard cells was examined immunohistochemically using an antiserum against the second phosphorylated threonine from the end of the H + -ATPase polypeptide (anti-pThr). Briefly, epidermal tissue was isolated from leaves of plants adapted to the dark using a commercially available blender in the dark, and incubated for 20 minutes with 10 μM pyrabactin or ABA (or solvent control) under a background red light of 50 μmol m -2 s -1 in incubation buffer containing 5 mM KCl, 50 μM CaCl2, and 10 mM MES-Tris (pH 6.15), and then 10 μmol m -2 s -1Blue light treatment was performed for 2.5 minutes. Epidermal tissues were fixed before and after blue light irradiation. After these treatments, epidermal fragments were collected on a nylon net sheet. The collected fragments were fixed with paraformaldehyde, attached to a coverslip, and then treated with 3% Driselase 20 (Kyowa Hakko Kogyo Co., Ltd., Tokyo) and 0.5% Macerozyme R-10 (Yakult Pharmaceutical Industry Co., Ltd., Tokyo) at 37 °C for 45 minutes. As described by Hayashi et al. (2011), after permeabilizing the cells with 3% TritonX-100 at room temperature for 30 minutes, phosphorylated H + -ATPase was visualized with anti-pThr diluted 1:1000 in PBS containing 3% BSA and Alexa Fluor488-labeled secondary antibody diluted 1:500 in PBS containing 3% BSA. Fluorescent images were observed with a fluorescence microscope (BX50, Olympus) equipped with LED and LDP light sources (U-LGPS, Olympus) and photographed using a CCD camera system (DP71, Olympus). Fluorescent intensity was quantified with ImageJ.

[0086] 1-7. Statistical Analysis Student's t-test and ANOVA using the Tukey-Kramer test or Dunnett's test were used to evaluate the significance of differences between datasets. Differences at the p < 0.05 level were considered significant.

[0087] 2. Results 2-1. Pyrabactin Does Not Inhibit Light-Induced Stomatal Aperture, but ABA Does Some ABA receptors have a high specific affinity for pyrabactin, an ABA agonist (Melcher et al., 2010; Okamoto et al., 2013). Using this unique affinity, we investigated the bioactivity of pyrabactin against two different stomatal responses, namely closure induction and aperture inhibition, to elucidate the differential contributions of ABA receptor isoforms and the potential of pyrabactin for the selective regulation of stomatal behavior.

[0088] When exogenous pyrabactin was applied to the entire excised leaf of wild-type Arabidopsis thaliana, stomatal closure and ABA induction were observed, consistent with previous studies (Okamoto et al., 2013) (Figs. 1A and B). When pyrabactin was applied to the isolated epidermis, a similar trend of response to that of the whole leaf was obtained, but the absolute aperture width was narrower in the purified epidermal specimens. The narrowing of the apertures in the isolated epidermis may be caused by the absence of the mesophyll effect that promotes stomatal opening during the pre-opening procedure (Fujita et al., 2013). The stomatal aperture width of the sextuple mutant was generally wider than that of the wild-type and quadruple mutants. This may be due to the influence of the action from the mesophyll mediated by PYL5 and / or PYL8. When the purified epidermis in dark adaptation was incubated under light conditions, stomatal opening from 0.61 × 0.04 μm to 1.42 × 0.07 μm was induced in the absence of pyrabactin. In contrast to ABA (Fig. 1D), the opening was not inhibited in the presence of 1 μM and 10 μM pyrabactin (Fig. 1C). Since the concentration of K + in the experimental buffer may affect stomatal opening, an opening inhibition assay was performed in the presence of 5 mM KCl (Figs. 1C and D). No significant inhibition of opening by 1 μM and 10 μM pyrabactin was observed, but the inhibition by ABA was significant and consistent with the 5 mM KCl condition.

[0089] In addition to the wild type, the effects of ABA and pyrabactin on stomatal movement were examined in two ABA receptor mutants, the pyr1 pyl1 pyl2 pyl4 quadruple mutant (Park et al., 2009) and the pyr1 pyl1 pyl2 pyl4 pyl5 pyl8 sextuple mutant (Gonzalez-Guzman et al., 2012). These mutants have defects in ABA-induced stomatal closure (Gonzalez-Guzman et al., 2012 and Figure 1B). In contrast to wild-type plants, pyrabactin did not induce stomatal closure in either mutant (Figure 1A). The same trend of results was obtained in isolated epidermis. Pyrabactin also did not inhibit light-induced stomatal opening in the quadruple and sextuple mutants as in the wild type, even when the KCl concentration in the assay solution was changed (Figure 1C). It is noteworthy that the inhibition of opening occurred more strongly in the sextuple mutant compared to the wild type (Figure 1D). This is probably because the stomatal apertures of the sextuple mutant were wider under 0 μM ABA conditions.

[0090] These results indicate that the biological activity of pyrabactin in the regulation of stomatal movement can be partially distinguished from that of ABA. The phenotype of the sextuple mutant suggests that the pyrabactin-binding ABA receptor is not sufficient for aperture inhibition.

[0091] 2-2. Regulation of I by Pyrabactin Kin Control The transport of K across the plasma membrane of guard cells is important for stomatal movement. Many studies have shown that ABA inhibits I in guard cells (Hedrich et al., 1989; Blatt et al., 1993). Here, the inventors examined the effect of pyrabactin on the amplitude of I in Arabidopsis guard cell protoplasts isolated by whole-cell patch clamp (Figure 2). Application of 50 μM pyrabactin, in contrast to 50 μM ABA (Yin et al., 2013), did not affect I in wild type. + transport is important for stomatal movement. Many studies have shown that ABA inhibits I in guard cells (Hedrich et al., 1989; Blatt et al., 1993). Here, the inventors examined the effect of pyrabactin on the amplitude of I in Arabidopsis guard cell protoplasts isolated by whole-cell patch clamp (Figure 2). Application of 50 μM pyrabactin, in contrast to 50 μM ABA (Yin et al., 2013), did not affect I in wild type. Kin transport in guard cells (Hedrich et al., 1989; Blatt et al., 1993). Here, the inventors examined the effect of pyrabactin on the amplitude of I in Arabidopsis guard cell protoplasts isolated by whole-cell patch clamp (Figure 2). Application of 50 μM pyrabactin, in contrast to 50 μM ABA (Yin et al., 2013), did not affect I in wild type. Kin transport in guard cells (Hedrich et al., 1989; Blatt et al., 1993). Here, the inventors examined the effect of pyrabactin on the amplitude of I in Arabidopsis guard cell protoplasts isolated by whole-cell patch clamp (Figure 2). Application of 50 μM pyrabactin, in contrast to 50 μM ABA (Yin et al., 2013), did not affect I in wild type. Kindid not inhibit. As shown in Figures 2B and 2C, the quadruple and sextuple mutants also demonstrated the lack of inhibitory effect of pyrabactin on I Kin It was noted that the current amplitude was greater in these mutants than in the wild type. This may be explained by the effect of ABA that reduces the expression of KAT1 and AKT1 genes in guard cells (Inoue et al., 2017), which is impaired in these ABA receptor mutants. This result suggests that PYR1 and PYL1, which are ABA receptors with high affinity for pyrabactin, do not mainly control the inactivation of I Kin by pyrabactin.

[0092] 2-3. Pyrabactin Inhibits Blue Light-Induced Phosphorylation of Cell Membrane H + -ATPase H + -ATPase activation is an important step in light-induced stomatal opening (Shimazaki et al., 1986). Phosphorylation of the second last threonine residue of H + -ATPase is a prominent feature of H + -ATPase activation (Kinoshita et al., 1999). The effect of pyrabactin on the phosphorylation of the second last threonine of the plasma membrane H + -ATPase in guard cells was examined by immunohistochemical staining using specific antiserum (Ying et al., 2013; Hayashi et al., 2011). In background red light, H + -ATPase was slightly phosphorylated in guard cells of the wild type and sextuple mutants (Figure 3). Illumination with blue light overlaid on background red light enhanced the phosphorylation of H + -ATPase in both the wild type and sextuple mutants (Figure 3). Addition of 10 μM pyrabactin completely inhibited the phosphorylation of H + -ATPase in the wild type and sextuple mutants such as ABA (Figure 3). Stomatal opening was not inhibited by pyrabactin, but H +-ATPase activation was inhibited by pyrabactin, which is worth noting. These results indicate that the ABA receptor that binds to pyrabactin is not sufficient to inhibit H + -ATPase activation, suggesting that some other PYL members that can bind to pyrabactin or an as-yet-unidentified part of the ABA receptor are involved in the inactivation of H + -ATPase. Although it has been established that the inactivation of the H + pump is essential for stomatal inhibition (Shimazaki et al., 1986; Takemiya et al., 2006), our results showed that the activation of H + -ATPase is not exclusively sufficient for stomatal induction.

[0093] 2-4. Regulation of Second Messenger Mobilization by Pyrabactin The production of reactive oxygen species (ROS), nitric oxide (NO), and cytosolic alkalinization are hypothesized to act as second messengers in ABA signaling in guard cells (Irving et al., 1992; Pei et al., 2000; Desikan et al., 2002). According to reports, ABA was unable to induce ROS production, NO production, and cytosolic alkalinization in the quadruple mutant (Ying et al., 2013). In the sextuple mutant, the mobilization of these second messengers by ABA treatment also did not occur (Figures 4A and B). Previous results have shown that H2O2 production occurs simultaneously with NO production and cytosolic alkalinization, suggesting a close relationship between the mobilization of these second messengers in ABA signaling. Here, the inventors addressed the possibility of differences in the regulation of these second messengers in ABA signaling using pyrabactin and ABA receptor mutants. Pyrabactin induced increases in the fluorescence levels of H2DCF, an indicator of H2O2; DAF, an indicator of NO; and BCECF, an indicator of pH, by 31 ± 10%, 24 ± 5.5%, and 40 ± 6%, respectively, in wild-type guard cells (Figures 4C and D). The inventors initially predicted from these results that the levels of H2O2, NO, and pH in mutant guard cells treated with pyrabactin would remain the same as those in the control treatment. However, in the quadruple mutant, the levels of H2O2, NO, and alkalinization decreased significantly to -37 ± 6%, -18 ± 1.8%, and -36 ± 8%, respectively (Figure 4D). Similarly, the H2O2 level decreased significantly to -28% - 10% (Figure 4D), but interestingly, the NO and alkalinization levels remained the same as in the control treatment in the sextuple mutant (Figure 4D). The inventors questioned whether these downregulations of second messenger mobilization by ABA treatment occurred in the quadruple mutant of a previous study (Ying et al., 2013). To address this, the inventors reexamined the published data.A slight decrease in the level of the fluorescent indicator by 10 μM ABA occurred on the surface of the guard cells of the quadruple mutant (Table S1). However, statistical analysis revealed that the p-values of the fluorescence decrease [93.6 ≤ 3.9% (mean ≤ standard deviation) for H2DCF and 96.6 ≤ 2.8% for DAF] were higher than 0.05 (p = 0.07 and 0.10 by Student's t-test, respectively). On the other hand, the BCECF fluorescence decreased significantly statistically (92.0 ≤ 3.9%, p = 0.04).

[0094]

Table 1

[0095] These results indicate that pyrabactin has a partially different effect from ABA on the mobilization and feedback downregulation of second messengers in guard cells, which can be observed in the mutant background (Figure 4). Probably, different sets of ABA receptors control the activation of ABA signaling involving feedback downregulation of second messengers in a complex manner.

[0096] 2-5. Involvement of ROS Generation in Aperture Inhibition ROS production, NO production, and cytoplasmic alkalization reactions showed a slight difference between ABA and pyrabactin (Figure 4). On the other hand, their effects on stomatal inhibition were clearly different (Figure 1A). These complex results led the inventors to further investigate the involvement of ROS in stomatal inhibition in more detail. Here, the inventors adopted molecular genetic and pharmacological approaches to investigate the involvement of ROS generation in the inhibition of light-induced stomatal opening. Diphenyleneiodonium (DPI) is an inhibitor of flavoproteins such as NADPH oxidase. Tyron (1,2-dihydroxybenzene-3,5-disulfonate) and N-acetylcysteine (NAC) can function as chemical scavengers of various biologically relevant oxidants with different substrate specificities. The reaction constant of tyron for O2·- is very high (5×10 8 M -1 s -1 )(Greenstock et al., 1975). On the other hand, the value of NAC for O2·- is low (6.8×10 -1 M -1 s -1 ), while the value of NAC for OH·- is very high (>10 10 M -1 s -1 )(Aruoma et al., 1989; Aldini et al., 2018).

[0097] ABA- and pyrabactin-induced stomatal closure was impaired in the NADPH oxidase double knockout mutant atrbohD / F (Figures S3A and S4A). DPI also strongly inhibited stomatal closure (Figure S3B). To further confirm the involvement of ROS in the closure induction process, we examined the effects of the ROS scavengers, catalase and NAC. Catalase partially but significantly inhibited ABA-induced stomatal closure (Figure S3C). Notably, ABA-induced stomatal closure was not or only slightly inhibited by NAC in wild-type plants (Figure S3D). Speculating on the specificity of the scavengers for ROS, the involvement of O2·− in closure induction is strongly suggested, while OH· is either not involved or only minimally involved.

[0098] Next, we examined the involvement of ROS in the inhibition of light-induced stomatal opening. In the atrbohD / F mutant, a defect in opening inhibition was observed (Figures 5A, S4B). Treatment with DPI, catalase and NAC abolished the inhibition of stomatal opening by 10 μM ABA (Figures 5C, D and E). These results indicate that the inhibition of light-induced stomatal opening is mediated by O2·− production by NADPH oxidase and subsequent OH· formation. This is somewhat inconsistent with our previous mention based on the observation of the lack of H2O2 accumulation in the cytosol (Ying et al., 2013).

[0099] To understand this discrepancy in the involvement of ROS in the aperture inhibition process, we examined the effects of DPI, tyrosine, and NAC on H2DCF fluorescence, which mainly detects H2O2 accumulation in the cytosol. Pretreatment with DPI and tyrosine inhibited the increase in H2DCF fluorescence (Figs. 5F and 5G). In contrast, NAC did not inhibit H2O2 production (Fig. 5H). This difference can be explained by the different reactivity of NAC to O2·- and OH·. Furthermore, pyrabactin did not induce H2O2 production in atrbohD / F (Fig. S4C). Considering previous studies (Jannat et al., 2011) together, these results strongly support that inducible H2O2 accumulation in the cytosol mediates the induction of stomatal closure and that NAC-sensitive OH· production plays an essential role in the inhibition of stomatal aperture.

[0100] 3. Discussion 3-1. Chemical Manipulation of ABA Response in Plants Targeting Specific ABA Receptors In this study, we examined the effects of pyrabactin and ABA on stomatal movement and signaling events in guard cells to explore the differential action of ABA receptors leading to stomatal closure induction and aperture inhibition. Using specific ABA agonists may potentially selectively regulate stomatal behavior. This approach is thought to be beneficial for enhancing crop water use efficiency under abiotic stress. Stomata open rapidly in the early morning to absorb CO2 quickly and close rapidly during the hot midday drought to prevent excessive water loss.

[0101] Piravactin induced stomatal closure in the same way as ABA (Figs. 1A and B, Supplementary Fig. S1), but, unlike ABA, did not inhibit stomatal opening (Figs. 1C and D, Supplementary Fig. S2). The methods were slightly different in the opening inhibition assay and the closure induction assay. Therefore, to compensate for potential methodological artifacts, we performed these assays using alternative experimental conditions. We tested two different methods for the closure assay. That is, we used whole excised leaves and purified epidermis as materials. The aperture of stomatal opening during pre-opening differed between the two methods. In the experiment with excised leaves, wider openings were obtained under control conditions compared to the experiment with epidermal fragments. This is thought to be due to the contribution of mesophyll factors that promote stomatal opening (Fujita et al., 2013). As a result, the same conclusion was obtained by either method. We performed two different conditions, namely high K + and low K + for the opening inhibition assay. Since stomatal opening is narrower in the whole leaf sample than in the epidermal preparation, the resolution of the opening assay may be lower. Generally, when the K + concentration in the stomatal assay solution is high, the width of the stomata becomes wider. Therefore, we examined stomatal opening under low K + (5 mM KCl) and high K + (50 mM KCl) conditions. The same conclusion was obtained under either condition. However, an obvious difference between the two conditions was found in the stomatal opening width of the sextuple mutant. The stomatal opening of the sextuple mutant under light was clearly wider than that of the wild type and the quadruple mutant. The mechanism of the sensitivity of the stomata to the high K + condition in the sextuple mutant is I in the guard cells KinIt may be due to the high expression of the KAT1 channel that is reactive to activity (Figure 2; Kwak et al., 2001; Takahashi et al., 2013). The lack of inhibitory effect of pyrabactin on stomatal aperture provides pharmacological evidence that the inhibition of stomatal opening and the induction of closure are at least partially regulated by different ABA receptor isoforms, as predicted by previous physiological studies (Anderson et al., 1994; Assmann et al., 1994; Schwartz et al., 1994) and genetic studies (Yin et al., 2013). The biochemical events leading to stomatal movement were regulated in different ways by pyrabactin. I Kin nactivation was not carried out by ABA receptors that recognize pyrabactin (Figure 2). In contrast, inactivation of H + -ATPase was carried out by ABA receptors that recognize pyrabactin (Figure 3). This clearly suggests that, as illustrated in Figure 6, the regulation of stomatal aperture is carried out by a combination of signaling pathways that are signaled under different ABA receptor isoforms.

[0102] The levels of intracellular ROS accumulation and NO accumulation are in a dynamic equilibrium between production and disappearance. pH regulation, together with the buffering capacity of the cytosol, involves H +It is achieved by regulating pumps, antiporters, and symporters. ROS and NO levels and cytosolic alkalinization are upregulated by ABA receptors that recognize pyrabactin (Figure 4). On the other hand, their downregulation appears to be controlled in a subtly different manner that is only detectable in the mutant background (Figure 4). This study emphasized that the feedback downregulation of H2O2, NO, and pH in ABA signaling is managed in different ways. The mechanism of pyrabactin action in quadruple and sextuple mutants of ABA receptors for the downregulation of second messengers remains to be further elucidated. The differences in ABA signaling events regulated under different ABA receptors in guard cells provide clues for the selective chemical regulation of plant abiotic and biotic responses related to ABA, such as transpiration rate regulation, CO2 uptake, air pollutant entry, or pathogen entry using chemically engineered ABA agonists (Vaidya et al., 2017). It also makes it possible to individually control stomatal behavior using appropriately synthesized ligands.

[0103] 3-2. ROS Generation in Stomatal Aperture Inhibition - Potential Target for Selective Regulation of Stomatal Behavior It has been established that ROS plays an important role in ABA-induced stomatal closure (Pei et al., 2000; Zhang et al., 2001; Kwak et al., 2003; Sirichandra et al., 2009). Jannat et al., (2011) argued that ROS production in guard cells is spatiotemporally distinct, and as a result, constitutive ROS elevation and induced ROS elevation play different roles in guard cell signaling. The production of NO is expected to be related to ROS generation in the process. The levels of induced H2O2 accumulation and induced NO accumulation were correlated (Bright et al., 2006; Yan et al., 2007; Jannat et al., 2020). H2O2 and NO generate peroxynitrite, continuously form nitrated cyclic GMP, and ultimately Ca 2+is thought to cause channel activation (Gayatri et al., 2013; Joudoi et al., 2013; Jannat et al., 2020). Ca 2+ elevations / vibrations are probably characteristic of the steady-state closure of stomatal apertures (Allen et al., 2001). In this model, the formation of nitrated cyclic nucleotide monophosphates should occur in the cytosol. Therefore, an elevation of H2O2 in the cytosol may be a prerequisite for the reaction.

[0104] Although the role of ROS in closure induction is well understood, its involvement in aperture inhibition is poorly understood. Yan et al. (2007) reported that exogenous application of H2O2 inhibited stomatal aperture and simultaneously induced NO production. They also showed that NO scavengers inhibited light-induced stomatal aperture. This pharmacological study suggests the involvement of an H2O2 elevation associated with NO production in stomatal aperture inhibition. Yamauchi et al. (2019) demonstrated genetic evidence that plant peroxisome-specific autophagy, so-called pexophagy, plays a role in regulating the basal ROS level in guard cells, and that an elevation of the ROS level in atg mutants with defects in pexophagy causes suppression of light-induced stomatal aperture. On the other hand, Yin et al. (2013) showed that the accumulation of H2O2 in the guard cell cytosol is not necessary for aperture inhibition.

[0105] In the present disclosure, the inventors used a pharmacological approach with DPI, an NADPH oxidase inhibitor, and tyrosine and NAC, ROS scavengers, and a genetic approach with atrbohD / F, an NADPH oxidase double mutant, to examine the involvement of ROS in aperture inhibition (Figure 5). It was shown that NADPH oxidase is clearly required for the ABA inhibition of stomatal aperture. However, it is noteworthy that ABA did not induce the accumulation of H2O2 and NO in guard cells in quadruple receptor mutants and sextuple ABA receptor mutants, while light-induced stomatal aperture was clearly impaired in these mutants. Furthermore, in wild type, pyrabactin induced the accumulation of H2O2 and NO in the cytosol but did not inhibit stomatal aperture. This discrepancy may be explained by differences in the reactive species of ROS involved in either aperture inhibition or closure induction. Tyrosine is a ROS scavenger that readily reacts with O2·- (Greenstock et al., 1975). On the other hand, NAC has very little reactivity with H2O2 and O2·-, but shows very high reactivity with OH· (Aruoma et al., 1989; Aldini et al., 2018). The ABA inhibition of stomatal aperture was inhibited by both tyrosine and NAC (Figures 5C and D). This indicates that the responsive ROS is OH· rather than O2·-. When examining the effects of tyrosine and NAC on H2O2 accumulation in guard cells evaluated by DCF fluorescence, a difference was observed: tyrosine inhibited H2O2 accumulation, but NAC did not (Figures 5F and G). Tyrosine-sensitive O2·- production is a prerequisite for ABA-induced H2O2 accumulation in guard cells, but NAC-sensitive OH· production is not critically important. Whether tyrosine and NAC act outside or inside the cell. The distribution of these chemicals predicted by hydrophobicity will provide information on the site of action of ROS in the signaling process.The LogP values of tyrosine and NAC are estimated to be -0.9 and 0.4, respectively (PubChem online database, https: / / pubchem.ncbi.nlm.nih.gov / compound / 9001 and https: / / pubchem.ncbi.nlm.nih.gov / compound / Acetylcysteine). Therefore, these scavenger agents are thought to be present in both the apoplast and the cytoplasmic sol of the border cells. Considering that O2·- is generated by NADPH oxidase and continuously converted to H2O2 in the apoplast and then flows into the cytoplasmic sol through the plasma membrane, tyrosine blocks the initial stage of ROS production in the apoplast. The cytoplasmic sol H2O2 accumulation (referred to as inducible H2O2) brought about by this process has been proposed to be involved in closure induction (Jannat et al., 2011). In the aperture inhibition signal in border cells, NAC-sensitive OH· production in the apoplast and subsequent H2O2 formation are considered important steps (Figure 6). This process in the apoplast may not affect the accumulation of H2O2 in the cytoplasmic sol.

[0106] The inventors have shown that ROS production is involved in the ABA response of border cells. On the other hand, there are several distinguishable features between aperture inhibition and closure induction. By designing chemicals selective for a subset of ABA receptors, it may be possible to control the ROS generation mode in plant cells. It has been shown that accelerating the aperture of stomata increases the water use efficiency of plants and the biomass production (Papanatsiou et al., 2019). Chemical control of stomata contributes to plant production under abiotic stress in field conditions when the aperture remains rapid while the closure part is sufficiently sensitive to ABA. Therefore, the regulation of ROS in border cells by plant stimulants targeting specific ABA receptors is one of the important targets of downstream signaling events in cells.

Claims

1. A plant growth regulator comprising an abscisic acid (ABA) agonist that induces stomatal closure and does not inhibit stomatal opening.

2. The plant growth regulator according to claim 1, which is used in combination with a stomatal opening promoting factor.

3. The plant growth regulator according to claim 1 or 2, wherein the ABA agonist is a non-ABA analog type.

4. The plant growth regulator according to claim 1 or 2, wherein the ABA agonist binds to a dimeric ABA receptor.

5. The plant growth regulator according to claim 1 or 2, wherein the ABA agonist is pyrabactin.

6. A plant growth regulation method comprising the step of supplying to a plant a plant growth regulator containing an ABA agonist that induces stomatal closure and does not inhibit stomatal opening.

7. The plant growth regulation method according to claim 6, further comprising the step of supplying to the plant a stomatal opening promoting factor.

8. A plant growth regulation device comprising means for supplying to a plant an ABA agonist that induces stomatal closure and does not inhibit stomatal opening.

9. The plant growth regulation device according to claim 8, further comprising means for supplying to the plant a stomatal opening promoting factor.

10. A plant growth regulation system comprising: (A) a measuring unit for measuring the degree of stomatal opening of a plant; (B) a first supply unit for supplying to the plant an ABA agonist that induces stomatal closure and does not inhibit stomatal opening; and (C) a second supply unit for supplying to the plant a stomatal opening promoting factor.

11. A first agent containing an ABA agonist that induces stomatal closure and does not inhibit stomatal opening; and a second agent containing a stomatal opening promoting factor. A plant growth regulation kit.

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