JP7805671B2 - Jasmonic acid-based anti-transpiration agents - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act Publication 1: Abstracts of the 249th Lecture of the Crop Science Society of Japan (2020), Publication date: March 26, 2020

This disclosure relates to transpiration suppression technology. More specifically, this disclosure relates to reversible transpiration suppression technology using jasmonic acids.

Climate change caused by global warming is a serious problem in the agricultural sector, and in paddy rice, problems include leaf damage and drought damage when transplanting seedlings, and reduced yield and quality due to hypertranspiration caused by high temperatures during ripening. One solution to this problem is to suppress hypertranspiration by applying anti-transpiration agents.

The present disclosure makes it possible to rapidly suppress transpiration in plants (e.g., rice) by applying jasmonic acids, preventing a decline in quality and yield due to planting damage, drought damage, and excessive transpiration caused by high temperatures during ripening. Without wishing to be bound by theory, the present disclosure solves the problem of physically closing stomata, making it difficult to reversibly increase transpiration again, and resulting in a decline in yield and quality depending on the application timing and weather conditions.

The chemicals used in this disclosure induce temporary stomatal closure and transpiration suppression upon application, but this is a reversible reaction, allowing for flexible transpiration suppression in response to changes in weather conditions, etc.

It is contemplated that one or more of the above-described features may be provided in combinations other than those explicitly stated. Still further embodiments and advantages of the present invention will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary.

This disclosure has solved a serious problem in the agricultural sector, which is climate change caused by global warming. For example, in paddy rice, it has solved the problems of reduced yield and quality due to leaf damage and drought damage when transplanting seedlings, and hypertranspiration caused by high temperatures during ripening. This disclosure solves the problem of suppressing hypertranspiration by applying an anti-transpiration agent. In particular, this disclosure quickly suppresses transpiration in plants such as rice by applying jasmonic acids such as prohydrojasmone, making it possible to prevent reductions in quality and yield due to planting damage, drought damage, and hypertranspiration caused by high temperatures during ripening.

The transpiration suppression effect of the present disclosure in plants such as rice is characterized by its reversibility, compared to existing transpiration suppressants.

Figure 1 shows the transpiration suppression effect of Microcrystalline Wax confirmed by thermal imaging. The thermal image (left) and visible image (right) of a plant 2 days (top) and 7 days (bottom) after spray application of 5-fold or 10-fold diluted Microcrystalline Wax are shown. Figure 2 shows the transpiration suppression effect of paraffin wax confirmed by thermal imaging. The thermal image (left) and visible image (right) of a plant 2 days (top) and 7 days (bottom) after spray application of 100-fold or 200-fold diluted paraffin wax are shown. Figure 3 shows the transpiration suppression effect of Microcrystal Wax confirmed by thermal imaging. The thermal image (left) and visible image (right) of a plant before (top) and 0.5 hours (bottom) after spray application of 5-fold or 10-fold diluted Microcrystal Wax are shown. Figure 4 shows the transpiration suppression effect of paraffin wax confirmed by thermal imaging. Thermal images (left) and visible images (right) of a plant before (top) and 0.5 hours (bottom) after spray application of 5-fold or 10-fold diluted paraffin wax are shown. Figure 5 shows the transpiration suppression effect of PDJ confirmed by thermal imaging. Thermal images (left) and visible images (right) of plants before (A, B), 0.5 hours after (C, D), 2 days after (E, F), and 7 days after (G, H) spray application of PDJ diluted 2,000-fold or 20,000-fold are shown. Figure 6 shows the results of RNA-seq analysis using PDJ-treated spikelets. The gene expression level of the control was set at 1 for each gene.

The present disclosure is described below. Throughout this specification, singular terms should be understood to include the plural form as well, unless otherwise specified. Therefore, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural form as well, unless otherwise specified. Furthermore, terms used in this specification should be understood to have the meaning commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the case of conflict, the present specification (including definitions) will take precedence.

(definition)
In this specification, "about" refers to plus or minus 10% of the numerical value that follows, unless otherwise specified.

As used herein, "transpiration suppression" refers to the suppression of water transpiration when applied to all or part of a plant (e.g., leaves, panicles, etc.) compared to when the compound is not applied. The transpiration suppression effect may be a direct result of the compound's components, or, if the component is a gene, it may be achieved by the gene product, etc., expressed as a result of application or incorporation into the plant.

As used herein, "reversible transpiration inhibition" means that the transpiration inhibition effect is reversible, i.e., once the transpiration inhibition effect is achieved, it can be restored to its original state (the transpiration inhibition effect disappears or is reduced).

In one embodiment, transpiration evaluated by the method for confirming transpiration suppression effect disclosed herein is suppressed to 80% or less, preferably 50% or less, more preferably 35% or less, and most preferably 20% or less, compared to transpiration in a plant not using the transpiration suppressing ingredient of the present invention. The transpiration suppressing ingredient may be in the form of a liquid, fine powder, paste, or the like, but other formulations may also be used as long as they do not impair the stability of the active ingredient contained in the transpiration suppressing ingredient of the present disclosure and can be used in the use examples described below.

As used herein, "jasmonic acids" includes jasmonic acid and jasmonic acid derivatives. Jasmonic acid includes free jasmonic acid and salts of jasmonic acid. Salts of jasmonic acid include, but are not limited to, sodium, potassium, magnesium, and calcium salts. Jasmonic acid derivatives include methyl jasmonate, prohydrojasmone, jasmonic acid glucoside, epijasmonic acid, methyl epijasmonate, dihydrojasmonic acid, tuberonic acid, and amino acid derivatives of jasmonic acid. Amino acid derivatives of jasmonic acid include isoleucine jasmonate, phenylalanine jasmonate, and valine jasmonate. These jasmonic acid-related compounds may be included individually as anti-transpiration components, or in any combination.

As used herein, "prohydrojasmone" is registered as propyl (1RS,2RS)-(3-oxo-2-pentylcyclopentyl)acetate containing 10±2% propyl (1RS,2SR)-(3-oxo-2-pentylcyclopentyl)acetate in the pesticide registration system and is known as a jasmonic acid derivative plant growth regulator, but for purposes of this disclosure, this ratio may be any. Prohydrojasmone is an artificially produced jasmonic acid derivative commonly used as a plant growth regulator for various fruits and rice seedlings. Prohydrojasmone has been confirmed to affect various biological processes in a manner similar to jasmonic acid. Prohydrojasmone promotes anthocyanin accumulation in grapes and apples, improves hand-picking efficiency in Satsuma mandarins, induces a defense response against armyworms in corn, and has effects on phenolic compounds, anthocyanin accumulation, and antioxidant activity in komatsuna and lettuce. It is also a growth regulator that inhibits germination and root growth in both indica and japonica rice varieties.

In this disclosure, the effects of prohydrojasmone (PDJ) on transpiration were investigated in comparison with commercially available microcrystalline wax and paraffin formulations. Takanari is a high-yielding indica variety that exhibits higher transpiration rates than common japonica varieties (Fukuda et al., 2018). In some examples of this disclosure, Takanari was used to demonstrate the activity of PDJ in preventing transpiration in rice. The results of these examples demonstrate that PDJ functions differently from commercially available formulations and can be used to reduce excessive transpiration in rice.

As used herein, "plant application" refers to the direct application of a target component to a plant. Plant application may involve primarily applying the target component to the plant, with small amounts of the component incidentally being applied to the environment. In plant application, for example, the effect intended by the present disclosure can be achieved by directly applying the target component to the plant. Examples of plant application methods include, but are not limited to, directly applying the anti-transpiration component, scattering the target component on the underside of the leaves, and, after ear emergence, applying the anti-transpiration component to the entire plant, including the ears. Specifically, plant application can be considered a concept that encompasses foliar spraying, floral spraying, flower spike spraying, tree spraying, and more.

As used herein, "environmental spraying" refers to spraying a target component into the environment surrounding a plant. Environmental spraying involves primarily spraying the target component into the environment, and may also involve spraying a small amount of the component onto the plant. In environmental spraying, for example, the effect intended by this disclosure can be achieved by spraying the target component into the environment. Examples of environmental spraying methods include spraying the plant's environment, but also include, but are not limited to, soil treatment in which the component is applied directly into the soil in advance, and base application in which the component is sprayed at the base of the plant. Environmental spraying can be considered a concept that includes water spraying, soil spraying, base spraying, irrigation water spraying, etc.

In this specification, "application" refers to the act of rubbing a drug or active ingredient onto the desired area.

In the present disclosure, any formulation that can be used with pesticides can be used, including, for example, dust, DL dust, FD (flow dust), granule, jumbo, dust granule, fine granule, fine granule, powder, wettable powder, flowable sol (SC), water dispersible granule, dry flowable water-soluble, water-soluble granule, emulsifiable concentrate, EW, liquid, ME liquid, oil, surf, aerosol, paste, fumigant, fumigant, microcapsule, and pack.

As used herein, "liquid" refers to a drug that is provided in a liquid state at room temperature.

As used herein, "dust" refers to a formulation in which the pesticide active ingredient is diluted with fine mineral powder such as clay, and anti-decomposition agents are added as needed, to form a fine powder of, for example, 45 μm or less. It refers to a formulation to be used as is. Formulations that use a bulking agent with a low content of fine powder of 10 μm or less to reduce drift (drift), and further use a flocculant to aggregate any fine powder present and make it less likely to drift, are also called DL dusts. DL stands for Drift Less. Formulations that are made into ultra-fine powder of 2 μm or less to increase the time they remain suspended in the air, and are designed to be sprayed from outside the greenhouse and distributed evenly throughout, are also called FD agents or flow dust.

In this specification, "granules" refers to chemicals that are solid at room temperature and supplied in granular form. Under the Agricultural Chemicals Control Act, these are fine granules with a particle size of 300 to 1700 μm, and are intended for use as is. They come in a variety of shapes depending on the manufacturing method, including types in which the pesticide active ingredient is kneaded into mineral matter such as clay (mainly cylindrical), and types in which the active ingredient is impregnated into porous mineral matter (mainly fine sand-like). Larger particle sizes also exist in pellet or tablet form. In this specification, "jumbo formulations" are registered granules under the Agricultural Chemicals Control Act. They are available as 50g tablets (tablets) or packs containing granules, tablets, or powder wrapped in 50g of water-soluble film. 10 to 20 of these formulations are dropped into rice paddies from the ridges per 10 ares. Because no spraying equipment is required, they are highly labor-saving. Most of them are herbicides.

As used herein, "wettable powder" refers to a powdery formulation that dissolves in water and is used by suspending it in water; it settles when the liquid is left to stand. As used herein, "flowable agent" refers to a formulation in which the active ingredient is broken down into fine particles (average particle size 1-5 μm) and dispersed in a liquid with an appropriate surfactant.

In this specification, "water-soluble" refers to water-soluble powder, granular, or other preparations that are dissolved in water for use. Because the active ingredient is completely soluble in water, the preparation does not precipitate.

In this specification, "emulsifiable concentrate" refers to an oily liquid formulation in which a poorly water-soluble pesticide active ingredient is dissolved in an organic solvent and an emulsifier is added. It is used in an emulsified state after diluting with water.

As used herein, "microcapsules" refers to formulations containing micro-sized capsules in which the target ingredient is coated with a polymer membrane or similar. Microencapsulation increases the durability of the drug's effectiveness while reducing inhalation toxicity, drug damage, and paint contamination. The formulation appears liquid, similar to a flowable formulation.

In this specification, "aerosol" refers to a spray agent contained in a can (cylinder) that sprays the desired ingredients using internal gas pressure. It has the advantage of being easy to use.

As used herein, the term "plant" is used in the broadest sense in the art to refer to an organism that engages in life phenomena, photosynthesizing, and lives without movement. Plants typically possess various characteristics, such as cell structure, proliferation (self-reproduction), growth, regulation, metabolism, and repair ability, and typically possess fundamental attributes of proliferation, which involves genetics governed by nucleic acids and metabolism governed by proteins. Plants may be cells of either angiosperms or gymnosperms, dicotyledonous or monocotyledonous plants, or herbaceous or woody plants. Examples of herbaceous plants include cereals, turfgrasses, and vegetables, while examples of woody plants include evergreen broadleaf trees and deciduous broadleaf trees. Specific examples of horticultural crops include, but are not limited to, rice, wheat, barley, corn, grapes, apples, pears, peaches, cherries, persimmons, citrus fruits, soybeans, green beans, strawberries, potatoes, cabbage, lettuce, tomatoes, cucumbers, eggplants, watermelons, sugar beets, spinach, snow peas, pumpkins, sugarcane, tobacco, bell peppers, sweet potatoes, taro, konjac, cotton, sunflowers, tulips, chrysanthemums, and lawn grass. Furthermore, the term "plant" as used herein refers to all parts of the plant. Preferably, such a plant is fertile. More preferably, such a plant is capable of producing seeds. It is understood that the compositions and methods of the present disclosure can be applied to any plant that has leaves.

As used herein, a "part of a plant" may refer to a specific part of a plant, such as a stem, leaf, root, seed, flower, or fruit, or may refer to a combination of multiple organs including stems, leaves, seeds, etc. A part of a plant may include above-ground parts (e.g., leaves, stems, and nodes, or leaves, stems, nodes, and panicles), underground parts, etc.

As used herein, "seed" refers to a substance that stores nutrients for the germination of young plants and is used for agricultural propagation. Specific examples include grains such as rice, corn, cottonseed, wheat, and barley; grass family grains such as pearl millet, foxtail millet, common millet, finger millet, barnyard millet, annual millet, sorghum, Job's tears, oats, and rye; sunflower seeds, pumpkin seeds, legumes, and rapeseed seeds. Alternatively, the present disclosure may also apply to the edible parts of crops (cereal seeds and fruit of fruit trees).

As used herein, "aerial parts" refers to parts of a plant that include leaves and stems during the vegetative growth period, and leaves, stems, inflorescences, and flowers during the reproductive growth period. For example, in grasses, the "aerial parts" during the vegetative growth period consist of leaves, stems, and nodes, while the "aerial parts" during the reproductive growth period consist of leaves, stems, nodes, and panicles (rachises and spikelets).

In the present disclosure, a plant may be an edible part of a plant other than a seed. For example, a plant part may be the fruit of a vegetable such as a tomato, cucumber, eggplant, snow pea, pumpkin, or bell pepper. Alternatively, a plant part may be a leafy vegetable such as spinach, mizuna, or nozawana. A plant part may also be the edible underground portion of a taro, potato, sweet potato, konjac, lotus root, or lily root. A plant or part thereof used in the present disclosure may also not be edible. A plant or part thereof may be a bulb such as a seed potato, lily, or tulip, or a seed bulb such as a rakkyo.

As used herein, "aquatic plants" refers to plants whose roots are submerged in water during their normal life cycle. Examples include floating plants, floating-leaf plants, submerged plants, emergent plants, and wetland plants. Freshwater, brackish, and saltwater plants are all acceptable, but freshwater is preferred. "Floating plants" refer to plants whose roots are exposed to the water rather than extending to the bottom, with the entire plant body floating on the water surface. Examples include plants of the Lemnaceae family, such as duckweed (Lemna aoukikusa) and duckweed (Lemna minor), and plants of the Pontederiaceae family, such as water hyacinth (Eichhornia crassipes). "Floating-leaf plants" refer to plants whose roots extend to the bottom and whose leaves float on or near the water surface. Examples include plants from the Nymphaeaceae family, such as water lily (Nymphaeatetragona) and water shield (Brasenia schreberi), plants from the Trapaceae family, such as water chestnut (Trapajaponica), and plants from the Menyanthaceae family, such as floating heart (Nymphoidespeltata). "Submerged plants" refer to plants that have roots on the water bottom and whose entire plant body is below the water surface. Examples include plants from the Hydrocharitaceae family, such as Hydrillaverticillata, plants from the Potamogetonaceae family, such as Potamogeton crispus, and algae from the Charophyceae family, such as Charabraunii.

As used herein, the term "emergent plants" refers to plants that have roots on the water bottom and upper parts of the plant body, such as leaves and stems, that extend above the water surface. Examples of such plants include plants of the Poaceae family, such as rice (Oryza sativa), waterweed (Zizania latifolia), and common reed (Phragmites australis), plants of the Nelumbonaceae family, such as lotus (Nelumbonucifera), plants of the Nymphaeaceae family, such as water lily (Nuphar japonicum), and plants of the Typhaceae family, such as cattail (Typhalatifolia).
As used herein, the term "hygrophyte" refers to a plant that lives in places where its roots can be submerged in water, such as wetlands or around rivers or ponds, but where the majority of the plant body, excluding the roots and rhizomes, is not submerged in water. Examples of such plants include plants of the Lythraceae family, such as Lythrum anceps, plants of the Orchidaceae family, such as Habenaria radiata, and plants of the Iridaceae family, such as Irispseudacorus.

As used herein, "stomatal closure" refers to the closure of stomata in a plant, and can be observed under a microscope.

As used herein, "planting pain" refers to damage that occurs when planting or transplanting a plant (e.g., rice or vegetables), and includes temporary cessation of growth or leaf drop due to roots being cut, etc.

In this specification, "dryness damage" refers to damage that occurs when the plant body does not receive the necessary amount of water due to weather conditions such as high temperatures and dryness, or when drought or light rain causes a lack of soil moisture and no measures such as irrigation are taken, without adequate rainfall at appropriate intervals.

As used herein, "harvest quality" refers to the quality of the desired agricultural product (e.g., rice).

As used herein, "yield (of a harvested product)" refers to the yield of the desired agricultural product (e.g., rice in the case of rice), and is usually expressed as the yield per planted area (e.g., per 10 ares in the case of rice).

(Preferred embodiment)
Preferred embodiments of the present disclosure will be described below. The embodiments provided below are provided for a better understanding of the present disclosure, and it is understood that the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that those skilled in the art can make appropriate modifications within the scope of the present disclosure by referring to the description in this specification. It is also understood that the following embodiments of the present disclosure can be used alone or in combination.

The embodiments described below are all comprehensive or specific examples. The numerical values, shapes, materials, components, component placement and connection configurations, steps, and step order shown in the following embodiments are merely examples and are not intended to limit the scope of the claims. Furthermore, among the components in the following embodiments, components that are not recited in an independent claim that represents the highest concept will be described as optional components.

(Anti-transpirant)
In this aspect, the present disclosure provides a composition for suppressing transpiration of a plant.

In one aspect, the present disclosure provides a composition for reversible transpiration suppression of a plant or a portion thereof, comprising jasmonic acids. In the present disclosure, application of jasmonic acids to a plant can suppress transpiration of the plant, preferably in a reversible manner. The composition of the present disclosure may be provided in a form that can be used immediately as a diluted solution, or may be provided as a concentrated solution or concentrate (also referred to herein as a stock solution) that is intended to be diluted at the time of use or before use. Therefore, when the composition of the present disclosure is provided as a stock solution, it can be provided as a diluted solution by diluting it with an appropriate dilution medium (e.g., water, an organic solvent (such as an alcohol), a mixture thereof, etc.).

The jasmonic acids used in the present disclosure may be jasmonic acid, a jasmonic acid derivative, or a mixture thereof (e.g., a mixture of jasmonic acid and one or more jasmonic acid derivatives, or a mixture of two or more different jasmonic acid derivatives). In a preferred embodiment, the jasmonic acid derivative includes prohydrojasmone. Alternatively, prohydrojasmone or its derivatives that may be used in the present disclosure may be methyl jasmonate, prohydrojasmone, isoleucine jasmonate, phenylalanine jasmonate, valine jasmonate, jasmonic acid glucoside, epijasmonic acid, methyl epijasmonate, etc. In a preferred embodiment, the jasmonic acid derivative is prohydrojasmone.

The present disclosure can be used for any plant, but is preferably directed to monocotyledonous plants. In a preferred embodiment, the present disclosure may be directed to cereals. Alternatively, the present disclosure may be directed to grass plants (e.g., rice, preferably paddy rice).

In one embodiment, the subject of the present disclosure may be an above-ground part, an edible part of a crop, or even a seed. The present disclosure can provide reversible transpiration suppression. If transpiration suppression is irreversible, the absorption of water and nutrient elements using the transpiration flow will also decrease, resulting in a decrease in crop yield and quality, so it is desirable that it be temporary and reversible.

The plants targeted by this disclosure may be any vegetation, but are preferably aquatic plants, and more preferably, may be, but are not limited to, emergent or wetland plants.

The composition of the present disclosure may take any dosage form, including dust, DL dust, FD (flow dust), granules, jumbo, dust granules, fine granules, fine granules, powder, wettable powder, flowable sol (SC), water dispersible granules, dry flowable water-soluble powder, water-soluble granules, emulsifiable concentrate, EW, liquid, ME liquid, oil, surf, aerosol, paste, fumigant, fumigant, microcapsules, or packs, and is preferably a liquid, granule, flowable, jumbo, emulsifiable concentrate, water dispersible granule, dust, microcapsules, or aerosol.

In one embodiment, the jasmonic acid may be prohydrojasmone. When prohydrojasmone is applied, it is particularly used to suppress transpiration for a period of 0.5 hours or more but less than 2 days after application.

Antitranspiration can be achieved by using natural or synthetic plant hormones and their derivatives, which promote adaptation to abiotic and biotic stresses such as heat and water deficit. Antitranspiration agents can be used to prevent excessive transpiration by closing stomata, reducing transpiration and thereby increasing leaf temperature under heat stress.

The technology disclosed herein addresses concerns about serious water shortages in agricultural production and food supply caused by global warming. Water shortages have been reported to affect crop production in Europe, India, South America, and the United States. Rising temperatures lead to increased transpiration in plants due to upregulation of stomatal conductance and decreased leaf water potential. The technology disclosed herein solves these problems. According to the technology disclosed herein, a solution for improving rice's tolerance to water shortages is to use an anti-transpiration agent to delay excessive transpiration. Commercially available microcrystalline wax and paraffin wax agents previously used for this purpose physically coat the leaf surface and reduce water loss, but have the drawback of being irreversible. However, the technology disclosed herein makes it possible to achieve reversible transpiration suppression.

In one embodiment, the present disclosure demonstrated the superiority of prohydrojasmone (PDJ) in suppressing transpiration in the indica rice cultivar Takanari compared to commercially available microcrystalline wax and paraffin formulations in greenhouse experiments. Microcrystalline wax and paraffin formulations suppressed transpiration by approximately 30-50% three hours after application, and the suppression of transpiration persisted for seven days after treatment, depending on the concentration and the agent used. However, PDJ suppressed transpiration by approximately 40-80% compared to the control 0.5 hours after treatment, and the effect persisted for one day. However, the effect was largely lost seven days after treatment. Therefore, PDJ affects transpiration differently than commercially available products and can be used to reduce excessive transpiration in rice, especially when initial, transient, and reversible suppression is needed.

In the present disclosure, the compositions of the present disclosure function to inhibit transpiration in a manner different from the physical inhibition caused by microcrystalline wax and paraffin wax agents. For example, the anti-transpiration components used in the present disclosure physically seal the stomata or leaf surface when applied, and this effect can disappear as soon as three hours or up to seven days after treatment, demonstrating short- to medium-term effects and also being reversible.

Jasmonates, such as their methyl ester (MeJA), have been shown to induce water stress tolerance in several plant species, including soybean and barley, and 12-oxo-phytodienoic acid (12-OPDA) is also known to function as a regulator of stomatal closure in Arabidopsis thaliana, along with ABA. Based on these reports, JA, its derivatives, and precursors play an important role in adaptation to drought stress through stomatal closure, although the effective concentration and timing vary depending on the compound and plant species.

The compositions of the present disclosure can be used at any concentration of the active ingredient, PDJ, etc., within the range of 0.01 to 1000 (μmol/L), preferably 0.1 to 500 (μmol/L), and more preferably 10 to 100 (μmol/L). For example, PDJ acts quickly and is active in relatively small amounts. The effects of PDJ, etc., can be observed 0.5 hours to 2 days after administration when administered at approximately 10 μmol/L.

The disclosed composition mediates a hormonal response, so its effects are reversible. Stomatal closure is controlled by various signals, including light, humidity, and sugar decomposition. The long-term inhibitory effect of external application of PDJ is unstable, and negative feedback control may be induced depending on the PDJ concentration and application timing.

The disclosed compositions can be used as inhibitors of excessive transpiration in target plants, such as rice, due to their early, transient, and reversible inhibition. These properties are important in adapting to transient heat stress, such as föhn events, and water shortages, where rapid recovery from suppressed transpiration is necessary for normal growth and yield.

According to the present disclosure, applying a plant hormone derivative as an anti-transpiration component can induce temporary stomatal closure and cause transpiration suppression. Because this transpiration suppression is a reversible reaction, effective transpiration suppression is possible by applying it flexibly in response to changes in weather conditions, etc.

This disclosure can be used, for example, in rice cultivation sites. This disclosure can also suppress hypertranspiration in response to sudden high temperature predictions, and can be used to suppress the translocation of heavy metals, etc.

(Method for suppressing transpiration of plants)
The present disclosure also provides a method for suppressing transpiration in a plant, wherein the suppression of transpiration is preferably reversible.

In one aspect, the present disclosure provides a method for suppressing transpiration of a plant, comprising the step of applying jasmonic acids to the plant. The method of the present disclosure can employ any of the embodiments and features described herein, particularly in the section (Anti-transpiration Agent), in combination as needed. In one embodiment, the present disclosure provides a composition containing jasmonic acids directly as a liquid that can be used as is (also referred to as a diluted liquid in the present disclosure), and the jasmonic acids may be applied as is. Alternatively, in another exemplary embodiment, when jasmonic acids are provided as a concentrated liquid, the present disclosure may include a step of diluting the jasmonic acids with an appropriate dilution medium (e.g., water, an organic solvent (such as an alcohol), a mixture thereof, etc.) at or before use.

Application in this disclosure can be any method, such as spraying, coating, or addition to the cultivation environment (also known as environmental spraying). In addition to spraying onto plants, environmental spraying can also be considered.

When spraying on plants, it is effective to spray on the underside of the leaves, which have many stomata. When spraying on the leaves, it may contain an adjuvant that will help the product be retained on the leaf surface. Such an adjuvant can include a surfactant.

When spraying in the environment, it is expected that the product will be absorbed primarily through the roots, and so it can be sprayed by hand from the product bag, by mechanical spraying using a manual sprayer, a power sprayer, an unmanned helicopter or a drone, or by soil treatment or application at the base of the plant.

(Reducing plant damage)
The composition or method of the present disclosure can reduce or eliminate planting pain in plants. Planting pain refers to damage that occurs when planting or transplanting plant (e.g., rice or vegetable) seedlings, and can cause roots to be cut, temporarily halting growth or causing leaves to fall off. However, the technology of the present disclosure can suppress or eliminate such damage.

To reduce or eliminate planting pain, it is effective to use it 0.5-24 hours before transplanting or planting. If drought, light rain, high temperatures, dryness, etc. are predicted before transplanting seedlings, using it before these events are predicted to occur can reduce or eliminate planting pain.

(Reduction of drought damage to plants)
The composition or method of the present disclosure can reduce or eliminate drought damage to plants. Drought damage refers to damage that occurs when plants (e.g., rice or vegetable seedlings) are short of water. Drought damage occurs when there is a lack of water, for example, due to drought, and occurs when an adequate amount of rain does not fall at appropriate intervals and measures such as irrigation are not taken. The composition or method of the present disclosure can suppress or eliminate such damage.

To reduce or eliminate drought damage, it is effective to use the product 0.5 to 24 hours before drought, light rain, high temperatures, dry weather, etc. are predicted. If drought, light rain, high temperatures, dry weather, etc. are predicted, it is possible to reduce or eliminate drought damage by using the product before these events are predicted to occur.

(Reducing the decline in quality or yield of harvested plants)
The compositions or methods of the present disclosure can also reduce or improve the quality or yield of harvested products. Suppression of declines in quality and yield can be confirmed by grain identification, the number of unhulled grains per panicle (the total number of unhulled grains divided by the number of panicles), and the weight of 1,000 grains of brown rice (calculated by counting the number of grains per 20 g of polished brown rice). When cultivated in an environment where planting damage or drought damage occurs, the use of the present disclosure can significantly reduce or improve declines in the quality and/or yield of harvested products (e.g., rice grains).

For paddy rice, during the reproductive growth period, particularly during the ripening period from one week before heading to three weeks after heading, if drought, light rain, high temperatures, dryness, or hot, dry winds (foehn winds) are predicted, applying the product 0.5 to 24 hours before the predicted occurrence of these events can reduce or eliminate any decline in harvest quality or yield. If the above weather conditions persist, it may be possible to apply it several times every 3-7 days.

(Pesticide use)
The compositions of the present disclosure can be used as inhibitors of excessive transpiration in plants (e.g., rice) due to their transient and reversible inhibition. The compositions of the present disclosure can be used during the early stages of plant growth. These properties are important for adapting to transient heat stress, such as föhn events, and water shortages. Transient and reversible inhibition is particularly useful because rapid recovery from suppressed transpiration is necessary for normal growth and harvest.

For example, if drought, light rain, high temperatures, or dryness is predicted before transplanting seedlings, applying the product 0.5 to 24 hours before transplanting can reduce drought damage that occurs at the time of transplanting. Furthermore, during the reproductive growth period, or in the case of paddy rice, particularly during the ripening period from one week before heading to three weeks after heading, if drought, light rain, high temperatures, dryness, or hot, dry winds (foehn winds) are predicted, applying the product 0.5 to 24 hours before these events are expected to occur can not only reduce declines in quality and yield, but may also improve quality and yield depending on the application conditions. If light rain, high temperatures, dryness, or hot, dry winds (foehn winds) are predicted throughout the cultivation period, spray application 0.5 to 24 hours before transplanting is recommended.

(Cultivation method)
In this specification, plants can be cultivated by any method known in the art. Plant cultivation methods are described, for example, in the Paddy Rice Cultivation Guide (Niigata Prefecture Agriculture, Forestry and Fisheries Department), etc., which can be referenced as appropriate in this disclosure, and the necessary descriptions (preferably the entirety) are incorporated herein by reference as needed.

(Note)
In this specification, "or" is used when "at least one or more" of the items listed in the sentence can be employed. The same applies to "alternative." When it is specified in this specification that "within a range of two values," the range includes the two values themselves.

All references cited herein, including scientific literature, patents, patent applications, and the like, are hereby incorporated by reference in their entirety to the same extent as if each were specifically set forth.

The present disclosure has been described above by illustrating preferred embodiments for ease of understanding. Below, the present disclosure will be described based on examples. However, the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present disclosure. Below, the present invention will be described in more detail using reference examples, examples, and test examples. However, these are provided for illustrative purposes only and do not limit the present disclosure. Note that the compound names shown in the following reference examples and examples do not necessarily conform to IUPAC nomenclature. Note that abbreviations may be used to simplify the description, but these abbreviations have the same meaning as described above. The scope of the present disclosure is not limited to the embodiments or examples specifically described herein, but is limited only by the claims.

Where necessary, the organisms used in the following examples were handled in accordance with the standards set by the Japanese government or the National Agriculture and Food Research Organization. Furthermore, while the reagents used were specifically those listed in the examples, equivalent products from other manufacturers (e.g., SIGMA-ALDRICH, Wako Pure Chemical Industries, Nacalai Tesques, Kanto Chemical) can also be used.

(Example 1: Rice: Comparison of conventional antidesiccant and PDJ)
In this example, a comparison is made between a conventional antidesiccant and PDJ.

Materials and Methods
(Plant growth conditions)
Seeds of the indica rice cultivar Takanari were sterilized with 5% ipconazole and 4.6% copper hydroxide and then germinated by soaking in 1/10 MS medium containing MS vitamins at 25.0°C under continuous white fluorescent light. Germinated seeds were grown in seedling medium containing nitrogen, phosphorus, and potassium for 10–14 days under the same conditions. Five seedlings were then transplanted into pots (150 mm × 150 mm) containing 350 g of soil. They were grown in a greenhouse under natural photoperiods of 25°C/20°C (day/night) until the maximum tillering stage, which was 5–6 weeks after the initial growth.

(Application of drugs)
To evaluate transpiration inhibition, 20 plants were sprayed with 100 ml of 2,000- or 20,000-fold diluted PDJ (5% PDJ by weight, final concentrations of 98.3 μM and 9.83 μM, respectively, jasmomate; Meiji Seika Pharma Co., Ltd., Japan), 5- or 10-fold diluted microcrystalline wax (Greener; 10% microcrystalline wax, Greener Co., Ltd., JP), or 100- or 200-fold diluted paraffin wax (Abion C; 36% paraffin wax, Avion Co., Ltd., Japan) using a plastic spray bottle according to the manufacturer's instructions. Control plants were sprayed with a 2,000-fold diluted solution of the solvent for PDJ or tap water for the other treatments.

(Measurement of transpiration rate)
Transpiration was measured using a porometer (AP4, Delta-T, UK) at 0.5 hours, 1 day, 2 days, and 7 days after PDJ treatment, and at 3 hours, 2 days, and 7 days after treatment with paraffin wax and microcrystalline wax. Thermal images were captured using a thermal camera (Thermal Shot F30S, NEC/Avio) at 0 hours, 0.5 hours, 2 days, and 7 days after PDJ treatment, and at 2 days and 7 days after treatment with paraffin wax and microcrystalline wax. The thermal images were analyzed using image analysis software (NS9500LT, Avio, Japan). Two independent, similar experiments were performed.

(result)
In this example, we first investigated experimental conditions using microcrystalline wax, a commonly used agent for suppressing transpiration in various plants, including rice. Stomatal conductance was measured using a porometer on expanded leaves of the indica rice cultivar Takanari, which had been sprayed with microcrystalline wax. Three hours after spraying with a 5-fold or 10-fold dilution of microcrystalline wax, transpiration was suppressed by approximately 50%. Regardless of the concentration of the agent, the effect persisted for two days. The effect was still observed seven days after treatment (Table 1).

The suppression of transpiration in foliage can also be monitored by foliage temperature (Fukuda et al., 2018, Iseki and Olaleye, 2020). To this end, thermal images were taken using a thermal camera. The foliage temperature of the control plants was also found to be lower than that of the plants sprayed with microcrystalline wax two days after treatment. The difference between the treatment and control groups was still evident seven days after treatment (Figure 1).

Regardless of the concentration, 100-fold or 200-fold diluted paraffin wax reduced the amount of evaporation by approximately 35% to 50% within 3 hours after spraying. This effect persisted for 2 days and was detectable even 7 days after treatment (Table 2).

However, the foliage temperatures in the thermal images taken 2 and 7 days after treatment were not significantly different from those in the control images (Figure 2).

In plants sprayed with paraffin wax, transpiration was clearly suppressed, although it could not be monitored by thermal imaging. The reason why foliage temperature failed to reflect the suppressive effect of the paraffin wax is unclear, but the presence of surfactants in the solvent may be a factor.

These chemicals physically coat the leaf surface, and their effects cannot be measured by the porometer until they dry to a waxy coating. Therefore, the initial response was not detected by the porometer. Therefore, thermal images of plants sprayed with microcrystalline wax or paraffin wax taken 0.5 hours later initially showed no clear difference from plants sprayed with these chemicals (Figures 3 and 4).

In this example, the differences between microcrystalline wax and paraffin wax were not clarified, but both agents were clearly able to inhibit transpiration in a greenhouse under natural light conditions.

Using the same method used to confirm the effects of microcrystalline wax and paraffin wax applications, the transpiration suppression effect of PDJ was investigated by applying 2,000- or 20,000-fold diluted PDJ solutions to fully expanded leaves. Regardless of the concentration, PDJ treatment reduced transpiration by approximately 40% to 80% 0.5 hours after spraying. Inhibition by PDJ was observed 1 day after spraying and disappeared 2 and 7 days after spraying with the 20,000-fold diluted solution. The inhibitory effect observed 1 day after spraying with the 2,000-fold diluted solution disappeared more quickly than with the 20,000-fold diluted solution (Table 3).

These results suggest that the concentration in the 20,000-fold diluted solution was sufficient to inhibit transpiration, and that the 2000-fold diluted solution may have induced a negative feedback response earlier than the 20,000-fold diluted solution. Therefore, the concentration of PDJ should be selected according to its intended use. Leaf temperature was monitored 0.5 and 3 hours, and 2 and 7 days after PDJ treatment. Although the difference in the concentration of PDJ sprayed was unclear, the leaf temperature of control plants was lower than that of PDJ-sprayed plants 0.5 hours after treatment. The effect of PDJ was not observed 2 and 7 days later. This is consistent with the stomatal conductance measurement data (Figure 5).

Compared to other chemicals, PDJ suppressed transpiration in rice plants earlier and more transiently. The results of this example suggest that PDJ inhibits transpiration in a manner different from the physical inhibition caused by commercially available microcrystalline wax and paraffin wax. These chemicals physically seal the stomata or leaf surface, and their effects were observed 3 hours after treatment and up to 7 days after treatment. These results indicate that the transpiration inhibition effects of microcrystalline wax and paraffin wax are not transient, but continue for approximately one week.

PDJ is a derivative of JA and therefore functions in a similar manner to JA in part. Classically, JA methyl ester (MeJA) induces water stress tolerance in several plant species, including soybean and barley (Horton, 1991; Wang et al., 2020). 12-oxo-phytodienoic acid (12-OPDA), a precursor of JA, functions as a regulator of stomatal closure in Arabidopsis thaliana, along with ABA (Savchenko et al., 2014). Given these reports, JA, its derivatives, and precursors play an important role in stomatal closure-mediated adaptation to drought stress, although the effective concentration and timing vary depending on the compound and plant species. PDJ acts rapidly and is active at relatively low doses. The effect was detected 0.5 hours after spraying a 20,000-fold diluted solution of PDJ (20,000-fold diluted solution (9.8 μM) of a 5% by weight PDJ solution), and was observed one day after spraying, but disappeared two days after spraying (Table 3, Figure 5).

Not surprisingly, hormonal responses are reversible, and stomatal closure is regulated by various signals, including light, humidity, and glycolysis (Daszkowska-Golec and Szarejko, 2013; Lawson and Matthews, 2020). Therefore, the long-term inhibitory effect of externally applied PDJ is unstable, and negative feedback regulation may be induced depending on the PDJ concentration.

In summary, PDJ can be used as an inhibitor of excessive transpiration in rice, particularly due to its early, transient, and reversible inhibition. These properties are important for adaptation to transient heat stress such as föhn events and water shortages. Meanwhile, early recovery from suppressed transpiration is necessary for normal growth and yield. However, MeJA has been reported to inhibit stomatal development in Arabidopsis (Deng et al., 2020).

(Example 2: Suppression of planting pain when transplanting seedlings)
(method)
If drought, light rain, high temperatures, or dryness is predicted before transplanting seedlings, spray PDJ diluted 20,000 times, or in some cases 2000 times, in the case of paddy rice, 0.5 to 24 hours before transplanting.
(result)
Spray application of PDJ can reduce planting pain when transplanting seedlings.

(Example 3: Suppression of yield and quality decline caused by hypertranspiration due to drought damage and high temperatures during ripening)
(method)
During the reproductive growth period, especially in the ripening period from one week before heading to three weeks after heading, if drought, light rain, high temperatures, dryness, or hot, dry winds (foehn winds) are predicted, spray PDJ diluted 20,000 times, or in some cases 2,000 times, 0.5 to 24 hours before the season. If the above weather conditions continue, spraying can be done several times every 3 to 7 days.
(result)
Spray application of PDJ can suppress yield and quality decline caused by excessive evaporation due to drought damage and high temperatures during ripening.

(Example 4: Suppression of rice leaf blight (Nagasaki Prefecture) and white head (Niigata Prefecture) caused by hot, dry winds (foehn))
(method)
If light rain, high temperatures, dryness, or hot, dry winds (foehn winds) are predicted throughout the growing season, spray PDJ diluted 20,000 times, or in some cases 2,000 times, 0.5 to 24 hours beforehand. If the above weather conditions continue, spray several times every 3 to 7 days.
(result)
Spray application of PDJ can suppress rice leaf blight and white head caused by hot, dry winds (foehn).

(Example 5: Agent)
The composition of the present disclosure can be provided in any formulation, for example, as a liquid formulation such as the following, which can be prepared by mixing the components shown.
Jasmomate Liquid Concentrate Propyl = 3 Oxo-2-pentyl Cyclopentyl Acetate
(Common name: Prohidojasmone) 1.0-10.0 w/v% (e.g., 5.0 w/v%)
1-propanol 30.0-40.0 w/v% (e.g., 33.0 w/v%)
Surfactant 25.0-35.0 w/v% (e.g., 30.0% w/v%)
Water remaining

An instruction sheet can be attached to instruct the user to use the solution at an appropriate dilution (e.g., 2000-fold dilution (e.g., PDJ final concentration 100 μM)).

Example 6: Gene expression analysis
Conventionally grown, commonly cultivated Hinohikari plants were transplanted into 1/5000a Wagner pots at the panicle differentiation stage, and on the seventh day after heading, 20 mL of 2.00-fold diluted PDJ (Jasmomate, Meiji Seika Pharma Co., Ltd., Japan) was sprayed into each pot. Total RNA was extracted from the spikelets, which were then left overnight in an artificially grown room set at 32/28°C (day/night), and RNAseq analysis was performed. Control plants were sprayed with a 2.000-fold diluted solution of solvent.

RNASeq analysis was performed as follows.
Total RNA was extracted from the spikelets, which were left overnight after PDJ treatment, and a library was prepared using the KAPA Stranded mRNA-Seq Kit (KAPABIOSYSTEMS). PCR amplification was performed using 14 cycles, and adapters from the Fast Gene Adapter Kit (Fast Gene) were used. The quality of the prepared library was confirmed using the Fragment Analyzer High Sensitivity NGS Fragment Analysis Kit (Advanced Analytical Technologies), and sequencing was performed using a NextSeq500 with 2x76bp conditions. The resulting reads were quality checked using Sickle (ver. 1.33), and reads with a length of less than 20 bases were removed. Reads with a fragment length of 30 bases or less and their paired reads were discarded. The filtered reads were mapped to the reference sequence (The Rice Annotation Project Database http://rapdb.dna.affrc.go.jp/download/irgsp1.html) using Hisat2 (ver. 2.1.0), and a bam file was generated using Samtools (ver. 1.3). Read sequences that mapped to genic regions were counted using featureCounts (ver. 1.5.0p3). After normalization using the iDEGES normalization method, differentially expressed genes were identified using DESeq.

The sequences of the primers used are as follows:
(Primer sequences used in library creation)
(Index Ligation)
Ad153_5T_1-index/5Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAATGAAGCGTTGCAACTCCTTGGCTCACA (SEQ ID NO: 1)
Ad153_5T_1-index/5Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAACGTGCGATCCCAACTCCTTGGCTCACA (SEQ ID NO: 2)
MGI Tech Co.,Ltd Tech. Support Center Field Application Support Team
Ad153_5T_1-index/5Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAATCGGAAGGCACAACTCCTTGGCTCACA (SEQ ID NO: 3)
Ad153_5T_1-index/5Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAACCGATGTCGCCAACTCCTTGGCTCACA (SEQ ID NO: 4)
Ad153_5T_1-index/5Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAAACTTAGAATGCAACTCCTTGGCTCACA (SEQ ID NO: 5)
Ad153_5T_1-index/5Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAATCCAAGCCTGCAACTCCTTGGCTCACA (SEQ ID NO: 6)
Ad153_5T_1-index/5Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAAAGACGATGATCAACTCCTTGGCTCACA (SEQ ID NO: 7)
Ad153_5T_1-index/5Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAAAGTCTCGTGTCAACTCCTTGGCTCACA (SEQ ID NO: 8)
Ad153Ω_Bottom_2:
TTGTCTTCCTAAGGAACGACATGGCTACGATCCGACTT (SEQ ID NO: 9)
(PCR amplification)
Ad153_PCR2_2: TGTGAGCCAAGGAGTTG (SEQ ID NO: 10)
Ad153_PCR2_1:/5Phos/GAACGACATGGCTACGA (SEQ ID NO: 11)

(Sequence)
Read 1 sequencing primer: GCTCACAGAACGACATGGCTACGATCCGACTT (SEQ ID NO: 12)
Read 2 sequencing primer: TTGTCTTCCTAAGACCGCTTGGCCTCCGACTT (SEQ ID NO: 13)

(adapter sequence)
Read 1 side: AAGTCGGAGGCCAAGCGGTCTTAGGAAGACAA (SEQ ID NO: 14)
Read 2 side: AAGTCGGATCGTAGCCATGTCGTTCTGTGAGCCAAGGAGTTG (SEQ ID NO: 15)

The results are shown in Figure 6. For each gene, the expression level of the control is shown as 1.
These results indicate that gene products related to transpiration are suppressed.

(Note)
While the present invention has been illustrated by way of preferred embodiments thereof, it is understood that the scope of the present invention should be construed solely in terms of the claims that follow. It is understood that the patents, patent applications, and other documents cited herein are incorporated by reference in their entirety as if the contents themselves were specifically set forth herein.

This disclosure can be used in agriculture, particularly in the cultivation of grain crops.

SEQ ID NO: 1: Primer sequence for index ligation SEQ ID NO: 2: Primer sequence for index ligation SEQ ID NO: 3: Primer sequence for index ligation SEQ ID NO: 4: Primer sequence for index ligation SEQ ID NO: 5: Primer sequence for index ligation SEQ ID NO: 6: Primer sequence for index ligation SEQ ID NO: 7: Primer sequence for index ligation SEQ ID NO: 8: Primer sequence for index ligation SEQ ID NO: 9: Primer sequence for index ligation SEQ ID NO: 10: Primer sequence for PCR amplification SEQ ID NO: 11: Primer sequence for PCR amplification SEQ ID NO: 12: Primer sequence for sequencing SEQ ID NO: 13: Primer sequence for sequencing SEQ ID NO: 14: Adapter sequence SEQ ID NO: 15: Adapter sequence

Claims (9)

A composition for reducing, eliminating or improving quality deterioration of a plant body due to planting pain , which is to be used 0.5 to 24 hours before transplanting a seedling , the composition containing jasmonic acids , the jasmonic acid being prohydrojasmone, and used so as to suppress transpiration for 0.5 hours or more but less than 2 days after application . The composition of claim 1 , wherein the plant is a monocotyledonous plant. The composition according to claim 1 or 2 , wherein the plant is a cereal. The composition according to any one of claims 1 to 3 , wherein the plant is a grass plant. The composition according to any one of claims 1 to 4 , wherein the plant body is an aquatic plant. The composition according to claim 1 , wherein the plant is an emergent plant or a wetland plant. The composition according to claim 1 , wherein the plant is rice. The composition according to any one of claims 1 to 7 , which is in a dosage form selected from the group consisting of a liquid, a granule, a flowable, a jumbo, an emulsifiable concentrate, a water dispersible granule, a dust, a microcapsule, and an aerosol. The composition according to any one of claims 1 to 8 , wherein the prohydrojasmone is used after being diluted to a concentration of 10 µM to 100 µM.