WO2017130630A1 - Agent for enhancing high-temperature resistance in plant, method for enhancing high-temperature resistance, agent for suppressing whitening, and dreb2a gene expression promoter - Google Patents

Abstract

A purpose of the present invention is to provide a means for enhancing high-temperature resistance in a plant. Provided is an agent for enhancing high-temperature resistance in a plant, said agent comprising allantoin as an active ingredient. Also provided is a method for enhancing high-temperature resistance in a plant, said method comprising a step for applying the aforesaid agent for enhancing high-temperature resistance to the plant.

Description

High temperature stress tolerance improver in plant, method for improving high temperature stress tolerance, whitening inhibitor, and DREB2A gene expression promoter

The present invention relates to a high-temperature stress tolerance improver in plants, a method for improving high-temperature stress tolerance, a whitening inhibitor, and a DREB2A gene expression promoter.

Plants are exposed to various environmental stresses such as high temperature and dryness. Since plants are difficult to move like animals, mechanisms have been developed to protect themselves from environmental stress.

A drought stress responsive element (DRE) is a sequence whose presence was confirmed in the Arabidopsis genome by promoter analysis of RD29A, which is one of water stress-inducible genes. DREB (DRE binding protein) is a transcription factor isolated as a protein that binds to DRE. Among the DREBs, DREB2A is a transcription factor of APETALA2 / ethylene-responsive element binding factor type (AP2 / ERF-type) and was isolated as a protein that recognizes DRE (Non-patent Document 3). Since DREB2A is strongly induced by dry and high salt concentration conditions, it is considered that DREB2A is a transcription factor that works under dry and high salt stress conditions (Non-Patent Document 1). The activity of DREB2A is controlled after translation, and the 30 amino acid region adjacent to the AP2 / ERF DNA binding domain is thought to play an important role in the post-translational control of the protein. In DREB2A, DREB2A CA lacking this region has activity, and Arabidopsis overexpressing DREB2A CA exhibits a fertile phenotype, and drought stress tolerance has been significantly improved (non-patent literature). 2).

Non-Patent Document 3 and Patent Document 1 report a mechanism by which DREB2A induces a target gene when a plant is subjected to high temperature stress. According to Non-Patent Document 3 and Patent Document 1, a mechanism has been proposed in which the protein DPB3-1 (or NF-YC10) interacts with DREB2A to promote induction of a high-temperature stress resistance gene by DREB2A. Yes. Non-Patent Document 3 shows that the interaction between DPB3-1 and DREB2A does not affect the expression of a drought stress-inducing gene.

Patent Document 2 describes that in a transformed plant into which the DREB2A gene has been introduced, the rooting rate is improved and the longevity of cut flowers is prolonged.

On the other hand, allantoin (5-ureidohydantoin) is an intermediate product generated in the process of decomposing nucleobase (purine base). In the plant, allantoin is produced from 5-hydroxyisouric acid by allantoin synthase (AS) and decomposed into allantoic acid by allantoinase (ALN). Non-Patent Document 4 reports that the aln-1 mutant strain in which Arabidopsis thaliana has disrupted the ALN gene and mutated to accumulate allantoin in the plant body has higher drought stress tolerance than the wild strain. .

Non-Patent Document 5 reports that growth is promoted when allantoin is given to Arabidopsis thaliana.

Patent Document 3 discloses a method for protecting a plant from stress by giving a ureido such as allantoin to the plant. Patent Document 3 discloses that plants are damaged by oxidative stress during environmental disturbances such as drought and cold, and that when the concentration of ureido is high, the scavenger pathway is promoted and the plants are protected from damage.

WO2013 / 111755 Japanese Patent No. 4219711 US2010 / 0333337

When a plant is subjected to high temperature stress, problems such as whitening of the plant, wilt, and leaf shrinkage may occur, and if the plant is excessively hot, the plant may not be able to survive. For this reason, the improvement of the high temperature stress tolerance in plants, such as agricultural products, is calculated | required.

As described above, allantoin is known to have an effect of improving stress tolerance such as drought stress tolerance and low temperature stress tolerance in plants. However, the relationship between high temperature stress tolerance and allantoin has not been investigated so far.

ラ ン ト Allantoin has been studied to improve drought stress tolerance in plants.

However, in plants, drought stress tolerance and high temperature stress tolerance are different properties, and since the mechanism of stress tolerance expression is different, there is no correlation between drought stress tolerance and high temperature stress tolerance. For example, in Non-Patent Document 3, as described above, interaction between DPB3-1 and DREB2A is necessary for induction of high-temperature stress tolerance by DREB2A, whereas this interaction is necessary for induction of drought stress tolerance. It is described as irrelevant. As is clear from this, even if a certain component can improve resistance to stress other than high temperature stress such as drought stress, it does not necessarily have an effect of improving high temperature stress resistance. Therefore, from the conventional knowledge that allantoin has an effect of improving tolerance to several stresses other than high-temperature stress in plants, the action of allantoin related to high-temperature stress tolerance cannot be predicted.

On the other hand, the expression of the DREB2A gene in plants contributes to improvement of various stress tolerances such as high temperature stress tolerance and drought stress tolerance as described above. It is also known that the expression of the DREB2A gene has advantageous effects such as an improved rooting rate and an extended flower life of cut flowers. A substance capable of promoting the expression of the DREB2A gene can exert these advantageous effects when applied to plants. However, no such material has been previously provided. As described in Patent Document 1, a transformed plant into which the DREB2A gene has been introduced can exert advantageous effects due to DREB2A, but because the transformed plant is a plant that does not exist in nature, it is difficult to use industrially. There's a problem. It is considered that the utility of a technique for improving the expression of the DREB2A gene in a plant by applying a naturally occurring substance to the plant is considered to be great.

Therefore, an object of the present invention is to provide means for improving tolerance to high temperature stress in plants. Another object of the present invention is to provide a means for suppressing plant whitening. Another object of the present invention is to provide a means for promoting the expression of the DREB2A gene in plants.

The present inventors have found that when allantoin is allowed to act on a plant, an unexpected effect of improving the high temperature stress tolerance of the plant, suppressing the whitening of the plant, and promoting the DREB2A gene in the plant occurs. The invention has been completed. Specifically, the present invention includes the following inventions.
(1) A high-temperature stress tolerance improver for improving the high-temperature stress tolerance of a plant, containing allantoin as an active ingredient.
(2) The high temperature stress according to (1) for suppressing one or more of whitening of the plant due to high temperature stress, wiping of the plant due to high temperature stress, and winding of a plant leaf due to high temperature stress. Tolerance improver.
(3) The high-temperature stress tolerance improving agent according to (1) or (2), which promotes the expression of the DREB2A gene in a plant.
(4) A method for improving the high temperature stress tolerance of a plant, comprising a step of applying the high temperature stress tolerance improving agent according to any one of (1) to (3) to the plant.
(5) The method according to (4), wherein in the step, the high-temperature stress tolerance improver is applied to a plant before being subjected to stress.
(6) A whitening inhibitor for suppressing plant whitening, which contains allantoin as an active ingredient.
(7) A DREB2A gene expression promoter for promoting the expression of the DREB2A gene in a plant, containing allantoin as an active ingredient.
(8) A method for suppressing whitening of a plant, comprising a step of applying the whitening inhibitor according to (6) to a plant.
(9) A method for promoting the expression of the DREB2A gene in a plant, comprising a step of applying the DREB2A gene expression promoting agent according to (7) to the plant.
(10) Use of allantoin to improve high temperature stress tolerance of plants.
(11) Use of allantoin to suppress plant whitening.
(12) Use of allantoin to promote the expression of the DREB2A gene in plants.

This specification includes the disclosure of Japanese Patent Application Nos. 2016-016383 and 2016-043503, which are the basis of the priority of the present application.

It is explanatory drawing of the division in 1 petri dish in Experiment 1. FIG. In Experiment 1, photographs of each petri dish after Arabidopsis thaliana was treated under each heat shock condition and grown for 1 week are shown. In Experiment 1, the survival rate when Arabidopsis thaliana is treated under each heat shock condition and grown for 1 week is shown. In Experiment 2, the photograph of the onion of the 3rd day after the heat shock processed for 1.5 hours at 45 degreeC is shown. The upper part of FIG. 3 is a photograph of an onion in the allantoin area (survival rate 33.3%), and the lower part of FIG. 3 is a photograph of the onion in the water area (survival rate 0%). In Experiment 3, a photograph of Komatsuna on the seventh day after heat shock treated at 45 ° C. for 1 hour is shown. The upper part of FIG. 4 is a photograph of Komatsuna in the allantoin area (survival rate 100%), and the lower part of FIG. 4 is a photograph of the Komatsuna in the water area (survival rate 33.3%). It is explanatory drawing of the division in 1 petri dish in the experiment 6. FIG. In Experiment 6, the photographs of each petri dish after growing Arabidopsis cultivated in a medium with each allantoin concentration under heat shock conditions and control conditions for 1 week are shown. In Experiment 6, the survival rate when Arabidopsis thaliana cultivated in a medium with each allantoin concentration was treated with heat shock conditions and control conditions and grown for one week is shown.

<1. Allantoin>
Allantoin is also called 5-ureidohydantoin, and has a structure in which a free form is represented by the following formula.

Allantoin has one asymmetric carbon (indicated by * in the formula), and there are (R) -allantoin and (S) -allantoin forms. The allantoin used in the present invention may be (R) -allantoin, (S) -allantoin, or a mixture thereof.

Allantoin may be in any form that can be used by plants, and can be in an appropriate form such as a free form or a solvate such as a hydrate. Moreover, 2 or more types of mixtures may be sufficient among forms, such as a free body and a hydrate.

<2. Plant>
In the present invention, the target plant to which the allantoin, the allantoin-containing composition, the high-temperature stress tolerance improver, the bleaching inhibitor or the DREB2A gene expression promoter is applied is not particularly limited and includes various plants such as dicotyledonous plants and monocotyledonous plants.

Examples of dicotyledonous plants include morning glory plants, Brassica plants, convolvulaceae plants, sweet potato plants, Arabidopsis plants, papaver plants, pteridophytes plants, chickweed plants, papaver plants, prunus plants, clover plants , Nominotsunuri genus plant, Oyafusuma genus plant, Waspionian genus plant, Hamachabe genus plant, Otsumexa genus plant, Shiotsume genus plant, Mantema genus plant, genus genus plant, Fusiflora genus plant, Nadesicoaceae plant, euphorbiaceae plant, Asteraceae plant, Pepperaceae plant, Cypridaceae plant, Willow family plant, Prunus plant plant, Walnut plant plant, Birch plant plant, Beech plant plant, Elmaceae plant, Mulberry plant plant, Nettle plant plant plant, Chrysanthemum plant plant, Papaver plant plant Plant, sandalwood family, mistletoe Plant, urchinaceae plant, japonicaceae plant, scorpionaceae plant, capeaceae plant, red crustacean plant, amaranthaceae plant, white-bellied plant, scorpionaceae plant, pokeweed plant, periwinkle plant, magnoliaceae plant, magnoliaceae plant , Corn borer plant, wigaceae plant, water lily family plant, pine family plant, buttercup plant, echaceae plant, barberry plant, camellia plant, camellia plant, camphor plant, poppy plant, cruciferous plant, rape Genus plant, genus genus plant, nematode plant, crassulaceae plant, saxifragaceae plant, toberaceae plant, witch plant, sycamore plant, rose family plant, legume plant, oxenaceae plant, plant family plant, leguminous plant plant , Anopheles family, Citrus family, Nigella family, Senda Plant, Himegi plant, Euphorbiaceae plant, Amagoaceae plant, Boxwood plant, Ganoderma plant, Dermatophyceae plant, Ursiaceae plant, Ilexaceae plant, Euonymaceae plant, Oleopteraceae plant, Black-faced plant family, Maple family plant , Cariaceae, Scarletaceae, Astraphyceae, Periwinkle, Cryptomaceae, Grape, Horaceae, Lindenaceae, Mallow, Aegiriaceae, Sarnaceae, Camellia, Hypericum Plant, Ezoaceae plant, Goryoaceae plant, Violet family plant, Hygienaceae plant, Brachyceae plant, Passifloraceae plant, Pleurotus plant, Cactiaceae plant, Dendrobaceae plant, Gumiaceae plant, Papilioceae plant, Pomegranate plant , Oleaceae, urchinaceae, membran Plant, cypress plant, red spider plant, arynotaceae plant, cedar plant, araceae plant, sericaceae plant, pachyderm plant, cranaceae plant, rhododendron plant, ivyaceae plant, azalea plant, azalea family plant , Primaceae plant, pine plant, oyster plant, cypress plant, rhododendron plant, oleaceae plant, oleaceae plant, gentian plant, oleander plant, genus plant, red oak plant, oleaceae plant, oleander Plant, Lamiaceae plant, Solanum plant, Solanum plantaceae plant, Sphagnum plantaceae plant, Sesame plant plant, Scorpion plant plant, Siwa tobacco plant plant, Raccoonaceae plant, Scarlet plant family plant, Nymphalidae plant, Scorpionaceae plant, Plantain plant , Rubiaceae, Honeysuckle, Lempuku C Department of the plant, can be applied valerianaceae plant, dipsacaceae plants, cucurbits, Campanulaceae plants, Asteraceae, and the like.

Monocotyledonous plants include, for example, duckweed plants, duckweed plants, cattleya plants, cymbidium plants, dendrobum plants, leek plants, phalaenopsis plants, banda plants, paphiopedilum plants, orchidaceae plants, gammaidae Plants, licorices, periwinkles, cicadaceae, euphorbiaceae, euphorbiaceae, stigmaceae, scorpionaceae, cyperaceae, palmaceae, taroaceae, asteraceae, cypressaceae, It is applied to scorpionaceae plants, rush family plants, sandalaceae plants, liliaceae plants, amaryllidaceae plants, genus genus plants, iris plants, scallops plants, ginger plants, cannaid plants, cynomolgus plants, etc. Can do.

The target plant is not limited to a wild type plant, and may be a mutant or a transformant.

<3. High temperature stress tolerance improving agent, method for improving high temperature stress tolerance>
One embodiment of the present invention is a high temperature stress tolerance improver for improving the high temperature stress tolerance of a plant, containing allantoin as an active ingredient.

One embodiment of the present invention is a method for improving the high temperature stress tolerance of a plant, comprising the step of applying the above-mentioned high temperature stress tolerance improving agent to a plant.

Here, the high temperature stress is stress caused by exposure of a plant to a temperature higher than the normal growth temperature, for example, 25 ° C. or higher, more specifically 30 ° C. or higher, and more specifically 50 This is stress caused by exposure of plants to temperatures below ℃. Although it does not specifically limit as the time per day when a plant body is exposed to the said high temperature, For example, 60 minutes or more, More specifically, 90 minutes or more are mentioned, More specifically, 600 minutes or less is mentioned.

The high-temperature stress tolerance improving agent and the high-temperature stress tolerance improving method of the present invention are effective for improving tolerance to stress received by plants exposed to a high-temperature environment, particularly in a high-temperature stress. It is.

Generally, when a plant is exposed to a high temperature stress environment, the plant is whitened (chlorosis), the plant is wilted, a physiological disorder such as a leaf is rolled up. According to the high temperature stress tolerance improving agent and the high temperature stress tolerance improving method of the present invention, it is expected that one or more of these physiological disorders can be suppressed. In the experiment described in the present specification, the high temperature stress tolerance improving agent and the high temperature stress tolerance enhancement method of the present invention are characterized in that, among the above physiological disorders caused by high temperature stress, at least the plant is whitened by high temperature stress (chlorosis), and high temperature stress. It has been confirmed that one or more of the plant withering and suppression of plant leaf rolling due to high temperature stress were suppressed.

In the high-temperature stress tolerance improving agent and high-temperature stress tolerance improvement method of the present invention, the mechanism of the high-temperature stress tolerance enhancement action by allantoin is not particularly limited, but as one possibility, high temperature is promoted by promoting the expression of the DREB2A gene in plants. The mechanism that stress tolerance improves is estimated. Regarding the promotion of the expression of the DREB2A gene, the following <5. DREB2A gene expression promoter and method for promoting the expression of DREB2A gene>

In the high temperature stress tolerance improving agent and the high temperature stress tolerance improving method of the present invention, as another possibility of the mechanism of the high temperature stress tolerance enhancement action by allantoin, the expression of the DREB2A gene is promoted in plants, and the heat shock transcription factor 3 ( It is estimated that the high temperature stress tolerance is improved by suppressing the expression of the HSF3) gene. According to Non-Patent Document 1, in Arabidopsis thaliana in which DREB2A CA is overexpressed, the expression level of the HSF3 gene is improved, and accordingly, high-temperature stress tolerance is improved. In the present invention, by giving allantoin to a plant, the expression level of the DREB2A gene is improved, while the expression level of the HSF3 gene is suppressed and high-temperature stress tolerance is improved, so that allantoin has been known so far. It is presumed that high temperature stress tolerance in plants is improved by a mechanism different from the mechanism. Regarding the suppression of the expression of the HSF3 gene, the following <5. DREB2A gene expression promoter and method for promoting the expression of DREB2A gene>

The high-temperature stress tolerance improver of the present invention may be any agent that contains allantoin and has an effect of improving the high-temperature stress tolerance of plants, and may be allantoin itself or a combination of allantoin and other components. The allantoin-containing composition may be used. The high temperature stress tolerance improving agent of the present invention can contain an effective amount of allantoin that improves high temperature stress tolerance in plants.

The high-temperature stress resistance improver of the present invention can be in any shape such as solid or liquid.

When the high-temperature stress tolerance improver of the present invention is an allantoin-containing composition, in addition to allantoin, it may further contain other components useful for plants and components necessary for formulation. Examples of other components include known fertilizer components. Components necessary for formulation include carriers, liquid media and the like.

When the high-temperature stress resistance improver of the present invention is an allantoin-containing composition, its production method is not particularly limited, and each component is mixed, or if it is a solid composition, it is pulverized, granulated, and dried as necessary. If it is a liquid composition, operations such as stirring and dispersion can be performed as necessary.

The method for improving the high temperature stress resistance of a plant according to the present invention includes a step of applying the above high temperature stress resistance improving agent to a plant. At this time, the target plant is a plant that needs to be improved in resistance to high temperature stress, for example, a plant cultivated in an environment that may be subjected to high temperature stress such as the above temperature condition. Specific plant types are as described above.

As a method of applying the high temperature stress tolerance improver of the present invention to plants, plants such as plant roots, stems, leaves, etc. in which the allantoin released from the high temperature stress tolerance improver of the present invention or the high temperature stress tolerance improver of the present invention is used. The method is not particularly limited as long as it can contact the body, and may be applied so that the high-temperature stress tolerance improver of the present invention is in direct contact with the plant body, or cultivation of soil or the like on which the plant body is established You may apply the high temperature stress tolerance improvement agent of this invention to a support | carrier. In the present invention, the high-temperature stress tolerance improver of the present invention can be applied to plants so that an effective amount of allantoin that improves high-temperature stress tolerance is applied in plants.

The timing for applying the high-temperature stress tolerance improver of the present invention to plants is not particularly limited, but preferably, the high-temperature stress tolerance improver of the present invention is applied to plants before being subjected to high-temperature stress. The high temperature stress tolerance improving agent of the present invention promotes the expression of the DREB2A gene. As described above, when the DREB2A gene is expressed, it is presumed that the expression of various stress resistance genes is induced. For this reason, it is estimated that the plant to which the high temperature stress tolerance improving agent of the present invention has been applied in advance before being subjected to high temperature stress is in a state in which high temperature stress tolerance has been improved in advance, and the survival rate when subsequently subjected to high temperature stress is high. The

When applying the high temperature stress tolerance improver of the present invention to a plant before being subjected to high temperature stress, the time when the high temperature stress tolerance improver of the present invention is applied to the plant is T1, and the plant begins to be exposed to high temperature stress. When T2 is T2, the time from T1 to T2 is preferably 0.5 to 10 days, preferably 0.5 to 9 days, preferably 0.5 to 8 days, preferably 0.5 to 7 days, preferably Is 0.5-6 days, preferably 0.5-5 days, preferably 0.5-4 days, preferably 0.5-3 days, preferably 0.5-2 days, preferably 0.5-days. 1.5 days. According to this embodiment, since the plant is exposed to high temperature stress in a state in which high temperature stress tolerance is improved by the high temperature stress tolerance improver of the present invention, the survival rate after exposure to high temperature stress is high. The high temperature stress tolerance improver of the present invention may be applied N times (N is 2 or more) to a plant before being subjected to high temperature stress. In that case, each time point T1 _n (n is It is preferable that the time from an integer of 1 to N) to T2 satisfy the above range. By applying the high temperature stress tolerance improver of the present invention a plurality of times, the high temperature stress tolerance of the plant can be further improved.

<4. Whitening Inhibitor, Method for Inhibiting Plant Whitening>
One embodiment of the present invention is a whitening inhibitor for suppressing plant whitening, which contains allantoin as an active ingredient.

One embodiment of the present invention is a method for suppressing plant whitening, which comprises the step of applying the whitening inhibitor to a plant.

Plant whitening, also called chlorosis, is mainly caused by high-temperature stress.

The plant that is subject to whitening suppression according to the present invention is a plant that requires whitening suppression, for example, a plant that is cultivated in an environment that may be subjected to high-temperature stress such as the above temperature conditions. Specific plant types are as described above.

The whitening inhibitor of the present invention only needs to contain allantoin and has an action of suppressing plant whitening, may be allantoin itself, or may contain allantoin and other components. It may be a composition. The whitening inhibitor of the present invention can contain an effective amount of allantoin that suppresses whitening in plants.

The whitening inhibitor of the present invention can be in any shape such as solid or liquid.

When the whitening inhibitor of the present invention is an allantoin-containing composition, the same allantoin-containing composition as described for the high-temperature stress resistance improver can be used.

As a method of applying the whitening inhibitor of the present invention to a plant, the whitening inhibitor of the present invention or the allantoin released from the whitening inhibitor of the present invention may come into contact with a plant body such as a plant root, stem or leaf. The method is not particularly limited as long as it can be applied, and may be applied so that the whitening inhibitor of the present invention is in direct contact with the plant body, or the whitening inhibitor of the present invention is applied to a cultivation carrier such as soil in which the plant body is fixed. May be applied. In this invention, the whitening inhibitor of this invention can be applied to a plant so that the effective amount of allantoin which suppresses whitening may be applied in a plant.

The timing for applying the whitening inhibitor of the present invention to plants is not particularly limited, but preferably, the whitening inhibitor of the present invention is applied to plants before being subjected to stress that causes whitening (for example, high-temperature stress). The whitening inhibitor of the present invention promotes the expression of the DREB2A gene. As described above, when the DREB2A gene is expressed, it is presumed that the expression of various stress resistance genes is induced. For this reason, the plant to which the whitening inhibitor of the present invention has been applied in advance before being subjected to stress is in a state in which stress tolerance has been improved in advance, and it is predicted that whitening when subsequently subjected to stress is effectively suppressed. .

When applying the whitening inhibitor of the present invention to a plant before being subjected to stress that causes whitening, the time when the whitening inhibitor of the present invention is applied to the plant is T1, and the plant begins to be exposed to the stress. When the time point is T2, the time from T1 to T2 is preferably 0.5 to 10 days, preferably 0.5 to 9 days, preferably 0.5 to 8 days, preferably 0.5 to 7 days, Preferably 0.5 to 6 days, preferably 0.5 to 5 days, preferably 0.5 to 4 days, preferably 0.5 to 3 days, preferably 0.5 to 2 days, preferably 0.5 ~ 1.5 days. According to this embodiment, since the plant is exposed to the stress in a state in which stress tolerance is improved by the whitening inhibitor of the present invention, whitening after being exposed to the stress is more effectively suppressed. The whitening inhibitor of the present invention may be applied N times (N is 2 or more) to the plant before receiving the stress. In this case, each time point T1 _n (n is 1 to N) at which the whitening inhibitor is applied. It is preferable that the time from T) to T2 satisfy the above range. By applying the whitening inhibitor of the present invention a plurality of times, plant whitening can be further suppressed.

<5. DREB2A Gene Expression Promoter, Method for Promoting DREB2A Gene Expression>
One embodiment of the present invention is a DREB2A gene expression promoter for promoting the expression of the DREB2A gene in plants, containing allantoin as an active ingredient.

One embodiment of the present invention is also a method of promoting the expression of the DREB2A gene in a plant, comprising the step of applying the above-mentioned DREB2A gene expression promoter to the plant.

These embodiments of the present invention contribute to the improvement of various stress tolerances such as high temperature stress tolerance and drought stress tolerance by promoting the expression of the DREB2A gene in plants. Moreover, there are advantageous effects such as improvement of the rooting rate in the target plant and extension of the flower life of cut flowers.

In the method of the present invention for promoting the expression of the DREB2A gene in a plant, the expression of the HSF3 gene is preferably suppressed.

In the present invention, gene expression includes a process of expressing mRNA using genomic DNA as a template (transcription) and / or a process of synthesizing protein using the mRNA as a template (translation). In the present invention, a gene is a nucleic acid containing a base sequence encoding a predetermined polypeptide, and typically refers to genomic DNA of a plant or mRNA generated using genomic DNA as a template.

Acceleration of the expression of the DREB2A gene in the present invention means that the expression level of the DREB2A gene is significantly increased as compared with a plant not applied with allantoin. The promotion of the expression of the DREB2A gene in a plant can be confirmed by detecting the increase in the amount of mRNA encoding the DREB2A polypeptide and / or the amount of the DREB2A polypeptide in the plant. . The degree of promotion of the expression of the DREB2A gene in the present invention is not particularly limited, but when the expression level of the DREB2A gene in a plant not applied with the DREB2A gene expression promoter of the present invention is 100, the expression of the DREB2A gene of the present invention The expression level of the DREB2A gene in the plant to which the promoter is applied is, for example, 120 or more, preferably 150 or more, preferably 200 or more.

<Suppression of HSF3 gene expression in the present invention means that the expression level of the HSF3 gene is significantly reduced as compared with plants not applied with allantoin. Suppression of HSF3 gene expression in plants can be confirmed by detecting a decrease in the amount of mRNA encoding the HSF3 polypeptide and / or a decrease in the amount of HSF3 polypeptide in the plant. . The degree of suppression of the expression of the HSF3 gene in the present invention is not particularly limited, but when the expression level of the HSF3 gene in a plant not applied with the DREB2A gene expression promoter of the present invention is 100, the expression of the DREB2A gene of the present invention The expression level of the HSF3 gene in the plant to which the promoter is applied is, for example, 90 or less, preferably 80 or less, preferably 75 or less.

The plant that is the target of promoting the expression of the DREB2A gene according to the present invention is a plant that needs to promote the expression of the gene. For example, there is a possibility of being subjected to high-temperature stress such as the above temperature condition or other stresses. It is a plant cultivated in a certain environment. Specific plant types are as described above.

The DREB2A gene expression promoter of the present invention may be any agent that contains allantoin and has an action of promoting the expression of the DREB2A gene in plants, and may be allantoin itself, or allantoin and other components may be used. It may be a combined allantoin-containing composition. The DREB2A gene expression promoter of the present invention can contain an effective amount of allantoin that promotes the expression of the DREB2A gene in plants. The DREB2A gene expression promoter of the present invention can also contain an effective amount of allantoin that suppresses the expression of the HSF3 gene in plants.

The DREB2A gene expression promoter of the present invention can be in any shape such as solid or liquid.

When the DREB2A gene expression promoter of the present invention is an allantoin-containing composition, the same allantoin-containing composition as described for the high-temperature stress resistance improver can be used.

As a method of applying the DREB2A gene expression promoter of the present invention to plants, plants such as plant roots, stems, leaves, etc., in which the DREB2A gene expression promoter of the present invention or the allantoin released from the DREB2A gene expression promoter of the present invention is used. The method is not particularly limited as long as it is a method capable of contacting the body, and the DREB2A gene expression promoter of the present invention may be applied directly to the plant body, or cultivation of soil or the like to which the plant body has been established. The DREB2A gene expression promoter of the present invention may be applied to the carrier. In the present invention, the DREB2A gene expression promoter of the present invention can be applied to a plant so that an effective amount of allantoin that promotes the expression of the DREB2A gene in the plant is applied. In the present invention, the DREB2A gene expression promoter of the present invention can be applied to a plant so that an effective amount of allantoin that suppresses the expression of the HSF3 gene in the plant is applied.

The timing of applying the DREB2A gene expression promoter of the present invention to plants is not particularly limited, but preferably, the DREB2A gene expression promoter of the present invention is applied to plants before being subjected to stress (for example, high temperature stress). The DREB2A gene expression promoter of the present invention promotes the expression of the DREB2A gene. As described above, when the DREB2A gene is expressed, it is presumed that the expression of various stress resistance genes is induced. For this reason, it is estimated that the plant to which the DREB2A gene expression promoter of the present invention was applied in advance before being stressed is in a state in which stress tolerance has been improved in advance, and the survival rate when stressed thereafter is high.

When the DREB2A gene expression promoter of the present invention is applied to a plant before being subjected to stress, the time point at which the DREB2A gene expression promoter of the present invention is applied to the plant is T1, and the time point at which the plant begins to be exposed to the stress When T2 is T2, the time from T1 to T2 is preferably 0.5 to 10 days, preferably 0.5 to 9 days, preferably 0.5 to 8 days, preferably 0.5 to 7 days, preferably Is 0.5-6 days, preferably 0.5-5 days, preferably 0.5-4 days, preferably 0.5-3 days, preferably 0.5-2 days, preferably 0.5-days. 1.5 days. According to this embodiment, since the plant is exposed to the stress in a state where stress tolerance is improved by the DREB2A gene expression promoter of the present invention, the survival rate after exposure to the stress is high. The DREB2A gene expression promoter of the present invention may be applied N times (N is 2 or more) to the plant before receiving the stress. In this case, each time point T1 _n (n Is preferably an integer of 1 to N) to T2 respectively. By applying the DREB2A gene expression promoter of the present invention a plurality of times, the stress resistance of the plant can be further improved.

In the present invention, the DREB2A gene refers to a database (http://www.ncbi.nlm.nih.gov/) provided by NCBI (National Center for Biotechnology Information, National Center for Biotechnology) and other databases. a gene comprising a base sequence encoding a polypeptide annotated as responsive element-binding protein 2A or dehydration-responsive element-binding protein 2A-like, and a base encoding a polypeptide having a function homologous to the polypeptide Refers to a gene that contains a sequence.

Encodes the polypeptide annotated as dehydration-responsive element-binding protein 2A or dehydration-responsive element-binding protein 2A-like in the database provided by NCBI (http://www.ncbi.nlm.nih.gov/) The gene containing the nucleotide sequence to be confirmed can be confirmed by the search result shown on the website of the following URL:
http://www.ncbi.nlm.nih.gov/gene/?term=dehydration-responsive+element-binding+protein+2A
For example, the Arabidopsis DREB2A gene (AGI code = AT5G05410) is as shown in SEQ ID NO: 1 as the base sequence of cDNA. A partial base sequence from the 189th base to the 1196th base in the base sequence shown in SEQ ID NO: 1 is a region encoding the DREB2A polypeptide.

In addition, depending on the target plant, a nucleotide sequence encoding a polypeptide having a function homologous to a polypeptide annotated as dehydration-responsive-element-binding protein 2A or dehydration-responsive element-binding protein 2A-like is included. Gene expression may be promoted.

Examples of the gene containing a base sequence encoding a polypeptide having a function homologous to the polypeptide annotated as dehydration-responsive element-binding protein 2A or dehydration-responsive element-binding protein 2A-like, for example,
(1) 1 or several, for example, 1 to 20, preferably 1 in the amino acid sequence of the Arabidopsis DREB2A polypeptide encoded by the partial base sequence from the 189th base to the 1196th base in the base sequence shown in SEQ ID NO: 1. -15, preferably 1-10, preferably 1-5, preferably 1-3, preferably 1 or 2, comprising amino acid sequences substituted, deleted, inserted and / or added And a gene comprising a base sequence encoding a polypeptide having a function homologous to the Arabidopsis DREB2A polypeptide, or (2) 60% or more, preferably 70% or more, preferably the amino acid sequence of the Arabidopsis DREB2A polypeptide 80% or more, preferably 85% or more, preferably 90% or more, preferably 95% or more, preferably Properly comprises an amino acid sequence with a sequence identity of 98% or more, and, the Arabidopsis DREB2A gene comprising a nucleotide sequence encoding a polypeptide having a polypeptide homologous to function but are not limited to these.

In the present invention, the sequence identity of amino acid sequences can be determined using methods well known to those skilled in the art, sequence analysis software, and the like. For example, when the amino acid sequence of the Arabidopsis DREB2A polypeptide is aligned with the two amino acid sequences by inserting gaps as necessary so that the degree of coincidence between the amino acid sequence and other amino acid sequences is maximized, Refers to the ratio (%) of the number of matched amino acid residues to the number of amino acid residues (including gap number when gaps are inserted), and can be determined using a protein search system by BLAST or FASTA (Karlin, S., et al., 1993, Proceedings of the National, Academic, Sciences, USA, 90, p. 5873-5877; Altschul, SF, et al., 1990, Journal of Molecular Biology, 21st. Pearson, WR et al., 1988, Proceedingsｉｎｇof the National Academic SciencesU.S.A., 85, pp. 2444-2448).

In the present invention, the HSF3 gene refers to heat stress in a database (http://www.ncbi.nlm.nih.gov/) provided by NCBI (National Center for Biotechnology Information, National Center for Biotechnology) and other databases. a gene comprising a base sequence encoding a polypeptide annotated as transcription factor A-1b or heat stress transcription factor A-1b-like, and a base sequence encoding a polypeptide having a function homologous to the polypeptide. Refers to the gene containing.

Base encoding a polypeptide annotated as heat stress transcription factor A-1b or heat stress transcription factor A-1b-like in the database provided by NCBI (http://www.ncbi.nlm.nih.gov/) The gene containing the sequence can be confirmed by the search results shown on the website at the following URL:
http://www.ncbi.nlm.nih.gov/gene/?term=heat+stress+transcription+factor+A-1b
For example, the HSF3 gene (AGI code = AT5G16820) of Arabidopsis thaliana is as shown in SEQ ID NO: 2 as the base sequence of cDNA. The partial base sequence from the 174th base to the 1619th base in the base sequence shown in SEQ ID NO: 2 is a region encoding the HSF3 polypeptide.

Depending on the target plant, a gene containing a base sequence encoding a polypeptide having a function homologous to a polypeptide annotated as heat stress transcription factor A-1b or heat stress transcription factor A-1b-like Expression may be suppressed.

Examples of the gene containing a base sequence encoding a polypeptide having a function homologous to the polypeptide annotated as heat stress transcription factor A-1b or heat stress transcription factor A-1b-like,
(1) In the amino acid sequence of the Arabidopsis thaliana HSF3 polypeptide encoded by the partial base sequence from the 174th base to the 1619th base in the base sequence shown in SEQ ID NO: 2, one or several, for example, 1 to 20, preferably 1, -15, preferably 1-10, preferably 1-5, preferably 1-3, preferably 1 or 2, comprising amino acid sequences substituted, deleted, inserted and / or added And a gene comprising a base sequence encoding a polypeptide having a function homologous to the Arabidopsis thaliana HSF3 polypeptide, or (2) 60% or more, preferably 70% or more, preferably the amino acid sequence of the Arabidopsis thaliana HSF3 polypeptide 80% or more, preferably 85% or more, preferably 90% or more, preferably 95% or more, preferably 98 It comprises an amino acid sequence having a sequence identity or more, and, the Arabidopsis HSF3 but gene comprising a nucleotide sequence encoding a polypeptide having a polypeptide homologous functions include, but are not limited thereto.

Hereinafter, the present invention will be described with reference to specific examples. However, the following specific examples do not limit the scope of the present invention.
<Experiment 1. Evaluation of high-temperature stress tolerance of allantoin-accumulating Arabidopsis>
1. Plant material For Arabidopsis thaliana (L.) Heynh., Accessum Columbia-0, the following three lines with different genetic backgrounds were used.
(1) Wild strain (WT)
(2) Allantoinase gene disruption strain (aln-1)
(3) Complementary strain of allantoinase gene disruption strain (aln-1 35S: ALN)
These plants are the same as those used in Non-Patent Document 4. The allantoinase gene-disrupted strain complement (aln-1 35S: ALN) is the same as that indicated in the above document as “35Spro: ALN / aln-1”.

The above (2) Allantoinase gene disruption strain (SALK_000325, Yang, J. and Han K.-H, Plant Physiology, 134: 1039-1049) was obtained from the Arabidopsis Biological Resource Center (OhiitiUsitOs). is there.

The (3) complement of the allantoinase gene-disrupted strain (aln-1 35S: ALN) is obtained by converting a DNA encoding the entire length of allantoinase derived from wild-type Arabidopsis thaliana into the above-mentioned aln-1 It was obtained by introduction. A specific preparation method is as described in Non-Patent Document 4.

2. Growth medium Mixed salt for Murashige and Skoog medium (Murashige T., F. Skoog F. 1962, A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiologia Plantarum 15: 473-497) and sucrose Vitamins and gellan gum (solidifying agent) were dissolved in MES [2- (N-morpholino) ethanesulfonic acid] buffer. The resulting solution was autoclaved and then dispensed into a deep sterile petri dish FX (Sansei Medical Equipment; 90 × 20 mm) in a volume of 25 mL and solidified in a clean bench to obtain a ½ MS solid medium. The obtained 1 / 2MS solid medium contains each component at the concentrations shown in Table 1.

3. Growth conditions and high-temperature stress treatment (1) Hundreds of mature seeds of the above plants were collected and placed in a 1.5 mL tube. The following operations (2) to (6) were performed in a clean bench.
(2) 1 mL of 2.5% (v / v) sodium hypochlorite was placed in a tube containing seeds, placed in a small swirling mixer (18 rotations / min) and sterilized for 10 minutes.
(3) After spin down, the sodium hypochlorite solution was discarded and 1 mL of sterilized water was added, and the operation of (2) was performed.
(4) Using the sterilized water, the operation of (3) was repeated 3 times to sufficiently wash the seeds.
(5) One petri dish of 1 / 2MS solid medium was radially divided into three sections, and nine seeds (one petri dish total of 27 grains) were sown in each section for each line (see FIG. 1A).
(6) The petri dish was placed in a clean bench with the lid open, the water around the seeds was evaporated (about 20-30 minutes), and the petri dish was sealed with surgical tape.
(7) Each petri dish was wrapped with aluminum foil one by one and subjected to low temperature treatment (4 ° C.) for 2 days to overcome sleep.
(8) The cells were transferred to a culture room and cultured for 7 days under conditions of 22 ° C. and long days (light irradiation for 16 hours under fluorescent lamps; 0.07 mmol photons m ⁻¹ s ⁻¹ ).
(9) The 7-day-old plant grown aseptically according to (8) above was placed in an incubator set at 45 ° C. in advance, and heat shock was applied for 75 minutes, 90 minutes, or 105 minutes in the dark. . In the control test, the growth at 22 ° C. was continued without applying heat shock.
(10) After the heat shock treatment, the petri dish was cooled in a 22 ° C. incubator for 10 to 15 minutes, and grown again for 1 week under the condition (8). If the damage is serious, leaf chlorosis will be observed from the third day. The survival rate was evaluated using this phenomenon.

The above test was performed twice.

4). Results A photograph of each petri dish after treatment under each heat shock condition and growth for 1 week is shown in FIG. 1B. The arrangement of plant lines in each petri dish shown in FIG. 1B is as shown in FIG. 1A. The numerical value in the petri dish indicates (number of surviving seedlings) / (number of germinated seedlings) in each section.

Fig. 2 shows the survival rate under each heat shock condition. The survival rate shown in FIG. 2 is the result of calculating the average value in two tests by obtaining the survival rate (%) in each condition based on the survival number result shown in FIG. 1B.

1B and 2 show that the allantoinase gene-disrupted strain (aln-1) is resistant to high-temperature stress. In the allantoinase gene-disrupted strain (aln-1), allantoin metabolism does not proceed in the plant body, so the high temperature stress tolerance observed in this experiment is caused by the high concentration of allantoin in the plant body. It was concluded that

In the plants damaged by high temperature stress, leaf chlorosis (whitening phenomenon) was observed.

<Experiment 2. Giving high temperature stress tolerance to monocotyledonous plants by allantoin application>
Place a 5cm side polypot on a balance dish BD-2 (As One), put 70ml vermiculite (Protolef), put 80ml culture soil (Takii seedling) on it, onion (variety: Neo Earth, Takii seedling) The seeds were sowed in 3 grains × 3 places, and further 20 ml of vermiculite was covered with the seeds. After that, tap water 50 ml × 2 times water supply was performed in a balance dish. A polypot (integrated with a balance dish) was placed on a bat and placed in an incubator at 22 ° C., 10000 Lux, 12 hours light period, 12 hours dark period, and cultivation was started. Let the cultivation start date be the 0th day of sowing. On the 5th day of sowing, germination was confirmed. On the 6th day of sowing, 3 individuals (1 plant per site) were left in each pot, and the remaining plants were thinned out.

On the 6th day of sowing, tap water (hereinafter referred to as “water zone”) or 1 mM allantoin aqueous solution (allantoin zone) 40 ml / pot was applied to each experimental zone. On the 11th day after sowing, 40 ml / pot of tap water was supplied to all pots. On the 13th day after sowing (onion is the second leaf stage of the main leaf), each bat was placed in a thermostatic chamber and exposed to 45 ° C. control for 1 hour, 1.5 hours, and 2 hours to give high temperature stress. The soil in the pot was kept in sufficient moisture before and after the high temperature stress. After completion of the high temperature stress treatment, it was returned to the incubator at 22 ° C. and cultivation was continued. On the second day after the heat shock, 40 ml / pot of water was supplied. Further, 40 ml / pot of water was supplied at intervals of 2 days.

Survival rate on the third day after the heat shock was 22.2% for the water zone, 66.7% for the allantoin zone after treatment for 1 hour, 0% for the water zone, 33.3% for the allantoin zone for 2 hours. By time treatment, it was 0% in the water zone and 11.1% in the allanto-in zone. The survival rate on the 8th day after the heat shock was the same result. From the above result, the high temperature stress tolerance imparting effect by application of allantoin was confirmed.

Here, “survival rate” indicates the ratio of the surviving plant to the number of plants subjected to heat shock treatment. In calculating the survival rate, individuals with physiological disorders (whitening, wilting, leaf winding) in the latest leaf and no longer growing were treated as non-viable individuals.

FIG. 3 shows a photograph of the plant on the third day after heat shock treated at 45 ° C. for 1.5 hours. The upper part of FIG. 3 is a photograph of a plant in the allantoin area (survival rate 33.3%), and the lower part of FIG. 3 is a photograph of the plant in the water area (survival rate 0%).

<Experiment 3. Addition of high temperature stress tolerance to dicotyledonous plants by allantoin application>
Place a 5cm side polypot on Balance Dish BD-2 (As One), put 70ml vermiculite (Protolef), put 80ml culture soil (Takii seedling) on top, Komatsuna (variety: Rakuten, Takii seedling) 3 seeds were sown in the center of the pot, and 20 ml of vermiculite was covered with the seed. After that, tap water 50 ml × 2 times water supply was performed in a balance dish. A polypot (integrated with a balance dish) was placed on a bat and placed in an incubator at 22 ° C., 10000 Lux, 12 hours light period, 12 hours dark period, and cultivation was started. Let the cultivation start date be the 0th day of sowing.

On the third day of sowing, germination was confirmed. On the 6th day after sowing, one individual was left in each pot, and the remaining individuals were thinned out. On the 6th day after sowing, 40 ml / pot of tap water or 1 mM allantoin aqueous solution was applied to each experimental group. On the 11th day after sowing, 40 ml / pot of tap water was supplied to all pots. On the 13th day after sowing, 40 ml / pot of tap water or 1 mM allantoin aqueous solution was applied to each experimental group. On the 14th day after sowing (Komatsuna is the second leaf stage of the main leaf), the bat was placed in a thermostatic chamber and exposed to 45 ° C. for 1 hour to give high temperature stress. The soil in the pot was kept in sufficient moisture before and after the high temperature stress. After completion of the high temperature stress treatment, it was returned to the incubator at 22 ° C. and cultivation was continued. On the second day after the heat shock, 40 ml / pot of water was supplied. Further, 40 ml / pot of water was supplied at intervals of 2 days.

The survival rate on the seventh day after the heat shock was 33.3% in the water zone and 100% in the allanto-in zone after 1 hour treatment. From the above result, the high temperature stress tolerance imparting effect by application of allantoin was confirmed.

Here, “survival rate” indicates the ratio of the surviving plant to the number of plants subjected to heat shock treatment. In calculating the survival rate, individuals with physiological disorders (whitening, wilting, leaf winding) in the latest leaf and no longer growing were treated as non-viable individuals.

FIG. 4 shows a photograph of the plant on the seventh day after heat shock treated at 45 ° C. for 1 hour. The upper part of FIG. 4 is a photograph of a plant body in the allantoin area (survival rate 100%), and the lower part of FIG. 4 is a photograph of the plant body in the water area (survival rate 33.3%).

<Experiment 4. Promotion of DREB2A gene expression in allantoin-accumulating plants>
1. Summary In order to investigate the effects of allantoin on the gene expression of Arabidopsis thaliana (L.) Heynh., Microarray raw data (NCBI Gene) of alan-1 mutants that accumulate allantoin by gene disruption of allantoinase Expression Omnibus accession number GSE44922) is standardized according to the method described in Konishi, T. (2004) Three-parameter longnormal distribution ubiquitously found in cDNA microarray data and its application to parametric data treatment.BMC Bioinformatics 5: 5. did. Applying two-dimensional analysis of variance to the standardized data obtained, and selecting genes whose transcript level fluctuated more than 3 times compared to wild-type strains under strict test conditions (P <0.001) (Konishi, T. (2011) Microarray test results should not be compensated for multiplicity of gene contents. BMC Syst. Biol. 5 (Suppl 2): S6). Using these genes, Virtual Ontology (version 1.3; http://virtualplant.bio.nyu.edu/cgi-bin/vpweb/) was used for gene ontology (GO) analysis on biological processes.

2. Details of Procedure Total RNA was extracted from seedlings of 2 weeks old wild and aln-1 mutants of Arabidopsis thaliana. Two independent biological samples were used for each genotype. Reverse transcription and labeling of RNA to cDNA and hybridization to Affymetrics ATH1 Gene Chips were performed according to the manufacturer's instructions. After washing the hybridized microarray, signals derived from hybridization were collected using Affymetrics GeneChip Scanner 3000 7G. According to this procedure, a total of four microarray raw data were obtained for each of the wild strain and the aln-1 mutant. This procedure is as described in Watanabe, S. et. Al., (2014) Plant Cell Environ. 37: 1022-1036.

These microarray raw data are registered and published under the accession number of NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/). These microarray data were standardized by a parametric method using a three-parameter lognormal distribution model using the SuperNORM service of Skylight Baotech Co., Ltd., and the expression level of each gene was converted into a z-score (see Konishi above) , T. (2004)). This standardization work enables comparison of gene expression levels between different microarrays. Standardized data is registered with the GSE73841 accession number.

The expression level of each gene was compared between the wild strain and the aln-1 mutant. A gene whose expression level of the aln-1 mutant was increased or decreased by 3 times or more was selected by two-way analysis of variance (ANOVA) under the strict test conditions (P <0.001) according to the method of Konishi, 2011. did. Use the BioMaps tool of VirtualPlant (version 1.3; http://virtualplant.bio.nyu.edu/cgi-bin/vpweb/) with default settings (Fisher exact probability test corrected for false discovery rate, P <0.01) According to Arabidopsis Genome Annotation (TAIR release 10; http://arabidopsis.org/), gene ontology (GO) analysis on biological processes was performed.

3. Results As a result of the above analysis, the expression level of the DREB2A gene (AGI code = AT5G05410, SEQ ID NO: 1) in the aln-1 mutant was 3.17 times that in the wild strain.

On the other hand, the expression level of heat shock transcription factor 3 gene (HSF3) (AGI code = AT5G16820, SEQ ID NO: 2) in the aln-1 mutant was 0.577 times that of the wild strain.

<Experiment 5. Promotion of DREB2A gene expression by cultivation in an allantoin-containing medium>
After sterilizing Arabidopsis wild seeds (Columbia-0) with 2.5% (v / v) sodium hypochlorite, experiment 1, 1/2 MS solid medium described in Table 1 (including 1 mM allantoin, control group is allantoin) None, 90 mm in diameter and 20 mm in height type petri dish, and seeded for 14 days under long day conditions (5000 Lux, 16 hours light period, 8 hours dark period incubator, 22 ° C.). Extraction of RNA from Arabidopsis leaves and stems using commercially available kits (NucleoSpin RNAII, MACHEREY-NAGEL), reverse transcription (ReverseTra Ace qPCR RT Master Mix, TOYOBO) and quantitative PCR (KAPA SYBR FAST qAP Master X . ACT2 (AGI code = AT3G18780) was used as a reference gene. As a result, the relative expression level of DREB2A (AGI code = AT5G05410, SEQ ID NO: 1) was 2.2 times that of the control group, and the relative expression level of HSF3 (AGI code = AT5G16820, SEQ ID NO: 2) was 0.7 times. Met.

<Experiment 6. Evaluation of high-temperature stress tolerance of Arabidopsis grown in an allantoin-containing medium>
1. Plant Material For Arabidopsis thaliana (L.) Heynh., Accession Columbia-0), Experiment 1. The wild strain (WT) described in 1. was used.

2. Growth medium Experiment 1, 1 / 2MS solid medium described in Table 1 (Allantin-added sections were further added with 10 μM, 100 μM, and 1000 μM allantoin, and allantoin-free sections were not added with allantoin, deep dish No. 903 VALMARK sterilized dish; Ina Optica).
3. Growth conditions and high-temperature stress treatment (1) Hundreds of mature seeds of the above plants were collected and placed in a 1.5 mL tube. The following operations (2) to (6) were performed in a clean bench.
(2) 1 mL of 2.5% (v / v) sodium hypochlorite was placed in a tube containing seeds, placed in a small swirling mixer (18 rotations / min) and sterilized for 10 minutes.
(3) After spin down, the sodium hypochlorite solution was discarded and 1 mL of sterilized water was added, and the operation of (2) was performed.
(4) Using the sterilized water, the operation of (3) was repeated 3 times to sufficiently wash the seeds.
(5) In the petri dish, the circular accommodating portion in plan view is divided into two semicircular sections by one partition wall extending in the diameter direction. One compartment of the petri dish contains an allantoin-free 1 / 2MS solid medium, the other compartment contains an allantoin-added 1 / 2MS solid medium, and each seed contains 15 seeds (30 dishes in total). (See FIG. 5A).
(6) The petri dish was placed in a clean bench with the lid open, the water around the seeds was evaporated (about 20-30 minutes), and the petri dish was sealed with surgical tape.
(7) Each petri dish was wrapped with aluminum foil one by one and subjected to low temperature treatment (4 ° C.) for 2 days to overcome sleep.
(8) The cells were transferred to a culture room and cultured for 7 days under conditions of 22 ° C. and long days (light irradiation for 16 hours under fluorescent lamps; 0.07 mmol photons m ⁻¹ s ⁻¹ ).
(9) The 7-day-old plant grown aseptically according to the above (8) was placed in an incubator set at 45 ° C. in advance, and heat shock was applied for 105 minutes in the dark. In the control test, the growth at 23 ° C. was continued without applying heat shock.
(10) After the heat shock treatment, the petri dish was cooled in a 23 ° C. incubator for 10 to 15 minutes, and grown again for 1 week under the condition (8) above. If the damage is serious, leaf chlorosis will be observed from the third day. The survival rate was evaluated using this phenomenon.

The above test was performed twice.

4). Results A photograph of each petri dish after treatment under each heat shock condition and growth for 1 week is shown in FIG. 5B. The arrangement of plant lines in each petri dish shown in FIG. 5B is as shown in FIG. 5A. The numerical value in the petri dish indicates (number of surviving seedlings) / (number of germinated seedlings) in each section.

Fig. 6 shows the survival rate under each heat shock condition. As for the survival rate shown in FIG. 6, the survival rate (%) in each condition was obtained based on the result of the survival number shown in FIG. 5B, and the survival rate was similarly obtained for another single test not shown in the photograph. It is the result of calculating the rate (%) and calculating the average value in the two tests.

From the test results shown in FIG. 5B and FIG. 6, it can be seen that Arabidopsis cultivated in the allantoin-containing medium is resistant to high temperature stress. In Arabidopsis thaliana cultivated in an allantoin-containing medium, it was concluded that the high temperature stress tolerance observed in this experiment was caused by the high concentration of allantoin in the plant.

In the plants damaged by high temperature stress, leaf chlorosis (whitening phenomenon) was observed.

All publications, patents and patent applications cited in this specification are incorporated herein by reference in their entirety.

Claims (7)

1. A high-temperature stress tolerance improver containing allantoin as an active ingredient for improving the high-temperature stress tolerance of plants.
2. The high-temperature stress tolerance improver according to claim 1 for suppressing one or more of whitening of a plant by high-temperature stress, wiping of a plant by high-temperature stress, and winding of a plant leaf by high-temperature stress. .
3. The high temperature stress tolerance improver according to claim 1 or 2, which promotes the expression of the DREB2A gene in a plant.
4. A method for improving the high temperature stress resistance of a plant, comprising a step of applying the high temperature stress resistance improving agent according to any one of claims 1 to 3 to the plant.
5. The method according to claim 4, wherein in the step, the high-temperature stress tolerance improver is applied to a plant before being subjected to stress.
6. A whitening inhibitor containing allantoin as an active ingredient to suppress plant whitening.
7. A DREB2A gene expression promoter for promoting the expression of the DREB2A gene in plants, containing allantoin as an active ingredient.

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