Plant environmental stress alleviator, plant growth promoter, plant DNA repair promoter, and method for producing same

Abstract

The purpose of the present disclosure is to provide a novel plant environmental stress alleviator, a plant growth promoter, and a plant DNA repair promoter that have an excellent effect of alleviating environmental stresses such as high temperatures.　The present disclosure provides a plant environmental stress alleviator, a plant growth promoter, and a plant DNA repair promoter containing: one or more neurotransmitters selected from choline esters, catecholamines, serotonin, and amino acids, and / or one or more acetylcholinesterase inhibitors selected from polyphenols, flavonoids, and organic acids, or a plant extract containing said inhibitors; and calcium ions. The present disclosure also provides a method for producing the aforementioned agents.

Description

Plant environmental stress reducer, plant growth promoter, plant DNA repair promoter, and method for producing the same

[0001] This disclosure relates to plant environmental stress reducers, plant growth promoters, plant DNA repair promoters, and methods for producing the same.

[0002] Climate change, including global warming, is increasing abiotic stress on plants, raising concerns about its impact on yield and quality. In Japan, temperatures rise significantly during the summer, causing high-temperature stress on plants. Under high-temperature stress, even if the soil moisture content is sufficient, germination rates decrease, early growth is inhibited, plants become leggy, and they are susceptible to diseases and pests. Furthermore, in greenhouse cultivation, nighttime temperatures do not drop easily, creating an environment where it is difficult to produce high-quality vegetables. Methods to address high-temperature stress include night-cooling seedling cultivation, high-altitude seedling cultivation, and shading cultivation, but sufficient methods have not yet been established.

[0003] In recent years, the use of biostimulant materials has attracted attention as a countermeasure against environmental stress on plants, such as high-temperature stress. Biostimulant materials currently available on the domestic and international markets are made from humus, seaweed, amino acids, minerals, and microbial materials. For example, L-arginine has been reported to promote plant growth under high-temperature conditions (see Non-Patent Literature 1).

[0004] On the other hand, fundamental knowledge about the link between plant high-temperature stress responses and DNA damage responses is also accumulating. It has been reported that under high temperatures, the degradation of the E3 ubiquitin ligase HOS1 is suppressed by HSP90 controlled by HSFA1, and the accumulation of HOS1 induces the transcription of DNA repair-related genes such as RECQ2, thereby contributing to the repair of heat-induced DNA damage (see Non-Patent Literature 2).

[0005] Matysiak, K. et al.(2020) Agronomy 10:769. doi: 10.3390 / agronomy10060769Dorn, A. et al.(2020) Nature plants, 6 (12), 1398-1399. doi: 10.5445 / IR / 1000129881

[0006] However, the effectiveness of existing biostimulant materials has not been sufficient to reliably mitigate high-temperature stress and other environmental stresses across different crop species and temperature conditions. Furthermore, it is not known that choline esters or acetylcholinesterase (AChE) inhibitors, when combined with calcium ions, are effective in promoting the repair of plant DNA damage. Therefore, there has been a need for the development of more useful and powerful environmental stress mitigators and plant growth promoters, as well as DNA repair promoters to protect plants from DNA damage caused by high-temperature stress and other environmental stresses.

[0007] The objective of this disclosure is to provide a novel plant environmental stress reliever, plant growth promoter, and DNA repair promoter that have excellent mitigating effects against environmental stresses such as high temperatures.

[0008] The inventors of this application, after various studies to solve the above problems, have found that applying an extract of eggplant residue (containing choline esters), which is one of the underutilized biomass materials, together with calcium ions to plants can confer tolerance to environmental stresses such as high temperatures to the plants, and that the effect is greater than that of conventional biostimulant materials. Furthermore, the inventors have found that using an extract of underutilized biomass containing neurotransmitters or AChE inhibitors other than eggplant residue, or using neurotransmitters or AChE inhibitors alone, yields effects equivalent to or better than those of eggplant residue, and that treatment with choline esters induces the expression of many genes involved in DNA repair, such as DNA repair-related genes, mismatch DNA repair-related genes, and DNA replication-related genes in lettuce. Specifically, the reactions occurring in the plant were confirmed by gene expression analysis (RNA sequencing; RNA-seq), and the genes related to acquiring high-temperature stress tolerance were identified. Furthermore, we found that these new materials have simpler manufacturing processes and are easier to use compared to conventional biostimulant materials. Based on these findings, this disclosure is complete.

[0009] In other words, this disclosure provides: (1) A plant environmental stress reliever comprising one or more neurotransmitters selected from choline esters, catecholamines, serotonin and amino acids, and / or one or more acetylcholinesterase inhibitors selected from polyphenols, flavonoids and organic acids, or a plant extract containing the same, and calcium ions. (2) The plant environmental stress reliever according to (1), wherein the environmental stress is high temperature stress. (3) The plant environmental stress reliever according to (1) or (2), wherein the plant extract is an extract of one or more plants selected from the group consisting of eggplant, tomato, rosemary, bamboo grass, mint, eucalyptus, thyme, lemon balm, pine, coconut, bamboo, plum, chayote, lettuce, soybean, cassava, asparagus, mango, hibiscus, dandelion, horsetail, jolokia, and cacao. (4) A plant environmental stress reliever according to any one of (1) to (3) above, further containing iron ions and one or more trace elements selected from the group consisting of boron, manganese, zinc, copper, and molybdenum. (5) A plant environmental stress reliever according to any one of (1) to (4) above, containing the neurotransmitter at a concentration of 0.00001 mM to 100 mM when applied to plants. (6) A plant environmental stress reliever according to any one of (1) to (5) above, containing the calcium ions at a concentration of 0.0001 mM to 100 mM when applied to plants. (7) A plant environmental stress reliever according to any one of (1) to (6) above, used by adding to a hydroponic solution, or by foliar spraying, soil injection, or irrigation. (8) A plant environmental stress reducer as described in any one of items (1) to (7) above, applicable to one or more plants selected from the group consisting of flowers, legumes, grains, root vegetables, leafy vegetables, fruit vegetables, and fruit trees.(9) A plant environmental stress reliever according to any one of items (1) to (8) above, applicable to one or more plants selected from the group consisting of lettuce, garland chrysanthemum, cabbage, spinach, tomato, bell pepper, eggplant, lisianthus, snow pea, peanut, soybean, carrot, radish, corn, sweet corn, rice, melon, watermelon, banana, mandarin orange, orange, lemon, grape, pineapple, pear, apple, peach, chestnut, cherry, persimmon, fig, walnut, raspberry, Elaeagnus, Nanking cherry, blueberry, cotton, coffee, and sugarcane. (10) A plant growth promoter containing one or more neurotransmitters selected from choline esters, catecholamines, serotonin, and amino acids, and / or one or more acetylcholinesterase inhibitors selected from polyphenols, flavonoids, and organic acids, or a plant extract containing the same, and calcium ions. (11) The plant growth promoter described in (10) above, which is used under temperature conditions higher than the optimal growth temperature of the crop. (12) The plant growth promoter described in (10) or (11) above, which enhances the expression of one or more genes selected from the group consisting of photosynthesis-related genes and G protein-related genes under normal temperature conditions. (13) A plant DNA repair promoter containing one or more neurotransmitters selected from choline esters, catecholamines, serotonin and amino acids, and / or one or more acetylcholinesterase inhibitors selected from polyphenols, flavonoids and organic acids, or a plant extract containing the same, and calcium ions. (14) The DNA repair promoter described in (13) above, which enhances the expression of one or more genes selected from the group consisting of DNA repair-related genes, mismatch DNA repair-related genes and DNA replication-related genes under temperature conditions higher than the optimal growth temperature of the crop.(15) A method for producing a plant environmental stress reliever according to any one of (1) to (9), comprising the step of mixing a plant extract obtained by extracting a plant containing a neurotransmitter and / or an acetylcholinesterase inhibitor using an extraction solvent, or a neurotransmitter and / or an acetylcholinesterase inhibitor with calcium ions in the presence of water, wherein the neurotransmitter is one or more selected from choline esters, catecholamines, serotonin and amino acids, and the acetylcholinesterase inhibitor is one or more selected from polyphenols, flavonoids and organic acids. (16) The method according to (15), wherein in the extraction step, the extraction solvent is water and the extraction conditions are 40 to 150°C for 5 to 60 minutes. (17) The method according to (15) or (16), wherein in the mixing step, iron ions and trace elements are further mixed. (18) A method for producing a plant growth promoter according to any one of (10) to (12), comprising the step of mixing a plant extract obtained by extracting a plant containing a neurotransmitter and / or an acetylcholinesterase inhibitor using an extraction solvent, or a neurotransmitter and / or an acetylcholinesterase inhibitor with calcium ions in the presence of water, wherein the neurotransmitter is one or more selected from choline esters, catecholamines, serotonin and amino acids, and the acetylcholinesterase inhibitor is one or more selected from polyphenols, flavonoids and organic acids. (19) A method for producing a DNA repair promoter according to (13) or (14), comprising the step of mixing a plant extract obtained by extracting a plant containing a neurotransmitter and / or an acetylcholinesterase inhibitor using an extraction solvent, or a neurotransmitter and / or an acetylcholinesterase inhibitor, with calcium ions in the presence of water, wherein the neurotransmitter is one or more selected from choline esters, catecholamines, serotonin, and amino acids, and the acetylcholinesterase inhibitor is one or more selected from polyphenols, flavonoids, and organic acids.

[0010] This disclosure describes how applying neurotransmitters and / or AChE inhibitors, or plant extracts such as eggplant containing these, together with calcium ions, can confer tolerance to environmental stresses such as high temperatures to plants, promote plant growth, and accelerate the repair of DNA damage caused by high temperature stress, with effects greater than those of conventional biostimulant materials. This effect is presumed to be due to the activation of the plant's inherent high temperature stress tolerance function by the combination of neurotransmitters such as ACh or AChE inhibitors contained in the plant extract and calcium ions, through the induction of expression of many genes involved in DNA repair, such as DNA repair-related genes, mismatch DNA repair-related genes, and DNA replication-related genes. However, the detailed mechanism has not yet been elucidated.

[0011] Thus, this disclosure provides a novel environmental stress reliever, plant growth promoter, and DNA repair promoter that have excellent mitigating effects against environmental stress such as high temperature. Specifically, this disclosure makes it possible to (a) reduce environmental stress such as high temperature stress, (b) promote plant growth, and (c) promote the repair of DNA damage caused by high temperature stress, etc., by applying a composition comprising a neurotransmitter and / or an AChE inhibitor, or a plant extract containing these, and calcium ions to a plant.

[0012] These are photographic images showing the high-temperature tolerance test using eggplant sprout extract and eggplant pulp extract (calcium-free) (Test Example 2). These are photographic images showing the high-temperature tolerance test using eggplant sprout extract and eggplant pulp extract (calcium 0.5 mM) (Test Example 2). These are photographic images showing the high-temperature tolerance test using eggplant sprout extract and eggplant pulp extract (calcium 1.0 mM) (Test Example 2). This is a graph showing the results of a high-temperature tolerance test (2 weeks) using eggplant sprout extract and eggplant pulp extract (Test Example 2). In the figure, the vertical axis represents yield (fresh weight (mg / plant)), the horizontal axis represents the treatment group, the bars represent the standard deviation, and different letters indicate a statistically significant difference at a significance level of 5% (LSD method). This is a graph showing the calcium concentration in young lettuce plants after the completion of the Test Example 2. In the figure, (A) shows the results with no added calcium, (B) shows the results with 0.5 mM calcium, and (C) shows the results with 1.0 mM calcium. In each graph, the vertical axis represents Ca concentration (%), and the horizontal axis represents yield (fresh weight (mg / plant)). This graph shows the results of a high-temperature tolerance test (2 weeks) using an environmental stress reliever made from plant extracts other than eggplant (Test Example 3). In the figure, the vertical axis represents relative yield (%), and the horizontal axis represents the treatment group. This graph shows the effect of various standard substances and calcium ions on reducing high-temperature stress (Test Example 4). In the figure, the vertical axis represents relative yield (%), and the horizontal axis represents the treatment group. This graph shows the temperature changes inside the greenhouse during the cultivation period of Test Example 5. This graph shows the results of a small-scale hydroponic cultivation test of leaf lettuce using the environmental stress reliever of this embodiment (Test Example 5). In the figure, the horizontal axis represents the number of days after transplanting (days), and the vertical axis represents the fresh weight per lettuce plant (g / plant) in each treatment group. This graph shows the temperature changes inside the greenhouse during the cultivation period of Test Example 6. This graph shows the results of a large-scale hydroponic cultivation test of leaf lettuce using the environmental stress reliever of this embodiment (Test Example 6). In the figure, the horizontal axis shows the variety name, white represents the control group (liquid fertilizer only), black represents the group with 0.01% eggplant residue solution added, gray represents the group with 0.1% eggplant residue solution added, and diagonal lines represent the group with 1% eggplant residue solution added. The vertical axis shows the fresh weight (g / plant) per lettuce plant in each treatment group. ＊This indicates a statistically significant difference compared to the control group (P<0.05, t-test, n=4). This graph shows the temperature changes during the cultivation period in Test Example 7. This graph shows the results of the open-field cultivation test of head lettuce using the environmental stress reliever of this embodiment (Test Example 7). In the figure, the horizontal axis shows each treatment group, white represents the control group (liquid fertilizer only), black represents the group with 0.1% eggplant residue solution added, and the vertical axis shows the fresh weight (g / plant) per lettuce plant in each treatment group. ＊ This indicates a significant difference between each treatment group and the control group (P<0.05, Tukey test, n=3). This is a schematic diagram to explain the plant height of two pairs of true leaves (length from the tip of one leaf to the tip of the other leaf) measured in Test Example 8. This is a graph showing the results of the hydroponic cultivation test of Eustoma grandiflorum 'F1 Blue Tea' using the environmental stress reliever of this embodiment (Test Example 8). In the figure, the horizontal axis represents the date, the vertical axis represents the plant height (cm), white circles represent the control group (liquid fertilizer only), and black circles represent the group with 0.1% eggplant residue solution added. ＊ This indicates a statistically significant difference compared to the control group (P<0.05, Tukey test, n=10). This graph shows the results of a hydroponic cultivation test of Eustoma grandiflorum 'F1 Mink Passion' using the environmental stress mitigating agent of this embodiment (Test Example 8). In the figure, the horizontal axis represents the date, and the vertical axis represents the plant height (cm). White circles indicate the control group (liquid fertilizer only), and black circles indicate the group with 0.1% eggplant residue solution added. ＊ This indicates a statistically significant difference compared to the control group (P<0.05, Tukey test, n=10). This graph shows the results of a hydroponic cultivation test of Eustoma 'Midnight' using the environmental stress mitigating agent of this embodiment (Test Example 8). In the figure, the horizontal axis represents the date, and the vertical axis represents the plant height (cm). White circles indicate the control group (liquid fertilizer only), and black circles indicate the group with 0.1% eggplant residue solution added. ＊ This indicates a statistically significant difference compared to the control group (P<0.05, Tukey test, n=10). This graph shows the results of a hydroponic cultivation test of Turkish bellflower "F1 Namida Plus" using the environmental stress mitigating agent of this embodiment (Test Example 8). In the figure, the horizontal axis represents the date, and the vertical axis represents the plant height (cm). White circles indicate the control group (liquid fertilizer only), and black circles indicate the group with 0.1% eggplant residue solution added. ＊indicates a significant difference from the control group (P<0.05, Tukey test, n = 10). It is a graph showing the results of the hydroponic cultivation test of Turkish carnation "F1 Mink Ocean" using the environmental stress reliever of this embodiment (Test Example 8). In the figure, the horizontal axis represents the date, the vertical axis represents the plant height (cm), white circles represent the control group (only liquid fertilizer), and black circles represent the group with 0.1% addition of eggplant residue mixture. ＊ indicates a significant difference from the control group (P<0.05, Tukey test, n = 10). It is a graph showing the results of the hydroponic cultivation test of Turkish carnation "F1 Veil (registered trademark) Type 3 Lavender" using the environmental stress reliever of this embodiment (Test Example 8). In the figure, the horizontal axis represents the date, the vertical axis represents the plant height (cm), white circles represent the control group (only liquid fertilizer), and black circles represent the group with 0.1% addition of eggplant residue mixture. ＊ indicates a significant difference from the control group (P<0.05, Tukey test, n = 10). It is a graph showing the results of the hydroponic cultivation test of Turkish carnation "F1 Prima (registered trademark) Type 1 Light Lavender" using the environmental stress reliever of this embodiment (Test Example 8). In the figure, the horizontal axis represents the date, the vertical axis represents the plant height (cm), white circles represent the control group (only liquid fertilizer), and black circles represent the group with 0.1% addition of eggplant residue mixture. ＊ indicates a significant difference from the control group (P<0.05, Tukey test, n = 10). It is a graph showing the results of the hydroponic cultivation test of Turkish carnation "Chris Heart" using the environmental stress reliever of this embodiment (Test Example 8). In the figure, the horizontal axis represents the date, the vertical axis represents the plant height (cm), white circles represent the control group (only liquid fertilizer), and black circles represent the group with 0.1% addition of eggplant residue mixture. ＊ indicates a significant difference from the control group (P<0.05, Tukey test, n = 10). It is a graph showing the change in indoor temperature during the cultivation period of Test Example 9. It is a graph showing the results of the soil cultivation house test of green bean using the environmental stress reliever of this embodiment (Test Example 9). In the figure, white represents the control group (only water), black represents the group with 0.1% addition of eggplant residue mixture, and the vertical axis represents the fresh weight of pods per plant (g / plant) in each treatment group. ＊This indicates a significant difference between each treatment group and the control group (P<0.05, Tukey test, n=30). This graph shows the results of a soil cultivation test of snow peas in a greenhouse using the environmental stress mitigating agent of this embodiment (Test Example 9). In the figure, white represents the control group (water only), black represents the group with 0.1% eggplant residue solution added, and the vertical axis shows the fresh above-ground weight per plant (kg / plant) in each treatment group. ＊ This indicates a significant difference between each treatment group and the control group (P<0.05, Tukey test, n=30). This graph shows precipitation and temperature during the cultivation period for cultivation example 2. This graph shows temperature changes and planting time during the cultivation period for test example 10. This graph shows the results of a comparative test with humus in open-field cultivation of leaf lettuce (test example 10). In the figure, the horizontal axis shows each treatment group, white is the control group (no application), gray is the conventional product (humus) treatment group, and black is the disclosed (0.1% eggplant residue mixture added) treatment group, and the vertical axis shows the fresh weight (g / plant) per lettuce plant in each treatment group. ＊ 'n' indicates a statistically significant difference, and 'ns' indicates no statistically significant difference (P<0.05, Tukey test, n=30). This graph shows the temperature changes during the cultivation period of Test Example 11. This graph shows the results of a comparative study with humus in open-field cultivation of head lettuce (Test Example 11). In the figure, the horizontal axis shows the variety, white is the control group (no application), gray is the conventional product (humus) treatment group, and black is the disclosed group (0.1% eggplant residue mixture added) treatment group, and the vertical axis shows the fresh weight (g / plant) per lettuce plant in each treatment group. ＊ 'n' indicates a statistically significant difference, and 'ns' indicates no statistically significant difference (P<0.05, Tukey test, n=32). This graph shows the temperature changes inside the greenhouse during the cultivation period of Test Example 12. This graph shows the results of a comparative test with humus in soil-based greenhouse tomato cultivation (Test Example 12). In the figure, the horizontal axis shows each treatment group, with white being the control group (no application), gray being the conventional product (humus) treatment group, and black being the disclosed group (0.1% eggplant residue mixture added). The vertical axis shows the total fruit weight per plant (g / plant) in each treatment group. ＊'n' indicates a statistically significant difference, and 'ns' indicates no statistically significant difference (P<0.05, Tukey test, n=9). This graph shows the results of a comparative study with room temperature in a small-scale hydroponic cultivation trial of lettuce (Test Example 13). In the figure, the horizontal axis shows each treatment group, white indicates the control group, black indicates the group with 0.1% eggplant residue extract added (this disclosure), and the vertical axis shows the fresh above-ground weight per plant (g / plant) in each treatment group. ＊ This indicates a statistically significant difference (P<0.05, t-test, n=8). This graph shows the results of a comparative study with room temperature in a small-scale hydroponic cultivation trial of lettuce (Test Example 13). In the figure, the horizontal axis shows each treatment group, white represents the control group, and black represents the group with 0.1% eggplant residue extract added (this disclosure). The vertical axis shows the Ca content (mg / plant) of the above-ground part of lettuce per plant in each treatment group (calculated on dry weight). ＊ This indicates a statistically significant difference (P<0.05, t-test, n=8). This graph shows the results of a comparative study with room temperature in a small-scale hydroponic tomato cultivation trial (Test Example 13). In the figure, the horizontal axis shows each treatment group, white represents the control group, black represents the group with 0.1% eggplant residue extract added (this disclosure), and the vertical axis shows the fresh above-ground weight (g / plant) per plant in each treatment group. ＊ This indicates a statistically significant difference (P<0.05, t-test, n=6). This is a schematic diagram illustrating the function of the calcium channel blocker in Test Example 14. This graph shows the results of a comparative test with room temperature using a calcium channel blocker (GdCl3) (Test Example 14). In the figure, the horizontal axis shows each treatment group, white indicates room temperature (25℃ / 18℃) and black indicates high temperature (35℃ / 25℃), and the vertical axis shows the fresh weight of above-ground parts per plant (g / plant) in each treatment group. ＊ This indicates a statistically significant difference (P<0.05 vs. room temperature (25°C / 18°C), Tukey test, n=4). This graph shows the results of a comparative study with room temperature using a calcium channel blocker (LaCl3) (Test Example 14). In the figure, the horizontal axis shows each treatment group, white represents room temperature (25°C / 18°C) and black represents high temperature (35°C / 25°C), and the vertical axis shows the fresh above-ground weight per plant (g / plant) in each treatment group. ＊This indicates a statistically significant difference (P<0.05 vs. room temperature (25℃ / 18℃), Tukey test, n=4). This graph shows the results of a soil cultivation test of lettuce in a greenhouse using the environmental stress mitigating agent of this embodiment (Test Example 15). In the figure, the horizontal axis shows each treatment group, white represents the control group and black represents the treatment group of this disclosure, and the vertical axis shows the fresh weight of roots per plant (g / plant) in each treatment group. ＊ This indicates a statistically significant difference (P<0.05, t-test, n=5). This graph shows the results of a radish culture test using the environmental stress mitigating agent of this embodiment (Test Example 16). In the figure, white represents the control group, black represents the 0.004% treatment group, and the vertical axis shows the average root length (cm) in each treatment group. ＊ This indicates a statistically significant difference compared to the control group (P<0.05, t-test, n=9). This graph shows the results of a radish culture test using the environmental stress mitigating agent of this embodiment (Test Example 16). In the figure, white represents the control group, black represents the 0.004% additive group, and the vertical axis shows the average hypocotyl length (cm) in each treatment group. ＊This indicates a significant difference compared to the control group (P<0.05, t-test, n=9). This figure shows the flow of gene function analysis in RNA-seq (Test Example 18). This figure shows the effect of eggplant extract and ACh treatment on lettuce yield (30 days after sowing) (Test Example 18). This is a heatmap showing the results of cluster analysis for gene expression values ​​(FPKM) of all samples (Test Example 18). This figure shows the results of principal component analysis (PCA) for gene expression values ​​(FPKM) of all samples (Test Example 18). This is a Volcano Plot of differentially expressed genes (DEGs) (eggplant extract vs control) under normal temperature conditions (Test Example 18). This is a Volcano Plot of differentially expressed genes (DEGs) (eggplant extract vs control) under high temperature conditions (Test Example 18). This is a Volcano Plot of differentially expressed genes (DEGs) (ACh vs control) under high temperature conditions (Test Example 18). This figure compares genes whose expression levels significantly increased in DEGs analysis (eggplant extract vs. ACh) (Test Example 18). This figure compares genes whose expression levels significantly increased in DEGs analysis of eggplant extract and ACh treatment under high temperature conditions (Test Example 18). This figure shows the DEGs analysis results of ACh and eggplant extract treatment under room temperature conditions (Test Example 18). This figure compares the number of genes whose expression levels significantly increased in DEGs analysis (Test Example 18). This plot shows the results of comparing the untreated group under room temperature and high temperature conditions in GO analysis (Test Example 18). This plot shows the results of comparing eggplant extract and ACh treatment under room temperature conditions in GO analysis (Test Example 18). This figure shows GO terms that were significantly activated by eggplant extract treatment under room temperature conditions (left) and high temperature conditions (right) (Test Example 18). This figure shows the mechanism of "ON" and "OFF" of the low molecular weight GTP switch by eggplant extract treatment (Test Example 18). This is a heat map analysis diagram of Ras GTP-related gene groups under high temperature conditions (Test Example 18). This figure compares the GO analysis results of eggplant extract (right) and ACh treatment (left) under room temperature conditions (Test Example 18). This figure plots the GO analysis results of the action of ACh under high temperature conditions (Test Example 18). This Venn diagram shows the GO terms (total 94) for eggplant extract treatment, the GO terms (total 52) for ACh treatment, and 47 common GO terms (Test Example 18).This figure shows the results of functional analysis and heatmap comparison of expression data for protein folding-related gene groups common to eggplant extract and ACh treatment under high temperature conditions (Test Example 18). This figure shows the results of heatmap comparison of expression data for ion channel-related gene groups such as glutamic acid (GLR) common to eggplant extract and ACh treatment under high temperature conditions (Test Example 18). This figure shows the results of heatmap comparison of expression data for G protein-related gene groups common to eggplant extract and ACh treatment under high temperature conditions (Test Example 18). This figure shows the results of heatmap comparison of expression data for Ca-binding protein-related gene groups common to eggplant extract and ACh treatment under high temperature conditions (Test Example 18). This figure shows the results of heatmap comparison of expression data for heat shock protein-related gene groups common to eggplant extract and ACh treatment under high temperature conditions (Test Example 18). This figure shows the results of heatmap comparison of expression data for heat shock protein-related gene groups common to eggplant extract and ACh treatment under high temperature conditions and room temperature conditions (Test Example 18). This figure shows the results of comparing expression data using heatmaps for peptide biosynthesis-related genes common to eggplant extract and ACh treatment under high-temperature conditions (Test Example 18). This figure shows the results of comparing expression data using heatmaps for translation-related genes common to eggplant extract and ACh treatment under high-temperature conditions (Test Example 18). This figure shows the results of comparing expression data using heatmaps for DNA repair-related genes common to eggplant extract and ACh treatment under high-temperature conditions (Test Example 18). This figure shows the results of comparing expression data using heatmaps for reactive oxygen species-related genes common to eggplant extract and ACh treatment under high-temperature conditions (Test Example 18). This figure shows the results of comparing expression data using heatmaps for mismatch DNA repair-related genes common to eggplant extract and ACh treatment under high-temperature conditions (Test Example 18). This figure shows the results of functional analysis and comparison of expression data using heatmaps for DNA replication-related genes common to eggplant extract and ACh treatment under high-temperature conditions (Test Example 18). This figure shows the results of functional analysis and comparison of expression data using heatmaps for reactive oxygen species-related genes common to eggplant extract and ACh treatment under high-temperature conditions (Test Example 18).This figure shows the results of functional analysis and heatmap comparison of expression data for amino acid biosynthesis-related gene groups common to eggplant extract and ACh treatment under high temperature conditions (Test Example 18). This figure shows the results of functional analysis and heatmap comparison of expression data for amide biosynthesis-related gene groups common to eggplant extract and ACh treatment under high temperature conditions (Test Example 18). This figure shows the results of functional analysis and heatmap comparison of expression data for mismatch DNA repair-related gene groups common to eggplant extract and ACh treatment under high temperature conditions (Test Example 18). This figure shows the results of functional analysis and heatmap comparison of expression data for cell stress response-related gene groups common to eggplant extract and ACh treatment under high temperature conditions (Test Example 18). This figure shows the mechanism inferred from RNA-seq analysis that is common to eggplant extract and ACh treatment (Test Example 18). This figure shows the measurement results of total polyphenol content in culture media prepared using multiple eggplant varieties (Test Example 18). This figure shows the relationship between GO terms common to eggplant extract and ACh treatment (Test Example 18). This figure shows the relationship between GO terms common to eggplant extract and ACh treatment (Test Example 18). This figure shows the mechanism by which eggplant extract treatment alleviates high-temperature stress in lettuce in GO analysis (Test Example 18).

[0013] The following provides a detailed explanation of this disclosure.

[0014] [Composition] The composition according to the present disclosure is characterized by containing a neurotransmitter and / or an acetylcholinesterase (AChE) inhibitor, or a plant extract containing them (hereinafter, these may be collectively referred to as "plant extracts, etc."), and calcium ions. Here, the "neurotransmitter" can be one or more compounds selected from the group consisting of choline esters such as acetylcholine, metacholine, carbachol, besanechol, and succinylcholine; catecholamines such as epinephrine, norepinephrine, and dopamine; serotonin; and amino acids such as glutamic acid, alanine, glycine, proline, leucine, threonine, phenylalanine, γ-aminobutyric acid (GABA), arginine, histidine, cysteine, and asparagine amino acids. Preferably, it is one or more choline esters selected from the group consisting of acetylcholine, metacholine, carbachol, besanechol, succinylcholine, pharmaceutically acceptable salts thereof, and hydrates thereof, particularly acetylcholine. Recent studies have revealed that plants possess many homologous molecules similar to neurotransmitters in the animal nervous system, such as GABA, serotonin, melatonin, dopamine, ACh, and glutamate (Tretyn and Kendrick, 1991; Kuklin and Conger, 1995; Odjakova and Hadjiivanova, 1997; Roshchina, 2001; Baluska et al., 2004; Brenner et al., 2006). However, the role of these molecules in plants remains unclear (Ramakrishna et al., 2011; Pelagio-Flores et al., 2011; Pelagio-Flores et al., 2011; Park and Back, 2012). These compounds may function as deterrents against predation, but they may also provide signaling functions to plants in some way and may be involved in mitigating environmental stress in plants.The concentration of the neurotransmitter is not particularly limited, but when applied to plants, for example, in the case of hydroponics (concentration in hydroponic solution), it can be 0.00001 mM to 100 mM, preferably 0.0001 mM to 100 mM, or 0.001 mM to 10 mM. Furthermore, when applied by foliar spraying, soil injection, or irrigation, the application concentration of the neurotransmitter can be, for example, 0.0001 mM to 100 mM, but is not limited thereto. In this specification, the AChE inhibitor can be one or more selected from polyphenols, flavonoids, and organic acids, preferably one or more selected from malic acid, fumaric acid, tocopherol, pharmaceutically acceptable salts thereof, and hydrates thereof. The concentration of the AChE inhibitor is not particularly limited, but when applied to plants, the concentration in hydroponics (concentration in the hydroponic solution) can be 0.001 mM to 100 mM, preferably 0.01 mM to 10 mM. Furthermore, the application concentration of the AChE inhibitor when applied by foliar spraying, soil injection, or irrigation can be, for example, 0.01 mM to 1 M, but is not limited to this.

[0015] In this specification, "plant extract" may refer to an extract, extract solution, or dried or purified product thereof obtained by extracting from a plant containing the neurotransmitter and / or AChE inhibitor with a suitable extraction solvent. Furthermore, since it contains components equivalent to those of the extract, the sap of the plant, its concentrate, dried or purified product, etc., are also included in "plant extract." The concentration of the plant extract is not particularly limited, but as a solid content mass ratio when applied to a plant, for example in the case of hydroponics (concentration in hydroponic solution), it can be 0.0001 ppb to 100 ppm, preferably 10 ppm to 100 ppm. Here, the "extraction solvent" is preferably water, hot water, alcohol (especially ethanol), aqueous alcohol (especially aqueous ethanol), or petroleum ether, but is not limited to these. The "dried product" is preferably a product that has been processed by crushing, pulverizing, or powdering, and a powder with a small particle size is particularly desirable.

[0016] Since the plants used as raw materials for "plant extracts" contain the aforementioned neurotransmitters and / or AChE inhibitors, for example, eggplant, broccoli, banana, coconut, mandarin orange, herbs (coriander, cumin, sage, lemongrass, mugwort, comfrey, sage, perilla, lemon balm, oregano, catnip, common thyme, thyme, dill, dark opal, basil, hyssop, peppermint, spearmint, lamb's ear), houttuynia cordata, lavender, marigold, grape, coffee (coffee tree), tea (tea tree), cacao, acacia, Japanese cedar, pine, sugarcane, mango, banana, papaya, avocado, apple, cherry, guava, olive, root vegetables (sweet potato, purple sweet potato (sweet potato containing a lot of purple pigment), potato, yam, taro (taro, shrimp taro, etc.), konjac, etc.), persimmon, mulberry, blueberry, poplar, ginkgo, chrysanthemum, sunflower, bamboo, citrus fruits (lemon, lime, orange, grapefruit, navel orange, yuzu, kumquat, kabosu, summer orange, hassaku, iyokan, lime, Satsuma mandarin, shikwasa, mandarin, etc.), strawberry Blackberries, cranberries, raspberries, bilberries, huckleberries, plums, peaches, Japanese apricots, pears, European pears, loquats, kiwifruit, mangosteen, shishito peppers, prunes, melons, dragon fruit, goji berries, blackcurrants, cashews, viburnum, pomegranates, acai, aronia, tomatoes, soybeans, black soybeans, adzuki beans, green beans, peanuts, black sesame, buckwheat, buckwheat, sesame, purple cabbage, lacquer tree, sumac, garland chrysanthemum, spinach, komatsuna, mitsuba, okra, butterbur, onions, molokhia, garlic, purple onions, asparagus, parsley, eucalyptus, u Do, Gymnema sylvestre, Senna, Dandelion, Horsetail, Ferns (Bracken, Royal Fern, etc.), Oak, Quercus acutissima, Maple, Redwood, Metasequoia, Hinoki cypress, Mallotus japonicus, Japanese angelica tree, Hydrangea macrophylla, Akebia quinata, Aralia cordata, Clethra barbinervis, Magnolia obovata, Actinidia arguta, Lindera umbellata, Lindera umbellata, Aralia cordata, Clerodendrum trichotomum, Magnolia obovata, Actinidia polygama, Banaba, Rooibos, Rhodiola rosea, Lycium barbarum, Pueraria lobata, Iron chinensis, Melbao, Chinese parasol tree, Suou, Brazilberry, Melinjo, Cherry blossom, Magnolia, Yerba mate, Rhizophora stylosa, Bruguiera gymnorrhiza, Rhizophora stylosa, Rhizophora stylosa, Prunus jamasakura, Nipa palm,The following plants can be used, or processed products thereof: Amanita rhombea, false mangrove, Barringtonia racemosa, burdock, turmeric, lotus root, shepherd's purse, pine, carrot, bell pepper, chili pepper, paprika, bamboo, bamboo shoot, goldenrod, cassava, dwarf bamboo, clove, ginkgo, goldenrod, radish, mustard greens, etc. Of the above plants, eggplant, tomato, rosemary, dwarf bamboo, mint, eucalyptus, thyme, lemon balm, pine, coconut, bamboo, plum, chayote, lettuce, soybean, cassava, asparagus, mango, hibiscus, dandelion, horsetail, jolokia, and cacao. The above plants may also be parts of the plant body such as stems, leaves, fruits, roots, seeds, outer skin, buds, flowers, and rhizomes. Two or more of these plants can also be used in combination. Furthermore, these plants are preferable from the perspective of reducing manufacturing costs and solving the food loss problem, as they represent unused biomass such as organic waste discharged from food processing plants and crop residues generated after harvest in agriculture.

[0017] In this specification, "calcium ion" can be a water-soluble salt of calcium and is not particularly limited as long as it produces the effects of this disclosure. The calcium ion may be, for example, an inorganic salt such as calcium sulfate or calcium chloride, or a hydrate thereof, or an organic acid salt such as calcium lactate, or a hydrate thereof, with calcium chloride being particularly preferred. The concentration of the calcium ion is not particularly limited, but as a concentration when applied to plants, for example in hydroponics (concentration in hydroponic solution), it can be 0.0001 mM to 100 mM, preferably 0.001 mM to 100 mM, more preferably 0.01 mM to 10 mM, even more preferably 0.1 mM to 1.0 mM, and particularly preferably 0.2 mM to 0.7 mM. Furthermore, the application concentration of calcium ions when applied by foliar spraying, soil injection, or irrigation can be, for example, 0.0001 mM to 100 mM, but is not limited thereto.

[0018] The compositions relating to this disclosure may, in addition to the plant extracts and calcium ions, contain iron ions and other nutrients necessary for crop growth, other biostimulants, other plant growth promoters, fungicides, insecticides, plant hormones, and other additives commonly used for agricultural purposes, provided that they do not inhibit the effects of this disclosure.

[0019] The "iron ions" can be water-soluble salts of iron(II) or iron(III), and are not particularly limited as long as they do not hinder the effects of this disclosure. The iron ions may be, for example, iron(II) chloride, iron(III) chloride, iron(II) sulfate, iron(III) sulfate, or hydrates thereof, with iron(II) chloride being particularly preferred. The concentration of the iron ions is not particularly limited, but as a concentration when applied to plants, for example in hydroponics (concentration in hydroponic solution), it can be 0.0001 mM to 100 mM, preferably 0.001 mM to 100 mM, more preferably 0.01 mM to 10 mM, even more preferably 0.1 mM to 1.0 mM, and particularly preferably 0.2 mM to 0.7 mM. Furthermore, the application concentration of iron ions when applied by foliar spraying, soil injection, or irrigation can be, for example, 0.0001 mM to 100 mM, but is not limited thereto.

[0020] The "other nutrients necessary for crop growth" that may be included in the compositions of this disclosure may be, for example, trace elements other than iron (one or more selected from the group consisting of boron, manganese, zinc, copper, and molybdenum) or macro elements (nitrogen, phosphorus, potassium), and the aforementioned trace elements are preferred but not limited thereto. Boron may be, for example, 0.001 mM to 10 mM boric acid or sodium borate; manganese may be, for example, 0.001 mM to 10 mM manganese sulfate, manganese nitrate, or manganese chloride; zinc may be, for example, 0.00001 mM to 10 mM zinc sulfate, zinc nitrate, or zinc chloride; copper may be, for example, 0.00001 mM to 10 mM copper sulfate, copper nitrate, or copper chloride; and molybdenum may be, for example, 0.000001 mM to 10 mM sodium molybdate or ammonium molybdate, and these may be hydrates, but are not limited thereto.

[0021] The plants to which the composition of the present disclosure can be applied are not particularly limited, and examples thereof include vegetables such as leafy vegetables, fruit vegetables, root vegetables, and flower vegetables, grains such as rice and wheat, flowers, fruit trees, beans, etc. Preferably, they are lettuce, shungiku, cabbage, spinach, tomato, eggplant, bell pepper, turkey mullein, green bean, soybean, carrot, daikon radish, corn, sweet corn, rice, melon, watermelon, banana, orange, lemon, grape, pineapple, pear, apple, peach, chestnut, kaki, fig, walnut, raspberry, goji berry, Japanese apricot, blueberry, cotton, coffee, sugarcane, etc. Further, the form of the composition of the present disclosure is not particularly limited and may be any form such as liquid, powder, granule, etc., but an aqueous solution form is preferred.

[0022] [Method for producing the composition] Next, the method for producing the composition according to the present disclosure will be described. The composition according to the present disclosure can be produced by mixing the plant extract or the like and the calcium ions in the presence of water. That is, the method for producing a composition containing a plant extract in one embodiment of the present disclosure includes: (1) a step of extracting a plant using an extraction solvent to obtain a plant extract; and (2) a step of mixing the plant extract and calcium ions in the presence of water. Further, the method for producing a composition containing a neurotransmitter and / or an AChE inhibitor instead of the plant extract in another embodiment of the present disclosure includes a step of mixing the neurotransmitter and / or the AChE inhibitor and calcium ions in the presence of water. Here, the form of the neurotransmitter and / or the AChE inhibitor used in the other embodiment is not particularly limited, and it may be an isolated or synthesized compound, or it may be a composition obtained by concentrating or purifying the plant extract by a known method.

[0023] The plant extract can be obtained by extracting the aforementioned plant using the aforementioned extraction solvent. The extraction conditions are not particularly limited, but the extraction temperature may be room temperature to 50°C, 50 to 70°C, 70 to 90°C, 90 to 110°C, 110 to 130°C, 130 to 150°C, room temperature to 150°C, 40 to 150°C, 50 to 150°C, 60 to 150°C, 70 to 150°C, 80 to 140°C, 90 to 140°C, 90 to 130°C, 100 to 130°C, 100 to 120°C, or 110 to 120°C. The extraction time is also not particularly limited, but for example, at room temperature (e.g., 10 to 35°C) it may be 1 to 48 hours or 2 to 24 hours, and under heating conditions of 40°C or higher it may be 5 to 60 minutes, 10 to 45 minutes, 15 to 30 minutes, or 15 to 25 minutes. The amount of extraction solvent used is not particularly limited, but it may be 1 / 20 to 20 parts by mass per 1 part by mass of plant material (on a dry basis). Furthermore, operations such as purification, concentration, and drying can be performed after extraction.

[0024] The mixing operation of the plant extract and calcium ions is carried out in the presence of water. Here, the presence of water means that the plant extract and calcium ions can react with water as the medium. The amount of water should be at least enough to allow mixing and stirring of the plant extract and calcium ions, and may be just enough to wet the mixture of plant extract and calcium ions. For example, the condition may be that there is 1 / 20 to 20 parts by mass of water per 1 part by mass of solid content of the plant extract. When using plant juice or extract in liquid form as the plant extract, or when using calcium ions in aqueous solution form, the two can be mixed directly without adding a new medium. Furthermore, the iron ions and / or trace elements may be mixed in the mixing step. The mixing operation of iron ions and / or trace elements may be carried out simultaneously with the mixing operation of plant extract and calcium ions, or as a separate step. If it is a separate step, there is no particular order in which the plant extract, calcium ions, iron ions, and trace elements are mixed. For example, the mixing step may sequentially include a step of mixing plant extracts and trace elements (and iron ions), and a step of further mixing calcium ions, or it may include a step of simultaneously mixing plant extracts, calcium ions, trace elements and / or iron ions. The mixing operation can be performed by simple stirring with a stirrer, but it can also be done with a mixer, a large stirring tank, a vortex, a shaker, etc. The temperature conditions during mixing are not particularly limited and can be, for example, around room temperature (e.g., 10 to 35°C) or under heated conditions (e.g., 40 to 60°C, 60 to 80°C, 80 to 100°C). The mixing time can be until the plant extracts and calcium ions are in sufficient contact and is not particularly limited, but can be, for example, 1 to 60 minutes, 5 to 50 minutes, 10 to 40 minutes, or 20 to 30 minutes.

[0025] The method for manufacturing the composition according to the present disclosure may further include a step of mixing other biostimulant materials, other plant growth promoters, fungicides, insecticides, plant hormone agents, and other additives generally used for agricultural materials. Further, it can include one or more steps necessary for formulation, such as sterilization treatment, concentration, powdering, granulation, etc., in any combination.

[0026] [Functions and Effects of the Composition] The composition of the present disclosure thus obtained is considered to have a function of turning on the switches of various stress tolerance mechanisms of plants, such as high-temperature tolerance, low-temperature tolerance, salt stress tolerance, and oxidative stress tolerance, which plants inherently possess. Further, it has been clarified that the composition of the present disclosure has a function of inducing the expression of many gene groups related to DNA repair, such as DNA repair-related gene groups, mismatch DNA repair-related gene groups, and DNA replication-related gene groups. Therefore, it is expected that the composition according to the present disclosure can artificially control the environmental stress tolerance acquisition mechanism of plants by applying this to plants. For example, when plant cells are exposed to environmental stress such as high temperature, the influx of calcium ions from the outside of the cell through ion channels is caused. The composition of the present disclosure is considered to activate intracellular calcium signal transduction by opening this ion channel and confer environmental stress tolerance to plants. Further, the composition of the present disclosure is considered to repair DNA damage caused by environmental stress and confer environmental stress tolerance to plants by upregulating the expression of many gene groups related to DNA repair. There is no particular limitation on the plants to which the composition of the present disclosure can be applied. Further, the composition of the present disclosure can promote the growth of plants under environmental stress by conferring environmental stress tolerance to plants. From the above, the composition of the present disclosure has functions as a "plant environmental stress reliever", a "plant growth promoter", and a "plant DNA repair promoter".

[0027] [Method of Use of the Composition] Next, the method of use of the composition of this disclosure will be described. The composition of this disclosure can be used by applying an effective amount to plants in the same manner as conventional fertilizers, plant growth promoters, and biostimulant materials. The effective amount may be an amount that produces an effect of mitigating environmental stress, promoting growth, or promoting the expression of DNA repair-related genes in plants. For example, in the case of hydroponics, a simple and preferred method of use is to add an appropriate amount of the composition of this disclosure to the hydroponic solution. Alternatively, for example, the composition of this disclosure may be sprayed or applied in an appropriate amount to the leaves of plants. Alternatively, for example, it can be used by drenching the soil in which plants are growing, treating seeds, or mixing it with other agricultural materials such as liquid fertilizers, plant growth promoters, and biostimulant materials. When adding the composition of this disclosure to hydroponic solutions or other agricultural materials, the amount and concentration added should be adjusted as appropriate so that the concentration of plant extracts, calcium ions, etc., is within the range of the application concentration described above, or so that an effect of mitigating environmental stress, promoting growth, or promoting the expression of DNA repair-related genes in plants is produced. Furthermore, the timing and number of times the composition of this disclosure is used can be appropriately set in accordance with the period when crops are susceptible to environmental stress, and are not particularly limited.

[0028] [Plant Environmental Stress Relief Agent] The plant environmental stress relief agent according to the first embodiment of this disclosure will be described below. Matters not specifically defined herein will follow the definitions of "composition" and other terms previously stated. In this specification, "environmental stress" means abiotic stress experienced by plants, and examples include high temperature stress, low temperature stress, salt stress, and oxidative stress. In this specification, "high temperature" refers to temperature conditions higher than the optimal growth temperature of the crop to which the agent is applied. By applying the environmental stress relief agent according to this embodiment to plants, environmental stress such as high temperature stress to plants can be alleviated. Whether or not the environmental stress of plants has been alleviated by the environmental stress relief agent of this embodiment can be evaluated by cultivating plants under specific environmental stress and comparing the yield of plants treated with the environmental stress relief agent with that of untreated plants. Specifically, if the yield of plants treated with the environmental stress relief agent is statistically significant (Tukey test, p<0.05, n≧3) compared to untreated plants during the same cultivation period, it can be evaluated that "the environmental stress of plants has been alleviated."

[0029] [Plant Growth Promoter] The plant growth promoter according to the second embodiment of this disclosure will be described below. Matters not specifically defined herein will be defined according to previously stated definitions, etc. By applying the plant growth promoter according to this embodiment to plants, plant growth can be promoted. There are no particular restrictions on the temperature conditions when applying the plant growth promoter, and the plant growth promoter of this embodiment can be used under both high temperature and normal temperature conditions. In this specification, "high temperature" refers to temperature conditions higher than the optimal growth temperature of the crop to be applied, and "normal temperature" refers to temperature conditions within the range of the optimal growth temperature of the crop to be applied. However, the plant growth promoter of this embodiment exhibits a higher effect under high temperature conditions. However, as will be described later in the examples, the composition according to this disclosure has been shown to increase the expression levels of photosynthesis-related gene groups and / or G protein-related gene groups in plants under normal temperature conditions. Here, the photosynthesis-related gene group includes, for example, the Photosystem II (PSII) related gene group such as PsbA, PsbB, PsbC, PsbD, PsbH, PsbO, PsbR, PsbS, PsbW, PsbY, PSB27A, Psb28; the Photosystem I (PSI) related gene group such as PsaC, PsaD, PsaE, PsaF, PsaG, PsaH, PsaK, PsaL, PsaN, PsaO, Psa2; and PNSL1, PNSL2, PNSL 3. The genes may be one or more genes selected from the following: PNS subunit-related genes of the chloroplast NDH complex, such as PNSL4, PNSL5, PNSB1, PNSB2, PNSB3, PNSB4, and PNSB5; antenna complex-related genes, such as LHCA5 and LHCA6; photoreceptor / photosignaling-related genes, such as PHOT1, PHOT2, RPT2, RPT3, CRY1, and CRY2; and assembly / stabilization factor-related genes, such as PAM68.The G protein-related gene group may be one or more genes selected from, for example, the small GTPase-related gene group such as RAC1, RAC2, ROPGEF1, ARAC7, Rab2B, RABA2a, RABA3, RB11C, RABH1b, RABH1e, RAN1A, RAN3, MIRO2; the membrane transport / protein delivery system GTPase-related gene group such as SRP54, TOC34_PEA; and the translation / ribosome-related GTP / ATP-binding protein-related gene group such as EF1A1, NOG1, NOA1, NSN1, OLA1, OBGM, BPG2, GTPase ERA-Like. Orthologs or paralogs of the above genes are also included in the above examples. From these findings, it is considered that the plant growth promoter of this embodiment has the effect of promoting plant growth even under normal temperature conditions. Whether or not plant growth has been promoted by the plant growth promoter of this embodiment can be evaluated by comparing the yield of cultivated plants with and without the plant growth promoter. Specifically, if the yield of plants treated with the plant growth promoter is statistically significant (Tukey test, p<0.05, n≧3) compared to untreated plants during the same cultivation period, then it can be evaluated that "plant growth has been promoted."

[0030] [Plant DNA Repair Accelerator] The plant DNA repair accelerator according to the third embodiment of this disclosure will be described below. Matters not specifically defined herein will follow the definitions and other conventions previously stated. By applying the plant DNA repair accelerator according to this embodiment to plants, the expression of DNA repair-related genes in the plants can be increased, thereby promoting the repair of DNA damage (particularly caused by environmental stress). There are no particular restrictions on the temperature conditions when applying the DNA repair accelerator, and the DNA repair accelerator of this embodiment can be used under both high-temperature and normal-temperature conditions. However, as will be described later in the examples, the DNA repair accelerator of this embodiment exhibits a higher effect under high-temperature stress conditions. Whether or not the repair of DNA damage in plants has been promoted by the DNA repair accelerator of this embodiment can be evaluated by measuring the expression levels of DNA repair-related genes in plants (i.e., one or more genes selected from the DNA repair-related gene group, the mismatch DNA repair-related gene group, and the DNA replication-related gene group) and comparing the results with and without treatment of the DNA repair accelerator. Here, "DNA repair-related gene group" means one or more genes selected from the DNA repair-related gene group selected from TFB4, POLH, FEN1, FPG, GEN1, TYDP1, RQL4A, MSL1, MRE11; the mismatch DNA repair-related gene group selected from MSH1, MSH2, MSH3, MSH4, MSH5, MSH6, MSH7; and the DNA replication-related gene group selected from SR160, POLA1, POLD2, MCM2, MCM3, MCM5, MCM6, MCM7, ORC2, RPA70, RFC1, RFC2, RFC3, THP2A, TEBICHI, RTEL1, FANCJ, PCNA, SWI3, POLε, PPO2. The gene names listed above are examples of names corresponding to genes in lettuce, and orthologs or paralogs in crops are also included in these examples. Specifically, if the RNA expression level of a particular gene among the DNA repair-related gene group increases statistically significantly (Wald test, padj<0.05, n≧3) in plants treated with a DNA repair accelerator compared to untreated plants, it can be evaluated that "the repair of DNA damage in plants was promoted (i.e., the expression level of the DNA repair-related gene group increased)."Gene expression levels are measured using next-generation sequencers (e.g., Illumina's NovaSeq). TM This can be done using X Plus or equivalent equipment.

[0031] The "DNA repair-related gene group" whose expression is enhanced by the composition of this disclosure is suggested to be a group of genes related to the acquisition of environmental stress tolerance (particularly high temperature stress tolerance) in plants, as shown in the examples described later. Therefore, this gene group is provided as a gene set useful for acquiring environmental stress tolerance (particularly high temperature stress tolerance) in plants. For example, it is thought that it will be possible to cultivate and develop plants with environmental stress tolerance (high temperature stress tolerance) by performing genetic modification to enhance the function of at least one gene from the above gene group in the plant body. In other words, this disclosure provides a gene set used to confer environmental stress tolerance to plants, and a method for breeding plants with environmental stress tolerance, which includes performing genetic modification on plants to enhance the function of one or more genes selected from the gene set. Here, the method for genetic modification can be any method known in the art, for example, traditional breeding methods, marker-assisted selection methods, or genetic recombination methods can be employed.

[0032] Furthermore, it has become clear that the composition of this disclosure has an effect of enhancing the expression of various gene groups, including not only the "DNA repair-related gene group" but also gene groups related to environmental stress response mechanisms such as high-temperature stress, and photosynthesis-related gene groups, as shown in the examples described later. Therefore, the composition of this disclosure also functions as a "gene expression enhancer" that enhances the expression of these genes.

[0033] The present disclosure will be described in detail below with reference to examples and comparative examples.

[0034] (Test Example 1) High Temperature Tolerance Test Using Lettuce Young Plants (Part 1) Many vegetables cultivated in Japan have low heat tolerance. Lettuce is also adapted to relatively cool weather conditions, and its optimal growing temperature is 18-23°C. In the cultivation of head lettuce at high temperatures, abnormal heads such as oversized heads, undersized heads, bamboo shoot-shaped heads, and heads with protruding midribs tend to occur, and the occurrence of physiological disorders (tip burn) due to Ca deficiency becomes a problem. Conventionally, tests to confirm the heat tolerance of crops based on these symptoms have taken several months. Therefore, the inventor of this application has developed a model experimental system to investigate whether lettuce has acquired heat tolerance by conducting a two-week high-temperature cultivation test, utilizing the fact that in lettuce young plants, the differentiation of flower buds and bolting stems is induced by high temperature and long-day conditions, and the stem elongates.

[0035] (1) Preparation of environmental stress mitigating agent using eggplant residue The eggplant residue used was: (a) 50g of eggplant flesh after removing the peel after harvest, (b) 20g of sprouts removed during eggplant cultivation management, (c) 20g of eggplant peel after harvest, and (d) 20g of eggplant root after harvest. Each of these eggplant residues was placed in a 1L beaker, 700mL of distilled water was added, and the mixture was heated at 120°C for 20 minutes. The supernatant was then filtered through filter paper, and boric acid (3g), manganese sulfate (2g), zinc sulfate (0.22g), copper sulfate (0.05g), sodium molybdate (0.01g), iron sulfate (15g), and calcium chloride (55g) were added to the filtrate (eggplant residue extract). Further distilled water was added to obtain 1L eggplant residue mixtures (a to d). Furthermore, eggplant residue mixtures without added calcium ions (a' to d') were prepared in the same manner as above, except that calcium chloride was not added, and these were used as comparative examples.

[0036] (2) Hydroponic cultivation test of young lettuce plants 1 mL each of the following nutrient solutions A to D was combined with 1 mL of any of the eggplant residue mixtures (a to d, a' to d') prepared above, and distilled water was added to prepare 1 L of hydroponic solution. The amount of nutrient solution used to prepare each hydroponic solution (1 L) is shown in Table 1. The concentrations of calcium and trace elements in each hydroponic solution were as follows: boric acid 0.05 mM, manganese sulfate 0.01 mM, zinc sulfate 0.001 mM, copper sulfate 0.0003 mM, sodium molybdate 0.0005 mM, iron sulfate 0.1 mM, and calcium chloride 0.5 mM. The solid content concentrations of the eggplant residue extract in each hydroponic solution were 50,000 mg / L for the pulp (a, a'), 20,000 mg / L for the sprouts (b, b'), 20,000 mg / L for the peel (c, c'), and 20,000 mg / L for the roots (d, d').

[0037] Nutrient solution A: Put 700 mL of distilled water and 202 g of KNO3 into a 1 L beaker, stir until dissolved, and then add more distilled water to make a total volume of 1 L. Nutrient solution B: Put 700 mL of distilled water and 178 g of KH2PO4 into a 1 L beaker, stir until dissolved, and then add more distilled water to make a total volume of 1 L. Nutrient solution C: Put 700 mL of distilled water and 44 g of NH4NO3 into a 1 L beaker, stir until dissolved, and then add more distilled water to make a total volume of 1 L. Nutrient solution D: Put 700 mL of distilled water and 489 g of MgSO4・7H2O into a 1 L beaker, stir until dissolved, and then add more distilled water to make a total volume of 1 L.

[0038] Each prepared hydroponic solution was placed in a 1.2L simple hydroponic growing container, and lettuce seedlings (10 days after sowing) were transplanted. The plants were then cultivated for 14 days under the following conditions: 12 hours of light / 35°C, 12 hours of darkness / 25°C, and 60% humidity. As a control group, a hydroponic solution prepared without the eggplant residue mixture (a-d, a'-d') was used. After 14 days of cultivation, the lettuce was harvested and the yield was measured.

[0039]

[0040] (3) Results and discussion are shown in Table 2. The values ​​in Table 2 represent the relative yield (%) in each treatment group, with the yield of each control group (nutrient solutions A to D only) set to 100%. From Table 2, it was shown that the treatment groups using hydroponic solution with added eggplant residue extract and calcium ions (calcium chloride) showed a significant increase in yield, ranging from 4 to 10.5 times, compared to the comparative example treatment group using hydroponic solution without added calcium ions. From these results, it was found that cultivating lettuce seedlings with the environmental stress mitigating agent of this embodiment added to the hydroponic solution greatly promotes growth under high temperature conditions (25°C to 35°C) and imparts high temperature stress tolerance.

[0041]

[0042] (Test Example 2) High Temperature Tolerance Test Using Young Lettuce Plants (Part 2) Next, we investigated the effect of changes in calcium concentration on mitigating high temperature stress.

[0043] (1) Preparation of environmental stress mitigating agent using eggplant residue As eggplant residue, 20g of sprouts removed during eggplant cultivation management and 50g of eggplant fruit (with skin) after harvest were used. Each of these eggplant residues was placed in a 1L beaker, 1000mL of distilled water was added, and the mixture was heated at 120°C for 20 minutes. The supernatant was then filtered through filter paper, and boric acid (3g), manganese sulfate (2g), zinc sulfate (0.22g), copper sulfate (0.05g), sodium molybdate (0.01g), iron sulfate (15g), and calcium chloride (55g or 110g) were added to the filtrate (eggplant residue extract). Further distilled water was added to obtain 1L each of eggplant sprout mixture and eggplant fruit mixture. Eggplant sprout mixture and eggplant fruit mixture without calcium ions were prepared in the same manner as above, except that calcium chloride was not added, and these were used as comparative examples.

[0044] (2) Hydroponic cultivation test of young lettuce plants 1 mL each of nutrient solutions A to D from Test Example 1 and 1 mL of either the eggplant sprout mixture or eggplant fruit mixture prepared above were combined, and distilled water was added to prepare 1 L of hydroponic solution. The amount of nutrient solution used to prepare each hydroponic solution (1 L) is shown in Table 3. The concentrations of calcium and trace elements in each hydroponic solution were 0.05 mM boric acid, 0.01 mM manganese sulfate, 0.001 mM zinc sulfate, 0.0003 mM copper sulfate, 0.0005 mM sodium molybdate, 0.1 mM iron sulfate, and 0.5 mM or 1.0 mM calcium chloride. The solid content concentration of the eggplant residue extract in each hydroponic solution was 20.000 mg / L for sprouts and 50.000 mg / L for fruit.

[0045] Each prepared hydroponic solution was placed in a 1.2L simple hydroponic growing container, and lettuce seedlings (10 days after sowing) were transplanted. The plants were then cultivated for 14 days under cultivation conditions of 12 hours of light / 35°C, 12 hours of dark / 25°C, and 60% humidity. As a control group, a hydroponic solution to which a predetermined amount of calcium chloride solution was added was used instead of the eggplant sprout mixture and eggplant fruit mixture. After 14 days of cultivation, the lettuce was harvested and the yield was measured, and the Ca concentration in the plants was measured using an ICP emission spectrometer.

[0046]

[0047] (3) Results and Discussion The results are shown in Table 4 and Figures 1A, B, C, 2, and 3. Figures 1A, B, and C are photographic images showing the high-temperature tolerance test. Figure 1A shows the treatment group with no calcium added, Figure 1B shows the treatment group with 0.5 mM calcium, and Figure 1C shows the treatment group with 1.0 mM calcium. Figure 2 is a graph showing the measured values ​​of lettuce yield, and Figure 3 is a graph showing the relationship between lettuce yield and calcium concentration. The values ​​in Table 4 and Figure 2 represent the relative yield (%) in each treatment group, with the yield of each control group set to 100%.

[0048] In Figure 1A (no calcium added), leaves yellowed in all treatment groups, showing symptoms of Ca deficiency due to heat damage. However, in Figure 1B (0.5 mM calcium), compared to the control group (untreated) without eggplant residue extract, the treatment group with eggplant residue extract showed no heat damage to the leaves and clearly promoted growth. In Figure 1C (1.0 mM calcium), no leaf damage was observed in the control group (untreated), but a clear growth-promoting effect was observed in the treatment group with eggplant residue extract.

[0049] Table 4 and Figure 2 show that the treatment groups using hydroponic solutions supplemented with eggplant residue extract and calcium ions (calcium chloride) showed a significant increase in yield, ranging from 2 to 6.5 times, compared to the control groups using hydroponic solutions without eggplant residue extract, at all calcium ion concentrations. In contrast, no significant difference was observed in the treatment groups without calcium ions compared to the control groups (nutrient solutions A to D only). These results indicate that cultivating lettuce seedlings with the environmental stress mitigating agent of this embodiment added to the hydroponic solution greatly promotes growth under high-temperature conditions (25°C to 35°C) and confers high-temperature stress tolerance.

[0050] Figures 3(B) and (C) show that calcium ion concentration increased in the treatment plots to which the environmental stress mitigating agent of this embodiment was applied. Furthermore, since higher calcium ion concentrations in plants tended to correlate with higher yields, it was suggested that the increase in calcium ion concentration was related to the acquisition of high-temperature tolerance. As mentioned above, it is thought that an increase in intracellular calcium ion concentration switches on various stress tolerance mechanisms in plants. Therefore, it was inferred that the environmental stress mitigating agent of this embodiment has the effect of mitigating various environmental stresses in addition to high-temperature stress.

[0051]

[0052] (Test Example 3) Comparison with Unused Biomass Other Than Eggplant Residue: The environmental stress mitigating agent of this embodiment was prepared using unused biomass other than eggplant residue, and its effects were compared.

[0053] (1) Preparation of environmental stress mitigating agents Various crop residues shown in Table 5 below were used. The amounts of each crop residue shown in Table 5 were placed in 1 L beakers, 700 mL of distilled water was added, and the mixture was heated at 120°C for 20 minutes. The supernatant was then filtered through filter paper to obtain the filtrate (crop residue extract). Boric acid (3 g), manganese sulfate (2 g), zinc sulfate (0.22 g), copper sulfate (0.05 g), sodium molybdate (0.01 g), iron sulfate (15 g), and calcium chloride (55 g) were added to this filtrate, and then distilled water was added to obtain 1 L of mixed crop residue solution.

[0054]

[0055] (2) Hydroponic cultivation test of young lettuce plants. 1 mL each of nutrient solutions A to D from Test Example 1 was combined with 1 mL of any of the mixed crop residue solutions prepared above, and distilled water was added to prepare 1 L of hydroponic solution. The solid content concentration of the crop residue extract in each hydroponic solution was 10,000 mg / L.

[0056] Each prepared hydroponic solution was placed in a 1.2L simple hydroponic growing container, and lettuce seedlings (10 days after sowing) were transplanted. The plants were then cultivated for 14 days under the following conditions: 12 hours light / 35°C, 12 hours dark / 25°C, and 60% humidity. As a control group, a hydroponic solution to which 1 mL of 0.5 M calcium chloride solution was added instead of the crop residue mixture was used. As a comparative example, a hydroponic solution to which humic substances (humic acid 0.3 g / L) and amino acids (glutamic acid 0.1 mM), which have been reported to have a high-temperature stress-relieving effect, were added instead of the crop residue mixture was used. After 14 days of cultivation, the lettuce was harvested and the yield was measured.

[0057] (3) Results and Discussion The results are shown in Table 6 and Figure 4. The values ​​in Table 6 and Figure 4 represent the relative yield (%) in each treatment group, with the yield of the control group set to 100%. From Table 6 and Figure 4, it was shown that the treatment groups using hydroponic solution supplemented with crop residue extract and calcium ions (calcium chloride) showed a significant increase in yield, ranging from 2 to 11 times, compared to the control group using hydroponic solution without the addition of crop residue extract. In particular, the environmental stress mitigators using eggplant, tomato, rosemary, bamboo grass, mint, eucalyptus, thyme, lemon balm, pine, coconut, bamboo, plum, chayote, lettuce, soybean, cassava, asparagus, mango, hibiscus, dandelion, horsetail, jolokia, and cacao showed a remarkable effect compared to conventional humus and amino acids. From these results, it was found that the environmental stress mitigators of this embodiment can impart high-temperature stress tolerance to plants even when using plant extracts other than eggplant.

[0058] **Conventional techniques used included humic substances (humic acid 0.3 g / L) and amino acids (glutamic acid 0.1 mM).**

[0059] (Test Example 4) High Temperature Stress Reduction Effect of Standard Substances The plant environmental stress mitigator in this embodiment is thought to activate intracellular Ca signaling and confer environmental stress tolerance to plants. Furthermore, Test Example 2 suggested a correlation between improved high temperature tolerance and increased Ca concentration in plants.Therefore, this test example aimed to investigate the environmental stress reduction effect when a standard substance known as a neurotransmitter or acetylcholinesterase inhibitor that causes the influx of Ca ions into cells is used together with calcium ions, in order to help elucidate the mechanism of this disclosure.

[0060] (1) Test Method 20g of each standard substance shown in Figure 5 was taken, and boric acid (3g), manganese sulfate (2g), zinc sulfate (0.22g), copper sulfate (0.05g), sodium molybdate (0.01g), iron sulfate (15g), and calcium chloride (55g) were added to it, and distilled water was added to obtain 1L of each standard substance mixture. Next, 1mL each of nutrient solutions A to D from Test Example 1 was combined with 1mL of any of the standard substance mixtures prepared above, and distilled water was added to prepare 1L of hydroponic solution. Each of the prepared hydroponic solutions was placed in a 1.2L simple hydroponic growing container, and lettuce seedlings (10 days after sowing) were planted in them and cultivated for 14 days under cultivation conditions of 12h light / 35℃, 12h dark / 25℃, and 60% humidity. As a control group (untreated), a hydroponic solution to which 1mL of 0.5M calcium chloride aqueous solution was added instead of the standard substance mixture was used. As a comparative example, hydroponic solutions were used to which GABA (final concentration 0.516 g / L), humic acid (final concentration 0.3 g / L), and alanine (final concentration 0.5 g / L), which have been conventionally reported to have high-temperature stress-relieving effects, were added instead of the standard substance. Lettuce was harvested after 14 days of cultivation and the yield was measured.

[0061] (2) Results and Discussion The results are shown in Figure 5. The values ​​in Figure 5 represent the relative yield (%) in each treatment group, with the yield of the control group (untreated) set to 100%. From Figure 5, it was shown that the treatment groups using malic acid, fumaric acid, acetylcholine, tocopherol, and succinic acid showed a significant increase in yield compared to the control group and the comparative example. Acetylcholine is a neurotransmitter known to be contained in many plants, and malic acid, fumaric acid, and tocopherol are all acetylcholinesterase inhibitors contained in plants. From these results, it was suggested that these acetylcholine and acetylcholinesterase inhibitors are likely to be the active ingredients in the environmental stress mitigator of this embodiment.

[0062] (Test Example 5) High Temperature Tolerance Test Using Young Lettuce Plants (Part 3) An environmental stress reliever made from eggplant residue was added to the hydroponic solution, and leaf lettuce was grown on a small scale using hydroponics to verify its effect on reducing high temperature stress.

[0063] (1) Preparation of an environmental stress mitigator using eggplant residue 500g of eggplant fruit (with skin) after harvest was used as the eggplant residue. This eggplant residue was placed in a 1L beaker, and trace elements (150g iron sulfate, 30g boric acid, 20g manganese sulfate pentahydrate, 2.2g zinc sulfate heptahydrate, 0.5g copper sulfate pentahydrate, 0.01g ammonium molybdate dihydrate) were added and mixed until completely homogeneous. Then, 1000mL of distilled water was added, and the mixture was heated at 120°C for 20 minutes. The supernatant was filtered through filter paper to obtain the filtrate (eggplant residue extract). 55g of calcium chloride was added to this filtrate, and then distilled water was added to obtain a 1L eggplant residue mixture. Next, the above eggplant residue mixture was added to a commercially available liquid fertilizer for hydroponics (OAT House A formula: OAT House No. 1 (1.5g / L) + OAT House No. 2 (1g / L), manufactured by OAT Agrio Co., Ltd.) in the ratios shown in Table 7 to obtain a hydroponic solution. The above-mentioned commercially available liquid fertilizer was used as a comparative example.

[0064]

[0065] (2) Small-scale hydroponic cultivation test of lettuce The prepared hydroponic solutions were placed in 14L hydroponic cultivation pipes (36 plants) and leaf lettuce seedlings (10 days after sowing, variety name "Sunny") were transplanted and cultivated in a greenhouse for 27 days. Figure 6 shows the temperature changes inside the greenhouse during the cultivation period. As shown in Figure 6, there were days when the maximum daytime temperature reached 50°C. The weight of the lettuce was measured every few days during the cultivation period.

[0066] (3) Results and discussion are shown in Figure 7 and Table 8. In Figure 7, the horizontal axis shows the number of days after transplanting, and the vertical axis shows the fresh weight per lettuce plant (g / plant) in each treatment group. From Figure 7 and Table 8, it was shown that in the treatment groups using hydroponic solutions with 0.1% and 1% eggplant residue mixture added, the yield increased by more than three times compared to the control group of the comparative example using hydroponic solutions without eggplant residue mixture. From these results, it was found that by adding the environmental stress reliever of this embodiment to the hydroponic solution during cultivation, the growth of lettuce seedlings under high temperature conditions is greatly promoted, and high temperature stress tolerance is conferred.

[0067] *:Number of days after planting

[0068] (Test Example 6) High Temperature Tolerance Test Using Young Lettuce Plants (Part 4) An environmental stress reliever made from eggplant residue was added to the hydroponic solution, and large-scale hydroponic cultivation of leaf lettuce was carried out to verify its effect on reducing high temperature stress.

[0069] (1) Preparation of an environmental stress reliever using eggplant residue The preparation of the eggplant residue mixture and the hydroponic solution obtained by adding it to a commercially available liquid fertilizer was carried out in the same manner as in Test Example 5. In each treatment group, as in Test Example 5, a commercially available liquid fertilizer (OAT House A formula) to which the eggplant residue mixture was added in the following proportions of 0.01%, 0.1%, and 1%, respectively, was used, while the comparative example (control group) used only the commercially available liquid fertilizer.

[0070] (2) Large-scale hydroponic cultivation test of lettuce. The prepared hydroponic solution (700L) was placed in each hydroponic cultivation bed (10m long, 328 plants) with a capacity of 700L. Leaf lettuce seedlings (10 days after sowing, varieties "Sunny", "Green Web", "Nishinabe", and "Greenspan") were transplanted and cultivated in a greenhouse for 28 days. Figure 8 shows the temperature changes inside the greenhouse during the cultivation period. As shown in Figure 8, there were days when the maximum daytime temperature reached 45°C. After the end of cultivation, the weight of the lettuce was measured.

[0071] (3) Results and discussion are shown in Figure 9 and Table 9. In Figure 9, the horizontal axis shows each treatment group, with white representing the control group (liquid fertilizer only), black representing the group with 0.01% eggplant residue solution added, gray representing the group with 0.1% eggplant residue solution added, and diagonal lines representing the group with 1% eggplant residue solution added. The vertical axis shows the fresh weight (g / plant) per lettuce plant in each treatment group. ＊ This indicates a significant difference between each treatment group and the control group (P<0.05, t-test, n=4). Figure 9 and Table 9 show that even in large-scale cultivation under high-temperature conditions, the addition of the eggplant residue mixture increased the fresh weight of each variety 28 days after transplanting to more than three times that of the control group. From these results, it was found that even in large-scale hydroponic cultivation, adding the environmental stress reliever of this embodiment to the hydroponic solution greatly promotes the growth of lettuce seedlings under high-temperature conditions and confers high-temperature stress tolerance.

[0072] *: P<0.05 vs control group (t test, n=4)

[0073] (Test Example 7) High Temperature Tolerance Test in Open-Field Cultivation of Heading Lettuce The effect of reducing high temperature stress was verified by cultivating heading lettuce in open fields using a liquid fertilizer containing an environmental stress reliever made from eggplant residue.

[0074] (1) Open-field cultivation test of young head lettuce seedlings Young head lettuce seedlings (varieties: "Perfection", "Marina", "Fuyuhikari", "Deus", "Summer Guy", "Cisco", "Casper", "Olympia") were transplanted into the field and cultivated in the open field for approximately 40 days (planting density: row width 60cm, plant spacing 30cm, 1 row). CDU compound phosphate ammonium fertilizer S555 (J-Cam Agri Co., Ltd.) was used, and the fertilizer application rate was N:P2O5:K2O ratio 15:15:15 (kg / 10a). Magcal (100kg / 10a; Shiraishi Calcium KK) was used as a soil conditioner. During the cultivation period, 0.1% of an eggplant residue mixture prepared in the same manner as in Test Example 5 was added to the irrigation water and injected into the soil twice a week (10L / 10a) using a liquid fertilizer injector. As a comparative example (control group), only irrigation water (10 L / 10a) was injected into the soil. Figure 10 shows the temperature changes during the cultivation period. As shown in Figure 10, there were days when the maximum daytime temperature reached 33°C. After the end of cultivation, the weight of the lettuce was measured.

[0075] (2) Results and discussion are shown in Figure 11 and Table 10. In Figure 11, the horizontal axis shows each treatment group, with white representing the control group (liquid fertilizer only) and black representing the group with 0.1% eggplant residue solution added. The vertical axis shows the fresh weight (g / plant) per lettuce plant in each treatment group. ＊ This indicates a significant difference compared to the control group in each treatment group (P<0.05, Tukey test, n=3). Figure 11 and Table 10 show that soil injection of 0.1% eggplant residue mixture alleviated high-temperature stress in head lettuce, resulting in a significantly higher fresh weight of lettuce compared to the control group. From these results, it was found that even in open-field cultivation, using the environmental stress mitigator of this embodiment greatly promotes the growth of lettuce seedlings under high-temperature conditions and confers high-temperature stress tolerance.

[0076] *: vs control group, P<0.05 (Tukey test, n=3)

[0077] (Test Example 8) Hydroponic cultivation test using lisianthus: An environmental stress reliever made from eggplant residue was added to the hydroponic solution, and lisianthus seedlings were grown hydroponically to verify the effect of promoting growth.

[0078] (1) Preparation of an environmental stress reliever using eggplant residue The preparation of the eggplant residue mixture and the hydroponic solution obtained by adding it to a commercially available liquid fertilizer was carried out in the same manner as in Test Example 5. Each treatment group used a commercially available liquid fertilizer (OAT House A formula) to which the eggplant residue mixture was added at a ratio of 0.1%, while the comparative example (control group) used only the above-mentioned commercially available liquid fertilizer.

[0079] (2) Hydroponic cultivation test of lisianthus The prepared hydroponic solution (700L) was placed in each 700L hydroponic cultivation bed (10m in length), and two pairs of lisianthus seedlings with true leaves (cell seedlings; varieties "F1 Blue Tea", "F1 Mink Passion", "F1 Namida Plus", "F1 Veil (registered trademark) Type 3 Lavender", "F1 Prima (registered trademark) Type 1 Light Lavender", "F1 Mink Ocean", "Midnight", and "Chris Hart") were planted and cultivated in a greenhouse for about 20 days. During the cultivation period, the height of the two pairs of true leaves (length from the tip of one leaf to the tip of the other leaf = length indicated by the arrow in Figure 12) was measured over time.

[0080] (3) Results and discussion are shown in Figures 13A to 13H. Figure 13A shows the results for "F1 Blue Tea", Figure 13B for "F1 Mink Passion", Figure 13C for "Midnight", Figure 13D for "F1 Namida Plus", Figure 13E for "F1 Mink Ocean", Figure 13F for "F1 Veil (registered trademark) Type 3 Lavender", Figure 13G for "F1 Prima (registered trademark) Type 1 Light Lavender", and Figure 13H for "Chris Hart". In Figures 13A to 13H, the horizontal axis represents the date, and the vertical axis represents the plant height (cm). White circles indicate the control group (liquid fertilizer only), and black circles indicate the group with 0.1% eggplant residue mixture added. ＊ This indicates a statistically significant difference compared to the control group (P<0.05, Tukey test, n=10).

[0081] Figures 13A to 13H show that adding 0.1% of the eggplant residue mixture to the hydroponic solution promotes the growth of lisianthus seedlings. Although there were differences in this effect among varieties, it is thought that this can be improved by managing the nutrient solution composition according to the variety. In addition, each treatment group showed an effect of promoting root growth compared to the control group. In the control group, tip burn (brown discoloration of leaf margins) due to calcium deficiency occurred in all varieties, but it was hardly observed in the treatment groups. Lisianthus, which blooms during the hottest part of summer, has a long seedling period (sown in December, transplanted in March, flowering in early July), which has resulted in high production costs. By cultivating lisianthus with the environmental stress mitigating agent of this embodiment added to the hydroponic solution, it is expected that the initial growth of lisianthus will be promoted, leading to a reduction in production costs.

[0082] (Test Example 9) High Temperature Tolerance Test Using Snow Peas The effect of reducing high temperature stress was verified by cultivating snow peas in a soil-based greenhouse using a liquid fertilizer containing an environmental stress reducer made from eggplant residue.

[0083] (1) Experimental cultivation of snow peas in a greenhouse using soil cultivation Young snow pea plants were transplanted into a field inside a greenhouse and cultivated in soil cultivation for 99 days (planting density: row width 80 cm, plant spacing 10 cm, 1 row). Fertilizer used was a fertilizer specifically for potatoes and beans (Central Green Co., Ltd.), and the fertilizer application rate was set to an N:P2O5:K2O ratio of 5:15:15 (kg / 10a). Magcal (100 kg / 10a; Shiraishi Calcium KK) was used as a soil conditioner. 0.1% of an eggplant residue mixture prepared in the same manner as in Experimental Example 5 was added to the irrigation water and used for irrigation once a week (10 L / 10a). As a comparative example (control group), irrigation was performed with water only (10 L / 10a). Figure 14 shows the temperature changes inside the greenhouse during the cultivation period. As shown in Figure 14, there were days when the maximum daytime temperature reached 42°C. After the end of cultivation, the weight of the pods and above-ground parts was measured.

[0084] (2) Results and discussion are shown in Figures 15A, 15B, and Table 11. In Figures 15A and 15B, the horizontal axis shows each treatment group, with white representing the control group (water only) and black representing the group with 0.1% eggplant residue solution added. In Figure 15A, the vertical axis shows the fresh weight of pods per plant (g / plant) in each treatment group, and in Figure 15B, the vertical axis shows the fresh weight of above-ground parts per plant (kg / plant) in each treatment group. ＊ The results show a significant difference between each treatment group and the control group (P<0.05, Tukey test, n=30). Figures 15A, 15B, and Table 11 show that the application of 0.1% eggplant residue solution alleviated high-temperature stress in snow peas, and the fresh weight of the above-ground parts was significantly higher compared to the control group. From these results, it was found that in legumes as well, cultivation using the environmental stress mitigator of this embodiment greatly promotes growth under high-temperature conditions and confers high-temperature stress tolerance.

[0085] *: vs control, P<0.05 (Tukey test, n=30)

[0086] (Cultivation Example 1) Open-field cultivation of carrots: Carrots were grown in the open field using an environmental stress reducer made from eggplant residue.

[0087] Carrot seeds (variety name "Tokinashi Gosun" (Sakata), variety name "DR. Carotene 5" (Takii), variety name "Koyo No. 2" (Takii)) were sown in the field and cultivated in open fields (planting density: row width 60cm, plant spacing 6cm, row spacing 10cm, 4 rows). CDU compound phosphate ammonium fertilizer S555 (J-Cam Agri Co., Ltd.) was used, with a fertilizer application rate of N:P2O5:K2O ratio of 5:15:15 (kg / 10a). Magcal (100kg / 10a; Shiraishi Calcium KK) was used as a soil conditioner. During the cultivation period, 0.1% of an eggplant residue mixture prepared in the same manner as in Experimental Example 5 was added to the irrigation water and injected into the soil twice a week (10L / 10a) using a soil injector.

[0088] (Cultivation Example 2) Open-field cultivation of soybeans was carried out using a liquid fertilizer containing an environmental stress reliever made from eggplant residue.

[0089] Soybean seedlings (variety name "Fukuyutaka") were transplanted into the field and grown in open fields (planting density: 30 cm between rows, 20 cm between plants). Each test plot was 60 m². 2 The experiment was conducted in four iterations using a 6m x 10m plot. CDU Compound Phosphate-Added Ammonium Egg S555 fertilizer (J-Cam Agri Co., Ltd.) was used, with an N:P2O5:K2O ratio of 15:15:15 (kg / 10a). Magcal (100kg / 10a; Shiraishi Calcium KK) was used as a soil conditioner. During the cultivation period, 0.1% of an eggplant residue mixture prepared in the same manner as in Experimental Example 5 was added to the irrigation water and sprayed once a week using a sprayer (5L / 60m²). 2 Foliar spraying was performed. Figure 16 shows the temperature and precipitation during the cultivation period.

[0090] (Test Example 10) Comparative Test with Humus (1) Humus is currently the most widely used biostimulant material in Europe. Humus is produced by processing raw materials mainly extracted from the ground overseas, making it expensive, and there are concerns about environmental damage during extraction. In this test example, the high-temperature stress reduction effect was compared by growing leaf lettuce in open fields using humus and the environmental stress reducer according to this embodiment.

[0091] (1) Preparation of the sample The eggplant residue mixture was prepared in the same manner as in Test Example 5. An environmental stress reliever obtained by adding 0.1% of the eggplant residue mixture to irrigation water was used for foliar application (10 L / 10 a) in the treatment plots of the disclosed invention. As a comparative example, humus (product name "Jiryoku no Moto", PIC Bio Co., Ltd.) was used for soil mixing (40 kg / 10 a).

[0092] (2) Open-field cultivation test of leaf lettuce seedlings Leaf lettuce seedlings (variety name "Flare Rouge") were transplanted into the field and cultivated in the open field for four seasons (planting density: row width 60cm, plant spacing 30cm, 1 row). CDU compound phosphorus ammonium fertilizer S555 (J-Cam Agri Co., Ltd.) was used as fertilizer, and the fertilizer application rate was N:P2O5:K2O ratio 15:15:15 (kg / 10a). Magcal (100kg / 10a; Shiraishi Calcium KK) was used as a soil conditioner. During the cultivation period, irrigation was carried out once a week (10L / 10a). In the treatment plots disclosed herein, the environmental stress mitigating agent prepared above was applied as a foliar spray once a week (10L / 10a) using a sprayer. On the other hand, in the conventional treatment plot (humus-based), foliar application was not performed, but humus was mixed into the soil before planting (40 kg / 10a). In the control plot, neither foliar application nor soil mixing was performed (no application). Figure 17 shows the temperature changes and planting timing during the cultivation period. As shown in Figure 17, the temperature rose in the latter half of the cultivation period, and cultivation was carried out in extremely hot conditions with temperatures exceeding 35°C. After the end of cultivation, the weight of the lettuce was measured.

[0093] (3) Results and discussion are shown in Figure 18 and Table 12. In Figure 18, the horizontal axis shows each treatment group, with white representing the control group (no treatment), gray representing the conventional product (humic) treatment group, and black representing the disclosed treatment group (0.1% eggplant residue mixture added). The vertical axis shows the fresh weight (g / plant) per lettuce plant in each treatment group. ＊ 'n' indicates a statistically significant difference, and 'ns' indicates no statistically significant difference (P<0.05, Tukey test, n=30). Figure 18 and Table 12 show that in all cultivation types, the fresh yield in the treatment area of ​​this disclosure was significantly higher than that of the treatment area of ​​the conventional product. Furthermore, while discoloration of new leaves, presumably due to high temperature damage, was observed in the treatment area of ​​this disclosure, no discoloration was observed. From these results, it was found that foliar application of the environmental stress reliever of this embodiment provides superior plant growth promotion and superior heat stress relief effects under high temperature conditions compared to humus.

[0094] *: vs conventional product, P<0.05 (Tukey test, n=30)

[0095] (Test Example 11) Comparative Test with Humus (2) In this test example, open-field cultivation of head lettuce was carried out using humus and the environmental stress mitigating agent according to this embodiment, and the effect of reducing high temperature stress was compared.

[0096] (1) Preparation of the sample The eggplant residue mixture was prepared in the same manner as in Test Example 5. An environmental stress reliever obtained by adding 0.1% of the eggplant residue mixture to irrigation water was used for soil injection (10 L / 10 a) in the treatment area of ​​the present disclosure. As a comparative example, the humus used in Test Example 10 was used for soil mixing (40 kg / 10 a).

[0097] (2) Open-field cultivation test of young head lettuce seedlings Young head lettuce seedlings (varieties: "Olympia", "Extra Early Cisco", "Excel", and "Patriot") were transplanted into the field and cultivated in the open field (planting density: row width 60cm, plant spacing 30cm, 1 row). CDU compound phosphorus-containing ammonium fertilizer S555 (J-Cam Agri Co., Ltd.) was used, and the fertilizer application rate was N:P2O5:K2O ratio 15:15:15 (kg / 10a). Magcal (100kg / 10a; Shiraishi Calcium KK) was used as a soil conditioner. During the cultivation period, irrigation was carried out twice a week (10L / 10a). In the treatment plots of this disclosure, the environmental stress mitigating agent prepared above was injected into the soil twice a week (10L / 10a) using a soil injector. On the other hand, in the conventional treatment plot (humus-based), no soil injection was performed; instead, humus was mixed into the soil before planting (40 kg / 10a). In the control plot, neither soil injection nor soil mixing was performed (no application). Figure 19 shows the temperature changes during the cultivation period. As shown in Figure 19, high temperatures of 30°C or higher persisted from the middle of the cultivation period. After the end of cultivation, the weight of the lettuce was measured.

[0098] (3) Results and discussion are shown in Figure 20 and Table 13. In Figure 20, the horizontal axis shows each treatment group, with white representing the control group (no treatment), gray representing the conventional product (humic) treatment group, and black representing the disclosed treatment group (0.1% eggplant residue mixture added). The vertical axis shows the fresh weight (g / plant) per lettuce plant in each treatment group. ＊The values ​​indicate a statistically significant difference, while ns indicates no significant difference (P<0.05, Tukey test, n=32). Figure 20 and Table 13 show that during high-temperature periods, the treatment plots of the disclosed invention yielded significantly higher yields than those of the conventional product. From these results, it was found that even when the environmental stress mitigating agent of this embodiment is injected into the soil, it exhibits superior plant growth promotion and high-temperature stress mitigation effects compared to humus under high-temperature conditions.

[0099] *: vs conventional product, P<0.05 (Tukey test, n=32)

[0100] (Test Example 12) Comparative Test with Humus (3) In this test example, the effect of reducing high-temperature stress was compared when cultivating tomatoes in a soil-based greenhouse using humus and the environmental stress reducer according to this embodiment.

[0101] (1) Preparation of the sample The eggplant residue mixture was prepared in the same manner as in Test Example 5. An environmental stress reliever obtained by adding 0.1% of the eggplant residue mixture to irrigation water was used for soil injection in the treatment area of ​​the present disclosure. As a comparative example, the humus used in Test Example 10 was used for soil mixing.

[0102] (2) Tomato soil cultivation test in greenhouses Tomato seedlings (variety names: "Reika", "Aiko", and "Frutica") were transplanted into the field and grown in open fields (planting density: row width 120 cm, plant spacing 25 cm, single row planting, single plant training). CDU compound phosphate ammonium fertilizer S555 (J-Cam Agri Co., Ltd.) was used, and the fertilizer application rate was N:P2O5:K2O ratio 25:25:25 (kg / 10a). Magcal (100 kg / 10a; Shiraishi Calcium KK) was used as a soil conditioner. During the cultivation period, in the treatment plots disclosed herein, the environmental stress reliever prepared above was injected into the soil twice a day (5 minutes each time, 133 mL / plant) using a soil injector. On the other hand, in the conventional (humic) treatment plots and the control plots, only irrigation water was injected into the soil in the same manner as above. In the conventional treatment plot (humic soil), humic soil (40g / plant) was mixed into the soil before planting. Table 14 shows the cultivation conditions for each treatment plot, and Figure 21 shows the temperature changes inside the greenhouse during the cultivation period. As shown in Figure 21, the first half of the cultivation period saw many extremely hot days with maximum temperatures of 35°C or higher, while the second half consisted of midsummer days with maximum temperatures of 30°C or higher. After the end of cultivation, the weight of the fruit was measured.

[0103]

[0104] (3) Results and discussion are shown in Figure 22 and Table 15. In Figure 22, the horizontal axis shows each treatment group, with white representing the control group (no application), gray representing the conventional product (humic) treatment group, and black representing the disclosed treatment group (0.1% eggplant residue mixture added). The vertical axis shows the total fruit weight per plant (g / plant) in each treatment group. ＊ The values ​​indicate a statistically significant difference, while ns indicates no significant difference (P<0.05, Tukey test, n=9). Figure 22 and Table 15 show that for all varieties, the treatment group in this disclosure had a significantly higher yield than the conventional product. From these results, it was found that, even in soil cultivation of tomatoes using the environmental stress reliever of this embodiment, a superior effect on promoting plant growth and a superior effect on mitigating high-temperature stress were observed compared to humus.

[0105] *: vs conventional product, P<0.05 (Tukey test, n=9)

[0106] (Test Example 13) Comparative Test with Room Temperature In this test example, in order to help elucidate the mechanism of the high-temperature stress mitigation effect of this embodiment, lettuce and tomatoes were grown hydroponically on a small scale under high-temperature conditions and room temperature conditions, and the effects on plant growth were compared.

[0107] (1) Preparation of Samples The eggplant residue extract was prepared in the same manner as in Test Example 5. Eggplant residue extract (final concentration 0.1%) and various nutrients were added to water in the composition shown in Table 8 to prepare a hydroponic solution, which was used in each treatment group. In the comparative example (control group), the same hydroponic solution as the treatment groups was used, except that eggplant residue extract was not added.

[0108] *Extract from eggplant fruit (500g / L)

[0109] (2) Small-scale hydroponic cultivation tests of lettuce and tomatoes under high-temperature and normal-temperature conditions. 3L of prepared hydroponic solution was placed in each of 0.3L (75mm x 75mm x 100mm, plant box containers) simple hydroponic cultivation devices. Leaf lettuce seedlings (variety name "Sunny") and tomato seedlings (variety name "Momotaro") were planted and cultivated in a growth chamber for 14 days. Normal-temperature conditions were 25°C, 12h light / 18°C, 12h dark, and high-temperature conditions were 35°C, 12h light / 25°C, 12h dark. After cultivation, the weight of the above-ground parts of the lettuce and tomatoes and the Ca content of the above-ground parts of the lettuce were measured. Ca content was measured using an ICP emission spectrometer.

[0110] (3) Results and Discussion The results are shown in Figures 23A, 23B, 24, Tables 17 and 18. In Figures 23A, 23B, and 24, the horizontal axis shows each treatment group, with white representing the control group and black representing the group with 0.1% eggplant residue extract added (this disclosure). In Figures 23A and 24, the vertical axis shows the fresh above-ground weight per plant (g / plant) in each treatment group, and in Figure 23B, the vertical axis shows the Ca content per lettuce plant per dry weight (mg / 100mg) in each treatment group. ＊ This indicates a statistically significant difference.

[0111] Figures 23A and 23B, and Table 17 show that under high-temperature conditions, using the environmental stress mitigator of this embodiment significantly increased both lettuce yield and Ca ion concentration, while under normal temperature conditions, no significant difference was observed compared to the control (P<0.05, t-test, n=8). This result indicates that using the environmental stress mitigator of this embodiment significantly promotes lettuce growth specifically under high-temperature conditions and confers high-temperature stress tolerance. Furthermore, since the Ca ion concentration of lettuce also increased under high-temperature conditions in the treatment group using the environmental stress mitigator, it is suggested that the increase in Ca ion concentration is related to the acquisition of high-temperature tolerance.

[0112] *: P<0.05 vs control group (t test, n=8)

[0113] Figure 24 and Table 18 show that, under high-temperature conditions, the above-ground weight of tomatoes significantly increased compared to the control when using the environmental stress reliever of this embodiment, whereas no significant difference was observed under normal temperature conditions (P<0.05, Tukey test, n=8). Regarding plant height, under high-temperature conditions, tomatoes tended to be taller when using the environmental stress reliever compared to the control, but there was little difference under normal temperature conditions (data not shown). These results indicate that, for tomatoes as well, using the environmental stress reliever of this embodiment significantly promotes growth under high-temperature conditions and confers high-temperature stress tolerance.

[0114] *: P < 0.05 vs control group (Tukey test, n=8)

[0115] (Test Example 14) Comparative Test with Room Temperature Using a Calcium Channel Blocker In this test example, in order to help elucidate the mechanism of the high-temperature stress mitigation effect of this embodiment, a calcium channel blocker and the environmental stress mitigator of this embodiment were added to the hydroponic solution, and lettuce was grown on a small scale under high-temperature and room-temperature conditions to compare the effects on plant growth. A calcium channel blocker is a substance that blocks calcium ion channels in the cell membrane, thereby inhibiting the inflow (or outflow) of calcium ions into the cell, as shown in Figure 25. In this test example, gadolinium (Gd) and lanthanum (La) were used as calcium channel blockers.

[0116] (1) Preparation of Samples The eggplant residue extract was prepared in the same manner as in Test Example 5. Eggplant residue extract (final concentration 0.1%), calcium channel blocker GdCl3 or LaCl3, and various nutrients were added to water in the compositions shown in Tables 19 and 20 to prepare hydroponic solutions, which were then used in each treatment group.

[0117] *Extract from eggplant fruit (500g / L)

[0118] *Extract from eggplant fruit (500g / L)

[0119] (2) Small-scale hydroponic cultivation tests of lettuce under high-temperature and normal-temperature conditions. 3L of prepared hydroponic solution was placed in each of 0.3L simple hydroponic cultivation devices, and leaf lettuce seedlings (variety name "Sunny") were planted and cultivated in a growth chamber for 14 days. The normal-temperature conditions were 25°C, 12h light / 18°C, 12h dark, and the high-temperature conditions were 35°C, 12h light / 25°C, 12h dark. After cultivation, the weight of the lettuce was measured.

[0120] (3) Results and discussion are shown in Figures 26, 27, and Table 21. In Figures 26 and 27, the horizontal axis represents each treatment group, with white representing room temperature (25°C / 18°C) and black representing high temperature (35°C / 25°C), and the vertical axis represents the fresh above-ground weight (g / plant) per plant in each treatment group. ＊The results indicate a statistically significant difference (P<0.05 vs. room temperature (25°C / 18°C), Tukey test, n=4). Figure 26 and Table 21 show that in the treatment plots where GdCl3 was added, lettuce yield was significantly lower under high temperature conditions compared to room temperature. On the other hand, Figure 27 and Table 21 show that in the treatment plots where LaCl3 was added, at La50mM, lettuce yield was significantly lower under high temperature conditions compared to room temperature, but below La50mM, no significant difference was observed between high and room temperature. These results indicate that the addition of calcium channel blockers prevents sufficient heat stress tolerance from being imparted by the environmental stress mitigator of this embodiment, suggesting that the influx of calcium ions into plant cells via calcium channels is involved in the environmental stress tolerance and growth-promoting effects of this embodiment.

[0121] *: vs room temperature, P<0.05 (Tukey test, n=4)

[0122] (Test Example 15) Root Growth Promotion Effect Test In this test example, in order to help elucidate the mechanism of the high-temperature stress mitigation effect according to this embodiment, lettuce was grown in a soil-based greenhouse using the environmental stress mitigating agent according to this embodiment, and a root growth promotion effect test was conducted.

[0123] (1) Preparation of the sample The eggplant residue mixture was prepared in the same manner as in Test Example 5. An environmental stress reliever obtained by adding 0.1% of the eggplant residue mixture to irrigation water was used for irrigation in the treatment area of ​​the present disclosure.

[0124] (2) Soil cultivation test of lettuce seedlings in a greenhouse. Fertilizer-containing potting soil (Kumiai Nippi Horticultural Potting Soil No. 1, Nippon Fertilizer Co., Ltd.; fertilizer contains 220 mg of nitrogen, 2775 mg of phosphorus, 220 mg of potassium, and 220 mg of magnesium per 1 kg) was placed in a 128-cell tray, and one seed of heading lettuce (variety name "Olympia") was sown in each cell. The seedlings were cultivated in a greenhouse for 3 weeks. During the cultivation period, the treatment group disclosed herein was irrigated with 1 L of the environmental stress reliever prepared above twice a week. In the control group, irrigation was carried out similarly using only irrigation water. The temperature inside the greenhouse during the cultivation period was 30°C or higher. After the end of cultivation, the roots were washed and the soil was removed, and then the weight of the roots was measured.

[0125] (3) Results and discussion are shown in Figure 28. In Figure 28, the horizontal axis shows each treatment group, with white representing the control group and black representing the treatment group disclosed herein, and the vertical axis shows the fresh weight of roots per plant (g / plant) in each treatment group. ＊ The result indicates a statistically significant difference (P<0.05, t-test, n=5). Figure 28 shows that cultivation using the environmental stress mitigator of this embodiment significantly promotes lettuce root growth compared to the control. Visual inspection also revealed that the roots in the treated area of ​​this disclosure were better developed than those in the control. These results suggest that the promotion of root growth is involved in the environmental stress tolerance and growth-promoting effects of this embodiment.

[0126] (Test Example 16) Radish Growth Promotion Effect Test An environmental stress reliever obtained by adding 0.1% eggplant residue mixture to commercially available MS medium (M5529-50L, Sigma) was used. In this test example, in order to help elucidate the mechanism of the high temperature stress relief effect according to this embodiment, cultivation was carried out in a medium in which the environmental stress reliever according to this embodiment was used at a low concentration and a medium solidifying agent (gellan gum) was added, and a root growth promotion effect test was conducted.

[0127] (1) Sample Preparation 500g of eggplant fruit (with skin) after harvesting was used as the eggplant residue. This eggplant residue was placed in a 1L beaker, 700mL of distilled water was added, and it was heated at 120°C for 20 minutes. The supernatant was then filtered through filter paper to obtain the filtrate (eggplant residue extract). Eggplant residue extract (final concentration 0.004%) and various nutrients were added to water in the composition shown in Table 22 to prepare a liquid culture medium, which was used in each treatment group. In the comparative example (control group), the same liquid culture medium as the treatment groups was used, except that eggplant residue extract was not added.

[0128] *Extract from eggplant fruit (500g / L)

[0129] (2) In a test container (75mm x 75mm x 100mm, plant box) for cultivating young radish plants in a culture medium, 0.3L of the liquid culture medium prepared above and 0.3% gellan gum were added and the medium was allowed to solidify. Radish seeds (variety name "Taibyo Sotofutori", Takii Seed Co., Ltd.) were sown on this medium and cultivated in the medium for 5 days at 25°C and 24 hours in the dark. After cultivation was completed, the culture medium was removed from the roots and the lengths of the roots and hypocotyls were measured.

[0130] (3) Results and Discussion The results are shown in Figures 29A, 29B, and Table 23. In Figures 29A and 29B, the horizontal axis shows each treatment group, with white representing the control group and black representing the 0.004% additive group. In Figure 29A, the vertical axis shows the average root length (cm) in each treatment group, and in Figure 29B, the vertical axis shows the average hypocotyl length (cm) in each treatment group. ＊ The result shows a statistically significant difference compared to the control group (P<0.05, t-test, n=9). Figures 29A and 29B, and Table 23 show that even at low concentrations, cultivation using the environmental stress reliever of this embodiment significantly promotes radish growth compared to the control group. Visual observation also confirmed that the radishes in the treatment group using the environmental stress reliever grew better than the control group. These results demonstrate that the environmental stress reliever of this embodiment can promote plant growth even at low concentrations and is also effective for root vegetables.

[0131] *: P<0.05 vs control (t test, n=9)

[0132] (Test Example 17) Test of Rooting Promotion Effect Using Cassava Remains In this test example, radish was grown in a culture medium to which a medium solidifying agent (gellan gum) was added, using an environmental stress reliever prepared using cassava residue, and the effect of promoting rooting was tested.

[0133] (1) Preparation of the sample 100 g of cassava leaves after harvesting was used as the cassava residue. This cassava residue was placed in a 1 L beaker, 700 mL of distilled water was added, and it was heated at 120°C for 20 minutes. The supernatant was then filtered through filter paper to obtain the filtrate (cassava residue extract). A liquid medium was prepared by adding the cassava residue extract (final concentration 0.004%) and various nutrients to 1 / 2 MS medium (MS medium with all component concentrations reduced to 1 / 2) according to the composition shown in Table 24, and this was used in each treatment group. In the comparative example (control group), the same liquid medium as the treatment group was used, except that the cassava residue extract was not added.

[0134] *Cassava leaf extract 100g / L

[0135] (2) In a test container (75mm x 75mm x 100mm, plant box) for cultivating young radish plants in a culture medium, 0.3L of the liquid culture medium prepared above and 0.3% gellan gum were added and the medium was allowed to solidify. Radish seeds (variety name "Taibyo Sotofutori", Takii Seed Co., Ltd.) were sown on this medium and cultivated in the medium for 5 days at 25°C and 24 hours in the dark. After cultivation, the roots in the culture medium were observed using a digital camera (OM-D E-M5, Olympus Corporation) equipped with a macro lens (M.ZUIKO DIGITAL ED 90mm F3.5 Macro IS PRO, OM Digital Solutions, 4x magnification) to examine the rooting process.

[0136] (3) Results and Discussion The results showed that the fine roots of the radishes grown in a culture medium containing cassava residue extract as an environmental stress reliever at a concentration of 0.004% were very well developed, whereas the fine root development of the radishes grown in a culture medium without cassava residue extract in the control group was not observed (data not shown). Visual inspection also confirmed that the growth of the hypocotyl and roots was significantly promoted in the 0.004% supplement group compared to the control group. These results indicate that, even when using cassava residue extract, the environmental stress reliever of this embodiment, similar to eggplant residue extract, can promote plant growth, especially root development, even at low concentrations, and is also effective for root vegetables.

[0137] (Test Example 18) Verification Test of the Mechanism of Acquiring High Temperature Stress Tolerance by RNA-seq Analysis Previous test examples have accumulated evidence that acetylcholine (ACh) is involved in mitigating high temperature stress in plants, but the molecular mechanism remains unclear. To verify this, in this test example, plants were treated with ACh alone, and the stress response gene induction activity was examined by RNA-seq analysis.

[0138] (1) Experimental Method (1-1) Preparation of Culture Solution ・Eggplant Extract Stock Solution: 50g of unripe eggplant fruit (off-grade) after harvest was placed in a 1L beaker, and distilled water was added to make a total volume of 1L. This was heated in an autoclave at 120°C for 20 minutes, and the supernatant was filtered through filter paper to obtain the "eggplant extract stock solution" (ACh content: 20mg / L = 0.137mM). ・Acetylcholine Stock Solution: 25mg of acetylcholine chloride was placed in a 1L beaker, and distilled water was added to make a total volume of 1L to make a "acetylcholine stock solution" (ACh content: 20mg / L = 0.137mM). ・Iron Stock Solution: 27.8g of ferrous sulfate heptahydrate was placed in a 1L beaker, and distilled water was added to make a total volume of 1L to make a "iron stock solution". ・Calcium Stock Solution: 30g of calcium chloride dihydrate was placed in a 1L beaker, and distilled water was added to make a total volume of 1L to make a "calcium stock solution". - Potassium and Phosphorus stock solution: 1.9g of potassium nitrate and 170g of potassium phosphate were placed in a 1L beaker, and distilled water was added to make a total volume of 1L, which was called the "potassium and phosphorus stock solution". - Magnesium and Nitrogen stock solution: 1.6g of ammonium nitrate and 370g of magnesium sulfate heptahydrate were placed in a 1L beaker, and distilled water was added to make a total volume of 1L, which was called the "magnesium and nitrogen stock solution". - Other trace element stock solution: 6.2g of boric acid, 22.3g of manganese sulfate tetrahydrate, 8.6g of zinc sulfate heptahydrate, 0.025g of copper sulfate pentahydrate, 0.25g of sodium molybdate dihydrate, and 0.025g of cobalt chloride hexahydrate were placed in a 1L beaker, and distilled water was added to make a total volume of 1L, which was called the "other trace element stock solution". Eggplant extract concentrate or ACh concentrate was added to distilled water at a dilution ratio of 1 / 1000, and iron concentrate, potassium / phosphorus concentrate, magnesium / nitrogen concentrate, and other trace element concentrates were added at a dilution ratio of 1 / 1000 each to prepare culture solutions for each treatment group. In the control group, culture solutions without eggplant extract concentrate or ACh concentrate were used. The final concentrations of each component in the culture solutions are shown in Table 25.

[0139] 〇: Additive, -: No additive

[0140] (1-2) Cultivation Method Lettuce (variety name "Olympia") seeds were sown in sponges filled with distilled water and maintained at room temperature conditions (25°C, 18 hours light / 18°C, 6 hours dark) for 10 days. Next, the lettuce seedlings were transferred to 1.2L hydroponic growing containers containing 1L of the culture solution for each treatment group, and cultivated for another 10 days at the same room temperature conditions (25°C / 18°C). After that, the hydroponic growing containers were moved to growth chambers (SANYO, MRL-350) set to either high temperature conditions (38°C, 18 hours light / 25°C, 6 hours dark) or room temperature conditions (25°C / 18°C) to begin temperature treatment. The cultivation period was 30 days after sowing. Five replicates were performed for the eggplant extract treatment group and the control group, and four replicates for the ACh treatment group. After the end of cultivation, the above-ground parts were harvested and the fresh mass (g / plant) was measured.

[0141] (1-3) RNA extraction and purification: Sampling for RNA-seq was performed 4 hours after the start of the light phase, 4 days after the start of the temperature treatment. The entire above-ground part of young lettuce plants was collected and rapidly frozen with liquid nitrogen. After the frozen samples were crushed using a multi-bead shocker (Yasui Kikai Co., Ltd.), total RNA was extracted by the Trizol method, and the RNA was purified using the RNeasy Plant Mini Kit (Qiagen Co., Ltd.) before being subjected to RNA-seq analysis.

[0142] (1-4) Sequencing and Quality Control The obtained RNA is converted into a library and scanned using an Illumina next-generation sequencer (NovaSeq). TMSequencing was performed using X Plus. The quality of the sequencing data was evaluated, and the Q score, error rate, GC content, etc., were examined. The quality evaluation results are shown in Table 26. Errors can occur in RNA-seq data due to various factors. The main factors include poor sequencing quality, read mapping errors, sample contamination, and data normalization errors. If the sequencing quality is low, unreliable data may be generated. If read mapping is not performed correctly, incorrect gene expression levels may be calculated. If the sample is contaminated, the analysis results may be significantly distorted. If the data is not properly normalized, accurate comparison of expression levels may not be possible. The sequencing data in this study showed a very low error rate. This means that the accuracy of the sequencing data is high, and the risk of incorrect mapping or analysis results is extremely low. In addition, the "Q score" (Q20 and Q30) in RNA-seq indicates how accurately the bases are read and is used as an indicator to evaluate the quality of sequencing data. The average Q20 of the data in Table 26 was 98.7%, which is higher than the generally high-quality value of 90% or more. The average Q30 was also high at 96.1%, which is higher than the generally high-quality range of 85-90% or more. These errors can be minimized by accurately executing the data analysis process and performing sufficient QC (quality control). The GC content of plants is generally in the range of 40-50%, and the GC content in Table 26 is approximately 45%, indicating that it is a gene with a balanced structure. In other words, this RNA, with its low error rate, high Q score, and balanced structure (GC content), allows for high-level RNA-seq analysis and demonstrates that analysis can be performed without any concerns regarding quality.

[0143]

[0144] (1-5) Expression Data Analysis To visualize the results of RNA-seq analysis, we performed expression data analysis of approximately 50,000 genes in lettuce with the aim of elucidating the mechanism of heat stress mitigation by eggplant extract treatment. Reads were matched and quantified based on known annotations for lettuce (Lactuca sativa), and normalized using FPKM to enable comparison of data between samples. Using DESeq2, we identified genes with changes in expression levels between time points. For visualization of the results, we used PCA (principal component analysis), heatmaps (row-direction Z-score homogenization), etc., to visualize changes in expression levels over time. In differential gene expression analysis (DEG), between-group comparisons were performed between treatment groups and controls under each temperature condition, and statistical analysis (Wald test) was performed using DESeq2, with padj (FDR correction) < 0.05 being considered significant. |log2(fold change)| ≥ 1.0 was used as a guideline for effect size, and a Volcano plot was used for visualization.

[0145] (1-6) Gene Function Analysis (GO Analysis) GO (BP / CC / MF) analysis was performed, and significant terms were extracted with an FDR (padj) < 0.05. Gene Ontology (GO) is a database that systematically organizes the "biological processes (BP)", "where in the cell they function (intracellular compartment; CC)", and "molecular functions (MF)" involved in genes and their products (e.g., proteins). The purpose of GO analysis is to analyze the vast amount of gene data obtained by RNA-seq and clarify the "biological functions" involved in genes that are particularly activated (or suppressed) under high-temperature stress conditions. In other words, the goal is to investigate "which genes are working a lot" using RNA-seq, then group and classify "what kind of function those genes are performing" using GO analysis, and identify the mechanisms and functions from the results. In this example, in order to clarify the mechanism by which lettuce acquires high-temperature stress tolerance, the reactions that occur in the plant when treated with eggplant extract / ACh were analyzed by dividing them into "upstream", "midstream", and "downstream" (Figure 30). The stage preceding intracellular signal transduction, involving ligands (ACh) and receptors, is called the "upstream" stage. In the "upstream" stage, we focused on "G proteins" and "receptors" involved in extracellular and intracellular signal transduction at the cell membrane. While the presence of ACh in plants is known, the existence of ACh receptors (AChRs) in plants has not been confirmed to date. In the "midstream" stage, we focused on the activation of intracellular signal transduction pathways. Based on previous test results, we hypothesize that stimulation of ion channels by ACh leads to an influx of calcium into the cell, thereby activating the signal transduction pathway. The basic intracellular signal transduction pathway consists of the activation and release of second messengers. By amplifying signals generated extracellularly and directing them to intended intracellular targets, it triggers transcription, translation, protein modification, enzyme activation, cellular metabolism, mitosis, apoptosis, and other processes. The resulting changes in cellular function are called the "downstream" stage. In the "downstream" stage, we analyzed changes in the cellular stress response.

[0146] (2) Results and Discussion (2-1) Lettuce Yield Figure 31 shows the results of comparing the lettuce yields of each treatment group, measured 30 days after sowing. Figure 31 shows the lettuce yield (fresh mass, unit: g / plant) for each treatment group under normal temperature and high temperature conditions. Under normal temperature conditions (25°C / 18°C), a significant growth-promoting effect of eggplant extract treatment (1.9 g / plant increase) was observed in lettuce. Under high temperature conditions (38°C / 25°C), there was no significant difference between eggplant extract treatment and ACh treatment, and both groups showed a significant increase in lettuce yield (3.2 g / plant increase) compared to the control group. No significant difference in growth was observed between the ACh treatment under high temperature conditions and the control group under normal temperature conditions (Tukey test, p<0.05, n=3).

[0147] (2-2) Heatmap Analysis The results of the heatmap analysis are shown in Figure 32. Figure 32 shows the overall results of the FPKM cluster analysis clustered using log2(FPKM+1) values ​​and represented as a heatmap. In the figure, the horizontal axis represents the treatment group and the vertical axis represents the gene. ＊＊A significant difference compared to the control group is indicated (Wald test, padj<0.05, n=5). Red (dark gray in the figure) indicates genes with high expression levels, black indicates genes with moderate expression levels, and green (light gray in the figure) indicates genes with low expression levels. The change in color from dark gray to light gray indicates the range from large to small log2(FPKM+1) values. Cluster analysis was performed on different gene sets (treatment groups), and genes with similar expression patterns were clustered. Mainstream hierarchical clustering was used to cluster the fpkm values ​​of genes and homogenize the rows (Z-scores). Genes or samples with similar expression patterns are grouped in the heatmap (Figure 32). The color of each grid reflects the value obtained after homogenizing the expression data rows (usually between -2 and 2), not the gene expression value itself. Therefore, the colors in the heatmap can only be compared horizontally (expression of the same gene in different treatment groups), not vertically (same treatment group). There is not only inter-group (treatment interval) clustering but also inter-sample clustering. In Figure 32, genes with similar expression patterns are located close together. This figure demonstrates that gene expression patterns differ between room temperature and high-temperature treatment. As an example, the gene Cyclic Nucleotide-Gated Ion Channel 2 (CNGC2), which is related to pathogen response and stress tolerance, is shown below Figure 32. It can be seen that the expression pattern of this gene also differs between room temperature and high-temperature treatment.

[0148] (2-3) Principal Component Analysis Principal component analysis (PCA) was performed on the gene expression values ​​(FPKM) of all samples. Figure 33 shows the results of the PCA analysis. PCA is often used to evaluate differences between groups (between treatments) and sample overlap within groups. PCA reduces dimensionality using linear algebra calculations and can extract principal components from tens of thousands of gene variables. The horizontal axis (PC1) represents the axis with the greatest data variance, and the vertical axis (PC2) reflects the next most important variable that explains the differences in gene expression between samples. In PCA, genes with similar expression patterns are generally plotted closer together. Creating a PCA plot shows how the samples are grouped. From Figure 33, it was found that gene expression in lettuce tends to differ between high temperature and room temperature. In particular, at high temperature, the gene expression patterns of eggplant extract and ACh treatment were shown to be similar. This suggests that ACh is likely the active ingredient in eggplant extract.

[0149] (2-4) Differential Gene Expression Analysis (DEG) After quantifying gene expression, statistical analysis of the expression data is necessary to screen for genes whose expression levels differ significantly under different conditions. As criteria for statistical screening, |log2(fold change)|≧1.0 and padj≦0.05 were used. These criteria are empirical values ​​commonly used in RNA-seq. Figures 34, 35, and 36 are graphs showing the ratio of change (fold change) and its statistical significance obtained when comparing the variation in gene expression under different conditions (high temperature and room temperature) and between groups (eggplant extract and control) using Volcano Plot. Figure 34 is a Volcano Plot of differentially expressed genes (DEGs) (eggplant extract vs control) under room temperature conditions. Figure 35 is a Volcano Plot of differentially expressed genes (DEGs) (eggplant extract vs control) under high temperature conditions. Figure 36 is a Volcano Plot of differentially expressed genes (DEGs) (ACh vs control) under high-temperature conditions. The horizontal axis of each figure represents the logarithmic expression ratio (log2(fold change)), where the right side (log2(fold change)>+1) represents genes whose expression level increased by more than double (upregulation), and the left side (log2(fold change)<-1) represents genes whose expression level decreased to half or less (downregulation). The vertical axis represents statistical significance (-log10(pvalue)), where the higher the value, the higher the statistical significance. Genes with large expression ratio changes and high statistical significance are located in the upper right or upper left of each figure. Analysis of these figures revealed that 252 genes (Figure 34) under normal temperature conditions and 474 genes (Figure 35) under high-temperature conditions showed a significant increase in expression levels after eggplant extract treatment. Furthermore, when comparing under high-temperature conditions, it was revealed that the expression levels of 474 genes (Figure 35) increased significantly with eggplant extract treatment, and 325 genes (Figure 36) increased significantly with Ach treatment. The following GO analysis was performed using the genes whose expression levels increased significantly compared to the control in each treatment.

[0150] Figure 37 is a bar graph showing genes whose expression was significantly increased by DESeq2 with log2(fold change) = 1.0 and adjusted p-value < 0.05. In the figure, the horizontal axis represents each treatment, and the vertical axis represents the number of genes. Black bars represent genes that differ between eggplant extract and ACh treatment, while red (gray) bars represent genes common to both eggplant extract and ACh treatment. Under room temperature conditions, 114 genes were significantly increased compared to the control with ACh treatment, and 215 genes with eggplant extract treatment. It became clear that eggplant extract treatment increased the expression of many genes. Furthermore, under high temperature conditions, the number of genes whose expression increased with each treatment increased even further. 325 genes were increased with ACh treatment, and 474 genes were increased with eggplant extract treatment. In addition, increased expression of many common genes (gray) was observed, so further detailed analysis was performed. Common genes under high-temperature conditions included those related to cation transport, glutamine receptor activity, ion channel proteins, LIM domain-containing protein WLIM1, S-type anion channel SLAH, cyclic nucleotide-gated ion channel 4, mechanosensitive ion channel protein 6, S-type anion channel SLAH1, S-type anion channel SLAH2, cyclic nucleotide-gated ion channel 4, vacuum iron transporter homolog 4, vacuum cation / proton exchanger 3, OsDREB1 genes (1B, 1D), HSP26-A, HSP11, HSP70, HSP90, HSP26, HSP7 proteins, and the hsfA-3 transcription factor.

[0151] Figure 38 shows a detailed comparison of genes whose expression levels significantly increased in DEGs analysis of eggplant extract and ACh treatment under high temperature conditions. The left side of Figure 38 is a graph showing the number of genes that specifically increased under eggplant extract and ACh treatment under high temperature conditions. In the figure, the horizontal axis represents each treatment, and the vertical axis represents the number of genes. In the bar graph, black represents genes that differ between eggplant extract and ACh treatment, and green (gray in the figure) represents genes common to both eggplant extract and ACh treatment. When comparing the two gene groups, it became clear that many genes were common. Genes whose expression levels differed statistically under the set conditions and whose fluctuations exceeded a threshold were screened as differentially expressed genes (DEGs). This can sometimes lead to the elucidation of biologically important processes and pathways. In the Venn diagram in the upper right of Figure 38, the two circles show the number of genes expressed independently in each treatment group, and the overlapping region of the circles shows the number of genes co-expressed in the two treatment groups. It became clear that approximately 40% of the genes whose expression levels significantly increased under eggplant extract and ACh treatment under high temperature conditions overlapped. One example of a common gene is osmotic response gene 1 (ROS1). The lower right of Figure 38 shows the expression level of ROS1 in RNA-seq heatmap analysis. Here, red (dark gray in the figure) indicates a gene with a high expression level, black indicates a gene with a moderate expression level, and green (light gray in the figure) indicates a gene with a low expression level. From this figure, it was shown that ROS1 is expressed in large quantities under high temperature conditions, and is particularly highly expressed in eggplant extract and ACh treatment. It is presumed that high ROS1 expression induces heat resistance by activating DNA repair components. Figure 39 shows the results of DEGs analysis for ACh and eggplant extract under room temperature conditions. The ratio of commonly expressed genes in ACh and eggplant extract was lower compared to high temperature conditions, but it was 16% for eggplant extract treatment and 36% for ACh treatment. Figure 40 shows the results of comparing the number of genes with increased expression under room temperature and high temperature conditions in eggplant extract or ACh treatment using DEGs analysis. The results of comparing room temperature and high temperature showed a significant decrease in common genes. This reflects the results of the heatmap in Figure 32 (showing different gene expression patterns at room temperature and high temperature). This demonstrates that different gene expressions are induced at room temperature and high temperature.

[0152] (2-5) Gene Function Analysis (GO Analysis) To investigate the effects of high temperature, gene function analysis (GO analysis) was performed on lettuce under high temperature and room temperature conditions in the untreated group (control). The results are shown in Table 27 and Figure 41. Figure 41 is a plot comparing the untreated group at room temperature and the untreated group at high temperature. When lettuce was exposed to high temperature, signal transduction, signaling, intercellular communication, and cellular responses to stimuli were significantly activated. However, ion channel-related genes and G protein-related genes were not activated. RNA-Seq analysis performs statistical tests on many genes, which can lead to an increase in the number of genes that are judged as significant by chance. Therefore, in order to reduce the false detection rate and obtain more reliable results, we used not only the p-value but also the q-value (padj), which is the p-value corrected by applying the concept of false detection rate (FDR) to the p-value. Even if p < 0.05 or less, if the q-value is high, it is affected by FDR and is not significant.

[0153]

[0154] Table 28 and Figure 42 show the results of GO analysis of eggplant extract and control groups under room temperature conditions. Under room temperature conditions, eggplant extract treatment activated genes related to GTPase activity, guanylate nucleotide binding, GTP binding, ribonucleoside binding, purine-ribonucleoside binding, guanylate-ribonucleotide binding, nucleoside binding, extramembrane components, photosynthetic system II oxygen-evolving complex, photosynthetic system II, oxidoreductase complexes, and thylakoid membranes (Table 28). Figure 42 shows that eggplant extract treatment under room temperature conditions significantly increased the activation of G protein-related genes. It was also revealed that eggplant extract treatment significantly increased the activation of photosynthesis-related genes (Figure 42 left). In plants, G proteins are thought to be involved in plant growth and development or signal transduction, but the detailed mechanism is not yet understood (Figure 42 right). G proteins (GTP-binding proteins) play an important role in plant signal transduction, but the relationship between G proteins and ion channels is not yet clear. For example, plants may regulate ion concentrations inside and outside cells by controlling the opening and closing of ion channels in response to specific stimuli. Furthermore, plants may possess mechanisms similar to "G protein-coupled receptors" found in animals. When these receptors recognize specific signals, they are expected to activate or deactivate ion channels, ultimately influencing cellular responses.

[0155]

[0156] Table 29 (Tables 29-1 and 29-2) shows the GO analysis results for eggplant extract and the control group under high-temperature conditions. A total of 94 GO terms were significantly extracted.

[0157] * Biological Process (BP): Biological processes such as metabolic pathways and signaling pathways. * Cellular Component (CC): Where in the cell it is located. * Molecular Function (MF): Molecular function such as enzyme activity and ligand binding.

[0158] Figure 43 shows the GO terms (fractions) that were significantly activated by eggplant extract treatment under room temperature conditions (left) and high temperature conditions (right). Common to both room temperature and high temperature conditions were G protein-related genes. Furthermore, photosynthesis-related genes were activated only under room temperature conditions (Figure 43 left). On the other hand, low-molecular-weight GTP-related genes and genes related to environmental stress response were activated only under high temperature conditions (Figure 43 right). Summarizing the results regarding the "upstream" (signaling pathway) mentioned above, it was confirmed that signaling pathways are mainly activated by eggplant extract treatment under high temperature conditions. It was also suggested that low-molecular-weight GTP is switched "ON" under high temperature conditions, activating the stress response pathway. Meanwhile, under room temperature conditions, the photosynthesis-promoting effect of eggplant extract treatment was confirmed.

[0159] Figure 44 illustrates the mechanism of "ON" and "OFF" switching of small GTP switches by eggplant extract treatment. As shown in Figure 43, GO analysis confirmed the activation of gene groups related to Ras GTP (GO:0017016), Ran GTP (GO:0008536), and small GTP (GO:0031267). These are known as "molecular switches." Molecular switches play an important role in plant signal transduction. They function as intracellular switches, turning specific signals "ON" or "OFF" within the cell, triggering appropriate responses such as intracellular signal transduction. The results of this analysis suggest that eggplant extract treatment turns "ON" high-temperature stress response-related signals. Figure 45 is a heat map analysis of Ras GTP-related gene groups under high-temperature conditions. ROPGEF1 (Rop Guanine Nucleotide Exchange Factor 1) gene expression was confirmed in both eggplant extract and ACh treatment. The Rop GTPase signaling pathway, which involves ROPGEF1, a molecule that activates Rop by converting GDP to GTP, is important for plant growth and development and may also be involved in other stress responses.

[0160] In animals, ACh is a neurotransmitter. On the other hand, while ACh is present in plants, which lack nerves, its role and receptors are not fully understood. Plant signal transduction involves calcium ions (Ca ２＋ It is believed that a unique mechanism mediated by ) is the dominant mechanism. Enhanced calcium absorption was confirmed by administration of eggplant extract. Furthermore, gene expression data obtained by RNA-seq suggested the presence of ACh receptors in lettuce. The basis for this is that when treated with eggplant extract, a group of genes related to G proteins (GTP) showed significantly higher expression levels in the cell membrane compared to the control group. In short, ACh receptors are present in the cell membrane, and the introduction of eggplant extract / ACh activates ion channels, thereby increasing calcium absorption within the cell. ２＋ It is thought that the influx of certain substances led to the lettuce acquiring heat stress tolerance. Furthermore, the very strong activation of low-molecular-weight GTP under heat stress conditions is intriguing. Current prior studies lack clear evidence as to whether low-molecular-weight GTP is directly involved in plant heat stress tolerance. Low-molecular-weight GTP plays a crucial role in signal transduction in animal cells, and may also be involved in intracellular transport and signal transduction in plants. Further research is needed to determine its specific relationship to the acquisition of heat stress tolerance in lettuce.

[0161] Figure 46 compares the GO analysis results of eggplant extract (right) and ACh treatment (left) under room temperature conditions. Similar to the eggplant extract, ACh treatment also activated G protein-related genes. While the activity of photosynthesis-related genes was lower than with eggplant extract treatment, activation of these genes by ACh treatment was confirmed. Figure 47 plots the GO analysis results of ACh's effects under high-temperature conditions. Under high-temperature conditions, ACh treatment activated DNA repair, protein biosynthesis, DNA replication, peptide biosynthesis, peptide metabolism, amide biosynthesis, and DNA metabolic pathways.

[0162] To identify biological processes (BP), cellular components (CC), and molecular functions (MF) from commonly expressed genes under high-temperature conditions of eggplant extract treatment and ACh treatment, GO analysis was performed to elucidate their common mechanisms. GO analysis allows for the extraction of characteristic GO terms based on statistical evaluation of a target gene group, such as differentially expressed genes or gene groups grouped by cluster analysis. This makes it possible to capture biological processes from expression levels. The results are shown in Figure 48 and Table 30 (Tables 30-1 and 30-2). Figure 48 is a Venn diagram showing GO terms (94 in total) for eggplant extract treatment, GO terms (52 in total) for ACh treatment, and 47 common GO terms. Table 30 shows a list of 47 biological processes (GO terms) obtained from GO analysis of common differentially expressed genes. It became clear that eggplant extract and ACh treatment promoted many common chemical processes. Among these common GO terms were DNA repair, cellular stress response, and protein folding, which could be understood as mechanisms of the high-temperature stress response in lettuce described in this disclosure. Table 30 also includes the P-value and padj, which indicate the significance of increased gene expression.

[0163]

[0164] * Biological Process (BP): Biological processes such as metabolic pathways and signaling pathways. * Cellular Component (CC): Where in the cell it is located. * Molecular Function (MF): Molecular function such as enzyme activity and ligand binding.

[0165] From the 47 common GO terms shown in Table 30, we identified common genes and performed functional analysis on the 10 common terms (Table 31) that best captured the high-temperature stress response. Table 31 also shows the results of Wald tests performed between each group. It became clear that processes important for acquiring high-temperature stress tolerance were statistically significantly concentrated in eggplant extract and ACh treatment.

[0166] ***: p<0.01, **: p<0.05, ns: no significant difference

[0167] Figure 49 shows the results of functional analysis and heatmap comparison of expression data for protein folding-related genes common to eggplant extract and ACh treatment under high-temperature conditions. Significant increase in expression of protein folding-related genes was observed by DESeq2, with log2(fold change) = 1.0 and adjusted p-value < 0.05 (Table 31). Protein folding is crucial in the high-temperature stress response. In high-temperature environments, proteins become more susceptible to denaturation, potentially impairing cellular function. To prevent this, plants activate proteins called molecular chaperones. RNA-seq analysis in this study showed no significant difference between eggplant extract and ACh treatment, with significantly increased expression compared to the control group. The Venn diagram in the upper left of Figure 49 shows 16 common genes whose expression increased under high-temperature conditions with eggplant extract and ACh treatment. GO analysis using these 16 common genes revealed significantly increased activity in the endoplasmic reticulum, intracellular membrane system, calcium binding, abnormal protein binding, heat shock protein binding, and chaperone binding (Wald test, p<0.05). Figure 49 (right) is a heatmap analysis of protein folding-related genes under high-temperature conditions. Red (dark gray in the figure) indicates genes with high expression levels, and green (light gray in the figure) indicates genes with low expression levels. It was shown that eggplant extract and ACh treatment increased the expression levels of protein folding-related genes compared to the control group.

[0168] Figure 50 shows the results of a heatmap comparison of expression data for ion channel-related genes such as glutamate (GLR) common to eggplant extract and ACh treatment under high-temperature conditions. Dark gray indicates genes with high expression levels, black indicates genes with moderate expression levels, and light gray indicates genes with low expression levels. For ion channel-related genes such as glutamate (GLR), there was no significant difference in expression levels (DESeq2 log2(fold change)=1.0, adjusted p-value<0.05), but the expression of several related genes increased with eggplant extract and ACh treatment. GLR ion channels are non-selective cation channels that are activated by amino acids such as glutamate, and increase Ca2+12+ ２＋ Promote inflow. This Ca ２＋ Signaling is known to be involved in plant defense responses and environmental stress responses, and may also be related to acute environmental changes such as high temperature stress. Activation of ion channel-related genes was confirmed in both eggplant extract and ACh treatment, suggesting that multiple genes related to "ion channels" are rapidly induced in cells sensing high temperature stress. In this study, activation of GLR ion channel-related genes common to both eggplant extract and ACh treatment was confirmed, but the activity of GLR ion channel-related genes tended to be lower in ACh treatment than in eggplant extract treatment.

[0169] Figure 51 shows the results of a heatmap comparison of expression data for G protein-related genes common to eggplant extract and ACh treatment under high-temperature conditions. Dark gray indicates genes with high expression levels, black indicates genes with moderate expression levels, and light gray indicates genes with low expression levels. Significant increase in expression was observed in the G protein-related genes using DESeq2, with log2(fold change) = 1.0 and adjusted p-value < 0.05. G proteins (GTP-binding proteins) are important molecules that play a role in signal transduction in conjunction with receptors on the cell membrane and are present not only in animals but also in plants. In plants, it has been suggested that G proteins are involved in the reception and transmission of stress signals in response to environmental stress (high temperature, drought, salinity, etc.). In the reception and transmission of stress signals, G proteins play a role in transmitting signals from receptors that sense environmental stress into the cell. This changes gene expression and metabolic pathways, improving stress tolerance. G proteins may function as upstream signaling factors in this network, and their application to the development of stress-tolerant varieties is expected. Previous studies have shown that eggplant extract and ACh treatment commonly increase the expression levels of many G protein-related genes. Plants utilize GTPases to regulate calcium signaling and enhance stress tolerance as a response to environmental stress. These processes demonstrate that GTP and calcium play essential roles in plant growth. Therefore, it is suggested that plant G proteins are important molecules involved in the reception, transmission, and response to environmental stress, and likely contribute to the acquisition of stress tolerance. In signal transduction, G proteins receive signals on the cell membrane and trigger various intracellular responses. In environmental stress responses, G proteins regulate the response to environmental stress and assist in cellular adaptation. This study demonstrated that eggplant extract and ACh treatment can improve the DNA damage response in plants caused by environmental stress.Therefore, the composition of this disclosure containing eggplant extract / ACh as an active ingredient is expected to improve crops, enhance their growth, increase their lifespan, and give them better properties, thereby contributing to global food stability. Plants cannot avoid various environmental stresses such as high and low temperatures, drought, pathogens, parasites, and soil deterioration, and in order for plants to protect their own lives, they have no choice but to respond to environmental stresses on the spot. Natural radiation and ultraviolet rays damage DNA, but many environmental stresses also cause DNA damage, inhibiting plant development and growth. Therefore, the DNA damage response is extremely important for plants to grow and survive in fluctuating environments.

[0170] Figure 52 shows the results of a heatmap comparison of expression data for Ca-binding protein-related genes common to eggplant extract and ACh treatment under high-temperature conditions. Dark gray indicates genes with high expression levels, black indicates genes with moderate expression levels, and light gray indicates genes with low expression levels. While there was no significant difference in expression levels for Ca-binding protein-related genes (DESeq2 log2(fold change)=1.0, adjusted p-value<0.05), the expression of several related genes increased with eggplant extract and ACh treatment. 2+ Binding protein (Ca 2+ Ca-binding proteins play a crucial role in plants acquiring heat stress tolerance. These proteins are essential for Ca-binding under high-temperature conditions. 2+ It acts as part of a mechanism that senses changes in ions and regulates the stress response. Regarding Ca-binding protein-related genes, there were few genes with increased expression in common between eggplant extract and ACh treatment, but further detailed research is needed on these.

[0171] Figure 53 shows the results of comparing heat map expression data of heat shock protein-related genes common to eggplant extract and ACh treatment under high-temperature conditions. Figure 54 shows the results of comparing heat map expression data of heat shock protein-related genes common to eggplant extract and ACh treatment under high-temperature and room-temperature conditions. In both figures, dark gray indicates genes with high expression levels, black indicates genes with moderate expression levels, and light gray indicates genes with low expression levels. While there were no significant differences in the heat shock protein-related gene group (DESeq2 log2(fold change)=1.0, adjusted p-value<0.05), the expression of many common related genes increased with eggplant extract and ACh treatment. It has been reported that HSF (Heat Shock Transcription Factor) plays a central role in the high-temperature stress response, inducing the expression of heat shock proteins (HSPs). In this study, a rapid increase in the expression of multiple HSP genes was observed in both the eggplant extract and ACh treatments compared to the control group (Figures 53 and 54). Furthermore, activation of the master transcription factor HsfA3 was also confirmed (Figure 54). Since HsfA3 is only activated when subjected to high-temperature stress, it became clear that the high-temperature treatment conditions in this study caused high-temperature stress to the lettuce.

[0172] Figure 55 shows the results of functional analysis and heatmap comparison of expression data for peptide biosynthesis-related genes common to eggplant extract and ACh treatment under high-temperature conditions. Peptide biosynthesis-related genes showed significant increased expression with DESeq2 log2(fold change)=1.0 and adjusted p-value<0.05. Peptides are responsible for long-range signal transduction within plants and function as important factors in regulating stress responses. It was revealed that peptide biosynthesis is involved in the acquisition of high-temperature stress tolerance in lettuce under eggplant extract and ACh treatment. The Venn diagram in the upper left of Figure 55 shows 58 common genes whose expression increased under high-temperature conditions with eggplant extract and ACh treatment. Gene function analysis using these 58 common genes showed significant activation of peptide metabolism, peptide biosynthesis, protein biosynthesis, RNA binding, and structural molecule activity (Wald test, p<0.05). Figure 55 right shows the heatmap analysis of peptide biosynthesis-related genes under high-temperature conditions. Red (dark gray in the figure) indicates genes with high expression levels, black indicates genes with moderate expression levels, and green (light gray in the figure) indicates genes with low expression levels. It was shown that the expression levels of peptide biosynthesis-related genes increased compared to the control group after eggplant extract and ACh treatment.

[0173] Figure 56 shows the results of functional analysis and heatmap comparison of expression data for translation-related genes common to eggplant extract and ACh treatment under high-temperature conditions. The expression of translation-related genes was significantly increased by DESeq2, with log2(fold change) = 1.0 and adjusted p-value < 0.05. Under high-temperature stress, specific mRNAs are selectively translated, and proteins necessary for the stress response (such as HSPs) are rapidly synthesized. Eggplant extract and ACh treatment activated the translational regulatory mechanism in lettuce in response to high-temperature stress, strengthening its defense against stress. The Venn diagram in the upper left of Figure 56 shows 58 common genes whose expression increased under high-temperature conditions with eggplant extract and ACh treatment. Gene function analysis using these 58 common genes revealed significant activation of peptide biosynthesis, peptide metabolism, amide biosynthesis, structural molecule activity, RNA binding, translation factor activity, and translation initiation factor activity (Wald test, p < 0.05). Figure 56 on the right shows the heatmap analysis of translation-related genes under high-temperature conditions. Red (dark gray in the figure) indicates genes with high expression levels, black indicates genes with moderate expression levels, and green (light gray in the figure) indicates genes with low expression levels. It was shown that eggplant extract and ACh treatment increased the expression levels of translation-related genes compared to the control group.

[0174] Figure 57 shows the results of functional analysis and heatmap comparison of expression data for DNA repair-related genes common to eggplant extract and ACh treatment under high-temperature conditions. DNA repair-related genes showed significant increased expression with DESeq2 log2(fold change)=1.0 and adjusted p-value<0.05. DNA repair is crucial in the plant's response to high-temperature stress. High-temperature stress promotes the generation of reactive oxygen species (ROS) within cells, which can cause DNA damage. To counteract this, plants activate DNA repair mechanisms to maintain genomic stability. Under high-temperature conditions, eggplant extract and ACh treatment induced increased activity of DNA repair-related genes in lettuce. The Venn diagram in the upper left of Figure 57 shows 16 common genes whose expression increased under high-temperature conditions with eggplant extract and ACh treatment. Gene function analysis using these 16 common genes revealed significantly increased activity in response to DNA damage, cellular stress response, and mismatch repair (Wald test, p<0.05). Figure 57 on the right shows the heatmap analysis of DNA repair-related genes under high-temperature conditions. Red (dark gray in the figure) indicates genes with high expression levels, black indicates genes with moderate expression levels, and green (light gray in the figure) indicates genes with low expression levels. It was shown that eggplant extract and ACh treatment increased the expression levels of DNA repair-related genes compared to the control group.

[0175] Figure 58 shows the results of a heatmap comparison of gene expression data for reactive oxygen species-related genes common to eggplant extract and ACh treatment under high-temperature conditions. Red (dark gray in the figure) indicates genes with high expression levels, black indicates genes with moderate expression levels, and green (light gray in the figure) indicates genes with low expression levels. ***: p<0.01, **: p<0.05, ns: no significant difference (Wald test, p<0.05). While there was no significant difference in expression levels of reactive oxygen species-related genes (DESeq2 log2(fold change)=1.0, adjusted p-value<0.05), the activity of reactive oxygen species (ROS) generation genes was lower and ROS metabolism gene activity was increased in both eggplant extract and ACh treatment. The SOD gene group, included in ROS metabolism genes, is known to play an important role as a defense mechanism against oxidative stress. In the control group, ROS generation gene activity was high, and ROS metabolism gene activity was lower than in eggplant extract and ACh treatment. In the control group, a large amount of ROS accumulated, suggesting that administration of eggplant extract and ACh induced ROS metabolic genes and protected cells from ROS.

[0176] Figure 59 shows the results of a heatmap comparison of expression data for mismatch DNA repair-related genes common to eggplant extract and ACh treatment under high-temperature conditions. Red (dark gray in the figure) indicates genes with high expression levels, black indicates genes with moderate expression levels, and green (light gray in the figure) indicates genes with low expression levels. The mismatch DNA repair-related genes were significantly increased in expression by DESeq2 with log2(fold change)=1.0 and adjusted p-value<0.05. The MSH gene group included in the mismatch DNA repair-related gene group also plays an important role in DNA mismatch repair (MMR) in humans. This MSH gene group maintains genetic stability by detecting and repairing base pair mismatches that occur during DNA replication. In this test example, it was suggested that mismatches (incorrect base pairs) occurring during DNA replication in lettuce were corrected, and genomic stability was maintained even under high-temperature conditions. ACh is a neurotransmitter that plays a very important role in the human body and is known to be involved in muscle contraction, memory and learning, and regulation of the autonomic nervous system. While the role of acetylcholine in plants differs from that in animals, it has several important functions: regulation of growth and development, stress response (when plants are exposed to environmental stress such as heat stress, ACh regulates the movement of calcium ions, aiding the stress response), and regulation of ion channels (ACh controls the opening and closing of ion channels, regulating the transport of substances). However, there have been no reports on DNA repair by ACh. This study has revealed that ACh has DNA repair function in plants. We believe that eggplant extract or ACh may provide a more effective and safer treatment and will play an important role in future medicine. To date, various plant-derived extracts have been reported as DNA repair agents, but no DNA repair agents with eggplant extract or ACh as the active ingredient are known. The development of new DNA repair drugs is extremely important, and is particularly needed in the treatment of cancer and genetic diseases. We believe that the findings of this disclosure apply to both animals and plants. Current medical research is working to gain a deeper understanding of the mechanisms of DNA repair and to develop new drugs that target them.Drugs that promote DNA repair are expected to be useful in treating or preventing genetic disorders and age-related problems.

[0177] Figure 60 shows the results of functional analysis and heatmap comparison of expression data for DNA replication-related genes common to eggplant extract and ACh treatment under high-temperature conditions. DNA replication-related genes showed significant increased expression with DESeq2 log2(fold change)=1.0 and adjusted p-value<0.05. High-temperature stress causes DNA damage, and in response, plants perform DNA repair, cell cycle arrest, and stress response protein synthesis. These mechanisms allow plants to minimize damage while continuing DNA replication even under high-temperature stress. The Venn diagram in the upper left of Figure 60 shows 30 common genes whose expression increased under high-temperature conditions with eggplant extract and ACh treatment. Gene function analysis using these 30 common genes revealed significantly increased activity in DNA-dependent DNA polymerase, DNA polymerase activity, nucleotidyl transferase activity, DNA replication initiation, and DNA recombination (Wald test, p<0.05). Figure 60 on the right shows the heatmap analysis of DNA replication-related genes under high-temperature conditions. Red (dark gray in the figure) indicates genes with high expression levels, black indicates genes with moderate expression levels, and green (light gray in the figure) indicates genes with low expression levels. It was shown that eggplant extract and ACh treatment increased the expression levels of DNA repair-related genes compared to the control group.

[0178] Figure 61 shows the results of functional analysis and heatmap comparison of expression data for DNA metabolism-related genes common to eggplant extract and ACh treatment under high-temperature conditions. DNA metabolism-related genes showed significant increased expression with DESeq2 log2(fold change)=1.0 and adjusted p-value<0.05. DNA metabolism in lettuce subjected to high-temperature stress under eggplant extract and ACh treatment differed from normal conditions, with specific response mechanisms (activation of DNA repair, activation of transcription factors) being activated. The Venn diagram in the upper left of Figure 61 shows 49 common genes whose expression increased under high-temperature conditions with eggplant extract and ACh treatment. Gene function analysis using these 49 common genes revealed significant activation of DNA-dependent DNA polymerase, DNA replication initiation, cellular DNA damage response, DNA repair, mismatch repair, and DNA polymerase activity (Wald test, p<0.05). Figure 61 on the right shows the heatmap analysis of DNA metabolism-related genes under high-temperature conditions. Red (dark gray in the figure) indicates genes with high expression levels, black indicates genes with moderate expression levels, and green (light gray in the figure) indicates genes with low expression levels. It was shown that eggplant extract and ACh treatment increased the expression levels of DNA metabolism-related genes compared to the control group.

[0179] Figure 62 shows the results of functional analysis and heatmap comparison of expression data for amino acid biosynthesis-related genes common to eggplant extract and ACh treatment under high-temperature conditions. The amino acid biosynthesis-related genes showed significant expression increases by DESeq2, with log2(fold change) = 1.0 and adjusted p-value < 0.05. Genes involved in plant amino acid biosynthesis also play an important role in the plant's response to heat stress. The METE (Methionine Synthase) gene is involved in methionine biosynthesis. Methionine functions as a precursor to the antioxidant glutathione and is an important substance in the antioxidant response under heat stress, helping to protect plants from oxidative stress. The HIS4 gene is involved in histidine biosynthesis and is important for DNA repair and protein stabilization under heat stress. Histidine is a component of histone proteins and is involved in the regulation of gene expression. These genes support various physiological processes necessary for plants to adapt to heat stress and survive. Increased expression of genes involved in the biosynthesis of amino acids important for the high-temperature stress response (valine, leucine, isoleucine, histidine, phenylalanine, tyrosine, tryptophan, arginine, and methionine) was confirmed. The Venn diagram in the upper left of Figure 62 shows a group of nine common genes whose expression increased under high-temperature conditions with eggplant extract and ACh treatment. Gene function analysis using these nine common genes revealed significant activation of cellular amino acid biosynthesis, cellular amino acid biosynthesis metabolism, organic acid biosynthesis, and organic acid metabolism (Wald test, p<0.05). The right side of Figure 62 shows a heatmap analysis of amino acid biosynthesis-related genes under high-temperature conditions. Red (dark gray in the figure) indicates genes with high expression levels, black indicates genes with moderate expression levels, and green (light gray in the figure) indicates genes with low expression levels. It was shown that the expression levels of amino acid biosynthesis-related genes increased compared to the control group with eggplant extract and ACh treatment.

[0180] Figure 63 shows the results of functional analysis and heatmap comparison of expression data for amide biosynthesis-related genes common to eggplant extract and ACh treatment under high-temperature conditions. The amide biosynthesis-related genes showed significant increased expression by DESeq2, with log2(fold change)=1.0 and an adjusted p-value<0.05. Induction of amide (putrescine, spermidine, spermine, etc.) biosynthesis genes contributes to plant growth and improved tolerance under high-temperature stress. Amides are thought to help stabilize cell membranes and reduce oxidative stress. The Venn diagram in the upper left of Figure 63 shows 58 common genes whose expression increased under high-temperature conditions with eggplant extract and ACh treatment. Gene function analysis using these 58 common genes revealed significant activation of translation, peptide biosynthesis, peptide metabolism, translation elongation, and translation initiation (Wald test, p<0.05). Figure 63 on the right shows the heatmap analysis of amide biosynthesis-related genes under high-temperature conditions. Red (dark gray in the figure) indicates genes with high expression levels, black indicates genes with moderate expression levels, and green (light gray in the figure) indicates genes with low expression levels. It was shown that eggplant extract and ACh treatment increased the expression levels of amide biosynthesis-related genes compared to the control group.

[0181] Figure 64 shows the results of functional analysis and heatmap comparison of expression data for mismatch DNA repair-related genes common to eggplant extract and ACh treatment under high-temperature conditions. The mismatch DNA repair-related genes showed significant increased expression with DESeq2 log2(fold change)=1.0 and adjusted p-value<0.05. Under high-temperature stress, damage occurs not only to proteins and cell membranes but also to DNA, particularly during replication, where incorrect base pairing can occur. If mismatch DNA repair (MMR) functions normally under these conditions, these errors can be efficiently corrected, thus improving cell viability. Furthermore, MMR may cooperate with other repair pathways as part of the damage response, and its importance is presumed to increase under high-temperature stress conditions. The Venn diagram in the upper left of Figure 64 shows 16 common genes whose expression increased under high-temperature conditions with eggplant extract and ACh treatment. Gene function analysis using these 16 common genes revealed significant activation of DNA repair, cellular DNA damage response, cellular stress response, and mismatch repair (Wald test, p<0.05). Figure 64 (right) shows a heatmap analysis of mismatch DNA repair-related genes under high-temperature conditions. Red (dark gray in the figure) indicates genes with high expression levels, black indicates genes with moderate expression levels, and green (light gray in the figure) indicates genes with low expression levels. It was shown that eggplant extract and ACh treatment increased the expression levels of mismatch DNA repair-related genes compared to the control group.

[0182] Figure 65 shows the results of functional analysis and heatmap comparison of expression data for cellular stress response-related genes common to eggplant extract and ACh treatment under high-temperature conditions. The cellular stress response-related genes showed significant increased expression with DESeq2 log2(fold change)=1.0 and adjusted p-value<0.05. In the GO analysis of this study, the expression of genes with functions related to "DNA damage response, DNA repair, and mismatch repair" increased as part of "cellular stress response." This suggests that eggplant extract and ACh treatment activate the DNA repair mechanism in lettuce. The Venn diagram in the upper left of Figure 65 shows 17 common genes whose expression increased under eggplant extract and ACh treatment under high-temperature conditions. Gene function analysis using these 17 common genes showed significant activation of DNA damage response, DNA repair, mismatch repair, mismatch DNA binding, and damaged DNA binding (Wald test, p<0.05). Figure 65 right shows the heatmap analysis of cellular stress response-related genes under high-temperature conditions. Red (dark gray in the figure) indicates genes with high expression levels, black indicates genes with moderate expression levels, and green (light gray in the figure) indicates genes with low expression levels. It was shown that eggplant extract and ACh treatment increased the expression levels of cellular stress response-related genes compared to the control group.

[0183] Figure 66 shows the hypothesized common mechanism between eggplant extract and ACh treatment based on RNA-seq analysis. Previous GO analysis results have shown that eggplant extract and ACh treatment significantly increase the expression of genes related to G proteins, Ca signaling, DNA repair, and protein folding. It was revealed that eggplant extract and ACh alleviate the inhibition of Ca absorption and activate intracellular Ca signaling, thereby promoting the folding of abnormal proteins in the endoplasmic reticulum. Furthermore, it was revealed that eggplant extract and ACh induce DNA repair-related genes and repair DNA damaged by reactive oxygen species in the cell nucleus. From these findings, it was hypothesized that eggplant extract alleviates high-temperature stress in lettuce. It was also suggested that it promotes photosynthesis at room temperature.

[0184] Table 32 summarizes the GO analysis conducted so far, showing the results of the analysis of 10 GO terms common to eggplant extract and ACh treatment. In this study, it was revealed that eggplant extract and ACh treatment activated many common genes under high-temperature stress conditions, conferring high-temperature stress tolerance to lettuce. The high degree of commonality in the genes whose expression was activated by eggplant extract and ACh treatment suggests that many of the effects of eggplant extract treatment are due to ACh. In particular, the commonality was very high for protein folding and DNA replication. For other fractions, it was suggested that more than 40% of the effects of eggplant extract treatment were due to ACh. This suggests that eggplant extract contains polyphenols (Figure 67) and amino acids other than ACh that influence the acquisition of high-temperature stress tolerance in lettuce. Polyphenols are thought to help the function of ACh in plants by suppressing the activity of acetylcholinesterase (AChE), an enzyme that breaks down ACh. In addition, the genes expressed by ACh treatment had a very high average commonality of 94% with the genes expressed by eggplant extract treatment. Figure 67 is a graph showing the total polyphenol content in a culture medium obtained by mixing eggplant extracts, extracted using the same method as in this test example from multiple eggplant varieties (immature fruits), with each component at the concentrations shown in Table 25, and measuring the result using the Folin-Ciocalteu method. Chlorogenic acid was used as the polyphenol standard for calculating the total polyphenol content. The measurement results are shown as chlorogenic acid equivalents per liter of culture medium.

[0185] common

[0186] Figures 68 and 69 illustrate the relationships between GO terms common to eggplant extract and ACh treatment. GO terms have a hierarchical structure, with broader GO terms encompassing narrower GO terms. Figures 68 and 69 visualize these relationships between GO terms. Broader GO terms are shown at the top of the figures, and narrower GO terms are shown at the bottom. Darker gray GO terms indicate high statistical significance, while lighter colors indicate lower statistical significance. When enrichment is observed in the lower part of the figures, i.e., in narrower GO terms, it is presumed that a more specific biological response occurred. In relation to the acquisition of high-temperature stress tolerance by eggplant extract and ACh treatment, processes related to "peptide biosynthesis," "DNA repair," "amino acid biosynthesis," "amide biosynthesis," and "protein folding" were found to be statistically significantly enriched. These findings suggest that lettuce acquired high-temperature stress tolerance through eggplant extract / ACh treatment (Figure 70). Figure 70 shows the flow of the high-temperature stress response reaction induced by eggplant extract / ACh treatment, as determined from the analysis results so far. The first response reaction is mediated by ion channel stimulation (Figure 70 left). The second response reaction is mediated by DNA damage repair (Figure 70 right).

[0187] In this study, eggplant extract and ACh treatment under high temperatures induced the expression of various common genes. The genes whose expression was induced by eggplant extract and ACh treatment promoted the following common biological phenomena, thereby enabling lettuce to acquire heat stress tolerance: protein folding, amino acid biosynthesis, peptide biosynthesis, amide biosynthesis, DNA replication, DNA metabolism, DNA repair, and translation. Furthermore, it is expected that by enhancing the function of the proteins identified above, it will be possible to develop varieties that are highly resistant to heat stress without inhibiting plant growth.

[0188] Furthermore, this study revealed that treatment with eggplant extract and ACh under high-temperature conditions significantly increased the expression of DNA repair-related genes (DNA repair, DNA replication, and mismatch DNA repair). Therefore, the composition of this disclosure can be applied to plant DNA repair accelerators (DNA repair-promoting pharmaceuticals) by enhancing the expression of these genes. The DNA repair accelerator of this disclosure may be applicable not only to plants but also to animals (humans and non-human animals). The development of new DNA repair drugs is extremely important and is particularly needed in the treatment of cancer and genetic diseases. Drugs that promote DNA repair are also expected to be useful in the treatment of genetic diseases and age-related problems. For example, there is a need for drugs that efficiently repair DNA damage by activating specific proteins or enzymes. The new DNA repair accelerator provided by this disclosure has the potential to provide more effective and safer treatments and is expected to play an important role in future medical care.

[0189] As shown in the above examples, by applying a composition containing neurotransmitter / AChE inhibitors or plant extracts containing them, along with calcium ions, to plants, it is possible to confer resistance to environmental stress to the plants while maintaining sufficient growth-promoting effects. Furthermore, the compositions of this disclosure can utilize unused biomass such as crop residues generated in agriculture and waste from food processing plants as raw materials. Therefore, this disclosure is applicable in agriculture and the food processing industry. Cross-reference of related applications

[0190] This application claims priority pursuant to Japanese Patent Application No. 2024-224430 filed with the Japan Patent Office on 19 December 2024 and Japanese Patent Application No. 2025-192755 filed with the Japan Patent Office on 12 November 2025, all of which disclosures thereof are incorporated herein by reference in their entirety.

Claims

1. A plant environmental stress reliever containing one or more neurotransmitters selected from choline esters, catecholamines, serotonin, and amino acids, and / or one or more acetylcholinesterase inhibitors selected from polyphenols, flavonoids, and organic acids, or a plant extract containing the same, and calcium ions.

2. The plant environmental stress mitigator according to claim 1, wherein the environmental stress is high temperature stress.

3. The plant environmental stress reliever according to claim 1, wherein the plant extract is an extract of one or more plants selected from the group consisting of eggplant, tomato, rosemary, bamboo grass, mint, eucalyptus, thyme, lemon balm, pine, coconut, bamboo, plum, chayote, lettuce, soybean, cassava, asparagus, mango, hibiscus, dandelion, horsetail, jolokia, and cacao.

4. The plant environmental stress mitigator according to claim 1, further containing iron ions and one or more trace elements selected from the group consisting of boron, manganese, zinc, copper, and molybdenum.

5. The plant environmental stress mitigator according to claim 1, comprising the neurotransmitter at a concentration of 0.00001 mM to 100 mM when applied to plants.

6. The plant environmental stress mitigator according to claim 1, comprising the calcium ions at a concentration of 0.0001 mM to 100 mM when applied to plants.

7. The plant environmental stress reliever according to claim 1, which is used by adding it to a hydroponic solution, or by foliar spraying, soil injection, or irrigation.

8. The plant environmental stress reliever according to claim 1, applicable to one or more plants selected from the group consisting of flowers, legumes, grains, root vegetables, leafy vegetables, fruit vegetables, and fruit trees.

9. The plant environmental stress reliever according to claim 1, applicable to one or more plants selected from the group consisting of lettuce, garland chrysanthemum, cabbage, spinach, tomato, bell pepper, eggplant, lisianthus, snow pea, peanut, soybean, carrot, radish, corn, sweet corn, rice, melon, watermelon, banana, mandarin orange, orange, lemon, grape, pineapple, pear, apple, peach, chestnut, cherry, persimmon, fig, walnut, raspberry, gooseberry, cherry plum, blueberry, cotton, coffee, and sugarcane.

10. A plant growth promoter containing one or more neurotransmitters selected from choline esters, catecholamines, serotonin, and amino acids, and / or one or more acetylcholinesterase inhibitors selected from polyphenols, flavonoids, and organic acids, or a plant extract containing the same, and calcium ions.

11. The plant growth promoter according to claim 10, which is used under temperature conditions higher than the optimal growth temperature of the crop.

12. The plant growth promoter according to claim 10, which enhances the expression of one or more genes selected from the group consisting of photosynthesis-related genes and G protein-related genes under normal temperature conditions.

13. A plant DNA repair promoter containing one or more neurotransmitters selected from choline esters, catecholamines, serotonin, and amino acids, and / or one or more acetylcholinesterase inhibitors selected from polyphenols, flavonoids, and organic acids, or a plant extract containing the same, and calcium ions.

14. The DNA repair promoter according to claim 13, which enhances the expression of one or more genes selected from the group consisting of DNA repair-related genes, mismatch DNA repair-related genes, and DNA replication-related genes, under temperature conditions higher than the optimal growth temperature of the crop.

15. A method for producing a plant environmental stress reliever according to claim 1, comprising the step of mixing a plant extract obtained by extracting a plant containing a neurotransmitter and / or an acetylcholinesterase inhibitor using an extraction solvent, or a neurotransmitter and / or an acetylcholinesterase inhibitor with calcium ions in the presence of water, wherein the neurotransmitter is one or more selected from choline esters, catecholamines, serotonin and amino acids, and the acetylcholinesterase inhibitor is one or more selected from polyphenols, flavonoids and organic acids.

16. The method according to claim 15, wherein the extraction solvent is water and the extraction conditions are 40 to 150°C for 5 to 60 minutes.

17. The method according to claim 15, wherein iron ions and trace elements are further mixed in the mixing step.

18. A method for producing a plant growth promoter according to claim 10, comprising the step of mixing a plant extract obtained by extracting a plant containing a neurotransmitter and / or an acetylcholinesterase inhibitor using an extraction solvent, or a neurotransmitter and / or an acetylcholinesterase inhibitor with calcium ions in the presence of water, wherein the neurotransmitter is one or more selected from choline esters, catecholamines, serotonin and amino acids, and the acetylcholinesterase inhibitor is one or more selected from polyphenols, flavonoids and organic acids.

19. A method for producing a DNA repair promoter according to claim 13, comprising the step of mixing a plant extract obtained by extracting a plant containing a neurotransmitter and / or an acetylcholinesterase inhibitor using an extraction solvent, or a neurotransmitter and / or an acetylcholinesterase inhibitor, with calcium ions in the presence of water, wherein the neurotransmitter is one or more selected from choline esters, catecholamines, serotonin, and amino acids, and the acetylcholinesterase inhibitor is one or more selected from polyphenols, flavonoids, and organic acids.

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