Plant growth promoter and method for growing grasses

Abstract

To provide a plant growth promoter and a method for raising Poaceae plants in which metal concentration in a plant body can be properly adjusted.SOLUTION: A plant growth promoter promotes growth of Poaceae plants, and contains Epigallocatechin gallate. By adding Epigallocatechin gallate to the plant growth promoter, reduction of a pectin methylation degree of roots is suppressed, and a pectin amount of roots can be increased more than WT.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a plant growth promoter and a method for growing grass plants. [Background technology]

[0002] Known techniques for promoting plant growth include techniques using plant hormones such as cytokinin, auxin, and gibberellin, and growth regulation techniques using gene modification. For example, Patent Document 1 describes a method for regulating growth by introducing into plant cells or plant tissues a recombinant vector containing a gene in which the sequence encoding a biomembrane-crossing protein has been modified and which is linked so as to be regulated by transcription and translation regulatory factors that can be expressed in plants, thereby changing the tolerance or accumulation of heavy metals and salt in the plant. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent No. 4648352 Summary of the Invention [Problem to be solved by the invention]

[0004] In plants, such as grasses, copper is involved in redox reactions and functions as a cofactor for various enzymes. Copper also plays an important role in photosynthesis, respiration, cell wall metabolism, lignification, and other processes. However, depending on the concentration of copper in the plant, it can affect these functions. For example, copper deficiency or excess in the plant can cause toxicity. In addition to copper, aluminum, lead, and strontium can also adversely affect growth depending on their concentrations. Therefore, to promote plant growth, it is necessary to appropriately regulate the metal concentrations in the plant.

[0005] The present invention has been made in view of the above, and aims to provide a plant growth promoter and a method for growing grasses that can appropriately adjust metal concentrations in plants. [Means for solving the problem]

[0006] In order to solve the above problems and achieve the object, the plant growth promoter of the present invention is a plant growth promoter that promotes the growth of grass plants, and contains epigallocatechin gallate.

[0007] The plant growth promoter according to the present invention is the above-mentioned plant growth promoter obtained by adding epigallocatechin gallate to water.

[0008] The plant growth promoter according to the present invention, in the above invention, contains 25 μM to 100 μM of epigallocatechin gallate.

[0009] The plant growth promoter according to the present invention, in the above invention, contains 25 μM to 50 μM of epigallocatechin gallate.

[0010] The method for growing a grass plant according to the present invention comprises cultivating the grass plant using a plant growth promoter that promotes the growth of the grass plant, the plant growth promoter containing epigallocatechin gallate. [Effects of the Invention]

[0011] The present invention has the effect of enabling appropriate adjustment of metal concentrations in a plant body. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is a diagram for explaining the cell wall of a plant. [Figure 2] FIG. 2 is a diagram illustrating the enzymes involved in pectin regulation. [Figure 3] FIG. 3 is a diagram (part 1) for explaining the demethylation of methylated pectin. [Figure 4] FIG. 4 is a diagram (part 2) for explaining the demethylation of methylated pectin. [Figure 5] FIG. 5 is a diagram illustrating the concentration effect of EGCG on PME enzyme activity. [Figure 6] FIG. 6 is a diagram illustrating the relationship between the concentration of EGCG and the elongation of rice roots. [Figure 7] FIG. 7 is a photograph (part 1) showing pectin staining of rice roots. [Figure 8] FIG. 8 is a diagram (part 2) showing pectin staining of rice roots. [Figure 9] FIG. 9 is a diagram illustrating the change in pectin amount due to the addition / non-addition of EGCG. [Figure 10] FIG. 10 is a diagram for explaining the growing point in rice. [Figure 11] FIG. 11 is a diagram illustrating the effect of copper on the root elongation of rice. [Figure 12] FIG. 12 is a diagram for explaining the relative amount of root elongation of rice plants at different copper concentrations. [Figure 13] FIG. 13 is a diagram illustrating the change in pectin amount depending on the copper concentration. [Figure 14] FIG. 14 is a diagram for explaining the relative amount of pectin contained in rice roots at different copper concentrations. [Figure 15] FIG. 15 is a diagram illustrating the relative amount of rice root elongation at different copper concentrations in Nipponbare and PG2-FOX. [Figure 16] FIG. 16 is a diagram illustrating the relative elongation of rice aboveground parts at different copper concentrations in Nipponbare and PG2-FOX. [Figure 17] FIG. 17 is a diagram illustrating the relative amounts of pectin accumulated in rice roots at different copper concentrations in Nipponbare and PG2-FOX. [Figure 18] FIG. 18 is a diagram for explaining the relative amount of root elongation of rice at different copper concentrations in Nipponbare and Kasalath. [Figure 19] FIG. 19 is a diagram illustrating the relative elongation of the above-ground parts of rice plants at different copper concentrations in Nipponbare and Kasalath. [Figure 20] FIG. 20 is a diagram illustrating the relative amounts of pectin accumulated in rice roots at different copper concentrations in Nipponbare and Kasalath. [Figure 21] FIG. 21 is a diagram illustrating the relative amount of rice root elongation at different copper concentrations in Nipponbare and PM3-FOX. [Figure 22] FIG. 22 is a diagram illustrating the relative elongation of rice aboveground parts at different copper concentrations in Nipponbare and PM3-FOX. [Figure 23] FIG. 23 is a diagram illustrating the relative amount of pectin accumulated in rice roots at different copper concentrations in Nipponbare and PM3-FOX. [Figure 24] FIG. 24 is a diagram illustrating the relative amount of rice root elongation at different copper concentrations when EGCG is added / not added to Nipponbare. [Figure 25] FIG. 25 is a diagram illustrating the relative elongation of rice aboveground parts at different copper concentrations when EGCG is added / not added to Nipponbare. [Figure 26] FIG. 26 is a diagram illustrating the relative amount of pectin accumulated in rice roots at different copper concentrations when EGCG is added / not added to Nipponbare. [Figure 27] FIG. 27 is a diagram illustrating the analysis of copper dynamics using PETIS. [Figure 28] FIG. 28 shows images of rice plants (Nipponbare and PM3-FOX) used in the analysis of copper dynamics using PETIS. [Figure 29] FIG. 29 shows the results of an analysis of copper dynamics using PETIS (0.3 h) in Nipponbare and PM3-FOX. [Figure 30] FIG. 30 shows the results of analysis of copper dynamics using PETIS (20.0 h) in Nipponbare and PM3-FOX. [Figure 31]FIG. 31 shows the results of analysis of copper dynamics using PETIS (50.0 h) in Nipponbare and PM3-FOX. [Figure 32] FIG. 32 shows the change in copper adsorption by Nipponbare and PG2-FOX under appropriate conditions. [Figure 33] FIG. 33 shows the change in copper adsorption by Nipponbare and PG2-FOX under excess conditions. [Figure 34] FIG. 34 shows the change in copper adsorption of Nipponbare and Kasalath under appropriate conditions. [Figure 35] FIG. 35 shows the change in copper adsorption of Nipponbare and Kasalath under excess conditions. [Figure 36] FIG. 36 shows the change in copper adsorption by Nipponbare and PM3-FOX under appropriate conditions. [Figure 37] FIG. 37 shows the change in copper adsorption by Nipponbare and PM3-FOX under excess conditions. [Figure 38] FIG. 38 shows the change in copper adsorption to Nipponbare with or without the addition of EGCG under optimal conditions. [Figure 39] FIG. 39 shows the change in copper adsorption to Nipponbare with or without the addition of EGCG under excess conditions (1.0 μM). [Figure 40] FIG. 40 shows the change in copper adsorption to Nipponbare with or without the addition of EGCG under excess conditions (2.5 μM). [Figure 41] FIG. 41 shows images of rice plants (with / without EGCG addition) used in the analysis of copper dynamics using PETIS. [Figure 42] FIG. 42 shows images visualizing elemental copper in rice plants (with / without EGCG addition) used in the analysis of copper dynamics using PETIS. [Figure 43] FIG. 43 is a diagram illustrating the amount of copper contained in roots per 1 μg of pectin in Nipponbare, PG2-FOX, PM3-FOX, Kasalath, and EGCG-added Nipponbare. DETAILED DESCRIPTION OF THE INVENTION

[0013] Hereinafter, modes for carrying out the present invention (hereinafter referred to as "embodiments") will be described with reference to the accompanying drawings.

[0014] (Embodiment) Fig. 1 is a diagram for explaining the cell wall of a plant. A cell of a land plant is composed of a nucleus, vacuole, chloroplast, Golgi apparatus, cytoplasm, and cell membrane, all of which are surrounded by a cell wall 10. In this embodiment, a grass plant belonging to the Poaceae family is targeted. An example of adding the agent to rice will be described below.

[0015] The cell wall 10 is composed of three polysaccharide components classified as cellulose 11, hemicellulose 12, and pectin 13. The cell wall 10 has a structure in which cellulose 11 is cross-linked by hemicellulose 12, and pectin 13 fills the gaps formed by cellulose 11 and hemicellulose 12. Cellulose 11, hemicellulose 12, and pectin 13 are synthesized in the Golgi apparatus.

[0016] Figure 2 is a diagram illustrating the enzymes involved in pectin regulation, and Figures 3 and 4 are diagrams illustrating the demethylation of methylated pectin. Pectin 13 is primarily composed of galacturonic acid 131. It is synthesized in cells by galacturonosyltransferase (GAUT) and consists of multiple galacturonic acid 131 molecules linked together. In addition, some of the galacturonic acid 131 molecules in pectin 13 are methylated. For example, the carboxyl group in galacturonic acid 131 is methylated with methyl group 132.

[0017] The pectin 13 released into the cell wall is then demethylated by pectin methylesterase (PME). The demethylated pectin 13 is converted to calcium ions 133 (Ca 2+) forms a cross-linked structure. Pectin 13 that has formed a cross-linked structure with calcium ions 133 gels and becomes more viscous than before the cross-linked structure was formed. Therefore, the viscosity of pectin 13 can be adjusted by controlling the formation of a cross-linked structure with calcium ions 133.

[0018] Here, as described above, the methylated pectin 13 is demethylated by pectin methylesterase (PME) 14 (see FIG. 3). In this case, demethylation can be suppressed by inhibiting the activity of PME14. In this embodiment, epigallocatechin gallate (EGCG) 15 is bound to PME14 to inhibit the activity of PME14 (see FIG. 4). By binding EGCG 15 to PME14, the demethylation activity of PME14 can be reduced.

[0019] EGCG15 is a type of catechin found in abundance in plants, particularly tea. EGCG15 can be obtained, for example, by extracting tea leaves with hot water. EGCG15 is added to grasses (rice in this case) during the root growth period, such as seed, germination including root formation, and root elongation. For the growth of grasses, EGCG is added to a hydroponic solution to prepare an EGCG aqueous solution containing 25 μM to 100 μM of EGCG. The EGCG concentration is more preferably 25 μM to 50 μM. The EGCG aqueous solution will be described as being prepared by adding EGCG to water, but various additives may be added in addition to water.

[0020] Here, the effect of EGCG15 on PME enzyme activity will be described. Figure 5 is a diagram illustrating the concentration effect of EGCG on PME enzyme activity. When the PME enzyme activity was examined for rice samples with and without EGCG added, as shown in Figure 5, it was found that, when the enzyme activity of the sample without EGCG (0 μM) was set at 100, the PME enzyme activity was reduced in both the sample with 25 μM EGCG added and the sample with 100 μM EGCG added.

[0021] Next, we will explain the effect of EGCG15 on rice root elongation. Figure 6 is a diagram illustrating the relationship between rice root elongation and EGCG concentration. When the amount of root elongation was examined for rice samples with and without EGCG added, as shown in Figure 6, the root elongation of the sample without EGCG (0 μM) was approximately 0.4 cm, while the root elongation of the sample with 50 μM EGCG added was approximately 0.75 cm. From these results, it can be said that the addition of EGCG promotes root elongation. In FIG. 6, * indicates p<0.05.

[0022] Next, the amount of pectin in rice roots with and without the addition of EGCG will be explained. Figures 7 and 8 are diagrams showing pectin staining of rice roots. Figure 7 shows pectin staining (ruthenium red staining) of roots without the addition of EGCG. Figure 8 shows pectin staining of roots with the addition of EGCG. In each figure, (a) shows the entire root, (b) shows the tip of the main root or branch root, and (c) shows the tip of a root hair. In Figures 7 and 8, the darker the color, the higher the amount of pectin. As shown in Figures 7 and 8, it can be said that the total amount of pectin in the roots was higher when EGCG was added. The white bar in the figure is a scale bar indicating a length of 100 μm.

[0023] Figure 9 is a diagram illustrating the change in pectin amount with or without the addition of EGCG. Figure 9 shows the results of quantifying the amount of uronic acid (μg / mg) in rice roots using the carbazole sulfate method. Galacturonic acid is an uronic acid derived from galactose, and by quantifying this uronic acid, the amount of pectin, which is a linear chain of galacturonic acid, can be quantified. As shown in Figure 9, the amount of uronic acid was higher in the case of adding EGCG than in the case of not adding it. From this result, it can be said that the total amount of pectin in the roots was higher in the case of adding EGCG.

[0024] As explained above, inhibiting the enzyme activity of PME increases the total pectin content in rice and promotes root elongation. Thus, a positive correlation between pectin content and root elongation was demonstrated. Therefore, controlling pectin content is necessary for root elongation, and inhibiting the enzyme activity of PME by adding EGCG makes it possible to control pectin content and promote root elongation.

[0025] Next, we will explain the role of copper in plants. Copper is one of the essential trace elements that is essential for normal plant growth. Copper is involved in redox reactions in plants and functions as a cofactor for various enzymes. Copper also plays an important role in photosynthesis, respiration, cell wall metabolism, lignification, etc.

[0026] However, copper becomes toxic when its content in a plant becomes deficient or excessive, so the copper concentration in the plant body must be strictly controlled. Copper is known to accumulate mainly in the cytoplasm, chloroplasts, vacuoles, and cell walls. Copper has a high affinity for pectin among divalent cations, and is known to bind well to pectin in the cell wall. Therefore, controlling copper concentrations at an appropriate level is important for the normal growth of plants.

[0027] Therefore, the effects of adding or not adding EGCG were examined using Nipponbare, PG2-FOX, PME3-FOX, and Kasalath. PG2-FOX and PME3-FOX are rice varieties that were modified based on Nipponbare. Nipponbare: Japonica rice. PG2-FOX: Polygalacturonase (PG), an enzyme that degrades pectin backbone, was overexpressed to reduce the pectin content of the cell wall. PME3-FOX: A PME-overexpressing mutant of Nipponbare, which has high calcium cross-linking of pectin (low degree of pectin methyl esterification). Kasalath: An indica rice variety with longer and thinner grains than Nipponbare, and a higher pectin content than Nipponbare.

[0028] <Cultivation method> Each rice plant was hydroponically grown in a 1.0 mM CaCl2 solution (pH 5.4). First, the plants were allowed to absorb water for 3 days, then hydroponically grown in the CaCl2 solution for 3 days. After hydroponics, they were treated with copper for 7 days. The copper concentration was varied and each treatment was carried out separately.

[0029] <Test Method> Measurement of elongation Using copper-treated rice plants, the root and aboveground growth rates were measured at each copper concentration. Quantitative determination of pectin content Images of the rice plants stained with ruthenium red were taken, and the amount of pectin was quantified based on the intensity of the stained color. Copper resistance Rice plants were grown under various copper concentrations in copper treatment, and root and shoot elongation was measured. The amount of pectin accumulated in the roots was quantified by pectin staining. Observation of copper dynamics in rice The dynamics of copper in rice were observed using a Positron-emitting Tracer Imaging System (PETIS), which uses radioisotopes to visualize the time course of the target element (copper in this case). Determination of copper and calcium in rice After copper treatment, the copper and calcium contents in the roots and aboveground parts of rice plants were quantified using ICP-AES (ICPS-8100).

[0030] FIG. 10 is a diagram for explaining the growing point in rice. Regarding the amount of elongation of rice, the boundary between the root and the above-ground part is the area P G and the region P G Measurements were taken with the lower part considered to be the roots and the upper part considered to be the above-ground part.

[0031] <Test Results> (Measurement of elongation amount) Figure 11 is a diagram illustrating the effect of copper on rice root elongation. Figure 12 is a diagram illustrating the relative amount of rice root elongation at various copper concentrations. Figures 11 and 12 show the results of copper treatment of Nipponbare at copper concentrations of 0 μM (no copper added), 0.1 μM, 1.0 μM, and 2.5 μM. Here, the amount of root elongation was confirmed for two samples at each concentration. Figure 12 shows the relative amount of root elongation at each copper concentration, with the amount of root elongation at 0 μM (no copper added) set to 100.

[0032] Nipponbare was tested with copper concentrations of 0 μM (no copper added), 0.1 μM, 1.0 μM, and 2.5 μM. Two samples were tested for root elongation at each concentration. It was confirmed that root elongation generally decreased as the copper concentration increased. The copper concentration that produced the greatest elongation was 0.1 μM. Based on these results, it can be said that the optimum copper concentration for rice root growth is 0.1 μM. Furthermore, 2.5 μM, where relative elongation decreased dramatically, is an excessive concentration.

[0033] (Quantitative determination of pectin content) Figure 13 illustrates the change in pectin content depending on copper concentration. Figure 13(a) shows a portion of a Nipponbare root stained for pectin at a copper concentration of 0 μM (no copper added), Figure 13(b) shows a portion of a Nipponbare root stained for pectin at a copper concentration of 0.1 μM, and Figure 13(a) shows a portion of a Nipponbare root stained for pectin at a copper concentration of 2.5 μM. As shown in Figure 13, when the copper concentrations were 0 μM and 0.1 μM (see Figures 13(a) and (b)), it can be seen that pectin accumulated in large amounts at the root tip. In contrast, when the copper concentration was 2.5 μM (see Figure 13(c)), it can be seen that pectin was absorbed from the root tip toward the base, accumulating throughout the root.

[0034] Figure 14 is a diagram illustrating the relative amount of pectin contained in rice roots at different copper concentrations. Figure 14 shows the relative amount of pectin at each copper concentration, with the amount of pectin in roots at a copper concentration of 0 μM (no copper added) set to 1. As shown in Figure 14, the amount of pectin increases as the copper concentration increases. From the results shown in Figures 13 and 14, it can be said that the amount of pectin in the roots increases as the copper concentration increases.

[0035] (copper resistance) Comparison between Nipponbare and PG2-FOX Fig. 15 is a diagram illustrating the relative root elongation amounts of rice plants at different copper concentrations in Nipponbare and PG2-FOX. Fig. 15 shows the relative root elongation amounts at each copper concentration, with the root elongation amount at a copper concentration of 0 µM (no copper added) set at 100. Fig. 16 is a diagram illustrating the relative amount of elongation of rice aboveground parts at different copper concentrations in Nipponbare and PG2-FOX. Fig. 16 shows the relative amount of elongation at each copper concentration, with the amount of elongation of aboveground parts at a copper concentration of 0 µM (no copper added) set to 100. FIG. 17 is a diagram illustrating the relative amounts of pectin accumulated in rice roots at different copper concentrations in Nipponbare and PG2-FOX.

[0036] 15 and 16, the elongation of roots and aboveground parts of Nipponbare was greater than that of PG2-FOX. Furthermore, the amount of pectin accumulated in roots was greater in Nipponbare (see FIG. 17). These results suggest that PG2-FOX, which has a low pectin content, is less tolerant to excess copper than Nipponbare.

[0037] Comparison between Nipponbare and Kasalath Fig. 18 is a diagram illustrating the relative root elongation of rice plants at different copper concentrations in Nipponbare and Kasalath. Fig. 18 shows the relative root elongation at each copper concentration, with the root elongation at a copper concentration of 0 µM (no copper added) set at 100. Fig. 19 is a diagram illustrating the relative elongation of rice aboveground parts at different copper concentrations in Nipponbare and Kasalath varieties. Fig. 19 shows the relative elongation at each copper concentration, with the elongation of aboveground parts at a copper concentration of 0 µM (no copper added) set to 100. FIG. 20 is a diagram illustrating the relative amounts of pectin accumulated in rice roots at different copper concentrations in Nipponbare and Kasalath.

[0038] As shown in Figures 18 and 19, the elongation of the roots and aboveground parts of Nipponbare and Kasalath is greater than that of Kasalath. It is also clear that the amount of pectin accumulated in the roots is greater in Kasalath (see Figure 20). These results suggest that Kasalath, which has a high pectin content, is more tolerant to excess copper than Nipponbare.

[0039] Comparison between Nipponbare and PME3-FOX Figure 21 is a diagram illustrating the relative root elongation of rice plants at different copper concentrations in Nipponbare and PME3-FOX. Figure 21 shows the relative root elongation at each copper concentration, with the root elongation at a copper concentration of 0 μM (no copper added) set at 100. Fig. 22 is a diagram illustrating the relative amount of elongation of rice aboveground parts at different copper concentrations in Nipponbare and PME3-FOX. Fig. 22 shows the relative amount of elongation at each copper concentration, with the amount of elongation at a copper concentration of 0 µM (no copper added) set to 100. FIG. 23 is a diagram illustrating the relative amounts of pectin accumulated in rice roots at different copper concentrations in Nipponbare and PME3-FOX.

[0040] 21 and 22, the elongation of roots and aboveground parts was greater in Nipponbare than in PME3-FOX. Furthermore, the amount of pectin accumulated in roots was greater in Nipponbare (see FIG. 23). These results suggest that PME3-FOX, which has a low degree of pectin methyl esterification and a small amount of pectin, has lower tolerance to excess copper than Nipponbare.

[0041] Comparison of Nipponbare with and without EGCG Figure 24 is a diagram illustrating the relative amount of root elongation of rice plants at various copper concentrations when EGCG was added or not added to Nipponbare. Figure 24 shows the relative amount of root elongation at each copper concentration, with the amount of root elongation at a copper concentration of 0 μM (no copper added) set to 100. Note that in Figure 24, the amount of root elongation at copper concentrations of 0.1 μM and 2.5 μM shows p<0.05, and the amount of root elongation at a copper concentration of 1.0 μM shows p<0.01. Figure 25 is a diagram illustrating the relative amount of elongation of rice aboveground parts at different copper concentrations when EGCG was added or not added to Nipponbare. Figure 25 shows the relative amount of elongation at each copper concentration, with the amount of elongation of aboveground parts at a copper concentration of 0 μM (no copper added) set to 100. Note that in Figure 25, the amount of elongation at copper concentrations of 1.0 μM and 2.5 μM shows p<0.01. FIG. 26 is a diagram illustrating the relative amount of pectin accumulated in rice roots at different copper concentrations when EGCG is added / not added to Nipponbare. 24 to 26 show the results of cultivating Nipponbare in a hydroponic solution containing 50 μM EGCG as the EGCG-added condition.

[0042] 24 and 25, the elongation of roots and aboveground parts was greater with the addition of EGCG than without the addition of EGCG. Furthermore, the amount of pectin accumulated in roots was greater with the addition of EGCG (see FIG. 26). These results suggest that the EGCG-added condition, in which the degree of pectin methyl esterification is high and the amount of pectin is large, is more tolerant to excess copper than the EGCG-free condition.

[0043] (Observation of copper dynamics in rice) In the observation of copper dynamics, a radioactive isotope ( 64 A fixed amount (90 kBq) of copper was added, and hydroponic solution was supplied from the bottom of the syringe so that the liquid level remained constant. Measurements were then carried out using PETIS for one week. Figure 27 is a diagram for explaining the analysis of copper dynamics using PETIS. As shown in Figure 27, rice samples were placed in a syringe, and measurements were carried out using PETIS while hydroponic solution was supplied from below. 64 Even if Cu is absorbed into the sample, 64 The total amount of Cu was kept constant. The copper concentration was set to 0.1 μM, 1.0 μM, and 2.5 μM, and observations were carried out under each condition.

[0044] Figure 28 shows images of rice (Nipponbare and PM3-FOX) used in the analysis of copper dynamics using PETIS. For the observation of copper dynamics, PETIS images were acquired with the roots facing downward and the aboveground parts facing upward, as shown in Figure 28. The PETIS images are 64 The color temperature is expressed as bluer as the copper concentration decreases, and redder as the concentration increases. PETIS images were also taken for samples of Nipponbare (WT) and PME3-FOX with copper concentrations of 0.1 μM, 1.0 μM, and 2.5 μM. The same procedure was used for the other types of comparisons below.

[0045] Below, we will explain the observation of copper dynamics with reference to PETIS images of Nipponbare and PM3-FOX. Figure 29 shows the results of analysis of copper dynamics using PETIS in Nipponbare and PM3-FOX (0.3 h). Figure 30 shows the results of analysis of copper dynamics using PETIS in Nipponbare and PM3-FOX (20.0 h). Figure 31 shows the results of analysis of copper dynamics using PETIS in Nipponbare and PM3-FOX (50.0 h). Note that the following PETIS images are expressed using the same color temperature as the color temperature scale bar shown in Figure 29.

[0046] As shown in Figures 29 to 31, in the case of PME3-FOX, after 20.0 hours, 64 Cu accumulates, and after 50 hours, in the sample with a copper concentration of 0.1 μM, 64 It was confirmed that Cu was transported to the aboveground part. On the other hand, at concentrations of 1.0 μM or more, 64 Cu was not observed being transported to the aboveground part. At this time, the number of cells at the base of the aboveground part was large, so 64 The fluorescence of Cu becomes stronger. Below, PETIS images were observed for Nipponbare, PG2-FOX, PME3-FOX, and Kasalath, with a copper concentration of 0.1 μM as the appropriate condition and 1.0 μM as the excessive condition.

[0047] Comparison between Nipponbare (WT) and PG2-FOX Figure 32 shows the change in copper adsorption by Nipponbare and PG2-FOX under optimal conditions. As shown in Figure 32, Nipponbare showed greater and faster copper adsorption to the roots under optimal conditions. It was also confirmed that copper was transported to the aboveground parts over time.

[0048] Figure 33 shows the change in copper adsorption by Nipponbare and PG2-FOX under excess copper conditions. As shown in Figure 33, the amount and speed of copper adsorption to roots under excess copper conditions was greater and faster for PG2-FOX.

[0049] From Figures 32 and 33, it can be said that the higher the amount of pectin, the faster and more copper is adsorbed to the roots.

[0050] Comparison between Nipponbare (WT) and Kasalath Figure 34 shows the change in copper adsorption in Nipponbare and Kasalath under optimal conditions. As shown in Figure 34, the amount and speed of copper adsorption to the roots under optimal conditions was greater and faster in Nipponbare. It was also confirmed that copper was transported to the aboveground parts over time, and the amount and speed of this transport was greater and faster in Nipponbare.

[0051] Figure 35 shows the change in copper adsorption in Nipponbare and Kasalath under excess conditions. As shown in Figure 35, the amount and speed of copper adsorption to the roots under excess conditions was greater and faster in Nipponbare. It was also confirmed that copper was transported to the aboveground parts over time, and the amount and speed of this transport were greater and faster in Nipponbare.

[0052] 34 and 35, it can be seen that when the amount of pectin is high, the amount and speed of copper adsorption to the roots, and the amount and speed of copper transport to the above-ground parts are slow and small.

[0053] Comparison between Nipponbare (WT) and PME3-FOX Figure 36 shows the change in copper adsorption by Nipponbare and PME3-FOX under optimal conditions. As shown in Figure 36, the amount and speed of copper adsorption to the roots under optimal conditions was greater and faster in Nipponbare. It was also confirmed that copper was transported to the aboveground parts over time, and the amount and speed of transport were greater and faster in Nipponbare.

[0054] Figure 37 shows the change in copper adsorption by Nipponbare and PME3-FOX under excess copper conditions. As shown in Figure 37, there was almost no difference in the amount or speed of copper adsorption to the roots under excess copper conditions. It was also confirmed that copper was transported to the aboveground part over time (see red box), and the amount and speed of transport were greater and faster with PME3-FOX.

[0055] From Figures 36 and 37, it can be said that when the degree of methyl esterification of pectin is low and the amount of pectin is small, the amount and speed of copper transport to the aboveground parts is high and large.

[0056] Comparison of Nipponbare with and without EGCG Figure 38 shows the change in copper adsorption in Nipponbare with or without the addition of EGCG under optimal conditions. As shown in Figure 38, the amount and speed of copper adsorption to the roots under optimal conditions was greater and faster under the EGCG-free condition. It was also confirmed that copper was transported to the aboveground part over time, and the amount and speed of transport were greater and faster under the EGCG-free condition.

[0057] Figure 39 shows the change in copper adsorption to Nipponbare with or without the addition of EGCG under excess conditions (1.0 μM). Figure 40 shows the change in copper adsorption to Nipponbare with or without the addition of EGCG under excess conditions (2.5 μM). As shown in Figures 39 and 40, the amount and speed of copper adsorption to roots under excess conditions was greater and faster under EGCG addition. It was also confirmed that copper was transported to the aboveground parts over time (see red frame), and the amount and speed of transport were greater and faster under EGCG non-addition.

[0058] 38 to 40, it can be said that when the degree of methyl esterification of pectin is high and the amount of pectin is large, the amount and speed of copper transport to the aboveground part is slow and small.

[0059] Figure 41 shows images of rice plants (with and without EGCG) used in the analysis of copper dynamics using PETIS. Figure 42 shows images visualizing elemental copper in rice plants (with and without EGCG) used in the analysis of copper dynamics using PETIS. As shown in Figure 42, EGCG-added rice plants (see Figure 41) treated with each copper concentration took up copper, but under excess conditions (here, 1.0 μM or higher), copper was not transported to the aboveground shoots. From these results, it can be said that in EGCG-added rice, copper absorbed through the roots was not transported to the aboveground shoots.

[0060] (Determination of copper in rice) FIG. 43 is a diagram illustrating the amount of copper contained in roots per 1 μg of pectin in Nipponbare, PG2-FOX, PM3-FOX, Kasalath, and EGCG-added Nipponbare. In addition, Figure 43 shows the results of cultivating Nipponbare in a hydroponic solution containing 50 µM EGCG as the EGCG-added condition.

[0061] As shown in Figure 43, PME3-FOX, which had a low root pectin content and a low degree of methyl esterification, accumulated more copper in its roots than Nipponbare (WT). On the other hand, EGCG-added Nipponbare, which had a high root pectin content and a high degree of methyl esterification, accumulated less copper in its roots than Nipponbare (WT). When the copper concentration was 0 μM (no addition) or 0.1 μM, no copper was detected when 1 μg of pectin was used.

[0062] These test results indicated that rice plants with altered cell wall pectin content and degree of methyl esterification showed differences in copper adsorption to roots, transport to aboveground shoots, and accumulation. High pectin content and a high degree of methyl esterification resulted in fewer calcium cross-links in pectin, leading to higher tolerance to excess copper. On the other hand, low pectin content and a low degree of methyl esterification resulted in more calcium cross-links in pectin, leading to lower tolerance to excess copper.

[0063] It was also revealed that rice changes the amount of pectin accumulated in the roots and its degree of methyl esterification depending on the copper concentration, thereby changing the dynamics of copper within the plant and mitigating the toxicity of excess copper.

[0064] Therefore, it can be said that rice has a system that responds to excess copper by increasing the amount of pectin accumulated in the roots, controlling the degree of methyl esterification, and adsorbing the toxic metal onto pectin, thereby suppressing its transport to the aboveground parts and mitigating its toxicity to the plant body.

[0065] Furthermore, it was shown that the addition of EGCG, which provides the effects of epigallocatechin gallate, is effective in controlling the amount of pectin and the degree of methyl esterification in the roots. EGCG inhibits the decrease in the degree of pectin methylation in the roots and increases the amount of pectin in the roots compared to WT (Nipponbare is used here as an example). This has the effect of promoting root growth and facilitating the mechanism for alleviating toxicity against excessive copper. According to this embodiment, by cultivating rice in a hydroponic solution containing added EGCG, the metal concentration in the plant body can be appropriately adjusted. Note that the above-mentioned effects are also effective for grass plants belonging to the Poaceae family, in addition to rice.

[0066] The plant growth promoters according to the above-described embodiments can promote growth and also suppress the toxicity of metals (e.g., heavy metals) contained in soil, thereby suppressing various disorders such as whitening and yellowing of young leaves, the generation of reactive oxygen species (ROS) that have oxygen-containing chemical reactivity, and excess symptoms such as induction of iron deficiency in leaves. Thus, use of the plant growth promoters according to the present embodiments is expected to make it possible to cultivate land that is unsuitable for cultivation and contains excessive heavy metals.

[0067] Although the embodiments for carrying out the present invention have been described above, the present invention should not be limited to only the above-described embodiments.

[0068] As described above, the present invention can include various embodiments within the scope of the technical idea described in the claims.

[0069] As described above, the plant growth promoter and the method for growing a grass plant according to the present invention are useful for appropriately adjusting metal concentrations in a plant body. [Explanation of symbols]

[0070] 10 cell wall 11 Cellulose 12 Hemicellulose 13 Pectin 14 Pectin methylesterase (PME) 15 Epigallocatechin gallate (EGCG) 131 Galacturonic acid 132 Methyl group 133 Calcium ions

Claims

1. A plant growth promoter that promotes the growth of grasses, Contains epigallocatechin gallate, Plant growth promoter.

2. The epigallocatechin gallate is added to water. The plant growth promoter according to claim 1.

3. Contains 25 μM to 100 μM of epigallocatechin gallate, The plant growth promoter according to claim 2.

4. Contains 25 μM to 50 μM of epigallocatechin gallate, The plant growth promoter according to claim 3.

5. Cultivating a grass family plant using a plant growth promoter that promotes the growth of the grass family plant, the plant growth promoter containing epigallocatechin gallate. How to grow grasses.

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