Plant heat-resistant domestication method for inhibiting excessive accumulation of H2O2

Abstract

The invention discloses a plant heat-resistant domestication method for inhibiting excessive accumulation of H2O2, which is characterized by comprising the following steps: (1) treating a plant at the temperature of 35 DEG C for 1 hour, and then treating the plant at the temperature of 25 DEG C for 1 day; (2) treating the plants at the high temperature of 45 DEG C for 1 hour, and then treating the plants at the temperature of 25 DEG C for 1 day. According to the heat-resistant domestication method provided by the invention, excessive accumulation of H2O2 content in a plant body in a heat stress response process can be remarkably inhibited and maintained in a normal range, the plant is promoted to obtain higher heat resistance, and the domestication method is also suitable for other plants except kiwi fruits.

Description

Technical Field

[0001] The invention relates to the technical field of plant cultivation, and in particular to a method for acclimating plants to heat resistance by inhibiting excessive accumulation of H2O2. Background Art

[0002] Plant leaves directly sense changes in ambient temperature. Their photosynthesis and transpiration, as the basis of plant growth and development, are extremely susceptible to temperature changes. Heat stress induces protein denaturation, programmed cell death in specific cells and tissues, especially plant leaves, leading to leaf burns, dryness and necrosis. In high temperature environments, plants are prone to water loss and wilting, which is mainly manifested by a decrease in leaf water content, resulting in a decrease in cell turgor pressure, which in turn causes symptoms such as leaf bending and wilting. This process is related to the increase in transpiration rate induced by high temperature, which exceeds the root water absorption rate, causing excessive water loss in the plant body, which has a serious impact on plant growth.

[0003] According to existing research progress, when encountering high temperature stress, plants will quickly produce a variety of physiological changes to respond to the stress and reduce the harm of high temperature stress to growth. Reactive oxygen species (ROS), including H2O2, ·O 2- and 1 O2, etc., play a dual role in plants: usually at low concentrations, ROS as an important signal molecule participates in regulating the activation of the antioxidant system to cope with oxidative damage; but at high concentrations, the accumulation of ROS leads to the oxidation of proteins, lipids, carbohydrates and DNA, causing programmed cell death and causing serious harm to plants. Existing research results have shown that the effect of heat stress on plant growth is related to the excessive accumulation of ROS in plants. Summary of the invention

[0004] The main purpose of the present invention is to provide a method for heat-resistant acclimation of plants which can inhibit excessive accumulation of H2O2.

[0005] To achieve the above object, the present invention provides a method for heat-resistant acclimation of plants for inhibiting excessive accumulation of H2O2, comprising the following steps:

[0006] (1) The plants were placed at 35°C for 1 hour and then at 25°C for 1 day.

[0007] (2) The plants were placed at 45°C for 1 hour and then at 25°C for 1 day.

[0008] Furthermore, the high temperature treatment processes of step (1) and step (2) are both performed under 1000 lux of light.

[0009] Furthermore, the recovery processes of step (1) and step (2) are both performed under 2000 lux of light.

[0010] The effects of the heat-resistant acclimation method of the present invention on plants, taking kiwi fruit plants as an example, are as follows:

[0011] Under suitable growth conditions, the H2O2 content in the plant is about 7μmol / g. However, when experiencing heat stress exceeding 35°C, the H2O2 content in the plant significantly increases to 15-20μmol / g, and the plant growth is seriously hindered or even dies.

[0012] For plants that have not been heat-acclimated, the growth potential cannot be restored after the 50°C heat stress is eliminated, and the leaves wilt and lose water until they dry up and die; however, for plants acclimated by the present invention, the growth potential is significantly restored after the 50°C heat stress is eliminated, the leaves stretch and turn green, and the stems become upright. Through the heat-resistant acclimation method provided by the present invention, the H2O2 content in the body of the plant can be about 7μmol / g when experiencing 50°C heat stress, which is not significantly different from that of the plant that has not experienced heat stress, and the heat resistance is significantly enhanced.

[0013] The beneficial effects of the present invention are embodied in:

[0014] The heat-resistant acclimation method provided by the present invention can significantly inhibit the excessive accumulation of H2O2 content in the plant during the heat stress response process, maintain it within a normal range, and promote the plant to acquire stronger heat resistance. The acclimation method is also applicable to other plants besides kiwifruit. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Photos of the effects of high temperature stress of different intensities on the growth of kiwifruit plants, (a) is a top view, and (b) is a front view.

[0016] Figure 2 Effects of different intensities of high temperature stress on the water content of plant leaves.

[0017] Figure 3 Effects of high temperature stress of different intensities on H2O2 content in plants.

[0018] Figure 4 Apparent changes in the growth of kiwifruit plants affected by different heat acclimation methods (top view).

[0019] Figure 5 Apparent changes in the growth of kiwifruit plants affected by different heat acclimation methods (front view).

[0020] Figure 6 Effects of different heat acclimation methods on H2O2 content in plants. DETAILED DESCRIPTION

[0021] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.

[0022] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial channels or can be obtained by existing known methods; unless otherwise specified, the methods used in the embodiments of the present invention are methods known to those skilled in the art. Among them, the plants used are kiwifruit plants, and the kiwifruit plant cultivation method is: select the young branches of 'Donghong' kiwifruit as explants, and after surface disinfection, inoculate them in 1 / 2MS culture medium; after the axillary buds germinate, cut them off from the explants and continue to inoculate them in 1 / 2MS culture medium, which is the primary generation tissue culture seedlings; expand the primary generation tissue culture seedlings to obtain tissue culture seedlings with consistent genetic background and sufficient quantity, inoculate them in 1 / 2MS culture medium, inoculate 3 plants per bottle, place them in a tissue culture room at 25°C, light (light intensity 2000lux): dark cycle of 16h:8h, and culture them until 5 leaves grow, and select tissue culture seedlings with consistent growth as plants in the following embodiments.

[0023] Example 1

[0024] Effects of different intensities of heat stress on plant growth and development

[0025] I. Research Methods

[0026] 1. Heat stress treatment

[0027] Under 1000 lux light, plants were treated with heat stress at 35℃, 45℃, and 55℃ for 1, 2, 3, and 4 hours respectively as experimental groups, and plants always cultured at 25℃ were used as control groups.

[0028] 2. Leaf water content determination

[0029] Immediately after heat stress treatment, leaves were collected into 5 mL cryovials and weighed to obtain fresh weight (FW). The leaves were then dried in a freeze dryer (Sentry 2.0, VirTis, America) for three days, and the dry weight (DW) was determined. Leaf water content (w, %) w = (FW-DW) / FW.

[0030] 3. Determination of H2O2 content

[0031] Immediately after heat stress treatment, leaves were collected into 5 mL cryovials and ground in liquid nitrogen to obtain fresh samples. The hydrogen peroxide (H2O2) content detection kit (Solarbio, China) was used to measure the H2O2 content in leaves. The basic principle is that H2O2 reacts with titanium sulfate to form a yellow titanium peroxide complex, which is measured by a spectrophotometer ( The characteristic absorption value of the complex at 415 nm was measured by ELISA basic; Eppendorf, Germany to calculate the H2O2 content.

[0032] II. Research Results

[0033] 1. Heat stress of different intensities can cause plants to lose water and wilt to varying degrees

[0034] The experimental group placed five-leaf kiwifruit seedlings with the same growth at 25℃, 35℃, 45℃, and 55℃ for 1h, 2h, 3h, and 4h. Compared with the control, as the treatment temperature increased and the treatment time prolonged, the curling degree of the leaves of the plants gradually increased, the leaves drooped more and more, and showed a wilting state due to water loss. Figure 1 shown.

[0035] 2. Heat stress of different intensities can cause plants to lose water to varying degrees

[0036] Heat stress caused a significant decrease in the water content of kiwifruit leaves. Compared with the control group under 25°C temperature conditions, the water content of plants treated at 35°C and 45°C for 1 hour did not change significantly, but the water content of kiwifruit leaves decreased significantly after 2, 3, and 4 hours of treatment. Treatment at 55°C for 1, 2, 3, and 4 hours caused the water content of leaves to decrease by 13.6%, 45.6%, 50.9%, and 70.8%, respectively. Figure 2 shown.

[0037] 3. Heat stress of different intensities can cause a significant increase in the H2O2 content of plants

[0038] After heat stress treatment, H2O2 content increased rapidly within 1 hour and continued to increase within 2 hours. For example, after 2 hours of treatment at 35℃, the H2O2 content in the leaves increased from 7.6μmol / g to 17.8μmol / g. Figure 3 shown.

[0039] Example 2

[0040] Effects of different combinations of plant heat tolerance acclimation methods to inhibit excessive H2O2 accumulation on H2O2 content in plants subjected to 50℃ heat stress

[0041] I. Research Methods

[0042] 1. Heat resistance acclimation treatment

[0043] Three heat-resistant acclimation treatment combinations were set up, namely: (1) 35℃-50℃, i.e., 35℃ high temperature treatment for 1h, 25℃ recovery treatment for 1d, and then the response ability to 50℃ heat stress was tested; (2) 45℃-50℃, i.e., 45℃ high temperature treatment for 1h, 25℃ recovery treatment for 1d, and then the response ability to 50℃ heat stress was tested; (3) 35℃-45℃-50℃, i.e., 35℃ high temperature treatment for 1h, 25℃ recovery treatment for 1d, 45℃ high temperature treatment for 1h, 25℃ recovery treatment for 1d, and then the response ability to 50℃ heat stress was tested. For each combination, 1000lux of light was given during the high temperature treatment process and 2000lux of light was given during the recovery treatment process. Finally, the plants' response ability to 1000lux, 50℃, and 1h of heat stress was tested.

[0044] The control group was 25℃-50℃, that is, the plants were not subjected to heat tolerance acclimation treatment and directly experienced heat stress of 1000lux, 50℃, and 1h.

[0045] 2. Determination of H2O2 content

[0046] After 50℃ heat stress treatment, leaves were immediately collected into 5mL cryovials and ground in liquid nitrogen to obtain fresh samples. The hydrogen peroxide (H2O2) content detection kit (Solarbio, China) was used to measure the H2O2 content in leaves. The basic principle is that H2O2 reacts with titanium sulfate to form a yellow titanium peroxide complex, which is measured by a spectrophotometer ( The characteristic absorption value of the complex at 415 nm was measured by ELISA basic; Eppendorf, Germany to calculate the H2O2 content.

[0047] II. Research Results

[0048] 1.35℃-45℃-50℃ heat acclimation treatment significantly enhances the heat tolerance of plants

[0049] As the control group, the leaves of plants that directly experienced 50℃ for 1h suffered from severe water loss and wilting; 7 days after the heat stress was eliminated, the plants were still unable to resume growth, and some plants even dried up and died. However, the leaves of plants that were heat-resistant and acclimated at 35℃-45℃-50℃ recovered to a stretched and green state 1 day after the heat stress was eliminated, and the stems were upright. Although a very small number of leaves of plants that were heat-acclimated at 35℃-50℃ and 45℃-50℃ recovered to green, their ability to recover growth was significantly lower than that of plants that were heat-acclimated at 35℃-45℃-50℃, see Figure 4 and Figure 5 Therefore, it can be proved that the heat-resistant acclimation treatment of 35°C-45°C-50°C of the present invention is the best method to enable plants to acquire stronger heat resistance.

[0050] 2. Heat tolerance acclimation treatment significantly inhibited excessive accumulation of H2O2 in plants

[0051] like Figure 6 As shown in Figure 2, as the control group, the plants directly subjected to 50℃, 1h heat stress had an increase in H2O2 content in the leaves to 15.8μmol / g. After heat acclimation at 35℃-50℃ and 45℃-50℃, the H2O2 content in the leaves of the plants increased to 13.8μmol / g and 16.2μmol / g, respectively, with no significant difference compared with the plants without heat acclimation treatment. Figure 6 The H2O2 content in the leaves of plants that were heat-acclimated at 35℃-45℃-50℃ was 7.2μmol / g, which was not significantly different from that of plants grown under suitable conditions.

[0052] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for acclimating plants to heat resistance by inhibiting excessive accumulation of H2O2, characterized in that: The following steps are involved: (1) The plants were placed at 35°C for 1 hour and then at 25°C for 1 day. (2) The plants were placed at 45°C for 1 hour and then at 25°C for 1 day.

2. The method for acclimating plants to heat resistance by inhibiting excessive accumulation of H2O2 as claimed in claim 1, characterized in that: During the high temperature treatment process of step (1) and step (2), 1000 lux of light is applied.

3. The method for acclimating plants to heat resistance by inhibiting excessive accumulation of H2O2 as claimed in claim 2, characterized in that: During the recovery process of step (1) and step (2), 2000 lux of light is applied.

4. The method for acclimating plants to heat resistance by inhibiting excessive accumulation of H2O2 as claimed in claim 1, 2 or 3, characterized in that: The plant is a kiwi fruit plant.

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