Application model and implementation method for improving nutrition stress of arabidopsis thaliana

Abstract

The invention belongs to the technical field of biology, and relates to plant nutrition stress, in particular to an application model and an implementation method for improving the nutrition stress of arabidopsis thaliana. The arabidopsis thaliana WT is Columbia type arabidopsis thaliana (Columbia / Col-0), and the used mutants are all mutated in the wild type plant. The applicant finds that hydrogen peroxide and potassium iodide have a good regulation effect on plant response nutrition stress through experiments, meanwhile, internal reasons are analyzed, according to the analysis result, the applicant provides a plant nutrition stress and active oxygen regulation and control model, and according to the model, a scheme for improving the resistance of plants under the nutrition stress is provided. By using the model and the corresponding processing method provided by the invention, the growth condition and chlorophyll content of the plant under nutrition stress can be effectively improved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to plant nutrient stress. Background Technology

[0002] Arable land resources face various nutrient problems, directly impacting agricultural output and food security. The imbalance of ions (such as sodium) in saline-alkali soil within arable land... + Excessively high content), low utilization rate of nutrients (such as calcium) 2+ K + At the same time, due to industrial development, a lot of heavy metals (such as Cd and Cr) have polluted the land, which not only has biological toxicity but also affects the nutrient absorption of plants.

[0003] Reactive oxygen species (ROS) are important signaling molecules in plants, participating in the regulation of various life processes such as seed germination, plant growth, reproduction, and stress resistance. Studies have shown that saline-alkali soil or heavy metal salts can stimulate plants to produce ROS signals, thereby enhancing plant resistance. In various crops, stress-induced ROS signals have been confirmed to be associated with the autophagy pathway. The autophagy pathway is a spontaneous, programmed monitoring and optimization system in organisms. Plant cells can clear intracellular pathologies through autophagy, while also recovering depleted nutrients and recombining them for reuse. Through autophagy, plants can effectively cope with soil nutrient deficiencies and clear damage caused by various factors. Therefore, autophagy can effectively increase plant yields while ensuring food security.

[0004] In summary, plants can generate reactive oxygen species (ROS) to regulate autophagy pathways in response to biotic and abiotic stresses. Hydrogen peroxide can eliminate ROS, and CN109295147A discloses that potassium iodide can increase astaxanthin yield under high light-induced conditions; however, the specific regulatory mechanisms and methods of existing oxidants in Arabidopsis autophagy remain unclear. Therefore, this study explored the regulatory mechanisms of ROS on plant autophagy pathways and examined their effects, aiming to propose a simple in vitro ROS regulation strategy to manipulate plant autophagy, thereby directly and effectively increasing crop yield and contributing to food security. Summary of the Invention

[0005] To achieve the above objectives, this invention proposes an application model and implementation method for improving Arabidopsis thaliana under nutritional stress.

[0006] The technical solution of this invention is implemented as follows:

[0007] On one hand, this invention discloses the application of hydrogen peroxide and / or potassium iodide in regulating plant nutrient stress and autophagy. When plants cope with long-term nutrient stress, they manipulate the level of reactive oxygen species by adjusting catalase activity, thereby using oxidative signaling to partially improve the plant's tolerance to nutrient stress.

[0008] The concentration of hydrogen peroxide is 0.01-1 mM; the concentration of potassium iodide is 0.01-2 mM.

[0009] Applying 0.01-1 mM hydrogen peroxide at the initial stage of long-term nutritional stress slowed down the chlorosis in Arabidopsis plants, increased the chlorophyll content in Arabidopsis plants, and increased the number of autophagosomes in Arabidopsis plants.

[0010] Applying 0.01-2 mM potassium iodide at the initial stage of long-term nutrient stress accelerated chlorosis in Arabidopsis plants, reduced chlorophyll content, and decreased the number of autophagosomes.

[0011] Applying 0.01-2 mM potassium iodide in the later stages of long-term nutrient stress slowed down the chlorosis of Arabidopsis plants, increased the chlorophyll content, and increased the number of autophagosomes.

[0012] Furthermore, the above-mentioned regulatory mechanisms can be utilized to enhance plant resistance by applying hydrogen peroxide exogenously in the early stages of plant nutrient stress, and to enhance plant resistance by applying potassium iodide exogenously in the later stages of plant nutrient stress.

[0013] Preferably, the plant is Arabidopsis thaliana.

[0014] Secondly, the present invention provides a method for improving plant resistance to nutrient stress, comprising the following steps: applying hydrogen peroxide exogenously in the early stage of long-term nutrient stress to slow down the chlorosis of Arabidopsis plants, increase chlorophyll content, and increase the number of autophagic vacuoles; and applying potassium iodide exogenously in the later stage of long-term nutrient stress to accelerate chlorosis and reduce the harm of plant nutrient stress.

[0015] The concentration of hydrogen peroxide used is 0.01-1 mM, 3 ml / plant, once a day; the concentration of potassium iodide used is 0.01-2 mM, 3 ml / plant, once or twice a day; the initial stage of long-term nutritional stress refers to 0-2 days of nutritional stress; the later stage of long-term nutritional stress refers to 8-10 days of nutritional stress.

[0016] Preferably, the plant mentioned above is Arabidopsis thaliana.

[0017] Thirdly, the present invention provides a regulatory model for observing plant nutrient stress and reactive oxygen species, which is constructed using the above-mentioned method for improving plant resistance to nutrient stress.

[0018] The specific steps are as follows: Four-week-old Arabidopsis thaliana planted in nutrient soil containing 1 / 3 vermiculite were subjected to 0 / 2 / 4 / 6 days of dark treatment, and DAB staining and CAT enzyme activity assays were performed.

[0019] This experiment first explored the relationship between nutrient stress and plant reactive oxygen species (ROS) metabolism, then examined the relationship between ROS and changes in plant autophagy flux, and finally investigated the effects of different endogenous and exogenous ROS on plant nutrient stress.

[0020] Plants accumulate reactive oxygen species (ROS) under nutrient stress, which simultaneously increases autophagy levels. In long-term nutrient stress, ROS exert positive signaling and negative damaging effects before and after stress. Exogenous application of hydrogen peroxide and potassium iodide revealed that in the early stages of nutrient stress response, hydrogen peroxide application increased oxidative signaling levels and enhanced plant resistance; while potassium iodide application in the early stages decreased oxidative signaling levels, causing oxidative damage. Later application of potassium iodide reduced oxidative damage caused by stress and increased plant resistance. We also used the autophagy mutants atg1abc and atg13ab as controls without autophagy. Endogenous ROS were detected using the high-hydrogen-peroxide mutant cat2 and the low-hydrogen-peroxide mutant rbohd / f. The cat2 mutant, due to its high hydrogen peroxide level, had a higher survival rate in the early stages, but its survival rate decreased in the later stages due to severe oxidative damage. The robhd / f mutant, with its low ROS content, had a lower survival rate in the early stages because it did not accumulate sufficient stress response substances in the early stages, resulting in poor survival in the later stages.

[0021] This indicates that we have preliminarily identified the mechanism by which reactive oxygen species (ROS) regulate autophagy in plants, and therefore we designed a scheme to apply it. We used hydrogen peroxide in the early stage and potassium iodide in the later stage. Through this simple in vitro, macroscopic treatment, we effectively reduced plant damage under nutrient stress. We also subjected the cat2 and rbohd / f mutants to oxidation or deoxidation treatments, and the results showed that they were consistent with our hypothesized pattern of ROS regulating autophagy. This indicates that our recommended method is effective for plants of different genotypes.

[0022] There is an interaction between reactive oxygen species and plant autophagy, and related findings have been reported in various crops (corn, soybean, apple, tomato, barley, rice, etc.). Considering that autophagy is highly conserved in eukaryotes, it can be inferred that this method can be used to increase yields in a variety of crops.

[0023] In summary, through molecular biology and botanical methods, we proposed a scientific law governing ROS-mediated autophagy in plants, and improved this law into a visualized regulatory model, obtaining a solution to effectively improve the growth and survival rate of plants (Arabidopsis thaliana) under nutritional stress. This also serves as a good example of translating basic research into applied research.

[0024] The present invention has the following beneficial effects:

[0025] This invention regulates plant activity through the in vitro application of hydrogen peroxide or potassium iodide. The required equipment is simple, requiring only a small spray bottle. It boasts high biosafety, as the application of common chemicals eliminates the need for genetic modification. Operation is straightforward, requiring only 2-3 ml of the chemical per seedling; large-scale production can be achieved using traditional agricultural tools such as pesticide sprayers, allowing for macroscopic regulation of microscopic plant expression. It exhibits strong applicability, as the autophagy process in plants is highly conserved, enabling its application to crops. The preliminary mechanisms of action of the chemicals (hydrogen peroxide and potassium iodide) are clearly defined, facilitating further improvements. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 The results of the analysis of plant growth status and reactive oxygen species levels in wild-type (WT) and endogenous reactive oxygen species high-content mutant (cat2) under nutrient stress are as follows: A shows the phenotypes of WT and cat2 under carbon deficiency stress for 0, 2, 4, and 6 days; B shows the DAB staining results of seedlings of WT and cat2 under carbon deficiency treatment for 0, 2, 4, and 6 days; C shows the DAB staining results of mature leaves of WT and cat2 under carbon deficiency treatment for 0, 2, 4, and 6 days; and D shows the catalase activity detection results of WT and cat2 under carbon deficiency treatment for 0, 2, 4, and 6 days.

[0028] Figure 2 Figure 1 shows the in vitro oxidation treatment analysis of wild-type autophagy-deficient mutants (atg1abc / atg13ab). In the figure, A represents the phenotype of WT, atg1abc, and atg13ab after applying hydrogen peroxide or potassium iodide under carbon deficiency stress; B represents the chlorophyll content of WT after applying hydrogen peroxide or potassium iodide under carbon deficiency stress; C represents the chlorophyll content of atg1abc mutant after applying hydrogen peroxide or potassium iodide under carbon deficiency stress; and D represents the chlorophyll content of atg13ab mutant after applying hydrogen peroxide or potassium iodide under carbon deficiency stress.

[0029] Figure 3 The graph shows the autophagy flux detection in wild-type WT. In the graph, A is a fluorescence image of autophagosomes in the roots of WT after applying hydrogen peroxide or potassium iodide under carbon deficiency stress. B is the statistical analysis of the number of autophagosomes in A. C is the Free-GFP detection of WT after applying hydrogen peroxide or potassium iodide under carbon deficiency stress.

[0030] Figure 4 This is a regulatory model of reactive oxygen species (ROS) and plant autophagy, and the application effect of the model's rules; where A is a schematic diagram of the changes in ROS and catalase activity in plants under continuous nutritional stress, and B is a diagram of the effect of using exogenous hydrogen peroxide or potassium iodide to regulate plant adaptation to long-term nutritional stress.

[0031] Figure 5 The effects of different oxidation levels on plant stress resistance after applying hydrogen peroxide and potassium iodide at different stages were investigated. A represents a phased diagram of nutrient stress before and after application; B represents the phenotypes of WT, cat2, and rbohd / f at the early stage of nutrient stress; C represents the phenotypes of WT, cat2, and rbohd / f at the late stage of nutrient stress; D represents the phenotypes of WT, cat2, and rbohd / f with hydrogen peroxide added at the early stage of nutrient stress; E represents the phenotypes of WT, cat2, and rbohd / f with potassium iodide added at the early stage of nutrient stress; F represents the phenotypes of WT, cat2, and rbohd / f with hydrogen peroxide added at the late stage of nutrient stress; and G represents the phenotypes of WT, cat2, and rbohd / f with potassium iodide added at the late stage of nutrient stress.

[0032] Figure 6 The following experiments demonstrate the interaction between plant CAT2 protein and ATG1a and ATG13a: A shows bimolecular fluorescence complementarity demonstrating pairwise interactions among CAT2, ATG1, and ATG13a; B shows yeast two-hybrid experiments demonstrating pairwise interactions among CAT2, ATG1, and ATG13a; C shows in vitro pull-down experiments demonstrating His-CAT2 and GST-ATG1a; D shows in vitro pull-down experiments demonstrating GST-ATG1a and MBP-ATG13a; and E shows in vitro pull-down experiments demonstrating GST-CAT2 and MBP-ATG13a. Detailed Implementation

[0033] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0034] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0035] Example 1: Phenotypic analysis of wild-type (WT) and cat2 homozygous mutants under nutritional stress

[0036] This invention first used a nutrient stress gradient treatment to observe the phenotype of wild-type and cat2 mutant Arabidopsis thaliana materials, and then measured hydrogen peroxide levels and catalase activity. The method is as follows:

[0037] (1) Phenotypic experiment of WT, cat2 plants

[0038] First, surface disinfection of WT and cat2 Arabidopsis seeds was performed using 75% ethanol for 5-10 minutes, followed by rinsing 8 times with sterile ultrapure water. After washing, the seeds were placed in a 4 ℃ refrigerator for sterile vernalization for 2-3 days, and then grown on 1 / 2 MS medium (an improved version of the MS medium invented by Murashige and Skoog) for 7 days. After 7 days, the mature Arabidopsis seedlings were carefully transferred with tweezers to nutrient soil containing 1 / 3 vermiculite, ensuring the roots and hypocotyls were buried below the soil layer while the cotyledons remained exposed. After watering, the seedlings were placed in a greenhouse (22 ℃; 16 hours of light at 10000 Lux; 8 hours of darkness) for 2 weeks. The prepared experimental materials were placed in a dark incubator every two days, repeated 3 times. On the sixth day, experimental materials with 0, 2, 4, and 6 levels of dark treatment were obtained, and photographs, diaminobenzidine (DAB) staining, and catalase activity detection were performed.

[0039] 1 / 2MS medium formulation: MS salt (final concentration 0.221 g / 100 ml), sucrose (final concentration 1 g / ml), agar (final concentration 1 g / ml), pH adjusted to 5.8 using potassium hydroxide.

[0040] DAB staining method: Dissolve DAB powder in PBS to a final concentration of 1 mg / mL. Place WT and cat2 seedlings, after being treated in the dark, into 2 ml centrifuge tubes, add 1 ml of DAB staining solution, and stain under light at room temperature for 40 min. Destain with 75% ethanol, and then photograph using a stereomicroscope.

[0041] Catalase activity assay: Take 20 WT and 20 cat2 seedlings each and place them in 1.5 ml centrifuge tubes. After quick freezing in liquid nitrogen, grind the sample with a grinder until it becomes a uniform white powder. Add 100 μl of CAT extraction buffer, incubate on ice for 10 min, and centrifuge using a refrigerated centrifuge (12000 rpm, 4 ℃, 15 min). After centrifugation, discard the supernatant as the extracted total CAT protein. Determine the protein concentration using a NanoDrop One micro-spectrophotometer (wavelength 280 nm) and record the results. Take a clean 1.5 ml centrifuge tube, add 10 μl of sample protein, then add 500 μl of 10 mM hydrogen peroxide dissolved in pH phosphate buffer, react at room temperature for 10 min, and use 10 μl of CAT protein extraction buffer as a blank control. Add 50 μl of the reaction solution to 150 μl of the colorimetric solution, then add 1 μl of peroxidase (POD) to the microplate wells and react in the dark for 10 min. After the reaction, detect the reaction using a microplate reader (wavelength 590 nm). Statistical analysis was performed. A standard curve was constructed by reacting multiple sets of hydrogen peroxide solutions of known concentrations with the reaction solution. The amount of hydrogen peroxide consumed in the sample was analyzed based on the standard curve, and the total catalase activity rate of the sample was obtained by dividing the result by the reaction time and the initial OD value.

[0042] The extraction buffer formulation is as follows: 20 mM Tris-HCl pH 7.4; 10% glycerol.

[0043] 0.1 M pH 5.6 phosphate buffer formulation: Dissolve 0.454 g disodium hydrogen phosphate and 4.42 g sodium dihydrogen phosphate in pure water and bring the volume to 200 ml.

[0044] Colorimetric solution formulation: 3.3 mM 3-methyl-2-benzothiazolinone hydrazone phosphate compound (MBTH) and 0.01 mM m-dimethylaminobenzoic acid (DABA) dissolved in 0.1 M pH 5.6 phosphate buffer.

[0045] POD formulation: 0.3 U / μL peroxidase (POD) dissolved in water.

[0046] Figure 1 In the A group, WT / cat2 plants were treated with nutrient stress for 0 / 2 / 4 / 6 days, respectively. The growth of WT plants gradually deteriorated under progressively increasing nutrient stress. The mutant cat2 showed a faster increase in reactive oxygen species compared to the wild type, and therefore its growth was slightly better than that of WT. Figure 1 In the B test, the hydrogen peroxide level of whole plant materials under gradient nutrient stress was detected. As the nutrient stress intensified, both wild-type and mutant showed a gradual increase in reactive oxygen species (ROS). Among them, the cat2 mutant had a higher ROS level and faster ROS accumulation compared to the wild-type. Figure 1 C represents the determination of reactive oxygen species (ROS) content in rosette leaves of plants under gradient nutrient stress, and the results are consistent with... Figure 1 Similar to B in the middle. Figure 1 The study measured catalase activity in plants under nutrient stress. In the wild-type plant, catalase activity showed a gradual increase, indicating that plants progressively enhance hydrogen peroxide scavenging efficiency as nutrient stress intensifies. The experiment demonstrates that nutrient stress causes oxidative damage alongside plant injury. Furthermore, during nutrient stress, plants regulate catalase activity to maintain ROS levels at a certain level.

[0047] Example 2: Oxidation treatment enhances the plant's resistance to nutrient stress

[0048] Following the method described in Example 1, Arabidopsis thaliana seedlings (WT, atg1abc, and atg13ab) were obtained after seven days of growth. Under aseptic conditions, the seedlings were carefully transferred to culture media containing sucrose (C-), sucrose (containing 0.1 mM hydrogen peroxide), and sucrose (containing 0.5 mM potassium iodide), respectively. Following the culture conditions in Example 1, the seedlings were first cultured under normal light for 1 day to allow them to recover. After recovery, they were wrapped in black plastic bags and placed in a dark incubator for 7 days. After the dark treatment, the seedlings were placed in a 3000 Lux light incubator for 7 days to allow them to slowly recover, during which time photographs were taken and chlorophyll content was recorded.

[0049] Sugar-free (C-) formulation: MS salt (final concentration 0.221 g / 100 ml), agar (final concentration 1 g / ml), pH adjusted to 5.8 with potassium hydroxide.

[0050] Chlorophyll content determination: Weigh the sample using a 0.01% balance and place it in a 1.5 ml centrifuge tube. Add 500 μl of 95% ethanol and incubate overnight at 4°C in the dark. Use an empty 95% ethanol tube as a blank control. After centrifugation at 12000 rpm, take 200 μl of the supernatant and measure the absorption peaks of OD663 and OD645 using a microplate reader.

[0051] Chlorophyll a = 12.72 * OD663 - 2.59 * OD645;

[0052] Chlorophyll b = 22.88 * OD645 - 4.67 * OD663;

[0053] Total chlorophyll = chlorophyll a + chlorophyll b;

[0054] Sample chlorophyll content = total chlorophyll content / sample mass.

[0055] like Figure 2The results showed that the sugar-free and dark treatments effectively inhibited plant growth. Under these conditions, the addition of hydrogen peroxide increased the chlorophyll content, while the addition of potassium iodide decreased the chlorophyll content. In vitro oxidation treatment analysis was performed on wild-type autophagy-deficient mutants (atg1abc / atg13ab). Figure 2 Figure A shows the results after treating three materials with nutrient stress, nutrient stress + 0.01-1 mM hydrogen peroxide, and nutrient stress + 0.01-2 mM potassium iodide. Applying hydrogen peroxide to the WT plant effectively improved plant growth and reduced chlorosis, while applying potassium iodide worsened plant growth and increased chlorosis. No significant difference was observed in the treatment of the autophagy mutant. Figure 2 B, C, and D represent the chlorophyll content determination results after treatment with the three materials, and the results support... Figure 2 The phenotype of plant A. Experiments show that exogenous application of hydrogen peroxide can indeed increase the autophagy level of plants, thereby improving their survival under nutritional stress and chlorophyll content.

[0056] Example 3: Effects of Endogenous ROS in Plants

[0057] To further explore the underlying principles of this model, the experimenters used WT, cat2, and rbohd / f for verification (e.g., Figure 5 Mature Arabidopsis thaliana plants that have grown normally in the soil for 4-5 weeks were subjected to continuous dark treatment for 0-8 days. During the dark treatment, water, hydrogen peroxide, and potassium iodide were applied simultaneously for observation. Applying the drugs during the first 0-3 days of darkness was considered the early dark treatment, and applying the drugs during the last 5-8 days of darkness was considered the late dark treatment.

[0058] like Figure 5 As shown, in the early stages of nutrient stress, plants spontaneously generate ROS as signaling molecules. The Cat2 mutant exhibits both high ROS levels and the effect of CAT2 protein deficiency, thus showing better growth compared to WT. The rbohd / f mutant has low ROS levels and the effect of RBOHD / F protein deficiency, therefore its growth is weaker than WT. Figure 5 (Medium B). Applying 0.1 mM hydrogen peroxide during this period can effectively compensate for the deficiency of rbohd / f caused by ROS deficiency. Due to the dual effect of cat2 itself, the applied hydrogen peroxide significantly increased the plant's oxidation level, causing oxidative stress to arrive earlier. Therefore, the growth status of cat2 is worse than that of WT. Figure 5 (D). During this period, applying potassium iodide to the three plants produced opposite results to rbohd / f, but cat2 still showed a better growth trend, which may indicate that the loss of CAT2 protein also plays a key role in this process. Figure 5 (E).

[0059] In the later stages of plant growth, the oxidative damage in the cat2 mutant gradually intensifies, resulting in weaker growth compared to the WT mutant. The rbohd / f mutant suffers partial damage due to the lack of oxidative signaling, and its growth eventually approaches that of the WT mutant. This may be because the absence of the rbohd / f protein also plays a role, although this role is currently unclear. Figure 5 (C) Applying hydrogen peroxide to plants at this stage showed significantly increased oxidative damage in the cat2 mutant, resulting in poorer growth compared to WT, while robhd / f was similar to WT. Applying potassium iodide significantly improved the oxidative damage in cat2, leading to better growth in the cat2 mutant compared to WT. For rbohd / f, late-stage potassium iodide application resulted in extremely low ROS signal levels throughout the entire growth period. Even though RBOHD / F had some impact, it could not reverse the overall poorer growth of the rbohd / f mutant.

[0060] Example 4: Oxidation treatment increases autophagy flux at both the subcellular and protein levels in plants.

[0061] Wild-type seedlings were obtained by sowing according to the method in Example 1. After 7 days of growth, some seedlings were transferred to a culture medium containing sucrose, sucrose containing concanavalin A (CA; final concentration 0.1 μM) and cultured under light for 24 h under sterile conditions. Other seedlings were transferred to a culture medium without sucrose, without sucrose containing 0.1 mM hydrogen peroxide, without sucrose containing 0.5 mM potassium iodide, without sucrose containing concanavalin A (CA; final concentration 0.1 μM), without sucrose containing 0.1 mM hydrogen peroxide and concanavalin A (CA; final concentration 0.1 μM), and without sucrose containing 0.5 mM potassium iodide and concanavalin A (CA; final concentration 0.1 μM) and treated in the dark for 24 h. The number of autophagic vesicles in the roots was observed and counted using a laser confocal microscope.

[0062] Immunoprecipitation assay: Samples were placed in clean centrifuge tubes and homogenized using a grinder. 100 μl of IP-Buffer was added, and the mixture was incubated on ice for 15 min, followed by centrifugation at 12000 rpm, 4 °C, for 15 min. The supernatant was collected for SDS-PAGE gel electrophoresis. After electrophoresis, the sample was transferred to a PVDF membrane (100 V, 60 min). The PVDF membrane was washed with PBST, then blocked with 5% skim milk (dissolved in PBST) for 1 hour, followed by three more washes with PBST. The membrane was then incubated with GFP antibody for 6 h. After primary antibody incubation, the sample was washed with PBST, followed by incubation with the corresponding GFP secondary antibody for 1 h, and then washed again with PBST. Finally, the sample was developed using a high-sensitivity luminescent solution and exposed using a gel imaging system.

[0063] The results are as follows Figure 3As shown, autophagy flux was detected in the wild type, with GFP-ATG8e being a biomarker for plant autophagosomes. The number of autophagosomes increased after nutrient stress, and the level of autophagosomes was higher after applying hydrogen peroxide compared to nutrient stress alone. However, the number of autophagosomes decreased after applying potassium iodide compared to nutrient stress alone. Figure 3 Figure B is a... Figure 3 Statistics on the number of autophagic vesicles in Figure A, and Figure 3 The results in Figure A are consistent. Figure 3 Figure C shows the changes in autophagy flux in plants at the protein level. GFP-ATG8e is degraded during autophagy, with the ATG8e portion being rapidly degraded or recycled. The foreign protein GFP degrades slowly and exists in a free state. Therefore, the change in autophagy flux can be translated using the free GFP / GFP-ATG8e ratio. Under nutrient stress, the GFP / GFP-ATG8e ratio in plants significantly increased, continuing to rise after the addition of hydrogen peroxide, and decreasing somewhat after the addition of potassium iodide compared to nutrient stress alone. The results indicate that plants exhibit a nutrient stress-insensitive phenotype after oxidative treatment, which is indeed due to the activation of intracellular autophagy flux. This clarifies the correlation between oxidation and autophagy.

[0064] Example 5: Experiment to verify the feasibility of the method of the present invention

[0065] According to Example 1, changes in hydrogen peroxide content and catalase activity under plant nutrient stress can be effectively observed. Therefore, we propose a patented model of the relationship between plant nutrient stress and reactive oxygen species regulation, such as... Figure 4 As shown in Figure A. Under normal growth conditions, plant reactive oxygen species (ROS) are kept at a low level to participate in normal metabolic activities. In the early stages of stress, ROS levels rise rapidly, enhancing oxidation signals, which helps improve plant resistance. In the middle stages of stress, ROS levels continue to rise, exceeding the highest oxidation signal level. ROS activates signals on one hand and causes oxidative damage on the other. In the later stages of stress, ROS levels are high, at which point only a small portion of ROS acts as an oxidation signal, while most of ROS has caused oxidative damage.

[0066] Figure 4Experiment B is an application experiment: Seven-day-old WT seedlings were obtained using the method in Example 1 and carefully transplanted into nutrient soil (prepared as in Example 1) using tweezers. The experiment began after the plants had grown normally for three weeks. The plant materials were divided into four groups: A, B, C, and D. Group A was kept under normal growth conditions and sprayed with an appropriate amount of pure water as control 1. Group B was kept in darkness and sprayed with an appropriate amount of pure water as control 2. Group C was sprayed with 0.1 mM hydrogen peroxide for 1-2 days in darkness and with pure water for 2-10 days. Group D was sprayed with 0.1 mM hydrogen peroxide for 1-2 days in darkness, with pure water for 2-8 days, and with 0.5 mM potassium iodide for 8-10 days. The number of sprays was twice per day.

[0067] Figure 4 The middle B diagram is based on Figure 4 Applying the principles outlined in Figure A, the nutrient-stressed group showed more severe damage compared to the nutrient-sufficient group. Spraying with reactive oxygen species in the early stages of nutrient stress effectively improved plant growth, while spraying with potassium iodide in the later stages effectively reduced oxidative damage. The fourth group demonstrates that using hydrogen peroxide to enhance signaling in the early stages and potassium iodide to clear damage in the later stages maximized plant survival under nutrient stress.

[0068] Based on the above experimental results, the model and implementation method proposed in this patent application can effectively improve the tolerance of groups CD under nutritional stress, with group D showing the best results.

[0069] Example 6: Verification of the interaction between CAT2 protein and plant autophagy genes

[0070] Download the cDNA sequences of CAT2, ATG1a, and ATG13a from the Tair website (https: / / www.arabidopsis.org / ), and ligate them into the pGADT7 and pGBKT7 vectors using ligases and restriction enzymes. Recombinant cloning was then performed using *E. coli* DH5α. Recombinant plasmids were extracted using a small-volume nucleic acid extraction kit and transformed into AH109 yeast competent cells. Screening was performed using a dual auxotrophic medium lacking tryptophan and leucine. Suitable positive colonies were selected, diluted 1, 10, and 100 times with sterile water, and then dropped onto a multi-auxotrophic medium lacking tryptophan, leucine, histidine, and adenine for observation and photography (28℃, 36 hours). Results showed that only yeast transformed with AD-CAT2 and BD-ATG1a or AD-CAT2 and BD-ATG13a could grow on the quadruple auxotrophic medium, demonstrating that CAT2 and ATG1a and ATG13a can interact within yeast. Figure 6 (B)

[0071] The above genes were constructed into the YNE / YCE vector, recombinantly cloned, and transformed into Agrobacterium GV3101. Positive plaques were screened using solid LB agar plates containing kanamycin and rifampin resistance. A suitable positive plaque was inoculated into liquid LB medium containing kanamycin and rifampin and cultured overnight (28℃, 180 r / min). When the bacterial OD reached 0.7, the cells were collected by centrifugation (3000 rpm, 5 min), resuspended in tobacco buffer, and injected into the abaxial surface of leaves of 3-4 week-old *Nicotiana benthamiana*. The injected tobacco was cultured in the dark for 12 hours, then transferred to a light-controlled culture chamber for 36 hours. The interaction was observed using a Zeiss 980 laser confocal microscope. The results showed that co-injection of CAT2-YNE with ATG1a-YCE and ATG13a-YCE could elicit green light, demonstrating that CAT2 can interact with ATG1a and ATG13a in plants. Figure 6 (A)

[0072] Tobacco Buffer formulation: 100 mM 2-morpholinoethanesulfonic acid (MES); 100 mM magnesium chloride; 200 mM acetylsuccinone (AS).

[0073] The above genes were constructed in pGEX4T and pET28a vectors, recombinantly cloned, and transformed into prokaryotic expression-type Escherichia coli DE3. Protein induction was performed using an appropriate concentration of IPTG, and protein purification was performed using GST-beads and forceps. Elution was performed using different gradients of imidazole, and the eluted imidazole solution was concentrated using a protein concentrator column and the buffer was replaced with PBS.

[0074] The proteins with GST and His tags obtained above were mixed, and a portion of the sample was used as input. The mixture was then incubated on ice for 4-8 hours. GST-beads were added to the reaction system, and after incubation for 2 hours, the mixture was carefully centrifuged to remove the supernatant. The mixture was then washed multiple times with PBS buffer (700g, 2min). Protein loading buffer was added to the precipitate, and SDS-PAGE electrophoresis was performed using the transfer conditions mentioned above. Multiple specific antibodies, including GST, His, and CAT2, were used for detection. The results showed that GST-ATG1a protein could pull down His-CAT2 and HIS-ATG13a in vitro, and GST-CAT2 protein could pull down His-ATG13a protein in vitro, demonstrating direct pairwise interactions among the three proteins. Figure 6 (CE).

[0075] In the later stages of plant growth, oxidative damage in the cat2 mutant gradually worsens, resulting in weaker growth compared to the WT mutant. The rbohd / f mutant, due to the loss of oxidative signaling, suffers partial damage, but its growth eventually approaches that of the WT mutant. This may be because the loss of the rbohd / f protein also plays a role, although this role is currently unclear (Figure C). Applying hydrogen peroxide to plants at this stage significantly exacerbates oxidative damage in the cat2 mutant, leading to poorer growth than the WT mutant, while the robhd / f mutant shows growth similar to the WT mutant. Applying potassium iodide significantly improves the oxidative damage in the cat2 mutant, resulting in better growth than the WT mutant. For the rbohd / f mutant, late-stage potassium iodide application leads to extremely low ROS signal levels throughout the entire growth period. Even though RBOHD / F has some influence, it cannot reverse the overall decline in the growth of the rbohd / f mutant.

[0076] The contrasting phenotypes of CAT2 at different stages suggest that CAT2 protein deficiency is the cause. Therefore, we screened a library using CAT2 protein and successfully identified the plant autophagy protein ATG1a. Considering that ATG1 is controlled by the regulatory subunit ATG13 during the initiation of autophagy in plants, we also verified the interaction between CAT2 and ATG13a proteins. Using bimolecular fluorescence complementation, yeast two-hybrid assays, and pull-down experiments, we successfully demonstrated that CAT2 protein can indeed directly interact with ATG1a and ATG13a proteins. This indicates that CAT2 protein itself plays a crucial regulatory role in plant ROS and nutrient stress regulation models.

[0077] In summary, through various in vitro and in vivo experiments, the researchers elucidated the correlation between reactive oxygen species and autophagy flux in plants using molecular biology, genetic engineering, and other methods. Furthermore, by applying this correlation, they effectively improved the plant's resistance to nutritional stress. Finally, they summarized the core methods and schemes of this application.

[0078] 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 principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of hydrogen peroxide and / or potassium iodide in regulating plant nutrient stress and autophagy.

2. Use of hydrogen peroxide and / or potassium iodide in modulating plant nutrient stress and autophagy according to claim 1, characterized in that: The concentration of hydrogen peroxide is 0.01-1 mM; the concentration of potassium iodide is 0.01-2 mM.

3. Use of hydrogen peroxide and / or potassium iodide in modulating plant nutrient stress and autophagy according to claim 1, characterized in that: The hydrogen peroxide is applied exogenously in the early stages of plant nutrient stress to enhance plant resistance.

4. Use of hydrogen peroxide and / or potassium iodide in modulating plant nutrient stress and autophagy according to claim 1, characterized in that: Potassium iodide, when applied exogenously in the early stages of plant nutrient stress, exacerbates autophagy; when applied exogenously in the later stages of plant nutrient stress, it enhances plant resistance.

5. Use of hydrogen peroxide and / or potassium iodide according to any one of claims 1 to 4 for regulating plant nutrient stress and autophagy, characterized in that: The plant in question is Arabidopsis thaliana.

6. A method for improving plant resistance to nutrient stress, characterized in that, The steps are as follows: apply hydrogen peroxide exogenously in the early stage of long-term nutrient stress, and apply potassium iodide exogenously in the later stage of long-term nutrient stress to reduce the harm of plant nutrient stress.

7. The method for improving plant resistance to nutrient stress according to claim 6, characterized in that: The concentration of hydrogen peroxide used is 0.01-1 mM, 3 ml / plant, once a day; the concentration of potassium iodide used is 0.01-2 mM, 3 ml / plant, once or twice a day.

8. The method for improving plant resistance to nutrient stress according to claim 6 or 7, characterized in that: The initial stage of long-term nutritional stress refers to 0-2 days of nutritional stress; the later stage of long-term nutritional stress refers to 8-10 days of nutritional stress.

9. The method for improving plant resistance to nutrient stress according to any one of claims 6-8, characterized in that: The plant in question is Arabidopsis thaliana.

10. A model for observing the regulation of plant nutrient stress and reactive oxygen species, characterized in that: The regulation model is constructed using the method described in claim 9.

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