A method of modulating the growth and defense balance of a plant

Abstract

The application provides a method for regulating the balance between plant growth and defense, which comprises the following steps: contacting an exogenous substance capable of being recognized by a plant and triggering an extracellular ROS burst with the plant, triggering the ROS burst and its signal transduction outside the plant, and then inducing the transcription reprogramming process of the plant, so that the plant produces systemic acquired adaptation and systemic acquired resistance, thereby coordinating the balance between plant growth and defense, promoting the growth, development and reproduction of the plant, and having a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of plant growth and development regulation technology, specifically, it relates to a method for regulating the balance between plant growth and defense. Background Technology

[0002] During their growth and development, plants often encounter various adverse environmental stresses, including abiotic stresses (such as changes in light intensity, extreme temperatures, water deficit, waterlogging, salinity, and physical damage) and biotic stresses caused by pathogens such as bacteria, fungi, and viruses. To cope with these changing environmental stresses, plants have evolved a series of stress response programs over a long period, prompting them to prioritize the allocation of their limited energy and carbon resources to defense mechanisms rather than growth (He et al., 2022). While beneficial for plant survival, active growth suppression is undesirable for plant production. Recent research has revealed the mutual regulation between defense and growth signals, often referred to as the growth-defense tradeoff. This allows plants to reprogram growth and defense signals at multiple levels, thereby reducing their sensitivity to environmental stresses and coordinating the allocation of energy supply between growth and defense (Zhang et al., 2020). Therefore, improving plant stress resistance while avoiding excessive defensive responses is of great significance for plant production.

[0003] Moderate environmental stress can improve plant growth. Plants can overcome the inherent conflict between growth and fluctuating environmental stress through so-called "systemically acquired adaptation" and "systemically acquired resistance" strategies (Hussain et al., 2023; Mittler et al., 2015). Increasing research demonstrates that using stress training methods, allowing plants to undergo moderate initial stress treatment, results in stronger resistance / tolerance to subsequent stress, i.e., the development of "stress memory" (Wang et al., 2021), which is an effective method. Treatment with plant growth regulators is also a conventional and effective means of improving plant stress resistance (e.g., CN201610486694.3, CN201611066258.7). Recent advancements in biotechnology have shown that manipulating the expression of key genes can also enhance plant stress resistance (e.g., CN201510364802.5, CN201811391287.X). However, in plant production, especially crop production, it is crucial to simultaneously enhance the plant's ability to adapt to multiple environmental stresses, but no relevant methods have been reported.

[0004] Studies have shown that plants trigger similar early signaling responses when subjected to abiotic stress. Among these signals, the burst of reactive oxygen species (ROS) from the apoplast is considered a crucial event, alerting plants to stress and altering their "normal" growth state to a "stressed" state (Mittler et al., 2022). ROS helps plants establish defense mechanisms and restore growth capacity by mediating stress perception, integrating environmental signals, and activating stress response pathways. It is evident that ROS, as signaling molecules, plays a vital role in plant responses to abiotic and biotic stresses. Therefore, artificially manipulating ROS signaling may be an important pathway to enhance plant stress resistance and regulate the balance between plant growth and defense. Summary of the Invention

[0005] The purpose of this invention is to provide a method for regulating the balance between plant growth and defense. This method induces the plant to actively adopt systemic adaptation and systemic resistance response by triggering the ROS burst in the extracellular space and the resulting ROS signal transduction process, thereby regulating the balance between plant growth and defense.

[0006] To achieve the objectives of this invention, in a first aspect, this invention provides a method for regulating the balance between plant growth and defense. This method involves contacting exogenous substances that can be recognized by plants and trigger an extracellular ROS burst with the plant. By triggering the extracellular ROS burst and its signal transduction, the method induces the transcriptional reprogramming process in the plant, enabling the plant to develop system-acquired adaptation and system-acquired resistance. This coordinates the balance between plant growth and defense, and promotes plant growth, development, and reproduction.

[0007] In this invention, the exogenous substance is a biologically derived macromolecule or an artificially synthesized macromolecule, and the surface of the macromolecule is modified with carboxyl groups.

[0008] Furthermore, the biologically derived macromolecules can be selected from long-chain fatty acids, oligosaccharides, or small peptides, such as hydroxydecanoic acid and Flagelin 22; the artificially synthesized macromolecules can be nanoscale carbon or silicon materials with good biocompatibility and non-toxicity, such as graphene oxide and carboxyl-enriched carbon nanoparticles.

[0009] In this invention, the defense includes defensive responses to biological and abiotic stresses.

[0010] Furthermore, the exogenous substance is added to the plant cultivation substrate or soil, or sprayed onto the surface of plant leaves, or subjected to seed soaking treatment to induce an extracellular ROS burst in the plant.

[0011] The extracellular ROS burst signal is rapidly transmitted to nearby extracellular and intracellular spaces, causing a systemic response in other parts of the plant, including the entire plant. This systemic response includes early ROS signal transduction and rapid callus deposition, as well as slower regulation of excessive defense responses.

[0012] Secondly, the present invention provides the application of the method in improving plant yield and stress resistance.

[0013] Thirdly, the present invention provides the application of the exogenous substance that can be recognized by plants and trigger an extracellular ROS burst in the preparation of plant fertilizer.

[0014] Fourthly, the present invention provides the application of the exogenous substance that can be recognized by plants and trigger an extracellular ROS burst in the preparation of plant growth promoters.

[0015] The plants described in this invention include, but are not limited to, Arabidopsis thaliana, rice, wheat, and corn.

[0016] This invention is the first to discover that by acting on plants with exogenous macromolecules containing free carboxyl groups, a ROS burst in plants can be triggered, thereby inducing systemically acquired adaptation and systemically acquired resistance in plants, thus coordinating the balance between plant growth and defense, improving plant dry matter production, yield and stress resistance, and has broad application prospects. Attached Figure Description

[0017] Figure 1 This is a schematic diagram illustrating the technical principle of the method for regulating plant growth and defense balance in this invention.

[0018] Figure 2 This is a schematic diagram illustrating ROS triggered by different substances in a preferred embodiment of the present invention.

[0019] Figure 3 This is a schematic diagram of ROS burst triggered by carboxyl-enriched nanocarbon dots in a preferred embodiment of the present invention.

[0020] Figure 4 This is a schematic diagram illustrating the binding of carboxyl-enriched carbon nanoparticles to the cell membrane in a preferred embodiment of the present invention.

[0021] Figure 5 In a preferred embodiment of the present invention, carboxyl-enriched carbon nanodots regulate the early defense response and late regulatory response of plants.

[0022] Figure 6 In a preferred embodiment of the present invention, carboxyl-enriched nanocarbon dots promoted dry matter production and seed yield in Arabidopsis thaliana under suitable conditions.

[0023] Figure 7In a preferred embodiment of the present invention, carboxyl-enriched nanocarbon dots promoted dry matter production and seed yield in Arabidopsis thaliana under high-temperature conditions.

[0024] Figure 8 In a preferred embodiment of the present invention, carboxyl-enriched nanocarbon dots promote dry matter production and grain yield in rice.

[0025] Figure 9 The synthesis and structural characterization of carboxyl-enriched carbon nanodots CNCD, CD200, and CD500 are presented in the preferred embodiments of the present invention.

[0026] In the picture, , This indicates that the differences between the different treatment groups are statistically significant. This indicates that P < 0.05. This indicates that P < 0.01. Detailed Implementation

[0027] This invention provides a method for regulating the balance between plant growth and defense.

[0028] The present invention adopts the following technical solution:

[0029] This invention provides a method for triggering ROS signaling using exogenous macromolecules, activating plant stress response pathways, and coordinating the balance between plant growth and defense. For example... Figure 1 As shown, exogenous macromolecules, when added to plant cultivation substrates or soil, or sprayed onto leaves, roots, or leaf cell membrane receptors, recognize the exogenous macromolecules, triggering the ROS burst process. The generated ROS signals, through early signal transduction, reprogram the plant's transcriptome, thereby inducing a rapid defense response and subsequent regulation of excessive defense, ultimately achieving a balance between plant growth and defense, and thus promoting plant growth, development, and reproduction.

[0030] Preferably, the exogenous macromolecular substance is a carbon nanoparticle with a large number of carboxyl groups on its surface. The average diameter of the carboxyl-enriched carbon nanoparticle is 2-7 nm, and its pH value ranges from 2.1 to 3.0 due to the large number of carboxyl groups it contains.

[0031] Preferably, the exogenous macromolecule has cell membrane binding activity, is recognized by various receptor kinases, and thus triggers ROS signaling, wherein the carboxyl group is the main structure recognized by receptor kinases.

[0032] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0033] Example 1: Macromolecules containing carboxyl groups trigger an extracellular ROS burst in plants.

[0034] Macromolecules containing carboxyl groups, including long-chain fatty acids (hydroxydecanoic acid, CAS No. 5393-81-7, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.), peptides (Flagelin 22, CAS No. 304642-91-9, purchased from Shanghai Maclean Biochemical Technology Co., Ltd.), graphene oxide (GO, catalog number G405797, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.), and carboxyl-enriched carbon nanoparticles (CNCD, purchased from Beijing Xinna International New Materials Co., Ltd.), were prepared into stock solutions with a concentration of 1.5 g / L. Second-round flushes of Arabidopsis thaliana true leaves, aged 20 days, were immersed in a solution containing the reactive oxygen species fluorescent dye H2DCFDA (5 μM). After vacuuming for 15 minutes, the leaves were removed, rinsed with deionized water, and placed on glass slides. 20 μL of hydroxydecanoic acid, Flagelin 22, GO, and CNCD diluted 1000 times were added to each slide, and the fluorescence intensity of the reactive oxygen species was observed under a laser confocal microscope. Figure 2 As shown, significant DCF fluorescence enhancement was observed 15-20 minutes after the introduction of exogenous macromolecules, with the strongest fluorescence observed in CNCD treatment with high carboxyl content. Furthermore, CNCD was treated at 200℃ to remove some carboxyl groups (CD200) and at an even higher temperature (500℃) to remove the vast majority of carboxyl groups (CD500). The resulting CD200-triggered ROS intensity was significantly weakened. Figure 2 The intensity of ROS triggered by CD500 was further weakened. Simultaneously, treatment with the same concentration of a small-molecule organic acid containing carboxyl groups (malic acid, CAS number 636-61-3, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) also failed to trigger ROS production in plants. These results indicate that exogenous substances capable of triggering plant ROS bursts are characterized by a large core and carboxyl groups on the surface. Carboxyl groups are key sites for plant recognition and initiation of ROS signals, and the higher the proportion of carboxyl groups, the higher the efficiency of ROS signal initiation.

[0035] To better illustrate the present invention, the following embodiments all use CNCD, the exogenous material with the best ROS triggering efficiency in Example 1, to illustrate the implementation scheme of the present invention.

[0036] The preparation methods of carboxyl-enriched carbon nanodots CNCD, CD200 and CD500 are described in Example 10.

[0037] Example 2: CNCD triggered an extracellular ROS burst and regulated subsequent intracellular ROS homeostasis.

[0038] To verify that CNCD can regulate plant ROS signaling, Arabidopsis thaliana leaves were used as material. Using luminol chemiluminescence, a clear ROS burst followed by elimination was observed in the CNCD-treated Arabidopsis thaliana leaves. Figure 3 (a) Further, using Arabidopsis thaliana leaves and root tips of Arabidopsis thaliana, rice, wheat, and maize as materials, they were immersed in deionized water containing the ROS-specific fluorescent dye H2DCFDA. After vacuum permeation for 15 minutes, CNCD was added to the plant materials (1.5 μg / mL), and the fluorescence intensity of ROS was observed under a laser confocal microscope. Figure 3 b and Figure 3 As shown in c, the treated blade ( Figure 3 (b) and root tip ( Figure 3 In step c), a fluorescence burst was observed. This indicates that CNCD, upon contact with the plant, can rapidly trigger an extracellular ROS burst. Subsequently, we performed DAB staining on Arabidopsis leaves 1-4 days after CNCD application (…). Figure 3 The content of d) and H2O2 ( Figure 3 The measurements in section e) revealed that the intracellular H2O2 level also underwent a process of initial increase followed by elimination. These results indicate that CNCD treatment can regulate the homeostasis of ROS in plant leaves, and this homeostasis regulation process occurs at the minute and day levels.

[0039] Example 3: CNCD binds to the extracellular space of the cell membrane and can be recognized by receptor-like kinases.

[0040] To verify that the mechanism by which CNCD triggers ROS burst is similar to that of ROS burst triggered by plant immunity, Arabidopsis leaves and protoplasts were used as materials. FITC-labeled CNCD (1.5 μg / mL) was added to the protoplasts and leaves. The leaves were vacuum-treated for 15 minutes, and the binding site of CNCD was observed under a laser confocal microscope to be outside the cell membrane. Figure 4 a and Figure 4 (b) indicates that CNCD may be recognized by receptor-like kinases / proteins on the plasma membrane surface, thereby binding outside the plasma membrane. Transcriptome data after 3 hours of treatment showed that most of the 161 receptor-like kinases and 44 receptor-like proteins on the plasma membrane surface of root and leaf tissues were upregulated, especially in root tissues where the upregulation was stronger. Figure 4 c and Figure 4 (d in the text). These results indicate that CNCD acts as an exogenous substance in plant cells, triggering ROS bursts through receptor recognition.

[0041] Example 4: CNCD induced early signal transduction events similar to those experienced during abiotic stress treatment.

[0042] Transcriptome sequencing was used to compare gene expression characteristics of Arabidopsis roots 3 and 6 hours after CNCD treatment. Differences were found in the expression of key genes involved in seven early stress response signal transduction events between roots and leaves. These included: key ROS-producing enzymes RBOHs; PIP1, an aquaporin that receives ROS from apoplasts into the cytoplasm; 4 / PIP2.1; and ROS-dependent activation of Ca2+. 2+ Calcium channels flowing from the apoplast and vacuoles into the cytoplasm via glutamate receptors GLR3.3 / GLR3.6; and Ca 2+ Coupled mitogen-activated protein kinase modules; H+ coordinating the accumulation of apoplast ROS + -ATPase; regulates the deposition of callose on plasmodesmata to control PDLP5 / PDLP8 and chloroplast retrograde signaling genes EX1 / EX2 in symplastic signaling. The above experimental results indicate that CNCD triggers ROS to transmit stress signals both short distances from extracellular to intracellular and long distances from roots to leaves.

[0043] Example 5: CNCD-triggered ROS signaling simultaneously modulates the early and late responses of plants to stress.

[0044] Transcriptome sequencing was used to compare gene expression characteristics of the aboveground parts of Arabidopsis thaliana roots after short-term (3 hours and 6 hours) and long-term (15 days and 30 days) treatment with CNCD. The results showed that the number of differentially expressed genes increased rapidly in the short-term treatment period (from 3 hours to 6 hours), from 496 to 5365, while the number of differentially expressed genes decreased sharply in the long-term treatment period (15 days and 30 days), to 216 and 203 respectively. Figure 5 (a) Furthermore, during short-term treatment, the proportions of upregulated and downregulated genes were relatively consistent, while after long-term treatment, the differentially expressed genes were almost all upregulated. More importantly, over 50% of these upregulated genes were transcription factors. GO analysis of differentially expressed genes showed that during short-term treatment, more genes were involved in responses to various biotic and abiotic stresses, as well as genes related to energy metabolism. Figure 5 (b) indicates that CNCD induced multiple stress responses in plants; however, after prolonged treatment, especially after 15 days, a significant portion of the genes were negatively regulated in response to plant abiotic stress. Figure 5 c) indicates that after prolonged CNCD treatment, plants begin to actively adjust their previously excessive defensive responses, allocating more energy for growth and development. This demonstrates a regulatory process that coordinates plant growth and defense. These results suggest that after CNCD treatment, the plant response evolves from a stress response to an active regulation of these stress responses.

[0045] Example 6: CNCD treatment promoted dry matter accumulation and seed yield in Arabidopsis thaliana.

[0046] To verify that CNCD-triggered ROS can coordinate plant growth and defense, we analyzed the dry matter accumulation and seed yield of Arabidopsis thaliana under two conditions: culture medium and soil pot cultivation. We found that adding 1.5 mg / L CNCD to MS medium... Figure 6 (a) or, when transplanting seedlings into pots after 20 days, water with water containing 1.5 mg / L CNCD. Figure 6 (b) in the above formulas can significantly increase the amount of dry matter accumulation. Figure 6 (c in 6 and d in 6), and the seed yield was significantly increased in all three independent trials ( Figure 6 (d)

[0047] Example 7: CNCD treatment improved dry matter accumulation and seed yield in Arabidopsis thaliana under heat stress.

[0048] To verify that CNCD-triggered ROS can coordinate plant growth and defense, we further analyzed the dry matter accumulation and seed yield of Arabidopsis thaliana under pot cultivation conditions under heat stress. After transplanting 20-day-old seedlings into soil, they were allowed to recover at 24℃ for 10 days before being moved to a 28℃ incubation room. Water containing 1.5 mg / L CNCD was applied twice, once every 7 days; at other times, only water was applied. Figure 7 Dry matter and seed yield were measured at maturity. It was found that under heat stress, the dry matter and seed yield of Arabidopsis treated with nano-carbon dots increased by 44.2% and 51.6%, respectively, compared to the control. This proportion was significantly higher than the results under suitable temperature conditions. Figure 6 (d)

[0049] Example 8: CNCD treatment improved dry matter accumulation and yield in maize.

[0050] Maize seeds (variety Zhengdan 958) were soaked in an aqueous solution containing 1.5 mg / L CNCD for 6 hours to induce seed inoculation, and then air-dried before sowing. Both a 1-year pot experiment and a 2-year field trial showed that CNCD soaking treatment increased the root-to-shoot ratio (11.31%-33.54%), increased dry matter accumulation at flowering and maturity / harvest stages by 10.18%-14.55%, and increased grain yield per plant by 7.52%-10.26% (Table 1).

[0051] Table 1. Effects of CNCD soaking on corn dry matter production and yield.

[0052]

[0053] Example 9: CNCD treatment improved dry matter accumulation and yield in rice.

[0054] Under potted conditions (volume 120cm×60cm×60cm), 30mg of CNCD was added to each pot of rice (variety Yongyou 4949) during topdressing after transplanting and seedling establishment. Other water and fertilizer management measures were carried out as normal in the experiment. For three consecutive years, the experiment showed that CNCD treatment significantly increased the dry matter accumulation and yield of rice. Dry matter increased by 12.51%-27.66% at the tillering stage, 8.38%-10.71% at the heading stage, and 8.57%-10.45% at the maturity stage; single-plant yield increased by 12.0%-19.09%. Figure 8 ).

[0055] Example 10: CNCD treatment improved the low-temperature germination tolerance and yield of wheat.

[0056] CNCD was added to a conventional wheat seed coating agent (purchased from Bayer Crop Science (China) Co., Ltd.) to achieve a final concentration of 70 μg / mL. Then, 10 mL of the seed coating agent was added per kilogram of wheat seeds, and the mixture was stirred thoroughly to coat the seeds. The coated seeds were then air-dried for later use. Germination tests were conducted in an incubator with four temperature treatments: 0, 5, 10, and 20°C. Additionally, in soil pots, the temperature was controlled at 10°C during the day and 5°C at night. After four weeks, the dry matter content of seedlings and roots was measured. The coating agent treatment significantly improved the low-temperature germination ability of wheat, and the dry matter accumulation at low temperatures also increased significantly (Table 2). In 2020, in Xinxiang, Henan Province, a very late sowing experiment was conducted on treated wheat seeds (Zhongmai 66) (November 20th, compared to the conventional early October). The yield of the plot increased by 11.2%, and yield composition analysis showed that the increase in yield mainly came from the increase in the number of grains per ear (Table 3).

[0057] Table 2. Effects of CNCD coating treatment on wheat germination rate and growth under low temperature.

[0058]

[0059] Table 3. Effects of CNCD coating treatment on yield and yield composition of very late-sown wheat.

[0060]

[0061] Example 11 Synthesis and structural characterization of carboxyl-enriched carbon nanodots (CNCD)

[0062] Carboxyl-enriched carbon nanodots (CNCDs) were prepared using an ultra-low voltage electrolysis method. Using graphite plates as electrodes and deionized water as a raw material, graphite was electrolyzed at a constant current (200 mA) under ambient temperature and pressure. Utilizing a relatively low electrolysis voltage (1-5V), a large number of oxygen-containing groups could be modified onto the surface of the exfoliated graphite carbon cores. Further fractionation to remove large insoluble particles resulted in sol-gel CNCDs with a diameter of 2-7 nm and a pH range of 2.1-2.4. High-resolution TEM confirmed that the carbon nanodots of the CNCDs were sp... 2 The structure, with its π bonds between carbon atoms, endows CNCD with an active ability to gain and lose electrons. FTIR and XPS characterization show that the surface of the CNCD nanocarbon core is modified with a large number of carboxyl, carbonyl, and hydroxyl groups, with a molar ratio of 1:0.5–0.6:0.6–0.7, while the carbon-oxygen atom ratio of the entire material is 1:2.0–2.2. CNCD in sol state was freeze-dried, and the resulting powder was calcined in a muffle furnace at 200°C or 500°C for 30 minutes to obtain CD200 and CD500, respectively. FTIR and XPS characterization also show that the carboxyl and oxygen atom contents of CD200 and CD500 are much lower than those of CNCD. Figure 9 ).

[0063] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

[0064] References:

[0065] 1.He,Z.Webster,S.He,SYGrowth-Defense Trade-Offs inPlants.Curr.Biol.32(2022)634-639.

[0066] 2. Zhang, H. Zhao, Y. Zhu, J.-K. Thriving under Stress: How Plants BalanceGrowth and the Stress Response. Dev. Cell. 55 (5) (2020) 529-543.

[0067] 3.Hussain,M.Shakoor,N.Adeel,M.Ahmad,MAZhou,H.Zhang,Z.Xu,M.Rui,Y.White,JCNano-Enabled Plant Microbiome Engineering for DiseaseResistance.Nano Today.48(2023)101752.

[0068] 4. Mittler, R. Blumwald, E. The Roles of ROS and ABA in Systemic AcquireAcclimation. Plant Cell. 27 (2015) 64-70.

[0069] 5. Wang Xiao, Cai Jian, Zhou Qin, Dai Tingbo, Jiang Dong. Research progress on the physiological mechanism of abiotic stress training improving crop stress tolerance. Chinese Agricultural Science. 54(2021):2287-301.

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Claims

1. A method for regulating the balance between plant growth and defense, characterized in that, Exogenous substances that can be recognized by plants and trigger extracellular ROS bursts are brought into contact with plants. By triggering extracellular ROS bursts and their signal transduction, the transcriptional reprogramming process of plants is induced, enabling plants to develop system-acquired adaptation and system-acquired resistance. This coordinates the balance between plant growth and defense, and promotes plant growth, development and reproduction. The exogenous substance is a synthetic macromolecule, and the surface of the macromolecule is modified with carboxyl groups. The macromolecules modified with carboxyl groups are carboxyl-enriched carbon nanoparticles. These carboxyl-enriched carbon nanoparticles are prepared by ultra-low voltage electrolysis. The preparation method is as follows: using a graphite plate as an electrode, deionized water is used as a raw material at room temperature and pressure, and graphite is electrolyzed at a constant current of 200mA. Using a relatively low electrolysis voltage of 1-5V, a large number of oxygen-containing groups are modified on the surface of the exfoliated graphite carbon core. After further fractionation to remove large insoluble particles, carboxyl-enriched carbon nanoparticles in a sol state are formed, with a diameter of 2-7nm and a pH value range of 2.1-2.

4. The plant is rice, wheat, or corn.

2. The method according to claim 1, characterized in that, The exogenous substance is added to the plant cultivation substrate, sprayed onto the surface of plant leaves, or subjected to seed soaking treatment to induce an extracellular ROS burst in plants.

3. The application of the method described in claim 1 or 2 in improving plant yield and stress resistance.

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