CN117296593A - Method for regulating and controlling plant growth and defense balance - Google Patents

Abstract

The invention provides a method for regulating and controlling plant growth and defense balance, which is characterized in that exogenous substances which can be identified by plants and trigger extracellular ROS burst are contacted with the plants, and the extracellular ROS burst and signal transduction thereof are triggered to induce the transcription reprogramming process of the plants, so that the plants generate system acquired adaptation and system acquired resistance, thereby coordinating the plant growth and defense balance, promoting the growth, development and propagation of the plants, and having wide application prospect.

Description

Method for regulating and controlling plant growth and defense balance

Technical Field

The invention belongs to the technical field of plant growth and development regulation and control, and particularly relates to a method for regulating and controlling plant growth and defense balance.

Background

Plants are often subjected to various adverse environmental stresses during their growth, including abiotic stresses (e.g., light intensity changes, extreme temperatures, water deficiency, 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 develop a series of stress response programs in long-term evolution that prompt plants to assign their limited energy and carbon resources preferentially to defense mechanisms rather than to growth (He et al, 2022). While beneficial to plant survival, positive growth inhibition is not desirable for plant production. Recent studies have revealed a mutual regulation between defense and growth signals, commonly referred to as a growth-defense tradeoff, which enables plants to reprogram growth and defense signals at multiple levels, thereby reducing their sensitivity to environmental stresses and coordinating energy supply distribution between growth and defense (Zhang et al 2020). Therefore, the plant stress resistance is improved, and the excessive defense reaction of the plant is avoided, so that the method has important significance for plant production.

Moderate environmental stress can improve plant growth, plants can overcome the inherent conflict between growing and fluctuating environmental stress by so-called "systemic adaptive" and "systemic acquired resistance" strategies (humiin et al 2023;Mittler et al, 2015). More and more studies have demonstrated that the development of so-called "stress memory" (Wang Xiao, etc., 2021) is an effective method for developing stress, which is a method for developing stress in plants which are subjected to a moderate stress treatment in the early stage and then exhibit a strong resistance to the stress that occurs again. Treatment with plant growth regulators is also a conventional and effective means of improving plant stress tolerance (e.g., CN201610486694.3, CN201611066258.7, etc.). Recent years of biotechnology progress indicate that the effect of improving the stress resistance of plants (such as CN201510364802.5, CN201811391287.X and the like) can be achieved by controlling the expression of key genes. However, in plant, especially crop, production, while improving its adaptability to various environmental stresses is of paramount importance, related methods have not been reported.

Studies have shown that plants trigger similar early signal response events when subjected to stress. Among these signals, reactive Oxygen Species (ROS) bursts from the extracellular body are considered an important signaling event that alerts plants to stress and changes their "normal" growth state to a "stress" state (Mittler et al, 2022). ROS help plants build defense mechanisms and restore growth capacity by mediating stress perception, integrating environmental signals, and activating stress response pathways. ROS are seen to play an important role as signal molecules in plants' responses to abiotic and biotic stresses. Therefore, by manually manipulating ROS signals, it may be an important way to improve plant stress resistance and regulate plant growth and defense balance.

Disclosure of Invention

The invention aims to provide a method for regulating and controlling plant growth and defense balance, which is a method for regulating and controlling plant growth and defense balance by triggering ROS burst outside a plant and ROS signal transduction process brought by the ROS burst, and inducing the plant to actively adapt to system acquired resistance and the system acquired resistance.

In order to achieve the object of the present invention, in a first aspect, the present invention provides a method for regulating the balance of plant growth and defense, comprising contacting a plant with an exogenous substance capable of being recognized by the plant and triggering an extracellular ROS burst, and inducing a transcription reprogramming process of the plant by triggering the extracellular ROS burst and signal transduction thereof, so that the plant generates systemic adaptive and systemic acquired resistance, thereby coordinating the balance of plant growth and defense, and promoting the growth, development and propagation of the plant.

In the invention, the exogenous substance is a macromolecular substance of biological origin or an artificially synthesized macromolecular substance, and the surface of the macromolecular substance is modified with carboxyl groups.

Further, the macromolecular substance of biological origin may be selected from long-chain fatty acids, oligosaccharides or small peptides, etc., such as hydroxy-decanoic acid, flugulin 22; the artificially synthesized macromolecular substance can be a carbon or silicon material with good biocompatibility and no toxicity, such as graphene oxide and carboxyl-rich nano carbon dots.

In the present invention, the defenses include defensive responses to biotic and abiotic stresses.

Further, the exogenous material is added to a plant cultivation substrate or soil, or sprayed on the surface of a plant leaf, or subjected to seed soaking treatment to induce an extracellular ROS burst in the plant.

The extracellular ROS burst signal is rapidly transmitted to nearby extracellular and intracellular components, resulting in a systemic response in other parts of the plant, including the whole plant. The systemic response includes early ROS signaling and rapid callus deposition etc. defensive responses, as well as slower regulation of excessive defensive responses.

In a second aspect, the invention provides the use of the method for increasing plant yield and stress tolerance.

In a third aspect, the present invention provides the use of said exogenous substance capable of being recognized by a plant and triggering an extracellular ROS burst in the preparation of a plant fertilizer.

In a fourth aspect, the present invention provides the use of said exogenous substance capable of being recognized by a plant and triggering an extracellular ROS burst in the preparation of a plant growth-promoting agent.

Plants of the invention include, but are not limited to, arabidopsis, rice, wheat, maize.

The invention discovers that the exogenous macromolecular substance with free carboxyl group acts on plants for the first time, can trigger the ROS burst of the plants, and further induces the system acquired adaptation and the system acquired resistance of the plants, thereby coordinating the growth and the defense balance of the plants, improving the dry matter production, the yield and the stress resistance of the plants, and has wide application prospect.

Drawings

FIG. 1 is a schematic diagram of the technical principle of the method for regulating plant growth and defense balance according to the invention.

FIG. 2 is a schematic diagram of the different species triggering ROS in a preferred embodiment of the present invention.

FIG. 3 is a schematic representation of the triggering of ROS burst by carboxyl-rich nanocarbon dots in a preferred embodiment of the present invention.

FIG. 4 is a schematic diagram showing the binding of the carboxyl-rich nanocarbon dot to the cytoplasmic membrane in accordance with the preferred embodiment of the present invention.

FIG. 5 shows the early defense response and the late regulatory response of plants modulated by the carboxylated nanocarbon dot-rich plants according to the preferred embodiment of the present invention.

FIG. 6 is a graph showing that the carboxylated nanocarbon dot-rich promotes dry matter production and seed yield under suitable conditions in Arabidopsis thaliana, in a preferred embodiment of the present invention.

FIG. 7 is a graph showing that the carboxylated nanocarbon dot-rich promotes the dry matter production and seed yield under high temperature conditions of Arabidopsis thaliana, in a preferred embodiment of the present invention.

FIG. 8 shows that the carboxylated nanocarbon preparation rich in the modified carbon dots promotes the dry matter production and the grain yield of rice in the preferred embodiment of the present invention

FIG. 9 is a schematic representation of the synthesis and structure of the carboxylated nanocarbon dot CNCD, CD200 and CD500 according to the preferred embodiment of the invention.

In the figure, the differences between the different treatment groups are statistically significant, P <0.05, P <0.01.

Detailed Description

The invention provides a method for regulating and controlling plant growth and defense balance.

The invention adopts the following technical scheme:

the invention provides a method for activating plant stress response paths and coordinating plant growth and defense balance by utilizing exogenous macromolecular substances to trigger ROS signals. As shown in figure 1, exogenous macromolecular substances are identified by adding into plant culture medium or soil or spraying onto leaves, root systems or cell membrane receptors of the leaves, so as to trigger the explosion process of ROS, and the generated ROS signals reprogram transcriptome of plants through early signal transduction, thereby inducing rapid defense reaction and subsequent regulation and control of excessive defense of the plants, finally achieving the purpose of balancing plant growth and defense, and further promoting the growth, development and propagation effects of the plants.

Preferably, the exogenous macromolecular substance is a nano carbon dot with a large number of carboxyl groups modified on the surface, the average diameter of the carboxyl-rich nano carbon dot is 2-7nm, and the pH value range is 2.1-3.0 due to the large number of carboxyl groups.

Preferably, the exogenous macromolecular substance has a cytoplasmic membrane binding activity, is recognized by a plurality of receptor-like kinases, and triggers ROS signals, and the carboxyl group is a main structure recognized by the receptor-like kinases.

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available. Example 1 macromolecular substances containing carboxyl groups trigger the burst of ROS outside the plant cell

The carboxyl group-containing macromolecular substance, including long-chain fatty acid (hydroxy-decanoic acid, CAS No. 5393-81-7, purchased from Shanghai Ala Biotechnology Co., ltd.), polypeptide (Flagelin 22, CAS No. 304642-91-9, purchased from Shanghai Milin Biotechnology Co., ltd.), graphene oxide (GO, cat# G405797, purchased from Shanghai Ala Biotechnology Co., ltd.), and carboxylated nanocarbon dot (CNCD, purchased from Beijing Xinna International New Material Co.) were formulated as a mother liquor at a concentration of 1.5G/L, respectively. The second round of arabidopsis thaliana leaves at the age of 20 days are immersed in a solution containing an active oxygen fluorescent dye H2DCFDA (5 mu M), vacuumized for 15 minutes, taken out, rinsed with deionized water, put on a glass slide, respectively dripped with 20 mu L of diluted 1000-fold hydroxy-decanoic acid, flagelin 22, GO and CNCD, and then the fluorescence intensity of active oxygen is observed under a laser confocal microscope. As shown in FIG. 2, a significant fluorescence enhancement of DCF was observed after dropping the exogenous macromolecular substance for 15-20 minutes, wherein the fluorescence of CNCD treatment with high carboxyl content was strongest. At the same time, CNCD was also treated at 200 ℃ to remove part of the carboxyl groups (CD 200) and at higher temperature (500 ℃) to remove the vast majority of the carboxyl groups (CD 500), resulting in a substantial decrease in the ROS intensity triggered by CD200 (fig. 2), while the ROS intensity triggered by CD500 was further decreased. At the same time, treatment with the same concentration of small organic acids containing carboxyl groups (malic acid, CAS No. 636-61-3, available from Shanghai Ala Biotechnology Co., ltd.) also failed to trigger the plants to produce ROS. These results indicate that exogenous substances capable of triggering the burst of ROS in plants are characterized by having larger nuclei with carboxyl groups on the surface, which are key sites for plants to recognize and initiate ROS signaling, and that the higher the ratio of carboxyl groups, the higher the efficiency of initiating ROS signaling.

To better illustrate the invention, the following examples all use the exogenous substance CNCD of example 1, which is the most efficient at triggering ROS, to illustrate embodiments of the invention.

The preparation method of the carboxylated nano carbon dots CNCD, CD200 and CD500 is shown in example 10.

Example 2CNCD triggered an outbreak of ROS outside the plasma membrane and regulated subsequent intracellular ROS homeostasis

To verify that CNCD can regulate plant ROS signaling, first arabidopsis leaves were used as material, and a clear ROS burst to elimination process was observed in arabidopsis leaves after CNCD treatment using luminol chemiluminescence (fig. 3 a). Further, arabidopsis leaves and root tips of Arabidopsis, rice, wheat and corn are taken as materials, soaked in deionized water containing ROS specific fluorescent dye H2DCFDA, vacuumized and permeated for 15 minutes, CNCD is added into plant materials (1.5 ug/mL), and then fluorescence intensity of ROS is observed by a laser confocal microscope. As shown in FIGS. 3b and 3c, both the treated leaf (FIG. 3 b) and root tip (FIG. 3 c) can observe the course of fluorescence bursts. Indicating that the CNCD can trigger the extracellular ROS burst process rapidly after contacting plants. Subsequently we DAB stained the arabidopsis leaves 1-4 days after CNCD spraying (fig. 3 d) and H ₂ O ₂ Is measured to find H in the cell (FIG. 3 e) ₂ O ₂ Is also subjected to a lift-off followed by elimination process. These results demonstrate that after CNCD treatment, ROS homeostasis in plant leaves can be regulated, which undergoes a minute-level and day-level homeostasis regulation process.

Example 3CNCD binding outside the cytoplasmic membrane and is recognized by receptor-like kinases

To verify that the mechanism of CNCD triggering ROS burst has a similar mechanism to that of plant immune triggered ROS burst, using arabidopsis thaliana leaves and protoplasts as materials, fluorescein Isothiocyanate (FITC) labeled CNCD (1.5 μg/mL) was added to the protoplasts and leaves, wherein the leaves were vacuumized for 15 min and the CNCD binding sites were observed under a laser confocal microscope outside the cytoplasmic membrane (fig. 4a and 4 b). Indicating 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 indicated that the 161 receptor-like kinases and 44 receptor-like proteins on the plasma membrane surface of root and leaf tissue, most of the gene expression was up-regulated, especially in root tissues, with more enhanced expression (fig. 4c and 4 d). These results demonstrate that CNCD acts as a foreign substance on plant cells, triggering ROS bursts with a receptor-recognized mechanism.

Example 4CNCD induces early signaling events similar to stress treatment

The transcriptome sequencing method is used for comparing the gene expression characteristics of the arabidopsis root system treated by the CNCD for 3 hours and 6 hours, and the difference between the key gene expression of 7 early signal transduction events of stress response in the root system and the leaf is found. Comprising the following steps: the key enzyme RBOHs for generating ROS; aquaporin PIP1 which accepts apoplast ROS into the cytoplasm; 4/PIP2.1; relying on ROS activation to cause Ca ²⁺ Calcium channel glutamate receptors GLR3.3/GLR3.6 that flow from the apoplast and vacuoles into the cytoplasm; with Ca ²⁺ A coupled mitogen-activated protein kinase module; h to coordinate accumulation of apoplast ROS ⁺ -atpase; regulating the deposition of callose on plasmodesmata to regulate the signal transmission of ROS in the symplast PDLP5/PDLP8 and chloroplast retrograde signal gene EX1/EX2. The experimental results show that the CNCD can transmit stress signals from outside to inside in a short distance through triggering ROS, and can also transmit stress signals from root to leaf in a long distance.

Example 5CNCD triggered ROS signaling simultaneously regulates early and late response processes in plants to stress

As a result of comparing the gene expression characteristics of the aerial parts of Arabidopsis roots treated with CNCD by means of transcriptome sequencing, it was found that the number of differentially expressed genes increased rapidly from 496 to 5365 in a short period of time (from 3 hours to 6 hours) after the treatment, and the number of differentially expressed genes decreased sharply after a long period of time (15 days and 30 days) to 216 and 203 respectively (FIG. 5 a), and that the proportion of genes up-and down-regulated during the short period of treatment was relatively uniform, whereas the differentially expressed genes after the long period of treatment were both substantially up-regulated, more importantly, more than 50% of the genes up-regulated were transcription factors. GO analysis of differentially expressed genes indicated that more genes at short treatment time were involved in responses to various biotic and abiotic stresses and genes associated with energy metabolism (fig. 5 b), indicating that CNCD induced multiple stress-resistant responses in plants; whereas after prolonged treatment, in particular after 15 days of treatment, a considerable part of the genes belongs to negative regulation of the plant stress response (fig. 5 c), indicating that after prolonged treatment CNCD the plant starts actively regulating the early hyper-defensive response to use more energy for growth. Shows a regulatory process that coordinates plant growth and defense. These results indicate that following CNCD treatment, the response of the plants underwent a process from stress responses to actively modulating these.

Example 6CNCD treatment promotes Arabidopsis dry matter accumulation and seed yield

To verify that CNCD triggered ROS can coordinate plant growth and defense, we analyzed the dry matter accumulation and seed yield of arabidopsis in both medium and soil potting, and found that the addition of 1.5mg/L CNCD to MS medium (fig. 6 a) or 1.5mg/L CNCD in water when 20 days seedlings were transplanted into soil for potting (fig. 6 b) could significantly increase the dry matter accumulation when watered (fig. 6c and 6 d), and the seed yield was significantly increased in 3 independent experiments (fig. 6 d).

Example 7CNCD treatment improved dry matter accumulation and seed yield under Arabidopsis heat stress

To verify that CNCD triggered ROS can coordinate plant growth and defense, we further analyzed the dry matter accumulation and seed yield of arabidopsis under heat stress conditions under potted planting conditions. After transplanting the seedlings for 20 days into the soil, the seedlings are recovered to grow for 10 days at 24 ℃, and then the potted plants are transplanted into a culture room at 28 ℃. Water containing 1.5mg/L of CNCD was poured once for 7 days for a total of two times, and only water was poured at other times (FIG. 7). Dry matter and seed yield were measured at maturity. It was found that the dry matter and seed yield of nanocarbon spot treated arabidopsis increased by 44.2% and 51.6% respectively over the control under heat stress. This ratio is much higher than the result of the appropriate temperature conditions (fig. 6 d).

Example 8CNCD treatment improved dry matter accumulation and yield in corn

Seeds of maize (variety Zhengdan 958) were inoculated with an aqueous solution containing 1.5mg/L of CNCD for 6 hours and then naturally dried for sowing. Both the potting test of 1 year and the field test of 2 years found that CNCD seed soaking treatment increased the root cap ratio (11.31% -33.54%), the dry matter accumulation in the flowering and mature harvest period increased by 10.18% -14.55%, and the individual seed yield increased by 7.52% -10.26% (table 1).

TABLE 1 influence of CNCD seed soaking on corn dry matter production and yield

Example 9CNCD treatment improves Rice Dry matter accumulation and yield

Under the condition of potting (volume is 120cm multiplied by 60 cm), 30mg of CNCD is added to each pot when the rice (variety Zhongyou 4949) is applied after transplanting and seedling-recovering, and other water and fertilizer measures are managed according to a normal test. The continuous 3 years experiments show that the CNCD treatment obviously improves the accumulation and yield of rice dry matters, and the dry matters are respectively increased by 12.51% -27.66%,8.38% -10.71% and 8.57% -10.45% in the tillering stage, the scion stage and the maturity stage; the yield of the single plant is increased by 12.0% -19.09% (FIG. 8).

Example 10CNCD treatment improves Low temperature germination resistance and yield of wheat

CNCD was added to a conventional wheat seed coating (available from bayer crop science (china) limited) to a final concentration of 70 μg/mL. Then 10mL of seed coating agent is added into each kilogram of wheat seeds, and the seeds are uniformly stirred for coating. Naturally airing the coated seeds for later use. Performing germination test in an incubator, and setting 4 temperature treatments at 0, 5, 10 and 20 ℃; in addition, under the condition of soil basin planting, the temperature is controlled at 10 ℃ in the daytime and 5 ℃ at night, and the dry matters of seedlings and roots are measured after 4 weeks. It was found that the coating agent treatment significantly improved the low temperature germination capacity of the wheat, and the accumulation of dry matter at low temperature was also significantly increased (table 2). 2020 in Henan New countryside, the treated wheat seeds were subjected to a very late sowing test (11 months 20 days, normally 10 months old) for increasing the cell yield by 11.2%, and the yield composition analysis showed that the improvement in yield was mainly derived from the increase in the spike number (Table 3).

TABLE 2 influence of CNCD coating treatment on wheat Low temperature germination and growth

TABLE 3 influence of CNCD coating treatment on yield and yield composition of very late wheat

EXAMPLE 11 Synthesis and structural characterization of carboxylated carbon nano-dots (CNCD)

The Carboxylated Nano Carbon Dots (CNCD) are prepared by an ultralow-pressure electrolysis method. Graphite plate is used as electrode, deionized water is used as raw material at normal temperature and pressure, and graphite is electrolyzed under constant current (200 mA). By using lower electrolysis voltage (1-5V), a large amount of oxygen-containing groups can be modified on the surface of the stripped graphite carbon core, and large insoluble particles are removed through further classification to form the CNCD in a sol state, wherein the diameter of the CNCD is 2-7nm, and the pH value is 2.1-2.4. High resolution TEM proves that the nano carbon core of CNCD is sp ² The structure, pi bond between carbon atoms endows the structure with active electron-withdrawing capability; FTIR and XPS characterization prove that the surface of the CNCD nano carbon core is modified with a large number of carboxyl, carbonyl and hydroxyl groups, the molar ratio is 1:0.5-0.6:0.6-0.7, and the carbon-oxygen atomic ratio of the whole material is 1:2.0-2.2. Freeze-drying the CNCD in a sol state, and respectively burning the obtained powdery substance at 200 ℃ or 500 ℃ in a muffle furnace for 30 minutes to obtain CD200 and CD500.FTIR and XPS characterization also showed that both CD200 and CD500 had much lower carboxyl and oxygen atom contents than CNCD (fig. 9).

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Reference is made to:

1.He,Z.Webster,S.He,S.Y.Growth-Defense Trade-Offs in Plants.Curr.Biol.32(2022)634–639.

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

3.Hussain,M.Shakoor,N.Adeel,M.Ahmad,M.A.Zhou,H.Zhang,Z.Xu,M.Rui,Y.White,J.C.Nano-Enabled Plant Microbiome Engineering for Disease Resistance.Nano Today.48(2023)101752.

4.Mittler,R.Blumwald,E.The Roles of ROS and ABA in Systemic Acquired Acclimation.Plant Cell.27(2015)64–70.

5. wang Xiao, cai Jian, zhou Qin, dai Tingbo, ginger east. Physiological mechanism for improving stress tolerance of crops by exercise of abiotic stress, chinese agricultural science 54 (2021): 2287-301.

6. A method for obviously enhancing the stress resistance of eggplant under low-temperature weak light stress is CN201610486694.3[ P ] 2016-09-28.

7. The plant growth regulator with high stress resistance and its preparation process and use are CN201611066258.7[ P ] 2021-06-15.

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Claims (6)

1. A method for regulating the balance of plant growth and defense, which is characterized in that exogenous substances which can be recognized by plants and trigger the explosion of extracellular ROS are contacted with the plants, and the transcription reprogramming process of the plants is further induced by triggering the explosion of extracellular ROS and signal transduction thereof, so that the plants generate systemic adaptive and systemic acquired resistance, thereby coordinating the balance of plant growth and defense and promoting the growth, development and propagation of the plants.

2. The method according to claim 1, wherein the exogenous material is a macromolecular material of biological origin or an artificially synthesized macromolecular material, the macromolecular surface being modified with carboxyl groups.

3. The method according to claim 2, wherein the macromolecular substance of biological origin is selected from long-chain fatty acids, oligosaccharides or small peptides; the synthesized macromolecular substance is a carbon or silicon material which has nanometer scale, good biocompatibility and no toxicity.

4. A method according to any one of claims 1-3, characterized in that the exogenous material is added to the plant cultivation substrate or soil, or sprayed onto the surface of the plant leaves, or subjected to seed soaking treatment, to induce extracellular ROS bursts in the plants.

5. Use of the method according to any one of claims 1-4 for increasing plant yield and stress resistance.

6. The method according to any one of claims 1 to 4 or the use according to claim 5, wherein the plant is selected from the group consisting of arabidopsis, rice, wheat, maize.