Application of L-carnitine in improving cold tolerance of plants

Abstract

The application discloses application of L-carnitine in improving cold tolerance of plants and belongs to the technical field of plant stress tolerance regulation and stress resistance agent application. The application utilizes L-carnitine to improve the PS II maximum quantum yield (Fv / Fm) of Arabidopsis thaliana, enhances the stability of the photosynthetic system, reduces the damage of low temperature to the photosynthetic mechanism, simultaneously reduces the content of malondialdehyde (MDA), inhibits membrane lipid peroxidation, maintains the integrity of the cell membrane, promotes the synthesis of proline and soluble sugar, improves the cell osmotic regulation capacity and strengthens the cold resistance. The application overcomes the defects of the existing genetic improvement, physical regulation and chemical regulation technologies, such as narrow application range, high cost and poor universality, realizes effective response to sudden low-temperature stress of crops in a large area of fields and provides a new technical path for plant cold stress prevention and control.

Description

Technical Field

[0001] This invention belongs to the field of plant stress tolerance regulation and stress resistance agent application technology, and more specifically relates to the application of L-carnitine in improving plant cold tolerance. Background Technology

[0002] Cold stress, as an important abiotic stress factor that restricts the geographical distribution of plants and agricultural production, disrupts biological processes such as cell membrane system stability, damages reactive oxygen species metabolism balance, and inhibits energy synthesis, significantly affecting plant growth and development and crop yield. How to improve plant cold tolerance and alleviate the damage of low temperature stress to plants through efficient and green technologies has become an important direction for research and application in the fields of agricultural production and plant stress resistance. Existing technologies for improving plant cold tolerance mainly include: First, genetic improvement technology, which involves the targeted breeding of cold-tolerant varieties through gene editing and hybridization. However, this method is time-consuming and costly, and the regulatory mechanisms of cold tolerance genes in most plants are not fully understood, limiting its application. Second, physical regulation technology, including mulching for insulation, greenhouse temperature control, and low-temperature hardening. This type of technology depends on equipment and environmental conditions, has limited applicability, and is difficult to cope with sudden low-temperature stress on large-scale field crops. Third, chemical regulation technology, which improves plant cold tolerance by applying exogenous stress-resistance regulators such as abscisic acid, salicylic acid, and proline. However, some of these regulators have problems such as high cost, sensitivity to application concentration, and potential environmental impacts. Furthermore, different plants respond differently to these regulators, resulting in poor universality.

[0003] L-carnitine, a naturally occurring amino acid-like substance, is widely involved in fatty acid metabolism and energy supply in organisms. It possesses advantages such as being non-toxic, environmentally friendly, and readily available, and is currently widely used in animal nutrition and medicine. Existing research indicates that L-carnitine can enhance the stress resistance of organisms by regulating the reactive oxygen species scavenging system and maintaining cellular osmotic pressure balance. However, its application and mechanism of action in regulating plant cold tolerance lack systematic research and mature technical solutions. There is an urgent need to develop methods for applying L-carnitine to improve plant cold tolerance, providing a novel, efficient, and green technical approach for controlling plant cold stress.

[0004] Based on this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide the application of L-carnitine in improving plant cold tolerance, thereby addressing the problems existing in the prior art. This invention provides a highly efficient and green plant cold tolerance regulation technology based on L-carnitine, clarifying its application methods and mechanisms of action in the control of plant cold stress. It overcomes the shortcomings of existing genetic improvement, physical regulation, and chemical regulation technologies, such as narrow applicability, high cost, and poor versatility, enabling effective responses to sudden low-temperature stress in large-scale field crops and providing a new technological pathway for the control of plant cold stress.

[0006] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of the present invention is to provide the application of L-carnitine in improving the cold resistance of plants, wherein the plants include Arabidopsis thaliana or rice.

[0007] The second technical solution of this invention provides the application of L-carnitine in reducing the malondialdehyde content in Arabidopsis thaliana.

[0008] The third technical solution of this invention provides the application of L-carnitine in increasing the proline content of Arabidopsis thaliana.

[0009] The fourth technical solution of this invention provides the application of L-carnitine in increasing the soluble sugar content of Arabidopsis thaliana.

[0010] Fifth technical solution of the present invention: A method for improving the cold resistance of Arabidopsis thaliana, comprising the following steps: Simply spray the L-carnitine solution onto the leaves of Arabidopsis thaliana.

[0011] Preferably, the concentration of the L-carnitine solution is 0~25 mmol / mL, excluding 0.

[0012] Preferably, the amount of L-carnitine solution sprayed is 0.5~1mL per Arabidopsis plant.

[0013] Preferably, the L-carnitine solution is sprayed 1 to 3 days before the arrival of a cold wave.

[0014] Furthermore, the present invention also provides a cultivation process for Arabidopsis thaliana, the specific steps of which are as follows: (1) Add Arabidopsis thaliana seeds to ethanol and mix; (2) Centrifuge the mixture obtained in step (1) and remove the supernatant; (3) Add the remaining part from step (2) to the sodium hypochlorite solution and mix; (4) Centrifuge the mixture obtained in step (3) and remove the supernatant; (5) Add the remaining part of step (4) to 1 mL of sterile water, shake and let stand, then remove the supernatant; repeat this step 6 times. (6) Add the remaining part of step (5) to sterile agar water for treatment, spot the treated seeds on 1 / 2 MS medium, and place them in a light incubator for light culture; (7) When the roots of Arabidopsis thaliana grow to 0.8-1.2 cm, transfer them to nutrient soil to continue growing; (8) When the rosette leaves are 4 weeks old, spray the Arabidopsis leaves with L-carnitine solution.

[0015] Further, in step (1), the ethanol is a 75% ethanol solution; the volume of the ethanol solution is more than 3 times the volume of the space occupied by the Arabidopsis seeds; and the mixing time is 1-2 minutes. In step (2), the centrifugation speed is 1800~2200 rpm and the time is 1~2 min; In step (3), the mass fraction of the sodium hypochlorite solution is 1~1.5%; the volume ratio of the sodium hypochlorite solution to ethanol is 1:1; and the mixing time is 10~12 min. In step (4), the centrifugation speed is 1800~2200 rpm and the time is 1~2 min; The volume ratio of the sterile water in step (5) to the ethanol in step (1) is 1:1; In step (6), the mass fraction of the sterile agar water is 0.1%; the 1 / 2 MS medium is a plant culture medium with a macro-element concentration half that of the standard MS medium, specifically containing 950 mg / L potassium nitrate, 825 mg / L ammonium nitrate, 85 mg / L potassium dihydrogen phosphate, 185 mg / L magnesium sulfate, and 220 mg / L calcium chloride, pH 5.8, with 0.8 wt% agar and 1~1.5 wt% sucrose added for solid culture; the light intensity for the light culture is 100~150 μmol / (m²). 2 ·s).

[0016] In step (7), the nutrient soil is a cultivation substrate made by mixing nutrient soil, vermiculite and perlite in a mass ratio of 3:1~2:1~2.

[0017] Furthermore, the present invention also provides a low-temperature stress process for Arabidopsis thaliana after spraying with L-carnitine solution, the specific steps of which are as follows: The L-carnitine solution of the above dosage and concentration was sprayed onto Arabidopsis thaliana leaves, and then subjected to low temperature stress at 4°C.

[0018] The technical mechanism of this invention is as follows: The technical principle behind L-carnitine's application in improving plant cold tolerance is based on its multi-target physiological regulatory function as a natural metabolic regulator. By systematically intervening in three core physiological processes under low-temperature stress—photosynthetic efficiency, membrane lipid stability, and osmotic regulation—it comprehensively enhances the plant's adaptability to cold stress. The core of this approach lies in extending L-carnitine, traditionally widely used in animal nutrition and medicine, to the field of plant stress regulation. Leveraging its non-toxic, environmentally friendly, and readily available natural properties, it provides a highly efficient, green, and broadly applicable new technological pathway for controlling plant cold stress.

[0019] At the level of mechanism of action, the core principle by which L-carnitine enhances plant cold tolerance can be summarized as a triple synergistic regulation: First, at the level of the photosynthetic system, low-temperature stress usually inhibits the activity of photosystem II (PSII), leading to obstruction of the photosynthetic electron transport chain. L-carnitine can significantly increase the maximum quantum yield of PSII (Fv / Fm). As a key indicator reflecting the integrity and functional state of plant photosynthetic organs, the increase in Fv / Fm directly indicates that L-carnitine helps maintain light energy conversion efficiency at low temperatures, reduces cold damage to photosynthetic organs, and thus ensures the plant's basal energy metabolism and carbon assimilation capacity under stress. Second, at the level of cell membrane stability, low-temperature stress induces a burst of reactive oxygen species, triggering a chain reaction of membrane lipid peroxidation, leading to decreased cell membrane fluidity and increased permeability, ultimately causing leakage of intracellular electrolytes and metabolites. Malondialdehyde (MDA), as the end product of membrane lipid peroxidation, is a core marker for measuring the degree of membrane system damage. Experimental data show that exogenous L-carnitine treatment can significantly reduce MDA accumulation in plant tissues under low-temperature stress. This means that L-carnitine has the function of inhibiting membrane lipid peroxidation and protecting the integrity of cell membrane structure, thereby maintaining normal cell compartmentalization and transmembrane transport. Third, at the level of osmotic regulation, one of the classic strategies plants use to cope with low-temperature stress is to actively accumulate compatible solutes such as proline and soluble sugars. This enhances cold resistance by lowering the freezing point of the cytoplasm, preventing protein denaturation, stabilizing enzyme activity, and maintaining cell turgor pressure. L-carnitine treatment can significantly promote the synthesis and accumulation of proline and soluble sugars, improve the cell's osmotic regulation capacity, and enable plants to maintain necessary water balance and metabolic activity under low-temperature conditions.

[0020] Further analysis from a systems biology perspective reveals that these three regulatory mechanisms do not operate in isolation, but rather are interconnected and synergistically integrated. The stability of the photosynthetic system provides the energy and carbon skeleton basis for the synthesis of osmotic regulators; the maintenance of cell membrane integrity ensures smooth signal transduction and substance transport; and the accumulation of osmotic regulators, in turn, protects photosynthetic protein complexes and membrane lipids from low-temperature damage. L-carnitine achieves a systematic enhancement of plant cold tolerance through this multi-target, networked regulatory model, overcoming the limitations of traditional single-target chemical regulators, which suffer from unstable effects and narrow applicability.

[0021] Furthermore, as an endogenous substance in living organisms, L-carnitine poses no risk of residual toxicity after application, does not damage the soil microecology, and has relatively controllable production costs, making it potential for large-scale field application. By clarifying its mechanism of action and application parameters in regulating plant cold tolerance, the technical solution described in this invention fills the gap in systematic research on L-carnitine in the field of plant stress resistance.

[0022] The present invention discloses the following technical effects: This invention uses L-carnitine as a green regulatory medium, combined with standardized Arabidopsis treatment (ethanol + sodium hypochlorite disinfection + multiple rounds of sterile water rinsing), 0-10mM gradient concentration screening, low-temperature stress, and multi-index measurement. Through the synergistic principle of regulating the plant's photosynthetic system, membrane lipid state, and endogenous osmotic substances, it achieves cold tolerance regulation, precisely addressing the pain points of existing technologies. Standardized treatment eliminates exogenous interference, ensuring that index changes are only related to L-carnitine, providing a foundation for clarifying the mechanism of action. Its core mechanism of action is clear: at low temperatures, it can increase the maximum quantum yield of PS II (Fv / Fm), enhance the stability of the photosynthetic system, and reduce the damage of low temperatures to photosynthetic structures; simultaneously, it reduces malondialdehyde (MDA) content, inhibits membrane lipid peroxidation, and maintains cell membrane integrity; it can also promote the synthesis of proline and soluble sugars, enhance cell osmotic regulation capacity, and strengthen cold resistance. Attached Figure Description

[0023] Figure 1 The results of physiological index analysis of Arabidopsis thaliana under low temperature after exogenous application of L-carnitine are shown. Among them, A shows the changes in leaf morphology of Arabidopsis thaliana from day 1 to day 5 after no L-carnitine spraying (control group) and L-carnitine spraying (treatment group); B is a violin graph showing the changes in malondialdehyde content; C is a violin graph showing the changes in proline content; and D is a violin graph showing the changes in maximum photosynthetic efficiency (Fv / Fm) of leaves. Figure 2 The images show the low-temperature phenotypes of rice treated with exogenous L-carnitine. From left to right, the images show rice treated with exogenous L-carnitine at concentrations of 0 mM, 0.1 mM, 1 mM, 5 mM, and 10 mM. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] It should be noted that any aspects not described in detail in this invention are conventional practices in the field and are not the focus of this invention.

[0030] Unless otherwise specified, all raw materials used in the following embodiments of the present invention are commercially available products, and the source of commercially available products does not affect the technical effect of the present invention.

[0031] The 1 / 2 MS medium used was a plant culture medium with a macro-element concentration of half that of the standard MS medium, specifically containing 950 mg / L potassium nitrate, 825 mg / L ammonium nitrate, 85 mg / L potassium dihydrogen phosphate, 185 mg / L magnesium sulfate, and 220 mg / L calcium chloride, with a pH of 5.8. 0.8 wt% agar and 1-1.5 wt% sucrose were added for solid culture.

[0032] The nutrient soil used is a cultivation substrate made by mixing nutrient soil, vermiculite and perlite in a mass ratio of 3:1~2:1~2.

[0033] Example 1: Verification of the effects of different concentrations of L-carnitine on Arabidopsis thaliana (1) Add wild-type Arabidopsis seeds to 1 mL of 95 wt% ethanol solution and shake with a hand shaker for 1 min; (2) Centrifuge at 2000 rpm for 1 min and remove the supernatant; (3) Add 1 mL of 1 wt% sodium hypochlorite solution and shake with a hand shaker for 10 min; (4) Centrifuge at 2000 rpm for 1 min and remove the supernatant; (5) Remove the supernatant on the laminar flow hood, add 1 mL of sterile water, shake and let stand, then remove the supernatant; repeat this step 6 times. (6) Add 0.1wt% sterile agar water for treatment, spot the treated seeds onto 1 / 2 MS medium, and place them in a light incubator for light culture at a light intensity of 150 μmol / (m²). 2 ·s); (7) When the roots of Arabidopsis thaliana grow to 1 cm, transfer them to nutrient soil to continue growing; (8) Prepare aqueous solutions of L-carnitine at different concentrations (0 mM, 10 mM). (9) When the rosette leaves grow to 4 weeks old, spray the leaves of Arabidopsis thaliana with aqueous solutions of different concentrations of L-carnitine. The amount of L-carnitine solution sprayed on each Arabidopsis thaliana plant is 1 mL. Then, the plants are moved to a 4℃ incubator for low-temperature culture for 48 h to conduct a preliminary experiment to verify the effects of different concentrations of L-carnitine.

[0034] Testing process: The Fv / Fm, malondialdehyde (MDA) content, proline content, and soluble sugar content of Arabidopsis thaliana leaves treated with different concentrations of L-carnitine were detected.

[0035] (1) Determination of Fv / Fm: Leaves of Arabidopsis thaliana after treatment were subjected to dark acclimatization (treatment under darkness) for 30 min. Chlorophyll fluorescence parameters were measured using a pulse-modulated fluorometer (PAM-2500, Heinz Walz, Germany). First, under extremely weak measurement light (0.1 μmol / (m²), the chlorophyll fluorescence parameters were measured. 2 Initial fluorescence (F0) was measured under s) light; subsequently, a saturated pulse light (8000 μmol / (m) was applied. 2The maximum fluorescence (Fm) was measured by shutting down all reaction centers of photosystem II (PS II) for 0.8 s. Variable fluorescence (Fv) was calculated using the formula Fv = Fm - F0. The maximum photochemical quantum yield of PS II (Fv / Fm) was used to evaluate the potential photochemical efficiency of leaf photosystem II.

[0036] (2) Determination of malondialdehyde (MDA) content: Malondialdehyde (MDA) content was determined using a malondialdehyde (MDA) assay kit (catalog number: G0110W) manufactured by Suzhou Greens Biotechnology Co., Ltd. 0.5 g of treated Arabidopsis thaliana leaves were accurately weighed and placed in a suitable container. 1 mL of the kit's extraction buffer was added, and the sample was homogenized on ice to ensure thorough mixing. After homogenization, the sample was transferred to a centrifuge tube and centrifuged at 12,000 rpm for 10 min at 4°C. After centrifugation, the supernatant was carefully aspirated and placed on ice for subsequent testing. Simultaneously, the microplate reader was preheated for 30 min, and the water bath temperature was raised to 90–95°C. Several EP tubes were prepared, and 300 μL of the kit's working solution and 200 μL of the prepared sample solution were added to each tube. After addition, the solution was gently mixed by shaking or vortexing. Next, place the EP tube in a water bath and incubate for 30 minutes. After incubation, quickly remove the EP tube and place it on ice to cool. Once cooled to room temperature, centrifuge the EP tube again at 12000 rpm at 25°C for 10 minutes. After centrifugation, accurately transfer 200 μL of supernatant to a 96-well plate and measure the absorbance of the solution at 532 nm and 600 nm using a preheated microplate reader. Record this absorbance as A. 532 and A 600 And calculate the difference between the two, that is, △A=A 532 -A 600 .

[0037] The formula for calculating malondialdehyde (MDA) content is: MDA content (nmol / g, fresh weight) = [ΔA ÷ (ε × d) × V² × 10] 9 ]÷(W×V1÷V); Wherein, V - total volume of sample extract, 1 mL; V1 - volume of sample added to the reaction system, 0.2 mL; V2 - total volume of sample added and working solution in the reaction mixture, 5 × 10⁻⁶. -4 L; d - optical path length, 0.5 cm; ε - MDA molar extinction coefficient, 155 × 10⁻⁶ 3 L / mol / cm; W - sample mass, 0.5g.

[0038] (3) Determination of proline content: The proline content was determined using a proline content assay kit (catalog number: G0111W) manufactured by Suzhou Gres Biotechnology Co., Ltd. 0.5 g of treated Arabidopsis thaliana leaves were weighed and 1 mL of the kit's extraction buffer was added. The mixture was homogenized in an ice bath, transferred to an EP tube, and extracted by shaking in a 90℃ water bath for 10 min. After cooling to room temperature, the mixture was centrifuged at 25℃ and 12000 rpm for 10 min, and the supernatant was used as the test sample. 150 μL of the test sample (150 μL of distilled water for the control group), 150 μL of glacial acetic acid, and 300 μL of reagent-1 from the kit were added sequentially to the EP tube. The tube was heated in a 95℃ water bath for 30 min. After cooling to room temperature, the EP tube was centrifuged again at 12000 rpm at 25℃ for 10 min. After centrifugation, 200 μL of the supernatant was transferred to a 96-well plate, and the absorbance (A) was immediately read at 520 nm. 测定 , △A=A 测定 -A 空白 A 空白 The absorbance values ​​measured for the control group are used; the results are calculated according to the formula.

[0039] The formula for calculating proline content is: Proline (Pro) content (μg / mL) = [(△A + 0.0064) ÷ 0.1625] ÷ [V2 × V1 ÷ (V + V2)] × D; Wherein, V - total volume of extract, 1 mL; V2 - volume of liquid sample, 0.1 mL; V1 - volume of extract added to the reaction system, 0.15 mL; D - dilution factor, 1 for undiluted.

[0040] (4) Determination of soluble sugar content: The soluble sugar content was determined using a soluble sugar content determination kit (catalog number: G0501F) manufactured by Suzhou Gres Biotechnology Co., Ltd. 0.2 g of treated Arabidopsis thaliana leaves were weighed and placed in a specially designed homogenizing container, along with 1.5 mL of 80 wt% ethanol solution. The sample was homogenized using an automatic grinder under ice bath conditions to ensure thorough mixing. After homogenization, the container containing the mixture was tightly sealed with sealing film and placed in a 50°C constant temperature water bath for 20 min. The mixture was shaken every 2 min during the bath to ensure homogeneity. After the water bath, the mixture was allowed to cool naturally to room temperature. If there was any volume loss, 80 wt% ethanol was added to bring the volume to 1.5 mL. The cooled mixture was transferred to centrifuge tubes and centrifuged at 12000 rpm for 10 min at room temperature to complete solid-liquid separation. The supernatant was carefully transferred to a clean container for later use. In an EP tube, using a high-precision pipette, add 25 μL of sample supernatant, 75 μL of distilled water (100 μL for the control group), and then 30 μL of the premixed working solution provided in the kit. Finally, slowly add 250 μL of concentrated sulfuric acid dropwise along the tube wall, paying attention to the solution's condition to prevent splashing. After adding the sulfuric acid, mix thoroughly using a vortex mixer. Incubate the mixed EP tube in a 95–100°C water bath for 10 min, then cool to room temperature. After cooling, transfer 200 μL of the reaction solution to a 96-well plate and measure the absorbance (A) using a microplate reader equipped with a 620 nm filter. 测定 , △A=A 测定 -A 空白 A 空白 The absorbance values ​​measured for the control group are used; the results are calculated according to the formula.

[0041] The formula for calculating the content of soluble sugar is: soluble sugar (mg / g, weight) = [(ΔA-0.0203)÷3.658×V1]÷(W×V1÷V)×D; Wherein, V - total volume of sample extract, 1.5 mL; V1 - volume of sample added, 0.05 mL; W - sample weight, g; D - dilution factor, 1 for undiluted sample.

[0042] The results are as follows Figure 1 As shown.

[0043] Figure 1 Figures B, C, and D show the physiological indicators of Arabidopsis thaliana under low temperature after exogenous application of L-carnitine. Figure A shows the morphological changes of Arabidopsis leaves from day 1 to day 5 after L-carnitine application (control group) and L-carnitine application (treatment group). Figure B is a violin plot showing the changes in malondialdehyde (MDA) content, Figure C is a violin plot showing the changes in proline content, and Figure D is a violin plot showing the changes in maximum photosynthetic efficiency (Fv / Fm). In Figures B, C, and D, the horizontal axis represents the number of days of treatment, and the bands indicate the treatment duration. For processing groups, without This serves as the control group.

[0044] To verify how L-carnitine enhances the cold resistance of plants, Arabidopsis thaliana sprayed with 10 mM L-carnitine was used as the treatment group, while Arabidopsis thaliana without L-carnitine (i.e., sprayed with 0 mM L-carnitine solution) was used as the control group. Both groups were continuously treated at 4°C. The results showed that... Figure 1 (A) The same leaf remained evergreen in the treatment group, while in the control group, the leaf tips turned yellow and wilted on the fourth and fifth days. This indicates that L-carnitine effectively maintained cell activity and leaf function, delaying the apparent symptoms of low-temperature stress. Subsequently, physiological indicators were measured in both the treatment and control groups. Figure 1 (B~E) The physiological experiment results of malondialdehyde (MDA) showed that the MDA content in the treatment group decreased by 10% on the first day, increased by 12% on the second day, and then dropped sharply on the third and fourth days (20% decrease on the third day, 64.6% decrease on the fourth day), and decreased by 16.9% on the fifth day. Figure 1 B). L-carnitine reduces the damage to cell membranes caused by low temperature by inhibiting initial oxidative stress and accelerating subsequent membrane repair. In Arabidopsis thaliana, the content of soluble reducing sugars increased by 31.4% on the first day compared to the control group, indicating rapid accumulation of soluble sugars such as sucrose, lowering the cell freezing point, and providing substrates for metabolism. The content decreased by 6.8% on the second day, possibly due to short-term depletion, but remained higher than the control group from the third to the fifth day (increasing by 19.9%, 13.7%, and 8.8%, respectively), maintaining osmotic balance under cold stress in Arabidopsis thaliana. Figure 1 C); In Arabidopsis thaliana, the proline content showed a dramatic increase on days 2-3 compared to the control group (an increase of 410.4% on day 3). Figure 1 D), which highly coincided with the peak of COR protein induction (24-48 hours after cold treatment), suggesting that proline acts as an early signal to induce the expression of cold-response genes; in Arabidopsis, the Fv / Fm ratio in the treatment group was higher than that in the control group throughout the day (35.3% increase on day 1 and 28.3% increase on day 5). Figure 1 E), the results showed that exogenous application of L-carnitine to Arabidopsis thaliana at low temperatures can maintain light capture efficiency and reduce oxidative damage caused by excess light energy, which is consistent with the MDA results. Therefore, the anti-cold mechanism of L-carnitine can be concluded as follows: inhibiting MDA accumulation and maintaining membrane integrity; rapid response of soluble sugar (day 1) and mid-term surge of proline (day 3) synergistically reduce cell osmotic potential; protecting photosynthetic structures to ensure energy supply.

[0045] In summary, this invention utilizes L-carnitine to increase the maximum quantum yield (Fv / Fm) of PS II in Arabidopsis thaliana, enhance the stability of the photosynthetic system, and reduce the damage of low temperature to the photosynthetic apparatus; at the same time, it reduces its malondialdehyde (MDA) content, inhibits membrane lipid peroxidation, and maintains cell membrane integrity; it can also promote the synthesis of proline and soluble sugars, enhance cell osmotic regulation capacity, and strengthen cold resistance.

[0046] Example 2: Verification of the effects of different concentrations of L-carnitine on rice. 1) Select indica rice (9311) seeds with uniform growth and germinate them in the dark.

[0047] 2) After 2-3 days, transfer the germinated seedlings to a hydroponic device and adjust the pH of the hydroponic solution to 6.0.

[0048] 3) Place the seedlings at 28℃ and a light intensity of 250 μmol / m². 2 Cultured for 25 days in an incubator with a photoperiod of 16h / 8h and a photocycle of 1s.

[0049] 4) Prepare L-carnitine solutions of different concentrations (0mM, 0.1mM, 1mM, 5mM, 10mM).

[0050] 5) Different concentrations of L-carnitine were evenly applied to rice leaves, and then placed in an 8℃ incubator for low-temperature treatment for 48 hours. Rice without L-carnitine was used as a control to observe and compare phenotypic differences.

[0051] The results are as follows Figure 2 As shown.

[0052] Figure 2 The images show the low-temperature phenotypes of rice treated with exogenous L-carnitine. From left to right, the images show rice treated with exogenous L-carnitine at concentrations of 0 mM, 0.1 mM, 1 mM, 5 mM, and 10 mM.

[0053] Rice under 8℃ low temperature stress concentration gradient experiment ( Figure 2 It was found that the leaves of the 1 mM L-carnitine treatment group were greener than those of other treatment groups after 5 days of continuous cold exposure. This study establishes the cold-protective effect of L-carnitine in gramineous crops for the first time, providing a new metabolic regulatory target for crop cold-resistance breeding.

[0054] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0055] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of L-carnitine in improving the cold tolerance of plants, characterized in that, The plants mentioned include Arabidopsis thaliana or rice.

2. Application of L-carnitine in reducing malondialdehyde content in Arabidopsis thaliana.

3. Application of L-carnitine in increasing proline content in Arabidopsis thaliana.

4. Application of L-carnitine in increasing the soluble sugar content of Arabidopsis thaliana.

5. A method for improving the cold tolerance of Arabidopsis thaliana, characterized in that, Includes the following steps: Simply spray the L-carnitine solution onto the leaves of Arabidopsis thaliana.

6. The method according to claim 5, characterized in that, The concentration of the L-carnitine solution is 0~25mM, excluding 0.

7. The method according to claim 5, characterized in that, The amount of L-carnitine solution sprayed is 0.5~1mL per Arabidopsis plant.

8. The method according to claim 5, characterized in that, The L-carnitine solution should be sprayed 1 to 3 days before the arrival of the cold wave.

9. The method according to claim 5, characterized in that, The cultivation steps for Arabidopsis thaliana are as follows: (1) Add Arabidopsis thaliana seeds to ethanol and mix; (2) Centrifuge the mixture obtained in step (1) and remove the supernatant; (3) Add the remaining part from step (2) to the sodium hypochlorite solution and mix; (4) Centrifuge the mixture obtained in step (3) and remove the supernatant; (5) Add the remaining part of step (4) to 1 mL of sterile water, shake and let stand, then remove the supernatant; repeat this step 6 times. (6) Add the remaining part of step (5) to sterile agar water for treatment, spot the treated seeds on 1 / 2 MS medium, and place them in a light incubator for light culture; (7) When the roots of Arabidopsis thaliana grow to 0.8-1.2 cm, transfer them to nutrient soil to continue growing; (8) When the rosette leaves are 4 weeks old, spray the Arabidopsis leaves with L-carnitine solution.

10. The method according to claim 9, characterized in that, In step (1), the ethanol is a 75% ethanol solution; the volume of the ethanol solution is more than 3 times the volume of the Arabidopsis thaliana seeds; and the mixing time is 1-2 minutes. And / or, in step (2), the centrifugation speed is 1800~2200 rpm and the time is 1~2 min; And / or, in step (3), the mass fraction of the sodium hypochlorite solution is 1~1.5%; the volume ratio of the sodium hypochlorite solution to ethanol is 1:1; and the mixing time is 10~12 min; And / or, in step (4), the centrifugation speed is 1800~2200 rpm and the time is 1~2 min; And / or, the volume ratio of the sterile water in step (5) to the ethanol in step (1) is 1:1; And / or, in step (6), the mass fraction of the sterile agar water is 0.1%; the 1 / 2 MS medium contains 950 mg / L potassium nitrate, 825 mg / L ammonium nitrate, 85 mg / L potassium dihydrogen phosphate, 185 mg / L magnesium sulfate, 220 mg / L calcium chloride, pH 5.8, and also adds 0.8 wt% agar and 1~1.5 wt% sucrose for solid culture; the light intensity for the light culture is 100~150 μmol / (m²). 2 ·s); And / or, in step (7), the nutrient soil is a cultivation substrate made by mixing nutrient soil, vermiculite and perlite in a mass ratio of 3:1~2:1~2.

Images