Method for improving stress resistance of secondary metabolites of tea trees

Abstract

The invention belongs to the technical field of agriculture, and particularly relates to a method for improving stress resistance of tea tree secondary metabolites. The method for improving the stress resistance of the secondary metabolites of the tea trees comprises the following steps: S1, preparing a plasma sodium lactate solution: at the temperature of 25 DEG C, irradiating and activating a sodium lactate Green injection through a plasma source to obtain the plasma sodium lactate solution; s2, leaf surface spraying: diluting a plasma sodium lactate solution by 25-100 times with ultrapure water, and uniformly spraying the diluted solution on the front and back surfaces of the tea tree leaves until the front and back surfaces of the leaves are completely wet and liquid flows down. Plasma sodium lactate affects stress-resistant physiology of young tea trees and enhances activity of the tea trees, secondary metabolites of the tea trees can be accumulated to resist reactive oxygen stress when the plasma sodium lactate acts on the tea trees, synthesis of amino acid and protein with the stress-resistant function is up-regulated to enhance stress resistance, and the stress resistance of the young tea trees is improved. A scientific basis can be provided for tea tree organism and environmental stress resistance regulation and control in a soil barren area.

Description

Technical Field

[0001] The invention belongs to the technical field of agriculture, and particularly relates to a method for improving the stress resistance of secondary metabolites of tea trees. Background Art

[0002] Plant stress resistance is the result of long-term natural selection and adaptation. In-depth exploration and practical research on plant stress resistance will not only help reveal how plants respond to environmental stress and explore the inherent laws of their ecological adaptation, but also provide a biological basis for responding to global climate change. In the current context of increasingly serious problems such as frequent extreme weather, soil degradation, and water shortages, improving crop stress resistance has become a key strategy for ensuring sustainable agricultural development. Therefore, analyzing the response and regulatory mechanisms of plants under adverse conditions has important theoretical and practical significance for breeding high-yield, high-quality, and stress-resistant crop varieties.

[0003] Tea (Camellia sinensis) is a perennial woody plant used for its leaves. Widely cultivated and consumed worldwide, tea has become an important economically cultivated plant. As a perennial cash crop, tea has a long life cycle and a wide range of cultivation cycles, constantly exposed to a variety of stresses, including drought, high and low temperatures, salinity, and pests and diseases. Therefore, the tea plant's long-term survival and continued economic output are inseparable from its ability to adapt to stresses. Studying the stress tolerance mechanisms of tea not only reveals how perennial woody plants coordinate physiological metabolism with stress responses in complex environments but also provides theoretical support for improving tea plant stress tolerance and maintaining tea quality. Furthermore, as one of the world's most popular beverages, tea holds significant economic, cultural, and social value in China and around the world. China is the world's largest tea producer and consumer, and the tea industry plays a vital role in promoting rural revitalization and increasing farmers' incomes. However, in recent years, climate change and changes in intensive tea plantation land management have led to environmental degradation in tea plantations, resulting in significant concerns about tea yield and quality. Therefore, conducting research on the stress resistance of tea trees will help improve the stability and sustainability of tea production, and has important practical significance for ensuring the development of the tea industry and maintaining the security of the ecological agricultural system.

[0004] Current research on tea plant resistance focuses on the mechanisms related to soil heavy metal stress and environmental cold tolerance. The physiological and biochemical mechanisms of tea plant aluminum tolerance have been extensively studied and can generally be divided into external elimination mechanisms and internal detoxification mechanisms. Research on tea plant cold tolerance has focused on enhancing the expression of drought-resistant genes, promoting cold tolerance through protein kinases, or utilizing cold-inducible genes. Cold acclimation has significantly enhanced tea plant frost tolerance. Symptoms of cold acclimation include increased production of reactive oxygen species, upregulation of antioxidant enzymes, altered plant hormone levels, increased metabolites such as γ-aminobutyric acid and soluble sugars, and accumulation of osmotic amino acids such as proline. However, research on tea plant resistance in poor soils has been neglected. As land resources become increasingly scarce, tea cultivation is gradually expanding into marginal and even infertile areas. In these soils, nutrient deficiencies, low microbial activity, and poor physical and chemical properties severely limit normal tea plant growth and leaf quality. Therefore, systematically exploring the physiological responses, adaptation mechanisms and methods of tea trees under poor soil conditions is of great significance for improving their survival ability and leaf production stability in low-fertility environments.

[0005] Current research on plant adaptation strategies in infertile soils focuses on root morphological adjustments, interactions with symbiotic microorganisms (such as mycorrhizal fungi), regulation of nutrient transporters, and accumulation of osmotic regulators. Exploring whether tea trees possess similar mechanisms and their unique regulatory networks will not only expand our understanding of the stress adaptation mechanisms of perennial woody crops but also provide technical and theoretical support for the development of low-input, high-efficiency, ecologically sound, and low-carbon tea garden management models.

[0006] Sodium lactate has an inhibitory effect on the growth of microorganisms and can be used as a food preservative. Plasma technology applied to agriculture is an environmentally friendly new technology. Low-temperature plasma has the advantages of high modification efficiency, simple operation, no waste and no by-products. Plasma-activated sodium lactate is a sodium lactate solution that is activated by plasma gas masses after high voltage electricity and inert gas are applied. There are many plasma-generated substances in the production process, such as superoxide and NO solvate, but most of them are transient substances. They react to form the final products H2O2 and NO2 - and NO3 − The peroxides and nitrogen oxides in plasma activation products can trigger a burst of endogenous reactive oxygen species in plants, placing them under oxidative stress and regulating related response mechanisms. When reactive oxygen species reach a certain concentration, they can promote plant growth. However, it is unclear whether the effect of plasma sodium lactate on tea plants helps to affect their resistance and quality.

[0007] Because young tea trees are sensitive to physiological resistance and have weaker resistance and yield than mature tea trees, newly planted young tea trees in soil-poor areas face severe environmental challenges, which will affect tea production in the area. The inventors of this application used one-year-old young tea trees as research subjects. By applying a diluted solution of plasma sodium lactate, they used targeted metabolomics to study and analyze the effects of plasma sodium lactate on the accumulation of secondary metabolites and the metabolism of resistant amino acids in tea leaves. They also explored its effects on the biomass and antioxidant capacity of young tea trees, hoping to provide a scientific basis for the regulation of biological and environmental stress resistance of tea trees in soil-poor areas.

[0008] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the Invention

[0009] The purpose of the present invention is to provide a method for improving the stress resistance of secondary metabolites of tea plants.

[0010] To achieve the above object, the present invention provides the following technical solutions: A method for improving the stress resistance of tea plant secondary metabolites, comprising the following steps: S1. Preparation of plasma sodium lactate solution At a temperature of 25°C, the sodium lactate Ringer's injection is activated by irradiation with a plasma source to obtain a plasma sodium lactate solution; S2. Foliar spray Dilute the plasma sodium lactate solution 25-100 times with ultrapure water, and spray it evenly on the front and back of the leaves of tea seedlings (1 year old) transplanted 1 week ago, until the front and back of the leaves are completely wet and liquid flows down.

[0011] Furthermore, in step S1, the mass concentration of the sodium lactate solution is 0.96%.

[0012] Furthermore, in step S1, the plasma source is connected to 60Hz AC 9kV high voltage electricity, the argon flow rate is 2L / min, and the distance between the plasma source and the sodium lactate solution is 3mm.

[0013] Furthermore, in step S1, 2 mL of the sodium lactate Ringer's injection is irradiated and activated each time, and the irradiation activation time is 5 minutes.

[0014] Furthermore, in step S2, spraying is performed once every 15-20 days, for a total of 4 times.

[0015] The present invention also provides the use of plasma sodium lactate solution in improving the secondary metabolites and stress resistance of tea trees.

[0016] Furthermore, the plasma sodium lactate solution is prepared by irradiating and activating the sodium lactate solution with a plasma source at a temperature of 25° C., thereby obtaining the plasma sodium lactate solution; wherein the mass concentration of the sodium lactate solution is 11.2%; The plasma source was connected to a 60Hz AC 9kV high voltage power supply, an argon flow rate of 2L / min, and a distance between the plasma source and the sodium lactate solution was 3mm; 2 mL of the sodium lactate solution was activated in each irradiation, and the irradiation activation time was 5 min.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This study used plasma-activated sodium lactate (PAL) to treat young tea plants and studied its effects on biomass, antioxidant activity, and secondary metabolite accumulation in greenhouse cultures. The results showed that: (1) Plasma sodium lactate increased the secondary metabolites of young tea plants and had a positive effect on the accumulation of ester and non-ester catechins. The accumulation of epicatechin gallate was increased by 4.68% by low concentration of plasma sodium lactate and by 6.93% by high concentration of plasma sodium lactate; the accumulation of epigallocatechin was increased by 2.46% by low concentration of plasma sodium lactate and by 4.84% by high concentration of plasma sodium lactate.

[0018] (2) Plasma sodium lactate changes the physiological resistance of secondary metabolites in young tea plants. Asparagine, histidine, phenylalanine, arginine, γ-aminobutyric acid and theanine participate in the resistance regulation of young tea plants, promote the growth of tea plants and participate in the synthesis of proteins related to toxicity alleviation and substance transport.

[0019] (3) Plasma sodium lactate can significantly increase the biomass of young tea trees, allowing them to obtain more resources and energy to cope with external environmental stress.

[0020] As can be seen, the plasma sodium lactate of the present invention affects the stress resistance of young tea plants and enhances their vitality. It causes the accumulation of secondary metabolites in tea plants to resist reactive oxygen species stress. Simultaneously, the synthesis of amino acids and proteins with stress resistance is upregulated, enhancing stress resistance and improving the stress resistance of young tea plants. This provides a scientific basis for regulating the biological and environmental stress resistance of tea plants in soil-poor areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The accumulation of major ester and non-ester catechins was normalized; Figure 2 It is a quantitative graph of free amino acids; Figure 3 Cluster heat map of amino acid accumulation compared with the blank experimental group; Figure 4 Cluster heat map of protein expression; Figure 5 The results of the effect of PAL on the biomass of young tea plants; Figure 6 a is the effect of PAL on superoxide dismutase in tea leaves; Figure 6 b shows the growth of young tea trees.

[0022] Description of main reference numerals: Figure 5 In the figure, a-dry weight of aboveground part of young tea plants; b-dry weight of underground part of young tea plants; c-height of young tea plants; d-number of leaves; e-leaf area. DETAILED DESCRIPTION

[0023] The following is a clear and complete description of the technical solution of the present invention. Obviously, the embodiments described are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making any creative efforts are within the scope of protection of the present invention.

[0024] 1. Materials and Methods 1.1 Materials and Reagents The tea tree variety is the main cultivated variety in Japan, "Yabukita", cultivated by JA Agricultural Cooperative in Shizuoka Prefecture, Japan. Plant individuals with the same strong roots and strong trees are selected. The selected tea trees need to be pruned to 12cm for subsequent transplanting experiments.

[0025] The cultivation soil is made of Akadama soil, Kanuma soil, and horticultural soil, which are mixed in a volume ratio of 1:1:2. After being sterilized under high pressure at 120kPa and 121°C for 1 hour, it is used to cultivate young tea trees.

[0026] Cultivation tools are 1 / 3*12π(52+7.52+5*7.5)cm 3 plastic basin.

[0027] Fertilizer was applied once. The slow-release ternary compound fertilizer (N:P:K=3:11:13) was applied once. 3 g was applied to each pot of young tea trees. The transplanted young tea trees were cultured in a greenhouse.

[0028] Sodium lactate was purchased from Otsuka Pharmaceutical Co., Ltd. (sodium lactate Ringer's injection, mass concentration 0.96%; per 1000 mL, containing 6.0 g of NaCl, 0.3 g of KCl, 0.2 g of CaCl₂ hydrate, and 3.1 g of L-C₃H₅NaO₃). Plasma lactate solution (PAL) was generated by irradiation at 25°C using a plasma source. The plasma source was connected to a 60 Hz AC 9 kV high voltage power source with an argon flow rate of 2 L / min. The distance between the plasma source and the sodium lactate injection was 3 mm. The irradiation time was 5 minutes, and 2 mL of sodium lactate injection was irradiated each time. The PAL solution was diluted 25-fold and 100-fold with ultrapure water, respectively, and these were designated as the high and low concentrations, respectively.

[0029] 1.2 Instruments and Equipment DY302 constant temperature drying oven, Yamato Scientific Co., Ltd., Japan; AB204-S precision balance, Mettler Toledo, Switzerland; U-5100 UV spectrophotometer, Yamato Scientific Co., Ltd., Japan; 5425 R high-speed refrigerated centrifuge, Eppendorf, USA; DISMIC-25CS membrane filter, ADVANTEC, Japan; Nanosep 10K filter column, Japan Pole Co., Ltd. Waters Acquity UPLC-MS liquid chromatography-mass spectrometry system, Waters Company, USA; Constant temperature water bath, Yamato Scientific Co., Ltd., Japan; Multiphor II Novablot Amersham Pharmacia electrophoresis unit, AmershamPharmacia Biotech, UK.

[0030] 1.3 Test methods 1.3.1 Plasma sodium lactate solution treatment On April 26, 2023, the young seedlings of tea trees (1 year old) that had been pruned were transplanted into plastic pots. One young tea tree was planted in each plastic pot and transferred to a greenhouse for cultivation. The growth conditions of each pot were kept consistent.

[0031] Three experimental groups were set up: (1) Control: no treatment was applied to the tea leaves; (2) PAL25: a 25-fold dilution of PAL was applied to the tea leaves; and (3) PAL100: a 100-fold dilution of PAL was applied to the tea leaves. Five replicates were set up for each experimental group.

[0032] PAL was applied via foliar spraying, with four applications of PAL dilutions during the three-month cultivation period, on May 3, May 24, June 14, and July 5, 2023. Biological and agronomic traits were surveyed after the cultivation period ended on July 21, 2023. Following the survey, tea shoot samples were randomly collected, placed in liquid nitrogen, and stored in a -80°C freezer for subsequent UPLC-MS analysis of secondary metabolites.

[0033] 1.3.2 Biological and agronomic traits Biomass dry weight: After the PAL treatment, the soil around the roots of the tea plants was removed and cleaned. The aboveground and underground parts of the tea plants were separated using scissors, and then dried in a dryer (100°C, 24 h). The dry matter mass was weighed. Three samples were collected for each experimental group, and the average value was taken.

[0034] Plant height: The height of the above-ground part of young tea trees was measured. Three samples were collected for each experimental group and the average value was taken.

[0035] Leaf number: Count the number of leaves of all samples in the experimental group. Each experimental group has 5 samples and take the average value.

[0036] Leaf area: Fifteen mature leaves from young tea plants were randomly selected from each treatment group. The longitudinal length from the tip to the base of the leaf, as well as the transverse width of the leaf, were measured. The obtained data were multiplied by the leaf area index and the average value was taken.

[0037] Tea leaf area Leaf length (cm) × leaf width (cm) × leaf area coefficient 0.7 1.3.3 Superoxide dismutase activity in tea leaves The activity of SOD superoxide dismutase reflects the reactive oxygen pressure that plants are subjected to. The higher the activity of the enzyme, the higher the reactive oxygen pressure that the plants are subjected to.

[0038] SOD activity was determined using the NBT method. 0.1 g of tea leaf and root samples frozen by liquid nitrogen were placed in a grinding bowl. A small amount of liquid nitrogen was poured into the bowl and the sample was quickly ground into a powder. 50 mM phosphate buffer (50 mM dipotassium hydrogen phosphate, 50 mM potassium dihydrogen phosphate, 100 mL of distilled water) was added and the extract was centrifuged (13,000 rpm, 4°C, 10 min, eppondoff centrifuge). The supernatant was used as the analysis sample.

[0039] Add 2.2 mL of 50 mM sodium carbonate buffer (50 mM sodium carbonate, 50 mM sodium bicarbonate, 200 mL of distilled water, pH 10.2), 0.1 mL each of 1.0 mM NBT solution, 4.0 mM xanthine, 3.0 mM EDTA solution, 0.15% BSA solution, and 6.0 units / mL xanthine oxidase (100-fold dilution), followed by 0.1 mL of the sample to be tested. Incubate at 25°C in the dark for 30 minutes. Terminate the reaction by adding 0.2 mL of 14 mM copper chloride solution. Measure the absorbance at 560 nm using a spectrophotometer. Repeat three times for the average value used to calculate SOD activity.

[0040] 1.3.4 Detection of accumulation of secondary metabolites in tea leaves Tea samples were rapidly frozen in liquid nitrogen and stored at −80°C. Before analysis, 1.2 g of the tea sample to be tested was placed in a grinding bowl. An appropriate amount of liquid nitrogen was poured into the bowl and the sample was rapidly ground into a powder. Six mL of ultrapure water was added to fully dissolve the sample to obtain a sample extract (10%, w / v). The extract was then centrifuged (13,000 rpm, 4°C, 10 min, eppondoff centrifuge). The supernatant was then filtered through a membrane (0.45 μm, DISMIC-25CS, ADVANTEC) using a Nanosep 10K filter. The supernatant after membrane filtration was centrifuged again (13,000 rpm, 4°C, 15 min, eppondoff centrifuge) to remove protein.

[0041] The samples were analyzed using UPLC-MS. The liquid chromatography-mass spectrometry conditions were as follows: an ACQUITY UPLCBEH C18 quadrupole column (1.7 μm, 2.1 × 100 mm, reversed-phase column, Waters) was used at 40°C. The mobile phase (A) consisted of a 0.1% formic acid solution, and the mobile phase (B) consisted of acetonitrile. A gradient elution (Table 1) was used over a 35-minute period, with a sample injection volume of 7.5 μL. The mass spectrometer (XevoQT of MS, Waters) was operated in negative ion mode (ES (-) Negative) with a resolving power range of 50–1000 (m / z).

[0042] Table 1 Chromatographic conditions 1.3.5 Determination of free amino acid content in tea Take 0.2g of the same frozen sample as used in the tea polyphenols determination experiment, place it in a grinding bowl, pour in an appropriate amount of liquid nitrogen, and quickly grind the sample into a powder. Add 3mL of 0.2N perchloric acid to fully dissolve it to extract the amino acids. Transfer it to a centrifuge tube, then rinse the grinding apparatus with 1mL of ultrapure water. Add the extract to the rinsed solution and centrifuge (4000rpm, 4℃, 10min, tabletop refrigerated centrifuge 2800). The pH of the obtained supernatant is adjusted to 4 with potassium bicarbonate. Then centrifuge again (13000rpm, 4℃, 5min, eppondoff refrigerated centrifuge), and the obtained supernatant is passed through a 0.45μm membrane filter as the analysis sample.

[0043] The sample was induced using the AccQ Tag Ultra Derivatization Kit. 50 μL of the sample was mixed with 350 μL of borate buffer (<5% sodium borate in water) and 100 μL of the derivatization reagent (9 g / L 6-aminoquinoline-N-hydroxyphosphate (AQC) in acetonitrile). The mixture was incubated at room temperature for 1 minute and then heated to 55°C for 10 minutes for derivatization.

[0044] The induction-treated samples were analyzed using UPLC-MS. The liquid chromatography conditions were as follows: an ACQUITY UPLC BEH C18 quadrupole column (1.7 μm, 2.1 × 100 mm, reversed-phase column) was used. The mobile phase (A) consisted of 0.1% formic acid in water and the mobile phase (B) was acetonitrile, with a gradient elution as shown in Table 2, and the analysis lasted 15 minutes. The sample injection volume was 5 μL, and the mass spectrometer was operated in negative ion mode (ES (-) Negative) with a resolution range of 50–1000 (m / z). The standard sample was an amino acid standard solution H-form spiked with aspartic acid, glutamine, γ-aminobutyric acid (GABA), citrulline, theanine, and tryptophan. The free amino acid standard solution analyzed contained 22 amino acids (Table 3).

[0045] Table 2 UPLC-MS mobile phase gradient Time (min) Flow rate (mL / min) Mobile phase A (%) Mobile phase B (%) curve initial 0.4 99.0 0.1 initial 12.0 0.4 50.0 50.0 7 13.0 0.4 99.9 0.1 6 15.0 0.4 99.9 0.1 6 Table 3 Retention time and mass-to-charge ratio of free amino acids name Time (min) Mass-to-charge ratio m / z (positive) Histidine 4.37 326.127 Asparagine 4.84 304.095 Arginine 4.94 345.141 Glutamine 5.21 317.098 Serine 5.28 276.259 Glycine 5.45 246.049 Aspartic acid 5.72 304.051 Citrulline 5.72 346.119 glutamate 5.89 318.057 Threonine 6.06 290.069 Theanine 6.28 345.152 Alanine 6.36 260.061 γ-aminobutyric acid 6.43 274.084 Proline 6.68 286.082 Lysine 7.17 487.173 Cystine 7.17 581.093 Tyrosine 7.8 352.071 Methionine 7.93 320.059 Valine 7.95 288.077 Isoleucine I 8.76 302.089 Leucine 8.88 302.089 Phenylalanine 9.13 336.072 1.3.6 Tea Tree Protein Analysis Dissolve 150uL of sample in 4x SDS sample buffer, add 650uL of methanol / chloroform (4:1) and stir for 10min, add 450μL of dd W and stir for another 5min, centrifuge at 15000rpm at 15℃ for 10min, take the middle layer pellet, wash with 1mL of 100% methanol and centrifuge, and only take the pellet.

[0046] The pellet was dried to remove methanol and then dissolved in 7 M urea, 2 M thiourea, 4% (w / v) 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonic acid (CHAPS), 1% (w / v) dithiothreitol (DTT), 2% (v / v) Pharmalyte™ carrier amphoteric lysate, and 1 mM benzamide, mixed with the sample solution, and used as the sample for two-dimensional electrophoresis.

[0047] Protein concentration was determined using the Bradford method (Bradford et al., Anal. Biochem., 1976, 72, 248). First, for isoelectric focusing (IEF), IPG strips were placed in a swelling solution consisting of 7 M urea, 2 M thiourea, 2% 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate (CHAPS), 1% dithiothreitol (DTT), and 1% Pharmalyte™ carrier amphotericin B at 20°C for 12-16 hours. IEF was performed at 20°C using an Amersham Biosciences Multiphore II system according to the manufacturer's manual.

[0048] IEF conditions were set from 150 V to 3500 V over 3 hours, with a final IEF time of 26 hours at 3500 V, reaching a maximum voltage of 96 kVh. Next, prior to SDS-PAGE analysis, the IPG strips were incubated in equilibration buffer (50 mM TrisCl, pH 6.8, 6 M urea, 2% SDS, 30% glycerol) containing 1% DTT for 10 hours, followed by incubation in equilibration buffer containing 2.5% DTT for 10 hours. The strips were then incubated in equilibration buffer containing 2.5% iodoacetamide for another 10 hours. The equilibrated strips were loaded onto SDS-PAGE gels (20 x 24 cm, 10-16%) and expanded at 20°C to a final voltage of 1.7 kVh using the Hoefer DALT 2D system (Amersham Biosciences). The proteins in the two-dimensional gel after two-dimensional electrophoresis were stained with alkaline silver by the method of Oakley et al. (Anal. Biochem. 1980, 105: 361-363), omitting the glutaraldehyde treatment step.

[0049] Alkali-silver-stained two-dimensional gels were scanned using an AGFA Duoscan T1200 scanner and saved as images to a computer via an extender. Quantitative analysis was performed using PDQuest software (version 7.0, BioRad) to determine expression changes in protein spots within the scanned images. The number of spots was normalized to the intensity of the total valid spots, and protein spots with expression changes greater than twofold compared to the control were selected.

[0050] 1.3.7 Data Statistics and Analysis Excel was used to organize and calculate the data, and Graphpad Prism 10 software was used to perform statistical analysis of all data using t-test and one-way analysis of variance. Graphs were also generated, and data were expressed as mean ± SEM. Cluster diagrams were generated using TBtools-II.

[0051] 2. Results and Analysis 2.1 Effect of PAL on the accumulation of secondary metabolites in young tea plants Polyphenols in the secondary metabolites of tea plants are an important quality indicator. Ester-type catechins and non-ester-type catechins are the main secondary metabolites in tea plants, accounting for 76% of the total tea catechins. Among them, epicatechin gallate (ECG) and epigallocatechin gallate (EGCG) account for a higher proportion. EGCG contributes more to the taste of tea than other ester-type catechins. The results of the effect of PAL on the accumulation of secondary metabolites in young tea plants are shown in Figure 1 .

[0052] Depend on Figure 1 It can be seen that the relative contents of ester catechins in young tea plants were compared by peak area normalization. PAL has a certain effect on the four main ester catechins, among which epicatechin gallate (ECG) and epigallocatechin (EGC) have a certain degree of increase, and low concentrations of PAL have a higher increase effect. Low concentrations of PAL have an increase effect on EGCG, while high concentrations of PAL reduce the accumulation of EGCG. There is no significant change in epicatechin (EC). 2.2 Effect of PAL on the accumulation of free amino acids in tea plants See the results Figure 2 and Figure 3 .

[0053] Depend on Figure 2 and Figure 3Measurements of the amino acid content of young tea plants revealed that low concentrations of PAL promoted the accumulation of most amino acids, increasing the accumulation of 14 free amino acids, with a particularly significant increase in citrulline (CIT), a highly effective free radical scavenger. Furthermore, gamma-aminobutyric acid (GABA), a non-protein amino acid involved in metabolism, was also elevated.

[0054] The accumulation of some protein-forming amino acids was downregulated, with significant differences in asparagine (Asn), histidine (His), phenylalanine (Phe), and arginine (Arg). These are all protein-forming amino acids and may be used to synthesize a variety of proteins. The non-protein amino acid theanine was also downregulated. Theanine is an important form of nitrogen storage and transport in tea plants and is often hydrolyzed for the synthesis of polyphenols. Its hydrolysis products include γ-aminobutyric acid. Theanine is also a key secondary metabolite regulating the physiological activities of tea plants. In the face of adversity, theanine may be used to reduce the accumulation of reactive oxygen species.

[0055] 2.3 Effects of PAL on protein metabolism in young tea plants See the results Figure 4 .

[0056] Depend on Figure 4 The downregulation of free amino acid accumulation revealed that protein metabolism analysis was performed to verify that some amino acids were being used for protein synthesis. A total of 122 different proteins were detected in the two-dimensional gel electrophoresis, with 45 proteins detected in the blank control group and 112 proteins detected in the PAL100-treated group. The PAL100-treated group showed upregulation of 101 proteins and downregulation of 8 proteins.

[0057] By comparing the molecular weight of the proteins with the tea plant proteins in the NCBI database, the proteins whose expression levels were increased by PAL were found (see Table 4).

[0058] The functions of the upregulated proteins include detoxification, oxidation mitigation, substance synthesis, plant growth, substance transport, etc. A0A7J7GJ58_CAMSI controls lignin degradation and detoxification of lignin-derived products.

[0059] A0A0K0NPR2_CAMSI destroys free radicals that are normally generated within cells and are toxic to biological systems.

[0060] A0A7J7GDX0_CAMSI is involved in the localization and / or fusion of transport vesicles with target membranes.

[0061] A0A7J7H8L9_CAMSI catalyzes the condensation of carbamoyl phosphate and aspartate to form carbamoyl aspartate and inorganic phosphate, which is a key step in the de novo biosynthesis of pyrimidine nucleotides.

[0062] A0A4S4EPI4_CAMSI controls auxin response factors (ARFs), which are transcription factors that bind specifically to the DNA sequence 5'-TGTCTC-3' within the auxin-responsive promoter element (Auxres).

[0063] A0A7J7HF40_CAMSI controls the capping of F-actin by a Ca 2+ They bind to the rapidly growing ends of actin filaments (barbed ends) in an independent manner, thereby preventing the exchange of subunits at these terminals. Unlike other capping proteins (such as gelatin and cleavage proteins), these proteins do not sever actin filaments.

[0064] A0A4S4EM09_CAMSI controls magnesium transporters and mediates magnesium influx. Clearly, the upregulated proteins are closely related to plant physiological growth and the mitigation of reactive oxygen species stress. However, the functions of most of these proteins remain unclear, as do the functions of the downregulated proteins.

[0065] Table 4 Proteins whose expression was upregulated No. SSP Molecular mass MR Name 2218 19774.403 A0A4S4CX76_CAMSI 2307 28108.753 A0A4S4F1V4_CAMSI 3113 16928.816 A0A7J7I058_CAMSI 3115 16467.134 A0A4S4DY46_CAMSI 3116 16352.339 A0A7J7G8I1_CAMSI 3204 22276.19 A0A7J7FQD5_CAMSI 3205 20473.186 A0A4S4CYT6_CAMSI 3206 18713.612 A0A4S4DRY0_CAMSI 3207 19171.898 A0A4S4EMV2_CAMSI 3302 29166.685 A0A4S4DY90_CAMSI 3303 27831.898 A0A4S4EKW4_CAMSI 3304 32290.218 A0A7J7I6X5_CAMSI 3305 31539.598 A0A7J7GA52_CAMSI 3403 34880.341 A0A7J7GAP3_CAMSI 3405 40994.331 A0A7J7GE60_CAMSI 3503 58917.347 A0A4S4ELQ3_CAMSI 3607 63987.907 A0A4S4ETX4_CAMSI 3611 62060.799 A0A7J7GJ58_CAMSI 4112 16067.135 A0A0K0NPR2_CAMSI 4114 14854.535 A0A7J7H8J9_CAMSI 4116 12679.785 A0A7J7GN04_CAMSI 4204 21508.467 A0A7J7HLL0_CAMSI 4306 30927.147 A0A4S4ENA8_CAMSI 4307 28085.688 A0A4S4DDQ3_CAMSI 4409 38964.775 A0A4S4DD65_CAMSI 4412 38316.36 A0A4S4D4M7_CAMSI 4513 60373.734 A0A7J7G6P6_CAMSI 5109 13341.103 A0A7J7FPY4_CAMSI 5111 14893.619 A0A7J7GX78_CAMSI 5205 21740.347 A0A4S4EB33_CAMSI 5207 23523.666 A0A7J7I7Q8_CAMSI 5210 18933.142 A0A7J7HYA0_CAMSI 5211 20139.973 A0A7J7H4B1_CAMSI 5313 29516.621 A0A7J7GDX0_CAMSI 5314 33189.381 A0A7J7G5B9_CAMSI 5315 27659.897 A0A7J7HFU0_CAMSI 5415 38765.549 A0A7J7H8L9_CAMSI 5416 39333.652 A0A7J7G3T4_CAMSI 5417 40994.331 A0A7J7GE60_CAMSI 5512 60533.691 A0A4S4EPI4_CAMSI 6109 16307.993 A0A7J7FP89_CAMSI 6110 17101.816 A0A7J7GEZ8_CAMSI 6112 13966.739 A0A7J7GXQ6_CAMSI 6209 26218.752 A0A7J7H1C0_CAMSI 6210 25516.914 A0A7J7HF40_CAMSI 6211 24264.505 A0A4S4D4Q2_CAMSI 6412 38545.609 A0A4S4E516_CAMSI 6413 38798.065 A0A7J7FPC0_CAMSI 6414 38155.701 A0A4S4D1W2_CAMSI 6415 38104.618 A0A4S4F074_CAMSI 6509 60014.076 A0A4S4DLY2_CAMSI 7201 20998.184 A0A7J7GDX4_CAMSI 7336 31895.828 A0A4S4DTU9_CAMSI 7337 32861.156 A0A4S4E1M1_CAMSI 8316 30493.143 A0A4S4ECL2_CAMSI 8514 58753.654 A0A4S4EM09_CAMSI 8516 58597.252 A0A7J7FX66_CAMSI 2.4 Effect of PAL on the biomass of young tea plants See the results Figure 5 .

[0066] Depend on Figure 5 It can be seen that the biomass data of young tea trees showed that both their above-ground biomass and underground biomass were increased by PAL, and low concentrations of PAL had better results ( Figure 5 a, 5b). Plant growth is affected by the concentration of reactive oxygen species. Low concentrations of reactive oxygen species promote growth, while excessive amounts inhibit it. This indicates that a 100-fold diluted concentration of PAL promotes tea plant growth more than a 25-fold diluted concentration.

[0067] Investigations into the height of young tea trees revealed that high concentrations of PAL (PAL25) had a stronger effect on increasing plant height ( Figure 5 c) When plants are in adversity, they tend to absorb more external resources, which is manifested here as increased plant height, which may be related to the tea tree stretching outward to obtain more light.

[0068] The results of leaf number and biomass dry weight of young tea plants are consistent ( Figure 5d). Although the leaf area of ​​the treated group increased compared to the blank control group, the increase in leaf area was not significant under the low concentration treatment, and only the high concentration treatment showed a significant increase ( Figure 5 e). This shows the same trend as plant height. The increase in leaf area can promote photosynthesis from the source end, helping plants accumulate more energy to cope with stress.

[0069] 2.5 Effect of PAL on superoxide dismutase in tea leaves See the results Figure 6 .

[0070] Depend on Figure 6 It can be seen that the activity of superoxide dismutase (SOD) can reflect the ability of cells to clear superoxide anions (O 2− ) ability. The higher the activity, the stronger the ability of the sample to remove superoxide anions, which indirectly indicates that the plant is under high reactive oxygen stress. The SOD activity in young tea leaves was measured, and the SOD activity of the treatment group was significantly reduced ( Figure 6 a), which indicates that the reactive oxygen stress in the treated group is lower than that in the blank control group. It is worth mentioning that the SOD activity test samples were collected after a long period of PAL treatment. Combined with the biomass results, the plants at this time reduced the toxic effects of reactive oxygen through physiological regulation, and therefore grew better than the blank control group ( Figure 6 b).

[0071] In summary, sodium lactate plasma affects the accumulation of secondary metabolites in tea plants, while the polyphenols produced by tea plants can chelate heavy metals, protecting them from the toxic effects of heavy metals and reactive oxygen species. This suggests that the effects of sodium lactate plasma enhance the secondary metabolism of tea plants and improve their resistance. Exogenous reactive oxygen species stimulate the production of endogenous reactive oxygen species in plants. In addition to the fact that reactive oxygen species can be directly sensed by reactive oxygen species sensors, which in turn regulate hormone synthesis, the influx of calcium ions also plays a corresponding regulatory role. Calcium ions act as a stress signal to regulate the cellular clearance of reactive oxygen species. To neutralize excess ROS, plants produce thiols, ascorbic acid, carotenoids, and flavonoids as antioxidant molecules, resulting in increased accumulation of catechins. At the same time, extracellular calcium ions further activate CDPK and MAPK cascade proteins. The activity of CDPK and MAPK stress response pathways leads to plant stress resistance, thereby promoting the expression of genes related to secondary metabolism, including phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), dihydroflavonol reductase (DFR), etc., all of which are involved in the synthesis of secondary metabolites. In addition, some reactive oxygen species can also directly activate CDPK and MAPK pathways. Ca 2+Bursts lead to the activation of CML proteins, which act through CAMTA transcription factors, resulting in stress resistance. CBL proteins interact with CBL-interacting protein kinases (CBL-CIPKs), leading to the production of reactive oxygen species in the presence of NADPH oxidase, thus providing a feedback mechanism for ROS production that supports stress resistance. CaM proteins regulate stress responses through MAPKs and GABA. Calcium-binding proteins further interact with the plant hormones IAA, ABA, GA, BR, and NO to regulate stress signaling.

[0072] By applying high and low concentrations of PAL to young tea trees and investigating their biological and agronomic traits, it was found that low concentrations of PAL had a stronger growth-promoting effect, and the exogenous reactive oxygen species it carried may activate the outbreak of endogenous reactive oxygen species in the plant body, thereby enabling the plant to enhance its own resistance and achieve the effect of promoting growth. The present application found that plasma sodium lactate changes the biomass and antioxidant properties of tea trees, among which low concentrations of plasma sodium lactate have a better growth-promoting effect. There may be multiple pathways for the plasma sodium lactate reagent to enhance the biological traits and agronomic shape of young tea trees. When plants face adversity, they will regulate physiological activities through multiple pathways to cope with adverse external effects. Calcium ion regulation mechanisms and MAPK cascade regulation are widely present in plants and animals. When under reactive oxygen stress, both play an important regulatory role.

[0073] Therefore, plasma sodium lactate affects the stress resistance of young tea plants and enhances their vitality. It causes the accumulation of secondary metabolites to resist reactive oxygen species stress. Simultaneously, the synthesis of stress-resistant amino acids and proteins is upregulated to enhance stress resistance, improving the stress resistance of young tea plants. This suggests that the application of exogenous plasma sodium lactate helps activate the resistance of young tea plants to abiotic stress.

[0074] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the invention and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the invention and various options and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for improving the stress resistance of secondary metabolites of tea plants, characterized in that: The following steps are involved: S1. Preparation of Plasma Sodium Lactate Solution At a temperature of 25°C, the sodium lactate Ringer's injection is activated by irradiation with a plasma source to obtain a plasma sodium lactate solution; S2. Foliar spray Dilute the plasma sodium lactate solution 25-100 times with ultrapure water, and spray it evenly on the front and back of the leaves of tea seedlings (1 year old) transplanted 1 week ago, until the front and back of the leaves are completely wet and liquid flows down.

2. The method for improving the stress resistance of tea plant secondary metabolites according to claim 1, characterized in that: In step S1, the mass concentration of the sodium lactate solution is 0.96%.

3. The method for improving the stress resistance of tea plant secondary metabolites according to claim 1, characterized in that: In step S1, the plasma source is connected to 60Hz AC 9kV high voltage electricity, the argon flow rate is 2L / min, and the distance between the plasma source and the sodium lactate solution is 3mm.

4. The method for improving the stress resistance of tea plant secondary metabolites according to claim 1, characterized in that: In step S1, 2 mL of the sodium lactate Green injection is irradiated and activated each time, and the irradiation activation time is 5 minutes.

5. The method for improving the stress resistance of tea plant secondary metabolites according to claim 1, characterized in that: In step S2, spraying is performed once every 15-20 days, for a total of 4 times.

6. Application of a plasma sodium lactate solution in improving the secondary metabolites and stress resistance of tea trees.

7. The use according to claim 6, characterized in that The plasma sodium lactate solution is prepared by irradiating and activating the sodium lactate solution with a plasma source at a temperature of 25° C., thereby obtaining the plasma sodium lactate solution; wherein the mass concentration of the sodium lactate solution is 11.2%; The plasma source was connected to a 60Hz AC 9kV high voltage power supply, an argon flow rate of 2L / min, and a distance between the plasma source and the sodium lactate solution was 3mm; 2 mL of the sodium lactate solution was activated in each irradiation, and the irradiation activation time was 5 min.

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