Application of methyl jasmonate in improving the content of cycloartenic acid in Cyclobalanopsis glauca under drought stress

Abstract

This invention discloses the application of methyl jasmonate in increasing the ferruginic acid content of *Ilex chinensis* under drought stress. This invention is the first to discover that foliar spraying of *Methyl jasmonate* on *Ilex chinensis* plants under drought stress can completely reverse the inhibitory effect of drought on ferruginic acid synthesis. Test results show that drought treatment alone reduces ferruginic acid content by more than 82%, while methyl jasmonate treatment alone can increase ferruginic acid content by approximately 50 times; combined spraying of methyl jasmonate under drought stress still increases ferruginic acid content by approximately 48 times. This invention also provides a method for significantly increasing ferruginic acid content under drought adversity, which is simple to operate and has stable effects, providing a new approach for the sustainable production of ferruginic acid.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the application of methyl jasmonate in increasing the ferric acid content of Ilex chinensis under drought stress. Background Technology

[0002] Rotunda Thunb., a plant belonging to the genus Ilex in the family Aquifoliaceae, contains rotundic acid, a pentacyclic triterpenoid compound, as its main active ingredient in its stem bark. Modern pharmacological studies have shown that rotundic acid possesses significant hepatoprotective, antitumor, anti-inflammatory, antidiabetic, and antioxidant activities, exhibiting high development value and commercial potential in pharmaceuticals, nutritional supplements, and cosmetics. With the increasing market demand for rotundic acid, its sustainable supply has become a major concern.

[0003] However, the acquisition of ferruginic acid mainly relies on the harvesting of wild *Ilex chinensis* resources, an unsustainable method that places enormous pressure on natural populations. Simultaneously, *Ilex chinensis* is highly susceptible to abiotic stresses such as drought during artificial cultivation. Drought stress not only severely inhibits photosynthesis, leading to oxidative damage and growth retardation, but also significantly reduces the synthesis and accumulation of secondary metabolites. Existing research indicates that under drought stress, the content of ferruginic acid in the stem bark of *Ilex chinensis* decreases dramatically (by more than 80%), severely restricting its industrial production.

[0004] Methyl jasmonate (MeJA) is an important natural signaling molecule in plants, playing a crucial role in regulating plant growth and development, responding to biotic and abiotic stresses, and controlling the synthesis of secondary metabolites. Exogenous application of MeJA has been shown to induce the accumulation of active components such as terpenes and flavonoids in various medicinal plants (such as Eucommia ulmoides, chrysanthemum, and jujube), and can also enhance the plant's tolerance to adverse conditions such as drought and salinity to a certain extent.

[0005] However, there are currently no research reports on the response of *Ilex chinensis* and its characteristic component, ferruginic acid, to methyl jasmonic acid under drought stress. Whether methyl jasmonic acid can effectively reverse the inhibitory effect of drought on ferruginic acid synthesis under drought conditions, and whether there is a synergistic effect between methyl jasmonic acid and drought stress, are also unreported. Existing technologies also do not provide a method that can simultaneously protect plant growth and significantly increase ferruginic acid content under drought stress.

[0006] Therefore, how to alleviate the inhibitory effect of drought stress on the growth of Ilex chinensis, and on this basis, even increase the yield of ferrous oleic acid in its stem bark, so as to achieve high-quality and high-yield cultivation under adverse conditions, is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide the application of methyl jasmonate in increasing the content of ferruginic acid in the bark of Ilex chinensis under drought stress.

[0008] This invention is achieved through the following technical solution:

[0009] Application of methyl jasmonate in increasing the ferric acid content of Ilex chinensis under drought stress.

[0010] Preferably, the application concentration of methyl jasmonate is 50-200 µM; more preferably, the application concentration of methyl jasmonate is 100 µM.

[0011] Preferably, the methyl jasmonate is applied by foliar spraying.

[0012] Preferably, the foliar spraying frequency is once every 3 to 5 days.

[0013] The present invention also provides a method for increasing the content of ferruginic acid in the bark of Ilex chinensis under drought stress, the method comprising applying methyl jasmonate to Ilex chinensis plants during drought stress.

[0014] Preferably, the application concentration of methyl jasmonate is 50~200 µM, and the application method is foliar spraying; more preferably, the application concentration of methyl jasmonate is 100 µM.

[0015] Preferably, the foliar spraying frequency is once every 3 to 5 days; more preferably, the foliar spraying is carried out once each on day 0, day 4 and day 8 of drought stress.

[0016] The present invention also provides the application of methyl jasmonate in the preparation of formulations for increasing the yield of ferruginic acid in the stem bark of Ilex chinensis under drought stress.

[0017] Preferably, the formulation is a foliar spray. The beneficial effects of this invention are:

[0018] 1. This invention demonstrates for the first time that methyl jasmonic acid can completely reverse the inhibitory effect of drought stress on the biosynthesis of ferromalonic acid, achieving stable yield under adverse conditions. UPLC-MS / MS analysis showed that drought treatment alone reduced ferromalonic acid content by more than 82%, while methyl jasmonic acid treatment alone increased ferromalonic acid content by approximately 50 times; even under drought stress, combined application of methyl jasmonic acid still increased ferromalonic acid content by approximately 48 times. This indicates that this invention can significantly increase ferromalonic acid yield under drought stress, overcoming the technical difficulty in the prior art of drought-induced significant reduction in ferromalonic acid production.

[0019] 2. Through system optimization, this invention has determined the preferred application concentration, application method and application frequency of methyl jasmonate, enabling those skilled in the art to easily reproduce the effects of this invention and stably increase the content of iron-containing ferrous acid under artificial cultivation conditions.

[0020] 3. This invention utilizes the property of methyl jasmonate to significantly increase the content of ferruginic acid under drought stress, enabling high yields of ferruginic acid even when artificially cultivating ferruginic acid in arid or semi-arid regions. This reduces the pressure of harvesting wild ferruginic acid resources, provides a new approach for the sustainable production of ferruginic acid, and has significant economic and ecological benefits.

[0021] Instruction manual illustrations

[0022] Figure 1 A bar chart showing the absolute peak area of ​​ferromalleic acid in each treatment group;

[0023] exist Figure 1 In the figure, the horizontal axis represents the control group (CK), drought group (D), methyl jasmonate group (MeJA), and drought + methyl jasmonate group (D+MeJA), and the vertical axis represents the peak area of ​​ferroic acid detected by UPLC-MS / MS. Data are expressed as mean ± standard error (SE), and different lowercase letters above the columns indicate significant differences between treatments (P < 0.05, Duncan multiple range test).

[0024] Figure 2 A bar chart showing the relative abundance of ferromalonic acid in each treatment group;

[0025] exist Figure 2 In the figure, the horizontal axis represents the control group (CK), drought group (D), methyl jasmonate group (MeJA), and drought + methyl jasmonate group (D+MeJA), and the vertical axis represents the percentage of the peak area of ​​ferroholic acid relative to the peak area of ​​the total detected terpenoid compounds. Data are expressed as mean ± standard error (SE), and different lowercase letters above the columns indicate significant differences between treatments (P < 0.05, Duncan multiple range test).

[0026] Figure 3 Bar graph showing the top 20 upregulated and downregulated metabolites with the largest fold difference in the drought + methyl jasmonate combined treatment group (D+MeJA) compared with the control group (CK);

[0027] exist Figure 3 In the graph, the horizontal axis represents the log2FC value, and the vertical axis represents the metabolite name. Detailed Implementation

[0028] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the present invention.

[0029] Example 1: Experiment on the effect of methyl jasmonate on increasing the content of ferruginic acid in the bark of Ilex ferruginaceus under drought stress

[0030] 1. Experimental materials

[0031] The test plants were annual Ilex rotunda Thunb. plants, purchased from Hangzhou Runtu Horticulture Technology Co., Ltd. (Yuhang District, Hangzhou, China). The plants were transplanted into pots with a rim width of 9 cm and a height of 12 cm. The cultivation substrate was soil:peat:sand = 1:1:1 (volume ratio). The plants were then subjected to one month of acclimatization cultivation in a greenhouse under the following conditions: temperature 22±2°C, photoperiod of 12 h light / 12 h dark, relative humidity 65–70%, and watering every two days. After acclimatization, plants with uniform growth and a height of approximately 45 cm were selected for the experiment.

[0032] Methyl jasmonate (MeJA, CAS No: 39924-52-2) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. A 300 mM MeJA stock solution was prepared with anhydrous ethanol, and then diluted to a 100 µM working solution with distilled water containing 0.1% (v / v) Tween-20 before use.

[0033] 2. Experimental Design

[0034] The Ilex cornuta plants were randomly divided into 4 treatment groups, with 6 biological replicates in each group:

[0035] Control group (CK): Normal watering (watering once every 3 days), foliar spraying with a solution containing an equal amount of Tween-20 and ethanol but without MeJA;

[0036] Drought group (D): Watering was completely stopped for 10 days; foliar spraying with a control solution without MeJA was applied.

[0037] MeJA group: Normal watering, and on days 0, 4 and 8 of the experiment, a 100 µM MeJA solution was sprayed on the leaves until the liquid ran down;

[0038] Drought + MeJA group (D+MeJA): Watering was completely stopped (for 10 days), and 100 µM MeJA solution was sprayed on the leaves on days 0, 4 and 8 of the experiment until the liquid ran down.

[0039] The experiment lasted for 10 days. After 10 days, when the drought-affected plants showed obvious drought symptoms such as wilting and leaf curling, three biological replicates were randomly selected from each group, and stem bark samples were collected. The samples were immediately frozen in liquid nitrogen and stored at -80°C for the detection of iron-containing ferrous acid content.

[0040] 3. Detection Method

[0041] 3.1 Determination of ferrous oleic acid content (UPLC-MS / MS)

[0042] The content of ferruginic acid in the stem bark of *Ilex chinensis* was determined by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). The specific steps are as follows:

[0043] 3.1.1 Sample Pretreatment: The freeze-dried stem bark tissue was ground into a fine powder. 30 mg of the powder was weighed and added to 600 µL of pre-cooled methanol:water (7:3, v / v) extraction buffer (containing 1 µg / mL internal standard 2-chlorophenylalanine). After vortexing and mixing, the mixture was ultrasonically extracted for 30 min in an ice-water bath. The sample was then centrifuged at 4 ℃ and 13,000 rpm for 20 min. The supernatant was filtered through a 0.22 µm filter membrane for analysis.

[0044] 3.1.2 Chromatographic conditions: The chromatographic column was an Agilent SB-C18 column (2.1 × 100 mm, 1.8 μm), and the column temperature was 40 ℃. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was acetonitrile. Gradient elution program: 0 ~ 9 min, 5% ~ 95% B. Flow rate: 0.35 mL / min;

[0045] 3.1.3 Mass spectrometry conditions: A QTRAP® 6500+ mass spectrometer was used, with an electrospray ionization (ESI) source. The positive ion mode (ESI+) had an ion spray voltage of 5500 V, and the negative ion mode (ESI-) had a voltage of -4500 V. The source temperature was 500 °C; the curtain gas pressure was 25 psi; and the nebulizer gases GS1 and GS2 were 50 psi and 60 psi, respectively. Data were acquired using multiple reaction monitoring (MRM) mode.

[0046] 3.1.4 Qualitative and Quantitative Analysis: Qualitative analysis was performed by comparing the retention time, parent ion-daughter ion pairs, and MS / MS spectra with those of ferromalleic acid standards. Relative quantification was performed using peak area, calculating the log2 fold change (log2FC) and fold change of each treatment group relative to the control group. A bar chart of the absolute peak area of ​​ferromalleic acid was then plotted using this data. Figure 1 Bar chart showing the relative abundance of ferromalleic acid as a percentage of total terpenes ( Figure 2 ).

[0047] 3.2 Determination of methyl jasmonate and abscisic acid content (UPLC-MS / MS)

[0048] To further verify whether exogenous foliar sprayed MeJA can be effectively absorbed by Ilex chinensis plants and transported to the stem bark (the main synthesis site of holly acid), and to explore the interaction between MeJA and drought stress signaling pathways, this example further detected the content of MeJA and abscisic acid (ABA) in the stem bark of each treatment group.

[0049] The determination was performed using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). The specific steps are as follows:

[0050] Sample pretreatment: Fresh, frozen *Ilex chinensis* stem bark was ground into powder in liquid nitrogen. 50 mg of powder was accurately weighed and added to 10 μL of a 100 ng / mL internal standard solution. 1 mL of a methanol / water / formic acid (15:4:1, v / v / v) extraction solvent was added, and the mixture was vortexed for 10 min. The mixture was then centrifuged at 4°C and 12000 r / min for 5 min. The supernatant was collected and concentrated to dryness. The residue was reconstituted with 100 μL of 80% methanol / water solution, filtered through a 0.22 μm filter membrane, and placed in a sample vial for analysis.

[0051] Chromatographic conditions: The column was a Waters ACQUITY UPLC HSS T3 C18 column (1.8 µm, 100 mm × 2.1 mm id); mobile phase A was ultrapure water (containing 0.04% acetic acid), and mobile phase B was acetonitrile (containing 0.04% acetic acid); gradient elution program: 0–1 min, 5% B; 1–8 min, 5% → 95% B; 8–9 min, 95% B; 9.1–12 min, 5% B; flow rate 0.35 mL / min; column temperature 40°C; injection volume 2 μL.

[0052] Mass spectrometry conditions: Electrospray ionization (ESI) was used in negative ion mode, with an ion spray voltage of -4500 V, a source temperature of 550°C, and a curtain gas pressure of 35 psi. Data were acquired using multiple reaction monitoring (MRM) mode. Qualitative analysis of methyl jasmonate and abscisic acid was based on the retention times and characteristic ion pairs of standards, while quantification was performed using the external standard method. Results are expressed as ng / g fresh weight.

[0053] 3.3 Metabolomics differential analysis (OPLS-DA and differential metabolite screening)

[0054] Extensive targeted metabolomics analysis was performed on all metabolite data obtained from the UPLC-MS / MS assays. Unit variance scaling was used to standardize the relative quantifications of metabolites. An orthogonal partial least squares discriminant analysis (OPLS-DA) model was constructed, and its effectiveness was validated using a permutation test of 200 iterations (p < 0.005). The variable projective importance (VIP) value for each metabolite was calculated, with VIP > 1.0 used as the screening criterion for significantly contributing metabolites. Simultaneously, differentially accumulated metabolites (DAMs) between the drought + MeJA combined treatment group and the control group were screened based on |log2FC| ≥ 1 and t-test p < 0.05. Based on the above analysis results, plotting... Figure 3 , Figure 3 This is a bar graph showing the top 20 upregulated and downregulated metabolites in the drought + methyl jasmonate combined treatment group (D+MeJA) compared to the control group (CK). The horizontal axis represents log2FC (log2 fold change), where positive values ​​indicate upregulation and negative values ​​indicate downregulation, and the bar length reflects the magnitude of the change; the vertical axis represents the metabolite name. The graph shows that rotundic acid ranked 17th, with a log2FC of 5.59, corresponding to an approximately 48-fold upregulation.

[0055] 4. Results

[0056] 4.1 Results of determination of iron-containing ferrous acid content

[0057] The UPLC-MS / MS detection results are shown in Table 1:

[0058] Table 1 UPLC-MS / MS Detection Results

[0059]

[0060] Figure 1 , Figure 2 The results are further presented in the form of a bar chart. Figure 1 The absolute peak area of ​​ferromalonic acid in each treatment group. Figure 2The figure shows the relative abundance of ferromalaginous acid (as a percentage of the total detected terpenoids). As shown in the figure, compared with the control group (CK), the ferromalaginous acid content in the drought-only treatment (D) was significantly reduced, with a peak area decrease of approximately 82% (p < 0.01); the ferromalaginous acid content in the MeJA-only treatment group (MeJA) was approximately 50.7 times higher than the control (log2FC = 5.66, p < 0.001); the ferromalaginous acid content in the drought + MeJA combined treatment group (D+MeJA) was approximately 48.3 times higher than the control (log2FC = 5.59, p < 0.001), showing no significant difference from the MeJA-only treatment group, but significantly higher than the drought group and the control group. These results indicate that methyl jasmonate can completely reverse the inhibitory effect of drought stress on ferromalaginous acid synthesis, achieving an approximately 48-fold over-accumulation of ferromalaginous acid even under drought conditions.

[0061] 4.2 Results of determination of methyl jasmonate and abscisic acid content

[0062] The results of the determination of MeJA and ABA content in the stem bark of Ilex chinensis in each treatment group are shown in Table 2.

[0063] Table 2. Content of methyl jasmonic acid and abscisic acid in the stem bark of *Ilex chinensis* in each treatment group

[0064]

[0065] As shown in Table 2:

[0066] Absorption and translocation of exogenous MeJA: Methyl jasmonic acid was detected only in the MeJA-sprayed treatment groups (MeJA group and D+MeJA group), with contents of 359.93 ng / g and 348.48 ng / g, respectively; while MeJA was not detected in the control group (CK) and the drought-only group (D). These results indicate that exogenous foliar-sprayed MeJA can be effectively absorbed by the leaves of *Ilex chinensis* and transported to the stem bark tissue via the plant's transport system, directly acting on the main sites of *Ilex chinensis* acid synthesis.

[0067] Co-activation of the ABA signaling pathway: ABA is a core signaling molecule in plant responses to drought stress. Compared with the control group (64.05 ng / g), drought treatment alone (D) increased ABA content to 794.78 ng / g, an increase of approximately 12.4 times; MeJA treatment alone (MeJA) had a relatively small effect on ABA content (103.7 ng / g), only slightly higher than the control group; while the combined treatment of drought and MeJA (D+MeJA) caused a sharp increase in ABA content to 3109.16 ng / g, an increase of approximately 48 times compared with the control group and approximately 3.9 times compared with the drought-only group. These results indicate that MeJA and drought stress have a significant co-activation effect on ABA accumulation, rather than a simple additive effect.

[0068] The above results indicate that exogenous foliar application of MeJA can be effectively absorbed by Ilex chinensis plants and transported to the stem bark tissue, directly acting on the main synthesis sites of ferruginic acid. It can also synergize with drought signaling pathways, significantly enhancing the plant's drought defense response. This mechanism provides direct physiological and molecular evidence to explain why "the combined application of MeJA under drought stress can still maintain an ultra-high level of ferruginic acid by approximately 48 times."

[0069] 4.3 Results of Metabolomics Differential Analysis

[0070] Figure 3 The results showed that, in the drought + MeJA combined treatment group compared with the control group, rotundic acid ranked 17th among the upregulated metabolites, with a log2FC of 5.59, an upregulation of about 48 times, which was much higher than most other metabolites.

[0071] This embodiment demonstrates that spraying 100 µM methyl jasmonate onto the leaves of Ilex chinensis plants under drought stress can significantly increase the content of holmonic acid in the stem bark, achieving unexpected technical results.

Claims

1. Application of methyl jasmonate in increasing the content of ferruginic acid in the bark of Ilex chinensis under drought stress.

2. The application according to claim 1, characterized in that: The application concentration of methyl jasmonate is 50~200 µM.

3. The application according to claim 1, characterized in that: The application concentration of methyl jasmonate is 100 µM.

4. The application according to claim 2 or 3, characterized in that: The methyl jasmonate is applied by foliar spraying.

5. The application according to claim 4, characterized in that: The foliar spraying frequency was once each on day 0, day 4, and day 8 of drought stress.

6. A method for increasing the content of ferruginic acid in the bark of *Ilex ferruginaceae* stems under drought stress, characterized in that: This includes applying methyl jasmonate to Ilex chinensis plants during drought stress.

7. The method according to claim 6, characterized in that: The methyl jasmonate was applied at a concentration of 100 µM by foliar spraying.

8. The method according to claim 7, characterized in that: The foliar spraying was performed once each on day 0, day 4, and day 8 of drought stress.

9. Application of methyl jasmonate in the preparation of formulations for increasing the yield of ferruginic acid in the stem bark of Ilex chinensis under drought stress.

10. The application according to claim 9, characterized in that: The formulation is a foliar spray.

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