Application of notoginseng saponin R1 in regulating plant root development

Abstract

The application discloses application of ginger-like notoginseng saponin R1 in regulation and control of plant root development and belongs to the technical field of biotechnology. The application finds that the natural triterpene saponin compound ginger-like notoginseng saponin R1 (ZR1) can significantly inhibit plant taproot elongation, shorten the length of root tip and elongation zone, regulate the expression of root tip stem cell regulation genes, affect auxin distribution and transport homeostasis, affect root tip active oxygen metabolism and antioxidant system, and realize precise regulation and control of plant root development. The application provides application of ZR1 in regulation and control of plant root development and preparation of plant growth regulators. ZR1 is a natural product and has the advantages of safety and environmental friendliness, and can be used in agricultural seedling raising, plant type regulation, stress resistance cultivation and basic researches of plant root development, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a new application of a natural triterpenoid saponin compound—Zingibroside R1, particularly its application in regulating plant root development and as a natural plant growth regulator. Background Technology

[0002] Plant roots are the core organs for plants to absorb water and nutrients, anchor the plant, and sense environmental stress. The development of the root system directly determines crop growth, stress resistance, and yield. Safe, efficient, and environmentally friendly plant growth regulators have irreplaceable application value in agricultural production, horticulture, and plant science research.

[0003] Currently, most plant growth regulators widely used in agricultural production are chemically synthesized products, which pose problems such as high residue risks, easy phytotoxicity, single target, and potential threats to non-target organisms. With the advancement of green agriculture, ecological agriculture, and sustainable development strategies, the development of novel plant growth regulators derived from natural products has become an important direction for industry development.

[0004] Ginger-like saponin R1 is a natural triterpenoid saponin compound. Existing research mainly focuses on its pharmacological activity, and there is no literature or patent that discloses its application in regulating plant root development or as a plant growth regulator. Summary of the Invention

[0005] This invention, through in-depth research, has for the first time discovered that zingibroside R1 (hereinafter referred to as ZR1) has significant inhibitory activity on plant root development. Using the model plant Arabidopsis thaliana as the research subject, it was confirmed that ZR1 can effectively inhibit the division, proliferation, and growth of root stem cells; the reduction in stem cell activity directly leads to limited longitudinal elongation of plant roots, morphologically manifested as a significant and precise "shortening of the taproot." ZR1 has a significant and precise regulatory effect on plant root development, and it has the potential to regulate plant root development and serve as a plant growth regulator that inhibits plant growth.

[0006] The purpose of this invention is to provide a new application of ZR1 in regulating plant root development and to provide a new natural compound for plant growth regulation.

[0007] In a first aspect, the present invention provides the application of ZR1 in regulating plant root development.

[0008] The regulation of plant root development includes one or more of the following: inhibiting the elongation of the taproot, shortening the length of the root tip and elongation zone, regulating the expression of regulatory genes of root tip stem cells, affecting the distribution and transport homeostasis of auxin, and affecting the metabolism of reactive oxygen species and the antioxidant system in the root tip.

[0009] The regulatory genes of the root tip stem cells include SHR, SCR, PLT1, PLT2, WOX5, etc.

[0010] Secondly, this invention provides the application of ZR1 in the preparation of plant growth regulators. In this application, ZR1 can be used alone or compounded with agriculturally acceptable carriers, excipients, or adjuvants to prepare plant growth regulator formulations such as aqueous solutions, solutions, or culture medium additives.

[0011] Thirdly, the present invention provides a plant growth regulator whose active ingredient comprises ZR1. Further, the plant growth regulator may also comprise an agriculturally acceptable carrier, excipient, or adjuvant.

[0012] In some embodiments, the plant growth regulator is a regulator that inhibits plant root development. Further, the plant growth regulator is a herbicide.

[0013] In some embodiments, the plant is a dicotyledonous plant. Further, the plant is a cruciferous plant. Even further, the plant includes Arabidopsis thaliana, shepherd's purse, and salt mustard, etc.

[0014] In some implementations, the concentration of ZR1 used is 7.5 μM to 10 μM.

[0015] Fourthly, the present invention provides the application of the plant growth regulator in regulating plant root development, crop seedling cultivation, or plant architecture.

[0016] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0017] This invention marks the first discovery that ginger-like notoginsenoside R1 (ZR1) possesses activity in regulating plant root development, expanding the application areas of this natural compound. ZR1 exhibits precise site of action and controllable concentration dependence, enabling root shortening and shaping, making it suitable for fine-tuning plant growth. ZR1 is a natural source, environmentally friendly, and aligns with the needs of green agriculture and ecological cultivation. ZR1 can be used as a material for plant root development research and can also be applied in agricultural scenarios such as crop seedling cultivation, plant architecture control, and stress-resistant cultivation. Attached Figure Description

[0018] Figure 1The effects of different triterpenoid saponins on Arabidopsis root growth were investigated. (A, H) represent the DMSO control, and (BG) and (IK) represent the treatments of Zingibroside R1, Notoginsenoside R1, Araloside A, 20(S)-Notoginsenoside R2, Ginsenoside R1, 20(R)-Notoginsenoside R2, Ginsenoside Rb2, Ginsenoside Rb3, and Ginsenoside Rf, respectively.

[0019] Figure 2 Effects of ZR1 on root phenotype and meristem activity in Arabidopsis thaliana. (A) Phenotypic images of Arabidopsis roots treated with 0 μM, 5 μM, 7.5 μM, and 10 μM ZR1. (B) Statistical analysis of root elongation in Arabidopsis thaliana after 7 days of ZR1 treatment, showing the concentration-dependent changes in root elongation. (C) Longitudinal sections of the meristematic and elongation zones of Arabidopsis thaliana root tips after 2 days of ZR1 treatment with 0 μM, 5 μM, 7.5 μM, and 10 μM. Red asterisks indicate the quiescent center (QC), red arrows indicate the boundaries between the meristematic and elongation zones, and black arrows indicate the boundaries between the elongation and maturity zones. Scale bar = 100 μM. (D) Quantitative analysis of the length of the meristematic zone in Arabidopsis thaliana root tips after 2 days of ZR1 treatment (concentration gradient as above). (E) Quantitative analysis of the length of the elongation zone in Arabidopsis thaliana root tips after 2 days of ZR1 treatment (concentration gradient as above). (F) Longitudinal sections of the meristematic and elongation zones of Arabidopsis root tips treated with 0 μM, 5 μM, 7.5 μM, and 10 μM ZR1 for 4 days. A red asterisk indicates the quiescent center (QC), a red arrow indicates the boundary between the meristematic and elongation zones, and a black arrow indicates the boundary between the elongation and maturity zones. Scale bar = 100 μM. (G) Quantitative analysis of the length of the meristematic zone in Arabidopsis root tips after 4 days of ZR1 treatment (concentration gradient as above). (H) Quantitative analysis of the length of the elongation zone in Arabidopsis root tips after 4 days of ZR1 treatment (concentration gradient as above).

[0020] Figure 3The effect of ZR1 on gene expression regulation in Arabidopsis root tip stem cells. (AD) represent the fluorescence intensity of pSHR::SHR-GFP after 2 days (A, B) and 4 days (C, D) of ZR1 treatment, respectively; (EH) represent the fluorescence intensity of pSCR::SCR-GFP after 2 days (E, F) and 4 days (G, H) of ZR1 treatment, respectively; (IL) represent the fluorescence intensity of pPLT1::PLT1-YFP after 2 days (I, J) and 4 days (K, L) of ZR1 treatment, respectively; (MP) represent the fluorescence intensity of pPLT2::PLT2-GFP after 2 days (M, N) and 4 days (O, P) of ZR1 treatment, respectively; (QT) represent the fluorescence intensity of pWOX5::GFP after 2 days (Q, R) and 4 days (S, T) of ZR1 treatment, respectively. Scale bar = 50 µm, n = 3.

[0021] Figure 4 The effects of ZR1 on auxin homeostasis and distribution in Arabidopsis thaliana were investigated. (AD) represent the fluorescence intensity of the auxin signal sensor DR5::GFP after 2 days (A, B) and 4 days (C, D) of ZR1 treatment, respectively; (EH) represent the expression of the auxin efflux vector marker PIN1::PIN1-GFP after 2 days (E, F) and 4 days (G, H) of ZR1 treatment, respectively; (IL) represent the expression of the auxin efflux vector marker PIN2::PIN2-GFP after 2 days (I, J) and 4 days (K, L) of ZR1 treatment, respectively; and (MP) represent the expression of the auxin efflux vector marker PIN4::PIN4-GFP after 2 days (M, N) and 4 days (O, P) of ZR1 treatment, respectively. Scale bar = 50 µm, n = 3.

[0022] Figure 5 Transcriptome analysis of Arabidopsis roots after ZR1 treatment. (A) Principal component analysis (PCA) of Arabidopsis roots 7 days after ZR1 treatment. n = 3. (B) Squared Pearson correlation coefficients (R²) between samples. (C) Visualization of differentially expressed genes among different ZR1 treatment groups using Venn diagrams. (D) DAB staining after ZR1 treatment. n = 3. (E) NBT staining after ZR1 treatment. n = 3. (F) Effects of ZR1 treatment on SOD, POD, CAT activities and GSH and GSSG contents. n = 3. (G) Expression analysis of key genes GR and GPX in the glutathione redox system after ZR1 treatment. n = 3.

[0023] Figure 6 GO enrichment analysis of differentially expressed genes for pairwise comparisons of 5 µM ZR1 versus 0 µM ZR1, 10 µM ZR1 versus 0 µM ZR1, and 10 µM ZR1 versus 5 µM ZR1. Detailed Implementation

[0024] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0026] Example 1: Effects of different triterpenoid saponins on root growth of Arabidopsis thaliana

[0027] 1. Plant Materials and Culture Conditions: Arabidopsis thaliana Col-0 ecotype was used as the wild-type material. Seeds were surface-sterilized with 75% ethanol and aseptically sown on 1 / 2 MS solid medium (pH 5.8) containing 1% sucrose. The culture dishes were vernalized at 4 ℃ for 2 days, and then transferred to a light incubator for further cultivation. The cultivation conditions were: temperature 22 ℃, photoperiod 16 h light / 8 h dark. When the taproot length of the seedlings reached approximately 1 cm, healthy seedlings with consistent growth were selected for subsequent drug treatment experiments.

[0028] 2. Drug preparation and processing methods: Weigh Zingibroside R1 (ZR1, CAS#80930-74-1), 20(R)-Notoginsenoside R2 (20R-NR2, CAS#948046-15-9), Notoginsenoside R1 (NR1, CAS#80418-24-2), Araloside A (AA, CAS#7518-22-1), 20(S)-Notoginsenoside R2 (20S-NR2, CAS#80418-25-3), Ginsenoside Rb2 (Rb2, CAS#11021-13-9), Ginsenoside Rb3 (Rb3, CAS#68406-26-8), Ginsenoside Rf (Rf, CAS#52286-58-5), Ginsenoside Ro (Ro, CAS#34367-04-9) standard was completely dissolved in dimethyl sulfoxide (DMSO) to prepare a high-concentration stock solution.

[0029] Arabidopsis thaliana Col-0 seedlings with roots approximately 1 cm in length were transferred to 1 / 2 MS medium containing different concentrations of treatments. The treatment concentrations for ZR1, 20R-NR2, NR1, AA, 20S-NR2, Rb2, Rb3, Rf, and Ro were 0, 100, and 200 μM, respectively.

[0030] 3. Phenotypic observation and root length measurement: Seven days after treatment with the above compounds, the root morphology of Arabidopsis thaliana in each treatment group was photographed and recorded using a high-definition digital camera. Results are shown below. Figure 1 Compared with the DMSO control, ZR1 showed a significant inhibitory effect on Arabidopsis root growth, which increased with increasing treatment concentration. In contrast, other triterpenoid saponins did not show a significant inhibitory effect on taproot growth at 100 μM and 200 μM.

[0031] Example 2: Effects of ZR1 on root phenotype and meristem activity in Arabidopsis thaliana

[0032] 1. Plant materials and culture conditions: Same as in Example 1.

[0033] 2. Drug preparation and treatment methods: The drug preparation was the same as in Example 1. Arabidopsis seedlings with roots approximately 1 cm in length were transferred to 1 / 2 MS solid medium containing different concentrations of ZR1 (0 μM, 5 μM, 7.5 μM, 10 μM) for continued vertical culture. Samples were taken on the 2nd, 4th, and 7th days after drug treatment to observe and record changes in root phenotype and root tip microstructure.

[0034] 3. Phenotypic observation and root length measurement: Seven days after ZR1 treatment, the overall root morphology of Arabidopsis thaliana in each treatment group was photographed and recorded using a high-definition digital camera; the elongation of the taproot of each seedling was accurately measured and statistically analyzed using ImageJ image processing software. Results are shown below. Figure 2 A and B: With increasing ZR1 concentration, the growth of the taproot of Arabidopsis thaliana was significantly inhibited, exhibiting a marked "root shortening" phenotype. Further statistical analysis of root elongation over 7 consecutive days revealed that low concentration 5 μM ZR1 treatment had little effect on root growth; however, when the concentration reached 7.5 μM and 10 μM, root elongation of Arabidopsis thaliana was significantly inhibited.

[0035] 4. Observation and Length Measurement of Root Tip Tissue Structure: Arabidopsis seedlings from each concentration group were collected on days 2 and 4 after ZR1 treatment. After clearing with a clearing solution (chloral hydrate:glycerol:water = 9:3:1), the seedlings were mounted on glass slides. Differential interference contrast microscopy (DIC) was used for observation, and the lengths of the meristematic zone and elongation zone were measured using ImageJ software. Results are shown below. Figure 2C~H: Quantitative analysis of ZR1 after 2 days of treatment showed that the length of both the growing zone and elongation zone of Arabidopsis root showed a significant dose-dependent decrease. Figure 2 C). The 10 μM treatment group showed the most significant morphological changes, with a 45.9% reduction in meristematic zone length and a 66.1% reduction in elongation zone length compared to the control group. Figure 2 D, E). Similarly, after 4 days of ZR1 treatment, the length of the rhizosphere and the number of cortical cells in Arabidopsis roots were significantly reduced (D, E). Figure 2 F, G, H).

[0036] 5. Data analysis was performed using GraphPad Prism 8 software. Data are expressed as mean ± standard deviation (SD). Comparisons among multiple groups were performed using one-way ANOVA combined with Tukey's multiple comparison test, with * < 0.05 and ** < 0.01.

[0037] Example 3: Effects of ZR1 on gene expression regulation in Arabidopsis root tip stem cells

[0038] 1. Plant materials and culture conditions: The transgenic line pSHR::SHR-GFP, a marker for Arabidopsis stem cells, was used. pSCR::SCR-GFP pPLT1::PLT1-YFP pPLT2::PLT2-GFP and pWOX5::GFP These were used as experimental materials. The sterilization and culture conditions were the same as in Example 1.

[0039] The construction of the Arabidopsis transgenic lines used in this embodiment and Example 4 is described in the following prior art:

[0040] [1] Clark NM, Hinde E, Winter CM, et al. Tracking transcription factor mobility and interaction in arabidopsis roots with fluorescencecorrelation spectroscopy [J]. Elife, 2016, 5.

[0041] [2] Wysocka-Diller JW, Helariutta Y, Fukaki H, et al. Molecularanalysis of scarecrow function reveals a radial patterning mechanism commonto root and shoot [J]. Development, 2000, 127(3): 595-603.

[0042] [3] Galinha C, Hofhuis H, Luijten M, et al. A plethora of proteins asdose-dependent master regulators of arabidopsis root development [J]. Nature,2007,449(7165):1053-1057.

[0043] [4] Blilou I, Xu J, Wildwater M, et al. The pin auxin effluxfacilitator network controls growth and patterning in Arabidopsis roots [J].Nature, 2005, 433(7021): 39-44.

[0044] [5] Sabatini S, Beis D, Wolkenfeldt H, et al. An auxin-dependentdistal organizer of pattern and polarity in the Arabidopsis root [J]. Cell, 1999, 99(5): 463-472.

[0045] [6] Benkova E, Michniewicz M, Sauer M, et al. Local, efflux-dependentauxin gradients as a common module for plant organ formation [J]. Cell, 2003,115(5): 591-602.

[0046] [7] Vieten A, Vanneste S, Wiśniewska J, et al. Functional redundancy of pin proteins is accompanied by auxin-dependent cross-regulation of pinexpression [J]. Development, 2005, 132(20): 4521-4531.

[0047] 2. Drug preparation and treatment method: The drug preparation is the same as in Example 1. After culturing in 1 / 2 MS medium until the root length is about 1 cm, the Arabidopsis thaliana marker seedlings are aseptically transferred to 1 / 2 MS medium treatment plates containing different concentrations (0 μM, 5 μM, 10 μM) of ZR1.

[0048] 3. Laser confocal fluorescence observation and quantitative analysis: Root tips were imaged using a laser confocal scanning microscope (Olympus FV3000RS) with a 20× objective. Fluorescence detection parameters were set as follows: GFP excitation wavelength 488 nm / emission wavelength 500–550 nm, YFP excitation wavelength 514 nm / emission wavelength 525–575 nm, and propidium iodide (PI) excitation wavelength 561 nm / emission wavelength 570–620 nm. ImageJ software was used for quantitative analysis of the fluorescence images, with the average fluorescence intensity value of the root tip QC representing the expression level of each marker gene. Results are shown below. Figure 3 After treatment with 10 μM ZR1 for 2 and 4 days, the fluorescence intensity of pSCR::SCR-GFP and pSHR::SHR-GFP in Arabidopsis root tips was significantly reduced. Figure 3 AH). In contrast, treatment with 5 μM ZR1 for 2 days only significantly reduced the fluorescence intensity of pSCR::SCR-GFP (AH). Figure 3 AH). Whether treated for 2 days or 4 days, 10 μM ZR1 significantly reduced the fluorescence intensity of pPLT1::PLT1-YFP and pPLT2::PLT2-GFP in Arabidopsis root tips (AH). Figure 3 Furthermore, after treatment with 5 μM ZR1 for 2 days, the fluorescence intensity of pPLT1::PLT1-YFP decreased significantly, while the fluorescence intensity of pPLT2::PLT2-GFP increased significantly. Figure 3 IP). After 4 days of ZR1 treatment, pWOX5::GFP showed significantly induced fluorescence intensity at concentrations of 5 μM and 10 μM ( Figure 3The above results indicate that ZR1 can inhibit the expression of genes related to root meristem maintenance and cell proliferation, such as SCR, SHR, PLT1, and PLT2, while inducing the expression of the root quiescent center marker gene WOX5. This suggests that ZR1 disrupts root tip meristem homeostasis and thus inhibits Arabidopsis root development.

[0049] Example 4: Effects of ZR1 on auxin homeostasis and distribution in Arabidopsis thaliana

[0050] 1. Plant materials and culture conditions: The transgenic line DR5::GFP, marked with Arabidopsis thaliana auxin, was used. pPIN1::PIN1-GFP pPIN2::PIN2-GFP and pPIN4::PIN4-GFP These were used as experimental materials. The sterilization and culture conditions were the same as in Example 1.

[0051] 2. Drug preparation and treatment method: The drug preparation is the same as in Example 1. After culturing in 1 / 2 MS medium until the root length is about 1 cm, the Arabidopsis thaliana marker seedlings are aseptically transferred to 1 / 2 MS medium treatment plates containing different concentrations (0 μM, 5 μM, 10 μM) of ZR1.

[0052] 3. Laser confocal fluorescence observation and quantitative analysis: The fluorescence detection parameters and quantitative analysis were set as in Example 3. Results are shown below. Figure 4 Two days after treatment with ZR1, the fluorescence signal of DR5::GFP in Arabidopsis roots did not change significantly. Figure 4 (A, B), while after 4 days of ZR1 treatment, DR5::GFP fluorescence was significantly reduced only at a concentration of 10 μM ( Figure 4 C, D).

[0053] To further elucidate the molecular mechanism by which ZR1 affects auxin distribution, the PIN-FORMED (PIN) family of key regulators of polar auxin transport was analyzed. The results showed that after 2 days of ZR1 treatment, PIN1, PIN2, and PIN4 all exhibited a significant decrease in fluorescence intensity at a concentration of 10 μM. Figure 4 E, F, I, J, M, N). After 4 days of ZR1 treatment, the fluorescence intensity of PIN1 decreased significantly only at a concentration of 10 μM. Figure 4 G, H), while other PIN family members did not show significant changes (G ... Figure 4 K, L, O, P).

[0054] The above results indicate that ZR1 can affect the auxin signaling state and polar transport processes in Arabidopsis roots, mainly by reducing DII-VENUS signaling and inhibiting the expression of some PIN proteins, with PIN1, PIN2, and PIN4 being affected to varying degrees in the early response. Since PIN protein-mediated polar auxin transport is crucial for the establishment of the auxin gradient at the root tip and root elongation, this suggests that ZR1 may inhibit Arabidopsis root development by disrupting auxin distribution and transport homeostasis.

[0055] Example 5: Effects of ZR1 on the Arabidopsis transcriptome and antioxidant system

[0056] 1. Sample Collection, RNA Extraction, Library Construction, and High-Throughput Sequencing: Root tip samples for transcriptome analysis were collected using a sterile double-edged scalpel, immediately flash-frozen in liquid nitrogen, and stored at -80°C for later use. Three independent biological replicates were set up for each treatment group. Approximately 200 mg of tissue powder, ground in liquid nitrogen, was used to extract total RNA according to the TRIzol™ reagent (ThermoFisher Scientific) instructions. The purity and concentration of RNA were determined using a NanoDrop 2000 spectrophotometer. 0.5 μg of total RNA was used to synthesize first-strand cDNA using a cDNA synthesis kit (Takara), followed by the addition of DNA polymerase I, dNTPs, and RNase H to synthesize second-strand cDNA. Double-stranded cDNA was purified using a PCR extraction kit, and sequencing libraries were constructed through end repair, A-tailing, and adapter ligation. The libraries were then subjected to high-throughput sequencing on an Illumina NovaSeq 6000 platform (Illumina, USA).

[0057] 2. DAB staining: Place the sample in 50 mM Tris-HCl buffer (pH 5.0) containing 1 mg / mL DAB and incubate at room temperature in the dark for 1 hour. NBT staining: Place the sample in 20 mM phosphate buffer (pH 6.1) containing 2 mM NBT and soak at room temperature for 20 minutes. After each staining step, rinse the sample three times with distilled water. The staining effect was observed and evaluated using DIC, and the images were analyzed using ImageJ software.

[0058] 3. Determination of Antioxidant Enzyme Activity and Glutathione Content: Root tip tissues from wild-type Arabidopsis seedlings treated with different concentrations of ZR1 (0, 5, 7.5, and 10 μM) for 7 days were collected, flash-frozen in liquid nitrogen, and thoroughly ground. Approximately 0.1 g of tissue powder was weighed for subsequent index determination. Catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) activities were detected using the corresponding CAT, POD, and SOD activity assay kits, respectively. Reduced glutathione (GSH) and oxidized glutathione (GSSG) contents were determined using the corresponding GSH and GSSG content assay kits, respectively. All commercially available kits were purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0059] 4. PCA clearly divided the samples into three distinct clusters, corresponding to the control group, the 5 μM ZR1 treatment group, and the 10 μM ZR1 treatment group, respectively. Figure 5 A). The Pearson correlation coefficient (R² > 0.98) among biological replicates within each treatment group demonstrates the high reproducibility of the data in this invention. Figure 5 B). Venn diagram analysis showed that 78 core DEGs were consistently regulated across all three comparison groups, and there was significant overlap of 660 DEGs between the two ZR1 treatment groups and the control group. Figure 5 C). To elucidate these DEG-related biological processes, gene ontology (GO) enrichment analysis was performed to identify functional categories. Comparative GO enrichment analysis showed that differentially expressed genes common to both ZR1 treatment groups and the control group were significantly enriched during hypoxia response (FDR < 0.05). Figure 6 ).

[0060] The above data indicate that ZR1 treatment creates a hypoxic microenvironment in root cells, which may trigger NADH accumulation and oxidative stress through excessive production of reactive oxygen species (ROS). To assess ROS accumulation in root tip meristems, DAB and NBT staining were performed after ZR1 treatment. The results showed that as the ZR1 treatment concentration increased, DAB staining gradually lightened, while NBT staining gradually deepened. This indicates that ZR1 treatment increased the concentration of superoxide anions (O2⁻) in the root tip while decreasing the concentration of hydrogen peroxide (H₂O₂). Figure 3 D, E). To investigate the cellular antioxidant response after ZR1 treatment, the activities of SOD, POD, and CAT, as well as the contents of GSH and GSSG, were measured. The results showed that the activities of key reactive oxygen species scavenging enzymes SOD, CAT, and POD were reduced, indicating that their ability to scavenge reactive oxygen species was impaired. Figure 5F). In plant hypoxia response, the GSH / GSSG balance is a key detoxification pathway. Treatment with 10 μM ZR1 significantly increased GSH while decreasing GSSG, leading to an increased GSH / GSSG ratio (F). Figure 5 F), which corresponds to the expression patterns of GR and GPX ( Figure 3 G). These findings indicate that ZR1 treatment impairs the ROS scavenging capacity of root tips by inactivating SOD, CAT, and POD, thereby blocking the conversion of superoxide to H2O2; this renders GPX-mediated GSH utilization redundant, leading to an increased GSH / GSSG ratio.

[0061] The above embodiments are only used to help illustrate the present invention. The implementation of the present invention is not limited to the above embodiments. Any other changes made without departing from the principle of the present invention should be considered as equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. Application of notoginsenoside R1 in regulating plant root development.

2. The application according to claim 1, characterized in that: The regulation of plant root development includes one or more of the following: inhibiting the elongation of the taproot, shortening the length of the root tip and elongation zone, regulating the expression of regulatory genes of root tip stem cells, affecting the distribution and transport homeostasis of auxin, and affecting the metabolism of reactive oxygen species and the antioxidant system in the root tip.

3. The application according to claim 2, characterized in that: The regulatory genes of the root tip stem cells include SHR, SCR, PLT1, PLT2, and WOX5.

4. The application according to claim 1, characterized in that: The concentration of the ginger-like notoginsenoside R1 used is 7.5 μM to 10 μM.

5. Application of ginger-like notoginsenoside R1 in the preparation of plant growth regulators.

6. The application according to claim 5, characterized in that: The plant growth regulator is a regulator that inhibits the development of plant roots.

7. The application according to claim 5, characterized in that: The plant growth regulator is a herbicide.

8. A plant growth regulator, characterized in that: The active ingredient includes ginger-like saponin R1.

9. The plant growth regulator according to claim 8, characterized in that: It also includes agriculturally acceptable carriers, additives, or auxiliaries.

10. The application of the plant growth regulator according to claim 8 or 9 in regulating plant root development, crop seedling cultivation, or plant architecture.

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