A method for delaying leaf senescence in seashore paspalum under salt stress
By using the NADPH oxidase inhibitor DPI to inhibit root-derived H2O2, combined with mass spectrometry and gene expression analysis, jasmonic acid synthesis was regulated, thus solving the problem of leaf senescence under salt stress in Paspalum notoginseng and achieving the effect of delaying leaf senescence.
Patent Information
- Application Number
- CN202311205797.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-19
- Publication Date
- 2025-11-14
- Estimated Expiration
- 2043-09-19
AI Technical Summary
The phenomenon of leaf senescence in Paspalum notoriety under salt stress has not been effectively addressed, and the regulatory role of root-derived H2O2 in jasmonic acid synthesis has not been reported.
By using the NADPH oxidase inhibitor diphenylthioide (DPI) to inhibit the production of root H2O2, combined with mass spectrometry and gene expression analysis, we studied its effect on jasmonic acid synthesis and explored the role of root H2O2 in delaying leaf senescence.
It effectively delayed the senescence process of seashore paspalum leaves under salt stress, significantly reduced the JA content in the leaves by regulating jasmonic acid synthesis, and maintained the healthy state of the leaves.
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Figure CN117256458B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of plant science, and to a method for delaying leaf senescence in Paspalum notatum under salt stress; specifically, it relates to a method for reducing jasmonic acid content in Paspalum notatum leaves under salt stress and delaying leaf senescence. Background Technology
[0002] When plants are subjected to environmental stress, they produce and accumulate reactive oxygen species (ROS). High concentrations of ROS are toxic to plant cells, but low concentrations act as signaling molecules to regulate plant responses to abiotic stresses. Existing research shows that plasma membrane NADPH oxidases, a type of transmembrane redox enzyme, play a crucial role in responding to abiotic stresses and regulating plant growth and development. Recent studies emphasize that plasma membrane NADPH oxidase-mediated H2O2 plays a key positive regulatory role in plant sensing and response to salt stress. Unlike sweet soil plants, halophytes have developed robust and sophisticated salt tolerance mechanisms through long-term evolution. Studies have shown that the roots of the halophyte *Paspalum notatum* actively absorb sodium... + The method promotes the production of root H2O2, thereby activating key salt tolerance strategies such as the antioxidant system and lipid membrane adaptive remodeling, to improve the salt tolerance of seashore paspalum. In addition, the latest research shows that root NADPH oxidase-mediated H2O2 also participates in regulating leaf senescence induced by the plant senescence hormone abscisic acid (ABA).
[0003] Jasmonic acid (JA) is a plant senescence hormone controlled by environmental factors and plays a positive regulatory role in leaf senescence. Salt stress induces the production and accumulation of endogenous JA in the roots, leading to an increase in JA content in leaves, chloroplast degradation, and promoting leaf senescence. However, it has not yet been reported whether root-derived H2O2 under salt-driven conditions participates in regulating the endogenous homeostasis of JA to delay leaf senescence. Summary of the Invention
[0004] To address the aforementioned problems, this invention proposes a method for delaying leaf senescence under salt stress in order to solve the problems in the prior art; the purpose of this invention is to demonstrate that H2O2 from the roots of Paspalum notatum delays leaf senescence under salt stress by affecting jasmonic acid synthesis.
[0005] The technical solution of this invention is as follows: A method for delaying leaf senescence of Paspalum notatum under salt stress, the specific operation steps of which are as follows:
[0006] Step (1), plant material culture and salt stress treatment; specifically;
[0007] Select two-month-old and uniformly grown puffin stolons and plant them in seedling cups with holes at the bottom; hydroponically cultivate in Hogrange solution at pH 6.0, changing the solution every 7 days;
[0008] The stolons of the puffin plant were placed in a plant culture box for salt treatment after 14 days of growth.
[0009] The plant cultivation box has a light cycle of 16h / 8h (light / dark), a temperature of 30℃ / 28℃ (day / night), a relative humidity of 70%, and a light intensity of 1000lx.
[0010] The salt-treated and control-treated *Paspalum notatum* materials were then cultured in hydroponic plastic containers with or without 400 mM NaCl. The NaCl concentration was gradually increased by 100 mM each day until the target concentration of 400 mM was reached. On the fifth day of salt treatment, samples were taken from both the salt-treated and control-treated *Paspalum notatum* materials. To study the NADPH oxidase-mediated root H2O2 reaction, 100 μM diphenyl iodine chloride (DPI) was added to the 400 mM NaCl solution.
[0011] Five days after treatment, roots were collected for subsequent analysis. Six biological replicates were set up for each treatment; the following experiments were conducted using 3 to 6 biological replicates.
[0012] Step (2), H2O2 content determination; specifically, the change in the root H2O2 content is determined using an H2O2 determination kit;
[0013] Step (3), DAB staining; specifically: the separated roots are placed in DAB staining solution with a concentration of 0.5 mg / ml and stained in the dark at room temperature (usually 25-30℃) for 2 to 6 hours; finally, after rinsing with pure water 5 times, the roots are photographed and stored at room temperature.
[0014] Step (4), determination of chlorophyll content and Fv / Fm value; specifically: the total chlorophyll content (Chl(a+b)) was determined by spectrophotometry;
[0015] After rinsing the leaves with distilled water, blot the surface moisture with filter paper. Quickly weigh out 3 portions of the chopped fresh leaves, 2g each, and grind them thoroughly into a paste in a mortar with a small amount of quartz sand. Then add 10ml of ethanol and continue grinding until the tissue turns white.
[0016] After filtering in a funnel, the filtrate was diluted to 100 ml with ethanol.
[0017] The absorbance was measured at 665 nm, 645 nm and 652 nm, respectively, with 95% ethanol as a blank control; Chl(a+b)=A625 / 35.5×V÷1000×W;
[0018] Step (5), lipidomics data determination and analysis: Specifically, approximately 100 mg of leaf and root samples were taken, added to liquid nitrogen, and ground into powder. Lipids were extracted using methanol:distilled water (1:1). A Nexera UHPLC LC-30A system (Shimadzu, Kyoto, Japan) and a TripleTOF5600+ (AB SCIEX) were used. TM Lipids were analyzed by LC-MS using a mass spectrometer. The column temperature was set to 40℃.
[0019] The mobile phase was 0.1% HCOOH-H2O (A)-acetonitrile (B), the flow rate was 0.3 mL / min, and gradient washing was used.
[0020] The TripleTOF5600+ (AB SCIEX) TM The ESI source conditions in the mass spectrometer were set as follows: Ion source Gas1 (Gas1): 50, Ion source Gas2 (Gas 2): 50, Curtain gas (CUR): 25, Source temperature: 500℃ / 450℃ (positive ion / negative ion), Ion Sapari voltage fluctuation (ISVF) 5500V / 4400V (positive ion / negative ion), TOF-MS scan range: 100-1200Da, TOF MS scan accumulation time 0.2s, Declustering potential (DP): ±60V;
[0021] LipidSearch software is used for automated lipid identification and relative quantification analysis (Thermo Fisher Scientific, CA, USA);
[0022] Step (6), endogenous hormone level analysis; specifically: 50 mg of fresh root system is frozen in liquid nitrogen and placed in a 2 ml plastic microtube for sample preparation and extraction;
[0023] The plant hormone content was detected using an AB SCIEX QTRAP 6500LC / MS / MS system.
[0024] Step (7): Analyze gene expression levels using real-time quantitative PCR technology; specifically: analyze the relative expression levels of four genes using real-time quantitative PCR; design and synthesize specific primers based on gene sequences;
[0025] 2.5 μL of total RNA was reverse transcribed into cDNA according to the Bio-Rad iScript cDNA Synthesis Kit instructions; PCR was performed using an ABI 7500 Real-Time PCR System (Applied Biosystems) and Premix EX-TaqTM II was able to run; PCR amplification was performed using a 20 μL volume, with 2.5 μL of cDNA working solution, 10 μL of PCR-mix, 5.5 μL of autoclaved deionized water, and 1 μL each of upstream and downstream primers added to each PCR reaction tube;
[0026] The PCR reaction conditions were set as follows: 95℃ pre-denaturation for 30 s, 1 cycle; 95℃ denaturation for 5 s; 60℃ annealing for 34 s; 72℃ extension for 45 s, 40 cycles;
[0027] The fluorescence signal was collected during the extension phase, and the Ct value was derived after the reaction was completed.
[0028] The relative expression level is calculated according to formula 2-ΔΔCt;
[0029] The α-actin gene was used as an internal reference gene for relative quantitative analysis of the treated and control materials, with three replicates for each sample.
[0030] Step (8), transcriptomics data and analysis; specifically: extracting total RNA from leaves and roots using a plant total RNA kit;
[0031] Transcriptome sequencing and paired-end sequencing of de novo cDNA library assembly were performed using the HiSeq 2000 (Illumina Technologies) platform;
[0032] Raw reads were quality assessed, and adapter sequences and low-quality bases were then filtered using Trimmomatic (v0.32). Clean reads were aligned with a reference genome using HISAT2 (v2.1.0); gene expression was normalized by calculating reads per kilobase per million mapped reads (RPKM).
[0033] Differentially expressed genes were identified using the DESeq2 package in R software, and statistically significant differences in gene expression were determined using a false discovery rate (FDR) ≤ 0.05 and |log2FC| ≥ 1 as thresholds.
[0034] GO and KEGG metabolic pathway analyses were performed to determine the biological roles and functions of differentially expressed genes.
[0035] RNA-seq reads are stored in the NCBI Sequence Reading Archive (SRA) (Bioproject accession number: PRJNA874860).
[0036] The beneficial effects of this invention are as follows: 1. Using diphenylthioiodine (DPI), a specific inhibitor of NADPH oxidase, to study the role of root-borne H2O2 is the first key technology for implementing this project. As a common and important root-borne NADPH oxidase inhibitor, 100 μM of DPI can completely inhibit the enzyme-mediated root-borne H2O2, facilitating the study of the effect of low root-borne H2O2 content on leaf senescence. 2. From the gene expression level, the influence of the presence or absence of root-borne H2O2 on the expression of genes related to jasmonic acid synthesis precursors is explored. 3. Omics technology and data analysis based on mass spectrometry are the first key technologies for implementing this project. Lipidomics, as one of the most important branches of metabolomics, can perform qualitative and quantitative analysis at the level of individual lipid species. Attached Figure Description
[0037] Figure 1 This is a schematic diagram of the role of root NADPH oxidase-mediated H2O2 in delaying the salt-induced leaf senescence process in Example 1 of the present invention; wherein, Figure AB is a schematic diagram of leaf senescence caused by the combined application of NaCl stress and DPI; Figure CD is a diagram of trypan blue staining results; Figure E is a diagram of the multiple of chlorophyll content in salt-treated leaves compared to the combined treatment of salt stress and DPI.
[0038] Figure 2 This is a schematic diagram illustrating the effects of exogenous DPI and salt treatment on the root JA and CK concentration levels in Example 2 of the present invention; wherein, Figure A is a schematic diagram showing the increase of JA and its metabolic derivatives in the roots by salt treatment + DPI treatment; Figure B is a schematic diagram showing the effect of salt treatment + DPI treatment on the root CK of Amur arugula using LC-MS-based endogenous hormone level analysis.
[0039] Figure 3 Figure 3 illustrates the effects of exogenous DPI and salt treatment on the concentrations of JA and CK in leaves in Example 3 of this invention. Figure A is a schematic diagram illustrating the effects of salt treatment + DPI treatment on the concentration of plant hormone CK in leaves using lipidomics technology. Figure B is a schematic diagram illustrating the effects of salt treatment + DPI treatment on the concentration of plant hormone JA in leaves using lipidomics technology.
[0040] Figure 4 This is a schematic diagram illustrating the changes in the types and contents of lipids in roots under salt stress under DPI treatment or exogenous addition of low concentration H2O2, as analyzed using lipidomics technology in Example 4 of the present invention. Figure A is a schematic diagram of the effect of exogenous addition of low concentration H2O2 on the synthesis of C18 fatty acids after salt treatment; Figure B is a schematic diagram showing how salt treatment + DPI treatment severely inhibited the expression level of the PvACC1 gene in roots.
[0041] Figure 5 This is a flowchart of the operation of the present invention. Detailed Implementation
[0042] To more clearly illustrate the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to the accompanying drawings:
[0043] like Figure 5 As shown, this invention utilizes diphenylthioide (DPI), a specific inhibitor of NADPH oxidase, to inhibit root-derived H2O2 produced under salt treatment conditions, and measures physiological and biochemical characteristics related to leaf senescence (including chlorophyll content and F2O2). v / F m The study also investigated the expression of leaf senescence-related genes and the changes in the content of jasmonic acid and its derivatives by mass spectrometry.
[0044] The specific steps of the method for delaying leaf senescence of Paspalum notatum under salt stress described in this invention are as follows:
[0045] 1. Plant material culture and salt stress treatment:
[0046] Runners of *Grin-UPG145*, approximately two months old and of uniform size and growth, were planted in seedling cups with holes at the bottom. They were hydroponically cultured in Hogrange solution at pH 6.0, with the solution changed every 7 days. The incubator was set with a 16h / 8h light / dark cycle, a temperature of 30℃ / 28℃ (day / night), a relative humidity of 70%, and a light intensity of 1000 lx. Salt treatment and control treatments were conducted in hydroponic plastic containers with or without 400 mM NaCl. The NaCl concentration was gradually increased by 100 mM daily until the target concentration of 400 mM was reached. On the fifth day of salt treatment, samples were taken from both salt-treated and control *Grin-UPG145*. To investigate NADPH oxidase-mediated root-derived H2O2, 100 μM diphenyl iodine chloride (DPI) was added to the 400 mM NaCl solution. After 5 days of treatment, roots were collected for subsequent analysis. Six biological replicates were set up for each treatment. The following experiment was performed using 3 to 6 biological replicates;
[0047] 2. Determination of H2O2 content:
[0048] The change in root source H2O2 content was determined using an H2O2 assay kit (product number: BC3590);
[0049] 3. DAB staining:
[0050] The isolated roots were placed in DAB staining solution with a concentration of 0.5 mg / ml and stained in the dark at room temperature for 2 to 6 hours. Finally, they were rinsed 5 times with pure water, photographed and stored at room temperature.
[0051] 4. Chlorophyll content and Fv / Fm value determination:
[0052] Total chlorophyll content (Chl(a+b)) was determined spectrophotometrically. After rinsing the leaves with distilled water and blotting them dry with filter paper, three 2g portions of freshly chopped leaves were quickly weighed and ground into a paste in a mortar with a small amount of quartz sand. 10ml of ethanol was added, and grinding continued until the tissue turned white. The paste was filtered through a funnel and the filtrate was brought to a final volume of 100ml with ethanol. The absorbance was measured at 665nm, 645nm, and 652nm, with 95% ethanol as a blank control. Chl(a+b) = A625 / 35.5 × V ÷ 1000 × W;
[0053] Fv / Fm values were determined using the Pocket PEA plant efficiency analyzer.
[0054] 5. Lipidomics data measurement and analysis:
[0055] Approximately 100 mg of leaf and root samples were taken, added to liquid nitrogen, and ground into powder. Lipids were extracted using methanol:distilled water (1:1). A Nexera UHPLC LC-30A system (Shimadzu) and a TripleTOF 5600+ (AB SCIEX) were used for extraction. TM Lipids were analyzed by LC-MS using a mass spectrometer; the column temperature was set to 40℃. The mobile phase was 0.1% HCOOH-H2O (A)-acetonitrile (B), the flow rate was 0.3 mL / min, and gradient washing was used.
[0056] The ESI source settings are as follows: Ion source Gas1 (Gas 1): 50, Ion source Gas2 (Gas 2): 50, Curtain gas (CUR): 25, Source temperature: 500℃ / 450℃ (positive ion / negative ion), Ion Saparilla voltage fluctuation (ISVF): 5500V / 4400V (positive ion / negative ion), TOF-MS scan range: 100-1200 Da, TOF MS scan accumulation time: 0.2 s, Declustering potential (DP): ±60V. LipidSearch software was used for automated lipid identification and relative quantification analysis (Thermo Fisher Scientific).
[0057] 6. Analysis of endogenous hormone levels:
[0058] 50 mg of fresh root system was frozen in liquid nitrogen and placed in a 2 ml plastic microtube for sample preparation and extraction;
[0059] The plant hormone content was detected using an AB SCIEX QTRAP 6500LC / MS / MS system.
[0060] 7. Real-time quantitative PCR technology for analyzing gene expression levels:
[0061] The relative expression levels of four genes were analyzed using real-time quantitative PCR. Specific primers were designed and synthesized based on the gene sequences.
[0062] 2.5 μL of total RNA was reverse transcribed into cDNA according to the Bio-Rad iScript cDNA Synthesis Kit instructions; PCR was performed using an ABI 7500 Real-Time PCR System (Applied Biosystems) and Premix EX-Taq TM II was able to run; PCR amplification was performed using a 20 μL volume, with 2.5 μL of cDNA working solution, 10 μL of PCR-mix, 5.5 μL of autoclaved deionized water, and 1 μL each of upstream and downstream primers added to each PCR reaction tube;
[0063] The PCR reaction conditions were set as follows: 95℃ pre-denaturation for 30 s, 1 cycle; 95℃ denaturation for 5 s; 60℃ annealing for 34 s; 72℃ extension for 45 s, 40 cycles;
[0064] The fluorescence signal was collected during the extension phase, and the Ct value was derived after the reaction was completed.
[0065] The relative expression level was calculated using formula 2-ΔΔCt. The α-actin gene was used as an internal reference gene for relative quantitative analysis of the treated and control materials, with three replicates for each sample.
[0066] Gene sequence information: α-actin forward (5′-CTTGCGTATGTGGCTCTTGA-3′) and reverse (5′-AGGGCATCTGAACCTCTCTG-3′); PvACC1 (Acetyl-CoA carboxylase1) forward (5′-GATAGTGAAATGTGGAACGACG-3′) and reverse (5′-TCAGCAAGATGCGAGAACC-3′); PvFAD3 (omega-3fatty acid desaturase)forward(5′-CACAGGACACACCATCAGAAC-3′)and reverse(5′-GAAGAAAGTGGGAGCCGC-3′); PvFAD7(omega-7fatty acid desaturase)forward(5′-AT3G11170TATCGTGGAAAGGAATGGAG-3′)and reverse(5′-GGATTTGAGGGAAAAGGTGA-3′); PvROBHD(respiratory burst oxidase homolog D)forward(5′-TCCCGAAGGACTTGGCTACATT-3′)and reverse(5′-CTGGCTTAGTGCTTGGCTTGTG-3′);
[0067] 8. Transcriptomics data and analysis:
[0068] Total RNA was extracted from leaves and roots using a plant total RNA kit.
[0069] Transcriptome sequencing and paired-end sequencing of de novo cDNA library assembly were performed using the HiSeq 2000 (Illumina Technologies) platform; raw reads were quality assessed and then adapter sequences and low-quality bases were filtered using Trimmomatic (v0.32); clean reads were aligned with a reference genome using HISAT2 (v2.1.0); gene expression was normalized by calculating reads per kilobase per million mapped reads (RPKM); differentially expressed genes were identified using the DESeq2 package in R software, and statistically significant differences in gene expression were determined using a false discovery rate (FDR) ≤ 0.05 and |log2 FC| ≥ 1 as thresholds; GO analysis and KEGG metabolic pathway analysis were performed to determine the biological roles and functions of differentially expressed genes; RNA-seq reads were stored in the NCBI Sequence Reading Archive (SRA) (Bioproject accession number: PRJNA874860).
[0070] This invention is the first to discover that H2O2 from the roots of Paspalum notatum under salt-driven conditions delays leaf senescence by participating in the regulation of jasmonic acid.
[0071] Example 1
[0072] Under salt stress, exogenous application of DPI significantly inhibited the H2O2 content in the roots;
[0073] However, under control conditions, DPI treatment did not affect the H2O2 content in the roots; it is necessary to first confirm that, under NaCl stress and DPI treatment, the application of the NADPH oxidase activity inhibitor DPI (100 μM) can inhibit the production of H2O2 from the roots. Figure 1 C-1D); Under 400 mM NaCl treatment, the H2O2 content in roots induced by salt stress decreased significantly, but no change in the H2O2 content in salt-free roots was observed, as shown by trypan blue staining. Figure 1 C-1D);
[0074] To determine whether root-derived NADPH oxidase-mediated H2O2 is needed to prevent salt-induced leaf senescence in *Paspalum notatum*, 100 μM MDPI should be used to block the production of root-derived H2O2.
[0075] Clearly, compared to plants treated with NaCl alone, the combined application of NaCl stress and DPI resulted in obvious symptoms of leaf senescence, due to significant yellowing and cell death. Figure 1 A-1B);
[0076] The combined treatment of salt stress and DPI significantly reduced leaf Fv / Fm and chlorophyll content; while the chlorophyll content of salt-treated leaves was 2-3 times that of the combined treatment. Figure 1 E);
[0077] The results showed that root-derived NADPH-dependent H2O2 was essential to prevent salt-induced leaf senescence in *Paspalum notoginseng*. Compared with salt treatment alone and the control treatment, salt stress plus DPI treatment not only led to leaf senescence symptoms such as leaf yellowing and cell death, but also significantly reduced chlorophyll fluorescence (F). v / F m Value) and total chlorophyll content (Chl(a+b)).
[0078] Example 2
[0079] Hormone level analysis was performed using LC / MS. The results showed that while salt stress significantly increased endogenous JA levels compared to the control, salt treatment combined with DPI treatment greatly increased the levels of JA and its metabolic derivatives (JA-IIE, OPC-6, and OPC-4) in roots. Figure 2 A);
[0080] The results showed that under salt stress, H2O2 mediated by root NADPH oxidase plays a key role in maintaining JA homeostasis and thus delaying leaf senescence.
[0081] The effects of salt treatment plus DPI treatment on root cause control (CK) of *Aquilaria sinensis* were determined using LC-MS-based analysis of endogenous hormone levels. Figure 2 B); Salt stress alone did not affect the level of CK in the roots of *Aquilaria sinensis*. Figure 2 B); however, CK levels were significantly reduced in roots treated with salt + DPI, especially tZOG and iP9G. Figure 2 B); Conversely, the levels of cZ and 2MeSeZR in the roots of the salt-treated + DPI-treated plants were significantly increased; the results indicate that NADPH oxidase-mediated H2O2 under salt-driven conditions plays a key role in maintaining the antagonistic interaction between JA and CK hormones to delay leaf senescence in Aristolochia arborescens.
[0082] Example 3
[0083] The effects of salt treatment plus DPI treatment on the concentrations of plant hormones JA and CK in leaves were analyzed using lipidomics. The results showed that salt stress plus DPI treatment significantly increased the jasmonic acid content in leaves, but had no effect on the CK content. Figure 3 );
[0084] The results suggest that the increase in root JA content due to root H2O2 deficiency may be an important factor leading to the significant increase in leaf JA content.
[0085] Example 4
[0086] Lipidomics was used to analyze the changes in the types and contents of lipids in roots under salt stress under DPI treatment or exogenous low-concentration H2O2 addition. The results showed that exogenous low-concentration H2O2 addition promoted the synthesis of C18 fatty acids after salt treatment. Figure 4 A); In addition, salt stress plus DPI treatment reduces the accumulation of seven important UFAs, including JA synthesis precursors such as oleic acid (FA 18:1), linoleic acid (FA 18:2), and α-linolenic acid (FA 18:3). Figure 4 A);
[0087] The relative expression levels of key enzyme-encoding genes related to unsaturated fatty acid synthesis were analyzed using qRT-PCR, including acetyl-CoA carboxylase ACC1 (Baud et al., 2003) and fatty acid synthases FAD3 (Browse et al., 1993) and FAD7 (Iba et al., 1993).
[0088] Compared with salt treatment alone, salt treatment combined with DPI treatment severely suppressed the expression level of the PvACC1 gene in roots. Figure 4 B); Conversely, salt treatment + DPI treatment significantly increased the expression level of the PvFAD7 gene, while the expression level of the PvFAD3 gene remained unchanged; the above results demonstrate that root H2O2 is involved in the biosynthesis of JA precursors.
Claims
1. A method for delaying leaf senescence in seashore paspalum under salt stress, characterized in that, The specific operating steps are as follows: Step (1), plant material culture and salt stress treatment; specifically; Select two-month-old and uniformly grown puffin stolons and plant them in seedling cups with holes at the bottom; then place them in a Hogrange solution with pH=6.0 for hydroponics, changing the solution every 7 days; After the runners of the puffin plant have grown for 14 days, they are placed in a plant culture box for salt treatment; The salt-treated and control-treated *Gnaphalium affine* materials were then placed in hydroponic plastic containers with or without 400 mM NaCl. The NaCl concentration was gradually increased by 100 mM each day until the target concentration of 400 mM was reached. On the fifth day of salt treatment, samples were taken from the salt-treated and control-treated Amur arborvitae materials respectively; to study the NADPH oxidase-mediated root source H2O2 experiment, 100 μM diphenyl iodine chloride was added to a 400 mM NaCl solution. After 5 days of treatment, the root system was collected for subsequent analysis; Six biological replicates were set up for each treatment; experiments were conducted using 3 to 6 biological replicates. The light cycle of the plant cultivation box is: light: 16h, darkness: 8h; Temperatures are: Daytime: 30℃, Nighttime: 28℃; Relative humidity: 70%, light intensity: 1000 lx; Step (2), H2O2 content determination; specifically, the change in the root H2O2 content is determined using an H2O2 determination kit; Step (3), DAB staining; specifically: the separated roots are placed in DAB staining solution with a concentration of 0.5 mg / ml and stained at room temperature in the dark for 2 to 6 hours; finally, they are rinsed 5 times with pure water, photographed and stored at room temperature; Step (4), determination of chlorophyll content and Fv / Fm value; specifically: the total chlorophyll content is determined by spectrophotometry: after rinsing the leaves with distilled water, the surface moisture is dried with filter paper, and three portions of chopped fresh leaves, each 2g, are weighed and ground into a paste in a mortar with quartz sand, and then 10ml of ethanol is added and ground until the tissue turns white. After filtration in a funnel, the filtrate was diluted to 100 ml with ethanol, and the absorbance was measured at 665 nm, 645 nm and 652 nm respectively. Using 95% ethanol as a blank control, then: Chl(a+b)=A625 / 35.5×V÷1000×W; Step (5), lipidomics data determination and analysis; specifically: take 100mg of leaf and root samples, add liquid nitrogen and grind into powder to extract lipids, and use Nexera UHPLC LC-30A system and TripleTOF5600+ mass spectrometer for LC-MS analysis of lipids; The column temperature was set to 40℃, the mobile phase was 0.1% HCOOH-H2O(A)-acetonitrile(B), the flow rate was 0.3 mL / min, and gradient washing was used. The ESI source conditions for the TripleTOF5600+ mass spectrometer are set as follows: Ion source Gas1: 50, Ion source Gas2: 50, curtain gas: 25, source temperature: 500℃ / 450℃, ion saparilla voltage fluctuation: 5500V / 4400V. TOF-MS scan range: 100-1200 Da, TOF MS scan accumulation time: 0.2 s, declustering potential: ±60 V; LipidSearch software is used for automated lipid identification and relative quantitative analysis; Step (6), endogenous hormone level analysis; specifically: 50 mg of fresh root system is frozen in liquid nitrogen and placed in a 2 ml plastic microtube for sample preparation and extraction; The plant hormone content was detected using an AB SCIEX QTRAP 6500LC / MS / MS system. Step (7): Analyze gene expression levels using real-time quantitative PCR; specifically, analyze the relative expression levels of four genes using real-time quantitative PCR. Specific primers were designed based on the gene sequence: 2.5 μL of total RNA was reverse transcribed into cDNA, and PCR was performed using an ABI 7500 Real-Time PCR System and... Premix EX-Taq TM II was able to run; PCR amplification was performed using a 20 μL volume, with 2.5 μL of cDNA working solution, 10 μL of PCR-mix, 5.5 μL of autoclaved deionized water, and 1 μL each of upstream and downstream primers added to each PCR reaction tube; The PCR reaction conditions were set as follows: 95℃ pre-denaturation for 30 s, 1 cycle; 95℃ denaturation for 5 s; 60℃ annealing for 34 s; 72℃ extension for 45 s, 40 cycles; The fluorescence signal was collected during the extension phase, and the Ct value was derived after the reaction was completed. The relative expression level is calculated according to formula 2-ΔΔCt; The α-actin gene was used as an internal reference gene for relative quantitative analysis of the treated and control materials, with three replicates for each sample. Step (8), transcriptomics data and analysis; specifically: total RNA was extracted from leaves and roots using a plant total RNA kit; transcriptome sequencing and paired-end sequencing of de novo cDNA library assembly were performed using the HiSeq2000 platform; raw reads were quality assessed and then trimmomatic was used to filter adapter sequences and low-quality bases; clean reads were aligned with the reference genome using HISAT2; Gene expression is standardized by calculating readings per kilobase per million mapping readings; Differentially expressed genes were identified using the DESeq2 package in R software, and statistically significant differences in gene expression were determined using a false discovery rate ≤0.05 and |log2 FC|≥1 as thresholds; GO analysis and KEGG metabolic pathway analysis were performed. RNA-seq reads are stored in the NCBI Sequence Reading Archive.