Application of H3K27me3 in regulating cotton response to potassium chloride stress

Abstract

This invention belongs to the field of plant genetic engineering technology, specifically relating to the application of H3K27me3 in regulating the cotton response to potassium chloride stress. By integrating CUT & Tag chromatin analysis and RNA-seq, this invention demonstrates that potassium chloride stress induces a reduction in H3K27me3 deposition across the entire genome, accompanied by characteristic stress phenotypes in cotton seedlings. Inhibition of H3K27me3 using RDS 3434 significantly improved KCl-induced physiological damage, confirming the functional correlation between this epigenetic marker and stress tolerance. Furthermore, virus-induced gene silencing confirmed that genes associated with H3K27me3 are important components of the cotton ion stress response network. Therefore, this invention elucidates the epigenetic landscape regulating adaptation to potassium chloride stress.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically involving the application of H3K27me3 in regulating the response of cotton to potassium chloride stress. Background Technology

[0002] Potassium (K) is one of the most important mineral nutrients in plants, widely involved in various physiological functions such as stomatal regulation, photosynthesis, and water absorption, and plays a crucial role in plant growth regulation, signal transduction, and stress response. Potassium (K) deficiency in soil limits crop yield. In cotton production, potassium (K) deficiency can lead to premature leaf senescence, yellowing, and defoliation, and reduce fiber yield and quality. Therefore, the widespread application of potassium fertilizers is used to improve cotton yield and fiber quality. However, excessive application of potassium fertilizers (mainly potassium chloride (KCl)) increases soil salinity, transforming it into an abiotic stress factor. Increased potassium chloride concentration in the soil increases osmotic pressure and decreases soil water potential, thereby limiting water absorption by plant roots. This reduction in water availability lowers cell turgor pressure, triggering osmotic stress, disrupting metabolic activities such as photosynthesis and nutrient transport, ultimately leading to leaf wilting and growth inhibition. Furthermore, high concentrations of potassium ions (KCl) can also cause adverse reactions. + It can indirectly cause ion toxicity by disrupting intracellular ion balance, mainly through competitive inhibition of calcium ions (Ca). 2+ ) and magnesium ions (Mg 2+ Essential cations such as potassium chloride (KCC) are present in cotton. This ion imbalance impairs vital cellular metabolic processes, promotes excessive accumulation of reactive oxygen species (ROS), and triggers oxidative stress, ultimately leading to damage to cell membranes and other macromolecules. In cotton, these physiological dysfunctions manifest as premature aging, leaf yellowing and defoliation, and a significant decline in fiber yield and quality. Therefore, elucidating the molecular mechanisms of cotton's response to potassium chloride stress is crucial for breeding stress-resistant varieties.

[0003] The plant response to potassium chloride stress involves multiple mechanisms. Plant cells cope with high external potassium chloride concentrations by regulating intracellular solute concentrations to adapt to changes in osmotic pressure. For example, plant cells may increase the accumulation of organic solutes such as sucrose, proline, or soluble sugars to maintain intracellular osmotic balance. KCl stress may increase the generation of reactive oxygen species (ROS), thereby triggering oxidative stress. Plant cells scavenge excess ROS by activating antioxidant enzyme systems (including superoxide dismutase, catalase, and glutathione reductase) to prevent damage to cell structure and function. Furthermore, one of the main mechanisms of plant response to potassium chloride stress is maintaining intracellular potassium ion balance. On the one hand, plants utilize KCl… + Transport proteins (such as HKT, KUP / KT, ​​AKT, etc.) regulate K +Plants utilize the absorption and transport of Na+ to cope with potassium chloride stress. On the other hand, plants can utilize Na+ located on their vacuolar membranes... + (K) + ) / H + The exchange protein (NHX) will exchange excess Na+ + or K + Potassium chloride is transported from the cytoplasm into the vacuoles to help maintain ionic stability and osmotic pressure in the cytoplasm. Plants also respond to potassium chloride stress through the activation of signaling pathways and the regulation of related gene expression. These complex strategies enable plants to maintain growth and development even in environments with high potassium chloride concentrations.

[0004] Epigenetics studies how gene function is altered through changes in chromatin state without changing nucleotide sequence. Many epigenetic mechanisms dynamically regulate chromatin structure, such as DNA methylation, histone modifications, chromatin remodeling, and non-coding RNA. Trimethylation of histone H3 at lysine 27 (H3K27me3) is a common and well-studied histone modification in plants. Polycomb repressor complex 2 (PRC2), composed of the core subunits EZH2, EED, and SUZ12, epigenetically silences gene expression through H3K27me3 deposition. (3-Bromo-4-methoxyphenyl)pentane-1,4-dien-3-one (RDS 3434), a potent EZH2 inhibitor, effectively reduces H3K27me3 levels in Arabidopsis seeds. In studies known to the inventors, histone H3K27me3 has been involved in plant responses to salt stress. In castor bean, H3K4me3 and H3K27me3 regulate the expression of the key salt-responsive gene RSM1, a MYB-related transcription factor involved in the abscisic acid (ABA)-mediated salt stress response. In soybean, the level of H3K27me3 is negatively correlated with the expression levels of certain salt-sensitive genes. The histone H3K27me3 demethylase JMJ can respond to salt stress by removing H3K27me3 modification. Overexpression of GmJMJ30-1 / 2 reduces H3K27me3 levels at the GmZF351 locus, enhances GmZF351 expression through histone demethylation, and confers salt stress tolerance to soybean. Decreased MSI1 levels or weakened EMF1 activity in Arabidopsis thaliana can reduce H3K27me3 modification at target gene sites, thereby enhancing tolerance to salt stress. Currently, research on the response of histone H3K27me3 modification to salt stress mainly focuses on sodium chloride stress, while no research has been reported on the response of H3K27me3 modification to potassium chloride stress. Summary of the Invention

[0005] The purpose of this invention is to provide the application of H3K27me3 in regulating the response of cotton to potassium chloride stress, and to provide technical support for breeding cotton varieties resistant to potassium chloride stress.

[0006] This invention provides the application of histone H3K27me3 in regulating the response of cotton to potassium chloride stress.

[0007] Preferred examples include the use of chemical agents that inhibit histone H3K27me3 deposition in improving cotton's tolerance to potassium chloride stress.

[0008] Preferably, the chemical reagent includes a histone H3K27me3 inhibitor.

[0009] Preferably, the histone H3K27me3 inhibitor includes RDS 3434.

[0010] Preferably, the concentration of RDS 3434 is 10~50 µM.

[0011] Preferably, the downstream genes regulated by histone H3K27me3 under potassium chloride stress include: GhGSH1 and / or GhSRM1 The GhGSH1 and GhSRM1 The login number is Ghir_D09_G02354 and Ghir_D09_G01343 Overexpression of the aforementioned gene in the cotton genome GhGSH1 and / or GhSRM1 Improve cotton's tolerance to potassium chloride stress.

[0012] Preferably, the cotton includes upland cotton.

[0013] Preferably, the potassium chloride stress includes potassium chloride-induced ion stress.

[0014] The present invention also provides a method for improving the tolerance of cotton to potassium chloride stress, comprising the following steps: Cotton seedlings were cultured using the histone H3K27me3 inhibitor RDS 3434.

[0015] Preferably, the cultivation includes hydroponics, and the hydroponic step includes culturing cotton after adding RDS 3434 to the hydroponic system, wherein the concentration of RDS 3434 in the hydroponic system is 10~50 µM.

[0016] Beneficial effects: This invention provides the application of histone H3K27me3 in regulating the cotton response to potassium chloride stress. It reveals a previously unexplored epigenetic mechanism mediated by H3K27me3 in cotton adaptation to potassium chloride stress. Specifically, by integrating CUT & Tag chromatin analysis and RNA-seq, this invention demonstrates that potassium chloride stress induces a reduction in H3K27me3 deposition across the entire genome, accompanied by characteristic stress phenotypes in cotton seedlings. Inhibition of H3K27me3 using RDS 3434 significantly improves KCl-induced physiological damage, confirming the functional correlation between this epigenetic marker and stress tolerance. Furthermore, this invention reveals a negative correlation between H3K27me3 enrichment and transcriptional activation in 48 genes, including two key salt tolerance regulators: glutathione synthase GhGSH1 and stress response mediator GhSRM1. Virus-induced gene silencing (VIGS) validates that these H3K27me3-related genes are important components of the cotton ion stress response network. As can be seen, this invention not only elucidates the epigenetic landscape regulating potassium chloride stress adaptation, but also identifies H3K27me3-mediated chromatin remodeling as a key regulatory layer in plant abiotic stress responses, providing new insights into epigenetic engineering strategies for developing stress-resistant cotton varieties. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 The results of Western blot analysis in Example 1; Figure 2 The following are the phenotypic results of cotton seedlings under potassium chloride stress and different concentrations of RDS 3434 treatment in Example 1; where A shows the phenotypic changes after treatment with 0 mM (simulated) or 150 mM potassium chloride following the application of different concentrations of the H3K27me3 inhibitor RDS 3434, with DMSO as a control, and 3 plants are shown in each group; B shows the H2O2 content measurement results; C shows the O2 content. - Content determination results; D represents MDA content determination results; E represents chlorophyll content determination results; each treatment group was compared with the control group. P <0.01; Figure 3 The K content in cotton seedlings in Example 1 is calculated by comparing the K content in the roots (A), stems (B), and leaves (C) of cotton seedlings under simulated conditions and potassium chloride treatment. + Na + Content K + / Na +Ratios; data are expressed as mean ± standard deviation (n=3); each treatment group is compared with the control group. P <0.05, P <0.01; Figure 4 To illustrate the characteristics of H3K27me3 in cotton roots under simulated and potassium chloride treatments in Example 2, (A) the genomic distribution of H3K27me3 peaks in different regions of cotton roots under simulated conditions and (B) potassium chloride treatment; (C) the whole genome coverage of H3K27me3 in roots under simulated conditions and KCl treatment; and (D) the distribution of H3K27me3 in all cotton genes (from the upstream region to the downstream region of 3-kb) in roots under simulated conditions and KCl treatment. Figure 5 This section shows the changes in gene expression under potassium chloride treatment in Example 3; where A is a volcano map of differentially expressed genes in KCl-treated roots and the Mock group; B is the number of upregulated and downregulated genes identified in the KCl-treated root tissue compared to the Mock group; C is KEGG analysis of upregulated genes; D is KEGG analysis of downregulated genes; E is transcriptional analysis of ion transport genes; F is... GhCHX25 and GhKEA2 In containing different concentrations of K + AP medium supplemented with K + Intake-deficient yeast mutant R5421; Figure 6 In Example 4, KCl treatment affected the expression levels of H3K27 methylation modification and salt-responsive genes; where A is... GhGSH1 and GhSRM1 The H3K27me3 pattern in roots treated with Mock and KCl is shown at the bottom, including exons (blue boxes) and introns (black lines), with arrows indicating the direction of transcription; B represents analysis using CUT&Tag-qPCR technology. GhGSH1 and GhSRM1 The H3K27me3 level was determined using the spike in DNA genes (HD101, Vazyme) as an internal control and IgG as a negative control; C represents the level obtained through RNA sequencing and qRT-PCR analysis. GhGSH1 and GhSRM1 level of expression GhUBQ7 Used as an internal reference ， The error bars represent the standard deviation of three repeated experiments. P <0.01, Student's t-test; D represents the result of analysis using CUT&Tag-qPCR technology. GhGSH1 and GhSRM1H3K27me3 levels were measured, with IgG used as a negative control; E represents the H3K27me3 levels analyzed by qRT-PCR. GhGSH1 and GhSRM1 GhUBQ7 was used as an internal reference to measure expression levels. ， Error bars represent the standard deviation of three repeated experiments; each treatment group is compared with the control group. P <0.01; Figure 7 TRV in Example 5: GhGSH1 and TRV: GhSRM1 The silencing efficiency of the plant; the error bar represents the standard deviation of three replicate experiments. P <0.01, Student's t-test; Figure 8 In Example 5 GhGSH1 The silencing effect reduced the tolerance of cotton seedlings to potassium chloride, where A was TRV:00 and TRV: GhGSH1 Phenotypic changes in cotton seedlings, with 3 plants shown in each group; B represents the H2O2 content measurement results; C represents O2. - Content determination results; D represents MDA content determination results; E represents chlorophyll content determination results; data are expressed as mean ± standard deviation (n = 6). P <0.01, Student's t-test; Figure 9 K in cotton seedlings in Example 5 + and Na + Content and K + / Na + Under Mock and potassium chloride treatment, TRV:00 and TRV: GhGSH1 The ratio of (A) roots, (B) stems, and (C) leaves of a plant; the error bar represents the standard deviation of three replicate experiments; P <0.05, P <0.01, Student's t-test; Figure 10 Silence in Example 5 GhSRM1 It will reduce the tolerance of cotton seedlings to potassium chloride. A represents TRV:00 and TRV: GhSRM1 Phenotypic changes in cotton seedlings, with 3 plants shown in each group; B represents the H2O2 content measurement results; C represents O2. -Content determination results; D represents MDA content determination results; E represents chlorophyll content determination results; data are expressed as mean ± standard deviation (n = 6). P <0.01, Student's t-test; Figure 11 K represents the ion content in the cotton seedlings in Example 5. + and Na + Content and K + / Na + Under Mock and potassium chloride treatment, TRV:00 and TRV: GhSRM1 The ratio of (A) roots, (B) stems, and (C) leaves of a plant; the error bar represents the standard deviation of three replicate experiments. P <0.05, P <0.01, Student's t-test. Detailed Implementation

[0019] This invention provides the application of histone H3K27me3 in regulating the response of cotton to potassium chloride stress.

[0020] In one embodiment, the application includes the use of a chemical agent that inhibits histone H3K27me3 deposition in improving the tolerance of cotton to potassium chloride stress. In one embodiment, the chemical agent includes a histone H3K27me3 inhibitor, more specifically, RDS 3434; in another embodiment, the concentration of RDS 3434 is 10–50 µM, more specifically, 10 µM, 30 µM, or 50 µM.

[0021] As one implementation method, the downstream genes regulated by histone H3K27me3 under potassium chloride stress include GhGSH1 and / or GhSRM1 The GhGSH1 and GhSRM1 The accession number in the Cotton Breeding Database (http: / / 222.88.152.130:1130 / ) is Ghir_D09_G02354 and Ghir_D09_G01343 Overexpression of the aforementioned gene in the cotton genome GhGSH1 and / or GhSRM1 Improve cotton's tolerance to potassium chloride stress.

[0022] In one embodiment, the cotton includes upland cotton. In another embodiment, the potassium chloride stress includes potassium chloride-induced ion stress.

[0023] This invention experimentally verified the effects of different concentrations of H3K27me3 inhibitors on cotton seedlings under potassium chloride stress. Subsequently, a whole-genome H3K27me3 map of cotton roots was constructed under normal and potassium chloride stress conditions. A combined analysis of differentially enriched genes with H3K27me3 modification in CUT&Tag data and differentially expressed genes in RNA-seq was performed. The results showed that among genes exhibiting both expression and H3K27me3 modification changes, changes in H3K27me3 were negatively correlated with changes in transcripts under potassium chloride stress. VIGS technology was used to analyze two important salt-responsive genes (…). GhGSH1 and GhSRM1 Functional validation was performed, confirming that histone H3K27me3 modification regulates the expression of salt-responsive genes and participates in the cotton response to potassium chloride stress.

[0024] The present invention also provides a method for improving the tolerance of cotton to potassium chloride stress, comprising the following steps: Cotton seedlings were cultured using the histone H3K27me3 inhibitor RDS 3434. As one implementation method, The cultivation includes hydroponics; as another embodiment, the hydroponic step includes adding RDS 3434 to the hydroponic system and then culturing the cotton, wherein the concentration of RDS 3434 in the hydroponic system is 10~50 µM, and may further be 10 µM, 30 µM or 50 µM.

[0025] Epigenetic modifications are increasingly recognized as key regulators of plant-environment interactions. While H3K27me3 modification is known to participate in various biological processes, including flowering and responses to abiotic stresses, its specific function in cotton under potassium chloride stress remains poorly understood. This invention reveals that chemically inhibiting H3K27me3 deposition using RDS 3434 significantly enhances the tolerance of cotton seedlings to KCl-induced ionic stress. Notably, this inhibitor did not affect normal plant growth.

[0026] The relief of stress symptoms was accompanied by a significant reduction in the accumulation of reactive oxygen species (ROS) and lipid peroxidation, while potassium ions (K+) increased. + Steady state is improved. Figure 2 and Figure 3 Ion homeostasis and antioxidant stress management are core mechanisms of plant salt tolerance. The results of this invention establish H3K27me3 as a key epigenetic regulator that synergistically regulates oxidative stress and ion homeostasis during cotton adaptation to potassium chloride stress. CUT & Tag analysis showed that under potassium chloride stress, the overall expression level of H3K27me3 in cotton seedlings decreased (…). Figure 4This is consistent with the responses of castor beans and rice to salt stress (Zheng et al., 2019; Han et al., 2020), but contrary to the increasing trend observed in soybeans (Sun et al., 2019). This finding, along with the previously documented decrease in H3K27me3 modification levels in Arabidopsis under cold and salt stress (Kwon et al., 2009; Sani et al., 2013), further confirms that the dynamic changes in H3K27me3 modification are species-specific and stress-type-specific.

[0027] This invention provides the first evidence that H3K27me3 modification plays a key regulatory role in potassium chloride stress tolerance in cotton. Chemically inhibiting H3K27me3 deposition reduces reactive oxygen species and malondialdehyde levels while restoring potassium ion homeostasis, thereby alleviating potassium chloride-induced stress. Integrated CUT&Tag and transcriptome analysis revealed dynamic changes in H3K27me3 and key stress-response genes (especially...) GhGSH1 and GhSRM1 Silencing these genes disrupts ion balance and exacerbates oxidative damage. This invention provides valuable insights into the epigenetic regulation of salt stress tolerance, enriches our understanding of salt tolerance networks, and lays a theoretical foundation for future applications of epigenetic editing technologies to enhance crop resistance.

[0028] In this field, research on the response of histone H3K27me3 modification to salt stress has mainly focused on sodium chloride stress, and some studies have been published. However, from a technical perspective, histone H3K27me3 modification has fundamental differences in its response to sodium chloride stress and potassium chloride stress, mainly in the following aspects: First, there are fundamental differences at the signal pathway level: Na + Stress primarily activates Ca 2+ Signal-dependent SOS pathway regulates Na + External discharge, while K + Stress is responded to through separate potassium channels and transporter systems, which differ in signal sensing, transduction, and effector mechanisms.

[0029] Second, there are significant differences at the gene regulation level: taking cotton as an example. GhKEA4 For example, silencing this gene under NaCl stress leads to increased salt sensitivity, but silencing it under high potassium stress can maintain more stable Na+ levels. + / K +The balance was disrupted, and potassium ion transport activity decreased. This shows that the same gene has completely different functions under the two stresses, directly proving that the molecular mechanisms are not equivalent (Reference: Li Y, Feng Z, Wei H, Cheng S, Hao P, Yu S, Wang H. Silencing of GhKEA4 and GhKEA12 Revealed Their Potential Functions Under Salt and Potassium Streesses in Upland Cotton. Front Plant Sci. 2021).

[0030] Third, there are opposing effects at the physiological phenotypic level: in Arabidopsis, treatment with 200 mM KCl resulted in complete failure to develop flowers, while treatment with 200 mM NaCl enabled normal flowering and pod formation (Reference: Pantha P, Oh DH, Longstreth D, Dassanayake M. Living with high potassium: Balance between nutrient acquisition and K-induced salt stress signaling. Plant Physiol. 2023). In oats, low concentrations of NaCl showed stronger inhibition, but high concentrations of KCl caused greater damage. + and K + This process exhibits antagonistic effects.

[0031] The above evidence suggests that "resistance to NaCl stress" is not the same as "resistance to KCl stress," and may even show the opposite trend.

[0032] In summary, although sodium chloride stress and potassium chloride stress both belong to salt stress, their mechanisms of action on plants are fundamentally different and cannot be simply equated. Sodium chloride stress involves both "sodium ion-specific toxicity" and "osmotic stress." Sodium ions are not essential elements for plants; high concentrations of sodium ions competitively inhibit potassium ion absorption and metabolism, and activate specific ion detoxification pathways such as SOS, leading to cellular metabolic disorders. In contrast, potassium chloride stress involves potassium ions, an essential macronutrient for plants. High concentrations of potassium chloride primarily cause osmotic stress and an imbalance in the absorption of calcium, magnesium, and other cations due to potassium ion excess, without involving sodium ion-specific toxicity mechanisms. The physiological and molecular responses induced by the two stresses differ significantly at the levels of plant phenotype, ion homeostasis regulation, and signal transduction. Therefore, studying them separately is a classic experimental strategy for decoupling "osmotic stress" and "ion-specific toxicity," which helps to accurately elucidate the independent functions of specific genes or epigenetic modifications in responding to different types of ion stress. Based on the above differences, current research results on H3K27me3 modification under sodium chloride stress cannot directly deduce or predict that it has the same mechanism of action or research results in potassium chloride stress response.

[0033] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0034] Materials and methods used in the following embodiments 1. Plant materials and treatment Upland cotton ( Gossypium hirsutum Cotton seedlings (cv. 'Zhongmian 113') were planted in vermiculite and, after the two cotyledons had fully expanded, were transferred to a hydroponic system containing Hoagland nutrient solution. These samples were placed in a greenhouse under the following conditions: 28°C, a 16-hour light / 8-hour dark photoperiod, and 60% relative humidity. Cotton seedlings with two true leaves were treated with either 0 mM (Mock) or 150 mM potassium chloride solution for 6 hours. Additionally, the H3K27me3 inhibitor RDS 3434 was added at concentrations of 10, 30, and 50 µM, with DMSO serving as a parallel control. Root tissue was collected for cut & tag experiments, with each treatment biologically replicated three times.

[0035] 2. Measurement of physiological parameters The hydrogen peroxide (H2O2) content and O2 content in leaves were determined using a hydrogen peroxide (H2O2) content detection kit (catalog number BC3595), a superoxide anion activity content detection kit (catalog number BC1295), a malondialdehyde (MDA) content detection kit (catalog number BC0025), and a chlorophyll detection kit (catalog number BC0995) (Beijing Solarbio Biotechnology Co., Ltd.). -Content, MDA content and chlorophyll content.

[0036] 3. Western blot analysis Extraction of root proteins from cotton seedlings was performed using an extraction buffer containing 1 mM PMSF and 10 mM DTT [120 mM Tris-HCl (pH 7.5) / 20% glycerol / 4% SDS]. Protein separation was performed by 12.5% ​​sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by wet transfer (200 V, 2 h) onto nitrocellulose membranes. The membranes were blocked for 1 h at room temperature with 1×Tris-buffered saline (containing Tween-20, TBST) with continuous shaking. Protein detection was performed using specific antibodies against H3K27me3 (Millipore, 07-449) and histone H3 (Proteintech, 17168-1-AP) as loading controls. Goat anti-rabbit IgG-HRP (HuaAn Biotechnology, HA1001) was used as a secondary antibody. Protein levels were visualized using the SuperFemto ECL chemiluminescence detection kit (Vazyme, E423-01) and signals were captured using an automated chemiluminescence imaging analysis system (5200, Tanon, Shanghai, China).

[0037] 4. Determination of ion content Roots, stems, and the second true leaf of cotton seedlings were collected and dried at 85℃ for 72 hours. 0.05 g of the powder sample was weighed, dissolved in 5 mL of concentrated nitric acid, then diluted 12-fold with deionized water and centrifuged. Na+ was determined using an inductively coupled plasma optical emission spectrometer (ICP-OES) system (model ICAP7400, Thermo Fisher Scientific, USA). + and K + content.

[0038] 5. Cut & Tag and Data Analysis Nuclear components were extracted from cotton roots using the CelLytic™ PN Isolation / Extraction Kit (Sigma-Aldrich, CELLYTPN1), strictly following the manufacturer's experimental protocol. H3K27me3-modified genome enrichment analysis was performed using the Hyperactive Universal CUT&Tag Assay Kit for Illumina (Vazyme, TD-903). Subsequently, the purified samples were sequenced on an Illumina Novaseq 6000 sequencing platform (Novogene, Beijing, China). Raw reads obtained from sequencing underwent basic quality control using FastQC (version 0.11.5) (de Sena Brandine and Smith, 2019). Adapter sequences in the raw data were pruned using fastp software (version 0.20.1) (Chen et al., 2018). Clean reads were aligned to the reference genome of Zhongmian 113 using BWA software (version 0.7.17) (Jung and Han, 2022; Hu et al., 2025). DeepTools was used to generate bw files for correlation analysis, and the CUT & Tag signal intensities in the upstream and downstream 3 kb regions of the genomic genome were calculated (Ramírez et al., 2014). The Integrative Genomics Viewer (IGV) software was used to visualize the CUT & Tag sequencing data (Thorvaldsdóttir et al., 2013). MACS2 software was used to identify relevant peaks, and the ChIPseeker package was used for peak annotation in different genomic regions (Yu et al., 2015; Jeon et al., 2020). Genes with differential H3K27me3 enrichment were defined as those with an absolute fold change ≥ 2 and a p-value < 0.05.

[0039] 6. RNA sequencing data analysis Raw RNA-seq reads (accession number PRJNA1034287) were downloaded from the NCBI Sequence Reading Archive. Clean reads were aligned to the Zhongmian 113 reference genome using HISAT2 and assembled and quantified using StringTie (Kim et al., 2015; Pertea et al., 2015; Hu et al., 2025). Gene expression levels were assessed by calculating the number of fragments per kilobase transcript per million aligned reads (FPKM) for each gene based on gene length. Differentially expressed genes were identified by DESeq2-based differential analysis (|Fold Change|≥2, corrected P-value (padj) <0.05) (Love et al., 2014), followed by KEGG pathway annotation (Kanehisa and Goto, 2000).

[0040] 7. Real-time quantitative PCR (qRT-PCR) and CUT&Tag-qPCR analysis For qRT-PCR, total RNA was extracted using the EASY spin Plus Complex Plant Kit (Aidlab, Beijing, China). First-strand cDNA synthesis used 1 μg RNA and HiScrip. ® II Q RT SuperMix was used for qPCR (Vazyme, Nanjing, China). qRT-PCR reactions were performed on a LightCycler 480 system (Roche, Basel, Switzerland) using ChamQ™ Universal SYBR qPCR Master Mix (Vazyme). The GhUBQ7 gene was used as an internal control gene.

[0041] In the CUT&Tag-qPCR experiments, DNA extracted using the Hyperactive pG-MNase CUT&RUN Assay Kit for PCR / qPCR (Vazyme, HD-101) was used as the qPCR template, and the procedure was strictly followed according to the manufacturer's instructions. IgG was used as a negative control. Each sample was subjected to three biological replicates. Relative expression levels were calculated using the comparison cycle threshold (Ct) method (Livak and Schmittgen, 2001). Primer details are shown in Table 1. GhGSH1 The forward and reverse primers for the gene are numbered SEQ ID NO:1 and SEQ ID NO:2, respectively. GhSRM1 The forward and reverse primers for the gene are numbered SEQ ID NO:3 and SEQ ID NO:4, respectively, and so on, numbered sequentially from top to bottom according to the gene name list, until... GhSRM1 The forward and reverse primers for the gene are numbered SEQ ID NO:13 and SEQ ID NO:14, respectively. Table 1 Primer Information

[0042] 8. Yeast complementarity test GhCHX25 (SEQ ID NO:15) and GhKEA2 The coding sequence of (SEQ ID NO:16) was constructed into the pYES2 expression vector, inserted between BamHI and EcoRI, and transformed into yeast strain R5421 (trk1Δ, trk2Δ) lacking endogenous TRK1 and TRK2, using yeast K + Transporter protein detection kit (Coolaber, Beijing, China). The empty pYES2 vector was used as a negative control, and AKT1 as a positive control (Han et al., 2016). Monoclonal yeast cells were cultured in SD / -Ura medium supplemented with 100 mM potassium chloride at 30°C until OD. 600 The OD value was reached at 1.0. Cells were then collected by centrifugation at 6,000 × g for 1 minute, washed three times with sterile double-distilled water, and resuspended in the same solution to OD. 600 The concentration was 0.2. Serial 10-fold dilutions were prepared and spotted onto AP medium plates containing different potassium ion concentrations. The plates were incubated at 30°C for 2–3 days to evaluate yeast growth under different potassium conditions.

[0043] GhCHX25 and GhKEA2 The encoded sequence is as follows:

[0044]

[0045] 9. Virus-induced gene silencing (VIGS) detection The specific fragment of the target gene was amplified using primers (Table 1), and the VIGS expression vector was constructed using homologous recombination. The recombinant plasmid TRV was then used. GhGSH1 and TRV: GhSRM1 Transformed into Agrobacterium tumefaciens (GV3101). A VIGS resuspension solution (1 M MgCl2 to prepare 1 M M MES, 200 mM AS solution) was used to resuspend the Agrobacterium, and the OD was adjusted. 600 The value was approximately 1.0. pTRV2 (empty vector) and TRV:CLA served as negative and positive controls, respectively. Subsequently, the solution was injected into the fully expanded cotyledons of cotton seedlings, and the seedlings were treated with 150 mM potassium chloride after they had grown two true leaves.

[0046] 10. Statistical Analysis Statistical analysis was performed using GraphPad Prism software (version 9.0). Comparisons between two experimental groups were conducted using a two-tailed Student's t-test; when comparing three or more experimental groups with a single control group, one-way ANOVA was performed first, followed by Dunnett's post-hoc test for multiple comparisons. A p-value less than 0.05 was considered acceptable. P A value <0.05 is considered statistically significant.

[0047] Example 1 RDS 3434 treatment alleviated potassium chloride stress in cotton seedlings. To investigate the effect of changes in H3K27me3 modification levels on cotton growth, cotton seedlings were treated with the inhibitor RDS 3434 at levels of 10 µM, 30 µM, and 50 µM to reduce H3K27me3 modification levels, with dimethyl sulfoxide (DMSO) as a control.

[0048] Figure 1 Western blot analysis showed that the RDS3434 inhibitor reduced the level of H3K27me3 in cotton seedlings. Meanwhile, through... Figure 2 The results showed that under simulated conditions, the application of the inhibitor had no significant effect on cotton growth. However, under 150 mM potassium chloride treatment, seedlings treated with the inhibitor exhibited less dehydration and wilting compared to the control group. Figure 2 (A) indicates that the reduction in H3K27me3 levels enhanced the tolerance of cotton seedlings to potassium chloride stress. Under potassium chloride treatment, the levels of hydrogen peroxide (H2O2) and superoxide anion (O2) in the leaves of plants treated with the inhibitor were significantly reduced. - The level of ) was significantly lower than that of the control group ( Figure 2The levels of B and C indicate a reduction in the accumulation of reactive oxygen species (ROS). Furthermore, the significantly decreased malondialdehyde (MDA) content suggests a decrease in the degree of lipid peroxidation in plant membranes. Figure 2 (D). However, the significant increase in chlorophyll content indicates that the cotton seedlings are in better growth condition. Figure 2 These results indicate that the application of H3K27me3 inhibitors does not affect the normal growth of cotton seedlings, but can enhance their tolerance to potassium chloride by reducing oxidative damage, suggesting that H3K27me3 is involved in the response of cotton to potassium chloride stress.

[0049] To investigate the effect of H3K27me3 levels on ion content, the K content in the roots of cotton seedlings treated with potassium chloride was analyzed. + and Na + Content determination was performed ( Figure 3 KCl treatment significantly increased potassium levels in the roots, stems, and leaves of cotton seedlings. + Content, while Na in the roots + The content of Na remains relatively constant in the stems and leaves. + The levels were significantly elevated. Compared with the DMSO control group, cotton seedlings treated with the RDS 3434 inhibitor showed significantly higher levels of K under potassium chloride treatment. + The content decreased significantly, while Na + The content increased. Subsequently, K, reflecting the plant's tolerance to potassium chloride, was calculated. + / Na + Ratio. Under potassium chloride treatment, K + / Na + The ratio increased significantly, especially in the roots. However, after treatment with the RDS 3434 inhibitor, this ratio decreased significantly compared to the DMSO control group. These results indicate that exogenous H3K27me3 inhibitors primarily regulate K... + / Na + To alleviate potassium chloride stress.

[0050] Example 2 Distribution of H3K27me3 modification sites in cotton roots under potassium chloride stress To further investigate the effect of H3K27me3 modification levels on the response of cotton seedlings to potassium chloride stress, this example conducted CUT&Tag experiments on cotton root tissues treated with 0 mM (mock group) and 150 mM potassium chloride. After quality control of the CUT&Tag sequencing data, the alignment rate with the reference genome consistently remained above 85%. This example analyzed the distribution of H3K27me3 modification sites in genomic regions (such as promoters, exons, introns, and intergenic regions). The results showed that H3K27me3 modification sites were mainly enriched in intergenic regions, promoter regions, and exon regions. Compared with the Mock treatment group ( Figure 4 In the study of potassium chloride stress, the number of H3K27me3 modification sites in intergenic regions increased, while the number of modification sites in promoters and exons decreased. Figure 4 (B). Subsequently, this embodiment performed a genome-wide analysis of H3K27me3 coverage and found that, compared with Mock, the coverage of H3K27me3 was lower under potassium chloride treatment (B). Figure 4 (C). This embodiment further analyzed the average CUT & Tag signal in the upstream and downstream 3 kb regions of the gene body. The results showed that, compared with the Mock group, the H3K27me3 modification in the gene body region was significantly reduced after potassium chloride treatment. Figure 4 (D). In summary, compared with the simulated treatment, the epigenetic marker H3K27me3 showed reduced enrichment in cotton root tissue treated with potassium chloride, indicating that potassium chloride stress induces changes in chromatin structure and histone modifications.

[0051] Example 3 KCl stress induces transcriptome reprogramming in cotton roots. To screen for genes associated with potassium chloride stress response, transcriptome analysis was performed on cotton roots treated with 0 mM and 150 mM potassium chloride, identifying 6629 differentially expressed genes (DEGs), including 2943 upregulated genes and 3686 downregulated genes. Figure 5 (A and B). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed distinct functional characteristics between upregulated and downregulated differentially expressed genes. Among the upregulated differentially expressed genes, the MAPK signaling pathway was significantly enriched in plants. Additional enriched pathways in the upregulated differentially expressed genes (DEGs) included galactose metabolism, alanine, aspartate, and glutamate metabolism, diurnal rhythm-plant pathways, and valine, leucine, and isoleucine degradation. Figure 5 Conversely, downregulated differentially expressed genes (DEGs) are associated with multiple biosynthetic processes, particularly the biosynthesis of phenylpropanes and flavonoids, pathways that are key to the production of antioxidant compounds that alleviate oxidative stress, and are also involved in DNA replication and glycerophospholipid metabolism. Figure 5 (D). Further exploration of RNA-seq data revealed that potassium chloride treatment induced the upregulation of various salt stress response genes, particularly those related to ion transport. Notably, this included... GhCHX25, GhACA11 GhACA7, GhCAX1, GhSULTR4 and GhKEA2 Isogenes ( Figure 5 E). CHX (cation / H) + Exchange body) and KEA (K + The efflux transporter gene family regulates K + / Na +Potassium ion transporters play an important role in balancing and maintaining ion homeostasis in plant cells. To assess the potassium ion transport activities of GhCHX25 and GhKEA2, they were expressed in the potassium-sensitive yeast mutant R5421 in this study. Growth experiments were conducted on arginine phosphate (AP) media supplemented with different potassium ion concentrations. Figure 5 As shown in Figure F, when the potassium ion concentration exceeded 500 µM, GhCHX25 and GhKEA2 partially recovered the growth of the empty vector, indicating that they play a functional role in potassium ion uptake in yeast. These results suggest that potassium chloride stress affects multiple biological processes at the transcriptional level and alters the expression patterns of several ion transport-related genes.

[0052] Example 4 Changes in H3K27me3 modification levels lead to alterations in gene expression levels. H3K27me3 is typically associated with transcriptional repression. To investigate whether changes in H3K27me3 modification under potassium chloride stress are related to gene expression, this study combined analysis of genes with differential H3K27me3 deposition in CUT&Tag data and differentially expressed genes (DEGs) in RNA-seq data. Under potassium chloride treatment, the H3K27me3 modification levels of 1096 genes changed. KEGG enrichment analysis showed that these genes were mainly enriched in phenylpropanoid biosynthesis, glutathione metabolism, ubiquinone, and other terpenoid-quinone biosynthesis. Among them, 48 genes showed the expected antagonistic relationship between H3K27me3 modification and gene expression. Specifically, 23 genes showed decreased H3K27me3 modification accompanied by upregulation of gene expression, while 25 genes showed increased H3K27me3 modification accompanied by downregulation of gene expression. Six genes were found to have... GhGSH1 ( Ghir_D09_ G02354 ), GhSRM1 ( Ghir_D09_G01343 ), GhSAUR71 ( Ghir_D12_G00340 ), GhFER-D ( Ghir_D13_ G01029 ), GhUVR8 ( Ghir_D13_G00874 )and GhFER - A ( Ghir_D13_G01028 This substance has been reported to be associated with salt stress. This embodiment pays particular attention to... GhGSH1 and GhSRM1 Both groups showed upregulated gene expression. The IGV peak diagram showed that the peak values ​​of these two genes after potassium chloride treatment were significantly lower than those in the Mock group (…). Figure 6 (A) showed a decreased level of H3K27me3 modification, a result that was also verified by CUT&Tag-qPCR detection. Figure 6(B). Detected by qRT-PCR. GhGSH1 and GhSRM1 The expression level was consistent with that of RNA-seq, and its expression level was upregulated after potassium chloride treatment. Figure 6 (C)

[0053] To further confirm the effect of histone modification on gene expression, this example examined the H3K27me3 modification levels and gene expression in cotton seedlings treated with 10 µM, 30 µM, and 50 µM inhibitors. GhGSH1 and GhSRM1 The expression level of H3K27me3 was found to be significantly reduced by the application of exogenous inhibitors. Figure 6 (middle D) and a significant increase in transcriptional levels ( Figure 6 (E). Overall, these findings provide strong evidence that dynamic changes in H3K27me3 modification are closely related to transcriptional reprogramming of salt stress response genes under potassium chloride stress, suggesting that this histone marker plays a key regulatory role in cotton adaptation to ionic stress.

[0054] Example 5 silence GhGSH1 or GhSRM1 It can reduce cotton's tolerance to potassium chloride. To verify GhGSH1 and GhSRM1 In this embodiment, the response function of cotton to potassium chloride stress was generated using virus-induced gene silencing (VIGS) technology. GhGSH1 Silence and GhSRM1 Silent cotton plants. qRT-PCR results showed that, compared to the control TRV:00, TRV: GhGSH1 and TRV: GhSRM1 In the plant GhGSH1 and GhSRM1 The expression was significantly reduced, confirming that... GhGSH1 and GhSRM1 Effective gene silencing ( Figure 7 ).

[0055] After treating cotton seedlings with 150 mM potassium chloride for three days, TRV: GhGSH1 The plants exhibited enhanced sensitivity, displaying more severe wilting and dehydration phenotypes than the control plants. Figure 8 (A). Physiological analysis shows that... GhGSH1 Gene silencing leads to H2O2 and O2 - The accumulation of MDA increased significantly, while chlorophyll content decreased. Figure 8 (B to E), indicating impaired management of oxidative stress. Furthermore, GhGSH1 K's silence disrupted + / Na+ steady state ( Figure 9 Similarly, TRV:Gh SRM1 The plants exhibited a similar hypersensitive response to potassium chloride treatment, showing accelerated wilting and dehydration. Figure 10 (A). Consistent with these observations, GhSRM1 Silence also leads to increased levels of reactive oxygen species (H2O2 and O2). - MDA content increased, chlorophyll decreased ( Figure 10 (B to E). GhSRM1 Silence leads to K under potassium chloride stress + / Na + Change in ratio ( Figure 11 Overall, these results indicate that... GhGSH1 and GhSRM1 By mitigating oxidative damage and maintaining ionic homeostasis, cotton is endowed with tolerance to potassium chloride stress.

[0056] References: Zheng D, Wang L, Chen L, Pan X, Lin K, Fang Y, Wang XE, Zhang W. Salt-responsive genes are differentially regulated at the chromatin levels between seedlings and roots in rice. Plant Cell Physiol. 2019:60(8):1790-1803. doi: https: / / doi.org / 10.1093 / pcp / pcz095; Han B, Xu W, Ahmed N, Yu A, Wang Z, Liu A. Changes and associations of genomic transcription and histone methylation with salt stress in castorbean. Plant Cell Physiol. 2020:61(6):1120-1133. doi: https: / / doi.org / 10.1093 / pcp / pcaa037; Sun L, Song G, Guo W, Wang W, Zhao H, Gao T, Lv Q, Yang X, Xu F, DongY, et al. Dynamic changes in genome-wide histone3 lysine27 trimethylation andgene expression of soybean roots in response to salt stress. Front. PlantSci. 2019:10:1031. doi: https: / / doi.org / 10.3389 / fpls.2019.01031; Kwon CS, Lee D, Choi G, Chung WI. Histone occupancy-dependent and -independent removal of H3K27 trimethylation at cold-responsive genes inArabidopsis. Plant J. 2009:60(1):112-121. doi: https: / / doi.org / 10.1111 / j.1365-313X.2009.03938.x; Sani E, Herzyk P, Perrella G, Colot V, Amtmann A. Hyperosmoticpriming of Arabidopsis seedlings establishes a long-term somatic memoryaccompanied by specific changes of the epigenome. Genome Biol. 2013:14(6):R59. doi: https: / / doi.org / 10.1186 / gb-2013-14-6-r59; de Sena Brandine G, Smith AD. Falco: high-speed FastQC emulation forquality control of sequencing data. F1000Res 2019:8:1874. doi: https: / / doi.org / 10.12688 / f1000research.21142.2; Chen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQpreprocessor. Bioinformatics 2018:34(17):i884-i890. doi: https: / / doi.org / 10.1093 / bioinformatics / bty560; Jung Y, Han D. BWA-MEME: BWA-MEM emulated with a machine learningapproach. Bioinformatics 2022:38(9):2404-2413. doi: https: / / doi.org / 10.1093 / bioinformatics / btac137; Hu G, Wang Z, Tian Z, Wang K, Ji G, Wang X, Zhang X, Yang Z, Liu X,Niu R, et al. A telomere-to-telomere genome assembly of cotton providesinsights into centromere evolution and short-season adaptation. Nat. Genet.2025:57(4):1031-1043. doi: https: / / doi.org / 10.1038 / s41588-025-02130-4; Ramírez F, Dündar F, Diehl S, Grüning BA, Manke T. deepTools: aflexible platform for exploring deep-sequencing data. Nucleic Acids Res.2014:42(Web Server issue):W187-191. doi: https: / / doi.org / 10.1093 / nar / gku365; Thorvaldsdóttir H, Robinson JT, Mesirov JP. Integrative GenomicsViewer (IGV): high-performance genomics data visualization and exploration.Brief Bioinform. 2013:14(2):178-192. doi: https: / / doi.org / 10.1093 / bib / bbs017; Yu G, Wang LG, He QY. ChIPseeker: an R / Bioconductor package for ChIPpeak annotation, comparison and visualization. Bioinformatics 2015:31(14):2382-2383. doi: https: / / doi.org / 10.1093 / bioinformatics / btv145; Jeon H, Lee H, Kang B, Jang I, Roh TY. Comparative analysis ofcommonly used peak calling programs for ChIP-Seq analysis. Genomics Inform.2020:18(4):e42. doi: https: / / doi.org / 10.5808 / GI.2020.18.4.e42; Kim D, Langmead B, Salzberg SL. HISAT: a fast spliced aligner withlow memory requirements. Nat. Methods 2015:12(4):357-360. doi: https: / / doi.org / 10.1038 / nmeth.3317; Pertea M, Pertea GM, Antonescu CM, Chang TC, Mendell JT, Salzberg SL.StringTie enables improved reconstruction of a transcriptome from RNA-seqreads. Nat. Biotechnol. 2015:33(3):290-295. doi: https: / / doi.org / 10.1038 / nbt.3122; Love MI, Huber W, Anders S. Moderated estimation of fold change anddispersion for RNA-seq data with DESeq2. Genome Biol. 2014:15(12):550. doi:https: / / doi.org / 10.1186 / s13059-014-0550-8; Kanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes.Nucleic Acids Res. 2000:28(1):27-30. doi: https: / / doi.org / 10.1093 / nar / 28.1.27; Livak KJ, Schmittgen TD. Analysis of relative gene expression datausing real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods2001:25(4):402-408. doi: https: / / doi.org / 10.1006 / meth.2001.1262; Han M, Wu W, Wu WH, Wang Y. Potassium transporter KUP7 is Involved in K(+) acquisition and translocation in Arabidopsis root under K(+)-limitedconditions. Mol. Plant 2016:9(3):437-446. doi: https: / / doi.org / 10.1016 / j.molp.2016.01.012.

[0057] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of histone H3K27me3 in regulating the response of cotton to potassium chloride stress.

2. The application according to claim 1, characterized in that, include: Application of chemical reagents that inhibit histone H3K27me3 deposition in improving cotton's tolerance to potassium chloride stress.

3. The application according to claim 2, characterized in that, The chemical reagents include histone H3K27me3 inhibitors.

4. The application according to claim 3, characterized in that, The histone H3K27me3 inhibitor includes RDS3434.

5. The application according to claim 4, characterized in that, The concentration of RDS 3434 is 10~50 µM.

6. The application according to claim 2, characterized in that, Downstream genes regulated by histone H3K27me3 under potassium chloride stress include GhGSH1 and / or GhSRM1 The GhGSH1 and GhSRM1 The login number is Ghir_D09_G02354 and Ghir_D09_G01343 Overexpression of the aforementioned gene in the cotton genome GhGSH1 and / or GhSRM1 Improve cotton's tolerance to potassium chloride stress.

7. The application according to any one of claims 1 to 6, characterized in that, The cotton includes upland cotton.

8. The application according to any one of claims 1 to 6, characterized in that, The potassium chloride stress includes potassium chloride-induced ion stress.

9. A method for improving the tolerance of cotton to potassium chloride stress, characterized in that, Includes the following steps: Cotton seedlings were cultured using the histone H3K27me3 inhibitor RDS 3434.

10. The method according to claim 9, characterized in that, The cultivation includes hydroponics, and the hydroponic step includes culturing cotton after adding RDS 3434 to the hydroponic system, wherein the concentration of RDS 3434 in the hydroponic system is 10~50 µM.

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