Use of histone methylation inhibitors in improving male fertility of cotton under high temperature stress

Abstract

This invention belongs to the field of crop breeding technology, specifically relating to the application of histone methylation inhibitors in improving male fertility in cotton under high-temperature stress. By applying histone methylation inhibitors to cotton buds, this invention reduces histone methylation expression in cotton anthers, avoiding male abortion caused by enhanced histone methylation under high-temperature stress, thereby improving male fertility in cotton under high-temperature stress, stabilizing cotton yield, and reducing yield reduction under high-temperature stress.

Description

Technical Field

[0001] This invention belongs to the field of crop breeding technology, specifically relating to the application of histone methylation inhibitors in improving male fertility in cotton under high temperature stress. Background Technology

[0002] Crops typically grow within a specific temperature range. Drastic low or high temperatures can disrupt their physiological processes, ultimately leading to yield loss, as seen in crops like Xu. [1] It was pointed out that rising temperatures will have an adverse impact on crop yields.

[0003] Cotton (Gossypium hirsutum) is one of the world's most important fiber and cash crops. It is planted in summer, but its yield is susceptible to adverse effects from high-temperature stress during the reproductive stage. [2]-[3] This leads to severe yield reduction in cotton. When temperatures exceed 35℃, cotton anthers exhibit male sterility phenotypes such as anther insufficiency and reduced pollen activity, resulting in significant yield losses. [4] Genetic breeding is one of the current methods to improve the fertility of cotton under high temperatures, but it has the drawback of a long improvement cycle. Summary of the Invention

[0004] The purpose of this invention is to provide the application of histone methylation inhibitors in improving the male fertility of cotton under high temperature stress. The histone methylation inhibitors can rapidly improve the male fertility of cotton under high temperature and stabilize cotton yield.

[0005] This invention provides the application of histone methylation inhibitors in improving male fertility in cotton under high temperature stress, wherein the high temperature is above 35°C.

[0006] Preferably, the histone includes cotton anther histone H3K27me3.

[0007] Preferably, the histone methylation inhibitor includes GSK343.

[0008] The present invention also provides a method for improving the male fertility of cotton at high temperatures, comprising: applying a histone methylation inhibitor to cotton buds; wherein the high temperature is above 35°C.

[0009] Preferably, the histone methylation inhibitor is used at an amount of 0.0002–0.004 μmol / flower bud.

[0010] Preferably, the application is performed by spraying an aqueous solution of histone methylation inhibitor dimethyl sulfoxide onto cotton buds; the concentration of the aqueous solution of histone methylation inhibitor dimethyl sulfoxide is 20–50 μM.

[0011] The amount of the dimethyl sulfoxide aqueous solution of the histone methylation inhibitor used is 10-80 μL / flower bud.

[0012] Preferably, the cotton buds include one or more of the following stages: tetrad stage, tapetum degradation stage, and anther dehiscence stage.

[0013] Preferably, the histone includes cotton anther histone H3K27me3.

[0014] Preferably, the histone methylation inhibitor includes GSK343.

[0015] The present invention also provides a pesticide containing a histone methylation inhibitor, wherein the effective concentration of the histone methylation inhibitor in the pesticide is 20–50 μM.

[0016] Beneficial effects:

[0017] This invention provides the application of histone methylation inhibitors in improving male fertility in cotton under high-temperature stress, where the high temperature is above 35°C. By applying histone methylation inhibitors to cotton buds, this invention reduces the expression of histone methylation, significantly decreasing male abortion caused by enhanced histone methylation under high-temperature stress, thereby improving male fertility in cotton under high-temperature stress, stabilizing cotton yield, reducing yield reduction under high-temperature stress, and achieving a shorter improvement cycle. Attached Figure Description

[0018] 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.

[0019] Figure 1 This is a technical roadmap for embodiments 1 to 3 of the present invention;

[0020] Figure 2 The sensitivity of cotton varieties “84021” (high temperature resistant) and “H05” (high temperature sensitive) in Example 1 to high temperatures;

[0021] Figure 3The image shows the whole genome modification diagrams of cotton anthers H3K4me3 and H3K27me3 under high temperature stress in Example 1. The labels are as follows: 8N-TDS: anthers of "84021" during the tapetal degradation stage at room temperature; 8H-TDS: anthers of "84021" during the tapetal degradation stage at high temperature; 8N-ADS: anthers of "84021" during the dehiscence stage at room temperature; 8H-ADS: anthers of "84021" during the dehiscence stage at high temperature; HN-TDS: anthers of "H05" during the tapetal degradation stage at room temperature; HH-TDS: anthers of "H05" during the tapetal degradation stage at high temperature; HN-ADS: anthers of "H05" during the dehiscence stage at room temperature; HH-ADS: anthers of "H05" during the dehiscence stage at high temperature.

[0022] Figure 4 To explore the optimal H3K27me3 inhibitor spraying concentration for the flower buds of the heat-sensitive cotton material "H05" under normal temperature conditions in Example 2;

[0023] Figure 5 The figure shows the pollen fertility-related phenotypic diagrams after spraying under the optimal concentration range of H 3K27me3 inhibitor in Example 3. The figures are explained as follows: T: tapetum; Msp: microspore; En: inner wall of the anther chamber; St: slit; PG: pollen grain. Detailed Implementation

[0024] This invention provides the application of histone methylation inhibitors in improving male fertility in cotton under high temperature stress; wherein the high temperature is above 35°C.

[0025] The histone described in this invention is preferably cotton anther histone H3K27me3; the histone methylation inhibitor is preferably GSK343.

[0026] This invention also provides a method for improving male fertility in cotton under high temperatures, comprising: applying a histone methylation inhibitor to cotton flower buds; wherein the high temperature is above 35°C. The preferred dosage of the histone methylation inhibitor is 0.0002–0.004 μmol / flower bud, more preferably 0.0025 μmol / flower bud. The preferred histone is cotton anther histone H3K27me3; the preferred histone methylation inhibitor is GSK343. Preferably, the histone methylation inhibitor is applied to the cotton flower buds 3 days before the high temperature is reached.

[0027] In this invention, the histone inhibitor is preferably sprayed onto cotton buds in the form of a dimethyl sulfoxide aqueous solution of the histone methylation inhibitor. The concentration of the dimethyl sulfoxide aqueous solution of the histone methylation inhibitor is preferably 20–50 μM, more preferably 30–50 μM, and even more preferably 50 μM. The preparation of the dimethyl sulfoxide aqueous solution of the histone methylation inhibitor is preferably as follows: the histone methylation inhibitor is dissolved in dimethyl sulfoxide, and then the resulting dimethyl sulfoxide solution of the histone methylation inhibitor is diluted with water to obtain the dimethyl sulfoxide aqueous solution of the histone methylation inhibitor.

[0028] The preferred amount of the dimethyl sulfoxide solution of the histone methylation inhibitor described in this invention is 10–80 μL / flower bud, more preferably 50 μL / flower bud.

[0029] The cotton buds described in this invention preferably include one or more of the following: the tetrad stage, the tapetum degradation stage, and the anther dehiscence stage; more preferably, cotton buds are in the tetrad stage or the tapetum degradation stage.

[0030] This invention discovers that high temperature enhances the methylation level of H3K27me3 histone in cotton germplasm, especially temperature-sensitive cotton germplasm, leading to male abortion in cotton. The invention also describes the use of H3K27me3 histone methylation inhibitors as exogenous spraying substances to improve cotton resistance under high temperature stress, significantly reduce pollen abortion, and stabilize cotton yield.

[0031] This invention also provides a pesticide containing a histone methylation inhibitor, wherein the effective concentration of the histone methylation inhibitor in the pesticide is 20–50 μM, preferably 50 μM. The excipients (solvents) in the pesticide of this invention preferably include dimethyl sulfoxide and water.

[0032] 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.

[0033] The technical route of the present invention is as follows: Figure 1 As shown.

[0034] Example 1

[0035] Constructing whole-genome modification maps of H3K4me3 and H3K27me3 in cotton anthers under high-temperature stress

[0036] Regarding the two cotton varieties "84021" (high temperature resistant) and "H05" (high temperature sensitive) materials previously discovered by the applicant ( Figure 2 At room temperature, both "84021" and "H05" exhibit anther dehiscence and pollen fertility. Figure 2(A, C, E, and G). After high temperature treatment, the heat-resistant strain "84021" exhibited normal anther dehiscence and fertile pollen grains ( Figure 2 (B and F), while the heat-sensitive strain "H05" exhibits anther insufficiency and pollen abortion ( Figure 2 (D and H).

[0037] Chromatin immunoprecipitation sequencing (ChIP-seq) was performed on anthers at both the TDS (taenia degradation stage, bud length 9–14 mm) and ADS (anther dehiscence stage, bud length >24 mm) conditions under normal temperature (NT) light exposure for 14 h, darkness for 10 h; daytime temperature 29–35 °C, nighttime temperature 25–27 °C) and high temperature (HT) light exposure for 14 h, darkness for 10 h; daytime temperature 38–40 °C, nighttime temperature 28–31 °C. [5] Two histone modification maps were obtained. Figure 3 (A and B). The modification peaks of H3K4me3 and H3K27me3 in the anthers at TDS and ADS stages of "84021" and "H05" were statistically analyzed. Figure 3 In studies conducted in sections C and D, it was found that the peak values ​​of H3K4me3 in the anthers of both the heat-tolerant strain "84021" and the heat-sensitive strain "H05" at both the TDS and ADS periods decreased by a similar magnitude under high temperatures. Figure 3 (C). The peak value of H3K27me3 varied inconsistently between "84021" and "H05". In the "84021" TDS and ADS periods, it changed from 21,212 and 34,486 at room temperature to 19,513 and 27,490 at high temperature in the anthers. Figure 3 In the high-temperature sensitive strain "H05", the peak values ​​of H3K27me3 in TDS and ADS changed from 31,134 and 30,239 under room temperature treatment to 31,340 and 31,413 under high temperature treatment, respectively. This result indicates that the peak value of H3K27me3 under high temperature treatment decreases in the high-temperature resistant strain "84021" but increases in the high-temperature sensitive strain "H05". Figure 3 (D).

[0038] Therefore, it is speculated that H3K27me3 may play a more important role in the high-temperature reaction of cotton anthers, and the increase in H3K27me3 level in the anthers of heat-sensitive varieties may be an important reason for anther abortion.

[0039] Example 2

[0040] Different concentrations of H3K27me3 inhibitor were sprayed onto the flower buds of the temperature-sensitive material "H05".

[0041] Under normal temperature conditions (14 hours of light, 10 hours of darkness; daytime 29–35°C, nighttime 25–27°C), the flower buds (including TS anthers and TDS anthers) of the heat-sensitive cotton material “H05” were sprayed with 0 μM, 20 μM, 50 μM and 150 μM of the H3K27me3 inhibitor GSK343 ​​(Selleck; catalog no. S7164). The flower buds included the tetrad stage (TS) anthers and the tapetum degradation stage (TDS) as detailed below.

[0042] The H3K27me3 inhibitor GSK343 ​​(manufactured by Selleck, catalog number S7164) was dissolved in DMSO and diluted with distilled water to 0 μM, 20 μM, 50 μM, and 150 μM, respectively. Then, at room temperature, the different concentrations of the H3K27me3 inhibitor solution were sprayed onto all buds at a dosage of 50 μL / flower bud. Three days later, the plants were treated with high temperature (14 h light, 10 h darkness; daytime 38–40 °C, nighttime 28–31 °C) for 7 days, designated as the experimental group. Plants cultivated under the same conditions and treated with distilled water served as the control group.

[0043] Pollen from the experimental and control groups was stained for TTC viability. The specific steps were as follows: 0.8% 2,3,5-triphenyltetrazol solution (TTC) was dissolved in phosphate buffer containing 0.061 M K₂HPO₄ and 0.038 M KH₂PO₄. 8 g / L TTC was then used, and the mixture was treated at 37°C in the dark for 40 min. The stained pollen was then observed under a microscope, and the results are as follows: Figure 4 As shown, the TTC viability staining results of pollen treated with 0 μM, 20 μM, 50 μM and 150 μM H3K27me3 inhibitor solutions are A and E, B and F, C and G, and D and H, respectively.

[0044] Depend on Figure 4 It was found that H3K27me3 inhibitors below 50 μM had little effect on pollen viability, while H3K27me3 inhibitors at 150 μM caused severe early bud drop. Figure 4 In the middle stage, the anthers of the flower buds do not dehisce (I). Figure 4 The inhibitors (D and H) and pollen inactivation were investigated. Therefore, the optimal spraying concentration was initially determined to be 50 μM or lower. This concentration of inhibitor solution can maintain the fertility of anthers and pollen in heat-sensitive cotton materials without causing early bud drop. 50 μM has a stronger effect than 20 μM, so 50 μM was selected as the optimal spraying concentration.

[0045] Example 3

[0046] Phenotypic analysis of flower buds of thermosensitive materials after spraying with the optimal concentration of H3K27me3 inhibitor under normal and high temperature conditions.

[0047] The temperature-sensitive material "H05" flower buds were subjected to control and treatment group experiments under both room temperature and high temperature conditions, and further phenotypic observations were conducted. The specific experimental setup was as follows: HN+water: 5 mL of water was sprayed onto "H05" flower buds at the TDS stage under room temperature conditions; HN+inhibitor: 5 mL of 50 μM H3K27me3 inhibitor (prepared as in Example 2) was sprayed onto "H05" flower buds under room temperature conditions; HH+water: 5 mL of water was sprayed onto "H05" flower buds under high temperature conditions; HH+inhibitor: 5 mL of 50 μM H3K27me3 inhibitor was sprayed onto "H05" flower buds under high temperature conditions.

[0048] First, the morphology of the treated anthers was observed using a Canon 70d HD high-definition digital camera. To prevent negative background interference, a black backdrop was used as the photograph background. The anthers were then subjected to TTC viability staining, and the results are as follows: Figure 5 As shown in Figure I.

[0049] Depend on Figure 5 The results show that under normal temperature conditions, there was no significant difference in anther dehiscence rate and pollen viability between the H3K27me3 inhibitor (IB, IF) and the control (IA, IE). However, under high temperature conditions, compared with the control group (water) (IC, IG), the anther dehiscence rate and pollen viability of "H05" anthers treated with 50 μM H3K27me3 inhibitor were both increased under high temperature stress (ID, IH).

[0050] Therefore, it can be concluded that at room temperature, 50 μM of the H3K27me3 inhibitor has no significant effect on filament length and pollen viability. Figure 5 In the case of IA, IB, IE, and IF, the filaments are shorter under HH+water conditions, and the pollen cannot be stained by TTC. Figure 5 (IC and IG); however, HH+ inhibitor treatment can maintain pollen viability similar to that under normal temperature conditions. Figure 5 (ID and IH).

[0051] Tissue dissection, staining, and imaging were performed according to the literature of Wu et al. [6]Specifically, after removing bracts and petals, the anthers were fixed in FAA fixative (50 mL anhydrous ethanol, 10 mL 37% formaldehyde solution, 5 mL acetic acid, diluted with water to 100 mL). Dehydration was performed using a series of ethanol solutions of different volume percentages (30%, 50%, 70%, 95%, and 100%). The tissues were embedded in epoxy resin and sectioned to a thickness of 10 μm. The anther sections were stained with 1% toluidine blue and 1% aniline blue solutions. Bright-field imaging of the samples was performed using a Zeiss Axio Scope A1 microscope.

[0052] Observation by sectioning showed no significant difference in anther degradation during the tapetum degradation (TDS) and anther dehiscence (ADS) stages at room temperature between spraying with H3K27me3 inhibitor and spraying with water (control group). Figure 5 II-A to II-D). Under high temperature conditions, the anther cytological observation results of the water-sprayed (control group) showed that the microspores were not completely filled during the tapetum degradation period (TDS), and the tapetum was not completely degraded; the anther dehiscence period (ADS) did not form a fissure, the anther chamber wall was thickened, and the pollen grain morphology was abnormal. Figure 5 II-E and II-G). However, spraying with the H3K27me3 inhibitor was able to maintain the integrity of pollen grains in "H05" under high temperature stress conditions ( Figure 5 (II-F and II-H).

[0053] Therefore, it can be concluded that at room temperature, there was no significant difference between spraying with H3K27me3 inhibitor and spraying with water (control group) during the tapetum degradation period (TDS) and anther dehiscence period (ADS). Figure 5 (II-A to II-D). It was found that after high-temperature stress, the pollen grains in the HH+water group lacked contents, exhibited abnormal development, and showed pollen shriveling, displaying a typical male sterility phenotype. Figure 5 Pollen grains treated with 50 μM H3K27me3 inhibitor were similar to those treated at room temperature (II-E and II-G); while pollen grains treated with 50 μM H3K27me3 inhibitor were similar to those treated at room temperature (II-E and II-G). Figure 5 Further studies (II-F and II-H) confirmed that a 50 μM H3K27me3 inhibitor can improve male fertility under high temperature.

[0054] 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.

[0055] References:

[0056] [1]Xu,Y.,Chu,C.and Yao,S.(2021)The impact ofhigh-temperature stresson rice:challenges and solutions.Crop J.9,963–976.

[0057] [2]Khan,A.H.,Min,L.,Ma,Y.,Wu,Y.,Ding,Y.,Li,Y.,Xie,S.et al.(2020)Highday and night temperatures distinctively disrupt fatty acid and jasmonic acidmetabolism,inducing male sterility in cotton.J.Exp.Bot.71,6128–6141.

[0058] [3]Ma,Y.,Min,L.,Wang,J.,Li,Y.,Wu,Y.,Hu,Q.,Ding,Y.et al.(2021)Acombination of genome-wide and transcriptome-wide association studies revealsgenetic elements leading to male sterility during high temperature stress incotton.New Phytol.231,165-181.

[0059] [4]Min,L.,Li,Y.,Hu,Q.,Zhu,L.,Gao,W.,Wu,Y.,Ding,Y.,Liu,S.,Yang,X.,andZhang,X.(2014).Sugar and auxin signaling pathways respond to high-temperaturestress during anther development as revealed by transcript profiling analysisin cotton.Plant physiology 164,1293-1308.

[0060] [5]Wang M,Wang P,Tu L,Zhu S,Zhang L,Li Z,Zhang Q,Yuan D,Zhang X.(2016).Multi-omics maps of cotton fibre reveal epigenetic basis for stagedsingle-cell differentiation.NucleicAcids Res.44(9):4067-407.

[0061] [6]Wu Y.,Li X.,Li Y.,Ma H.,Chi H.,Ma Y.,Yang J.,Xie S.,Zhang R.,LiuL.,Su X.,Lv R.,Khan,A.H.,Kong J.,Guo X.,Lindsey K.,Min L.,Zhang X.(2022).Degradation of de-esterified pectin / homogalacturonan by thepolygalacturonase GhNSP is necessary for pollen exine formation and malefertility in cotton.Plant Biotechnol J.20(6):1054-1068.

Claims

1. The application of histone methylation inhibitors in improving male fertility in cotton under high-temperature stress, characterized in that, The high temperature is above 35°C; the histone is cotton anther histone H3K27me3; The histone methylation inhibitor is GSK343; the dosage of the histone methylation inhibitor is 0.0002~0.004 μmol / flower bud.

2. A method for improving male fertility in cotton under high temperature, characterized in that, include: The histone methylation inhibitor was applied to cotton flower buds; the high temperature was above 35°C; the histone was cotton anther histone H3K27me3; the dosage of the histone methylation inhibitor was 0.0002~0.004 μmol / flower bud; the histone methylation inhibitor was GSK343.

3. The method according to claim 2, characterized in that, The application method is as follows: spraying an aqueous solution of dimethyl sulfoxide, a histone methylation inhibitor, onto cotton buds; the concentration of the aqueous solution of dimethyl sulfoxide, the histone methylation inhibitor, is 20-50 μM. The amount of the dimethyl sulfoxide aqueous solution of the histone methylation inhibitor used is 10~80 μL / flower bud.

4. The method according to claim 2 or 3, characterized in that, The cotton buds include one or more of the following stages: tetrad stage, tapetum degradation stage, and anther dehiscence stage.

Images