High-temperature stress in crops: male sterility, yield loss and potential remedy approaches.

Global food security is one of the utmost essential challenges in the 21st century in providing enough food for the growing population while coping with the already stressed environment. High temperature (HT) is one of the main factors affecting plant growth, development and reproduction and causes male sterility in plants. In male reproductive tissues, metabolic changes induced by HT involve carbohydrates, lipids, hormones, epigenetics and reactive oxygen species, leading to male sterility and ultimately reducing yield. Understanding the mechanism and genes involved in these pathways during the HT stress response will provide a new path to improve crops by using molecular breeding and biotechnological approaches. Moreover, this review provides insight into male sterility and integrates this with suggested strategies to enhance crop tolerance under HT stress conditions at the reproductive stage.

N6-methyladenosine RNA modification regulates cotton drought response in a Ca2+ and ABA-dependent manner.

N6 -methyladenosine (m6 A) is the most prevalent internal modification present in mRNAs, and is considered to participate in a range of developmental and biological processes. Drought response is highly regulated at the genomic, transcriptional and post-transcriptional levels. However, the biological function and regulatory mechanism of m6 A modification in the drought stress response is still poorly understood. We generated a transcriptome-wide m6 A map using drought-resistant and drought-sensitive varieties of cotton under different water deficient conditions to uncover patterns of m6 A methylation in cotton response to drought stress. The results reveal that m6 A represents a common modification and exhibit dramatic changes in distribution during drought stress. More 5'UTR m6 A was deposited in the drought-resistant variety and was associated with a positive effect on drought resistance by regulating mRNA abundance. Interestingly, we observed that increased m6 A abundance was associated with increased mRNA abundance under drought, contributing to drought resistance, and vice versa. The demethylase GhALKBH10B was found to decrease m6 A levels, facilitating the mRNA decay of ABA signal-related genes (GhZEP, GhNCED4 and GhPP2CA) and Ca2+ signal-related genes (GhECA1, GhCNGC4, GhANN1 and GhCML13), and mutation of GhALKBH10B enhanced drought resistance at seedling stage in cotton. Virus-induced gene silencing (VIGS) of two Ca2+ -related genes, GhECA1 and GhCNGC4, reduced drought resistance with the decreased m6 A enrichment on silenced genes in cotton. Collectively, we reveal a novel mechanism of post-transcriptional modification involved in affecting drought response in cotton, by mediating m6 A methylation on targeted transcripts in the ABA and Ca2+ signalling transduction pathways.

High temperature induces male sterility via MYB66-MYB4-Casein kinase I signaling in cotton.

High temperature (HT) causes male sterility and decreases crop yields. Our previous works have demonstrated that sugar and auxin signaling pathways, Gossypium hirsutum Casein kinase I (GhCKI), and DNA methylation are all involved in HT-induced male sterility in cotton. However, the signaling mechanisms leading to distinct GhCKI expression patterns induced by HT between HT-tolerant and HT-sensitive cotton anthers remain largely unknown. Here, we identified a GhCKI promoter (ProGhCKI) region that functions in response to HT in anthers and found the transcription factor GhMYB4 binds to this region to act as an upstream positive regulator of GhCKI. In the tapetum of early-stage cotton anthers, upregulated expression of GhMYB4 under HT and overexpressed GhMYB4 under normal temperature both led to severe male sterility phenotypes, coupled with enhanced expression of GhCKI. We also found that GhMYB4 interacts with GhMYB66 to form a heterodimer to enhance its binding to ProGhCKI. However, GhMYB66 showed an expression pattern similar to GhMYB4 under HT but did not directly bind to ProGhCKI. Furthermore, HT reduced siRNA-mediated CHH DNA methylations in the GhMYB4 promoter, which enhanced the expression of GhMYB4 in tetrad stage anthers and promoted the formation of the GhMYB4/GhMYB66 heterodimer, which in turn elevated the transcription of GhCKI in the tapetum, leading to male sterility. Overall, we shed light on the GhMYB66-GhMYB4-GhCKI regulatory pathway in response to HT in cotton anthers.

Degradation of de-esterified pctin/homogalacturonan by the polygalacturonase GhNSP is necessary for pollen exine formation and male fertility in cotton.

The pollen wall exine provides a protective layer for the male gametophyte and is largely composed of sporopollenin, which comprises fatty acid derivatives and phenolics. However, the biochemical nature of the external exine is poorly understood. Here, we show that the male sterile line 1355A of cotton mutated in NO SPINE POLLEN (GhNSP) leads to defective exine formation. The GhNSP locus was identified through map-based cloning and confirmed by genetic analysis (co-segregation test and allele prediction using the CRISPR/Cas9 system). In situ hybridization showed that GhNSP is highly expressed in tapetum. GhNSP encodes a polygalacturonase protein homologous to AtQRT3, which suggests a function for polygalacturonase in pollen exine formation. These results indicate that GhNSP is functionally different from AtQRT3, the latter has the function of microspore separation. Biochemical analysis showed that the percentage of de-esterified pectin was significantly increased in the 1355A anthers at developmental stage 8. Furthermore, immunofluorescence studies using antibodies to the de-esterified and esterified homogalacturonan (JIM5 and JIM7) showed that the Ghnsp mutant exhibits abundant of de-esterified homogalacturonan in the tapetum and exine, coupled with defective exine formation. The characterization of GhNSP provides new understanding of the role of polygalacturonase and de-esterified homogalacturonan in pollen exine formation.

Rapid Identification of Pollen- and Anther-Specific Genes in Response to High-Temperature Stress Based on Transcriptome Profiling Analysis in Cotton.

Anther indehiscence and pollen sterility caused by high temperature (HT) stress have become a major problem that decreases the yield of cotton. Pollen- and anther-specific genes play a critical role in the process of male reproduction and the response to HT stress. In order to identify pollen-specific genes that respond to HT stress, a comparative transcriptome profiling analysis was performed in the pollen and anthers of Gossypium hirsutum HT-sensitive Line H05 against other tissue types under normal temperature (NT) conditions, and the analysis of a differentially expressed gene was conducted in the pollen of H05 under NT and HT conditions. In total, we identified 1111 pollen-specific genes (PSGs), 1066 anther-specific genes (ASGs), and 833 pollen differentially expressed genes (DEGs). Moreover, we found that the late stage of anther included more anther- and pollen-specific genes (APSGs). Stress-related cis-regulatory elements (CREs) and hormone-responsive CREs are enriched in the promoters of APSGs, suggesting that APSGs may respond to HT stress. However, 833 pollen DEGs had only 10 common genes with 1111 PSGs, indicating that PSGs are mainly involved in the processes of pollen development and do not respond to HT stress. Promoters of these 10 common genes are enriched for stress-related CREs and MeJA-responsive CREs, suggesting that these 10 common genes are involved in the process of pollen development while responding to HT stress. This study provides a pathway for rapidly identifying cotton pollen-specific genes that respond to HT stress.

Cytochrome P450 mono-oxygenase CYP703A2 plays a central role in sporopollenin formation and ms5ms6 fertility in cotton.

The double-recessive genic male-sterile (ms) line ms5 ms6 has been used to develop cotton (Gossypium hirsutum) hybrids for many years, but its molecular-genetic basis has remained unclear. Here, we identified the Ms5 and Ms6 loci through map-based cloning and confirmed their function in male sterility through CRISPR/Cas9 gene editing. Ms5 and Ms6 are highly expressed in stages 7-9 anthers and encode the cytochrome P450 mono-oxygenases CYP703A2-A and CYP703A2-D. The ms5 mutant carries a single-nucleotide C-to-T nonsense mutation leading to premature chain termination at amino acid 312 (GhCYP703A2-A312aa ), and ms6 carries three nonsynonymous substitutions (D98E, E168K, and G198R) and a synonymous mutation (L11L). Enzyme assays showed that GhCYP703A2 proteins hydroxylate fatty acids, and the ms5 (GhCYP703A2-A312aa ) and ms6 (GhCYP703A2-DD98E,E168K,G198R ) mutant proteins have decreased enzyme activities. Biochemical and lipidomic analyses showed that in ms5 ms6 plants, C12-C18 free fatty acid and phospholipid levels are significantly elevated in stages 7-9 anthers, while stages 8-10 anthers lack sporopollenin fluorescence around the pollen, causing microspore degradation and male sterility. Overall, our characterization uncovered functions of GhCYP703A2 in sporopollenin formation and fertility, providing guidance for creating male-sterile lines to facilitate hybrid cotton production and therefore exploit heterosis for improvement of cotton.

An enhanced photosynthesis and carbohydrate metabolic capability contributes to heterosis of the cotton (Gossypium hirsutum) hybrid 'Huaza Mian H318', as revealed by genome-wide gene expression analysis.

BACKGROUND: Heterosis has been exploited for decades in different crops due to resulting in dramatic increases in yield, but relatively little molecular evidence on this topic was reported in cotton. RESULTS: The elite cotton hybrid variety 'Huaza Mian H318' (H318) and its parental lines were used to explore the source of its yield heterosis. A four-year investigation of yield-related traits showed that the boll number of H318 showed higher stability than that of its two parents, both in suitable and unsuitable climate years. In addition, the hybrid H318 grew faster and showed higher fresh and dry weights than its parental lines at the seedling stage. Transcriptome analysis of seedlings identified 17,308 differentially expressed genes (DEGs) between H318 and its parental lines, and 3490 extremely changed DEGs were screened out for later analysis. Most DEGs (3472/3490) were gathered between H318 and its paternal line (4-5), and only 64 DEGs were found between H318 and its maternal line (B0011), which implied that H318 displays more similar transcriptional patterns to its maternal parent at the seedling stage. GO and KEGG analyses showed that these DEGs were highly enriched in photosynthesis, lipid metabolic, carbohydrate metabolic and oxidation-reduction processes, and the expression level of these DEGs was significantly higher in H318 relative to its parental lines, which implied that photosynthesis, metabolism and stress resistances were enhanced in H318. CONCLUSION: The enhanced photosynthesis, lipid and carbohydrate metabolic capabilities contribute to the heterosis of H318 at the seedling stage, and establishes a material foundation for subsequent higher boll-setting rates in complex field environments.

A combination of genome-wide and transcriptome-wide association studies reveals genetic elements leading to male sterility during high temperature stress in cotton.

Global warming has reduced the productivity of many field-grown crops, as the effects of high temperatures can lead to male sterility in such plants. Genetic regulation of the high temperature (HT) response in the major crop cotton is poorly understood. We determined the functionality and transcriptomes of the anthers of 218 cotton accessions grown under HT stress. By analyzing transcriptome divergence and implementing a genome-wide association study (GWAS), we identified three thermal tolerance associated loci which contained 75 protein coding genes and 27 long noncoding RNAs, and provided expression quantitative trait loci (eQTLs) for 13 132 transcripts. A transcriptome-wide association study (TWAS) confirmed six causal elements for the HT response (three genes overlapped with the GWAS results) which are involved in protein kinase activity. The most susceptible gene, GhHRK1, was confirmed to be a previously uncharacterized negative regulator of the HT response in both cotton and Arabidopsis. These functional variants provide a new understanding of the genetic basis for HT tolerance in male reproductive organs.

Disrupted Genome Methylation in Response to High Temperature Has Distinct Affects on Microspore Abortion and Anther Indehiscence.

High-temperature (HT) stress induces male sterility, leading to yield reductions in crops. DNA methylation regulates a range of processes involved in plant development and stress responses, but its role in male sterility under HT remains unknown. Here, we investigated DNA methylation levels in cotton (Gossypium hirsutum) anthers under HT and normal temperature (NT) conditions by performing whole-genome bisulfite sequencing to investigate the regulatory roles of DNA methylation in male fertility under HT. Global disruption of DNA methylation, especially CHH methylation (where H = A, C, or T), was detected in an HT-sensitive line. Changes in the levels of 24-nucleotide small-interfering RNAs were significantly associated with DNA methylation levels. Experimental suppression of DNA methylation led to pollen sterility in the HT-sensitive line under NT conditions but did not affect the normal dehiscence of anther walls. Further transcriptome analysis showed that the expression of genes in sugar and reactive oxygen species (ROS) metabolic pathways were significantly modulated in anthers under HT, but auxin biosynthesis and signaling pathways were only slightly altered, indicating that HT disturbs sugar and ROS metabolism via disrupting DNA methylation, leading to microspore sterility. This study opens up a pathway for creating HT-tolerant cultivars using epigenetic techniques.

Proteomic analysis reveals that sugar and fatty acid metabolisms play a central role in sterility of the male-sterile line 1355A of cotton.

Cotton (Gossypium spp.) is one of the most important economic crops and exhibits yield-improving heterosis in specific hybrid combinations. The genic male-sterility system is the main strategy used for producing heterosis in cotton. To better understand the mechanisms of male sterility in cotton, we carried out two-dimensional electrophoresis (2-DE) and label-free quantitative proteomics analysis in the anthers of two near-isogenic lines, the male-sterile line 1355A and the male-fertile line 1355B. We identified 39 and 124 proteins that were significantly differentially expressed between these two lines in the anthers at the tetrad stage (stage 7) and uninucleate pollen stage (stage 8), respectively. Gene ontology-based analysis revealed that these differentially expressed proteins were mainly associated with pyruvate, carbohydrate, and fatty acid metabolism. Biochemical analysis revealed that in the anthers of line 1355A, glycolysis was activated, which was caused by a reduction in fructose, glucose, and other soluble sugars, and that accumulation of acetyl-CoA was increased along with a significant increase in C14:0 and C18:1 free fatty acids. However, the activities of pyruvate dehydrogenase and fatty acid biosynthesis were inhibited and fatty acid ß-oxidation was activated at the translational level in 1355A. We speculate that in the 1355A anther, high rates of glucose metabolism may promote fatty acid synthesis to enable anther growth. These results provide new insights into the molecular mechanism of genic male sterility in upland cotton.

High day and night temperatures distinctively disrupt fatty acid and jasmonic acid metabolism, inducing male sterility in cotton.

High temperature stress is an inevitable environmental factor in certain geographical regions. To study the effect of day and night high temperature stress on male reproduction, the heat-sensitive cotton line H05 was subjected to high temperature stress. High day/normal night (HN) and normal day/high night (NH) temperature treatments were compared with normal day/normal night (NN) temperature as a control. At the anther dehiscence stage, significant differences were observed, with a reduction in flower size and filament length, and sterility in pollen, seen in NH more than in HN. A total of 36 806 differentially expressed genes were screened, which were mainly associated with fatty acid and jasmonic acid (JA) metabolic pathways. Fatty acid and JA contents were reduced more in NH than HN. Under NH, ACYL-COA OXIDASE 2 (ACO2), a JA biosynthesis gene, was down-regulated. Interestingly, aco2 CRISPR-Cas9 mutants showed male sterility under the NN condition. The exogenous application of methyl jasmonate to early-stage buds of mutants rescued the sterile pollen and indehiscent anther phenotypes at the late stage. These data show that high temperature at night may affect fatty acid and JA metabolism in anthers by suppressing GhACO2 and generate male sterility more strongly than high day temperature.

Laccase GhLac1 Modulates Broad-Spectrum Biotic Stress Tolerance via Manipulating Phenylpropanoid Pathway and Jasmonic Acid Synthesis.

Plants are constantly challenged by a multitude of pathogens and pests, which causes massive yield and quality losses annually. A promising approach to reduce such losses is to enhance the immune system of plants through genetic engineering. Previous work has shown that laccases (p-diphenol:dioxygen oxidoreductase, EC 1.10.3.2) function as lignin polymerization enzymes. Here we demonstrate that transgenic manipulation of the expression of the laccase gene GhLac1 in cotton (Gossypium hirsutum) can confer an enhanced defense response to both pathogens and pests. Overexpression of GhLac1 leads to increased lignification, associated with increased tolerance to the fungal pathogen Verticillium dahliae and to the insect pests cotton bollworm (Helicoverpa armigera) and cotton aphid (Aphis gosypii). Suppression of GhLac1 expression leads to a redirection of metabolic flux in the phenylpropanoid pathway, causing the accumulation of JA and secondary metabolites that confer resistance to V. dahliae and cotton bollworm; it also leads to increased susceptibility to cotton aphid. Plant laccases therefore provide a new molecular tool to engineer pest and pathogen resistance in crops.

microRNAs involved in auxin signalling modulate male sterility under high-temperature stress in cotton (Gossypium hirsutum).

Male sterility caused by long-term high-temperature (HT) stress occurs widely in crops. MicroRNAs (miRNAs), a class of endogenous non-coding small RNAs, play an important role in the plant response to various abiotic stresses. To dissect the working principle of miRNAs in male sterility under HT stress in cotton, a total of 112 known miRNAs, 270 novel miRNAs and 347 target genes were identified from anthers of HT-insensitive (84021) and HT-sensitive (H05) cotton cultivars under normal-temperature and HT conditions through small RNA and degradome sequencing. Quantitative reverse transcriptase-polymerase chain reaction and 5'-RNA ligase-mediated rapid amplification of cDNA ends experiments were used to validate the sequencing data. The results show that miR156 was suppressed by HT stress in both 84021 and H05; miR160 was suppressed in 84021 but induced in H05. Correspondingly, SPLs (target genes of miR156) were induced both in 84021 and H05; ARF10 and ARF17 (target genes of miR160) were induced in 84021 but suppressed in H05. Overexpressing miR160 increased cotton sensitivity to HT stress seen as anther indehiscence, associated with the suppression of ARF10 and ARF17 expression, thereby activating the auxin response that leads to anther indehiscence. Supporting this role for auxin, exogenous Indole-3-acetic acid (IAA) leads to a stronger male sterility phenotype both in 84021 and H05 under HT stress. Cotton plants overexpressing miR157 suppressed the auxin signal, and also showed enhanced sensitivity to HT stress, with microspore abortion and anther indehiscence. Thus, we propose that the auxin signal, mediated by miRNAs, is essential for cotton anther fertility under HT stress.

Promoters of Arabidopsis Casein kinase I-like 2 and 7 confer specific high-temperature response in anther.

KEY MESSAGE: (1) We systematically analyze the promoter activities of AtCKLs in various tissues; (2) AtCKL2 and AtCKL7 were expressed in early developmental anthers under high temperature (HT) conditions; (3) AtMYB24 may function as a positive regulator of AtCKL2 and AtCKL7 expression under HT. High temperature (HT) can seriously impede plant growth and development, causing severe loss of crop production. In Arabidopsis, AtCKL genes show high similarity to GhCKI, a gene reported to disrupt tapetal programmed cell death in cotton. However, most of AtCKL genes are not well characterized. Here, we systematically analyzed the expression patterns of AtCKLs in various tissues. The expression of AtCKL2 and AtCKL7 was induced in early anther development under HT, which is similar to the case of GhCKI. In silico analysis of AtCKL2 and AtCKL7 promoters indicated that four types of transcription factors (TFs) (MADS, NAC, WRKY and R2R3-MYB) might bind to AtCKL2 and AtCKL7 promoters. Furthermore, three MADS, three NAC, one WRKY, and three R2R3-MYB TFs were up-regulated in stage 1-8 anthers and three R2R3-MYB TFs were up-regulated in stage 9-10 anthers under HT, implying the important roles of R2R3-MYB genes in the response of anthers to HT. Among the R2R3-MYB genes, AtMYB24 showed the similar expression as AtCKL2 and AtCKL7 in the anthers under HT. Additionally, yeast one-hybrid and dual-luciferase reporter system assays verified that AtMYB24 could bind to AtCKL2 and AtCKL7 promoters and activate the expression of these two genes. In brief, this study provides the overall expression profiles of AtCKLs, useful information for unraveling the molecular mechanism of AtCKL2 and AtCKL7 gene expression in early anther development under HT, and important clues for elucidating the mechanism of transcriptional regulation of CKI genes in plant anther under HT, which are critical to the reduction of crop yield loss resulting from HT.

High efficient multisites genome editing in allotetraploid cotton (Gossypium hirsutum) using CRISPR/Cas9 system.

Gossypium hirsutum is an allotetraploid with a complex genome. Most genes have multiple copies that belong to At and Dt subgenomes. Sequence similarity is also very high between gene homologues. To efficiently achieve site/gene-specific mutation is quite needed. Due to its high efficiency and robustness, the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has exerted broad site-specific genome editing from prokaryotes to eukaryotes. In this study, we utilized a CRISPR/Cas9 system to generate two sgRNAs in a single vector to conduct multiple sites genome editing in allotetraploid cotton. An exogenously transformed gene Discosoma red fluorescent protein2(DsRed2) and an endogenous gene GhCLA1 were chosen as targets. The DsRed2-edited plants in T0 generation reverted its traits to wild type, with vanished red fluorescence the whole plants. Besides, the mutated phenotype and genotype were inherited to their T1 progenies. For the endogenous gene GhCLA1, 75% of regenerated plants exhibited albino phenotype with obvious nucleotides and DNA fragments deletion. The efficiency of gene editing at each target site is 66.7-100%. The mutation genotype was checked for both genes with Sanger sequencing. Barcode-based high-throughput sequencing, which could be highly efficient for genotyping to a population of mutants, was conducted in GhCLA1-edited T0 plants and it matched well with Sanger sequencing results. No off-target editing was detected at the potential off-target sites. These results prove that the CRISPR/Cas9 system is highly efficient and reliable for allotetraploid cotton genome editing.

LEAFY COTYLEDON1-CASEIN KINASE I-TCP15-PHYTOCHROME INTERACTING FACTOR4 Network Regulates Somatic Embryogenesis by Regulating Auxin Homeostasis.

Somatic embryogenesis (SE) is an efficient tool for the propagation of plant species and also, a useful model for studying the regulatory networks in embryo development. However, the regulatory networks underlying the transition from nonembryogenic callus to somatic embryos during SE remain poorly understood. Here, we describe an upland cotton (Gossypium hirsutum) CASEIN KINASE I gene, GhCKI, which is a unique key regulatory factor that strongly affects SE. Overexpressing GhCKI halted the formation of embryoids and plant regeneration because of a block in the transition from nonembryogenic callus to somatic embryos. In contrast, defective GhCKI in plants facilitated SE. To better understand the mechanism by which GhCKI regulates SE, the regulatory network was analyzed. A direct upstream negative regulator protein, cotton LEAFY COTYLEDON1, was identified to be targeted to a cis-element, CTTTTC, in the promoter of GhCKI. Moreover, GhCKI interacted with and phosphorylated cotton CINCINNATA-like TEOSINTE BRANCHED1-CYCLOIDEA-PCF transcription factor15 by coordinately regulating the expression of cotton PHYTOCHROME INTERACTING FACTOR4, finally disrupting auxin homeostasis, which led to increased cell proliferation and aborted somatic embryo formation in GhCKI-overexpressing somatic cells. Our results show a complex process of SE that is negatively regulated by GhCKI through a complex regulatory network.

Single-Cell Transcriptome Atlas and Regulatory Dynamics in Developing Cotton Anthers.

Plant anthers are composed of different specialized cell types with distinct roles in plant reproduction. High temperature (HT) stress causes male sterility, resulting in crop yield reduction. However, the spatial expression atlas and regulatory dynamics during anther development and in response to HT remain largely unknown. Here, the first single-cell transcriptome atlas and chromatin accessibility survey in cotton anther are established, depicting the specific expression and epigenetic landscape of each type of cell in anthers. The reconstruction of meiotic cells, tapetal cells, and middle layer cell developmental trajectories not only identifies novel expressed genes, but also elucidates the precise degradation period of middle layer and reveals a rapid function transition of tapetal cells during the tetrad stage. By applying HT, heterogeneity in HT response is shown among cells of anthers, with tapetal cells responsible for pollen wall synthesis are most sensitive to HT. Specifically, HT shuts down the chromatin accessibility of genes specifically expressed in the tapetal cells responsible for pollen wall synthesis, such as QUARTET 3 (QRT3) and CYTOCHROME P450 703A2 (CYP703A2), resulting in a silent expression of these genes, ultimately leading to abnormal pollen wall and male sterility. Collectively, this study provides substantial information on anthers and provides clues for heat-tolerant crop creation.

Dissecting the Superior Drivers for the Simultaneous Improvement of Fiber Quality and Yield Under Drought Stress Via Genome-Wide Artificial Introgressions of Gossypium barbadense into Gossypium hirsutum.

Global water scarcity and extreme weather intensify drought stress, significantly reducing cotton yield and quality worldwide. Drought treatments are conducted using a population of chromosome segment substitution lines generated from E22 (G. hirsutum) and 3-79 (G. barbadense) as parental lines either show superior yields or fiber quality under both control and drought conditions. Fourteen datasets, covering 4 yields and 4 quality traits, are compiled and assessed for drought resistance using the drought resistance coefficient (DRC) and membership function value of drought resistance (MFVD). Genome-wide association studies, linkage analysis, and bulked segregant analysis are combined to analyze the DR-related QTL. A total of 121 significant QTL are identified by DRC and MFVD of the 8 traits. CRISPR/Cas9 and virus-induced gene silencing techniques verified DRR1 and DRT1 as pivotal genes in regulating drought resistant of cotton, with hap3-79 exhibiting greater drought resistance than hapE22 concerning DRR1 and DRT1. Moreover, 14 markers with superior yield and fiber quality are selected for drought treatment. This study offers valuable insights into yield and fiber quality variations between G. hirsutum and G. barbadense amid drought, providing crucial theoretical and technological backing for developing cotton varieties resilient to drought, with high yield and superior fiber quality.

Histone H3 lysine 27 trimethylation suppresses jasmonate biosynthesis and signaling to affect male fertility under high temperature in cotton.

High-temperature (HT) stress causes male sterility in crops, thus decreasing yields. To explore the possible contribution of histone modifications to male fertility under HT conditions, we defined the histone methylation landscape for the marks histone H3 lysine 27 trimethylation (H3K27me3) and histone H3 lysine 4 trimethylation (H3K4me3) by chromatin immunoprecipitation sequencing (ChIP-seq) in two differing upland cotton (Gossypium hirsutum) varieties. We observed a global disruption in H3K4me3 and H3K27me3 modifications, especially H3K27me3, in cotton anthers subjected to HT. HT affected the bivalent H3K4me3-H3K27me3 modification more than either monovalent modification. We determined that removal of H3K27me3 at the promoters of jasmonate-related genes increased their expression, maintaining male fertility under HT in the HT-tolerant variety at the anther dehiscence stage. Modulating jasmonate homeostasis or signaling resulted in an anther indehiscence phenotype under HT. Chemical suppression of H3K27me3 deposition increased jasmonic acid contents and maintained male fertility under HT. In summary, our study provides new insights into the regulation of male fertility by histone modifications under HT and suggests a potential strategy for improving cotton HT tolerance.

Regulatory controls of duplicated gene expression during fiber development in allotetraploid cotton.

Polyploidy complicates transcriptional regulation and increases phenotypic diversity in organisms. The dynamics of genetic regulation of gene expression between coresident subgenomes in polyploids remains to be understood. Here we document the genetic regulation of fiber development in allotetraploid cotton Gossypium hirsutum by sequencing 376 genomes and 2,215 time-series transcriptomes. We characterize 1,258 genes comprising 36 genetic modules that control staged fiber development and uncover genetic components governing their partitioned expression relative to subgenomic duplicated genes (homoeologs). Only about 30% of fiber quality-related homoeologs show phenotypically favorable allele aggregation in cultivars, highlighting the potential for subgenome additivity in fiber improvement. We envision a genome-enabled breeding strategy, with particular attention to 48 favorable alleles related to fiber phenotypes that have been subjected to purifying selection during domestication. Our work delineates the dynamics of gene regulation during fiber development and highlights the potential of subgenomic coordination underpinning phenotypes in polyploid plants.