Soil Cd2+ pollution threatens agricultural safety. Branched-chain amino acid (BCAA) metabolism is involved in stress responses, but its role in cotton Cd2+ tolerance remains unclear. Here, 16 GhBCAT genes were identified in Gossypium hirsutum, among which GhBCAT12 was significantly induced by Cd2+ stress. Silencing GhBCAT12 increased Cd2+ sensitivity, causing growth inhibition, oxidative damage, and cell death. Metabolomic analysis revealed the accumulation of leucine, isoleucine, and valine, with decreased acetyl-CoA, indicating that GhBCAT12 mediates BCAA degradation. This study reveals a novel pathway in which GhBCAT12-mediated BCAA degradation provides acetyl-CoA precursors for the tricarboxylic acid (TCA) cycle, sustaining ATP production, which in turn powers vacuolar Cd2+ sequestration. These findings provide a new target for improving heavy metal tolerance in crops.

Soil salinization poses a significant threat to global cotton production. Enhancing salt tolerance in cotton requires a deep understanding of its underlying molecular mechanisms. Lignin, a crucial component of the plant cell wall, plays a vital role in stress adaptation, with Cinnamoyl-CoA Reductase (CCR) serving as the key rate-limiting enzyme in its biosynthesis. However, the function of CCR genes in cotton remains largely unexplored. Here, we conducted a genome-wide analysis and identified 34 CCR genes in Gossypium hirsutum. Among them, GhCCR24 was highly induced by salt stress, particularly in roots. Virus-induced gene silencing (VIGS) of GhCCR24 resulted in compromised plant growth, reduced lignin deposition, and thinner secondary cell walls. Under salt stress, GhCCR24-silenced plants exhibited severe oxidative damage, ion homeostasis disruption (elevated Na* / K* ratio), and a significant decline in salt tolerance. Our findings demonstrate that GhCCR24 acts as a core positive regulator of cotton's response to salt stress by mediating lignin biosynthesis, which reinforces cell walls and thereby maintains ion and oxidative homeostasis. This study provides a valuable gene resource for the genetic improvement of salt-tolerant cotton.

DNA methylation is a stable epigenetic modification with essential roles in plant drought response. It is known that methyltransferase mutant is necessary for the regulation of methylation variations, but this epigenetic molecular mechanism based on methyltransferase mutant in responding to drought stress was still unclear in cotton. In this study, we aim to decipher the epigenetic code of drought response regulated by methyltransferase gene GhDMT9 in cotton, providing valuable information for the molecular research of drought resistance in cotton. We successfully created the first cotton methyltransferase mutant ghdmt9 using CRISPR/Cas9 method and performed methylation variations analysis with whole-genome bisulfite sequencing (WGBS) and transcriptome analysis based on ghdmt9 mutant. In addition, specific antibody of methyltransferase GhDMT9 was prepared and used for Chromatin Immunoprecipitation (ChIP-seq) analysis. The results indicated that ghdmt9 mutant interpreted approximately 2.06% methylation variations under drought stress. Demethylation variations, mainly derived from the CHG and CHH contexts, were closely correlated with drought response. Whether at normal growth stage or under drought stress, the number of up-regulated genes induced by demethylation variations was apparently higher than the number of down-regulated genes, especially genes regulating lipids and lipid-like molecules and hormone-related genes. In addition, fiber quality of ghdmt9 mutant was obviously better than that of wild type (WT). Interestingly, a transcription factor lsh (lysine-specific histone) was found to interact with methyltransferase gene GhDMT9 to activate its hyper-methylation function of target genomic regions by ChIP-seq analysis. Overall, our results extend our understanding of the epigenetic regulation of methyltransferase GhDMT9 in drought response and contribute to further investigations of the epigenetic mechanisms underlying abiotic stresses in cotton.

INTRODUCTION:Soil salinization and alkalization are major challenges to global agricultural productivity and food security. Ascorbic acid (AsA) is an essential antioxidant that helps plants mitigate various abiotic stresses. However, the genetic mechanisms underlying AsA's role in enhancing alkaline stress tolerance remain poorly understood. OBJECTIVES:The objective of this study was to determine whether GhIMP10D, a gene involved in AsA biosynthesis, enhances alkaline stress tolerance in cotton and Arabidopsis by modulating AsA accumulation and cell wall integrity. METHODS:We performed gene identification and functional analysis using overexpression and silencing techniques in Arabidopsis, rice, and cotton. GhIMP10D, a gene involved in the AsA biosynthesis pathway, was studied for its response to alkaline stress. The role of the bHLH transcription factor GhbHLH48 in regulating GhIMP10D was also explored. RESULTS:Our findings showed that overexpression of GhIMP10D resulted in increased AsA production, reduced reactive oxygen species (ROS), and enhanced cell wall integrity in the tested plants. In contrast, silencing GhIMP10D compromised alkaline stress adaptation. We further identified that GhbHLH48 directly activates GhIMP10D by binding to its promoter's G-box element. Manipulating GhbHLH48 levels altered AsA, lignin, and cellulose content, which affected ROS balance and cell wall biosynthesis. CONCLUSION:The GhbHLH48-GhIMP10D regulatory module plays a crucial role in AsA biosynthesis and the maintenance of cell wall integrity under alkaline stress. These findings contribute to a better understanding of AsA signaling pathways and cell wall formation in response to alkaline stress, offering potential strategies for enhancing plant stress tolerance.

Salinized soil is a potential resource for agricultural development, and cotton can improve and rehabilitate saline-alkali soil. Therefore, identifying, analyzing, and validating salt tolerance genes in cotton are of great significance for remediating saline-alkali land. Flavonol synthase (FLS) plays a key role in flavonol biosynthesis in plant, and it is also important in plant growth, development, and abiotic stress tolerance. However, the function of the FLS gene in cotton under salt stress remains unclear. In this study, the function of GhFLS1 under salt stress was analyzed through combined transcriptomic and metabolomic, exogenous quercetin, 3-3’diaminobenzadine (DAB) and 2-aminoethyl diphyenylborate (DPBA) staining in cotton, and the generation and characterization of GhFLS1-overexpressing lines in Arabidopsis and cotton. Differentially expressed genes and differentially accumulated metabolites were significantly enriched in the flavonoid metabolic pathway. DAB and DPBA staining revealed significant reactive oxygen species (ROS) and flavonol accumulation in cotton leaves under salt stress. Exogenous quercetin application enhanced the salt stress tolerance of cotton. GhFLS1 expression was significantly upregulated in response to salt stress. GhFLS1-overexpressing Arabidopsis exhibited enhanced salt stress tolerance, and ROS accumulation was significantly reduced in GhFLS1-overexpressing cotton under salt stress. This study presents the first functional characterization of a cotton FLS gene in response to salt stress by overexpression in Arabidopsis and cotton. We integrated transcriptomic and metabolomic, identifying quercetin as the key downstream metabolite, and validated its protective role through exogenous application. These results establish a functional link from GhFLS to quercetin-mediated ROS homeostasis, providing a candidate gene and a metabolic marker for breeding salt-tolerant cotton.

Alkaline stress causes significant adverse effects that slows down the growth of plants and lowers the yield of crops; hence, it is a major challenge in cotton farming. Spermidine (Spd), a vital polyamine, plays a significant role in enhancing plant resistance to stress caused by various abiotic factors. The molecular mechanism of Spd biosynthesis and especially the role of spermidine synthase (SPDS) in tolerance of alkaline stress in cotton is, however, little known. In this study, a systematic comparative analysis of SPDS-associated genes was performed across four representative cotton cultivars (Gossypium spp.), followed by preliminary functional characterization through promoter cis-acting element profiling. Virus-induced gene silencing (VIGS) was utilized to disrupt GhSPDS11-mediated Spd biosynthesis. Under alkaline stress, GhSPDS11-silenced seedlings exhibited 29.14% and 11.12% reductions in superoxide dismutase (SOD) and catalase (CAT) activities, 31.57% and 15.16% decreases in soluble sugar and proline (Pro) content, along with 42.38% and 38.66% increases in malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) compared to controls. Concurrently, silenced plants showed 44.87% fewer open stomata and significant declines in Spd content, relative water content, and biomass. These results indicate the key importance of Spd, which is composed of GhSPDS11, in improving alkali tolerance in cotton. This research gives good information regarding the molecular processes that take part in the tolerance of cotton to the saline-alkaline soils, and that GhSPDS11 could be a good genetic target in cotton enhancement in this tough agro-climatic condition.

Citrate synthase (CS) catalyzes the biosynthesis of citrate (CA), a tricarboxylic acid metabolite that serves as a pivotal biochemical regulator in plant cellular metabolism. This investigation conducted systematic genome-wide identification of CS family, integrating structural, phylogenetic relationships, collinearity analysis, cis-acting elements and expression analysis. GhCS6 protein is localized in the mitochondria. The pivotal cadmium-responsive regulatory candidate gene GhCS6 was identified, followed by its silencing mediation through virus-induced gene silencing (VIGS), silenced plants displaying phenotypic evidence of enhanced Cd2+ susceptibility. Simultaneously, CA content in the silenced plants was also significantly decreased, which reduced the chelating capacity for Cd2+ and consequently exacerbates the accumulation of Cd2+. The silenced plants showed an imbalance in the reactive oxygen species (ROS) scavenging system, as evidenced by lower superoxide dismutase (SOD) activity and a marked increase in H2O2 content, causing increased oxidative damage. This oxidative stress further compromised the integrity of cellular membranes, resulting in elevated malondialdehyde (MDA) content and compromised cellular structure. These changes ultimately impaired photosynthetic efficiency and plant growth and development. This study contributes significantly to understanding the function of the GhCS6 in plant Cd2+ tolerance, and identifies potential gene targets for breeding Cd2+ tolerant cotton varieties.

This study provides a comprehensive analysis of the impact of DNA methylation in cotton under salt stress conditions, elucidating its effects on gene expression and biological processes. Here, we determined the structures of the DNA methylation landscape across the cotton genome subjected to salt stress using whole-genome bisulfite sequencing (WGBS) and RNA-seq methodologies. We identified 4938 differentially methylated regions (DMRs) correlated with alterations in gene expression. Salt stress induced significant shifts in DNA methylation patterns, particularly in CHH contexts, suggesting context-dependent epigenetic regulation. DMRs were found to be implicated in diverse biological processes and pathways, encompassing protein metabolism, cellular homeostasis, starch and sucrose metabolism, and plant hormone signaling, all pivotal for cotton's adaptation to salt stress. Furthermore, RNA-seq analysis confirmed the impact of DNA methylation on gene expression, uncovering 9642 salt stress-responsive differentially expressed genes (DEGs). These DEGs exhibited enrichment in pathways such as carbohydrate metabolism, cell wall synthesis, and defense response, underscoring the intricate interplay between methylation and gene regulation in stress response. Moreover, the study investigated the role of the key DNA methyltransferase gene GhDMT7 in modulating cotton's response to salt stress, revealing that its downregulation enhanced cotton's salt tolerance, potentially attributed to decreased DNA methylation levels, reduced membrane damage, and enhanced antioxidant capacity. These findings elucidate the role of DNA methylation in abiotic stress resilience and provide insights for crop improvement.

Introduction: Ascorbic acid (AsA) is involved in plant responses to various abiotic stresses. However, its specific function in alkaline stress tolerance remains poorly understood. The L-galactono-1,4-lactone dehydrogenase (GLDH) gene is crucial for AsA synthesis, yet the precise role of GLDH in modulating plant resistance to alkaline stress has not been comprehensively characterized. Objectives: To investigate the role of GLDH genes in enhancing tolerance to alkaline stress. Methods: Bioinformatics analysis of the GLDH gene family members was conducted, and an evolutionary tree was constructed using MEGA software. Cis-acting elements and gene structures were analyzed using TBtools. Gene expression levels were quantified by qRT-PCR, while the function of the GhGLDH35A gene was validated through VIGS (Virus-induced gene silencing) in cotton, heterologous overexpression in Arabidopsis thaliana, and complementation assays in yeast. Results: Our study investigated the effects of salt-alkaline stress on cotton and found that alkaline stress caused significantly more severe damage than salt stress. The GLDH family genes were identified and analyzed, revealing a high degree of evolutionary conservation. Most GhGLDH genes exhibited a positive response to alkaline stress and were regulated by light. Among them, GhGLDH35A, which is highly expressed within the GLDH family, was found to play a key role in conferring tolerance to alkaline stress. Subcellular localization analysis indicated that GhGLDH35A is localized in the mitochondria. Silencing of GhGLDH35A in cotton resulted in reduced tolerance to alkaline stress, disruption of ROS homeostasis, and impairment of photosynthesis and stomatal function. Conversely, overexpression of GhGLDH35A in Arabidopsis enhanced alkaline stress resistance by elevating AsA levels, increasing antioxidant enzyme activities to enhance ROS scavenging, sustaining photosynthesis, and promoting stomatal closure. Furthermore, heterologous expression of GhGLDH35A in yeast also improved its tolerance to alkaline stress. Conclusions: GhGLDH35A positively regulates alkaline stress tolerance by enhancing antioxidant defenses and regulating stomatal movement.

Cadmium (Cd2+) contamination threatens plant viability and human health by disrupting cellular homeostasis and metabolic processes. Investigating the molecular mechanism underlying Cd2+ tolerance in plants is necessary to remediate Cd2+-contaminated soil. This study presents an integrated physiological, metabolomic, and transcriptomic analysis of the roots, stems, and leaves in response to Cd2+ stress. The study found that Cd2+ accumulation was significantly lower in Cd2+-tolerant cotton. Under 4 mM Cd2+ stress, the Cd2+ content in cotton increased significantly, accompanied by elevated levels of malondialdehyde (MDA), proline (Pro), and hydrogen peroxide (H2O2), as well as noticeable damage to the cellular ultrastructure. Metabolomic profiling analysis revealed that Cd2+ stress significantly affected the distribution of lipids, amino acids, and organic acids in different tissues. The metabolic pathways of alanine, aspartate, and glutamate are closely associated with Cd2+ stress, and the induced elevation of GABA levels plays a crucial role in cotton’s adaptation to Cd2+ stress. Exogenous GABA application significantly enhances Cd2+ tolerance in cotton by reducing Cd2+ accumulation and decreasing the content of Pro, MDA, and H2O2. Silencing of the γ-aminobutyric acid (GABA) biosynthetic gene glutamate decarboxylase (GhGAD6) resulted in increased Cd2+ sensitivity, demonstrating that GABA alleviates Cd2+ toxicity in cotton through reducing Cd2+ accumulation and scavenging ROS. These findings elucidate the molecular basis of Cd2+ stress tolerance in plants and provide a key for the effective strategy of enhancing Cd2+ tolerance in cotton.

Silicon plays a dual role in plant growth. However, excessive application of sodium silicate (Na2SiO3), commonly utilised Si-based fertiliser, can adversely affect plant development. In the present study, a pretreatment concentration of 20 mM Na2SiO3 was used to investigate its effect on the growth and development of cotton during the germination and three-leaf stages. The radicle necrosis rates of 84 upland cotton genotypes were assessed. RNA-seq analysis revealed 9098 differentially expressed genes (DEGs). Gene Ontology (GO) analysis revealed the enrichment of DEGs associated with various stimuli and stress responses. Concurrently, Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis identified the regulation of DEGs linked to the plant MAPK signalling pathway, lipid metabolism-related pathways, carotenoid biosynthesis pathway, plant hormone signal transduction, and secondary metabolite biosynthesis under Na2SiO3 stress. Notably, key genes within the carotenoid biosynthesis pathway were upregulated, suggesting that this pathway plays a significant role in mitigating oxidative damage. This study demonstrates that under saline-alkali stress conditions, excessive exogenous application of Na2SiO3 exacerbates toxicity in cotton plants. These findings provide a theoretical foundation for understanding the mechanisms underlying the response of cotton to Na2SiO3 stress and inform the judicious use of Si fertilisers.

D-lactate dehydrogenase (D-LDH) is a key enzyme in the pyruvate metabolic pathway and is crucial for plant growth, development, and stress tolerance. This study provides a systematic analysis of the genetic characteristics, evolutionary history, cis-acting elements, and expression patterns of the D-LDH gene family in cotton with 14 D-LDH genes were discovered. Phylogenetic analysis showed that D-LDH family genes could be clustered into 2 clades. The D-LDH genes of the four cotton species show a closer relationship to those of T. cacao compared to other plant species. Structure analysis showed that GhDLDH genes from the same subgroup exhibited similar genetic structure. The D-LDH family members predominantly include elements associated with hormones, the light response, the drought response, stress and the temperature response. GhDLDH1 was significantly upregulated following exposure to various stress treatments. The quantitative real-time PCR (qRT-PCR) results demonstrated that most GhDLDH genes responded positively to salt stress, with GhDLDH1 exhibited the peak transcriptional activity. Subcellular localization analysis revealed that GhDLDH1 is specifically localized within the chloroplast. Compared with those of pYL156 plants, pYL156: GhDLDH1 plants presented significantly reductions in pyruvate (PA), lactic acid (LA), glutathione (GSH), lactic dehydrogenase (LDH), and chlorophyll levels, with a significant increase in the methylglyoxal (MG) content under salt stress. GhDLDH1 gene silencing in cotton seedlings resulted in heightened sensitivity to NaCl stress, as evidenced by trypan blue staining. Plants in which the GhDLDH1 gene was silenced exhibited lower chloroplast levels compared with pYL156 plants, and the structure was less coherent. Potential interactions were revealed between the D-LDH protein and the chloroplast-associated proteins PKP1 and PKP3. And the qRTPCR data revealed altered expression levels of GhPKP1 and GhPKP3 following GhDLDH1 gene silencing. In conjunction with the observed changes in chloroplast number and morphology, these findings suggest that GhDLDH1 may be a key regulator of chloroplast function. This study serves as a valuable reference for investigating the interaction between the D-LDH gene and chloroplast-associated genes, as well as their regulatory effects on cotton salt tolerance.

Flavonoid 3′-hydroxylase synthesis gene-GHCYP706A7, enhanced cotton resistance to alkali stress by scavenging ROS to regulate anthocyanin synthesis. Anthocyanins are a class of flavonoids that play a significant role in mediating plant responses to adverse environmental conditions. Flavonoid 3′-hydroxylase (F3′H), a member of the cytochrome P-450 (CYP) family, is a pivotal enzyme involved in the biosynthesis of anthocyanins. The present study identified 398 CYPs in the Gossypium hirsutum genome, of which GHCYP706A7 was responsible for F3′H synthesis and its ability to respond to alkaline stress. GHCYP706A7 suppression through virus-induced gene silencing (VIGS) diminished tolerance to alkali stress in cotton, evidenced by significantly reduced anthocyanin synthesis, markedly decreased antioxidant capacity, notable increases in reactive oxygen species, severe cellular damage, and observably decreased stomatal opening. The cumulative effects of these physiological disruptions ultimately manifest in cotton wilting and fresh weight decline. These findings lay a foundation for further investigations into the role of CYPs in regulating anthocyanin synthesis and responding to alkali stress.

Tryptophan decarboxylase (TDC) is the rate-limiting enzyme in the biosynthesis of melatonin and plays a crucial role in melatonin production in plants. Melatonin (MT), a multifunctional indoleamine compound, plays a pivotal role in plant responses to abiotic stress. Substantial evidence has demonstrated that MT can significantly enhance plant tolerance to drought stress. However, the molecular mechanisms underlying MT-mediated drought stress responses in plants remain unclear. This study systematically analyzed the evolutionary relationships of the TDC gene family in four cotton species using bioinformatics approaches, including phylogenetic analysis, chromosomal localization, gene structure, conserved motifs, cis-acting elements, synteny, and expression patterns. Functional characterization of GhTDC5 in Gossypium hirsutum was performed using virus-induced gene silencing (VIGS), with the following key findings: gene silencing efficiency reached 67.5

The rapid evolution of pesticide resistance imposes great pressure on food production. However, how resistance alleles arise and spread across field populations remains largely understood. Here, we study the evolutionary trajectories of resistance alleles in Tetranychus urticae, a rapidly evolving pest. We sequence the genomes of 258 T. urticae females collected from China. Combined with global reference genomic data, we examine the evolutionary origin(s) of 18 mutations across 10 target-site genes and analyze the global population genetic structure using genome-wide SNPs. Our findings reveal a striking prevalence of multiple independent origins of resistance mutations, with only two of 18 mutations showing an apparent single origin. Population structure and haplotype analyses point to an important role of gene flow in the spread of resistance alleles. Selection analyses reveal pesticide-driven sweeps affecting genetic diversity. These findings advance our understanding of the rapid adaptation of arthropod herbivores to extreme selective pressure.

Despite the rapid advances in sequencing technology, limited genomic resources are currently available for phytophagous spider mites, which include many important agricultural pests. One of these pests is Tetranychus piercei (McGregor), a serious banana pest in East Asia exhibiting remarkable tolerance to high temperature. In this study, we assembled a high-quality genome of T. piercei using a combination of PacBio long reads and Illumina short reads sequencing. With the assistance of chromatin conformation capture technology, 99.9% of the contigs were anchored into three pseudochromosomes with a total size of 86.02 Mb. Repetitive elements, accounting for 14.16% of this genome (12.20 Mb), are predominantly composed of long-terminal repeats (30.7%). By combining evidence of ab initio prediction, transcripts, and homologous proteins, we annotated 11,881 protein-coding genes. Both the genome and proteins have high BUSCO completeness scores (>94%). This high-quality genome, along with reliable annotation, provides a valuable resource for investigating the high-temperature tolerance of this species and exploring the genomic basis that underlies the host range evolution of spider mites.

Soil salinization is a global constraint that significantly hampers agricultural production, with cotton being an important cash crop that is not immune to its detrimental effects. The rhizosphere microbiome plays a critical role in plant health and growth, which assists plants in resisting adverse abiotic stresses including soil salinization. This study explores the impact of soil salinization on cotton, including its effects on growth, yield, soil physical and chemical properties, as well as soil bacterial community structures. The results of β-diversity analysis showed that there were significant differences in bacterial communities in saline-alkali soil at different growth stages of cotton. Besides, the more severity of soil salinization, the more abundance of Proteobacteria, Bacteroidota enriched in rhizosphere bacterial composition where the abundance of Acidobacteriota exhibited the opposite trend. And the co-occurrence network analysis showed that soil salinization affected the complexity of soil bacterial co-occurrence network. These findings provide valuable insights into the mechanisms by which soil salinization affects soil microorganisms in cotton rhizosphere soil and offer guidance for improving soil salinization using beneficial microorganisms.

The Oxidative Balance Score (OBS) was created to evaluate an individual’s overall antioxidant status. The objective of this study was to examine the association between OBS and abdominal aortic calcification (AAC) among individuals aged ≥ 40 years. This population-based cross-sectional study used data from the National Health and Nutrition Examination Survey in 2013–2014 and included adults aged ≥ 40 years. Survey-weighted multivariable logistic and restricted cubic spline models were used to assess the association between OBS and AAC. Among 2520 participants, 744 were diagnosed with AAC (weighted percentage, 28.13

The diet breadth of generalist herbivores when compared to specialists tends to be associated with greater transcriptional plasticity. Here, we consider whether it may also contribute to variation in host range among two generalists with different levels of polyphagy. We examined two related polyphagous spider mites with different host ranges, Tetranychus urticae (1200 plants) and Tetranychus truncatus (90 plants). Data from multiple populations of both species domesticated on common beans and transferred to new plant hosts (cotton, cucumber, eggplant) were used to investigate transcriptional plasticity relative to population-based variation in gene expression. Compared to T. truncatus, T. urticae exhibited much higher transcriptional plasticity. Populations of this species also showed much more variable expression regulation in response to a plant host, particularly for genes related to detoxification, transport, and transcriptional factors. In response to the different plant hosts, both polyphagous species showed enriched processes of drug/xenobiotics metabolism, with T. urticae orchestrating a relatively broader array of biological pathways. Through co-expression network analysis, we identified gene modules associated with host plant response, revealing shared hub genes primarily involved in detoxification metabolism when both mites fed on the same plants. After silencing a shared hub CYP gene related to eggplant exposure, the performance of both species on the original bean host improved, but the fecundity of T. truncatus decreased when feeding on eggplant. The extensive transcriptomic variation shown by T. urticae might serve as a potential compensatory mechanism for a deficiency of hub genes in this species. This research points to nuanced differences in transcriptomic variability between generalist herbivores.

BACKGROUND:DNA methylation is an important epigenetic mode of genomic DNA modification and plays a vital role in maintaining epigenetic content and regulating gene expression. Cytosine-5 DNA methyltransferase (C5-MTase) are the key enzymes in the process of DNA methylation. However, there is no systematic analysis of the C5-MTase in cotton so far, and the function of DNMT2 genes has not been studied.METHODS:In this study, the whole genome of cotton C5-MTase coding genes was identified and analyzed using a bioinformatics method based on information from the cotton genome, and the function of GhDMT6 was further validated by VIGS experiments and subcellular localization analysis.RESULTS:33 C5-MTases were identified from three cotton genomes, and were divided into four subfamilies by systematic evolutionary analysis. After the protein domain alignment of C5-MTases in cotton, 6 highly conserved motifs were found in the C-terminus of 33 proteins involved in methylation modification, which indicated that C5-MTases had a basic catalytic methylation function. These proteins were divided into four classes based on the N-terminal difference, of which DNMT2 lacks the N-terminal regulatory domain. The expression of C5-MTases in different parts of cotton was different under different stress treatments, which indicated the functional diversity of cotton C5-MTase gene family. Among the C5-MTases, the GhDMT6 had a obvious up-regulated expression. After silencing GhDMT6 with VIGS, the phenotype of cotton seedlings under different stress treatments showed a significant difference. Compared with cotton seedlings that did not silence GhDMT6, cotton seedlings silencing GhDMT6 showed significant stress resistance.CONCLUSION:The results show that C5-MTases plays an important role in cotton stress response, which is beneficial to further explore the function of DNMT2 subfamily genes.