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.

Abstract The VIM gene family plays a vital role in the growth, development, and stress responses of plants, while the molecular mechanism of VIM genes in responding to salt stress is remain unclear. This study analyzed the phylogenetic relationships, chromosomal localization, conserved motifs, gene structure, cis-acting elements, and gene expression patterns of the VIM gene family in four cotton varieties. A total of 29, 29, 17, and 14 members were identified in Gossypium hirsutum (G.hirsutum), Gossypium barbadense(G.barbadense), Gossypium arboreum (G.arboreum), and Gossypium raimondii (G. raimondii), respectively. Chromosomal localization indicated the maturity and evolution of this gene family. Analysis of gene structure and conserved motifs revealed a conserved pattern among GhVIMs genes. Furthermore, cis-acting element analysis demonstrated the presence of stress-responsive elements, hormone-responsive elements, and growth-related elements in the promoter region of GhVIMs genes, highlighting their significant role in plant growth and stress response. Additionally, gene expression analysis revealed different expression patterns and tissue specificity of GhVIMs genes under abiotic stress. To better understand the functional role of GhVIM28, GhVIM28 gene was silenced using virus-induced gene silencing, and the results indicated that GhVIM28 acts as a salt-tolerant negative regulator.

The first fruit branch node (FFBN) and height of the first fruit branch node (HFFBN) are two important traits related to plant architecture and early maturation in upland cotton. Several studies have been performed to elucidate the genetic basis of FFBN and HFFBN in cotton using biparental and natural populations. In this study, by using 9,244 SNP linkage disequilibrium block (SNPLDB) loci from 315 upland cotton accessions, we carried out restricted two‐stage multilocus and multiallele genome-wide association studies (RTM‐GWASs) and identified promising haplotypes/alleles of the four stable and true major SNPLDB loci that were significantly associated with FFBN and HFFBN. Additionally, a meta-QTL analysis was conducted on 274 original QTLs reported in 27 studies, and 40 MQTLs for FFBN and HFFBN were detected. Through the integration of RTM-GWAS and meta‑QTL analyses, two stable and true major SNPLDBs (LDB_5_15144433 and LDB_16_37952328) that were distributed in the two MQTLs were identified. Ultimately, 142 genes were annotated in the two genomic regions, and three candidate genes for FFBN and HFFBN were identified in the genomic region (A05:14.64–15.64 Mb) by RNA-Seq and qRT‒PCR. The results of virus-induced gene silencing (VIGS) experiments indicated that GhE6was a key gene related to HFFBN and that GhDRM1 and GhGES were important genes associated with early flowering in upland cotton. These findings will aid in the future identification of molecular markers and genetic resources for developing elite early-maturing cultivars with ideal plant characteristics.

Vitamin C, also referred to as ascorbic acid (AsA), is recognized for its capacity to cure and avert scurvy, and it is crucial for regular human growth and development. In various crops, AsA participates in stress response mechanisms mediated by abscisic acid and has been discovered to have a crucial function in the morphogenesis, growth, development, and production of male gametes in plants. GDP-D-mannose 3′,5′-epimerase (GME) is essential in the synthesis of vitamin C. Our research identified 91, 83, 51, and 46 genes, respectively, found in G. barbadense (GbGMEs), G. hirsutum (GhGMEs), G. arboretum (GaGMEs), and G. raimondii (GrGMEs). Plants resulting from VIGS infection with GhGME31D clearly showed yellowing, water loss and wilting of leaves and black spots on stems. Measurement of MDA and AsA levels indicated that the plants were more damaged. This indicates that AsA has a substantial impact on plant growth and development.

The VIM (belonged to E3 ubiquitin ligase) gene family is crucial for plant growth, development, and stress responses, yet their role in salt stress remains unclear. We analyzed phylogenetic relationships, chromosomal localization, conserved motifs, gene structure, cis-acting elements, and gene expression patterns of the VIM gene family in four cotton varieties. Our findings reveal 29, 29, 17, and 14 members in Gossypium hirsutum (G.hirsutum), Gossypium barbadense (G.barbadense), Gossypium arboreum (G.arboreum), and Gossypium raimondii (G. raimondii), respectively, indicating the maturity and evolution of this gene family. motifs among GhVIMs genes were observed, along with the presence of stress-responsive, hormone-responsive, and growth-related elements in their promoter regions. Gene expression analysis showed varying patterns and tissue specificity of GhVIMs genes under abiotic stress. Silencing GhVIM28 via virus-induced gene silencing revealed its role as a salt-tolerant negative regulator. This work reveals a mechanism by which the VIM gene family in response to salt stress in cotton, identifying a potential negative regulator, GhVIM28, which could be targeted for enhancing salt tolerance in cotton. The objective of this study was to explore the evolutionary relationship of the VIM gene family and its potential function in salt stress tolerance, and provide important genetic resources for salt tolerance breeding of cotton.

Caffeic acid-3-O-methyltransferase (COMT) serves as the final pivotal enzyme in melatonin biosynthesis and plays a crucial role in governing the synthesis of melatonin in plants. This research used bioinformatics to analyze the phylogenetic relationships, gene structure, and promoter cis-acting elements of the upland cotton COMT gene family members, which it identified as the key gene GhCOMT33D to promote melatonin synthesis and responding to Cd2+ stress. After silencing GhCOMT33D through virus-induced gene silencing (VIGS), cotton seedlings showed less resistance to Cd2+ stress. Under Cd2+ stress, the melatonin content in the silenced plants significantly decreased, while ROS, MDA, and proline accumulated in the plant cells. The stomatal aperture of the leaves was reduced, hindering normal photosynthesis, leading to cotton leaves withering and yellowing, and epidermal cells becoming twisted and deformed, with a large number of gaps appearing. The non-silenced plants had a significantly higher melatonin content and were in better condition, providing important evidence for further research on how plant melatonin enhances the Cd2+ resistance of cotton and its regulatory mechanisms.

Cotton yield is affected by drought stress. We performed widely targeted metabolomics to explore metabolic changes under drought stress in the roots and leaves of two cotton varieties differing in drought tolerance-XLZ8 as sensitive and G95079 as tolerant variety. A total of 537 metabolites were detected at different stages of drought stress. Abscisic acid (ABA) was significantly enriched in all stages of drought stress in both roots and leaves of XLZ8. Amino acids showed enrichment in leaves of G95079 in the early stage of drought stress. Flavonoids were significantly enriched in the leaves of the two varieties in the late stage of drought stress. In roots, nucleotides and their derivatives were highly enriched in XLZ8 in the late stage of drought stress. The biosynthetic pathways of kynurenine and polyamine and glycerophospholipid metabolism may play an important role in cotton drought tolerance by scavenging reactive oxygen species (ROS) and reconstructing cell membranes. Spraying appropriate concentrations of proline, kynurenine, S-adenosylmethionine, glucose and 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine (lyso PC18:1) significantly increased the survival rate of cotton seedlings under drought stress. We speculated that ABA-dependent and ABA-independent metabolic changes may lead to differences in drought tolerance among cotton varieties.

MicroRNAs (miRNAs) regulate target gene expression to modulate plant growth, development, and biotic and abiotic stress response at the post-transcriptional level. Ammopiptanthus mongolicus , an ecologically important desert plant, is increasingly used as a model for studying stress tolerance in plants. The miRNA-mediated gene regulatory network might remarkably contribute to the high stress tolerance of A . mongolicus . However, a genome-wide identification of miRNAs and their targets is still lacking in A . mongolicus . In this study, 170 conserved and 156 non-conserved miRNAs were identified in A . mongolicus . We experimentally identified 298 miRNA-target pairs from the degradome data. Quantitative real-time polymerase chain reaction analyses identified 28 drought-responsive miRNAs in leaves and 15 in roots. Some characteristics of the miRNA-mediated regulatory network were found in A . mongolicus . Multiple miRNAs, including 2 newly identified non-conserved miRNAs, miR-P11 and miR-P14, generated from the precursors of miR169, were found to be involved in drought stress response. Further, miR2118 and miR858 participated in drought stress response by up-regulating OZF1 gene and certain MYB genes that were involved in the regulation of flavonol biosynthesis in A . mongolicus . The findings of this study might provide new insights for understanding the functions of miRNA in stress response in plants.

Ammopiptanthus mongolicus (Maxim. Ex Kom.) Cheng f., a relic tree of the Tertiary period, plays a critical role in maintaining desert ecosystems in the Mid-Asia region. Genome-scale gene expression profiling studies will provide deep insight into the molecular mechanism underlying the drought tolerance of A. mongolicus. In the present study, we investigated the transcriptional changes induced by drought treatment in A. mongolicus leaves by establishing a comprehensive transcriptome database and then performing a Digital Gene Expression (DGE) analysis using Solexa sequencing technology. A comprehensive transcriptome database was obtained by assembling the Illumina unigenes with expressed sequence tags (EST) available publicly, and other high throughput sequencing data. To analyze the dynamic and complicated gene regulation network during PEG6000-induced drought treatment in leaves of A. mongolicus, a time-course gene expression analysis was performed using tag-based DGE technology, which identified 437, 1,247 and 802 differentially expressed transcripts in 1, 24 and 72 h drought stress libraries, respectively. GO and KEGG analyses revealed hormone signal transduction and phenylpropanoid biosynthesis were enriched during drought treatment. A batch of drought-regulated transcription factor transcripts were identified, including the subsets of HD-ZIP, bZIP, WRKY, AP2/ERF and bHLH family members, which may play roles in drought response in A. mongolicus. The sequence collection assembled in the present study represents one of the most comprehensive transcriptome databases for A. mongolicus currently. The differentially expressed transcripts identified in our study provide a good start for identifying the key genes in stress response and performing functional analysis to reveal their roles in stress adaptation in planta.

Glutathione peroxidases (GPX) catalyze the reduction of H2O2 or organic hydroperoxides to water or corresponding alcohols using reduced glutathione, which plays an essential role in ROS (reactive oxygen species) homeostasis and stress signaling. Thellungiella salsuginea (Eutrema salsugineum), a relative of Arabidopsis thaliana, displays an extremely high level of tolerance to salt, drought, cold and oxidative stresses. The enzymatic antioxidant systems may contribute to the stress tolerance of T. salsuginea. In the present study, we aimed at understanding the roles of the antioxidant enzymes in T. salsuginea by focusing on the GPX family. We identified the eight GPX genes in T. salsuginea, and the structure of the N-terminal domains indicated their putative chloroplastic, mitochondrial and cytoplasmic location. The exon-intron organization of these genes exhibited a conserved pattern among plant GPX genes. Multiple environmental stresses and hormone response related cis-acting elements were predicted in the promoters of TsGPX genes. The gene and protein expression profiles of TsGPXs in response to high level of salinity and osmotic stresses, in leaves and roots of T. salsuginea were investigated using real-time RT-PCR and western blotting analysis. Our result showed that different members of the GPX gene family were coordinately regulated under specific environmental stress conditions, and supported the important roles of TsGPXs in salt and drought stress response in T. salsuginea.

Ammopiptanthus mongolicus, a woody plant growing in the desert, plays a vital role in vegetation maintaining and restoration in the arid region in northwest China. The plant exhibits an extremely high tolerance to abiotic stress such as drought and freezing stresses, and it has been used as an ideal model for abiotic stress tolerance research in trees. MicroRNA (miRNA) is a class of approximately 21nt endogenous non-protein-coding small RNA, which plays an important role in plant growth, development and responses to environmental stresses. By now, a large number of miRNAs have been reported in many plant species, but no studies describing A.mongolicus miRNA were published. In the present study, the types, expression levels, and putative target genes of conserved miRNAs in seedlings of A. mongolicus were analyzed using small RNA deep sequencing technology and bioinformatics methods. Nineteen conserved miRNAs, which belong to 10 miRNA families, were identified, with abundance ranging from 55 to 1920269 reads. Target prediction analysis determined the target genes of 14 conserved miRNAs. The functional classification analysis indicated that the conserved miRNAs participate in the development and environmental response by regulating the biological processes including the transcription regulation, hormone signal transduction, metabolisms and stress resistance.