GhVIM28, a negative regulator identified from VIM family genes, positively responds to salt stress in cotton.

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.

Genome-wide characterization of DNA methyltransferase family genes implies GhDMT6 improving tolerance of salt and drought on cotton.

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.

Enhancing stimulation of cyaniding, GhLDOX3 activates reactive oxygen species to regulate tolerance of alkalinity negatively in cotton.

Anthocyanins belong to flavonoid secondary metabolites that act as plant pigments to give flowers and fruits different colors and as "scavengers" of reactive oxygen species (ROS) to protect plants from abiotic and biotic stresses. Few studies linked anthocyanins to alkaline resistance so far. In this study, anthocyanin synthesis-related gene leucoanthocyanidin dioxygenase (LDOX) was screened as a candidate gene to explore its relationship with alkali stress. The results found that pYL156: GhLDOX3 lines treated with 50 mM Na2CO3 (pH 11.11) for 24 h showed a significant increase in peroxidase (POD) activity, a decrease in total anthocyanin content and an increase in cyanidin content and a decrease in ROS accumulation compared to pYL156. The overexpressed (OE) lines, ldox mutant and wild-type (WT) lines in Arabidopsis were treated with 50 mM Na2CO3, 100 mM Na2CO3 and 150 mM Na2CO3 for 8 d, respectively. The wilted degree of the OE lines was more severe than WT lines, and less severe in the mutant lines in the 150 mM Na2CO3 treatment. After treatment, the expression levels of AtCAT and AtGSH genes related to antioxidant system in OE lines were significantly lower than in WT, and the expression levels of AtCAT and AtGSH in mutant lines were significantly higher than in WT. In conclusion, the above results suggest GhLDOX3 played a negative regulatory role in the mechanism of resisting Na2CO3 stress. Therefore, it can be considered in cotton breeding to improve the alkali tolerance of cotton by regulating the expression of related genes.

GhIMP10D, an inositol monophosphates family gene, enhances ascorbic acid and antioxidant enzyme activities to confer alkaline tolerance in Gossypium hirsutum L.

BACKGROUND: Inositol monophosphates (IMP) are key enzymes in the ascorbic acid (AsA) synthesis pathways, which play vital roles in regulating plant growth and development and stresses tolerance. To date, no comprehensive analysis of the expression profile of IMP genes and their functions under abiotic stress in cotton has been reported. RESULTS: In this study, the genetic characteristics, phylogenetic evolution, cis-acting elements and expression patterns of IMP gene family in cotton were systematically analyzed. A total of 28, 27, 13 and 13 IMP genes were identified in Gossypium hirsutum (G. hirsutum), Gossypium barbadense (G. barbadense), Gossypium arboreum (G. arboreum), and Gossypium raimondii (G. raimondii), respectively. Phylogenetic analysis showed that IMP family genes could cluster into 3 clades. Structure analysis of genes showed that GhIMP genes from the same subgroup had similar genetic structure and exon number. And most GhIMP family members contained hormone-related elements (abscisic acid response element, MeJA response element, gibberellin response element) and stress-related elements (low temperature response element, defense and stress response element, wound response element). After exogenous application of abscisic acid (ABA), some GhIMP genes containing ABA response elements positively responded to alkaline stress, indicating that ABA response elements played an important role in response to alkaline stress. qRT-PCR showed that most of GhIMP genes responded positively to alkaline stress, and GhIMP10D significantly upregulated under alkaline stress, with the highest up-regulated expression level. Virus-induced gene silencing (VIGS) experiment showed that compared with 156 plants, MDA content of pYL156:GhIMP10D plants increased significantly, while POD, SOD, chlorophyII and AsA content decreased significantly. CONCLUSIONS: This study provides a thorough overview of the IMP gene family and presents a new perspective on the evolution of this gene family. In particular, some IMP genes may be involved in alkaline stress tolerance regulation, and GhIMP10D showed high expression levels in leaves, stems and roots under alkaline stress, and preliminary functional verification of GhIMP10D gene suggested that it may regulate tolerance to alkaline stress by regulating the activity of antioxidant enzymes and the content of AsA. This study contributes to the subsequent broader discussion of the structure and alkaline resistance of IMP genes in cotton.

GhCYS2 governs the tolerance against cadmium stress by regulating cell viability and photosynthesis in cotton.

Cysteine, an early sulfur-containing compound in plants, is of significant importance in sulfur metabolism. CYS encodes cysteine synthetase that further catalyzes cysteine synthesis. In this investigation, CYS genes, identified from genome-wide analysis of Gossypium hirsutum bioinformatically, led to the discovery of GhCYS2 as the pivotal gene responsible for Cd2+ response. The silencing of GhCYS2 through virus-induced gene silencing (VIGS) rendered plants highly susceptible to Cd2+ stress. Silencing GhCYS2 in plants resulted in diminished levels of cysteine and glutathione while leading to the accumulation of MDA and ROS within cells, thereby impeding the regular process of photosynthesis. Consequently, the stomatal aperture of leaves decreased, epidermal cells underwent distortion and deformation, intercellular connections are dramatically disrupted, and fissures manifested between cells. Ultimately, these detrimental effected culminating in plant wilting and a substantial reduction in biomass. The association established between Cd2+ and cysteine in this investigation offered a valuable reference point for further inquiry into the functional and regulatory mechanisms of cysteine synthesis genes.

Changes in terpene biosynthesis and submergence tolerance in cotton.

BACKGROUND: Flooding is among the most severe abiotic stresses in plant growth and development. The mechanism of submergence tolerance of cotton in response to submergence stress is unknown. RESULTS: The transcriptome results showed that a total of 6,893 differentially expressed genes (DEGs) were discovered under submergence stress. Gene Ontology (GO) enrichment analysis showed that DEGs were involved in various stress or stimulus responses. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that DEGs related to plant hormone signal transduction, starch and sucrose metabolism, glycolysis and the biosynthesis of secondary metabolites were regulated by submergence stress. Eight DEGs related to ethylene signaling and 3 ethylene synthesis genes were identified in the hormone signal transduction. For respiratory metabolism, alcohol dehydrogenase (ADH, GH_A02G0728) and pyruvate decarboxylase (PDC, GH_D09G1778) were significantly upregulated but 6-phosphofructokinase (PFK, GH_D05G0280), phosphoglycerate kinase (PGK, GH_A01G0945 and GH_D01G0967) and sucrose synthase genes (SUS, GH_A06G0873 and GH_D06G0851) were significantly downregulated in the submergence treatment. Terpene biosynthetic pathway-related genes in the secondary metabolites were regulated in submergence stress. CONCLUSIONS: Regulation of terpene biosynthesis by respiratory metabolism may play a role in enhancing the tolerance of cotton to submergence under flooding. Our findings showed that the mevalonate pathway, which occurs in the cytoplasm of the terpenoid backbone biosynthesis pathway (ko00900), may be the main response to submergence stress.

Characterization and gene expression patterns analysis implies BSK family genes respond to salinity stress in cotton.

Identification, evolution, and expression patterns of BSK (BR signaling kinase) family genes revealed that BSKs participated in the response of cotton to abiotic stress and maintained the growth of cotton in extreme environment. The steroidal hormone brassinosteroids (BR) play important roles in different plant biological processes. This study focused on BSK which were downstream regulatory element of BR, in order to help to decipher the functions of BSKs genes from cotton on growth development and responses to abiotic stresses and lean the evolutionary relationship of cotton BSKs. BSKs are a class of plant-specific receptor-like cytoplasmic kinases involved in BR signal transduction. In this study, bioinformatics methods were used to identify the cotton BSKs gene family at the cotton genome level, and the gene structure, promoter elements, protein structure and properties, gene expression patterns and candidate interacting proteins were analyzed. In the present study, a total of 152 BSKs were identified by a genome-wide search in four cotton species and other 11 plant species, and phylogenetic analysis revealed three evolutionary clades. It was identified that BSKs contain typical PKc and TPR domains, the N-terminus is composed of extended chains and helical structures. Cotton BSKs genes show different expression patterns in different tissues and organs. The gene promoter contains numerous cis-acting elements induced by hormones and abiotic stress, the hormone ABA and Cold-inducing related elements have the highest count, indicating that cotton BSK genes may be regulated by various hormones at different growth stages and involved in the response regulation of cotton to various stresses. The expression analysis of BSKs in cotton showed that the expression levels of GhBSK06, GhBSK10, GhBSK21 and GhBSK24 were significantly increased with salt-inducing. This study is helpful to analyze the function of cotton BSKs genes in growth and development and in response to stress.

Combined transcriptomic and metabolomic analyses elucidate key salt-responsive biomarkers to regulate salt tolerance in cotton.

BACKGROUND: Cotton is an important industrial crop and a pioneer crop for saline-alkali land restoration. However, the molecular mechanism underlying the cotton response to salt is not completely understood. METHODS: Here, we used metabolome data and transcriptome data to analyze the salt tolerance regulatory network of cotton and metabolic biomarkers. RESULTS: In this study, cotton was stressed at 400 m M NaCl for 0 h, 3 h, 24 h and 48 h. NaCl interfered with cotton gene expression, altered metabolite contents and affected plant growth. Metabolome analysis showed that NaCl stress increased the contents of amino acids, sugars and ABA, decreased the amount of vitamin and terpenoids. K-means cluster analysis of differentially expressed genes showed that the continuously up-regulated genes were mainly enriched in metabolic pathways such as flavonoid biosynthesis and amino acid biosynthesis. CONCLUSION: The four metabolites of cysteine (Cys), ABA(Abscisic acid), turanose, and isopentenyladenine-7-N-glucoside (IP7G) were consistently up-regulated under salt stress, which may indicate that they are potential candidates for cotton under salt stress biomarkers. Combined transcriptome and metabolome analysis revealed accumulation of cysteine, ABA, isopentenyladenine-7-N-glucoside and turanose were important for salt tolerance in cotton mechanism. These results will provide some metabolic insights and key metabolite biomarkers for salt stress tolerance, which may help to understanding of the metabolite response to salt stress in cotton and develop a foundation for cotton to grow better in saline soil.

Systematic analysis and expression of Gossypium 2ODD superfamily highlight the roles of GhLDOXs responding to alkali and other abiotic stress in cotton.

BACKGROUND: 2-oxoglutarate-dependent dioxygenase (2ODD) is the second largest family of oxidases involved in various oxygenation/hydroxylation reactions in plants. Many members in the family regulate gene transcription, nucleic acid modification/repair and secondary metabolic synthesis. The 2ODD family genes also function in the formation of abundant flavonoids during anthocyanin synthesis, thereby modulating plant development and response to diverse stresses. RESULTS: Totally, 379, 336, 205, and 204 2ODD genes were identified in G. barbadense (Gb), G. hirsutum (Gh), G. arboreum (Ga), and G. raimondii (Gb), respectively. The 336 2ODDs in G. hirsutum were divided into 15 subfamilies according to their putative functions. The structural features and functions of the 2ODD members in the same subfamily were similar and evolutionarily conserved. Tandem duplications and segmental duplications served essential roles in the large-scale expansion of the cotton 2ODD family. Ka/Ks values for most of the gene pairs were less than 1, indicating that 2ODD genes undergo strong purifying selection during evolution. Gh2ODDs might act in cotton responses to different abiotic stresses. GhLDOX3 and GhLDOX7, two members of the GhLDOX subfamily from Gh2ODDs, were significantly down-regulated in transcription under alkaline stress. Moreover, the expression of GhLDOX3 in leaves was significantly higher than that in other tissues. These results will provide valuable information for further understanding the evolution mechanisms and functions of the cotton 2ODD genes in the future. CONCLUSIONS: Genome-wide identification, structure, and evolution and expression analysis of 2ODD genes in Gossypium were carried out. The 2ODDs were highly conserved during evolutionary. Most Gh2ODDs were involved in the regulation of cotton responses to multiple abiotic stresses including salt, drought, hot, cold and alkali.

Raffinose degradation-related gene GhAGAL3 was screened out responding to salinity stress through expression patterns of GhAGALs family genes.

A-galactosidases (AGALs), the oligosaccharide (RFO) catabolic genes of the raffinose family, play crucial roles in plant growth and development and in adversity stress. They can break down the non-reducing terminal galactose residues of glycolipids and sugar chains. In this study, the whole genome of AGALs was analyzed. Bioinformatics analysis was conducted to analyze members of the AGAL family in Gossypium hirsutum, Gossypium arboreum, Gossypium barbadense, and Gossypium raimondii. Meanwhile, RT-qPCR was carried out to analyze the expression patterns of AGAL family members in different tissues of terrestrial cotton. It was found that a series of environmental factors stimulated the expression of the GhAGAL3 gene. The function of GhAGAL3 was verified through virus-induced gene silencing (VIGS). As a result, GhAGAL3 gene silencing resulted in milder wilting of seedlings than the controls, and a significant increase in the raffinose content in cotton, indicating that GhAGAL3 responded to NaCl stress. The increase in raffinose content improved the tolerance of cotton. Findings in this study lay an important foundation for further research on the role of the GhAGAL3 gene family in the molecular mechanism of abiotic stress resistance in cotton.

GhAAO2 was observed responding to NaHCO3 stress in cotton compared to AAO family genes.

BACKGROUND: Abscisic acid (ABA) is an important stress hormone, the changes of abscisic acid content can alter plant tolerance to stress, abscisic acid is crucial for studying plant responses to abiotic stress. The abscisic acid aldehyde oxidase (AAO) plays a vital role in the final step in the synthesis of abscisic acid, therefore, understanding the function of AAO gene family is of great significance for plants to response to abiotic stresses. RESULT: In this study, 6, 8, 4 and 4 AAO genes were identified in four cotton species. According to the structural characteristics of genes and the traits of phylogenetic tree, we divided the AAO gene family into 4 clades. Gene structure analysis showed that the AAO gene family was relatively conservative. The analysis of cis-elements showed that most AAO genes contained cis-elements related to light response and plant hormones. Tissue specificity analysis under NaHCO3 stress showed that GhAAO2 gene was differentially expressed in both roots and leaves. After GhAAO2 gene silencing, the degree of wilting of seedlings was lighter than that of the control group, indicating that GhAAO2 could respond to NaHCO3 stress. CONCLUSIONS: In this study, the AAO gene family was analyzed by bioinformatics, the response of GhAAO gene to various abiotic stresses was preliminarily verified, and the function of the specifically expressed gene GhAAO2 was further verified. These findings provide valuable information for the study of potential candidate genes related to plant growth and stress.

Genome-wide identification, evolution and function analysis of UGTs superfamily in cotton.

Glycosyltransferases mainly catalyse the glycosylation reaction in living organisms and widely exists in plants. UGTs have been identified from G. raimondii, G. arboreum and G. hirsutum. However, Genome-wide systematic analysis of UGTs superfamily have not been studied in G. barbadense. 752 UGTs were identified from four cotton species and grouped into 18 clades, of which R was newly discovered clades. Most UGTs were clustered at both ends of the chromosome and showed a heterogeneous distribution. UGT proteins were widely distributed in cells, with the highest distribution in chloroplasts. UGTs of the same clade shared similar intron/exon structural features. During evolution, the gene family has undergone strong selection for purification. UGTs were significantly enriched in "transcriptional activity (GO:0016758)" and "metabolic processes (GO:0008152)". Genes from the same clade differed in function under various abiotic stresses. The analysis of cis-acting element and qRT-PCR may indicate that GHUGTs play important roles in plant growth, development and abiotic stress. We further found that GHUGT74-2 plays an important role under submergence. The study broadens the understanding of UGTs in terms of gene characteristics, evolutionary processes, and gene function in cotton and provides a new way to systematically and globally understand the structure-function relationship of multigene families in the evolutionary process.

Genome-wide identification of GAD family genes suggests GhGAD6 functionally respond to Cd2+ stress in cotton.

Glutamate decarboxylase (GAD) mainly regulated the biosynthesis of γ-aminobutyric acid (GABA) and played an important role in plant growth and stress resistance. To explore the potential function of GAD in cotton growth, the genome-wide identification, structure, and expression analysis of GAD genes were performed in this study. There were 10, 9, 5, and 5 GAD genes identified in G. hirsutum, G. barbadense, G. arboreum, and G. raimondii, respectively. GAD was divided into four clades according to the protein motif composition, gene structure, and phylogenetic relationship. The segmental duplication was the main way of the GAD gene family evolution. Most GhGADs respond to abiotic stress. Clade Ⅲ GAD was induced by Cd2+ stress, especially GhGAD6, and silencing GhGAD6 would lead to more serious Cd2+ poisoning in cotton. The oxidative damage caused by Cd2+ stress was relieved by increasing the GABA content. It was speculated that the decreased expression of GhGAD6 reduced the content of GABA in vivo and caused the accumulation of ROS. This study will further expand our understanding of the relationship between the evolution and function of the GhGAD gene family and provide new genetic resources for cotton breeding under environmental stress and phytoremediation.

A high-quality assembled genome and its comparative analysis decode the adaptive molecular mechanism of the number one Chinese cotton variety CRI-12.

BACKGROUND: Gossypium hirsutum L. is the most widely cultivated cotton species, and a high-quality reference genome would be a huge boost for researching the molecular mechanism of agronomic traits in cotton. FINDINGS: Here, Pacific Biosciences and Hi-C sequencing technologies were used to assemble a new upland cotton genome of the No. 1 Chinese cotton variety CRI-12. We generated a high-quality assembled CRI-12 genome of 2.31 Gb with a contig N50 of 19.65 Mb, which was superior to previously reported genomes. Comparisons between CRI-12 and other reported genomes revealed 7,966 structural variations and 7,378 presence/absence variations. The distribution of the haplotypes among A-genome (Gossypium arboreum), D-genome (Gossypium raimondii), and AD-genome (G. hirsutum and Gossypium barbadense) suggested that many haplotypes were lost and recombined in the process of polyploidization. More than half of the haplotypes that correlated with different tolerances were located on chromosome D13, suggesting that this chromosome may be important for wide adaptation. Finally, it was demonstrated that DNA methylation may provide advantages in environmental adaptation through whole-genome bisulfite sequencing analysis. CONCLUSIONS: This research provides a new reference genome for molecular biology research on Gossypium hirsutum L. and helps decode the broad environmental adaptation mechanisms in the No. 1 Chinese cotton variety CRI-12.

Molecular structures and functional exploration of NDA family genes respond tolerant to alkaline stress in Gossypium hirsutum L.

BACKGROUND: The internal NAD(P)H dehydrogenase (NDA) gene family was a member of the NAD(P)H dehydrogenase (ND) gene family, mainly involved in the non-phosphorylated respiratory pathways in mitochondria and played crucial roles in response to abiotic stress. METHODS: The whole genome identification, structure analysis and expression pattern of NDA gene family were conducted to analyze the NDA gene family. RESULTS: There were 51, 52, 26, and 24 NDA genes identified in G. hirsutum, G. barbadense, G. arboreum and G. raimondii, respectively. According to the structural characteristics of genes and traits of phylogenetic tree, we divided the NDA gene family into 8 clades. Gene structure analysis showed that the NDA gene family was relatively conservative. The four Gossypium species had good collinearity, and segmental duplication played an important role in the evolution of the NDA gene family. Analysis of cis-elements showed that most GhNDA genes contained cis-elements related to light response and plant hormones (ABA, MeJA and GA). The analysis of the expression patterns of GhNDA genes under different alkaline stress showed that GhNDA genes were actively involved in the response to alkaline stress, possibly through different molecular mechanisms. By analyzing the existing RNA-Seq data after alkaline stress, it was found that an NDA family gene GhNDA32 was expressed, and then theGhNDA32 was silenced by virus-induced gene silencing (VIGS). By observing the phenotype, we found that the wilting degree of silenced plants was much higher than that of the control plant after alkaline treatment, suggesting that GhNDA32 gene was involved in the response to alkaline stress. CONCLUSIONS: In this study, GhNDAs participated in response to alkaline stress, especially NaHCO3 stress. It was of great significance for the future research on the molecular mechanism of NDA gene family in responding to abiotic stresses.

Molecular structures and functional exploration of NDA family genes respond tolerant to alkaline stress in Gossypium hirsutum L

BACKGROUND: The internal NAD(P)H dehydrogenase (NDA) gene family was a member of the NAD(P)H dehydrogenase (ND) gene family, mainly involved in the non-phosphorylated respiratory pathways in mitochondria and played crucial roles in response to abiotic stress. METHODS: The whole genome identification, structure analysis and expression pattern of NDA gene family were conducted to analyze the NDA gene family. RESULTS: There were 51, 52, 26, and 24 NDA genes identified in G. hirsutum, G. barbadense, G. arboreum and G. raimondii, respectively. According to the structural characteristics of genes and traits of phylogenetic tree, we divided the NDA gene family into 8 clades. Gene structure analysis showed that the NDA gene family was relatively conservative. The four Gossypium species had good collinearity, and segmental duplication played an important role in the evolution of the NDA gene family. Analysis of cis-elements showed that most GhNDA genes contained cis-elements related to light response and plant hormones (ABA, MeJA and GA). The analysis of the expression patterns of GhNDA genes under different alkaline stress showed that GhNDA genes were actively involved in the response to alkaline stress, possibly through different molecular mechanisms. By analyzing the existing RNA-Seq data after alkaline stress, it was found that an NDA family gene GhNDA32 was expressed, and then theGhNDA32 was silenced by virus-induced gene silencing (VIGS). By observing the phenotype, we found that the wilting degree of silenced plants was much higher than that of the control plant after alkaline treatment, suggesting that GhNDA32 gene was involved in the response to alkaline stress. CONCLUSIONS: In this study, GhNDAs participated in response to alkaline stress, especially NaHCO3 stress. It was of great significance for the future research on the molecular mechanism of NDA gene family in responding to abiotic stresses.

Insight Between the Epigenetics and Transcription Responding of Cotton Hypocotyl Cellular Elongation Under Salt-Alkaline Stress.

Gossypium barbadense is a cultivated cotton not only known for producing superior fiber but also for its salt and alkaline resistance. Here, we used Whole Genome Bisulfite Sequencing (WGBS) technology to map the cytosine methylation of the whole genome of the G. barbadense hypocotyl at single base resolution. The methylation sequencing results showed that the mapping rates of the three samples were 75.32, 77.54, and 77.94%, respectively. In addition, the Bisulfite Sequence (BS) conversion rate was 99.78%. Approximately 71.03, 53.87, and 6.26% of the cytosine were methylated at CG, CHG, and CHH sequence contexts, respectively. A comprehensive analysis of DNA methylation and transcriptome data showed that the methylation level of the promoter region was a positive correlation in the CHH context. Saline-alkaline stress was related to the methylation changes of many genes, transcription factors (TFs) and transposable elements (TEs), respectively. We explored the regulatory mechanism of DNA methylation in response to salt and alkaline stress during cotton hypocotyl elongation. Our data shed light into the relationship of methylation regulation at the germination stage of G. barbadense hypocotyl cell elongation and salt-alkali treatment. The results of this research help understand the early growth regulation mechanism of G. barbadense in response to abiotic stress.

Genome-wide expression analysis of phospholipase A1 (PLA1) gene family suggests phospholipase A1-32 gene responding to abiotic stresses in cotton.

Cotton is the most important crop for the production of natural fibres used in the textile industry. High salinity, drought, cold and high temperature represent serious abiotic stresses, which seriously threaten cotton production. Phospholipase AS has an irreplaceable role in lipid signal transmission, growth and development and stress events. Phospholipase A can be divided into three families: PLA1, PLA2 and pPLA. Among them, the PLA1 family is rarely studied in plants. In order to study the potential functions of the PLA1 family in cotton, the bioinformatics analysis of the PLA1 family was correlated with cotton adversity, and tissue-specific analysis was performed. Explore the structure-function relationship of PLA1 members. It is found that the expression of GbPLA1-32 gene is affected by a variety of environmental stimuli, indicating that it plays a very important role in stress and hormone response, and closely associates the cotton adversity with this family. Through further functional verification, we found that virus-induced GbPLA1-32 gene silencing (VIGS) caused Gossypium barbadense to be sensitive to salt stress. This research provides an important basis for further research on the molecular mechanism of cotton resistance to abiotic stress.

Genome-wide identification and function analysis of HMAD gene family in cotton (Gossypium spp.).

BACKGROUND: The abiotic stress such as soil salinization and heavy metal toxicity has posed a major threat to sustainable crop production worldwide. Previous studies revealed that halophytes were supposed to tolerate other stress including heavy metal toxicity. Though HMAD (heavy-metal-associated domain) was reported to play various important functions in Arabidopsis, little is known in Gossypium. RESULTS: A total of 169 G. hirsutum genes were identified belonging to the HMAD gene family with the number of amino acids ranged from 56 to 1011. Additionally, 84, 76 and 159 HMAD genes were identified in each G. arboreum, G. raimondii and G. barbadense, respectively. The phylogenetic tree analysis showed that the HMAD gene family were divided into five classes, and 87 orthologs of HMAD genes were identified in four Gossypium species, such as genes Gh_D08G1950 and Gh_A08G2387 of G. hirsutum are orthologs of the Gorai.004G210800.1 and Cotton_A_25987 gene in G. raimondii and G. arboreum, respectively. In addition, 15 genes were lost during evolution. Furthermore, conserved sequence analysis found the conserved catalytic center containing an anion binding (CXXC) box. The HMAD gene family showed a differential expression levels among different tissues and developmental stages in G. hirsutum with the different cis-elements for abiotic stress. CONCLUSIONS: Current study provided important information about HMAD family genes under salt-stress in Gossypium genome, which would be useful to understand its putative functions in different species of cotton.

Genome-wide identification of CK gene family suggests functional expression pattern against Cd2+ stress in Gossypium hirsutum L.

Choline kinase (CK) gene plays an important role in plants growth, development and resistance to stress. It mainly regulates the biosynthesis of phosphatidylcholine. This study aims to explore the structure-function relationship, and to provide a framework for functional validation and biochemical characterization of various CK genes. Our analysis showed that 87 CK genes were identified in cotton and 7 diploid plants, of which 43 genes encode CK proteins in 4 cotton species, and 13 genes were identified in Gossypium hirsutum. Most of GhCK genes are affected by the abiotic stress conditions, indicating the importance of CK proteins for plant development and response to abiotic stress. RT-qPCR analysis showed the tissue specificity of GhCK genes in response to Cd2+ and other abiotic stresses. Under Cd2+ stress, the expression level of GhCK gene family members has undergone different changes. The expression level of GhCK5 was enhanced, indicating that Cd2+ stress caused the increase of phosphatidylcholine content, which in turn reacted on the plant cell membrane, finally reached the absorption of Cd2+ into plant cells to repair Cd2+ the purpose of contaminated soil. This study will further broaden our understanding of the association between evolution and function of the GhCK gene family.