Background: The cotton crop is universally considered as protein and edible oil source besides the major contributor of natural fiber and is grown in tropical and subtropical regions around the world Unpredicted environmental stresses are becoming significant threats to sustainable cotton production, ultimately leading to a substantial irreversible economic loss. Mitogen-activated protein kinase(MAPK) is generally considered essential for recognizing environmental stresses through phosphorylating downstream signal pathways and plays a vital role in numerous biological processes.Results: We have identified 74 MAPK genes across cotton, 41 from G. hirsutum, 19 from G. raimondii, whereas 14 have been identified from G. arboreum. The MAPK gene-proteins have been further studied to determine their physicochemical characteristics and other essential features. In this perspective, characterization, phylogenetic relationship,chromosomal mapping, gene motif, cis-regulatory element, and subcellular localization were carried out. Based on phylogenetic analysis, the MAPK family in cotton is usually categorized as A, B, C, D, and E clade. According to the results of the phylogenic relationship, cotton has more MAPKS genes in Clade A than Clade B. The cis-elements identified were classified into five groups(hormone responsiveness, light responsiveness, stress responsiveness, cellular development, and binding site). The prevalence of such elements across the promoter region of these genes signifies their role in the growth and development of plants. Seven GHMAPK genes(GH＿A07G1527, GH＿D02G1138,GH＿D03G0121, GH＿D03G1517, GH＿D05G1003, GH＿D11G0040, and GH＿D12G2528) were selected, and specific tissue expression and profiling were performed across drought and salt stresses. Results expressed that six genes were upregulated under drought treatment except for GH＿D11G0040 which is downregulated. Whereas all the seven genes have been upregulated at various hours of salt stress treatment.Conclusions: RNA sequence and qPCR results showed that genes as di erentially expressed across both vegetative and reproductive plant parts. Similarly, the qPCR analysis showed that six genes had been upregulated substantially through drought treatment while all the seven genes were upregulated across salt treatments. The results of this study showed that cotton GHMPK3 genes play an important role in improving cotton resistance to drought and salt stresses. MAPKs are thought to play a significant regulatory function in plants' responses to abiotic stresses according to various studies. MAPKs' involvement in abiotic stress signaling and innovation is a key goal for crop species research, especially in crop breeding.

Background: Nucleobase–ascorbate transporters(NAT), synonymously called nucleobase–cation symporter 2(NCS2) proteins, were earlier reported to be involved in plant growth, development and resistance to stress. Previous studies concluded that s a polymorphic SNP associated with NAT12 was significant di erent between salt-tolerant and salt-sensitive materials of upland cotton. In current study, a comprehensive analysis of NAT family genes was conducted for the first time in cotton.Results: In this study, we discovered 32, 32, 18, and 16 NAT genes in Gossypium hirsutum, G. barbadense, G. raimondii and G. arboreum, respectively, which were classified into four groups(groups I–IV) based on the multiple sequence analysis. These GhNAT genes were unevenly distributed on At and Dt sub-genome in G. hirsutum. Most GhNAT members in the same group had similar gene structure characteristics and motif composition. The collinearity analysis revealed segmental duplication as well as tandem duplication contributing to the expansion of the GhNATs. The analysis of cis-acting regulatory elements of GhNATs showed that the function of GhNAT genes in cotton might be related to plant hormone and stress response. Under di erent conditions, the expression levels further suggested the GhNAT family genes were associated with plant response to various abiotic stresses. GhNAT12 was detected in the plasma membrane. And it was validated that the GhNAT12 gene played an important role in regulating cotton resistance to salt and drought stress through the virus-induced gene silencing(VIGS) analysis.Conclusions: A comprehensive analysis of NAT gene family was performed in cotton, including phylogenetic analysis, chromosomal location, collinearity analysis, motifs, gene structure and so on. Our results will further broaden the insight into the evolution and potential functions of NAT genes in cotton. Current findings could make significant contribution towards screening more candidate genes related to biotic and abiotic resistance for the improvement in cotton.

Background:Cotton is a valuable economic crop and the main significant source of natural fiber for textile industries globally.The effects of drought and salt stress pose a challenge to strong fiber and large-scale production due to the ever-changing climatic conditions.However,plants have evolved a number of survival strategies,among them is the induction of various stress-responsive genes such as the ribosomal protein large(RPL) gene.The RPL gene families encode critical proteins,which alleviate the effects of drought and salt stress in plants.In this study,comprehensive and functional analysis of the cotton RPL genes was carried out under drought and salt stresses.Results:Based on the genome-wide evaluation,26,8,and 5 proteins containing the RPL14 B domain were identified in Gossypiumhirsutum,G.raimondii,and G.arboreum,respectively.Furthermore,through bioinformatics analysis,key cis-regulatory elements related to RPL 148 genes were discovered.The Myb binding sites(MBS),abscisic acid-responsive element(ABRE),CAAT-box,TATA box,TGACG-motif,and CGTCA-motif responsive to methyl jasmonate,as well as the TCA-motif responsive to salicylic acid,were identified.Expression analysis revealed a key gene,Gh_D01G0234(RPL14B),with significantly higher induction levels was further evaluated through a reverse genetic approach.The knockdown of Gh_D01G0234(RPL14B) significantly affected the performance of cotton seedlings under drought/salt stress conditions,as evidenced by a substantial reduction in various morphological and physiological traits.Moreover,the level of the antioxidant enzyme was significantly reduced in VIGS-plants,while oxidant enzyme levels increased significantly,as demonstrated by the higher malondialdehyde concentration level.Conclusion:The results revealed the potential role of the RPL 148 gene in promoting the induction of antioxidant enzymes,which are key in oxidizing the various oxidants.The key pathways need to be investigated and even as we exploit these genes in the developing of more stress-resilient cotton germplasms.

Background: Cotton fibers are single-celled extensions of the seed epidermis, a model tissue for studying cytoskeleton. Tubulin genes play a critical role in synthesizing the microtubules(MT) as a core element of the cytoskeleton. However, there is a lack of studies concerning the systematic characterization of the tubulin gene family in cotton. Therefore, the identification and portrayal of G. hirsutum tubulin genes can provide key targets for molecular manipulation in cotton breeding.Result: In this study, we investigated all tubulin genes from different plant species and identified 98 tubulin genes in G. hirsutum. Phylogenetic analysis showed that tubulin family genes were classified into three subfamilies. The protein motifs and gene structure of α-, β-tubulin genes are more conserved compared with γ-tubulin genes. Most tubulin genes are located at the proximate ends of the chromosomes. Spatiotemporal expression pattern by transcriptome and q RT-PCR analysis revealed that 12 α-tubulin and 7 β-tubulin genes are specifically expressed during different fiber development stages. However, Gh.A03 G027200, Gh.D03 G169300, and Gh.A11 G258900 had differential expression patterns at distinct stages of fiber development in varieties J02508 and ZRI015.Conclusion: In this study, the evolutionary analysis showed that the tubulin genes were divided into three clades.The genetic structures and molecular functions were highly conserved in different plants. Three candidate genes,Gh.A03 G027200, Gh.D03 G169300, and Gh.A11 G258900 may play a key role during fiber development complementing fiber length and strength.

Background: Cotton is an important commercial crop for being a valuable source of natural fiber. Its production has undergone a sharp decline because of abiotic stresses, etc. Drought is one of the major abiotic stress causing significant yield losses in cotton. However, plants have evolved self-defense mechanisms to cope abiotic factors like drought, salt, cold, etc. The evolution of stress responsive transcription factors such as the trihelix, a noduleinception-like protein(NLP), and the late embryogenesis abundant proteins have shown positive response in the resistance improvement to several abiotic stresses.Results: Genome wide identification and characterization of the effects of Light-Harvesting Chloro a/b binding(LHC) genes were carried out in cotton under drought stress conditions. A hundred and nine proteins encoded by the LHC genes were found in the cotton genome, with 55, 27, and 27 genes found to be distributed in Gossypium hirsutum, G. arboreum, and G. raimondii, respectively. The proteins encoded by the genes were unevenly distributed on various chromosomes. The Ka/Ks(Non-synonymous substitution rate/Synonymous substitution rate) values were less than one, an indication of negative selection of the gene family. Differential expressions of genes showed that majority of the genes are being highly upregulated in the roots as compared with leaves and stem tissues. Most genes were found to be highly expressed in MR-85, a relative drought tolerant germplasm.Conclusion: The results provide proofs of the possible role of the LHC genes in improving drought stress tolerance,and can be explored by cotton breeders in releasing a more drought tolerant cotton varieties.

Background: Calmodulin(CaM) is one of the most important Ca~(2+) signaling receptors because it regulates diverse physiological and biochemical reactions in plants. CaM functions by interacting with CaM-binding proteins(CaMBPs) to modulate Ca~(2+) signaling. IQ domain(IQD) proteins are plant-specific CaMBPs that bind to CaM by their specific CaM binding sites.Results: In this study, we identified 102 GhIQD genes in the Gossypium hirsutum L. genome. The GhIQD gene family was classified into four clusters(I, II, III, and IV), and we then mapped the GhIQD genes to the G. hirsutum L.chromosomes. Moreover, we found that 100 of the 102 GhIQD genes resulted from segmental duplication events,indicating that segmental duplication is the main force driving GhIQD gene expansion. Gene expression pattern analysis showed that a total of 89 GhIQD genes expressed in the elongation stage and second cell wall biosynthesis stage of the fiber cells, suggesting that GhIQD genes may contribute to fiber cell development in cotton. In addition, we found that 20 selected GhIQD genes were highly expressed in various tissues. Exogenous application of MeJA significantly enhanced the expression levels of GhIQD genes.Conclusions: Our study shows that GhIQD genes are involved in fiber cell development in cotton and are also widely induced by MeJA. Thw results provide bases to systematically characterize the evolution and biological functions of GhIQD genes, as well as clues to breed better cotton varieties in the future.

Background:DNA methylation is an important epigenetic factor that maintains and regulates gene expression.The mode and level of DNA methylation depend on the roles of DNA methyltransferase and demethylase,while DNA demethylase plays a key role in the process of DNA demethylation.The results showed that the plant's DNA demethylase all contained conserved DNA glycosidase domain.This study identified the cotton DNA demethylase gene family and analyzed it using bioinformatics methods to lay the foundation for further study of cotton demethylase gene function.Results:This study used genomic information from diploid Gossypium raimondii JGI(D),Gossypium arboreum L.CRI(A),Gossypium hirsutum L.JGI(AD1) and Gossypium barbadebse L NAU(AD2) to Arabidopsis thaliana.Using DNA demethylase genes sequence of Arabidopsis as reference,25 DNA demethylase genes were identified in cotton by BLAST analysis.There are 4 genes in the genome D,5 genes in the genome A,10 genes in the genome AD1,and 6 genes in the genome AD2.The gene structure and evolution were analyzed by bioinformatics,and the expression patterns of DNA demethylase gene family in Gossypium hirsutum L were analyzed.From the phylogenetic tree analysis,the DNA demethylase gene family of cotton can be divided into four subfamilies:REPRESSOR of SILENCING 1(ROS1),DEMETER(DME),DEMETER-LIKE 2(DML2),and DEMETER-LIKE3(DML3).The sequence similarity of DNA demethylase genes in the same species was higher,and the genetic relationship was also relatively close.Analysis of the gene structure revealed that the DNA demethylase gene family members of the four subfamilies varied greatly.Among them,the number of introns of ROS1 and DME subfamily was larger,and the gene structure was more complex.For the analysis of the conserved domain,it was known that the DNA demethylase family gene member has an endonuclease Ⅲ(END03 c) domain.Conclusion:The genes of the DNA demethylase family are distributed differently in different cotton species,and the gene structure is very different.High expression of ROS1 genes in cotton were under abiotic stress.The expression levels of ROS1 genes were higher during the formation of cotton ovule.The transcription levels of ROS1 family genes were higher during cotton fiber development.

Background: RING-H2 finger E3 ligase(RH2FE3) genes encode cysteine-rich proteins that mediate E3 ubiquitin ligase activity and degrade target substrates. The roles of these genes in plant responses to phytohormones and abiotic stresses are well documented in various species, but their roles in cotton fiber development are poorly understood. To date, genome-wide identification and expression analyses of Gossypium hirsutum RH2FE3 genes have not been reported.Methods: We performed computational identification, structural and phylogenetic analyses, chromosomal distribution analysis and estimated K_a/K_s values of G. hirsutum RH2FE3 genes. Orthologous and paralogous gene pairs were identified by all-versus-all BLASTP searches. We predicted cis-regulatory elements and analyzed microarray data sets to generate heatmaps at different development stages. Tissue-specific expression in cotton fiber, and hormonal and abiotic stress responses were determined by quantitative real time polymerase chain reaction(qRT-PCR) analysis.Results: We investigated 140 G. hirsutum, 80 G. arboreum, and 89 G. raimondi putative RH2FE3 genes and their evolutionary mechanisms and compared them with orthologs in Arabidopsis and rice. A domain-based analysis of the G.hirsutum RH2FE3 genes predicted conserved signature motifs and gene structures. Chromosomal localization showed the genes were distributed across al G. hirsutum chromosomes, and 60 duplication events(4 tandem and 56 segmental duplications) and 98 orthologs were detected. cis-elements were detected in the promoter regions of G. hirsutum RH2FE3 genes. Microarray data and qR T-PCR analyses showed that G. hirsutum RH2FE3 genes were strongly correlated with cotton fiber development. Additional y, almost al the identified genes were up-regulated in response to phytohormones(brassinolide, gibberel ic acid(GA), indole-3-acetic acid(IAA), and salicylic acid(SA)) and abiotic stresses(cold, heat,drought, and salt).Conclusions: The genome-wide identification, comprehensive analysis, and characterization of conserved domains and gene structures, as wel as phylogenetic analysis, cis-element prediction, and expression profile analysis of G. hirsutum RH2FE3 genes and their roles in cotton fiber development and responses to plant hormones and abiotic stresses are reported here for the first time. Our findings wil contribute to the genome-wide analysis of putative RH2FE3 genes in other species and lay a foundation for future physiological and functional research on G. hirsutum RH2FE3 genes.