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Background: Cotton, being extensively cultivated, holds immense economic significance as one of the most prominent crops globally. The SET (Su(var), E, and Trithorax) domain-containing protein is of significant importance in plant development, growth, and response to abiotic stress by modifying the lysine methylation status of histone. However, the comprehensive identification of SET domain genes (SDG) have not been conducted in upland cotton (Gossypium hirsutum L.). Results: A total of 229 SDGs were identified in four Gossypium species, including G. arboretum, G. raimondii, G. hirsutum, and G. barbadense. These genes could distinctly be divided into eight groups. The analysis of gene structure and protein motif revealed a high degree of conservation among the SDGs within the same group. Collinearity analysis suggested that the SDGs of Gossypium species and most of the other selected plants were mainly expanded by dispersed duplication events and whole genome duplication (WGD) events. The allopolyploidization event also has a significant impact on the expansion of SDGs in tetraploid Gossypium species. Furthermore, the characteristics of these genes have been relatively conserved during the evolution. Cis-element analysis revealed that GhSDGs play a role in resistance to abiotic stresses and growth development. Furthermore, the qRT-PCR results have indicated the ability of GhSDGs to respond to salt stress. Co-expression analysis revealed that GhSDG51 might co-express with genes associated with salt stress. In addition, the silencing of GhSDG51 in cotton by the virus-induced gene silencing (VIGS) method suggested a potential positive regulatory role of GhSDG51 in salt stress. Conclusions: The results of this study comprehensively analyze the SDGs in cotton and provide a basis for understanding the biological role of SDGs in the stress resistance in upland cotton. [ABSTRACT FROM AUTHOR]

Background: Phloem protein 2 (PP2) proteins play a vital role in the Phloem-based defense (PBD) and participate in many abiotic and biotic stress. However, research on PP2 proteins in cotton is still lacking. Results: A total of 25, 23, 43, and 47 PP2 genes were comprehensively identified and characterized in G.arboretum, G.raimondii, G.barbadense, and G.hirsutum. The whole genome duplication (WGD) and allopolyploidization events play essential roles in the expansion of PP2 genes. The promoter regions of GhPP2 genes contain many cis-acting elements related to abiotic stress and the weighted gene co-expression network analysis (WGCNA) analysis displayed that GhPP2s could be related to salt stress. The qRT-PCR assays further confirmed that GhPP2-33 could be dramatically upregulated during the salt treatment. And the virus-induced gene silencing (VIGS) experiment proved that the silencing of GhPP2-33 could decrease salt tolerance. Conclusions: The results in this study not only offer new perspectives for understanding the evolution of PP2 genes in cotton but also further explore their function under salt stress. [ABSTRACT FROM AUTHOR]

Soil salinization conditions seriously restrict cotton yield and quality. Related studies have shown that the DUF4228 proteins are pivotal in plant resistance to abiotic stress. However, there has been no systematic identification and analysis of the DUF4228 gene family in cotton and their role in abiotic stress. In this study, a total of 308 DUF4228 genes were identified in four Gossypium species, which were divided into five subfamilies. Gene structure and protein motifs analysis showed that the GhDUF4228 proteins were conserved in each subfamily. In addition, whole genome duplication (WGD) events and allopolyploidization might play an essential role in the expansion of the DUF4228 genes. Besides, many stress-responsive (MYB, MYC) and hormone-responsive (ABA, MeJA) related cis-elements were detected in the promoters of the DUF4228 genes. The qRT-PCR results showed that GhDUF4228 genes might be involved in the response to abiotic stress. VIGS assays and the measurement of relative water content (RWC), Proline content, POD activity, and malondialdehyde (MDA) content indicated that GhDUF4228-67 might be a positive regulator of cotton response to salt stress. The results in this study systematically characterized the DUF4228s in Gossypium species and will provide helpful information to further research the role of DUF4228s in salt tolerance. [ABSTRACT FROM AUTHOR]

As one of the most important factors in alternative splicing (AS) events, serine/arginine-rich (SR) proteins not only participate in the growth and development of plants but also play pivotal roles in abiotic stresses. However, the research about SR proteins in cotton is still lacking. In this study, we performed an extensive comparative analysis of SR proteins and determined their phylogeny in the plant lineage. A total of 169 SR family members were identified from four Gossypium species, and these genes could be divided into eight distinct subfamilies. The domain, motif distribution and gene structure of cotton SR proteins are conserved within each subfamily. The expansion of SR genes is mainly contributed by WGD and allopolyploidization events in cotton. The selection pressure analysis showed that all the paralogous gene pairs were under purifying selection pressure. Many cis-elements responding to abiotic stress and phytohormones were identified in the upstream sequences of the GhSR genes. Expression profiling suggested that some GhSR genes may involve in the pathways of plant resistance to abiotic stresses. The WGCNA analysis showed that GhSCL-8 co-expressed with many abiotic responding related genes in a salt-responding network. The Y2H assays showed that GhSCL-8 could interact with GhSRs in other subfamilies. The subcellular location analysis showed that GhSCL-8 is expressed in the nucleus. The further VIGS assays showed that the silencing of GhSCL-8 could decrease salt tolerance in cotton. These results expand our knowledge of the evolution of the SR gene family in plants, and they will also contribute to the elucidation of the biological functions of SR genes in the future. [ABSTRACT FROM AUTHOR]

The membrane attack complex/perforin (MACPF) domain-containing proteins are involved in the various developmental processes and in responding to diverse abiotic stress. The function and regulatory network of the MACPF genes are rarely reported in Gossypium spp. We study the detailed identification and partial functional verification of the members of the MACPF family. Totally, 100 putative MACPF proteins containing complete MACPF domain were identified from the four cotton species. They were classified into three phylogenetic groups and underwent multifold pressure indicating that selection produced new functional differentiation. Cotton MACPF gene family members expanded mainly through the whole-genome duplication (WGD)/segmental followed by the dispersed. Expression and cis- acting elements analysis revealed that MACPFs play a role in resistance to abiotic stresses, and some selected GhMACPFs were able to respond to the PEG and cold stresses. Co-expression analysis showed that GhMACPFs might interact with valine-glutamine (VQ), WRKY, and Apetala 2 (AP2)/ethylene responsive factor (ERF) domain-containing genes under cold stress. In addition, silencing endogenous GhMACPF26 in cotton by the virus-induced gene silencing (VIGS) method indicated that GhMACPF26 negatively regulates cold tolerance. Our data provided a comprehensive phylogenetic evolutionary view of Gossypium MACPFs. The MACPFs may work together with multiple transcriptional factors and play roles in acclimation to abiotic stress, especially cold stress in cotton. [ABSTRACT FROM AUTHOR]

Background: B-BOX (BBX) proteins are zinc-finger transcription factors with one or two BBX domains and sometimes a CCT domain. These proteins play an essential role in regulating plant growth and development, as well as in resisting abiotic stress. So far, the BBX gene family has been widely studied in other crops. However, no one has systematically studied the BBX gene in cotton. Results: In the present study, 17, 18, 37 and 33 BBX genes were detected in Gossypium arboreum, G. raimondii, G. hirsutum and G. barbadense, respectively, via genome-wide identification. Phylogenetic analysis showed that all BBX genes were divided into 5 main categories. The protein motifs and exon/intron structures showed that each group of BBX genes was highly conserved. Collinearity analysis revealed that the amplification of BBX gene family in Gossypium spp. was mainly through segmental replication. Nonsynonymous (Ka)/ synonymous (Ks) substitution ratios indicated that the BBX gene family had undergone purification selection throughout the long-term natural selection process. Moreover, transcriptomic data showed that some GhBBX genes were highly expressed in floral organs. The qRT-PCR results showed that there were significant differences in GhBBX genes in leaves and shoot apexes between early-maturing materials and late-maturing materials at most periods. Yeast two-hybrid results showed that GhBBX5/GhBBX23 and GhBBX8/GhBBX26 might interact with GhFT. Transcriptome data analysis and qRT-PCR verification showed that different GhBBX genes had different biological functions in abiotic stress and phytohormone response. Conclusions: Our comprehensive analysis of BBX in G. hirsutum provided a basis for further study on the molecular role of GhBBXs in regulating flowering and cotton resistance to abiotic stress. [ABSTRACT FROM AUTHOR]

The potassium transporter family HAK/KUP/KT is a large group of proteins that are important in plant potassium transport and plays a crucial role in plant growth and development, especially in economic crops. Although HAK/KUP/KT genes have been identified in many species, research on these genes in cotton is still quite rare. In this study, in total, 21, 24, 45, and 44 HAK/KUP/KT genes were identified in Gossypium arboreum , Gossypium raimondii , Gossypium hirsutum , and Gossypium barbadense , respectively. Phylogenetic analysis showed that these genes were divided into four clusters. The G. hirsutum gene promoters contained diverse cis -regulatory elements, such as drought-responsive elements, low temperature-responsive elements, and other elements. The RNA-seq data and qRT-PCR results showed that HAK/KUP/KT genes had different expression patterns in fiber development. The qRT-PCR results of drought and NaCl treatment indicated that HAK/KUP/KT genes might play important roles in abiotic stress responses. These results will provide molecular insights into potassium transporter research in cotton. [ABSTRACT FROM AUTHOR]

WRKY transcription factors (TFs) play essential roles in the plant response to leaf senescence and abiotic stress. However, the WRKY TFs involved in leaf senescence and stress tolerance in cotton (Gossypium hirsutum L.) are still largely unknown. In this study, a WRKY gene, GhWRKY91 , was isolated and thoroughly characterized. Transcriptional activity assays showed that GhWRKY91 could activate transcription in yeast. The expression pattern of GhWRKY91 during leaf senescence, and in response to abscisic acid (ABA) and drought stress was evaluated. β-Glucuronidase (GUS) activity driven by the GhWRKY91 promoter in transgenic Arabidopsis was reduced upon exposure to ABA and drought treatments. Constitutive expression of GhWRKY91 in Arabidopsis delayed natural leaf senescence. GhWRKY91 transgenic plants exhibited increased drought tolerance and presented delayed drought-induced leaf senescence, as accompanied by reinforced expression of stress-related genes and attenuated expression of senescence-associated genes (SAGs). Yeast one-hybrid (Y1H) assays and electrophoretic mobility shift assays (EMSAs) revealed that GhWRKY91 directly targets GhWRKY17 , a gene associated with ABA signals and reactive oxygen species (ROS) production. A transient dual-luciferase reporter assay demonstrated that GhWRKY91 activated the expression of GhWRKY17. Our results suggest that GhWRKY91 might negatively regulate natural and stress-induced leaf senescence and provide a foundation for further functional studies on leaf senescence and the stress response in cotton. [ABSTRACT FROM AUTHOR]

The glycosyltransferase (GT) 47 family is involved in the biosynthesis of xylose, pectin and xyloglucan and plays a significant role in maintaining the normal morphology of the plant cell wall. However, the functions of GT47s are less well known in cotton. In the present study, a total of 53, 53, 105 and 109 GT47 genes were detected by genome-wide identification in Gossypium arboreum , G. raimondii , G. hirsutum and G. barbadense , respectively. All the GT47s were classified into six major groups via phylogenetic analysis. The exon/intron structure and protein motifs indicated that each branch of the GT47 genes was highly conserved. Collinearity analysis showed that GT47 gene family expansion occurred in Gossypium spp. mainly through whole-genome duplication and that segmental duplication mainly promoted GT47 gene expansion within the A and D subgenomes. The Ka/Ks values suggested that the GT47 gene family has undergone purifying selection during the long-term evolutionary process. Transcriptomic data and qRT-PCR showed that GhGT47 genes exhibited different expression patterns in each tissue and during fiber development. Our results suggest that some genes in the GhGT47 family might be associated with fiber development and the abiotic stress response, which could promote further research involving functional analysis of GT47 genes in cotton. [ABSTRACT FROM AUTHOR]

Cold stress can significantly affect the development, yield, and quality of crops and restrict the geographical distribution and growing seasons of plants. Aquaporins are the main channels for water transport in plant cells. Abiotic stresses such as cold and drought dehydrate cells by changing the water potential. In this study, we cloned a gene GhTIP1;1-like encodes tonoplast aquaporin from the transcriptome database of cotton seedlings after cold stress. Expression analysis showed that GhTIP1;1-like not only responds to cold stress but was also induced by heat, drought and salt stress. Subcellular localization showed that the protein was anchored to the vacuole membrane. Promoter deletion analysis revealed that a MYC motif within the promoter region of GhTIP1;1-like were the core cis-elements in response to low temperature. Virus-induced gene silencing (VIGS) and histochemical staining indicate that GhTIP1;1-like plays a positive role in plant cold tolerance. Overexpression of GhTIP1;1-like in Arabidopsis delayed the senescence process and enhanced the cold tolerance of transgenic plants. Compared with the wild type, the soluble protein concentration and peroxidase activity of the transgenic lines under cold stress were higher, while the malondialdehyde content was lower. In addition, the expression levels of cold-responsive genes were significantly increased in transgenic plants under cold stress. Our results indicate that GhTIP1;1-like could respond to different abiotic stresses and be positively involved in regulating the cold tolerance of cotton. [ABSTRACT FROM AUTHOR]

Background: Valine-glutamine (VQ) motif-containing proteins play important roles in plant growth, development and abiotic stress response. For many plant species, the VQ genes have been identified and their functions have been described. However, little is known about the origin, evolution, and functions (and underlying mechanisms) of the VQ family genes in cotton. Results: In this study, we comprehensively analyzed the characteristics of 268 VQ genes from four Gossypium genomes and found that the VQ proteins evolved into 10 clades, and each clade had a similar structural and conservative motif. The expansion of the VQ gene was mainly through segmental duplication, followed by dispersal. Expression analysis revealed that many GhVQs might play important roles in response to salt and drought stress, and GhVQ18 and GhVQ84 were highly expressed under PEG and salt stress. Further analysis showed that GhVQs were co-expressed with GhWRKY transcription factors (TFs), and microRNAs (miRNAs) could hybridize to their cis-regulatory elements. Conclusions: The results in this study broaden our understanding of the VQ gene family in plants, and the analysis of the structure, conserved elements, and expression patterns of the VQs provide a solid foundation for exploring their specific functions in cotton responding to abiotic stresses. Our study provides significant insight into the potential functions of VQ genes in cotton. [ABSTRACT FROM AUTHOR]

Upland cotton (Gossypium hirsutum L.) is the main natural fiber crop worldwide and is an essential source of seed oil and biofuel products. Many abiotic stresses, such as drought and salinity, constrain cotton production. Thioredoxins (TRXs) are a group of small ubiquitous proteins that are widely distributed among organisms. TRXs play a crucial role in regulating diverse functions during plant growth and development. In the present study, a novel GhTRX134 gene was characterized and overexpressed in Arabidopsis and silenced in cotton under drought stress. Furthermore, the proline content and enzyme activity levels were measured in transgenic plants and wild-type (Wt) plants under drought and salt stress. The results revealed that the overexpression of GhTRX134 enhanced abiotic stress tolerance. When GhTRX134 was silenced, cotton plants become more sensitive to drought. Taken together, these findings confirmed that the overexpression of GhTRX134 improved drought and salt tolerance in Arabidopsis plants. Therefore, the GhTRX134 gene can be transformed into cotton plants to obtain transgenic lines for more functional details. [ABSTRACT FROM AUTHOR]

Cotton is an important textile industry raw material crops, which plays a critical role in the development of society. MADS transcription factors (TFs) play a key role about the flowering time, flower development, and abiotic stress responses in plants, but little is known about their functions on abiotic stress in cotton. In this study, a MIKCC subfamily gene from cotton, GhFYF (FOREVER YOUNG FLOWER), was isolated and characterized. Our data showed that GhFYF localized to the nucleus. A β-glucuronidase (GUS) activity assay revealed that the promoter of GhFYF was mainly expressed in the flower and seed of ProGhFYF :: GUS transgenic A. thaliana plants. The GUS staining of flowers and seeds was deepened after drought, salt treatment, and the expression level of the GUS gene and corresponding stress genes AtERD10 , AtAnnexin1 are up-regulated in the inflorescence. Overexpression GhFYF in A. thaliana could promote the seed germination and growth under different salt concentrations, and determin the proline content. Yeast two-hybrid (Y2H) assays showed that GhFYF interacted with the HAD-like protein GhGPP2, which has responds to abiotic stress. Our findings indicate that GhFYF is involved in abiotic stress responses, especially for salt stress. This work establishes a solid foundation for further functional analysis of the GhFYF gene in cotton. [ABSTRACT FROM AUTHOR]

NDR1/HIN1-like (NHL) proteins widely exist in the plant kingdom, and play an essential role in resistance to both biotic and abiotic stress. However, the study of NHL family in cotton is still lacking. In the present study, a total of 40, 46, 79, and 78 NHL genes were detected by genome-wide identification in G. arboreum , G. raimondii , G. barbadense , and G. hirsutum, respectively. By analyzing the duplication pattern, the whole genome duplication (WGD) and allotetraploidization events play a vital role in the expansion of GhNHLs. The analysis of gene structure, conserved protein motif, and Ka/Ks values suggest that the structure and function might remain conserved during the evolution. The cis -acting elements analysis revealed that the GhNHL genes could respond to the abiotic stress, and the further transcriptome analysis and qRT-PCR assay indicated that GhNHLs might play a role in the cold stress tolerance. The WGCNA (Weighted correlation network analysis) analysis showed that GhNHL69 co-express with many transcription factors that are related to cold stress. By performing virus-induced gene silencing (VIGS) assays, the GhNHL69 silencing plant exhibited more severe dehydration and injury compared to the control groups. Additionally, we further found the expression of many abiotic genes was inhibited in the GhNHL69 silenced plants. This study gained insight into NHL members in cotton and mined a gene related to cold stress and lays the important basis for further study on NHL genes related to cold stress tolerance. • NHL genes were systematically identified and analyzed in Gossypium species. • The whole genome duplication (WGD) and allotetraploidization events play a vital role in the expansion of GhNHLs. • GhNHLs play key roles during cold stress tolerance. • The GhNHL69 gene modulates cold resistance in cotton. [ABSTRACT FROM AUTHOR]

• GhAIFs are highly expressed in the initiation and elongation phases of the fibers. • Exogenous hormones can significantly induce GhAIFs expression. • The majority of GhAIFs actively respond to cold stress. • GhAIFs were more abundantly expressed in cold resistant than sensitive materials. ATBS1 INTERACTING FACTORs (AIFs) are atypical basic helix-loop-helix (bHLH) proteins play important roles in the growth and development of plants. There are no comprehensive reports on the genomic identification and functional analysis of AIFs in cotton. In this study, 8, 11, 16, and 15 AIFs were identified in Gossypium arboreum L., Gossypium raimondii L., Gossypium hirsutum L., and Gossypium barbadense L., respectively. AIFs were also screened in other species: 3 in Theobroma cacao L., 4 in Populus trichocarpa C. and 5 in Zea mays L. Based on a neighbour-joining phylogenetic tree, 66 AIF proteins identified in these species were divided into 3 groups. Multiple sequence alignment analysis showed that the AIF protein sequences were relatively conserved. Analysis of gene chromosome position and gene replication showed that AIFs amplification in cotton might be due to fragment and tandem replication. And cold stress response and plant hormone cis-elements were identified in the promoter regions of GhAIFs. Published transcriptional data showed that GhAIFs were differentially expressed in various tissues, especially in floral organs and fibers. qRT-PCR results showed that most of GhAIFs were highly expressed at the fiber initiation stage. Analysis of the expression of selected GhAIFs under abiotic stress (GA, IAA, SA, ABA, and 4 °C treatment) suggested that GhAIFs might be involved in the regulation of abiotic stress. The comprehensive analysis of the AIF gene family in upland cotton provides a foundation for further studies on the function of atypical bHLH-GhAIFs family members and screening of fiber development, and stress-resistant candidate genes in cotton. [ABSTRACT FROM AUTHOR]