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Abstract Transcription Factors (TFs) are key regulators of how plants grow and develop. Among the diverse TF families, the Glabrous-enhancer binding protein (GeBP) family plays a key role in trichome initiation and leaf development. The specific roles of GeBP TFs in plants remain largely unexplored, although GeBP transcription factors play important roles in plants. This study identified 16 GhGeBP genes in Gossypium hirsutum, ranging from 534 bp (GhGeBP14) to 1560 bp (GhGeBP2). Phylogenetic analysis grouped 16 GhGeBP genes clustered into three subgroups, unevenly distributed across 14 chromosomes. Analysis of the cis-acting elements revealed 408 motifs in the 2 kb upstream regions of the promoters, including stress-responsive, phytohormone-responsive, and light-responsive elements. Tissue-specific expression analysis showed 8 GhGeBP genes were highly expressed across all tissues, while GhGeBP4 and GhGeBP12 were down-regulated under conditions of drought, salt, cold, and heat stress. A genome-wide association study (GWAS) identified GhGeBP4 was associated with fiber micronaire (FM) and fiber strength (FS), while GhGeBP9 was linked to the node of the first fruiting branch (NFFB) and flowering time (FT). Haplotype analysis revealed that GhGeBP4-HAP2 exhibited higher fiber quality traits, while GhGeBP9-HAP2 was associated with early maturity. The results of this study offer significant insights that are worthy of further investigation into the role of the GhGeBP gene family in G. hirsutum and promising targets for marker-assisted selection strategies in cotton breeding programs, particularly for improving fiber quality and early maturity traits.

Abstract Background Cotton serves as a primary source of natural fibers crucial for the textile industry. However, environmental elements such as drought have posed challenges to cotton cultivation, resulting in adverse impacts on both production and fiber quality. Improving cotton’s resilience to drought could mitigate yield losses and foster the expansion of cotton farming. Rab7 protein, widely present in organisms, controls the degradation and recycling of cargo, and has a potential role in biotic and abiotic tolerance. However, comprehensive exploration of the Rab7 gene family in Gossypium remains scarce. Results Herein, we identified a total of 10, 10, 20, and 20 Rab7 genes through genome-wide analysis in Gossypium arboreum, Gossypium raimondii, Gossypium hirsutum, and Gossypium barbadense, respectively. Collinearity analysis unveiled the pivotal role of whole genome or segmental duplication events in the expansion of GhRab7s. Study of gene architecture, conserved protein motifs, and domains suggested the conservation of structure and function throughout evolution. Exploration of cis-regulatory elements revealed the responsiveness of GhRab7 genes to abiotic stress, corroborated by transcriptome analysis under diverse environmental stresses. Notably, the greatly induced expression of GhRab7B3-A under drought treatment prompted us to investigate its function through virus-induced gene silencing (VIGS) assays. Silencing GhRab7B3-A led to exacerbated dehydration and wilting compared with the control. Additionally, inhibition of stomatal closure, antioxidant enzyme activities and expression patterns of genes responsive to abiotic stress were observed in GhRab7B3-A silenced plants. Conclusions This study sheds light on Rab7 members in cotton, identifies a gene linked to drought stress, and paves the way for additional investigation of Rab7 genes associated with drought stress tolerance.

Abstract Background Monoacylglycerol lipase (MAGL) genes belong to the alpha/beta hydrolase superfamily, catalyze the terminal step of triglyceride (TAG) hydrolysis, converting monoacylglycerol (MAG) into free fatty acids and glycerol. Results In this study, 30 MAGL genes in upland cotton have been identified, which have been classified into eight subgroups. The duplication of GhMAGL genes in upland cotton was predominantly influenced by segmental duplication events, as revealed through synteny analysis. Furthermore, all GhMAGL genes were found to contain light-responsive elements. Through comprehensive association and haplotype analyses using resequencing data from 355 cotton accessions, GhMAGL3 and GhMAGL6 were detected as key genes related to lipid hydrolysis processes, suggesting a negative regulatory effect. Conclusions In summary, MAGL has never been studied in upland cotton previously. This study provides the genetic mechanism foundation for the discover of new genes involved in lipid metabolism to improve cottonseed oil content, which will provide a strategic avenue for marker-assisted breeding aimed at incorporating desirable traits into cultivated cotton varieties.

Abstract 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.

Cytochrome P450 proteins belong to one of the largest families of enzyme proteins in plants and play important roles in plant growth and development and the stress response. In our previous studies, a cytochrome P450 gene, GhCYP94C1 (cytochrome P450 94C1), was functionally characterized as a positive regulator of seed germination, main root elongation and early flowering. However, whether the gene has other potential functions remains to be further explored. In our study, expression analysis showed that GhCYP94C1 was highly expressed in roots and was suppressed by drought treatment. Endogenous silencing of GhCYP94C1 via virus-induced gene silencing (VIGS) increased drought resistance in cotton plants, which was accompanied by the upregulated expression of the abscisic acid (ABA) biosynthesis gene nine-cis-epoxycarotenoid dioxygenase 9 (GhNCED9) during drought stress. Our findings suggested that GhCYP94C1 may play an important role in drought resistance. Combined with previous research results, the present results provide a theoretical basis for future breeding of new cotton varieties with early maturation and drought resistance.

Abstract 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.

Summary: The 5′-deoxyadenosine deaminase (DADD), a member of the amidohydrolase family regulates biological purine metabolism. In this study, bioinformatic analysis, overexpression and knockdown of GhdadD gene were detected to identify its potential role in drought and salt stress tolerance. The results revealed that GhdadD was induced by ABA, Auxin, MBS and light responsive elements. In transgenic Arabidopsis, seed germination rate and root length were increased under drought or salt stress. GhdadD overexpressed seedlings resulted in higher plant height, less leaf damage and lower ion permeability. The expression of osmotic stress and ABA-responsive genes were up regulated. While in GhdadD-silenced cotton seedlings, CAT, SOD activity and soluble sugar content were reduced, MDA content was increased, and the stoma opening was depressed under drought or salt stress. Some osmics stress marker genes were also up regulated. These data indicating that GhdadD enhanced plant resistance to drought and salt stress through ABA pathways.

Seed vigor (SV) is a crucial trait determining the quality of crop seeds. Currently, over 80% of China’s cotton-planting area is in Xinjiang Province, where a fully mechanized planting model is adopted, accounting for more than 90% of the total fiber production. Therefore, identifying SV-related loci and genes is crucial for improving cotton yield in Xinjiang. In this study, three seed vigor-related traits, including germination potential, germination rate, and germination index, were investigated across three environments in a panel of 355 diverse accessions based on 2,261,854 high-quality single-nucleotide polymorphisms (SNPs). A total of 26 significant SNPs were detected and divided into six quantitative trait locus regions, including 121 predicted candidate genes. By combining gene expression, gene annotation, and haplotype analysis, two novel candidate genes (Ghir_A09G002730 and Ghir_D03G009280) within qGR-A09-1 and qGI/GP/GR-D03-3 were associated with vigor-related traits, and Ghir_A09G002730 was found to be involved in artificial selection during cotton breeding by population genetic analysis. Thus, understanding the genetic mechanisms underlying seed vigor-related traits in cotton could help increase the efficiency of direct seeding by molecular marker-assisted selection breeding.

Abstract 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 differentially 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.

Abstract Background The node of the first fruiting branch (NFFB) is an important precocious trait in cotton. Many studies have been conducted on the localization of quantitative trait loci (QTLs) and genes related to fiber quality and yield, but there has been little attention to traits related to early maturity, especially the NFFB, in cotton. Results To identify the QTL associated with the NFFB in cotton, a BC4F2 population comprising 278 individual plants was constructed. The parents and two DNA bulks for high and low NFFB were whole genome sequenced, and 243.8 Gb of clean nucleotide data were generated. A total of 449,302 polymorphic SNPs and 135,353 Indels between two bulks were identified for QTL-seq. Seventeen QTLs were detected and localized on 11 chromosomes in the cotton genome, among which two QTLs (qNFFB-Dt2–1 and qNFFB-Dt3–3) were located in hotspots. Two candidate genes (GhAPL and GhHDA5) related to the NFFB were identified using quantitative real-time PCR (qRT-PCR) and virus-induced gene silencing (VIGS) experiments in this study. Both genes exhibited higher expression levels in the early-maturing cotton material RIL182 during flower bud differentiation, and the silencing of GhAPL and GhHDA5 delayed the flowering time and increased the NFFB compared to those of VA plants in cotton. Conclusions Our study preliminarily found that GhAPL and GhHDA5 are related to the early maturity in cotton. The findings provide a basis for the further functional verification of candidate genes related to the NFFB and contribute to the study of early maturity in cotton.

Calcium-Dependent Protein Kinases (CDPKs) involved in regulating downstream components of calcium signaling pathways play a role in tolerance to abiotic stresses and seed development in plants. However, functions of only a few cotton CDPKs have been clarified at present. In this study, 80 conserved CDPKs in Gossypium hirsutum L. were identified and characterized, which was divided into four subgroups. Among them, the transcript level of GhCDPK60 was significantly upregulated under drought and several hormone treatments. And we found that the expression levels of several stress-inducible genes down-regulated in GhCDPK60-silence cotton and up-regulated in GhCDPK60-overexpressing Arabidopsis. In addition, physiological analyses demonstrated that GhCDPK60 improved drought stress tolerance by improving the osmotic adjustment ability and reducing the accumulation of reactive oxygen species (ROS) in plants. These findings broaden our understanding of the biological roles of GhCDPK60 and mechanisms underlying drought stress tolerance in cotton.

Abstract 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 nodule-inception-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.

Abstract 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.

Boll weight (BW) is a key determinant of yield component traits in cotton, and understanding the genetic mechanism of BW could contribute to the progress of cotton fiber yield. Although many yield-related quantitative trait loci (QTLs) responsible for BW have been determined, knowledge of the genes controlling cotton yield remains limited. Here, association mapping based on 25,169 single-nucleotide polymorphisms (SNPs) and 2,315 insertions/deletions (InDels) was conducted to identify high-quality QTLs responsible for BW in a global collection of 290 diverse accessions, and BW was measured in nine different environments. A total of 19 significant markers were detected, and 225 candidate genes within a 400 kb region (± 200 kb surrounding each locus) were predicted. Of them, two major QTLs with highly phenotypic variation explanation on chromosomes A08 and D13 were identified among multiple environments. Furthermore, we found that two novel candidate genes (Ghir_A08G009110 and Ghir_D13G023010) were associated with BW and that Ghir_D13G023010 was involved in artificial selection during cotton breeding by population genetic analysis. The transcription level analyses showed that these two genes were significantly differentially expressed between high-BW accession and low-BW accession during the ovule development stage. Thus, these results reveal valuable information for clarifying the genetic basics of the control of BW, which are useful for increasing yield by molecular marker-assisted selection (MAS) breeding in cotton.

Abstract 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 Background Histone deacetylases (HDACs) catalyze histone deacetylation and suppress gene transcription during various cellular processes. Within the superfamily of HDACs, RPD3/HDA1-type HDACs are the most studied, and it is reported that RPD3 genes play crucial roles in plant growth and physiological processes. However, there is a lack of systematic research on the RPD3/HDA1 gene family in cotton. Results In this study, genome-wide analysis identified 9, 9, 18, and 18 RPD3 genes in Gossypium raimondii, G. arboreum, G. hirsutum, and G. barbadense, respectively. This gene family was divided into 4 subfamilies through phylogenetic analysis. The exon-intron structure and conserved motif analysis revealed high conservation in each branch of the cotton RPD3 genes. Collinearity analysis indicated that segmental duplication was the primary driving force during the expansion of the RPD3 gene family in cotton. There was at least one presumed cis-element related to plant hormones in the promoter regions of all GhRPD3 genes, especially MeJA- and ABA-responsive elements, which have more members than other hormone-relevant elements. The expression patterns showed that most GhRPD3 genes had relatively high expression levels in floral organs and performed higher expression in early-maturity cotton compared with late-maturity cotton during flower bud differentiation. In addition, the expression of GhRPD3 genes could be significantly induced by one or more abiotic stresses as well as exogenous application of MeJA or ABA. Conclusions Our findings reveal that GhRPD3 genes may be involved in flower bud differentiation and resistance to abiotic stresses, which provides a basis for further functional verification of GhRPD3 genes in cotton development and a foundation for breeding better early-maturity cotton cultivars in the future.

Abstract Background Multiple C2 domains and transmembrane region proteins (MCTPs) may act as transport mediators of other regulators. Although increased number of MCTPs in higher plants implies their diverse and specific functions in plant growth and development, only a few plant MCTPs have been studied and no study on the MCTPs in cotton has been reported. Results In this study, we identified 31 MCTPs in G. hirsutum, which were classified into five subfamilies according to the phylogenetic analysis. GhMCTPs from subfamily V exhibited isoelectric points (pIs) less than 7, whereas GhMCTPs from subfamily I, II, III and IV exhibited pIs more than 7.5, implying their distinct biological functions. In addition, GhMCTPs within subfamily III, IV and V exhibited more diverse physicochemical properties, domain architectures and expression patterns than GhMCTPs within subfamily I and II, suggesting that GhMCTPs within subfamily III, IV and V diverged to perform more diverse and specific functions. Analyses of conserved motifs and pIs indicated that the N-terminus was more divergent than the C-terminus and GhMCTPs’ functional divergence might be mainly contributed by the N-terminus. Furthermore, yeast two-hybrid assay indicated that the N-terminus was responsible to interact with target proteins. Phylogenetic analysis classified multiple N-terminal C2 domains into four subclades, suggesting that these C2 domains performed different molecular functions in mediating the transport of target proteins. Conclusions Our systematic characterization of MCTPs in G. hirsutum will provide helpful information to further research GhMCTPs’ molecular roles in mediating other regulators’ transport to coordinate growth and development of various cotton tissues.

Senescence in plants is a complex trait, which is controlled by both genetic and environmental factors and can affect the yield and quality of cotton. However, the genetic basis of cotton senescence remains relatively unknown. In this study, we reported genome-wide association studies (GWAS) based on 185 accessions of upland cotton and 26,999 high-quality single-nucleotide polymorphisms (SNPs) to reveal the genetic basis of cotton senescence. To determine cotton senescence, we evaluated eight traits/indices. Our results revealed a high positive correlation (r>0.5) among SPAD value 20 days after topping (SPAD20d), relative difference of SPAD (RSPAD), nodes above white flower on topping day (NAWF0d), nodes above white flower 7 days after topping (NAWF7d), and number of open bolls on the upper four branches (NB), and genetic analysis revealed that all traits had medium or high heritability ranging from 0.53 to 0.86. Based on a multi-locus method (FASTmrMLM), a total of 63 stable and significant quantitative trait nucleotides (QTNs) were detected, which represented 50 genomic regions (GWAS risk loci) associated with cotton senescence. We observed three reliable loci located on chromosomes A02 (A02_105891088_107196428), D03 (D03_37952328_38393621) and D13 (D13_59408561_60730103) because of their high repeatability. One candidate gene (Ghir_D03G011060) was found in the locus D03_37952328_38393621, and its Arabidopsis thaliana homologous gene (AT5G23040) encodes a cell growth defect factor-like protein (CDF1), which might be involved in chlorophyll synthesis and cell death. Moreover, qRT-PCR showed that the transcript level of Ghir_D03G011060 was down-regulated in old cotton leaves, and virus-induced gene silencing (VIGS) indicated that silencing of Ghir_D03G011060 resulted in leaf chlorosis and promoted leaf senescence. In addition, two candidate genes (Ghir_A02G017660 and Ghir_D13G021720) were identified in loci A02_105891088_107196428 and D13_59408561_60730103, respectively. These results provide new insights into the genetic basis of cotton senescence and will serve as an important reference for the development and implementation of strategies to prevent premature senescence in cotton breeding programs.

The caleosin (CLO) protein family displays calcium-binding properties and plays an important role in the abiotic stress response. Here, a total of 107 CLO genes were identified in 15 plant species, while no CLO genes were detected in two green algal species. Evolutionary analysis revealed that the CLO gene family may have evolved mainly in terrestrial plants and that biological functional differentiation between species and functional expansion within species have occurred. Of these, 56 CLO genes were identified in four cotton species. Collinearity analysis showed that CLO gene family expansion mainly occurred through segmental duplication and whole-genome duplication in cotton. Sequence alignment and phylogenetic analysis showed that the CLO proteins of the four cotton species were mainly divided into two types: H-caleosins (class I) and L-caleosins (class II). Cis-acting element analysis and quantitative RT–PCR (qRT–PCR) suggested that GhCLOs might be regulated by abscisic acid (ABA) and methyl jasmonate (MeJA). Moreover, transcriptome data and qRT–PCR results revealed that GhCLO genes responded to salt and drought stresses. Under salt stress, gene-silenced plants (TRV: GhCLO06) showed obvious yellowing and wilting, higher malondialdehyde (MDA) content accumulation, and significantly lower activities of superoxide dismutase (SOD) and peroxidase (POD), indicating that GhCLO06 plays a positive regulatory role in cotton salt tolerance. In gene-silenced plants (TRV: GhCLO06), ABA-related genes (GhABF2, GhABI5, and GhNAC4) were significantly upregulated after salt stress, suggesting that the regulation of salt tolerance may be related to the ABA signaling pathway. This research provides an important reference for further understanding and analyzing the molecular regulatory mechanism of CLOs for salt tolerance.

The K+ efflux antiporter (KEA) mediates intracellular K+ and H+ homeostasis to improve salt tolerance in plants. However, the knowledge of KEA gene family in cotton is largely absent. In the present study, 8, 8, 15, and 16 putative KEA genes were identified in Gossypium arboreum, G. raimondii, G. hirsutum, and G. barbadense, respectively. These KEA genes were classified into three subfamilies, and members from the same subfamilies showed similar motif compositions and gene structure characteristics. Some hormone response elements and stress response elements were identified in the upstream 2000 bp sequence of GhKEAs. Transcriptome data showed that most of the GhKEAs were highly expressed in roots and stems. The quantificational real-time polymerase chain reaction (qRT-PCR) results showed that most of the GhKEAs responded to low potassium, salt and drought stresses. Virus-induced gene silencing (VIGS) experiments demonstrated that under salt stress, after silencing genes GhKEA4 and GhKEA12, the chlorophyll content, proline content, soluble sugar content, peroxidase (POD) activity and catalase (CAT) activity were significantly decreased, and the Na+/K+ ratio was extremely significantly increased in leaves, leading to greater salt sensitivity. Under high potassium stress, cotton plants silenced for the GhKEA4 could still maintain a more stable Na+ and K+ balance, and the activity of transporting potassium ions from roots into leaves was reduced silenced for GhKEA12. Under low potassium stress, silencing the GhKEA4 increased the activity of transporting potassium ions to shoots, and silencing the GhKEA12 increased the ability of absorbing potassium ions, but accumulated more Na+ in leaves. These results provided a basis for further studies on the biological roles of KEA genes in cotton development and adaptation to stress conditions.

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.

Cotton is one of the most important fiber crops globally. Despite this, various abiotic stresses, including drought, cause yield losses. We used transcriptome profiles to investigate the co-expression patterns of gene networks associated with drought stress tolerance. We identified three gene modules containing 3,567 genes highly associated with drought stress tolerance. Within these modules, we identified 13 hub genes based on intramodular significance, for further validation. The yellow module has five hub genes (Gh_A07G0563, Gh_D05G0221, Gh_A05G3716, Gh_D12G1438, and Gh_D05G0697), the brown module contains three hub genes belonging to the aldehyde dehydrogenase (ALDH) gene family (Gh_A06G1257, Gh_A06G1256, and Gh_D06G1578), and the pink module has five hub genes (Gh_A02G1616, Gh_D12G2599, Gh_D07G2232, Gh_A02G0527, and Gh_D07G0629). Based on RT-qPCR results, the Gh_A06G1257 gene has the highest expression under drought stress in different plant tissues and it might be the true candidate gene linked to drought stress tolerance in cotton. Silencing of Gh_A06G1257 in cotton leaves conferred significant sensitivity in response to drought stress treatments. Overexpression of Gh_A06G1257 in Arabidopsis also confirms its role in drought stress tolerance. L-valine, Glutaric acid, L-proline, L-Glutamic acid, and L-Tryptophan were found to be the most significant metabolites playing roles in drought stress tolerance. These findings add significantly to existing knowledge of drought stress tolerance mechanisms in cotton.

Photoperiod is an important external factor that regulates flowering time, the core mechanism of which lies in the circadian clock-controlled expression of FLOWERING LOCUS T (FT) and its upstream regulators. However, the roles of the circadian clock in regulating cotton flowering time are largely unknown. In this study, we cloned two circadian clock genes in cotton, GhLUX1 and GhELF3. The physicochemical and structural properties of their putative proteins could satisfy the prerequisites for the interaction between them, which was proved by yeast two-hybrid (Y2H) and Bimolecular Fluorescent Complimentary (BiFC) assays. Phylogenetic analysis of LUXs and ELF3s indicated that the origin of LUXs was earlier than that of ELF3s, but ELF3s were more divergent and might perform more diverse functions. GhLUX1, GhELF3, GhCOL1, and GhFT exhibited rhythmic expression and were differentially expressed in the early flowering and late-flowering cotton varieties under different photoperiod conditions. Both overexpression of GhLUX1 and overexpression of GhELF3 in Arabidopsis delayed flowering probably by changing the oscillation phases and amplitudes of the key genes in the photoperiodic flowering pathway. Both silencing of GhLUX1 and silencing of GhELF3 in cotton increased the expression of GhCOL1 and GhFT and resulted in early flowering. In summary, the circadian clock genes were involved in regulating cotton flowering time and could be the candidate targets for breeding early maturing cotton varieties.

The early flowering 4 (ELF4) family members play multiple roles in the physiological development of plants. ELF4s participated in the plant biological clock’s regulation process, photoperiod, hypocotyl elongation, and flowering time. However, the function in the ELF4s gene is barely known. In this study, 11, 12, 21, and 22 ELF4 genes were identified from the genomes of Gossypium arboreum, Gossypium raimondii, Gossypium hirsutum, and Gossypium barbadense, respectively. There ELF4s genes were classified into four subfamilies, and members from the same subfamily show relatively conservative gene structures. The results of gene chromosome location and gene duplication revealed that segmental duplication promotes gene expansion, and the Ka/Ks indicated that the ELF4 gene family has undergone purification selection during long-term evolution. Spatio-temporal expression patterns and qRT-PCR showed that GhELF4 genes were mainly related to flower, leaf, and fiber development. Cis-acting elements analysis and qRT-PCR showed that GhELF4 genes might be involved in the regulation of abscisic acid (ABA) or light pathways. Silencing of GhELF4-1 and GhEFL3-6 significantly affected the height of cotton seedlings and reduced the resistance of cotton. The identification and functional analysis of ELF4 genes in upland cotton provide more candidate genes for genetic modification.

Abstract Background Cotton fiber length and strength are both key traits of fiber quality, and fiber strength (FS) is tightly correlated with secondary cell wall (SCW) biosynthesis. The three-amino-acid-loop-extension (TALE) superclass homeoproteins are involved in regulating diverse biological processes in plants, and some TALE members has been identified to play a key role in regulating SCW formation. However, little is known about the functions of TALE members in cotton (Gossypium spp.). Results In the present study, based on gene homology, 46, 47, 88 and 94 TALE superfamily genes were identified in G. arboreum, G. raimondii, G. barbadense and G. hirsutum, respectively. Phylogenetic and evolutionary analysis showed the evolutionary conservation of two cotton TALE families (including BEL1-like and KNOX families). Gene structure analysis also indicated the conservation of GhTALE members under selection. The analysis of promoter cis-elements and expression patterns suggested potential transcriptional regulation functions in fiber SCW biosynthesis and responses to some phytohormones for GhTALE proteins. Genome-wide analysis of colocalization of TALE transcription factors with SCW-related QTLs revealed that some BEL1-like genes and KNAT7 homologs may participate in the regulation of cotton fiber strength formation. Overexpression of GhKNAT7-A03 and GhBLH6-A13 significantly inhibited the synthesis of lignocellulose in interfascicular fibers of Arabidopsis. Yeast two-hybrid (Y2H) experiments showed extensive heteromeric interactions between GhKNAT7 homologs and some GhBEL1-like proteins. Yeast one-hybrid (Y1H) experiments identified the upstream GhMYB46 binding sites in the promoter region of GhTALE members and defined the downstream genes that can be directly bound and regulated by GhTALE heterodimers. Conclusion We comprehensively identified TALE superfamily genes in cotton. Some GhTALE members are predominantly expressed during the cotton fiber SCW thicking stage, and may genetically correlated with the formation of FS. Class II KNOX member GhKNAT7 can interact with some GhBEL1-like members to form the heterodimers to regulate the downstream targets, and this regulatory relationship is partially conserved with Arabidopsis. In summary, this study provides important clues for further elucidating the functions of TALE genes in regulating cotton growth and development, especially in the fiber SCW biosynthesis network, and it also contributes genetic resources to the improvement of cotton fiber quality.

Abstract Background Improving the yield and fiber quality of upland cotton is a goal of plant breeders. However, increasing the yield and quality of cotton fibers is becoming more urgent. While the growing human population needs more cotton fiber, climate change is reducing the amount of land on which cotton can be planted, or making it difficult to ensure that water and other resources will be available in optimal quantities. The most logical means of improving yield and quality is understanding and manipulating the genes involved. Here, we used comparative transcriptomics to explore differences in gene expression between long- and short-fiber cotton lines to identify candidate genes useful for cotton improvement. Results Light and electron microscopy revealed that the initial fiber density was significantly greater in our short-fiber group (SFG) than in our long-fiber group (LFG). Compared with the SFG fibers, the LFG fibers were longer at all developmental stages. Comparison of the LFG and SFG transcriptomes revealed a total of 3538 differentially expressed genes (DEGs). Notably, at all three developmental stages examined, two expression patterns, consistently downregulated (profile 0) and consistently upregulated (profile 7), were identified, and both were significantly enriched in the SFG and LFG. Twenty-two DEGs known to be involved in fiber initiation were detected in profile 0, while 31 DEGs involved in fiber elongation were detected in profile 7. Functional annotation suggested that these DEGs, which included ERF1, TUA2, TUB1, and PER64, affect fiber elongation by participating in the ethylene response, microtubule synthesis, and/or the peroxidase (POD) catalytic pathway. qRT-PCR was used to confirm the RNA sequencing results for select genes. Conclusions A comparison of SFG and LFG transcription profiles revealed modest but important differences in gene expression between the groups. Notably, our results confirm those of previous studies suggesting that genes involved in ethylene, tubulin, and POD pathways play important roles in fiber development. The 22 consistently downregulated DEGs involved in fiber initiation and the 31 consistently upregulated genes involved in fiber elongation are seemingly good candidate genes for improving fiber initiation and elongation in cotton.

Abstract Background Many BURP domain-containing proteins, which are unique to plants, have been identified. They performed diverse functions in plant development and the stress response. To date, only a few BURP domain-containing genes have been studied, and no comprehensive analysis of the gene family in cotton has been reported. Results In this study, 18, 17 and 30 putative BURP genes were identified in G. raimondii (D5), G. arboreum (A2) and G. hirsutum (AD1), respectively. These BURP genes were phylogenetically classified into eight subfamilies, which were confirmed by analyses of gene structures, motifs and protein domains. The uneven distribution of BURPs in chromosomes and gene duplication analysis indicated that segmental duplication might be the main driving force of the GhBURP family expansion. Promoter regions of all GhBURPs contained at least one putative stress-related cis-elements. Analysis of transcriptomic data and qRT-PCR showed that GhBURPs showed different expression patterns in different organs, and all of them, especially the members of the RD22-like subfamily, could be induced by different stresses, such as abscisic acid (ABA) and salicylic acid (SA), which indicated that the GhBURPs may performed important functions in cotton’s responses to various abiotic stresses. Conclusions Our study comprehensively analyzed BURP genes in G. hirsutum, providing insight into the functions of GhBURPs in cotton development and adaptation to stresses.

Cotton fiber is the most important natural raw material for the textile industry, and fiber length (FL) is one of the most important traits in cotton. Quantitative trait locus (QTL) mapping based on high-density genetic maps is an efficient approach to identify genetic regions for FL. In our study, two backcrossed inbred lines (BILs) were chosen as parents to construct a high-density genetic map in F2 which was used to fine map FL QTL in F2:3 population. The genetic map had a total size of 3462.8 cM, containing 9182 single-nucleotide polymorphisms (SNPs) based on genotyping-by-sequencing. Two FL related stable QTL were identified on two chromosomes (qFL-A08–1 on A08 and qFL-D03–1 on D03), and qFL-A08–1 was confirmed by a meta-analysis. Utilizing previously obtained RNA-seq data for the two BILs and qRT-PCR analysis, two candidate genes annotated as cytochrome b5 (CB5, Gh_A08G1729) and microtubule end-binding 1C (EB1C, Gh_D03G0232) that may regulate FL during the fiber elongation stage were identified. In addition, nine recombination hotspots in this population were found. The results of this study will provide an important foundation for further studies on the molecular and genetic regulation of fiber elongation. Keywords: Gossypium, Single-nucleotide polymorphism, Fiber length, Quantitative trait locus

The cotton fiber is the most important raw material for the textile industry and an ideal model system for studying cell elongation. However, the genetic variation of fiber elongation in relation to miRNA is poorly understood. A high-throughput comparative RNA-seq of two lines differing in fiber length (FL) from a backcross inbred line (BIL) population of G. hirsutum × G. barbadense revealed differentially expressed (DE) miRNAs and their targets in rapidly elongating fibers. A real-time quantitative PCR analysis was further performed to validate the results. A total of 463 (including 47 DE) miRNAs were identified, and seven DE miRNAs were co-localized with seven FL quantitative trait loci (QTL) identified in the G. hirsutum × G. barbadense population. Of 82 (including 21 DE) targets identified, nine (including one DE) were also co-localized with the seven FL QTL. The relationship between the allopolyploid and its diploid ancestral species with respect to miRNAs and their targets was also characterized. These results will facilitate the understanding of the molecular genetic mechanism of fiber elongation with regards to miRNAs in cotton. Keywords: Allopolyploid Gossypium, Fiber elongation, miRNA, QTL hotspots

Abstract Background Premature senescence can reduce the yield and quality of crops. WRKY transcription factors (TFs) play important roles during leaf senescence, but little is known about their ageing mechanisms in cotton. Results In this study, a group III WRKY TF, GhWRKY27, was isolated and characterized. The expression of GhWRKY27 was induced by leaf senescence and was higher in an early-ageing cotton variety than in a non-early-ageing cotton variety. Overexpression of GhWRKY27 in Arabidopsis promoted leaf senescence, as determined by reduced chlorophyll content and elevated expression of senescence-associated genes (SAGs). Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that GhWRKY27 interacted with an MYB TF, GhTT2. Putative target genes of GhWRKY27 were identified via chromatin immunoprecipitation followed by sequencing (ChIP-seq). Yeast one-hybrid (Y1H) assay and electrophoretic mobility shift assay (EMSA) revealed that GhWRKY27 binds directly to the promoters of cytochrome P450 94C1 (GhCYP94C1) and ripening-related protein 2 (GhRipen2–2). In addition, the expression patterns of GhTT2, GhCYP94C1 and GhRipen2–2 were identified during leaf senescence. Transient dual-luciferase reporter assay indicated that GhWRKY27 could activate the expression of GhCYP94C1 and GhRipen2–2. Conclusions Our work lays the foundation for further study of the functional roles of WRKY genes during leaf senescence in cotton. In addition, our data provide new insights into the senescence-associated mechanisms of WRKY genes in cotton.

Glycerol-3-phosphate acyltransferases (GPATs), critical for multiple biological processes like male fertility, have been extensively characterized. However, their precise functions and underlying regulatory mechanism in cotton anther development are unclear. This research demonstrated the importance of GhGPAT12/25 (a paralogs pair on A12/D12 sub-chromosome of cotton) to regulate the degradation of tapetum, anther cuticle formation, and pollen exine development. GhGPAT12 and GhGPAT25 exhibited specifically detected transcripts in tapetum and pollen exine during the early anther developmental stages. GhGPAT12/25 are sn-2 glycerol-3-phosphate acyltransferases and can transfer the acyl group of palmitoyl-CoA to glycerol-3-phosphate (G3P). CRISPR/Cas9-mediated knockout identified the functional redundancy of GhGPAT12 and GhGPAT25. Knockout of both genes caused completely male sterility associated with abnormal anther cuticle, swollen tapetum, and inviable microspores with defective exine and irregular unrestricted shape. RNA-seq analysis showed that the loss of function of GhGPAT12/25 affects the processes of wax metabolic, glycerol monomer biosynthesis, and transport. Consistently, cuticular waxes were dramatically reduced in mutant anthers. Yeast one-hybrid system (Y1H), virus-induced gene silencing (VIGS), and dual-luciferase (LUC) assays illustrated that GhMYB80s are likely to directly activate the expression of GhGPAT12/25. This study provides important insights for revealing the regulatory mechanism underlying anther development in cotton.

In the process of growth and development, cotton exhibits premature senescence under various abiotic stresses, impairing yield and fiber quality. NAC (NAM, ATAF1,2, and CUC2) protein widely distributed in land plants, play the critical role in responding to abiotic stress and regulating leaf senescence. We have identified and functional analyzed a NAM domain gene GhNAC82 in upland cotton, it was located on the A11 chromosome 4,921,702 to 4,922,748 bp, only containing one exon. The spatio-temporal expression pattern analysis revealed that it was highly expressed in root, torus, ovule and fiber development stage. The results of qRT-PCR validated that GhNAC82 negatively regulated by salt stress, drought stress, H2O2 stress, IAA treatment, and ethylene treatment, positively regulated by the ABA and MeJA treatment. Moreover, heterologous overexpression of GhNAC82 results in leaf premature senescence and delays root system development in Arabidopsis thaliana. The phenotype of delayed-senescence was performed after silencing GhNAC82 by VIGS in premature cotton. Taken together, GhNAC82 was involved in different abiotic stress pathways and play important roles in negatively regulating leaf premature senescence.

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.

The sodium/hydrogen antiporter (NHX) gene family with the Na+/H+ exchange protein domain is a transporter of sodium and hydrogen ions and plays an important role in the response of plants to salt stress. Studying the response of cotton to salt stress through comprehensive identification and analysis of NHX genes in several species and their roles in salt tolerance mechanisms is of great significance. In this study, 23, 24, 12, and 12 NHX genes were identified from Gossypium hirsutum (Gh), G. barbadense, G. arboreum and G. raimondii, respectively. Phylogenetic analysis showed that these genes were mainly divided into three clades with significant subcellular localization, namely, endosome (Endo-class), plasma membrane (PM-class) and vacuole (Vac-class). By analyzing the structure of NHX genes and proteins, each branch of the NHX gene family was found to be structurally conserved, and collinearity analysis showed that NHX genes were mainly expressed through whole genome and segmental duplication. The non-synonymous (Ka)/synonymous (Ks) values showed that the NHX gene family experienced strong purifying selection during long-term evolution. Cis-acting element analysis showed that the NHX gene family may be related to the regulation of abscisic acid (ABA) and methyl jasmonate (MeJA) hormones. Additionally, transcriptomic data analysis and qRT-PCR showed that GhNHXs exhibited different expression patterns in each tissue and under different salinities. These results provide an important reference for us to further understand and analyze the molecular regulation mechanism of cotton NHX genes.

Abstract Background In upland cotton (Gossypium hirsutum L.), genotypes with the same mature fiber length (FL) might possess different genes and exhibit differential expression of genes related to fiber elongation at different fiber developmental stages. However, there is a lack of information on the genetic variation influencing fiber length and its quantitative trait loci (QTLs) during the fiber elongation stage. In this study, a subset of upland cotton accessions was selected based on a previous GWAS conducted in China and grown in multiple environments to determine the dynamic fiber length at 10, 15, 20, and 25 days post-anthesis (DPA) and maturity. The germplasm lines were genotyped with the Cotton 63 K Illumina single-nucleotide polymorphism (SNP) array for GWAS. Results A total of 25, 38, 57, 89 and 88 SNPs showed significant correlations with fiber length at 10, 15, 20 and 25 DPA and maturity, respectively. In addition, 60 more promising SNPs were detected in at least two tests and two FL developmental time points, and 20 SNPs were located within the confidence intervals of QTLs identified in previous studies. The fastest fiber-length growth rates were obtained at 10 to 15 DPA in 69 upland cotton lines and at 15 to 20 DPA in 14 upland cotton accessions, and 10 SNPs showed significant correlations with the fiber-length growth rate. A combined transcriptome and qRT-PCR analysis revealed that two genes (D10G1008 and D13G2037) showed differential expression between two long-fiber genotypes and two short-fiber genotypes. Conclusions This study provides important new insights into the genetic basis of the time-dependent fiber-length trait and reveals candidate SNPs and genes for improving fiber length in upland cotton.

Abstract Background Pectin is a major component and structural polysaccharide of the primary cell walls and middle lamella of higher plants. Pectate lyase (PEL, EC 4.2.2.2), a cell wall modification enzyme, degrades de-esterified pectin for cell wall loosening, remodeling and rearrangement. Nevertheless, there have been few studies on PEL genes and no comprehensive analysis of the PEL gene family in cotton. Results We identified 53, 42 and 83 putative PEL genes in Gossypium raimondii (D5), Gossypium arboreum (A2), and Gossypium hirsutum (AD1), respectively. These PEL genes were classified into five subfamilies (I-V). Members from the same subfamilies showed relatively conserved gene structures, motifs and protein domains. An analysis of gene chromosomal locations and gene duplication revealed that segmental duplication likely contributed to the expansion of the GhPELs. The 2000 bp upstream sequences of all the GhPELs contained auxin response elements. A transcriptomic data analysis showed that 62 GhPELs were expressed in various tissues. Notably, most (29/32) GhPELs of subfamily IV were preferentially expressed in the stamen, and five GhPELs of subfamily V were prominently expressed at the fiber elongation stage. In addition, qRT-PCR analysis revealed the expression characteristics of 24 GhPELs in four pollen developmental stages and significantly different expression of some GhPELs between long- and short-fiber cultivars. Moreover, some members were responsive to IAA treatment. The results indicate that GhPELs play significant and functionally diverse roles in the development of different tissues. Conclusions In this study, we comprehensively analyzed PELs in G. hirsutum, providing a foundation to better understand the functions of GhPELs in different tissues and pathways, especially in pollen, fiber and the auxin signaling pathway.

Abstract Background WRKY transcription factors (TFs) participate in various physiological processes of plants. Although WRKY genes have been well studied in model plants, knowledge of the functional roles of these genes is still extremely limited in cotton. Results In this study, a group IId WRKY gene from cotton, GhWRKY42, was isolated and characterized. Our data showed that GhWRKY42 localized to the nucleus. A transactivation assay in yeast demonstrated that GhWRKY42 was not a transcriptional activator. A β-glucuronidase (GUS) activity assay revealed that the promoter of GhWRKY42 showed fragment deletion activity in Nicotiana tabacum and was mainly expressed in the roots, stems and leaves of ProGhWRKY42::GUS transgenic Arabidopsis plants. Quantitative real-time PCR (qRT-PCR) analysis indicated that GhWRKY42 was up-regulated during leaf senescence and was induced after exposure to abiotic stresses. Constitutive expression of GhWRKY42 in Arabidopsis led to a premature aging phenotype, which was correlated with an increased number of senescent leaves, reduced chlorophyll content and elevated expression of senescence-associated genes (SAGs). In addition, virus-induced gene silencing (VIGS) was used to silence the endogenous GhWRKY42 gene in cotton, and this silencing reduced plant height. Conclusions Our findings indicate that GhWRKY42 is involved in abiotic stress responses, premature leaf senescence and stem development. This work establishes a solid foundation for further functional analysis of the GhWRKY42 gene in cotton.

Abstract Background Small auxin-up RNA (SAUR) gene family is the largest family of early auxin response genes in higher plants, which have been implicated in the regulation of multiple biological processes. However, no comprehensive analysis of SAUR genes has been reported in cotton (Gossypium spp.). Results In the study, we identified 145, 97, 214, and 176 SAUR homologous genes in the sequenced genomes of G. raimondii, G. arboreum, G. hirsutum, and G. barbadense, respectively. A phylogenetic analysis revealed that the SAUR genes can be classified into 10 groups. A further analysis of chromosomal locations and gene duplications showed that tandem duplication and segmental duplication events contributed to the expansion of the SAUR gene family in cotton. An exon-intron organization and motif analysis revealed the conservation of SAUR-specific domains, and the auxin responsive elements existed in most of the upstream sequences. The expression levels of 16 GhSAUR genes in response to an exogenous application of IAA were determined by a quantitative RT-PCR analysis. The genome-wide RNA-seq data and qRT-PCR analysis of selected SAUR genes in developing fibers revealed their differential expressions. The physical mapping showed that 20 SAUR genes were co-localized with fiber length quantitative trait locus (QTL) hotspots. Single nucleotide polymorphisms (SNPs) were detected for 12 of these 20 genes between G. hirsutum and G. barbadense, but no SNPs were identified between two backcross inbred lines with differing fiber lengths derived from a cross between the two cultivated tetraploids. Conclusions This study provides an important piece of genomic information for the SAUR genes in cotton and lays a solid foundation for elucidating the functions of SAUR genes in auxin signaling pathways to regulate cotton growth.

Abstract Background Cotton (Gossypium spp.) fibers are single-celled elongated trichomes, the molecular aspects of genetic variation in fiber length (FL) among genotypes are currently unknown. In this study, two backcross inbred lines (BILs), i.e., NMGA-062 (“Long”) and NMGA-105 (“Short”) with 32.1 vs. 27.2 mm in FL, respectively, were chosen to perform RNA-Seq on developing fibers at 10 days post anthesis (DPA). The two BILs differed in 4 quantitative trait loci (QTL) for FL and were developed from backcrosses between G. hirsutum as the recurrent parent and G. barbadense. Results In total, 51.7 and 54.3 million reads were obtained and assembled to 49,508 and 49,448 transcripts in the two genotypes, respectively. Of 1 551 differentially expressed genes (DEGs) between the two BILs, 678 were up-regulated and 873 down-regulated in “Long”; and 703 SNPs were identified in 339 DEGs. Further physical mapping showed that 8 DEGs were co-localized with the 4 FL QTL identified in the BIL population containing the two BILs. Four SNP markers in 3 DEGs that showed significant correlations with FL were developed. Among the three candidate genes encoding for proline-rich protein, D-cysteine desulfhydrase, and thaumatin-like protein, a SNP of thaumatin-like protein gene showed consistent correlations with FL across all testing environments. Conclusions This study represents one of the first investigations of positional candidate gene approach of QTL in cotton in integrating transcriptome and SNP identification based on RNA-Seq with linkage and physical mapping of QTL and genes, which will facilitate eventual cloning and identification of genes responsible for FL QTL. The candidate genes may serve as the foundation for further in-depth studies of the molecular mechanism of natural variation in fiber elongation.

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.

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.

Cotton (Gossypium spp.) is the most important natural fiber crop in the world. The R2R3-MYB gene family is a large gene family involved in many plant functions including cotton fiber development. Although previous studies have reported its phylogenetic relationships, gene structures, and expression patterns in tetraploid G. hirsutum and diploid G. raimondii, little is known about the sequence variation of the members between G. hirsutum and G. barbadense and their involvement in the natural quantitative variation in fiber quality and yield. In this study, a comprehensive genome-wide comparative analysis was performed among the four Gossypium species using whole genome sequences, i.e., tetraploid G. hirsutum (AD1) and G. barbadense (AD2) as well as their likely ancestral diploid extants G. raimondii (D5) and G. arboreum (A2), leading to the identification of 406, 393, 216, and 213 R2R3-MYB genes, respectively. To elucidate whether the R2R3-MYB genes are genetically associated with fiber quality traits, 86 R2R3-MYB genes were co-localized with quantitative trait loci (QTL) hotspots for fiber quality and yield, including 42 genes localized within the fiber length QTL hotspots, in interspecific G. hirsutum × G. barbadense populations. There were 20 interspecific nonsynonymous single-nucleotide polymorphism (SNP) sites between the two tetraploid cultivated species, of which 16 developed from 11 R2R3-MYB genes were significantly correlated with fiber quality and yield in a backcross inbred population (BIL) of G. hirsutum × G. barbadense in at least one of the four field tests. Taken together, these results indicate that the sequence variation in these 11 R2R3-MYB genes is associated with the natural variation (i.e., QTL) in fiber quality and yield. Moreover, the functional SNPs of five R2R3-MYB allele pairs from the AD1 and AD2 genomes were significantly correlated with the gene expression related to fiber quality in fiber development. The results will be useful in further elucidating the role of the R2R3-MYB genes during fiber development.

Fiber length (FL) is an important fiber quality trait in cotton. Although many fiber quality quantitative trait loci (QTL) responsible for FL have been identified, most cannot be applied to breeding programs, mainly due to unstable environments or large confidence intervals. In this study, we combined a genome-wide association study (GWAS) and linkage mapping to identify and validate high-quality QTLs responsible for FL. For the GWAS, we developed 93,250 high-quality single-nucleotide polymorphism (SNP) markers based on 355 accessions, and the FL was measured in eight different environments. For the linkage mapping, we constructed an F2 population from two extreme accessions. The high-density linkage maps spanned 3,848.29 cM, with an average marker interval of 1.41 cM. In total, 14 and 13 QTLs were identified in the association and linkage mapping analyses, respectively. Most importantly, a major QTL on chromosome D03 identified in both populations explained more than 10% of the phenotypic variation (PV). Furthermore, we found that a sucrose synthesis-related gene (Gh_D03G1338) was associated with FL in this QTL region. The RNA-seq data showed that Gh_D03G1338 was highly expressed during the fiber development stage, and the qRT-PCR analysis showed significant expression differences between the long fiber and short fiber varieties. These results suggest that Gh_D03G1338 may determine cotton fiber elongation by regulating the synthesis of sucrose. Favorable QTLs and candidate genes should be useful for increasing fiber quality in cotton breeding.

Cotton production is hampered by a variety of abiotic stresses that wreak havoc on the growth and development of plants, resulting in significant financial losses. According to reports, cotton production areas have declined around the world as a result of the ongoing stress. Therefore, plant breeding programs are concentrating on abiotic stress-tolerant cotton varieties. Mitogen-activated protein kinase (MAPK) cascades are involved in plant growth, stress responses, and the hormonal signaling pathway. In this research, three abiotic stresses (cold, drought, and salt) were analyzed on GhMPK3 transformed Arabidopsis plants. The transgenic plant’s gene expression and morphologic analysis were studied under cold, drought, and salt stress. Physiological parameters such as relative leaf water content, excised leaf water loss, chlorophyll content, and ion leakage showed that overexpressed plants possess more stable content under stress conditions compared with the WT plants. Furthermore, GhMPK3 overexpressed plants had greater antioxidant activities and weaker oxidant activities. Silencing GhMPK3 in cotton inhibited its tolerance to drought stress. Our research findings strongly suggest that GhMPK3 can be regarded as an essential gene for abiotic stress tolerance in cotton plants.

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.

WRKY transcription factors have diverse functions in regulating stress response, leaf senescence, and plant growth and development. However, knowledge of the group IId WRKY subfamily in cotton is largely absent. This study identified 34 group IId WRKY genes in the Gossypium hirsutum genome, and their genomic loci were investigated. Members clustered together in the phylogenetic tree had similar motif compositions and gene structural features, revealing similarity and conservation within group IId WRKY genes. During the evolutionary process, 14 duplicated genes appeared to undergo purification selection. Public RNA-seq data were used to examine the expression patterns of group IId WRKY genes in various tissues and under drought and salt stress conditions. Ten highly expressed genes were identified, and the ten candidate genes revealed distinct expression patterns under drought and salt treatments by qRT-PCR analysis. Among them, Gh_A11G1801 was used for functional characterization. GUS activity was differentially induced by various stresses in Gh_A11G1801p::GUS transgenic Arabidopsis plants. The virus-induced gene silencing (VIGS) of Gh_A11G1801 resulted in drought sensitivity in cotton plants, which was accompanied by elevated malondialdehyde (MDA) content and reduced catalase (CAT) content. Taken together, these findings obtained in this study provide valuable resources for further studying group IId WRKY genes in cotton. Our results also enrich the gene resources for the genetic improvements of cotton varieties that are suitable for growth in stressful conditions.

Nitric oxide (NO) is an important signaling molecule with diverse physiological functions in plants. It is therefore important to characterize the downstream genes and signal transduction networks modulated by NO. Here, we identified 1,932 differentially expressed genes (DEGs) responding to NO in upland cotton using high throughput tag sequencing. The results of quantitative real-time polymerase chain reaction (qRT-PCR) analysis of 25 DEGs showed good consistency. Gene Ontology (GO) and KEGG pathway were analyzed to gain a better understanding of these DEGs. We identified 157 DEGs belonging to 36 transcription factor (TF) families and 72 DEGs related to eight plant hormones, among which several TF families and hormones were involved in stress responses. Hydrogen peroxide and malondialdehyde (MDA) contents were increased, as well related genes after treatment with sodium nitroprusside (SNP) (an NO donor), suggesting a role for NO in the plant stress response. Finally, we compared of the current and previous data indicating a massive number of NO-responsive genes at the large-scale transcriptome level. This study evaluated the landscape of NO-responsive genes in cotton and identified the involvement of NO in the stress response. Some of the identified DEGs represent good candidates for further functional analysis in cotton.

WRKY transcription factors play important roles in plant defense, stress response, leaf senescence, and plant growth and development. Previous studies have revealed the important roles of the group IIa GhWRKY genes in cotton. To comprehensively analyze the group IIa GhWRKY genes in upland cotton, we identified 15 candidate group IIa GhWRKY genes in the Gossypium hirsutum genome. The phylogenetic tree, intron-exon structure, motif prediction and Ka/Ks analyses indicated that most group IIa GhWRKY genes shared high similarity and conservation and underwent purifying selection during evolution. In addition, we detected the expression patterns of several group IIa GhWRKY genes in individual tissues as well as during leaf senescence using public RNA sequencing data and real-time quantitative PCR. To better understand the functions of group IIa GhWRKYs in cotton, GhWRKY17 (KF669857) was isolated from upland cotton, and its sequence alignment, promoter cis-acting elements and subcellular localization were characterized. Moreover, the over-expression of GhWRKY17 in Arabidopsis up-regulated the senescence-associated genes AtWRKY53, AtSAG12 and AtSAG13, enhancing the plant's susceptibility to leaf senescence. These findings lay the foundation for further analysis and study of the functions of WRKY genes in cotton.