Cotton Fiber Development Research Articles

Genome-Wide Identification of R2R3-MYB Transcription Factors Regulating Secondary Cell Wall Thickening in Cotton Fiber Development.

MYB proteins represent one of the largest transcription factor (TF) families in plants, some of which act as key transcriptional regulators of secondary cell wall (SCW) biosynthesis. Cotton (Gossypium hirsutum) fiber is thought to be an ideal single-cell model to study cell elongation and SCW biosynthesis. However, little knowledge regarding the TFs controlling fiber SCW biosynthesis, particularly for R2R3-MYBs is known. By far, no comprehensive genome-wide analysis of the secondary wall-associated R2R3-MYBs has been reported in cultivated tetraploid upland cotton. In this study, we identified 419 R2R3-MYB genes by systematically examining the cotton genome. A combination of phylogenetic, RNA-seq and co-expression analyses indicated that 36 R2R3-MYBs were either preferentially or highly expressed in 20 day post anthesis (dpa) fibers and are putative SCW regulators. Among these MYB genes, 22 MYBs are homologs of known SCW MYB proteins and the other 14 MYBs are novel proteins without prior reported SCW biosynthesis-related functions. Finally, we highlighted on the roles of two MYBs named GhMYB46_D13 and GhMYB46_D9, both of which displayed the highest expression in 20 dpa fibers. Expression of GhMYB46_D13 or GhMYB46_D9 individually in Arabidopsis resulted in ectopic SCW deposition in transgenic plants. Furthermore, both GhMYB46_D13 and GhMYB46_D9 were able to activate the cotton fiber SCW cellulose synthase gene promoters. Thus, we have identified 36 R2R3-MYBs as potential SCW regulators in cotton fibers that represent strong candidates for further functional studies during fiber development and SCW thickening.

Genome-wide analysis of cotton GH3 subfamily II reveals functional divergence in fiber development, hormone response and plant architecture

BackgroundAuxin-induced genes regulate many aspects of plant growth and development. The Gretchen Hagen 3 (GH3) gene family, one of three major early auxin-responsive families, is ubiquitous in the plant kingdom and its members function as regulators in modulating hormonal homeostasis, and stress adaptations. Specific Auxin-amido synthetase activity of GH3 subfamily II genes is reported to reversibly inactivate or fully degrade excess auxin through the formation of amino acid conjugates. Despite these crucial roles, to date, genome-wide analysis of the GH3 gene family has not been reported in cotton.ResultsWe identified a total of 10 GH3 subfamily II genes in G. arboreum, 10 in G. raimondii, and 20 in G. hirsutum, respectively. Bioinformatic analysis showed that cotton GH3 genes are conserved with the established GH3s in plants. Expression pattern analysis based on RNA-seq data and qRT-PCR revealed that 20 GhGH3 genes were differentially expressed in a temporally and spatially specific manner, indicating their diverse functions in growth and development. We further summarized the organization of promoter regulatory elements and monitored their responsiveness to treatment with IAA (indole-3-acetic acid), SA (salicylic acid), GA (gibberellic acid) and BL (brassinolide) by qRT-PCR in roots and stems. These hormones seemed to regulate the expression of GH3 genes in both a positive and a negative manner while certain members likely have higher sensitivity to all four hormones. Further, we tested the expression of GhGH3 genes in the BR-deficient mutant pag1 and the corresponding wild-type (WT) of CCRI24. The altered expression reflected the true responsiveness to BL and further suggested possible reasons, at least in part, responsible for the dramatic dwarf and shriveled phenotypes of pag1.ConclusionWe comprehensively identified GH3 subfamily II genes in cotton. GhGH3s are differentially expressed in various tissues/organs/stages. Their response to IAA, SA, BL and GA and altered expression in pag1 suggest that some GhGH3 genes might be simultaneously involved in multiple hormone signaling pathways. Taken together, our results suggest that members of the GhGH3 gene family could be possible candidate genes for mechanistic study and applications in cotton fiber development in addition to the reconstruction of plant architecture.

Genome-Wide Analysis of LRR-RLK Gene Family in Four Gossypium Species and Expression Analysis during Cotton Development and Stress Responses.

Leucine-rich repeat receptor-like kinases (LRR-RLKs) have been reported to play important roles in plant growth, development, and stress responses. However, no comprehensive analysis of this family has been performed in cotton (Gossypium spp.), which is an important economic crop that suffers various stresses in growth and development. Here we conducted a comprehensive analysis of LRR-RLK family in four Gossypium species (Gossypium arboreum, Gossypium barbadense, Gossypium hirsutum, and Gossypium raimondii). A total of 1641 LRR-RLK genes were identified in the four Gossypium species involved in our study. The maximum-likelihood phylogenetic tree revealed that all the LRR-RLK genes were divided into 21 subgroups. Exon-intron organization structure of LRR-RLK genes kept relatively conserved within subfamilies and between Arabidopsis and Gossypium genomes. Notably, subfamilies XI and XII were found dramatically expanded in Gossypium species. Tandem duplication acted as an important mechanism in expansion of the Gossypium LRR-RLK gene family. Functional analysis suggested that Gossypium LRR-RLK genes were enriched for plant hormone signaling and plant-pathogen interaction pathways. Promoter analysis revealed that Gossypium LRR-RLK genes were extensively regulated by transcription factors (TFs), phytohormonal, and various environmental stimuli. Expression profiling showed that Gossypium LRR-RLK genes were widely involved in stress defense and diverse developmental processes including cotton fiber development and provides insight into potential functional divergence within and among subfamilies. Our study provided valuable information for further functional study of Gossypium LRR-RLK genes.

Characterization of bHLH/HLH genes that are involved in brassinosteroid (BR) signaling in fiber development of cotton (Gossypium hirsutum)

BackgroundBasic helix-loop-helix/helix-loop-helix (bHLH/HLH) transcription factors play important roles in plant development. Many reports have suggested that bHLH/HLH proteins participate in brassinosteroid (BR) hormone signaling pathways to promote cell elongation. Cotton fibers are single-cells and derived from seed surface. To explore the roles of bHLH/HLH proteins in cotton fiber development progress by modulating BR signaling pathway, we performed a systematic analysis of the bHLH/HLH gene family in upland cotton (Gossypium hirsutum) genome.ResultsIn this study, we identified 437 bHLH/HLH genes in upland cotton (G. hirsutum) genome. Phylogenetic analysis revealed that GhbHLH/HLH proteins were split into twenty six clades in the tree. These GhbHLH/HLH genes are distributed unevenly in different chromosomes of cotton genome. Segmental duplication is the predominant gene duplication event and the major contributor for amplification of GhbHLH/HLH gene family. The GhbHLH/HLHs within the same group have conserved exon/intron pattern and their encoding proteins show conserved motif composition. Based on transcriptome data, we identified 77 GhbHLH/HLH candidates that are expressed at relatively high levels in cotton fibers. As adding exogenous BR (brassinolide, BL) or brassinazole (Brz, a BR biosynthesis inhibitor), expressions of these GhbHLH/HLH genes were up-regulated or down-regulated in cotton fibers. Furthermore, overexpression of GhbHLH282 (one of the BR-response genes) in Arabidopsis not only promoted the plant growth, but also changed plant response to BR signaling.ConclusionCollectively, these data suggested that these GhbHLH/HLH genes may participate in BR signaling transduction during cotton fiber development. Thus, our results may provide a valuable reference data as the basis for further studying the roles of these bHLH/HLH genes in cotton fiber development.

Genome-wide analysis of BES1 genes in Gossypium revealed their evolutionary conserved roles in brassinosteroid signaling.

Brassinosteroids (BRs), which are essential phytohormones for plant growth and development, are important for cotton fiber development. Additionally, BES1 transcription factors are critical for BR signal transduction. However, cotton BES1 family genes have not been comprehensively characterized. In this study, we identified 11 BES1 genes in G. arboreum, 11 in G. raimondii, 16 in G. barbadense, and 22 in G. hirsutum. The BES1 sequences were significantly conserved in the Arabidopsis thaliana, rice, and upland cotton genomes. A total of 94 BES1 genes from 10 different plant species were divided into three clades according to the neighbor-joining and minimum-evolution methods. Moreover, the exon/intron patterns and motif distributions were highly conserved among the A. thaliana and cotton BES1 genes. The collinearity among the orthologs from the At and Dt subgenomes was estimated. Segmental duplications in the At and Dt subgenomes were primarily responsible for the expansion of the cotton BES1 gene family. Of the GhBES1 genes, GhBES1.4_At/Dt exhibited BL-induced expression and was predominantly expressed in fibers. Furthermore, Col-0/mGhBES1.4_At plants produced curled leaves with long and bent petioles. These transgenic plants also exhibited decreased hypocotyl sensitivity to brassinazole and constitutive BR induced/repressed gene expression patterns. The constitutive BR responses of the plants overexpressing mGhBES1.4_At were similar to those of the bes1-D mutant.

Zinc Finger-Homeodomain Transcriptional Factors (ZHDs) in Upland Cotton (Gossypium hirsutum): Genome-Wide Identification and Expression Analysis in Fiber Development.

Zinc finger-homeodomain (ZHD) genes encode a family of plant-specific transcription factors that not only participate in the regulation of plant growth and development but also play an important role in the response to abiotic stress. The ZHD gene family has been studied in several model plants, including Solanum lycopersicum, Zea mays, Oryza sativa, and Arabidopsis thaliana. However, a comprehensive study of the genes of the ZHD family and their roles in fiber development and pigmentation in upland cotton has not been completed. To address this gap, we selected a brown fiber cultivar for our study; brown color in cotton is one of the most desired colors in the textile industry. The natural colored fibers require less processing and little dying, thereby eliminating dye costs and chemical residues. Using bioinformatics approaches, we identified 37 GhZHD genes from Gossypium hirsutum and then divided these genes into seven groups based on their phylogeny. The GhZHD genes were mostly conserved in each subfamily with minor variations in motif distribution and gene structure. These genes were largely distributed on 19 of the 26 upland cotton chromosomes. Among the Gossypium genomes, the paralogs and orthologs of the GhZHD genes were identified and further characterized. Furthermore, among the paralogs, we observed that the ZHD family duplications in Gossypium genomes (G. hirsutum, G. arboreum, and G. raimondii) were probably derived from segmental duplication or genome-wide duplication (GWD) events. Through a combination of qRT-PCR and proanthocyanidins (PA) accumulation analyses in brown cotton fibers, we concluded that the candidate genes involved in early fiber development and fiber pigment synthesis include the following: GhZHD29, GhZHD35, GhZHD30, GhZHD31, GhZHD11, GhZHD27, GhZHD18, GhZHD15, GhZHD16, GhZHD22, GhZHD6, GhZHD33, GhZHD13, GhZHD5, and GhZHD23. This study delivers insights into the evolution of the GhZHD genes in brown cotton, serves as a valuable resource for further studies, and identifies the conditions necessary for improving the quality of brown cotton fiber.

Highly Efficient Targeted Gene Editing in Upland Cotton Using the CRISPR/Cas9 System.

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) gene editing system has been shown to be able to induce highly efficient mutagenesis in the targeted DNA of many plants, including cotton, and has become an important tool for investigation of gene function and crop improvement. Here, we developed a simple and easy to operate CRISPR/Cas9 system and demonstrated its high editing efficiency in cotton by targeting-ALARP, a gene encoding alanine-rich protein that is preferentially expressed in cotton fibers. Based on sequence analysis of the target site in the 10 transgenic cottons containing CRISPR/Cas9, we found that the mutation frequencies of GhALARP-A and GhALARP-D target sites were 71.4–100% and 92.9–100%, respectively. The most common editing event was deletion, but deletion together with large insertion was also observed. Mosaic mutation editing events were detected in most transgenic plants. No off-target mutation event was detected in any the 15 predicted sites analyzed. This study provided mutants for further study of the function of GhALARP in cotton fiber development. Our results further demonstrated the feasibility of use of CRISPR/Cas9 as a targeted mutagenesis tool in cotton, and provided an efficient tool for targeted mutagenesis and functional genomics in cotton.

Genome-wide identification and expression analyses of TCP transcription factor genes in Gossypium barbadense

Sea-island cotton (Gossypium barbadense) has drawn great attention in the textile industry for its comprehensive resistance and superior fiber properties. However, the mechanisms involved in fiber growth and development are unclear. As TCP transcription factors play important roles in plant growth and development, this study investigated the TCP family genes in G. barbadense (GbTCP). We identified 75 GbTCP genes, of which 68 had no introns. Phylogenetic analyses categorized the GbTCP transcription factors into 11 groups. Genomic analyses showed that 66 genes are located on 21 chromosomes. Phylogenetic analyses of G. arboreum, G. raimondii, G. hirsutum, G. barbadense, Theobroma cacao, Arabidopsis thaliana, Oryza sativa, Sorghum bicolor, and Zea mays, Picea abies, Sphagnum fallax and Physcomitrella patens, categorized 373 TCP genes into two classes (Classes I and II). By studying the structures of TCP genes in sea-island cotton, we identified genes from the same evolutionary branches that showed similar motif patterns. qRT-PCR results suggested that the GbTCPs had different expression patterns in fibers at various developmental stages of cotton, with several showing specific expression patterns during development. This report helps lay the foundation for future investigations of TCP functions and molecular mechanisms in sea-island cotton fiber development.

Hydrogen Peroxide Generation of Copper/Ascorbate Formulations on Cotton: Effect on Antibacterial and Fibroblast Activity for Wound Healing Application.

Greige cotton (unbleached cotton) is an intact plant fiber that retains much of the outer cotton fiber layers. These layers contain pectin, peroxidases, and trace metals that are associated with hydrogen peroxide (H2O2) generation during cotton fiber development. When greige cotton is subjected to a nonwoven hydroentanglement process, components of the outer cotton fiber layers are retained. When hydrated, this fabric can generate H2O2 (5–50 micromolar). This range has been characterized as inducing accelerated wound healing associated with enhanced cell signaling and the proliferation of cells vital to wound restoration. On the other hand, H2O2 levels above 50 micromolar have been associated with bacteriostatic activity. Here, we report the preparation and hydrogen peroxide activity of copper/ascorbate formulations, both as adsorbed and in situ synthesized analogs on cotton. The cooper/ascorbate-cotton formulations were designed with the goal of modulating hydrogen peroxide levels within functional ranges beneficial to wound healing. The cotton/copper formulation analogs were prepared on nonwoven unbleached cotton and characterized with cotton impregnation titers of 3–14 mg copper per gram of cotton. The copper/ascorbate cotton analog formulations were characterized spectroscopically, and the copper titer was quantified with ICP analysis and probed for peroxide production through assessment with Amplex Red. All analogs demonstrated antibacterial activity. Notably, the treatment of unbleached cotton with low levels of ascorbate (~2 mg/g cotton) resulted in a 99 percent reduction in Klebsiella pneumoniae and Staphylococcus aureus. In situ synthesized copper/ascorbate nanoparticles retained activity and did not leach out upon prolonged suspension in an aqueous environment. An assessment of H2O2 effects on fibroblast proliferation are discussed in light of the copper/cotton analogs and wound healing.

Unraveling Cotton Fiber Development Using Fiber Mutants in the Post‐Genomic Era

Cotton (Gossypium spp.) fibers are unicellular trichomes that differentiate from ovule epidermal cells. Cotton fiber development is divided into four distinct yet overlapping stages: initiation, elongation, secondary cell wall (SCW) biosynthesis, and maturation. There are numerous naturally occurring and man‐made fiber mutants that display aberrant phenotypes ranging from fiberless to extremely short fiber, and to immature fiber. These mutants have provided cotton researchers an excellent model system to study fiber growth and development. During the past two decades, much advancement in the understanding of fiber development has been achieved through comparative analyses of fiber mutants and wild‐type cotton lines using a variety of technologies such as transcriptomic analysis and genetic mapping. The causative genes of five mutations related to fiber initiation, elongation, or SCW assembly have been identified. Lint and fuzz fiber initiations are regulated by MYBMIXTA‐like transcription factors along with many other genes. Cytoskeleton actins play a critical role in fiber elongation. Genes that are involved in biological processes such as transporting osmoticum or loosening cell walls are highly expressed during the elongation stage. Production of large amounts of cellulose at the SCW stage requires a sustainable supply of energy and carbohydrate precursors. Plant hormones affect fiber development. In this paper, we review progress in the elucidation of fiber development mechanisms based on analyzing fiber mutants and summarize genes and mechanisms that are critical to fiber development. We identify research gaps that should be future research priorities and provide a future perspective.

Multi-Locus Genome-Wide Association Studies of Fiber-Quality Related Traits in Chinese Early-Maturity Upland Cotton.

Early-maturity varieties of upland cotton are becoming increasingly important for farmers to improve their economic benefits through double cropping practices and mechanical harvesting production in China. However, fiber qualities of early-maturing varieties are relatively poor compared with those of middle- and late- maturing ones. Therefore, it is crucial for researchers to elucidate the genetic bases controlling fiber-quality related traits in early-maturity cultivars, and to improve synergistically cotton earliness and fiber quality. Here, multi-locus genome-wide association studies (ML-GWAS) were conducted in a panel consisting of 160 early-maturing cotton accessions. Each accession was genotyped by 72,792 high-quality single nucleotide polymorphisms (SNPs) using specific-locus amplified fragment sequencing (SLAF-seq) approach, and fiber quality-related traits under four environmental conditions were measured. Applying at least three ML-GWAS methods, a total of 70 significant quantitative trait nucleotides (QTNs) were identified to be associated with five objective traits, including fiber length (FL), fiber strength (FS), fiber micronaire (FM), fiber uniformity (FU) and fiber elongation (FE). Among these QTNs, D11_21619830, A05_28352019 and D03_34920546 were found to be significantly associated with FL, FS, and FM, respectively, across at least two environments. Among 96 genes located in the three target genomic regions (A05: 27.95 28.75, D03: 34.52 35.32, and D11: 21.22 22.02 Mbp), six genes (Gh_A05G2325, Gh_A05G2329, Gh_A05G2334, Gh_D11G1853, Gh_D11G1876, and Gh_D11G1879) were detected to be highly expressed in fibers relative to other eight tissues by transcriptome sequencing method in 12 cotton tissues. Together, multiple favorable QTN alleles and six candidate key genes were characterized to regulate fiber development in early-maturity cotton. This will lay a solid foundation for breeding novel cotton varieties with earliness and excellent fiber-quality in the future.

Comprehensive analysis of NAC transcription factors uncovers their roles during fiber development and stress response in cotton

BackgroundTranscription factors operate as important switches of transcription networks, and NAC (NAM, ATAF, and CUC) transcription factors are a plant-specific family involved in multiple biological processes. However, this gene family has not been systematically characterized in cotton.ResultsHere we identify a large number of genes with conservative NAC domains in four cotton species, with 147 found in Gossypium arboreum, 149 in G. raimondii, 267 in G. barbadense and 283 in G. hirsutum. Predicted membrane-bound NAC genes were also identified. Phylogenetic analysis showed that cotton NAC proteins clustered into seven subfamilies and homologous protein pairs showed similar characteristics. Evolutionary property analysis revealed that purifying selection of NAC genes occurred between diploid and polyploid cotton species, and variation analysis showed GhNAC genes may have been subjected to selection and domestication. NAC proteins showed extensive transactivation and this was dependent on the C-terminus. Some development and stress related cis-elements were enriched in the promoters of GhNAC genes. Comprehensive expression analysis indicated that 38 GhNAC genes were candidates for involvement in fiber development, and 120 in stress responses. Gene co-expression network analysis revealed relationships between fiber-associated NAC genes and secondary cell wall (SCW) biosynthesis genes.ConclusionsNAC genes were identified in diploid and tetraploid cotton, revealing new insights into their evolution, variation and homology relationships. Transcriptome analysis and co-expression network indicated roles for GhNAC genes in cotton fiber development and stress response, and NAC genes may prove useful in molecular breeding programmes.

GhHUB2, a ubiquitin ligase, is involved in cotton fiber development via the ubiquitin-26S proteasome pathway.

Cotton fibers, which are extremely elongated single cells of epidermal seed trichomes and have highly thickened cell walls, constitute the most important natural textile material worldwide. However, the regulation of fiber development is not well understood. Here, we report that GhHUB2, a functional homolog of AtHUB2, controls fiber elongation and secondary cell wall (SCW) deposition. GhHUB2 is ubiquitously expressed, including within fibers. Overexpression of GhHUB2 in cotton increased fiber length and SCW thickness, while RNAi knockdown of GhHUB2 resulted in shortened fibers and thinner cell walls. We found that GhHUB2 interacted with GhKNL1, a transcriptional repressor predominantly expressed in developing fibers, and that GhHUB2 ubiquitinated and degraded GhKNL1 via the ubiquitin-26S proteasome pathway. GhHUB2 negatively regulated GhKNL1 protein levels and lead to the disinhibition of genes such as GhXTH1, Gh1,3-β-G, GhCesA4, GhAGP4, GhCTL1, and GhCOBL4, thus promoting fiber elongation and enhancing SCW biosynthesis. We found that GhREV-08, a transcription factor that participates in SCW deposition and auxin signaling pathway, was a direct target of GhKNL1. In conclusion, our study uncovers a novel function of HUB2 in plants in addition to its monoubiquitination of H2B. Moreover, we provide evidence for control of the fiber development by the ubiquitin-26S proteasome pathway.

Genome-wide characterization, identification, and expression analysis of the WD40 protein family in cotton.

WD40 repeat proteins are largely distributed across the plant kingdom and play an important role in diverse biological activities. In this work, we performed genome-wide identification, characterization, and expression level analysis of WD40 genes in cotton. A total of 579, 318, and 313 WD40 genes were found in Gossypium hirsutum, G. arboreum, and G. raimondii, respectively. Based on phylogenetic tree analyses, WD40 genes were divided into 11 groups with high similarities in exon/intron features and protein domains within the group. Expression analysis of WD40 genes showed differential expression at different stages of cotton fiber development (0 and 8 DPA) and cotton stem. A number of miRNAs were identified to target WD40 genes that are significantly involved in cotton fiber development during the initiation and elongation stages. These include miR156, miR160, miR162, miR164, miR166, miR167, miR169, miR171, miR172, miR393, miR396, miR398, miR2950, and miR7505. The findings provide a stronger indication of WD40 gene function and their involvement in the regulation of cotton fiber development during the initiation and elongation stages.

Genome-wide identification and expression analysis of Gossypium RING-H2 finger E3 ligase genes revealed their roles in fiber development, and phytohormone and abiotic stress responses

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

The phosphatidylinositol synthase gene (GhPIS) contributes to longer, stronger, and finer fibers in cotton.

Cotton fibers are the most important natural raw material used in textile industries world-wide. Fiber length, strength, and fineness are the three major traits which determine the quality and economic value of cotton. It is known that exogenous application of phosphatidylinositols (PtdIns), important structural phospholipids, can promote cotton fiber elongation. Here, we sought to increase the in planta production of PtdIns to improve fiber traits. Transgenic cotton plants were generated in which the expression of a cotton phosphatidylinositol synthase gene (i.e., GhPIS) was controlled by the fiber-specific SCFP promoter element, resulting in the specific up-regulation of GhPIS during cotton fiber development. We demonstrate that PtdIns content was significantly enhanced in transgenic cotton fibers and the elevated level of PtdIns stimulated the expression of genes involved in PtdIns phosphorylation as well as promoting lignin/lignin-like phenolic biosynthesis. Fiber length, strength and fineness were also improved in the transgenic plants as compared to the wild-type cotton, with no loss in overall fiber yield. Our data indicate that fiber-specific up-regulation of PtdIns synthesis is a promising strategy for cotton fiber quality improvement.

Genomic Comparison of the P-ATPase Gene Family in Four Cotton Species and Their Expression Patterns in Gossypium hirsutum

Plant P-type H+-ATPase (P-ATPase) is a membrane protein existing in the plasma membrane that plays an important role in the transmembrane transport of plant cells. To understand the variety and quantity of P-ATPase proteins in different cotton species, we combined four databases from two diploid cotton species (Gossypium raimondii and G. arboreum) and two tetraploid cotton species (G. hirsutum and G. barbadense) to screen the P-ATPase gene family and resolved the evolutionary relationships between the former cotton species. We identified 53, 51, 99 and 98 P-ATPase genes from G. arboretum, G. raimondii, G. barbadense and G. hirsutum, respectively. The structural and phylogenetic analyses revealed that the gene structure was consistent between P-ATPase genes, with a close evolutionary relationship. The expression analysis of P-ATPase genes showed that many P-ATPase genes were highly expressed in various tissues and at different fiber developmental stages in G. hirsutum, suggesting that they have potential functions during growth and fiber development in cotton.

Effects of potassium deficiency on the enzymatic changes in developing cotton fibers

Although effects of potassium (K) on cotton growth have been explored extensively, the effects of K deficiency on the physiological changes closely related to cotton fiber development are lacking. Thus, a 2-year field experiment was conducted with two cotton cultivars (Simian 3 and Siza 3) under 0 kg K2O ha−1 (K deficiency) and 300 kg K2O ha−1 (K sufficiency). The results showed that tonoplast adenosine triphosphatase (V-ATPase), pyrophosphatase (PPase), plasma membrane H+-ATPase (PM H+-ATPase), phosphoenolpyruvate carboxylase (PEPC), sucrose synthesis (SuSy) and vacuolar invertase (V-INV) were highly sensitive to K deficiency. The decreases in those enzymes resulted in low malate and soluble sugar contents, which together with low K concentration declined the driving force for fiber elongation, leading to significantly lower fiber length in the 0 kg K2O ha−1 treatment. The activity of sucrose phosphate synthase (SPS) was obviously increased by K deficiency before 20 days post anthesis (DPA), which could partly explain the acceleration of fiber cellulose synthesis and the increase in fiber strength in the 0 kg K2O ha−1 treatment in the early stage. However, SPS activity was decreased by K deficiency after 20 DPA and SuSy activity was reduced by K deficiency at any sampling date, resulting in low fiber strength in the end. Compared with Simian 3, the enzymes V-ATPase, PPase, PM H+-ATPase, PEPC and SuSy during fiber elongation stage were more sensitive to K deficiency in Siza 3, and the enzymes SuSy and SPS during fiber-thickening stage were more sensitive to K deficiency in Siza 3, which were the important reasons causing greater decreases in final fiber length and final fiber strength for Siza 3 than Simian 3 under K deficiency.

Genome-wide comparative and evolutionary analysis of Calmodulin-binding Transcription Activator (CAMTA) family in Gossypium species

The CAMTA gene family is crucial in managing both biotic and abiotic stresses in plants. Our comprehensive analysis of this gene family in cotton resulted in the identification of 6, 7 and 9 CAMTAs in three sequenced cotton species, i.e., Gossypium arboreum, Gossypium raimondii, and Gossypium hirsutum, respectively. All cotton CAMTAs were localized in the nucleus and possessed calmodulin-binding domain (CaMBD) as identified computationally. Phylogenetically four significant groups of cotton CAMTAs were identified out of which, Group II CAMTAs experienced higher evolutionary pressure, leading to a faster evolution in diploid cotton. The expansion of cotton CAMTAs in the genome was mainly due to segmental duplication. Purifying selection played a significant role in the evolution of cotton CAMTAs. Expression profiles of GhCAMTAs revealed that GhCAMTA2A.2 and GhCAMTA7A express profoundly in different stages of cotton fiber development. Positive correlation between expression of these two CAMTAs and fiber strength confirmed their functional relevance in fiber development. The promoter region of co-expressing genes network of GhCAMTA2A.2 and GhCAMTA7A showed a higher frequency of occurrence of CAMTA binding motifs. Our present study thus contributes to broad probing into the structure and probable function of CAMTA genes in Gossypium species.

Inhibition of Heat Shock proteins HSP90 and HSP70 induce oxidative stress, suppressing cotton fiber development

Cotton fiber is a specialized unicellular structure useful for the study of cellular differentiation and development. Heat shock proteins (HSPs) have been shown to be involved in various developmental processes. Microarray data analysis of five Gossypium hirsutum genotypes revealed high transcript levels of GhHSP90 and GhHSP70 genes at different stages of fiber development, indicating their importance in the process. Further, we identified 26 and 55 members of HSP90 and HSP70 gene families in G. hirsutum. The treatment of specific inhibitors novobiocin (Nov; HSP90) and pifithrin/2-phenylethynesulfonamide (Pif; HSP70) in in-vitro cultured ovules resulted in a fewer number of fiber initials and retardation in fiber elongation. The molecular chaperone assay using bacterially expressed recombinant GhHSP90-7 and GhHSP70-8 proteins further confirmed the specificity of inhibitors. HSP inhibition disturbs the H2O2 balance that leads to the generation of oxidative stress, which consequently results in autophagy in the epidermal layer of the cotton ovule. Transmission electron microscopy (TEM) of inhibitor-treated ovule also corroborates autophagosome formation along with disrupted mitochondrial cristae. The perturbations in transcript profile of HSP inhibited ovules show differential regulation of different stress and fiber development-related genes and pathways. Altogether, our results indicate that HSP90 and HSP70 families play a crucial role in cotton fiber differentiation and development by maintaining cellular homeostasis.