Cotton is a globally significant economic crop. Brassinosteroids (BRs) are crucial to cotton development. This study systematically analyzed the BR synthase gene family in four cotton species and identified 60 BR genes: 20 in Gossypium hirsutum (GhBRs), 20 in G. barbadense (GbBRs), 10 in G. arboreum (GaBRs), and 10 in G. raimondii (GrBRs). The analysis was extended to chromosomal localization, evolutionary relationships, domain features, and cis-regulatory elements in the promoter regions of BR synthase genes. The results showed that the BR synthase genes were evenly distributed across different subgenomes and chromosomes. Bioinformatics analyses revealed high conservation of amino acid sequences, secondary structures, and conserved domains among the subfamily members, which is closely linked to their pivotal roles in the BR biosynthesis pathway. Cis-element distribution analysis of the BR synthase genes further underscored the complexity of BR gene expression regulation, which is influenced by multiple factors, including plant hormones, abiotic stress, and transcription factors. Expression profiling of GhBRs genes in various cotton tissues and developmental stages highlighted the key roles of GhROT3-1 and GhDET2-1 in fiber elongation and initiation, respectively. Protein-protein interactions and transcription factor analyses further elucidated the regulatory mechanisms of GhROT3-1 and GhDET2-1 in cotton growth and development. This study lays a theoretical foundation for understanding the role of the BR signaling pathway in cotton development, facilitating molecular breeding.

Cotton is the most widely planted fiber crop in the world, and improving cotton fiber quality has long been a research hotspot. The development of cotton fibers is a complex process that includes four consecutive and overlapping stages, and although many studies on cotton fiber development have been reported, most of the studies have been based on cultivars that are promoted in production or based on lines that are used in breeding. Here, we report a phenotypic evaluation of Gossypium hirsutum based on immature fiber mutant (xin w 139) and wild-type (Xin W 139) lines and a comparative transcriptomic study at seven time points during fiber development. The results of the two-year study showed that the fiber length, fiber strength, single-boll weight and lint percentage of xin w 139 were significantly lower than those of Xin W 139, and there were no significant differences in the other traits. Principal component analysis (PCA) and cluster analysis of the RNA-sequencing (RNA-seq) data revealed that these seven time points could be clearly divided into three different groups corresponding to the initiation, elongation and secondary cell wall (SCW) synthesis stages of fiber development, and the differences in fiber development between the two lines were mainly due to developmental differences after twenty days post anthesis (DPA). Differential expression analysis revealed a total of 5131 unique differentially expressed genes (DEGs), including 290 transcription factors (TFs), between the 2 lines. These DEGs were divided into five clusters. Each cluster functional category was annotated based on the KEGG database, and different clusters could describe different stages of fiber development. In addition, we constructed a gene regulatory network by weighted correlation network analysis (WGCNA) and identified 15 key genes that determined the differences in fiber development between the 2 lines. We also screened seven candidate genes related to cotton fiber development through comparative sequence analysis and qRT-PCR; these genes included three TFs (GH_A08G1821 (bHLH), GH_D05G3074 (Dof), and GH_D13G0161 (C3H)). These results provide a theoretical basis for obtaining an in-depth understanding of the molecular mechanism of cotton fiber development and provide new genetic resources for cotton fiber research.

The β-amylase (BAM) gene family encodes important enzymes that catalyze the conversion of starch to maltose in various biological processes of plants and play essential roles in regulating the growth and development of multiple plants. So far, BAMs have been extensively studied in Arabidopsis thaliana (A. thaliana). However, the characteristics of the BAM gene family in the crucial economic crop, cotton, have not been reported. In this study, 27 GhBAM genes in the genome of Gossypium hirsutum L (G. hirsutum) were identified by genome-wide identification, and they were divided into three groups according to sequence similarity and phylogenetic relationship. The gene structure, chromosome distribution, and collinearity of all GhBAM genes identified in the genome of G. hirsutum were analyzed. Further sequence alignment of the core domain of glucosyl hydrolase showed that all GhBAM family genes had the glycosyl hydrolase family 14 domain. We identified the BAM gene GhBAM7 and preliminarily investigated its function by transcriptional sequencing analysis, qRT-PCR, and subcellular localization. These results suggested that the GhBAM7 gene may influence fiber strength during fiber development. This systematic analysis provides new insight into the transcriptional characteristics of BAM genes in G. hirsutum. It may lay the foundation for further study of the function of these genes.

Three carbon-chain extension genes associated with fatty acid synthesis in upland cotton (Gossypium hirsutum), namely GhKAR, GhHAD, and GhENR, play important roles in oil accumulation in cotton seeds. In the present study, these three genes were cloned and characterized. The expression patterns of GhKAR, GhHAD, and GhENR in the high seed oil content cultivars 10H1014 and 10H1041 differed somewhat compared with those of 10H1007 and 2074B with low seed oil content at different stages of seed development. GhKAR showed all three cultivars showed higher transcript levels than that of 2074B at 10-, 40-, and 45-days post anthesis (DPA). The expression pattern of GhHAD showed a lower transcript level than that of 2074B at both 10 and 30 DPA but a higher transcript level than that of 2074B at 40 DPA. GhENR showed a lower transcript level than that of 2074B at both 15 and 30 DPA. The highest transcript levels of GhKAR and GhENR were detected at 15 DPA in 10H1007, 10H1014, and 10H1041 compared with 2074B. From 5 to 45 DPA cotton seed, the oil content accumulated continuously in the developing seed. Oil accumulation reached a peak between 40 DPA and 45 DPA and slightly decreased in mature seed. In addition, GhKAR and GhENR showed different expression patterns in fiber and ovule development processes, in which they showed high expression levels at 20 DPA during the fiber elongation stage, but their expression level peaked at 15 DPA during ovule development processes. These two genes showed the lowest expression levels at the late seed maturation stage, while GhHAD showed a peak of 10 DPA in fiber development. Compared to 2074B, the oil contents of GhKAR and GhENR overexpression lines increased 1.05~1.08 folds. These results indicated that GhHAD, GhENR, and GhKAR were involved in both seed oil synthesis and fiber elongation with dual biological functions in cotton.

UNASSIGNED: Upland cotton (Gossypium hirsutum) is the main source of natural fiber in the global textile industry, and thus its fiber quality and yield are important parameters. In this study, comparative transcriptomics was used to analyze differentially expressed genes (DEGs) due to its ability to effectively screen candidate genes during the developmental stages of cotton fiber. However, research using this method is limited, particularly on fiber development. The aim of this study was to uncover the molecular mechanisms underlying the whole period of fiber development and the differences in transcriptional levels.
UNASSIGNED: Comparative transcriptomes are used to analyze transcriptome data and to screen for differentially expressed genes. STEM and WGCNA were used to screen for key genes involved in fiber development. qRT-PCR was performed to verify gene expression of selected DEGs and hub genes.
UNASSIGNED: Two accessions of upland cotton with extreme phenotypic differences, namely EZ60 and ZR014121, were used to carry out RNA sequencing (RNA-seq) on fiber samples from different fiber development stages. The results identified 704, 376, 141, 269, 761, and 586 genes that were upregulated, and 1,052, 476, 355, 259, 702, and 847 genes that were downregulated at 0, 5, 10, 15, 20, and 25 days post anthesis, respectively. Similar expression patterns of DEGs were monitored using short time-series expression miner (STEM) analysis, and associated pathways of DEGs within profiles were investigated. In addition, weighted gene co-expression network analysis (WGCNA) identified five key modules in fiber development and screened 20 hub genes involved in the development of fibers.
UNASSIGNED: Through the annotation of the genes, it was found that the excessive expression of resistance-related genes in the early fiber development stages affects the fiber yield, whereas the sustained expression of cell elongation-related genes is critical for long fibers. This study provides new information that can be used to improve fibers in newly developed upland cotton genotypes.

Cotton is a valuable cash crop in many countries. Cotton fiber is a trichome that develops from a single epidermal cell and serves as an excellent model for understanding cell differentiation and other life processes. Alternative splicing (AS) of genes is a common post-transcriptional regulatory process in plants that is essential for plant growth and development. The process of AS during cotton fiber formation, on the other hand, is mainly unknown. A substantial number of multi-exon genes were discovered to be alternatively spliced during cotton fiber formation in this study, accounting for 23.31% of the total number of genes in Gossypium hirsutum. Retention intron (RI) is not necessarily the most common AS type, indicating that AS genes and processes during fiber development are very temporal and tissue-specific. When compared to fiber samples, AS is more prevalent at the fiber initiation stages and in the ovule, indicating that development stages and tissues use different AS strategies. Genes involved in fiber development have gone through stage-specific AS, demonstrating that AS regulates cotton fiber development. Furthermore, AS can be regulated by trans-regulation elements such as splicing factor and cis-regulation elements such as gene length, exon numbers, and GC content, particularly at exon-intron junction sites. Our findings also suggest that increased DNA methylation may aid in the efficiency of AS, and that gene body methylation is key in AS control. Finally, our research will provide useful information about the roles of AS during the cotton fiber development process.

Cotton has been domesticated independently four times for its fiber, but the genomic targets of selection during each domestication event are mostly unknown. Comparative analysis of the transcriptome during cotton fiber development in wild and cultivated materials holds promise for revealing how independent domestications led to the superficially similar modern cotton fiber phenotype in upland (G. hirsutum) and Pima (G. barbadense) cotton cultivars. Here we examined the fiber transcriptomes of both wild and domesticated G. hirsutum and G. barbadense to compare the effects of speciation versus domestication, performing differential gene expression analysis and coexpression network analysis at four developmental timepoints (5, 10, 15, or 20 days after flowering) spanning primary and secondary wall synthesis. These analyses revealed extensive differential expression between species, timepoints, domestication states, and particularly the intersection of domestication and species. Differential expression was higher when comparing domesticated accessions of the two species than between the wild, indicating that domestication had a greater impact on the transcriptome than speciation. Network analysis showed significant interspecific differences in coexpression network topology, module membership, and connectivity. Despite these differences, some modules or module functions were subject to parallel domestication in both species. Taken together, these results indicate that independent domestication led G. hirsutum and G. barbadense down unique pathways but that it also leveraged similar modules of coexpression to arrive at similar domesticated phenotypes.

Comparative transcriptome analysis of fiber tissues between Gossypium barbadense and Gossypium hirsutum could reveal the molecular mechanisms underlying high-quality fiber formation and identify candidate genes for fiber quality improvement. In this study, 759 genes were found to be strongly upregulated at the elongation stage in G. barbadense, which showed four distinct expression patterns (I-IV). Among them, the 346 genes of group IV stood out in terms of the potential to promote fiber elongation, in which we finally identified 42 elongation-related candidate genes by comparative transcriptome analysis between G. barbadense and G. hirsutum. Subsequently, we overexpressed GbAAR3 and GbTWS1, two of the 42 candidate genes, in Arabidopsis plants and validated their roles in promoting cell elongation. At the secondary cell wall (SCW) biosynthesis stage, 2275 genes were upregulated and exhibited five different expression profiles (I-V) in G. barbadense. We highlighted the critical roles of the 647 genes of group IV in SCW biosynthesis and further picked out 48 SCW biosynthesis-related candidate genes by comparative transcriptome analysis. SNP molecular markers were then successfully developed to distinguish the SCW biosynthesis-related candidate genes from their G. hirsutum orthologs, and the genotyping and phenotyping of a BC3F5 population proved their potential in improving fiber strength and micronaire. Our results contribute to the better understanding of the fiber quality differences between G. barbadense and G. hirsutum and provide novel alternative genes for fiber quality improvement.

Cotton fiber provides the predominant plant textile in the world, and it is also a model for plant cell wall biosynthesis. The development of the single-celled cotton fiber takes place across several overlapping but discrete stages, including fiber initiation, elongation, the transition from elongation to secondary cell wall formation, cell wall thickening, and maturation and cell death. During each stage, the developing fiber undergoes a complex restructuring of genome-wide gene expression change and physiological/biosynthetic processes, which ultimately generate a strikingly elongated and nearly pure cellulose product that forms the basis of the global cotton industry. Here, we provide an overview of this developmental process focusing both on its temporal as well as evolutionary dimensions. We suggest potential avenues for further improvement of cotton as a crop plant.

The natural environment of plants comprises a complex set of biotic and abiotic stresses, and plant responses to these stresses are complex as well. Plant proteomics approaches have significantly revealed dynamic changes in plant proteome responses to stress and developmental processes. Thus, we reviewed the recent advances in cotton proteomics research under changing environmental conditions, considering the progress and challenging factors. Finally, we highlight how single-cell proteomics is revolutionizing plant research at the proteomics level. We envision that future cotton proteomics research at the single-cell level will provide a more complete understanding of cotton\'s response to stresses.