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.

E3 ligases are key enzymes required for protein degradation. Here, we identified a C3H2C3 RING domain-containing E3 ubiquitin ligase gene named GhATL68b. It is preferentially and highly expressed in developing cotton fiber cells and shows greater conservation in plants than in animals or archaea. The four orthologous copies of this gene in various diploid cottons and eight in the allotetraploid G. hirsutum were found to have originated from a single common ancestor that can be traced back to Chlamydomonas reinhardtii at about 992 million years ago. Structural variations in the GhATL68b promoter regions of G. hirsutum, G. herbaceum, G. arboreum, and G. raimondii are correlated with significantly different methylation patterns. Homozygous CRISPR-Cas9 knockout cotton lines exhibit significant reductions in fiber quality traits, including upper-half mean length, elongation at break, uniformity, and mature fiber weight. In vitro ubiquitination and cell-free protein degradation assays revealed that GhATL68b modulates the homeostasis of 2,4-dienoyl-CoA reductase, a rate-limiting enzyme for the β-oxidation of polyunsaturated fatty acids (PUFAs), via the ubiquitin proteasome pathway. Fiber cells harvested from these knockout mutants contain significantly lower levels of PUFAs important for production of glycerophospholipids and regulation of plasma membrane fluidity. The fiber growth defects of the mutant can be fully rescued by the addition of linolenic acid (C18:3), the most abundant type of PUFA, to the ovule culture medium. This experimentally characterized C3H2C3 type E3 ubiquitin ligase involved in regulating fiber cell elongation may provide us with a new genetic target for improved cotton lint production.

In this study, the whole HD-Zip family members of G. hirsutum were identified, and GhHDZ76 was classified into the HD-Zip IV subgroup. GhHDZ76 was predominantly expressed in the 0-5 DPA of fiber development stage and localized in the nucleus. Overexpression of GhHDZ76 significantly increased the length and density of trichomes in Arabidopsis thaliana. The fiber length of GhHDZ76 knockout lines by CRISPR/Cas9 was significantly shorter than WT at the early elongation and mature stage, indicating that GhHDZ76 positively regulate the fiber elongation. Scanning electron microscopy showed that the number of ovule surface protrusion of 0 DPA of GhHDZ76 knockout lines was significantly lower than WT, suggesting that GhHDZ76 can also promote the initiation of fiber development. The transcript level of GhWRKY16, GhRDL1, GhEXPA1 and GhMYB25 genes related to fiber initiation and elongation in GhHDZ76 knockout lines were significantly decreased. Yeast two-hybrid and Luciferase complementation imaging (LCI) assays showed that GhHDZ76 can interact with GhWRKY16 directly. As a transcription factor, GhHDZ76 has transcriptional activation activity, which could bind to L1-box elements of the promoters of GhRDL1 and GhEXPA1. Double luciferase reporter assay showed that the GhWRKY16 could enhance the transcriptional activity of GhHDZ76 to pGhRDL1, but it did not promote the transcriptional activity of GhHDZ76 to pGhEXPA1. GhHDZ76 protein may also promote the transcriptional activity of GhWRKY16 to the downstream target gene GhMYB25. Our results provided a new gene resource for fiber development and a theoretical basis for the genetic improvement of cotton fiber quality.

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.

Cotton fiber holds immense importance as the primary raw material for the textile industry. Consequently, comprehending the regulatory mechanisms governing fiber development is pivotal for enhancing fiber quality. Our study aimed to construct a regulatory network of competing endogenous RNAs (ceRNAs) and assess the impact of non-coding RNAs on gene expression throughout fiber development. Through whole transcriptome data analysis, we identified differentially expressed genes (DEGs) regulated by non-coding RNA (ncRNA) that were predominantly enriched in phenylpropanoid biosynthesis and the fatty acid elongation pathway. This analysis involved two contrasting phenotypic materials (J02-508 and ZRI015) at five stages of fiber development. Additionally, we conducted a detailed analysis of genes involved in fatty acid elongation, including KCS, KCR, HACD, ECR, and ACOT, to unveil the factors contributing to the variation in fatty acid elongation between J02-508 and ZRI015. Through the integration of histochemical GUS staining, dual luciferase assay experiments, and correlation analysis of expression levels during fiber development stages for lncRNA MSTRG.44818.23 (MST23) and GhKCR2, we elucidated that MST23 positively regulates GhKCR2 expression in the fatty acid elongation pathway. This identification provides valuable insights into the molecular mechanisms underlying fiber development, emphasizing the intricate interplay between non-coding RNAs and protein-coding genes.

Plant height (PH) is an important factor affecting bast fiber yield in jute. Here, we report the mechanism of dwarfism in the \'Guangbaai\' (gba) of jute. The mutant gba had shorter internode length and cell length compared to the standard cultivar \'TaiZi 4\' (TZ4). Exogenous GA3 treatment indicated that gba is a GA-insensitive dwarf mutant. Quantitative trait locus (QTL) analysis of three PH-related traits via a high-density genetic linkage map according to re-seq showed that a total of 25 QTLs were identified, including 13 QTLs for PH, with phenotypic variation explained ranging from 2.42 to 74.16%. Notably, the functional mechanism of the candidate gene CoGID1a, the gibberellic acid receptor, of the major locus qPHIL5 was evaluated by transgenic analysis and virus-induced gene silencing. A dwarf phenotype-related single nucleotide mutation in CoGID1a was identified in gba, which was also unique to the dwarf phenotype of gba among 57 cultivars. Cogid1a was unable to interact with the growth-repressor DELLA even in the presence of highly accumulated gibberellins in gba. Differentially expressed genes between transcriptomes of gba and TZ4 after GA3 treatment indicated up-regulation of genes involved in gibberellin and cellulose synthesis in gba. Interestingly, it was found that up-regulation of CoMYB46, a key transcription factor in the secondary cell wall, by the highly accumulated gibberellins in gba promoted the expression of cellulose synthase genes CoCesA4 and CoCesA7. These findings provide valuable insights into fiber development affected by endogenous gibberellin accumulation in plants.

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.