Brown cotton is a major cultivar of naturally colored cotton, and brown cotton fibers (BCFs) are widely utilized as raw materials for textile industry production due to their advantages of being green and dyeing-pollution-free. However, the mechanisms underlying the pigmentation in fibers are still poorly understood, which significantly limits their extensive applications in related fields. In this study, we conducted a multidimensional comparative analysis of the transcriptomes and metabolomes between brown and white fibers at different developmental periods to identify the key genes and pathways regulating the pigment deposition. The transcriptomic results indicated that the pathways of flavonoid biosynthesis and phenylpropanoid biosynthesis were significantly enriched regulatory pathways, especially in the late development periods of fiber pigmentation; furthermore, the genes distributed in the pathways of PAL, CHS, F3H, DFR, ANR, and UFGT were identified as significantly up-regulated genes. The metabolic results showed that six metabolites, namely (−)-Epigallocatechin, Apiin, Cyanidin-3-O-glucoside, Gallocatechin, Myricetin, and Poncirin, were significantly accumulated in brown fibers but not in white fibers. Integrative analysis of the transcriptomic and metabolomic data demonstrated a possible regulatory network potentially regulating the pigment deposition, in which three MYB transcription factors promote the expression levels of flavonoid biosynthesis genes, thereby inducing the content increase in (−)-Epigallocatechin, Cyanidin-3-O-glucoside, Gallocatechin, and Myricetin in BCFs. Our findings provide new insights into the pigment deposition mechanism in BCFs and offer references for genetic engineering and breeding of colored cotton materials.

Background: Green cotton fibers (GCFs) are valued for their natural coloration and eco-friendly properties, but their pigmentation mechanisms remain unclear, limiting their wider application in the textile industry. This study aims to uncover the key regulatory genes and metabolic pathways involved in GCF coloration. Methods: We conducted transcriptome and metabolome profiling of green and white cotton fibers at different developmental stages to identify differences in gene expression and metabolite accumulation related to pigmentation. Results: Transcript analysis revealed significant enrichment in α-linolenic acid metabolism, flavonoid biosynthesis and phenylpropane metabolism pathways during late pigmentation stages. Key genes in methyl jasmonate (MeJA) biosynthesis and flavonoid biosynthesis (LOX, JMT, ANS, C4H, DFR, F3H) were upregulated. The MYB transcription factor showed the most significant increase during fiber development. Metabolomic analysis identified 12 metabolites that accumulated specifically in green fibers. MeJA treatment promoted the expression of MYB genes and flavonoid biosynthesis genes (DFRs, ANSs, F3H, C4H), as well as the accumulation of Luteolin, Gallocatechin, Cyanidin and Chrysanthemum metabolites. Conclusions: Our study demonstrates that MeJA-mediated flavonoid biosynthesis, regulated by MYB transcription factors, is the central pathway controlling pigment deposition in GCFs. These findings provide valuable insights for developing improved colored cotton materials.

Background/Objectives: The TOPLESS (TPL) and TOPLESS-related (TPR) proteins represent a highly conserved class of transcriptional co-repressors in plants, playing pivotal roles in modulating growth, development, and stress responses through the repression of key transcriptional regulators. However, a comprehensive genome-wide analysis of the TPL/TPR gene family and its involvement in stress responses remains unexplored in cotton. Methods: In this study, 60 TPL/TPR genes were identified from the genomes of ten Gossypium species via bioinformatics approaches, and their protein physicochemical properties, gene structures, phylogenetic relationships, cis-regulatory elements, and expression profiles were characterized. Results: Chromosomal localization and collinearity analyses revealed that segmental duplication events have contributed to the expansion of the TPL/TPR gene family. Further examination of exon–intron architectures and conserved motifs highlighted strong evolutionary conservation within each TPL/TPR subgroup. Expression profiling demonstrated that TPL/TPR genes exhibit tissue-specific expression patterns, with particularly high transcript abundance in floral organs (e.g., petals and stigmas). Cis-element analysis suggested their potential involvement in multiple stress-responsive pathways. Notably, GhTPL3 showed high constitutive expression across various tissues and under stress conditions, with the most pronounced up-regulation under salt stress. Functional validation via Virus-Induced Gene Silencing (VIGS) confirmed that GhTPL3 silencing significantly impairs cotton salt stress tolerance, underscoring its critical role in abiotic stress adaptation. Conclusions: Our findings provide novel insights into the functional diversification and regulatory mechanisms of the TPL/TPR family in cotton, offering a valuable genetic resource for breeding stress-resilient cotton varieties.

Histidine triad (HIT) family proteins contain a conserved histidine triad motif and play key roles in fungal metabolism and pathogenicity. This study focused on VD9136, a member of the HIT family in Verticillium dahliae, aiming to elucidate its biological function and mechanism underlying its role in cotton pathogenesis. A systematic investigation of the VD9136 gene in V. dahliae was conducted using bioinformatics analysis, gene knockout, genetic complementation, and pathogenicity assays. The results showed that VD9136 protein consists of 136 amino acids and is a stable, neutral, and weakly hydrophilic protein that lacks transmembrane domains and signal peptides; it is localized to the extracellular space via a non-classical secretion pathway. Its secondary structure is predominantly composed of α-helices and random coils. Phylogenetic analysis revealed that VD9136 is closely related to VliHIT, a homologous protein from V. longisporum, the pathogen responsible for Verticillium wilt in rapeseed. The promoter region of VD9136 contains multiple cis-acting elements, including light-responsive, hormone-responsive, and stress-responsive elements, indicating that its transcription may be regulated by multiple signaling pathways. VD9136 was significantly upregulated during the early stage of cotton infection (6–24 h post-inoculation). Pathogenicity assays demonstrated that V. dahliae knockout mutants lacking VD9136 exhibited a significant reduction in virulence, as evidenced by a lower disease index, decreased fungal biomass within plant tissues, and attenuated vascular browning in cotton plants. The pathogenic phenotype was successfully restored in genetic complementation strains. This study identified VD9136 as a key regulatory factor in the pathogenic process of V. dahliae, and its loss of function reduces the pathogenicity of V. dahliae. The findings provide a theoretical basis for elucidating the pathogenic mechanism of cotton Verticillium wilt and for developing corresponding prevention and control strategies.