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.

Purple-grained wheat (Triticum aestivum L.) is an important germplasm source in crop breeding. Anthocyanin biosynthesis in the pericarps of purple-grained wheat is largely light-dependent; however, the regulatory mechanisms underlying light-induced anthocyanin accumulation in the wheat pericarp remain unknown. Here we determined that anthocyanins rapidly accumulate in the pericarps of the purple-grained wheat cultivar Heixiaomai 76 (H76) at 16 days after pollination under light treatment. Using transcriptome sequencing, differential gene expression analysis, and phylogenetic analysis, we identified two key genes involved in light signaling in wheat: ELONGATED HYPOCOTYL 5-7A (TaHY5-7A) and B-BOX-3B (TaBBX-3B). TaHY5-7A and TaBBX-3B were highly expressed in purple-grained wheat pericarps. The heterologous expression of TaHY5-7A partially restored the phenotype of the Arabidopsis (Arabidopsis thaliana) hy5 mutant, resulting in increased anthocyanin accumulation and a shortened hypocotyl. The heterologous expression of TaBBX-3B in wild-type Arabidopsis had similar effects. TaHY5-7A and TaBBX-3B were nucleus-localized, consistent with a function in transcription regulation. However, TaHY5-7A, which lacks a transactivation domain, was not sufficient to activate the expression of PURPLE PERICARP-MYB 1 (TaPpm1), the key anthocyanin biosynthesis regulator in purple pericarps of wheat. TaHY5-7A physically interacted with TaBBX-3B in yeast two-hybrid and bimolecular fluorescence complementation assays. Additionally, TaHY5-7A, together with TaBBX-3B, greatly enhanced the promoter activity of TaPpm1 in a dual luciferase assay. Overall, our results suggest that TaHY5-7A and TaBBX-3B collaboratively activate TaPpm1 expression to promote light-induced anthocyanin biosynthesis in purple-pericarp wheat.

Lipid transfer proteins (LTPs) play crucial regulatory roles in plant growth, development, and abiotic stress responses. Stipagrostis pennata is a species of grass widely distributed in arid and semi-arid regions, particularly adapted to desert and steppe environments. Under extreme drought conditions, it exhibits a variety of physiological and morphological adaptation mechanisms, making it an important species for studying plant drought tolerance. Recently, LTPs have been found to exhibit upregulated expression under drought stress in plants such as wheat and tobacco, enhancing their drought tolerance. However, the functional role of LTPs in S. pennata remains unexplored. In this study, the SpLTP1 gene was isolated from S. pennata via molecular cloning, encoding a 116-amino acid protein. Phylogenetic analysis revealed that this protein contains a highly conserved nsLTP1 (cd01960) domain and has high sequence similarity with LTPs of Setaria viridis, Setaria italica, Musa acuminata and Phragmites australis. qRT-PCR revealed that SpLTP1 was highly expressed and dynamically regulated under drought, suggesting its potential role in root rhizosheath formation and drought tolerance. To investigate SpLTP1 function, SpLTP1-overexpressing (SpLTP1-OE) and complementation (SpLTP1-atltp) Arabidopsis lines were generated using the floral dip method, in comparison with the existing wild-type (WT) and the LTP-deficient mutant (atltp). Drought stress phenotyping and physiological assays indicated that SpLTP1 likely enhances drought tolerance by elevating antioxidant enzyme activities and osmolyte accumulation. Comparative transcriptome analysis of SpLTP1-OE and WT plants further suggested that SpLTP1 modulates critical pathways, including phenylpropanoid biosynthesis, zeatin biosynthesis, and plant hormone signal transduction, thereby influencing plant growth and stress adaptation. These findings not only provide novel insights into the molecular mechanisms by which SpLTP1 regulates rhizosheath development in S. pennata but also establish a foundation for deciphering its role in extreme drought adaptation.

Allene oxide cyclase (AOC) is an integral enzyme in jasmonic acid (JA) biosynthesis and a key regulator of plant stress responses. However, the regulatory mechanisms and enzymatic activities of cotton AOC under stress responses remain poorly understood. In our research, using BLASTP and conserved domain validation, we identified 68 AOC gene members across 10 Gossypium species. Phylogenetic, gene-structure, and syntenic analyses showed high evolutionary conservation of AOC genes, accompanied by lineage-specific expansion. Promoter sequence analysis indicated that the AOCs contained abundant cis-elements associated with hormone signaling, abiotic-stress response, and transcription factor (TF) regulation. Expression profiling revealed tissue-specific and stress-responsive expression patterns among GhAOC genes, with GhAOC3A showing the most pronounced upregulation under chilling stress. GhAOC3A was identified as a chloroplast-localized protein based on the subcellular localization assay. GhAOC3A overexpression enhanced cotton chilling tolerance with elevated JA accumulation, stronger antioxidant defense, stable osmotic homeostasis, and retained chlorophyll content, and VIGS results provided supportive evidence for the function of GhAOC3A in chilling tolerance. Among the predicted TFs that could regulate the expression of GhAOC3A, GhWRKY65 was further identified and confirmed to bind to the W-box element and activate GhAOC3A transcription. Overexpression of GhWRKY65 significantly enhanced cotton chilling tolerance, as well as elevated GhAOC3A expression and increased JA accumulation. Conversely, silencing GhWRKY65 compromised cotton chilling tolerance. Collectively, these findings suggest that the GhWRKY65-GhAOC3A regulatory module positively modulates cotton chilling tolerance and JA levels. Our study provides insights into the transcriptional network associated with plant stress response and offers effective candidates for breeding chilling-tolerant cotton materials.

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.