As key enzymes in the gibberellin (GA) biosynthesis pathway, GAoxs function as regulators of bioactive GA levels and plant architecture, yet little is understood about GAoxs in Gossypium. In this study, 78 GAox genes identified in four cotton species were divided into three subgroups: GA2ox, GA3ox, and GA20ox. Syntenic relationships of GAoxs in Gossypium suggested that divergencies in gene function may be attributed to whole-genome duplication during evolution. Cis-acting element analysis suggested that the GbGAox genes might participate in plant growth, development, and hormone responses. Moreover, transcriptome analysis was performed to characterize the molecular response of the exogenous GA₃ application. It was found that DEGs (differentially expressed genes) are widely involved in cell division and cell wall modification, in which the most XTH (xyloglucan endotransglucosylase/hydrolase) and GAox genes responded actively to the exogenous GA₃ treatment. Some transcription factors and protein kinases cooperated with those GbGAoxs in response to GA₃. These findings underlie the biological function of GAox genes and their responses to GA₃ in regulating plant growth in Gossypium barbadense.

The initial number of fibers and fiber length affect fiber yield and quality in cotton (Gossypium hirsutum). The mechanism of fiber initiation centers on the transcription factor GhMYB25-like, and the involvement of other regulatory factors remains unclear. Here, we established a GhMYB25-like-based interaction network that regulates fiber initiation, and focused on the interacting protein AGAMOUS-like 4 (GhAGL4), which is a MIKC-MADS transcription factor, and other MIKC-MADS family members. Genome-wide identification of the cotton MIKC-MADS family members identified two highly co-expressed genes, GhAGL1 and GhAGL4, which are predominantly expressed in ovules at 0 days post-anthesis (DPA). CRISPR/Cas9-mediated loss of function of GhAGL1 or GhAGL4 led to a decrease in fiber initials and impeded fiber elongation, with the Ghagl1 Ghagl4 double mutant exhibiting a stronger phenotype. GhAGL1 and GhAGL4 interact in vitro and in vivo. DNA affinity purification and sequencing (DAP-seq) combined with RNA sequencing identified CCCH zinc finger 1 (GhCZF1) as a downstream gene of GhAGL1 and GhAGL4. Interestingly, GhMYB25-like enhances the ability of GhAGL1-GhAGL4 heterodimers to activate downstream genes. During the early fiber elongation stage, GhCZF1 activates the expression of the xyloglucan endotransglycosylase/hydrolase (XTH) genes GhXTH16 and GhXTH23 to regulate fiber elongation. Our findings reveal a molecular mechanism involved in fiber initiation and early elongation, providing a foundation for enhancing cotton fiber yield and quality.

Mitogen-activated protein kinase kinase kinases (MAPKKKs) are pivotal upstream regulators of MAPK cascades, integrating signals that coordinate plant development and stress responses. However, the specific functions of MAPKKKs, particularly within the MEKK subfamily, in mediating cotton resistance to Verticillium wilt and Fusarium wilt remain poorly characterized. To address this, we conducted a systematic, cross-species analysis of the MAPKKK family in four key cotton species: Gossypium arboreum, Gossypium barbadense, Gossypium hirsutum, and Gossypium raimondii. Genome-wide identification and phylogenetic analysis revealed 660 MAPKKK genes, classifying them into the MEKK, Raf, and ZIK subfamilies. Evolutionary analysis indicated that Whole-Genome Duplication (WGD) events were the primary driver of family expansion. Promoter cis-element and Gene Ontology (GO) enrichment analyses implicated these genes in hormone signaling and stress adaptation. Expression profiling demonstrated functional modularity, with distinct members responding specifically to cold stress or cooperatively to drought and salt stresses. Upon pathogen infection, members diverged into regulatory modules associated with immune homeostasis, tissue-specific defense, and core signaling potentially governing systemic acquired resistance (SAR). The temporal expression patterns of core candidate genes were validated by qRT-PCR. This study provides, for the first time, a comprehensive evolutionary and functional framework for the MEKK subfamily within the cotton MAPKKK family. It reveals the conserved and divergent roles of this subfamily in stress adaptation and identifies key candidate genes for breeding disease-resistant cotton varieties.

G. barbadense represents an indispensable germplasm resource for high-quality textile fiber and disease resistance; nevertheless, systematic information regarding its ZRT/IRT-like protein (ZIP) gene family remains limited. Here, a total of 46 GbZIP genes were identified across the G. barbadense genome. Comprehensive bioinformatic investigations revealed uneven chromosomal distribution and confirmed that segmental/whole-genome duplications, supplemented by localized tandem duplications, drove family expansion. Members clustered within the same phylogenetic clades shared conserved motif organization and gene architecture, while promoter regions harbored abundant cis-acting elements associated with phytohormone and stress signaling. Transcriptome profiling indicated distinct expression patterns across vegetative/reproductive tissues, fiber and ovule developmental stages, and diverse abiotic stress conditions (cold, hot, drought, and salt). Quantitative Real-Time PCR (qRT-PCR) further validated that several GbZIP candidates exhibited temporal expression variations upon exposure to cadmium toxicity, V. dahliae infection, and combined Cd-V. dahliae stress. Specifically, GbZIP13, GbZIP18, GbZIP27, and GbZIP36 displayed prominent broad-spectrum responses to all three stress conditions, whereas GbZIP16, GbZIP29, and GbZIP30 showed stress-specific regulatory divergence. Overall, this study aims to systematically analyze the evolutionary characteristics and expression patterns of the GbZIP family, and to specifically evaluate the response differences under Cd stress, V. dahliae stress, and combined stress, in order to identify potential key candidate genes.

The translocase of the inner membrane (TIM) family plays an essential role in mediating the transport of nuclear-encoded precursor proteins across the inner mitochondrial membrane and regulating cellular energy metabolism. Sea-island cotton (Gossypium barbadense) is valued for its superior fiber quality, yet the composition, evolution, and expression patterns of its TIM genes remain unclear. To address this, we performed a genome-wide analysis of the TIM family in G. barbadense, using the diploid cotton G. arboreum, and G. raimondii and the allotetraploid G. hirsutum as comparative references. A total of 44 GbTIM genes were identified in G. barbadense and characterized through phylogenetic, structural, collinearity, promoter, and expression analyses. The TIM family expanded during cotton polyploidization, with tetraploid species containing approximately twice as many genes as diploids. Phylogenetic analysis categorized TIM proteins into seven subfamilies, with Group VI representing the largest clade. Promoter regions were enriched in light- and phytohormone-responsive elements. Interspecific synteny analysis demonstrated extensive collinearity between G. barbadense and its diploid ancestors but limited collinearity with G. hirsutum, indicating divergence among tetraploids. Transcriptomic profiling revealed highly spatiotemporal expression patterns. Gbar_A11G001280 was abundantly expressed during fiber initiation and upregulated in low-lint-percentage germplasm, whereas Gbar_D07G010770 was continuously expressed throughout fiber development. This study clarifies the evolutionary lineage of the TIM family across four cotton species, with a primary focus on G. barbadense, and provides candidate genes for molecular breeding of high-quality fiber. Functional validation of these candidate genes is recommended in future studies.

Cotton (Gossypium spp.) is a major industrial crop supplying raw materials for the global textile, chemical, and pharmaceutical sectors. Phytochromes (PHYs) are key photoreceptors regulating plant growth and environmental adaptation, yet their functions in the complex allotetraploid genome of upland cotton (G. hirsutum) remain poorly understood. Here, we identified 29 PHY genes across four Gossypium species and performed comprehensive genomic and evolutionary analyses. We focused on GhPHYB1, a AD₁-Dt PHYB homolog exhibiting light-inducible and stem-predominant expression. Virus-induced gene silencing (VIGS) revealed that GhPHYB1 negatively regulates vegetative elongation by modulating the GhPIF4 signaling axis. Additionally, GhPHYB1-silenced plants showed enhanced drought sensitivity, with reduced proline content, elevated malondialdehyde (MDA) and H₂O₂ levels, and impaired photosynthesis efficiency (F_v/F_m). Heterologous expression in yeast further confirmed its role in enhancing osmotic stress tolerance. Collectively, these findings establish GhPHYB1 as a dual regulator of plant architecture and drought tolerance, offering a promising target for molecular breeding of industrial cotton varieties.