Protein palmitoylation is an ability of the frame of the cell marker protein is one of the most notable reversible changes after translation. However, studies on protein palmitoylation in cotton have not yet been performed. In our current research, the PAT gene family was systematically identified and bioinformatically analyzed in G. arboreum, G. raimondii, G. barbadense and G. hirsutum, and 211 PAT genes were authenticated and classified into six subfamilies. Sixty-nine PAT genes were identified in upland cotton, mainly at the ends of its the 26 chromosomes of upland cotton. The majority of these genes are located in the nucleus of the plant. Gene structure analysis revealed that each member encodes a protein that which contains at least one DHHC structural domain. Cis-acting element analysis indicated that GhPATs genes are mainly involved in hormone production, light response and stress response. Gene expression pattern analysis indicated that most GhPATs genes were differentially expressed upon induction by pathogenic bacteria, drought, salt, hot and cold stresses, and some GhPATs could be induced by multiple abiotic stresses simultaneously. GhPATs genes showed different expression patterns in tissue-specific assays and were found to be preferentially expressed in roots, followed by expression in stems and leaves. Virus-induced gene silencing (VIGS) experiments showed that cotton was significantly less resistant to Verticillium dahliae when GhPAT27 was silenced. We conclude that the GhPAT27 gene, which mediates S-palmitoylation acetylation, may be involved in the regulation of upland cotton resistance to Verticillium wilt (VW). Overall, this work has provided a fundamental framework for understanding the latent capabilities of GhPATs and a solid foundation for molecular breeding and plant pathogen resistance in cotton.

DUSPs, a diverse group of protein phosphatases, play a pivotal role in orchestrating cellular growth and development through intricate signaling pathways. Notably, they actively participate in the MAPK pathway, which governs crucial aspects of plant physiology, including growth regulation, disease resistance, pest resistance, and stress response. DUSP is a key enzyme, and it is the enzyme that limits the rate of cell metabolism. At present, complete understanding of the DUSP gene family in cotton and its specific roles in resistance to Verticillium wilt (VW) remains elusive. To address this knowledge gap, we conducted a comprehensive identification and analysis of four key cotton species: Gossypium arboreum, Gossypium barbadense, Gossypium hirsutum, and Gossypium raimondii. The results revealed the identification of a total of 120 DUSP genes in the four cotton varieties, which were categorized into six subgroups and randomly distributed at both ends of 26 chromosomes, predominantly localized within the nucleus. Our analysis demonstrated that closely related DUSP genes exhibited similarities in terms of the conserved motif composition and gene structure. A promoter analysis performed on the GhDUSP gene promoter revealed the presence of several cis-acting elements, which are associated with abiotic and biotic stress responses, as well as hormone signaling. A tissue expression pattern analysis demonstrated significant variations in GhDUSP gene expression under different stress conditions, with roots exhibiting the highest levels, followed by stems and leaves. In terms of tissue-specific detection, petals, leaves, stems, stamens, and receptacles exhibited higher expression levels of the GhDUSP gene. The gene expression analysis results for GhDUSPs under stress suggest that DUSP genes may have a crucial role in the cotton response to stress in cotton. Through Virus-Induced Gene Silencing (VIGS) experiments, the silencing of the target gene significantly reduced the resistance efficiency of disease-resistant varieties against Verticillium wilt (VW). Consequently, we conclude that GH_A11G3500-mediated bispecific phosphorylated genes may serve as key regulators in the resistance of G. hirsutum to Verticillium wilt (VW). This study presents a comprehensive structure designed to provide an in-depth understanding of the potential biological functions of cotton, providing a strong foundation for further research into molecular breeding and resistance to plant pathogens.

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.

The protein phosphatase 2C (PP2C) gene family plays vital roles in plant growth and stress responses, yet remains inadequately characterized in cotton, particularly in Gossypium barbadense renowned for its superior fiber quality. Here, we identified 152 GbPP2Cs in Gossypium barbadense through genome-wide analysis and comparative genomics with three related cotton species (G. arboreum, G. raimondii, and G. hirsutum), identifying 105, 105, and 204 GbPP2Cs, respectively. GbPP2Cs show uneven chromosomal distribution with notable clustering on A05, evolutionary conservation in gene structure and motif composition, and predominant nuclear/chloroplast localization. Phylogenetic analysis classified them into 15 subfamilies showing conserved evolution. Protein enrichment revealed 15 GbPP2Cs involved in mitogen-activated protein kinase (MAPK) and hormone signaling pathways. Expression profiling revealed distinct members responsive to biotic/abiotic stresses, fiber development stages, and maturity. Notably, we discovered potential pleiotropic regulators including two genes (Gbar_D13G012000 and Gbar_A13G012360) co-regulating lint percentage and disease resistance, GbAIP1 coordinating fiber length-strength trade-off, and GbPP2C59 as a maturity negative candidate. These findings provide valuable genetic resources for cotton improvement.