Growth Regulating Factors (GRFs) are plant-specific transcription factors that play crucial roles in regulating growth and development throughout the plant life cycle. A total of 34 Gossypium hirsutum GRF family genes were identified at the genome-wide level, which were unevenly distributed on 19 chromosomes, and were predicted to be mainly localized in the nucleus and plasma membrane. The number of GRF family genes varied greatly among different species, and they were categorized into four subfamilies (I–IV) according to their phylogenetic relationships. The G. hirsutum GRF genes possessed specific highly conserved structural domains, Trp-Arg-Cys motif (WRC) and Gln, Leu, Gln motif (QLQ), and structural analysis of the genes revealed that they contained 1–23 exons, and most of them contained UTRs. Intraspecies covariance analysis revealed that the GRF genes expanded in G. hirsutum by segmental duplication. The promoter region of the G. hirsutum GRF gene contained a large number of adversity stress response elements, as well as a small number of hormone response elements and growth and development-related response elements. Transcriptome data showed that the expression of G. hirsutum GRF genes was significantly higher in leaves than in other tissues, and some GRF genes responded to a variety of abiotic stresses. Additionally, transcriptomic sequencing revealed significantly higher expression levels of GhGRFs (e.g., GhGRF13/14/18) in embryonic callus (EC) compared to non-embryonic callus (NEC). This differential expression was validated by RT-qPCR, which confirmed that GhGRF13/14/16/20 were significantly upregulated in EC relative to NEC. These findings provide valuable candidate genes and molecular insights for improving G. hirsutum regeneration efficiency and yield-related traits through genetic manipulation, thereby accelerating the molecular breeding of elite G. hirsutum varieties.

Vascular Plant One-Zinc finger (VOZ) transcription factors are pivotal regulators of plant growth and stress adaptation, yet their functional roles in Gossypium hirsutum, a key fiber crop, remain poorly characterized. In this study, we systematically identified six VOZ genes in G. hirsutum and conducted a comprehensive analysis of their phylogenetic relationships, genomic distribution, promoter architecture, and expression profiles. Phylogenetic classification placed the GhVOZ proteins into three distinct clades, and chromosomal localization revealed that family expansion was likely driven by segmental duplication events. Promoter analysis uncovered an abundance of stress-related cis-regulatory elements, suggesting a potential role in abiotic stress signaling. Consistent with this, expression profiling demonstrated that GhVOZ1/3, GhVOZ2/4/5, and GhVOZ6 were specifically induced under drought, salt, and cold stress, respectively, with qRT-PCR further confirming their tissue-specific dynamic regulation under salt treatment. Furthermore, the GhVOZ family exhibited stage-specific expression patterns during somatic embryogenesis. GhVOZ1, GhVOZ3, and GhVOZ4 were upregulated at the early induction phase, implicating them in the initiation of cell reprogramming. In contrast, GhVOZ2 and GhVOZ4 showed sustained expression in embryogenic callus at later stages, suggesting a role in maintaining embryogenic competence, whereas GhVOZ5—preferentially expressed in non-embryogenic callus—may act as a repressor of embryogenesis. Synteny analysis further highlighted evolutionary conservation and subgenomic divergence of VOZ genes in G. hirsutum. Collectively, these findings establish GhVOZs as key regulators integrating abiotic stress response and somatic embryogenesis, providing a genetic framework for future functional studies and crop improvement.

MLO (Mildew Resistance Locus O) genes encode seven-transmembrane proteins that function as critical regulators of powdery mildew resistance and abiotic stress responses. Despite their established importance, the MLO gene family in Gossypium hirsutum L. has not been systematically investigated under salt stress conditions. Here, we performed genome-wide identification of 46 GhMLO members using Hidden Markov Model and BLAST searches based on the latest cotton genome assembly. Phylogenetic analysis classified these genes into four distinct subfamilies. Transmembrane topology and conserved domain analyses revealed that all GhMLO proteins contain typical MLO domains and transmembrane structures, maintaining high structural similarity with dicotyledonous model plants. Synteny analysis demonstrated that the expansion of the GhMLO family was primarily driven by segmental and tandem duplications. Integration of transcriptomic data from the COTTONOMICS database revealed tissue-specific expression patterns, with higher transcript abundance in receptacles, stems, and roots, but lower levels in stamens and petals. Salt, drought, and cold stress treatments induced upregulation of GhMLO family members, with most genes showing increased expression over time. RT-qPCR analysis validated that five candidate GhMLO genes were significantly upregulated under salt stress. In summary, this study provides a comprehensive genome-wide characterization of the GhMLO gene family, elucidating their phylogenetic relationships and expression dynamics, which establishes a theoretical basis for identifying key regulatory genes involved in abiotic stress responses and offers novel genetic resources for improving stress tolerance in cotton molecular breeding.

The S-locus-specific glycoprotein/EP1 gene family encodes a class of epidermis-specific secretory glycoproteins involved in plant responses to abiotic stress. However, their role in cotton (Gossypium hirsutum) drought response remains unclear. Here, we identified 12 GhEP1 genes from the G. hirsutum genome, clustered into three subfamilies. Structural predictions indicated that most GhEP1 proteins are hydrophilic and localised to the outer side of the plasma membrane or nucleus, with a few containing transmembrane domains. Collinearity analysis revealed that segmental and tandem duplications drove family expansion, with a high degree of collinearity conservation among dicotyledonous plants. Transcriptomic data showed generally low GhEP1 expression across tissues, but GhEP1-2, GhEP1-6, GhEP1-7 and GhEP1-8 responded significantly to NaCl, PEG-induced drought and cold stresses. qRT-PCR further confirmed that the expression of most GhEP1 genes was significantly up-regulated in roots under drought stress, while GhEP1-3 was induced in leaves, indicating tissue-specific regulatory divergence. This study systematically characterises the GhEP1 family and provides potential genetic resources for improving drought tolerance in cotton.

In this study, we performed the first genome-wide identification and characterization of the cinnamoyl-CoA reductase (CCR) gene family in upland cotton (Gossypium hirsutum), focusing on its potential association with stem strength. We identified 76 GhCCR genes and classified them into four subfamilies. We then analyzed their evolutionary relationships, conserved domains, synteny, promoter cis-elements, and expression patterns. All GhCCR proteins possess the NADB_Rossmann superfamily domain, and family expansion appears to have been driven mainly by segmental and tandem duplications. A small number of GhCCR genes showed relatively high expression in leaf, pistil, and torus tissues, while genes such as GhCCR3/9/10 exhibited elevated transcript levels under abiotic stress conditions. RT-qPCR results indicated that three candidate GhCCR genes (GhCCR25, GhCCR52 and GhCCR64) were significantly more highly expressed in multiple tissues of the stiff-stem line JY-25 than in the soft-stem line JR-15. Together, these findings suggest that GhCCR genes may contribute to the regulation of growth, development, and stress adaptation in G. hirsutum. However, direct biochemical or genetic validation is required to confirm their functional roles in lignin biosynthesis and stem rigidity.

Verticillium wilt, caused by Verticillium dahliae, is a major constraint on cotton production. The GASA (Gibberellic Acid-Stimulated in Arabidopsis) proteins are cysteine-rich peptides induced by gibberellin signaling; however, their systemic role in upland cotton (Gossypium hirsutum) resistance to Verticillium wilt remains elusive. Here, we identified 40 GhGASA members from the G. hirsutum genome, which were clustered into three subfamilies. All members possessed a typical gibberellin-regulated domain, and segmental duplication was the primary driver of family expansion. Transcriptomic analyses revealed tissue-specific expression, with high abundance in pistils and roots, and distinct responsive patterns to salt, drought, and cold stresses. Quantitative real-time PCR (qRT-PCR) showed that multiple GhGASA genes were significantly upregulated following V. dahliae inoculation, with transcript levels being much higher in roots and stems than in leaves. Notably, GhGASA17, harboring ethylene-responsive elements, was identified as a key candidate involved in the defense response. Overall, this study provides a comprehensive characterization of the GhGASA gene family, offering a theoretical foundation for understanding its regulatory functions in Verticillium wilt resistance and delivering potential genetic resources for molecular breeding in cotton.