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The Actin Depolymerizing Factor (ADF) protein, highly conserved among eukaryotes, is essential for plant growth, development, and stress responses. Cotton, a vital economic crop with applications spanning oilseed, textiles, and military sectors, has seen a limited exploration of its ADF gene family. This research has identified 118 unique ADF sequences across four principal cotton species: Gossypium hirsutum L., Gossypium barbadense Linn, Gossypium raimondii, and Asiatic cotton. The study found that the structural domains and physicochemical properties of these proteins are largely uniform across species. The ADF genes were classified into four subfamilies with a notable expansion in groups III and IV due to tandem and chromosomal duplication events. A thorough analysis revealed a high degree of conservation in gene structure, including exon counts and the lengths of introns and exons, with the majority of genes containing three exons, aligning with the characteristics of the ADF family. RNA-seq analysis uncovered a spectrum of responses by GhADFs to various abiotic stresses with GhADF19 showing the most significant reaction. Virus-induced gene silencing (VIGS) experiments were conducted to assess the role of GhADF19 in plant growth under abiotic stress. The results demonstrated that plants with silenced GhADF19 exhibited significantly slower growth rates and lower dry weights when subjected to cold, salt, and drought stress compared to the control group. This marked reduction in growth and dry weight under stress conditions highlights the potential importance of GhADF19 in stress tolerance mechanisms. [ABSTRACT FROM AUTHOR]