The Fruit-Weight 2.2-Like (FWL) gene family plays crucial roles in determining organ size and stress responsiveness in plants. However, the FWL family members remain largely unexplored in Gossypium species. In this study, we identified and characterized the FWL family in Gossypium hirsutum to investigate their gene expansion, functional evolution, and potential association with key agronomic traits. A total of 51 GhFWLs were identified, and their gene structures, domain compositions, and phylogenetic relationships were comprehensively analyzed. Expression profiling under heat, cold, salt, and drought stress conditions revealed that most GhFWLs participate in abiotic stress response pathways. Haplotype-based association analysis revealed significant associations for 14 GhFWLs with fiber-related traits, suggesting their potential functions in regulating fiber development. Furthermore, silencing the family member GhMCA1 provided supporting evidence for its role in salt stress tolerance. Our findings provided insightful information about the FWL gene family in G. hirsutum and highlighted candidate genes for improving fiber-related traits and stress tolerance through molecular breeding approaches.

Enhancing cotton fiber quality is essential for improving textile production. The transition from fiber elongation to secondary cell wall (SCW) synthesis is a critical developmental phase that determines both fiber length and strength. Although MYB transcription factors regulate the process, their precise functions remain incompletely characterized. In this study, we employed genome-wide identification and expression screening, together with candidate gene association analysis, to identify GhMYS1. This gene, selected from 186 cotton 1R-MYB transcription factors, is specifically expressed during the fiber elongation stage (5-15 days post-anthesis (DPA)). CRISPR/Cas9-mediated knockout of GhMYS1 significantly reduced fiber length, increased cell wall thickness, and raised the micronaire value compared to the control. Concurrently, expression of SCW synthesis-related genes GhCESA4, GhCESA8, and GhCOBRA4D was markedly up-regulated, accompanied by a pronounced increase in cellulose content. Yeast one-hybrid (Y1H), electrophoretic mobility shift (EMSA), and luciferase (LUC) assays demonstrated that GhHOX3 binds to the L1-box motif in the GhMYS1 promoter and activates its transcription in fibers and trichomes. The nuclear-localized GhMYS1 protein contains a C-terminal EAR motif with transcriptional repression activity. Through its N-terminal MYB domain, GhMYS1 binds directly to the promoters of GhCESA4 and GhCESA8, repressing their transcription and thereby delaying the onset of SCW deposition to facilitate fiber elongation. We propose a GhHOX3-GhMYS1-GhCESA4/8 regulatory module that governs the transition from fiber elongation to SCW synthesis. This study provides valuable genetic resources for molecular design breeding of cotton fiber quality and offers insights into the function of 1R-MYB transcription factors in fiber development.