AbstractBackgroundIn plants, 14-3-3 proteins, also called GENERAL REGULATORY FACTORs (GRFs), encoded by a large multigene family, are involved in protein–protein interactions and play crucial roles in various physiological processes. No genome-wide analysis of theGRFgene family has been performed in cotton, and their functions in flowering are largely unknown.ResultsIn this study, 17, 17, 31, and 17 GRFgenes were identified inGossypium herbaceum,G. arboreum,G. hirsutum, andG. raimondii, respectively, by genome-wide analyses and were designated asGheGRFs,GaGRFs,GhGRFs, andGrGRFs, respectively. A phylogenetic analysis revealed that these proteins were divided into ε and non-ε groups. Gene structural, motif composition, synteny, and duplicated gene analyses of the identifiedGRFgenes provided insights into the evolution of this family in cotton.GhGRFgenes exhibited diverse expression patterns in different tissues. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that the GhGRFs interacted with the cotton FLOWERING LOCUS T homologue GhFT in the cytoplasm and nucleus, while they interacted with the basic leucine zipper transcription factor GhFD only in the nucleus. Virus-induced gene silencing inG. hirsutumand transgenic studies inArabidopsisdemonstrated thatGhGRF3/6/9/15repressed flowering and thatGhGRF14promoted flowering.ConclusionsHere, 82GRFgenes were identified in cotton, and their gene and protein features, classification, evolution, and expression patterns were comprehensively and systematically investigated. The GhGRF3/6/9/15 interacted with GhFT and GhFD to form florigen activation complexs that inhibited flowering. However, GhGRF14 interacted with GhFT and GhFD to form florigen activation complex that promoted flowering. The results provide a foundation for further studies on the regulatory mechanisms of flowering.