Branch architecture and flowering time are separately regulated by GhFL and GhSP2 in cotton.
Crop flowering, a critical aspect of plant growth, is influenced by genetic and environmental factors. The indeterminate growth of cotton can lead to asynchronous flowering and boll setting. This study identified and characterized a determinate growth mutant, dt2, in Gossypium arboreum (G. arboreum) Shixiya 1 (SXY1) through EMS mutagenesis. The dt2 mutant exhibited main axis termination and shoot apical meristem (SAM) transition into flowers. Map-based cloning revealed a single nucleotide mutation in the coding sequence (CDS) of GaFL (Ga07G0556), a FLORICAULA/LEAFY homolog responsible for the determinate growth phenotype. Virus-induced gene silencing (VIGS) and protein interaction assays, including bimolecular fluorescence complementation, luciferase imaging, and GST pull-down, were employed to functionally characterize GaFL and its Gossypium hirsutum (G. hirsutum) homolog GhFL (Gh_A07G051000). These integrated approaches demonstrated the critical roles of both proteins in cotton development. The results indicated that GhFL interacts with GhSP2 (Gh_D09G150100), both of which negatively influence flowering time in cotton. Furthermore, a reduction of GhSP2 expression resulted in increased GhJAZ5 (Gh_D06G087500) expression while overexpression of GhJAZ5 in cotton promoted early flowering. This research uncovers the genetic basis of determinate growth and provides insights into the mechanisms of flowering regulation in cotton. These findings have significant implications for breeding strategies to improve cotton plant architecture.