Cis-regulatory architecture downstream of FLOWERING LOCUS T underlies quantitative control of flowering

The FLOWERING LOCUS T (FT) gene is a central integrator of floral induction in Arabidopsis thaliana, with expression tightly regulated by complex transcriptional networks. Using CRISPR/Cas9 genome editing, we dissected the functional architecture of the FT downstream region and reveal that a 2.3-kb region immediately downstream of the FT coding sequence containing the Block E enhancer is essential for proper FT expression and flowering. Fine-scale deletions revealed a 63-bp core module with adjacent CCAAT- and G-boxes, whereas other conserved motifs had minor, context-dependent effects. We also uncovered a cryptic CCAAT-box module that becomes active when repositioned, coinciding with increased transcription factor binding and local chromatin accessibility, indicating that enhancer function is governed by local chromatin and motif context. The cis-regulatory logic revealed here provides insights into manipulating gene expression through the architecture and spatial arrangement of enhancer elements, potentially applicable beyond flowering genes or plant species.