Abstract Background The FLOWERING LOCUS T ( FT ) gene is a central integrator of floral induction in Arabidopsis thaliana , with its precise expression controlled by complex transcriptional networks. While upstream regulatory regions are well-studied, the role of downstream cis -regulatory elements in modulating FT expression remains poorly characterized. Results Systematic dissection of the FT downstream region in its native chromosomal context using CRISPR/Cas9-mediated genome editing provides genetic evidence that a 2.3-kb sequence, encompassing the Block E enhancer immediately adjacent to the FT coding sequence, is essential for proper FT expression and timely flowering. Fine-scale deletions within Block E reveal that a 63-bp sequence containing one CCAAT-box and one G-box, both closely spaced, forms a core functional module, whereas other conserved motifs contribute modestly in a context-dependent manner. Strikingly, a cryptic CCAAT-box module downstream of Block E that becomes active when repositioned. This coincides with increased transcription factor occupancy and local chromatin accessibility. Conclusions Our work reveals that quantitative FT expression and flowering time are governed by the spatial organization and chromatin context of downstream cis -regulatory elements. The positional sensitivity and modular logic of these elements provide framework for understanding and engineering quantitative gene regulation through targeted cis-regulatory design, a concept broadly applicable across diverse developmental systems.
Zhou et al. (Mon,) studied this question.