Functional Dissection of cis-Regulatory Elements Controlling the Rice Florigen Gene RFT1

The florigen gene RFT1 plays a critical role in regulating flowering time under artificial long-day (LD) conditions and functions prior to another florigen gene, Hd3a , under natural conditions in temperate regions of rice cultivation. Thus, RFT1 is important for the adaptation of rice to temperate areas of Asia. Compared with Hd3a , RFT1 is less strongly repressed under LD conditions. However, the cis -regulatory mechanisms governing RFT1 expression remain poorly understood. Here, we generated a series of CRISPR-Cas9 deletion mutants spanning an approximately 10 kb noncoding region surrounding RFT1 . Expression and heading-date analyses identified multiple functional cis -regulatory regions involved in RFT1 activation. Combined analyses using mutants of the upstream Ghd7 regulator and Ehd1 -overexpressing lines demonstrated that these regions function downstream of Ehd1 and Ghd7. Further fine-scale deletion and transient reporter assays revealed the importance of two Ehd1-binding sites among many candidate sites. Our results support a multilayered cis-regulatory architecture at the RFT1 locus and suggest evolutionary divergence in the regulation of FT orthologs between rice and Arabidopsis , as canonical CORE and CCAAT motifs appear to play only limited roles in RFT1 regulation. Together, these findings provide a mechanistic basis for the quantitative tuning of flowering time in rice cultivated in temperate regions.