Mapping of the FD binding site in the AP1 promoter. Mapping of the FD binding site in the AP1 promoter. A, Normalized reads from six ChIP-seq experiments mapped on the AP1 locus. Horizontal bars below the peaks indicate regions of significant enrichment. The position of the summit is indicated for each peak. The C-box previously implicated in FD binding is located upstream (to the right) of peak summits in all six experiments. B, Nucleotide sequence encompassing the six peak summits shows several palindromic regions representing putative binding sites of FD on the AP1 promoter. The distance between the closest potential FD binding site under the ChIP-seq peaks and the C-box is 92 bp. Black triangles indicate the summits of the six separate ChIP-seq experiments. Putative FD binding sites are underlined and numbered from 1 to 4. C, EMSA of the phosphomimic version of FD-C (FD-C T282E) in combination with 14-3-3v, FT, and TFL1 using the four putative binding sites reported in (B). Free probes are not visible because gels were running longer to maximize the distance between shifted probes. Colored squares indicate shifted probes. D, Comparison of the probes used for EMSA: the G-box in SEP3 promoter (Fig. 2) and the binding site 2 in AP1 promoter. The putative FD binding site (site 2) in the AP1 promoter, which is also conserved in SEP3 promoter where it overlaps with the G-box, is marked in red. Silvio Collani et al. Plant Physiol. 2019;180:367-380 ©2019 by American Society of Plant Biologists