Figure 4: FLP and ARF7 are direct regulators of PIN3 expression.

(a) Schematic presentation of the PIN3 promoter (1.8 kb upstream of the translational start site at position (+1) with indication of the regions targeted for ChIP analysis (PIN3_P1 and PIN3_P2), the fragments used for yeast-one-hybrid (FR1-5) and the presence of auxin response elements (AuxREs, black triangles) and FLP binding sites (FBS, grey triangles) in fragment FR2. (b, c) Yeast-one-hybrid analysis of the interaction of (b) FLP with PIN3 promoter fragments (FR1-5), FBS1, a mutated FBS1 (mFBS1) and FBS2, and (c) of ARF7 with PIN3 promoter fragment FR2, its internal AuxREs (AuxRE1-3) and corresponding mutated versions (mAuxRE1-3). Left is yeast grown on SD–Leu medium in absence of Aureobasidin A (AbA). Right are the corresponding yeast strains in a dilution series grown on SD–Leu medium in the presence of 100 ng ml⁻¹ AbA, a concentration where no autoactivation was detected for any of the strains. (d,e) Enrichment of the indicated DNA fragments (PIN3_P1, and PIN3_P2) following ChIP using anti-GR antibodies with (d) proFLP::FLP-GR/flp-1 myb88 or (e) proARF7::ARF7-GR/arf7 arf19 treated for 6 h with or without DEX. A fragment from the ACTIN2 promoter was tested as a negative control. Data are presented as means ± s.d. **P<0.01, ***P<0.001 (Student’s t-test). The experiment was repeated three times with similar results.