Media pH and Fe availability control the expression of coumarin-related genes. Plants were directly germinated for 14 d on either Fe-deficient media (with 120 µM ferrozine) or media supplemented with either Fe-EDTA (40 µM), FeEDDHA (40 µM), FeCl3 (40 µM), or precipitated Fe(OH)3 (40 µM) at pH 5.5 or pH 7.0. A, Expression of MYB72, BGLU42, PDR9, F6′H1, COSY, CYP82C4, and S8H in roots. Gene expression was determined using the ΔCT method with elongation factor 1 alpha as an internal control. Each bar represents the mean ± se of three independent experiments. Distinct letters above the bar graph indicate significant differences (P < 0.05) in two-way ANOVA followed by Tukey’s HSD test. B, Expression of S8H protein (∼40 kDa) in root samples. Actin (∼45 kDa) was used as loading control. Per lane, 20 µg of total protein were loaded. An image representative of three independent experiments is shown.