Figure 4.

Coumarin Biosynthesis Is Affected in the bhlh121 Loss-of-Function Mutants.

(A) Relative expression of IRT1 and FRO2 (high-affinity Fe uptake system).

(B) Relative expression of F6’H1, S8H, and CYP82C4 (coumarin biosynthesis).

(C) Relative expression of BGLU42 and PDR9 (coumarin secretion).

(D) Relative expression of MYB10 and MYB72 (transcriptional control of coumarin biosynthesis and secretion).

(A) to (D) Relative expression was determined by RT-qPCR in 1-week-old Arabidopsis seedlings grown on Fe-sufficient or Fe-deficient medium. Means within each condition with the same letter are not significantly different according to one-way ANOVA followed by post hoc Tukey test, P < 0.05 (n = 3 technical repeats from one representative experiment). Error bars show ±sd. Each experiment (biological repeat) comprised pooled RNA extracted from ∼30 seedlings and was independently repeated three times.

(E) (Top) Schematic representation of the coumarin biosynthetic pathway. (Bottom) Representative absorbance chromatograms obtained at 338 nm for root extracts of Arabidopsis wild type (WT), cyp8c24-1, f6’h1-1, and bhlh121-2 mutant seedlings grown for 7 d on half-strength Murashige and Skoog medium agar plates at pH 5.5. +Fe, control; −Fe, Fe deficiency. Numbered peaks correspond to sideretin-glycosides (1 and 2), scopolin: scopoletin glycoside (3), sideretin (4), and fraxin: fraxetin glycoside (5).