AP39-induced stomatal closure requires complex IV activity. (A), Scheme showing the sites of action of the different mETC inhibitors; Epidermal strips from the abaxial side of five to six weeks old Arabidopsis Col-0 leaves were floated in OB (5 mM MES, 50 mM KCl, pH = 6.1) for 3 h in the light and then they were treated with Dimethyl sulfoxide (DMSO) 0.01% (v/v) (Mock), 100 nM AP39, or they were pre-incubated for 30 min with the different mETC inhibitors: (B) 1 µM Rotenone (Rtn), (C) 100 µM 2-TTFA, (D) 50 µM Antimycin A (AA) or (E) 1 mM potassium cyanide (KCN), and subsequently treated or not with AP39 for 90 min. The values of stomatal aperture are expressed in microns (µm) and are represented as points in the boxplots where the box is bound by the 25th to 75th percentile. The line in the middle is the median, the darker point is the mean and whiskers span 10th to 90th percentile. The black points represent outliers. Data is the result from at least three independent assays. Letters indicate statistical differences among treatments (Tukey's Method, confidence level = 0.95). IMS, intermembrane space.