Figure 5.

ROS generation is required for the hy1- and ABI4-mediated Arabidopsis stomatal closure. A, Confocal analysis of ROS production in the stomata of the wild type and hy1-100, abi4, and hy1-100/abi4 mutants upon 10 μm ABA for 20 min. ROS production of the wild type at 0 min was regarded as 100%. B, Histochemical detection of ABA-induced hydrogen peroxide (H₂O₂) and superoxide radical production. Four-week-old seedling leaves of each ecotype were treated with 10 μm ABA for 2 h and stained with DAB or NBT (left). Bar = 1 cm. Randomly selected leaves were used for quantification (right), separately taking values of corresponding untreated control lines (−ABA) as 100%. C, Stomatal aperture (n = 50 from three independent experiments). Arabidopsis leaves of each ecotype were treated with ABA (10 μm) or H₂O₂ (100 μm) in MES-KCl buffer for 2 h after 0.5 h of pretreatment with diphenyleneiodonium (DPI; 50 μm), catalase (CAT; 60 units mL⁻¹), or ascorbic acid (AsA; 100 μm). Data are means ± se from at least three independent experiments. Differences among treatments were analyzed by one-way ANOVA, taking P < 0.05 as significant according to Tukey’s multiple range test. D, Representative images are shown. Bar = 10 μm.