FIGURE 1.

ALA inhibits ABA-induced stomatal closure. (A) Effects of different concentrations of ALA on ABA-induced stomatal closure. Isolated epidermal strips of wild-type Arabidopsis were incubated at 25°C in CO₂-free MES-KCl buffer without ABA (Control), or containing 10 μM ABA and different concentrations of ALA under light (240 μmol m^-2 s^-1), and stomatal apertures were determined after 2 h. (B) Time courses of stomatal responses to 0.5 mg L^-1 ALA, 10 μM ABA, or 10 μM ABA + 0.5 mg L^-1 ALA, respectively. Different letters on the same time point indicate significant differences at P < 0.01. (C,D) Guard cell viability observed by fluorescence microscopy. Isolated epidermal strips of wild-type Arabidopsis were incubated at 25°C in either buffer (Control), or containing 10 μM ABA, 0.5 mg L^-1 ALA, 10 μM ABA + 0.5 mg L^-1 ALA for 2 h under light (240 μmol m^-2 s^-1), respectively, and then loaded with 0.25 μM fluorescein diacetate for 5 min. Images (C) obtained with a Nikon-TE300 digital camera depict one representative picture from three independent experiments. The upper image is in bright field and the lower image is in fluorescence. Scale bar: 100 μm. Cell viability (D) was quantified by counting the percentage of fluorescent guard cells relative to total guard cells in the bright field. (E,F) Levulinic acid (LA) inhibits ABA-induced stomatal closure. Isolated epidermal strips of wild-type Arabidopsis were incubated at 25°C in either buffer (Control), or containing 10 μM ABA, 1 mM LA (an analog of ALA which can block ALA metabolism), 10 μM ABA + 1 mM LA for 1 h under light (240 μmol m^-2 s^-1), respectively, and then images (E) were recorded and stomatal apertures (F) were determined. Scale bar: 10 μm. Values in (A,B,F) are the means of 90 measurements ± SE from three independent experiments. Different small letters represent significant differences among treatments (P < 0.01).