Figure 2.

ABA Induces Vacuolar Acidification in Fava Bean Stomatal Guard Cells.

(A) to (D) Vacuolar acidification as observed using acridine orange (AO). Guard cells were stained with 50 μM AO for 100 min before ABA treatment and the fluorescence ratio of red (R) to green (G) emissions was obtained using a Zeiss LSM510 Meta microscope. Representative R/G ratio images obtained at the indicated time points are shown ([A] to [C]). The R/G ratio is displayed in pseudocolor. Regions with the highest R/G ratio (more acidic) are in white, while those with the lowest R/G ratio are in dark blue. Bars = 10 μm.

(D) Vacuolar acidification was quantified as the mean values of R/G ratios in vacuolar lumens. The results are from three independent experiments (mean ± se, 30 ≤ n ≤ 44). A.U., arbitrary unit.

(E) to (G) Vacuolar acidification observed with LysoSensor Green DND-189 (lysosensor). Guard cells were stained with lysosensor (4 μM) for 20 min before analysis. Representative confocal images of guard cells stained with lysosensor without (0 min) or with (30 and 60 min) 10 μM ABA treatment are presented. Bars = 10 μm.

(H) The extent of vacuolar acidification was quantified as the mean values of green fluorescence intensity of lysosensor in vacuolar lumens. Higher values indicate more acidic conditions. The results are from six independent experiments (mean ± se, 71 ≤ n ≤ 84).