Measurement of pH in Guard Cells Using a Confocal Laser Scanning Microscope

Abstract

Two experiments were conducted to establish a method to measure pH in guard cells in situ for analysis of the relationship between pH changes in guard cells and stomatal aperture changes. To measure pH in guard cells of potato plants (Solanum tuberosum L. cv. Benimaru), ratio imaging was conducted using a confocal laser scanning microscope system with SNARF-1/AM as a pH fluorescent probe. Emitted fluorescence was divided into two ranges (580 and 640 nm) and the ratio of fluorescent intensities at 580 and 640 nm (F580/F640) was calculated. In vivo and in vitro calibrations showed an increase in the F580/F640 ratio with decrease in pH. In experiment 1, the relationship between stomatal aperture and pH in guard cells was investigated when the leaf disc was bathed in low pH solution (citric acid solution: pH 2.7). It was observed that stomata opened and F580 increased. Ratio imaging revealed that stomata with guard cells, which were initially at pH 7.0, opened as pH in the guard cells decreased. In experiment 2, to measure pH in guard cells for an attached leaf, 4.4 mM SNARF-1/AM solution was injected into the stem of a potato plant. However, the pH in the guard cells could not be measured because of low fluorescence intensity.