SELECTIVE ACCUMULATION OF THE FLUORESCENT PH INDICATOR, BCECF, IN VACUOLES OF MAIZE ROOT-HAIR CELLS

The vacuolar-type H+-ATPase localized on the tonoplast membrane is believed to regulate acidity within the vacuolar lumen. However, the ability to monitor vacuolar pH in situ can be difficult because of the use of destructive assays based on cell sap pH or accumulation of weak bases, or elicition of wound responses after impaling cells with a pH-sensing microelectrode. The goal of our present study was to identify a fluorescent pH probe that is localized in vacuoles of (Zea mays L.) root cells. When excised corn root segments were exposed to the ester derivative of 2',7' -bis-2-(carboxyethyl)-5-(and-6)-carboxy-fluorescein (BCECF) for 30 to 60 min, the root-hair cells preferentially became fluorescent. The distribution of the fluorescence within cells followed closest to that of the vacuole when fluorescent and phase contrast images were compared. The ratio of the fluorescence intensities at 535 nm using excitation wavelengths of 440 and 490 nm varied directly with pH between 4.5 and 7.5. The ratio of fluorescent intensities of BCECF-labeled root-hair cells indicated that the majority of the dye was localized in a compartment with an average pH of 5.8. The above data indicated that the BCECF was localized in the vacuoles of root-hair cells from maize seedlings, and the changes in the fluorescence ratio of BCECF may provide a means of monitoring changes of vacuolar pH in situ.