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 bel ieved to regulate acidity within the vacuolar lumen. However, the abil ity 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 ba ses, or elicition of wound responses after impaling cells with a pH-se nsing microelectrode. The goal of our present study was to identify a fluorescent pH probe that is localized in vacuoles of (Zea mays L.) ro ot cells. When excised corn root segments were exposed to the ester de rivative of 2',7' bis-2-(carboxyethyl)-5-(and-6)-carboxy-fluorescein ( BCECF) for 30 to 60 min, the root-hair cells preferentially became flu orescent. The distribution of the fluorescence within cells followed c losest to that of the vacuole when fluorescent and phase contrast imag es 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-la beled root-hair cells indicated that the majority of the dye was local ized in a compartment with an average pH of 5.8. The above data indica ted that the BCECF was localized in the vacuoles of root-hair cells fr om maize seedlings, and the changes in the fluorescence ratio of BCECF may provide a means of monitoring changes of vacuolar pH in situ.