Determining Apoplastic pH Differences in Pea Roots by Use of the Fluorescent Dye Fluorescein

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Abstract
The uptake of the fluorescent dye fluorescein (uranin) was used to determine apoplastic pH differences in pea roots. Fluorescein, an acidic dye (pKa≊4·9), is able to pass cell membranes in its undissociated form. The uptake was studied in roots of young and older pea seedlings growing in solution or in sand, respectively. Following 16 h of incubation in an unbuffered 0·01% (w/v) fluorescein solution with a pH of 7·9, the dye only accumulated in cells of the root apex, as was visible under ultraviolet irradiation. Dye accumulation was prevented when the proton buffering strength of the incubation medium was increased. Under more acidic conditions (pH 5·0) it also accumulated in the root base. Fluorescein uptake was strongly suppressed by CCCP, reduced by DNP and stimulated by KCl. The results indicate that net proton extrusion activity is the main mechanism for generating a low apoplastic pH. Cross-sections of roots of seedlings, incubated in the unbuffered fluorescein-containing medium, showed discriminative dye uptake by the different root tissues and cell types, suggesting that net proton extrusion activity may vary with the cell type and its location in the root. The method can be used to localize root regions, tissues, and cells inside the root, with a relatively low apoplastic pH.

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