Cytoplasmic calcium levels in protoplasts from the cap and elongation zone of maize roots

Calcium has been implicated as a key component in the signal transduction process of root gravitropism. We measured cytoplasmic free calcium in protoplasts isolated from the elongation zone and cap of primary roots of light-grown, vertically oriented seedlings of Zea mays L. Protoplasts were loaded with the penta-potassium salts of fura-2 and indo-1 by incubation in acidic solutions of these calcium indicators. Loading increased with decreasing pH but the pH dependence was stronger for indo-1 than for fura-2. In the case of fura-2, loading was enhanced only at the lowest pH (4.5) tested. Dyes loaded in this manner were distributed predominantly in the cytoplasm as indicated by fluorescence patterns. As an alternative method of loading, protoplasts were incubated with the acetoxymethylesters of fura-2 and indo-1. Protoplasts loaded by this method exhibited fluorescence both in the cytoplasm and in association with various organelles. Cytoplasmic calcium levels measured using spectrofluorometry, were found to be 160 +/- 40 nM and 257 +/- 27 nM, respectively, in populations of protoplasts from the root cap and elongation zone. Cytoplasmic free calcium did not increase upon addition of calcium to the incubation medium, indicating that the passive permeability to calcium was low.