Add time:08/10/2019         Source:sciencedirect.com

Previous attempts to measure cytoplasmic Ca2+ in plant cells using the new generation of fluorescent probes, indo-1 and fura-2, have been unsuccessful. We investigated the use of indo-1 and fura-2 to measure cytoplasmic Ca2+ in barley aleurone protoplasts and found that indo-1 could be successfully used when it was loaded into protoplasts in the Ca2+-sensitive form. The acetoxymethyl esters of both dyes accumulated in aleurone protoplasts, but fura-2 was sequestered in the vacuole and indo-1 was not adequately hydrolyzed. We developed a non-disruptive method for loading the Ca2+-sensitive form of indo-1 into aleurone protoplasts in mildly acidic solutions. Using this approach, protoplasts accumulate indo-1 in a pH-dependent manner. The accumulated dye is Ca2+-sensitive, it is not sequestered in vacuoles or the endomembrane system, and it is not rapidly secreted. Fluorescence from indo-1 in individual cells was quenched by Mn2+ in the presence of digitonin. We estimate the cytoplasmic Ca2+ concentration in aleurone protoplasts to be approximately 250 nM. The Ca2+ ionophore, ionomycin does not induce changes in the fluorescence of protoplasts loaded with indo-1, but fluorescence changes could be induced by changes in extracellular Ca2+ in the presence of digitonin. We conclude that the strategy of loading indo-1 at acidic pH provides a useful means of measuring cytoplasmic Ca2+ in the barley aleurone that may also be applicable to other types of plant cells.

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