Measurement of cytoplasmic calcium in aleurone protoplasts using indo-1 and fura-2.
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.
Department of Botany, University of California, Berkeley 94720.
D S
DS
Bush
R L
RL
Jones
D001572:Benzofurans; D002118:Calcium / Q000032:analysis*; D003593:Cytoplasm / Q000032:analysis; D005456:Fluorescent Dyes; D005470:Fluorometry; D016257:Fura-2; D001467:Hordeum; D006863:Hydrogen-Ion Concentration; D007211:Indoles; D011523:Protoplasts / Q000032:analysis* / Q000201:enzymology
D001572:Benzofurans; D005456:Fluorescent Dyes; D007211:Indoles; C048960:indo-1; D002118:Calcium; D016257:Fura-2