Determination of intracellular Ca2+ in cells of intact wheat roots:: loading of acetoxymethyl ester of Fluo-3 under low temperature

Loading of Ca2+-sensitive fluorescent probes into plant cells is an essential step to measuring activities of cytoplasmic free Ca2+ ions with a fluorescent imaging technique. A major barrier to the loading of the fluorescent probes into plant cells using the acetoxymethyl (AM) esters of the Ca2+-sensitive dyes is the presence of cell-wall associated esterases. These esterases hydrolyse the esterified form of the fluorescent probes, rendering the probes membrane-impermeable. A novel non-invasive loading protocol was described in this paper to load the Ca2+-sensitive fluorescent probe Fluo-3/AM ester into apical cells of intact wheat roots by incubating the roots in Fluo-3/AM ester solution at 4 degrees C for 2 h followed by 2-h incubation in the dye-free solution at 20 degrees C. The incubation at low temperature inhibited extracellular hydrolysis of Fluo-3/AM ester but had less effect on diffusion of membrane-permeable Fluo-3/AM ester across the plasma membrane, because hydrolysis of Fluo-3/AM ester by extracellular esterases is a chemical process (high Q(10)), while diffusion of Fluo-3/AM across the plasma membrane is a physical process (low Q(10)). The Fluo-3/AM ester, accumulated in the root cells during the low temperature incubation, was then cleaved by intracellular esterases during the incubation at 20 degrees C, releasing the membrane-impermeable Ca2+-sensitive Fluo-3 in the cytoplasm. The root cells loaded with Fluo-3 showed strong intracellular fluorescence under confocal microscopy. The fluorescence from the root cells was sensitive to the Ca2+ ionophore and hydrogen peroxide, indicating that the intracellular fluorescence was due to intracellular Ca2+ ions.