DETERMINATION OF INTRACELLULAR CA2- LOADING OF ACETOXYMETHYL ESTER OFFLUO-3 UNDER LOW-TEMPERATURE( IN CELLS OF INTACT WHEAT ROOTS )

Loading of Ca2+-sensitive fluorescent probes into plant cells is an es sential step to measuring activities of cytoplasmic free Ca2+ ions wit h a fluorescent imaging technique. A major barrier to the loading of t he fluorescent probes into plant cells using the acetoxymethyl (AM) es ters of the Ca2+-sensitive dyes is the presence of cell-wall associate d esterases. These esterases hydrolyse the esterified form of the fluo rescent 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 int act 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 soluti on at 20 degrees C. The incubation at low temperature inhibited extrac ellular hydrolysis of Fluo-3/AM ester but had less effect on diffusion of membrane-permeable Fluo-3/AM ester across the plasma membrane, bec ause hydrolysis of Fluo-3/AM ester by extracellular esterases is a che mical process (high Q(10)), while diffusion of Fluo-3/AM across the pl asma membrane is a physical process (low Q(10)). The Fluo-3/AM ester, accumulated in the root cells during the low temperature incubation, w as then cleaved by intracellular esterases during the incubation at 20 degrees C, releasing the membrane-impermeable Ca2+-sensitive Fluo-3 i n the cytoplasm. The root cells loaded with Fluo-3 showed strong intra cellular fluorescence under confocal microscopy. The fluorescence from the root cells was sensitive to the Ca2+ ionophore and hydrogen perox ide, indicating that the intracellular fluorescence was due to intrace llular Ca2+ ions.