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

Abstract

SummaryLoading 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°C for 2 h followed by 2‐h incubation in the dye‐free solution at 20°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 Q10), while diffusion of Fluo‐3/AM across the plasma membrane is a physical process (low Q10). 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°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.