Cytoplasmic calcium measurements in intact higher plant cells: Results from fluorescence ratio imaging of fura-2

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Abstract
Roots of seedling tomato (Lycopersicon esculenturn) and oilseed rape (Brassica napus) plants were grown on agarose slopes formed on microscope slides. Root hairs were used for microelectrode insertions, for observations of the rate of cytoplasmic streaming and for measurements of fluorescence of the calcium indicator fura-2. The resting potential across the plasma membrane of root hair cells was between −140 and −160mV in both species. Insertion of microelectrodes caused immediate cessation of cytoplasmic streaming; 1–4 min later streaming restarted and quickly recovered its normal velocity of 1–3 μms−1. Once the electrode had been inserted, current pulses had no obvious effect on streaming. Thus, fura-2 injected by iontophoresis was quickly distributed throughout the length of the hair and the epidermal cell body. Injections were made into the cap of cytoplasm at the tip of the hair. Movement of fura-2 from the cytoplasm into the vacuole was observed; this was marked by a great increase in its fluorescence after excitation at 350 nm and occurred after about 15–30 min in tomato, but more rapidly (5–10 min) in oilseed rape hairs. No fluorescence was associated with the cell walls; this was demonstrated by plasmolysing the cells. The distribution and concentration of Ca2+ was estimated from the ratio of fura-2 fluorescence excited at 350 and 385 nm and from digital image analysis. Resting levels of calcium in the cytoplasm near the tip were in the range 30–90 nM, while much higher levels, which eventually saturated the fura-2 response (>5μM), were seen in vacuoles once the indicator had crossed the tonoplast. This distribution was rapidly perturbed by the addition of the calcium channel blocker, verapamil, to the outer solution. There was a return to the original distribution as the verapamil was diluted. Separate experiments showed that verapamil inhibited cytoplasmic streaming over a time-course similar to these perturbations in cytoplasmic Ca2+ and caused a small depolarization (by approx. 30 mV) of the membrane potential. The application of 3x10−5 or 3 × 10−4 M-N-ethyl maleimide to the hairs strongly and irreversibly depolarized the plasma membrane electric potential, caused cytoplasmic streaming to stop or slow down and resulted in a very rapid rise in cytoplasmic Ca2+, which became uniformly distributed with saturating 350:385 nm ratios.