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Use of co-loaded Fluo-3 and Fura Red fluorescent indicators for studying the cytosolic Ca(2+)concentrations distribution in living plant tissue.

A method for visualisation of cytosolic [Ca(2+)] distribution was applied to living plant tissue. A mixture of the fluorescent probes Fluo-3 and Fura Red was used. The emitted fluorescence was scanned simultaneously in two channels with a laser-scanning confocal microscope and rationing was performed. The homogeneity of the Fluo-3/Fura Red concentration ratio throughout the tissue after AM-ester loading was proven. In vitro calibration permitted conversion of Fluo-3/Fura Red fluorescence ratios to [Ca(2+)] values. Apparent K(D)of 286 nM, R(min)of 0.43 and R(max)of 18 were calculated. The in vivo determination of extreme ratio values was performed by permeabilizing the plasmalemma for Ca(2+)with a ionophore and manipulating the extracellular [Ca(2+)]. The resultant R(minv)of 1.33 and R(maxv)of 2.69 for vegetative apices, and R(mini)of 1.26 and R(maxi)of 3.45 for apices induced to flowering, suggested incomplete equalization of extra- and intracellular Ca(2+)levels in these experiments. In Chenopodium rubrum, the cytosolic [Ca(2+)] patterns of apical tissue obtained using Fluo-3 and Fura Red were significantly different between vegetative apices and apices after photoperiodic flower induction. This methodological approach may also be helpful for studying cytosolic [Ca(2+)] distribution in other living plant tissues.