Use of co-loaded Fluo-3 and Fura Red fluorescent indicators for studying the cytosolic Ca2+concentrations distribution in living plant tissue†

Abstract

A method for visualisation of cytosolic [Ca2+] 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 [Ca2+] values. Apparent KDof 286 nM, Rminof 0.43 and Rmaxof 18 were calculated. The in vivo determination of extreme ratio values was performed by permeabilizing the plasmalemma for Ca2+with a ionophore and manipulating the extracellular [Ca2+]. The resultant Rminvof 1.33 and Rmaxvof 2.69 for vegetative apices, and Rminiof 1.26 and Rmaxiof 3.45 for apices induced to flowering, suggested incomplete equalization of extra- and intracellular Ca2+levels in these experiments. In Chenopodium rubrum, the cytosolic [Ca2+] 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 [Ca2+] distribution in other living plant tissues.