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.
Walczysko
P
P
Institute of Biology II, University of Freiburg, Freiburg, Germany. walczysk@uni-freiburg.de
Wagner
E
E
Albrechtová
J T
JT
D000814:Aniline Compounds / Q000494:pharmacology*; D001572:Benzofurans / Q000494:pharmacology*; D000001:Calcimycin / Q000031:analogs & derivatives* / Q000494:pharmacology; D002118:Calcium / Q000032:analysis*; D019663:Chenopodiaceae / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D003600:Cytosol / Q000737:chemistry; D005456:Fluorescent Dyes / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology*; D007476:Ionophores / Q000494:pharmacology; D018519:Meristem / Q000166:cytology / Q000187:drug effects; D018613:Microscopy, Confocal; D017440:Photoperiod; D014966:Xanthenes / Q000494:pharmacology*
D000814:Aniline Compounds; D001572:Benzofurans; D005456:Fluorescent Dyes; D007093:Imidazoles; D007476:Ionophores; D014966:Xanthenes; C082877:fura red; C059715:Fluo-3; D000001:Calcimycin; C033170:4-bromo-A-23187; D002118:Calcium