CYTOSOLIC CA2+-CONCENTRATIONS AND DISTRIBUTIONS IN RHIZOIDS OF CHARA-FRAGILIS DESV DETERMINED BY RATIO ANALYSIS OF THE FLUORESCENT-PROBE INDO-1

Rhizoids of Chara fragilis Desv. were iontophoretically loaded with the Ca2+-sensitive ratio dye indo-1. After loading, the rhizoids regained their preinjection-membrane potential within 2 to 5 min and survived the procedure for more than 24 h, but their growth in length was permanently inhibited. Microfluorimetric measurements of the indo-1 fluorescence-ratio showed spontaneous fluctuations of the cytoplasmic Ca2+-concentration, usually declining from high values after loading to 425 +/- 80 nM (+/- SD, n = 7) as determined by in-vitro calibration. Increasing the extracellular K+-concentration (0.1 mM to 10 mM) or Ca2+-concentration (1 mM to 10 mM) led to increases of 100 to 200 nM in cytoplasmic Ca2+-concentration. The spatial distribution of cytosolic Ca2+ in the rhizoid tips was visualised in ratio images computed from low-light video-pictures. These images showed a faily homogeneous distribution of Ca2+ throughout the tip cytoplasm with concentrations being in the same range as determined by microfluorimetry. A tip-to-base gradient in cytoplasmic Ca2+, thought to be a prerequisite for cell polarity and tip growth, was found in only 1 out of 16 successfully microinjected cells. Additionally, a progressive compartmentalization of the fluorochrome indo-1, probably in the proplastids and the very abundant endoplasmic reticulum of the rhizoids, was observed.