Cytosolic Ca2+-Concentrations and Distributions in Rhizoids of Chara fragilis Desv. Determined by Ratio Analysis of the Fluorescent Probe Indo-1

Rhizoids of Charafragilis 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 fairly 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.