Cytosolic Ca<sup>2+</sup>‐Concentrations and Distributions in Rhizoids of Chara fragilis Desv. Determined by Ratio Analysis of the Fluorescent Probe Indo‐1
Hodick D., Gilroy S., Fricker MD., Trewavas AJ.
Rhizoids of Charafragilis Desv. were iontophoretically loaded with the Ca 2+ ‐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 Ca 2+ ‐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 Ca 2+ ‐concentration (1 mM to 10 mM) led to increases of 100 to 200 nM in cytoplasmic Ca 2+ ‐concentration. The spatial distribution of cytosolic Ca 2+ in the rhizoid tips was visualised in ratio images computed from low‐light video‐pictures. These images showed a fairly homogeneous distribution of Ca 2+ throughout the tip cytoplasm with concentrations being in the same range as determined by microfluorimetry. A tip‐to‐base gradient in cytoplasmic Ca 2+ , 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. 1991 Deutsche Botanische Gesellschaft/German Botanical Society