PLANT MITOCHONDRIAL ELECTRICAL POTENTIAL MONITORED BY FLUORESCENCE QUENCHING OF RHODAMINE-123

The suitability of the fluorescent dye rhodamine 123 for qualitative a nd quantitative determinations of the electrical potential difference (Delta Psi) in isolated pea (Pisum sativum L.) stem mitochondria was e valuated. A fluorescence quenching of rhodamine 123, as a consequence of dye uptake, occurred following mitochondria energization by both ex ternal and internal substrates. This quenching was associated to the g eneration of Delta Psi, because it was completely released by uncouple rs and respiratory inhibitors. The conversion of the proton gradient ( Delta pH) into Delta Psi, induced by nigericin or a permeant weak acid (phosphate), increased the quenching. The uptake of the probe was acc ompanied by 40 % of unspecific binding in coupled, but not in uncouple d, mitochondria. Rhodamine 123 quenching varied linearly with a K+-dif fusion potential. ADP induced a transient and cyclic change of fluores cence which was associated to ATP synthesis. Consequently, rhodamine 1 23 did not influence oxygen consumption by mitochondria in both state 4 and 3, thus indicating that, at the concentrations assayed, the prob e was not toxic. It is concluded that rhodamine 123, followed by fluor escence quenching, is a suitable probe to study the energetics of isol ated plant mitochondria.