biologia plantarum

Biologia plantarum 41:193-201, 1998 | DOI: 10.1023/A:1001898027218

Plant mitochondrial electrical potential monitored by fluorescence quenching of rhodamine 123

E. Braidot¹, E. Petrussa¹, F. Macrì¹, A. Vianello¹

¹ Department of Biology and Agro-Industrial Economics, Section of Plant Biology, University of Udine, Via Cotonificio 108, Udine, Italy

The suitability of the fluorescent dye rhodamine 123 for qualitative and quantitative determinations of the electrical potential difference (ΔΨ) in isolated pea (Pisum sativum L.) stem mitochondria was evaluated. A fluorescence quenching of rhodamine 123, as a consequence of dye uptake, occurred following mitochondria energization by both external and internal substrates. This quenching was associated to the generation of ΔΨ, because it was completely released by uncouplers and respiratory inhibitors. The conversion of the proton gradient (ΔpH) into ΔΨ, induced by nigericin or a permeant weak acid (phosphate), increased the quenching. The uptake of the probe was accompanied by 40 % of unspecific binding in coupled, but not in uncoupled, mitochondria. Rhodamine 123 quenching varied linearly with a K+-diffusion potential. ADP induced a transient and cyclic change of fluorescence which was associated to ATP synthesis. Consequently, rhodamine 123 did not influence oxygen consumption by mitochondria in both state 4 and 3, thus indicating that, at the concentrations assayed, the probe was not toxic. It is concluded that rhodamine 123, followed by fluorescence quenching, is a suitable probe to study the energetics of isolated plant mitochondria.

Keywords: membrane potential; mitochondria; Pisum sativum L.

Subjects: membrane potential; mitochondrial electrical potential; Pisum arvense; rhodamine 123, fluorescence quenching

Published: March 1, 1998  Show citation