Flow Cytometric Analysis of Rhodamine 123 Fluorescence during Modulation of the Membrane Potential in Plant Mitochondria

Abstract

The fluorescent dye rhodamine 123, which selectively accumulates in mitochondria based on the membrane potential, was used with flow cytometry to evaluate variations in activity of mitochondria isolated from plant tissues. In the presence of succinate and ATP, potato (Solanum tuberosum L.) tuber mitochondrial activity was affected by metabolic inhibitors and compounds that modify the membrane potential. The more uniform the mitochondrial population, the higher the observed membrane potential. The reactive population corresponds to the proportion of intact mitochondria (94-97%) defined by classic methods. Changes in the light-scattering properties are more related to internal modifications affecting the inner membrane-matrix system of the mitochondria during metabolic modulation than to specific volume change or outer membrane surface modifications. We tested our approach using an Arum maculatum preparation that contains three different types of mitochondria and demonstrated the validity of the light-scatter measurements to distinguish the a, ,B, and y mitochondria and to measure their ability to built up a membrane potential in the presence of succinate. These results demonstrate clearly that flow cytometric techniques using rhodamine 123 can be employed to study the activity in isolated plant mitochondria.Fluorescent probes have been applied as optical indicators ofthe membrane potential differences in several types ofcells, isolated organelles, and lipid vesicles (1,4,20,31). The technique relies on membrane potential-dependent partitioning of charged lipophilic dye molecules across the membrane. Changes in membrane potential result in changes in the intensity of dye fluorescence, termed "redistribution signals" (4). Lipophilic cationic dyes have been used successfully to measure changes in the membrane potential of in situ or isolated mitochondria of yeast cells (9,20), several kinds of animal cells (27), and, more recently, plant cells and protoplasts (13,15,26). In plant mitochondria, the dyes most widely used for mitochondria are either derivatives of rhodamine or cyanine dyes developed by Waggoner (30, 31). The laser dye, Rh 123,' has been extensively employed as a fluorescent stain of mitochondria in living cells (27). Because Rh 123 is an aromatic cation, it has been assumed to distribute itself electrophoretically into the mitochondrial matrix in response to AI.At high concentrations, Rh 123 has toxic side effects, but Emaus et al. (6) have recently demonstrated that, at concentrations that do not inhibit mitochondrial function, Rh 123 is indeed a sensitive and specific probe of AI in isolated mitochondria. Agents known to depolarize or deenergize mitochondria, such as uncouplers (valinomycin) and respiratory inhibitors (KCN, SHAM), decreased Rh123 fluorescence of mitochondria in cultured cells, whereas nigericin, which collapses ApH and raises AI, increases fluorescence. For Rh 123, the energy-linked changes were accompanied by dye uptake into the matrix space and the concentration ratio in-to...

Flow Cytometry of Spinach Chloroplasts

“…In contrast with the most common observations with whole cells, the FALS seems to reflect the density (internal structure) of the particles rather than their size in subcellular particles. A similar phenomenon was encountered in the case of both animal and plant mitochondria (27,30).…”

Analysis of cell cycle activity and population dynamics in heterogeneous plant cell suspensions using flow cytometry

“…Methods have been developed in recent years based on plant protoplast, nuclear and pollen suspensions (Brown and Bergounioux, 1989;Galbraith et al, 1983Galbraith et al, , 1984Harkins and Galbraith, 1987). Current applications of flow cytometry to plant cells include DNA and RNA analysis, detection of transgene expression, karyotyping, cell counting, studies of chloroplasts, cell membranes and cell wall regeneration, evaluation of mitochondrial activity, measurement of secondary metabolite accumulation, and selection of particular cells or sub-cellular organelles of interest (Adamse, 1990;Bergounioux et al, 1992;Conia and Muller, 1989;Galbraith, 1989;Galbraith et al, 1995;Nicoloso et al, 1994;Petit, 1992;Sakamoto et al, 1994;Schröder and Petit, 1992). Methods for BrdU labeling of plant cells were first described by Levi et al (1987) to determine the proportion of S-phase cells in pea root meristems using microscope photometry.…”

Changes in Respiratory Mitochondrial Machinery and Cytochrome and Alternative Pathway Activities in Response to Energy Demand Underlie the Acclimation of Respiration to Elevated CO2 in the Invasive Opuntia ficus-indica

“…Plant mitochondrial heterogeneity depends on plants species, tissue, ontogeny, cellular type, and energetic state of mitochondria and they are often associated with other organelles and structures (Logan and Leaver, 2000). Contrary to TEM, confocal microscopy: (1) eliminates interferences from chloroplasts regardless of mitochondrial localization within the cytoplasm; (2) provides a global three-dimensional cell view so that mitochondrial morphology and mitochondrial-in-cell position is considered; and (3) Rhodamine 123 stains only metabolically functional mitochondria (Petit, 1992).…”

“…Active mitochondria were stained by using Rhodamine 123 (Sigma-Aldrich), which selectively accumulates in mitochondria based on the membrane potential (Petit, 1992). Fresh tissue samples were incubated for 12 min in 250 mM Rhodamine 123 (Sigma Aldrich) at 37°C in the dark (previous trial studies indicated that less incubation time stained less brightly mitochondria and more incubation time quickly toxified tissues, and the staining rapidly disappeared).…”