Pontentiometric Cyanine Dyes Are Sensitive Probes for Mitochondria in Intact Plant Cells

Abstract

Selected fluorescent dyes were tested for uptake by mitochrondria in intact cells of barley, maize, and onion. The cationic cyanine dye 3,3'-diheptyloxacarbocyanine iodide [DiOC7(3)j accumulated in mitochondria within 15 to 30 minutes without appreciable staining of other protoplasmic constituents. The number, shape, and movement of the fluorescent mitochondria could be seen readily, and the fluorescence intensity of the mitochondria could be monitored with a microscope photometer. Fluorescence was eliminated in 1 to 5 minutes by the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) indicating that maintenance of dye concentration was dependent on the inside-negative transmembrane potential maintained by functional mitochondria. Fluorescence of prestained mitochondria was enhc within 5 to 10 minutes after addition of 0.1 minlimolar kinetin to cells. The fluorescence in kinetintreated cells was dissipated by CCCP. These results suggest that kinetin interacted with respiratory processes resulting in higher potential across the mitochondrial membrane.Fluorescent probes have been applied as optical indicators of membrane potential differences in several types of cells, isolated organelles, and lipid vesicles (2,6,29,30). The technique relies on potential-dependent partitioning of charged lipophillic dye molecules across the membrane. Changes in membrane potential result in changes in intensity of dye fluorescence, termed 'redistribution signals' (6).Potentiometric dyes have been used successfully to measure changes in membrane potential of mitochondria within or isolated from cells of yeasts (15,24), several kinds of animal cells (3,7,8,13,14,16,25), and recently, in cells or protoplasts of higher plants (18,26 (30) and co-workers for use as potentiometric probes. Through the use ofrespiratory inhibitors and ionophores and ofothers means, the fluorescence intensity of these dyes has been shown to be related to mitochondrial membrane potential (2, 6, 8, 13.).We have recently found that certain cyanine dyes gave fluorescence to mitochondria of haustoria within living barley epidermal cells infected with Erysiphe graminis (4). The mitochondrial fluorescence was rapidly dissipated by the protonophores CCCP2 or DNP indicating that maintenance of dye concentration within the mitochondria was dependent on membrane potential. Several of the cyanines tested were observed also to stain mitochondria of host epidermal cells, which prompted us to screen various cyanines and other dyes used elsewhere as mitochondrial probes for ability to stain higher plant mitochondria. Our objectives were to learn which dyes were most effective with cells of higher plants, to learn if fluorescence of stained plant mitochondria could be monitored photometrically (as had been possible for haustorial mitochondria), and to learn if fluorescence was related to mitochondrial membrane potential. We also measured the effect of kinetin on mitochondrial fluorescence. Our interest in kinetin grew out of the fact that kinetin had interf...

Pulsing of Membrane Potential in Individual Mitochondria: A Stress-Induced Mechanism to Regulate Respiratory Bioenergetics in Arabidopsis

“…During a pulse the GFP fluorescence of the mitochondrion remained constant ( Figures 1B and 1C), showing that the mitochondrion had not simply moved out of the focal plane of the microscope. Pulses were not an artifact caused by the presence of TMRM because the same phenomenon was also observed with the chemically distinct mitochondrial membrane potential dye 3,39-dihexyloxacarbocyanine [DiOC 6 (3)] (Matzke and Matzke, 1986;Liu et al, 1987) (see Supplemental Figure 1 online). Although pulses were rare events under control conditions, a first pulse for any given mitochondrion was often followed by another pulse within seconds or minutes.…”

Correlation of Rapid Cell Death with Metabolic Changes in Fungus-Infected, Cultured Parsley Cells

“…It is known that exogenously applied kinetin promotes or facilitates rapid cell death in infected plant (Chen and Heath, 1991) and callus tissue (Miller et al, 1984) but the mechanism for this effect is obscure. One important clue, however, is the observation that low concentrations of kinetin activated respiration of suspended soybean callus cells (Moore and Miller, 1972), suspension-cultured maize cells, and epidermal cells of barley and onion (Liu et al, 1987).…”

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

“…The FALS (Table III) (11)(12)(13), at much higher concentrations (8,19). This low probe concentration is possible because of the optical advantages of the flow cytometer compared with conventional fluorometers.…”

“…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).…”