Single-cell, real-time measurements of extracellular oxygen and proton fluxes from Spirogyra grevilleana

We have adapted the self-referencing microelectrode technique to allow sensitive and noninvasive measurement of oxygen fluxes around single cells. The self-referencing technique is based on the translational movement of a selective microelectrode through the gradient next to the cell wall or membrane. The electrode is moved at a known frequency and between known points. The differential electrode output values are converted into a directional measurement of flux by the Fick equation. By coupling the newly developed oxygen-selective self-referencing electrochemical microelectrode (SREM-O-2) system with self-referencing ion-selective proton measurements (SRIS-H+) we have characterized oxygen and proton fluxes from a single cell of the filamentous green alga Spirogyra gre illeana (Hass.). Oxygen showed a net efflux and protons showed a net influx when the cell was illuminated. These photosynthesis-dependent fluxes were found to be spatially associated with the chloroplasts and were sensitive to treatment with dichlorophenyldimethylurea. In the dark the directions of oxygen and proton fluxes were reversed. This oxygen influx was associated with mitochondrial respiration and was reduced by 78% when the cells was treated with 0.5 mM KCN. The residual cyanide-resistant respiration was inhibited by the application of 5 mM salicylhydroxamic acid, an inhibitor of the alternative oxidase. Similarly the cytochrome pathway was also inhibited by the presence of 20 mu M NO, while the cyanide-resistant alternative oxidase was not. These results demonstrate the use of the newly developed SREM-O-2 system to measure and characterize metabolic fluxes at a level of sensitivity that allows for subcellular resolution. These measurements, in conjunction with SERIS-H+ measurements, have led to new insights in our understanding of basic cellular physiology in plant cells.