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

We have adapted the self-referencing microelectrode technique to allow sens itive and noninvasive measurement of oxygen fluxes around single cells. The self-referencing technique is based on the translational movement of a sel ective microelectrode through the gradient next to the cell wall or membran e. The electrode is moved at a known frequency and between known points. Th e differential electrode output values are converted into a directional mea surement of flux by the Fick equation. By coupling the newly developed oxyg en-selective self-referencing electrochemical microelectrode (SREM-O-2) sys tem with self-referencing ion-selective proton measurements (SRIS-H+) we ha ve characterized oxygen and proton fluxes from a single cell of the filamen tous green alga Spirogyra gre illeana (Hass.). Oxygen showed a net efflux a nd protons showed a net influx when the cell was illuminated. These photosy nthesis-dependent fluxes were found to be spatially associated with the chl oroplasts 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-re sistant respiration was inhibited by the application of 5 mM salicylhydroxa mic acid, an inhibitor of the alternative oxidase. Similarly the cytochrome pathway was also inhibited by the presence of 20 mu M NO, while the cyanid e-resistant alternative oxidase was not. These results demonstrate the use of the newly developed SREM-O-2 system to measure and characterize metaboli c 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 cel ls.