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

Single-cell, real-time measurements of extracellular oxygen and proton fluxes fromSpirogyra grevilleana
Single-cell, real-time measurements of extracellular oxygen and proton fluxes fromSpirogyra 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-O2) system with self-referencing ionselective proton measurements (SRIS-H+) we have characterized oxygen and proton fluxes from a single cell of the filamentous green algaSpirogyra 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 M NO, while the cyanide-resistant alternative oxidase was not. These results demonstrate the use of the newly developed SREM-O2 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.
alternative oxidase, photosynthesis, respiration, self-referencing microelectrode, spirogyra gre illeana, vibrating probe
0033-183X
80-88
Porterfield, D.M.
90790fa2-b4d8-4d76-94b6-cb50591c1c2a
Smith, P.J.S.
003de469-9420-4f12-8f0e-8e8d76d28d6c
Porterfield, D.M.
90790fa2-b4d8-4d76-94b6-cb50591c1c2a
Smith, P.J.S.
003de469-9420-4f12-8f0e-8e8d76d28d6c

Porterfield, D.M. and Smith, P.J.S. (2000) Single-cell, real-time measurements of extracellular oxygen and proton fluxes fromSpirogyra grevilleana. Protoplasma, 212 (1-2), 80-88. (doi:10.1007/BF01279349).

Record type: Article

Abstract

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-O2) system with self-referencing ionselective proton measurements (SRIS-H+) we have characterized oxygen and proton fluxes from a single cell of the filamentous green algaSpirogyra 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 M NO, while the cyanide-resistant alternative oxidase was not. These results demonstrate the use of the newly developed SREM-O2 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.

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Published date: 2000
Keywords: alternative oxidase, photosynthesis, respiration, self-referencing microelectrode, spirogyra gre illeana, vibrating probe

Identifiers

Local EPrints ID: 190515
URI: http://eprints.soton.ac.uk/id/eprint/190515
ISSN: 0033-183X
PURE UUID: a3383dde-da13-416f-a2de-0c53bfdb4227
ORCID for P.J.S. Smith: ORCID iD orcid.org/0000-0003-4400-6853

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Date deposited: 17 Jun 2011 10:46
Last modified: 21 Nov 2021 03:04

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Contributors

Author: D.M. Porterfield
Author: P.J.S. Smith ORCID iD

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