Advertisement

Protoplasma

, Volume 212, Issue 1–2, pp 80–88 | Cite as

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

  • D. M. Porterfield
  • P. J. S. Smith
Original Papers

Summary

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 μM 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.

Keywords

Alternative oxidase Photosynthesis Respiration Self-referencing microelectrode Spirogyra gre illeana Vibrating probe 

Abbreviations

SRIS

self-referencing ion selective

SREM

self-referencing electrochemical microelectrode

ICP

inductive coupled plasma spectroscopy

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Armstrong W (1994) Polarographic oxygen electrodes and their use in plant aeration studies. Proc R Soc Edinburg 102: 511–527Google Scholar
  2. Atkins CA, Graham D (1971) Light-induced pH changes by cells ofChlamydomonas reinhardtii: dependence on CO2 uptake. Biochim Biophys Acta 226: 481–485Google Scholar
  3. Badger MR, Price GD (1994) The role of carbonic anhydrase in photosynthesis. Annu Rev Plant Physiol 45: 369–392.Google Scholar
  4. Bates TE, Loesch A, Burnstock G, Clark JB (1996) Mitochondrial nitric oxide synthase: a ubiquitous regulator of oxidative phosphorylation? Biochem Biophys Res Commum 218: 40–44Google Scholar
  5. Davies PW, Brink F (1942) Microelectrodes for measuring local oxygen tension in animal tissues. Rev Sci Instr 13: 524–533Google Scholar
  6. Degenhardt J, Larsen PB, Howell SH, Kochian LV (1998) Aluminum resistance in theArabidopsis mutant alr-104 is caused by an aluminum-induced increase in rhizosphere pH. Plant Physiol 117: 19–27Google Scholar
  7. Dennis DT, Turpin DH (1990) Plant physiology, biochemistry and molecular biology. Wiley, New YorkGoogle Scholar
  8. Denny P, Weeks DC (1970) Effects of light and bicarbonate on membrane potential inPotamogeton schweinfurthii (Benn.). Ann Bot 34: 483–496Google Scholar
  9. de Visser R, Blacquiere T (1984) Inhibition and Stimulation of root respiration in Pisum and Plumbago by hydroxamate. Plant Phyiol 75: 813–817Google Scholar
  10. Dutta A, Popel AS (1995) A theoretical analysis of intracellular oxygen diffusion. J Theor Biol 175: 433–445Google Scholar
  11. Felle H, Bertl A (1986) The fabrication of H+-selective liquid membrane micro-electrodes for use in plant cells. J Exp Bot 37: 1416–1428Google Scholar
  12. Freidman MN, Robinson SW, Gerhadt GA (1996) O-phenylenediamine-modified carbon fiber electrodes for the detection of nitric oxide. Anal Chem 68: 2621–2628Google Scholar
  13. Garcia JL (1976) Nitric oxide production in rice soils. Ann Microbiol 127: 401–414Google Scholar
  14. Gnaiger E, Forstner H (1983) Polarographic oxygen sensors. Springer, Berlin Heidelberg New York TokyoGoogle Scholar
  15. Hodges TK (1973) Ion absorption by plant roots. Adv Agron 25: 163–207Google Scholar
  16. Huppe HC, Turpin DH (1994) Integration of carbon and nitrogen metabolism in plants and algal cells. Annu Rev Plant Physiol 45: 577–607Google Scholar
  17. Jaffe LF, Nuccitelli R (1974) An ultrasensitive vibrating probe for measuring steady extracellular currents. J Gell Biol 63: 614–628Google Scholar
  18. James DE (1978) Culturing algae. Carolina Biological Company, Burlington, NCGoogle Scholar
  19. Kochian LV, Shaffe JE, Kühtreiber WM, Jaffe LF, Lucas WJ (1992) Use of an extracellular, ion-selective vibrating microelectrode system for the quantification of K+, H+, and Ca2+ fluxes in maize roots and suspension cells. Planta 188: 601–610Google Scholar
  20. Kühtreiber WM, Jaffe LF (1990) Detection of extracellular calcium gradients with a calcium-specific vibrating electrode. J Cell Biol 110: 1565–1573Google Scholar
  21. Lüttge U, Higinbotham N (1979) Transport in plants. Springer, New York Heidelberg BerlinGoogle Scholar
  22. McClure PR, Kochian LV, Spanswick RM, Shaff JE (1990) Evidence of cotransport of nitrate and protons in maize roots. Plant Physiol 93: 281–289Google Scholar
  23. Millar AH, Day DA (1996) Nitric oxide inhibits the cytochrome oxidase but not the alternative oxidase of plant mitochondria. FEBS Lett 398: 155–158Google Scholar
  24. Neuman J, Levine RP (1971) Reversible pH changes in cells ofChlamydomonas reinhardtii resulting from CO2 fixation in the light and its evolution in the dark. Plant Physiol 47: 700–704Google Scholar
  25. Novacky A, Fischer E, Ullrich-Eberius CI, Lüttge U, Ullrich WR (1978) Membrane potential changes during transport of glycine as a neutral amino acid and nitrate inLemna gibba. FEBS Lett 88: 264–267Google Scholar
  26. Okazaki Y, Tazawa M, Iwasaki N (1994) Light-induced changes in cytosolic pH in leaf cells ofEgeria densa: measurements with pH-sensitive microelectrodes. Plant Cell Physiol 35: 943–950Google Scholar
  27. Raghavendra AS, Yin ZH, Heber U (1993) Light dependent pH changes in leaves of C4 plants: comparison of the light response to carbon dioxide and oxygen with that of C3 plants. Planta 189: 278–287Google Scholar
  28. Ross E (1938) The effects of sodium cyanide and methylene blue on oxygen consumption byNitella da ata. Am J Bot 25: 458–463Google Scholar
  29. Sargent DF, Taylor CPS (1972) Terminal oxidases ofChlorella pyrenoidosa. Plant Physiol 49: 775–778Google Scholar
  30. Schneiderman G, Goldstick TK (1978) Oxygen electrode design criteria and performance characteristics: recessed cathode. J Appl Physiol 45: 145–154Google Scholar
  31. Silver IA (1967) Problems in the investigation of tissue oxygen microenvironment. In: Reneau D (ed) Chemical engineering in medicine. American Chemical Society, Washington, DC, pp 343–351Google Scholar
  32. Smith PJS, Sanger RH, Jaffe LF (1994) The vibrating Ca2+ electrode: a new technique for detecting plasma membrane regions of Ca2+ influx and efflux. Methods Cell Biol 40: 115–134Google Scholar
  33. Syrett PJ (1951) The effect of cyanide on the respiration and the oxidative assimilation of glucose byChlorella ulgaris. Ann Bot 15: 473–492Google Scholar
  34. Thaler M, Simonis W, Schönknecht G (1992) Light-dependent changes of the cytoplasmic H+ and Cl activity in the green algaeEremosphaera irdis. Plant Physiol 99: 103–110Google Scholar
  35. Torkelson JD, Lynnes JA, Weger HG (1995) Extracellular peroxidase mediated oxygen consumption inChlamydomonas reinhardtii (Chlorophyta). J Phycol 31: 562–567Google Scholar
  36. Trebst AV, Tsujimoto HY, Arnon DI (1958) Separation of light and dark phases in the photosynthesis of isolated chloroplasts. Nature 182: 351–355Google Scholar
  37. Ullrich WR (1987) Nitrate and ammonium uptake in green algae and higher plants: mechanism and relationship with nitrate metabolism. In: Ullrich WR, Aparicio PJ, Syrett PJ, Castillo F (eds) Inorganic nitrogen metabolism. Springer, Berlin Heidelberg New York Tokyo, pp 32–38Google Scholar
  38. —, Novacky A (1981) Nitrate-dependent membrane potential changes and their induction inLemna gibba. Plant Sci Lett 22: 211–217Google Scholar
  39. Webster DA, Hackett DP (1965) Respiratory chain of colorless algae I: Chlorophyta and Euglenophyta. Plant Physiol 41: 1091–1100Google Scholar
  40. Weger HG, Dasgupta R (1993) Regulation of alternative pathway respiration inChlamydomonas reinhardtii (Chlorophyceae). J Phycol 29: 300–308Google Scholar
  41. —, Guy RD, Turpin DH (1990) Cytochrome and alternative pathway respiration in green algae. Plant Physiol 93: 356–360Google Scholar
  42. —, Lynnes JA, Torkelson JD (1996) Characterization of extracellular oxygen consumption by the green algaeSelenastrum minutum. Physiol Plant 96: 268–274Google Scholar
  43. Whalen WJ (1974) Some problems with an intracellular pO2 electrode. Adv Exp Med Biol 50: 39–41Google Scholar
  44. —, Riley J, Nair P (1967) A microelectrode for measuring intracellular PO2. J Appl Physiol 23: 798–801Google Scholar
  45. Yin ZH, Neimanis S, Heber U (1990) Light-dependent pH changes in the leaves of C3 plants II: effects of CO2 and O2 on the cytosolic and vacuolar pH. Planta 182: 253–261Google Scholar
  46. —, Heber U, Raghavendra AS (1993) Light induced pH changes in leaves of C4 plants: comparison of cytosolic alkalization and vacuolar acidification with that of C3 plants. Planta 189: 267–277Google Scholar

Copyright information

© Springer-Verlag 2000

Authors and Affiliations

  • D. M. Porterfield
    • 1
  • P. J. S. Smith
    • 2
  1. 1.Department of Biological Sciences105 Schrenk Hall, University of Missouri-RollaRollaUSA
  2. 2.Marine Biological LaboratoryBioCurrents Research CenterWoods Hole

Personalised recommendations