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Self-referencing optrodes for measuring spatially resolved, real-time metabolic oxygen flux in plant systems

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Abstract

The ability to non-invasively measure metabolic oxygen flux is a very important tool for physiologists interested in a variety of questions ranging from basic metabolism, growth/development, and stress adaptation. Technologies for measuring oxygen concentration near the surface of cells/tissues include electrochemical and optical techniques. A wealth of knowledge was gained using these tools for quantifying real-time physiology. Fiber-optic microprobes (optrodes) have recently been developed for measuring oxygen in a variety of biomedical and environmental applications. We have adopted the use of these optical microsensors for plant physiology applications, and used the microsensors in an advanced sensing modality known as self-referencing. Self-referencing is a non-invasive microsensor technique used for measuring real-time flux of analytes. This paper demonstrates the use of optical microsensors for non-invasively measuring rhizosphere oxygen flux associated with respiration in plant roots, as well as boundary layer oxygen flux in phytoplankton mats. Highly sensitive/selective optrodes had little to no hysteresis/calibration drift during experimentation, and an extremely high signal-to-noise ratio. We have used this new tool to compare various aspects of rhizosphere oxygen flux for roots of Glycine max, Zea mays, and Phaseolus vulgaris, and also mapped developmentally relevant profiles and distinct temporal patterns. We also characterized real-time respiratory patterns during inhibition of cytochrome and alternative oxidase pathways via pharmacology. Boundary layer oxygen flux was also measured for a phytoplankton mat during dark:light cycling and exposure to pharamacological inhibitors. This highly sensitive technology enables non-invasive study of oxygen transport in plant systems under physiologically relevant conditions.

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References

  • Alderman J, Hynes J, Floyd SM, Kruger J, O’Connor R, Papkovsky DB (2004) A low-volume platform for cell-respirometric screening based on quenched-luminescence oxygen sensing. Biosens Bioelectron 19(11):1529–1535

    Article  CAS  PubMed  Google Scholar 

  • Armstrong W, Webb T, Darwent M, Beckett PM (2009) Measuring and interpreting respiratory critical oxygen pressures in roots. Ann Botany 103:281–293

    Article  Google Scholar 

  • Arnholdt-Schmitt B, Costa JH, de Melo DF (2006) Aox—a functional marker for efficient cell reprogramming under stress? Trends Plant Sci 11:281–287

    Article  CAS  PubMed  Google Scholar 

  • Bailey-Serres J, Voesenek LACJ (2008) Flooding stress: acclimations and genetic diversity. Ann Rev Plant Biol 59:313–339

    Article  CAS  Google Scholar 

  • Borgens RB (1984) Endogenous ionic currents traverse intact and damaged bone. Science 225:478–482

    Article  CAS  PubMed  Google Scholar 

  • Borisov SM, Wolfbeis OS (2008) Optical biosensors. Chem Rev 108:423–461

    Article  CAS  PubMed  Google Scholar 

  • Buerk DG (2004) Oxygen sensing. Meth Enzymol 381:665–690

    Article  CAS  PubMed  Google Scholar 

  • Chatni MR, Porterfield DM (2009) Self-referencing optrode technology for non-invasive real-time measurement of biophysical flux and physiological sensing. Analyst 134:2224–2232

    Article  CAS  PubMed  Google Scholar 

  • Chatni MR, Maier DE, Porterfield DM (2009) Optimization of oxygen sensitive optical dye membrane polymers for fluorescent lifetime based physiological biosensing. Sens Actuat B Chem 141:471–477

    Article  Google Scholar 

  • Chen Z, Newman I, Zhou M, Mendham N, Zhang G, Shabala SN (2005) Screening plants for salt tolerance by measuring K+ flux; a case study for barley. Plant Cell Environ 28:1230–1246

    Article  CAS  Google Scholar 

  • Cloutier M, Chen J, Tatge F, McMurray-Beaulieu V, Perriera M, Jolicoeur M (2009) Kinetic metabolic modelling for the control of plant cells cytoplasmic phosphate. J Theor Biol 259(1):118–131

    Article  CAS  PubMed  Google Scholar 

  • Darwent MJ, Armstrong W, Armstrong J, Beckett PM (2003) Exploring the radial and longitudinal aeration of primary maize roots by means of clark-type oxygen microelectrodes. Russ J Plant Physiol 50(6):722–732

    Article  CAS  Google Scholar 

  • De Dobbeleer C, Cloutier M, Fouilland M, Legros R, Jolicoeur MA (2006) High-rate perfusion bioreactor for plant cells. Biotechnol Bioeng 95(6):1126–1137

    Article  PubMed  Google Scholar 

  • Dodds WK, Biggs BJF, Lowe RL (1999) Photosynthesis-irradiance patterns in benthic microalgae: variations as a function of assemblage thickness and community structure. J Phycol 35(1):42–53

    Article  Google Scholar 

  • Feijo JA, Sainhas J, Holdaway-Clarke T, Cordeiro MS, Kunkel JG, Hepler PK (2001) Cellular oscillations and the regulation of growth: the pollen tube paradigm. Bioassays 23:86–94

    Article  CAS  Google Scholar 

  • Ferris MJ, Hirsch CF (1991) Method for isolation and purification of cyanobacteria. Appl Environ Microbiol 57(5):1448–1452

    CAS  PubMed  Google Scholar 

  • Gilliham M, Sullivan W, Tester M, Tyerman SD (2006) Simultaneous flux and current measurement from single plant protoplasts reveals a strong link between K+ fluxes and current, but no link between Ca2 + fluxes and current. Plant J 46:134–144

    Article  CAS  PubMed  Google Scholar 

  • Gradmann D, Slayman CL (1975) Oscillations of an electrogenic pump in the plasma membrane of Neurospora. J Membr Biol 23:181–212

    Article  CAS  PubMed  Google Scholar 

  • Gupta KJ, Zabalza A, van Dongen JT (2009) Regulation of respiration when the oxygen availability changes. Physiol Plant 137:383–391

    Article  CAS  PubMed  Google Scholar 

  • Holdaway-Clarke TL, Hepler PK (2003) Control of pollen tube growth: role of ion gradients and fluxes. New Phytol 159:539–563

    Article  CAS  Google Scholar 

  • Jaffe LF, Nuccitelli R (1974) An ultrasensitive vibrating probe for measuring steady extracellular currents. J Cell Biol 63:614–628

    Article  CAS  PubMed  Google Scholar 

  • Kochian LV, Shaff 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 maize suspension cells. Planta 188:601–610

    Article  CAS  Google Scholar 

  • Kühl M, Jørgensen BB (1992) Spectral light measurements in microbenthic phototrophic communities with a fiber-optic microprobe coupled to a sensitive diode array detector. Limnol Oceanogr 37:1813–1823

    Article  Google Scholar 

  • Kühtreiber WM, Jaffe LF (1990) Detection of extracellular calcium gradients with a calcium-specific vibrating electrode. J Cell Biol 110:1565–1573

    Article  PubMed  Google Scholar 

  • Kupper H, Setlik I, Hlasek M (2004) A versatile chamber for simultaneous measurements of oxygen exchange and fluorescence in filamentous and thallous algae as well as higher plants. Photosynthetica 42(4):579–583

    Article  Google Scholar 

  • Laan P, Tosserams M, Huys P, Bienfait HF (1991) Oxygen uptake by roots of rumex species at different temperatures—the relative importance of diffusive resistance and enzyme kinetics. Plant Cell Environ 14(2):235–240

    Article  CAS  Google Scholar 

  • Lamboursain L, St-Onge F, Jolicoeur M (2002) A lab-built respirometer for plant and animal cell culture. Biotechnol Prog 18(6):1377–1386

    Article  CAS  PubMed  Google Scholar 

  • Land SC, Portefield DM, Sanger RH, Smith PJS (1999) The self-referencing oxygen-selective microelectrode: detection of transmembrane oxygen flux from single cells. J Exp Biol 202:211–218

    CAS  PubMed  Google Scholar 

  • Lee SK, Okura I (1997) Photostable optical oxygen sensing material: platinum tetrakis(pentafluorophenyl)porphyrin immobilized in polystyrene. Anal Comm 34(6):185–188

    Article  CAS  Google Scholar 

  • Mancoso S, Boselli M (2002) Characterisation of the oxygen fluxes in the division, elongation and mature zone of Vitis roots: influence of oxygen availability. Planta 214:767–774

    Article  Google Scholar 

  • Mancuso S, Papeschi G, Marras AM (2000) A polarographic, oxygen-selective, vibrating-microelectrode system for the spatial and temporal characterization of transmembrane oxygen fluxes in plants. Planta 211:384–389

    Article  CAS  PubMed  Google Scholar 

  • McLamore ES, Porterfield DM, Banks MK (2009) Non-invasive self-referencing electrochemical sensors for quantifying real time biophysical flux in biofilms. Biotechnol Bioeng 102:791–799

    Article  CAS  PubMed  Google Scholar 

  • McLamore ES, Mohanty S, Shi S, Claussen J, Jedlicka SS, Rickus JL, Porterfield DM (2010a) A self-referencing glutamate biosensor for measuring real time neuronal glutamate flux. J Neurosci Methods 189:14–22

    Article  CAS  PubMed  Google Scholar 

  • McLamore ES, Stensberg M, Yale G, Ochoa-Acuna H, Sepulveda M, Sun X, Akkus O, Porterfield DM (2010b) A difference imaging technique for monitoring real time changes in morphology within the cell, tissue, and organism spatial domain. Proc SPIE 7674(14):1–9

    Google Scholar 

  • Mclntosh L (1994) Molecular biology of the alternative oxidase. Plant Physiol 105:781–786

    Article  Google Scholar 

  • Miller HL, Dunton KH (2007) Stable isotope (C-13) and O2 micro-optode alternatives for measuring photosythesis in seaweeds. Mar Ecol Prog Ser 329:85–97

    Article  CAS  Google Scholar 

  • O’Riordan TC, Buckley D, Ogurtsov VI, O’Connor R, Papkovsky DB (2000) A cell viability assay based on monitoring respiration by optical oxygen sensing. Anal Biochem 278:221–227

    Article  PubMed  Google Scholar 

  • Ober ES, Sharp RE (1996) A microsensor for direct measurement of O2 partial pressure within plant tissues. J Exp Bot 47(296):447–454

    Article  CAS  Google Scholar 

  • Polidoros AN, Mylona PV, Arnholdt-Schmitt B (2009) Aox gene structure, transcript variation and expression in plants. Physiol Plant 137(4):342–353

    Article  CAS  PubMed  Google Scholar 

  • Porterfield DM (2002) The use of microsensors for studying the physiological activity of plant roots. In: Waisel Y, Eshel A, Kafkafi U (eds) Plant roots: the hidden half, 3rd edn. Marcel Decker, New York, pp 333–347

    Google Scholar 

  • Porterfield DM (2007) Measuring metabolism and biophysical flux in the tissue, cellular and sub-cellular domains: recent developments in self-referencing amperometry for physiological sensing. Biosens Bioelectron 22:1186–1196

    Article  CAS  PubMed  Google Scholar 

  • Porterfield DM, Smith PJS (2000) Single-cell, real-time measurements of extracellular oxygen and proton fluxes from Spirogyra grevilleana. Protoplasma 212:80–88

    Article  CAS  Google Scholar 

  • Porterfield DM, Trimarchi JR, Keefe DL, Smith PJS (1998) Characterization of oxygen and calcium fluxes from early mouse embryos and oocytes. Biol Bull 195:208–209

    Article  CAS  PubMed  Google Scholar 

  • Porterfield DM, Kuang A, Smith PJS, Crispi ML, Musgrave ME (1999) Oxygen-depleted zones inside reproductive structures of Brassicaceae: implications for oxygen control of seed development. Can J Bot 77:1439–1446

    Article  CAS  PubMed  Google Scholar 

  • Porterfield DM, Corkey RF, Sanger RH, Tornheim K, Smith PJS, Corkey BE (2000) Oxygen consumption oscillates in single clonal pancreatic β-cells (HIT). Diabetes 49:1511–1516

    Article  CAS  PubMed  Google Scholar 

  • Porterfield DM, Rickus JL, Kopelman R (2007) Non-Invasive approaches to measuring respiratory patterns using a pttfpp based, phase-lifetime, self-referencing oxygen optrode. Proc Soc Photonics (SPIE) 6380:1–8

    Google Scholar 

  • Porterfield DM, McLamore ES, Banks MK (2009) Microsensor technology for measuring H+ flux in buffered media. Sens Actuat B Chem 136:383–387

    Article  Google Scholar 

  • Richard P (2003) The rhythm of yeast. FEMS Microbiol Rev 791:1–11

    Google Scholar 

  • Ryan PR, Ditomaso JM, Kochian LV (1993) Aluminum toxicity in roots—an investigation of spatial sensitivity and the role of the root cap. J Exp Bot 44:437–446

    Article  CAS  Google Scholar 

  • Sanchez BC, Ochoa-Acuña H, Porterfield DM, Sepúlveda MS (2008) Oxygen flux as an indicator of physiological stress in fathead minnow (Pimephales promelas) embryos: a real-time biomonitoring system of water quality. Environ Sci Technol 42:7010–7017

    Article  CAS  PubMed  Google Scholar 

  • Serrano M, Robatzek S, Torres M, Kombrink E, Somssich IE, Robinson M, Schulze-Lefert P (2007) Chemical interference of pathogen-associated molecular pattern-triggered immune responses in Arabidopsis reveals a potential role for fatty-acid synthase type II complex-derived lipid signals. J Biol Chem 282(9):6803–6811

    Article  CAS  PubMed  Google Scholar 

  • Shabala SN, Newman IA (1997) Proton and calcium flux oscillations in the elongation region correlate with root nutation. Physiol Plant 100(4):917–926

    Article  CAS  PubMed  Google Scholar 

  • Shabala SN, Newman IA (1998) Osmotic sensitivity of Ca2+ and H+ transporters in corn roots: effect on fluxes and their oscillations in the elongation region. J Membr Biol 161:45–54

    Article  CAS  PubMed  Google Scholar 

  • Shabala SN, Shabala L (2002) Kinetics of net H+, Ca2+, K+, Na+, NH4+, and Cl fluxes associated with post-chilling recovery of plasma membrane transporters in Zea mays leaf and root tissues. Physiol Plant 114:47–56

    Article  CAS  PubMed  Google Scholar 

  • Shabala SN, Newman IA, Morris J (1997) Oscillations in H+ and Ca2+ ion fluxes around the elongation region of corn roots and effects of external pH. Plant Physiol 113:111–118

    CAS  PubMed  Google Scholar 

  • Shabala SN, Newman I, Whittington J, Juswono U (1998) Protoplast ion fuxes: their measurement and variation with time, position and osmoticum. Planta 204:146–152

    Article  CAS  Google Scholar 

  • Shabala L, Ross T, McMeekin T, Shabala S (2006) Non-invasive microelectrode ion flux measurements to study adaptive responses of microorganisms to the environment. FEMS Microbiol Rev 30:472–486

    Article  CAS  PubMed  Google Scholar 

  • Shi K, Hu WH, Dong DK, Zhou YH, Yu JQ (2007) Low O2 supply is involved in the poor growth in root-restricted plants of tomato (Lycopersicon esculentum Mill.). Environ Exp Bot 61(2):181–189

    Article  CAS  Google Scholar 

  • Siedow JN, Umbach AL (2000) The mitochondrial cyanide-resistant oxidase: structural conservation amid regulatory diversity. Biochim Biophys Acta 1459:432–439

    Article  CAS  PubMed  Google Scholar 

  • Verslues PE, Ober ES, Sharp RE (1998) Root growth and oxygen relations at low water potentials. Impact of oxygen availability in polyethylene glycol solutions. Plant Physiol 116:1403–1412

    Article  CAS  PubMed  Google Scholar 

  • Wolfbeis OS (2004) Fiber-optic chemical sensors and biosensors. Anal Chem 76:3269–3284

    Article  CAS  PubMed  Google Scholar 

  • Zitova A, O’Mahony FC, Cross M, Davenport J, Papkovsky DB (2009) Toxicological profiling of chemical and environmental samples using panels of test organisms and optical oxygen respirometry. Environ Toxicol 24(2):116–127

    Article  CAS  PubMed  Google Scholar 

  • Zuberi M, Liu-Snyder P, Ul Haque A, Porterfield DM, Borgens RB (2008) Large naturally-produced electric currents and voltage traverse damaged mammalian spinal cord. J Biol Eng 2:17–26

    Article  PubMed  Google Scholar 

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Acknowledgments

The authors would like to thank the National Science Foundation for funding this research.

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Correspondence to Eric S. McLamore.

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McLamore, E.S., Jaroch, D., Chatni, M.R. et al. Self-referencing optrodes for measuring spatially resolved, real-time metabolic oxygen flux in plant systems. Planta 232, 1087–1099 (2010). https://doi.org/10.1007/s00425-010-1234-6

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