Abstract
In leaves, the functioning of many key proteins under conditions promoting oxidative stress depends to a large extent on the redox potential of the glutathione couple. Routine measurements of the glutathione pool in leaves are destructive and labor-intensive processes that tend to underestimate the redox state. Therefore, a challenge for plant scientists is to develop a tool capable of measuring the redox state of the glutathione couple spatially (at different levels of resolution) and temporally in tissues and subcellular compartments in vivo. This chapter highlights the possibilities of using redox-sensitive green fluorescence proteins (roGFPs) as real-time redox reporters for use in intact plants and focuses on practical assessments of using such bioindicators in different leaf cell types subjected to environmental change. The advantages and shortcomings of different GFP variants are discussed along with the choice of system for leaves and possible approaches to overcoming some of the problems. We consider roGFP1-12 as an ideal candidate for developing a redox reporter system in whole plants because it has several advantages over the other variants, with dual excitation peaks allowing a ratiometric approach, insensitivity to pH and halide ions, increased response times for real-time measurements, and appropriate emission wavelengths for use in leaves. We conclude that when using roGFP1-12 with specific cell promotors, it would be possible to target distinct cell compartments and tissues and monitor changes in glutathione redox state to determine the effects of reactive oxygen species on specific cellular components.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Apel, K. and Hirt, H. (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Ann Rev Plant Biol 55, 373–399.
Hanson, G. T., Aggelers, R., Oglesbees, D., Cannon, M., Capaldis, R. A., Tsien, R. Y., and Remington, S. J. (2004) Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators. J Biol Chem 279, 13044–13053.
LeMoan, N., Clement, G., Le Maout, S., Tacnet, F., and Toledano, M. B. (2006) The Saccharomyces cerevisiae proteome of oxidized protein thiols. J Biol Chem 281, 10420–10430.
Mullineaux, P. M., Karpinski, S., and Baker, N. R. (2006) Spatial dependence for hydrogen peroxide-directed signalling in light-stressed plants. Plant Physiol 141, 346–350.
Bechtold, U., Karpinski, S., and Mullineaux, P. M. (2005) The influence of the light environment and photosynthesis on oxidative signalling responses in plant-biotrophic pathogen interactions. Plant Cell Environ 28, 1046–1055.
Gapper, C. and Dolan, L. (2006) Control of plant development by reactive oxygen species. Plant Physiol 141, 341–345.
Torres, M. A., Jones, J. D.G., and Dangl, J. L. (2006) Reactive oxygen species signalling in response to pathogens. Plant Physiol 141, 373–378.
Fryer, M. J., Ball, L., Oxborough, K., Karpinski, S., Mullineaux, P. M., and Baker, N. R. (2003) Control of Ascorbate Peroxidase 2 expression by hydrogen peroxide and leaf water status during excess light stress reveals a functional organisation of Arabidopsis leaves. Plant J 33, 691–705.
Ushio-Fukai, M. (2006) Localizing NADPH oxidase-derived ROS. Science STKE. www.stke.org/cgi/content/full/2006/349/re8
Mullineaux, P. M. and Rausch, T. (2005) Glutathione, photosynthesis and the redox regulation of stress-responsive gene expression. Photos Res 86, 459–474.
Noctor, G., Gomez, L., Vanacker, H., and Foyer, C. H. (2002) Interactions between biosynthesis, compartmentation and transport in the control of glutathione homeostasis and signalling. J Exp Bot 53, 1283–1304.
Jaing, K., Schwarzer, C., Lally, E., Zhang, S., Ruzin, S., Machen, S., Remington, J., and Feldman, L. (2006) Expression and characterization of a redox-sensing green fluorescence protein (reduction-oxidation-sensitive green fluorescence protein) in Arabidopsis. Plant Physiol 141, 397–403.
Fricker, M., Runions, J., and Moore, I. (2006) Quantitative fluorescence microscopy: From art to science. Ann Rev Plant Biol 57, 79–109.
Hartmann, T. N., Fricker, M. D., Rennenberg, H., and Meyer, A. J. (2003) Cell-specific measurement of cytosolic glutathione in poplar leaves. Plant Cell Environ 26, 965–975.
Gutierrez-Alcala, G., Gotor, C., Meyer, A. J., Fricker, M., Vega, J. M., and Romero, C. (2000) Glutathione biosynthesis in Arabidopsis trichome cells. Proc Natl Acad Sci USA 97, 11108–11113.
Chalfie, M. and Kain, S. (2005) Green Fluorescence Protein. Properties, Applications and Protocols. Methods of Biochemical Analysis. Wiley, London/New York.
Ostergaard, H., Henriksen, A., Hansen, F. G., and Winther, J. R. (2001) Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein. EMBO J 20, 5853–5862.
Haseloff, J., Siemering, K. R., Prasher, D. C., and Hodge, S. (1997) Removal of a cryptic intron and subcellular localization of green fluorescence protein are required to mark transgenic Arabidopsis plants brightly. Proc Natl Acad Sci USA 94, 2122–2127.
Ostergaard, H., Tachibana, C., and Winther, J. R. (2004) Monitoring disulfide bond formation in the eukaryotic cytosol. J Cell Biol 166, 337–345.
Dooley, C. T., Dore, T. M., Hanson, G. T., Jackson, W. C., Remington, S. J., and Tsien, R. Y. (2004) J Biol Chem 279, 22284–22293.
Canon, M. B. and Remington, S. J. (2006) Re-engineering redox-sensitive green fluorescence protein for improved response rate. Protein Sci 15, 45–57.
Hanson, R. E., Ostergaard, H., and Winther, J. R. (2005) Increasing the reactivity of an artificial dithiol-disulfide pair through modification of the electrostatic milieu. Biochemistry 41, 5899–5906.
Remington, S. J. (2000) Structural basis for understanding spectral variations in green fluorescence protein. Bioluminescence and chemiluminescence, Methods Enzymol 305, 196–211.
Griesbeck, O., Baird, G. S., Campbell, R. E., Zacharias, D. A., and Tsien, R. Y. (2001) Reducing the environmental sensitivity of yellow fluorescent protein. J Biol Chem 276, 29188–29194.
Zhou, X., Carranco, R., Vitha, S., and Hall, T. C. (2005) The dark side of green fluorescent protein. New Phytol 168, 313–322.
Vogelmann, T. V. and Evans, J. R. (2002) Profiles of light absorption and chlorophyll within spinach leaves from chlorophyll fluorescence. Plant Cell Environ 25, 1313–1323.
Ramonell, K. M., Kuang, A., Porterfield, D. M., Crispi, M. L., Xiao, Y., McClure, G., and Musgrave, M. E. (2001) Influence of atmospheric oxygen on leaf structure and starch deposition in Arabidopsis thaliana. Plant Cell Environ 24, 419–428.
Alonso, R., Elvira, S., Castillo, J., and Gimeno, B. S. (2001) Interactive effects of ozone and drought stress on pigments and activities of antioxidative enzymes in Pinus halepensis. Plant Cell Environ 24, 905–916.
Araki, N., Kusumi, K., Masamoto, K., Niwa, Y., and Iba, K. (2000) Temperature-sensitive Arabidopsis mutant deficient in 1-deoxy-D-xylulose-5-phosphate synthase within the plastid non-mevalonate pathway of isoprenoid biosynthesis. Physiol. Plantarum. 108, 19–24.
Bieza, K. and Lois, R. (2001) An Arabidopsis mutant tolerant to lethal ultraviolet-B levels shows constitutively elevated accumulation of flavinoids and other phenolics. Plant Physiol 126, 1105–1111.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Mullineaux, P.M., Lawson, T. (2008). Measuring Redox Changes In Vivo in Leaves: Prospects and Technical Challenges. In: Hancock, J.T. (eds) Redox-Mediated Signal Transduction. Methods in Molecular Biology™, vol 476. Humana Press. https://doi.org/10.1007/978-1-59745-129-1_5
Download citation
DOI: https://doi.org/10.1007/978-1-59745-129-1_5
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-58829-842-3
Online ISBN: 978-1-59745-129-1
eBook Packages: Springer Protocols