Abstract
Continuous control of metabolism and development is a key feature of life and is of particular importance under stress conditions. While under normal conditions most cellular compartments maintain a reducing environment, the cellular redox state can be influenced by external factors. Redox changes might in turn be employed as part of a signalling cascade leading to molecular responses to adverse situations. To enable dynamic measurements of the cellular redox poise in vivo, reduction-oxidation sensitive GFP (roGFP) can be expressed in plant cells and observed by confocal microscopy. When imaged by confocal microscopy this probe exhibits significant opposing shifts in the fluorescence intensities excited at 488 and 405 nm upon formation of an intramolecular disulfide bridge, which enables ratiometric analysis. The formation of the disulfide bridge is directly responsive to the redox state of the glutathione redox buffer within the subcellular compartment to which roGFP is targeted.
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References
Schiirmann, P. and Jacquot, J. -P. (2000) Plant thioredoxin systems revisited. Annu. Rev. Plant Physiol. Plant Mol. Biol. 51, 371–1OO.
Buchanan, B. B. and Balmer, Y. (2005) Redox regulation: a broadening horizon. Annu. Rev. Plant Biol. 56,187–220.
Buchanan, B. B. (1980) Role of light in the regulation of chloroplast enzymes. Annu. Rev. Plant Physiol. 31, 341–374.
Scheibe, R ( 1991) Redox-modulation of chloroplast enzymes: a common principle for individual control. Plant Physiol. 96,1–3.
Foyer, C. H. and Noctor, G. (2005) Redox homeostasis and antioxidant signaling: a metabolic interface between stress perception and physiological responses. Plant Cell 17,1866–1875.
Mittler, R, Vanderauwera, S., Gollery, M., and Van Breusegem, F. (2004) Reactive oxygen gene network of plants. Trends Plant Sci. 9, 490–498.
Meyer, A. J. and Fricker, M. D. (2008) Imaging thiol-based redox processes in live cells. In: Sulfur Metabolism in Phototrophic Organisms, R. Hell, C. Dahl, D. Knaff, and T. Leustek, Eds., Dordrecht: Springer, pp. 483–501.
Hanson, G. T., Aggeler, R, Oglesbee, D., Cannon, M., Capaldi, R. A., Tsien, R. Y., and Remington, S. J. (2004) Investigating mitochondrial redox potential with redox- sensitive green fluorescent protein indicators./. Biol. Chem. 279,13044–13053.
Dooley, C. T., Dore, T. M., Hanson, G. T., Jackson, W. C., Remington, S. J., and Tsien, R. Y. (2004) Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators. J. Biol. Chem. 279,22284–22293.
Meyer, A. J., Brach, T., Marty, L., Kreye, S., Rouhier, N., Jacquot, J.-P., and Hell, R. (2007) Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the redox potential of the cellular glutathione redox buffer. Plant/., 52, 973–986.
Elsliger, M., Wachter, R, Hanson, G., Kallio, K., and Remington, S. (1999) Structural and spectral response of green fluorescent protein variants to changes in pH. Biochemistry 38, 5296–5301.
Remington, S., and Hanson, G. T. (2006) Oxidation reduction sensitive green fluorescent protein variants. Patent US 7,015,310 B2.
Hansen, R, Østergaard, H., andWinther, J. (2005) Increasing the reactivity of an artificial dithiol-disulfide pair through modification of the electrostatic milieu. Biochemistry 44,5899–5906.
Cannon, M. B. and Remington, S. J. (2006) Re-engineering redox-sensitive green fluorescent protein for improved response rate. Protein Sci. 15, 45–57.
Haseloff, J., Siemering, K. R, Prasher, D. C., and Hodge, S. (1997) Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proc. Natl. Acad. Sci. USA94:, 2122–2127.
Chiu, W., Niwa, Y., Zeng, W., Hirano, T., Kobayashi, H., and Sheen, J. (1996) Engineered GFP as a vital reporter in plants. Curr. Biol. 6, 325–330.
Jiang, K., Schwarzer, C., Lally, E., Zhang, S., Ruzin, S., Machen, T., Remington, S. J., and Feldman, L. (2006) Expression and characterization of a redox-sensing green fluorescent protein (reduction-oxidation- sensitive green fluorescent protein) in Arabidopsis. Plant Physiol. 141, 397–403.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Meyer, A., Brach, T. (2009). Dynamic Redox Measurements with Redox-Sensitive GFP in Plants by Confocal Laser Scanning Microscopy. In: Pfannschmidt, T. (eds) Plant Signal Transduction. Methods in Molecular Biology, vol 479. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-289-2_6
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DOI: https://doi.org/10.1007/978-1-59745-289-2_6
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-58829-943-7
Online ISBN: 978-1-59745-289-2
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