Abstract
Mitochondrial physiology sets the basis for function of the organelle and vice versa. While a limited range of in vivo parameters, such as oxygen consumption, has been classically accessible for measurement, a growing collection of fluorescent protein sensors can now give insights into the physiology of plant mitochondria. Nevertheless, the meaningful application of these sensors in mitochondria is technically challenging and requires rigorous experimental standards. Here we exemplify the application of three genetically encoded sensors to monitor glutathione redox potential, pH, and calcium in the matrix of mitochondria in intact plants. We describe current methods for quantitative imaging and analysis in living root tips by confocal microscopy and discuss methodological limitations.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Baradaran R, Berrisford JM, Minhas GS et al (2013) Crystal structure of the entire respiratory complex I. Nature 494:443–448
Schwarzländer M, Finkemeier I (2013) Mitochondrial energy and redox signaling in plants. Antioxid Redox Signal 18:2122–2144
Schwarzländer M, Fricker MD, Sweetlove LJ (2009) Monitoring the in vivo redox state of plant mitochondria: effect of respiratory inhibitors, abiotic stress and assessment of recovery from oxidative challenge. Biochim Biophys Acta 1787:468–475
Poburko D, Santo-Domingo J, Demaurex N (2011) Dynamic regulation of the mitochondrial proton gradient during cytosolic calcium elevations. J Biol Chem 286:11672–11684
Loro G, Drago I, Pozzan T et al (2012) Targeting of Cameleons to various subcellular compartments reveals a strict cytoplasmic/mitochondrial Ca2+ handling relationship in plant cells. Plant J 71:1–13
Imamura H, Nhat KP, Togawa H et al (2009) Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators. Proc Natl Acad Sci U S A 106:15651–15656
Albrecht SC, Sobotta MC, Bausewein D et al (2014) Redesign of genetically encoded biosensors for monitoring mitochondrial redox status in a broad range of model eukaryotes. J Biomol Screen 19:379–386
Schwarzländer M, Logan DC, Fricker MD et al (2011) The circularly permuted yellow fluorescent protein cpYFP that has been used as a superoxide probe is highly responsive to pH but not superoxide in mitochondria: implications for the existence of superoxide ‘flashes’. Biochem J 437:381–387
Behera S, Krebs M, Loro G et al (2013) Ca2+ imaging in plants using genetically encoded Yellow Cameleon Ca2+ indicators. Cold Spring Harb Protoc 2013:700–703
Schwarzländer M, Logan DC, Johnston IG et al (2012) Pulsing of membrane potential in individual mitochondria: a stress-induced mechanism to regulate respiratory bioenergetics in Arabidopsis. Plant Cell 24:1188–1201
Loro G, Costa A (2013) Imaging of mitochondrial and nuclear Ca2+ dynamics in Arabidopsis roots. Cold Spring Harb Protoc 2013:781–785
Costa A, Candeo A, Fieramonti L et al (2013) Calcium dynamics in root cells of Arabidopsis thaliana visualized with selective plane illumination microscopy. PLoS One 8:e75646
Schwarzländer M, Fricker MD, Müller C et al (2008) Confocal imaging of glutathione redox potential in living plant cells. J Microsc 231:299–316
Logan DC, Knight MR (2003) Mitochondrial and cytosolic calcium dynamics are differentially regulated in plants. Plant Physiol 133:21–24
Gutscher M, Pauleau AL, Marty L et al (2008) Real-time imaging of the intracellular glutathione redox potential. Nat Methods 5:553–559
Logan DC, Leaver CJ (2000) Mitochondria-targeted GFP highlights the heterogeneity of mitochondrial shape, size and movement within living plant cells. J Exp Bot 51:865–871
Fricker MD, May M, Meyer AJ et al (2000) Measurement of glutathione levels in intact roots of Arabidopsis. J Microsc 198:162–173
Zechmann B, Mauch F, Sticher L et al (2008) Subcellular immunocytochemical analysis detects the highest concentrations of glutathione in mitochondria and not in plastids. J Exp Bot 59:4017–4027
Marty L, Siala W, Schwarzländer M et al (2009) The NADPH-dependent thioredoxin system constitutes a functional backup for cytosolic glutathione reductase in Arabidopsis. Proc Natl Acad Sci U S A 106:9109–9114
Dooley CT, Dore TM, Hanson GT et al (2004) Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators. J Biol Chem 279:22284–22293
Ostergaard H, Tachibana C, Winther JR (2004) Monitoring disulfide bond formation in the eukaryotic cytosol. J Cell Biol 166:337–345
Meyer AJ, Brach T, Marty L et al (2007) Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the redox potential of the cellular glutathione redox buffer. Plant J 52:973–986
Nagai T, Sawano A, Park ES et al (2001) Circularly permuted green fluorescent proteins engineered to sense Ca2+. Proc Natl Acad Sci U S A 98:3197–3202
Wang W, Fang H, Groom L et al (2008) Superoxide flashes in single mitochondria. Cell 134:279–290
Nagai T, Yamada S, Tominaga T et al (2004) Expanded dynamic range of fluorescent indicators for Ca2+ by circularly permuted yellow fluorescent proteins. Proc Natl Acad Sci U S A 101:10554–10559
Filippin L, Abad MC, Gastaldello S et al (2005) Improved strategies for the delivery of GFP-based Ca2+ sensors into the mitochondrial matrix. Cell Calcium 37:129–136
Schwarzländer M, Wagner S, Ermakova YG et al (2014) The ‘mitoflash’ probe cpYFP does not respond to superoxide. Nature 514(7523):E12–E14. doi:10.1038/nature13858
Acknowledgement
M.S. was supported by the Deutsche Forschungsgemeinschaft through the Emmy Noether Programme (SCHW1719/1-1).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Wagner, S. et al. (2015). Analysis of Plant Mitochondrial Function Using Fluorescent Protein Sensors. In: Whelan, J., Murcha, M. (eds) Plant Mitochondria. Methods in Molecular Biology, vol 1305. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2639-8_17
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2639-8_17
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2638-1
Online ISBN: 978-1-4939-2639-8
eBook Packages: Springer Protocols