Abstract
Anthocyanins are intrinsically fluorescent pigments that accumulate in plant vacuoles. We have developed a platform to analyze the fluorescence decay of anthocyanins by fluorescence lifetime imaging microscopy (FLIM), under in vitro and in vivo conditions. Fluorescence lifetime of a fluorophore can be influenced by temperature, pH, oxygen concentration, and other environmental conditions. Within plant cells, the anthocyanin fluorescence lifetime correlates with distinct subcellular compartments. Vacuolar anthocyanins exhibit shorter fluorescence lifetime than the cytoplasmic pool. Consistent with these observations, lower pH of anthocyanins solutions correlated with shorter fluorescence lifetimes. We discuss here the use of FLIM as a tool for analyzing the subcellular distribution of anthocyanins and estimating variation in vacuolar pH in intact cells.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Marty F (1999) Plant vacuoles. Plant Cell 11:587–599
Roberts JKM, Ray PM, Wade-Jardetzky N, Jardetzky O (1980) Estimation of cytoplasmic and vacuolar pH in higher plant cells by 31P NMR. Nature 283:870–872
Krebs M, Beyhl D, Gorlich E, Al-Rasheid KAS, Marten I, Stierhof YD, Hedrich R, Schumacher K (2010) Arabidopsis V-ATPase activity at the tonoplast is required for efficient nutrient storage but not for sodium accumulation. Proc Natl Acad Sci U S A 107:3251–3256
Otegui MS, Herder R, Schulze J, Jung R, Staehelin LA (2006) The proteolytic processing of seed storage proteins in Arabidopsis embryo cells starts in the multivesicular bodies. Plant Cell 18:2567–2581
Martiniere A, Bassil E, Jublanc E, Alcon C, Reguera M, Sentenac H, Blumwald E, Paris N (2013) In vivo intracellular pH measurements in tobacco and Arabidopsis reveal an unexpected pH gradient in the endomembrane system. Plant Cell 25:4028–4043
Tang R-J, Liu H, Yang Y, Yang L, Gao X-S, Garcia VJ, Luan S, Zhang H-X (2012) Tonoplast calcium sensors CBL2 and CBL3 control plant growth and ion homeostasis through regulating V-ATPase activity in Arabidopsis. Cell Res 22:1650–1665
Harborne JB, Williams CA (2000) Advances in flavonoid research since 1992. Phytochemistry 55:481–504
Grotewold E (2006) The genetics and biochemistry of floral pigments. Annu Rev Plant Biol 57:761–780
Chanoca A, Kovinich N, Burkel B, Stecha S, Bohorquez-Restrepo A, Ueda T, Eliceiri KW, Grotewold E, Otegui MS (2015) Anthocyanin vacuolar inclusions form by a microautophagy mechanism. Plant Cell 27:2545–2559
Poustka F, Irani NG, Feller A, Lu Y, Pourcel L, Frame K, Grotewold E (2007) A trafficking pathway for anthocyanins overlaps with the endoplasmic reticulum-to-vacuole protein-sorting route in Arabidopsis and contributes to the formation of vacuolar inclusions. Plant Physiol 145
Pourcel L, Irani NG, Lu Y, Riedl K, Schwartz S, Grotewold E (2010) The formation of anthocyanic vacuolar inclusions in Arabidopsis thaliana and implications for the sequestration of anthocyanin pigments. Mol Plant 3:78–90
Faraco M, Spelt C, Bliek M, Verweij W, Hoshino A, Espen L, Prinsi B, Jaarsma R, Tarhan E, de Boer AH, Di Sansebastiano GP, Koes R, Quattrocchio FM (2014) Hyperacidification of vacuoles by the combined action of two different P-ATPases in the tonoplast determines flower color. Cell Rep 6:32–43
Reguera M, Bassil E, Tajima H, Wimmer M, Chanoca A, Otegui MS, Paris N, Blumwald E (2015) pH regulation by NHX-type antiporters is required for receptor-mediated protein trafficking to the vacuole in Arabidopsis. Plant Cell 27:1200–1217
Chang CW, Sud D, Mycek MA (2007) Fluorescence lifetime imaging microscopy. Methods Cell Biol 81:495–524
Chanoca A, Burkel B, Kovinich N, Grotewold E, Eliceiri KW, Otegui MS (2016) Using fluorescence lifetime microscopy to study the subcellular localization of anthocyanins. Plant J 88:895–903
So PTC, Dong CY (2001) Fluorescence spectrophotometry. In eLSJohn Wiley & Sons Ltd, Chichester
Lakowicz JR (2006) Principles of fluorescence spectroscopy. Springer
Lakowicz JR, Szmacinski H, Nowaczyk K, Johnson ML (1992) Fluorescence lifetime imaging of free and protein-bound NADH. Proc Natl Acad Sci U S A 89:1271–1275
Nelson BK, Cai X, Nebenfuhr A (2007) A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J 51:1126–1136
Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682
Saslowsky DE, Dana CD, Winkel-Shirley B (2000) An allelic series for the chalcone synthase locus in Arabidopsis. Gene 255:127–138
Lange H, Shropshire W, Mohr H (1971) An analysis of phytochrome-mediated anthocyanin synthesis. Plant Physiol 47:649–655
Mancinelli AL, Yang C-PH, Lindquist P, Anderson OR, Rabino I (1975) Photocontrol of anthocyanin synthesis: III. The action of streptomycin on the synthesis of chlorophyll and anthocyanin. Plant Physiol 55:251–257
Lindoo SJ, Caldwell MM (1978) Ultraviolet-B radiation-induced inhibition of leaf expansion and promotion of anthocyanin production: lack of involvement of the low irradiance Phytochrome system. Plant Physiol 61:278–282
Acknowledgments
This work was supported by NSF grant MCB-1048847 to EG and MSO.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Chanoca, A., Burkel, B., Grotewold, E., Eliceiri, K.W., Otegui, M.S. (2018). Imaging Vacuolar Anthocyanins with Fluorescence Lifetime Microscopy (FLIM). In: Pereira, C. (eds) Plant Vacuolar Trafficking. Methods in Molecular Biology, vol 1789. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7856-4_10
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7856-4_10
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7855-7
Online ISBN: 978-1-4939-7856-4
eBook Packages: Springer Protocols