Imaging Vacuolar Anthocyanins with Fluorescence Lifetime Microscopy (FLIM)
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.
Key wordsFluorescence lifetime microscopy (FLIM) Anthocyanins Arabidopsis thaliana Plant vacuole pH
This work was supported by NSF grant MCB-1048847 to EG and MSO.
- 3.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 107:3251–3256CrossRefPubMedPubMedCentralGoogle Scholar
- 10.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 145Google Scholar
- 12.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 6:32–43CrossRefPubMedGoogle Scholar
- 17.Lakowicz JR (2006) Principles of fluorescence spectroscopy. SpringerGoogle Scholar
- 20.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 9:676–682CrossRefPubMedGoogle Scholar