title: Using fluorescence lifetime microscopy to study the subcellular localization of anthocyanins.
abstract: Anthocyanins are flavonoid pigments that accumulate in most seed plants. They are synthesized in the cytoplasm but accumulate inside the vacuoles. Anthocyanins are pigmented at the lower vacuolar pH, but in the cytoplasm they can be visualized based on their fluorescence properties. Thus, anthocyanins provide an ideal system for the development of new methods to investigate cytoplasmic pools and association with other molecular components. We have analyzed the fluorescence decay of anthocyanins by fluorescence lifetime imaging microscopy (FLIM), in both in vitro and in vivo conditions, using wild-type and mutant Arabidopsis thaliana seedlings. Within plant cells, the amplitude-weighted mean fluorescence lifetime (τm) correlated with distinct subcellular localizations of anthocyanins. The vacuolar pool of anthocyanins exhibited shorter τmthan the cytoplasmic pool. Consistently, lowering the pH of anthocyanins in solution shortened their fluorescence decay. We propose that FLIM is a useful tool for understanding the trafficking of anthocyanins and, potentially, for estimating vacuolar pH inside intact plant cells.
authors: Department of Botany, University of Wisconsin-Madison, 430 Lincoln Drive, Madison, WI, 53706, USA.|Laboratory of Molecular and Cellular Biology, University of Wisconsin-Madison, 1525 Linden Drive, Madison, WI, 53706, USA.
authors: Alexandra
authors: A
authors: Chanoca
authors: Laboratory for Optical and Computational Instrumentation (LOCI), University of Wisconsin-Madison, 1675 Observatory Drive, Madison, WI, 53706, USA.
authors: Brian
authors: B
authors: Burkel
authors: Center for Applied Plant Sciences (CAPS), Department of Molecular Genetics and Department of Horticulture and Crop Science, The Ohio State University, 012 Rightmire Hall, 1060 Carmack Rd, Columbus, OH, 43210, USA.
authors: Nik
authors: N
authors: Kovinich
authors: Center for Applied Plant Sciences (CAPS), Department of Molecular Genetics and Department of Horticulture and Crop Science, The Ohio State University, 012 Rightmire Hall, 1060 Carmack Rd, Columbus, OH, 43210, USA.
authors: Erich
authors: E
authors: Grotewold
authors: Laboratory of Molecular and Cellular Biology, University of Wisconsin-Madison, 1525 Linden Drive, Madison, WI, 53706, USA.|Laboratory for Optical and Computational Instrumentation (LOCI), University of Wisconsin-Madison, 1675 Observatory Drive, Madison, WI, 53706, USA.
authors: Kevin W
authors: KW
authors: Eliceiri
authors: Department of Botany, University of Wisconsin-Madison, 430 Lincoln Drive, Madison, WI, 53706, USA.|Laboratory of Molecular and Cellular Biology, University of Wisconsin-Madison, 1525 Linden Drive, Madison, WI, 53706, USA.|Department of Genetics, University of Wisconsin-Madison, 425 Henry Mall, Madison, WI, 53706, USA.
authors: Marisa S
authors: MS
authors: Otegui
mesh_terms: D000872:Anthocyanins / Q000378:metabolism*; D017360:Arabidopsis / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008856:Microscopy, Fluorescence / Q000379:methods*
chemical_list: D000872:Anthocyanins; D029681:Arabidopsis Proteins
keywords: 
  Arabidopsis thaliana
  ; anthocyanins; fluorescence lifetime microscopy; imaging; specialized metabolites; technical advance