Using Fluorescence Lifetime Microscopy to Study the Subcellular Localization of Anthocyanins

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 invitro and invivo conditions, using wild-type and mutant Arabidopsis thaliana seedlings. Within plant cells, the amplitude-weighted mean fluorescence lifetime ((m)) correlated with distinct subcellular localizations of anthocyanins. The vacuolar pool of anthocyanins exhibited shorter (m) than 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. Significance Statement Plants accumulate thousands of compounds derived from primary and specialized metabolic pathways. Analytical tools have been developed to accurately measure such metabolites in extracts, but it is still a significant challenge to establish their location inside plant cells and to determine how they interact with proteins or with the subcellular environment. Here we use anthocyanins to show that fluorescence lifetime imaging microscopy is a useful tool for understanding anthocyanin trafficking and potentially for estimating vacuolar pH inside intact plant cells. We suggest that this approach can also be applied to other autofluorescent plant metabolites.