Esculetin and esculin (esculetin 6-O-glucoside) occur as inclusions and are differentially distributed in the vacuole of palisade cells in Fraxinus ornus leaves: A fluorescence microscopy analysis

The location of individual coumarins in leaves of Fraxinus ornus acclimated at full solar irradiance wasestimated using their specific UV- and fluorescence spectral features. Using a combination of UV-inducedfluorescence and blue light-induced fluorescence of tissues stained with diphenylborinic acid 2-aminoethylester,in wide field or confocal laser scanning microscopy, we were able to visualize the distributionof esculetin and esculetin 6-O-glucoside (esculin) in palisade cells. Coumarins are not uniformly distributedin the cell vacuole, but accumulate mostly in the adaxial portion of palisade cells. Our study indeedshows, for the first time, that coumarins in palisade cells accumulate as vacuolar inclusions, as previouslyreported in the pertinent literature only for anthocyanins. Furthermore, esculetin and esculin have a differentvacuolar distribution: esculetin largely predominates in the first 15 lm from the adaxial epidermis.This leads to hypothesize for esculetin and esculin different transport mechanisms from theendoplasmic reticulum to the vacuole as well as potentially different roles in photoprotection. Our studyopen to new experiments aimed at exploring the mechanisms that deliver coumarins to the vacuole usingdifferent fluorescence signatures of coumarin aglycones and coumarin glycosides.