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

• Inter- and intra-cellular location of coumarins in Fraxinus ornus leaves is shown.
• The fluorescence technique used distinguishes between esculin and esculetin.
• Coumarins in palisade cells accumulate as vacuolar inclusions.
• Esculetin preferentially accumulates in the adaxial leaf layers.
• The differential role of coumarins in photoprotection is discussed.

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

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