Flavonoid Distribution in Tissues of Phillyrea latifolia L. Leaves as Estimated by Microspectrofluorometry and Multispectral Fluorescence Microimaging

• Published in: Photochemistry and photobiology 2002-09, Vol.76 (3), p.350-360
• Main Authors: Agati, Giovanni, Galardi, Carlotta, Gravano, Elisabetta, Romani, Annalisa, Tattini, Massimiliano
• Format: Article
• Language: English
• Subjects: Flavonoids - metabolism; Oleaceae - metabolism; Photosensory and Circadian Biology; Plant Leaves - metabolism; Spectrometry, Fluorescence - methods; Ultraviolet Rays
• ISSN: 0031-8655; 1751-1097
• DOI: 10.1562/0031-8655(2002)076<0350:FDITOP>2.0.CO;2

A new method for detecting the tissue-specific distribution of flavonoids has been developed by coupling microspectrofluorometry and multispectral fluorescence microimaging techniques. Fluorescence responses of cross sections taken from 1 year old Phillyrea latifolia leaves exposed to full (sun leaves) or 15% (shade leaves) solar radiation in a coastal area of Southern Tuscany were analyzed. Fluorescence spectra of different tissue layers, each normalized at its fluorescence maximum, that were stained or not stained with Naturstoff reagent A (in ethanol), under excitation with UV light (λexc = 365 nm) or blue light (λexc = 436 nm) were recorded. The shape of the fluorescence spectra of tissue layers from shade and sun leaves differed only under UV excitation. The fluorescence of stained cross sections from sun and shade leaves as well as from different layers of sun leaves received a markedly different contribution from the blue (470 nm) and the yellow-red (580 nm) wavebands. Such changes in tissue fluorescence signatures were related to light-induced changes of extractable caffeic acid derivatives and flavonoid glycosides, namely quercetin 3-O-rutinoside and luteolin 7-O-glucoside. Wall-bound phenolics, i.e. hydroxycinnamic acids (p-coumaric, ferulic and caffeic acid) and flavonoids (apigenin and luteolin derivatives), did not substantially differ between sun and shade leaves. A Gaussian deconvolution analysis of fluorescence spectra was subsequently performed to estimate the contribution of flavonoids (emitting at 600 nm, F600 [red fluorescence contribution = signal integrated over a Gaussian band centered at about 600 nm]) relative to the tissue fluorescence (Ftot [total fluorescence = signal integrated over the whole fluorescence spectrum]). The F600/Ftot ratios sharply differed between analogous tissues of sun and shade leaves, as well as among tissue layers within each leaf type. A highly resolved picture of the tissue flavonoid distribution was finally provided through a fluorescence microimaging technique by acquiring fluorescence images at the blue (fluorescence at about 470 nm [F470]) and yellow-red (fluorescence at about 580 nm [F580]) wavelengths and correcting the F580 image for the contribution of nonflavonoids to the fluorescence at 580 nm. Monochrome images were elaborated by adequate computing functions to visualize the exclusive accumulation of flavonoids in different layers of P. latifolia leaves. Our data show that in shade leaves flavo