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

Abstract

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 flavonoids almost exclusively occurred in the adaxial epidermal layer. In sun leaves flavonoids largely accumulated in the adaxial epidermal and subepidermal cells and followed a steep gradient passing from the adaxial epidermis to the inner spongy layers. Flavonoids also largely occurred in the abaxial epidermal cells and constituted the exclusive class of phenylpropanoids synthesized by the cells of glandular trichomes. The proposed method also allowed for the discrimination of the relative abundance of hydroxycinnamic derivatives and flavonoids in different...

The biosynthesis of flavonoids is enhanced similarly by UV radiation and root zone salinity in L. vulgare leaves

“…6B). We note here that (1): cell-wall conjugated phenolics (possibly including flavonoids, as previously reported to occur in another member of the Oleaceae family, Agati et al, 2002) may have contributed in part to F 580 in the mesophyll cells, although to a similar degree in PAR30 and PAR100 leaves (wall-bound phenolics did not substantially differ in differentially irradiated leaves); and (2) the strong F 580 intensity from the abaxial epidermal layers, which also include the leaf surfaces, originates from both the leaf angle (which was steep in PAR100 leaves) and the fluorescence due to flavonoids secreted by the glandular trichomes, which are mostly distributed on the abaxial surface in L. vulgare (Tattini et al, 2005;Agati and Tattini, 2010).…”

“…By contrast, wall-bound conjugated phenolics contributed to F 580 , as phenolics, when "associated" with cellular matrixes (and not dissolved in the cellular milieu) are capable of UV-and blue light-induced fluorescence (even auto-fluorescence, McNally et al, 2003;Tattini et al, 2007;Agati and Tattini, 2010). Wall-bound phenolics, which were estimated as reported previously (Agati et al, 2002), differed less than 15% in differentially irradiated leaves.…”

Total flavonoid concentrations of bryophytes from Tianmu Mountain, Zhejiang Province (China): Phylogeny and ecological factors

“…The results were same with pteridophytes [41]. The stress environment such as dimmer sunshine would promote the synthesis of flavonoids [47]. Light quality has been found that could alter flavonoid production [48–49].…”

Morphology and glandular activity of unicellular trichomes of Epilobium hirsutum

“…2A). To test if the glandular trichomes secrete flavonoic compounds, plant material was sprayed with 1 % aminoethyldiphenylborinate in methanol (Naturstoffreagenz A, Sigma-Aldrich, St. Louis, USA) (Jork et al 1989) and observed under excitation by blue radiation (450 -490 nm) (Agati et al 2002). In the trichome exudates no fluorescence that could be related to the flavonoids was detected ( Fig.…”