Experiments were conducted on Phillyrea latifolia plants grown under a dense overstorey of Pinus pinea (shade plants) or on seashore dunes (sun plants) in a coastal area of Tuscany (42m 46h N, 10m 53h E). Total integrated photon flux densities averaged 1n67 and 61n4 m mol m −# d −" for shade and sun sites, respectively. A leaf morphological-structural analysis, a qualitative and quantitative analysis of phenylpropanoids of leaf tissue and leaf surface, and a histochemical localization of flavonoids were conducted. The area of sun leaves reached 57% of that of shade leaves, whereas leaf angle (β), sclerophylly index (ratio of leaf d. wt : leaf area), and trichome frequency (trichome number mm −# ) were markedly greater in leaves exposed to full solar radiation than in leaves acclimated to shade. The total thickness of sun leaves was 78% higher than that of shade leaves, mostly owing to a greater development of both palisade parenchyma and spongy mesophyll. The concentration, but not the composition, of leaf tissue phenylpropanoids varied significantly between sun and shade leaves, with a marked increase in flavonoid glycosides in sun leaves. Flavonoids occurred almost exclusively in the upper epidermal cells of shade leaves. By contrast, flavonoids largely accumulated in the upper and lower epidermis, as well as in the mesophyll tissue of leaves that were acclimated to full sunlight. Flavonoid glycosides were found exclusively in the secretory products of glandular trichomes of P. latifolia leaves exposed to high levels of light ; luteolin 7-Oglucoside and quercetin 3-O-rutinoside were the major constituents. By contrast, verbascoside and an unidentified caffeic acid derivative constituted 72% of total phenylpropanoids secreted by glandular trichomes of shade leaves, whereas they were not detected in glandular trichomes of sun leaves. These findings suggest that the light-induced synthesis of flavonoids in glandular trichomes of P. latifolia probably occurs in situ and concomitantly inactivates other branch pathways of the general phenylpropanoid metabolism. This is the first report of the key role of glandular trichomes and of flavonoid glycosides in the integrated mechanisms of acclimation of P. latifolia to excess light.

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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 (lambdaexc = 365 nm) or blue light (lambdaexc = 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 diffe...

Sclerophylly and synthesis of phenolic compounds are active responses of plants subjected to environmental stress (drought, low nutrient supply, u.v.-B radiation, ozone). Here we describe the morphological and histochemical alterations occurring in field-grown leaves of Fagus sylvatica L. from three sites located along an ecological gradient : from a site in cool and protected conditions to one located on a mountain ridge, where the trees grow on a thin layer of soil and are exposed to the wind and to intense solar radiation in summer. The morphological data show that, as the ecological conditions of the stand worsen, individual leaf surface decreases, while the thickness of the leaves and their specific d. wt (i.e. d. wt per unit leaf area) increases. Histochemical and ultrastructural tests show a marked increase of phenolics during the course of the year. These substances, present primarily in the leaves of trees growing in stress conditions, have been identified mainly as tannins. They accumulate in the vacuoles, especially those of the upper epidermal layer and the palisade mesophyll ; at a later stage they appear to be solubilized in the cytoplasm and retranslocated, eventually impregnating the outer wall of the epidermal cells amidst the cellulose fibrils, where they cluster together and form an electron-opaque layer between the wall and the cuticle. Observation of the epidermal cells also reveals that the outer cell wall is thicker. The paper discusses the roles of secondary metabolites in protection and detoxification processes ; the possible ecological significance of these alterations in the ecophysiology of beech trees.

This study was carried out in the summer of 2001 at the Lattecaldo open‐top chamber research facility (Canton Ticino, southern Switzerland). The aim of this research study was to examine the behaviour of Fraxinus excelsior, Prunus avium and Viburnum lantana seedlings grown in charcoal‐filtered (CF) (∼ 50% of the ambient O3) and non‐filtered (NF) (∼ 92% of the ambient O3) air open‐top chambers. Investigations included the assessment of visible foliar symptom development, anatomical and ultrastructural analysis of symptoms, measurements of direct chlorophyll a fluorescence (fluorescence findings were processed by means of the JIP test) and leaf gas exchange measurements. The three species displayed different foliar symptoms. In F. excelsior, symptoms consisted of punctiform stipples with necrotic cells (hypersensitive response, HR), whereas, in P. avium and V. lantana, reddening developed, revealing the accumulation of anthocyanins. In V. lantana, symptoms appeared earlier than in the other species; in F. excelsior, symptoms developed more rapidly and led to premature leaf abscission. In F. excelsior, at least at the beginning, the onset of symptoms was combined with an enhanced photosynthetic efficiency (compensation mechanism), whereas, in P. avium and V. lantana, this efficiency progressively decreased. The fluorescence parameters most closely connected to ozone stress were as follows: a reduction in performance index (PIABS) and active reaction centres (RC/CS0), and an increase in the variable fluorescence relative to 30 ms (VI) and of the dissipation processes. Dissipation is a form of defence mechanism against oxidative stress and is related to the role of the deactivated reaction centres (the silent centres) as well as anthocyanins. Symptom development correlated in all three species with the reduction in reaction centres. Symptomatic leaves had a lower net photosynthetic rate (Pn). Net photosynthesis correlated with the reduction in VI, which suggests an accumulation of reduced plastoquinone, produced in the luminous phase of photosynthesis, which was not capable of reaching the dark phase reactions.

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Identification and quantification of flavonol glycosides and secoiridoids was carried out on leaves of Ligustrum vulgare L. (Oleaceae) by means of HPLC-DAD and HPLC-MS analysis. In addition to previously reported secoiridoids (oleuropein, ligustaloside A, ligustaloside B, and ligstroside) four kaempferol glycosides (kaempferol 3-O-glucoside 7-O-rhamnoside, kaempferol 3, 7-O-dirhamnoside, kaempferol 3-O-rhamnoside, and kaempferol 3-O-glucoside) and two quercetin glycosides (quercetin 3-O-glucoside 7-O-rhamnoside and quercetin 3,7-O-dirhamnoside) were present in leaves of L. vulgare L. Although secoiridoids accounted for nearly the 76% of the total leaf polyphenols content (with ligustaloside A as the main component), kaempferol glycosides were also accumulated in the leaves of L. vulgare L. to a relatively high extent (23%). Contribution of quercetin derivatives was minor under our experimental conditions. Our findings suggest that flavonol glycosides may have a central role in both the ecology and the biology of L. vulgare L.

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...