Massimiliano Tattini

Functional roles of flavonoids in photoprotection: new evidence, lessons from the past.

We discuss on the relative significance of different functional roles potentially served by flavonoids in photoprotection, with special emphasis to their ability to scavenge reactive oxygen species (ROS) and control the development of individual organs and whole plant. We propose a model in which chloroplast-located flavonoids scavenge H2O2 and singlet oxygen generated under excess light-stress, thus avoiding programmed cell death. We also draw a picture in which vacuolar flavonoids in conjunction with peroxidases and ascorbic acid constitute a secondary antioxidant system aimed at detoxifying H2O2, which may diffuse out of the chloroplast at considerable rates and enter the vacuole following excess light stress-induced depletion of ascorbate peroxidase. We hypothesize for flavonols key roles as developmental regulators in early and current-day land-plants, based on their ability to modulate auxin movement and auxin catabolism. We show that antioxidant flavonoids display the greatest capacity to regulate key steps of cell growth and differentiation in eukaryotes. These regulatory functions of flavonoids, which are shared by plants and animals, are fully accomplished in the nM concentration range, as likely occurred in early land plants. We therefore conclude that functions of flavonoids as antioxidants and/or developmental regulators flavonoids are of great value in photoprotection. We also suggest that UV-B screening was just one of the multiple functions served by flavonoids when early land-plants faced an abrupt increase in sunlight irradiance.

Stress-induced flavonoid biosynthesis and the antioxidant machinery of plants

There is a growing body of evidence that flavonoids do not primarily function as UV-B screening pigments in photoprotection. Recent findings support the idea that excess light stress, irrespective of the relative proportions of the solar wavebands reaching the leaf surface, up-regulates the biosynthesis of dihydroxy B-ring-substituted flavonoid glycosides, as a consequence of and aimed at countering the generation of ROS. Intriguingly, the very conditions that lead to the inactivation of antioxidant enzymes can also up-regulate the biosynthesis of antioxidant flavonoids, which suggests flavonoids constituting a secondary ROS-scavenging system in plants exposed to severe/prolonged stress conditions. H2O2 may diffuse out of the chloroplast at considerable rates and be transported to the vacuole, the storing site for flavonoids, by tonoplast intrinsic proteins, under severe excess light conditions. We suggest that the unanticipated key role of the vacuole in the ROS homeostasis might be mediated by flavonoids.

Flavonoids as Antioxidants and Developmental Regulators: Relative Significance in Plants and Humans

Phenylpropanoids, particularly flavonoids have been recently suggested as playing primary antioxidant functions in the responses of plants to a wide range of abiotic stresses. Furthermore, flavonoids are effective endogenous regulators of auxin movement, thus behaving as developmental regulators. Flavonoids are capable of controlling the development of individual organs and the whole-plant; and, hence, to contribute to stress-induced morphogenic responses of plants. The significance of flavonoids as scavengers of reactive oxygen species (ROS) in humans has been recently questioned, based on the observation that the flavonoid concentration in plasma and most tissues is too low to effectively reduce ROS. Instead, flavonoids may play key roles as signaling molecules in mammals, through their ability to interact with a wide range of protein kinases, including mitogen-activated protein kinases (MAPK), that supersede key steps of cell growth and differentiation. Here we discuss about the relative significance of flavonoids as reducing agents and signaling molecules in plants and humans. We show that structural features conferring ROS-scavenger ability to flavonoids are also required to effectively control developmental processes in eukaryotic cells.

Antioxidant Activity of Galloyl Quinic Derivatives Isolated from P. lentiscus Leaves

The antioxidant properties of galloyl quinic derivatives isolated from Pistacia lentiscus L. leaves have been investigated by means of Electron Paramagnetic Resonance spectroscopy (EPR) and UV-Vis spectrophotometry. Antioxidant properties have been also estimated using the biologically relevant LDL test. The scavenger activities of gallic acid, 5- O -galloyl, 3,5- O -digalloyl, 3,4,5- O -trigalloyl quinic acid derivatives, have been estimated against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, superoxide ( O 2 m ) radical, and hydroxyl (OH) radical. On the whole, the scavenger activity raised as the number of galloyl groups on the quinic acid skeleton increased. The half-inhibition concentrations (IC 50 ) of di- and tri-galloyl derivatives did not exceed 30 w M for all the tested free radicals. All the tested metabolites strongly reduced the oxidation of low-density lipoproteins (LDL), following a trend similar to that observed for the scavenger ability against OH radical.

Photosynthetic limitations and volatile and non‐volatile isoprenoids in the poikilochlorophyllous resurrection plant Xerophyta humilis during dehydration and rehydration

We investigated the photosynthetic limitations occurring during dehydration and rehydration of Xerophyta humilis, a poikilochlorophyllous resurrection plant, and whether volatile and non-volatile isoprenoids might be involved in desiccation tolerance. Photosynthesis declined rapidly after dehydration below 85% relative water content (RWC). Raising intercellular CO(2) concentrations during desiccation suggest that the main photosynthetic limitation was photochemical, affecting energy-dependent RuBP regeneration. Imaging fluorescence confirmed that both the number of photosystem II (PSII) functional reaction centres and their efficiency were impaired under progressive dehydration, and revealed the occurrence of heterogeneous photosynthesis during desiccation, being the basal leaf area more resistant to the stress. Full recovery in photosynthetic parameters occurred on rehydration, confirming that photosynthetic limitations were fully reversible and that no permanent damage occurred. During desiccation, zeaxanthin and lutein increased only when photosynthesis had ceased, implying that these isoprenoids do not directly scavenge reactive oxygen species, but rather protect photosynthetic membranes from damage and consequent denaturation. X. humilis was found to emit isoprene, a volatile isoprenoid that acts as a membrane strengthener in plants. Isoprene emission was stimulated by drought and peaked at 80% RWC. We surmise that isoprene and non-volatile isoprenoids cooperate in reducing membrane damage in X. humilis, isoprene being effective when desiccation is moderate while non-volatile isoprenoids operate when water deficit is more extreme.

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 layers of the P. latifolia leaves.

Drought stress has contrasting effects on antioxidant enzymes activity and phenylpropanoid biosynthesis in Fraxinus ornus leaves: an excess light stress affair?

The experiment was conducted using Fraxinus ornus plants grown outside under full sunlight irradiance, and supplied with 100% (well-watered, WW), 40% (mild drought, MD), or 20% (severe drought, SD) of the daily evapotranspiration demand, with the main objective of exploring the effect of excess light stress on the activity of antioxidant enzymes and phenylpropanoid biosynthesis. Net CO₂ assimilation rate at saturating light and daily assimilated CO₂ were significantly smaller in SD than in WW and MD plants. Xanthophyll-cycle pigments supported nonphotochemical quenching to a significantly greater extent in SD than in MD and WW leaves. As a consequence, the actual efficiency of PSII (Φ(PSII)) was smaller, while the excess excitation-energy in the photosynthetic apparatus was greater in SD than in WW or MD plants. The concentrations of violaxanthin-cycle pigments relative to total chlorophyll (Chl(tot)) exceeded 200 mmol mol⁻¹ Chl(tot) in SD leaves at the end of the experiment. This leads to hypothesize for zeaxanthin a role not only as nonphotochemical quencher, but also as chloroplast antioxidant. Reductions in ascorbate peroxidase and catalase activities, as drought-stress progressed, were paralleled by greater accumulations of esculetin and quercetin 3-O-glycosides, both phenylpropanoids having effective capacity to scavenge H₂O₂. The drought-induced accumulation of esculetin and quercetin 3-O-glycosides in the vacuoles of mesophyll cells is consistent with their putative functions as reducing agents for H₂O₂ in excess light-stressed leaves. Nonetheless, the concentration of H₂O₂ and the lipid peroxidation were significantly greater in SD than in MD and WW leaves. It is speculated that vacuolar phenylpropanoids may constitute a secondary antioxidant system, even on a temporal basis, activated upon the depletion of primary antioxidant defences, and aimed at keeping whole-cell H₂O₂ within a sub-lethal concentration range.

Identification and Quantitation of Polyphenols in Leaves of Myrtus communis L.

SummaryA liquid-solid extraction and purification procedure (LSE) was developed to identify and quantify polyphenols in the leaf tissue ofMyrtus communis L. Identification and quantitation of individual compounds was performed using HPTLC, HPLC-DAD and HPLC-MS analysis. Leaves ofMyrtus communis L. contain small amounts of phenolic acids (caffeic, ellagic and gallic acids) and quercetin derivatives (quercetin 3-O-galactoside and quercetin 3-O-rhamnoside), whereas catechin derivatives (epigallocatechin, epigallocatechin 3-O-gallate, epicatechin 3-O-gallate) and myricetin derivatives (myricetin 3-O-galactoside, myricetin 3-O-rhamnoside) are present in large amounts. This is the first report on the occurrence of galloyl-derivatives of catechin and gallo-catechin inMyrtus communis L. leaves.

Flavonoids as Antioxidants in Plants Under Abiotic Stresses

Flavonoids make a relevant contribution to the response mechanisms of higher plants to a plethora of abiotic stresses. In addition to the long-reported functions as screeners of damaging short-wave solar radiation, flavonoids have been suggested as playing key functions as antioxidants in stressed plants, by inhibiting the generation and reducing reactive oxygen species (ROS) once formed. The ROS-scavenging properties of flavonoids are restricted to few structures, namely, the dihydroxy B-ring-substituted flavonoid glycosides. This structure–activity relationship conforms to the well-known stress-induced preferential biosynthesis of dihydroxy B-ring-substituted both flavones and flavonols. These flavonoids, especially the derivatives of quercetin, have been shown to greatly affect the movement of auxin at intra- and intercellular levels, and hence to tightly regulate the development of individual organs and the whole plant. The effectiveness of flavonoids to inhibit the activity of the auxin efflux facilitator proteins tightly depends on the chemical features that confer the antioxidant potential. In this review article, we discuss about (1) the effect of different abiotic stresses on the accumulation of individual flavonoids, (2) the potential role served by antioxidant flavonoids in the antioxidant machinery of plants exposed to severe stress conditions, and (3) the function of flavonoids as developmental regulators.

Isoprenoids and phenylpropanoids are part of the antioxidant defense orchestrated daily by drought-stressed Platanus × acerifolia plants during Mediterranean summers

The hypothesis was tested that isoprenoids and phenylpropanoids play a prominent role in countering photooxidative stress, following the depletion of antioxidant enzyme activity in plants exposed to severe drought stress under high solar irradiance and high temperatures. Platanus × acerifolia, a high isoprene-emitting species, was drought-stressed during summer (WS) and compared with unstressed controls (WW). Water relations and photosynthetic parameters were measured under mild, moderate, and severe drought stress conditions. Volatile and nonvolatile isoprenoids, antioxidant enzymes, and phenylpropanoids were measured with the same time course, but in four different periods of the day. Drought severely inhibited photosynthesis, whereas it did not markedly affect the photochemical machinery. Isoprene emission and zeaxanthin concentration were higher in WS than in WW leaves, particularly at mild and moderate stresses, and during the hottest hours of the day. The activities of catalase and ascorbate peroxidase steeply declined during the day, while the activity of guaiacol peroxidase and the concentration of quercetin increased during the day, peaking in the hottest hours in both WW and WS plants. Our experiment reveals a sequence of antioxidants that were used daily by plants to orchestrate defense against oxidative stress induced by drought and associated high light and high temperature. Secondary metabolites seem valuable complements of antioxidant enzymes to counter oxidative stress during the hottest daily hours.