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Flavonoids have the potential to serve as antioxidants in addition to their function of UV screening in photoprotective mechanisms. However, flavonoids have long been reported to accumulate mostly in epidermal cells and surface organs in response to high sunlight. Therefore, how leaf flavonoids actually carry out their antioxidant functions is still a matter of debate. Here, the distribution of flavonoids with effective antioxidant properties, i.e. the orthodihydroxy B-ring-substituted quercetin and luteolin glycosides, was investigated in the mesophyll of Ligustrum vulgare leaves acclimated to contrasting sunlight irradiance.
In the first experiment, plants were grown at 20 % (shade) or 100% (sun) natural sunlight. Plants were exposed to 100 % sunlight irradiance in the presence or absence of UV wavelengths, in a second experiment. Fluorescence microspectroscopy and multispectral fluorescence microimaging were used in both cross sections and intact leaf pieces to visualize orthodihydroxy B-ring-substituted flavonoids at inter- and intracellular levels. Identification and quantification of individual hydroxycinnamates and flavonoid glycosides were performed via HPLC-DAD.
Quercetin and luteolin derivatives accumulated to a great extent in both the epidermal and mesophyll cells in response to high sunlight. Tissue fluorescence signatures and leaf flavonoid concentrations were strongly related. Monohydroxyflavone glycosides, namely luteolin 4′-O-glucoside and two apigenin 7-O-glycosides were unresponsive to changes in sunlight irradiance. Quercetin and luteolin derivatives accumulated in the vacuoles of mesophyll cells in leaves growing under 100 % natural sunlight in the absence of UV wavelengths.
The above findings lead to the hypothesis that flavonoids play a key role in countering light-induced oxidative stress, and not only in avoiding the penetration of short solar wavelengths in the leaf.
The idea that flavonoids may counter oxidative damage, in addition to attenuating the highly energetic UV-B wavelengths reaching sensitive targets in a leaf, in response to high solar irradiance, may be inferred from several lines of evidence.
Firstly, it is unlikely that the widely reported UV-B-induced increase in the flavonoid to hydroxycinnamate ratio (Ollson et al., 1999; Burchard et al., 2000; Tattini et al., 2004) depends on their relative abilities to absorb the UV-B wavelengths. Hydroxycinnamic acid derivatives (εmax between 310 and 325 nm) have a greater ability to absorb over the UV-B waveband with respect to most flavonoids (εmax > 330 nm and εmin around 300 nm; Sheahan, 1996; Harborne and Williams, 2000; Tattini et al., 2004).
Secondly, in high light-treated leaves, the biosynthesis of orthodihydroxy B-ring-substituted flavonoids is strongly favoured over that of its monohydroxy B-ring-substituted counterparts (Markham et al., 1998; Ryan et al., 1998; Gould et al., 2000; Tattini et al., 2004, 2005). Nevertheless, the molar extinction coefficient, over the 300–390-nm waveband, of the monohydroxy flavone apigenin 7-O-rutinoside (12·2 mm−1 cm−1) considerably exceeds that of the orthodihydroxy flavonol quercetin 3-O-rutinoside (9·8 mm−1 cm−1; Tattini et al., 2004).
Finally, UV-B radiation is not a pre-requisite for flavonoid biosynthesis (Christie and Jenkins, 1996; Brosché and Strid, 1999; Jenkins et al., 2001; Ibdah et al., 2002). An increase in the concentration of leaf flavonoids was observed in grapevine in response to visible light (Kolb et al., 2001), and the very same flavonoids increased in Lemma gibba in response to both natural sunlight and visible light and excess copper ions (Babu et al., 2003). Bilger et al. (2007) observed an increase in UV-screening compounds in response to low-temperature stress in the absence of UV radiation.
These findings lead to the hypothesis that flavonoids may actually serve a key function to counter UV-induced oxidative stress (Close and McArthur, 2002; Winkel-Shirley, 2002; Tattini et al., 2005), as concluded by Landry et al. (1995): ‘Arabidopsis thaliana responds to UV-B as to an oxidative stress, and sunscreen compounds reduce the oxidative damage caused by UV-B’. Orthodihydroxy B-ring-substituted flavonoids have the potential to effectively inhibit the generation of, as well as to quench, reactive oxygen species (ROS), and structural/antioxidant relationships are well-established (Saskia et al., 1996; Rice-Evans et al., 1997; Brown et al., 1998; Nguyen et al., 2006). It has been suggested previously that metal-chelating and quenching properties, not just the UV-screening features of flavonoids, need to be considered to conclusively explain their roles in land-plant evolution (Swain, 1986; Cooper-Driver and Bhattacharya, 1998; Cockell and Knowland, 1999).
However, it is a matter of fact that flavonoids accumulate mostly in surface organs and epidermal cells because of high-light stress (Schnitzler et al., 1996; Fishback et al., 1997; Tattini et al., 2000, 2007). Yamasaki et al. (1997) proposed that orthodihydroxy B-ring-substituted flavonoids help quench hydrogen peroxide freely diffusing from the mesophyll to enter the vacuoles of the epidermal cells (i.e. serving as substrates for class III peroxidases; Takahama and Oniki, 1997; Pérez et al., 2002). By contrast, few data are available on light-induced accumulation of ‘potentially antioxidant’ flavonoids in chlorophyll-containing tissues, which is a pre-requisite for flavonoids to actually carry out antioxidant functions within the sites of ROS generation (Gould et al., 2002; Kytridis and Manetas, 2006; Agati et al., 2007).
To gain new insights on the complex issue of the multiple functional roles, particularly the antioxidant one, of ‘UV-absorbing’ flavonoids in photoprotection, experiments were performed aimed at visualizing them in ROS-generating cells of leaves exposed to different solar irradiances. For this purpose, ‘new’ techniques of fluorescence microspectroscopy and multispectral fluorescence microimaging were used. Leaves of Ligustrum vulgare, acclimated to (a) 20 % or 100 % natural sunlight and (b) to full sunlight irradiance by cutting or not cutting the UV waveband, were studied. The distribution of hydroxycinnamates and flavonoids was estimated in the leaves paying special emphasis to the mesophyll tissues. Finally, the intracellular distribution of orthodihydroxy B-ring-substituted flavonoids in mesophyll cells was analysed using confocal laser scanning microscopy (CLSM) analysis in intact leaves. As far as is known this is the first report on this subject.
In the first experiment, Ligustrum vulgare L. plants were grown outside under 20 % (shade) or 100 % natural sunlight (sun) over a 5-week period. Plants at the full-sun site received a daily dose of 12·1 and 1·05 MJ m−2 and 19·4 KJ m−2 in the PAR (photosynthetic active radiation over 400–700 nm), UV-A and UV-B wavebands, respectively. Mean daily doses of 2·4 and 0·19 MJ m−2 and 3·6 KJ m−2 in the PAR, UV-A and UV-B wavebands, respectively, were detected at the shade site.
In the second experiment, L. vulgare plants were grown in greenhouses constructed with the roof and walls made from plastic foil with specific transmittances, over an 8-week experimental period. The greenhouses were north–south exposed, with small shielded openings below the front roof at the north-east and north-west corners to permit air circulation. Solar UV radiation was excluded by LEE 226 UV foils (LEE Filters, Andover, UK) in the PAR100 treatment, whereas in the PAR100 + UV treatment plants were grown under a 100-μm ETFE fluoropolymer film (NOWOFLON® ET-6235; NOWOFLON Kunststoffprodukte GmbH & Co. KG, Siegsdorf, Germany). Control plants were grown under 25 % PAR irradiance (PAR25), which was obtained by adding a proper black polyethylene frame to the LEE 226 UV foil. UV irradiance (280–400 nm) and PAR inside the greenhouses were measured with a SR9910-PC double-monochromator spectroradiometer (Macam Photometric Ltd, Livingstone, UK) and a calibrated Li-190 quantum sensor (Li-Cor Inc., Lincoln, NE, USA), respectively. Total UV irradiance was 24·5 or 1·1 W m−2 in the PAR100 + UV and PAR100 treatments, respectively, at midday on a clear day (PAR was 1450 ± 46 µmol m−2 s−1 at both sites).
Hydroxycinnamates and flavonoids were extracted, identified and quantified by HPLC-DAD as reported previously in Tattini et al. (2004, 2005). Hydroxycinnamates were p-coumaric acid and echinacoside (a caffeic glycoside ester), and flavonoids were identified as the orthodihydroxy B-ring-substituted quercetin 3-O-rutinoside and luteolin 7-O-glucoside, and the monohydroxy B-ring-substituted luteolin 4′-O-glucoside and apigenin 7-O-glycosides (both glucoside and rutinoside; Tattini et al., 2004). P-coumaric acid was calibrated at 310 nm, echinacoside at 330 nm and flavonoids at 350 nm, using calibration curves of individual compounds operating in the range 0–30 µg. Echinacoside was isolated with semi-preparative HPLC using protocols previously reported in Romani et al. (2000, 2002). The extinction coefficient spectra of 40 µm phenolic solutions (authentic standards purchased from Extrasynthese, Lyon, Nord, Genay, France) with or without the addition of 200 µm of 2-amino ethyl diphenyl boric acid [0·1 %, w/v, Naturstoff reagent (NR)] in phosphate buffer (pH 6·8) with addition of 1 % NaCl (w/v), were recorded using a diode array spectrophotometer (HP8453, Agilent, Les Ulis, France). Fluorescence spectra of 10 µm solutions (in phosphate buffer, pH 6·8) of echinacoside, luteolin 7-O-glucoside and quercetin 3-O-rutinoside with the addition of 50 µm NR were recorded under UV- (λexc = 365 ± 5 nm) and blue-light excitation (λexc = 488 ± 5 nm). A 1 × 1 cm quartz cuvette was horizontally positioned on the sample-holder of a Diaphot epifluorescence microscope (Nikon, Japan) equipped with a high-pressure mercury lamp (HBO 100 W; OSRAM, The Netherlands). Fluorescence spectra were recorded with a CCD multichannel spectral analyser, described in detail in the next section.
Fluorescence microspectroscopy and multispectral fluorescence microimaging were performed on cross-sections (approx. 100 µm thick) cut from leaves with a vibratory microtome (Vibratome 1100 Plus; St Louis, MO, USA), and stained with NR. The multispectral fluorescence microscope unit has been detailed previously (Tattini et al., 2004; Agati et al., 2007). Fluorescence spectra were recorded using the 365-nm excitation wavelength, which was selected using a 10-nm bandwidth interference filter 365FS10-25 (Andover Corporation, Salem, NH, USA) and an ND 400 Nikon dichroic mirror. Fluorescence spectra (over the 400–800-nm waveband) were measured with a CCD multichannel spectral analyser (PMA 11-C5966; Hamamatsu, Photonics Italia, Arese, Italy) connected to the microscope through an optical fibre bundle, with a ×40 Plan Fluor objective. Fluorescence imaging (on a 404 × 404 µm area) was performed at 580 ± 5 nm using both the 365- and 488-nm excitation wavelengths using a ×10 objective, as described previously (Agati et al., 2007).
CLSM analysis was conducted on a leaf-half infiltrated with approx. 100 µL of NR solution using a plastic syringe without the needle, through a pinhole (made with a 100 µL pipette tip), on the upper leaf end. A leaf piece of approx. 5 × 5 mm, was cut at a distance of 4–5 mm from the pinhole, mounted in the staining buffer, and observed from the adaxial surface. Images were recorded using a Leica TCS SP5 confocal microscope (Leica Microsystems CMS, Wetzlar, Germany) equipped with an acusto-optical beam splitter, and an upright microscope stand (DMI6000). A 246 × 246 µm area was imaged using a ×63 objective (HCX PL APO lambda blue 63·0 × 1·40 OIL UV), and image spatial calibration was 0·5 µm pixel−1. The pinhole was set to one ‘Airy unit’. A microscope was used in the sequential scan mode to detect (a) flavonoids: λexc = 488 nm, λem over the 560–600 nm spectral band; and (b) chlorophyll: λexc = 514 nm, λem over the 670–750 nm spectral band. Fluorescence spectra of NR-stained mesophyll cells were recorded (over the 500–640 nm waveband, using the Leica LAS-AF software package) through measurements in the λ-scan mode with a detection window of 10 nm.
UV spectra of p-coumaric acid and apigenin derivatives, which have a monohydroxy substitution in the benzene ring or in the B-ring of the flavonoid skeleton, respectively, did not undergo a bathochromic shift upon treatment with Naturstoff reagent, in contrast to that observed with the orthodihydroxy structures (Fig. 1A, B). We propose the adduct formation for echinacoside (in blue) and for the orthodihydroxy B-ring-substituted luteolin 7-O-glucoside (in red) upon reaction with NR. It is likely that the same adduct is formed after reaction of quercetin 3-O-rutinoside with the fluorescence enhancer.
The fluorescence spectra of the mesophyll, not only those of the epidermal tissues, greatly differed between UV-excited cross-sections of L. vulgare leaves acclimated to contrasting sunlight irradiance (Fig. 2). The fluorescence intensity of the adaxial epidermis in sun leaves (λem = 573 nm) was three times greater than the fluorescence intensity in shade leaves (λem = 559 nm; Fig. 2A). Fluorescence spectra of the abaxial epidermises differed mostly for the shape, more than for the intensity, when comparing shade (λem = 470 nm) and sun leaves (λem = 562 nm). The fluorescence intensity of ‘sunny’ mesophyll tissues (both palisade and spongy parenchyma tissues) was more than four times greater than the fluorescence intensity of ‘shade’ mesophyll tissues (Fig. 2B).
These findings are consistent with the concentration and composition of the ‘soluble’ phenylpropanoid pool detected in shade or sun L. vulgare leaves. Indeed, the concentration of the ‘highly fluorescent’ (NR-treated) orthodihydroxy-substituted compounds (Fig. 3A), i.e. echinacoside (+75 %), and particularly luteolin 7-O-glucoside (+275 %) and quercetin 3-O-rutinoside (+520 %), mostly varied because of high sunlight (Table 1). In contrast, the concentrations of p-coumaric, apigenin 7-O-glycosides and luteolin 4′-O-glucoside, which have negligible fluorescence yields upon staining with NR (Agati et al., 2002), decreased on average by 20 % passing from shade to sun leaves (Table 1).
However, note that changes in tissue anatomy and in the tissue-specific content in wall-bound phenolics may contribute considerably to tissue fluorescence signatures under UV-light excitation. Furthermore, the phenolic content varies considerably among different leaf tissues (Fig. 4) and, hence, the whole-leaf phenolic concentration is unlikely to relate to tissue-specific fluorescence signatures.
The mesophyll distribution of UV-responsive phenylpropanoids, i.e. quercetin 3-O-rutinoside, luteolin 7-O-glucoside and echinacoside, was visualized by exciting cross-sections with UV- (λexc = 365 ± 5 nm; Fig. 4B) or blue-light (λexc = 488 ± 5 nm; Fig. 4A), and recording fluorescence at 580 nm (F580). First, note that only orthodihydroxy B-ring-substituted flavonoids have detectable fluorescence yields when treated with the fluorescence enhancer (NR) and excited with far blue-light (Fig. 3B), as previously reported (Sheahan and Rechnitz, 1993; Sheahan et al., 1998). It is assumed that flavonoids are dissolved in the cellular milieu. Indeed, the monohydroxy B-ring-substituted kaempferol 3-O-glucoside (astragalin) is autofluorescent when conjugated to the epidermal cell walls or ‘dissolved’ in lipid bilayers, and quercetin non-covalently bound to proteins emits in the green-yellow waveband under the 488-nm excitation wavelength (Strack et al., 1988; Bondar et al., 1998; Nifli et al., 2007; Tattini et al., 2007).
Flavonoids largely occurred in the adaxial cells of the palisade parenchyma of sun leaves, and their tissue-specific distribution did not markedly differ between UV-excited (λexc = 365 ± 5 nm; Fig. 4B) and blue light-excited cross-sections (λexc = 488 ± 5 nm; Fig. 4A). These data are consistent with a preferential accumulation of quercetin and luteolin derivatives in the adaxial palisade cells, as echinacoside, if present in appreciable amounts, would have enhanced greatly F580 emitted from those cells (fluorescence intensity of echinacoside is greater than that of luteolin 7-O-glucoside and above all of quercetin 3-O-rutinoside, under UV-excitation; Fig. 3A). Changes in the relative intensities of the 365- and 488-nm excitation wavelengths may have partially contributed to slight variations in F580 between UV- and blue light-excited cross-sections (Tattini et al., 2004). Finally, it is noted that fluorescence quenching, as a consequence of re-absorption and dimer annhiliation processes (Ferrer et al., 2003; Rodríguez et al., 2004), which is of increasing significance as tissue flavonoid concentration increases, is more likely to have underestimated, rather than enhanced, light-induced increase in F580.
Quercetin and luteolin glycosides did not accumulate in the mesophyll of leaves acclimated to 25 % natural sunlight in the absence of UV wavelengths (PAR25; Fig. 5C). Noticeably, the green-yellow fluorescence due to these orthodihydroxy flavonoids did not vary substantially in intensity when comparing the adaxial palisade cells of leaves exposed to 100 % natural sunlight in presence (PAR100 + UV; Fig. 5A) or absence (PAR100; Fig. 5B) of UV radiation. These findings are consistent with the leaf concentrations of quercetin 3-O-rutinoside and luteolin 7-O-glucoside that increased from 1·1 ± 0·17 µmol g−1 d. wt under PAR25 (means ± s.d., n = 4) to 8·6 ± 0·8 and 11·3 ± 1·9 µmol g−1 d. wt, under PAR100 or PAR100 + UV treatments, respectively. The fluorescence spectrum of blue-light excited (λexc = 488 nm) adaxial palisade cells in PAR100 leaves, which peaked at 565 nm (Fig. 5D), conclusively confirmed the occurrence of these orthodihydroxy B-ring-substituted flavonoids in the cell vacuole (Fig. 5A, B). Chlorophyll-induced attenuation of the excitation wavelength is unlikely to have affected the imaging of flavonoid distribution, as chlorophyll fluorescence did not differ in the adaxial palisade cells of leaves exposed to contrasting sunlight irradiance (Fig. 5A–C).
In the present experiments, the ‘new’ techniques of fluorescence micro-spectroscopy and multispectral fluorescence microimaging, aimed to visualize leaf flavonoids at inter- and intracellular levels, were used to give new insights on their antioxidant functions in photoprotective mechanisms.
It is shown that antioxidant flavonoids occurred to a great extent in the adaxial (both epidermal and mesophyll) cells of leaves acclimated to high sunlight. This finding, taken together with the preferential distribution of hydroxycinnamates (potentially, the best UV-B screening compounds; Landry et al., 1995; Sheahan, 1996; Harborne and Williams, 2000) in deeper leaf tissues (Fig. 4; Ollson et al., 1999; Tattini et al., 2004), suggest that the functional roles of flavonoids in photoprotective mechanisms, do not depend merely on their UV-absorbing features (Markham et al., 1998; Gould et al., 2000).
Conclusive evidence is offered on the mesophyll distribution of the ‘antioxidant’ quercetin 3-O-rutinoside and luteolin 7-O-glucoside in leaves of L. vulgare exposed to natural sunlight in the presence or absence of UV irradiance. These compounds are vacuolar, which conforms to the findings of Neill et al. (2002) and of Kytridis and Manetas (2006) on the vacuolar compartmentation of ‘flavonoid anthocyanins’ in the mesophyll cells of Elatostema rugosum and Cistus creticus, respectively. It is suggested that mechanical pressure on the tonoplast membrane, using the present infiltration technique, allowed NR to enter the vacuole of mesophyll cells, although the pH gradient across the tonoplast would drive its exclusion from the vacuole (Sheahan et al., 1998). Our fluorescence imaging avoided the generation of artefacts during cross-sectioning (Hutzler et al., 1998), and conclusively localized the flavonoids in ROS-generating cells (Gould et al., 2002; Kytridis and Manetas, 2006). Hence, major criticisms on the localization/functional relationship of flavonoids in photoprotection, which mostly concerns their almost exclusive occurrence in epidermal cells (Yamasaki et al., 1997), have been addressed in the present experiment. With the multispectral fluorescence micro-imaging it was not possible to visualize the distribution of flavonoids in other cellular compartments (e.g. the cytoplasm and the chloroplasts; Sheahan et al., 1998; Agati et al., 2007), probably due to the relatively low resolution of fluorescence imaging.
We hypothesize that flavonoids with a catechol group in the B-ring may quench free radicals and hydrogen peroxide (i.e. serving as substrates for class III peroxidases; Yamasaki et al., 1997; Pérez et al., 2002; Pourcel et al., 2006) in mesophyll cells suffering from severe high-light stress, not only to help scavenge H2O2 freely diffusing from them to enter the vacuole of the epidermal ones. These scavenger activities against H2O2 have been previously reported to be effectively served by orthodihydroxy substituted hydroxycinnamates, like echinacoside (Grace et al., 1998; Tattini et al., 2004). As a consequence, the differential tissue-specific distribution of orthodihydroxy B-ring-substituted flavonoids and echinacoside in high-light exposed leaves may merit a deep comment. Here it is highlighted that the flavonoid skeleton, and not only the presence of the catechol group in the B-ring, confers to flavonoids a greater ability to inhibit the generation of free radicals as compared with other phenylpropanoids. Metal-chelating properties are enhanced by the presence of the C = O group in the C-ring of the flavonoid skeleton, and metal-flavonoid complexes mimic superoxide dismutase activity (Morel et al., 1998; Kostyuk et al., 2004). As a consequence, the overall scavenger activity of ‘antioxidant’ flavonoids may exceed that of ‘antioxidant’ hydroxycinnamates. Also it is not excluded that the light-induced biosynthesis of caffeic derivatives from p-coumaric acid, as observed in the present experiment, may have enhanced lignin biosynthesis, more than increasing the concentration of soluble hydroxycinnamic intermediates in highly irradiated cells. This process may be of key significance in cells suffering from high light-induced oxidative damage, i.e. the adaxial palisade parenchyma cells. In wounded tissues of Arabidopsis thaliana, the unusual substrate used by CYP98A3, a 3′-hydroxylase of phenolic esters, which synthesizes caffeic from p-coumaric acid, was to give priority to the synthesis of flavonoids (Schoch et al., 2001), which further corroborates the idea of an involvement of flavonoids in both preventing and repairing light-induced oxidative damage. Nevertheless, the present experimental data do not allow the issue of tissue-specific accumulation of different phenylpropanoid classes because of high sunlight to be conclusively addressed. This matter has, however, great physiological and biochemical significance, and merits further and in-depth investigation.
The accumulation of mesophyll orthodihydroxy B-ring-substituted flavonoids in leaves acclimated to natural sunlight in the absence of UV radiation, adds further evidence for an important role of flavonoids in countering the oxidative stress generated under excess-light conditions, not only to attenuate the highly energetic UV wavelengths from reaching ROS-generating cells. We hypothesize that UV stress (Gerhardt et al., 2008) does not differ from other stressful agents, of both biotic and abiotic origin (Wojtaszek, 1997; Schoch et al., 2001; Roberts and Paul, 2006; Agati et al., 2007; Lillo et al., 2008), in up-regulating the phenylpropanoid branch pathway leading to the biosynthesis of antioxidant flavonoids.
We thank Stefano Mancuso (Dipartimento di Ortoflorofrutticoltura, Università di Firenze) for his decisive contribution in CLSM analyses, Zoran G. Cerovic (Laboratoire d'Ecologie Systématique et Evolution, CNRS – Université Paris-Sud) for help in extinction coefficient measurements, and D. Grifoni and F. Sabatini (IBIMET-CNR, Italy) for performing spectroradiometric detections. G.S. is supported by a grant from Ente Cassa di Risparmio di Firenze.