Chloroplast-located flavonoids can scavenge singlet oxygen

Authors

  • Giovanni Agati,

    1. Istituto di Fisica Applicata ‘Nello Carrara’, IFAC, Consiglio Nazionale delle Ricerche, Via Madonna del Piano 10, I-50019, Sesto F.no, Firenze, Italy;
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  • Paolo Matteini,

    1. Istituto di Fisica Applicata ‘Nello Carrara’, IFAC, Consiglio Nazionale delle Ricerche, Via Madonna del Piano 10, I-50019, Sesto F.no, Firenze, Italy;
    2. Dipartimento di Chimica Organica ‘Ugo Schiff’, Università di Firenze, Via della Lastruccia 13, I-50019 Sesto F.no, Firenze, Italy;
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  • Andrea Goti,

    1. Dipartimento di Chimica Organica ‘Ugo Schiff’, Università di Firenze, Via della Lastruccia 13, I-50019 Sesto F.no, Firenze, Italy;
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  • Massimiliano Tattini

    1. Istituto per la Valorizzazione del Legno e delle Specie Arboree, IVALSA, Consiglio Nazionale delle Ricerche, Via Madonna del Piano 10, I-50019, Sesto F.no, Firenze, Italy
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Author for correspondence: Massimiliano Tattini Tel: +39 0555225 692 Fax: +39 0555225 656 Email: m.tattini@ivalsa.cnr.it

Summary

  • • The hypothesis was tested that flavonoids may scavenge singlet oxygen (1O2) in mesophyll cells of Phillyrea latifolia exposed to excess-light stress.
  • • In cross-sections taken from leaves developed at 10% (shade) or 100% (sun) solar irradiance, we evaluated the excess photosynthetically active radiation (PAR)-induced accumulation of 1O2 in mesophyll cells by imaging the fluorescence quenching of the specific 1O2 probe N-[2-(diethylamino)ethyl]-N-[(2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrol-3-yl)methyl]-5-(dimethylamino)-1-naphthalenesulfonamide (DanePy). The intracellular location of flavonoids was also analyzed using three-dimensional deconvolution microscopy.
  • • Photo-induced quenching of DanePy fluorescence was markedly greater in the mesophyll of shade leaves than in that of sun leaves, the former showing a negligible accumulation of mesophyll flavonoids. The photo-induced generation of 1O2 was inversely related to the content of flavonoids in the mesophyll cells of sun leaves. Flavonoids were located in the chloroplasts, and were likely associated with the chloroplast envelope.
  • • Here we provide relevant evidence for the potential scavenger activity of chloroplast-located flavonoids against 1O2 and new insights into the photo-protective role of flavonoids in higher plants.

Introduction

Flavonoids have long been recognized as playing multiple roles in the response mechanisms of higher plants to stressful agents of different origin, ranging from defense against pathogens and predators (Dixon & Paiva, 1995; Harborne & Williams, 2000; McNally et al., 2003) to the protection of sensitive targets in the leaf from highly damaging short solar wavelengths (Reuber et al., 1996; Ryan et al., 1998; Burchard et al., 2000; Jordan, 2002; Bassman, 2004). The wide distribution of flavonoids on the leaf surface (Wollenweber & Dietz, 1981; Onyilagha & Grotewold, 2004) and in the epidermal cells (Schnabl et al., 1986; Hutzler et al., 1998) may constitute an effective shield against the penetration of ultraviolet B (UV-B) and UV-A radiation, because of their optical properties (Tattini et al., 2004).

More recently, flavonoids have been hypothesized to perform antioxidant functions in tissues exposed to a wide range of environmental stressors (Reuber et al., 1996; Schoch et al., 2001; Agati et al., 2002; Babu et al., 2003; Pearse et al., 2005). These suggestions are supported both by the preferential accumulation of di-hydroxy B-ring substituted flavonoid glycosides as compared to their mono-hydroxy B-ring substituted counterparts (Ollson et al., 1998; Ryan et al., 1998; Hofmann et al., 2000, 2003) and by their large accumulations in mesophyll cells, in response to both UV-B and high solar radiation (Ollson et al., 1999; Tattini et al., 2000; Agati et al., 2002). Flavonoids have been shown to effectively scavenge free radicals in both in vitro and ex vivo experiments (for review articles, see Rice-Evans et al., 1996, 1997), as a result of their ability to quench unpaired electrons (Sichel et al., 1992; Yokozawa et al., 1998; Tattini et al., 2004) and/or to inhibit the generation of free radicals (e.g. through their copper (Cu2+) ion-chelating properties; Brown et al., 1998).

However, it is still a matter of debate how flavonoids (mostly occurring as flavonoid glycosides) may actually carry out their antioxidant functions in plant cells, because of their predominant vacuolar distribution in most species (Hutzler et al., 1998; Neill & Gould, 2003; Pearse et al., 2005). It is widely accepted that hydrogen peroxide (H2O2) may freely diffuse from cellular organelles into the vacuole (even in epidermal cells), where flavonoids serve as substrates for class III peroxidases, which reduce H2O2 by forming relatively stable phenoxyl radicals (Yamasaki et al., 1997; Takahama, 2004; Pearse et al., 2005). Flavonoids have been shown, however, to occur in both the cytosolic and the apoplastic spaces in rye mesophyll cells (Anhalt & Weissenböck, 1992), and cytosolic anthocyanins in Lactuca sativa and Pseudowintera colorata effectively scavenge superoxide anion (Neill & Gould, 2003) and H2O2 (Gould et al., 2002), respectively. Moreover, flavonoids were first detected several decades ago in the chloroplast fractions of several species (Oettmeier & Heupel, 1972; Saunders & McClure, 1976; Ruzieva et al., 1980), and chloroplasts are capable of flavonoid biosynthesis (Zaprometov & Nikolaeva, 2003).

Recently, in Vaccinium spp., flavonoids have been hypothesized to be associated with chloroplasts, and therefore to be optimally located to function as scavengers of highly reactive oxygen (Semerdjieva et al., 2003). The association of flavonoids with chloroplasts might help to address the complex issue of their functional roles (Ollson et al., 1998; Tattini et al., 2005), particularly in the protection of the photosynthetic apparatus from photo-induced generation of 1O2 (Hideg et al., 1994; Hideg et al., 1998, 2000). Flavonoids have been reported to quench 1O2 in vitro (Tournaire et al., 1993; Nagai et al., 2005) and to protect isolated chloroplasts of Triticum aestivum from photo-oxidation (Chauhan et al., 1992).

Here, we report data obtained in experiments designed to evaluate the in vivo scavenger activity of flavonoids against 1O2. We analyzed leaves of Phillyrea latifolia plants adapted to shade (approx. 10% solar irradiance) or to full sunlight (100%) in a coastal area of South Tuscany, Italy. It has been shown that flavonoids do not accumulate in the mesophyll cells of shade leaves (Tattini et al., 2000; Agati et al., 2002), whereas they were found to occur in high quantities in the mesophyll of sun leaves, following a steep gradient from the adaxial palisade to the inner spongy parenchymal tissues (Tattini et al., 2000, 2005; Agati et al., 2002). We used cross-sections (as paradermal sections made difficult the analysis of the various mesophyll layers (data not shown and Gould et al., 2002)) taken from shade- or sun-adapted leaves, and exposed to excess photosynthetically active radiation (PAR), to analyze the tissue-specific and intracellular distributions of (i) flavonoids, using two- and three-dimensional deconvolution (McNally et al., 1999) fluorescence microscopy, respectively; and (ii) 1O2, by coupling microspectrofluorometry and multispectral fluorescence microimaging of the specific 1O2 probe N-[2-(diethylamino)ethyl]-N-[(2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrol-3-yl)methyl]-5-(dimethylamino)-1-naphthalenesulfonamide (DanePy) (Hideg et al., 1998, 2002).

Materials and Methods

Plant material and growing conditions

Fourteen-month-old leaves, which were fully developed (and labeled) in May 2003, were sampled from Phillyrea latifolia L. plants growing either on seashore dunes and exposed to 100% solar irradiance (sun leaves) or under a dense overstory of Pinus pinea and exposed to approximately 10% full solar radiation (shade leaves) in south Tuscany (42°45′N, 10°54′E), at the end of June 2004. The distance between the shade and the sunny sites was ≤ 50 m. Solar irradiance in the UV and PAR wavebands was estimated using an SUV100 scanning spectroradiometer (Biospherical Instruments, San Diego, CA, USA) and a Li-Cor 1800 (Li-Cor Inc., Lincoln, NE, USA) portable spectroradiometer equipped with a remote cosine sensor, respectively. Measurements were conducted on a total of 130 d (both clear (70%) and cloudy) from May 2003 until the sampling period. Irradiance was then integrated on a 14-month basis, and averaged 5.36, 257.9, 2896.8 MJ m−2 in the UV-B, UV-A, and PAR wavebands, respectively, at the sun site. Corresponding values at the shade site were 0.55, 26.8, and 297.4 MJ m−2 in the UV-B, UV-A, and PAR wavebands, respectively.

Basic leaf morphological traits, i.e. leaf area, leaf mass per area (LMA), leaf thickness, and both total chlorophyll (Chltot) and carotenoid (Car) leaf contents were determined as previously described (Tattini et al., 2005).

Cross-sectioning and functionality of photosystem II (PSII) photochemistry

Shoots were immersed in distilled water and sealed in plastic bags, and cross-sections (approx. 100-µm-thick) were cut with a 1100 Plus vibratory microtome (Vibratome, St Louis, MO, USA) from 14-month-old leaves within 6 h of sampling. A preliminary test conducted on cross-sections (Evan's blue test; see Dai et al., 1996) showed that leaf cells were still viable after 6 h after sampling. The functionality of the photosynthetic apparatus in leaf cross-sections was evaluated by measuring the actual quantum yield of PSII photochemistry, i.e. ΦPSII, at 5-min intervals over a 20-min period (the period over the which excess PAR was imposed), following the protocols previously reported in Genty et al. (1989) and Genty & Meyer (1995). Briefly, ΦPSII was calculated as 1 − (Fs/inline image), where Fs and inline image are the chlorophyll (Chl) fluorescence yields, measured at steady state just before and during a saturating light pulse, respectively (Genty et al., 1989). Imaging of Chl fluorescence at 680 nm on blue-light excited (λexc = 436 ± 5 nm) cross-sections allowed estimation of ΦPSII (Genty & Meyer, 1995). In detail, images of Fs and inline image were obtained on cross-sections exposed to 77.2 µmol m−2 s−1 for 20 s and to 7688 µmol m−2 s−1 for 200 ms, respectively. ΦPSII did not vary for > 8% in both shade and sun leaves over a 20-min period.

Chemicals

2,5-Dihydro-2,2,5,5-tetramethyl-1H-pyrrole-3-carboxamide, methyltrioxorhenium (MTO), N,N-diethyl-ethylenediamine (H2NCH2CH2NEt2) and urea-hydroperoxide (UHP) were purchased from Acros Chimica (Geel, Belgium). 5-Dimethylamino-1-naphtalensulfonyl chloride (dansyl chloride) was obtained from Alfa Aesar (Karlsruhe, Germany). Diphenylborinic acid 2-amino-ethylester, NaClO, Rose Bengal (RB), dimethylsulfoxide, sodium bis(2-methoxyethoxy)aluminium hydride (Red-Al), methanesulfonyl chloride (MsCl), dichloromethane (CH2Cl2), triethylamine (Et3N), and all other solvents were purchased from Sigma (St Louis, MO, USA). Phosphate-buffered saline (PBS) solution, i.e. phosphate buffer at pH 6.8 with the addition of 1% (weight/volume (w/v)) NaCl, was used to prepare all solutions for both in vitro and in vivo experiments.

Synthesis and in vitro properties of the 1O2 fluorescent probe (DanePy)

The synthesis of DanePy was performed following the seven-step procedure reported in the Supplementary material (Appendices S1–S3 and Fig. S1), by improving the synthesis protocols proposed by Rozantzev & Krinitzkaya (1965), Hankovszky et al. (1980), Hideg et al. (1980), Kálai et al. (1998), and Murray & Iyanar (1998). DanePy is both a fluorescent and a spin probe, which reacts with 1O2 to produce the corresponding nitroxide radical, DanePyO, the fluorescence of which is then quenched by an energy transfer from the ‘donor’ dansyl to the ‘acceptor’ nitroxide moiety (Kálai et al., 1998; Hideg et al., 2002). The quenching of DanePy fluorescence has therefore been reported to be related to the content of 1O2 (Hideg et al., 2002). DanePy has been shown to be suitable for the detection of 1O2 in plants, as its diethylaminoethyl side-chain allows the probe to penetrate tissues easily (Kálai et al., 1998).

The effectiveness of the DanePy synthesized here for the detection of 1O2 was preliminarily tested in vitro using both a physical and a chemical method of 1O2 generation. First, 1O2 was generated photo-chemically by adding the photo-sensitizer RB (from a 0.1% stock solution in dimethylsulfoxide) to DanePy in PBS to final concentrations of 0.05 and 0.25 mm, respectively. The blank solution consisted of 0.25 mm DanePy in PBS. Secondly, 1O2 was produced by mixing 0.25 mm (final concentration) sodium hypochlorite (NaOCl), 0.25 mm H2O2 and 0.25 mm DanePy in PBS. The blank solution consisted of H2O2 and DanePy in PBS, both at a concentration of 0.25 mm. In both experiments, sample and blank solutions were then exposed to white light (at a radiation power density of 96 mW cm−2, as quantified using a Nova power meter coupled to a 2A thermal head (Ophir Optronics Ltd, Jerusalem, Israel) from a fiber-optics Xenon lamp (LQX 1800; Linos Photonics, Milford, MA, USA), over a 40-min period. The fluorescence spectra of the sample and blank solutions were recorded under UV excitation (λexc = 365 nm) using the microspectro-fluorometry equipment described in the next section, with a 1 × 1 cm quartz cuvette placed over the stage of an inverted epifluorescence microscope.

Microspectrofluorometry and multispectral fluorescence microimaging of flavonoids and 1O2

The tissue-specific distributions of flavonoids and 1O2 were estimated in 100-µm-thick cross-sections of leaf fresh material, which were stained in 0.1% (w/v) diphenylborinic acid 2-amino-ethylester (Naturstoff reagent (NR)) or in 0.2 mm DanePy (both in PBS), respectively, over a 4-min period. All measurements were performed using an inverted epifluorescence microscope (Diaphot, Nikon, Japan) equipped with a high-pressure mercury lamp (HBO 100 W; Osram, Augsberg, Germany) as the light source. The excitation wavelengths were selected using 10-nm bandwidth interference filters, 365FS10-25 and 488FS10-25 (Andover Corporation, Salem, NH, USA), coupled to ND400 and ND510 (Nikon) dichroic mirrors, for the excitation wavelengths λexc = 365 and λexc = 488 nm, respectively. Fluorescence spectra were recorded with a CCD multichannel spectral analyzer (PMA 11-C5966; Hamamatsu, Photonics Italia, Arese, Italy), connected to the microscope through an optical fiber bundle (1 mm in diameter), using a ×40 Plan Fluor (Nikon) objective. The fluorescence signal (from a 490-µm2 spot) was integrated over a 2-s period, and fluorescence spectra were corrected for the transmission properties of both optics and filters. Fluorescence images were acquired using a slow-scan cooled CCD camera (Chroma CX260; DTA, Cascina, Italy) equipped with a Kodak KAF261E, 512 × 512 pixel detector, and elaborated as previously described (Agati et al., 2002; Tattini et al., 2004). The image spatial calibration, using the ×10 Plan Fluor (Nikon) objective, was 0.79 µm pixel−1.

The tissue-specific distribution of flavonoids was evaluated using fluorescence images, acquired at 580 nm (selected using a 10-nm bandwidth interference filter, 580FS10-25; Andover Corporation), of blue light-excited (λexc = 488 nm) cross-sections (Hutzler et al., 1998; Tattini et al., 2004). DanePy fluorescence was imaged at 546 nm (using a 10-nm bandwidth interference filter, 546FS10-25; Andover Corporation), in UV-excited (λexc = 365 nm) cross-sections stained with DanePy. Finally, the tissue distribution of Chl autofluorescence was analyzed from fluorescence images acquired at 680 nm (selected using a 10-nm bandwidth interference filter, 680FS10-25; Andover Corporation), under both UV (λexc = 365 nm) and blue-light (λexc = 436 nm) excitation. The tissue fluorescence profiles produced by DanePy, flavonoids, and Chl were then measured following the protocol previously reported in Tattini et al. (2004). In detail, profiles of normalized (to relative maximum values, except for the quenching of DanePy fluorescence, which originated from normalized images; see Eqn 1) fluorescence intensity for DanePy, flavonoid and Chl, over the whole leaf depth or along the longitudinal axis of the cross-sections, were computed from 404 × 404 µm fluorescence images, by averaging the profiles of fluorescence intensity of 512 columns or 512 rows of pixels (image-pro plus software; Media Cybernetics, Silver Spring, MD, USA).

Finally, the intracellular distribution of flavonoids and Chl in the mesophyll was determined using three-dimensional deconvolution fluorescence microscopy (McNally et al., 1999). Image acquisition and reconstruction were performed with a DeltaVision RT platform (Applied Precision, Issaquah, WA, USA), using an Olympus IX71 inverted microscope (Olympus, Melville, NY, USA) equipped with an Olympus 60×/1.4 NA oil immersion objective lens, and mercury-arc illumination coupled to a COOLSNAP_HQ/ICX285 CCD camera (Photometrix, Tucson, AZ, USA). The fluorescence of NR-stained tissues was sequentially recorded (in two channels) using a polychroic mirror under the following experimental conditions: (a) λexc = 490 ± 20 nm (Chroma Technology Corp., Brattleboro, VT, USA) and λem = 608 ± 10 nm (Quanta System, Milano, Italy) to visualize flavonoids; (b) λexc = 405 ± 10 nm (405FS10-25; Andover Corporation) and λem = 685 ± 40 nm (Chroma Technology) to image Chl fluorescence. A total of 80 optical sections, taken at 0.2-µm intervals along the z-axis, were acquired moving from outside the sample, and the stack of images was then deconvoluted to remove the contribution of out-of-focus fluorescence. The effective pixel size was 0.11 × 0.11 µm.

Induction of photo-oxidative stress and DanePy fluorescence quenching in vivo

DanePy-stained cross-sections were mounted on a glass microscope slide, with the coverslip sealed with paraffin to avoid sample dehydration, and exposed to excess PAR, over a 20-min period, to induce photo-oxidation. Excess-PAR treatment was carried out by irradiating cross-sections at approx. 2800 µmol m−2 s−1, mostly consisting of the 436-nm Hg emission line provided by the epifluorescence microscope excitation source (and selected using a 400-nm GG400 long-pass filter (Schott Glas, Mainz, Germany) coupled to a Nikon ND510 dichroic mirror at 510 nm). The irradiation light was focused by a ×10 Plan Fluor objective over a 0.78-mm2 area (as estimated by a micrometer calibration slide (Reichert-Jung, Heerbrugg, Switzerland)). The excess-PAR-induced photoinhibition was then monitored by comparing the ΦPSII values of light-treated and control samples (Hideg et al., 2002). Fluorescence spectra and images (404 × 404 µm in size) were acquired before (control) and at the end of the light treatment (light-treated), by changing the set of excitation filters, but avoiding movement of the sample. This experimental set-up allowed the sequential analysis of DanePy and flavonoid fluorescence (after specimen staining with NR), over the same tissue portion. DanePy and flavonoid fluorescence was also analyzed in two serial cross-sections, to check for photochemical-induced changes in flavonoid fluorescence, which did not occur in our experiment (data not shown).

The actual light-induced quenching of DanePy fluorescence was then calculated as:

image(Eqn 1)

(inline image and inline image, the fluorescence images at 546 nm of UV-excited cross-sections before (t0) and after (t1) a 20-min exposure to excess PAR, respectively.) The normalization of fluorescence images as proposed in Eqn 1 has two major advantages with respect to calculation of the quenching of DanePy fluorescence simply as inline image. First, wound-induced (as a result of cross-sectioning) generation of 1O2 at t0, which may differ between sun and shade leaves or among different mesophyll tissues, is taken into account. Secondly, the proposed normalization procedure also considers the potential changes in inline image, because of differential penetration of the fluorescence probe among tissue layers or individual cells.)

Experimental design and data analysis

The experimental design was completely randomized, with five replicate plants at both the shade and the sun sites. Unless otherwise stated, measurements were conducted on five replicate leaves sampled from five plants at each sampling site. Leaves were measured for their size and, after cross-sectioning, LMA and photosynthetic pigment content were estimated on two 0.28-cm2 leaf discs per replicate. Data were subjected to one-way analysis of variance (ANOVA). Mean values (from five replicate cross-sections) of the actual efficiency of PSII photochemistry (ΦPSII) were subjected to a two-way ANOVA (with site and light treatment as factors, with their interactions). Changes in the shape and intensity of DanePy fluorescence spectra as a result of the generation of 1O2 in vitro (through both photochemical (RB) and chemical (NaOCl) methods) were evaluated in triplicate experiments. The 1O2-induced quenching of DanePy fluorescence intensity did not differ by > 6% between replicate experiments. Fluorescence imaging of DanePy, Chl, and flavonoids was performed on five replicate cross-sections taken from shade and sun leaves, respectively, and representative images are presented (fluorescence yields, particularly from the palisade parenchymal tissues, did not differ by > 15% between replicate measurements). The relationship between the tissue flavonoid fluorescence (% of normalized fluorescence intensity) and the quenching of DanePy fluorescence in photo-oxidized tissues was investigated by recording the relative fluorescence profiles at 50-µm intervals over the whole leaf depth in sun and shade leaves, respectively. Data of flavonoid and DanePy fluorescence were fitted using a linear regression equation. Because the fluorescence signal attributable to mesophyll flavonoids was negligible (i.e. near to the sensitivity limit of the spectral analyzer) in shade leaves (see Fig. 5g for details), fitting of DanePy and flavonoid fluorescence was carried out only for sun leaves. Similarly, differences in DanePy fluorescence quenching (intensity profiles) between shade and sun leaves were not tested for their statistical significance, as marked changes in tissue anatomy should have greatly altered the DanePy fluorescence yields (McClendon & Fukshansky, 1990).

Figure 5.

Fluorescence imaging and microspectroscopy of sun (a–d) and shade (e–g) leaves of Phillyrea latifolia following photo-oxidative stress (blue light at 2800 µmol m−2 s−1). (a, e) Normalized fluorescence images, acquired at 546 nm, of N-[2-(diethylamino)ethyl]-N-[(2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrol-3-yl)methyl]-5-(dimethylamino)-1-naphthalenesulfonamide (DanePy)-stained cross-sections taken from sun (a) and shade (e) leaves, respectively, showing the photo-induced quenching of DanePy fluorescence. Images were calculated as reported in Eqn 1 (see the Materials and Methods section) on the basis of the change in DanePy fluorescence before (t0) and after the light treatment (t1 = 20-min excess photosynthetically active radiation (PAR)), normalized to DanePy fluorescence at t0. (c, f) Images in (c) and (f) are the recombinations of fluorescence images for flavonoids (yellow) with those for the quenching of DanePy (blue) in sun and shade leaves, respectively. (b, g) Profiles of DanePy fluorescence quenching (blue) and flavonoid fluorescence (yellow) over the whole leaf depth (i.e. from y0 to y1 in (c) and (f)) of sun (b) and shade (g) leaves, following the vertical arrow in (c). (d) The horizontal profile (from x0 to x1 in (c)) of DanePy quenching and flavonoid fluorescence of adjacent cells located in the adaxial palisade parenchymal layer of sun leaves. Bars: (a) 100 µm; (e) 50 µm.

Results and Discussion

DanePy as 1O2 probe in vitro and DanePy fluorescence signatures in vivo

The effectiveness of DanePy, synthesized in our experiment, for the detection of singlet oxygen was preliminarily estimated in vitro by physical (Fig. 1a) and chemical (data not shown) methods, using fluorescence microspectroscopy. The emission spectrum of UV-excited (λexc = 365 nm) DanePy peaked at approx. 585 nm, and neither the fluorescence yield nor the shape of the emission spectrum was affected by the excess-PAR treatment (Fig. 1a, upper curves). The addition of RB to DanePy decreased the fluorescence intensity of DanePy and, in addition, shifted the fluorescence spectrum to longer wavelengths (maximum fluorescence peaked at approx. 600 nm; Fig. 1a, lower curves). In fact, RB has been shown to appreciably reabsorb the 520–570-nm wavelengths (Stiel et al., 1996). The fluorescence intensity of DanePy in the presence of RB was decreased by approx. 30% because of excess PAR, as the energy transfer from the photo-sensitizer to molecular oxygen actually generated 1O2 (Neckers, 1989). A decrease (by approx. 50%) in DanePy fluorescence intensity was also observed when 1O2 was produced by adding NaOCl to the DanePy + H2O2 solution (data not shown).

Figure 1 (a) Fluorescence spectra of a 0.25 mm N‐[2‐(diethylamino)ethyl]‐N‐[(2,5‐dihydro‐2,2,5,5‐tetramethyl‐1H‐pyrrol‐3‐yl)methyl]‐5‐(dimethylamino)‐1‐naphthalenesulfonamide (DanePy) solution (in phosphate‐buffered saline solution (PBS)) without (thin upper curves) or with (thick lower curves) the addition of 50 µm Rose Bengal (RB) under UV‐light excitation (λexc = 365 nm). Solutions were either irradiated at 96 mW cm−2 (dashed lines) or kept in the dark (solid lines), over a 40‐min period. Each curve represents the mean of three replicate experiments (1O2‐induced quenching of DanePy fluorescence intensity did not vary by > 8% between replicate measurements). (b) Fluorescence spectra of palisade parenchyma in Phillyrea latifolia sun leaves, under UV‐light excitation. Cross‐sections were stained (solid line) or not (dashed line) with 0.2 mm DanePy. Measurements were carried out on the second layer of palisade parenchyma tissue (at a depth of approx. 120 µm from the adaxial epidermis). The fluorescence spectra are the means of five replicate cross‐sections.

Figure 1 (a) Fluorescence spectra of a 0.25 mm N-[2-(diethylamino)ethyl]-N-[(2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrol-3-yl)methyl]-5-(dimethylamino)-1-naphthalenesulfonamide (DanePy) solution (in phosphate-buffered saline solution (PBS)) without (thin upper curves) or with (thick lower curves) the addition of 50 µm Rose Bengal (RB) under UV-light excitation (λexc = 365 nm). Solutions were either irradiated at 96 mW cm−2 (dashed lines) or kept in the dark (solid lines), over a 40-min period. Each curve represents the mean of three replicate experiments (1O2-induced quenching of DanePy fluorescence intensity did not vary by > 8% between replicate measurements). (b) Fluorescence spectra of palisade parenchyma in Phillyrea latifolia sun leaves, under UV-light excitation. Cross-sections were stained (solid line) or not (dashed line) with 0.2 mm DanePy. Measurements were carried out on the second layer of palisade parenchyma tissue (at a depth of approx. 120 µm from the adaxial epidermis). The fluorescence spectra are the means of five replicate cross-sections.

The UV-excited fluorescence spectrum of DanePy in vivo was that resulting from the fluorescence signal of palisade cells stained with 0.2 mm DanePy (Fig. 1b). The shape of the DanePy fluorescence signal in vivo markedly differed from the corresponding spectrum in vitro, the former showing a peak of maximum fluorescence at a shorter wavelength (550 nm; Fig. 1b) than the latter (585 nm; Fig. 1a). We suggest that the autofluorescence attributable to both wall-bound (ferulic and caffeic acid derivatives; Harris & Hartley, 1976; Morales et al., 1996; Agati et al., 2002) and soluble hydroxycinnamates (mostly verbascoside; Agati et al., 2002; Tattini et al., 2004) may have been partially responsible for the hypsochromatic shift of DanePy fluorescence in the palisade cells of P. latifolia leaves. Similar suggestions have previously been made by Kálai et al. (1998) and Hideg et al. (2002) to explain the fluorescence signatures of DanePy in thylakoid membranes and in whole spinach (Spinacia oleracea) leaves, respectively. It is possible that changes in pH between in vitro (PBS at pH 6.8) and in vivo experiments may also have affected the shape of DanePy fluorescence spectra (Valeur, 2002).

Morpho-anatomical features, photosynthetic pigment content, and fluorescence characteristics of sun and shade P. latifolia leaves

Shade leaves were significantly greater in size and much thinner (with a significantly smaller LMA) than sun leaves (Table 1). Furthermore, the mesophyll of sun leaves consisted of three to four layers of long, closely packed palisade parenchyma cells (see also Tattini et al., 2000), whereas the mesophyll of shade leaves consisted of just a single layer of short palisade cells (Fig. 2). Total Chl (Chltot) did not differ between sun and shade leaves when expressed on a leaf area basis, but was smaller in the former when expressed on a leaf dry weight basis (Table 1). The extent to which an increase in sunlight irradiance decreased the leaf carotenoid (Car) content on a dry weight basis was still significant in P. latifolia, but the Car content was significantly greater in sun than in shade leaves when expressed on a leaf area basis (Table 1). Finally, leaf flavonoids, which were estimated using fluorescence images at 580 nm (Hutzler et al., 1998; Tattini et al., 2005) of blue-excited cross-sections, largely occurred, following a steep gradient from adaxial to abaxial tissues, in the mesophyll cells of sun leaves (Fig. 2d; Agati et al., 2002; Tattini et al., 2005), whereas they were almost exclusively located in the epidermis of shade leaves (Fig. 2h; Tattini et al., 2000).

Table 1.  Morphological features and photosynthetic pigment content of Phillyrea latifolia leaves sampled at the shade (10% solar irradiance) or the sun (100%) site
ParameterSampling site
ShadeSun
  1. Measurements were taken on 14-month-old leaves sampled at the end of June. Data are means ± standard deviation (n = 5), and those not followed by the same letter are significantly different at P ≤ 0.05, using the least significant difference (LSD) test.

  2. Car, carotenoid; Chltot, total chlorophyll; DW, dry weight; LMA, leaf mass per area; ns, not significant.

Leaf size (cm2)  6.7 ± 0.8 a  3.7 ± 0.5 b
LMA (mg DW cm−2) 12.6 ± 1.1 b 24.9 ± 1.3 a
Leaf thickness (µm)229.8 ± 14.7 b442.5 ± 19.9 a
Chltot (mg g−1 DW)  4.8 ± 0.6 a  2.5 ± 0.3 b
Chltot (µg cm−2) 60.5 ± 5.8 62.5 ± 7.7 ns
Car (mg g−1 DW)  1.0 ± 0.1 a  0.7 ± 0.1 b
Car (µg cm−2) 12.7 ± 0.9 b 17.5 ± 1.5 a
Figure 2.

Multispectral fluorescence microimaging of Phillyrea latifolia leaves that developed at the sun (a–d) or shade (e–h) site. (a, c, e, g) Fluorescence images at 546 nm of cross-sections stained with N-[2-(diethylamino)ethyl]-N-[(2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrol-3-yl)methyl]-5-(dimethylamino)-1-naphthalenesulfonamide (DanePy) (0.2 mm in phosphate-buffered saline solution (PBS)), excited with ultraviolet (UV) light (λexc = 365 nm) before (a, e) and after (c, g) a 20-min period of excess photosynthetically active radiation (PAR) treatment (at 2800 µmol m−2 s−1; mostly blue light at 436 ± 5 nm). (b, f) Chlorophyll fluorescence images at 680 nm of UV-excited cross-sections taken from sun (b) or shade (f) leaves. (d, h) Flavonoid fluorescence images at 580 nm of Naturstoff reagent (NR)-stained cross-sections taken from sun (d) and shade (h) leaves, under blue-light excitation at 488 nm. Bar, 100 µm.

We suggest that the strikingly different mesophyll contents in UV-absorbing compounds in sun (Fig. 2d) and shade (Fig. 2h) leaves were largely responsible for the intensities of DanePy (Fig. 2a,e) and Chl (Fig. 2b,f) fluorescence in the corresponding cross-sections under UV-light excitation (λexc = 365 nm). In fact, DanePy fluorescence, as evaluated from images recorded at 546 nm, was much weaker in the adaxial mesophyll cells (which had the greatest accumulation of flavonoids; Figs 2a, 3a) than in the abaxial mesophyll cells in sun leaves, whereas it appeared to be more equally distributed in the mesophyll of shade leaves (but note that the strongest DanePy fluorescence likely originated from the vascular bundles; Fig. 2e). Analogously, Chl fluorescence mostly originated from mesophyll tissues located at a greater distance from the adaxial surface in sun leaves (Figs 2b, 3a), but did not appreciably vary among mesophyll cells in shade leaves (Fig. 2f). We argue that flavonoids may have effectively reduced the intensity of UV light actually available to excite both DanePy and Chl. Indeed, the intensity of Chl fluorescence did not vary by > 10–15% over the entire mesophyll (Fig. 3b), when cross-sections taken from sun leaves were excited with blue light (flavonoids do not absorb at 436 nm), as previously reported to occur in analogous tissues of Acer platanoides (McCain et al., 1993).

Figure 3.

Intensity profiles of N-[2-(diethylamino)ethyl]-N-[(2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrol-3-yl)methyl]-5-(dimethylamino)-1-naphthalenesulfonamide (DanePy), flavonoid and chlorophyll (Chl) fluorescence, each normalized to the relative maximum value, throughout the leaf depth of a Phillyrea latifolia sun leaf. Profiles were computed from the fluorescence images reported in Fig. 2(b,d) by averaging the intensity of 512 columns of pixels (each individual row of pixels vs the leaf depth). (a) DanePy (solid line) and Chl (dotted line) fluorescence recorded at 546 and 680 nm, respectively, under ultraviolet (UV) excitation. (b) Flavonoid fluorescence at 580 nm (thick dashed line) and Chl fluorescence at 680 nm, under UV (λexc = 365 nm, thin dotted line) or blue-light (λexc = 436 nm, solid line) excitation. Chl fluorescence depression at a depth of 180–240 µm was attributable to the presence of vascular bundles (see Fig. 2b). ep, epidermal layer; palisade, palisade parenchyma; spongy, spongy parenchyma.

Finally, we note that the nearly identical distributions of DanePy and Chl fluorescence in the mesophyll of P. latifolia leaves confirm that DanePy was widely distributed in the chloroplasts (Hideg et al., 2001, 2002). Nevertheless, DanePy fluorescence appeared to originate also from the vacuolar compartment (Fig. 2), although photo-micrograph resolution does not allow conclusive intracellular compartmentation of DanePy to be established.

On the relation between flavonoids and photo-induced quenching of DanePy

Cross-sections irradiated with blue light (mostly at 436 nm) at 2800 µmol m−2 s−1, over a 20-min period, underwent photoinhibition, as ΦPSII of light-treated cross-sections was significantly smaller than that of controls (Table 2). Furthermore, the excess-PAR-induced decrease in ΦPSII was slightly greater in shade (−34%) than in sun (−27%) leaves, the latter also showing a smaller ΦPSII (15%), before the onset of the excess-light treatment (Table 2). Photoinhibition of photosynthesis has previously been reported to generate 1O2 in leaves and thylakoid membranes (Hideg et al., 1994, 1995, 2001), as molecular oxygen interacts with the triplet state of Chl (which results from the transfer of excess energy to Chl) to form 1O2 (Krieger-Liszkay, 2005).

Table 2.  Changes in the efficiency of photosystem II (PSII) photochemistry (ΦPSII) in cross-sections taken from shade and sun Phillyrea latifolia leaves exposed or not exposed to excess photosynthetically active radiation (PAR) treatment, over a 20-min period
Leaf typeLight treatmentΦPSII
  1. The excess-PAR treatment was applied by irradiating (at 2800 µmol m−2 s−1) cross-sections with blue light (mostly consisting of the 436-nm line provided by an Hg lamp mounted on an epi-fluorescence microscope). ΦPSII was calculated as 1 − (Fs/inline image). Fs and inline image are the chlorophyll fluorescence yields (measured at steady state) before and during a saturating light pulse, respectively (see the Materials and Methods section for details). Data are means ± standard deviation (n = 5), and those not followed by the same letter are significantly different at P ≤ 0.05, using the least significant difference (LSD) test.

ShadeControl0.658 ± 0.025 a
Sun 0.558 ± 0.031 b
ShadeExcess PAR0.432 ± 0.041 c
Sun 0.409 ± 0.027 c

In turn, the reaction of 1O2 with DanePy produces the slightly fluorescent nitroxide radical DanePyO, which is responsible for the quenching of DanePy fluorescence (Fryer et al., 2002; Hideg et al., 2002), and allows 1O2 in plant tissues to be imaged. Our data show that DanePyO was generated upon photo-oxidation in P. latifolia leaves, as the intensity of DanePy fluorescence decreased greatly when leaf cross-sections, of both sun (Fig. 2a) and shade (Fig. 2e) leaves, were exposed to the excess-PAR treatment (Fig. 2c,g). For example, the emission spectra of photo-oxidized palisade cells, in both sun and shade leaves, in addition to decreasing in intensity were shifted (by 15 nm) towards the shorter wavelengths, as compared with untreated cells (Fig. 4). These data are consistent with (i) the shorter emission wavelength of DanePyO with respect to DanePy (Hideg et al., 2002; Kálai et al., 2002), and (ii) the greater contribution of blue-green autofluorescence to the fluorescence spectrum of photo-oxidized tissues stained with DanePy (Fig. 4a,b), the intensity of which decreased because of the partial conversion of DanePy to DanePyO. Finally, we note that the much greater yield of DanePy fluorescence in shade (+400%) than in sun palisade cells in controls (Fig. 4) offers additional evidence of an inverse relation between the mesophyll flavonoid content and the intensity of UV light actually available to excite the 1O2 probe.

Figure 4.

Ultraviolet (UV)-excited fluorescence spectrum of sun (a) and shade (b) palisade parenchyma tissue, stained with 0.2 mm N-[2-(diethylamino)ethyl]-N-[(2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrol-3-yl)methyl]-5-(dimethylamino)-1-naphthalenesulfonamide (DanePy), before (solid line) and after (dashed line) a 20-min period of excess photosynthetically active radiation (PAR) treatment (blue light at 2800 µmol m−2 s−1). Spectra are the means of five replicate measurements.

The extent to which photo-oxidation decreased DanePy fluorescence (i.e. the quenching of DanePy fluorescence) in the mesophyll tissues of sun and shade P. latifolia leaves is shown in Fig. 5, on the basis of normalized fluorescence images (Eqn 1) acquired at 546 nm. The quenching of DanePy fluorescence, i.e. following photo-induced generation of 1O2 (Hideg et al., 1998), was much greater in shade leaves (on average 69%; Fig. 5e,g) than in sun leaves (32%; Fig. 5a,b). Quenching of DanePy fluorescence did not appreciably vary throughout the mesophyll of shade leaves (which had a negligible flavonoid content), but substantially increased passing from adaxial (20%) to abaxial (45%; solid line in Fig. 5b) mesophyll tissues in sun leaves. The flavonoid/DanePy quenching relationship has been also visualized using a false-color image recombination (Fig. 5c,f), obtained by superimposing the fluorescence images of flavonoid (colored in yellow) and DanePy-quenching (colored in blue) distributions. We detected a significant (r2 = 0.979) inverse linear regression (y = 41.92 − 0.22x, where y is the quenching of DanePy fluorescence) between the tissue flavonoid content (estimated on normalized fluorescence images at 580 nm of blue light-excited, λexc = 488 nm cross-sections) and the quenching of DanePy (through imaging fluorescence at 546 nm of UV-excited cross-sections following Eqn 1), when the relative fluorescence signals were integrated over leaf depths of 50 µm, following the vertical arrow in Fig. 5c (data not shown, but see Fig. 5b). Notably, the quenching of DanePy was greatest (note the blue arrows) in adjacent adaxial palisade cells (along x0 to x1 in Fig. 5c) with the lowest flavonoid content (yellow arrows) in sun P. latifolia leaves (Fig. 5d).

In our experiment, the quenching of DanePy fluorescence almost exclusively depended on the photo-induced generation of 1O2 (see also Hideg et al., 1998), as changes in DanePy fluorescence did not occur in P. latifolia cross-sections kept in the dark over a 20-min period (data not shown). Nevertheless, we cannot exclude the possibility that photo-induced generation of H2O2 might have also partially contributed to the quenching of DanePy fluorescence. This hypothesis may actually relate vacuolar H2O2 to the apparent initial distribution (Fig. 2) and the photo-induced quenching of DanePy (Fig. 5) in the cell vacuole. We note, however, that DanePy reacts with 1O2 at a rate 1 order of magnitude higher than with H2O2 (Kálai et al., 1998), and DanePy fluorescence was not quenched by a 250 µm H2O2 solution (data not shown, but see the method of 1O2 generation using NaOCl + H2O2 in the Materials and Methods section). Therefore, our data support the conclusion that both the tissue and cellular contents of flavonoids inversely relate to the photo-induced generation of 1O2.

Flavonoids as scavengers of 1O2 in vivo

To scavenge the highly reactive (1O2 is several-fold more reactive than H2O2 and the superoxide anion; see Halliwell, 1995) and, hence, short-diffusing 1O2, flavonoids have to be located within or near the site of 1O2 production, i.e. the chloroplasts. The complex issue of the functional/localization relationship of flavonoids in P. latifolia mesophyll cells has been investigated in the present experiment, using three-dimensional deconvolution microscopy (McNally et al., 1999). Fluorescence images concomitantly acquired at 608 (λexc = 490 nm) and 685 nm (λexc = 405 nm) of NR-stained cross-sections, optically sectioned at 0.2-µm intervals moving from outside the sample, were recombined to (concomitantly) visualize flavonoids (colored yellow in Fig. 6) and Chl (colored red in Fig. 6).

Figure 6.

Three-dimensional deconvolution microscopy analysis of palisade parenchyma cells in sun leaves of Phillyrea latifolia. Cross-sections were stained with 0.1% (weight/volume) Naturstoff reagent, and fluorescence was sequentially recorded in two channels under the following conditions: (a) λexc = 490 ± 20 nm; λem = 608 ± 10 nm to visualize flavonoids; (b) λexc = 405 ± 10 nm; λem = 685 ± 40 nm to visualize chlorophyll (Chl). Images (a–c) are the recombination of flavonoid (yellow) and Chl (red) fluorescence. Images were recorded following optical sectioning (at 0.2-µm intervals along the z-axis), and the stack of images was then deconvoluted to remove the contribution of out-of-focus fluorescence. (d) Flavonoid and Chl localization in an individual mesophyll cell. The image shows an optical section taken at a depth of 2.9 µm, together with x–z (bottom inset) and y–z (right inset) orthogonal projections. Bars: (a) 20 µm; (d) 5 µm.

Fluorescence imaging shows that flavonoids were actually associated with chloroplasts (likely with the chloroplast envelope; Fig. 6a–c), as shown in the internal view of a mesophyll cell, together with its xz (bottom inset) and yz (right inset) projections, shown in Fig. 6(d). Vacuolar fluorescence attributed to flavonoids was not detected in our experiment, although we do not know to what extent NR is able to enter the vacuole in mesophyll cells (Sheahan & Cheong, 1998). Our data therefore confirm previous reports of a chloroplastic location of flavonoids (Saunders & McClure, 1976; Charriere-Ladreix & Tissut, 1981; Semerdjieva et al., 2003; Zaprometov & Nikolaeva, 2003), and lead us to conclude that flavonoids appeared to be optimally located to scavenge the short-diffusing 1O2 generated by the photosynthetic apparatus in P. latifolia leaves under photoinhibition. Moreover, we note that flavonoids detectable in our experiment (i.e. NR staining and excitation of stained tissues with blue light at 488 nm; Hutzler et al., 1998; Sheahan & Cheong, 1998; Tattini et al., 2005) were the di-hydroxy B-ring substituted quercetin and luteolin derivatives (Agati et al., 2002; Tattini et al., 2005), which are the most effective quenchers of 1O2 (in vitro and ex vivo; Chauhan et al., 1992; Tournaire et al., 1993; Nagai et al., 2005).

Nevertheless, other metabolites, such as tocopherols and carotenoids, have been shown to have the potential to quench 1O2 (Krieger-Liszkay, 2005). Carotenoids have been also reported to additionally quench the triplet state of Chl in the antenna, and hence to inhibit the photo-sensitized production of 1O2 (Peterman et al., 1995). Therefore, a greater content of tocopherols and carotenoids with respect to the content of the 1O2-generating pigment, i.e. Chl (Logan et al., 1998; Garcia-Plazaola et al., 1999; Tattini et al., 2005), may have contributed greater 1O2 scavenger ability to mesophyll cells of sun leaves than to those of shade leaves. In our experiment, the carotenoid to Chl ratio increased from 0.21 in shade to 0.28 in sun leaves of P. latifolia (Table 1). Moreover, chloroplast-localized isoprene (Logan et al., 2000), the content of which has been also reported to be greater in sun than in shade leaves (Affek & Yakir, 2002), may have conferred a greater 1O2 scavenger ability to sun than to shade mesophyll tissues in P. latifolia (Loreto & Velikova, 2001; Velikova et al., 2004).

Therefore, the ecophysiological significance of flavonoids as quenchers of 1O2 cannot be fully assessed by simply comparing mesophyll tissues of sun and shade leaves, which may markedly differ in the content of 1O2 scavengers other than flavonoids. However, strong evidence for the scavenger activity of flavonoids against 1O2 in vivo comes from the analysis of mesophyll tissues in sun leaves (Fig. 5). We speculate that the amount of both carotenoids and isoprene relative to that of Chl may have differed in mesophyll tissues located at different distances from the adaxial epidermis (Fig. 5b,c), but should have been rather constant in adjacent cells in the adaxial palisade parenchymal layer (Fig. 5d). As a consequence, our data strongly suggest that flavonoids may effectively play a role in the overall 1O2 scavenging system in vivo, likely by complementing the action of other antioxidant compounds, under severe conditions of excess-light stress. The extent to which individual antioxidant compounds may contribute to scavenging 1O2 under natural conditions needs to be fully assessed in future experiments.

Acknowledgements

We are greatly indebted to Professor Éva Hideg (University of Pécs, Hungary), who provided us with the basic procedure of DanePy synthesis. We also thank Elcomind, Milano, Italy, particularly Dr G. Guzzi, for the use of the DeltaVision RT platform. Finally, our thanks are extended to Professor Laura Morassi Bonzi, for her hospitality at Centro Microscopie Elettroniche, Area della Ricerca – CNR, Firenze, Italy.

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