agati et al (2007) chloroplast located flavonoids

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77

Research

Blackwell Publishing Ltd

Chloroplast-located flavonoids can scavenge

singlet oxygen

Giovanni Agati

1

, Paolo Matteini

1,2

, Andrea Goti

2

and Massimiliano Tattini

3

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;

3

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

Summary

• The hypothesis was tested that flavonoids may scavenge singlet oxygen (

1

O

2

) inmesophyll cells of

Phillyrea latifolia

exposed to excess-light stress.• In cross-sections taken from leaves developed at 10% (shade) or 100% (sun) solarirradiance, we evaluated the excess photosynthetically active radiation (PAR)-inducedaccumulation of

1

O

2

in mesophyll cells by imaging the fluorescence quenching of thespecific

1

O

2

probe

N-

[2-(diethylamino)ethyl]-

N

-[(2,5-dihydro-2,2,5,5-tetramethyl-1

H

-pyrrol-3-yl)methyl]-5-(dimethylamino)-1-naphthalenesulfonamide (DanePy). Theintracellular location of flavonoids was also analyzed using three-dimensionaldeconvolution microscopy.• Photo-induced quenching of DanePy fluorescence was markedly greater in themesophyll of shade leaves than in that of sun leaves, the former showing a negligibleaccumulation of mesophyll flavonoids. The photo-induced generation of

1

O

2

was inversely related to the content of flavonoids in the mesophyll cells of sunleaves. Flavonoids were located in the chloroplasts, and were likely associated withthe chloroplast envelope.• Here we provide relevant evidence for the potential scavenger activity ofchloroplast-located flavonoids against

1

O

2

and new insights into the photo-protectiverole of flavonoids in higher plants.

Key words:

DanePy, flavonoids, free radical scavengers, multispectral fluorescencespectroscopy and three-dimensional fluorescence microscopy,

1

O

2

fluorescentprobe,

Phillyrea latifolia

, singlet oxygen.

New Phytologist

(2007)

174

: 77–89

© The Authors (2007). Journal compilation ©

New Phytologist

(2007)

doi

: 10.1111/j.1469-8137.2007.01986.x

Author for correspondence:

Massimiliano Tattini Tel: +39 0555225 692 Fax: +39 0555225 656 Email: [email protected]

Received:

2 October 2006

Accepted:

15 November 2006

Introduction

Flavonoids have long been recognized as playing multipleroles in the response mechanisms of higher plants to stressfulagents of different origin, ranging from defense againstpathogens and predators (Dixon & Paiva, 1995; Harborne &Williams, 2000; McNally

et al

., 2003) to the protection ofsensitive targets in the leaf from highly damaging short solarwavelengths (Reuber

et al

., 1996; Ryan

et al

., 1998; Burchard

et al

., 2000; Jordan, 2002; Bassman, 2004). The wide distri-bution 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 ofultraviolet B (UV-B) and UV-A radiation, because of theiroptical properties (Tattini

et al

., 2004).More recently, flavonoids have been hypothesized to

perform antioxidant functions in tissues exposed to a widerange 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 preferen-tial accumulation of di-hydroxy B-ring substituted flavonoidglycosides as compared to their mono-hydroxy B-ring substi-tuted counterparts (Ollson

et al

., 1998; Ryan

et al

., 1998;

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Hofmann

et al

., 2000, 2003) and by their large accumula-tions in mesophyll cells, in response to both UV-B and highsolar radiation (Ollson

et al

., 1999; Tattini

et al

., 2000; Agati

et al

., 2002). Flavonoids have been shown to effectivelyscavenge free radicals in both

in vitro

and

ex vivo

experiments(for review articles, see Rice-Evans

et al

., 1996, 1997), as aresult 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 theircopper (Cu

2+

) ion-chelating properties; Brown

et al

., 1998).However, it is still a matter of debate how flavonoids

(mostly occurring as flavonoid glycosides) may actually carryout their antioxidant functions in plant cells, because of theirpredominant vacuolar distribution in most species (Hutzler

et al

., 1998; Neill & Gould, 2003; Pearse

et al

., 2005). It iswidely accepted that hydrogen peroxide (H

2

O

2

) may freelydiffuse from cellular organelles into the vacuole (even in epi-dermal cells), where flavonoids serve as substrates for class IIIperoxidases, which reduce H

2

O

2

by forming relatively stablephenoxyl radicals (Yamasaki

et al

., 1997; Takahama, 2004;Pearse

et al

., 2005). Flavonoids have been shown, however, tooccur in both the cytosolic and the apoplastic spaces in ryemesophyll cells (Anhalt & Weissenböck, 1992), and cytosolicanthocyanins in

Lactuca sativa

and

Pseudowintera colorata

effectively scavenge superoxide anion (Neill & Gould, 2003)and H

2

O

2

(Gould

et al

., 2002), respectively. Moreover, flavo-noids were first detected several decades ago in the chloroplastfractions of several species (Oettmeier & Heupel, 1972;Saunders & McClure, 1976; Ruzieva

et al

., 1980), andchloroplasts are capable of flavonoid biosynthesis (Zaprometov& Nikolaeva, 2003).

Recently, in

Vaccinium

spp., flavonoids have been hypoth-esized to be associated with chloroplasts, and therefore to beoptimally located to function as scavengers of highly reactiveoxygen (Semerdjieva

et al

., 2003). The association of flavonoidswith chloroplasts might help to address the complex issue oftheir functional roles (Ollson

et al

., 1998; Tattini

et al

., 2005),particularly in the protection of the photosynthetic apparatusfrom photo-induced generation of

1

O

2

(Hideg

et al

., 1994;Hideg

et al

., 1998, 2000). Flavonoids have been reported toquench

1

O

2

in vitro

(Tournaire

et al

., 1993; Nagai

et al

., 2005)and to protect isolated chloroplasts of

Triticum aestivum

fromphoto-oxidation (Chauhan

et al

., 1992).Here, we report data obtained in experiments designed to

evaluate the

in vivo

scavenger activity of flavonoids against

1

O

2

. We analyzed leaves of

Phillyrea latifolia

plants adaptedto shade (approx. 10% solar irradiance) or to full sunlight(100%) in a coastal area of South Tuscany, Italy. It has beenshown that flavonoids do not accumulate in the mesophyllcells of shade leaves (Tattini

et al

., 2000; Agati

et al

., 2002),whereas they were found to occur in high quantities in themesophyll of sun leaves, following a steep gradient fromthe 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 ofthe various mesophyll layers (data not shown and Gould

et al

.,2002)) taken from shade- or sun-adapted leaves, and exposedto excess photosynthetically active radiation (PAR), to analyzethe tissue-specific and intracellular distributions of (i) flavo-noids, using two- and three-dimensional deconvolution(McNally

et al

., 1999) fluorescence microscopy, respectively;and (ii)

1

O

2

, by coupling microspectrofluorometry andmultispectral fluorescence microimaging of the specific

1

O

2

probe

N-

[2-(diethylamino)ethyl]-

N


H

-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 (andlabeled) 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 solarradiation (shade leaves) in south Tuscany (42

°

45

′

N, 10

°

54

′

E),at the end of June 2004. The distance between the shade andthe sunny sites was

≤

50 m. Solar irradiance in the UV andPAR wavebands was estimated using an SUV100 scanningspectroradiometer (Biospherical Instruments, San Diego, CA,USA) and a Li-Cor 1800 (Li-Cor Inc., Lincoln, NE, USA)portable spectroradiometer equipped with a remote cosinesensor, respectively. Measurements were conducted on a totalof 130 d (both clear (70%) and cloudy) from May 2003 untilthe sampling period. Irradiance was then integrated on a 14-month basis, and averaged 5.36, 257.9, 2896.8 MJ m

−

2

in theUV-B, UV-A, and PAR wavebands, respectively, at the sunsite. 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 perarea (LMA), leaf thickness, and both total chlorophyll (Chltot)and carotenoid (Car) leaf contents were determined as previouslydescribed (Tattini et al., 2005).

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

Shoots were immersed in distilled water and sealed in plasticbags, and cross-sections (approx. 100-µm-thick) were cut witha 1100 Plus vibratory microtome (Vibratome, St Louis, MO,USA) from 14-month-old leaves within 6 h of sampling. Apreliminary test conducted on cross-sections (Evan’s blue test;see Dai et al., 1996) showed that leaf cells were still viable after6 h after sampling. The functionality of the photosyntheticapparatus in leaf cross-sections was evaluated by measuringthe actual quantum yield of PSII photochemistry, i.e. ΦPSII, at

© The Authors (2007). Journal compilation © New Phytologist (2007) www.newphytologist.org New Phytologist (2007) 174: 77–89

Research 79

5-min intervals over a 20-min period (the period over the whichexcess PAR was imposed), following the protocols previouslyreported in Genty et al. (1989) and Genty & Meyer (1995).Briefly, ΦPSII was calculated as 1 − (Fs/ ), where Fs and are the chlorophyll (Chl) fluorescence yields, measured at steadystate just before and during a saturating light pulse, respectively(Genty et al., 1989). Imaging of Chl fluorescence at 680 nmon blue-light excited (λexc = 436 ± 5 nm) cross-sectionsallowed estimation of ΦPSII (Genty & Meyer, 1995). In detail,images of Fs and were obtained on cross-sections exposedto 77.2 µmol m−2 s−1 for 20 s and to 7688 µmol m−2 s−1 for200 ms, respectively. ΦPSII did not vary for > 8% in bothshade 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) werepurchased from Acros Chimica (Geel, Belgium). 5-Dimethylamino-1-naphtalensulfonyl chloride (dansyl chloride)was obtained from Alfa Aesar (Karlsruhe, Germany). Diphenyl-borinic acid 2-amino-ethylester, NaClO, Rose Bengal (RB),dimethylsulfoxide, sodium bis(2-methoxyethoxy)aluminiumhydride (Red-Al), methanesulfonyl chloride (MsCl), dichloro-methane (CH2Cl2), triethylamine (Et3N), and all other solventswere purchased from Sigma (St Louis, MO, USA). Phosphate-buffered saline (PBS) solution, i.e. phosphate buffer at pH 6.8with the addition of 1% (weight/volume (w/v)) NaCl, wasused to prepare all solutions for both in vitro and in vivoexperiments.

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 synthesisprotocols proposed by Rozantzev & Krinitzkaya (1965),Hankovszky et al. (1980), Hideg et al. (1980), Kálai et al.(1998), and Murray & Iyanar (1998). DanePy is both afluorescent and a spin probe, which reacts with 1O2 toproduce the corresponding nitroxide radical, DanePyO, thefluorescence of which is then quenched by an energy transferfrom the ‘donor’ dansyl to the ‘acceptor’ nitroxide moiety(Kálai et al., 1998; Hideg et al., 2002). The quenching ofDanePy fluorescence has therefore been reported to be relatedto the content of 1O2 (Hideg et al., 2002). DanePy has beenshown to be suitable for the detection of 1O2 in plants, as itsdiethylaminoethyl side-chain allows the probe to penetratetissues easily (Kálai et al., 1998).

The effectiveness of the DanePy synthesized here for thedetection of 1O2 was preliminarily tested in vitro using both aphysical and a chemical method of 1O2 generation. First, 1O2

was generated photo-chemically by adding the photo-sensitizerRB (from a 0.1% stock solution in dimethylsulfoxide) toDanePy in PBS to final concentrations of 0.05 and 0.25 mM,respectively. The blank solution consisted of 0.25 mM DanePyin PBS. Secondly, 1O2 was produced by mixing 0.25 mM (finalconcentration) sodium hypochlorite (NaOCl), 0.25 mM

H2O2 and 0.25 mM DanePy in PBS. The blank solution con-sisted of H2O2 and DanePy in PBS, both at a concentrationof 0.25 mM. In both experiments, sample and blank solutionswere then exposed to white light (at a radiation power densityof 96 mW cm−2, as quantified using a Nova power meter cou-pled to a 2A thermal head (Ophir Optronics Ltd, Jerusalem,Israel) from a fiber-optics Xenon lamp (LQX 1800; LinosPhotonics, Milford, MA, USA), over a 40-min period. Thefluorescence spectra of the sample and blank solutions wererecorded under UV excitation (λexc = 365 nm) using themicrospectro-fluorometry equipment described in the nextsection, with a 1 × 1 cm quartz cuvette placed over the stageof an inverted epifluorescence microscope.

Microspectrofluorometry and multispectral fluorescence microimaging of flavonoids and 1O2

The tissue-specific distributions of flavonoids and 1O2 wereestimated 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. Allmeasurements were performed using an inverted epifluo-rescence microscope (Diaphot, Nikon, Japan) equipped witha high-pressure mercury lamp (HBO 100 W; Osram, Augsberg,Germany) as the light source. The excitation wavelengths wereselected using 10-nm bandwidth interference filters, 365FS10-25 and 488FS10-25 (Andover Corporation, Salem, NH, USA),coupled to ND400 and ND510 (Nikon) dichroic mirrors, forthe excitation wavelengths λexc = 365 and λexc = 488 nm,respectively. Fluorescence spectra were recorded with aCCD multichannel spectral analyzer (PMA 11-C5966;Hamamatsu, Photonics Italia, Arese, Italy), connected to themicroscope through an optical fiber bundle (1 mm in diameter),using a ×40 Plan Fluor (Nikon) objective. The fluorescencesignal (from a 490-µm2 spot) was integrated over a 2-s period,and fluorescence spectra were corrected for the transmissionproperties of both optics and filters. Fluorescence imageswere acquired using a slow-scan cooled CCD camera(Chroma CX260; DTA, Cascina, Italy) equipped with aKodak KAF261E, 512 × 512 pixel detector, and elaborated aspreviously 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 evaluatedusing fluorescence images, acquired at 580 nm (selectedusing a 10-nm bandwidth interference filter, 580FS10-25;Andover Corporation), of blue light-excited (λexc = 488 nm)

′Fm ′Fm

′Fm

New Phytologist (2007) 174: 77–89 www.newphytologist.org © The Authors (2007). Journal compilation © New Phytologist (2007)

Research80

cross-sections (Hutzler et al., 1998; Tattini et al., 2004). DanePyfluorescence was imaged at 546 nm (using a 10-nm bandwidthinterference filter, 546FS10-25; Andover Corporation), inUV-excited (λexc = 365 nm) cross-sections stained with DanePy.Finally, the tissue distribution of Chl autofluorescence wasanalyzed from fluorescence images acquired at 680 nm (selectedusing a 10-nm bandwidth interference filter, 680FS10-25;Andover Corporation), under both UV (λexc = 365 nm) andblue-light (λexc = 436 nm) excitation. The tissue fluorescenceprofiles produced by DanePy, flavonoids, and Chl were thenmeasured following the protocol previously reported inTattini et al. (2004). In detail, profiles of normalized (to rela-tive maximum values, except for the quenching of DanePyfluorescence, which originated from normalized images; seeEqn 1) fluorescence intensity for DanePy, flavonoid and Chl,over the whole leaf depth or along the longitudinal axis of thecross-sections, were computed from 404 × 404 µm fluores-cence images, by averaging the profiles of fluorescence inten-sity 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-dimensionaldeconvolution fluorescence microscopy (McNally et al., 1999).Image acquisition and reconstruction were performed with aDeltaVision RT platform (Applied Precision, Issaquah, WA,USA), using an Olympus IX71 inverted microscope (Olym-pus, Melville, NY, USA) equipped with an Olympus 60×/1.4NA oil immersion objective lens, and mercury-arc illumina-tion 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 experimentalconditions: (a) λexc = 490 ± 20 nm (Chroma TechnologyCorp., 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 Chlfluorescence. A total of 80 optical sections, taken at 0.2-µmintervals along the z-axis, were acquired moving from outsidethe sample, and the stack of images was then deconvoluted toremove the contribution of out-of-focus fluorescence. Theeffective 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 glassmicroscope slide, with the coverslip sealed with paraffin toavoid sample dehydration, and exposed to excess PAR, overa 20-min period, to induce photo-oxidation. Excess-PARtreatment was carried out by irradiating cross-sections atapprox. 2800 µmol m−2 s−1, mostly consisting of the 436-nmHg emission line provided by the epifluorescence microscopeexcitation source (and selected using a 400-nm GG400 long-

pass filter (Schott Glas, Mainz, Germany) coupled to a NikonND510 dichroic mirror at 510 nm). The irradiation light wasfocused 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-inducedphotoinhibition was then monitored by comparing the ΦPSIIvalues of light-treated and control samples (Hideg et al.,2002). Fluorescence spectra and images (404 × 404 µm insize) were acquired before (control) and at the end of the lighttreatment (light-treated), by changing the set of excitationfilters, but avoiding movement of the sample. This experi-mental set-up allowed the sequential analysis of DanePy andflavonoid fluorescence (after specimen staining with NR),over the same tissue portion. DanePy and flavonoid fluore-scence was also analyzed in two serial cross-sections, to checkfor photochemical-induced changes in flavonoid fluorescence,which did not occur in our experiment (data not shown).

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

Eqn 1

( and , the fluorescence images at 546 nm of UV-excited cross-sections before (t0) and after (t1) a 20-minexposure to excess PAR, respectively.) The normalization offluorescence images as proposed in Eqn 1 has two majoradvantages with respect to calculation of the quenching ofDanePy fluorescence simply as . First, wound-induced (as a result of cross-sectioning) generation of 1O2 att0, which may differ between sun and shade leaves or amongdifferent mesophyll tissues, is taken into account. Secondly,the proposed normalization procedure also considers thepotential changes in , because of differential penetration ofthe fluorescence probe among tissue layers or individual cells.)

Experimental design and data analysis

The experimental design was completely randomized, withfive replicate plants at both the shade and the sun sites. Unlessotherwise stated, measurements were conducted on fivereplicate 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 ontwo 0.28-cm2 leaf discs per replicate. Data were subjected toone-way analysis of variance (ANOVA). Mean values (fromfive replicate cross-sections) of the actual efficiency of PSIIphotochemistry (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 fluorescencespectra as a result of the generation of 1O2 in vitro (throughboth photochemical (RB) and chemical (NaOCl) methods)were evaluated in triplicate experiments. The 1O2-induced

% quenching of DanePy fluorescence

( )/

=

× −1000 1 0

546 546 546F F Ft t t

Ft0

546 Ft1

546

F Ft t0 1

546 546 −

Ft0

546


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quenching of DanePy fluorescence intensity did not differ by> 6% between replicate experiments. Fluorescence imaging ofDanePy, Chl, and flavonoids was performed on five replicatecross-sections taken from shade and sun leaves, respectively,and representative images are presented (fluorescence yields,particularly from the palisade parenchymal tissues, did notdiffer by > 15% between replicate measurements). Therelationship between the tissue flavonoid fluorescence (% ofnormalized fluorescence intensity) and the quenching ofDanePy fluorescence in photo-oxidized tissues was investi-gated by recording the relative fluorescence profiles at 50-µmintervals over the whole leaf depth in sun and shade leaves,respectively. Data of flavonoid and DanePy fluorescencewere fitted using a linear regression equation. Because thefluorescence signal attributable to mesophyll flavonoids wasnegligible (i.e. near to the sensitivity limit of the spectralanalyzer) in shade leaves (see Fig. 5g for details), fitting ofDanePy and flavonoid fluorescence was carried out onlyfor sun leaves. Similarly, differences in DanePy fluorescencequenching (intensity profiles) between shade and sun leaveswere not tested for their statistical significance, as marked changesin tissue anatomy should have greatly altered the DanePyfluorescence yields (McClendon & Fukshansky, 1990).

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 estimatedin vitro by physical (Fig. 1a) and chemical (data not shown)methods, using fluorescence microspectroscopy. The emissionspectrum of UV-excited (λexc = 365 nm) DanePy peaked atapprox. 585 nm, and neither the fluorescence yield nor theshape of the emission spectrum was affected by the excess-PAR treatment (Fig. 1a, upper curves). The addition of RB toDanePy decreased the fluorescence intensity of DanePy and,in addition, shifted the fluorescence spectrum to longer wave-lengths (maximum fluorescence peaked at approx. 600 nm;Fig. 1a, lower curves). In fact, RB has been shown toappreciably reabsorb the 520–570-nm wavelengths (Stielet al., 1996). The fluorescence intensity of DanePy in thepresence of RB was decreased by approx. 30% because of excessPAR, as the energy transfer from the photo-sensitizer tomolecular oxygen actually generated 1O2 (Neckers, 1989). Adecrease (by approx. 50%) in DanePy fluorescence intensitywas also observed when 1O2 was produced by adding NaOClto the DanePy + H2O2 solution (data not shown).

The UV-excited fluorescence spectrum of DanePy in vivowas that resulting from the fluorescence signal of palisade cellsstained with 0.2 mM DanePy (Fig. 1b). The shape of theDanePy fluorescence signal in vivo markedly differed from thecorresponding 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 theautofluorescence attributable to both wall-bound (ferulic andcaffeic acid derivatives; Harris & Hartley, 1976; Moraleset al., 1996; Agati et al., 2002) and soluble hydroxycinna-mates (mostly verbascoside; Agati et al., 2002; Tattini et al.,2004) may have been partially responsible for the hypsochro-matic shift of DanePy fluorescence in the palisade cells ofP. latifolia leaves. Similar suggestions have previously beenmade by Kálai et al. (1998) and Hideg et al. (2002) to explain

Fig. 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.


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the fluorescence signatures of DanePy in thylakoid membranesand in whole spinach (Spinacia oleracea) leaves, respectively.It is possible that changes in pH between in vitro (PBS atpH 6.8) and in vivo experiments may also have affected theshape 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 tofour layers of long, closely packed palisade parenchyma cells(see also Tattini et al., 2000), whereas the mesophyll of shadeleaves consisted of just a single layer of short palisade cells(Fig. 2). Total Chl (Chltot) did not differ between sun andshade leaves when expressed on a leaf area basis, but wassmaller in the former when expressed on a leaf dry weightbasis (Table 1). The extent to which an increase in sunlightirradiance decreased the leaf carotenoid (Car) content on adry weight basis was still significant in P. latifolia, but the Carcontent was significantly greater in sun than in shade leaveswhen expressed on a leaf area basis (Table 1). Finally, leafflavonoids, which were estimated using fluorescence images at580 nm (Hutzler et al., 1998; Tattini et al., 2005) of blue-excited cross-sections, largely occurred, following a steepgradient from adaxial to abaxial tissues, in the mesophyll cellsof sun leaves (Fig. 2d; Agati et al., 2002; Tattini et al., 2005),whereas they were almost exclusively located in the epidermisof shade leaves (Fig. 2h; Tattini et al., 2000).

We suggest that the strikingly different mesophyll contentsin UV-absorbing compounds in sun (Fig. 2d) and shade

(Fig. 2h) leaves were largely responsible for the intensities ofDanePy (Fig. 2a,e) and Chl (Fig. 2b,f ) fluorescence in thecorresponding cross-sections under UV-light excitation (λexc =365 nm). In fact, DanePy fluorescence, as evaluated fromimages recorded at 546 nm, was much weaker in the adaxialmesophyll cells (which had the greatest accumulation offlavonoids; Figs 2a, 3a) than in the abaxial mesophyll cells insun leaves, whereas it appeared to be more equally distributedin the mesophyll of shade leaves (but note that the strongestDanePy fluorescence likely originated from the vascularbundles; Fig. 2e). Analogously, Chl fluorescence mostly origi-nated from mesophyll tissues located at a greater distancefrom the adaxial surface in sun leaves (Figs 2b, 3a), but did notappreciably vary among mesophyll cells in shade leaves(Fig. 2f ). We argue that flavonoids may have effectivelyreduced the intensity of UV light actually available to exciteboth DanePy and Chl. Indeed, the intensity of Chl fluores-cence did not vary by > 10–15% over the entire mesophyll(Fig. 3b), when cross-sections taken from sun leaves wereexcited with blue light (flavonoids do not absorb at 436 nm),as previously reported to occur in analogous tissues of Acerplatanoides (McCain et al., 1993).

Finally, we note that the nearly identical distributions ofDanePy and Chl fluorescence in the mesophyll of P. latifolialeaves confirm that DanePy was widely distributed in thechloroplasts (Hideg et al., 2001, 2002). Nevertheless, DanePyfluorescence appeared to originate also from the vacuolarcompartment (Fig. 2), although photo-micrograph resolutiondoes not allow conclusive intracellular compartmentation ofDanePy 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, underwentphotoinhibition, as ΦPSII of light-treated cross-sections wassignificantly smaller than that of controls (Table 2). Further-more, the excess-PAR-induced decrease in ΦPSII was slightlygreater in shade (−34%) than in sun (−27%) leaves, the latteralso showing a smaller ΦPSII (–15%), before the onset of theexcess-light treatment (Table 2). Photoinhibition of photo-synthesis has previously been reported to generate 1O2 inleaves and thylakoid membranes (Hideg et al., 1994, 1995,2001), as molecular oxygen interacts with the triplet state ofChl (which results from the transfer of excess energy to Chl)to form 1O2 (Krieger-Liszkay, 2005).

In turn, the reaction of 1O2 with DanePy produces theslightly fluorescent nitroxide radical DanePyO, which isresponsible for the quenching of DanePy fluorescence (Fryeret al., 2002; Hideg et al., 2002), and allows 1O2 in planttissues to be imaged. Our data show that DanePyO wasgenerated upon photo-oxidation in P. latifolia leaves, as theintensity of DanePy fluorescence decreased greatly when leaf

Table 1 Morphological features and photosynthetic pigment content of Phillyrea latifolia leaves sampled at the shade (10% solar irradiance) or the sun (100%) site

Parameter

Sampling site

Shade Sun

Leaf size (cm2) 6.7 ± 0.8 a 3.7 ± 0.5 bLMA (mg DW cm−2) 12.6 ± 1.1 b 24.9 ± 1.3 aLeaf thickness (µm) 229.8 ± 14.7 b 442.5 ± 19.9 aChltot (mg g−1 DW) 4.8 ± 0.6 a 2.5 ± 0.3 bChltot (µg cm−2) 60.5 ± 5.8 62.5 ± 7.7 nsCar (mg g−1 DW) 1.0 ± 0.1 a 0.7 ± 0.1 bCar (µg cm−2) 12.7 ± 0.9 b 17.5 ± 1.5 a

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


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cross-sections, of both sun (Fig. 2a) and shade (Fig. 2e) leaves,were exposed to the excess-PAR treatment (Fig. 2c,g). Forexample, the emission spectra of photo-oxidized palisade cells,in both sun and shade leaves, in addition to decreasing inintensity were shifted (by 15 nm) towards the shorter wave-lengths, as compared with untreated cells (Fig. 4). These dataare consistent with (i) the shorter emission wavelength ofDanePyO with respect to DanePy (Hideg et al., 2002; Kálaiet al., 2002), and (ii) the greater contribution of blue-greenautofluorescence to the fluorescence spectrum of photo-

oxidized tissues stained with DanePy (Fig. 4a,b), the intensityof which decreased because of the partial conversion ofDanePy to DanePyO. Finally, we note that the much greateryield of DanePy fluorescence in shade (+400%) than in sunpalisade cells in controls (Fig. 4) offers additional evidence ofan inverse relation between the mesophyll flavonoid contentand the intensity of UV light actually available to excite the1O2 probe.

The extent to which photo-oxidation decreased DanePyfluorescence (i.e. the quenching of DanePy fluorescence) in

Fig. 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.


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the mesophyll tissues of sun and shade P. latifolia leaves isshown in Fig. 5, on the basis of normalized fluorescenceimages (Eqn 1) acquired at 546 nm. The quenching of DanePyfluorescence, i.e. following photo-induced generation of 1O2(Hideg et al., 1998), was much greater in shade leaves (onaverage 69%; Fig. 5e,g) than in sun leaves (32%; Fig. 5a,b).Quenching of DanePy fluorescence did not appreciably varythroughout the mesophyll of shade leaves (which had a negli-gible flavonoid content), but substantially increased passingfrom adaxial (20%) to abaxial (45%; solid line in Fig. 5b)

Fig. 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.

Fig. 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.

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 type Light treatment ΦPSII

Shade Control 0.658 ± 0.025 aSun 0.558 ± 0.031 bShade Excess PAR 0.432 ± 0.041 cSun 0.409 ± 0.027 c

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/ ). Fs and 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.

′Fm′Fm


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mesophyll tissues in sun leaves. The flavonoid/DanePy quench-ing relationship has been also visualized using a false-colorimage recombination (Fig. 5c,f ), obtained by superimposingthe fluorescence images of flavonoid (colored in yellow)and DanePy-quenching (colored in blue) distributions. Wedetected a significant (r2 = 0.979) inverse linear regression(y = 41.92 − 0.22x, where y is the quenching of DanePyfluorescence) between the tissue flavonoid content (estimatedon normalized fluorescence images at 580 nm of blue light-excited, λexc = 488 nm cross-sections) and the quenchingof DanePy (through imaging fluorescence at 546 nm ofUV-excited cross-sections following Eqn 1), when the relativefluorescence signals were integrated over leaf depths of 50 µm,following the vertical arrow in Fig. 5c (data not shown, butsee Fig. 5b). Notably, the quenching of DanePy was greatest(note the blue arrows) in adjacent adaxial palisade cells (alongx0 to x1 in Fig. 5c) with the lowest flavonoid content (yellowarrows) in sun P. latifolia leaves (Fig. 5d).

In our experiment, the quenching of DanePy fluorescencealmost exclusively depended on the photo-induced generationof 1O2 (see also Hideg et al., 1998), as changes in DanePy flu-orescence did not occur in P. latifolia cross-sections kept in thedark over a 20-min period (data not shown). Nevertheless, wecannot exclude the possibility that photo-induced generationof H2O2 might have also partially contributed to the quenchingof DanePy fluorescence. This hypothesis may actually relatevacuolar H2O2 to the apparent initial distribution (Fig. 2)and the photo-induced quenching of DanePy (Fig. 5) in thecell vacuole. We note, however, that DanePy reacts with 1O2at a rate 1 order of magnitude higher than with H2O2 (Kálaiet al., 1998), and DanePy fluorescence was not quenched bya 250 µM H2O2 solution (data not shown, but see the methodof 1O2 generation using NaOCl + H2O2 in the Materials andMethods section). Therefore, our data support the conclu-sion that both the tissue and cellular contents of flavonoidsinversely relate to the photo-induced generation of 1O2.

Fig. 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.


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Flavonoids as scavengers of 1O2 in vivo

To scavenge the highly reactive (1O2 is several-fold morereactive than H2O2 and the superoxide anion; see Halliwell,1995) and, hence, short-diffusing 1O2, flavonoids have to belocated within or near the site of 1O2 production, i.e. thechloroplasts. The complex issue of the functional/localizationrelationship of flavonoids in P. latifolia mesophyll cells hasbeen 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 fromoutside the sample, were recombined to (concomitantly)visualize flavonoids (colored yellow in Fig. 6) and Chl(colored red in Fig. 6).

Fluorescence imaging shows that flavonoids were actuallyassociated with chloroplasts (likely with the chloroplast enve-lope; Fig. 6a–c), as shown in the internal view of a mesophyllcell, together with its x–z (bottom inset) and y–z (right inset)projections, shown in Fig. 6(d). Vacuolar fluorescence attri-buted to flavonoids was not detected in our experiment,although we do not know to what extent NR is able to enterthe vacuole in mesophyll cells (Sheahan & Cheong, 1998).Our data therefore confirm previous reports of a chloroplasticlocation of flavonoids (Saunders & McClure, 1976; Charriere-Ladreix & Tissut, 1981; Semerdjieva et al., 2003; Zaprometov& Nikolaeva, 2003), and lead us to conclude that flavonoidsappeared to be optimally located to scavenge the short-diffusing1O2 generated by the photosynthetic apparatus in P. latifolialeaves under photoinhibition. Moreover, we note that fla-vonoids detectable in our experiment (i.e. NR staining andexcitation of stained tissues with blue light at 488 nm; Hutzleret al., 1998; Sheahan & Cheong, 1998; Tattini et al., 2005)were the di-hydroxy B-ring substituted quercetin and luteolinderivatives (Agati et al., 2002; Tattini et al., 2005), which arethe 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 andcarotenoids, have been shown to have the potential to quench1O2 (Krieger-Liszkay, 2005). Carotenoids have been alsoreported to additionally quench the triplet state of Chl in theantenna, and hence to inhibit the photo-sensitized produc-tion of 1O2 (Peterman et al., 1995). Therefore, a greatercontent of tocopherols and carotenoids with respect to thecontent of the 1O2-generating pigment, i.e. Chl (Logan et al.,1998; Garcia-Plazaola et al., 1999; Tattini et al., 2005), mayhave contributed greater 1O2 scavenger ability to mesophyllcells of sun leaves than to those of shade leaves. In our experi-ment, the carotenoid to Chl ratio increased from 0.21 inshade to 0.28 in sun leaves of P. latifolia (Table 1). Moreover,chloroplast-localized isoprene (Logan et al., 2000), the con-tent of which has been also reported to be greater in sun thanin shade leaves (Affek & Yakir, 2002), may have conferred a

Fig. 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.


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greater 1O2 scavenger ability to sun than to shade mesophylltissues in P. latifolia (Loreto & Velikova, 2001; Velikova et al.,2004).

Therefore, the ecophysiological significance of flavonoidsas quenchers of 1O2 cannot be fully assessed by simply com-paring mesophyll tissues of sun and shade leaves, which maymarkedly differ in the content of 1O2 scavengers other thanflavonoids. However, strong evidence for the scavenger activ-ity of flavonoids against 1O2 in vivo comes from the analysisof mesophyll tissues in sun leaves (Fig. 5). We speculate thatthe amount of both carotenoids and isoprene relative to thatof Chl may have differed in mesophyll tissues located at dif-ferent distances from the adaxial epidermis (Fig. 5b,c), butshould have been rather constant in adjacent cells in the adaxialpalisade parenchymal layer (Fig. 5d). As a consequence, ourdata strongly suggest that flavonoids may effectively play arole in the overall 1O2 scavenging system in vivo, likely bycomplementing the action of other antioxidant compounds,under severe conditions of excess-light stress. The extent towhich individual antioxidant compounds may contribute toscavenging 1O2 under natural conditions needs to be fullyassessed in future experiments.

Acknowledgements

We are greatly indebted to Professor Éva Hideg (University ofPécs, Hungary), who provided us with the basic procedure ofDanePy synthesis. We also thank Elcomind, Milano, Italy,particularly Dr G. Guzzi, for the use of the DeltaVision RTplatform. Finally, our thanks are extended to Professor LauraMorassi Bonzi, for her hospitality at Centro MicroscopieElettroniche, Area della Ricerca – CNR, Firenze, Italy.

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Supplementary Material

The following supplementary material is available for thisarticle online:

Appendix S1

General protocol for the synthesis of DanePy

Appendix S2

Details of the synthesis of 3-(aminocarbonyl)-2,5-dihydro-2,2,5,5-tetramethyl-1

H

-pyrrol-1-yloxy (2)

Appendix S3

Details of the synthesis of

N-

[2-(diethylamino)-ethyl]-

N


H

-pyrrol-3-yl)-methyl]-5-(dimethylamino)-1-naphthalenesulfonamide(DanePy, 8), and its chemical characterization

Fig. S1

A schematic protocol for the synthesis of DanePy (8).

This material is available as part of the online article from http://www.blackwell-synergy.com/doi/abs/10.1111/j.1469-8137.2007.01986.x (This link will take you to the article abstract.)

Please note: Blackwell Publishing are not responsible forthe content or functionality of any supplementary materialssupplied by the authors. Any queries (other than aboutmissing material) should be directed to the correspondingauthor for the article.

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agati et al (2007) chloroplast located flavonoids

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