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Environmental and Experimental Botany 70 (2011) 88–95

Contents lists available at ScienceDirect

Environmental and Experimental Botany

journa l homepage: www.e lsev ier .com/ locate /envexpbot

he impact of UV-radiation on the physiology and biochemistry of Ligustrumulgare exposed to different visible-light irradiance

ucia Guidia, Elena Degl’Innocenti a, Damiano Remorinib, Stefano Biricolti c, Alessio Fini c,rancesco Ferrini c, Francesco Paolo Nicesec, Massimiliano Tattinid,∗

Dipartimento di Biologia delle Piante Agrarie, Università di Pisa, Via del Borghetto 80, I-56124, Pisa, ItalyDipartimento di Difesa e Coltivazione delle Specie Legnose ‘G. Scaramuzzi’, Università di Pisa, Via del Borghetto 80, I-56124, Pisa, ItalyDipartimento di Ortoflorofrutticoltura, Università di Firenze, Viale delle Idee 30, I-50019, Sesto. F.no, Firenze, ItalyIstituto per la Protezione delle Piante, Consiglio Nazionale delle Ricerche, Via Madonna del Piano 10, I-50019, Sesto. F.no, Firenze, Italy

r t i c l e i n f o

rticle history:eceived 15 March 2010eceived in revised form 3 June 2010ccepted 6 August 2010

eywords:ntioxidant enzymesxcess lightlavonoidsxidative damageolyphenolsSII photochemistry

a b s t r a c t

We have conducted a UV-exclusion experiment in which plants grew at 35 or 100% sunlight irradiance,in the presence or in the absence of UV-irradiance, with the aim of exploring the effects of visible-lightand UV-irradiance on the physiology and biochemistry of Ligustrum vulgare, a world-wide distributedshrub, which inhabits partially shaded areas of the Mediterranean Basin. We measured relevant phys-iological and biochemical traits, namely: (i) leaf expansion and leaf area expansion rates; (ii) the netCO2 assimilation rate and the PSII photochemistry; (iii) the concentrations of soluble carbohydrates andphotosynthetic pigments; (iv) the activities of superoxide dismutase (SOD), catalase (CAT) and ascorbateperoxidase (APX); (v) the share of assimilated carbon recovered in individual polyphenols; (vi) the leafoxidative damage. UV-irradiance had a relatively minor impact on most examined traits, as comparedwith the effect of visible-light irradiance. UV-induced variations in plant growth and net CO2 assimila-tion rate were minor. Maximal (Fv/Fm) and actual (˚PSII) efficiencies of PSII photochemistry varied to agreater extent because of visible-light than UV-irradiance, and full-sun leaves had smaller Fv/Fm and ˚PSII

than the partially shaded ones. The conversion state of violaxanthin-cycle pigments was either largelyincreased by visible-light or unaffected by UV-radiation, as also observed for the activities of antioxi-dant enzymes (with the exception of SOD). In contrast, UV-radiation greatly enhanced the allocation ofcarbon to polyphenols, particularly flavonoids, irrespective of visible-light irradiance. Lipid peroxidation

and protein oxidation were superior in UV-treated leaves growing under partial shading, whereas leafoxidative damage was unaffected by UV-radiation in full-sun leaves. We explain the differential UV-induced oxidative damage in partially shaded or full-sun leaves, on the basis of visible-light-inducedbiochemical adjustments, aimed at avoiding the generation and reducing reactive oxygen forms (ROS).These adjustments included an increase in (1) violaxanthin-cycle pigments, particularly antheraxanthinand zeaxanthin, relative to chlorophyll; (2) antioxidant enzyme activities and flavonoid concentration,

ibit t

which may effectively inh

. Introduction

Most species world-wide, particularly those inhabitingediterranean-type ecosystems are faced with severe “excess-

ight” stress, as the concomitant action of high temperature andater shortage, which occurs frequently during the summer

eason, reduces greatly the usage of high fluxes of radiant energyn photosynthetic processes (Chaves et al., 2009; Li et al., 2009).n addition, short solar UV-wavelengths (particularly in the90–315 nm, UV-B spectral region) have the potential to dele-

∗ Corresponding author. Tel.: +39 055 4574038; fax: +39 055 4574017.E-mail address: m.tattini@ipp.cnr.it (M. Tattini).

098-8472/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.envexpbot.2010.08.005

he generation and quench ROS once formed.© 2010 Elsevier B.V. All rights reserved.

teriously affecting the DNA integrity (Britt, 1999), in addition toimpairing most metabolic processes as a consequence of reactiveoxygen species (ROS) generation (Stratmann, 2003; Jenkins, 2009).

However, UV-B-radiation may have a relatively minor impact onthe physiology and biochemistry of plants growing under naturalconditions (Caldwell et al., 1995; Ballaré et al., 2001; Searles et al.,2001; Rousseaux et al., 2004). The deleterious effects of short solarwavelengths on a plant’s performance have been likely exagger-ated in most experiments conducted in growth chambers, in which

plants have been exposed to unrealistic proportions of the varioussolar wavelengths [i.e., very low photosynthetic active radiation(PAR, over the 400–700 nm waveband) to UV ratio, Booij-James etal., 2000; Alexieva et al., 2001]. Indeed, both UV-A (315–390 nm)and blue-light have long been reported to greatly attenuate the

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amaging effects of UV-B wavelengths on the DNA integrity (Britt,999), and ameliorate the detrimental effects of UV-B radiation onplant’s performance (Schntizler et al., 1999; Adamse et al., 1994).e also note that the UV-B radiation, although it has the greatest

otential of damaging the cellular homeostasis, does account foress than 2% of total UV-radiation under natural conditions at midatitudes (Ballaré, 2003), and it is much less penetrating than theV-A wavelengths in sensitive leaf targets (Vogelmann, 1993).

It has been also questioned whether UV-B irradiance deleteri-usly affects photosynthetic rates and PSII photochemistry undereld conditions, as UV-B-induced damage to PSII depends greatlyn the concomitant irradiance over the 400–700 nm wavebandAllen et al., 1998; Levizou and Manetas, 2001; Searles et al., 2001;aul and Gwynn-Jones, 2003). There were no changes in both Fv/Fm

the maximal efficiency of PSII photochemistry) and ˚PSII (the effi-iency of PSII photochemistry in light conditions) in plants exposedo UV-B irradiance as high as 32–40 kJ m−2 d−1, when PAR irradi-nce exceeded 450–500 �mol m−2 s−1 (Nogués et al., 1995; Allent al., 1997). UV-B radiation had a significant effect on stomatalonductance, but did not markedly reduce the net CO2 assimila-ion rate and Fv/Fm in Arabidopsis (Poulsen et al., 2006). On theontrary, UV-B irradiance as low as 1.24 �mol m−2 s−1 had a greatmpact on the degradation of D1 and D2 PSII proteins, when plantsrew at PAR irradiance in the range 50–200 �mol m−2 s−1 (Booij-ames et al., 2000). Similarly, the intensity of PAR irradiance to

hich UV-exposed plants are faced with, has been reported ear-ier to control the activities of enzymes and the concentration of

etabolites devoted to countering photo-oxidative damage (Raot al., 1996). Catalase (CAT) and ascorbate peroxidase (APX) activi-ies did not vary in Vitis vinifera (Berli et al., 2010), but were stronglyepressed in soybean (Xu et al., 2008) in response to UV-B radiation.V-radiation did not enhance the lipid peroxidation in Gunneraagellanica under field conditions (Giordano et al., 2004), whereas

reatly affected the antioxidant enzyme activities and markers ofxidative damage in growth-chamber experiments (Mackerness etl., 1998; Alexieva et al., 2001).

High PAR, which occurs in concomitance with other stressfulgents under natural conditions (e.g., heat and/or drought), haseen shown to activate a wide-range of biochemical adjustments,

ikely as a consequence of ROS production, to preserve the cellularomeostasis from the oxidative damage driven by an enhancement

n UV-irradiance. For example, antioxidant flavonoids accumulaten response to high PAR (Babu et al., 2003; Kotilainen et al., 2008;gati et al., 2009) in the absence of UV-radiation, in agreementith early findings of the upregulation of the expression of chal-

one synthase (CHS), the enzyme involved in the first committedtep of flavonoid biosynthesis, by blue-light (for a review arti-le, see Jenkins, 2009). Further, non-structural carbohydrates, likeannitol in Olea europaea and Ligustrum vulgare (Oleaceae), the

oncentration of which greatly increased in response to excess lightGuidi et al., 2008; Melgar et al., 2009), have the potential to scav-nging the highly reactive hydroxyl radical (Shen et al., 1997), inddition to help limiting the generation of ROS (Valderrama et al.,006).

To date, there is still an uncertainty on the extent to which UV-adiation affects the physiology, and particularly the biochemistryf most species under field conditions, and, to address this complexssue, UV-exclusion experiments have to be performed (Ballaré etl., 2001; Caldwell et al., 2003). Here the hypothesis was testedf a greater UV-induced damage in plants growing under partialhading than in full sun, because of the high light-induced acti-

ation of antioxidant defense systems in the leaf. Therefore, weonducted a UV-exclusion experiment in which the effects of bothV-radiation and visible-light irradiance were examined on rele-ant physiological and biochemical-related traits in L. vulgare (ahrub of the Oleaceae family inhabiting partially shaded areas of

rimental Botany 70 (2011) 88–95 89

the Mediterranean Basin). We grew plants in outside, under partialshading (35% full sunlight) or fully exposed to sunlight irradiance,in the absence or in the presence of UV-radiation, by keeping con-stant the ratio of PAR to UV. Measurements were conducted of(i) growth, gas exchange, soluble carbohydrates; (ii) chlorophyll afluorescence-derived parameters and photosynthetic pigment con-centration; (iii) the activity of antioxidant enzymes and the leafoxidative damage; (iv) the share of “newly assimilated carbon”(Tattini et al., 2004) devoted to polyphenol biosynthesis.

2. Materials and methods

2.1. Plant material, growth conditions and plant growth

Self-rooted one-year-old L. vulgare plants (32.7 ± 3.6 leaf pairs,mean ± standard deviation, n = 10) were grown outdoors (in 4.5 Lpots with artificial substrate consisting of coarse sand:pumice:peat,20:40:40, v:v:v) in a greenhouse under 45% full sunlight irradianceduring four weeks. Then plants were transferred in screenhouses(3 m × 2 m × 2 m, length × width × height) constructed with roofand walls using plastic foils of specific transmittance, over a four-week experimental period. The screenhouses were N-S exposed,with small-shielded openings below the front roof at the NEand NW corners, to permit air circulation. Plants, which wereirrigated three times a week until complete leaching of thesubstrate, were exposed to 35 or 100% solar irradiance (actu-ally plastic films reduced by approx. 10% outside irradiance)in the absence (referred as to PAR35/PAR100 plants through-out the paper) or in the presence of UV-irradiance (referredas to PAR35UV/PAR100UV). Solar UV-radiation was excludedby LEE # 226 UV foil (LEE Filters, Andover, UK, which fullyexcluded solar wavelengths in the range of 240–380 nm, andtransmitted 3% of UV-radiation in the 380–390 nm range) in thePAR35/PAR100 treatments, whereas in the PAR35UV/PAR100UVtreatments plants grew under a 100 �m ETFE fluoropolymer film(NOWOFLON® ET-6235, NOWOFLON® Kunststoffprodukte GmbH& Co. KG, Siegsdorf, Germany). The PAR35 irradiance was obtainedby adding a proper black polyethylene frame to the LEE 226UV foil. UV-irradiance (280–400 nm) and PAR inside the green-houses, were measured by a SR9910-PC double-monochromatorspectroradiometer (Macam Photometric Ltd., Livingstone, UK),and a calibrated Li-190 quantum sensor (Li-Cor Inc., Lincoln, NE,USA), respectively. UV-radiation was on average 59.2 or 19.4, andPAR1228 or 403 �mol m−2 s−1 in the PAR100UV or PAR35UV treat-ments, respectively, on a daily basis. UV-irradiance in PAR100 orPAR35 treatments, reached 0.6 or 0.2 �mol m−2 s−1, respectively.Temperature maxima/minima were measured daily with Tiny-tag Ultra2 data loggers (Gemini Dataloggers, UK) and averaged29.5/15.2 ◦C or 27.7/16.3 ◦C in 100% or 35% sunlight screenhouses,respectively.

Growth was estimated in terms of the leaf expansion rate (LER,number d−1), i.e., the daily rate of newly fully expanded leaves, andthe leaf areas expansion rate, i.e., the newly developed leaf area ona daily basis (cm2 d−1).

2.2. Gas exchange, PSII photochemistry, photosynthetic pigmentsand soluble carbohydrates

Net CO2 assimilation at saturating light (Asat, i.e., at 600 and>900 �mol m−2 s−1 over the PAR waveband in PAR35 or PAR100-

treated leaves, respectively) was performed on medial leaves atroom temperature (75% relative humidity), using a portable Li-Cor6400 (Li-Cor Inc.) infrared gas analyzer operating at 34 ± 0.5 MPaambient CO2. A modulated Chl a fluorescence analysis was con-ducted on dark-adapted (over a 40-min period) leaves using

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PAM-2000 fluorometer (Walz, Effeltrich, Germany) connectedo a Walz 2030-B leaf-clip holder through a Walz 2010-F tri-urcated fiber optic, under laboratory conditions. The maximumfficiency of photosystem II (PSII) photochemistry was calculated asv/Fm = (Fm − F0)/Fm, where Fv is the variable fluorescence and Fm ishe maximum fluorescence of dark-adapted (over a 40-min period)eaves. The minimal fluorescence, F0, was measured using a mod-lated light pulse <1 �mol m−2 s−1, to avoid appreciable variableuorescence. Fm and F ′

m were determined at 20 kHz using a 0.8-saturating light pulse of white light at 8000 �mol m−2 s−1. F ′

m, i.e.,he maximum fluorescence in light conditions, was determined at00 or 900 �mol m−2 s−1 of photons over the PAR waveband, i.e., at

ight intensities at which saturation of photosynthesis occurred forhade and sun leaves, respectively (data not shown). PhotosystemI quantum yield in the light (˚PSII) and nonphotochemical quench-ng [qNP = (Fm/F ′

m) − 1] were estimated using the saturation pulseethod described in Schreiber et al. (1986), and calculated accord-

ng to Bilger and Björkman (1990), respectively. The excitationressure on PSII, (1 − qP), qP being the coefficient of photochemicaluenching, was calculated after Schreiber et al. (1995).

Violaxanthin-cycle pigments were identified and quantified viaPLC-DAD as reported in Remorini et al. (2009). Total chlorophylloncentration was measured spectrophotometrically as reported inichtenthaler (1987). Glucose and mannitol were separated, iden-ified and quantified as reported in Tattini et al. (1996) using aPLC-RI unit.

.3. Antioxidant enzymes and oxidative damage

Antioxidant enzyme activities were measured in fresh leafaterial, which was extracted as described in Di Cagno et al.

2001). Superoxide dismutase (SOD; EC 1.15.1.1) activity was mea-ured photometrically at 560 nm, based on the inhibition by SODf nitroblue tetrazolium (NBT) reduction. One unit of SOD wasefined as the amount needed to bring about a 50% inhibitionf the NBT reduction state (Beyer and Fridovich, 1987). CatalaseCAT; EC 1.11.1.6) activity was measured photometrically at 270 nmCakmak and Marschner, 1992) by determining the rate of conver-ion of H2O2 to O2. Total ascorbate peroxidase (APX; EC 1.11.1.11)ctivity was measured as the decrease in absorbance at 290 nm,esulting from ascorbate oxidation (Nakano and Asada, 1981).xidative damage was estimated in terms of lipid peroxidationnd protein oxidation, by measuring the concentration of malon-ialdehyde (MDA) and carbonyl groups, respectively, as recentlyeported in detail in Remorini et al. (2009). Measurements of MDAere performed as in Hodges et al. (1999), which takes into account

he possible influence of interfering compounds in the assay forhe 2-thiobarbituric acid (TBA)-reactive substances. Protein oxida-ion was estimated using the carbonyl assay of Reznick and Packer1994), as modified by Di Cagno et al. (2001).

.4. Quantification of polyphenols and calculation of CO2-basedolyphenol accumulation

Identification and quantification of polyphenols were con-ucted as previously reported in Romani et al. (2000) and inattini et al. (2004), using HPLC-DAD and HPLC-MS analyses.olyphenols in L. vulgare basically consisted of secoiridoids (oleu-opein, ligustaloside A and B, ligstroside), hydroxycinnamic aciderivatives (p-coumaric and caffeic acid derivatives) and flavonoid

lycosides (apigenin, luteolin, and quercetin derivatives). The CO2-ased polyphenol accumulation (PolyCO2 ) was calculated using thequation previously reported in Tattini et al. (2004):

olyCO2 = (Polyt1− Polyt0

)/(mol−1 CO2 m−2)t0 ÷ t1 (1)

imental Botany 70 (2011) 88–95

where Polyt1− Polyt0

is the increment in polyphenol concentra-tion, on a leaf area basis, during the experimental period (t0 ÷ t1),and mol CO2 m−2 is the CO2 assimilated over the whole experi-mental period. Total assimilated CO2 was estimated as reported inTattini et al. (2004), by daily measurements of CO2 assimilation rateand night-time respiration rates, which were conducted at weeklyintervals, during the experiment.

2.5. Experimental design and statistics

The experimental design was a complete random with five repli-cate screenhouses (3 × 2 × 2 m, length × width × height) per eachlight treatment, and plant position was changed at 10-day-intervalsduring the whole experimental period. There were 10 plants in eachscreenhouse. All measurements were performed on five replicateplants (one plant from each screenhouse) with the exception ofgrowth-related features, which were determined on 10 replicateplants (two plants from each screenhouse). Data were analyzedfor statistical significance using a one-way (light treatment as fac-tor) Analysis of Variance (ANOVA), whereas those of time-courseexperiments were analyzed using a two-way ANOVA with lighttreatment and sampling time as fixed factors, with their interactionfactor.

3. Results

3.1. Growth, gas exchange, photosynthetic pigments andnon-structural carbohydrates

UV-irradiance did not affect the leaf expansion rate and had aminor impact on the rate leaf area expansion (on average −15%)as compared with the effect of the visible-light treatment (−76%,Table 1). There was a significant effect of the UV-treatment on therate of leaf area expansion in plants growing under partial shading,as a consequence of UV-induced reduction in leaf lamina size. Thesedata are to be considered with some cautions to estimating thegrowth of the above-ground organs, as sunlight irradiance eitherenhanced the dry to fresh weight ratio of the shoot (from 0.37 to0.41) or depressed the leaf lamina size (from 12 to 7.5 cm2, data notshown, but see Table 1).

On the whole, UV-irradiance had a minor effect on the netCO2 assimilation rate at saturating light (Asat), which was slightlysuperior in PAR100 than in PAR35 leaves. Instead the net CO2 assim-ilated over the whole experimental period was slightly smaller (onaverage −17%) in UV-treated than in untreated leaves. L. vulgareresponded to an increase in sunlight irradiance, by significantlydecreasing (−41%) the concentration of chlorophyll, and dramat-ically increasing (+160%) the concentration of violaxanthin-cyclepigments relative to total chlorophyll concentration. Visible sun-light steeply enhanced (+145%) the concentration of zeaxanthinand antheraxanthin relative to the violaxanthin concentration,this ratio being unaffected by UV-radiation in both full-sunand partially shaded leaves. PAR100 leaves had significantlygreater concentrations of glucose (+41%) and mannitol (+43%)than the PAR35 counterparts, irrespective of UV-irradiance(Table 1).

3.2. PSII photochemistry

UV-induced decline in Fv/Fm was greater in PAR100 than in

PAR35 leaves early during the stress period, but the recovery inFv/Fm was complete in UV-treated leaves growing in full sunlight,at the end of the experiment (Fig. 1A). By contrast the actualefficiency of PSII photochemistry, ˚PSII, decreased significantlyin PAR35 leaves after 10 days of treatment with UV-radiation,

L. Guidi et al. / Environmental and Experimental Botany 70 (2011) 88–95 91

Table 1Growth, net CO2 assimilation rates, leaf photosynthetic pigments and soluble carbohydrates, in Ligustrum vulgare plants exposed to 35 or 100% solar irradiance in the absence(−UV) or in the presence (+UV) of UV-radiation, over a four-week period.

Trait 35% 100%

−UV +UV −UV +UV

LER (leaf number d−1) 1.1 ± 0.2 a 1.1 ± 0.2 a 0.4 ± 0.1 b 0.4 ± 0.1 bLAER (cm2 d−1) 13.6 ± 0.8 a 11.2 ± 0.6 b 3.0 ± 0.3 c 2.9 ± 0.4 c

Asat (�mol m−2 s−1) 10.4 ± 0.7 b 10.2 ± 1.5 b 12.7 ± 1.4 a 11.2 ± 1.9 abCO2

tot (mol m−2) 8.7 ± 0.8 c 7.4 ± 1.2 d 13.8 ± 1.0 a 11.8 ± 1.3 b

Chltot (�mol g−1 DW) 5.46 ± 0.55 a 5.28 ± 0.32 a 3.13 ± 0.22 b 3.17 ± 0.28 bVAZ/Chltot (mol mol−1) 0.07 ± 0.01 b 0.08 ± 0.01 b 0.19 ± 0.02 a 0.20 ± 0.01 a(Z + A)/(V + A + Z) 0.14 ± 0.01 b 0.15 ± 0.02 b 0.36 ± 0.03 a 0.35 ± 0.04 a

Glucose (mM) 214.5 ± 26 b 223.7 ± 19 b 310.8 ± 17 a 307.2 ± 21 aMannitol (mM) 76.3 ± 8 b 81.8 ± 6 b 118.0 ± 11 a 117.6 ± 14 a

LER is the number of fully expanded leaves on a daily basis; LAER is the newly developed leaf area on a daily basis; Asat is the net CO2 assimilation rate at saturating light( band io heraxp res, nV using

bmNsmqPeei(ewt(

FLdo(F

which was measured at 600 or >900 �mol m−2 s−1 of photons over the PAR wavever the whole experimental period; Chltot, total chlorophyll; V, violaxanthin; A, antigments. Data are the means ± standard deviation (n = 10 for growth-related featualues in a row not accompanied by the same letter differ significantly for P < 0.05,

ut did not vary in PAR35 leaves at the end of the experi-ent in the absence or in the presence of UV-radiation (Fig. 1B).onphotochemical quenching, which operated to a substantially

uperior degree (+20%) in PAR100 than in PAR35 leaves, increasedore in full sun than in partially shaded leaves as a conse-

uence of the UV-treatment (Fig. 1C). The reduction state ofSII centers (1 − qP), which may be taken to estimate the excessxcitation energy reaching the photosynthetic apparatus (Tattinit al., 2005), was similar in PAR35 and PAR100 leaves growingn the absence of UV-irradiance (Fig. 1D), but sharply greater

+20%) in partially shaded than in full-sun leaves concomitantlyxposed to UV-radiation. Interestingly, in full-sun leaves (1 − qP)as smaller in the presence than in the absence of UV-radiation,

he reverse being observed in leaves growing under partial shadingFig. 1D).

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ig. 1. Maximal (Fv/Fm, A) and actual (˚PSII , B) efficiency of PSII photochemistry, nonphotigustrum vulgare exposed to 35% (grey bars) or 100% sunlight irradiance (open bars) in theeviation, n = 5, and those, at each sampling date, not accompanied by the same letter, sigf a two-way ANOVA (total error degree of freedoms = 39) for the effects of light treatmlight × time), on chlorophyll fluorescence-derived parameters is the following: Fv/Fm: F

light × time = 38.6**; qNP: Flight = 19.9**, Ftime = 18.8**, Flight × time = 4.7*; 1 − qP: Flight = 71.4**, F

n 35 or 100% sunlight exposed leaves, respectively); CO2tot is the CO2 assimilated

anthin; Z, zeaxanthin; (Z + A)/(V + A + Z) is the conversion state of violaxanthin-cycle= 15 for Asat, n = 5 for CO2

tot, photosynthetic pigments and soluble carbohydrates).a least significant difference (LSD) test.

3.3. Antioxidant enzymes, polyphenols, and oxidative damage

On the whole the activities of enzymes devoted to the removalof the reactive oxygen forms, particularly the first line of defenseagainst the generation of the superoxide anion (SOD) were sig-nificantly greater in PAR100 than in PAR35 leaves (Fig. 2A). ThePAR-induced increase in SOD activity was observed early duringthe experimental period (on average +295% at day 10), espe-cially in leaves additionally exposed to UV-irradiance (+370%).UV-irradiance enhanced the SOD activity to a very similar degree

(+30%) in PAR35 or PAR100 leaves (Fig. 2A). The activities of CAT(+27%, Fig. 2B) and APX (+31%, Fig. 2C) significantly increased inresponse to visible-light, whereas UV-irradiance either depressed(−14% for CAT and −12% for APX) or unaffected the activity ofboth enzymes in PAR35 or PAR100 leaves, respectively (Fig. 2B and

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ochemical quenching (qNP, C), and excitation pressure on PSII (1 − qP, D) in leaves ofabsence or in the presence (hutched bars) of UV-radiation. Data are mean ± standardnificantly differ for P < 0.05 using a least significant difference (LSD) test. Summaryent (light) and sampling time (time) as fixed factors, with their interaction factorlight = 160.1**, Ftime = 1.6 n.s., Flight × time = 26.1**; ˚PSII: Flight = 119.5**, Ftime = 1.1 n.s.,

time = 0.8 n.s., Flight × time = 5.2*. **, P < 0.001; *, P < 0.05; n.s., not significant.

92 L. Guidi et al. / Environmental and Experimental Botany 70 (2011) 88–95

302010

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Fig. 2. Time-course in the activities of superoxide dismutase (SOD, A), catalase (CAT,B), and ascorbic peroxidase (APX) in Ligustrum vulgare leaves growing at 35% (closedlines) or 100% sunlight irradiance (dotted lines) in the absence (open symbols)or in the presence (closed symbols) of UV-radiation. Bar graphs in the insets arethe means ± standard deviation (n = 15), by pooling together data from time-coursemeasurements, and those not accompanied by the same letter significantly differfor P < 0.05 using a least significant difference (LSD) test. Summary of a two-wayANOVA (total error degree of freedoms = 59) with light treatments (light) and timeofF*

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POLYPHENOLS FLAVONOIDSSECOIRIDOIDS HYDROXYCINN

PAR35

PAR35UV

PAR100

PAR100UV

a

aa

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Fig. 3. The CO2-based accumulation of polyphenols (i.e., the increase in polyphe-nol concentration normalized to the assimilated during the experimental period) inleaves of Ligustrum vulgare exposed to 35 or 100% sunlight irradiance in the absenceor in the presence of UV-radiation. Data are the means ± standard deviation (n = 5),and bars not accompanied by the same letter differ significantly for P < 0.05, using aleast significant difference (LSD) test. Polyphenols denote the total polyphenol con-

respectively) than the corresponding partially shaded leaves, butUV-radiation did not affect the membrane lipid peroxidation andoxidation of proteins in leaves growing at full sunlight (Fig. 4). Incontrast, markers of oxidative damage increased steeply (+135%

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Fig. 4. The lipid peroxidation, estimated in terms of malondialdehyde concentra-

f exposure (time) as fixed factors, with their interaction factor (light × time) is theollowing: SOD: Flight = 52.0**, Ftime = 51.6**, Flight × time = 156.6**; CAT: Flight = 23.1**,time = 24.7**, Flight × time = 21.2**; APX: Flight = 32.1**, Ftime = 82.4**; Flight × time = 25.2**.*, P < 0.001.

). There was a highly significant interaction effect of light treat-ents × exposure time on the activities of all antioxidant enzymes.PAR and UV-treatments mostly affected the amount of “newly

ssimilated” carbon recovered in polyphenols, as the CO2-basedccumulation of polyphenols was 50% greater in PAR100 than inAR35 leaves, in the presence or in the absence of UV-irradianceFig. 3). The PAR and UV-irradiance also significantly altered the

olyphenol composition, as both treatments increased steeply theavonoid to other polyphenols ratio, from 0.17 in PAR35 to 0.63

n PAR100 leaves, and from 0.24 in PAR-treated to 0.54 in PARUV-reated leaves. Noticeably, hydroxycinnamic acid derivatives, i.e.,-coumaric and caffeic acid derivatives, were unresponsive to UV-

centration. Secoiridoids include oleuropein, ligustaloside A and B, and ligstroside.Hydroxycinnamic include p-coumaric and caffeic acid derivatives (i.e., echinaco-side and verbascoside). Flavonoids include the 3-O-glycosides of quercetin, and the7-O-glycosides of both luteolin and apigenin.

irradiance, despite these phenylpropanoids are very effective UV-attenuators.

Sun leaves in the absence of UV-radiation underwent greateroxidative damage (+31% or +38% for MDA or carbonyl groups,

tion, and the protein oxidation, estimated in terms of the concentration of carbonylgroups, in leaves of Ligustrum vulgare exposed to 35% (grey bars) or 100% sun-light irradiance (open bars) in the absence or in the presence (hutched bars) ofUV-radiation. Data are the means ± standard deviation (n = 5), and those not accom-panied by the same letter differ significantly for P < 0.05 using a least significantdifference (LSD) test.

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nd +42% for MDA and carbonyl groups, respectively) because ofhe UV-treatment in leaves growing at 35% sunlight irradiance.

. Discussion

Data of the present experiment draw a comprehensive picturen the impact of UV-irradiance on relevant physiological and bio-hemical traits in L. vulgare growing under field conditions, and addome insights on the role of the visible-light irradiance in mediatinghe effects of UV-radiation on the complex antioxidant machineryand oxidative damage) in this species.

Firstly, we show that the growth of L. vulgare was affectedreatly by PAR irradiance, whereas UV-radiation had a relativelyinor impact on the number and the size of fully expanded leaves,

articularly in plants growing in full sunlight. These findings, whichre consistent with previous reports on a relatively minor signifi-ance of short solar wavelengths in inhibiting plant growth underealistic sunlight irradiance (Kyparissis et al., 2001; Searles et al.,001; Caldwell et al., 2003; Stratmann, 2003), suggest that mostata concerning the UV-induced effects on plant growth should bee-evaluated on the basis of the intensity of visible-light at whichlants have been actually exposed (Caldwell et al., 1995; Ballaré,003). The steep decrease in leaf expansion rate and leaf areaxpansion rate as a consequence of high visible-light (both traitseing poorly affected by UV-radiation, Frohnmeyer and Staiger,003) may have depended on an altered auxin movement dueo the light-induced accumulation of flavonoids. Flavonoids haveeen long reported to inhibit the auxin movement in vivo (Brownt al., 2001) and control the canopy development, thus allowinglants growing in full sunlight to exposing few, but thick leaveso the detrimental effects of short solar wavelengths (broadly self-hading, Jansen, 2002).

Light-induced effects on growth-related features are consis-ent with those observed on gas exchange performances, whichere also poorly affected by UV-irradiance (Schntizler et al., 1999;ogués and Baker, 2000; Bassman and Robberecht, 2006). It isoted that net assimilation rate at saturating light varied little inartially shaded or full-sun leaves, so that PAR100 plants actu-lly suffered from excess-light stress in our experiment (Li et al.,009). The steep light-induced increase in the concentration oflucose and mannitol may have down-regulated net photosyn-hesis (Paul and Foyer, 2001) but, at the same time, would haveikely conferred a greater capacity to full-sun leaves to counteringV-induced oxidative damage with respect to the partially shadedounterparts (Shen et al., 1997; Valderrama et al., 2006).

The extent to which L. vulgare suffered from excess light inur experiment is consistent with the ecological distribution ofhe species in the Mediterranean Basin, which is actually lim-ted to the partially shaded areas (Tattini et al., 2004). Indeed,he dramatic reduction in chlorophyll concentration passing fromAR35 to PAR100 leaves is a common response of ‘sun-sensitive’pecies (Greenberg et al., 1997), and strongly depresses the radi-tion use efficiency to photosynthetic processes. A decrease inhlorophyll content is likely to be considered as an ultimateechanism of avoidance against the penetration of photons in

he visible region of the solar spectrum, which operates greatlyn species whose surface morphology is ineffective to avoidingffectively the penetration of radiant energy in the leaf (Tattinit al., 2005). Visible-light enhancements in both the relative (tohltot) concentration of violaxanthin-cycle pigments and the con-

ersion state of violaxanthin are of great significance, as theyould have likely conferred PAR100 leaves a greater capacity torotect the chloroplast from photo-oxidative damage (Havaux etl., 2007) as compared with the PAR35 counterparts, in additiono dissipating effectively excess energy through nonphotochem-

rimental Botany 70 (2011) 88–95 93

ical quenching mechanisms (Demmig-Adams and Adams, 1996).It has been suggested that a certain fraction of VAZ is appar-ently free in chloroplast membranes (i.e., not bound to thelight-harvesting antenna pigment-protein complexes) at VAZ con-centrations greater than 0.05 mol mol−1 Chltot, as actually occurredin our experiment (Niinemets et al., 2003).

Visible-light more than UV-irradiance affected the efficiency ofPSII photochemistry, but did not irreversible damage the photosyn-thetic apparatus. Fv/Fm declined early, but greatly recovered afterfour weeks of treatment with high visible-light and UV-irradiance,indicating that PSII acclimated to changing sunlight environments,in the presence or in the absence of the UV-wavelengths. Thisrecovery in Fv/Fm was accompanied by an efficient nonphotochem-ical quenching mechanism (which was likely responsible for adecline in the actual efficiency of PSII photochemistry, ˚PSII, ascompared with PAR35 leaves), aimed at dissipating excess exci-tation energy in the chloroplast, operating in both PAR100, andespecially in PAR100UV-treated leaves (see the sharp increasein VAZ/Chltot ratio, Logan et al., 1998). This enhancement inqNP driven by UV-radiation would have likely preserved fromdamage the photosynthetic apparatus in PAR100UV leaves to agreater extent than in PAR100 leaves (Bolink et al., 2001). Actu-ally, chlorophyll photobleaching in response to UV-irradiance wasnot observed in our experiment. On the other side, in shade leaves,nonphotochemical quenching mechanisms appeared less effectivein preserving the photosynthetic apparatus from the detrimentaleffect of UV-radiation, and exposed UV-treated leaves to a greaterexcess excitation pressure (1 − qP) than the full-sun counterparts(although neither PAR35 nor PAR100 plants suffered from severeexcess-radiant energy in the sense proposed by Demmig-Adams,1990). Our findings lead to the hypothesis that the impact of UV-irradiance on PSII photochemistry depends not only upon the UVto PAR ratio (Allen et al., 1998) but, possibly, on the actual intensityof visible-light irradiance at which plants grow.

As a consequence of the increase in excess excitation pressureon the chloroplasts, UV-treated leaves should have suffered moreat the shade than at the sun site from the enhanced ROS produc-tion, unless an effective system aimed to detoxifying ROS operated.In PAR100 plants, however, enzymes devoted to the removal ofsuperoxide anion and the SOD-induced H2O2 generation, operatedto a superior degree than in PAR35 plants. PAR-induced effects onthe antioxidant enzyme activities were pronounced early duringthe treatment period, and would have likely protected sun leavesfrom the so-called “oxidative burst” more effectively than the shadecounterparts. There was a clear effect of the exposure-time andvisible-light irradiance in modulating the effects of UV-irradianceon the activities of antioxidant enzymes, which may help explain-ing the conflicting results coming from experiments conductedunder controlled (Alexieva et al., 2001) or in field conditions (Renet al., 2006; Agrawal and Rathore, 2007).

UV-irradiance had the major impact of increasing the biosyn-thesis of polyphenols (Caldwell et al., 1999; Searles et al., 2001).The finding that flavonoids mostly increased in response to UV-irradiance may be interpreted in terms of UV-induced oxidativedamage (Jenkins, 2009), not only as a consequence of a great fluxof short solar wavelengths reaching and penetrating the leaves.Indeed, hydroxycinnamates were unresponsive to UV-radiation,although they are the most effective UV-attenuators (particularly inthe 290–315 nm UV-B region) as compared with the other phenyl-propanoids detected in leaves of L. vulgare (Harborne and Williams,2000; Tattini et al., 2004). It has been previously shown (Tattini

et al., 2004; Agati et al., 2009) that UV-inducible flavonoids in L.vulgare leaves are the antioxidant quercetin and luteolin deriva-tives, not the effective UV-attenuators apigenin 7-O-glycosides.UV-radiation has long been reported to preferentially enhance thebiosynthesis of dihydroxy B-ring substituted flavonols or flavones,

9 Exper

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he former having a steeply greater ability to inhibiting the gen-ration and quench free radicals, not a superior ability to absorbhe UV-wavelengths, than the monohydroxy B-ring-substitutedavonoid structures (Markham et al., 1998; Ryan et al., 1998; Tattinit al., 2005).

The finding that PolyphenolCO2 (i.e., the increase in total pheno-ic concentration normalized to total assimilated CO2) did not differ,ut the carbon allocated to flavonoids steeply increased passingrom PAR35UV to PAR100 plants is remarkable, and agree with thelue-light-induced increase in the expression of CHS, the enzyme

nvolved in the first committed step of the flavonoid biosynthesisJenkins, 2009). The increase in soluble carbohydrate concentrationecause of PAR irradiance may have both supplied carbon skeletonsor the biosynthesis of polyphenols and up-regulated the expres-ion of genes of the flavonoid biosynthetic pathway (Koch, 1996).inally, it is conceivable that in PAR100 plants, in the presence orn the absence of UV-radiation, the biosynthesis of flavonoids wasp-regulated by common oxidative signals, aimed to counteringxidative damage, as a consequence of excess light (Mackerness etl., 2001; Agati et al., 2007). This hypothesis is consistent with theery similar degree to which lipid peroxidation and protein oxida-ion occurred in full-sun leaves in the absence or in the presencef UV-radiation. The large UV-induced increase in carbon recov-red in flavonoids did not effectively protect UV-treated plants athe shade site from oxidative damage. It has been previously shownhat the UV-induced accumulation of antioxidant flavonoids in par-ially shaded leaves of L. vulgare was confined to the epidermal cells,.e., far from centers of ROS generation (Agati et al., 2009). As a con-equence, epidermal quercetin and luteolin derivatives would haveikely preserved just the epidermal cells, not the leaf interior, fromhoto-oxidative damage (Stafford, 1991).

. Conclusions

Visible sunlight irradiance had a markedly greater impact thanV-irradiance in most physiological and biochemical-related traits

n L. vulgare. The great extent of visible-light-induced phenotypiclasticity is consistent with L. vulgare being a species typicallyvolved in partially shaded areas. The finding that the intensityf visible sunlight irradiance, more than the UV to PAR ratio, wasesponsible for light-induced changes in shoot and leaf develop-ent is interesting, although the extent to which UV-radiation

ffects the growth performances of this species cannot be conclu-ively addressed from the results of our short-term experiments.

PAR irradiance also modulated the UV-induced changes in thectivity of antioxidant enzymes, especially during the early stagesf sunlight treatments: this likely allowed sun leaves to counteringhe UV-induced oxidative burst, and preserving leaves from oxida-ive stress to a greater degree than the partially shaded leaves.his interesting issue may merit further investigation, especiallyevoted to estimate the time-induced changes in enzyme activ-

ty, which has been only partially explored in our experiment.ther visible-light-induced biochemical adjustments likely servedkey role in mitigating the UV-induced oxidative damage in plantsrowing in full sunlight: (a) an increase in the conversion statef violaxanthin-cycle pigments together with (b) an increase inhe concentrations of glucose and mannitol, and (c) the upregu-ation of the biosynthesis of the flavonoid structures with effectiventioxidant properties.

Our findings are consistent with previous reports on a rela-

ively minor effect of UV-irradiance on a plant’s performance at

id-latitudes, and lead to the hypothesis that physiological andiochemical-related features in “shade” or “full-sun” environmentsre mostly controlled by visible- more than UV-irradiance. Ourxperiment has been conducted, however, on a short-term basis,

imental Botany 70 (2011) 88–95

and future investigations are needed to assess the actual role ofvisible-light and UV-radiation on the ecology of this species.

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