Induction of Phlorotannins During UV Exposure Mitigates Inhibitionof Photosynthesis and DNA Damage in the Kelp Lessonia nigrescens

Ivan Gomez*1 and Pirjo Huovinen1,2

1Universidad Austral de Chile, Instituto de Biologıa Marina, Valdivia, Chile2Centro i�mar, Universidad de Los Lagos, Puerto Montt, Chile

Received 18 February 2010, accepted 20 June 2010, DOI: 10.1111/j.1751-1097.2010.00786.x

ABSTRACT

Phlorotannins of brown algae are multifunctional compounds

with putative roles in herbivore deterrence, antioxidation and as

primary cell wall components. Due to their peripheral localiza-

tion and absorption at short wavelengths, a photoprotective role

is suggested. We examined the induction of phlorotannins by

artificial UV radiation in the intertidal kelp Lessonia nigrescens

and whether they attenuate the inhibition of photosynthesis and

DNA damage, two major detrimental effects of UV. The soluble

and cell wall-bound fractions of phlorotannins were quantified in

blades collected in summer and winter. Major findings were that

(1) the synthesis of phlorotannins (both forms) was induced by

UV only in summer; (2) the induction was fast (within 3 days);

and (3) there was a positive relationship between of the contents

of insoluble phlorotannins and the suppression of photoinhibition

and DNA damage, measured as formation of cyclobutane

pyrimidine dimers and 6-4 photoproducts. Overall, the photo-

protective role of phlorotannins appears to respond to an

interplay between the external UV stimulus, seasonal acclima-

tion and intrinsic morpho-functional processes. In summer, when

algae are naturally exposed to high UV irradiances, soluble

phlorotannins are induced, while their transition to insoluble

phlorotannins could be related with the growth requirements, as

active blade elongation occurs during this season.

INTRODUCTION

Phlorotannins (polymers of phloroglucinol; 1,3,5-trihydroxy-

benzene) are phenolic compounds that are found exclusively inbrown algae in which they can account for up to 25% of thedry weight (1,2). In cells, the soluble fraction is sequestered in

physodes due to their capacity to form complexes withmacromolecules through hydrophobic interactions. For a longtime, the phlorotannins located in physodes were considered tobe strictly inducible secondary metabolites and associated

mainly with antiherbivory defense (3,4), similar to thecondensed tannins and flavonols of higher plants (5). How-ever, phlorotannins are essential components in the polymer-

ization of the cell wall (insoluble cell wall-bound fraction) andcan be rapidly allocated during wound-healing and sealingafter amputation of thallus regions (6), thus acting as primary

structural compounds during growth.

Due to their localization at the periphery of the cells andtissues and their absorbance maximum in the UV region(maximal absorption between 200 and 300 nm, i.e. well in the

UV-C and shorter UV-B wavelengths), their role as sunscreensubstance against harmful solar radiation has been suggested,similar as reported for mycosporine-like amino acids and

coumarins from other groups of macro- and microalgae (7–12), and various UV-screening substances from plants (e.g.flavonoids) (13,14). However, the assessment of a potentialrole of phlorotannins as UV screens is difficult as the transient

soluble fraction and the constitutive insoluble fraction appar-ently can have different functional roles depending on theirlocation in the cell (15).

Consequently they can respond to different environmentaltriggers as has been demonstrated in cell wall-bound phenoliccompounds of Antarctic mosses (16). Some existing experi-

mental evidence suggests that phlorotannins may play a role inprocesses associated with UV tolerance in brown algae.Increase of phlorotannins in response to UV-B radiation inAscophyllum nodosum has been reported (17). Phlorotannins

have been shown to protect Fucus embryos from harmful UV-B radiation, thus being an important survival factor duringthese especially vulnerable life cycle stages (18–20). In juvenile

kelp sporophytes, such as Alaria esculenta, Sacchoriza derma-todea and Saccharina latissima, increasing thallus thicknessand opacity associated with a higher availability of cell-bound

UV-absorbing compounds were reported to minimize UVeffects on growth and DNA damage (21,22). In the giant kelpMacrocystis integrifolia, UV-B exposure has been shown to

increase tissue concentration of phlorotannins and also toenhance their exudation to the surrounding water, suggesting aUV-B shielding mechanism providing photoprotection toadult thalli and propagules (23). In other kelps, such as

Saccharina and Nereocystis, a combination of high UV-B andhigh CO2 can stimulate synthesis of phlorotannins (24).Nevertheless, the ecophysiological bases underlying UV pro-

tection by phlorotannins and whether and to what extent theyactually protect the key metabolic functions (e.g. photosyn-thesis) and reduce UV injury (e.g. DNA damage) are poorly

understood. Hitherto, evidence relating the UV induction ofphlorotannins with any beneficial effect on UV-sensitivemolecules and metabolic processes is lacking.

Based on the environmental context and the photobio-

logical responses of intertidal algae from southern Chile, it iswell established that midday summer levels of solar irradi-ance are stressful to a number of macroalgae (25,26). In the

*Corresponding author email: igomezo@uach.cl (Ivan Gomez)� 2010TheAuthors. JournalCompilation.TheAmericanSociety of Photobiology 0031-8655/10

Photochemistry and Photobiology, 2010, 86: 1056–1063

1056

South Pacific kelp Lessonia nigrescens, which inhabits thewave battered intertidal zone, some aspects of phlorotanninsand their potential role as herbivore deterrent have beenstudied (27,28). Recently, the impact of the interaction of

contaminants (Cu), nutrients and UV radiation on thecontent of phlorotannins of L. nigrescens was reported (29).Although Lessonia is in general more sensitive to UV

radiation than the upper intertidal seaweeds such as Ulvaand Porphyra, decreases in e.g. chlorophyll fluorescencebased maximum quantum yield of photosystem II (Fv ⁄Fm)

normally do not exceed 40% even under exposure to highmidsummer UV irradiances (26,30,31). This remarkabletolerance to current solar UV-B radiation raises a question

whether UV-shielding substances (such as phlorotannins)prevent an exacerbated damage on key components of thephotosynthetic apparatus, e.g. photosystem II and theelectron transport machinery.

In this study, the following questions were addressed: (1)how efficient are phlorotannins to withstand or mitigate themetabolic effects of UV radiation? and (2) does the expression

of such a photoprotective strategy provide ecophysiologicalbenefits? The experimental approach was based on theexamination of the induction of soluble and insoluble phloro-

tannins of L. nigrescens upon exposure to UV radiation inlaboratory conditions. Because of their localization in the cellperiphery, we hypothesize that the cell wall-bound phlorotan-nins have a UV-screening function additional to their primary

structural function, thus increasing relative to the solublefraction. The concentrations of these compounds were corre-lated with inhibition of photosynthesis and DNA damage, two

well-known harmful effects of UV-B radiation reported inalgae (32,33). To test the morpho-functional regulation of thephlorotannin induction, exposures to UV radiation included

algal material collected in summer (growth season) and inwinter (no growth).

MATERIAL AND METHODS

Sampling and maintenance of the algal material. Six young individuals(holdfast diameter between 10 and 20 cm) of the intertidal Lessonianigrescens Bory were collected at the coast of Valdivia, Chile (39�51¢S,73�23¢W) in winter (May) and summer (January). Lessonia nigrescensis a perennial kelp that can live several years. In southern Chilean,sporophytes of Lessonia (up to 2–3 m length) are reproductive all yearround, with the highest frequencies of mature fronds occurring inautumn and winter. Thus, size classes of holdfast diameter <20 cm(young individuals) can be found at all seasons (34). Observed growthrates of L. nigrescens in summer are around 0.4% per day, whereas inwinter <0.1% per day (unpublished data). In the laboratory, algaewere cleaned from epiphytes using filtered sea water and maintainedovernight under constant illumination provided by fluorescent lamps(120 lmol m)2 s)1; Osram L36 W ⁄ 640). Disks with a diameter of8 mm were punched from the longitudinal axis of blades (between 20and 40 cm length) of six different algae using a cork borer andmaintained for 24 h before starting the experiments so as to standard-ize the initial physiological status of the algal samples. Preliminaryassays indicated no clear patterns in intrablade contents of phloro-tannins and thus, disks from the middle part of the blade were used. Inthe experiments, the disks were maintained in Petri dishes containing150 mL of filtered seawater (0.45 lm) in a thermoregulated water bath(13–15�C in winter and 15–17�C in summer) under vigorous aeration.Although there is no evidence of marked physiological changes inalgae from southern Chile experiencing these low seasonal temperaturevariations, the ambient temperatures of each season were used to avoidpossible thermal stress of winter algae. The seawater was changedevery 12 h.

Exposure to UV radiation. Algal disks (30 disks per replicate) wereincubated in Petri dishes under three irradiation conditions usingphotosynthetically active radiation (PAR) (Osram), UV-A (Q-panel340, USA) and UV-B (Q-Panel 313, USA) lamps. For each irradiationtreatment, three replicates were used. A stress factor caused by diskexcision (6) was ruled out by a constant monitoring of the physiolog-ical status by measuring photochemical quenching of disks (see below)and the use of controls in each experimental series. To set threedifferent UV treatments, the dishes were covered with cut-off filters: (1)PAR + UV-A + UV-B = Ultraphan 295 (Digefra, Germany) forfull spectrum; (2) PAR + UV-A = Folex 320 (Folex, Germany), tocut-off wavelengths <320 nm; and (3) PAR = Ultraphan 395 (Dig-efra, Germany), which cuts off UV wavelengths. Irradiances under thedifferent combinations of lamps and cut-off filters were 2.4 and8.8 W m)2 for UV-B and UV-A, respectively, whereas PAR was120 lmol photons m)2 s)1. The algal disks were exposed to theseirradiation conditions during 2, 6, 24, 48 and 72 h. The experimentalirradiances were weighted using the action spectrum for photoinhibi-tion of photosynthesis as normalized to unity at 300 nm (35) to obtainbiologically effective UV doses (BED). The levels of UV-B in theexperiments were in the range of cloudless summer days in the studysite in southern Chile (26). The radiation measurements were carriedout using a Li-1400 data logger (LI-COR Biosciences, USA) fitted witha Li-190-S quantum sensor for visible irradiance and two semisphericalUV-A ⁄UV-B detectors (Walz, Germany). At each time interval, threedisks from each treatment were used for the determination ofphlorotannins, three for the determination of DNA damage and sixfor measurements of maximum quantum yield of photosynthesis (seebelow). In both seasons, algae were exposed to the summer doses ofUV-B and UV-A as described above.

Localization of phlorotannins. The spatial distribution of phloro-tannins within the tissues was investigated by light microscopy aftervanillin–HCl staining (10% vanillin and 90% of a mixture of ethanoland HCl) giving an orange color (36). The ultrastructural localizationof physodes was examined through transmission electron microscopy(Philips 300, The Netherlands) in samples primarily fixed in 2.5%glutaraldehyde and with postfixation with 1% osmium tetroxide. Forstaining, uranyl acetate and lead citrate were used.

Extraction of phlorotannins. Phlorotannins were determined usingthe Folin–Ciocalteu method as described by Koivikko et al. (37) withmodifications (mainly of the algal weight, extract volumes andincubation times). For the determination of soluble phlorotannins(cytosol fraction), dry algal material (100 mg) was homogenized withliquid nitrogen in a mortar. Phlorotannins were extracted (at leastthree times for 24 h) using 10 mL of 70% acetone. After shakingovernight at 4�C and centrifugation for 10 min at 2600 g, 500 mL ofsupernatant (separately from each extraction) was mixed with 2.5 mLof distilled water, 2 mL of 20% NaCO3 and 1 mL of 2 N Folin–Ciocalteu reagent. The samples were incubated in the darkness for45 min at room temperature, centrifuged at 2250 g (3 min) and theabsorbance was read at 750 nm. The total content of solublephlorotannins in the sample was finally obtained by adding togetherthe readings from the different extractions.

The cell wall-bound fraction of phlorotannins was quantified usingan alkaline method (38) modified by Koivikko et al. (34). Theprecipitated fraction from the first extraction was successivelyextracted using a series of solvents: methanol, H2O–methanol, acetoneand diethyl ether. After drying at 60�C for at least 1 h, the totalinsoluble residue, remaining after the extraction and washing ofsoluble phlorotannins, was suspended in 8 mL of 1 MM aqueous NaOHsolution and stirred for 17 h at room temperature. Samples werecentrifuged (5 min at 2400 g), and 1 mL aliquots were neutralized with100 lL of H3PO4. The alkaline treatment was repeated four times andthe aliquots of each extraction were analyzed separately.

Determination of chlorophyll fluorescence. In vivo chlorophyll fluo-rescence of photosystem II (PSII) was measured using a computer-aided portable pulse amplitude modulation fluorometer (PAM-2000;Walz, Germany). Samples were incubated for 20 min in the dark andmeasured for optimal quantum yield of fluorescence (Fv ⁄Fm) (30).

Determination of DNA damage. Cyclobutane pyrimidine dimers(CPDs) and (6-4) photoproducts (6-4 PPs) were determined using anELISA method (39) with modifications. The sample (5 mg) washomogenized in a mortar with liquid N2. DNA was isolated using apurification kit (Easy-DNA Kit; Invitrogen), and quantified with

Photochemistry and Photobiology, 2010, 86 1057

reference to spectrophotometric absorbance at 260 nm. Heat-dena-tured DNA was applied into microtiter well plates and dried. Theplates were washed with phosphate buffered saline with TWEEN(PBS-T) buffer to remove nonbound DNA, and blocked with bovineserum albumin solution. The blocking solution was removed bywashing with PBS-T. A primary antibody (monoclonal antibodiesMC-062 and MC-082 to recognize CPDs and 6-4 PPs, respectively)was added and the plates were incubated for 30 min at 37�C. Afterwashing, the second antibody (monoclonal rabbit antimouse antibodyconjugated with horse radish peroxidase) was added and incubated for30 min at 37�C. For detection, o-phenylenediamine–H2O2 was usedand the color formation stopped with 2 MM H2SO4. Absorbance of areaction mixture at 492 nm in an ELISA reader was used as relativevalue of DNA damage.

Statistical treatment. Response variables after the exposure treat-ments were compared using two-way ANOVA with UV treatmentsand exposure time as factors followed by a Tukey HSD post hocanalysis when differences were detected (STATISTICA 7.0; Statsoft,Inc., USA). ANOVA assumptions (homogeneity of variances andnormal distribution) were examined using the Levene and Shapiro-Wilk W tests, respectively. The covariation of soluble vs. cell wall-bound fractions as well as photoinhibition and DNA damage were alsoexamined in previously arcsine-transformed percentage data usingmultivariate analysis (MANOVA ⁄MANCOVA) with the Wilks’Lambda (Rao’s R) as a multivariate F. Multivariate homogeneitywas tested using Box M, whereas normality was assessed for eachdependent variable similar as in the one-way ANOVA. Statisticalsignificance was set to P < 0.05.

RESULTS

At cellular level, the phlorotannins were abundant and

enclosed in large physodes, which normally were arranged atthe periphery of the cell (Fig. 1A). At tissue level, the highestcontents of phlorotannins were mainly located in the most

external cell layers, particularly in cortex and meristoderm(Fig. 1B). In summer, both soluble and insoluble phlorotan-nins of L. nigrescens were strongly induced in disks ofsporophytes (P < 0.05; ANOVA–Tukey HSD). A significant

effect of UV exposure and exposure time on the induction ofphlorotannins was detected (P < 0.001; two-way ANOVA;Fig. 2 and Table 1). Soluble phlorotannins increased over six-

fold after a 72 h exposure. The maximal increase underPAR + UV-A + UV-B treatment was detected after 24 h,whereas under PAR + UV-A, the highest content of phloro-

tannins was measured after 48 h (P < 0.05; Tukey HSD,Fig. 2). In treatments deprived of UV radiation (PAR alone),the induction of phlorotannins was comparatively low(P > 0.05; ANOVA).

Decreases in photosynthesis measured as Fv ⁄Fm were lowerin winter compared with summer. In summer, Fv ⁄Fm decreasedunder PAR + UV-A + UV-B from the initial 0.7 to 0.4 after

48 h (P < 0.05; Tukey HSD; Fig. 3). In the case of DNAdamage, CPDs and 6-4 PPs increased with time of exposure,with the highest absorbances at 492 nm for 6-4 PPs determined

in sample disks exposed to PAR + UV-A + UV-B in winter.In general, PAR alone did not have significant negative effecton photosynthesis or on DNA at any season (Fig. 3).

Low contents of cell wall-bound phlorotannins were relatedto higher photoinhibition of photosynthesis in winter algaldisks exposed to summer UV doses. In summer, this patternwas reversed (Fig. 4). Under PAR + UV-A + UV-B treat-

ment after 72 h (equivalent to UV doses [BED] close to 700 kJm)2), insoluble phlorotannins increased by 110%, whereasphotosynthesis decreased only by 17% and DNA damage

measured as CPDs and 6-4 PPs was 13% and 25%, respec-tively. In the exposure deprived of UV-B (PAR + UV-Atreatment), induction of phlorotannins was high in algae

collected in summer (similar to or higher than underUV-A + UV-B), but in winter the induction did not exceed10% relative to the initial value. In both seasons, decrease inphotosynthesis under PAR + UV-A exposure was compara-

tively low (<15%) (data not shown). In winter, when theinsoluble phlorotannins induction did not exceed 16%, theinhibition of photosynthesis in PAR + UV-A + UV-B treat-

ment reached values close to 36%. The DNA damagemeasured as CPDs in this season increased to 50% alreadyafter 2 h exposure and the 6-4 PPs increased up to 60% after

72 h (Fig. 4).A resolution using MANOVA ⁄ANCOVA indicated that

insoluble phlorotannins covaried significantly with the soluble

fraction and the UV target variables inhibition of photosynthe-sis and DNA damage (thymine dimers) on the base of seasonand exposure time (P < 0.0001; MANOVA Wilks-Lambda,Table 2).

Figure 1. Localization of phlorotannins. (A) Electron microscopy image of a cortex cell of Lessonia nigrescens showing abundance of physodes; (B)cross-section of a blade stained with vanillin-HCl indicating accumulation of phlorotannins in the outer cell layers. Chl = chloroplast;phy = physodes; med = medulla; cor = cortex; mer = meristoderm.

1058 Ivan Gomez and Pirjo Huovinen

DISCUSSION

In this study, four major findings can be outlined: (1)the induction of phlorotannins in algal disks occurred underexposure to high UV radiation during the period when

sporophytes are actively growing; (2) the induction occurredwithin 3 days, and was always higher in the solublethan in the insoluble fraction; (3) both UV-A and UV-B

radiation induced phlorotannins; and (4) a positive relation

between induction of phlorotannins and reduction in theinhibition of photosynthesis and DNA damage was

observed.

Induction of phlorotannins by UV radiation

The results of this study point to a complex interplaybetween the induction of soluble phlorotannins enclosed inphysodes and their subsequent deposition in the cell-wall

Table 1. Summary of factorial ANOVA indicating the main and interaction effects of UV exposure and growth season on contents of two fractionsof phlorotannins in Lessonia nigrescens.

Dependent variable Factor d.f. MS F P-value

Solublephlorotannins

Season (A) 1 1234.77 2156.52UV treatment (B) 2 477.73 834.73 <0.0001Time exposure (C) 4 56.85 99.28 <0.0001A · B 2 460.16 803.66 <0.0001A · C 4 71.84 125.47 <0.0001B · C 8 20.55 35.88 <0.0001A · B · C 8 12.29 21.47 <0.0001Error 60 0.57 2156.52 <0.0001

Cell wall-boundphlorotannins

Season (A) 1 0.47 1.10 >0.05UV treatment (B) 2 39.54 91.22 <0.0001Time exposure (C) 4 19.45 44.88 <0.0001A · B 2 29.79 68.71 <0.0001A · C 4 4.35 10.05 <0.0001B · C 8 3.34 7.70 <0.0001A · B · C 8 2.07 4.77 <0.001Error 60 0.43

Results of MANOVA for estimation of covariation between soluble and cell wall-bound phlorotannins in each species is also indicated (see text fordetails).

Figure 2. Effect of UV exposure on the content of soluble and cell wall-bound phlorotannins in Lessonia nigrescens. The results from two-wayANOVA (factors: UV treatments and exposure time) are detailed in Table 2. Values are mean ± SE; n = 3–6.

Photochemistry and Photobiology, 2010, 86 1059

matrix. Phlorotannins were induced as a response to high

UV radiation when sporophytes of L. nigrescens wereactively growing, but in winter no induction was detected,which is in contrast with previous studies on other brownalgae (40,41). These findings suggest that phlorotannin

induction forms part of the suite of efficient acclimationmechanisms that exhibits L. nigrescens to cope with theharsh intertidal environment in summer (31). This appar-

ently is triggered not only by enhanced solar radiation, butalso by metabolic requirements, e.g. endogenous signals

from an enhanced lamina elongation via the intercalary

meristem.Our results also open new questions with respect to the

balance between transient and constitutive phlorotannins. Theoptimal defense model, which suggests that chemical defenses

are produced in direct proportion to the risk, appears to beapplicable to the situation in L. nigrescens as the phlorotanninswould be produced at a direct expense of other functions

(28,41,42). In the case of UV-induced increase of phlorotanninsin this species, high initial costs could be compensated through

Figure 3. Effect of UV exposure on photosynthesis (as Fv ⁄Fm) and DNA damage (as absorbance of cyclobutane pyrimidine dimers [CPDs] and 6–4photoproducts [PPs] at 492 nm) in Lessonia nigrescens in winter and summer. Time exposure for photosynthesis was up to 48 h. Values aremean ± SE; n = 3–6.

Figure 4. Induction of phlorotannins versus photosynthesis and DNA damage in Lessonia nigrescens exposed for 48 h to UV radiation in winterand summer. Data are percentage of PAR + UV-A ) UV-B treatment relative to initial control and correspond to calculations based in data fromFig. 3. Values are mean ± SE; n = 3–6.

1060 Ivan Gomez and Pirjo Huovinen

their transformation and subsequent bound in the cell wall (15).Thus, although insoluble phlorotannins can be present in lower

amounts than the soluble fraction (37), they can be important ina series of conformational processes in the cell wall (6,15,42).

The increase in the cell wall fraction observed inL. nigrescens was a consequence of the induction of soluble

phlorotannins in response to UV radiation. Cellular cyclesand processes of biomass formation (e.g. growth) finally settheir concentration in the cell wall. At tissue level, phloro-

tannins (soluble and insoluble) are mostly located in the outercortex and meristoderm layers (Fig. 1). Therefore, a putativeUV photoprotection may be regarded as a secondary

function derived from a peripheral localization causing aninterception of UV photons. The question whether increaseof insoluble phlorotannins in response to environmentalfactors can occur independently of the induction of the

soluble phlorotannins could not be addressed. It is knownthat the cell wall-bound phenols may have many functionalroles as has been reported in terrestrial plants (43). Cell

wall-bound compounds, such as ferulic esters can act asUV-screening substances (16,44) and as strong antioxidants(45), which has important implications for growth processes

of outer cell layers (46). In the case of algae, antioxidantcapacity has been determined in soluble phlorotannins ofAscophyllum nodosum, Sargassum muticum, Laminaria hyper-

borea (47) and L. nigrescens (29). Taking into account thatantioxidation mechanisms are broadly extended in macroal-gae as a response to UV radiation (33), a possible role ofinsoluble phlorotannins as reactive oxygen species scavengers

may not be ruled out.

Photoprotective role of phlorotannins

The decreased inhibition of photosynthesis and lower DNA

damage in the presence of high levels of insoluble phloro-tannins was an important confirmation that intracellularincreases in phlorotannins prevent harmful effects of UV

radiation on key physiological targets. The uniform distribu-tion of insoluble phlorotannins in the cell suggests that theyact as a physical barrier for UV wavelengths protecting thewhole suite of intracellular components, including nuclear

DNA and the photosynthetic machinery. Taking into accountthat phlorotannins exhibit absorption maxima at 200 and265 nm (1), the putative shielding capacity of phlorotannins

would be more efficient in the case of DNA damage (causedmainly by UV-B wavelengths) (48) than, e.g. photosynthesis,

which normally is affected also by wavelengths in the UV-Aregion (35). However, evidence from dinoflagellates indicatesthat some photosynthetic processes (e.g. carbon fixation) canbe affected in a higher extent than DNA at similar UV doses

(49).In our study, the accumulation of CPDs and 6-4 PPs was

not linear during the course of UV exposure as at some point

the amount of DNA damage did not increase anymore or evendecreased. Apparently, photoreactivation repair of DNAdamage by UV-A and PAR during the exposures (50) could

take place, explaining partly this pattern. However, consis-tently with other studies, shielding of DNA by UV-screeningsubstances appears to be a major component contributing tothe decrease of CPDs (51), in a similar way as has been

reported in other algal groups and plants. For example, inRhodophyta, an association between the accumulation ofUV-screening substances and a minimized effect of UV-B

radiation on DNA has been reported (32). In the Antarcticmoss Ceratodon purpureus, the different contents of cell wall-bound UV-screening phenolic compounds between species

have been regarded as a factor explaining their different UVsensitivity, particularly DNA damage (16).

The inhibition of photosynthesis reported in this study in

blades collected in winter was not induced by high PAR, asin the experiments this factor was maintained low to unmaskthe effect of UV-B. However, under high PAR in the field,photoinhibition of photosynthesis, a down-regulation mecha-

nism to quench excess solar energy, is operating simulta-neously with phlorotannins to minimize harmful effects of UV.Other factors, such as temperature, which have been reported

to affect DNA repair processes in higher plants (52) and somered algae (50), likely were not determinant in the decrease ofphotosynthesis or accumulation of CPDs ⁄ 6-4 PPs in this

study, which is in accordance with other studies (32,51). Itmust be emphasized that L. nigrescens is subject to lowseasonal changes of water temperature (2–3�C) and thus, thedifference in the UV sensitivity between individuals collected in

winter and summer is mostly triggered by changes in solarradiation (26).

Overall, the results of this study outline important ecolog-

ical implications, especially for intertidal species which areexposed to high levels of solar radiation. The UV radiation

Table 2. Summary of bifactorial MANOVA ⁄MANCOVA for estimation of covariation between insoluble (cell wall-bound) phlorotannins andsoluble phlorotannins and physiological variables’ photoinhibition of photosynthesis and DNA damage.

Dependent variables Factor Effect d.f. Error d.f. Wilk’s value F P-value

Insoluble phlorotannins versusSoluble phlorotannins Season (A) 2 15 0.004 2003.23 <0.0001

Exposure time (B) 6 30 0.025 26.29 <0.0001A · B 6 30 0.041 19.53 <0.0001

Inhibition of photosynthesis Season (A) 2 15 0.022 321.80 <0.0001Exposure time (B) 6 30 0.036 21.24 <0.0001A · B 6 30 0.068 14.03 <0.0001

DNA damage (CPDs) Season (A) 2 15 0.029 250.22 <0.0001Exposure time (B) 6 30 0.054 16.44 <0.0001A · B 6 30 0.130 8.86 <0.0001

Arcsine-transformed data correspond to the percentage values from PAR + UV-A + UV-B treatment relative to PAR control for series of 2, 5, 6,24 and 48 h exposure measured in summer and winter (see text for details).

Photochemistry and Photobiology, 2010, 86 1061

conditions in southern Chile (40�S) show a strong seasonalvariation and the risk of UV damage could be up to 37 timeshigher in summer than in winter. In all, the results of this studyare among the first ones indicating that phlorotannins act as

UV photoprotective substances preventing damage of keyphysiological processes and molecules and raise further ques-tions related to at which extent these responses are expressed

under natural conditions.

Acknowledgements—This study was financed by Conicyt (Fondecyt

No 1060503 to I.G) and Conicyt-World Bank (IPA09 to P.H.). The

authors thank M. Orostegui and G. Butendieck for assistance during

laboratory analysis. We thank G. Alvial and O. Garrido for logistic

support and histological analyses.

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