University of Groningen

Nutrient limitation and high irradiance acclimation reduce PAR and UV-induced viability lossin the Antarctic diatom Chaetoceros brevis (Bacillariophyceae)van de Poll, Willem; van Leeuwe, Maria; Roggeveld, J; Buma, Anita

Published in:Journal of Phycology

DOI:10.1111/j.1529-8817.2005.00105.x

IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.

Document VersionPublisher's PDF, also known as Version of record

Publication date:2005

Link to publication in University of Groningen/UMCG research database

Citation for published version (APA):van de Poll, W. H., van Leeuwe, M. A., Roggeveld, J., & Buma, A. G. J. (2005). Nutrient limitation and highirradiance acclimation reduce PAR and UV-induced viability loss in the Antarctic diatom Chaetoceros brevis(Bacillariophyceae). Journal of Phycology, 41(4), 840-850. DOI: 10.1111/j.1529-8817.2005.00105.x

CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).

Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.

Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.

Download date: 27-03-2018

NUTRIENT LIMITATION AND HIGH IRRADIANCE ACCLIMATION REDUCEPAR AND UV-INDUCED VIABILITY LOSS IN THE ANTARCTIC DIATOM

CHAETOCEROS BREVIS (BACILLARIOPHYCEAE)1

Willem H. van de Poll,2 Maria A. van Leeuwe, Jan Roggeveld, and Anita G. J. Buma

Department of Marine Biology, Center for Ecological and Evolutionary Studies, University of Groningen,

P.O. Box 14, 9750 AA Haren, The Netherlands

The effects of high PAR (400–700 nm), UVA (315–400 nm), and UVB (280–315 nm) radiation on via-bility and photosynthesis were investigated forChaetoceros brevis Schutt. This Antarctic marine di-atom was cultivated under low, medium, and highirradiance and nitrate, phosphate, silicate, and ironlimitation before exposure to a simulated surfaceirradiance (SSI) treatment, with and without UVBradiation. Light-harvesting and protective pigmentcomposition and PSII parameters were determinedbefore SSI exposure, whereas viability was meas-ured by flow cytometry in combination with a via-bility stain after the treatment. Recovery of PSIIefficiency was measured after 20 h in dim light in aseparate experiment. In addition, low and high ir-radiance acclimated cells were exposed outdoorsfor 4 h to assess the effects of natural PAR, UVA, andUVB on viability. Low irradiance acclimated cellswere particularly sensitive to photo induced viabil-ity loss, whereas no viability loss was found afteracclimation to high irradiance. Furthermore, nutri-ent limitation reduced sensitivity to photo inducedviability loss, relative to nutrient replete conditions.No additional viability loss was found after UVBexposure. Sunlight exposed cells showed no addi-tional UVB effect on viability, whereas UVA andPAR significantly reduced the viability of low irra-diance acclimated cells. Recovery of PSII functionwas nearly complete in cultures that survived thelight treatments. Increased resistance to high irra-diance coincided with an increased ratio betweenprotective- and light-harvesting pigments before theSSI treatment, demonstrating the importance ofnonphotochemical quenching by diatoxanthin forsurvival of near-surface irradiance. We concludethat a sudden transfer to high irradiance can befatal for low irradiance acclimated C. brevis.

Key index words: Antarctic diatoms; flow cytometry;irradiance acclimation; nutrient limitation; oxida-tive stress; photosynthesis; pigments; UVA radia-tion; UVB radiation; viability

Abbreviations: MAA, mycosporine-like amino acids;rETRmax, relative maximal photosynthetic electron

transport rate; ROS, reactive oxygen species; SSI,simulated surface irradiance

Phytoplankton productivity and community com-position in the Southern Ocean depends on many abi-otic factors, such as the availability of iron, silicate, andlight quality and quantity (De Baar et al. 1990, Cullenand MacIntyre 1991, Agustı and Duarte 2000, Sarmientoet al. 2003). Together with the seasonal and diurnalcycle, weather conditions and wind-driven verticalmixing impose dramatic changes on the irradianceconditions of phytoplankton. These changes can beabrupt or slow and can last for seconds up to weeks.Deeper in the water column, light can be limiting,which requires investment in maximizing light absorp-tion and photosynthetic efficiency. However, when re-siding close to the surface, algae can experienceirradiances that exceed the requirement for photosyn-thesis by 10- to 20-fold. Here, protective de-excitationmechanisms become important. Apart from high PAR(400–700 nm), phytoplankton can encounter harmfullevels of UVA (315–400 nm) and UVB (280–315 nm)radiation in the upper part of the water column, thelatter of which can be enhanced by stratospheric ozonedepletion in the Southern Hemisphere (WMO 2002).The degree of exposure depends on absorption char-acteristics of the water column and the near-surfaceresidence time of the algae. It has been shown thathigh PAR, UVA, and UVB can exercise a negative ef-fect on the productivity of phytoplankton assemblagesfrom the Southern Ocean (Smith et al. 1992, Nealeet al. 1994). Effects of excessive PAR and UVA are pri-marily mediated in the chloroplast, where light is ab-sorbed and reactive oxygen species (ROS) are formedwhen chl becomes an alternative electron acceptor af-ter over-saturation of the photosynthetic electrontransport chain in the presence of oxygen (Salin1987, Asada and Takahashi 1987). The reaction cen-ter of the PSII protein–pigment complex and enzymesfrom the Calvin cycle are highly sensitive to ROS. Ad-ditional UVB exposure increases ROS formation anddamage to PSII, whereas reduction of the RUBISCOpool and DNA damage are also reported (Schofieldet al. 1995, Lesser 1996a,b, Lesser et al. 1996,Malanga et al. 1997, Rijstenbil et al. 2000, Rijstenbil2001, Buma et al. 2001). Because UVB coincides with

1Received 13 September 2004. Accepted 30 April 2005.2Author for correspondence: e-mail w.h.van.de.poll@rug.nl.

840

J. Phycol. 41, 840–850 (2005)r 2005 Phycological Society of AmericaDOI: 10.1111/j.1529-8817.2005.00105.x

high UVA and PAR, UVB-induced stress always co-oc-curs with UVA and PAR stress.

Survival of these unpredictable irradiance condi-tions is mediated by tolerance mechanisms that oper-ate on very short (seconds and minutes) and longer(hours and days) time scales. Diatoms respond imme-diately to excessive irradiance by de-epoxidation of di-adinoxanthin to diatoxanthin, which allows dissipationof absorbed energy as heat, upstream of the photosys-tems (Olaizola and Yamamoto 1994, Lavaud et al.2002). Furthermore, ROS can be detoxified byenzymatic and nonenzymatic antioxidants. Increasedantioxidant capacity was found in Chlamydomonasreinhardtii, benthic diatoms, dinoflagellates, and mac-roalgae in response to high irradiance and chronic UVexposure (Lesser et al. 1996, Rijstenbil et al. 2000,Rossa et al. 2002, Trebst et al. 2002). On a time scaleof days, light absorption can be adjusted to the pre-vailing irradiance conditions by balancing antenna size,photosystems, and the activity of the dark reactions(Falkowski and LaRoche 1991). Over a similar tempo-ral scale, some phytoplankton species accumulate mi-crosporine-like amino acids (MAAs), which reduce UVexposure of cellular components (Karentz et al. 1991a,Helbling et al. 1996). Moreover, efficient repair path-ways for UVB-induced DNA damage that operatewithin hours are presumably present in all algae(Karentz et al. 1991b, Buma et al. 2003).

Much of the current knowledge on phytoplanktonUV sensitivity is based on experiments documentingthe effects on photosynthesis after short PAR and UVradiation exposure (Cullen et al. 1992, Neale et al.1994, Boucher and Prezelin 1996). Action spectra forinhibition of photosynthesis showed that the acclimat-ion state of algae influences PAR and UV sensitivity inassemblages from the Southern Ocean. High irradi-ance acclimation reduced light sensitivity comparedwith samples that were obtained from a low irradianceenvironment (Neale et al. 1994). Furthermore, a dis-crepancy in UV sensitivity between algae from the fieldand those cultivated in the laboratory was observed,with the latter being less sensitive (Lesser et al. 1996,Neale et al. 1998). Nutrient limitation, and particularlynitrate limitation, has been associated with increasedvulnerability to photoinhibition by PAR and UV indinoflagellates and diatoms (Cullen and Lesser 1991,Lesser et al. 1994, Litchman et al. 2002, Shelly et al.2002). Nutrient availability has a pronounced effect onpigment composition and photosynthetic performanceand consequently may affect light sensitivity of algae(Geider et al. 1993). Furthermore, low iron concentra-tions restrain phytoplankton productivity in vast partsof the Southern Ocean, possibly by reducing photo-synthetic efficiency (Geider and LaRoche 1994, DeBaar et al. 1995, Van Oijen et al. 2004). However,how iron limitation affects sensitivity to the high irra-diance periodically experienced near the surface ispresently unclear. It was shown that a considerablepart of a phytoplankton assemblage from the NorthAtlantic could be classified as nonviable, suggesting

that viability loss due to suboptimal conditions may bean important variable in the field (Veldhuis et al.2001). Here, we explore if near-surface PAR and UVirradiance affect phytoplankton viability. Experimentsare described where viability and photosynthesis of thesmall (� 5mm) Antarctic diatom, Chaetoceros brevis,were used as indicators for UV and high PAR sensitiv-ity after cultivation under various irradiance andnutrient conditions.

MATERIALS AND METHODS

Three series of experiments were performed to gain insightinto the light sensitivity of C. brevis. In the first and second ex-periments, C. brevis was cultivated under seven conditions be-fore 4 h of simulated surface irradiance (SSI) exposure. Thefirst experimental series focused on viability loss during thetime course of the SSI treatment. Here, cultures were dilutedto 5.6 � 104 cells �mL�1. In the second series, PSII efficiencywas determined before and 20 h after SSI exposure, whereasviability was measured directly after exposure. In this exper-imental series, the concentration was adjusted to9 � 105 cells �mL�1 to facilitate fluorescence measurements.In the third experimental series, viability was determined dur-ing 4 h of sunlight exposure of low and high irradiance accli-mated C. brevis (diluted to 5.6 � 104 cells �mL�1).

Cultivation conditions. Chaetoceros brevis (strain CCMP 163)was grown in a batch regime under three irradiance levels andfour nutrient limitation conditions in 100–300 mL F/2-en-riched autoclaved seawater of 35 psu (unless stated otherwise)at 41 C under a 16:8-h light:dark cycle. Low (6mmol pho-tons �m� 2 � s�1) and medium irradiance (75mmol pho-tons �m� 2 � s�1) conditions were created in a refrigeratedcabinet equipped with 12-W biolux lamps (Osram GmbH,Munich, Germany), whereas a higher irradiance level(220mmol photons �m�2 � s�1) was achieved with ten 36-W Os-ram biolux lamps in combination with a temperature-control-led water bath. Irradiance was measured in air with an LI-250light meter with a flat sensor (LI-COR, Lincoln, NE, USA).

All nutrient limitation experiments were performed undermedium irradiance. Nitrate and phosphate limitation was im-posed by growing the algae in autoclaved seawater where, re-spectively, nitrate and phosphate were omitted. Silicatelimitation was induced by suspending cells in silicate-free arti-ficial seawater (Veldhuis and Admiraal 1987) in polycarbonatevessels after harvesting by mild centrifugation. Iron limitationwas achieved by growing the cells for 4 weeks in seawater fromthe Southern Ocean in polycarbonate vessels. Seawater andnutrients (except Si stock) were passed over a Chelex-100 col-umn (Chelex, Rochester, NY, USA) to remove iron, and EDTAwas added (10mM final concentration) to bind remaining iron.Iron limitation cultures were handled in a clean room to pre-vent contamination, and seawater was refreshed every 7–10days. After the experiments, 10 nM iron (final concentration)was added to the iron-limited cultures, and growth and fluo-rescence parameters were measured 4 days after iron additionto test whether iron was the limiting factor.

Growth under medium irradiance was monitored for 10days before SSI exposure. Medium irradiance acclimatedC. brevis was used for low and high irradiance and nutrientlimitation experiments. After the transfer to low and high irra-diance, growth was followed for 16 and 10 days, respectively, toaccumulate sufficient biomass for the SSI experiments andother measurements. For the nutrient limitation experiments,cells were considered nutrient limited when exponentialgrowth stopped, that is, cell division was inhibited. At thispoint the SSI exposure experiments were performed. Growthinhibition was observed after 10 and 7 days under nitrate- and

EFFECTS OF PAR AND UV ON C. BREVIS 841

phosphate-limiting conditions, respectively. Cell numbers insilicate free medium were monitored for 4–5 days, and for 7days for iron limitation. Chlorophyll fluorescence measure-ments and pigment samples were taken from the cultures onthe same day as SSI exposure. For these parameters andgrowth rate measurements, three individually growing cul-tures were used as replicates.

Simulated surface irradiance exposure. Light was provided bya combination of two UVA 340 lamps (Q-panel, Cleveland,OH, USA) and a 250-W Philips MHN-TD powertone lamp(Philips, Aachen, Germany), resulting in 350 W �m�2 PAR,42 W �m�2 UVA, and 0.47 W �m�2 UVB, as was measuredwith a MACAM SR9910 double monochromator scanningspectroradiometer (Macam Photometrics, Livingston, UK)and a cosine sensor in air (Fig. 1). The PAR correspondedto 1580 mmol photons �m�2 � s�1 when measured with a LI-COR LI-250 light meter and a flat sensor. The purpose of thistreatment was to mimic the sunlight algae can experiencenear the water surface, that is, moderate UVB radiation incombination with a high UVA and PAR background. The al-gae were exposed from below in a polystyrene box with a(UV) transparent bottom from PMMA (Vink NV, Heist opden Berg, Netherlands). Inside the box, algae were placedeither on a WG 305 or a WG 335 filter (Schott, Mainz,Germany) to discriminate between UVB effects and thoseof PAR and UVA (Fig. 1). Using the WG 305 filter, biologi-cally effective UVB irradiance was 0.041 and 0.084 W �m�2

when weighted by the DNA damage action spectrum (Setlow1974) and the generalized plant damage action spectrum(Caldwell 1971), respectively (both normalized to 1 at300 nm). The water temperature of the algae was maintainedat 41 C by regularly adding ice to the box for the duration ofthe exposure and by cooling the lamps with a fan.

Viability during SSI exposure. In the first series of experi-ments, the algae were diluted and transferred to 1.5-mLquartz vessels (diameter 0.5 cm, length 7.5 cm, Boelen et al.1999) that were sealed with Parafilm. Twenty-one of thesevessels were prepared for each cultivation condition, whichwere exposed for 0, 1, 2, and 4 h to the two spectral condi-tions, with three replicates per condition per time point. Af-ter exposure, the cells were incubated with Sytox Greenviability stain (Molecular Probes, Eugene, OR, USA) (see be-low). In the second series of experiments, a higher cell con-centration was used and the cells were exposed to 4 h SSI inlarger quartz vessels (see below). In both experiments, un-exposed samples were used as controls.

Viability during sunlight exposure. During the third series,low (6 mmol photons �m�2 � s� 1) and high (220 mmol pho-tons �m�2 � s�1) irradiance acclimated cells were exposed to4 h of sunlight around noon in Groningen on a clear day(11:30 a.m. to 3:30 p.m. on 9 August 2004) in 1.5-mL quartzvessels. Viability samples were harvested after 2 and 4 h ofexposure. The algae were cooled to approximately 61 C in acryostat controlled water bath and were covered by Ultra-phan URUV farblos (Digefra GmbH, Munich, Germany),Folex 320 (Dreireich, Germany), and UBT 300 foil (Digefra)to discriminate between PAR, PARþUVA, andPARþUVAþUVB, respectively. This experiment was car-ried out with two replicate cultures.

Flow cytometry. Cell counts and viability estimates wereperformed on a Coulter XL-MCL flow cytometer (BeckmanCoulter, Miami, FL, USA) with a 15-mW argon ion laser (ex-citation at 488 nm). Forward scatter, side scatter (estimatesfor particle shape, size, and density), and red fluorescence(4660 nm, originating from chl a autofluorescence) signalswere used to separate C. brevis from other particles. For cellcounts, the sample volume was determined by taking theweight before and after measurement on an analytical bal-ance.

Viable and nonviable cells were distinguished and quanti-fied after incubating the sample with Sytox Green (10mLof 100 times diluted stock solution, Molecular Probes) for 1 hon ice in darkness (performed after Veldhuis et al. 2001).This dye selectively stains DNA of permeable cells, whichcan be detected as green fluorescence (520 � 15 nm) whenexcited at 488 nm. Cells with at least 20 times enhanced greenfluorescence after Sytox incubation were considered nonvia-ble. Earlier tests had shown that viable and nonviable (0.1%formalin fixed cells) C. brevis could be separated in this way.Membrane permeability has been acknowledged as a late ir-reversible stage before lyses (Kroemer et al. 1995, Naganuma1996). Our tests confirmed that Sytox staining detects deadcells, as the number of nonviable cells remained constantduring 24 h in dim light after high irradiance exposure ofC. brevis (i.e. cells do not recover from this condition; resultsnot shown).

Chlorophyll fluorescence after SSI exposure. Information onPSII efficiency was gathered during the second experimentalseries. The algae (1.35 � 108 cells in 15 mL) were transferredto a quartz cuvette (12 � 5 cm surface), resulting in an ap-proximately 4-mm layer. Two replicate cuvettes per irradi-ance condition were placed in the box (see above) andexposed to 4 h of SSI. Afterward, the algae were allowed torecover for 20 h under dim light (10 mmol pho-tons �m�2 � s�1) at 41 C, before fluorescence measurementswere performed (after Van Oijen et al. 2004). Algae weretransferred to a filter (GF/F, 113 mm diameter, Whatman,Maidstone, UK) by mild vacuum. Filtration was stopped be-fore the filter dried, and the filter was placed in a cooledseawater cuvette (51 C). Chlorophyll fluorescence parameterswere measured with a PAM 2000 pulse amplitude modulatedfluorometer (Walz, Effeltrich, Germany). Maximal quantumyield of PSII was determined after 5 min of dark acclimation,and afterward a rapid light curve was recorded (0 to430 mmol photons �m�2 � s�1, 30 s each) from the same sam-ple. Relative maximal photosynthetic electron transport rate(rETRmax) and saturating irradiance for electron transportwere calculated from the rapid light curve as described byVan Oijen et al. (2004). Maximal quantum yield is a measurefor photosynthetic efficiency, whereas ETRmax is indicativefor the activity of the Calvin cycle and alternative electronsinks such as the Mehler reaction (Bischof et al. 2002, Miyakeand Asada 2003). Fluorescence parameters of SSI exposedalgae were compared with untreated controls that wereplaced in dim light.

FIG. 1. Spectral irradiance in the PARþUVAþUVB andPARþUVA treatments during SSI exposure. Irradiance isshown on a logarithmic scale to highlight differences betweentreatments.

WILLEM H. VAN DE POLL ET AL.842

Pigments. Pigment samples (30–75 mL) were taken beforeSSI exposure. The samples were filtered through GF/F filters(Whatman), immediately frozen in liquid nitrogen, andstored at � 801 C. Filters were extracted in 90% acetone for48 h after freeze drying. The pigments were separated andquantified according to Kraay et al. (1992) by HPLC, whichconsisted of a Waters 2690 separation module, 996 photodi-ode array detector, and a C18 5 mm DeltaPak reversed-phasecolumn (Waters, Milford, MA, USA). Three replicates wereanalyzed for each condition. Pigment data were expressed aspg per cell.

Statistics. Differences between groups of data were testedfor significance using one-way analysis of variance or multi-ple analyses of variance. Post-hoc tests were performed tofurther specify differences. Differences were consideredsignificant at Po0.05.

RESULTS

Growth rates. The cultivation conditions resulted indifferent growth rates of C. brevis (Table 1). Highestgrowth rates were found under medium irradiancecultivation, whereas high and low irradiance inhibit-ed growth. Exponential growth was observed underlow, medium, and high irradiance, whereas growthrates declined after some time under nitrate- andphosphate-limited conditions. No growth was ob-served in silicate-free medium. Instead, cell numbersdeclined up to 21% over a 5-day period. Iron limita-tion also reduced growth rates. After iron addition,normal growth was observed in all cultures (resultsnot shown).

Viability during simulated surface exposure. Clustersof viable and nonviable cells were readily separatedby the Sytox-specific green fluorescence signal. Thegreen fluorescence was on average enhanced 200-fold in nonviable cells, raising the fluorescence levelfrom 0.2 in viable cells to approximately 100 fluores-cence units in nonviable cells (not shown).

Differences in the number of nonviable cells wereobserved before SSI exposure after cultivation undervarious conditions. After cultivation in high irradiance,10% of the cells were nonviable at the start of the firstexperiment, whereas lower numbers of nonviable cellswere found in the second and third experiments (Figs.2 and 4). Ten percent and 20% of silicate-limited cells

were nonviable before SSI exposure for the first andsecond experiment, respectively. Other cultivationconditions displayed lower levels of nonviable cells.

The cultivation conditions resulted in cells with dif-ferent sensitivity to SSI-induced viability loss (Figs. 2and 3). Because differences in viability between UVBexposure and UVB exclusion treatments were not ob-served (multiple analyses of variance, P 5 0.33), datawere pooled. Low irradiance cultivated cells were most

TABLE 1. Growth rates and photosynthetic parameters based on chl fluorescence of PSII from Chaetoceros brevis during threeirradiance and four nutrient limitation conditions.

Condition Growth (d–1) Fv/Fm rETRmax Ek

Medium light 0.56 � 0.03 0.534 � 0.018 45.9 � 8.0 91 � 15High light 0.44 � 0.01 0.447 � 0.027 45.2 � 3.2 106 � 17Low light 0.21 � 0.01 0.725 � 0.005 40.2 � 3.7 50 � 5N limitation 0.20 � 0.03a 0.498 � 0.005 31.6 � 3.6 65 � 21P limitation 0.12 � 0.02a 0.425 � 0.016 22.8 � 6.0 77 � 11Si limitation 0b 0.436 � 0.023 16.6 � 0.51 34 � 2Fe limitation 0.17 � 0.085 0.251 � 0.020 8.32 � 2.5 30 � 12

Values are means � SD shown for three replicates. Ek, irradiance where electron transport saturates; Fv/Fm, maximal quantumyield.

aNonexponential growth, growth rate over the last time interval.bCell numbers declined.

FIG. 2. First experimental series. Viability loss of Chaetocerosbrevis (cells with Sytox staining, expressed as a percentage of totalcells) during SSI after cultivation under several irradiance (A)and nutrient limitation conditions (B). (A) Low (triangle), medi-um (square), and high (diamond) irradiance. (B) Nitrate (dia-mond), phosphate (triangle), silicate (hexagon), and iron (circle)limitation. Each data point represents the mean and SD of sixreplicates (PARþUVA and PARþUVAþUVB pooled), exceptthe control (n 5 3).

EFFECTS OF PAR AND UV ON C. BREVIS 843

sensitive. A significantly increased number of nonvia-ble cells was detected after 1 h of SSI (45% of all cells),whereas the entire population was nonviable after 2 h.Similarly, all cells were nonviable after 4 h of SSI in thesecond experiment that was performed at a higher celldensity (Fig. 3). Medium irradiance grown cells wereless sensitive than low irradiance grown cells. For theformer, increases in nonviable cells were not significantafter 1 h of SSI, but nonviable cells comprised 16% and100% of the examined cells after 2 and 4 h of SSI ex-posure, respectively. When performed at a higher celldensity in the second experiment, 30% of the cellswere nonviable after 4 h of SSI. No significant increases

in nonviable cells were observed for high irradianceacclimated cells in either experiment. Similarly, no sig-nificant increases in nonviable cells were observed afterSSI exposure of cells cultivated under nitrate, phos-phate, and silicate limitation in both experiments.Iron-limited cultures showed increased numbers ofnonviable cells after 2 h, whereas on average 65% ofthe cells were nonviable after 4 h of SSI in the first ex-periment. High variability was observed among repli-cates for viability data of iron-limited cultures, with twocultures having a significantly higher number of non-viable cells (84%) after 4 h of SSI than the third rep-licate (32%), when comparing pooled data for

FIG. 3. Second experimentalseries. (A and B) Viability loss ofChaetoceros brevis after 4 h of SSIafter growth under low, medi-um, and high irradiance (A) andphosphate (P-lim), nitrate (N-lim), silicate (Si-lim), and iron(Fe-lim) limitation (B). Meanand SD are shown for two rep-licates of untreated controls,PARþUVA, and PARþUVAþUVB exposed samples. (C andD) Ratio between diadino- anddiatoxanthin (DDþDT) and chla and fucoxanthin (Chl aþFuco) as an indication for pro-tective relative to light-harvest-ing pigments in cultures grownunder low, medium, and highirradiance (C) and phosphate (P-lim), nitrate (N-lim), silicate (Si-lim), and iron (Fe-lim) limitation(D). Mean and SD are shown forthree replicates. (E and F) Max-imal quantum yield of PSIIafter SSI exposure followed by20 h of recovery in dim light(expressed as a percentage ofuntreated controls of each con-dition) in cultures grown underlow, medium, and high irradi-ance (E) and phosphate (P-lim),nitrate (N-lim), silicate (Si-lim),and iron (Fe-lim) limitation (F).Mean and SD are shown for tworeplicates; bars indicated withasterisks represent unreliablemeasurements due to low fluo-rescence.

WILLEM H. VAN DE POLL ET AL.844

PARþUVA and PARþUVAþUVB exposed cells, re-spectively. However, no significant increases in nonvi-able cells were observed after 4 h in the secondexperiments.

Viability during sunlight exposure. Two hours of sun-light caused significant viability loss in low irradianceacclimated cells that were exposed to PAR (5% non-

viable) and PARþUV radiation (35% nonviable,Fig. 4). Viability loss was not significantly differentfor PARþUVA and PARþUVAþUVB exposedcells. More than 97% of low irradiance acclimatedcells were nonviable after 4 h of sunlight. No signif-icant increase in nonviable cells was observed in highirradiance acclimated cells after 2 and 4 h of sunlightexposure.

Other flow cytometry parameters. Red fluorescence asmeasured by flow cytometry was significantly differ-ent for cells from all cultivation conditions (Table 2).Red fluorescence decreased with increasing irradi-ance and further decreased in phosphate-, nitrate-,and iron-limited cultures. Silicate limitation caused adoubling of red fluorescence per cell compared withmedium light grown nutrient-replete cells. The SSItreatment initiated a rapid decline in red fluores-cence for cells from all cultivation conditions. Thedecline in red fluorescence was not different forPARþUVAþUVB and PARþUVA exposed cells(not shown).

Forward scatter and side scatter showed little varia-tion among irradiance and nutrient conditions, withthe exception of silicate limitation. Here, scatter pa-rameters doubled compared with silicate-replete con-ditions. Cells that were classified nonviable after Sytoxstaining also exhibited a marked (on average 38%)decline of the forward scatter signal.

Pigments. Cultivation conditions caused significantdifferences in the pigment composition of C. brevis.When compared with growth under medium irradi-ance (Table 3), the light-harvesting pigments chl a,chl c2, and fucoxanthin increased during low irradi-ance cultivation ( � 200% of medium irradiance),whereas chl c2 and fucoxanthin were significantlylower after high irradiance cultivation ( � 50% ofmedium irradiance). The cellular diadino-þdi-atoxanthin pool increased significantly duringgrowth under high irradiance (230% of medium ir-radiance) but was not different from medium lightafter low light cultivation. Furthermore, 52% of thediadino-þdiatoxanthin pool was de-epoxidized dur-ing high irradiance cultivation, compared with

FIG. 4. Third experimental series. Viability loss of after 2 and4 h of sunlight in high (A) and low (B) irradiance acclimatedChaetoceros brevis when exposed to PAR, PARþUVA, andPARþUVAþUVB. Control samples are shown at t 5 0 h.Mean and SD are shown for two replicates.

TABLE 2. Flow cytometry parameters for medium light (ML), low light (LL), and high light (HL) grown and nutrient-limitedChaetoceros brevis before and after 4 h of SSI exposure.

ML LL HL P(-) N(-) Si(-) Fe(-)

Forward scatterBefore 43 � 3 38 � 3 44 � 6 46 � 3 43 � 1 84 � 4 42 � 124 h SSI 25 � 6 25 � 1 45 � 1 45 � 1 43 � 1 80 � 5 22 � 6

Side scatterBefore 5.4 � 0.5 4.9 � 0.5 6.7 � 1 7.9 � 0.3 7.2 � 0.2 14 � 0.4 8.1 � 44 h SSI 4.0 � 0.7 4.8 � 0.1 6.5 � 0.2 7.4 � 1.5 7.1 � 0.3 13.4 � 2 6.6 � 1

Red fluorescenceBefore 40 � 0.2 65 � 5 25 � 4 23 � 0.6 17 � 1 68 � 3 36 � 64 h SSI 2.5 � 0.3 2.5 � 0.3 7.4 � 0.9 5.4 � 1 4.3 � 0.3 16 � 2 5.4 � 4

Forward scatter and side scatter give information on particle size, volume, density, and shape, whereas red fluorescence originatesfrom cellular chl. Means � SD are shown for data from the first experiment. Data from UVB exposed and unexposed cells werepooled. Phosphate, P(-); nitrate, N(-); silicate, Si(-); and iron limitation, Fe(-).

EFFECTS OF PAR AND UV ON C. BREVIS 845

approximately 1.7% for medium and low irradiancegrown cells. When compared with nutrient-repleteconditions under medium irradiance, nitrate- andphosphate-limited cells contained significantly lesslight-harvesting chl a, chl c2, and fucoxanthin( � 45% of medium light), whereas changes in cellu-lar diadino-þdiatoxanthin were not significant. Cel-lular chl a and chl c2 from silicate and iron-limitedcultures were not different from nutrient-repleteconditions, but fucoxanthin was significantly lowerunder iron limitation (71% of medium light). Cellulardiadino-þdiatoxanthin increased significantly undersilicate limitation (228%), and 18% was de-epoxi-dized. The increase in diadino-þdiatoxanthin wasnot significant for iron-limited cultures.

The changes in pigment composition resulted in asignificant change in the ratio between pigments with aprotective function (diadino-þdiatoxanthin) and light-harvesting pigments (chl aþ fucoxanthin) (Fig. 3). Thisratio decreased in low irradiance grown cells (�30% ofmedium irradiance) and increased in high irradiancegrown cells (320% of medium irradiance). When com-pared with medium irradiance grown cells, nutrientlimitation caused a significant increase in this ratio( � 185% of medium irradiance).

Chlorophyll fluorescence. Maximal quantum yield ofPSII (Fv/Fm) significantly decreased with growth un-der increasing irradiance, whereas differences inrETRmax were not significant (Table 1). Photosynthe-sis of low irradiance grown cells saturated at a lowerirradiance than that of medium and high irradiancegrown cells (50 vs. 97 mmol photons �m� 2 � s� 1, re-spectively). Differences in Fv/Fm and rETRmax werenot significant between nitrate-limited and -repleteconditions, whereas these parameters were signifi-cantly reduced under all other nutrient-limited con-ditions. Iron limitation caused the most pronounceddecreases in Fv/Fm and rETRmax and photosyntheticelectron transport saturated at low irradiance(30 mmol photons �m�2 � s� 1) when compared withnutrient-replete cells. After iron addition to iron-lim-ited cultures, fluorescence parameters became iden-tical to those of medium irradiance grown cells(results not shown).

After SSI exposure, followed by 20 h in dim light,low and medium irradiance cultivated cells showedextremely low fluorescence values compared with the

other samples (Fig. 3). After 20 h of dim light, Fv/Fm ofSSI-exposed high irradiance and nitrate-limited cul-tures was significantly lower than that of untreatedcontrols for these conditions (88% and 65% of control,respectively), whereas this was not significant for otherconditions (Fig. 3). Relative ETRmax was not signifi-cantly different from the untreated controls for anycondition (not shown). Significant differences in Fv/Fm

and rETRmax between PARþUVAþUVB andPARþUVA exposed cells were not observed after20 h of recovery (Fig. 3).

DISCUSSION

Here we demonstrated that surface irradiance ex-posure can have a pronounced effect on the Antarcticdiatom C. brevis. Apart from reversible inhibition ofphotosynthesis, surface irradiance exposure potential-ly caused rapid viability loss. Furthermore, our exper-iments showed that irradiance and nutrient conditionsmediate differences in sensitivity to near-surface irra-diance exposure in this small diatom. Acclimation tolow irradiance reduced survival during SSI, whereassurvival was enhanced by nutrient limitation relative tonutrient-replete conditions. Viability loss was probablymediated by photo-induced oxidative stress that wasimposed by the sudden transfer to SSI or sunlight.Over-reduction of the photosynthetic electron trans-port chain by high irradiance promotes the formationof super oxide, which can be transformed into a hy-droxyl radical via hydrogen peroxide in the (Haber-Weiss) Fenton reaction. These radicals can initiate alipid peroxidation chain reaction that can cause loss ofmembrane integrity, which is a characteristic of non-viable cells (Dix and Aikens 1993). Specifically, poly-unsaturated fatty acids that are abundant in thylakoidmembranes of low irradiance acclimated phytoplank-ton are vulnerable to lipid peroxidation (Thompsonet al. 1990, Hideg and Vass 1996, Mock and Kroon2002a,b). De-epoxidation of diadinoxanthin can pre-vent over-reduction of the photosynthetic electrontransport chain, because this enhances energy dissipa-tion as heat in the light-harvesting antennae. Thisprocess constitutes a general feature of photoprotec-tion in diatoms, comparable with the violaxanthin-antheraxanthin-zeaxanthin cycle in higher plants(Olaizola et al. 1994, Lavaud et al. 2004). In agree-

TABLE 3. Pigment composition of Chaetoceros brevis, expressed as pg per cell.

Condition Chl a Chl c2 Fucoxanthin DiadinoþDiato Diato (%)

Medium light 0.117 � 0.016 0.035 � 0.004 0.091 � 0.013 0.031 � 0.006 1.65 � 0.31High light 0.099 � 0.017 0.020 � 0.003 0.051 � 0.007 0.071 � 0.015 52.23 � 0.49Low light 0.298 � 0.024 0.065 � 0.004 0.200 � 0.013 0.021 � 0.002 1.75 � 0.38N limitation 0.051 � 0.009 0.013 � 0.002 0.028 � 0.004 0.022 � 0.005 6.21 � 2.43P limitation 0.070 � 0.014 0.003 � 0.001 0.044 � 0.012 0.031 � 0.007 2.75 � 1.77Si limitation 0.110 � 0.009 0.029 � 0.016 0.081 � 0.032 0.065 � 0.029 19.46 � 5.00Fe limitation 0.124 � 0.012 0.023 � 0.002 0.066 � 0.007 0.039 � 0.006 0.59 � 1.00

Means � SD are shown for three replicates. The last column shows diatoxanthin as a percentage of the total diadino–diatoxanthinpool.

WILLEM H. VAN DE POLL ET AL.846

ment with observations for other diatoms, de-epoxi-dation of diadinoxanthin to diatoxanthin was observedwithin minutes during SSI exposure (not shown),which coincided with a sharp decline of maximal quan-tum yield of PSII ( � 2% of control, not shown). Inaddition, a marked decline in cellular red fluorescencewas observed, irrespective of the cultivation conditions.This may indicate quenching of chl a fluorescence bydiatoxanthin, but also chloroplast contraction, PSII in-activation, or cellular loss of chl a by oxidative stress ora combination of these, making the interpretation ofthis flow cytometer signal difficult (Neale et al. 1989,Demers et al. 1991).

Cultivation under different irradiance conditionscaused changes in pigmentation that affected the ratiobetween protective and light-harvesting pigments. Be-cause C. brevis was cultivated in a batch regime, chang-es in cell number inevitably influenced the irradiancein the culture over time. Therefore, the irradiance re-ceived by the algae that were used for SSI exposurewas probably lower than that stated in Materials andMethods. Low irradiance grown cells contained morethan twice the amount of chl a and fucoxanthin anddisplayed a 36% increased maximal quantum yield ofPSII compared with medium irradiance grown cells,demonstrating a highly expanded light-harvesting ca-pacity and photosynthetic efficiency. However, cellularconcentrations of protective pigments remained un-changed. Not surprisingly, low irradiance acclimatedcells were extremely sensitive to SSI and sunlight.Nonviable cells were observed within 1–2 h, showingthat photodestruction of these cells proceeds at highrates at near surface irradiance. In contrast, high irra-diance acclimation enhanced the resistance to surfaceirradiance, and a 4-h exposure did not increase thenumber of nonviable cells. Lower sensitivity corre-sponded to a doubled cellular diadino-þdiatoxanthinpool and a reduced light-harvesting pigment concen-tration, causing a highly increased ratio betweenprotective and light-harvesting pigments.

Nutrient limitation reduced growth, maximal quan-tum yield, and rETRmax, regardless of the limiting nu-trient. Furthermore, nutrient limitation reducedsensitivity to photo-induced viability loss in C. brevis.All nutrient-limited cells shared an increased ratio be-tween protective and light-harvesting pigments. Dur-ing nitrate, phosphate, and iron limitation, this wascaused by a reduction in light-harvesting pigments,whereas cellular concentrations of protective pigmentsremained relatively unchanged, underlining their im-portance for survival of near-surface irradiance (Lavaudet al. 2002). Differences in pigmentation and photo-synthetic responses were observed between the specificnutrient limitation conditions. In iron-limited cells, thereduction in maximal quantum yield did not coincidewith a large reduction in light-harvesting pigments, aswas observed during phosphate and nitrate limitation.This agrees with previous observations for iron-limitedC. brevis and Phaeocystis sp., where minor reductionswere observed under saturating light conditions (Van

Leeuwe and Stefels 1998, Van Oijen et al. 2004). How-ever, the pronounced reduction in growth ratesshowed that C. brevis was severely iron limited in com-parison with other studies (Timmermans et al.2001a,b). A subtle increase in the ratio between pro-tective- and light-harvesting pigments significantly in-creased resistance to photo-induced loss of viabilityduring iron limitation. The degree of viability loss ofiron-limited cells was different for the two experimen-tal series. This may be related to differences in cellconcentration during SSI exposure. Presumably, ahigher cell concentration increased self-shading andconsequently decreased individual cell exposure andphoto-induced viability loss in the second experimen-tal series. A similar reduction was observed for medi-um irradiance acclimated cells when the experimentwas performed at a higher cell concentration.

Silicate limitation does not directly affect photosyn-thesis but limits the ability of diatoms to construct newfrustules. In anticipation of division, the cells double inorganelles and size, as was indicated by the scatter sig-nals from the flow cytometer. Red fluorescence alsodoubled, in contrast to cellular chl a content, which wasnot different from normal cells. We presume that thisphenomenon was caused by a reduced package effect(self-shading of pigments) of chl a, after being dividedamong the newly synthesized chloroplasts (Greeneet al. 1991). In addition, this may explain the increasedconcentration of protective pigments. A reduced pack-age effect of chl a increases the relative light absorptionand may require active photoprotection by de-epoxi-dation of diadinoxanthin. Growth arrest due to silicatelimitation appeared to be stressful for C. brevis, because10%–20% of the cells were nonviable after 4–5 days insilicate-free medium. However, this viability loss wasnot related to irradiance stress because SSI exposuredid not increase the number of nonviable cells. Rather,this may reflect the absolute requirement for silicate ofdiatoms, although we cannot completely exclude thatviability loss was caused by the centrifugation of thecells.

In the current experiments, UVB exposure in ad-dition to UVA and PAR did not change the resultscompared with treatments where UVB was omitted.Effects of UVA and PAR were not considered sepa-rately during SSI exposure. However, experiments insunlight showed that viability loss was enhanced byUVA radiation in low irradiance acclimated cells,whereas no additional UVB effect was observed. Thisdemonstrates that UVA radiation can have a pro-nounced effect on viability, as was previously foundfor photosynthesis (Turcsanyi and Vass 2000). No sig-nificant UVB-related changes were observed in thenumber of nonviable cells and in efficiency parametersof PSII after 20 h of recovery. Therefore, UVB did notinteract with the effects of PAR and UVA in C. brevis.

This implies that low irradiance acclimated andnutrient-limited cells did not have increased UVBsensitivity compared with high irradiance acclimatedcells in terms of viability loss. This contradicts some

EFFECTS OF PAR AND UV ON C. BREVIS 847

previous studies on photosynthesis, which showed lowlight acclimated and nitrate-limited cells were moresensitive to UV exposure (Cullen and Lesser 1991,Neale et al. 1994). Both stimulation and reversal of de-epoxidation of diadinoxanthin have been reported forUVB-exposed Phaeodactylum tricornutum (Goss et al.1999, Mewes and Richter 2002). However, these ex-periments were conducted at high UVB irradianceswith a relatively low UVA and PAR background. In ourexperiments, no evidence was obtained for increasedlight sensitivity as a result of UVB exposure, and weassume that deviations from previous results can beattributed to differences in UV radiation and PAR.During SSI and sunlight exposure, photosynthesis wascompletely impaired by thermal energy dissipation orROS-induced damage, and these processes wereclearly dominated by PAR and UVA.

Chaetoceros brevis does not produce significantamounts of MAAs, as the characteristic absorptionpeak was not found in methanol extracts from thisspecies (results not shown). Nitrate limitation increasedUVB sensitivity of photosynthesis of two dinoflagel-lates, probably by reduced MAA production (Litchmanet al. 2002). On the other hand, no interaction be-tween nitrate limitation and UVB exposure was foundfor growth of Phaeodactylum tricornutum (Behrenfeldet al. 1994). Apparently, C. brevis has a relatively highresistance to periodic UVB exposure without the pro-tection of MAAs and can efficiently repair UVB-in-duced DNA damage (unpublished data).

We have shown that photo-induced viability lossmay occur in the Antarctic diatom C. brevis when rel-atively low irradiance acclimated cells are suddenly ex-posed to near surface irradiance. Although the abrupttransition from very low to 200-fold increased irradi-ance is arguably not natural, wind-induced verticalmixing can bring about changes from 1% to surfaceirradiance in 30 min, which leaves little time for accli-mation (Denman and Gargett 1983). Consequently,dramatic irradiance changes are not unusual forphytoplankton. Veldhuis et al. (2001) demonstratedthat up to 50% of a population of small eukaryotes insamples from the North Atlantic was nonviable in theupper part of the photic zone, in contrast to samplesfrom greater depths where the nonviable fraction com-prised only 10% of the population. Further experi-ments are needed to determine whether naturalphytoplankton assemblages are sensitive to photo-in-duced viability loss. Obviously, the irradiance exposureof individual cells depends on the density (self-shad-ing) of the algae, as was observed in our experiments,and on other factors that influence the attenuation ofirradiance in water (Barros et al. 2003). Weather con-ditions, ice cover, season, the diurnal cycle, and watertransport imposed by mixing determines the rate ofchange in irradiance experienced by the algae. Thesedynamic irradiance conditions are in sharp contrast tothe light:dark cycle that was used in our experiments.However, it was shown previously that there was littlevariation in pigment composition of C. brevis when

grown under different dynamic irradiance regimesthat mimic mixing of the water column (Van Leeuweet al. 2005). Apparently, pigment composition was notregulated by peak irradiances but by the average irra-diance received by the algae. It appears that pigmen-tation is the main determinant in photo-inducedviability loss in C. brevis, with a central role for the dia-dino-þdiatoxanthin pool. A high ratio of protectiverelative to light-harvesting pigments allowed high ir-radiance acclimated and most nutrient-limited cells tosurvive prolonged near-surface irradiance exposure,without apparent damage after 20 h of recovery in dimlight. Because phytoplankton species have diverse pig-ment composition, effects of near-surface exposuremay be species specific. Therefore, photo-inducedviability loss may affect species composition andabundance in the upper part of the photic zone.

We thank Han van der Strate and Monika Tyl for their helpwith silicate limitation experiments.

Agustı, S. & Duarte, C. 2000. Experimental induction of a largephytoplankton bloom in Antarctic coastal waters. Mar. Ecol.Prog. Ser. 206:73–85.

Asada, K. & Takahashi, M. 1987. Production and scavenging ofactive oxygen in photosynthesis. In Kyle, D. J., Osmond, C. &Arntzen, C. J. [Eds.] Photoinhibition. Elsevier, New York,pp. 227–97.

Barros, M. P., Pedersen, M., Colepicolo, P. & Snoeijs, P. 2003. Self-shading protects phytoplankton communities against H2O2-induced oxidative damage. Aquat. Microb. Ecol. 30:275–82.

Behrenfeld, M. J., Lee II, H. & Small, L. F. 1994. Interactions be-tween nutritional status and long-term responses to ultravio-let-B radiation stress in a marine diatom. Mar. Biol. 118:523–30.

Bischof, K., Peralta, G., Krabs, G., van de Poll, W. H., Wiencke, C.,Perez-Llorens, J. L. & Breeman, A. M. 2002. Effects of solarUV-B radiation on canopy formation of natural Ulva commu-nities from Southern Spain. J. Exp. Bot. 53:2411–21.

Boelen, P., Obernosterer, I., Vink, A. A. & Buma, A. G. J. 1999.Attenuation of biologically effective UV radiation in tropicalAtlantic waters measured with a biochemical DNA dosimeter.Photochem. Photobiol. 69:34–40.

Boucher, N. P. & Prezelin, B. B. 1996. An in situ biological weight-ing function for UV inhibition of phytoplankton carbon fixa-tion in the Southern Ocean. Mar. Ecol. Prog. Ser. 144:223–36.

Buma, A. G. J., Boelen, P. & Jeffrey, W. H. 2003. UVR-inducedDNA damage in aquatic organisms. In Helbling, W. & Zag-arese, H. [Eds.] UV Effects in Aquatic Organisms and Ecosystems.The Royal Society of Chemistry, Cambridge, UK, pp. 291–320.

Buma, A. G. J., de Boer, M. K. & Boelen, P. 2001. Depth distribu-tions of DNA damage in Antarctic marine phyto- and bacte-rioplankton exposed to summertime UV radiation. J. Phycol.37:200–8.

Caldwell, M. M. 1971. Solar UV irradiation and the growth anddevelopment of higher plants. In Giese, A. C. [Eds.] Photophys-iology. Vol. 6.. Academic Press, New York, pp. 131–77.

Cullen, J. J. & Lesser, M. P. 1991. Inhibition of photosynthesis byultraviolet radiation as a function of dose and dosage rate:results for a marine diatom. Mar. Biol. 111:183–90.

Cullen, J. J. & MacIntyre, G. J. 1991. Behavior, physiology and theniche of depth-regulating phytoplankton. In Anderson, D. M.,Cembella, A. D. & Hallegraf, G. M. [Eds.] The PhysiologicalEcology of Harmful Algal Blooms. Springer-Verlag, Heidelberg,pp. 559–80.

WILLEM H. VAN DE POLL ET AL.848

Cullen, J. J., Neale, P. J. & Lesser, P. 1992. Biological weightingfunction for the inhibition of phytoplankton photosynthesis byultraviolet radiation. Science 258:646–50.

De Baar, H. J. W., Buma, A. G. J., Nolting, R. F., Cadee, G. C.,Jacques, G. & Trequer, P. 1990. On iron limitation of theSouthern Ocean: experimental observations in the Weddeland Scotia seas. Mar. Ecol. Prog. Ser. 65:105–22.

De Baar, H. J. W., de Jong, J. T. M., Bakker, D. C. E., Loscher, B.M., Veth, C., Bathmann, U. & Smetacek, V. 1995. Importanceof iron for plankton blooms and carbon dioxide draw down inthe Southern Ocean. Nature 373:412–5.

Demers, S., Roy, S., Gagnon, R. & Vignault, C. 1991. Rapid light-induced changes in cell fluorescence and in xanthophyll-cyclepigments of Alexandrium excavata (Dinophyceae) and Thalassiosirapseudonana (Bacillariophyceae): a photo-protection mechanism.Mar. Ecol. Prog. Ser. 76:185–93.

Denman, K. L. & Gargett, A. E. 1983. Time and space scales ofvertical mixing and advection of phytoplankton in the upperocean. Limnol. Oeanogr. 28:801–15.

Dix, T. A. & Aikens, J. 1993. Mechanisms and biological relevanceof lipid peroxidation initiation. Chem. Res. Toxicol. 6:2–18.

Falkowski, P. G. & LaRoche, J. 1991. Acclimation to spectral irra-diance in algae. J. Phycol. 27:8–14.

Geider, R. J. & LaRoche, J. 1994. The role of iron in phytoplanktonphotosynthesis, and the potential for iron-limitation of prima-ry productivity in the sea. Photosynth. Res. 39:275–301.

Geider, R. J., LaRoche, J., Greene, R.M & Olaizola, M. 1993. Re-sponse of the photosynthetic apparatus of Phaeodactylumtricornutum (Bacillariophyceae) to nitrate, phosphate, or ironstarvation. J. Phycol. 29:755–66.

Goss, R., Mewes, H. & Wilhelm, C. 1999. Stimulation of the di-adinoxanthin cycle by UV-B radiation in the diatom.Phaeodactylum tricornutum. Photosynth. Res. 59:73–80.

Greene, R. M., Geider, R. J. & Falkowski, P. G. 1991. Effect of ironlimitation on photosynthesis in a marine diatom. Limnol.Oceanogr. 36:1772–82.

Helbling, E. W., Chalker, B. E., Dunlap, W. C., Holm-Hansen, O. &Villafane, V. E. 1996. Photoacclimation of Antarctic marinediatoms to solar ultraviolet radiation. Mar. Biol. 204:85–101.

Hideg, E. & Vass, I. 1996. UV-B induced free radical production inplant leaves and isolated thylacoid membranes. Plant Sci.115:251–60.

Karentz, D., Cleaver, J. E. & Mitchell, D. L. 1991a. Cell survivalcharacteristics and molecular responses of Antarctic phyto-plankton to ultraviolet-B radiation. J. Phycol. 27:326–41.

Karentz, D., McEuen, F. S., Land, M. C. & Dunlap, W. C. 1991b.Survey of mycosporine-like amino acids compounds in Ant-arctic marine organisms: potential protection from ultravioletexposure. Mar. Biol. 108:157–66.

Kraay, G. W., Zapata, M. & Veldhuis, M. J. W. 1992. Separation ofchlorophylls c1, c2 and c3 of marine phytoplankton by re-versed-phase-C18-high-performance-liquid-chromatography.J. Phycol. 28:708–12.

Kroemer, G., Petit, P., Zamzami, N., Vayssiere, J. L. & Mignote, B.1995. The biochemistry of programmed cell death. FASEB J.9:1277–87.

Lavaud, J., Rousseau, B. & Etienne, A. L. 2004. General features ofphotoprotection by energy dissipation in planktonic diatoms(Bacillariophyceae). J. Phycol. 40:130–7.

Lavaud, J., Rousseau, B., van Gorkom, H. J. & Etienne, A. L. 2002.Influence of the diadinoxanthin pool size on photoprotectionin the marine planktonic diatom Phaeodactilum tricornutum.Plant Physiol. 129:1398–406.

Lesser, M. P. 1996a. Acclimation of phytoplankton to UV-Bradiation: oxidative stress and photoinhibition of photosyn-thesis are not prevented by UV-absorbing compounds in thedinoflagellate Prorocentrum micans. Mar. Ecol. Prog. Ser. 132:287–97.

Lesser, M. P. 1996b. Elevated temperatures and ultraviolet radia-tion cause oxidative stress and inhibit photosynthesis in sym-biotic dinoflagellates. Limnol. Oceanogr. 41:271–83.

Lesser, M. P., Cullen, J. J. & Neale, P. J. 1994. Carbon uptake in amarine diatom during acute exposure to ultraviolet B radia-

tion: relative importance of damage and repair. J. Phycol.30:183–92.

Lesser, M. P., Neale, P. J. & Cullen, J. J. 1996. Acclimation of Ant-arctic phytoplankton to ultraviolet radiation: ultraviolet-ab-sorbing compounds and carbon fixation. Mol. Mar. Biol.Biotechnol. 5:314–25.

Litchman, E., Neale, P. J. & Banaszak, A. T. 2002. Increased sen-sitivity to ultraviolet radiation in nitrogen-limited dinoflagel-lates: Photoprotection and repair. Limnol. Oceanogr. 47:86–94.

Malanga, G., Calmanovici, G. & Puntarulo, S. 1997. Oxidativedamage to chloroplasts from Chlorella vulgaris exposed toultraviolet-B radiation. Physiol. Plant. 101:455–62.

Mewes, H. & Richter, M. 2002. Supplementary ultraviolet-B radi-ation induces a rapid reversal of the diadinoxanthin cycle inthe strong light-exposed diatom Phaeodactylum tricornutum.Plant Physiol. 130:1527–35.

Miyake, C. & Asada, K. 2003. The water-water cycle. In Larkum, A.W. D., Dougles, S. E. & Raven, J. A. [Eds.] Advances in Photo-synthesis and Respiration: Photosynthesis in Algae. Kluwer Aca-demic Publisher, The Netherlands, pp. 183–204.

Mock, T. & Kroon, B. M. A. 2002a. Photosynthetic energy conver-sion under extreme conditions. I. Important role of lipids asstructural modulators and energy sink under N-limitedgrowth in Antarctic sea ice diatoms. Phytochemistry 61:41–51.

Mock, T. & Kroon, B. M. A. 2002b. Photosynthetic energy conver-sion under extreme conditions. II. The significance of lipidsunder light limited growth in Antarctic sea ice diatoms. Phyto-chemistry 61:53–60.

Naganuma, T. 1996. Differential enumeration of intact and dam-aged planktonic bacteria based on cell membrane integrity.J. Aquat. Ecosyst. Health 5:217–22.

Neale, P. J., Banaszak, A. T. & Jarriel, C. R. 1998. Ultraviolet sun-screens in Gymnodinium sanguineum (Dinophyceae): my-cosporine-like amino acids protect against inhibition ofphotosynthesis. J. Phycol. 34:928–38.

Neale, P. J., Cullen, J. J. & Yentsch, C. M. 1989. Bio-optical infer-ences from chlorophyll a fluorescence: What kind of fluores-cence is measured in flow cytometry? Limnol. Oceanogr.34:1739–48.

Neale, P. J., Lesser, M. P. & Cullen, J. J. 1994. Effects of ultravioletradiation on the photosynthesis of phytoplankton in the vicin-ity of McMurdo station, Antarctica. Antarct. Res. Ser. 62:125–42.

Olaizola, M., LaRoche, J., Kolber, Z. & Falkowski, P. G. 1994. Non-photochemical fluorescence quenching and the diadinoxan-thin cycle in a marine diatom. Photosynth. Res. 41:357–70.

Olaizola, M. & Yamamoto, H. Y. 1994. Short-term response of thediadinxanthin cycle and fluorescence yield to high irradiancein Cheatoceros muelleri (Bacilariophyceae). J. Phycol. 30:606–12.

Rijstenbil, J. W. 2001. Effects of periodic, low UVA radiation on cellcharacteristics and oxidative stress in the marine planktonicdiatom Ditylum brightwellii. Eur. J. Phycol. 36:1–8.

Rijstenbil, J. W., Coelho, S. M. & Eijsackers, M. 2000. A method forthe assessment of light-induced oxidative stress in embryos offucoid algae via confocal laserscan microscopy. Mar. Biol.137:763–74.

Rossa, M. M., de Oliveira, M. C., Okamoto, O. K., Lopes, P. F. &Colepicolo, P. 2002. Effects of visible light on superoxide di-smutase (SOD) activity in the red alga Gracilariopsis tenuifrons(Gracelariales, Rodophyta). J. Appl. Phycol. 14:151–7.

Salin, M. L. 1987. Toxic oxygen species and protective systems ofthe chloroplast. Physiol. Plant. 72:686–9.

Sarmiento, J. L., Gruber, N., Brzezinski, M. A. & Dunne, J. P. 2003.High latitude controls of thermocline nutrients and low lati-tude biological productivity. Nature 427:56–60.

Schofield, O., Kroon, B. M. A. & Prezelin, B. B. 1995. Impact ofultraviolet-B radiation on photosystem II activity and its rela-tionship to the inhibition of carbon fixation rates for Antarcticice algae communities. J. Phycol. 31:703–15.

Setlow, R. B. 1974. The wavelengths in sunlight effective in pro-ducing skin cancer: a theoretical analysis. Proc. Natl. Acad. Sci.USA 71:3363–6.

Shelly, K., Heraud, P. & Beardall, J. 2002. Nitrogen limitation inDunaliella tertiolecta (Chlorophyceae) leads to increased suscep-

EFFECTS OF PAR AND UV ON C. BREVIS 849

tibility to damage by UV-B radiation but also increased repaircapacity. J. Phycol. 38:713–20.

Smith, R. C., Prezelin, B. B., Baker, K. S., Bridigare, R. R., Bouc-her, N. P., Coley, T., Karentz, D., MacIntyre, S., Menzies, H. A.,Ondrusek, M., Wan, Z. & Waters, K. J. 1992. Ozone depletion:ultraviolet radiation and phytoplankton biology in Antarcticwaters. Science 255:952–9.

Thompson, P. A., Harrison, P. J. & Whyte, J. N. C. 1990. Influenceof irradiance on the fatty acid composition of phytoplankton.J. Phycol. 26:278–88.

Timmermans, K. R., Davey, M. S., van der Wacht, B., Snoek, J.,Geider, R. J., Veldhuis, M. J. W. & Gerringga, L. J. A. 2001a.Co-limitation by iron and light of Chaetoceros brevis, C. dichaetaand C. calcitrans (Bacillariophyceae). Mar. Ecol. Prog. Ser. 217:287–97.

Timmermans, K. R., Gerringa, L. J. A., de Baar, H. J. W., van derWagt, B., Veldhuis, M. J. W., de Jong, J. T. M. & Croot, P. L.2001b. Growth rates of large and small Southern Ocean dia-toms in relation to availability of iron in natural seawater.Limnol. Oceanogr. 46:260–6.

Trebst, A., Depka, B. & Hollander-Czytko, H. 2002. A specific rolefor tocopherol and chemical singlet oxygen quenchers in themaintenance of photosystem II structure and function inChlamydomonas reinhardtii. FEBS Lett. 516:156–60.

Turcsanyi, E. & Vass, I. 2000. Inhibition of photosynthetic electrontransport by UV-A radiation targets the photosystem II com-plex. Photochem. Photobiol. 72:513–20.

Van Leeuwe, M. A. & Stefels, J. 1998. Effects of iron and light stresson the biochemical composition of Antarctic Phaeocystis Sp.(Prymnesiophyceae). II. Pigment composition. J. Phycol. 34:496–503.

Van Leeuwe, M. A., van Sikkelerus, B., Gieskes, W. W. C. & Stefels,J. 2005. Taxon-specific differences in photoacclimation tofluctuating irradiance in an Antarctic diatom and a greenflagellate. Mar. Ecol. Prog. Ser. 288:9–19.

Van Oijen, T., Van Leeuwe, M. A., Gieskes, W. W. C. & de Baar, H.J. W. 2004. Effects of iron limitation on photosynthesis andcarbohydrate metabolism in the Antarctic diatom Chaetocerosbrevis (Bacillariophyceae). Eur. J. Phycol. 39:161–71.

Veldhuis, M. J. W. & Admiraal, W. 1987. The influence of phos-phate depletion on the growth and colony formation ofPhaeocystis pouchetii. Mar. Biol. 95:47–54.

Veldhuis, M. J. W., Kraay, G. W. & Timmermans, K. R. 2001. Celldeath in phytoplankton: correlation between changes in mem-brane permeability, photosynthetic activity, pigmentation andgrowth. Eur. J. Phycol. 36:167–77.

WMO Report No. 44. Updated 2002. Scientific assessment ofozone depletion: 1998. www.WMO.ch/indexflash.html

WILLEM H. VAN DE POLL ET AL.850