BIOLOGICAL WEIGHTING FUNCTIONS FOR UV INHIBITION OF PHOTOSYNTHESIS IN THE KELP LAMINARIA HYPERBOREA...

BIOLOGICAL WEIGHTING FUNCTIONS FOR UV INHIBITION OF PHOTOSYNTHESISIN THE KELP LAMINARIA HYPERBOREA (PHAEOPHYCEAE)1

Harlan L. Miller III 2,3

The University of Texas at Austin, Marine Science Institute, 750 Channel View Drive, Port Aransas, Texas 78373, USA

Patrick J. Neale

Smithsonian Environmental Research Center, P. O. Box 28, Edgewater, Maryland 21037, USA

and Kenneth H. Dunton

The University of Texas at Austin, Marine Science Institute, 750 Channel View Drive, Port Aransas, Texas 78373, USA

Different wavelengths of sunlight either drive orinhibit macroalgal production. Ultraviolet radiation(UVR) effectively disrupts photosynthesis, but sinceUVR is rapidly absorbed in coastal waters, macroal-gal photoinhibition and tolerance to UVR dependon the depth of attachment and acclimation state ofthe individual. The inhibition response to UVR isquantified with a biological weighting function(BWF), a spectrum of empirically derived weightsthat link irradiance at a specific wavelength to over-all biological effect. We determined BWFs for shal-low (0 m, mean low water [MLW]) and deep (10 m)Laminaria hyperborea (Gunnerus) Foslie collectedoff the island of Finnøy, Norway. For each replicatesporophyte, we concurrently measured both O2

evolution and 13C uptake in 48 different light treat-ments, which varied in UV spectral composition andirradiance. The relative shape of the kelp BWF wasmost similar to that of a land plant, and the abso-lute spectral weightings and sensitivity were typicallyless than phytoplankton, particularly in the ultra-violet radiation A (UVA) region. Differences inBWFs between O2 and 13C photosynthesis andbetween shallow (high light) and deep (low light)kelp were also most significant in the UVA. Becauseof its greater contribution to total incident irradi-ance, UVA was more important to daily loss of pro-duction in kelp than ultraviolet radiation B (UVB).Photosynthetic quotient (PQ) also decreased withincreased UVR stress, and the magnitude of PQdecline was greater in deepwater kelp. Significantly,BWFs assist in the comparison of biologicalresponses to experimental light sources versus insitu sunlight and are critical to quantifying kelpproduction in a changing irradiance environment.

Key index words: acclimation; biological weight-ing function; carbon-13; Laminaria hyperborea;

oxygen; ozone depletion; photoinhibition; photo-synthesis; photosynthetic quotient; UV radiation

Abbreviations: BWF, biological weighting function;DIC, dissolved inorganic carbon; DO, dissolvedoxygen; MAA, mycosporine-like amino acids;PAM, pulse-amplitude-modulated fluorescence;PCA, principal component analysis; PE, photosyn-thesis versus irradiance; SD, standard deviation;SE, standard error; UVA, ultraviolet radiation A;UVB, ultraviolet radiation B; UVC, ultravioletradiation C; UVR, ultraviolet radiation

Attenuation of downwelling sunlight through theatmosphere and ocean and the biological responseto UVR are two processes that must be well parame-terized to quantitatively assess whether solar UVradiation affects marine organisms. In phototrophs,photosynthesis and inhibition of photosynthesis areboth functions of irradiance (quantity) and spectraldistribution of sunlight (quality). Broadband PARdoes not necessarily characterize the quality of lightphotosynthetically utilizable by a photosyntheticorganism, particularly underwater. Similarly, broad-band measures of UVR (i.e., UVA and UVB) obfus-cate the wavelength dependency of UVphotoinhibition. The relationship between light andphotoinhibition is better quantified with a BWF: aset of spectral weights that accounts for the wave-length dependency of the photobiological processand properly scales the exposure spectra to theeffective biological response (Neale and Kieber2000).

In the simplest case, because an equal exposureto broadband UVB is more effective than UVA atinhibiting photosynthesis, a BWF would give agreater weight to UVB in inducing inhibition in fullspectrum sunlight. This simplified weighting func-tion is, however, inadequate for full solar spectrumor ozone depletion models because the generalweight assigned to broadband UVB does not

1Received 31 January 2008. Accepted 16 February 2009.2Present address: Algenol Biofuels Inc., www.algenolbiofuels.com3Author for correspondence: e-mail [email protected].

J. Phycol. 45, 571–584 (2009)� 2009 Phycological Society of AmericaDOI: 10.1111/j.1529-8817.2009.00694.x

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provide information on wavelength dependency ofinhibition within the UVB spectral range (i.e., lessstratospheric ozone allows more high-energy, short-wavelength photons within the UVB range to reachthe earth’s surface). A more sophisticated BWF canbe generated if a greater number of spectral treat-ments are included in the photosynthetic experi-ments. Spectral weights at 1 nm resolution can thenbe inferred using either nonlinear regression of anassumed exponential response function (Rundel1983) or statistically parameterized using one to sev-eral principal components that describe the spectralirradiance treatments (Cullen et al. 1992, Cullenand Neale 1997, Neale 2000).

BWF weights are defined under polychromaticlight treatments. This is an important distinctionbetween the BWF and a traditional action spectrumdetermined using monochromatic light (Jones andKok 1966, Caldwell 1971). In a BWF experiment,irradiance treatments are created with a series ofcutoff filters that selectively remove shorter wave-lengths of light, and the polychromatic treatmentsare designed to incorporate multiple light-regulatedphysiological processes. In kelp, for instance, bluelight enhances recovery and survival after UVC(253 nm) exposure [Han and Kain (Jones) 1992,1993]. More specifically, Karsten et al. (1998a) andlater Franklin et al. (2001) demonstrated that PAR,in particular blue light, and UVR interact to inducesynthesis of UV-absorbing mycosporine-like aminoacids in the red alga Chondrus crispus. Interactiveprocesses would not be reflected in spectral weight-ing functions based on monochromatic light, butthe BWF approach accounts for the net effect oflight and is more representative of natural condi-tions.

Understanding of the temporal response to UVexposure is fundamental to the predictive power ofthe BWF model. The time-dependency of UV-induced inhibition is evaluated with an exposureresponse curve (Coohill 1994). From these curves, itcan be determined whether the biological responseresults from cumulative radiant exposure or fromthe instantaneous exposure rate (Cullen and Lesser1991), that is, whether reciprocity is satisfied. If theeffect is essentially irreversible over the timescale ofinterest, reciprocity is upheld, and the response is afunction of cumulative UV exposure (J Æ m)2). If,however, organisms possess mechanisms that cancounteract UV damage, then the rate of photosyn-thesis under UV exposure declines until it reaches asteady state, reflecting a balance between damageand repair (Lesser et al. 1994). In this case, reci-procity fails, and photosynthetic inhibition is pro-portional to maximum exposure rate (mW Æ m)2)(Cullen et al. 1992). For example, an irradiance-dependent BWF was appropriate for temperate (i.e.,relatively high natural UV) phytoplankton (Banas-zak and Neale 2001), but Antarctic (i.e., relativelylow UV) phytoplankton show little short-term ability

to counteract UV inhibition, implying that inhibi-tion is a function of cumulative exposure (Nealeet al. 1998b).

Current understanding of the effect of UVR onseaweed genetics, physiology, and production isreviewed by Bischof et al. (2006). Seaweeds adaptto the prevailing light environment at their attach-ment depth, and the response to both high-lightand UVR stress is correlated with vertical distribu-tion (Larkum and Wood 1993, Dring et al.1996a,b, Franklin and Forster 1997, Hanelt et al.1997b, Karsten et al. 2001, Johannsson and Snoeijs2002, Bischof et al. 2006, Roleda et al. 2006). Ses-sile at their growing site, kelp (Laminariales, Pha-eophyta) individuals are restricted to specific lighthistories and radiant UV exposure. The rapidattenuation of UVR relative to PAR by seawater,typical of coastal waters with high concentrationsof dissolved organic matter (Tedetti and Sempere2006), protects kelps deeper in the water columnfrom high-intensity UV. Consequently, macroalgalsensitivity to UV exposure depends on attachmentdepth, and intertidal and upper subtidal seaweedshave more effective UV-counteracting mechanisms.In brown algae, phlorotannin production is induc-ible by UVR, and its role as a sunscreen isimplied (Pavia and Brock 2000, Swanson and Dru-ehl 2002, Fairhead et al. 2005). Also, photoaccli-mated algae employ efficient protein repair andantioxidant systems after UV damage has occurred(Vincent and Neale 2000, Aguilera et al. 2002,Hader et al. 2002). Thus, macroalgae have thecapacity to acclimate to changes in sunlight, andreciprocity is likely depth-dependent since deep-water algae receive little appreciable UVR.

Kelp populations from boreal Norway (north ofAlesund) were studied because of their excep-tional productivity along the Atlantic coast. Stand-ing crops of the dominant kelp, L. hyperborea,approach 40 kg fresh weight (fwt) Æ m)2 (Sjøtunet al. 1995). Warmer seawater temperatures alongNorwegian coastlines impede winter ice formation,and without winter ice scour, kelp grows into theupper subtidal where there is greater potential forUVR exposure. Kelp beds throughout the variouscoastal islands and fjords extend from the inter-tidal to 30 m (Sjøtun et al. 1995), and we hypo-thesize that individual L. hyperborea are wellacclimated to the quantity and quality of sunlightincident at their attachment depth.

The purpose of this study was to experimentallydetermine and compare BWFs for L. hyperborea col-lected from the eulittoral and from a subtidaldepth (10 m) without appreciable UVR (Eyvindand Højerslev 2001). Three separate experimentswere conducted. First, in order to correctly formu-late the shape of the PAR response in the BWFmodel, short-duration photosynthesis-PAR irradi-ance (PE) experiments were performed in an oxy-gen electrode chamber. Second, the temporal

572 HARLAN L. MILLER III ET AL.

component of BWF was addressed in an exposureresponse experiment with surface-collected kelpand was otherwise inferred for deeper-water kelp.Finally, the BWF was quantified from photosynthe-sis experiments, which varied in spectral treatmentand where we concurrently measured oxygen evo-lution and carbon uptake. Weightings were param-eterized using both irradiance-based andcumulative exposure models, and the spectralshapes of the BWF responses were then comparedbetween the two measures of photosynthesis andbetween kelp collection depths.

MATERIALS AND METHODS

Site description, kelp collection, and tissue preparation. Photo-synthetic experiments were conducted at a field laboratory onthe island of Finnøy, Norway, which was located <1 km from anextensive L. hyperborea kelp forest (Fig. 1). The populationextends subtidally from mean low water (MLW = 0 m) to�20 m into a tidal channel. BWF components were deter-mined experimentally during three field excursions to thearea. PAR-only photosynthetic experiments were performed inSeptember 1999 (results in Fig. S1 in the supplementarymaterial). The temporal aspect of the BWF (the exposureresponse curve) was investigated in October 2000, and the BWFwas determined in March ⁄ April 2001.

Adult kelp sporophytes with lamina >0.5 m in length wereharvested 1 d prior to photosynthetic experiments. Subtidalkelps were collected via SCUBA, and divers took care to removeindividuals with holdfasts intact to minimize stress prior toexperimentation. Kelps were kept in fine mesh collection bagsand placed in dark ice chests for the short trip back to the fieldlaboratory. Whole kelp thalli were stored overnight in under-water collection bags suspended in a nearby protected boatbasin.

Photosynthetic tissue samples excised at least 10 cm from thebasal, meristematic region of the lamina and 1 cm away fromblade margins were used in the three photosynthesis experi-ments. Samples were maintained under dim laboratory light(estimated 50 lmol photons Æ m)2 Æ s)1) in temperature-con-trolled seawater at the ambient seawater temperature of theseason. Margins of the cut kelp tissues initially producedmucilage, and we observed that the wound response ceased

within 1–2 h. The incubation bath was replaced frequently withfresh seawater.

Pigment extraction and thallus absorbance. Thallus opticalproperties were compared in kelp tissues collected from 0 mand 10 m depth. Living kelp blade samples were transported(<3 d) in a dark, temperature-controlled container to theSmithsonian Environmental Research Center, Maryland. Thalliwere not visibly damaged during transport, though changes inoptical properties were uncontrolled. Whole-thallus sectionswere immersed in 5�C seawater in a UV transparent cuvette,and absorbance spectra were measured on a Cary 400 spectro-photometer (Varian Inc., Palo Alto, CA, USA) using the frostedsides of the cuvette as a diffuser (Shibata 1958).

Exposure response curves. Time-course experiments to deter-mine the photosynthetic response to UVA and UVB exposurewere performed using L. hyperborea collected from 0 m (MLW).Tissue samples of 12 cm2 were excised from the first lamina andsecured in a chamber built from UV transparent acrylic. The8 mm fiber optic probe of a DIVING-PAM fluorometer (HeinzWalz GmbH, Effeltrich, Germany) was inserted at a 60� angleand at a fixed distance to the thallus. Incubation seawater wasconstantly stirred with a magnetic stir bar, and temperature wasmaintained at 12�C–13�C (ambient October seawater temper-ature) by placing the sample chamber in a large-volume waterbath, also constructed of UV transparent acrylic (Laird Plastics,Austin, TX, USA). Illumination was provided by a 150 W xenonlamp (Schoeffel Instruments, Westwood, NJ, USA). Relativephotosynthesis was quantified as the quantum yield of PSIIelectron transport (i.e., DF ⁄ F¢m). Each spectral treatment(UVB+UVA+PAR, UVA+PAR, and PAR-only) was replicatedwith tissue excised from three L. hyperborea individuals.

Exposure response time courses were designed in fourphases. In a pretreatment phase, thalli were dark acclimatedfor 15 min, then PAR-only irradiance was incrementallyincreased to the exposure intensity in four 10 min intervals(32, 82, 171, and 270 lmol photons Æ m)2 Æ s)1). The value ofDF ⁄ F¢m was recorded every 30 s, and steady-state yield wasachieved at each light increment. Light levels were manipu-lated with neutral density filters, and UVR was removed with aSchott GG400 long-pass cutoff filter (Schott Glass Technolo-gies Inc., Elmsford, NY, USA). Second, spectral exposureduring the next 60 min was controlled with WG305, WG320,or WG400 nm long-pass cutoff filters to obtain, respectively,nominal UVB+UVA+PAR, UVA+PAR, and PAR-only spectraltreatments. The filter suffix is the wavelength with nominal50% transmittance, so, for example, there is a minorcomponent of UVB in the WG320 treatment. PAR irradiancesfor all spectral exposures were similar to the maximumPAR-only treatment in the pretreatment phase, �270lmol photons Æ m)2 Æ s)1. Third, photosynthetic recovery inthe light was monitored for 60 min with PAR-only irradiance,again maintained at 270 lmol photons Æ m)2 Æ s)1. Finally, thechamber was covered with black cloth, and recovery wasmonitored in the dark for 60 min. Optimal quantum yield,Fv ⁄ Fm, was measured every 5 min in the dark recovery phase,and dark recovery was monitored overnight for one replicatefrom each spectral treatment. Fluorescence yields were thennormalized to an average of the last 10 measurements in thefinal PAR-only treatment just prior to the exposure phase.Reciprocity and the dynamics of UV photodamage (k) andphysiological repair (r) during exposure were assessed usingan exponential function and data from the UVB+UVA+PARtreatment (Neale 2000):

Y ðtÞY0¼ 1

r þ kðr þ k eð�ðrþkÞtÞÞ ð1Þ

where Y ⁄ Y0 is DF ⁄ F¢m yield normalized to PAR-only DF ⁄ F¢m.BWF: experimental measurement. Photosynthesis and UV-

dependent photoinhibition in L. hyperborea from two depthsFig. 1. Laminaria hyperborea collection site and field laboratory

location.

LAMINARIA HYPERBOREA BWF 573

(0 m and 10 m, MLW) were determined in a spectral incuba-tor, the photoinhibitron (Neale and Fritz 2002), which wasmodified from phytoplankton applications to be used with kelptissue disks (Miller and Dunton 2007). The photoinhibitronallows for polychromatic light treatments that differ in bothspectral composition and irradiance. The apparatus consists of(1) an aluminum block bored to hold eighty 20 mL quartz-bottomed incubation cuvettes, (2) a continuous-flow waterbath to control temperature, (3) and a 2500 W xenon lamplight source. A central mirror reflects light up through theapparatus to illuminate kelp tissue in individual incubationcuvettes. Along the beam path, light passes through (1) a waterbath with a UV-transparent Plexiglas bottom; (2) one of eightlong-pass filters (WG280, WG295, WG305, WG320, WG335, andGG395 [Schott Glass Technologies Inc.] and LG350 andLG370 [Spectra-Physics, Waltham, MA, USA]) with nominal50% transmittance at indicated wavelengths (nm); and (3) acellulose acetate shield that removes UVR below 290 nm.Within each spectral treatment, irradiance was varied usingneutral density screens. Photosynthesis in L. hyperborea wasmeasured at six light irradiances within each of eight UV filtersfor a total of 48 light treatments per 1.5 h incubation.Temperature was maintained within the cuvettes at 5 ± 1�C(the ambient in situ sea temperature) by adding ice as neededto a large volume (� 50 L) reservoir and by continuouslycycling this water through the water bath of the apparatus.

Spectral irradiance in each of the 48 light treatments wasdetermined with a scanning spectroradiometer system (Nealeand Fritz 2002). Light was collected from each incubation cellwith a quartz fiber-optic probe fitted with a diffuser designedfor PAR and UV measurement, and the probe was connected toan Acton Research monochromator (SP-401, Princeton Instru-ments, Acton, MA, USA) with a photomultiplier tube (BurleTechnologies, Lancaster, PA, USA) and computer interface.The spectroradiometer was calibrated at the field locationusing a National Institute of Standards and Technology–traceable, 1000 W quartz-halogen lamp operated at a currentof 7.9000 (±0.0001) A (Xantrex [Burnaby, BC, Canada] powersupply, with current monitored by HP3457A multimetermeasuring across a 0.1 ohm shunt). The lowest wavelength ofdetectable irradiance in experimental light treatments was286 nm. In addition, PAR irradiances in all light cells weremonitored prior to each replicate incubation using a QSL-100(Biospherical Instruments, San Diego, CA, USA) 4-p quantumsensor.

Photosynthetic measurements were determined for kelp disksexcised from the first blade, that is, the newly formed bladematerial produced that winter ⁄ spring. The blade tissue in thisregion was visually uniform and consistent in density. For eachcomplete incubation, 51 tissue disks (48 light treatments andthree dark treatments) were cut with a brass coring device(1.8 cm diameter), and disks were placed in a temperature-controlled seawater container for 1 h. The BWF experiment wasreplicated with five individual kelp plants from both 0 m and10 m depth intervals. To avoid potential variation due to naturalacclimation to field conditions over weekly timescales, all five0 m replicates were completed first in a 3 d period followed byfive 10 m plants on the subsequent 2 d.

Photosynthesis was quantified by concurrent measurementsof oxygen evolution and H13CO3

� uptake. In brief, several daysprior to incubations, 20 L of filtered seawater was enrichedwith NaH13CO3 [see Miller and Dunton (2007) for details,including amount added and effect on total DIC]. The carboylid was left loose, and the carboy was occasionally shaken toallow the dissolved inorganic carbon (DIC) concentration topartially equilibrate with the atmosphere. Before incubations,13C-enriched seawater was brought to experimental tempera-ture (5�C), and dissolved oxygen (DO) was stripped with N2 for8 min to decrease DO to about 20%–30% air saturation. Water

samples were taken in 70 mL serum bottles, fixed with 0.2 mLof saturated HgCl2 solution, and stored in a refrigerator at 4�Cfor later analysis of initial

PCO2 and d13C.

Photoinhibitron cuvettes were filled with O2-stripped and13C-enriched seawater, and a kelp disk was suspended in thecuvette about 1 cm from the bottom. The tissue disk was heldin place between two silicone O-rings crosshatched with thintransparent thread. Suspending the disk in this way assuredperpendicular orientation of the disk to the irradiance beamand allowed for unrestricted water motion around the disk.After removing air bubbles, cuvettes were sealed with modifiedsilicon stoppers that were fitted with small, 1.5 V DC electricmotors and magnetic stir bars to create water motion in thecuvettes (Miller and Dunton 2007). Motors were powered witha B&K Precision 20 A DC power source (B&K Precision Corp.,Yorba Linda, CA, USA; model 1688), and the speed of themagnetic stir bars was adjusted by controlling the voltage of thepower source.

DO concentrations were determined in the treatmentseawater with a PreSens Microx TX micro-optode oxygensystem (Presens Precision Sensing GmbH, Regensberg, Ger-many) using a needle-type optode. DO measurements weremade both before and after incubations. Oxygen-evolutionrates were corrected for nonphotosynthetic changes in DOassessed in blank cuvettes, that is, cuvettes without the kelptissue. Kelp tissue disks were quickly removed from cuvettesbefore final oxygen measurements, after which disks were thenrinsed twice to remove excess 13C label and dried at 60�C. 13C-amended seawater and tissue samples were shipped to Univer-sity of Texas, Marine Science Institute (UTMSI), for isotopicanalysis on a Finnigan MAT DeltaPlus continuous flow isotoperatio mass spectrometer (Thermo Fisher Scientific, Waltham,MA, USA) attached to a Carlo Erba elemental analyzer (NC2500, Carlo Erba, Milan, Italy). Oxygen-evolution and carbon-uptake photosynthetic rates were calculated using equationsdescribed in Miller and Dunton (2007). Both measures wereinterpreted as gross photosynthesis since dark respiration wasadded to oxygen evolution and light treatments were suffi-ciently short to assume minimal carbon turnover (see discus-sion in Miller and Dunton 2007).

BWF: statistical evaluation. BWFs were statistically deter-mined for L. hyperborea based on carbon and oxygen photosyn-thesis from kelp collected at 0 m and 10 m depth. BWFcalculations began with an underlying model describingpotential photosynthesis, Ppot, as a saturating function ofirradiance. UV inhibition is estimated as a fraction of Ppot, andthe parameterization of the BWF is sensitive to the overall shapeof the PE function. Four photosynthetic equations werecompared using PE data collected from L. hyperborea sporo-phytes at three depths. The Michaelis-Menten equation bestreproduced the broad transition zone between the light-limitingand light-saturating regions of the curve (Fig. S1 and Miller2002). Therefore, gross Ppot was calculated using the followingequation (see Table 1 for symbol notation and units):

Ppot ¼PmaxEPAR

Ek þ EPARð2Þ

where Pmax is the maximum potential rate of photosynthesis,particular to either carbon or oxygen units, and Ek is PARirradiance at nominal light saturation.

If photosynthesis is inhibited in the presence of UVR, thenthe magnitude decrease from Ppot is determined with a secondexpression that depends on the kinetics of the exposureresponse curve (Neale 2000). In the case when steady-stateinhibition is achieved (i.e., active repair processes counteractUV damage), the mean rate of photosynthetic production (P)is described using an irradiance-based model (the BWFE-PEmodel):


P ¼ Ppot1

1þ E�inh

� �ð3Þ

If photosynthesis consistently declines over the exposuretime period (i.e., reciprocity is upheld), then an inhibitorymodel based on cumulative exposure is used (the BWFH-PEmodel):

P ¼ Ppot1� exp�H �inh

H �inh

� �ð4Þ

Though L. hyperborea likely possesses repair and ⁄ or protec-tion mechanisms, particularly in shallow kelp accustomed tohigh irradiance environments, both the BWFE-PE and BWFH-PE models were chosen to predict the response of the kelppopulation to ozone depletion. The natural response is likelybetween these two extremes and is dependent on the lighthistory and collection depth of the alga. Biologically effectivefluence rate (E�inh) was determined from 286 to 400 nm:

E�inh ¼X400 nm

k¼286 nm

eEðkÞEðkÞDk ð5Þ

and biologically effective cumulative exposure (H �inh) was for-mulated as follows:

H �inh ¼X400 nm

k¼286 nm

eHðkÞH ðkÞDk ð6Þ

where H kð Þ ¼R T

0 E kð Þdt(J Æ m)2 Æ nm)1) is the total radiantexposure during the incubation period, T. eE(k) (eq. 5) andeH(k) (eq. 6) are wavelength-specific weightings for UV inhi-bition of photosynthesis and are calculated below. PARphotoinhibition (i.e., ePAR) was omitted in the calculation ofboth E�inh and H �inh as it was shown to be negligible (Fig. S1and Miller 2002).

The experiment produced 48 oxygen- and 48 carbon-basedvalues of P per kelp replicate, and BWF models were param-eterized from photosynthetic data and the measured spectralirradiance in each individual cuvette (Neale et al. 1998a). Thebiological weights, either eE(k) or eH(k), for each UV wave-length were determined using the Rundel method, whichassumes the natural log of the BWF is a polynomial (Rundel1983, Cullen and Neale 1997, Neale 2000). Only two terms of

the polynomial (constant and linear) were required in fittingthe kelp BWF:

e kð Þ ¼ C exp� a0þa1kð Þ ð7Þ

where C = 1 is a proportionality constant with units(mW Æ m)2))1 or (J Æ m)2))1. Coefficients ai of the BWF, as wellas photosynthetic parameters Pmax and Ek, were estimated withnonlinear regression techniques implemented in a MATLABv.6.1 application (Mathworks Inc., Natick, MA, USA). Fits wereperformed on the pooled data of all replicates from eachdepth. Small, but significant, variations in Pmax between repli-cates were accounted for by fitting a separate Pmax for eachreplicate. Standard errors of e(k) were calculated from approx-imate variances and covariances of ai using propagation oferrors (Cullen and Neale 1997). Photosynthetic responses tospectral irradiance were also determined using principalcomponent analysis (Cullen et al. 1992, Neale 2000), but theinternal spectral structure within e(k) derived with the PCAmethod was not statistically justified over the simpler Rundelmethod (Cullen and Neale 1997).

RESULTS

Optical properties of the thallus. Kelp thalli from10 m were noticeably more translucent than kelpoccurring in the upper subtidal. We compared abso-lute absorbance of whole-thallus spectra andobserved that depending on wavelength, deepwaterkelp absorbed 35%–60% less light than shallow-water kelp. On an areal basis, 0 m kelp containsmore chl a than deep kelp, 433 ± 6 versus371 ± 8 mg Æ m)2, respectively. Chl a:fucoxanthinratios increased little with depth from 1.8 to 2.1,but chl a:chl c decreased from 8.2 at the surface to4.6 at depth (Miller 2002). When spectra were nor-malized to their respective blue chl peaks (430 nm),it was apparent that shallow kelp also absorbed rela-tively more radiation in the blue-green, violet, UVA,and most UVB wavelengths (Fig. 2). Photoprotec-tive pigments absorbing in these wavelengths, for

Table 1. Symbol notation and abbreviations.

Symbol Description Units

a0 First coefficient for polynomial in Rundel weighting function Dimensionlessa1 Second coefficient for polynomial in Rundel weighting function nm)1

C Proportionality constant (mW Æ m)2))1 or (J Æ m)2))1

E�inh Biologically effective fluence rate for inhibition of photosynthesis DimensionlesseE(k) Biological weighting as a function of UV irradiance (mW Æ m)2))1

eH(k) Biological weighting as a function of cumulative radiant exposure (J Æ m)2))1

E(k) Spectral irradiance, scalar underwater in irradiance model mW Æ m)2 Æ nm)1

EPAR Photosynthetically active radiation (400–700 nm) lmol photons Æ m)2 Æs)1

Ek Photosynthesis saturation irradiance W Æ m)2

H(k) Radiant exposure J Æ m)2 Æ nm)1

H �inh Biologically effective cumulative exposure for photosynthesis inhibition DimensionlessP Photosynthesis (e.g., mean rate during experimental exposures) lmol O2 Æ m)2 Æ s)1 or lmol C Æ m)2 Æ s)1

Pmax Maximum rate of photosynthesis in absence of inhibition lmol O2 Æ m)2 Æ s)1 or lmol C Æ m)2 Æ s)1

Ppot Potential photosynthesis in absence of inhibition lmol O2 Æ m)2 Æ s)1 or lmol C Æ m)2 Æ s)1

T Duration of experiment sUVR Ultraviolet irradiance at 220–440 nm; solar UVR (290–400 nm) mW Æ m)2

UVA Ultraviolet irradiance at 320–400 nm mW Æ m)2

UVB Ultraviolet irradiance at 280–400 nm mW Æ m)2

UVC Ultraviolet irradiance at 220–280 nm mW Æ m)2


example, b-carotene in the visible and mycosporine-glycine and phlorotannin in the UVB (Fig. 2 inset),potentially contribute to the spectral differencesbetween the two depth collections.

Temporal exposure response. Choice of weightingfunction based on irradiance (BWFE-PE) or cumula-tive exposure (BWFH-PE) depends on the temporalkinetics of photodamage and repair processes dur-ing UVR exposure. After 20 min of exposure,UVB+UVA+PAR caused definite, but moderate, pho-toinhibition in 0 m L. hyperborea (Fig. 3A). WhenUVB+UVA+PAR exposure data were comparedusing a kinetic equation (eq. 1), which assumes thatrepair processes are proportional to damage, thensteady-state balance between repair and UVR dam-age would occur after about 1 h and result in anapproximate 20% decrease in photosynthetic effi-ciency (R2 = 0.83). Both UVA+PAR and UVB+UVA+PAR exposed thalli recovered at parallel rates in thepresence of PAR (Fig. 3B). Long-term recovery inthe dark was clearly biphasic with the duration ofthe initial, fast phase less for the UVB+UVA+PARtreatments than UVA+PAR and PAR (Fig. 3C). Thisincreased the time for recovery to 90% of initialoptimal quantum yield from 3 h in the PAR-onlyexperimental treatment to nearly 6 h when thalliwere exposed to UVB.

L. hyperborea BWF. It was discovered after fieldexperiments that polychromatic light treatments inthe photoinhibitron were deficient of expectedshort-wavelength UVB radiation (Fig. 4, cf. Cullenet al. 1992, Neale et al. 1994, Neale and Fritz 2002).This finding was likely due to errant mirror configu-

ration in the photoinhibitron. The spectrumachieved in the UVB range was similar to naturalsunlight at noon and under 300 D.U. column ozone(Fig. 4). Thus, the incubation did not bracket thefull range at short wavelengths, but the Rundelmethod extrapolated the BWF [i.e., eE(k) andeH(k)] to the shorter wavelengths for which therewas little treatment variation. There is no reason tosuspect a specific bias in spectral weightings atshorter wavelengths, since Rundel estimates at thesewavelengths are in any case constrained by the over-all slope (a1), the estimate of which is definedmainly by the response to the intermediatewavelengths that have greatest effective irradiance

Fig. 2. Thallus absorbance spectra, normalized to its bluechl peak at 430 nm, for Laminaria hyperborea collected from 0 mand 10 m. Data are �x ± SD, n = 5. Inset: (1) difference spectrumbetween 0 m and 10 m kelp, (2) phlorotannin absorbancespectrum (Henry and van Alstyne 2004), and (3) mycosporine-glycine absorbance spectrum (Dunlap et al. 1986). ABS,absorbance.

Fig. 3. Quantum yield time course measured by PAM fluo-rometry during (A) exposure to UV+PAR (�x, n = 3), (B) postex-posure recovery under PAR-only exposure (�x, n = 3), and (C)subsequent overnight recovery in the dark (n = 1) for Laminariahyperborea collected from 0 m. Fluorescence yield data in (A) and(B) are normalized to DF ⁄ F¢m in the PAR-only treatments duringthe time trial. Fv ⁄ Fm values in dark recovery phase (C) are nor-malized to optimal quantum yield assessed in dark-adapted thalliprior to experiment. Solid lines in (C) are fits (R2 > 0.99) to thebiphasic equation of the form 1 ) [p1exp()k1t) ) p2 exp()k2t)],where k1, k2 are the rates of recovery and p1, p2 are the changesin normalized Fv/Fm during the fast and slow phases, respectively.Though rate constants k1 and k2 were similar (0.025 min)1 and0.003 min)1), p1 decreased from 0.30 to 0.19, and p2 increasedfrom 0.18 to 0.33, respectively, for PAR-only and UVB+UVA+PARtreatments. PAM, pulse-amplitude-modulated fluorescence; UVA,ultraviolet radiation A; UVB, ultraviolet radiation B.


(Cullen and Neale 1997). Nonetheless, the statisticalpower for estimating the effect of exposure is lowerin the UVB region (as indicated by large confidenceintervals), and so these weights should be inter-preted conservatively.

A total of eight BWFs for UV inhibition of photo-synthesis are presented for the kelp L. hyperborea(Table 2). BWFs differed between carbon and oxygenmetabolisms, collection depth, and choice of weight-ing model (BWFE-PE or BWFH-PE). Irrespective ofinhibitory model chosen, photosynthesis decreasedappreciably when kelp were exposed to increasingamounts of weighted UVR (Fig. 5, points), and 10 mkelp exhibited a greater UV-inhibitory response than0 m kelp. The proportion of variances (R2) explainedby the Rundel method was similar in BWFE-PE andBWFH-PE (Table 2) models. In general, R2 valueswere less in 0 m kelp and when photosynthesis wasmeasured as oxygen evolution.

Spectrally, UVB wavelengths were two to threeorders of magnitude more effective at inhibitingL. hyperborea photosynthesis than UVA (Fig. 6). Abso-lute sensitivity of the inhibition weightings variedspectrally depending on photosynthetic measure-ment and collection depth, and overall spectral slope(a1) was similar for both BWFE-PE and BWFH-PEmodels (Table 2). Inhibition weightings for kelp col-lected at 10 m depth were greater than that of 0 mkelp in the UVA region between 330 and 370 nm(Fig. 6), yet both collections had a response of similarmagnitude in the UVB. This was also the case for theBWFH weightings (results not shown, but seeTable 2). BWFs derived from oxygen had consistentlysmaller intercepts (a0) and slopes (a1) than BWFsderived from carbon measures of photosynthesis(Table 2). As a result, each pair of BWFs showed acrossover pattern, with carbon assimilation more sen-sitive to UVB but oxygen evolution more sensitive toUVA (Fig. 6 for BWFE, similar results for BWFH notshown). However, these differences were not statisti-cally significant for 0 m kelp. On the other hand,there was less absolute difference for O2- and C-basedBWFs from 10 m, but the increase in oxygen photo-synthetic inhibition at UVA wavelengths was signifi-cant (Fig. 6). The BWFs are significantly differenteven though the corresponding slopes and interceptsare not because of the high covariance between theestimates (correlation 0.75–0.85). Estimated confi-dence intervals for the weightings also differedspectrally with decreased model confidence in thelong-wavelength UVA and short-wavelength UVBregions. This spectral variance was most pronouncedin oxygen-based measurements of inhibition.

The general spectral shape and relative magni-tude of L. hyperborea BWFs were most similar to thepolychromatic weighting function developed forinhibition of photosynthesis in Rumex patientia(Polygonaceae, Fig. 7A). Action spectra based onmonochromatic light tended to either underestimate(Avena) or overestimate (isolated spinach chloro-plasts) UV inhibition, compared to the kelp

Fig. 4. Spectral treatments in the photoinhibitron resultingfrom Schott long-pass cutoff filters. Values are normalized to irradi-ance at 400 nm. Sunlight spectral distribution was generated withSTAR radiative transfer model (Ruggaber et al. 1994) and superim-posed for comparison. Sunlight was modeled for clear-sky irradi-ance at noon on 7 April 2001 with 300 D.U. column ozone (65� N).

Table 2. Laminaria hyperborea: Gross photosynthesis and photoinhibition parameters determined with least squares analysisof experimental results using either the BWFE-PE or BWFH-PE models. The coefficient of determination (R2) is shown forP ⁄ Ppot analysis in Figure 5. Photosynthesis and R2 values are similar for E�inh and H �inh models. Pmax is mean of replicatePmax’s with SE of the mean. All other values are mean with SE calculated using propagation of errors (n = 5). Ek conver-sion to W Æ m)2 is 0.227 J Æ lmol)1 for xenon lamp.

Depth (m) Measure Model Pmax (lmol Æ m)2 Æ s)1) Ek (lmol photons Æ m)2 Æ s)1) a0 a1 R2

0 C E�inh 1.75 (0.09) 101 (13) 13.59 (7.84) 0.074 (0.024) 0.69

H �inh 12.45 (7.85) 0.073 (0.023)

O2 E�inh 2.03 (0.16) 88 (15) 4.82 (8.31) 0.047 (0.024) 0.65

H �inh 3.25 (8.61) 0.045 (0.025)

10 C E�inh 1.41 (0.08) 92 (11) 7.14 (4.54) 0.052 (0.013) 0.87

H �inh 4.27 (4.60) 0.047 (0.013)

O2 E�inh 1.87 (0.17) 97 (14) 3.05 (4.23) 0.039 (0.012) 0.88

H �inh )0.95 (4.26) 0.031 (0.012)


responses, for wavelengths greater than the normali-zation wavelength (300 nm). Phytoplankton fromLake Bonney (Antarctica) and a high-sensitivity phy-toplankton assemblage in the Rhode River (Mary-land) were in general an order of magnitude moresensitive than kelp across the UV spectrum (Fig. 7B),though irradiance-based kelp BWFs were comparableto a low sensitivity Rhode River assemblage. On acumulative-exposure basis, phytoplankton from theWeddell Sea Confluence were more than five timesmore sensitive to increases in UVB than Norwegiankelp (Fig. 7C).

UV stress and the photosynthetic quotient. Since bothoxygen evolution and carbon uptake were measuredconcurrently for the same tissue sample and irradi-ance treatment, it was possible to evaluate theresponse in the photosynthetic quotient withincreasing UV exposure. We defined photosyntheticquotient (PQ) in this analysis as the molar ratio ofgross oxygen evolution to gross carbon assimilationduring photosynthesis and considered how this mea-sure varied as a function of unweighted UVR(Fig. 8). The BWFs showed a consistent pattern ofhigher sensitivity of oxygen evolution in the UVA

Fig. 5. Photosynthetic respon-se of Laminaria hyperborea to wei-ghted experimental UV radiationbased on an irradiance model(BWFE-PE). Gross oxygen evolu-tion and carbon fixation (points)were measured for kelp collectedfrom 0 m and 10 m depths (�x,n = 5). Values are displayed nor-malized to potential photosynthe-sis. Expected P ⁄ Ppot (line) isshown for each. See Table 2 forR2. BWF, biological weightingfunction; PE, phototsynthesis ver-sus irradiance.

Fig. 6. Comparison of BWFfor UV photoinhibition in Lami-naria hyperborea. The BWF spectralshape is compared for both 0 mand 10 m collections (Depth, leftcolumn) and between carbonuptake and oxygen evolution(Measurement type, right col-umn). BWFH-PE results are simi-lar to BWFE-PE model datapresented. Shaded areas are 95%confidence intervals. BWF, bio-logical weighting function; PE,phototsynthesis versus irradiance.


compared to carbon fixation (Fig. 6). This findingimplies that PQ should decrease with increasing UVexposure to filtered xenon arc irradiance, butsince the weights were not significantly different for

oxygen evolution versus carbon assimilation, wedecided to examine this possibility directly. In theabsence of UVR stress, the mean PQ for 10 m kelpwas 1.30, and using a one-tailed t-test (t = )42.1,P << 0.01) assuming equal variances (F46,46 = 1.19,P = 0.57), the 10 m PQ was significantly greaterthan 0 m samples (PQ = 1.18). The PQ from bothcollections significantly declined with UVR expo-sure; that is, the variables were negatively correlated(r0m = )0.55, P << 0.01; r10m = )0.82, P << 0.01),and trend line slopes were significantly less thanzero (one-tailed t-test: t0m = )4.45, P << 0.01;t10m = )9.95, P << 0.01). Further, as variances in10 m and 0 m slopes were similar (F46,46 =1.22,P = 0.50), a one-tailed t-test assuming equalvariances suggested that PQ declined more rapidlyin 10 m kelp (t = 29.8, P << 0.01). This trend is con-sistent with the higher weights for UV inhibition ofoxygen evolution by 10 m kelp.

DISCUSSION

The BWF is an empirical formulation that assignsspectral weights to account for the wavelengthdependency of UV effects on a biological process.Here, polychromatic weightings for photoinhibitionare provided for both high-light-acclimated (0 m)and low-light-acclimated (10 m) L. hyperborea, and

Fig. 7. Comparison of kelp BWFs with published plant andphytoplankton BWFs and BWFs fitted with different models. (A)Relative inhibition normalized to 300 nm: inhibition of Hill reac-tion in isolated spinach chloroplasts (monochromatic, Jones andKok 1966), general plant damage (monochromatic, Caldwell1971, redrawn from Caldwell et al. 1986), UV inhibition ofphotosynthesis in Rumex patientia (Rundel method, differentialeffectiveness, eq. 13, Rundel 1983), UV inhibition of Avena sativagrowth (Flint and Caldwell 2003), 0 m Laminaria hyperborea(Rundel method, carbon-based H �inh model), 10 m L. hyperborea(Rundel method, carbon-based H �inh model). (B) Irradiance-dependent BWFs (carbon-based E�inh models): phytoplanktonfrom Lake Bonney, Antarctica (PCA method, redrawn from Nealeet al. 1994), spring and winter phytoplankton assemblages intemperate Rhode River, Maryland, estuary (PCA method,Banaszak and Neale 2001), 0 m L. hyperborea (Rundel method),and 10 m L. hyperborea (Rundel method), mostly diatom commu-nity collected from McMurdo Sound, Antarctica, and maintainedin outdoor culture (PCA method, redrawn from Neale et al.1994, Banaszak and Neale 2001). (C) Cumulative exposure-dependent BWFs (carbon-based H �rminh models): natural intactphytoplankton colonies (Rundel method) or homogenizedsamples (PCA method) from Weddell–Scotia Confluence (Nealeet al. 1998b), Antarctica, 0 m L. hyperborea (Rundel method), and10 m L. hyperborea (Rundel method). BWF, biological weightingfunction; PCA, principal component analysis; WSC, Weddell SeaConfluence.

Fig. 8. Decrease in the photosythetic quotient with increased(unweighted) UVR exposure in Laminaria hyperborea collectedfrom (A) 0 m and (B) 10 m depth. Observed data points are�x ± SE (n = 5), and the trend was modeled with a linear regres-sion (line) with ±95% confidence intervals of the fit (dottedlines). Regression equations include parameter error in parenthe-ses. PQ, photosynthetic quotient; UVR, ultraviolet radiation.


the functions are applicable to oxygen- or carbon-derived photosynthesis studies. Empirical BWFs arealso presented assuming both irradiance-based andcumulative-exposure response models.

The importance of the BWF is that it enables (1)intercomparison of results obtained using differenttypes of polychromatic exposures, including solarsimulators in the laboratory and natural sunlight,and (2) extrapolation of experimental results tomodel simulations of ozone depletion (Cullen andNeale 1997, Neale 2000, Miller 2002). In two deep-water red algae, UVA exposures that were requiredto inhibit Fv ⁄ Fm by 50% were lower under artificialsunlamps (Dring et al. 1996b) than total exposurein natural sunlight (Dring et al. 2001). The authorsattributed this discrepancy to spectral differences inpeak irradiance between laboratory sunlamps andoutdoor sunlight, and the inconsistency in biologi-cally effective exposure would be reconciled with anappropriate weighting function. To date, fewresearchers have applied spectral weightings in stud-ies on UV effects on macroalgae. Those studies thathave relied on action spectra of Caldwell (1971) forgeneral plant damage (e.g., Bischof et al. 2000) or anormalized version of the Jones and Kok (1966)photoinhibition spectrum for isolated spinach chlo-roplasts (e.g., Forster and Luning 1996, Gomezet al. 2001, 2005, Huovinen et al. 2006). Morerecently, a new general plant action spectrum hasbeen presented based on the growth response ofAvena (Flint and Caldwell 2003). Our results showthat these action spectra are not suitable for quanti-fying the effects of a changing light climate on kelpphysiology for several reasons. Most importantly, thecontribution of UVA relative to UVB differs fromthe shape of the L. hyperborea BWFs; that is, Avenaspectrum has too little weight, and the Jones andKok spectrum has too much weight in the UVA (thegeneral plant spectrum is not defined in the UVA).Also, the Jones and Kok relationship assumes reci-procity, which was demonstrated for the Hill reac-tions they used to measure photosynthesis. Finally,these action spectra predict only the relative biologi-cal response to varied irradiance, so a quantitativeprediction requires knowledge of the biologicaleffect for at least one weighted exposure (Cullenand Neale 1997).

Relative shapes of the L. hyperborea BWFs are mostsimilar to the land plant Rumex patientia, with 10 mkelp slightly more sensitive to UVA, relative to UVBat 300 nm, than R. patientia, and 0 m kelp less sensi-tive to UVA. The magnitude of the spectral weightsis greater in the UVA above 350 nm in all phyto-plankton BWFs considered. Phytoplankton are verti-cally mixed in the ocean and acclimate to the netlight environment. In contrast, kelps are anchoredat depth, and the relative insensitivity of surfacekelp to UVA likely results from long-term acclima-tion to ambient light experienced near the sea sur-face. Kelps from deeper water are more susceptible

to UVR stress, but the spectral differences in weigh-tings appear to be related to UVA tolerance. Theonly phytoplankton community compared that wasmore tolerant of UVB than kelp was a diatom-domi-nated assemblage from McMurdo Sound, Antarctica.In this study, phytoplankton were allowed to adjustto a high UVB climate as cultures were acclimatedin shallow outdoor chambers for several weeksunder ozone-depletion conditions prior to BWFexperiments (Neale et al. 1994).

The relative contribution of UVA and UVB tophotoinhibition depends on the light field and thespectral response. In L. hyperborea, UVB weightingsare in general several orders greater than UVA, butthe magnitude of a photoinhibitory response alsodepends on the spectral irradiance received at thealgal thallus. For instance, under 300 D.U. columnozone, daily photosynthetic production at the sur-face is potentially reduced 10% (Fig. 9; 0 m kelp,BWFE-PE model). If UVB wavelengths are removedfrom the calculation, photosynthesis is still inhibited8%. This finding suggests that under non-ozone-depletion scenarios, UVA contributes significantlymore to kelp photoinhibition than UVB. The pointis emphasized if reciprocity (BWFH-PE model) isassumed for 10 m kelp using the same irradiancemodel. In this case, UVA accounts for 38% reduc-tion in potential daily photosynthesis, whereas

Fig. 9. Ratio of UV-inhibited, daily integrated photosynthesisto potential daily integrated photosynthesis under different col-umn ozone conditions and different irradiance weightingschemes. Underwater irradiance at 30 min time intervals duringthe day was modeled using STAR (Ruggaber et al. 1994) andassuming idealized conditions: a clear-sky in April at the studysite, a flat sea surface of constant depth (0.5 m MLW, no tidalvariation), and locally measured water column attenuation (Miller2002). Potential photosynthesis was calculated using the Michael-is-Menten relationship, and UV-inhibition was modeled using thefour different BWFs determined from carbon-based measure-ments of Laminaria hyperborea photosynthesis. Variation in columnozone results in modest production losses relative to 600 D.U.,irrespective of BWF model and acclimation state. BWF, biologicalweighting function; MLW, mean low water.


UVA+UVB increases total inhibition to only 42%.Clearly, the much greater amount of solar irradi-ance in the UVA dominates the relatively low bio-logical weightings in the region compared to that inthe UVB. Significant UVB photoinhibition is likelyonly a near-surface phenomenon since UVB is atten-uated more rapidly than UVA in coastal waters(Tedetti and Sempere 2006).

Kelps, as other benthic macroalgae, are perma-nently anchored in their light habitat, and there islittle doubt that algae exposed to full sunlight nearthe surface are able to adapt to ambient UV stress.Kelp individuals from depth are generally more sen-sitive to high light and UV stress than intertidal andupper-subtidal algae (Larkum and Wood 1993,Dring et al. 1996b, Hanelt et al. 1997a, Johannssonand Snoeijs 2002, Bischof et al. 2006). For example,in transplantation experiments with subarctic Saccha-rina latissima (=Laminaria saccharina) and L. digitata,subtidal algae showed increased inhibition andlonger recovery in UVA and UVB treatments (Ha-nelt et al. 1997b). Twenty-four-hour percent recov-ery of optimum quantum yield after a 4 h exposureto PAR+UVA+UVB decreased incrementally withS. latissima collection depth until 7–9 m (Bischofet al. 1998). Photosynthetic response to UV was afunction of depth in Norwegian L. hyperborea as well.Calculations based on the weighting functions pre-dict that single-day photosynthesis is 7%–12% less indeepwater sporophytes, depending on BWF model,when both surface and deepwater kelps are exposedto the same near-surface simulated irradiance(Fig. 9, 300 D.U.). Surface and deep L. hyperboreahave similar weights in the UVB region, but in 10 mkelp, biological weights were greater in the UVAregion. Apparent photosynthetic decreases in 10 mkelp, relative to 0 m kelp, were a result of increasedinhibition in the UVA and not necessarily a greaterresponse to UVB.

Depth differences in UV response are importantas they are evidence, albeit circumstantial, of thepresence of active repair and ⁄ or protection mecha-nisms in algae. Tolerance to UVR is an acclimationprocess tuned to maximize growth (Roleda et al.2006), and physiological development of thesemechanisms correlates with light history of the alga.High-light algae with daily exposure to UVR neces-sarily invest resources into pigment production anddamage repair enzymes that mitigate potential pho-toinhibition and photodamage. This investmentconstitutes energy expenditure otherwise availablefor allocation to growth and reproduction. Presum-ably, the expenditure is less than production lostwith photoinhibition [and structural and DNA dam-age as well, see Bischof et al. (2006) and Roledaet al. (2007)]. In the less varied light environmentdeeper in the water column, algae with little expo-sure to high light and UVR allocate less energy andnutrient resources to UVR stress response and areless efficient at ameliorating damage if suddenly

introduced to UVR. Individuals of a kelp specieswith a wide depth range adopt the optimal strategyin their light realm, and kelps nearer the moststressful light climate at the surface retain the abilityto acclimate to a changing irradiance field. Thetimescale of acclimation to moderately heightenedUVR appears to be on the order of days to weeksbut varies with kelp species and depends on thallusage and season (Bischof et al. 1999, 2002, Brouweret al. 2000, Roleda et al. 2006).

Evidence showing minor differences in thallusabsorbance indicates that photoprotective pigmentsare probably not very important contributors tothe lower sensitivity of shallow material (Fig. 2).The exposure response in our study, however, sug-gests that repair and recovery are active in 0 mL. hyperborea. Steady state between UV-inhibitionand repair was achieved about 1 h after exposure toUVB+UVA+PAR, and potential quantum yield wasrecovered after �6 h in darkness (Fig. 3). In con-trast, steady-state quantum yield occurred within15 min in the laboratory-cultured dinoflagellateGymnodinium sanguineum, and inhibition progressedfrom initial exposure to steady state without hystere-sis (Neale et al. 1998a). The slower rates observedfor L. hyperborea suggest that repair capability ismodest, even in 0 m kelp. In situ, blade undulationsdue to wave and water motion potentially result inless overall UV exposure and lessened ability torespond to direct, one-sided exposure, as providedin the experiment.

If balance occurs between damage and repair ontimescales less than the total time of interest, reci-procity fails (Lesser et al. 1994). Production shouldthen be modeled with an irradiance-based BWF(BWFE-PE). A BWFE-PE model may not, however,be valid for algae from deeper in the water, orfrom other environments with typically less incidentUVR, such as polar latitudes (Neale 2000). Forinstance, as a percent of control, Laminaria solidun-gula from >7 m showed a nearly complete collapsein photosynthetic activity during prolonged UVexposure (Bischof et al. 2000). In addition, photo-synthetic oxygen evolution was almost completelyarrested in subtidal Saccharina latissima and L. soli-dungula from Spitsbergen after 6 h sunlamp expo-sure (Aguilera et al. 1999). In cases of reciprocity,photosynthesis in the presence of UVR is best simu-lated with respect to total cumulative UV exposure(BWFH-PE).

Due to logistic constraints, we were not able tocomplete an exposure response experiment fordeepwater kelp, and without more detailed kineticwork and longer duration experiments at bothdepths, the choice of BWF model is inconclusive. AsUVR photoinhibition and repair ⁄ recovery are likelytemperature-dependent processes (Sobrino andNeale 2007), it is also significant that the exposureresponse curve was produced in September andBWF experiments were performed in April.


Nevertheless, surface kelps at least partially have thecapability to counteract UV damage at hourly time-scales and are able to recover photosynthetic func-tion in darkness. When irradiance varies slowly,rates can be assumed to be near steady state, and anirradiance (BWFE-PE) model would be appropriate.Given the apparent slow rates of repair, however,steady state may not be achieved in more variablelight regimes (e.g., due to cloudiness and waveaction), and it may be necessary to implement atime varying prediction using a defined repair rate.This step can be done using the ‘‘R model’’ (Hiriartet al. 2002), but such an analysis requires morekinetic data than that obtained in this study.Indeed, recent work has shown that the R modelperforms better than the H model for extendedexposures of WSC phytoplankton to moderate UVR(Fritz et al. 2008). Therefore, we report the photo-synthetic response to UVR using both BWFE-PE andBWFH-PE functions and emphasize that thesebracket the full range of response dependence onrepair rate (Fig. 9). Actual response probably liessomewhere in between these two predictions.

The effect of UVR on the photosynthetic quo-tient is further evidence of depth-dependent physi-ology in kelps (Fig. 8). In this study, the average PQwas greater in kelp collected from deeper water.The PQ declined with increasing UVR in both shal-low- and deepwater L. hyperborea, but the responsewas nearly three times more severe in the deep sam-ples. The PQ response possibly results from anincrease in oxygen consumption with UVR stressand not a relative increase in carbon fixation.Increased respiration, in addition to light-enhancedrespiration (Ekelund 2000), could account forgreater oxygen consumption during UV stress.Increased oxygen consumption could result fromincreased cellular metabolism and protein synthesisassociated with repair of damaged reaction centerproteins in PSII and RUBISCO (Vincent and Neale2000), but other studies with kelp and macroalgaesuggest that no significant increases in dark respira-tion occur under UV stress (Larkum and Wood1993, Clendennen et al. 1996, Aguilera et al. 1999).Reduction in PQ could otherwise be attributed toother cellular and photochemical processes thatconsume O2, including (1) increased oxygenaseactivity of RUBISCO, that is, photorespiration; (2)O2 uptake and transformation to H2O2 in the Meh-ler reaction or other antioxidant activities in thechloroplast (Aguilera et al. 2002); and (3) UV pho-tolysis of dissolved organic matter to produce oxy-gen radicals and H2O2 in incubation seawater(Neale and Kieber 2000, Vincent and Neale 2000).Regardless of the underlying process, the BWFs cap-ture both the differential response of O2- and C-based photosynthesis and the depth-dependentdecline in PQ (Fig. 6).

Ultimately, investigators are interested in theeffect of increased UVR on primary productivity and

the ecological consequences of production losses totrophic connections and ecosystem function. Thesequestions generally require a modeling approachand knowledge of solar irradiance, how it propa-gates through the atmosphere and sea, and the bio-logical response to incident light. The L. hyperboreaBWFs provide an empirical description of UV pho-toinhibition applicable for springtime populationsat high-light and low-light acclimation states. Evenwith a simplified irradiance model without tides, therelationship allows for the estimation that near-sur-face (0.5 m) kelp production is potentiallydecreased 6% under 100 D.U. column ozone com-pared to 600 D.U. (Fig. 9), a modest amount con-sidering the depth-integrated production of thekelp forest as a whole. Yet the biological response isexpected to change with time and space. Significantquestions remain that concern the variation inBWFs with thallus age and location, life-historystage, season and microclimate, air exposure at lowtide, nutrient status, and induction of protectionand repair processes in a changing light climate(Davison et al. 2007, Roleda et al. 2007).

We extend our great appreciation to S. Fredriksen and thekelp team from the University of Oslo, Norway, and our spe-cial thanks to R. Myklebust and his gracious crew at FinnøySjøhus. We also acknowledge the contributions of J. Lempa,E. Tang, A. Banaszak, K. Jackson, and J. Keller and criticalreadings by J. Brandes, E. Buskey, T. Villareal, H. Alexander,M. Scanlin, and C. Aumack. A. Stangelmayer of PreSens isappreciated for the generous loan of the Microx TX oxygensystem. J. Biedenbach donated instrument time on the IOAnalytical TOC analyzer. This research was funded in part bythe National Science Foundation (OPP-9622483), the Smith-sonian Institute predoctoral fellowship program, and theEnvironmental Science Institute Summer Fellowship programat the University of Texas at Austin.

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Supplementary Material

The following supplementary material is avail-able for this article:

Figure S1. Comparison of four photosynthesismodels for the kelp Laminaria hyperborea collectedfrom (A) 0 m (mean low water, MLW), (B) 5 m,and (C) 15 m.

This material is available as part of the onlinearticle.

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