Vol. 364: 97-106, 2008doi: 10.3354/meps07588

MARINE ECOLOGY PROGRESS SERIES Mar Ecol Prog Ser Published July 29

OPENJVCCESS

Blue light regulation of host pigment in reef-building corals

Cecilia D'Angelo1, Andrea Denzel1, Alexander Vogt1, Mikhail V. Matz2, Franz Oswald3, Anya Salih4, G. Ulrich Nienhaus5,6, Jörg Wiedenmann1,7*

'institute of General Zoology and Endocrinology, and in stitu te of Biophysics,University of Ulm, Albert Einstein A llee 11, 89069 Ulm, Germany

2Integrative Biology, University of Texas in Austin, 1 University Station C0930, Austin, Texas 78712, USA 3Department of Internal Medicine I, Robert Koch Strasse 8, 89081 Ulm, Germany

4Confocal Bio-Imaging Facility, University of Western Sydney, Hawkesbury Campus, Locked Bag 1797, Penrith South DC,New South Wales 1797, Australia

6Department of Physics, University of Illinois at Urbana-Champaign, 1110 West Green, Urbana, Illinois 61801, USA

7Present address: National Oceanography Centre, University of Southampton, Waterfront Campus, European Way,Southampton S014 3ZH, UK

ABSTRACT: Reef-building corals harbor an astounding diversity of colorful GFP (green fluorescent protein)-like proteins. These pigm ents can easily be detected and thus may serve as intrinsic optical m arkers of physiological condition, provided that the determ inants that control their expression are well understood. H ere we have analyzed the effect of light on the regulation of major classes of GFP- like pigm ents in corals of the taxa Acroporidae, M erulinidae and Pocilloporidae. Pigment levels in the tissues of all studied species w ere observed to be tightly controlled by light. Two groups could be d is­tinguished by their distinctly different light-dependent regulation. The low-threshold group contains mainly cyan fluorescent proteins; they are expressed in considerable am ounts at very low light in ten­sities, and their tissue content increases w ith light to a maximum at a photon flux of 400 pmol n r 2 s_1. The high-threshold group includes green and red fluorescent proteins as well as non-fluorescent chromoproteins. These pigm ents are essentially absent in corals grown under very low light, but their tissue content increases in proportion to photon flux densities >400 pmol n r 2 s~T The enhancem ent of coral pigm entation is primarily dependent on the blue com ponent of the spectrum and regulated at the transcriptional level. The specific regulation patterns suggest complex functions of GFP-like proteins related to the photobiology of reef corals. Moreover, the distinct response of coral coloration to light climate promises that the pigm ent com plem ent can also be predicted in natural habitats. Our results stress the potential of GFP-like proteins as intrinsic m arkers of physiological processes, as well as overall health, in corals.

KEY WORDS: Fluorescent protein • Coral • Light-induced protein expression • Fluorescence • GFP • G reen fluorescent protein • RFP • Red fluorescent protein • Light sensing • Coral health

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INTRODUCTION

Coral reefs cover an area of 123 million km2 of tropical oceans and are am ong the most complex ecosystems in the world (Schuhmacher 1976). Hermatypic corals are primarily responsible for the formation of m odern reefs. They owe their success in oligotrophic w aters to the

light-dependent symbiosis w ith unicellular dinoflagel- late algae called zooxanthellae (Falkowski et al. 1984). Excessive light exposure, however, leads to photoinhibi­tion of the symbionts and the generation of harmful reac­tive oxygen species, especially at elevated water tem per­atures (Gleason & Wellington 1993, Brown 1997, Coles & Brown 2003, Lesser 2006). Oxidative stress can trigger

’ C o rresp o n d in g author. Email: J .W iedenm ann@ soton .ac.uk © In ter-R esearch 2008 • w w w .in t-res.com

98 M ar Ecol P rog Ser 364: 97-106 , 2008

expulsion of zooxanthellae by the host, w hich results in bleaching of the coral tissue (Brown 1997, Coles & Brown 2003, Smith et al. 2005, Lesser 2006). Mass mortality of bleached corals is frequently observed and contributes to the global degradation of coral reefs (Hughes et al.2003, Donner et al. 2007).

Corals can acclimate to strong-light environm ents by controlling the type and/or num ber of harbored algal cells and their pigm ent content, and by adjusting the complement of UV-screening, mycosporin-like amino acids (MAAs) and antioxidant molecules (Falkowski & Dubinsky 1981, H oegh-G uldberg & Smith 1989, Igle- sias-Prieto & Trench 1994, Shick et al. 1995, Rowan et al. 1997, Richier et al. 2005). A photoprotective func­tion has also been suggested for the host pigm ents that are mainly responsible for the intense bluish, green, or reddish hues of many anthozoans living in symbiosis with zooxanthellae (Kawaguti 1944, Kawaguti 1969, W iedenm ann et al. 1999, Salih et al. 2000). However, g reen fluorescent protein (GFP)-like proteins are not restricted to symbiotic anthozoans but can also be found in azooxanthellate species from dim-light hab i­tats (W iedenmann et al. 2004, Schnitzler et al. 2008). These pigm ents are proteins related to the GFP from Aequorea victoria. Fluorescent proteins (FPs) emit light in the color range from cyan to red (W iedenmann 1997, M atz et al. 1999, W iedenm ann et al. 2000, 2002,2004, Dove et al. 2001, Shagin et al. 2004, W ieden­m ann & N ienhaus 2006, Oswald et al. 2007), w hereas chromoproteins (CPs) display bright purple to blue col­ors but are non-fluorescent (W iedenmann et al. 1999, 2000, Lukyanov et al. 2000, Dove et al. 2001, Shagin et al. 2004). In contrast to GFP, its anthozoan homologues usually form hom otetram ers (Nienhaus et al. 2003,2005, 2006a,b). In recent years, proteins from the GFP family have becom e powerful tools in biomedical research. They have been em ployed as reporters of gene expression, variations of intracellular conditions (Griesbeck 2004), developm ental processes, as protein labels in living cells (W iedenmann & N ienhaus 2006) and, most recently, in super-resolution im aging beyond the diffraction barrier (Shaner et al. 2007).

The high expression levels of GFP-like proteins in coral tissue (Oswald et al. 2007) and their advanta­geous photophysical properties m ake these pigm ents extrem ely prom ising as easily accessible indicators of the health of th reatened coral reefs in a globally changing environm ent. To this end, a deep under­standing of the regulation of the pigm ent levels is of utmost importance. As yet, only very few reports have focused on the influence of habitat conditions on the expression levels of GFP-like proteins. Montastrea annularis and M. faveolata from shallow w ater habitats did not contain more FPs than specim ens from greater depths (Mazei et al. 2003). In Montastrea spp. and

Anem onia spp., expression levels of fluorescent and non-fluorescent GFP-like proteins w ere found to be genetically determ ined rather than m odulated by the light environm ent (Kelmanson & M atz 2003, Leuteneg- ger et al. 2007b, Oswald et al. 2007). Down-regulation of GFP-like proteins was most recently reported in response to stressful environm ental conditions includ­ing elevated w ater tem peratures (Leutenegger et al. 2007b, Smith-Keune & Dove 2007).

In contrast, we recently noticed that the green fluo­rescence of Acropora nobilis from the Heron Island reef flat was more intense in light-exposed parts of the branches, pointing to a light-dependent regulation of the pigm ent content. Likewise, a pink morph of Pocil­lopora damicornis showed an increase in pigm entation upon exposure to high light intensity (Takabayashi & H oegh-G uldberg 1995). The latter findings motivated us to systematically explore the expression of GFP-like proteins in reef corals upon exposure to light of vary­ing intensity and color, using 5 different species of her- matypic corals (Acropora millepora, A. pulchra, M on­tipora digitata, Hydnophora grandis, and Seriatopora hystrix) from the taxa Acroporidae, M erulinidae and Pocilloporidae. Here we establish that tissue p igm en­tation is indeed m odulated by light intensity in all these species, and that blue light triggers the regu la­tion at the transcriptional level.

MATERIALS AND METHODS

Coral material. Coral specimens w ere acquired via the G erm an aquarium trade and kept in artificial sea­w ater at 25°C and a 12 h light:12 h dark cycle in the sea w ater facility at the University of Ulm. Replicate colonies w ere incubated for at least 6 w k under m oder­ate light intensity before being subjected to experim en­tation. All regeneration of replicates and experim ents w ere perform ed w ithin the same tank, ensuring that the specim ens experienced identical w ater conditions.

Light treatments and spectroscopic analysis. The exposure of replicate coral colonies to different light con­ditions w as perform ed as described by Leutenegger et al. (2007a). Briefly, colonies w ere split into 4 groups con­sisting of 6 colonies each and exposed to different in ten­sities of white light at 80, 100, 400 or 700 pmol photons n r 2 s-1, provided by a metal halide lamp (Aqua Light), for 6 wk. Afterwards, tissue fluorescence was excited by blue (450 nm) or green (530 nm) light; photographs w ere taken w ith a Cam edia C-730 Ultra Zoom digital compact cam era (Olympus) through a yellow long pass filter (Nightsea) or a 550 nm long pass glass filter (Schott). Fluorescence spectra of each specim en w ere m easured using a Varian Cary Eclipse fluorescence spectrom eter (Varian) equipped with a fiber optic probe.

D 'A ngelo e t ai.: B lue lig h t re g u la tio n of p ig m en t ex p ress io n in corals 99

Illumination with different colors was achieved using lighting filters (Lee Filters) transmitting at -450 nm (band pass 'Zenith Blue', 80 nm full w idth at half maximum, FWHM), -512 nm (band pass, 'Dark G reen', 80 nm FWHM) and >580 nm (long pass, Primary Red (see Sup­plem entary Material, Appendix 1, available at: www.int- res.com/articles/suppl/m364p97_app.pdf). The spectra w ere decom posed using MATLAB® (Mathworks). In all experim ents using color filters, the specimens experi­enced a total photon flux of 200 pmol n r 2 s_1 w ithin the spectral range of 400-700 nm for 6 wk. The fluorescence of prim ary polyps of Acropora millepora was m easured with a modified experim ental setup as described in the Supplem entary M aterial, A ppendix 2.

C leared coral tissue extracts w ere p repared from different individuals directly after spectroscopic char­acterization of the animal tissue as described by L eutenegger et al. (2007a) and Oswald et al. (2007). Total protein content w as m easured using the BCA m ethod (Pierce). Proteins (10 pg total) w ere separated by sodium dodecyl sulphate polyacrylamide gel e lec­trophoresis (SDS-PAGE) and transferred to polyvinyl- diene difluoride (PVDF) m em branes (Amersham). The differential regulation of GFP-like protein content was further analyzed by w estern blotting, using an an ti­body raised against coral FPs (Oswald et al. 2007).

Tissue extraction, protein content and western blot analyses. C leared coral tissue extracts w ere prepared as described by Leutenegger et al. (2007a) and Oswald et al. (2007). Total protein content was m easured using the BCA m ethod (Pierce). Proteins (10 pg total) w ere separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinyldiene difluoride (PVDF) m em branes (Amer­sham). The differential regulation of GFP-like protein content was further analyzed by w estern blotting, using an antibody raised against coral FPs (Oswald et al. 2007). Protein extracts w ere p repared from different individuals directly after spectroscopic characteriza­tion of the animal tissue.

RNA preparation and RT-PCR. RNA w as prepared from tissues of Acropora millepora and A. pulchra after exposure to blue, g reen or red light for 6 wk. Using primers specific for amilFP484, amilF597, apulFP483 and apulCP584, fragm ents w ith the correct length of -700 base pairs w ere amplified.

For transcript analyses, approxim ately 100 m g of coral tissue was quickly frozen in liquid N2. Total RNA was extracted using RNAqueous® (Ambion). The RNA concentration of each sample was determ ined spectro­scopically; the RNA quality was analyzed on an ag a ­rose gel. cDNA was produced from 1 pg of RNA using 100 units of M oloney m urine leukem ia virus (MMLV) reverse transcriptase (Promega) in the presence of 8 mM dNTPs, 16 U RNasin (Promega) and 500 ng of

oligo-dT prim er (Promega). Fragm ents encoding GFP- like proteins and the housekeeping genes GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and A m E S (Acropora millepora expressed sequence, Gen- Bank accession num ber DY587236, Kortschak et al. 2003) w ere amplified from 100 ng of cDNA (primer sequences are given in A ppendix 3). The num ber of PCR cycles was individually optimized to ensure that the samples w ere analyzed during exponential am pli­fication. Equal amounts (10 pi) of each PCR reaction w ere visualized on ethidium brom ide-stained agarose gels and photographed w ith the Bio-Rad Fluor-S Mul- tilm ager (Bio-Rad). The intensity of every transcript band was quantified using Adobe Photoshop 5.0 Soft­w are (Adobe Systems) and norm alized to the 2 an a ­lyzed housekeeping genes. The amplified FP cDNAs (amilFP484/497/512//597, apulFP483 and apulCP584) w ere cloned and the encoded proteins w ere character­ized as described by W iedenm ann et al. (2005).

To study the response of corals to a changed light stimulus, specimens w ere incubated for 6 w k under red light and subsequently transferred to blue light, after which samples w ere taken over a period of 8 h. Following this, 2 colonies w ere m aintained in red light and analyzed as a negative control and 2 w ere m ain­tained in blue light for another 4 w k and their transcript levels analyzed as a positive control.

Statistical analysis. The software ANALYSE IT for Microsoft Excel, Version 1.73 (Microsoft) w as used for statistical analyses. Using Student's t-test (2 indepen­dent groups), 2-tailed p-values <0.05 w ere considered statistically significant. Highly significant differences w ere assum ed for p-values <0.01. The probability val­ues and sample num bers used for statistical analysis are listed in A ppendix 4.

RESULTS AND DISCUSSION

Identification of GFP-like proteins in coral tissue

The 5 coral species studied contained a variety of pigm ents ranging from cyan to red, which showed e lu ­tion profiles in size exclusion chrom atography typical of GFP-like proteins (W iedenmann et al. 2005). The protomers of the tetram eric assemblies have a m olecu­lar mass of -26 kDa, as determ ined by SDS-PAGE and immunoblot analysis using an antiserum raised against coral fluorescent proteins (Oswald et al. 2007). Further evidence of the GFP-like nature of these pigm ents was provided by cloning and sequencing of 2 rep resen ­tatives of Acropora pulchra and 6 of A. millepora (Table 1). The proteins amilFP484, amilFP512 and amilFP597 displayed >97% amino acids identical to those of other, previously sequenced GFP-like proteins

100 M ar Ecol P rog Ser 364: 97-106 , 2008

T able 1. H ydnophora grandis, Seriatopora hystrix , A lontipora digitata, A cropora pu lchra , a n d A . m illepora. S pec tra l p ro p e rtie s of g re e n f lu o rescen t p ro te in (G FP)-like p ro te in . 7Abs/ExA Em: position ot the absorption (Abs) or excitation (Ex) m axim um given as w ave­leng th X (nm)/Position ot the em ission (Em) m axim um given as w avelength (nm); p ro tein nam e: proteins w ere nam ed according to Cox et al. (2007); C hrom ophore structure: basic structural features w ere ded u ced from the absorption of the d en atu red p rotein in 0.1 N N aO H (Gross et al. 2000, Osw ald e t al. 2007); CFP: cyan fluorescent protein; CP: chrom oprotein; OFP: orange fluorescent protein;

RFP: red fluorescent pro tein

Pigm entclass

7 | -,//-| i, (nm)

Proteinnam e

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GenBank accession no.

Hydnophora grandis CFP 443/492a hgraFP492 GFP -Seriatopora hystrix CP 562a shysCP562 dsRed -Alontipora digitata CFP 470/486a mdigFP486 GFP -

GFP 508/514a mdigFP514 GFP -OFP 556/572a mdigFP572 dsRed -

Acropora pulchra CFP 420/483b apulFP483 GFP apulFP483 EU709806CP 584b apulCP584 dsRed apulCP584 EU709807

Acropora millepora CFP 420/484b amilFP484 GFP amilFP484 EU709808GFP 477/497b amilFP497 GFP amilFP4.97 EU709809GFP 500/512b amilFP512 GFP amilFP512 EU709810RFP 558/597b amilFP597 dsRed amilFP5.97 EU709811

Peak positions w ere de term ined irom spectra aof GFP-iike proteins puriiied irom tissue extracts by size exclusion chrom atography, or bof puriiied proteins recom binantly expressed in Escherichia coli

from A. millepora (Cox et al. 2007) (GenBank accession num bers DQ206400, AY646073 and AY646070). The identified pigm ents represent major color classes of GFP-like proteins, nam ely cyan, green, orange and red fluorescent proteins and also non-fluorescent pink or purple-blue chromoproteins (Table 1).

Effect of light intensity on GFP-like protein expression

Coral pigm entation was analyzed after subjecting colonies of each species for 6 w k to 4 different light intensities (photon flux densities), w hich we classify as very low (80 pmol n r 2 s_1), low (100 pmol n r 2 s_1), m od­erate (400 pmol n r 2 s_1) and high (700 pmol n r 2 s_1). As indicated by the gain of w et w eight of coral colonies, grow th rates under low light reached 20 to 40% of the rates determ ined for specimens under m oderate light (data not shown). Consequently, colonies from low light treatm ents w ere smaller than their counterparts grown under m oderate light (Fig. 1A-E).

Colonies illuminated with low-intensity light appeared brownish, w hereas those growing under m oderate light displayed distinctive hues ranging from blue over green to reddish. The increase in pigm entation of m oderate-light-treated specimens was striking w hen the tissue fluorescence was excited w ith blue or green light (Fig. 1A-E).

The am ount of pigm ents was quantified by m easur­ing the emission of FPs directly on the corals or by collecting absorption spectra of size-fractionated tis­sue extracts. In 4 cases, varying light treatm ents w ere observed to m odulate the spectral am plitude w ithout changing the overall shape (Fig. IF —I). An exception was the change in the ratio of the 2 em is­sion bands at -490 and -510 nm of Acropora m ille­pora upon light intensity variation (Fig. 1J). These overall emission spectra taken from tissues can be decom posed using the spectra of the 3 recom binant proteins amilFP484, amilFP497 and amilFP512 (Tablet) cloned from one of the individuals used in the experiments.

Fig. 1. H ydnophora grandis, Seriatopora hystrix, A lontipora digitata, A cropora pulchra , a n d A. m illepora. Effect of ligh t in tensity on th e content of g reen fluorescen t p ro te in (GFP)-like proteins: (A-E) Im ages of corals k e p t for 6 w k in p h o to n flux d ensities of 100 pm ol rrT2 s_1 (low light, L) or 400 pm ol rrT2 s_1 (m oderate light, M). All spec ies w e re p h o to g rap h e d u n d e r w h ite lig h t illum ination (upper panels). Lower p an e ls in (A,C,D) a n d m idd le p a n e l in (E) depict tissue fluo rescence im ag ed u n d e r b lu e ligh t excitation. Lower p an e l in (E) show s re d tissu e flu o rescen ce excited b y g re e n light. (F -J) F luo rescence an d /o r ab so rp tion (Abs) sp ec tra a fte r lig h t exposure (m ean + SD from 12 to 33 m easu rem en ts). M axim um p e a k in every g ra p h w as se t to 100 w h ile th e o thers w e re n o rm alized a cco rd ­ingly. F luo rescence w as m ea su re d d irectly on th e rep lica te colonies, w h e reas th e ch rom opro tein absorp tion sp ec tra of S. hystr ix (G) an d A . pulchra (I) w e re de te rm in ed from th e p e a k fraction of size-fractionated tissue extracts an d norm alized to th e to ta l p ro te in con­ten t of th e sam ples. T he photon fluxes of th e trea tm en ts (pmol r r r 2 s_1) a re given in th e keys. (K-O) T issue fluorescence an d /o r abso rp ­tion at th e p e a k w av elen g th (nm) specified in th e key of e ach graph. C yan an d g re en fluorescence in tensities in (O) re p re se n t resu lts from th e spectral decom position. "H ig h ly significant difference (p < 0.01, in d ep en d en t t-test) com pared to th e closest low er photon flux

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Based on the response to light intensity, 2 different groups of proteins could be distinguished: a low- threshold group that includes all cyan fluorescent proteins (CFPs) in the present study, mdigFP514, mdigFP572 and amilFP497, and a high-threshold group consisting of amilFP512, amilFP597, and all CPs from Acropora pulchra and Seriatopora hystrix. Proteins of the high-threshold group w ere essentially absent w hen the light intensity fell below 100 pmol photons n r 2 s_1, and they accum ulated nearly in proportion to light in ­tensity up to 700 pmol photons n r 2s% In contrast, rep ­resentatives of the low-threshold group w ere expressed in considerable amounts at low light intensity, reaching their highest expression levels at a photon flux of <400 pmol photons n r 2 s_1 and either saturating or even decreasing at higher light intensity. These results dem onstrate unam biguously that the expression of GFP-like proteins in these corals is tightly controlled by light. Consequently, a variety of genes encoding GFP- like proteins regulated by environm ental conditions can be clearly distinguished from another set constitu- tively expressed in the coral tissue (Kelmanson & Matz 2003, Leutenegger et al. 2007b, Oswald et al. 2007).

Effect of light color on GFP-like protein expression

Exposure of replicate colonies of each species to red, g reen or blue light at a photon flux of 200 pmol n r 2 s_1 showed that corals incubated w ith blue light had the highest amounts of GFP-like proteins. Pigments of the low-threshold group w ere consistently found in corals growing in red light and reached >50% of the ex ­pression levels achieved w ith green light irradiation (Appendix 5). In contrast, representatives of the high- threshold group w ere essentially undetectable in corals grown under red light, and green-light-exposed speci­m ens did not reach 50 % of the levels observed under blue light.

W estern blot analyses to further characterize the dif­ferential regulation of GFP-like protein content showed that, in all exam ined coral species, the antibody revealed the -26 kD a-band typical of GFP-like pro­teins (Fig. 2A-F). The observed band intensities fur­ther confirmed that the concentrations of FPs or CPs w ere always highest for b lue-light-treated specimens.

The differences in pigm ent content betw een corals incubated under low and m oderate light are similar to those betw een green and blue-light-exposed speci­m ens (Appendix 5). The m etal halide lamps used to illuminate the corals emit -50 % of the photons in the blue spectral region. Therefore, corals subjected to low or m oderate light treatm ent experienced a photon flux of -50 or -200 pmol n r 2 s_1 of blue light, respectively. The green filter utilized in our experim ents still trans­

mits -25% of the photons, corresponding to a photon flux of -50 pmol n r 2 s-1, in the blue region of the spec­trum (Appendix 1). Therefore, the light-driven accum u­lation of GFP-like proteins observed upon g reen light exposure is likely due to residual blue light passing the green filter. The expression of GFP-like proteins in m em bers of the low-threshold group under red light suggests that a constitutive basal expression exists.

Regulation of GFP-like protein expression in corals

Sem iquantitative RT-PCR was perform ed to d e te r­mine w hether the pigm ent content was regulated at the transcriptional level. As expected from the similarity betw een cyan and green FPs from Acropora millepora that w ere sequenced earlier (Cox et al. 2007), PCR with amilFP484 primers also led to amplification of cDNAs encoding amilFP497 and amilFP512 (Fig. 3A,B). Low transcription levels of the amilFP484/497/512 group and amilFP597 w ere detected in A. millepora exposed to red light. A major increase in the am ount of FP tran ­scripts occurred w ith green light, and a further incre­m ent w ith blue light exposure, indicating a regulation of the tissue pigm ent content at the transcriptional level. The transcript of the low-threshold protein apul- FP483 was already present in red-light-illum inated A. pulchra-, its am ount increased to a maximum level for green-light-exposed specimens. This result agrees well w ith the small changes in tissue fluorescence due to proteins of the low-threshold group for illumination w ith blue and green light. In contrast, the transcript level of the high-threshold protein apulCP584 was significantly higher upon exposure to blue light.

RT-PCR analysis was performed to determ ine the time the corals require to increase transcript levels in re ­sponse to a changed light stimulus. FP transcripts accu­m ulated at very low levels at time point 0 (i.e. im m edi­ately before exposure to blue light, Fig. 3C,D) and in specimens treated for another 8 h w ith red light. In con­trast, transcript levels w ere significantly increased after 8 h of exposure to blue light. However, after 8 h of stimulation, the maximal transcript level was not yet reached, as shown by comparison with the levels in the positive control group after 4 w k of blue-light exposure. This rather slow increase suggests that the accum ula­tion of pigm ents is a long-term adaptive process.

Fluorescence in primary polyps of Acropora m illepora

Further investigations w ere conducted to test w hether the presence of endosymbiotic algae is mandatory for the regulation of GFP-like proteins. Examination of azoo- xanthellate primary polyps (exposed to red or blue light

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RL GL BL RL GL

Fig. 2. H ydnophora grandis, Seriatopora hystrix, A lontipora d ig ­itata, A cropora pulchra , an d A. millepora. Effect of ligh t en erg y (color) on th e accum ula tion of g re e n fluorescen t p ro te in (GFP)- like proteins. R eplicate colonies w ere exposed to re d (RL), g reen (GL) or b lu e lig h t (BL) (photon flux of 200 pm ol m -2 s-1). Left g ra p h of e ac h p a n e l gives th e fluo rescence em ission or ab so rp ­tion (Abs) of fluo rescen t p ro te in s (FPs) a n d ch rom atopro te ins (CPs), respectively (corresponding w aveleng ths in n m ind icated in th e keys). Bars rep re sen t th e m ean (+SD) of 12 to 33 m ea su re ­m ents. Significant (*p < 0.05) or h ig h ly significant (**p < 0.01) differences, as calculated by indep en d en t t-tes t b e tw ee n GL and RL exposed specim ens (asterisk on th e G L bars), or b e tw ee n BL a n d GL exp o sed spec im ens (asterisks on th e BL bars). R ight g rap h of each p an e l displays th e resu lts of w este rn blot analyses of tissu e ex tracts p re p a re d a t th e e n d of th e experim en t. T h ree in d e p e n d e n t m easu rem en ts of th e op tical density of th e b a n d s specific for G FP-like p ro te in s (insets) w e re m ade . D a ta a re m ean s (+SD), w ith v a lu es from BL ex p o su re se t to 100 a n d all

o th er d a ta n o rm alized accord ing ly

im m ediately after metamorphosis) by fluorescence m i­croscopy after 5 d of illumination revealed a significant increase of tentacle fluorescence in the green spectral region (Fig. 4). This suggests that photoreceptors of the host are involved in the regulation of pigm ent levels. Perception of blue-green light by the ectodermal, zoo­xanthellae-free cells of M ontastrea cavernosa and Eus­milia fastigiata was dem onstrated earlier (Gorbunov & Falkowski 2002), and only very recently, cryptochromes w ere identified in Acropora millepora, providing p u ta­tive blue-light photoreceptors (Levy et al. 2007).

Implications for biological function

Among the known FPs and CPs, only the absorption properties of CFPs spectrally m atch the major absorp­tion band of chlorophyll a and c at -430 nm, m aking them suitable for effective shielding of the photosyn­thetic system of the zooxanthellae. Our results have shown that the amounts of these proteins in coral tissue increased at light intensities > 80 pmol m-2 s-1 and satu­rated at -400 pmol m-2 s-1. This response is in striking agreem ent with photosynthesis-irradiance curves of zooxanthellae from different coral species that show a similar increase and saturation at 400 pmol m-2 s-1 (Falkowski et al. 1990, Smith et al. 2005). However, the detrim ental effects of excessive light levels becom e ap ­parent once photosynthesis is saturated and the gener­ation of reactive oxygen species (ROS) increases (Smith et al. 2005, Lesser 2006). If CFPs and other m em bers of the low-threshold group w ere exclusively involved in a photoprotective function, an upregulation of these pro­teins should be expected, especially at photon fluxes higher than 400 pmol m-2 s-1. Our data do not favor such a scenario, so these pigm ents might also play other roles in the interaction of the coral host and its algal symbionts. Possible functions might include sensing of

104 M ar Ecol P rog Ser 364: 97-106 , 2008

amilFP597amilFP

484/497/512GAPDH

2.5-,

QOCD>ma)cc

2.0

1.5

1.0

0.5

0.0

----- --

□ amilFP597 □amilFP484/497/512

BapulCP584apulFP483

GAPDH3.5-

3 .0-

I § 2-5-O è 2 . 0 -Q. ro

• -i c;•q; OC 1 *5-

1 .0 -

0.5-

0 .0 -

□ apulCP584□ apulFP483

RL GL BL RL GL BL

1.6-1

1.4.

1.2-2 Qo O 1.0-.o. ID> 0.81 m

(DCC 0.6-

0.4.

0.2-0.0

Time (h)0 2 4 8BLRL

amilFP597amiiFP

484/497/512GAPDH

I iamilFP597 □ amilFP

484/497/512

JZLJL

2 4Time (h)

D

S o§ 5

Q_ ™CDcr

1 . 6 .

1.4.

1 . 2 .

1 . 0 .

0 . 8 .

0 . 6 .

0.4.

0 . 2 -

0.0

Time (h)0 2 4 8BLRL

apulCP5841 apulFP483|

GAPDH

□apu!CP584□apuiFP483

Time (h)Fig. 3. A cropora pu lchra a n d A cropora m illepora. S em i-quan tita tive RT-PCR analysis of g re e n fluo rescen t p ro te in (GFP)-like p ro ­te in transcrip ts: (A,B) S em i-q u an tita tiv e analysis of tran scrip t levels a fte r trea tm e n t w ith re d (RL), g re e n (GL) or b lu e ligh t (BL) (photon flux of 200 pm ol m 2 s-1). R ep resen ta tiv e e th id ium b ro m id e -s ta in ed gels in th e u p p e r p a r t of th e p a n e ls show th e b a n d s c o rresp o n d in g to am plified tran scrip ts of G FP-like p ro te in s a n d th e h o u se k e e p in g g e n e GAPDH. As gel analysis of tran scrip t lev ­els of th e h o u se k e ep in g g e n e A m E S y ie ld ed re su lts com parab le to GAPDH, th e re sp ec tiv e p a n e ls w e re om itted for sp ace reasons. D a ta a re m ean s (+ SD) of th e in ten sitie s of re p lica te b a n d s from 6 in d e p e n d e n t m easu rem en ts , n o rm alized to th e co rresp o n d in g b a n d s of th e h o u se k e ep in g g e n es G A P D H a n d A m E S. O p en a n d filled circles d ep ic t v a lu es o b ta in ed b y n o rm aliza tion to G APD H a n d to A m E S , respec tive ly . (C,D) A . m illepora a n d A . pu lchra sp ec im ens tran s fe rre d from RL to BL (tim e p o in t 0 h). Sam ples w e re ta k e n after 2, 4 a n d 8 h of BL ex p o su re a n d u se d for RT-PCR analysis. C ontrol sp ec im ens in cu b a ted for 8 h u n d e r RL a n d 4 w k

u n d e r BL w e re p ro cessed in paralle l. OD: optical density, *’h ig h ly sign ifican t d ifferen ces (p < 0.01, in d e p e n d e n t i-test)

photosynthetic activity of zooxanthellae, support of algal productivity by the addition or removal of rate- limiting substances, or accum ulation of the pigm ents for amino acid or nitrogen storage purposes. A close spatial association with zooxanthellae required for such functions was previously observed for certain coral FPs (Mazei et al. 2003, Oswald et al. 2007).

The light-induced upregulation of GFP-like proteins from the high-threshold group resem bles the increas­

ing accum ulation of well known photoprotectants such as MAAs, ß-carotene, xanthophylls or m elanins (Shick et al. 1995, Bandaranayake 2006). However, none of the m em bers of the high-threshold group absorbs light at w avelengths that would provide an efficient shield­ing of the photosynthetic apparatus of the zooxanthel­lae. The screening effect could be enhanced if these proteins w ere to act as acceptors in fluorescence reso­nance energy transfer (FRET) (Gilmore et al. 2003, Cox

D 'A ngelo e t ai.: B lue lig h t re g u la tio n of p ig m en t ex p ress io n in corals 105

250-

200 -

150-

100 -

50-

0-Red light Blue light

Fig. 4. A cropora m illepora. U pregu lation of g re en fluorescen t p ro te in (GFP)-like p ro te in expression in p rim ary polyps. F luorescence p h o to m icrographs of ten tac les tre a te d w ith (A) re d a n d (B) b lu e light. (C) G re en flu o rescen ce (m ean + SD) of ten tac les from re d an d b lu e lig h t- trea te d po lyps from 28 in d e p e n d e n t m easu rem en ts . " H ig h ly sign ifican t d ifferences (p < 0.01, in d e p e n d e n t t-test)

et al. 2007). Alternatively, protection from harmful light effects could also involve an as yet undiscovered function, for instance ROS scavenging (Bou-Abdallah et al. 2006) or antimicrobial defense of corals becoming vulnerable under light stress. Interestingly, in the case of Acropora pulchra, the strong accum ulation of apulCP584 was limited to the tips of the branches. The apical polyps are essentially free of zooxanthellae, but are the areas w ith the highest grow th rates (Fang et al. 1989). The constrained localization of some FPs in zooxanthellae-free parts of certain corals (e.g. skeletal ridges) has been noticed earlier (Mazei et al. 2003). Therefore, GFP-like proteins, particularly rep resen ta­tives of the high-threshold group, could play a role in metabolic pathw ays related to coral growth.

Coral color as an indicator of environmental conditions

In this work, w e have established the tight regu la­tion of GFP-like proteins in corals from different taxo­nomic groups in response to the intensity and spectral composition of the irradiating light. Consequently, the pigm ent com plem ent of coral species that express rep ­resentatives of both high- and low-threshold groups, might serve as a predictable 'ratiometric' indicator of light conditions in the habitat. In conclusion, coral p ig ­m entation appears promising as a sensitive indicator of environm ental conditions. Future work will further refine our know ledge about the influence of diverse environm ental factors on the expression levels of GFP- like proteins in reef-building corals.

A ck n o w led g e m e n ts . T he au tho rs th a n k A. K obitski, Institu te of B iophysics, U niversity of Ulm, for decom position of spec tra , an d V. B eltran Ram irez, A. Reyes a n d D. J. M iller, Jam es Cook U niversity, Tow nsville, A ustra lia , for ex p ert advice on h a n ­d ling coral larv ae. This w o rk w as su p p o rted b y th e D eu tsche F orsch u n g sg em ein sch aft (SFB497/B9 to F.O., SFB497/D2 an d SFB569/A4 to G.U.N., an d W Í1990/2-1 to J.W.), F luo rescence

A pplications in B iotechnology an d Life Sciences, A u stra lia (to A.S. et al.), N ationa l In stitu tes of H e a lth (GM 66243 to M.M.) a n d th e D eu tsch er A k ad em isch er A u stau sch D ienst (to J.W.).

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S u b m itted : F ebruary 8, 2008: A ccep ted : M a y 27, 2008 Proofs re c e ive d fro m author(s): J u ly 16, 2008