It’s cheap to be colorful Anthozoans show a slow turnover of GFP-like proteins Alexandra Leutenegger 1, *, Cecilia D’Angelo 1, *, Mikhail V. Matz 2 , Andrea Denzel 1 , Franz Oswald 3 , Anya Salih 4 , G. Ulrich Nienhaus 5,6 and Jo ¨ rg Wiedenmann 1 1 Institute of General Zoology and Endocrinology, University of Ulm, Germany 2 Integrative Biology, University of Texas in Austin, TX, USA 3 Department of Internal Medicine I, University of Ulm, Germany 4 Electronic Microscopy Unit, University of Sydney, Australia 5 Institute of Biophysics, University of Ulm, Germany 6 Department of Physics, University of Illinois at Urbana-Champaign, IL, USA The vivid blue, green, pink, orange or red hues of anthozoans are mainly due to fluorescent proteins (FPs) and nonfluorescent chromoproteins (CPs) [1–14]. These pigments are homologs of green fluorescent protein (GFP), which acts as secondary emitter in the bioluminescence reaction in Aequorea victoria [15]. For GFP-like proteins in nonbioluminescent anthozoans, a photoprotective function has been suggested [2,16–19]; the underlying mechanism, however, remains con- troversial [20,21]. Whereas some FPs have spectral properties that appear to be inappropriate for photo- protecting tissue by modulating the intracellular light climate [20], other FPs are spectrally well suited to fluorescence energy transfer and dissipation of light energy via radiative and nonradiative pathways [19,21–23]. Alternatively, an antioxidant function has recently been suggested [24]. The distinct tissue-specific expression of FPs, the occurrence in anthozoans from habitats without light stress, and the separate evolutionary histories of differently colored FPs and CPs tend to support multiple specific functions for these proteins [3,5,12,19,25–27]. GFP-like proteins contribute a surprisingly high fraction to the overall soluble protein content of FP-expressing tissues in anthozoans, ranging from 4.5% in the coral Montastrea cavernosa to over 7% in Keywords coral pigments; green fluorescent protein; photoconversion; protein half-life; protein metabolism Correspondence J. Wiedenmann, Institute of General Zoology and Endocrinology, University of Ulm, Albert Einstein-Allee 11, 89069 Ulm, Germany Fax: +49 731 502 2581 Tel: +49 731 502 2591 ⁄ 2584 E-mail: joerg.wiedenmann@uni-ulm.de *These authors contributed equally to this work (Received 17 February 2007, revised 10 March 2007, accepted 12 March 2007) doi:10.1111/j.1742-4658.2007.05785.x Pigments homologous to the green fluorescent protein (GFP) contribute up to  14% of the soluble protein content of many anthozoans. Maintenance of such high tissue levels poses a severe energetic penalty to the animals if protein turnover is fast. To address this as yet unexplored issue, we estab- lished that the irreversible green-to-red conversion of the GFP-like pig- ments from the reef corals Montastrea cavernosa (mcavRFP) and Lobophyllia hemprichii (EosFP) is driven by violet–blue radiation in vivo and in situ. In the absence of photoconverting light, we subsequently tracked degradation of the red-converted forms of the two proteins in coral tissue using in vivo spectroscopy and immunochemical detection of the post-translational peptide backbone modification. The pigments displayed surprisingly slow decay rates, characterized by half-lives of  20 days. The slow turnover of GFP-like proteins implies that the associated energetic costs for being colorful are comparatively low. Moreover, high in vivo stability makes GFP-like proteins suitable for functions requiring high pigment concentrations, such as photoprotection. Abbreviations chl., chlorophyll; CP, chromoprotein; FP, fluorescent protein; GFP, green fluorescent protein. 2496 FEBS Journal 274 (2007) 2496–2505 ª 2007 The Authors Journal compilation ª 2007 FEBS Lobophyllia hemprichii and up to 14% in Acropora nobilis (Wiedenmann, unpublished data) [27]. These results suggest that the animals face considerable energy costs maintaining such high expression levels, at least, if protein turnover is fast. To date, no kinetic data are available on the degradation of GFP-like pro- teins in anthozoans. In vitro, GFP and its homologs are known to be extraordinarily resistant to heat, detergents, chaotropes, reducing agents, extremes of pH and protease activity [3,15]. This stability results from the rigid b-can fold conserved among different members of this protein family, which comprises 11 antiparallel b-sheets surrounding a central helix [6,28–36]. The chromophore resides near the geometric center of the molecule and is stabilized by the sur- rounding protein scaffold. In contrast to GFP, which forms dimers only at concentrations > 1 mg Æ mL )1 [15,37], its anthozoan homologs are most often seen to form tightly packed tetramers, which might further sta- bilize the molecular structures. Degradation of recom- binant GFP in cultured mouse cells follows first-order kinetics with a half-life of  26 h [38]. In dividing human embryonic kidney cells, we found that the red fluorescence of the anthozoan EosFP was detectable for more than 9 days, whereas in Xenopus laevis embryos the protein could be tracked for more than 14 days [6,39,40]. EosFP from L. hemprichii and Kaede from Trachy- phyllia geoffroyii belong to the green-to-red photocon- vertible FPs [41,42]. They exhibit similar green fluorescence emission spectra, peaking at 516 and 518 nm, respectively, and following irradiation with  400 nm light, the proteins can be irreversibly switched to emit red fluorescence, with peak emissions at 581 and 582 nm, respectively. Light-driven photo- conversion involves cleavage of the polypeptide chain into two fragments of  20 and  8 kDa [6,29,40,42]. Therefore, the amount of both fragments is indicative of the amount of the red form. Cleavage occurs on a submillisecond time scale, as shown for single EosFP molecules [6]. Analysis of the crystal structure of EosFP showed that the b-can fold and the tetrameric protein assembly remain unperturbed by internal frag- mentation [29]. Proteins belonging to the green-to-red photoconverting GFP-like proteins have also been found as major colorants in the scleractinian corals M. cavernosa, Scolymia cubensis, Catalaphyllia jardinei, the corallimorpharian Ricordia florida and the alcyo- narian Dendronepthya sp. [10,12,27]. An unusual, green-to-orange variant was recently described from an orange color morph of L. hemprichii [27]. Photoconversion offers a strategy to precisely and noninvasively monitor protein degradation in the cell. Cells expressing one of these photoactivatable proteins are irradiated with activating light at a specific time, and subsequently, the red form can be monitored inde- pendently of the cellular pool of newly synthesized, green fluorescent pigment. Consequently, degradation of the photoconverted molecules can be followed via the decay in red fluorescence. Obviously, light expo- sure has to be such that photoconversion is avoided and photobleaching is minimized. In this study, we used this approach to measure the half-lives of green- to-red photoconvertible proteins in corals in order to obtain insights into GFP-like protein turnover in anthozoans. To this end, we first had to verify that green-to-red conversion of mcavRFP and EosFP occurs only via photoinduction and not in any other way in the tissues of the corals they originate from, M. cavernosa and L. hemprichii. Results and Discussion Photoconversion in vivo and in situ To measure protein degradation in red-converted forms of mcavRFP and EosFP by monitoring the decay in red fluorescence in the tissues of red morphs of M. caver- nosa and L. hemprichii , we first had to ensure that green-to-red conversion is solely light-driven in the ani- mals and cannot be mediated by any other mechanism. To this end, colonies of both species were exposed to a photon flux of 100 lmolÆm )2 Æs )1 or kept in the dark for 30 days. In light-exposed M. cavernosa colon- ies, the green fluorescence of the contracted tentacles shines through the overlaid red-fluorescent tissue, resulting in a yellowish fluorescence of the polyp cen- ters, whereas the polyps and the coenosarc show a bright red fluorescence (Fig. 1A). In contrast, the coe- nosarc of colonies kept in the dark fluoresce only in green (Fig. 1A). This difference in the fluorescence images correlates with the different shapes of the emis- sion spectra. In light-exposed animals, the red fluores- cence at 582 nm is more than three times as intense as the green emission at 516 nm (Fig. 1C). In contrast, colonies incubated in the dark display a slightly higher green than red fluorescence (Fig. 1C). A similar color change was observable for L. hem- prichii. Animals exposed to light showed red fluores- cence after excitation with blue light (Fig. 1B). In dark-treated corals, the red fluorescence intensity at 581 nm was reduced compared with the green fluores- cence at 516 nm and, consequently, the fluorescence of the animals appears yellowish (Fig. 1B,D). Tissue extracts of M. cavernosa and L. hemprichii from light and dark treatments were subjected to A. Leutenegger et al. Turnover of GFP-like proteins FEBS Journal 274 (2007) 2496–2505 ª 2007 The Authors Journal compilation ª 2007 FEBS 2497 AB CD E G KL HI F J M Turnover of GFP-like proteins A. Leutenegger et al. 2498 FEBS Journal 274 (2007) 2496–2505 ª 2007 The Authors Journal compilation ª 2007 FEBS immunoblot analysis using a polyclonal antiserum raised against mcavRFP⁄ EosFP [27]. In light-exposed M. cavernosa, a strong band corresponding to the  20 kDa fragment of the photoconverted, red form is clearly visible. The nonconverted, green fluorescent form appears only as a comparatively weak band cor- responding to a molecular mass of  25 kDa (Fig. 1E). Conversely, the  25 kDa band is more pronounced than the  20 kDa fragment for specimens kept in the dark, suggesting that reduction of the red-converted form is accompanied by an accumulation of the non- converted green form of mcavRFP. This observation is in good agreement with the color change seen in the animals (Fig. 1A). Similarily, in L. hemprichii, the reduction in red tissue fluorescence is correlated with a decrease in the intensity of the  20 kDa fragment (Fig. 1F). However, no accumulation of nonconverted protein was observed for this species. Following dark treatment, a colony of M. cavernosa was exposed to  400 nm light. Within 3 h, the fluores- cence of the coenosarc and polyps changed from green to red (Fig. 1G–I) and, accordingly, the fluorescence spectra show a relative increase in the 582 nm peak compared with the 516 nm maximum (Fig. 1J). The majority of eggs and, subsequently, embryos of M. cavernosa show predominantly green fluorescence peaking at 516 nm when kept in the dark following their release from the mother colony (Fig. 1K). Irradi- ation with violet–blue light on the fluorescence micro- scope induces photoconversion and gives rise to red fluorescence with a maximal emission at 582 nm (Fig. 1K–M). It is interesting to note that the ratio of green-to-red fluorescence in M. cavernosa eggs is a faithful indicator of the exposure of the mother colony to violet light around 400 nm. In L. hemprichii, no photoconversion was noticed in the dark-treated animals upon irradiation with violet light, which is in good agreement with the lack of a visible accumulation of the nonconverted green form of EosFP. However, a small amount of green-to-red photoconversion was seen when irradiating macerated samples of ectodermal tissue with 366 nm light on the fluorescence microscope (data not shown). To examine whether light intensity affects the level of FP photoconversion in M. cavernosa tissues, we compared animals kept in either weak or strong light for 4 weeks. Whereas the tissue content of zooxanthel- lae pigments from the different light climates shows considerable changes [27], the photograph of the red fluorescence of colonies in Fig. 2A reveals that the red tissue fluorescence remains identical. Both emis- sion spectra show maxima at 516 and 582 nm (Fig. 2B), and the overall emission from both peaks and thus the pigment content of strong- and weak- light treated animals are also identical (Fig. 2C). In accord with these results, immunoblot analysis revealed identical amounts of mcavRFP, as judged by the intensity of the diagnostic  20 kDa fragment (Fig. 2D). From the identical results obtained with animals exposed to weak and strong light, we con- clude that the pool of green-to-red photoconverting proteins in M. cavernosa is completely converted at a photon flux of 100 lmolÆm )2 Æs )1 . Therefore, emission at 516 nm may arise from the presence of another FP, the so-called long-wave GFP described previously for M. cavernosa [12,27]. We note that, under our experimental conditions, light intensity does not regu- late FP expression levels. This is in good agreement with our finding that mcavRFP mRNA can be detec- ted in tissue even after 4 weeks of dark treatment (data not shown). Also, the tissue content of GFP in M. cavernosa and M. faveolata is not significantly altered in a depth-dependent light gradient [20]. Taken together, our results provide clear evidence that green- to-red conversion of mcavRFP and EosFP is a light- driven process in vivo and in situ. Fig. 1. Effect of prolonged darkness on the presence of GFP-like proteins in M. cavernosa and L. hemprichii (A–F). Fluorescence images of (A) M. cavernosa and (B) L. hemprichii after 30 days with and without light. Fluorescence was excited with blue light and photographed through a yellow long-pass filter. Average fluorescence spectra of the coenosarcs of (C) M. cavernosa and (D) L. hemprichii after light and dark treatment. Spectra were normalized to 1 at the maximum of the green form at 516 nm. Error bars indicate standard deviations calcula- ted from six independent measurements. Immunoblot analyses of tissue extracts isolated from (E) M. cavernosa and (F) L. hemprichii after light and dark treatment. For each treatment, four replicate samples are shown. Dark-treated animals show a reduced content of the  20 kDa fragment indicative of the red form of green-to-red converting proteins. Only M. cavernosa shows an increase in the intensity of the 25 kDa band in the dark. Photoconversion of mcavRFP in situ (G–M). A colony of M. cavernosa was kept in the dark for 30 days and then continuously irradiated with  400 nm light. Fluorescence images were taken (G) at the start of the experiment, after (H) 1 and (I) 3 hours. Fluorescence emission spectra of the coenosarc were measured at the same time intervals (J). Six independent spectra were aver- aged and normalized to 1 at 516 nm. Error bars indicate the standard deviations. (K) Fluorescence micrograph of M. cavernosa embryos. The two yellowish embryos were previously photoconverted on the fluorescence microscope and mixed with unconverted embryos. The yel- low color derives from an increased amount of red fluorescence, as is apparent from (L), where the image was taken in the red channel of the fluorescence microscope. (M) Fluorescence spectra of the embryos recorded during photoconversion. Over time, the red fluorescence at 582 nm increases relative to the green fluorescence at 516 nm. A. Leutenegger et al. Turnover of GFP-like proteins FEBS Journal 274 (2007) 2496–2505 ª 2007 The Authors Journal compilation ª 2007 FEBS 2499 Kinetic analysis of protein degradation To determine the half-lives of the red forms of GFP- like proteins mcavRFP and EosFP in situ, colonies of M. cavernosa and L. hemprichii were kept in the dark for 30 days so as to prevent light-induced photocon- version of newly synthesized green fluorescent proteins. During this time, red tissue fluorescence, and thus pro- tein content, was monitored spectrometrically. We found that red-converted mcavRFP and EosFP decay very slowly, with half-lives of 20 ± 2 days (Fig. 3A,B). By contrast, control animals, which were exposed to light for the same time interval, displayed constant overall red fluorescence of the tissue. Prolonged darkness is stressful for the corals because the light-deprived symbiont reduces the transfer of photosynthetic products to the host. Indeed, we observed a partial loss of zooxanthellae during this time (data not shown). To assess the influence of this stressful condition on the protein degradation kinetics, we investigated the response of different colonies of M. cavernosa to different light colors (red, green and blue) at a constant photon flux of 200 lmolÆm )2 Æs )1 A B DC Fig. 2. Independence of red tissue fluorescence emission from the treatment with different light intensities. For 4 weeks, different col- onies of M. cavernosa were exposed to weak (WL) or strong (SL) light. (A) Red tissue fluorescence of representative colonies kept under weak and strong light photographed through a Schott filter glass (550 nm long-pass). Fluorescence was excited by irradiation with 530 nm light. (B) Emission spectra of the tissue fluorescence after weak and strong light treatment with excitation at 460 nm. The graphs show averages of 12 independent measurements, error bars indicate standard deviations. (C) Comparison of the fluores- cence intensity of the coral tissue recorded at the green (516 nm) and red (582 nm) emission peaks. (D) Mean optical density for the  20 kDa band corresponding to the red-emitting form of mcavRFP as deduced from immunoblotting analysis. The immunoblot is shown as an inset. The error bars show the standard deviations of four independent tissue extracts from colonies incubated under weak and strong light, respectively. Fig. 3. Kinetics of red fluorescence emission determined in situ on (A) M. cavernosa (mcavRFP) and (B) L. hemprichii (EosFP). The dia- grams show the decay in tissue fluorescence at 582 nm (mcavRFP) and 581 nm (EosFP) over 25 days. The diagrams show the medians of 12 measurements per time point; error bars display the first and third quartiles. Data from dark-treated animals were fitted to expo- nential decays. No significant changes in fluorescence intensity were detected for light-treated animals. By contrast, significant dif- ferences (P<0.01) between animals from dark and light treat- ments were determined at day 25. Turnover of GFP-like proteins A. Leutenegger et al. 2500 FEBS Journal 274 (2007) 2496–2505 ª 2007 The Authors Journal compilation ª 2007 FEBS for 5 weeks. Blue light (k max ¼ 450 nm) is effective for both photosynthesis of zooxanthellae and photocon- version of GFP-like proteins. Green (k max ¼ 512 nm) and red (k max > 580 nm) light can be utilized for pho- tosynthesis of zooxanthellae due to absorption by either the carotenoid peridinin (green light) or chloro- phyll (red light), but both colors are essentially ineffec- tive for photoconversion of mcavRFP. During the 5-week experiment, tissue fluorescence in corals exposed to different light colors was measured using a fiber-optic probe coupled to the fluorescence spectrometer. At the end of the experiment, coral A B CD Fig. 4. Effects of blue, green and red light treatment on red fluorescence of mcavRFP determined on M. cavernosa. (A) Fluorescence ima- ges with blue light excitation, and photographed through a yellow long-pass filter (Nightsea) after 37 days. (B) Emission spectra of the coe- nosarc (k ex ¼ 460 nm), measured for animals maintained under experimental light conditions for 37 days. The graphs show the average spectra and standard deviations for 12 independent measurements. (C) Immunoblot analysis of the red-emitting form of mcavRFP. Average optical density and standard deviations were calculated for the  20 kDa band of the red form, taking measurements from four independent, zooxanthellae-free tissue extracts per light treatment at the end of the experiment. The inset shows the corresponding immunoblot results. (D) Time dependence of the red tissue fluorescence intensity, determined over 37 days at 582 nm for colonies treated with red, green or blue light. Symbols represent the median values and error bars the first and third quartile for every time point, calculated from 12 independ- ent measurements per light treatment. Fluorescence of colonies irradiated with blue light remained constant during the experiment (P<0.01). The data for colonies irradiated with red and green were fitted with single-exponential decay functions (red and green curves, respectively). At day 37, significant differences indicated by asterisks were determined compared with the initial fluorescence (P<0.01). Also, the red fluorescence of animals treated with green light is significantly lower (P<0.01) than that of animals exposed to red light. A. Leutenegger et al. Turnover of GFP-like proteins FEBS Journal 274 (2007) 2496–2505 ª 2007 The Authors Journal compilation ª 2007 FEBS 2501 fluorescence was photographed and tissue samples were removed for immunoblot analysis. Colonies exposed to blue light revealed bright orange fluores- cence (Fig. 4A). In contrast, colonies illuminated with red light fluoresced greenish-yellow, whereas red fluor- escence was essentially absent in animals kept under green light. As expected, these differences are due to varying mcavRFP content, as deduced from decreased tissue fluorescence at 582 nm and the lower amounts of the  20 kDa fragment which represents the photo- converted protein in tissue extracts (Fig. 4B,C). The tissue content of chlorophyll a (chl. a) from zooxant- hellae was identical for blue- and green-light-treated colonies ( 7.0ngchl.aÆlg )1 total protein), t he amount of algal pigment, however, was reduced under red light ( 3.0 ng chl. aÆlg )1 total protein), indicating less fav- orable light conditions. Overall, red tissue fluorescence remained unaltered during 5-week exposure to blue light (Fig. 4D). In contrast, we observed mcavRFP degradation in animals kept under green and red light. The half-lives of mcavRFP for the respective light conditions were calculated by fitting the tissue fluorescence emission data collected at 582 nm to exponential decay func- tions. The variability in half-life values was estimated from the maximal deviation of the individual time points. We obtained half-lives of mcavRFP of 13 ± 2 days in green light and 19 ± 2 days in red light. The latter is most similar to the value obtained for dark- treated animals, indicating only a minor, if any, effect of red light on mcavRFP degradation. Upon exposure to green light, however, the half-life was shortened. Green light excites the chromophores efficiently, and thus, the observed shortened half-life may indicate that photobleaching of the chromophores also destabilizes the overall protein. However, an influence by other pigments completely disconnected from the FPs may also play a role. Indeed, it is well known that light of shorter wavelengths induces photodamage in living cells [44,45]. Nevertheless, the GFP-like proteins proved to be extraordinarily stable in the coral tissue even under damaging light conditions. Therefore, the stability of the tetrameric proteins observed in vitro is indicative of stability in vivo and in situ. This stability is very beneficial for maintaining high concen- trations of protein with minimal metabolic effort, which is a requirement for functional roles such as photoprotection. Conclusions Green-to-red conversion of GFP-like proteins is a photoinduced process driven by violet–blue light in scleractinian corals. Red pigments are retained in the tissues for many days, with half-lives of up to  20 days. The slow turnover of GFP-like proteins in anthozoans implies that the energetic cost of maintain- ing a high pigment concentration in the tissue is com- paratively low. The exceptionally high stability of GFP-like proteins in vivo and in situ makes them well suited to fulfill functions that require a high protein concentration, for example, protection from potentially damaging light intensities. Experimental procedures Collection and maintenance of coral colonies Specimens of M. cavernosa were collected in Key West, Florida under Florida Keys National Marine Sanctuary permit number 2003-053-A1 and adapted to aquarium con- ditions at the Whitney Laboratory (St Augustine, FL). L. hemprichii was purchased via the German aquarium trade. Colonies for experimentation were kept in artificial seawater at 25 ± 1 °C under a 12 h light ⁄ dark cycle in the Sea Water Facility of the Department of General Zoology and Endocrinology at the University of Ulm. Experiments involving varying light environments were always per- formed within one tank, exposing all animals under study to identical water conditions. Spectroscopy and photoconversion of coral pigments in situ Coral colonies growing in a photon flux of 100 lmolÆ m )2 Æs )1 were split into two groups, one of which (the con- trol group) remained illuminated, while the other was kept in complete darkness. After 4 weeks, fluorescence of the colonies was excited by using a hand-held blue light lamp (Nightsea, Andover, MA) or a metal halide lamp (Osram, Danvers, MA) equipped with a 530 nm bandpass filter glass (Schott, Mainz, Germany), and photographs were taken with a Camedia C-730 Ultra Zoom Digital Compact Camera (Olympus, Hamburg, Germany) through a yellow long-pass filter (Nightsea) or a 550 nm long-pass glass filter (Schott). Fluorescence spectra were measured in the coenosarc regions of the polyps by using a Varian Cary Eclipse fluorometer (Varian, Palo Alto, CA), equipped with a fiber-optic probe. A constant spacing between the head of the optical fiber and the tissue was ensured by means of a 4 mm spacer tip. After the spectroscopic measurements, tissue samples were collected for immunoblot analysis. Photoconversion in live corals was induced by irradiation with a blacklight blue fluorescent light source Sylvania 18 W (Osram) for 3 h at a photon flux of < 30 lmolÆm )2 Æs )1 . During the course of irradiation, the change in fluorescence was documented Turnover of GFP-like proteins A. Leutenegger et al. 2502 FEBS Journal 274 (2007) 2496–2505 ª 2007 The Authors Journal compilation ª 2007 FEBS photographically and spectrometrically. Alternatively, tissue samples were frozen to kill the cells. Subsequently, they were thawed on an object slide and photoconverted on the fluorescence microscope as described [6]. Also, embryos of M. cavernosa were photoconverted on a fluorescent micro- scope (MZ FL III, Leica Microsystems Inc., Wetzlar, Ger- many) by using a blue filter, while spectra of the embryos were collected using a USB2000 spectrometer (Ocean Optics, Dunedin, FL) equipped with a fiber optic probe that was coupled to the eyepiece of the microscope. To assess the influence of the light intensity on photoconver- sion, different colonies of M. cavernosa growing under weak light conditions (photon flux 100 lmolÆm )2 Æs )1 ) were divided into two groups, one of which continued with weak light exposure, while the other group was gradually adapted to a photon flux of 400 lmolÆm )2 Æs )1 (strong light). After 4 weeks’ exposure to strong light, tissue fluorescence was measured and protein extracts were prepared for immuno- blot analysis. Protein extraction, immunoblot analysis and determination of chlorophyll content Preparation of tissue extracts and immunoblot analysis was performed as described previously [27]. Protein concentra- tion was determined by the BCA TM protein assay (Pierce, Rockford, IL). Zooxanthellae pigments were extracted and quantified as outlined previously [27]. Determination of FP half-lives To measure the kinetics of degradation of the red-fluorescent proteins, colonies of M. cavernosa and L. hemprichii were either kept under weak light or transferred to total darkness. Changes in the intensity of red tissue fluorescence were deter- mined spectrometrically, as describe above, at intervals of 3–5 days. For M. cavernosa, a total of 18 replicate colonies derived from three different mother colonies was studied. In the case of L. hemprichii, we examined six replicate colonies. For each time-point, 12–24 independent measurements were taken and averaged from different colonies. In parallel, colonies of M. cavernosa were exposed for a total of 5 weeks to blue, green and red light, each with a photon flux of 200 lmolÆm )2 Æs )1 . The different colors were produced by filtering light from metal halide lamps with lighting filters (Lee Filters, Andover, UK). The filters allowed maximal light transmission at  450 ± 40 (FWHM) nm (band pass, ‘Zenith Blue’),  512 ± 40 (FWHM) nm (band pass, ‘Dark Green’) and > 580 nm (long pass, Primary Red). During the experimental period, 24 fluorescence spectra were collected from the tissues of the colonies at five different time points. At the end of the experiment, tissue extracts were prepared and subjected to immunoblot analysis. Statistical analysis The software analyse it for Microsoft Excel, Version 1.73 (Microsoft, Redmond, CA), was used for statistical analy- ses. P -values of < 0.01 obtained from t-test (two dependent groups) and Mann–Whitney U-test (two independent groups) were considered statistically significant. Acknowledgements The work was supported by the Deutsche Forschungs- gemeinschaft (SFB 497 ⁄ B9 to FO, SFB 497 ⁄ D2 to GUN, and Wi1990 ⁄ 2-1 to JW), Landesforschungsschwerpunkt Baden-Wu ¨ rttemberg (to JW and GUN), the ARC ⁄ NHMRC Network FABLS Australia (collaborat- ive grant to AS et al.), and IDP Education Australia (Australia–Europe Scholarship to AL). The authors acknowledge technical help of Florian Schmitt (Univer- sity of Ulm) during the dark treatment experiment. References 1 Wiedenmann J (1997) Die Anwendung eines orange flu- oreszierenden Proteins und weiterer farbiger Proteine und der zugeho ¨ renden Gene aus der Artengruppe Anemonia sp. (sulcata) Pennant, (Cnidaria, Anthozoa, Actinaria) in Gentechnologie und Molekularbiologie. Offenlegungsschrift DE 197(18), 640 [Deutsches Patent– und Markenamt, 1–18.] 2 Wiedenmann J, Ro ¨ cker C & Funke W (1999) The morphs of Anemonia aff. sulcata (Cnidaria, Anthozoa) in particular consideration of the ectodermal pigments. 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The authors acknowledge. corals M. cavernosa, Scolymia cubensis, Catalaphyllia jardinei, the corallimorpharian Ricordia florida and the alcyo- narian Dendronepthya sp. [10,12,27]. An unusual, green -to- orange variant was recently