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Screening by coral green fluorescent protein (GFP)-like chromoproteins supports a role in photoprotection of zooxanthellae

Abstract

Green fluorescent protein (GFP)-like pigments are responsible for the vivid colouration of many reef-building corals and have been proposed to act as photoprotectants. Their role remains controversial because the functional mechanism has not been elucidated. We provide direct evidence to support a photoprotective role of the non-fluorescent chromoproteins (CPs) that form a biochemically and photophysically distinct group of GFP-like proteins. Based on observations of Acropora nobilis from the Great Barrier Reef, we explored the photoprotective role of CPs by analysing five coral species under controlled conditions. In vitro and in hospite analyses of chlorophyll excitation demonstrate that screening by CPs leads to a reduction in chlorophyll excitation corresponding to the spectral properties of the specific CPs present in the coral tissues. Between 562 and 586 nm, the CPs maximal absorption range, there was an up to 50 % reduction of chlorophyll excitation. The screening was consistent for established and regenerating tissue and amongst symbiont clades A, C and D. Moreover, among two differently pigmented morphs of Acropora valida grown under identical light conditions and hosting subclade type C3 symbionts, high CP expression correlated with reduced photodamage under acute light stress.

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Acknowledgments

The study was funded by NERC (NE/I01683X/1 & NE/I012648/1 to JW; studentship to EGS), DFG (Wi1990/2-1 to JW), DAAD (Heron Island Fieldwork to JW), European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement n. 311179 to JW and ARC (Discovery grant to AS et al.). We acknowledge the assistance of D. Owsianka (University of Southampton, LED array construction) and F. Schmitt (University of Ulm, field sampling) and thank Tropical Marine Centre (London) and Tropic Marine (Wartenberg) for sponsoring of the NOCS Coral Reef Laboratory. We would also like to thank the two anonymous reviewers and A. Banaszak for their helpful comments.

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Communicated by Biology Editor Dr. Anastazia Banaszak

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338_2012_994_MOESM1_ESM.tif

ESM Figure S1. Analysis of blue and brown morphs of Acropora nobilis from the Great Barrier Reef. (a) Photograph of the two morphs side-by-side on the reef. The white arrow indicates a light exposed branch whereas the dark arrow shows a branch that is subject to self shading within the blue morph colony. (b, c) Mean zooxanthellae densities (b) and pigment concentrations (c) from five replicate colonies collected at 1.5 m. Error bars indicate the ±SD of the mean (TIFF 1678 kb)

338_2012_994_MOESM2_ESM.tif

ESM Figure S2. Optical properties of the light sources used in this study. The normalised spectral output of the metal halide burners used for growing the aquarium corals and the amber LEDs used for the high irradiance experiments. The absorption spectrum for avalCP580 is shown to demonstrate the overlap between the LED output and the absorption of the CP (TIFF 8770 kb)

338_2012_994_MOESM3_ESM.tif

ESM Figure S3. Absorption spectra of CPs isolated and purified from the corals Seriatopora hystrix (shysCP562), Montipora foliosa (mfolCP577), Acropora valida (avalCP580), Acropora nobilis (anobCP582), Acropora polystoma (apolCP584) and Acropora millepora (amilCP586). Spectra are normalised to a peak height of 1.0 (TIFF 171 kb)

338_2012_994_MOESM4_ESM.tif

ESM Figure S4. Localisation of avalCP580 in the purple morph of Acropora valida. (a) Micrograph of a cross section of an A. valida tip showing the localisation of the purple chromoprotein. (b) Fluorescent image of the corresponding cross section showing the presence of ectodermal green fluorescent proteins (green) and chlorophyll fluorescence from the zooxanthellae (red) (TIFF 3353 kb)

338_2012_994_MOESM5_ESM.tif

ESM Figure S5. Characterisation of chromoproteins in growth regions. (a) Photograph of two Montipora foliosa morphs expressing (right) and not expressing (left) chromoproteins in the colony margins. (b, c) Mean estimated absorbance spectra of the brown (b) and purple morphs (c) at the centre and growth margins of the colonies. Dotted lines indicate the ±SD of 5 independent measurements. (d, e) Excitation difference spectra for the brown (d) and purple (e) morphs. The absorption spectra of mfolCP577 are shown for reference (TIFF 1799 kb)

338_2012_994_MOESM6_ESM.tif

ESM Figure S6. Reflectance spectra of regenerating zones in Acropora polystoma. The solid lines show the mean reflectance of the high and low light regeneration zones with the standard deviations of 5 measurements shown by the dotted lines. The absorption spectrum of apolCP584 is shown for comparison (TIFF 394 kb)

338_2012_994_MOESM7_ESM.tif

ESM Figure S7. Excitation and emission properties of the brown and purple colour morphs of Acropora valida. (a, b) Excitation-emission plot for the brown (a) and purple (b) morphs. The excitation spectra are sampled at 1 nm intervals and the emission spectra are sampled every 5 nm. (c) Difference spectrum peak values across the chlorophyll emission range. The mean peak wavelength of the difference spectrum is 579 nm with a standard deviation of 1 nm and the respective CP absorption peak is at 580 nm (TIFF 671 kb)

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Smith, E.G., D’Angelo, C., Salih, A. et al. Screening by coral green fluorescent protein (GFP)-like chromoproteins supports a role in photoprotection of zooxanthellae. Coral Reefs 32, 463–474 (2013). https://doi.org/10.1007/s00338-012-0994-9

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Keywords

  • Green fluorescent protein (GFP)-like proteins
  • Chromoproteins
  • Function
  • Photoprotection
  • Growth
  • Coral-zooxanthellae symbiosis