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1
Fluorescent pigments in corals are photoprotective.
Anya Salih*,†, Anthony Larkum*, Guy Cox†, Michael Kühl‡ and Ove Hoegh-Guldberg*§
* School of Biological Sciences, A08, The University of Sydney, NSW, Australia.
† Electron Microscope Unit, F09, The University of Sydney, NSW, Australia.
‡ Marine Biological Laboratory, University of Copenhagen, Denmark.
§ Current address: Centre for Marine Studies, University of Queensland, St Lucia, QLD 4072, Australia
All reef-forming corals depend on the photosynthesis of their dinoflagellate algal
symbiont and are therefore restricted to the photic zone. In this zone light intensity
declines over several decades of intensity from high and damaging levels
(accompanied by UV radiation) at the surface to extreme shade conditions at the
lower limit1. The ability of corals to tolerate this range implies effective mechanisms
for light acclimation and adaptation2. Here we show that the fluorescent pigments3-9
(FPs) of corals provide a photobiological system for regulating the light environment
of coral host tissue. Previous studies have suggested that under low light, FPs may
enhance light availability4,5. We now present evidence that in excessive sunlight, FPs
are photoprotective through dissipation of excess energy at wavelengths of low
photosynthetic activity, as well as by reflection of visible and infra-red light by FP-
containing chromatophores. We also show that FPs enhance the resistance to mass
bleaching of corals during periods of heat stress, which has implications for the
impact of environmental stress on the diversity of reef-building corals.
The bright colours of corals (Scleractinia) and other Anthozoa are due to pigments of
animal-host origin3, many of which are intensely fluorescent under UVA and blue light, with
emission maxima at 420-620 nm4-9 (Fig. 1). In many corals distinct morphs are found which
differ greatly in the concentration of FPs. FPs are part of a group of coral pigments for which
the generic term "pocilloporins" has been proposed9,11. It includes both brightly coloured, low
fluorescence forms and highly fluorescent forms9 described here. Both types of pigments are
partially homologous to Green Fluorescent Protein (GFP)8,9, first found in the luminescent
jelly-fish Aequorea10 and widely used in cell biology. While the function of GFP in a
luminescent system is known, the role of similar FPs in non-luminescent anthozoans has
hitherto been unclear6,11,12.
We surveyed the distribution of fluorescent corals on the Great Barrier Reef (GBR)
(see methods) and found that 124 spp of 56 genera in 16 sampled families contained
fluorescent morphs, often found growing side by side with non-fluorescent morphs. Colour
polymorphism is typical of corals12,13, but many FPs are invisible in daylight, therefore this
widespread abundance was not previously known. The highest numbers of fluorescent morphs
were recorded at the shallowest sites; thus 97% of sampled reef flat corals at Heron Island,
southern GBR, contained medium or high FP concentrations. Moreover, their relative
concentration was significantly higher in sun-exposed as compared to their shaded colony
parts (P<0.001, T-test). We therefore explored an early suggestion3 that fluorescent pigments
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2
in shallow water corals might function in photoprotection, by comparing fluorescent and non-
fluorescent morphs.
We found that the emission maxima of FPs ranged from blue to green to red (Fig 1),
which is consistent with studies done on the isolated FP proteins of corals9. The shorter
wavelength FPs were more abundant. Microscopically we identified 2 broad groups: FPs
bound within 0.2-8µm fluorescent pigment granules (FPGs) as reported previously3,5,7 and
inter- or intracellular FPs, not enclosed in granules (CFPs). Significantly, the majority of corals
contained multiple FPG and CFP types. Correspondences between emission and excitation
maxima of FPs which occur in close association (Fig 1, e-f) suggest that energy
transformation to longer, non-photosynthetically active wavelengths might in some cases be a
sequential process, with the fluorescence of one pigment exciting another, as expected from
spectra of isolated proteins8,9. We demonstrated that this process could occur by comparing
fluorescence of green FPGs (excitation max. 482.5 nm) alone and mixed with blue FPGs
(excitation max. 382.5 nm). Only weak green fluorescence is seen under 330-380nm
excitation; addition of blue FPGs emitting at 480nm enhanced green fluorescence intensity 4 to
7-fold. (The effect was strongly dependent on distance, with maximal enhancement when blue
and green granules were less than 10µm apart.) The final energy spill would then (depending
on the pigments involved) lie between the two major peaks of the coral photosynthetic action
spectrum and hence be relatively inactive in photosynthesis (Fig 1f). This would be the inverse
counterpart of the process of light transfer to photosynthesis which others have proposed in
light limited habitats4-5 (Fig 1 g)
High light causes photodamage and photoinhibition14 in coral symbionts15-19. We
hypothesized that FPs may reduce the susceptibility to photoinhibition of fluorescent corals by
filtering out damaging UVA and excessive PAR. We compared the degree of day-time
photoinhibition in a polymorphic intertidal species, Acropora palifera, by exposing replicate
sub-colonies made from green fluorescent, brown medium fluorescent and beige non-
fluorescent mother colonies to full sunlight and monitored photosynthesis by chlorophyll
fluorescence analysis with a pulse amplitude modulation (PAM) fluorometer19,20. As
expected15,16, corals showed pronounced photoinhibition during periods of peak irradiance;
non-fluorescent morphs, however, were significantly more photoinhibited and recovered to
pre-inhibition rates slower than fluorescent morphs (P<0.001, ANOVA) (Fig. 2a). Similar
measurements with other polymorphic species (Acropora nobilis, Pocillopora damicornis,
Goniastrea retiformis) also indicated that FPs are correlated with reduced photoinhibition.
Since high solar radiation is a factor in the widely observed mass bleaching of
corals17,21,22, FPs might affect susceptibility to bleaching. Bleaching occurs as a consequence
of damage to dinoflagellate photosynthesis caused by combined effects of thermal stress and
sunlight18,19,22; consequently the dinoflagellates either degrade or are expelled from the host.
During the severe 1998 GBR mass bleaching event, we sampled 21 common coral species
affected by bleaching to varying degrees and found a significant correlation (r2 = 0.9471;
P<0.0001) between bleaching resistance (i.e., high tissue dinoflagellate biomass) and the
concentration of FPs within the tissue (Fig. 2b).
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Light scattering is an important factor linked to the sun screening function of
FPs. We measured spectral reflective properties of coral tissues with fiber-optic
microprobes23,24 positioned over specific parts of single coral polyps. The highly reflective bare
coral skeleton was used as a reflection standard (100%). FPs greatly modified the surface light
environment not only by their emissions but also by light scattering and reflectance, which was
higher in areas with high FPG concentrations (Fig. 3a). White pigmented regions of tissues,
formed by dense layers of FP chromatophores, had 60-100% reflectivity (Fig. 3, a-b). The
most pigmented, and most reflective, parts of colonies: (i) branch tips and colony edges and
(ii) the oral disk/cone and tentacle tips, which on polyp retraction form a sun-screening polyp
‘plug’7 (Fig. 4c), correspond to known areas of highest cell division and areas immediately
above reproductive organs, respectively. This distribution points to a photoprotective role of
FPs in screening sensitive coral tissues as well as symbionts.
Our observations also indicate that corals actively vary the areal density of
pigment chromatophores via polyp expansion/contraction. During expansion more light
penetrates into the tissues through the gaps between FPGs. Under high light, polyp contraction
leads to denser concentration of tissue FPGs and cytoplasmic FPs, forming a thicker and a
quasi continuous FP layer, acting as an effective sunscreen (Fig. 3, b-c) by light scattering and
by radiant fluorescence energy transfer from shorter to longer wavelengths. We also found
that FP-containing polyps of shade-adapted and high light-adapted corals exhibited
differences in spectral reflectivity. Shade-adapted polyps absorbed most of the incident light,
in line with previous observations25, while high light-adapted polyps were generally 20-100%
more reflective (Fig. 3b).
What are the causes of such different tissue optical properties of shade- and
high light-adapted corals? The 3-D cellular localization of FPs in corals showed a clear
difference in the distribution of FPs relative to the layers of endosymbionts7. In high light-
acclimated corals, FPs are localized above the endosymbionts (Fig. 4 a-c) and are in a
position to screen them from excess sunlight. In shade-adapted corals from light-limited
habitats, FPs are localized endodermally, among or below the layers of endosymbionts (Fig.
4d), consistent with their proposed function of light enhancement for photosynthesis via
wavelength-transformation and back-scattering4,5.
The results presented above all suggest that FPs reduce the photoinhibitory
effect of high levels of solar radiation, which in conjunction with thermal stress leads to
bleaching. These findings improve our understanding of the causes of observed inter- and
intraspecific variability in bleaching17,26 and may provide an insight into how changing global
climatic conditions will influence the species diversity and rate of change of coral reef
communities22.
In conclusion, our study provides a new and more complete understanding of
the role of coral FPs, which appear to be involved in regulation of the internal light
microenvironment of coral tissues. The evidence presented here indicates that the role of FPs
in photoprotection in shallow water, hitherto neglected, is at least as significant as the function
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of light capture in deep water previously assigned to them. Dinoflagellate photosynthesis is
vulnerable to both UV27,28 and high levels of PAR16-19. While accessory pigments in
dinoflagellates can dissipate excess PAR as heat29, FPs can dissipate excess light energy via
fluorescence and light scattering. FPs may also supplement UV-screening by mycosporin-like
amino acids (MAAs)30 since some FPs can transform absorbed UVA radiation to longer non-
actinic wavelengths via fluorescence. By screening chlorophylls and peridinin from high levels
of solar radiation and by absorbing UVA, FPs thereby decrease the likelihood of irreversible
photoinhibition, photooxidation and subsequent coral bleaching. By changing their optical
properties with the help of these GFP-like pigments, coral polyps are able to optimise the
photosynthetic activity of their tissues for the better survival of the organism.
Methods
Survey sites, sampling and manipulations: Surveys of fluorescent corals were made at the
inter-tidal lagoon, reef flat and inner and outer slope of Heron Island (23o26’S, 151o55’E) and
One Tree Island (OTI) (23o30’S, 152o06’E), and at several GBR mid-shelf-reefs (1-20m
depths). Corals were sampled by chiselling pieces from replicate colonies (n = 3-6). Each
sample was broken in two, and, subsequently, one subsample was frozen and the other was
chemically fixed as described previously7 for microscopy. Polymorphic Acropora palifera
colonies used in photoinhibition experiment were collected from the OTI lagoon from ~1m
depth. Three colonies of each colour morph were broken into replicate sub-colonies and fixed
in horizontal position in flowing seawater (27-28oC) with one side exposed to sunlight and the
other shaded. Controls were kept at 50-80µmol photons m-2s-1. During the March, 1998
bleaching event samples were taken at Coats (17o28’S, 146o30’E) and Cayley (18o30’S,
147oE) mid-shelf reefs from 1-6m depths. Replicate samples (n=3-6) were taken from species
selected by susceptibility to bleaching: 9 bleached (including non fluorescent Acropora nobilis
morph;); 5 partially bleached (including fluorescent A. nobilis); 7 unbleached.
Microscopy: Frozen, glutaradehyde-fixed and live coral samples were analysed by
fluorescence widefield and confocal (CLSM) microscopy as described previously7.
Fluorescence characteristics of FPs were not substantially affected by glutaraldehyde fixation.
Confocal imaging used 488nm excitation, with detection at 520-550 nm (FPs) and >585nm
(chlorophyll). 3-D reconstruction from optical sections was done with VoxelView Ultra 2.1.2
(Vital Images, USA). A fluorescence microscope fitted with cooled CCD camera (PCO
Sensicam) was used to test energy transfer from blue to green FPGs, both extracted from
Plesiastrea versipora. Intensity (in the green) was measured as the ratio of emission excited
at 330-380nm to the emission excited at 450-490nm, thereby compensating for differences
between granules. Relative FP concentrations in light- and shade-samples as well as in post-
bleaching samples were measured, semi-quantitatively, by CLSM as the fluorescence intensity
per µm2 of imaged coral surface (replicate 3-6 colonies / specimen). Zooxanthellae were
extracted from samples, their biomass cm-2 of coral surface was determined microscopically
and correlated to the relative concentration of FPs in surface tissue as determined by CLSM.
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Coral surface areas were measured as described previously18.
Spectroscopy: Fluorescence excitation and emission spectra of FPGs isolated by
homogenization and repeated centrifugation of coral tissues in phosphate buffer (0.1M, pH
7.2) were determined by a Perkin Elmer Luminescence Spectrometer S50B. All spectra were
normalized to their peaks. Reflectance spectra were measured on live corals in seawater by a
tapered (40µm tip) fibre-optic field radiance microprobe23 positioned ~100µm above the coral
surface. The microprobe was connected to a fibre-optic diode-array spectrometer
(Hamamatsu PMA-11) with a 300-800nm spectral range. A micromanipulator was used to
position the microprobe tip above specific single polyp regions, as viewed under a dissection
microscope. Samples were illuminated by a UV-VIS metal halide light source via a 1 mm
quartz fibre equipped with a collimator at the output end. Spectra of reflected light from the
corals were normalized to the spectrum of reflected light from a reflectance standard in order
to obtain reflectance spectra corrected for the spectral composition of incident light
(normalized reflectance). Data was subsequently expressed as percentages of surface
downwelling radiance reflected from cleaned coral skeleton (relative reflectance).
Active fluorescence measurements: Throughout the day (06:00, 09:00, 12:00, 14:00,
18:00) photoinhibition of light-exposed and shaded portions of sub-colonies (n=3 per morph),
and shaded controls, was measured as decrease in the maximal potential quantum yield
(Fv/Fm) of PSII by a pulse amplitude modulation fluorometer (DIVING-PAM)19,20 after 30
min dark-adaption. Photosynthetically active radiation (400-700nm) during the experiment
was measured by LI-190SA quantum sensor.
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under embargo until publication date, December 14th. Copyright © Salih et al, 2000
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hermatypic coral from the Gulf of Elat. Nature 289, 172-174 (1981)
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We acknowledge the Great Barrier Reef Marine Park Authority, especially J. Oliver, R. Berkelmans, M.
Russell and U. Engelhart for financial and logistic support. This work was also supported by an Australian
Research Council (ARC) SPIRT PhD award supporting A. Salih and ARC grants to O. Hoegh-Guldberg
and AWD Larkum. M. Kühl acknowledges the financial support of the Danish Natural Science Research
Council. We thank the staff of Heron and One Tree Island research stations for assistance during
fieldwork.
Correspondence and requests for materials should be addressed to: Anya Salih,
Electron Microscope Unit FO9, University of Sydney, NSW 2006, Australia
anya@emu.usyd.edu.au.
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under embargo until publication date, December 14th. Copyright © Salih et al, 2000
8
Figure 1 Main types of fluorescent pigments in coral polyps found in blue, green,
yellow and red combinations with overlapping excitation and emission spectra. a-
b, Mainly blue, in Acropora nobilis. c-d, Mainly green, in Pocillopora damicornis.
e-f, Emissions of outer blue/green and underlying yellow FPs in 'sun' Porites
cylindrica. Coral photosynthetic action spectrum24 (red line) shows that much of
the energy is emitted at wavelengths not usable in photosynthesis. g. Sub-surface
red FPs in green Montipora digitata. Arrow, red FPs in mesenterial filaments.
Scale bars 0.5mm
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Figure 2 Photoinhibition and bleaching responses of corals. a, Maximal potential
quantum yield (Fv/Fm) of dinoflagellates in green highly fluorescent (GF), brown
medium fluorescent (BF) and non fluorescent (NF) Acropora palifera. Results are
means ± S.E. for 3 sub-colonies x morph x 2 tanks x time interval. b,
Dinoflagellates cm-2 and relative concentration of fluorescent pigments µm-2 of
sampled corals: ¡ - bleached, s - part-bleached, l - unbleached. Inset - enlarged
section of graph marked in square.
Figure 3 Apparent reflectance. a, Plesiastrea versipora - tissues lacking FPs (1);
tissue with blue FPs overlying skeletal ridge (2); contracted tentacle with green
FPGs (3); blue FPGs in expanded (4) and contracted (5) oral disc; dense blue
FPGs in white oral disc of shallow-water Platygyra daedalea (6). b, Intertidal
yellow Porites cylindrica - 'shade' expanded (7); contracted (8); and 'sun'
expanded (11), contracted (12) tentacles; edge of polyp calyx (9); septal skeleton
with thin tissue (10). Dips in spectra are due to absorption by photosynthetic
pigments. Spectral peaks due to FPs emission and reflection.
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Figure 4 Reconstruction of serial confocal sections through tissues of 'sun' and
'shade' corals. a, FPs in chromatophores above endosymbionts in polyp tentacle
of 'sun' intertidal Goniopora tenuidens. b, FPG-filled chromatophores with long
extensions enveloping endosymbionts. Bars - 50µm and 10µm, respectively. c,
Retracted tentacles with dense FPGs form a 'plug' over polyp. Bar - 1 mm. d,
Endodermal FPs below dinoflagellates in 'shade' Lobophyllia corymbosa. Bar -
50µm. FPs shown in green/yellow, symbiotic dinoflagellates in red. Arrows - FP
chromatophores. Letter captions: Ec - ectoderm (epidermis), M - mesogloea; En -
endoderm (gastrodermis)