Báo cáo khoa học: It’s cheap to be colorful Anthozoans show a slow turnover of GFP-like proteins potx

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:
*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.
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