Diversity and evolution of coral fluorescent proteins

GFP-like fluorescent proteins (FPs) are the key color determinants in reef-building corals (class Anthozoa, order Scleractinia) and are of considerable interest as potential genetically encoded fluorescent labels. Here we report 40 additional members of the GFP family from corals. There are three major paralogous lineages of coral FPs. One of them is retained in all sampled coral families and is responsible for the non-fluorescent purple-blue color, while each of the other two evolved a full complement of typical coral fluorescent colors (cyan, green, and red) and underwent sorting between coral groups. Among the newly cloned proteins are a "chromo-red" color type from Echinopora forskaliana (family Faviidae) and pink chromoprotein from Stylophora pistillata (Pocilloporidae), both evolving independently from the rest of coral chromoproteins. There are several cyan FPs that possess a novel kind of excitation spectrum indicating a neutral chromophore ground state, for which the residue E167 is responsible (numeration according to GFP from A. victoria). The chromoprotein from Acropora millepora is an unusual blue instead of purple, which is due to two mutations: S64C and S183T. We applied a novel probabilistic sampling approach to recreate the common ancestor of all coral FPs as well as the more derived common ancestor of three main fluorescent colors of the Faviina suborder. Both proteins were green such as found elsewhere outside class Anthozoa. Interestingly, a substantial fraction of the all-coral ancestral protein had a chromohore apparently locked in a non-fluorescent neutral state, which may reflect the transitional stage that enabled rapid color diversification early in the history of coral FPs. Our results highlight the extent of convergent or parallel evolution of the color diversity in corals, provide the foundation for experimental studies of evolutionary processes that led to color diversification, and enable a comparative analysis of structural determinants of different colors.     

GFP-like proteins as ubiquitous metazoan superfamily: evolution of functional features and structural complexity

Homologs of the green fluorescent protein (GFP), including the recently described GFP-like domains of certain extracellular matrix proteins in Bilaterian organisms, are remarkably similar at the protein structure level, yet they often perform totally unrelated functions, thereby warranting recognition as a superfamily. Here we describe diverse GFP-like proteins from previously undersampled and completely new sources, including hydromedusae and planktonic Copepoda. In hydromedusae, yellow and nonfluorescent purple proteins were found in addition to greens. Notably, the new yellow protein seems to follow exactly the same structural solution to achieving the yellow color of fluorescence as YFP, an engineered yellow-emitting mutant variant of GFP. The addition of these new sequences made it possible to resolve deep-level phylogenetic relationships within the superfamily. Fluorescence (most likely green) must have already existed in the common ancestor of Cnidaria and Bilateria, and therefore GFP-like proteins may be responsible for fluorescence and/or coloration in virtually any animal. At least 15 color diversification events can be inferred following the maximum parsimony principle in Cnidaria. Origination of red fluorescence and nonfluorescent purple-blue colors on several independent occasions provides a remarkable example of convergent evolution of complex features at the molecular level.     

Spectral Diversity of Fluorescent Proteins from the Anthozoan <Emphasis Type="Italic">Corynactis californica</Emphasis>

Color morphs of the temperate, nonsymbiotic corallimorpharian Corynactis californica show variation in pigment pattern and coloring. We collected seven distinct color morphs of C. californica from subtidal locations in Monterey Bay, California, and found that tissue– and color–morph-specific expression of at least six different genes is responsible for this variation. Each morph contains at least three to four distinct genetic loci that code for these colors, and one morph contains at least five loci. These genes encode a subfamily of new GFP-like proteins, which fluoresce across the visible spectrum from green to red, while sharing between 75% to 89% pairwise amino-acid identity. Biophysical characterization reveals interesting spectral properties, including a bright yellow protein, an orange protein, and a red protein exhibiting a “fluorescent timer” phenotype. Phylogenetic analysis indicates that the FP genes from this species evolved together but that diversification of anthozoan fluorescent proteins has taken place outside of phylogenetic constraints, especially within the Corallimorpharia. The discovery of more examples of fluorescent proteins in a non-bioluminescent, nonsymbiotic anthozoan highlights possibilities of adaptive ecological significance unrelated to light regulation for algal symbionts. The patterns and colors of fluorescent proteins in C. californica and similar species may hold meaning for organisms that possess the visual pigments to distinguish them. Christine E. Schnitzler and Robert J. Keenan contributed equally to this work. Data deposition footnote: The GenBank () accession numbers for the genes and gene products discussed in this paper are: ccalRFP1 (AY823226); ccalYFP1 (AY823227); ccalRFP2 (DQ065851); ccalGFP1 (DQ065852); ccalOFP1 (DQ065853); ccalGFP3 (DQ899732)     

It's cheap to be colorful. Anthozoans show a slow turnover of GFP-like proteins

Pigments homologous to the green fluorescent protein (GFP) contribute up to approximately 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 established that the irreversible green-to-red conversion of the GFP-like pigments 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 approximately 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.     

Aposematic coloration does not deter corallivory by fish on the coral <Emphasis Type="Italic">Montastraea cavernosa</Emphasis>

Predation on corals by visual predators is a significant source of partial or total mortality on coral reefs, and corals have evolved strategies, including chemical defenses, to deter predation. One mechanism that organisms use to communicate the presence of chemical defenses is aposematic coloration, or the display of bright coloration as a warning to visual predators such as fish. Corals exhibit multiple colors, and it has been hypothesized that one role for this variability in coloration is as an aposematic warning of adverse palatability. Here, we test green and orange color morphs of the Caribbean coral Montastraea cavernosa for the presence of chemical defenses and whether their differences in coloration elicited different feeding responses. While M. cavernosa is chemically defended, there is no difference in feeding deterrence between color morphs; thus, the different color morphs of this coral species do not appear to represent an example of aposematic coloration.     

Key Amino Acid Residues Responsible for the Color of Green and Yellow Fluorescent Proteins from the Coral Polyp Zoanthus sp.

Site-directed mutagenesis was used to study the structural basis of color diversity of fluorescent proteins by the example of two closely related proteins from one organism (coral polyp Zoanthus sp.), one of which produces green and the other, yellow fluorescence. As a result, the following conversions of emission colors were performed: from yellow to green, from yellow to a dual color (yellow and green), and from green to yellow. The saltatory character of the spectral transitions and the manifestation of the dual-color fluorescence suggest that chemically different fluorophores are responsible for the green and yellow fluorescence. The simultaneous presence of three residues, Gly63, Lys65, and Asp68, is necessary for the efficient formation of the yellow rather than green fluorophore.     

Contributions of host and symbiont pigments to the coloration of reef corals

For a variety of coral species, we have studied the molecular origin of their coloration to assess the contributions of host and symbiont pigments. For the corals Catalaphyllia jardinei and an orange-emitting color morph of Lobophyllia hemprichii, the pigments belong to a particular class of green fluorescent protein-like proteins that change their color from green to red upon irradiation with approximately 400 nm light. The optical absorption and emission properties of these proteins were characterized in detail. Their spectra were found to be similar to those of phycoerythrin from cyanobacterial symbionts. To unambiguously determine the molecular origin of the coloration, we performed immunochemical studies using double diffusion in gel analysis on tissue extracts, including also a third coral species, Montastrea cavernosa, which allowed us to attribute the red fluorescent coloration to green-to-red photoconvertible fluorescent proteins. The red fluorescent proteins are localized mainly in the ectodermal tissue and contribute up to 7.0% of the total soluble cellular proteins in these species. Distinct spatial distributions of green and cyan fluorescent proteins were observed for the tissues of M. cavernosa. This observation may suggest that differently colored green fluorescent protein-like proteins have different, specific functions. In addition to green fluorescent protein-like proteins, the pigments of zooxanthellae have a strong effect on the visual appearance of the latter species.     

The molecular properties and applications of Anthozoa fluorescent proteins and chromoproteins

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its fluorescent homologs from Anthozoa corals have become invaluable tools for in vivo imaging of cells and tissues. Despite spectral and chromophore diversity, about 100 cloned members of the GFP-like protein family possess common structural, biochemical and photophysical features. Anthozoa GFP-like proteins are available in colors and properties unlike those of A. victoria GFP variants and thus provide powerful new fluorophores for molecular labeling and intracellular detection. Although Anthozoa GFP-like proteins provide some advantages over GFP, they also have certain drawbacks, such as obligate oligomerization and slow or incomplete fluorescence maturation. In the past few years, effective approaches for eliminating some of these limitations have been described. In addition, several Anthozoa GFP-like proteins have been developed into novel imaging agents, such as monomeric red and dimeric far-red fluorescent proteins, fluorescent timers and photoconvertible fluorescent labels. Future studies on the structure of this diverse set of proteins will further enhance their use in animal tissues and as intracellular biosensors.     

Key amino acid residues responsible for the color of green and yellow fluorescent proteins from the coral polyp Zoanthus

Site-directed mutagenesis was used to study the structural basis of color diversity of fluorescent proteins by the example of two closely related proteins from one organism (coral polyp Zoanthus sp.), one of which produces green and the other, yellow fluorescence. As a result, the following conversions of emission colors were performed: from yellow to green, from yellow to a dual color (yellow and green), and from green to yellow. The saltatory character of the spectral transitions and the manifestation of the dual-color fluorescence suggest that chemically different fluorophores are responsible for the green and yellow fluorescence. The simultaneous presence of three residues, Gly63, Lys65, and Asp68, is necessary for the efficient formation of the yellow rather than green fluorophore. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.     

Cloning of anthozoan fluorescent protein genes

Many cnidarians display vivid fluorescence under proper lighting conditions. In general, these colors are due to the presence of fluorescent proteins similar to the green fluorescent protein (GFP) originally isolated from the hydrozoan medusa Aequorea victoria (Cnidaria: Hydrozoa). To optimize the search for new fluorescent proteins (FPs), a technique was developed that allows for the rapid cloning and screening of FP genes without the need for a prior knowledge of gene sequence. Using this method, four new FP genes were cloned, a green from Montastraea cavernosa (Anthozoa: Scleractinia: Faviidae), a cyan from Pocillopora damicornis (Anthozoa: Scleractinia: Pocilloporidae), a cyan from Discosoma striata (Anthozoa: Corallimorpharia), and a red from a second Discosoma species. Two additional green FPs were cloned, one from M. cavernosa and one from its congener Montastraea faveolata, from purified cDNA using PCR primers designed for the first M. cavernosa green FP. Each FP has recognizable amino acid sequence motifs that place them conclusively in the GFP protein family. Mutation of these products using a low-stringency PCR protocol followed by screening of large numbers of bacterial colonies allowed rapid creation of mutants with a variety of characteristics, including changes in color, maturation time, and brightness. An enhanced version of the new red FP, DspR1+, matures faster at 30 degrees C than the commercially available DsRed but matures slower than DsRed at 37 degrees C. One of the M. cavernosa green FPs, McaG2, is highly resistant to photobleaching and has a fluorescence quantum yield approximately twice that of EGFP-1.     

Spectral diversity among the members of the family of Green Fluorescent Protein in hydroid jellyfish (Cnidaria, Hydrozoa)

The cDNAs encoding the genes of new proteins homologous to the well-known Green Fluorescent Protein (GFP) from the hydroid jellyfish Aequorea victoria were cloned. Two green fluorescent proteins from one un-identified anthojellyfish, a yellow fluorescent protein from Phialidium sp., and a nonfluorescent chromoprotein from another unidentified anthojellyfish were characterized. Thus, a broad diversity of GFP-like proteins among the organisms of the class Hydrozoa in both spectral properties and primary structure was shown.     

Adaptive Evolution of Multicolored Fluorescent Proteins in Reef-Building Corals

Here we investigate the evolutionary scenarios that led to the appearance of fluorescent color diversity in reef-building corals. We show that the mutations that have been responsible for the generation of new cyan and red phenotypes from the ancestral green were fixed with the help of positive natural selection. This fact strongly suggests that the color diversity is a product of adaptive evolution. An unexpected finding was a set of residues arranged as an intermolecular binding interface, which was also identified as a target of positive selection but is nevertheless not related to color diversification. We hypothesize that multicolored fluorescent proteins evolved as part of a mechanism regulating the relationships between the coral and its algal endosymbionts (zooxanthellae). We envision that the effect of the proteins’ fluorescence on algal physiology may be achieved not only through photosynthesis modulation, but also through regulatory photosensors analogous to phytochromes and cryptochromes of higher plants. Such a regulation would require relatively subtle, but spectrally precise, modifications of the light field. Evolution of such a mechanism would explain both the adaptive diversification of colors and the coevolutionary chase at the putative algae-protein binding interface in coral fluorescent proteins. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Rasmus Neilsen]     

Natural animal coloration can Be determined by a nonfluorescent green fluorescent protein homolog

It is generally accepted that the colors displayed by living organisms are determined by low molecular weight pigments or chromoproteins that require a prosthetic group. The exception to this rule is green fluorescent protein (GFP) from Aequorea victoria that forms a fluorophore by self-catalyzed protein backbone modification. Here we found a naturally nonfluorescent homolog of GFP to determine strong purple coloration of tentacles in the sea anemone Anemonia sulcata. Under certain conditions, this novel chromoprotein produces a trace amount of red fluorescence (emission lambda(max) = 595 nm). The fluorescence demonstrates unique behavior: its intensity increases in the presence of green light but is inhibited by blue light. The quantum yield of fluorescence can be enhanced dramatically by single amino acid replacement, which probably restores the ancestral fluorescent state of the protein. Other fluorescent variants of the novel protein have emission peaks that are red-shifted up to 610 nm. They demonstrate that long wavelength fluorescence is attainable in GFP-like fluorescent proteins.     

ALLOPATRIC DIVERGENCE AND SPECIATION IN CORAL REEF FISH: THE THREE‐SPOT DASCYLLUS,DASCYLLUS TRIMACULATUS,SPECIES COMPLEX

Long pelagic larval phases and the absence of physical barriers impede rapid speciation and contrast the high diversity observed in marine ecosystems such as coral reefs. In this study, we used the three‐spot dascyllus (Dascyllus trimaculatus) species complex to evaluate speciation modes at the spatial scale of the Indo‐Pacific. The complex includes four recognized species and four main color morphs that differ in distribution. Previous studies of the group using mitochondrial DNA revealed a noncongruence between color morphs and genetic groupings; with two of the color morphs grouped together and one color morph separated into three clades. Using extensive geographic sampling of 563 individuals and a combination of mitochondrial DNA sequences and 13 nuclear microsatellites, we defined population/species boundaries and inferred different speciation modes. The complex is composed of seven genetically distinct entities, some of which are distinct morphologically. Despite extensive dispersal abilities and an apparent lack of barriers, observed genetic partitions are consistent with allopatric speciation. However, ecological pressure, assortative mating, and sexual selection, were likely important during periods of geographical isolation. This study therefore suggests that primarily historical factors later followed by ecological factors caused divergence and speciation in this group of coral reef fish.     

Discovery of androecium color polymorphism in Epimedium pubescens with habitat preference of anther/pollen color in the genus

Aims Interspecific and intraspecific variation in flower color in natural populations provides an opportunity for us to understand the evolution and maintenance of diversity of floral traits. Compared to corolla color, little is known about the color polymorphism of sexual organs in flowering plants. To explore evolutionary transitions of androecium color and polymorphism within species, interspecific and intraspecific variation in androecium (anther and pollen) color in the genus Epimedium (Berberidaceae) was investigated.Methods To explore the geographical patterns of anther/pollen color variation in Epimedium species, data of 45 species were collected and their phylogeny was constructed based on available DNA sequences. To investigate whether intraspecific variation in androecium color relates to habitat preference, three environmental factors were measured in the field population of Epimedium pubescens in northeastern Sichuan, China, which plants had green or yellow androecia. Vegetative and reproductive traits of this species were compared between the two color morphs.Important findings Androecium (anther and pollen) color polymorphism in field populations of Epimedium pubescens is reported here where nine populations are monomorphic with a green androecium but three populations are dimorphic with individuals having either a green or a yellow androecium. Inflorescence stalk height, stalk diameter, leaf number, flower number and spur length (as well as spur and nectar volume) were not significantly different between two morphs. Compared to the yellow morph, the green morph had relatively larger leaves and anthers, but smaller sepals. The green morph produced more pollen and larger seeds, but the same number of ovules. Seed set was not significantly different between green and yellow morph. Investigations of environmental factors in the color dimorphic populations of E. pubescens indicated that the green morph was more likely to occur in habitats with relatively lower light intensity. The distribution survey of 45 Epimedium species showed that species with a green androecium tended to appear at lower elevations. Comparative phylogenetic analysis showed that transitions from yellow to a green androecium or to androecial color dimorphism occurred at least seven times. This genus, characterized by anther color diversity and containing some species with anther color polymorphism, provides a model system in which to study the evolution and maintenance of colorful sexual organs in flowering plants.     

Structural basis for the fast maturation of Arthropoda green fluorescent protein

Since the cloning of Aequorea victoria green fluorescent protein (GFP) in 1992, a family of known GFP-like proteins has been growing rapidly. Today, it includes more than a hundred proteins with different spectral characteristics cloned from Cnidaria species. For some of these proteins, crystal structures have been solved, showing diversity in chromophore modifications and conformational states. However, we are still far from a complete understanding of the origin, functions and evolution of the GFP family. Novel proteins of the family were recently cloned from evolutionarily distant marine Copepoda species, phylum Arthropoda, demonstrating an extremely rapid generation of fluorescent signal. Here, we have generated a non-aggregating mutant of Copepoda fluorescent protein and solved its high-resolution crystal structure. It was found that the protein beta-barrel contains a pore, leading to the chromophore. Using site-directed mutagenesis, we showed that this feature is critical for the fast maturation of the chromophore.     

Photo-induced peptide cleavage in the green-to-red conversion of a fluorescent protein

Green fluorescent protein from the jellyfish (Aequorea GFP) and GFP-like proteins from coral species encode light-absorbing chromophores within their protein sequences. A coral fluorescent protein, Kaede, contains a tripeptide, His(62)-Tyr(63)-Gly(64), which acts as a green chromophore that is photoconverted to red. Here, we present the structural basis for the green-to-red photoconversion. As in Aequorea GFP, a chromophore, 4-(p-hydroxybenzylidene)-5-imidazolinone, derived from the tripeptide mediates green fluorescence in Kaede. UV irradiation causes an unconventional cleavage within Kaede protein between the amide nitrogen and the alpha carbon (Calpha) at His(62) via a formal beta-elimination reaction, which requires the whole, intact protein for its catalysis. The subsequent formation of a double bond between His(62)-Calpha and -Cbeta extends the pi-conjugation to the imidazole ring of His(62), creating a new red-emitting chromophore, 2-[(1E)-2-(5-imidazolyl)ethenyl]-4-(p-hydroxybenzylidene)-5-imidazolinone. The present study not only reveals diversity in the chemical structure of fluorescent proteins but also adds a new dimension to posttranslational modification mechanisms.     

Floral Color Polymorphism and Reproductive Success in Annatto (Bixa orellana L.)

Floral color polymorphism of annatto (Bixa orellana L.) offers a wide range of colors that are maintained in the population by either pollinators or non pollinator agents of selection. In the present study, maintenance of different floral colors was analyzed in relation to reproductive success of Bixa orellana. The different floral petal colors (white, amaranth rose, petunia purple or cobalt violet) were determined from selected plants with reflectance spectrophotometry. Phenotypic measures of other floral traits, female reproductive success, seed set, seed output and seed weight also revealed variation between different floral morphs. Records on seed set varied significantly for different floral color morphs. Maximum fruit maturation (58 %) was observed in amaranth rose and least fruit maturation (25 %) in the white morph. Seed set data indicates pollinators’ preference for more intensely colored flowers. This preference may be due to ability of the pollinators to distinguish the morphs through differentially reduced sensitivity at the green wavelengths. In flowers which received fewer insect visits, polymorphism might be maintained by self fertilization. The color morphs showed differences in Random Amplified Polymorphic DNA (RAPD) profile indicating a genetic basis for floral color variation and consequent differences in seed set. Out of 88 bands generated with nine operon primers, 70 were polymorphic. The present study provides valuable information on the influence of petal color on maternal fitness in B. orellana.     

Novel fluorescent protein from Hydnophora rigida possess cyano emission

Currently, a broad diversity of fluorescent proteins among marine organisms range from cyano-red emissions. Fluorescent proteins differ in their DNA sequences from green fluorescent protein (GFP). We identified cDNA encoding the gene of a new protein from the reef coral Hydnophora rigida of the Merulinidae family. Both the spectral properties and putative primary sequence of the protein has been determined. The cloned cDNA encode peptide we call HriCFP is comprised of 134 amino acids. It has characteristics of a cyano fluorescent protein (HriCFP) and its sequence is markedly different from known GFP from the hydroid jellyfish Aequorea victoria. HriCFP was cloned, expressed, purified and exist as monomer. The peptide mass finger print on the purified protein confirmed identity of HriCFP.