Dynamic Regulation of Fluorescent Proteins from a Single Species of Coral

To gain a better understanding of the natural function of fluorescent proteins, we have undertaken quantitative analyses of these proteins in a single species of coral, Montastraea cavernosa, residing around Turneffe atoll, on the Belizean Barrier Reef. We identified at least 10 members of a fluorescent protein family in this species, which consist of 4 distinct spectral classes. As much as a 10-fold change in the overall expression of fluorescent proteins was observed from specimen to specimen, suggesting that fluorescent proteins are dynamically regulated in response to environmental or physiological conditions. We found that the expression of some proteins was inversely correlated with depth, and that groups of proteins were coordinately expressed. There was no relationship between the expression of fluorescent proteins and the natural coloration of the Montastraea cavernosa specimens in this study. These findings have implications for current hypotheses regarding the properties and natural function of fluorescent proteins.     

Fluorescent protein candidate genes in the coral Acropora digitifera genome

The vivid coloration of corals depends on fluorescent proteins that include cyan (CFP), green (GFP) and red (RFP) fluorescent proteins, and a non-fluorescent blue/purple chromoprotein. We examined how many genes encoding fluorescent proteins are present in the recently sequenced genome of the coral Acropora digitifera. Based on molecular phylogenetic analysis, we found one, five, one, and three candidate genes for CFP, GFP, RFP, and chromoprotein, respectively. The CFP and GFP genes are clustered in a ~80-kb-long genomic region, suggesting that they originated from an ancestral gene by tandem duplication. Since CFP and GFP possess the same chromophore, the gene clustering may provide the first genomic evidence for a common origin of the two proteins. Comparison between the fluorescent protein genes of closely related coral species suggests an expansion of chromoprotein genes in the A. digitifera genome, and of RFP genes in the A. millepora genome. The A. digitifera fluorescent protein genes are expressed during embryonic and larval developmental stages and in adults, suggesting that the genes play a variety of roles in coral physiology.     

Molecular basis and evolutionary origins of color diversity in great star coral Montastraea cavernosa (Scleractinia: Faviida)

Natural pigments are normally products of complex biosynthesis pathways where many different enzymes are involved. Corals and related organisms of class Anthozoa represent the only known exception: in these organisms, each of the host-tissue colors is essentially determined by a sequence of a single protein, homologous to the green fluorescent protein (GFP) from Aequorea victoria. This direct sequence-color linkage provides unique opportunity for color evolution studies. We previously reported the general phylogenetic analysis of GFP-like proteins, which suggested that the present-day diversity of reef colors originated relatively recently and independently within several lineages. The present work was done to get insight into the mechanisms that gave rise to this diversity. Three colonies of the great star coral Montastraea cavernosa (Scleractinia, Faviida) were studied, representing distinct color morphs. Unexpectedly, these specimens were found to express the same collection of GFP-like proteins, produced by at least four, and possibly up to seven, different genetic loci. These genes code for three basic colors-cyan, green, and red-and are expressed differently relative to one another in different morphs. Phylogenetic analysis of the new sequences indicated that the three major gene lineages diverged before separation of some coral families. Our results suggest that color variation in M. cavernosa is not a true polymorphism, but rather a manifestation of phenotypic plasticity (polyphenism). The family level depth of its evolutionary roots indicates that the color diversity is adaptively significant. Relative roles of gene duplication, gene conversion, and point mutations in its evolution are discussed.     

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.     

Red young leaves have less mechanical defence than green young leaves

In many plants, leaves that are young and/or old (senescent) are not green. One adaptive hypothesis proposed that leaf color change could be a warning signal reducing insect attack. If leaf coloration involves less herbivory, it remains unclear why leaves in many species are constantly green. To examine whether green leaves reduce herbivory by physical defense as an alternative to the supposed warning signal of red leaves, we conducted comparative analyses of leaf color and protective tissues of 76 woody species in spring. The protective features (trichomes, enhanced cuticle and multiple epidermis) and the distribution of red pigments within leaves were examined in both young and mature leaves. We observed that redness was more frequent in young leaves than in senescent leaves. Compared to 36 species with red young leaves, 40 species with green young leaves showed a significantly higher incidence of enhanced cuticle and trichomes in both phylogenetic and non‐phylogenetic analyses. The phylogenetic analysis indicated that the multiple origins of mechanical protection were generally associated with loss of red coloration. Our finding of relatively poor mechanical protection in red young leaves provides additional evidence for the adaptive explanation of leaf color change.     

不同叶色三叶海棠叶片成色色素分析

结合民族植物学和药理学的研究方法,对西双版纳地区傣族、哈尼族和基诺族等3个少数民族民间利用番石榴（Psidium guajava）、余甘子（Phyllanthus emblica）和水柳（Homonoia riparia）的传统知识进行调查研究及体外抗菌活性实验。结果表明：番石榴和余甘子在村寨中较为常见,当地少数民族将其种植于庭院中,常作为果蔬食用,食用番石榴嫩叶可缓解拉肚子的症状,治疗腹痛、腹泻。水柳生长在水边,傣族会将其叶作为腌酸鱼的配料之一。根据文献记载,番石榴、余甘子和水柳的叶部位作为药使用时,常煎水外洗,治疗皮肤瘙痒。对这3种药用植物叶部位采用80%乙醇浸泡制备的提取物进行体外抗菌实验,结果显示番石榴、余甘子和水柳3种药用植物对金黄色葡萄球菌和大肠埃希菌均有较好的抑菌和杀菌活性,其最小抑菌浓度MIC在98~390 μg·mL 1之间,最小杀菌浓度MBC在98~781 μg·mL 1之间。番石榴和水柳叶对铜绿假单胞菌有一定抑菌和杀菌活性,其MIC和MBC范围均为6 250~12 500 μg·mL 1。由此可见,这3种药用植物的民间利用具有一定的合理性和药用开发价值。     

Simultaneous detection of DsRed2-tagged and EGFP-tagged human beta-interferons in the same single cells

The red fluorescent protein DsRed2 is a useful fusion tag for various proteins, together with the enhanced green fluorescent protein (EGFP). These chromoproteins have spectral properties that allow simultaneous distinctive detection of tagged proteins in the same single cells by dual color imaging. We used them for tagging a secretory protein, human interferon-beta (IFN-beta). Expression plasmids for human IFN-beta tagged with DsRed2 or with EGFP at the carboxyl terminal were constructed and their coexpression was examined in Mardin-Darby canine kidney epithelial cells. Although maturation of DsRed2 for coloration was slow and the color intensity was weak compared with EGFP, low temperature treatment (20 degrees C) allowed DsRed2-tagged human IFN-beta to be detected in the cells using color imaging. Consequently, the two chimeric proteins were shown to be colocalized in the same single cells by dual color confocal microscopy. This approach will be useful for investigating subcellular localization of not only cell resident proteins but also secretory proteins.     

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.     

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.     

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.     

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.     

Patterns of fluorescent protein expression in Scleractinian corals

Biofluorescence exists in only a few classes of organisms, with Anthozoa possessing the majority of species known to express fluorescent proteins. Most species within the Anthozoan subgroup Scleractinia (reef-building corals) not only express green fluorescent proteins, they also localize the proteins in distinct anatomical patterns.We examined the distribution of biofluorescence in 33 coral species, representing 8 families, from study sites on Australia's Great Barrier Reef. For 28 of these species, we report the presence of biofluorescence for the first time. The dominant fluorescent emissions observed were green (480-520 nm) and red (580-600 nm). Fluorescent proteins were expressed in three distinct patterns (highlighted, uniform, and complementary) among specific anatomical structures of corals across a variety of families. We report no significant overlap between the distribution of fluorescent proteins and the distribution of zooxanthellae. Analysis of the patterns of fluorescent protein distribution provides evidence that the scheme in which fluorescent proteins are distributed among the anatomical structures of corals is nonrandom. This targeted expression of fluorescent proteins in corals produces contrast and may function as a signaling mechanism to organisms with sensitivity to specific wavelengths of light.     

The influence of carotenoid acquisition and utilization on the maintenance of species-typical plumage pigmentation in male American goldfinches (Carduelis tristis) and northern cardinals (Cardinalis cardinalis).

Birds display a tremendous variety of carotenoid-based colors in their plumage, but the mechanisms underlying interspecific variability in carotenoid pigmentation remain poorly understood. Because vertebrates cannot synthesize carotenoids de novo, access to pigments in the diet is one proximate factor that may shape species differences in carotenoid-based plumage coloration. However, some birds metabolize ingested carotenoids and deposit pigments that differ in color from their dietary precursors, indicating that metabolic capabilities may also contribute to the diversity of plumage colors we see in nature. In this study, we investigated how the acquisition and utilization of carotenoids influence the maintenance of species-typical plumage pigmentation in male American goldfinches (Carduelis tristis) and northern cardinals (Cardinalis cardinalis). We supplemented the diet of captive goldfinches with red carotenoids to determine whether males, which are typically yellow in color, were capable of growing red plumage. We also deprived cardinals of red dietary pigments to determine whether they could manufacture red carotenoids from yellow precursors to grow species-typical red plumage. We found that American goldfinches were able to deposit novel pigments in their plumage and develop a striking orange appearance. Thus, dietary access to pigments plays a role in determining the degree to which goldfinches express carotenoid-based plumage coloration. We also found that northern cardinals grew pale red feathers in the absence of red dietary pigments, indicating that their ability to metabolize yellow carotenoids in the diet contributes to the bright red plumage that they display.     

Predation on snakes of Argentina: Effects of coloration and ring pattern on coral and false coral snakes

The occurrence of coral snake coloration among unrelated venomous and non‐venomous snake species has often been explained in terms of warning coloration and mimicry. In Argentina, no field tests have been conducted to confirm this mimetic association between one venomous coral species (Micrurus phyrrocryptus, Elapidae) and two non‐venomous snake species with a similar color pattern (Lystrophis pulcher and Oxyrhopus rhombifer, Colubridae). The aims of this work were to test for the possible aposematic or cryptic function of the ring pattern and coloration of coral snakes and false coral snakes from central Argentina, and to analyse whether the pattern is effective throughout the year. Predation on snakes was estimated by using non‐toxic plasticine replicas of ringed venomous and non‐venomous snakes and unbanded green snakes placed along transects in their natural habitat during the dry and rainy season. Ringed color pattern was attacked by predators despite the background color. One of the replica types was attacked more than expected during the dry season, suggesting that both shape and width of rings may influence the choice by predators. The reaction of predators towards replicas that mimic snake species with ringed patterns is independent of the geographical region, and we can conclude that mimicry characteristics are quite general when the true models are present in the area.     

Countergradient variation and secondary sexual color: phenotypic convergence promotes genetic divergence in carotenoid use between sympatric anadromous and nonanadromous morphs of sockeye salmon (Oncorhynchus nerka)

Genetically distinct anadromous (sockeye) and nonanadromous (kokanee) morphs of the Pacific salmon, Oncorhynchus nerka, develop identical, brilliant red color at maturity during sympatric breeding in freshwater streams. The marine and lacustrine environments they occupy prior to maturity, however, appear to differ in the availability of dietary carotenoid pigments necessary to produce red coloration. We tested the hypothesis that kokanee, which occupy carotenoid-poor lakes, are more efficient at using the dietary pigments than are sockeye, which occupy the more productive North Pacific Ocean. In a 2-year controlled breeding study, flesh and skin color of mature and immature crosses fed a low-carotenoid diet were quantified with both a chromameter and by chemical extraction of carotenoid pigments. Results revealed striking countergradient variation in carotenoid use, with kokanee approximately three times more efficient at sequestering the pigments to the flesh musculature than similar age sockeye. This difference translated into virtually nonoverlapping differences between pure crosses in secondary sexual color at maturity, when the pigments are mobilized and transported to the skin. Kokanee crosses turned pinkish red over most of their body, whereas sockeye turned olive green. The olive green was similar to the breeding color of residuals in the wild, the progeny of anadromous sockeye that remain in fresh water and are believed to have given rise to kokanee on numerous independent occasions. Reciprocal hybrids were similar to each other and intermediate to the pure crosses, indicating additive genetic inheritance. Mate choice trials with sockeye males in the wild showed the ancestral morph strongly preferred red over green models. These results suggest a preference for red mates maintained in nonanadromous breeding populations drove the reevolution of the red phenotype in kokanee via more efficient use of dietary carotenoid pigments. This is a novel, yet hidden, mechanism by which sexual selection promotes the genetic differentiation of these sympatric populations.     

A natural fluorescent protein that changes its fluorescence during maturation

The gene of a new red fluorescent protein zoan2RFP from a coral polyp Zoanthus sp., a homologue of the known green fluorescent protein from the Aequorea victoria jellyfish, was cloned. At early maturation stages, zoan2RFP exhibits a green fluorescence, which then turns into the red one. A similar phenomenon was recently reported for the E5 mutant of the red fluorescent coral protein DsRed. Zoan2RFP differs from E5 by faster maturation kinetics and the complete disappearance of green fluorescence in the mature protein. Naturally occurring proteins of this type can be considered as intermediate forms between the green and red fluorescent proteins, which are formed during the microevolution of fluorescent proteins.     

A Natural Fluorescent Protein That Changes Its Fluorescence Color during Maturation

The gene of a new red fluorescent protein zoan2RFP from coral polyp Zoanthus sp., a homologue of the known green fluorescent protein from the jellyfish Aequorea victoria, was cloned. At early stages of maturation, zoan2RFP exhibits green fluorescence, which then turns to the red one. A similar phenomenon was recently reported for the E5 mutant of the red fluorescent coral protein DsRed. Zoan2RFP differs from E5 by faster maturation kinetics and the complete disappearance of green fluorescence in the mature protein. Naturally occurring proteins of this type can be considered as intermediate forms between the green and red fluorescent proteins, which are formed during the microevolution of fluorescent proteins.     

Exploring plant endomembrane dynamics using the photoconvertible protein Kaede

Photoactivatable and photoconvertible fluorescent proteins capable of pronounced light‐induced spectral changes are a powerful addition to the fluorescent protein toolbox of the cell biologist. They permit specific tracking of one subcellular structure (organelle or cell subdomain) within a differentially labelled population. They also enable pulse–chase analysis of protein traffic. The Kaede gene codes for a tetrameric protein found in the stony coral Trachyphyllia geoffroyi, which emits green fluorescence that irreversibly shifts to red following radiation with UV or violet light. We report here the use of Kaede to explore the plant secretory pathway. Kaede versions of the Golgi marker sialyl‐transferase (ST‐Kaede) and of the vacuolar pathway marker cardosin A (cardA‐Kaede) were engineered. Several optical devices enabling photoconversion and observation of Kaede using these two constructs were assessed to optimize Kaede‐based imaging protocols. Photoconverted ST‐Kaede red‐labelled organelles can be followed within neighbouring populations of non‐converted green Golgi stacks, by their gradual development of orange/yellow coloration from de novo synthesis of Golgi proteins (green). Results highlight some aspects on the dynamics of the plant Golgi. For plant bio‐imaging, the photoconvertible Kaede offers a powerful tool to track the dynamic behaviour of designated subpopulations of Golgi within living cells, while visualizing the de novo formation of proteins and structures, such as a Golgi stack.     

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.     

Ornamental non-carotenoid red feathers of wild burrowing parrots

Bird plumage colors have the potential to indicate individual quality, condition, health, immunocompetence, or the extend of parental care. Color intensity of feathers has been found to correlate with parameters of individual quality, condition, parental care and breeding success. Psittaciformes are well known for their colorful plumage but the significance of parrot coloration is still poorly understood. Red colors are very common in many parrot species. They are produced by at least four non-carotenoid-based pigments (linear polyenal structure). In the present study, we investigated a collection of red abdominal feathers of a marked population of wild Burrowing Parrots Cyanoliseus patagonus in Patagonia, Argentina. The aims of this study were to investigate the ecological significance of the recently described non-carotenoid-based red pigments of Psittaciformes, and the relationships between objectively assessed plumage color and body size, body condition, breeding success and nestling growth in wild Psittaciformes. We found that sexes differed in plumage coloration (sexual dichromatism), that plumage color was a good predictor of female body condition and male size, and we identified the red coloration of the abdominal patch as a signal of individual quality and parental investment.