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1.
Sci Total Environ ; 715: 136951, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32014776

RESUMO

The frequency and intensity of marine heatwaves that result in coral bleaching events have increased over recent decades and led to catastrophic losses of reef-building corals in many regions. The high-latitude coral assemblages at Lord Howe Island, which is a UNESCO listed site is the world southernmost coral community, were exposed to successive thermal anomalies following a fast phase-transition of the record-breaking 2009 to 2010 warm pool El Niño in the Central Pacific to a strong La Niña event in late 2010. The coral community experienced severe and unprecedented consecutive bleaching in both 2010 and 2011. Coral health surveys completed between March 2010 and September 2012 quantified the response and recovery of approximately 43,700 coral colonies to these successive marine heatwaves. In March 2010, coral bleaching ranged from severe, with 99% of colonies bleached at some shallow lagoon sites, to mild at deeper reef slope sites, with only 17% of individuals affected. Significant immediate mortality from thermal stress was evident during the peak of the bleaching event. Overall, species in the genera Pocillopora, Stylophora, Seriatopora and Porites were the most affected, while minimal bleaching and mortality was recorded among members of other coral families (e.g. Acroporidae, Dendrophyllidae & Merulinidae). Surviving corals underwent a subsequent, but much less intense, thermal anomaly in 2011 that led to a disproportionate bleaching response among susceptible taxa. While this observation indicates that the capacity of thermally susceptible high-latitude corals to acclimatize to future ocean warming may be limited, particularly if bleaching events occur annually, our long-term survey data shows that coral cover at most sites recovered to pre-bleaching levels within three years in the absence of further thermal anomalies.

2.
J Phys Chem Lett ; 8(12): 2548-2554, 2017 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-28530831

RESUMO

Fluorescent proteins (FPs) are indispensable markers for two-photon imaging of live tissue, especially in the brains of small model organisms. The quantity of physiologically relevant data collected, however, is limited by heat-induced damage of the tissue due to the high intensities of the excitation laser. We seek to minimize this damage by developing FPs with improved brightness. Among FPs with the same chromophore structure, the spectral properties can vary widely due to differences in the local protein environment. Using a physical model that describes the spectra of FPs containing the anionic green FP (GFP) chromophore, we predict that those that are blue-shifted in one-photon absorption will have stronger peak two-photon absorption cross sections. Following this prediction, we present 12 blue-shifted GFP homologues and demonstrate that they are up to 2.5 times brighter than the commonly used enhanced GFP (EGFP).


Assuntos
Proteínas de Fluorescência Verde/química , Temperatura Alta , Espectrometria de Fluorescência , Cor , Corantes Fluorescentes , Proteínas Luminescentes/química , Modelos Moleculares , Fótons
3.
Appl Environ Microbiol ; 83(13)2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28455341

RESUMO

To investigate the function of 2-methylhopanoids in modern cyanobacteria, the hpnP gene coding for the radical S-adenosyl methionine (SAM) methylase protein that acts on the C-2 position of hopanoids was deleted from the filamentous cyanobacterium Nostoc punctiforme ATCC 29133S. The resulting ΔhpnP mutant lacked all 2-methylhopanoids but was found to produce much higher levels of two bacteriohopanepentol isomers than the wild type. Growth rates of the ΔhpnP mutant cultures were not significantly different from those of the wild type under standard growth conditions. Akinete formation was also not impeded by the absence of 2-methylhopanoids. The relative abundances of the different hopanoid structures in akinete-dominated cultures of the wild-type and ΔhpnP mutant strains were similar to those of vegetative cell-dominated cultures. However, the ΔhpnP mutant was found to have decreased growth rates under both pH and osmotic stress, confirming a role for 2-methylhopanoids in stress tolerance. Evidence of elevated photosystem II yield and NAD(P)H-dependent oxidoreductase activity in the ΔhpnP mutant under stress conditions, compared to the wild type, suggested that the absence of 2-methylhopanoids increases cellular metabolic rates under stress conditions.IMPORTANCE As the first group of organisms to develop oxygenic photosynthesis, Cyanobacteria are central to the evolutionary history of life on Earth and the subsequent oxygenation of the atmosphere. To investigate the origin of cyanobacteria and the emergence of oxygenic photosynthesis, geobiologists use biomarkers, the remnants of lipids produced by different organisms that are found in geologic sediments. 2-Methylhopanes have been considered indicative of cyanobacteria in some environmental settings, with the parent lipids 2-methylhopanoids being present in many contemporary cyanobacteria. We have created a Nostoc punctiforme ΔhpnP mutant strain that does not produce 2-methylhopanoids to assess the influence of 2-methylhopanoids on stress tolerance. Increased metabolic activity in the mutant under stress indicates compensatory alterations in metabolism in the absence of 2-methylhopanoids.


Assuntos
Nostoc/metabolismo , Triterpenos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Concentração de Íons de Hidrogênio , Isomerismo , Metilação , Nostoc/química , Nostoc/genética , Nostoc/crescimento & desenvolvimento , Osmose , Triterpenos/química
4.
J Exp Bot ; 68(12): 3191-3204, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28338729

RESUMO

Regulation of root cell K+ is essential for acclimation to low oxygen stress. The potential roles of GORK (depolarization-activated guard cell outward-rectifying potassium) channels and RBOHD (respiratory burst oxidase homologue D) in plant adaptive responses to hypoxia were investigated in the context of tissue specificity (epidermis versus stele; elongation versus mature zone) in roots of Arabidopsis. The expression of GORK and RBOHD was down-regulated by 2- to 3-fold within 1 h and 24 h of hypoxia treatment in Arabidopsis wild-type (WT) roots. Interestingly, a loss of the functional GORK channel resulted in a waterlogging-tolerant phenotype, while rbohD knockout was sensitive to waterlogging. To understand their functions under hypoxia stress, we studied K+, Ca2+, and reactive oxygen species (ROS) distribution in various root cell types. gork1-1 plants had better K+ retention ability in both the elongation and mature zone compared with the WT and rbohD under hypoxia. Hypoxia induced a Ca2+ increase in each cell type after 72 h, and the increase was much less pronounced in rbohD than in the WT. In most tissues except the elongation zone in rbohD, the H2O2 concentration had decreased after 1 h of hypoxia, but then increased significantly after 24 h of hypoxia in each zone and tissue, further suggesting that RBOHD may shape hypoxia-specific Ca2+ signatures via the modulation of apoplastic H2O2 production. Taken together, our data suggest that plants lacking functional GORK channels are more capable of retaining K+ for their better performance under hypoxia, and that RBOHD is crucial in hypoxia-induced Ca2+ signalling for stress sensing and acclimation mechanism.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas , NADPH Oxidases/genética , Canais de Potássio/genética , Potássio/metabolismo , Aclimatação , Anaerobiose , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , NADPH Oxidases/metabolismo , Especificidade de Órgãos , Raízes de Plantas/metabolismo , Canais de Potássio/metabolismo
5.
Plant Physiol ; 167(3): 963-71, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25588734

RESUMO

The transfer of water from phloem into xylem is thought to mitigate increasing hydraulic tension in the vascular system of trees during the diel cycle of transpiration. Although a putative plant function, to date there is no direct evidence of such water transfer or the contributing pathways. Here, we trace the radial flow of water from the phloem into the xylem and investigate its diel variation. Introducing a fluorescent dye (0.1% [w/w] fluorescein) into the phloem water of the tree species Eucalyptus saligna allowed localization of the dye in phloem and xylem tissues using confocal laser scanning microscopy. Our results show that the majority of water transferred between the two tissues is facilitated via the symplast of horizontal ray parenchyma cells. The method also permitted assessment of the radial transfer of water during the diel cycle, where changes in water potential gradients between phloem and xylem determine the extent and direction of radial transfer. When injected during the morning, when xylem water potential rapidly declined, fluorescein was translocated, on average, farther into mature xylem (447 ± 188 µm) compared with nighttime, when xylem water potential was close to zero (155 ± 42 µm). These findings provide empirical evidence to support theoretical predictions of the role of phloem-xylem water transfer in the hydraulic functioning of plants. This method enables investigation of the role of phloem tissue as a dynamic capacitor for water storage and transfer and its contribution toward the maintenance of the functional integrity of xylem in trees.


Assuntos
Floema/metabolismo , Caules de Planta/metabolismo , Árvores/metabolismo , Água/metabolismo , Xilema/metabolismo , Transporte Biológico , Eucalyptus/anatomia & histologia , Eucalyptus/ultraestrutura , Corantes Fluorescentes/metabolismo , Fatores de Tempo , Madeira/anatomia & histologia , Madeira/ultraestrutura
6.
Methods Appl Fluoresc ; 1(3): 35001, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24040513

RESUMO

The phasor global analysis algorithm is common for fluorescence lifetime applications, but has only been recently proposed for spectral analysis. Here the phasor representation and fingerprinting is exploited in its second harmonic to determine the number and spectra of photo-activated states as well as their conversion dynamics. We follow the sequence of photo-activation of proteins over time by rapidly collecting multiple spectral images. The phasor representation of the cumulative images provides easy identification of the spectral signatures of each photo-activatable protein.

7.
Protist ; 164(2): 237-44, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23063731

RESUMO

Scleractinian corals occur in symbiosis with a range of organisms including the dinoflagellate alga, Symbiodinium, an association that is mutualistic. However, not all symbionts benefit the host. In particular, many organisms within the microbial mucus layer that covers the coral epithelium can cause disease and death. Other organisms in symbiosis with corals include the recently described Chromera velia, a photosynthetic relative of the apicomplexan parasites that shares a common ancestor with Symbiodinium. To explore the nature of the association between C. velia and corals we first isolated C. velia from the coral Montipora digitata and then exposed aposymbiotic Acropora digitifera and A. tenuis larvae to these cultures. Three C. velia cultures were isolated, and symbiosis was established in coral larvae of both these species exposed to all three clones. Histology verified that C. velia was located in the larval endoderm and ectoderm. These results indicate that C. velia has the potential to be endosymbiotic with coral larvae.


Assuntos
Alveolados/fisiologia , Antozoários/parasitologia , Simbiose , Alveolados/isolamento & purificação , Animais , Histocitoquímica , Larva/parasitologia , Microscopia
8.
Methods Appl Fluoresc ; 1(3): 035001, 2013 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-29148446

RESUMO

The phasor global analysis algorithm is common for fluorescence lifetime applications, but has only been recently proposed for spectral analysis. Here the phasor representation and fingerprinting is exploited in its second harmonic to determine the number and spectra of photo-activated states as well as their conversion dynamics. We follow the sequence of photo-activation of proteins over time by rapidly collecting multiple spectral images. The phasor representation of the cumulative images provides easy identification of the spectral signatures of each photo-activatable protein.

9.
J Exp Bot ; 63(16): 5829-41, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22915750

RESUMO

The rise in atmospheric [CO(2)] is associated with increasing air temperature. However, studies on plant responses to interactive effects of [CO(2)] and temperature are limited, particularly for leaf structural attributes. In this study, Eucalyptus saligna plants were grown in sun-lit glasshouses differing in [CO(2)] (290, 400, and 650 µmol mol(-1)) and temperature (26 °C and 30 °C). Leaf anatomy and chloroplast parameters were assessed with three-dimensional confocal microscopy, and the interactive effects of [CO(2)] and temperature were quantified. The relative influence of leaf structural attributes and chemical properties on the variation of leaf mass per area (LMA) and photosynthesis within these climate regimes was also determined. Leaf thickness and mesophyll size increased in higher [CO(2)] but decreased at the warmer temperature; no treatment interaction was observed. In pre-industrial [CO(2)], warming reduced chloroplast diameter without altering chloroplast number per cell, but the opposite pattern (reduced chloroplast number per cell and unchanged chloroplast diameter) was observed in both current and projected [CO(2)]. The variation of LMA was primarily explained by total non-structural carbohydrate (TNC) concentration rather than leaf thickness. Leaf photosynthetic capacity (light- and [CO(2)]-saturated rate at 28 °C) and light-saturated photosynthesis (under growth [CO(2)] and temperature) were primarily determined by leaf nitrogen contents, while secondarily affected by chloroplast gas exchange surface area and chloroplast number per cell, respectively. In conclusion, leaf structural attributes are less important than TNC and nitrogen in affecting LMA and photosynthesis responses to the studied climate regimes, indicating that leaf structural attributes have limited capacity to adjust these functional traits in a changing climate.


Assuntos
Dióxido de Carbono/metabolismo , Ecossistema , Eucalyptus/metabolismo , Fotossíntese , Folhas de Planta/anatomia & histologia , Folhas de Planta/metabolismo , Mudança Climática , Eucalyptus/anatomia & histologia , Eucalyptus/química , Cinética , Nitrogênio/metabolismo , Folhas de Planta/química , Temperatura Ambiente
10.
Methods Mol Biol ; 872: 217-33, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22700414

RESUMO

The discovery of multicolored fluorescent proteins (FPs), in reef corals, that are close relatives of the green fluorescent protein (GFP) has led to what is now viewed as the second GFP revolution. Numerous GFP-type proteins, termed "reef FPs," have been cloned from reef organisms and many possess new colors, novel molecular characteristics, protein chemistry and many display unusual photophysical properties. Although some FPs have certain disadvantageous properties, such as the tendency to oligomerize or have slow maturation rates, reef FPs have been developed into versatile probes for cell biology and imaging applications. Screening of natural sources for novel GFP-type proteins continues to be valuable due to the need to expand the range of spectral colors, brightness, monomeric or dimeric states, faster maturation states, and photoactivity. Confocal imaging, coupled with microspectral detection, provides a rapid technique for in vivo characterization of FPs with desirable spectral and photoactive properties.


Assuntos
Recifes de Corais , Proteínas de Fluorescência Verde , Microscopia de Fluorescência
11.
J Phycol ; 48(6): 1320-7, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27009985

RESUMO

A new habitat and a new chlorophyll (Chl) d-containing cyanobacterium belonging to the genus Acaryochloris are reported in this study. Hyperspectral microscopy showed the presence of Chl d-containing microorganisms in epiphytic biofilms on a red alga (Gelidium caulacantheum) colonizing the pneumato-phores of a temperate mangrove (Avicennia marina). The presence of Chl d was further proven by high performance liquid chromatography (HPLC)-based pigment analysis and by confocal imaging of cultured cells. Enrichment of mangrove biofilm samples under near-infrared radiation (NIR) yielded the new Acaryochloris sp. MPGRS1, which was closely related in terms of 16S rRNA gene sequence to an isolate from the hypertrophic Salton Sea, USA. The new isolate used Chl d as its major photopigment; Chl d and Chl a contents were ~98% and 1%-2% of total cellular chlorophyll, respectively. These findings expand the variety of ecological niches known to harbor Chl d-containing cyanobacteria and support our working hypothesis that such oxyphototrophs may be ubiquitous in habitats depleted of visible light, but with sufficient NIR exposure.

12.
PLoS One ; 6(7): e20841, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21750703

RESUMO

BACKGROUND: The calcifying siphonalean green alga, Halimeda macroloba is abundant on coral reefs and is important in the production of calcium carbonate sediments. The process by which new green segments are formed over-night is revealed here for the first time. METHODOLOGY/PRINCIPAL FINDINGS: Growth of new segments was visualised by epifluorescence and confocal microscopy and by pulse amplitude modulation (PAM) fluorimetry. Apical colourless proto-segments were initiated on day 1, and formed a loose network of non-calcified, non-septate filaments, containing no chloroplasts. Rapid greening was initiated at dusk by i) the mass movement of chloroplasts into these filaments from the parent segment and ii) the growth of new filaments containing chloroplasts. Greening was usually complete in 3-5 h and certainly before dawn on day 2 when the first signs of calcification were apparent. Mass chloroplast movement took place at a rate of ∼0.65 µm/s. Photosynthetic yield and rate remained low for a period of 1 to several hours, indicating that the chloroplasts were made de novo. Use of the inhibitors colchicine and cytochalasin d indicated that the movement process is dependent on both microtubules and microfilaments. SIGNIFICANCE: This unusual process involves the mass movement of chloroplasts at a high rate into new segments during the night and rapid calcification on the following day and may be an adaptation to minimise the impact of herbivorous activity.


Assuntos
Carbonato de Cálcio/metabolismo , Clorófitas/crescimento & desenvolvimento , Clorófitas/metabolismo , Cloroplastos/metabolismo , Clorofila/metabolismo , Clorófitas/efeitos dos fármacos , Colchicina/farmacologia , Citocalasinas/farmacologia , Corrente Citoplasmática/efeitos dos fármacos , Fluorometria/métodos , Cinética , Microscopia Confocal , Microscopia de Fluorescência , Oxigênio/metabolismo , Moduladores de Tubulina/farmacologia
13.
Antimicrob Agents Chemother ; 55(3): 1204-10, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21199928

RESUMO

Many studies have examined the evolution of bacterial mutants that are resistant to specific antibiotics, and many of these focus on concentrations at and above the MIC. Here we ask for the minimum concentration at which existing resistant mutants can outgrow sensitive wild-type strains in competition experiments at antibiotic levels significantly below the MIC, and we define a minimum selective concentration (MSC) in Escherichia coli for two antibiotics, which is near 1/5 of the MIC for ciprofloxacin and 1/20 of the MIC for tetracycline. Because of the prevalence of resistant mutants already in the human microbiome, allowable levels of antibiotics to which we are exposed should be below the MSC. Since this concentration often corresponds to low or trace levels of antibiotics, it is helpful to have simple tests to detect such trace levels. We describe a simple ultrasensitive test for detecting the presence of antibiotics and genotoxic agents. The test is based on the use of chromogenic proteins as color markers and the use of single and multiple mutants of Escherichia coli that have greatly increased sensitivity to either a wide range of antibiotics or specific antibiotics, antibiotic families, and genotoxic agents. This test can detect ciprofloxacin at 1/75 of the MIC.


Assuntos
Antibacterianos/farmacologia , Cor , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos
14.
PLoS One ; 3(7): e2680, 2008 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-18648549

RESUMO

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.


Assuntos
Antozoários/metabolismo , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Animais , Evolução Biológica , Clonagem Molecular , Evolução Molecular , Regulação da Expressão Gênica , Variação Genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/metabolismo , Fases de Leitura Aberta , Filogenia , Probabilidade , Espectrofotometria/métodos
15.
FEBS J ; 274(10): 2496-505, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17419724

RESUMO

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.


Assuntos
Antozoários/química , Proteínas Luminescentes/metabolismo , Pigmentos Biológicos/metabolismo , Animais , Antozoários/efeitos da radiação , Cor , Escuridão , Cinética , Luz , Proteínas Luminescentes/efeitos da radiação , Pigmentos Biológicos/efeitos da radiação , Espectrometria de Fluorescência
16.
Microsc Res Tech ; 70(3): 243-51, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17279514

RESUMO

Corals, like many other coelenterates, contain fluorescent pigments that show considerable homology with the well known green fluorescent protein of the jellyfish Aequoria. In corals, unlike jellyfish, multiple proteins are present and the range of excitations and emissions suggest the possibility of energy transfer. The occurrence of Förster resonant energy transfer (FRET) between fluorescent proteins in corals has already been reported and time-resolved spectra have shown the effect on fluorescent lifetime, but without any spatial resolution. Lifetime confocal microscopy offers lower time resolution but excellent spatial resolution. Lifetimes of the isolated A. millepora pigments amilFP490, amilFP504, and amilFP593 (names indicate emission peaks) were 2.8, 2.9, and 2.9 ns, respectively. In the coral sample, imaging the entire emission spectrum from 420 nm, the mean lifetime was reduced to 1.5 ns, implying that FRET was occurring. Looking just at the fluorescence from FRET donors the lifetime was even shorter, at 1.3 ns, supporting this interpretation. In contrast, no reduction in lifetime is seen in the coral Euphyllia ancora, where the pigment distribution also suggests that the pigments are unlikely to be involved in photoprotection. This study set out to determine the extent of FRET between pigments in two corals, Acropora millepora and Euphyllia, ancora which differ in the arrangement of their pigments and hence possibly in pigment function.


Assuntos
Antozoários/metabolismo , Fluorescência , Proteínas Luminescentes/análise , Microscopia de Fluorescência/métodos , Animais , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Fluorescência Verde/análise , Pigmentos Biológicos/análise
17.
FEBS J ; 274(4): 1102-9, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17244197

RESUMO

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.


Assuntos
Antozoários/metabolismo , Pigmentação/fisiologia , Pigmentos Biológicos/química , Simbiose , Animais , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Imunoquímica , Proteínas Luminescentes/química , Proteínas Luminescentes/metabolismo , Ficoeritrina/química , Especificidade da Espécie , Espectrometria de Fluorescência
18.
Proc Natl Acad Sci U S A ; 101(45): 15905-10, 2004 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-15505211

RESUMO

A gene encoding a fluorescent protein from the stony coral Lobophyllia hemprichii has been cloned in Escherichia coli and characterized by biochemical and biophysical methods. The protein, which we named EosFP, emits strong green fluorescence (516 nm) that changes to red (581 nm) upon near-UV irradiation at approximately 390 nm because of a photo-induced modification involving a break in the peptide backbone next to the chromophore. Single-molecule fluorescence spectroscopy shows that the wild type of EosFP is tetrameric, with strong Forster resonance coupling among the individual fluorophores. We succeeded in breaking up the tetramer into AB and AC subunit dimers by introducing the single point mutations V123T and T158H, respectively, and the combination of both mutations yielded functional monomers. Fusion constructs with a variety of proteins were prepared and expressed in human cells, showing that normal biological functions were retained. The possibility to locally change the emission wavelength by focused UV light makes EosFP a superb marker for experiments aimed at tracking the movements of biomolecules within the living cell.


Assuntos
Proteínas Luminescentes/química , Proteínas Luminescentes/efeitos da radiação , Animais , Antozoários/química , Antozoários/genética , Cor , Genes Reporter , Proteínas Luminescentes/genética , Dados de Sequência Molecular , Estrutura Molecular , Mutagênese Sítio-Dirigida , Fotoquímica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/efeitos da radiação , Espectrometria de Fluorescência , Raios Ultravioleta
19.
Photochem Photobiol ; 77(5): 515-23, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12812294

RESUMO

Light is absorbed by photosynthetic algal symbionts (i.e. zooxanthellae) and by chromophoric fluorescent proteins (FP) in reef-building coral tissue. We used a streak-camera spectrograph equipped with a pulsed, blue laser diode (50 ps, 405 nm) to simultaneously resolve the fluorescence spectra and kinetics for both the FP and the zooxanthellae. Shallow water (<9 m)-dwelling Acropora spp. and Plesiastrea versipora specimens were collected from Okinawa, Japan, and Sydney, Australia, respectively. The main FP emitted light in the blue, blue-green and green emission regions with each species exhibiting distinct color morphs and spectra. All corals showed rapidly decaying species and reciprocal rises in greener emission components indicating Förster resonance energy transfer (FRET) between FP populations. The energy transfer modes were around 250 ps, and the main decay modes of the acceptor FP were typically 1900-2800 ps. All zooxanthellae emitted similar spectra and kinetics with peak emission (approximately 683 nm) mainly from photosystem II (PSII) chlorophyll (chl) a. Compared with the FP, the PSII emission exhibited similar rise times but much faster decay times, typically around 640-760 ps. The fluorescence kinetics and excitation versus emission mapping indicated that the FP emission played only a minor role, if any, in chl excitation. We thus suggest the FP could only indirectly act to absorb, screen and scatter light to protect PSII and underlying and surrounding animal tissue from excess visible and UV light. We conclude that our time-resolved spectral analysis and simulation revealed new FP emission components that would not be easily resolved at steady state because of their relatively rapid decays due to efficient FRET. We believe the methods show promise for future studies of coral bleaching and for potentially identifying FP species for use as genetic markers and FRET partners, like the related green FP from Aequorea spp.


Assuntos
Antozoários/química , Clorofila/química , Eucariotos/química , Proteínas Luminescentes/química , Animais , Transferência de Energia , Fluorescência , Espectrometria de Fluorescência
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