Ultraviolet-B photodestruction of a light-harvesting complex.

Cyanobacteria are important contributors to global photosynthesis in both marine and terrestrial environments. Quantitative data are presented on UV-B-induced damage to the major cyanobacterial photosynthetic light harvesting complex, the phycobilisome, and to each of its constituent phycobiliproteins. The photodestruction quantum yield, phi295 nm, for the phycobiliproteins is high (approximately 10(-3), as compared with approximately 10(-7) for visible light). Energy transfer on a picosecond time scale does not compete with photodestruction. Photodamage to phycobilisomes in vitro and in living cells is amplified by causing dissociation and loss of function of the complex. In photosynthetic organisms, UV-B damage to light-harvesting complexes may significantly exceed that to DNA.

FM1-43 dye ultrastructural localization in and release from frog motor nerve terminals.

Previous work has shown that the fluorescent styryl dye FM1-43 stains nerve terminals in an activity-dependent fashion. This dye appears to label the membranes of recycled synaptic vesicles by being trapped during endocytosis. Stained terminals can subsequently be destained by repeating nerve stimulation in the absence of dye; the destaining evidently reflects escape of dye into the bathing medium from membranes of exocytosing synaptic vesicles. In the present study we tested two key aspects of this interpretation of FM1-43 behavior, namely: (i) that the dye is localized in synaptic vesicles, and (ii) that it is actually released into the bathing medium during destaining. To accomplish this, we first photolyzed the internalized dye in the presence of diaminobenzidine. This created an electron-dense reaction product that could be visualized in the electron microscope. Reaction product was confined to synaptic vesicles, as predicted. Second, using spectrofluorometry, we quantified the release of dye liberated into the medium from tubocurarine-treated nerve-muscle preparations. Nerve stimulation increased the amount of FM1-43 released, and we estimate that normally a stained synaptic vesicle contains a few hundred molecules of the dye. The key to the successful detection of released FM1-43 was to add the micelle-forming detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), which increased FM1-43 quantum yield by more than two orders of magnitude.

The rate constant of photoinhibition, measured in lincomycin-treated leaves, is directly proportional to light intensity.

Pumpkin leaves grown under high light (500-700 micromol of photons m-2.s-1) were illuminated under photon flux densities ranging from 6.5 to 1500 micromol.m-2.s-1 in the presence of lincomycin, an inhibitor of chloroplast protein synthesis. The illumination at all light intensities caused photoinhibition, measured as a decrease in the ratio of variable to maximum fluorescence. Loss of photosystem II (PSII) electron transfer activity correlated with the decrease in the fluorescence ratio. The rate constant of photoinhibition, determined from first-order fits, was directly proportional to photon flux density at all light intensities studied. The fluorescence ratio did not decrease if the leaves were illuminated in low light in the absence of lincomycin or incubated in darkness in the presence of lincomycin. The constancy of the quantum yield of photoinhibition under different photon flux densities strongly suggests that photoinhibition in vivo occurs by one dominant mechanism under all light intensities. This mechanism probably is not the acceptor side mechanism characterized in the anaerobic case in vitro. Furthermore, there was an excellent correlation between the loss of PSII activity and the loss of the D1 protein from thylakoid membranes under low light. At low light, photoinhibition occurs so slowly that inactive PSII centers with the D1 protein waiting to be degraded do not accumulate. The kinetic agreement between D1 protein degradation and the inactivation of PSII indicates that the turnover of the D1 protein depends on photoinhibition under both low and high light.

Relationship between photosynthetic electron transport and pH gradient across the thylakoid membrane in intact leaves.

Under conditions (0.2% CO2; 1% O2) that allow high rates of photosynthesis, chlorophyll fluorescence was measured simultaneously with carbon assimilation at various light intensities in spinach (Spinacia oleracea) leaves. Using a stoichiometry of 3 ATP/CO2 and the known relationship between ATP synthesis rate and driving force (Delta pH), we calculated the light-dependent pH gradient (Delta pH) across the thylakoid membrane in intact leaves. These Delta pH values were correlated with the photochemical (qP) and nonphotochemical (qN) quenching of chlorophyll fluorescence and with the quantum yield of photosystem II (phiPSII). At Delta pH > 2.1 all three parameters (qP, qN, and phiPSII) changed very steeply with increasing DeltapH (decreasing pH in the thylakoid). The observed pH dependences followed hexacooperative titration curves with slightly different pKa values. The significance of the steep pH dependences with slightly different pKa values is discussed in relation to the regulation of photosynthetic electron transport in intact leaves.

Photodissociation of a (mu-peroxo)(mu-hydroxo)bis[bis(bipyridyl)-cobalt(III)] complex: a tool to study fast biological reactions involving O2.

Upon photolysis at 355 nm, dioxygen is released from a (mu-peroxo)(mu-hydroxo)bis[bis(bipyridyl)cobalt-(III)] complex in aqueous solutions and at physiological pH with a quantum yield of 0.04. The [Co(bpy)2(H2O)2]2+ (bpy = bipyridyl) photoproduct was generated on a nanosecond or faster time scale as determined by time-resolved optical absorption spectroscopy. A linear correspondence between the spectral changes and the oxygen production indicates that O2 is released on the same time scale. Oxyhemoglobin was formed from deoxyhemoglobin upon photodissociation of the (mu-peroxo) (mu-hydroxo)bis[bis(bipyridyl)cobalt(III)] complex, verifying that dioxygen is a primary photoproduct. This complex and other related compounds provide a method to study fast biological reactions involving O2, such as the reduction of dioxygen to water by cytochrome oxidase.

Propriedades fotoquímicas de alguns complexos de ferro(II) e rênio(I)

Neste trabalho, foram estudadas as propriedades fotoquímica e/ou fotofísica de alguns compostos de coordenação de rênio(l) e ferro(I I). A irradiação dos complexos fac-[Re(CO)3(NN)(trans-L)]+, NN= 4,7-difenil-1,10- fenantrolina (ph2phen) ou 5-cloro-1,10-fenantrolina (Clphen) e L = 1,2-bis(4-piridil)etileno (bpe) ou 4-estirilpiridina (stpy), em acetonitrila ou em filme de poli(metacrilato de metila) (PMMA) resulta em variações espectrais condizentes com a fotoisomerização trans-cis do ligante coordenado. A determinação dos rendimentos quânticos para a fotorreação pela variação espectral resultou em valores aparentes, uma vez que o reagente e o fotoproduto absorvem na mesma região. Para a determinação do rendimento quântico real, Φreal, utilizou-se a técnica de 1H RMN, na qual os sinais do fotoproduto e do reagente são observados em regiões distintas com diferentes constantes de acoplamento. Os valores de Φreal obtidos para fac-[Re(CO)3h(NN)(trans-bpe)]+ (ph2phen: Φ313= 0,43 ± 0,03; Φ365= 0,44 ± 0,02; Φ404= 0,43 ± 0,02; Clphen: Φ313= 0,56 ± 0,03; Φ365= 0,55 ± 0,04; Φ404= 0,57 ± 0,06) são independentes do comprimento de onda de irradiação, indicando a existência de um único canal para a população do estado excitado 3ILtrans-bpe. Por outro lado, para fac-[Re(CO)3(NN)(trans-stpy)]+, os valores de Φreal sob irradiação a 404 nm são menores que os determinados para os demais comprimentos de onda de irradiação (ph2phen: Φ313= 0,60 ± 0,05; Φ365= 0,64 ± 0,09; Φ404= 0,42 ± 0,03; Clphen: Φ313= 0,52 ± 0,05; Φ365= 0,58 ± 0,02; Φ404= 0,41 ± 0,06), indicando que, a energias maiores, em que o Iigante absorve significativamente, deve existir a contribuição de outro canal para a população do estado excitado 3ILtrans-stpy. A eficiência do fotoprocesso foi avaliada por meio da substituição dos ligantes NN e/ou L, e a diferença nos valores de Φreal entre os complexos deve estar relacionada principalmente com as distintas eficiências de cruzamento intersistemas. o fotoprocesso altera as propriedades fotofísicas desses complexos. Os isômeros trans apresentam fraca ou nenhuma emissão a 298 K, enquanto os fotoprodutos, fac-[Re(CO)3(NN)(cis-L)]+, apresentam intensa luminescência dominada pelo estado excitado 3MLCTRe→NN, que é sensivel à rigidez do meio. A reatividade fotoquímica dos pentacianoferratos(II) [Fe(CN)5 (NN)]3-, NN= 2aminobenzilamina (aba), 2-aminobenzamida (ab), 2-(dimetilaminometil)-3-hidroxipiridina (dmampy), 2-aminometilpiridina (ampy), 2-aminoetilpiridina (aepy) ou 2-(2metilaminoetil) piridina (maepy), também foi investigada. A irradiação desses complexos resulta na fotossubstituição do CN-, a qual só pode ser detectada quando o ligante possui um segundo grupo coordenante nas proximidades da esfera de coordenação. Os rendimentos quânticos da fotossubstituição são dependentes do comprimento de onda de irradiação (Φ313= 0,13 ± 0,01; Φ334= 0,091 ± 0,001; Φ365= 0,056 ± 0,002; Φ404= 0,022 ± 0,002; Φ436= 0,015 ± 0,001, por exemplo, para NN = aba) e indicam a existência de canais distintos pelos quais a fotorreação ocorre ou as diferentes eficiências de cruzamento intersistema para a população do estado excitado reativo. A eficiência do fotoprocesso também depende do Iigante utilizado (λirr= 365 nm: Φaba= 0,056, Φab= 0,14, Φampy= 0,046, Φaepy= 0,066, Φmaepy= 0,069 e Φdmampy= 0,12). Na série das diaminas, o rendimento quântico é maior para [Fe(CN)5(ab)]3-, que possui dois sítios para ocorrer o fechamento do anel. Na série das aminopiridinas, observa-se a influência do comprimento da cadeia na eficiência do fechamento do anel. A presença de metilas ligadas ao nitrogênio alifático deve ter pouca ou nenhuma influência na eficiência do fotoprocesso.

Photogenerated o-Azaxylylenes: Mechanistic Studies and Synthetic Applications

This research sets out to build upon excited state o-azaxylylene cycloaddition. The mechanism behind the excitation and cycloaddition process of photogenerated o-azaxylylenes was determined experimentally. Time-correlated single-photon counting, steady-state spectroscopy, triplet quenching experiments, and quantum yield studies provided evidence suggesting that excited state intramolecular proton transfer is followed by intersystem crossing and stepwise addition to the tethered unsaturated pendant. In keeping with the principles of diversity oriented synthesis, a modular approach was taken to gain access to a diverse array of N,O,S-Polyheterocycles which were modified postphotochemically via Suzuki coupling to yield fused biaryls. Cycloaddition products, outfitted with halogens in the aromatic ring of the o-azaxylylene, proved to be reactive with a variety of boronic acids resulting in a rapid growth in structural complexity. A novel procedure was developed that utilized multiple o-azaxylylene cores in a photochemical cascade transformation yielding complex scaffolds of unprecedented topology. The photoprecursors were produced in a one-pot two-step sequence from commercially available starting materials, and upon irradiation yield structures containing up to five fused hetrocyclic rings, and showed complete diastereoselectivity.

Net community production, Thorium-234 activity and particle flux during the Southern Ocean iron fertilization experiment LOHAFEX

A closed eddy core in the Subantarctic Atlantic Ocean was fertilized twice with two tons of iron (as FeSO4), and the 300 km**2 fertilized patch was studied for 39 days to test whether fertilization enhances downward particle flux into the deep ocean. Chlorophyll a and primary productivity doubled after fertilization, and photosynthetic quantum yield (FV/FM) increased from 0.33 to >0.40. Silicic acid (<2 µmol/L) limited diatoms, which contributed <10% of phytoplankton biomass. Copepods exerted high grazing pressure. This is the first study of particle flux out of an artificially fertilized bloom with very low diatom biomass. Net community production (NCP) inside the patch, estimated from O2:Ar ratios, averaged 21 mmol POC/m**2/d, probably ±20%. 234Th profiles implied constant export of ~6.3 mmol POC/m**2/d in the patch, similar to unfertilized waters. The difference between NCP and 234Th-derived export partly accumulated in the mixed layer and was partly remineralized between the mixed layer and 100 m. Neutrally buoyant sediment traps at 200 and 450 m inside and outside the patch caught mostly <1.1 mmol POC/m**2/d, predominantly of fecal origin; flux did not increase upon fertilization. Our data thus indicate intense flux attenuation between 100 and 200 m, and probably between the mixed layer and 100 m. We attribute the lack of fertilization-induced export to silicon limitation of diatoms and reprocessing of sinking particles by detritus feeders. Our data are consistent with the view that nitrate-rich but silicate-deficient waters are not poised for enhanced particle export upon iron addition.

Seawater carbonate chemistry and photosynthetic response of Emiliania huxleyi (CS-369) to UV radiation and elevated temperature duirng experiments, 2011

Changes in calcification of coccolithophores may affect their photosynthetic responses to both, ultraviolet radiation (UVR, 280-400 nm) and temperature. We operated semi-continuous cultures of Emiliania huxleyi (strain CS-369) at reduced (0.1 mM, LCa) and ambient (10 mM, HCa) Ca2+ concentrations and, after 148 generations, we exposed cells to six radiation treatments (>280, >295, >305, >320, >350 and >395 nm by using Schott filters) and two temperatures (20 and 25 °C) to examine photosynthesis and calcification responses. Overall, our study demonstrated that: (1) decreased calcification resulted in a down regulation of photoprotective mechanisms (i.e., as estimated via non-photochemical quenching, NPQ), pigments contents and photosynthetic carbon fixation; (2) calcification (C) and photosynthesis (P) (as well as their ratio) have different responses related to UVR with cells grown under the high Ca2+ concentration being more resistant to UVR than those grown under the low Ca2+ level; (3) elevated temperature increased photosynthesis and calcification of E. huxleyi grown at high Ca2+concentrations whereas decreased both processes in low Ca2+ grown cells. Therefore, a decrease in calcification rates in E. huxleyi is expected to decrease photosynthesis rates, resulting in a negative feedback that further reduces calcification.

Adenylate concentration and energy charge in different thallus regions and after UV radiation in brown, red and green algae

A method was developed to extract adenine nucleotides AMP, ADP, and ATP from marine macroalgal tissue to gain information on the cellular energy charge. Quantification was carried out by high performance liquid chromatography (HPLC). Three species from the rocky shore of the island of Helgoland (German Bight) were examined: Laminaria saccharina (Phaeophyta), Chondrus crispus (Rhodophyta), and Ulva lactuca (Chlorophyta). In L. saccharina and C. crispus, the adenylate energy charge (AEC) was determined in different thallus regions. AEC varied in relation to tissue age and function. Higher AEC values typically occurred in thallus regions with meristematic activity. Furthermore, L. saccharina and U. lactuca were exposed to UV-A and elevated UV-B radiation. The AEC was calculated and the maximal quantum yield of photosystem II (Fv/Fm) was determined as indicators for UV stress. In both species, the AEC remained at high values (0.72 ± 0.04), while Fv/Fm dropped rapidly. The results show that the photosynthesis of the phaeophyte is more resistant to UV radiation than the chlorophyte.

Combined effects of coral reef foraminifera Marginopora vertebralis and Heterostegina depressa in a multi-fractorial experiment

Warming and changes in ocean carbonate chemistry alter marine coastal ecosystems at an accelerating pace. The interaction between these stressors has been the subject of recent studies on reef organisms such as corals, bryozoa, molluscs, and crustose coralline algae. Here we investigated the combined effects of elevated sea surface temperatures and pCO2 on two species of photosymbiont-bearing coral reef Foraminifera: Heterostegina depressa (hosting diatoms) and Marginopora vertebralis (hosting dinoflagellates). The effects of single and combined stressors were studied by monitoring survivorship, growth, and physiological parameters, such as respiration, photochemistry (pulse amplitude modulation fluorometry and oxygen production), and chl a content. Specimens were exposed in flow-through aquaria for up to seven weeks to combinations of two pCO2 (~790 and ~490 µatm) and two temperature (28 and 31 °C) regimes. Elevated temperature had negative effects on the physiology of both species. Elevated pCO2 had negative effects on growth and apparent photosynthetic rate in H.depressa but a positive effect on effective quantum yield. With increasing pCO2, chl a content decreased in H. depressa and increased in M. vertebralis. The strongest stress responses were observed when the two stressors acted in combination. An interaction term was statistically significant in half of the measured parameters. Further exploration revealed that 75 % of these cases showed a synergistic (= larger than additive) interaction between the two stressors. These results indicate that negative physiological effects on photosymbiont-bearing coral reef Foraminifera are likely to be stronger under simultaneous acidification and temperature rise than what would be expected from the effect of each of the stressors individually.

Phototoxicity of photosystem II (PSII) herbicides to coral

Recent reports of contamination of the Great Barrier Reef Marine Park by herbicides used in antifouling paints and in agriculture have caused concern over the possible effects on corals in nearshore areas. Pulse-Amplitude Modulated (PAM) chlorophyll fluorescence techniques were used to examine changes in the maximum effective quantum yield (ΔF/Fm′) of symbiotic dinoflagellates within the host tissues (in hospite) of the coral Seriatopora hystrix exposed to a number of Photosystem II (PSII) inhibiting herbicides in short-term toxicity tests. The concentration of herbicide required to reduce ΔF/Fm′ by 50% (median effective concentration [EC50]) differed by over 2 orders of magnitude: Irgarol 1051 (0.7 μg l-1) > ametryn (1.7 μg l-1) > diuron (2.3 μg l-1) > hexazinone (8.8 μg l -1) > atrazine (45 μg l-1) > simazine (150 μg l-1) > tebuthiuron (175 μg l-1) > ionynil (> 1 mg l-1). Similar absolute and relative toxicities were observed with colonies of the coral Acropora formosa (Irgarol 1051 EC50: 1.3 μg l-1, diuron EC50: 2.8 μg l-1), Time-course experiments indicated that ΔF/Fm′ was rapidly reduced (i.e. within minutes) in S. hystrix exposed to Irgarol 1051 and diuron. On return to fresh running seawater, ΔF/Fm′ recovered quickly in diuron-exposed corals (i.e. in minutes to hours), but slowly in corals exposed to Irgarol 1051 (i.e. hours to days). Time-course experiments indicated that the effects of diuron (3 μg l-1) on S. hystrix were inversely related to temperature over the range 20 to 30 °C, although initially the effects were less at the lower temperatures. Repeated exposure to pulses of Irgarol 1051 (daily 2 h exposure to 30 μg l -1 over 4 d) resulted in a 30% decrease in the density of symbiotic dinoflagellates in the tissues of S. hystrix.

Absorption and fluorescence spectroscopy of rhodamine 6G in titanium dioxide nanocomposites

A comparison has been made between the spectroscopic properties of the laser dye rhodamine 6G (R6G) in mesostructured titanium dioxide (TiO2) and in ethanol. Steady-state excitation and emission techniques have been used to probe the dye-matrix interactions. We show that the TiO2-nanocomposite studied is a good host for R6G, as it allows high dye concentrations, while keeping dye molecules isolated, and preventing aggregation. Our findings have important implications in the context of solid state dye-lasers and microphotonic device applications. (C) 2003 Elsevier B.V. All rights reserved.

Cell death and degeneration in the symbiotic dinoflagellates of the coral Stylophora pistillata during bleaching

Rising sea temperatures are increasing the incidences of mass coral bleaching (the dissociation of the coral-algal symbiosis) and coral mortality. In this study, the effects of bleaching (induced by elevated light and temperature) on the condition of symbiotic dinoflagellates (Symbiodinium sp.) within the tissue of the hard coral Stylophora pistillata (Esper) were assessed using a suite of techniques. Bleaching of S. pistillata was accompanied by declines in the maximum potential quantum yield of photosynthesis (F-v/F-m, measured using pulse amplitude modulated [PAM] fluorometry), an increase in the number of Sytox-green-stained algae (indicating compromised algal membrane integrity and cell death), an increase in 2',7'-dichlorodihydrofluroscein diacetate (H(2)DCFDA)stained algae (indicating increased oxidative stress), as well as ultrastructural changes (vacuolisation, losses of chlorophyll, and an increase in accumulation bodies). Algae expelled from S. pistillata exhibited a complete disorganisation of cellular contents; expelled cells contained only amorphous material. In situ samples taken during a natural mass coral bleaching event on the Great Barrier Reef in February 2002 also revealed a high number of Sytox-labelled algae cells in symbio. Dinoflagellate degeneration during bleaching seems to be similar to the changes resulting from senescence-phase cell death in cultured algae. These data support a role for oxidative stress in the mechanism of coral bleaching and highlight the importance of algal degeneration during the bleaching of a reef coral.

Testing the 'photoinhibition' model of coral bleaching using chemical inhibitors

Explants of the hard coral Seriatopora hystrix were exposed to sublethal concentrations of the herbicide diuron DCMU (N'-(3,4-dichlorophenyl,-N,N-dimethylurea)) and the heavy metal copper. Pulse amplitude modulated (PAM) chlorophyll fluorescence techniques were used to assess the effects on the photosynthetic efficiency of the algal symbionts in the tissue (in Symbio), and chlorophyll fluorescence and counts of symbiotic algae (normalised to surface area) were used to assess the extent of coral bleaching. At 30 mug DCMU l(-1), there was a reduction in both the maximum effective quantum yield (DeltaF/F-m') and maximum potential quantum yield (F-v/F-m) of the algal symbionts in symbio. Corals subsequently lost their algal symbionts and discoloured (bleached), especially on their upper sunlight-exposed surfaces. At the same DCMU concentration but under low light (5% of growth irradiance), there was a marked reduction in DeltaF/F-m' but only a slight reduction in F-v/F-m and slight loss of algae. Loss of algal symbionts was also noted after a 7 d exposure to concentrations as low as 10 mug DCMU l(-1) under normal growth irradiance, and after 14 d exposure to 10 mug DCMU l(-1) under reduced irradiance. Collectively the results indicate that DCMU-induced bleaching is caused by a light-dependent photoinactivation of algal symbionts, and that bleaching occurs when F-v/F-n, (measured 2 h after sunset) is reduced to a value of less than or equal to 0.6. Elevated copper concentrations (60 mug Cu l(-1) for 10 h) also induced a rapid bleaching in S. hystrix but without affecting the quantum yield of the algae in symbio. Tests with isolated algae indicated that substantially higher concentrations (300 mug Cu l(-1) for 8 h) were needed to significantly reduce the quantum yield. Thus, copper-induced bleaching occurs without affecting the algal photosynthesis and may be related to effects on the host (animal). It is argued that warm-water bleaching of corals resembles both types of chemically induced bleaching, suggesting the need for an integrated model of coral bleaching involving the effect of temperature on both host (coral) and algal symbionts.