Dan Tchernov

Genes encoding A-type flavoproteins are essential for photoreduction of O2 in cyanobacteria.

O(2) photoreduction by photosynthetic electron transfer, the Mehler reaction, was observed in all groups of oxygenic photosynthetic organisms, but the electron transport chain mediating this reaction remains unidentified. We provide the first evidence for the involvement of A-type flavoproteins that reduce O(2) directly to water in vitro. Synechocystis sp. strain PCC 6803 mutants defective in flv1 and flv3, encoding A-type flavoproteins, failed to exhibit O(2) photoreduction but performed normal photosynthesis and respiration. We show that the light-enhanced O(2) uptake was not due to respiration or photorespiration. After dark acclimation, photooxidation of P(700) was severely depressed in mutants Deltaflv1 and Deltaflv3 but recovered after light activation of CO(2) fixation, which gives P(700) an additional electron acceptor. Inhibition of CO(2) fixation prevented recovery but scarcely affected P(700) oxidation in the wild-type, where the Mehler reaction provides an alternative route for electrons. We conclude that the source of electrons for O(2) photoreduction is PSI and that the highly conserved A-type flavoproteins Flv1 and Flv3 are essential for this process in vivo. We propose that in cyanobacteria, contrary to eukaryotes, the Mehler reaction produces no reactive oxygen species and may be evolutionarily related to the response of anaerobic bacteria to O(2).

Flow enhances photosynthesis in marine benthic autotrophs by increasing the efflux of oxygen from the organism to the water

Worldwide, many marine coastal habitats are facing rapid deterioration due in part to human-driven changes in habitat characteristics, including changes in flow patterns, a factor known to greatly affect primary production in corals, algae, and seagrasses. The effect of flow traditionally is attributed to enhanced influx of nutrients and dissolved inorganic carbon (DIC) across the benthic boundary layer from the water to the organism however, here we report that the organism’s photosynthetic response to changes in the flow is nearly instantaneous, and that neither nutrients nor DIC limits this rapid response. Using microelectrodes, dual-pulse amplitude-modulated fluorometry, particle image velocimetry, and real time mass-spectrometry with the common scleractinian coral Favia veroni, the alga Gracilaria cornea, and the seagrass Halophila stipulacea, we show that this augmented photosynthesis is due to flow-driven enhancement of oxygen efflux from the organism to the water, which increases the affinity of the RuBisCO to CO2. No augmentation of photosynthesis was found in the absence of flow or when flow occurred, but the ambient concentration of oxygen was artificially elevated. We suggest that water motion should be considered a fundamental factor, equivalent to light and nutrients, in determining photosynthesis rates in marine benthic autotrophs.

Sustained net CO2 evolution during photosynthesis by marine microorganism

BACKGROUND Many aquatic photosynthetic microorganisms possess an inorganic-carbon-concentrating mechanism that raises the CO2 concentration at the intracellular carboxylation sites, thus compensating for the relatively low affinity of the carboxylating enzyme for its substrate. In cyanobacteria, the concentrating mechanism involves the energy-dependent influx of inorganic carbon, the accumulation of this carbon--largely in the form of HCO3(-)-in the cytoplasm, and the generation of CO2 at carbonic anhydrase sites in close proximity to the carboxylation sites. RESULTS During measurements of inorganic carbon fluxes associated with the inorganic-carbon-concentrating mechanism, we observed the surprising fact that several marine photosynthetic microorganisms, including significant contributors to oceanic primary productivity, can serve as a source of CO2 rather than a sink during CO2 fixation. The phycoerythrin-possessing cyanobacterium Synechococcus sp. WH7803 evolved CO2 at a rate that increased with light intensity and attained a value approximately five-fold that for photosynthesis. The external CO2 concentration reached was significantly higher than that predicted for chemical equilibrium between HCO3- and CO2, as confirmed by the rapid decline in the CO2 concentration upon the addition of carbonic anhydrase. Measurements of oxygen exchange between water and CO2, by means of stable isotopes, demonstrated that the evolved CO2 originated from HCO3- taken up and converted intracellularly to CO2 in a light-dependent process. CONCLUSIONS We report net, sustained CO2 evolution during photosynthesis. The results have implications for energy balance and pH regulation of the cells, for carbon cycling between the cells and the marine environment, and for the observed fractionation of stable carbon isotopes.

Apoptosis and the selective survival of host animals following thermal bleaching in zooxanthellate corals.

During the past several decades, numerous reports from disparate geographical areas have documented an increased frequency of “bleaching” in reef-forming corals. The phenomenon, triggered by increased sea surface temperatures, occurs when the cnidarian hosts digest and/or expel their intracellular, photosynthetic dinoflagellate symbionts (“zooxanthellae” in the genus Symbiodinium). Although coral bleaching is often followed by the death of the animal hosts, in some cases, the animal survives and can be repopulated with viable zooxanthellae. The physiological factors determining the ability of the coral to survive bleaching events are poorly understood. In this study, we experimentally established that bleaching and death of the host animal involve a caspase-mediated apoptotic cascade induced by reactive oxygen species produced primarily by the algal symbionts. In addition, we demonstrate that, although some corals naturally suppress caspase activity and significantly reduce caspase concentration under high temperatures as a mechanism to prevent colony death from apoptosis, even sensitive corals can be prevented from dying by application of exogenous inhibitors of caspases. Our results indicate that variability in response to thermal stress in corals is determined by a four-element, combinatorial genetic matrix intrinsic to the specific symbiotic association. Based on our experimental data, we present a working model in which the phenotypic expression of this symbiont/host relationship places a selective pressure on the symbiotic association. The model predicts the survival of the host animals in which the caspase-mediated apoptotic cascade is down-regulated.

Soft Robotic Grippers for Biological Sampling on Deep Reefs.

Abstract This article presents the development of an underwater gripper that utilizes soft robotics technology to delicately manipulate and sample fragile species on the deep reef. Existing solutions for deep sea robotic manipulation have historically been driven by the oil industry, resulting in destructive interactions with undersea life. Soft material robotics relies on compliant materials that are inherently impedance matched to natural environments and to soft or fragile organisms. We demonstrate design principles for soft robot end effectors, bench-top characterization of their grasping performance, and conclude by describing in situ testing at mesophotic depths. The result is the first use of soft robotics in the deep sea for the nondestructive sampling of benthic fauna.

Passive Entry of CO2 and Its Energy-dependent Intracellular Conversion to HCO in Cyanobacteria Are Driven by a Photosystem I-generated ΔμH+

CO2 entry intoSynechococcus sp. PCC7942 cells was drastically inhibited by the water channel blocker p-chloromercuriphenylsulfonic acid suggesting that CO2 uptake is, for the most part, passive via aquaporins with subsequent energy-dependent conversion to HCO 3 − . Dependence of CO2uptake on photosynthetic electron transport via photosystem I (PSI) was confirmed by experiments with electron transport inhibitors, electron donors and acceptors, and a mutant lacking PSI activity. CO2 uptake was drastically inhibited by the uncouplers carbonyl cyanide m-chlorophenylhydrazone (CCCP) and ammonia but substantially less so by the inhibitors of ATP formation arsenate and N, N,-dicyclohexylcarbodiimide (DCCD). Thus a ΔμH+ generated by photosynthetic PSI electron transport apparently serves as the direct source of energy for CO2 uptake. Under low light intensity, the rate of CO2 uptake by a high-CO2-requiring mutant ofSynechococcus sp. PCC7942, at a CO2concentration below its threshold for CO2 fixation, was higher than that of the wild type. At saturating light intensity, net CO2 uptake was similar in the wild type and in the mutant IL-3 suggesting common limitation by the rate of conversion of CO2 to HCO 3 − . These findings are consistent with a model postulating that electron transport-dependent formation of alkaline domains on the thylakoid membrane energizes intracellular conversion of CO2 to HCO 3 − .

Extracellular Production and Degradation of Superoxide in the Coral Stylophora pistillata and Cultured Symbiodinium

Background Reactive oxygen species (ROS) are thought to play a major role in cell death pathways and bleaching in scleractinian corals. Direct measurements of ROS in corals are conspicuously in short supply, partly due to inherent problems with ROS quantification in cellular systems. Methodology/Principal Findings In this study we characterized the dynamics of the reactive oxygen species superoxide anion radical (O2 −) in the external milieu of the coral Stylophora pistillata. Using a sensitive, rapid and selective chemiluminesence-based technique, we measured extracellular superoxide production and detoxification activity of symbiont (non-bleached) and aposymbiont (bleached) corals, and of cultured Symbiodinium (from clades A and C). Bleached and non-bleached Stylophora fragments were found to produce superoxide at comparable rates of 10−11–10−9 mol O2 − mg protein−1 min−1 in the dark. In the light, a two-fold enhancement in O2 − production rates was observed in non-bleached corals, but not in bleached corals. Cultured Symbiodinium produced superoxide in the dark at a rate of . Light was found to markedly enhance O2 − production. The NADPH Oxidase inhibitor Diphenyleneiodonium chloride (DPI) strongly inhibited O2 − production by corals (and more moderately by algae), possibly suggesting an involvement of NADPH Oxidase in the process. An extracellular O2 − detoxifying activity was found for bleached and non-bleached Stylophora but not for Symbiodinium. The O2 − detoxifying activity was partially characterized and found to resemble that of the enzyme superoxide dismutase (SOD). Conclusions/Significance The findings of substantial extracellular O2 − production as well as extracellular O2 − detoxifying activity may shed light on the chemical interactions between the symbiont and its host and between the coral and its environment. Superoxide production by Symbiodinium possibly implies that algal bearing corals are more susceptible to an internal build-up of O2 −, which may in turn be linked to oxidative stress mediated bleaching.

UPTAKE, EFFLUX, AND PHOTOSYNTHETIC UTILIZATION OF INORGANIC CARBON BY THE MARINE EUSTIGMATOPHYTE NANNOCHLOROPSIS SP.1

Uptake, efflux and utilization of inorganic carbon were investigated in the marine eustigmatophyte Nannochloropsis sp. grown under an air level of CO2. Maximal photosynthetic rate was hardly affected by raising the pH porn 5.0 to 9.0. The apparent photosynthetic affinity for dissolved inorganic carbon (DIC) was 35 μM DIC between pH 6.5 to 9.0, but increased approximately threefold at pH 5.0 suggesting that HCO3‐ was the main DIC species used from the medium. No external carbonic anhydrase (CA) activity could be detected by the pH drift method. However, application of ethoxyzolamide (an inhibitor of CA) resulted an a significant inhibition of photosynthetic O2 evolution and carbon utilization, suggesting involvement of internal CA or CA‐like activity in DIC utilization. Under high light conditions, the rate of HCO3− uptake and its internal conversion to CO2 apparently exceeded the rate of carbon fixation, resulting in a large leak of CO2 from the cells to the external medium. When the cells were exposed to low DIC concentrations, the ratio of internal to external DIC concentration was about eight. On the other hand, in the presence of 2 mM DIC, conditions prevailing in the marine environment, the internal concentration of DIC was only 50% higher than the external one.

The Covert World of Fish Biofluorescence: A Phylogenetically Widespread and Phenotypically Variable Phenomenon

The discovery of fluorescent proteins has revolutionized experimental biology. Whereas the majority of fluorescent proteins have been identified from cnidarians, recently several fluorescent proteins have been isolated across the animal tree of life. Here we show that biofluorescence is not only phylogenetically widespread, but is also phenotypically variable across both cartilaginous and bony fishes, highlighting its evolutionary history and the possibility for discovery of numerous novel fluorescent proteins. Fish biofluorescence is especially common and morphologically variable in cryptically patterned coral-reef lineages. We identified 16 orders, 50 families, 105 genera, and more than 180 species of biofluorescent fishes. We have also reconstructed our current understanding of the phylogenetic distribution of biofluorescence for ray-finned fishes. The presence of yellow long-pass intraocular filters in many biofluorescent fish lineages and the substantive color vision capabilities of coral-reef fishes suggest that they are capable of detecting fluoresced light. We present species-specific emission patterns among closely related species, indicating that biofluorescence potentially functions in intraspecific communication and evidence that fluorescence can be used for camouflage. This research provides insight into the distribution, evolution, and phenotypic variability of biofluorescence in marine lineages and examines the role this variation may play.

Locally accelerated growth is part of the innate immune response and repair mechanisms in reef-building corals as detected by green fluorescent protein (GFP)-like pigments

Homologs of the green fluorescent protein (GFP) are a prevalent group of host pigments responsible for the green, red and purple-blue colours of many reef-building corals. They have been suggested to contribute to the striking coloration changes of different corals species in response to wounding and infestation with epibionts/parasites. In order to elucidate the physiological processes underlying the potentially disease-related colour changes, we have analysed spatial and temporal expression patterns of GFP-like proteins and other biomarkers in corals from the Red Sea, the Arabian/Persian Gulf and Fiji both in their natural habitat and under specific laboratory conditions. The expression of distinct GFP-like proteins and the growth marker proliferating cell nuclear antigen was upregulated in growing branch tips and margins of healthy coral colonies as well as in disturbed colony parts. Furthermore, phenoloxidase activity increased in these proliferating tissues. It is thus demonstrated that locally accelerated growth is part of the innate immune response and repair mechanisms in reef-building corals and, moreover, these processes can be detected utilizing the excellent biomarker properties of GFP-like proteins. Finally, the results of this work suggest an additional vulnerability of corals in predicted future scenarios of increased ocean acidification, warming and eutrophication that are anticipated to reduce coral growth capacity.