CARBONIC ANHYDRASE IN THE MARINE DIATOM THALASSIOSIRA WEISSFLOGII (BACILLARIOPHYCEAE)1

The zinc metalloenzyme carbonic anhydrase plays a critical role in inorganic carbon acquisition in marine diatoms, thus conferring on zinc a key role in oceanic carbon cycling. As a first step in determining the location and function of carbonic anhydrase (CA) in Bacillariophyceae, we purified and partially sequenced CA from T. weissflogii (Gru) Fryxell et Hasle (TWCA1) and cloned the corresponding cDNA (twca1). The twca1 sequence is different from other known algal carbonic anhydrase genes, and encodes a protein of roughly 34 kDa. The amino terminal amino acids sequenced from purified TWCA1 are 72 residues downstream of the putative starting methionine predicted by twca1. This difference may be due to the presence of a short-lived signal sequence designed to guide the enzyme to the correct cellular location. The absence of any homology between TWCA1 and previously sequenced CAs from Chlorophyceae may indicate either convergent evolution or that carbon acquisition represents a fundamental physiological difference among algal phyla.     

Synthesis of 2‐amino‐3‐cyanopyridine derivatives and investigation of their carbonic anhydrase inhibition effects

The conversion of carbon dioxide (CO₂) and bicarbonate (HCO₃⁻) to each other is very important for living metabolism. Carbonic anhydrase (CA, E.C.4.2.1.1), a metalloenzyme familly, catalyzes the interconversion of these ions (CO₂ and HCO₃⁻) and are very common in living organisms. In this study, a series of novel 2‐amino‐3‐cyanopyridines supported with some functional groups was synthesized and tested as potential inhibition effects against both cytosolic human CA I and II isoenzymes (hCA I and II) using by Sepharose‐4B‐l ‐tyrosine‐sulfanilamide affinity chromatography. The structural elucidations of novel 2‐amino‐3‐cyanopyridines were achieved by NMR, IR, and elemental analyses. K _i values of the novel synthesized compounds were found in range of 2.84–112.44 μM against hCA I and 2.56–31.17 μM against hCA II isoenzyme. While compound 7d showed the best inhibition activity against hCA I (K _i: 2.84 μM), the compound 7b demonstrated the best inhibition profile against hCA II isoenzyme (K _i: 2.56 μM).     

Emerging roles for carbonic anhydrase in mesophyll conductance and photosynthesis

Carbonic anhydrase (CA) is an abundant protein in most photosynthesizing organisms and higher plants. This review paper considers the physiological importance of the more abundant CA isoforms in photosynthesis, through their effects on CO₂ diffusion and other processes in photosynthetic organisms. In plants, CA has multiple isoforms in three different families (α, β and γ) and is mainly known to catalyze the CO₂ equilibrium. This reversible conversion has a clear role in photosynthesis, primarily through sustaining the CO₂ concentration at the site of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco). Despite showing the same major reaction mechanism, the three main CA families are evolutionarily distinct. For different CA isoforms, cellular localization and total gene expression as a function of developmental stage are predicted to determine the role of each family in relation to the net assimilation rate. Reaction–diffusion modeling and observational evidence support a role for CA activity in reducing resistance to CO₂ diffusion inside mesophyll cells by facilitating CO₂ transfer in both gas and liquid phases. In addition, physical and/or biochemical interactions between CAs and other membrane‐bound compartments, for example aquaporins, are suggested to trigger a CO₂‐sensing response by stomatal movement. In response to environmental stresses, changes in the expression level of CAs and/or stimulated deactivation of CAs may correspond with lower photosynthetic capacity. We suggest that further studies should focus on the dynamics of the relationship between the activity of CAs (with different subcellular localization, abundance and gene expression) and limitations due to CO₂ diffusivity through the mesophyll and supply of CO₂ to photosynthetic reactions.     

CO2‐CONCENTRATING MECHANISMS OF THE POTENTIALLY TOXIC DINOFLAGELLATE PROTOCERATIUM RETICULATUM (DINOPHYCEAE,GONYAULACALES)1

The low CO₂ concentration in seawater poses severe restrictions on photosynthesis, especially on those species with form II RUBISCO. We found that the potentially toxic dinoflagellate Protoceratium reticulatum Clap. et J. Lachm. possesses a form II RUBISCO. To cast some light on the mechanisms this organism undergoes to cope with low CO₂ availability, we compared cells grown at atmospheric (370 ppm) and high (5000 ppm) CO₂ concentrations, with respect to a number of physiological parameters related to dissolved inorganic carbon (DIC) acquisition and assimilation. The photosynthetic affinity for DIC was about one order of magnitude lower in cells cultivated at high [CO₂]. End‐point pH‐drift experiments suggest that P. reticulatum was not able to efficiently use HCO₃^? under our growth conditions. Only internal carbonic anhydrase (CA) activity was detected, and its activity decreased by about 60% in cells cultured at high [CO₂]. Antibodies raised against a variety of algal CAs were used for Western blot analysis: P. reticulatum extracts only cross‐reacted with anti‐ß‐CA sera, and the amount of immunoreactive protein decreased in cells grown at high [CO₂]. No pyrenoids were observed under all growth conditions. Our data indicate that P. reticulatum has an inducible carbon‐concentrating mechanism (CCM) that operates in the absence of pyrenoids and with little intracellular CO₂ accumulation. Calculations on the impact of the CA activity to photosynthetic growth [CO₂] suggest that it is an essential component of the CCM of P. reticulatum and is necessary to sustain the photosynthetic rates observed at ambient CO₂.     

CO2‐concentrating mechanisms in three southern hemisphere strains of Emiliania huxleyi

Rising global CO₂ is changing the carbonate chemistry of seawater, which is expected to influence the way phytoplankton acquire inorganic carbon. All phytoplankton rely on ribulose‐bisphosphate carboxylase oxygenase (RUBISCO) for assimilation of inorganic carbon in photosynthesis, but this enzyme is inefficient at present day CO₂ levels. Many algae have developed a range of energy demanding mechanisms, referred to as carbon concentrating mechanisms (CCMs), which increase the efficiency of carbon acquisition. We investigated CCM activity in three southern hemisphere strains of the coccolithophorid Emiliania huxleyi W. W. Hay & H. P. Mohler. Both calcifying and non‐calcifying strains showed strong CCM activity, with HCO₃^? as a preferred source of photosynthetic carbon in the non‐calcifying strain, but a higher preference for CO₂ in the calcifying strains. All three strains were characterized by the presence of pyrenoids, external carbonic anhydrase (CA) and high affinity for CO₂ in photosynthesis, indicative of active CCMs. We postulate that under higher CO₂ levels cocco‐lithophorids will be able to down‐regulate their CCMs, and re‐direct some of the metabolic energy to processes such as calcification. Due to the expected rise in CO₂ levels, photosynthesis in calcifying strains is expected to benefit most, due to their use of CO₂ for carbon uptake. The non‐calcifying strain, on the other hand, will experience only a 10% increase in HCO₃^?, thus making it less responsive to changes in carbonate chemistry of water.     

Regulation of gene expression is associated with tolerance of the Arctic copepod Calanus glacialis to CO2‐acidified sea water

Ocean acidification is the increase in seawater pCO₂ due to the uptake of atmospheric anthropogenic CO₂, with the largest changes predicted to occur in the Arctic seas. For some marine organisms, this change in pCO₂, and associated decrease in pH, represents a climate change‐related stressor. In this study, we investigated the gene expression patterns of nauplii of the Arctic copepod Calanus glacialis cultured at low pH levels. We have previously shown that organismal‐level performance (development, growth, respiration) of C. glacialis nauplii is unaffected by low pH. Here, we investigated the molecular‐level response to lowered pH in order to elucidate the physiological processes involved in this tolerance. Nauplii from wild‐caught C. glacialis were cultured at four pH levels (8.05, 7.9, 7.7, 7.5). At stage N6, mRNA was extracted and sequenced using RNA‐seq. The physiological functionality of the proteins identified was categorized using Gene Ontology and KEGG pathways. We found that the expression of 151 contigs varied significantly with pH on a continuous scale (93% downregulated with decreasing pH). Gene set enrichment analysis revealed that, of the processes downregulated, many were components of the universal cellular stress response, including DNA repair, redox regulation, protein folding, and proteolysis. Sodium:proton antiporters were among the processes significantly upregulated, indicating that these ion pumps were involved in maintaining cellular pH homeostasis. C. glacialis significantly alters its gene expression at low pH, although they maintain normal larval development. Understanding what confers tolerance to some species will support our ability to predict the effects of future ocean acidification on marine organisms.     

PHOTOSYNTHETIC INORGANIC CARBON ACQUISITION IN AN ACID‐TOLERANT,FREE‐LIVING SPECIES OF COCCOMYXA (CHLOROPHYTA)1

The processes of CO₂ acquisition were characterized for the acid‐tolerant, free‐living chlorophyte alga, CPCC 508. rDNA data indicate an affiliation to the genus Coccomyxa, but distinct from other known members of the genus. The alga grows over a wide range of pH from 3.0 to 9.0. External carbonic anhydrase (CA) was detected in cells grown above pH 5, with the activity increasing marginally from pH 7 to 9, but most of the CA activity was internal. The capacity for HCO₃^? uptake of cells treated with the CA inhibitor acetazolamide (AZA), was investigated by comparing the calculated rate of uncatalyzed CO₂ formation with the rate of photosynthesis. Active bicarbonate transport occurred in cells grown in media above pH 7.0. Monitoring CO₂ uptake and O₂ evolution by membrane‐inlet mass spectrometry demonstrated that air‐grown cells reduced the CO₂ concentration in the medium to an equilibrium concentration of 15 μM, but AZA‐treated cells caused a drop in extracellular CO₂ concentration to a compensation concentration of 27 μM at pH 8.0. CO₂‐pulsing experiments with cells in the light indicated that the cells do not actively take up CO₂. An internal pool of unfixed inorganic carbon was not detected at the CO₂ compensation concentration, probably because of the lack of active CO₂ uptake, but was detectable at times before compensation point was reached. These results indicate that this free‐living Coccomyxa possesses a CO₂‐concentrating mechanism (CCM) due to an active bicarbonate‐uptake system, unlike the Coccomyxa sp. occurring in symbiotic association with lichens.     

Biochemical characterization of the δ-carbonic anhydrase from the marine diatom Thalassiosira weissflogii,TweCA

Abstract

Diatom genome sequences clearly reveal the presence of different systems for HCO₃^? uptake. Carbon-concentrating mechanisms (CCM) based on HCO₃^? transport and a plastid-localized carbonic anhydrase (CA, EC 4.2.1.1) appear to be more probable than the others because CAs have been identified in the genome of many diatoms. CAs are key enzymes involved in the acquisition of inorganic carbon for photosynthesis in phytoplankton, as they catalyze efficiently the interconversion between carbon dioxide and bicarbonate. Five genetically distinct classes of CAs exist, α-, β-, γ-, δ- and ζ and all of them are metalloenzymes. Recently we investigated for the first time the catalytic activity and inhibition of the δ-class CA from the marine diatom Thalassiosira weissflogii, named TweCA. This enzyme is an efficient catalyst for the CO₂ hydration and its inhibition profile with sulfonamide/sulfamate and anions have also been investigated. Here, we report the detailed biochemical characterization and chemico-physical properties of the δ-CA of T. weissflogii. The δ-CA encoding gene was cloned and expressed in Artic Express cells and the recombinant protein purified to homogeneity. Interesting to note that TweCA has no intrinsic esterase activity with 4-nitrophenyl acetate (pNpA) as substrate although the phylogenetic analysis showed that δ-CAs are closer to the α-CAs than to the other classes of such enzymes.     

Predictable ecological response to rising CO2 of a community of marine phytoplankton

Rising atmospheric CO₂ and ocean acidification are fundamentally altering conditions for life of all marine organisms, including phytoplankton. Differences in CO₂ related physiology between major phytoplankton taxa lead to differences in their ability to take up and utilize CO₂. These differences may cause predictable shifts in the composition of marine phytoplankton communities in response to rising atmospheric CO₂. We report an experiment in which seven species of marine phytoplankton, belonging to four major taxonomic groups (cyanobacteria, chlorophytes, diatoms, and coccolithophores), were grown at both ambient (500 μatm) and future (1,000 μatm) CO₂ levels. These phytoplankton were grown as individual species, as cultures of pairs of species and as a community assemblage of all seven species in two culture regimes (high‐nitrogen batch cultures and lower‐nitrogen semicontinuous cultures, although not under nitrogen limitation). All phytoplankton species tested in this study increased their growth rates under elevated CO₂ independent of the culture regime. We also find that, despite species‐specific variation in growth response to high CO₂, the identity of major taxonomic groups provides a good prediction of changes in population growth and competitive ability under high CO₂. The CO₂‐induced growth response is a good predictor of CO₂‐induced changes in competition (R² > .93) and community composition (R² > .73). This study suggests that it may be possible to infer how marine phytoplankton communities respond to rising CO₂ levels from the knowledge of the physiology of major taxonomic groups, but that these predictions may require further characterization of these traits across a diversity of growth conditions. These findings must be validated in the context of limitation by other nutrients. Also, in natural communities of phytoplankton, numerous other factors that may all respond to changes in CO2, including nitrogen fixation, grazing, and variation in the limiting resource will likely complicate this prediction.     

TAPP analogs containing β3‐homo‐amino acids: synthesis and receptor binding

β‐Amino acids containing α,β‐hybrid peptides show great potential as peptidomimetics. In this paper, we describe the synthesis and affinity to μ‐opioid and δ‐opioid receptors of α,β‐hybrids, analogs of the tetrapeptide Tyr‐ d ‐Ala‐Phe‐Phe‐NH₂ (TAPP). Each amino acid was replaced with an l ‐ or d ‐β³‐h‐amino acid. All α,β‐hybrids of TAPP analogs were synthesized in solution and tested for affinity to μ‐opioid and δ‐opioid receptors. The analog Tyr‐β³h‐ d ‐Ala‐Phe‐PheNH₂ was found to be as active as the native tetrapeptide. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

PHYTOPLANKTON COMMUNITY COMPOSITION AND GENE EXPRESSION OF FUNCTIONAL GENES INVOLVED IN CARBON AND NITROGEN ASSIMILATION1

A functional gene microarray was developed and used to investigate phytoplankton community composition and gene expression in the English Channel. Genes encoding the CO₂‐fixation enzyme RUBISCO (rbcL) and the nitrate assimilation enzyme nitrate reductase (NR) representing several major groups of phytoplankton were included as oligonucleotide probes on the “phytoarray.” Five major groups of eukaryotic phytoplankton that possess the Type 1D rbcL gene were detected, both in terms of presence (DNA) and activity (rbcL gene expression). Changes in relative signal intensity among the Type 1D rbcL probes indicated a shift from diatom dominance in the spring bloom to dominance by haptophytes and flagellates later in the summer. Because of the limitations of a smaller database, NR probes detected fewer groups, but due to the greater diversity among known NR sequences, NR probes provided higher phylogenetic resolution than did rbcL probes and identified two uncultivated diatom phylotypes as the most abundant (DNA) and active (NR gene expression) in field samples. Unidentified chlorophytes and the diatom Phaeodactylum tricornutum Bohlin were detected at both the DNA and cDNA (gene expression) levels. The reproducibility of the array was evaluated in several ways, and future directions for further improvement of probe development and sensitivity are outlined. The phytoarray provides a relatively high‐resolution, high‐throughput approach to assessing phytoplankton community composition in marine environments.     

Active transport of CO2 by three species of marine microalgae

The occurrence of an active CO₂ transport system and of carbonic anhydrase (CA) has been investigated by mass spectrometry in the marine, unicellular rhodophyte Porphyridium cruentum (S.F. Gray) Naegeli and two marine chlorophytes Nannochloris atomus Butcher and Nannochloris maculata Butcher. Illumination of darkened cells incubated with 100 μM H¹³CO₃^? caused a rapid initial drop, followed by a slower decline in the extracellular CO₂ concentration. Addition of bovine CA to the medium raised the CO₂ concentration by restoring the HCO₃^?–CO₂ equilibrium, indicating that cells were taking up CO₂ and were maintaining the CO₂ concentration in the medium below its equilibrium value during photosynthesis. Darkening the cell suspensions caused a rapid increase in the extracellular CO₂ concentration in all three species, indicating that the cells had accumulated an internal pool of unfixed inorganic carbon. CA activity was detected by monitoring the rate of exchange of ¹⁸O from ¹³C¹⁸O₂ into water. Exchange of ¹⁸O was rapid in darkened cell suspensions, but was not inhibited by 500 μM acetazolamide, a membrane‐impermeable inhibitor of CA, indicating that external CA activity was not present in any of these species. In all three species, the rate of exchange was completely inhibited by 500 μM ethoxyzolamide, a membrane‐permeable CA‐inhibitor, showing that an intracellular CA was present. These results demonstrate that the three species are capable of CO₂ uptake by active transport for use as a carbon source for photosynthesis.     

Regulation of carbonic anhydrase expression by zinc, cobalt, and carbon dioxide in the marine diatom Thalassiosira weissflogii

TWCA1 is the major Zn-requiring isoform of carbonic anhydrase (CA) in the marine diatom Thalassiosira weissflogii. We have examined the roles that trace metals and CO(2) play in the regulation of TWCA1 expression over ranges of concentrations that bracket those encountered in the marine environment. Both steady-state levels of TWCA1 and the kinetics of induction were measured by western analysis. TWCA1 levels correlated well with cellular CA activity levels. TWCA1 was induced at a low CO(2) concentration but the level of induction, as determined by western analysis, was dependent on the availability of Zn. Co effectively substituted for Zn in regulating TWCA1 expression and promoting TWCA1 activity. Upon shift from low to high CO(2), the concentration of TWCA1 decreased. The expression of TWCA1 is diel cycle regulated, and cellular TWCA1 decreased during the dark phase. These results provide the basis for studying the expression of CA in field populations and, taken together with previous radiolabeling studies, provide strong evidence of in vivo metal substitution of Co for Zn in a CA. Our data also support the conclusion that TWCA1 plays a central role in carbon acquisition in T. weissflogii.     

FUNCTIONAL GENETIC DIVERGENCE IN HIGH CO2 ADAPTED EMILIANIA HUXLEYI POPULATIONS

Predicting the impacts of environmental change on marine organisms, food webs, and biogeochemical cycles presently relies almost exclusively on short‐term physiological studies, while the possibility of adaptive evolution is often ignored. Here, we assess adaptive evolution in the coccolithophore Emiliania huxleyi, a well‐established model species in biological oceanography, in response to ocean acidification. We previously demonstrated that this globally important marine phytoplankton species adapts within 500 generations to elevated CO₂. After 750 and 1000 generations, no further fitness increase occurred, and we observed phenotypic convergence between replicate populations. We then exposed adapted populations to two novel environments to investigate whether or not the underlying basis for high CO₂‐adaptation involves functional genetic divergence, assuming that different novel mutations become apparent via divergent pleiotropic effects. The novel environment “high light” did not reveal such genetic divergence whereas growth in a low‐salinity environment revealed strong pleiotropic effects in high CO₂ adapted populations, indicating divergent genetic bases for adaptation to high CO₂. This suggests that pleiotropy plays an important role in adaptation of natural E. huxleyi populations to ocean acidification. Our study highlights the potential mutual benefits for oceanography and evolutionary biology of using ecologically important marine phytoplankton for microbial evolution experiments.     

Direct Growth of Flower‐Like δ‐MnO2 on Three‐Dimensional Graphene for High‐Performance Rechargeable Li‐O2 Batteries

A challenge still remains to develop high‐performance and cost‐effective air electrode for Li‐O₂ batteries with high capacity, enhanced rate capability and long cycle life (100 times or above) despite recent advances in this field. In this work, a new design of binder‐free air electrode composed of three‐dimensional (3D) graphene (G) and flower‐like δ‐MnO₂ (3D‐G‐MnO₂) has been proposed. In this design, graphene and δ‐MnO₂ grow directly on the skeleton of Ni foam that inherits the interconnected 3D scaffold of Ni foam. Li‐O₂ batteries with 3D‐G‐MnO₂ electrode can yield a high discharge capacity of 3660 mAh g^?1 at 0.083 mA cm^?2. The battery can sustain 132 cycles at a capacity of 492 mAh g^?1 (1000 mAh g_carbon ^?1) with low overpotentials under a high current density of 0.333 mA cm^?2. A high average energy density of 1350 Wh Kg^?1 is maintained over 110 cycles at this high current density. The excellent catalytic activity of 3D‐G‐MnO₂ makes it an attractive air electrode for high‐performance Li‐O₂ batteries.     

Acclimation to ocean acidification during long‐term CO2 exposure in the cold‐water coral Lophelia pertusa

Ocean acidity has increased by 30% since preindustrial times due to the uptake of anthropogenic CO₂ and is projected to rise by another 120% before 2100 if CO₂ emissions continue at current rates. Ocean acidification is expected to have wide‐ranging impacts on marine life, including reduced growth and net erosion of coral reefs. Our present understanding of the impacts of ocean acidification on marine life, however, relies heavily on results from short‐term CO₂ perturbation studies. Here, we present results from the first long‐term CO₂ perturbation study on the dominant reef‐building cold‐water coral Lophelia pertusa and relate them to results from a short‐term study to compare the effect of exposure time on the coral's responses. Short‐term (1 week) high CO₂ exposure resulted in a decline of calcification by 26–29% for a pH decrease of 0.1 units and net dissolution of calcium carbonate. In contrast, L. pertusa was capable to acclimate to acidified conditions in long‐term (6 months) incubations, leading to even slightly enhanced rates of calcification. Net growth is sustained even in waters sub‐saturated with respect to aragonite. Acclimation to seawater acidification did not cause a measurable increase in metabolic rates. This is the first evidence of successful acclimation in a coral species to ocean acidification, emphasizing the general need for long‐term incubations in ocean acidification research. To conclude on the sensitivity of cold‐water coral reefs to future ocean acidification further ecophysiological studies are necessary which should also encompass the role of food availability and rising temperatures.