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Symbiotic dinoflagellate algae residing inside coral tissues supply the host with the majority of their energy requirements through the translocation of photosynthetically fixed carbon. The algae, in turn, rely on the host for the supply of inorganic carbon. Carbon must be concentrated as CO2 in order for photosynthesis to proceed, and here we show that the coral host plays an active role in this process. The host-derived symbiosome membrane surrounding the algae abundantly expresses vacuolar H(+)-ATPase (VHA), which acidifies the symbiosome space down to pH ∼ 4. Inhibition of VHA results in a significant decrease in average H(+) activity in the symbiosome of up to 75% and a significant reduction in O2 production rate, a measure of photosynthetic activity. These results suggest that host VHA is part of a previously unidentified carbon concentrating mechanism for algal photosynthesis and provide mechanistic evidence that coral host cells can actively modulate the physiology of their symbionts.

Photosynthesis is crucial for life but is a slow process because the CO2 concentration near the principal carbon-assimilation enzyme RuBisCO is extremely low. Very few plants and algae perform a carbon-concentrating mechanism (CCM) to overcome the insufficiency, which are classified into biophysical and biochemical (C4) mechanism. The enzyme CA catalyzes the reversible dehydration of HCO3- to CO2 in biophysical CCMs and its active site contains a Zn2+. In this study, we hypothesized that Zn2+ availability can impact CCMs and therefore investigated the effect of Zn2+ availability on photosynthetic metabolism in a unicellular marine diatom Phaeodactylum tricornutum. P. tricornutum has a sequenced genome and can conduct both biophysical and C4 CCMs. We observed that Zn2+ has a significant effect on cell growth rate but no significant interference on intracellular metabolism, suggesting no essential compensation of C4 CCMs for biophysical CCMs even at low CA activity anticipated at low Zn2+ concentration.

We investigated the catalytic activity and inhibition of the beta-class carbonic anhydrase (CA, EC 4.2.1.1) CahB1, from the relict cyanobacterium Coleofasciculus chthonoplastes (previously denominated Microcoleus chthonoplastes). The enzyme showed good activity as a catalyst for the CO2 hydration, with a k(cat) of 2.4 x 10(5) s (1) and a k(cat)/K-m of 6.3 x 10(7) M (1) s (1). A range of inorganic anions and small molecules were investigated as inhibitors of CahB1. Perchlorate and tetrafluoroborate did not inhibit the enzyme (K(I)s >200 mM) whereas selenate and selenocyanide were ineffective inhibitors too, with K(I)s of 29.9-48.61 mM. The halides, pseudohalides, carbonate, bicarbonate, trithiocarbonate and a range of heavy metal ions-containing anions were submillimolar-millimolar inhibitors (K(I)s in the range of 0.15-0.90 mM). The best CahB1 inhibitors were N,N-diethyldithiocarbamate, sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, with K(I)s in the range of 8-75 mu M, whereas acetazolamide inhibited the enzyme with a K-I of 76 nM. This is the first kinetic and inhibition study of a cyanobacterial CA. As these enzymes are widespread in many cyanobacteria, being crucial for the carbon concentrating mechanism which assures substrate to RubisCO for the CO2 fixation by these organisms, a detailed kinetic/inhibition study may be essential for a better understanding of this superfamily of metalloenzymes and for potential biotechnological applications in biomimetic CO2 capture processes. (C) 2014 Elsevier Ltd. All rights reserved.

International audience; Zinc is an essential nutrient because of its role in catalysis and in protein stabilization, but excess zinc is deleterious. We distinguished four nutritional zinc states in the alga Chlamydomonas reinhardtii: toxic, replete, deficient, and limited. Growth is inhibited in zinc-limited and zinc-toxic cells relative to zinc-replete cells, whereas zinc deficiency is visually asymptomatic but distinguished by the accumulation of transcripts encoding ZIP family transporters. To identify targets of zinc deficiency and mechanisms of zinc acclimation, we used RNA-seq to probe zinc nutrition-responsive changes in gene expression. We identified genes encoding zinc-handling components, including ZIP family transporters and candidate chaperones. Additionally, we noted an impact on two other regulatory pathways, the carbon-concentrating mechanism (CCM) and the nutritional copper regulon. Targets of transcription factor Ccm1 and various CAH genes are up-regulated in zinc deficiency, probably due to reduced carbonic anhydrase activity, validated by quantitative proteomics and immunoblot analysis of Cah1, Cah3, and Cah4. Chlamydomonas is therefore not able to grow photoautotrophically in zinc-limiting conditions, but supplementation with 1% CO2 restores growth to wild-type rates, suggesting that the inability to maintain CCM is a major consequence of zinc limitation. The Crr1 regulon responds to copper limitation and is turned on in zinc deficiency, and Crr1 is required for growth in zinc-limiting conditions. Zinc-deficient cells are functionally copper-deficient, although they hyperaccumulate copper up to 50-fold over normal levels. We suggest that zinc-deficient cells sequester copper in a biounavailable form, perhaps to prevent mismetallation of critical zinc sites.

Many photosynthetic organisms have developed inorganic carbon (Ci) concentrating mechanisms (CCMs) that increase the CO2 concentration within the vicinity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). Several CCMs, such as four carbon (C4) and crassulacean acid metabolism (CAM), bicarbonate accumulation systems and capsular structures around RubisCO have been described in great detail. These systems are believed to have evolved several times as mechanisms that acclimate organisms to unfavourable growth conditions. Based on recent experimental evidence we propose the occurrence of another more general CCM system present in all plants. This basal CCM (bCCM) is supposed to be composed of mitochondrial carbonic anhydrases (a β-type carbonic anhydrase and the γ-type carbonic anhydrase domain of the mitochondrial NADH dehydrogenase complex) and probably further unknown components. The bCCM is proposed to reduce leakage of CO2 from plant cells and allow efficient recycling of mitochondrial CO2 for carbon fixation in chloroplasts. Fil: Zabaleta, Eduardo Julian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata; Argentina Fil: Martin, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata; Argentina Fil: Braun, Hans Peter. Leibniz Universität Hannover; Alemania

The fluxes of CO 2 and oxygen during photosynthesis by cell suspensions of Tessellaria volvocina and Mallomonas papillosa were monitored mass spectrometrically. There was no rapid uptake of CO 2, only a slow drawdown to compensation concentrations of 26 lM for T. volvocina and 18 lM for M. papillosa, when O 2 evolution ceased, indicating a lack of active bicarbonate uptake by the cells. Darkening of the cells after a period of photosynthesis did not cause rapid release of CO 2, indicating the absence of an intracellular inorganic carbon pool. However, upon darkening a brief burst of CO 2 was observed similar to the post-illumination burst characteristic of C3 higher plants. Treatment of the cells of both species with the membrane-permeable carbonic anhydrase inhibitor ethoxyzolamide had no adverse effect on photosynthetic rate, but stimulated the dark CO 2 burst indicating the dark oxidation of a compound formed in the light. In the absence of any active accumulation of inorganic carbon photosynthesis in these species should be inhibited by O 2. This was investigated in four synurophyte species T. volvocina, M. papillosa, Synura petersenii, and Synura uvella: Photosynthetic O 2 evolution rates in all four algae, measured by O 2 electrode, were significantly higher (40-50%) in media at low O 2 (4%) than in airequilibrated (21% O 2) media, indicating an O 2 inhibition of photosynthesis (Warburg effect) and thus the occurrence of photorespiration in these species. © Springer Science+Business Media B.V. 2011.

The activities of extra and intracellular carbonic anhydrases (CA) were studied in the microalgae Tetraselmis gracilis (Kylin) Butcher (Chlorophyta, Prasinophyceae) growing in laboratory cultivation. During ten days of batch cultivation, daily determinations of pH, cell number, enzymatic activity, and total dissolved inorganic carbon (DIC), as well as its main species, CO2 and HCO3-, were performed. Enzymatic activity increased as the growing cell population depleted inorganic carbon from the medium. Carbon dioxide concentration decreased quickly, especially in the third day of cultivation, when a significant increase of the intracellular enzymatic activity was observed. Bicarbonate concentration had its largest decrease in the cultivation medium in the fourth day, when the activity of the extracellular enzyme had its largest increase, suggesting its use by the alga through CA activity. After the fourth cultivation day, half of the cultures were aerated with CO2-free atmospheric air, which caused an increase in the total and external activity of the enzyme, although, in this condition, the stationary growth phase began earlier than in cultures aerated with atmospheric air. The pH of the media was measured daily, increasing from the first to the fourth day, and remaining almost constant until the end of the cultivation. Algal material transferred to the dark lost all enzymatic activity. As atividades da Anidrase Carbônica (AC) extra e intracelular foram estudadas na microalga marinha Tetraselmis gracilis (Kylin) Butcher (Chlorophyta, Prasinophyceae) crescendo em cultivos laboratoriais. Durante dez dias de cultivo, determinações diárias do pH, número de células, atividades enzimáticas, carbono inorgânico total dissolvido (CID) e suas principais espécies CO2 e HCO3- foram feitas. A atividade enzimática aumentou na medida em que a população celular em crescimento retirava carbono inorgânico do meio de cultivo. A concentração de dióxido de carbono decresceu rapidamente, especialmente no terceiro dia do cultivo, quando um significante aumento na atividade enzimática intracelular foi observado. A concentração de bicarbonato teve seu maior decréscimo no meio de cultivo no quarto dia, quando a atividade da enzima extracelular teve seu maior aumento, sugerindo seu uso pela alga através da atividade da AC. Após o quarto dia de cultivo, metade das culturas passou a ser aerada com ar atmosférico sem CO2, o que causou um aumento na atividade total e externa da enzima, fazendo com que esses cultivos entrassem na fase estacionária do crescimento antes que aqueles aerados com ar atmosférico normal. O pH do meio foi medido diariamente, aumentando desde o primeiro até o quarto dia e permanecendo quase constante até o fim do cultivo. Material algal transferido para o escuro perdeu toda a atividade enzimática.

Inorganic carbon (Ci) is the major sink for photosynthetic reductant in organisms capable of oxygenic photosynthesis. In the absence of abundant Ci, the cyanobacterium Synechocystis sp. strain PCC6803 expresses a high affinity Ci acquisition system, the CO2-concentrating mechanisms (CCM), controlled by the transcriptional regulator CcmR and the metabolites NADP+ and α-ketoglutarate, which act as co-repressors of CcmR by modulating its DNA binding. The CCM thus responds to internal cellular redox changes during the transition from Ci-replete to Ci-limited conditions. However, the actual changes in the metabolic state of the NADPH/NADP+ system that occur during the transition to Ci-limited conditions remain ill-defined. Analysis of changes in the redox state of cells experiencing Ci limitation reveals systematic changes associated with physiological adjustments and a trend towards the quinone and NADP pools becoming highly reduced. A rapid and persistent increase in F0 was observed in cells reaching the Ci-limited state, as was the induction of photoprotective fluorescence quenching. Systematic changes in the fluorescence induction transients were also observed. As with Chl fluorescence, a transient reduction of the NADPH pool (‘M’ peak), is assigned to State 2→State 1 transition associated with increased electron flow to NADP+. This was followed by a characteristic decline, which was abolished by Ci limitation or inhibition of the Calvin–Benson–Bassham (CBB) cycle and is thus assigned to the activation of the CBB cycle. The results are consistent with the proposed regulation of the CCM and provide new information on the nature of the Chl and NADPH fluorescence induction curves.

Bicarbonate species in the aqueous phase is the primary source for CO2 for the growth of microalgae. The potential of carbon dioxide (CO2) fixation by Chlorella pyrenoidosa in enriched bicarbonate medium was evaluated. In the present study, effects of parameters such as pH, sodium bicarbonate concentration and inoculum size were assessed for the removal of CO2 by C. pyrenoidosa under mixotrophic condition. Central composite design tool from response surface methodology was used to validate statistical methods in order to study the influence of these parameters. The obtained results reveal that the maximum removal of CO2 was attained at pH 8 with sodium bicarbonate concentration of 3.33 g/l, and inoculum size of 30 %. The experimental results were statistically significant with R 2 value of 0.9527 and 0.960 for CO2 removal and accumulation of chlorophyll content, respectively. Among the various interactions, interactive effects between the parameters pH and inoculum size was statistically significant (P