External Carbonic Anhydrase Research Articles

The Freshwater Cyanobacterium Synechococcus elongatus PCC 7942 Does Not Require an Active External Carbonic Anhydrase.

Under standard laboratory conditions, Synechococcus elongatus PCC 7942 lacks EcaASyn, a periplasmic carbonic anhydrase (CA). In this study, a S. elongatus transformant was created that expressed the homologous EcaACya from Cyanothece sp. ATCC 51142. This additional external CA had no discernible effect on the adaptive responses and physiology of cells exposed to changes similar to those found in S. elongatus natural habitats, such as fluctuating CO2 and HCO3- concentrations and ratios, oxidative or light stress, and high CO2. The transformant had a disadvantage over wild-type cells under certain conditions (Na+ depletion, a reduction in CO2). S. elongatus cells lacked their own EcaASyn in all experimental conditions. The results suggest the presence in S. elongatus of mechanisms that limit the appearance of EcaASyn in the periplasm. For the first time, we offer data on the expression pattern of CCM-associated genes during S. elongatus adaptation to CO2 replacement with HCO3-, as well as cell transfer to high CO2 levels (up to 100%). An increase in CO2 concentration coincides with the suppression of the NDH-14 system, which was previously thought to function constitutively.

The requirement for external carbonic anhydrase in diatoms is influenced by the supply and demand for dissolved inorganic carbon.

Photosynthesis by marine diatoms contributes significantly to the global carbon cycle. Due to the low concentration of CO2 in seawater, many diatoms use extracellular carbonic anhydrase (eCA) to enhance the supply of CO2 to the cell surface. While much research has investigated how the requirement for eCA is influenced by changes in CO2 availability, little is known about how eCA contributes to CO2 supply following changes in the demand for carbon. We therefore examined how changes in photosynthetic rate influence the requirement for eCA in three centric diatoms. Modeling of cell surface carbonate chemistry indicated that diffusive CO2 supply to the cell surface was greatly reduced in large diatoms at higher photosynthetic rates. Laboratory experiments demonstrated a trend of an increasing requirement for eCA with increasing photosynthetic rate that was most pronounced in the larger species, supporting the findings of the cellular modeling. Microelectrode measurements of cell surface pH and O2 demonstrated that individual cells exhibited an increased contribution of eCA to photosynthesis at higher irradiances. Our data demonstrate that changes in carbon demand strongly influence the requirement for eCA in diatoms. Cell size and photosynthetic rate will therefore be key determinants of the mode of dissolved inorganic carbon uptake.

Cooperation of an external carbonic anhydrase and HCO3- transporter supports underwater photosynthesis in submerged leaves of the amphibious plant Hygrophila difformis.

HCO3- can be a major carbon resource for photosynthesis in underwater environments. Here we investigate the underlying mechanism of uptake and membrane transport of HCO3- in submerged leaves of Hygrophila difformis, a heterophyllous amphibious plant. To characterize these mechanisms, we evaluated the sensitivity of underwater photosynthesis to an external carbonic anhydrase (CA) inhibitor and an anion exchanger protein inhibitor, and we attempted to identify components of the mechanism of HCO3- utilization. We evaluated the effects of the external CA inhibitor and anion exchanger protein inhibitor on the NaHCO3 response of photosynthetic O2 evolution in submerged leaves of H. difformis. Furthermore, we performed a comparative transcriptomic analysis between terrestrial and submerged leaves. Photosynthesis in the submerged leaves was decreased by both the external CA inhibitor and anion exchanger protein inhibitor, but no additive effect was observed. Among upregulated genes in submerged leaves, two α-CAs, Hdα-CA1 and Hdα-CA2, and one β-carbonic anhydrase, Hdβ-CA1, were detected. Based on their putative amino acid sequences, the α-CAs are predicted to be localized in the apoplastic region. Recombinant Hdα-CA1 and Hdβ-CA1 showed dominant CO2 hydration activity over HCO3- dehydration activity. We propose that the use of HCO3- for photosynthesis in submerged leaves of H. difformis is driven by the cooperation between an external CA, Hdα-CA1, and an unidentified HCO3- transporter.

Inorganic carbon utilization: A target of silver nanoparticle toxicity on a submerged macrophyte

Submerged macrophytes play an important role in the global carbon cycle through diversified pathways of inorganic carbon (Ci) utilization distinct from terrestrial plants. However, the effects of silver nanoparticles (AgNPs), an emerging contaminant, were unknown on the Ci utilization of submerged macrophytes. In Ottelia alismoides, the only known submerged macrophyte with three pathways of Ci utilization, before absorption, AgNPs inhibited the external carbonic anhydrase activity thus reducing the capacity of the plant to use HCO3−. After entering the plant, AgNPs mainly aggregated at the cell wall and in the chloroplast. The internalized AgNPs inhibited ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco) activity blocking CO2 fixation and disturbed C4 and crassulacean acid metabolism (CAM) by inhibiting phosphoenolpyruvate carboxylase (PEPC), pyruvate phosphate dikinase (PPDK), and NAD-dependent malic enzyme (NAD-ME) activities to alter intracellular malate biosynthesis and decarboxylation. Overall, our findings indicate that the Ci utilization of the submerged macrophyte is a target of AgNPs toxicity that might affect the carbon cycle in aquatic systems.

Adaptation of inorganic carbon utilization strategies in submerged and floating leaves of heteroblastic plant Ottelia cordata

During the life history, the aquatic heteroblastic plant undergoes submerged-leaf and floating/emerged-leaf phases, with dramatic changes in their surrounding environments. However, little is known about the physiological and biochemical changes of the two types of leaves adapting to submerged and aerial environments. In the present study, a heteroblastic plant Ottelia cordata was chosen to investigate the variations in morphology, anatomy, physiology, and biochemistry between the two types of leaves. The results showed that the floating leaf developed stomate to absorb atmospheric CO 2 for photosynthesis to adapt to the aerial habitat, while the submerged leaf showed longer, narrower, and thinner to maximize inorganic carbon uptake underwater. Through external carbonic anhydrase catalysis, both types of leaves could use HCO 3 - as an extra carbon source, and it was stronger in submerged leaves. Besides HCO 3 - use, the δ 13 C and the activity of C4 process key enzymes supported that the submerged leaf could perform C4 metabolism, while the floating leaves only carried on C3 metabolism. This is the first time to report an aquatic plant transiting from the C4 process underwater to the C3 process in the air. Based on the discrimination of stable carbon isotope, in the field, the main carbon source was atmospheric CO 2 for floating leaves and the free CO 2 underwater for submerged leaves. Additionally, HCO 3 - could approximately support 16% carbon source for underwater photosynthesis, which was the second important carbon source for submerged leaves. All our data indicated the heteroblastic aquatic plant adapts to underwater and aerial environments in morphology, anatomy, physiology, and biochemistry for completing life history and maintaining the population in the field. • Morphology and anatomy differed between submerged and floating leaves of O. cordata. • Both types of leaves could use HCO 3 - via external CA mediation for photosynthesis. • Submerged leaf performed C4 metabolism, floating leaf only carried on C3 metabolism. • Atmospheric CO 2 was the main carbon source for floating leaf. • Soluble CO 2 and HCO 3 - underwater were the two main carbon sources for submerged leaf.

Bicarbonate-use by aquatic macrophytes allows a reduction in photorespiration at low CO2 concentrations

Submerged macrophytes face the effects of the CO2 deficiency underwater that could result in higher photorespiration, nearly half of the submerged macrophytes examined to date have developed HCO3− use. In this study, the effects of HCO3− use on photorespiration were investigated in four submerged macrophytes, Cabomba caroliniana, Ceratophyllum demersum, Egeria densa and Ottelia alismoides. We used pH-drift experiments to show that three species, C. demersum, E. densa and O. alismoides, had the ability to use HCO3− (final pH exceeded 10) while C. caroliniana did not (final pH less than 9.5). The external carbonic anhydrase inhibitor, acetazolamide (AZA), was chosen to inhibit HCO3- use for oxygen evolution experiments under high CO2 and low CO2 conditions. The results showed that adding AZA significantly reduced oxygen production rate under low CO2 in the three HCO3- users, C. demersum, E. densa and O. alismoides. In addition, the activity of photorespiration-related enzymes, glycolate oxidase and catalase, significantly increased under AZA treatment in HCO3− users. However, for C. caroliniana, the CO2-only user, photorespiration and its related enzymes activities did not change under AZA addition.

Cell size influences inorganic carbon acquisition in artificially selected phytoplankton.

Cell size influences the rate at which phytoplankton assimilate dissolved inorganic carbon (DIC), but it is unclear whether volume-specific carbon uptake should be greater in smaller or larger cells. On the one hand, Fick's Law predicts smaller cells to have a superior diffusive CO2 supply. On the other, larger cells may have greater scope to invest metabolic energy to upregulate active transport per unit area through CO2 -concentrating mechanisms (CCMs). Previous studies have focused on among-species comparisons, which complicates disentangling the role of cell size from other covarying traits. In this study, we investigated the DIC assimilation of the green alga Dunaliella tertiolecta after using artificial selection to evolve a 9.3-fold difference in cell volume. We compared CO2 affinity, external carbonic anhydrase (CAext ), isotopic signatures (δ13 C) and growth among size-selected lineages. Evolving cells to larger sizes led to an upregulation of CCMs that improved the DIC uptake of this species, with higher CO2 affinity, higher CAext and higher δ13 C. Larger cells also achieved faster growth and higher maximum biovolume densities. We showed that evolutionary shifts in cell size can alter the efficiency of DIC uptake systems to influence the fitness of a phytoplankton species.

Identification and Characterization of a Periplasmic α-Carbonic Anhydrase (CA) in the Gametophytes of Saccharina japonica (Phaeophyceae).

Periplasmic or external carbonic anhydrases (CAs) have been well accepted as playing a crucial role in the acquisition of dissolved inorganic carbon; however, no cytological evidence or molecular information on these enzymes has been reported in seaweeds to date. In this study, the full-length cDNA sequence coding for a putative periplasmic Sjα-CA2 was cloned from the gametophytes of Saccharina japonica, an industrial brown seaweed. It was 1,728bp in length and included a 263-bp 5'-untranslated region (UTR), a 577-bp 3'-UTR, and an 888-bp open reading frame encoding a protein precursor consisting of 295 amino acids. The mature protein, after removal of a predicted 28-residue signal peptide, was composed of 267 amino acids with a relative molecular weight of 29.27kDa. Multisequence alignment and phylogenetic analysis indicated that it was a member of the α-CA family. Enzyme activity assays showed that the recombinant Sjα-CA2 in Escherichia coli possessed CO2 hydration and esterase activities, thus identifying this gene Sjα-CA2 in function. Immunogold electron microscopic observations with the prepared anti-Sjα-CA2 polyclonal antibody illustrated that Sjα-CA2 was located in periplasmic space of the kelp gametophyte cells. Quantitative real-time PCR results revealed that the transcription of Sjα-CA2 was induced by elevated levels, but it was little changed while the kelp gametophytes were subjected to elevated CO2 concentrations. This study suggests that the periplasmic Sjα-CA2 might play a role in adapting to elevated environmental levels of by dehydration of to generate CO2 , which could be readily taken up by S. japonica gametophytes.

Resilience of the temperate coral Oculina arbuscula to ocean acidification extends to the physiological level

Both juvenile and adult life stages of the temperate scleractinian coral Oculina arbuscula are resilient to the effects of moderate ocean acidification (OA) in contrast to many tropical corals in which growth and calcification rates are suppressed. Here, potential mechanisms of resilience to OA related to photosynthetic physiology and inorganic carbon processing were studied in adult O. arbuscula colonies. After exposing colonies to ambient and elevated carbon dioxide (CO2) treatments for 7 weeks, photosynthetic performance was characterized using photosynthesis versus irradiance experiments, chlorophyll fluorescence kinetics, and algal pigment content. Inorganic carbon-processing capabilities were assessed by measurement of internal and external carbonic anhydrase activity of the coral host, internal carbonic anhydrase activity of symbiotic algae, and the reliance of photosynthesis on external carbonic anhydrase. Photosynthetic physiology was unaffected by OA ruling out the possibility that resilience was mediated by increased photosynthetic energy supply. Carbonic anhydrase activities were maintained at elevated CO2 suggesting no major rearrangements of the inorganic carbon-processing machinery, but this could be a sign of resilience since tropical corals often down-regulate carbonic anhydrases at high CO2. The general lack of effect of ocean acidification on these physiological traits suggests other characteristics, such as maintenance of calcifying fluid pH and ability to acquire energy from heterotrophy, may be more important for the resilience of O. arbuscula to OA.

Different mechanisms of bicarbonate use affect carbon isotope composition in Ottelia guayangensis and Vallisneria denseserrulata in a karst stream

Abstract Macrophytes live in extremely different carbon environments compared to terrestrial plants. In response to bicarbonate dominated inorganic carbon in natural aquatic systems, the ability for HCO3− use has been developed in many macrophytes to alleviate a limitation by CO2 in aquatic systems. In the present study, all present macrophytes in a karst stream were investigated, and we found that seven of nine species were able to use HCO3− and two species at the source of the karst stream were restricted to CO2 use. Two different inhibitors were used to determine the HCO3− use mechanisms in Ottelia guanyangensis and Vallisneria denseserrulata, the only species coexisting from up- to down-stream. External carbonic anhydrase (CAext) mediated HCO3− use was present in O. guanyangensis up-stream, and then O. guanyangensis shifted to a combined mechanism of CAext and anion exchange (AE) mediated HCO3− use down-stream. In these two species, high HCO3− concentration induced the ability to use HCO3−, but high CO2 concentration inhibited this ability. From up-stream to down-stream, the decreasing CO2 induced increasing proportion of HCO3− use that led to changes in the δ13C in O. guanyangensis. However, a constant CAext and AE mediated HCO3− use caused a relatively stable δ13C in V. denseserrulata from river source to 330 m and then increased the proportion of HCO3− use at the stream outlet associated with the change in δ13C.

External α-carbonic anhydrase and solute carrier 4 are required for bicarbonate uptake in a freshwater angiosperm.

The freshwater monocot Ottelia alismoides is the only known species to operate three CO2-concentrating mechanisms (CCMs): constitutive bicarbonate (HCO3-) use, C4 photosynthesis, and facultative Crassulacean acid metabolism, but the mechanism of HCO3- use is unknown. We found that the inhibitor of an anion exchange protein, 4,4'-diisothio-cyanatostilbene-2,2'-disulfonate (DIDS), prevented HCO3- use but also had a small effect on CO2 uptake. An inhibitor of external carbonic anhydrase (CA), acetazolamide (AZ), reduced the affinity for CO2 uptake but also prevented HCO3- use via an effect on the anion exchange protein. Analysis of mRNA transcripts identified a homologue of solute carrier 4 (SLC4) responsible for HCO3- transport, likely to be the target of DIDS, and a periplasmic α-carbonic anhydrase 1 (α-CA1). A model to quantify the contribution of the three different pathways involved in inorganic carbon uptake showed that passive CO2 diffusion dominates inorganic carbon uptake at high CO2 concentrations. However, as CO2 concentrations fall, two other pathways become predominant: conversion of HCO3- to CO2 at the plasmalemma by α-CA1 and transport of HCO3- across the plasmalemma by SLC4. These mechanisms allow access to a much larger proportion of the inorganic carbon pool and continued photosynthesis during periods of strong carbon depletion in productive ecosystems.

A multistressor model of carbon acquisition regulation for macroalgae in a changing climate

AbstractIt is widely hypothesized that noncalcifying macroalgae will be more productive and abundant in increasingly warm and acidified oceans. Macroalgae vary greatly in the magnitudes and interactions of responses of photosynthesis and growth to multiple stressors associated with climate change. A knowledge gap that exists between the qualitative “macroalgae will benefit” hypothesis and the variable outcomes observed is regulation of physiological mechanisms that cause variation in the magnitudes of change in primary productivity, growth, and their covariation. In this context, we developed a model to quantitatively describe physiological responses to coincident variation in temperature, carbonate chemistry and light supply in a representative bicarbonate‐using marine macroalga. The model is based on Ulva spp., the best understood dissolved inorganic carbon uptake mechanism among macroalgae, with data enabling synthesis across all parameters. At boundary layer pH < 8.7 most inorganic carbon is taken up through the external carbonic anhydrase (CAext) mechanism under all conditions of photosynthetic photon flux density, temperature, and boundary layer thickness. Each 0.1 unit decline in pH causes a 20% increase in the fraction of diffusive uptake of CO2 thereby lessening reliance on active transport of bicarbonate. Modeled downregulation of anion exchange‐mediated active bicarbonate transport associated with a 0.4 unit decline in pH under ocean acidification is consistent with enhanced growth up to 4% per day without increasing photosynthetic rate. The model provides a means to quantify magnitudes of change in productivity under factorial combinations of changing temperature, pCO2, and light supply anticipated as climate changes.

Ultrastructural evolution, physiological traits, and carbon and nitrogen assimilation–related gene expression are compatible with the developmental transitions of parthenogenesis in Pyropia haitanensis (Bangiales, Rhodophyta)

Pyropia haitanensis has a typical parthenogenetic behavior under unisexual culture condition. In this study, ultrastructural evolution and physiological traits of photosynthesis and the expression characteristics of key enzyme genes related to carbon and nitrogen assimilation pathways at different developmental stages of parthenogenesis were analyzed. Ultrastructural observations showed that the gametophytic vegetative cells had cell wall, pyrenoids, abundant chloroplasts, and rarely scattered floridean starch grains. However, the appearance of gathered chloroplasts, plastoglobuli, large lipid droplets, pyrenoids encircled by numerous plastoglobuli, and an increased number of floridean starch grains in the cells during parthenogenesis provide neutral lipid and energy resources that are compatible with the developmental transitions. Physiologically, vegetative gametophytes possessed the highest Fv/Fm and Chl. a content and the lowest C/N ratio. However, compared with vegetative cell proliferation stage, the activities of RubisCO (ribulose bisphosphate carboxylase) and external CA (carbonic anhydrase) increased at early stages of parthenogenesis, and Fv/Fm and activities of PEPC (phosphoenolpyruvate carboxylase) and PEPCK (phosphoenolpyruvate carboxykinase) remained higher at later stages of parthenogenesis, suggesting that dynamic photosynthetic carbon fixation occurs in parthenogenesis. Quantitative real-time PCR analysis showed that selective regulation of key enzyme genes related to carbon and nitrogen assimilation pathways maintained stable carbon and nitrogen metabolism during developmental transitions of parthenogenesis. Moreover, during parthenogenesis, photosynthetic carbon fixation pathway potentially changed from C3 pathway at female gametophyte stage to C4 pathway at parthenosporophyte stage, which is similar to that of gametophytes and sporophytes in sexual reproduction. Meanwhile, the ammonia assimilation involved GDH pathway (glutamate dehydrogenase pathway) and GS/GOGAT cycle (glutamine synthetase/glutamate synthase cycle) in female gametophyte stage, while GS/GOGAT cycle was dominant in parthenosporophyte stage, suggesting that GS/GOGAT cycle may play an important role in the carbon and nitrogen balance during the developmental transitions of parthenogenesis. These results provide important information for revealing the adaptation strategy of parthenogenesis in Pyropia.

Effects of inorganic carbon concentration and pH on carbonic anhydrase activity of gametophytes of Saccharina japonica

Carbonic anhydrase (CA) was considered to be an important component of carbon concentrating mechanism (CCM) of algae. It was an inducible enzyme. Environmental factors, especially dissolved inorganic carbon and pH, were known to affect CA activity. Effects of inorganic carbon (CO2 and HCO3−) and pH on CA activity of gametophytes of Saccharina japonica were evaluated in this study. Under high-CO2 condition (3% CO2), the activity of external CA (CAext) was significantly decreased (P < 0.05) from 33.92 REA/g FW to 27.69 REA/g FW. In contrast, the internal CA (CAint) and total CA activities were elevated significantly (p < 0.01) from 36.83 REA/g FW to 48.80 REA/g FW, and from 70.75 REA/g FW to 76.49 REA/g FW, respectively. Addition of an appropriate concentration of HCO3− to the medium, CAint and total CA activities were promoted significantly, although the activity of CAext was inhibited significantly (P < 0.05). In higher HCO3− concentration (up to 1680 mg/L), the activity of CAint was promoted to 60.81REA/g FW, which was twice that of the control. Higher activities of CAint and total CA were induced at low pH. At pH 6.0, the highest activities of CAint (110.85 REA/g FW) and total CA (128.17 REA/g FW) were induced. However, at pH 9.0, the activities of CAint and total CA were reduced to the lowest of 23.31REA/g FW and 42.19 REA/g FW, respectively. This is the first report about the successful detection of CAext activity of gametophytes of S. japonica. The results would provide data for the analysis of Ci acquisition and transport mechanism in S.japonica gametophytes.

Photosynthesis and light-dependent proton pumps increase boundary layer pH in tropical macroalgae: A proposed mechanism to sustain calcification under ocean acidification

Ocean acidification (OA) projections predict ocean pH to decline between 0.2 and 0.4 by 2100 with potential negative consequences for marine calcifiers without acclimation or adaption strategies to accomodate greater [H+] in seawater. Biotic control of calcified reef macroalgae thalli surface diffusive boundary layer (DBL) chemistry may overcome low pH in seawater as one strategy to accommodate OA conditions. To investigate this strategy, we examined surface DBL O2 and pH dynamics in five calcifying macroalgae (Halimeda, Udotea, Jania, Neogoniolithon, crustose coralline algae [CCA]) from the Florida Reef Tract under ambient (8.1) and low (7.65) pH using microsensors (100 μm) at the thalli surface in a flow-through flume. The role of photosynthesis and photosystem II (PSII)-independent proton pumps in controlling DBL pH were examined. Four of the five macroalgae exhibited a strong positive linear relationship between O2 production and increasing pH in the first 15–30 s of irradiance. Once a quasi-steady-state O2 concentration was reached (300 s), all species had DBL pH that were higher (0.02–0.32) than bulk seawater. The DBL pH increase was greatest at low pH and dependent on PSII. Some evidence was found for a light-dependent, but PSII-independent, proton pump. High DBL Δ pH upon illumination was likely in response to carbon concentrating mechanisms (CCMs) for photosynthesis. CCMs may be a HCO3−–H+ symport, OH– antiport or other DIC transport system, accompanied by proton efflux. HCO3– dehydration by external carbonic anhydrase (CAext) also produces OH– that can neutralize H+ in the DBL. CO2 or HCO3– uptake for photosynthesis may also engage H+/OH– fluxes as part of intracellular acid-base regulation changing DBL pH. A higher Δ pH within the DBL at low pH could be accounted for by greater CO2 diffusion and/or lower efficiencies in exporting cellular H+ across a lower concentration gradient, and/or a more efficient removal of H+ by CAext-driven dehydration of HCO3−. In the dark, Δ pH was less than in the light as these dynamics were primarily due to photosynthesis. We present a conceptual model of inorganic carbon uptake and ion transport pathways, as well as other processes associated with photosynthesis that drive DBL Δ pH and sustain tropical macroalgal calcification in the light under OA. In the dark, unless PSII-independent proton pumps are present, which do not appear to be ubiquitous amongst species, acidification processes likely dominate, resulting in CaCO3 net dissolution, particularly under OA conditions.

Physiological differences in photosynthetic inorganic carbon utilization between gametophytes and sporophytes of the economically important red algae Pyropia haitanensis

Despite the lifecycle of the red seaweed Pyropia/Porphyra being extensively studied, photosynthesis and the CO2 concentrating mechanisms (CCMs) have comparatively not been well studied between the gametophyte phase (leafy thallus) and the sporophyte phase (filamentous conchocelis). This study found that the net photosynthetic rates of the thalli were about twice that of the conchocelis of Py. haitanensis, and the maximal quantum yield of photosystem II (Fv/Fm) and thallus respiration were significantly higher and lower than the conchocelis, respectively. Additionally, two phases did not show significant differences in phycocyanin (PC) content, but significantly higher contents of phycoerythrin (PE), allophycocyanin (APC), and chlorophyll α (Chl α) were observed in the thalli compared with the conchocelis. The external carbonic anhydrase (eCA) activity of the thalli was also higher than that of the conchocelis, but no differences in internal CA (iCA) activity were detected between the two phases. As for carbon acquisition for photosynthesis, the major carbon source of the thalli was seawater HCO3−, which was absorbed via eCA-catalyzed conversion to CO2. Conversely, the conchocelis used HCO3− transporters to directly absorb seawater HCO3−, which contributed equally to photosynthesis compared with the eCA- or iCA-mediated pathway. The discovery of different CCMs between the thallus and conchocelis phases could further our understanding of lifecycle regulation in Pyropia. In addition, a decrease in pH from 9 to 6 significantly increased the photosynthetic rates of both the thalli and conchocelis by about 100% and 600%, respectively. Thus, both phases have potential for carbon capture from flue gases and carbon sequestration.

Insights into carbon acquisition and photosynthesis in Karenia brevis under a range of CO2 concentrations

Karenia brevis is a marine dinoflagellate commonly found in the Gulf of Mexico and important both ecologically and economically due to its production of the neurotoxin brevetoxin, which can cause respiratory illness in humans and widespread death of marine animals. K. brevis strains have previously shown to be sensitive to changes in CO2, both in terms of growth as well as toxin production. Our study aimed to understand this sensitivity by measuring underlying mechanisms, such as photosynthesis, carbon acquisition, and photophysiology. K. brevis (CCFWC-126) did not show a significant response in growth, cellular composition of carbon and nitrogen, nor in photosynthetic rates between pCO2 concentrations of 150, 400, or 780 µatm. However, a strong response in its acquisition of inorganic carbon was found. Half saturation values for CO2 increased from 1.5 to 3.3 µM, inorganic carbon preference switched from HCO3− to CO2 (14–56% CO2 usage), and external carbonic anhydrase activity was downregulated by 23% when comparing low and high pCO2. We conclude that K. brevis must employ an efficient and regulated CO2 concentrating mechanism (CCM) to maintain constant carbon fixation and growth across pCO2 levels. No statistically significant correlation between CO2 and brevetoxin content was found, yet a positive trend with enhanced pCO2 was detected. This study is the first explaining how this socioeconomically important species is able to efficiently supply inorganic carbon for photosynthesis, which can potentially prolong bloom situations. This study also highlights that elevated CO2 concentrations, as projected for a future ocean, can affect underlying physiological processes of K. brevis, some of which could lead to increases in cellular brevetoxin production and therefore increased impacts on coastal ecosystems and economies.

Carbon acquisition mechanisms in Halophila johnsonii and Thalassia testudinum

Mechanisms for carbon uptake in the small-bodied Halophila johnsonii and large-bodied Thalassia testudinum were compared using photosynthesis measurements (oxygen flux) with, and without, the extracellular carbonic anhydrase inhibitor acetazolamide (AZ) and TRIS buffer. Our results indicated T. testudinum and H. johnsonii both utilize external membrane-bound carbonic anhydrase to facilitate dehydration of HCO3− into CO2(aq). T. testudinum also utilizes an active proton (H+) pump to create localized H+ gradients within the boundary layer and it derives a larger portion of photosynthetic carbon from bicarbonate than H. johnsonii. Stable carbon isotope composition (δ13C) of T. testudinum and H. johnsonii leaves under high and reduced irradiance were also compared to evaluate photosynthetic carbon use. Isotope ratios of H. johnsonii were significantly more negative than T. testudinum at a high-light field site (−10.44 ± 0.26‰ vs −9.25 ± 0.25‰). In contrast, there was no significant difference in isotope ratios between species for plants maintained in a greenhouse under reduced photosynthetically active radiation. This suggests photosynthesis of T. testudinum is more carbon limited or more reliant on bicarbonate for photosynthesis than H. johnsonii under high-light conditions. Although photosynthesis of both species is expected to increase in response to greater CO2(aq) availability, ocean acidification and its CO2(aq) enrichment of seawater may have greater benefits for H. johnsonii compared to T. testudinum, whose photosynthesis relies more heavily on HCO3− use.

Carbonic anhydrase activity in seaweeds: overview and recommendations for measuring activity with an electrometric method, using Macrocystis pyrifera as a model species

Carbonic anhydrase (CA) plays an important physiological role in all biological systems by accelerating the interconversion of CO2 and HCO3−. In algae, CA is essential for photosynthesis: external CA (CAext) dehydrates HCO3−, enhancing the supply of CO2 to the cell surface, and internal CA (CAint) interconverts HCO3− and CO2 to maintain the inorganic carbon (Ci) pool and supply CO2 to RuBisCO. We first conducted a literature review comparing the conditions in which CA extraction and measurement have been carried out, using the commonly used Wilbur–Anderson method. We found that the assay has been widely modified since its introduction in 1948, mostly without being optimized for the species tested. Based on the review, an optimized protocol for measuring CA in Macrocystis pyrifera was developed, which showed that the assay conditions can strongly affect CA activity. Tris–HCl buffer gave the highest levels of CA activity, but phosphate buffer reduced activity significantly. Buffers containing polyvinylpyrrolidone (PVP) and dithiothreitol (DTT) stabilized CA. Using the optimized assay, CAext and CAint activities were readily measured in Macrocystis with higher precision compared to the non-optimized method. The CAint activity was 2 × higher than CAext, which is attributed to the Ci uptake mechanisms of Macrocystis. This study suggests that the CA assay needs to be optimized for each species prior to experimental work to obtain both accurate and precise results.

Dynamic changes in carbonate chemistry in the microenvironment around single marine phytoplankton cells

Photosynthesis by marine diatoms plays a major role in the global carbon cycle, although the precise mechanisms of dissolved inorganic carbon (DIC) uptake remain unclear. A lack of direct measurements of carbonate chemistry at the cell surface has led to uncertainty over the underlying membrane transport processes and the role of external carbonic anhydrase (eCA). Here we identify rapid and substantial photosynthesis-driven increases in pH and [CO32−] primarily due to the activity of eCA at the cell surface of the large diatom Odontella sinensis using direct simultaneous microelectrode measurements of pH and CO32− along with modelling of cell surface inorganic carbonate chemistry. Our results show that eCA acts to maintain cell surface CO2 concentrations, making a major contribution to DIC supply in O. sinensis. Carbonate chemistry at the cell surface is therefore highly dynamic and strongly dependent on cell size, morphology and the carbonate chemistry of the bulk seawater.