Carbon-concentrating Mechanism Research Articles

Cyanophycin mediates the accumulation and storage of fixed carbon in non-heterocystous filamentous cyanobacteria from coniform mats.

Thin, filamentous, non-heterocystous, benthic cyanobacteria (Subsection III) from some marine, lacustrine and thermal environments aggregate into macroscopic cones and conical stromatolites. We investigate the uptake and storage of inorganic carbon by cone-forming cyanobacteria from Yellowstone National Park using high-resolution stable isotope mapping of labeled carbon (H13CO3 −) and immunoassays. Observations and incubation experiments in actively photosynthesizing enrichment cultures and field samples reveal the presence of abundant cyanophycin granules in the active growth layer of cones. These ultrastructurally heterogeneous granules rapidly accumulate newly fixed carbon and store 18% of the total particulate labeled carbon after 120 mins of incubation. The intracellular distribution of labeled carbon during the incubation experiment demonstrates an unexpectedly large contribution of PEP carboxylase to carbon fixation, and a large flow of carbon and nitrogen toward cyanophycin in thin filamentous, non-heterocystous cyanobacteria. This pattern does not occur in obvious response to a changing N or C status. Instead, it may suggest an unusual interplay between the regulation of carbon concentration mechanisms and accumulation of photorespiratory products that facilitates uptake of inorganic C and reduces photorespiration in the dense, surface-attached communities of cyanobacteria from Subsection III.

Differential effects of ocean acidification on carbon acquisition in two bloom-forming dinoflagellate species.

Dinoflagellates represent a cosmopolitan group of phytoplankton with the ability to form harmful algal blooms. Featuring a Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) with very low CO2 affinities, photosynthesis of this group may be particularly prone to carbon limitation and thus benefit from rising atmospheric CO2 partial pressure (pCO2) under ocean acidification (OA). Here, we investigated the consequences of OA on two bloom-forming dinoflagellate species, the calcareous Scrippsiella trochoidea and the toxic Alexandrium tamarense. Using dilute batch incubations, we assessed growth characteristics over a range of pCO2 (i.e. 180–1200 µatm). To understand the underlying physiology, several aspects of inorganic carbon acquisition were investigated by membrane-inlet mass spectrometry. Our results show that both species kept growth rates constant over the tested pCO2 range, but we observed a number of species-specific responses. For instance, biomass production and cell size decreased in S. trochoidea, while A. tamarense was not responsive to OA in these measures. In terms of oxygen fluxes, rates of photosynthesis and respiration remained unaltered in S. trochoidea whereas respiration increased in A. tamarense under OA. Both species featured efficient carbon concentrating mechanisms (CCMs) with a CO2-dependent contribution of HCO3− uptake. In S. trochoidea, the CCM was further facilitated by exceptionally high and CO2-independent carbonic anhydrase activity. Comparing both species, a general trade-off between maximum rates of photosynthesis and respective affinities is indicated. In conclusion, our results demonstrate effective CCMs in both species, yet very different strategies to adjust their carbon acquisition. This regulation in CCMs enables both species to maintain growth over a wide range of ecologically relevant pCO2.

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

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.

Enhanced production of a lutein-rich acidic environment microalga

This study was aimed at increasing productivity of a novel lutein-rich acidic environment microalga, Coccomyxa onubensis, based on efficient inorganic carbon use. Productivity was determined based on dry weight data; inorganic carbon concentration mechanisms were determined by means of carbonic anhydrase activity; carotenoids were extracted with methanol and measured by HPLC techniques. The existence of carbon concentration mechanisms and conditions that might lead to use them for addressing increased productivity of C. onubensis was studied. Best growth and carbon uptake capacity occurred at acidic pH, proving acid-tolerant behaviour of C. onubensis. Incubation in air followed by shift to high carbon conditions enhanced carbon-use efficiency in terms of growth rate and biomass productivity, based on the action of both carbonic anhydrase activities. Lutein accumulated in the microalga at high concentrations above 5-6 g kg(-1) dry weight and did not depend on inorganic carbon conditions. Consequently, repeated cycles of air incubation and high CO2 incubation of C. onubensis might become a suitable tool to perform production processes of lutein-enriched biomass. This study intends to show that acidic environment microalgae can be produced at similar productivities of nonextreme microalgae, with the added advantage of their growth in highly selective culture medium. Particularly, it is applied to C. onubensis which accumulates lutein at commercially relevant concentrations.

Insights into the industrial growth of cyanobacteria from a model of the carbon‐concentrating mechanism

With the advent of modern bioengineering tools, photosynthetic organisms are increasingly being engineered to produce chemicals from CO2 sources, thereby creating a potential route of sustainable chemical production. Cyanobacteria have evolved a carbon‐concentrating mechanism (CCM) that enables growth at low‐environmental carbon concentrations. However at high‐carbon concentrations these benefits may not outweigh synthesis costs. Here, mass transport and kinetic modeling analyses were performed on two species of cyanobacteria as well as a hypothetical no‐CCM mutant. Modeling results correlated with published experimental data. Three conclusions were drawn from the analysis. Carboxysome geometry was unimportant due to the fast relative rate of diffusion of carbon species. Interspecies variations were largely due to active transporters. The no‐carboxysome cell approaches the wild‐type at 10% CO2. Therefore, in high CO2 environments the carboxysome and active bicarbonate transporters provide no benefit and a metabolic advantage could be achieved by eliminating the energy‐intensive CCM proteins. © 2014 American Institute of Chemical Engineers AIChE J, 60: 1269–1277, 2014

Isolation and characterization of mutants defective in the localization of LCIB, an essential factor for the carbon-concentrating mechanism in Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii acclimates to low-CO2 (LC) conditions by actively transporting inorganic carbon (Ci) into the cell, resulting in an increase in photosynthetic efficiency. This mechanism is called the carbon-concentrating mechanism (CCM), and soluble protein LCIB is essential for the CCM. LCIB is localized in the vicinity of pyrenoid, a prominent structure in the chloroplast, under LC conditions in the light. In contrast, in the dark or in high-CO2 conditions, where the CCM is inactive, LCIB diffuses away from the pyrenoid. Although the functional importance of LCIB for the CCM has been shown, the significance and mechanism of the change in suborganellar localization of LCIB remain to be elucidated. In this study, we screened 13,000 DNA-tagged mutants and isolated twelve aberrant LCIB localization (abl) mutants under LC conditions. abl-1 and abl-3 with dispersed and speckled localization of LCIB in the chloroplast showed significant decreases in Ci affinity, Ci accumulation, and CO2 fixation. Ten abl mutants (abl-1, abl-3, abl-4, abl-5, abl-6, abl-7, abl-8, abl-9, abl-11, and abl-12) showed not only aberrant LCIB localization but also reduced pyrenoid sizes. Moreover, three abl mutants (abl-10, abl-11, and abl-12) showed the increased numbers of pyrenoids per cell. These results suggested that the specific LCIB localization could be related to pyrenoid development.

The Operation of Two Decarboxylases, Transamination, and Partitioning of C4 Metabolic Processes between Mesophyll and Bundle Sheath Cells Allows Light Capture To Be Balanced for the Maize C4 Pathway

The C4 photosynthesis carbon-concentrating mechanism in maize (Zea mays) has two CO2 delivery pathways to the bundle sheath (BS; via malate or aspartate), and rates of phosphoglyceric acid reduction, starch synthesis, and phosphoenolpyruvate regeneration also vary between BS and mesophyll (M) cells. The theoretical partitioning of ATP supply between M and BS cells was derived for these metabolic activities from simulated profiles of light penetration across a leaf, with a potential 3-fold difference in the fraction of ATP produced in the BS relative to M (from 0.29 to 0.96). A steady-state metabolic model was tested using varying light quality to differentially stimulate M or BS photosystems. CO2 uptake, ATP production rate (JATP; derived with a low oxygen/chlorophyll fluorescence method), and carbon isotope discrimination were measured on plants under a low light intensity, which is considered to affect C4 operating efficiency. The light quality treatments did not change the empirical ATP cost of gross CO2 assimilation (JATP/GA). Using the metabolic model, measured JATP/GA was compared with the predicted ATP demand as metabolic functions were varied between M and BS. Transamination and the two decarboxylase systems (NADP-malic enzyme and phosphoenolpyruvate carboxykinase) were critical for matching ATP and reduced NADP demand in BS and M when light capture was varied under contrasting light qualities.

Genome-Wide Identification of Regulatory Elements and Reconstruction of Gene Regulatory Networks of the Green Alga Chlamydomonas reinhardtii under Carbon Deprivation

The unicellular green alga Chlamydomonas reinhardtii is a long-established model organism for studies on photosynthesis and carbon metabolism-related physiology. Under conditions of air-level carbon dioxide concentration [CO2], a carbon concentrating mechanism (CCM) is induced to facilitate cellular carbon uptake. CCM increases the availability of carbon dioxide at the site of cellular carbon fixation. To improve our understanding of the transcriptional control of the CCM, we employed FAIRE-seq (formaldehyde-assisted Isolation of Regulatory Elements, followed by deep sequencing) to determine nucleosome-depleted chromatin regions of algal cells subjected to carbon deprivation. Our FAIRE data recapitulated the positions of known regulatory elements in the promoter of the periplasmic carbonic anhydrase (Cah1) gene, which is upregulated during CCM induction, and revealed new candidate regulatory elements at a genome-wide scale. In addition, time series expression patterns of 130 transcription factor (TF) and transcription regulator (TR) genes were obtained for cells cultured under photoautotrophic condition and subjected to a shift from high to low [CO2]. Groups of co-expressed genes were identified and a putative directed gene-regulatory network underlying the CCM was reconstructed from the gene expression data using the recently developed IOTA (inner composition alignment) method. Among the candidate regulatory genes, two members of the MYB-related TF family, Lcr1 (Low-CO 2 response regulator 1) and Lcr2 (Low-CO 2 response regulator 2), may play an important role in down-regulating the expression of a particular set of TF and TR genes in response to low [CO2]. The results obtained provide new insights into the transcriptional control of the CCM and revealed more than 60 new candidate regulatory genes. Deep sequencing of nucleosome-depleted genomic regions indicated the presence of new, previously unknown regulatory elements in the C. reinhardtii genome. Our work can serve as a basis for future functional studies of transcriptional regulator genes and genomic regulatory elements in Chlamydomonas.

Ocean acidification alleviates low-temperature effects on growth and photosynthesis of the red alga Neosiphonia harveyi (Rhodophyta)

This study aimed to examine interactive effects between ocean acidification and temperature on the photosynthetic and growth performance of Neosiphonia harveyi. N. harveyi was cultivated at 10 and 17.5 °C at present (~380 µatm), expected future (~800 µatm), and high (~1500 µatm) pCO2. Chlorophyll a fluorescence, net photosynthesis, and growth were measured. The state of the carbon-concentrating mechanism (CCM) was examined by pH-drift experiments (with algae cultivated at 10 °C only) using ethoxyzolamide, an inhibitor of external and internal carbonic anhydrases (exCA and intCA, respectively). Furthermore, the inhibitory effect of acetazolamide (an inhibitor of exCA) and Tris (an inhibitor of the acidification of the diffusive boundary layer) on net photosynthesis was measured at both temperatures. Temperature affected photosynthesis (in terms of photosynthetic efficiency, light saturation point, and net photosynthesis) and growth at present pCO2, but these effects decreased with increasing pCO2. The relevance of the CCM decreased at 10 °C. A pCO2 effect on the CCM could only be shown if intCA and exCA were inhibited. The experiments demonstrate for the first time interactions between ocean acidification and temperature on the performance of a non-calcifying macroalga and show that the effects of low temperature on photosynthesis can be alleviated by increasing pCO2. The findings indicate that the carbon acquisition mediated by exCA and acidification of the diffusive boundary layer decrease at low temperatures but are not affected by the cultivation level of pCO2, whereas the activity of intCA is affected by pCO2. Ecologically, the findings suggest that ocean acidification might affect the biogeographical distribution of N. harveyi.

Regulation of cyclic electron flow in Chlamydomonas reinhardtii under fluctuating carbon availability

The chloroplast must rapidly and precisely adjust photosynthetic ATP and NADPH output to meet changing metabolic demands imposed by fluctuating environmental conditions. Cyclic electron flow (CEF) around photosystem I is thought to contribute to this adjustment by providing ATP in excess of that supplied by linear electron low, balancing chloroplast energy budget when relative demand for ATP is high. We assessed the kinetics and energy production of CEF activation in Chlamydomonas reinhardtii under rapid changes of organic and inorganic carbon availability. Comparisons of transient electric field and chlorophyll fluorescence measurements indicated CEF was activated under conditions where ATP demand is expected to be high, consistent with a role in balancing the cellular ATP/NADPH budget under fluctuating environmental or metabolic conditions. CEF activation was not correlated with antenna state transitions, both in wild-type and the state transition mutant stt7-9, suggesting that CEF is rapidly regulated by allosteric or redox modulators. Comparing the CEF under ambient and high CO2 conditions suggests an increase in required energy output of approximately 1ATP/CO2 fixed, nearly sufficient to power proposed mechanistic models for the carbon-concentrating mechanism. Additionally, we see three-fold higher CEF rates in cells under steady-state conditions than cells under similar conditions with inhibited photosystem II, and up to five times higher in cells with severe depletion of inorganic carbon, implying that CEF has larger energetic capacity than predicted from some previous work.

Acclimation to low light by C4 maize: implications for bundle sheath leakiness

C4 plants have a biochemical carbon concentrating mechanism (CCM) that increases CO2 concentration around ribulose bisphosphate carboxylase oxygenase (Rubisco) in the bundle sheath (BS). Under limiting light, the activity of the CCM generally decreases, causing an increase in leakiness, (Φ), the ratio of CO2 retrodiffusing from the BS relative to C4 carboxylation processes. Maize plants were grown under high and low light regimes (respectively HL, 600 versus LL, 100 μE m(-2) s(-1) ). Short-term acclimation of Φ was compared from isotopic discrimination (Δ), gas exchange and photochemistry. Direct measurement of respiration in the light, and ATP production rate (JATP ), allowed us use a novel approach to derive Φ, compared with the conventional fitting of measured and predicted Δ. HL grown plants responded to decreasing light intensities with the well-documented increase in Φ. Conversely, LL plants showed a constant Φ, which has not been observed previously. We explain the pattern by two contrasting acclimation strategies: HL plants maintained a high CCM activity at LL, resulting in high CO2 overcycling and increased Φ; LL plants acclimated by down-regulating the CCM, effectively optimizing scarce ATP supply. This surprising plasticity may limit the impact of Φ-dependent carbon losses in leaves becoming shaded within developing canopies.

Regulation of the carbon-concentrating mechanism in the cyanobacterium Synechocystis sp. PCC6803 in response to changing light intensity and inorganic carbon availability

Photosynthetic organisms possess regulatory mechanisms to balance the various inputs of photosynthesis in a manner that minimizes over-excitation of the light-driven electron transfer apparatus, while maximizing the reductive assimilation of inorganic nutrients, most importantly inorganic carbon (Ci). Accordingly, the regulatory interactions coordinating responses to fluctuating light and responses to Ci availability are of fundamental significance. The inducible high affinity carbon-concentrating mechanism (CCM) in the cyanobacterium Synechocystis sp. PCC6803 has been studied in order to understand how it is integrated with the light and dark reactions of photosynthesis. To probe genetic regulatory mechanisms, genomic DNA microarrays were used to survey for differences in the expression of genes in response to a shift to high light conditions under conditions of either high or low Ci availability. Discrepancies in published experiments exist regarding the extent to which genes for the CCM are upregulated in response to high light treatment. These discrepancies may be due to critical differences in Ci availability existing during the different high light experiments. The present microarray experiments reexamine this by comparing high light treatment under two different Ci regimes: bubbling with air and bubbling with air enriched with CO2. While some transcriptional responses such as the downregulation of antenna proteins are quite similar, pronounced differences exist with respect to the differential expression of CCM and affiliated genes. The results are discussed in the context of a recent analysis revealing that small molecules that are intermediates of the light and dark reaction photosynthetic metabolism act as allosteric effectors of the DNA-binding proteins which modulate the expression of the CCM genes.

A modified pH drift assay for inorganic carbon accumulation and external carbonic anhydrase activity in microalgae

Threat of global warming due to carbon dioxide (CO2) emissions has stimulated research into carbon sequestration and emissions reduction technologies. Alkaline scrubbing allows CO2 to be captured as bicarbonate, which can be photochemically fixed by microalgae. The carbon concentrating mechanism (CCM), of which external carbonic anhydrase is a key component, allows organisms to utilise this bicarbonate. In order to select a suitable strain for this application, a screening tool is required. The current method for determining carbonic anhydrase activity, the Wilbur and Anderson assay, was found to be unsuitable as a screening tool as the associated error was unacceptably large and tests on whole cells were inconclusive. This paper presents the development of a new, whole cell assay to measure inorganic carbon uptake and external carbonic anhydrase activity, based on classical pH drift experiments. Spirulina platensis was successfully used to develop a correlation between the specific carbon uptake (C) and the specific pH change (dpH). The relationship is described by the following: C[mmol C (g dry algae)−1 h−1] = 0.064 × (dpH). Inhibitor and salt dissociation tests validated the activity and presence of external carbonic anhydrase and allowed correlation between the Wilbur and Anderson assay and the new whole cell assay. Screening tests were conducted on S. platensis, Scenedesmus sp., Chlorella vulgaris and Dunaliella salina that were found to have carbon uptake rates of 5.76, 5.86, 3.86 and 2.15 mmol C (g dry algae)−1 h−1, respectively. These results corresponded to the species' known bicarbonate utilisation abilities and validated the use of the assay as a screening tool.

Colimitation of the unicellular photosynthetic diazotroph Crocosphaera watsonii by phosphorus, light, and carbon dioxide

We describe interactive effects of total phosphorus (total P = 0.1–4.0 µmol L−1; added as H2NaPO4), irradiance (40 and 150 µmol quanta m−2 s−1), and the partial pressure of carbon dioxide (; 19 and 81 Pa, i.e., 190 and 800 ppm) on growth and CO2‐ and dinitrogen (N2)‐fixation rates of the unicellular N2‐fixing cyanobacterium Crocosphaera watsonii (WH0003) isolated from the Pacific Ocean near Hawaii. In semicontinuous cultures of C. watsonii, elevated positively affected growth and CO2‐ and N2‐fixation rates under high light. Under low light, elevated positively affected growth rates at all concentrations of P, but CO2‐ and N2‐fixation rates were affected by elevated only when P was low. In both high‐light and low‐light cultures, the total P requirements for growth and CO2‐ and N2‐fixation declined as increased. The minimum concentration (Cmin) of total P and half‐saturation constant (Kʽ) for growth and CO2‐ and N2‐fixation rates with respect to total P were reduced by 0.05 µmol L−1 as a function of elevated . We speculate that low P requirements under high resulted from a lower energy demand associated with carbon‐concentrating mechanisms in comparison with low‐ cultures. There was also a 0.10 µmol L−1 increase in Cmin and Kʽ for growth and N2 fixation with respect to total P as a function of increasing light regardless of concentration. We speculate that cellular P concentrations are responsible for this shift through biodilution of cellular P and possibly cellular P uptake systems as a function of increasing light. Changing concentrations of P, CO2, and light have both positive and negative interactive effects on growth and CO2‐, and N2‐fixation rates of unicellular oxygenic diazotrophs like C. watsonii.

Inorganic C utilization and C isotope fractionation by pelagic and sea ice algal assemblages along the Antarctic continental shelf

Physiological characteristics of inorganic C uptake were examined in Southern Oceanice algae and phytoplankton assemblages. Ice algal and phytoplankton assemblages were largelydominated by diatoms and Phaeocystis antarctica, and showed a high capacity for HCO3−utilization, with direct HCO3− transport accounting for ~60% of total inorganic C uptake. Extracellularcarbonic anhydrase (eCA) was detectable in all samples, but with significantly lower activityin sea ice algae. Neither HCO3− transport nor eCA activity was related to the in situ partial pressureof CO2 (pCO2) or taxonomic composition of samples. The half-saturation constant (KS) for inorganicC ranged from ~100 to 5000 μM, and showed significantly more variability among sea ice algaethan phytoplankton assemblages. For the phytoplankton assemblages, there were significant positivecorrelations between in situ pCO2 and KS (higher C substrate affinity in low pCO2 waters), andalso between KS and maximum C uptake rates (Vmax). In contrast, KS and Vmax in sea-ice algal assemblageswere not correlated to each other, or to any other measured variables. The C isotopecomposition of particulate organic carbon (δ13C-POC) in the phytoplankton assemblages showedmodest variability (range −30 to −24.6‰) and was significantly correlated to the ratio of inferredgrowth rates (derived from Vmax) and in situ CO2 concentrations, but not to any measured C uptakeparameters. δ13C-POC in sea ice algal samples (range −25.7 to −12.9‰) was significantly heavierthan in the phytoplankton assemblages, and not correlated to any other variables. Our results provideevidence for the widespread occurrence of carbon-concentrating mechanisms in SouthernOcean sea ice algae and phytoplankton assemblages.

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.

Sensitivity of Antarctic phytoplankton species to ocean acidification: Growth, carbon acquisition, and species interaction

Despite the fact that ocean acidification is considered to be especially pronounced in the Southern Ocean, little is known about CO2‐dependent physiological processes and the interactions of Antarctic phytoplankton key species. We therefore studied the effects of CO2 partial pressure (PCO2) (16.2, 39.5, and 101.3 Pa) on growth and photosynthetic carbon acquisition in the bloom‐forming species Chaetoceros debilis, Pseudo‐nitzschia subcurvata, Fragilariopsis kerguelensis, and Phaeocystis antarctica. Using membrane‐inlet mass spectrometry, photosynthetic O2 evolution and inorganic carbon (Ci) fluxes were determined as a function of CO2 concentration. Only the growth of C. debilis was enhanced under high PCO2. Analysis of the carbon concentrating mechanism (CCM) revealed the operation of very efficient CCMs (i.e., high Ci affinities) in all species, but there were species‐specific differences in CO2‐dependent regulation of individual CCM components (i.e., CO2 and uptake kinetics, carbonic anhydrase activities). Gross CO2 uptake rates appear to increase with the cell surface area to volume ratios. Species competition experiments with C. debilis and P. subcurvata under different PCO2 levels confirmed the CO2‐stimulated growth of C. debilis observed in monospecific incubations, also in the presence of P. subcurvata. Independent of PCO2, high initial cell abundances of P. subcurvata led to reduced growth rates of C. debilis. For a better understanding of future changes in phytoplankton communities, CO2‐sensitive physiological processes need to be identified, but also species interactions must be taken into account because their interplay determines the success of a species.

Zinc Deficiency Impacts CO2 Assimilation and Disrupts Copper Homeostasis in Chlamydomonas reinhardtii

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.

Dissecting the impact of CO2 and pH on the mechanisms of photosynthesis and calcification in the coccolithophore Emiliania huxleyi

Coccolithophores are important calcifying phytoplankton predicted to be impacted by changes in ocean carbonate chemistry caused by the absorption of anthropogenic CO2 . However, it is difficult to disentangle the effects of the simultaneously changing carbonate system parameters (CO2 , bicarbonate, carbonate and protons) on the physiological responses to elevated CO2 . Here, we adopted a multifactorial approach at constant pH or CO2 whilst varying dissolved inorganic carbon (DIC) to determine physiological and transcriptional responses to individual carbonate system parameters. We show that Emiliania huxleyi is sensitive to low CO2 (growth and photosynthesis) and low bicarbonate (calcification) as well as low pH beyond a limited tolerance range, but is much less sensitive to elevated CO2 and bicarbonate. Multiple up-regulated genes at low DIC bear the hallmarks of a carbon-concentrating mechanism (CCM) that is responsive to CO2 and bicarbonate but not to pH. Emilianiahuxleyi appears to have evolved mechanisms to respond to limiting rather than elevated CO2 . Calcification does not function as a CCM, but is inhibited at low DIC to allow the redistribution of DIC from calcification to photosynthesis. The presented data provides a significant step in understanding how E.huxleyi will respond to changing carbonate chemistry at a cellular level.

Breakdown of the coral-algae symbiosis: towards formalising a linkage between warm-water bleaching thresholds and the growth rate of the intracellular zooxanthellae

Abstract. Impairment of the photosynthetic machinery of the algal endosymbiont ("zooxanthellae") is the proximal driver of the thermal breakdown of the coral-algae symbiosis ("coral bleaching"). Yet, the initial site of damage, and early dynamics of the impairment are still not well resolved. In this perspective essay, I consider further a recent hypothesis which proposes an energetic disruption to the carbon-concentrating mechanisms (CCMs) of the coral host, and the resultant onset of CO2-limitation within the photosynthetic "dark reactions" as a unifying cellular mechanism. The hypothesis identifies the enhanced retention of photosynthetic carbon for zooxanthellae (re)growth following an initial irradiance-driven expulsion event as a strong contributing cause of the energetic disruption. If true, then it implies that the onset of the bleaching syndrome and setting of upper thermal bleaching limits are emergent attributes of the coral symbiosis that are ultimately underpinned by the characteristic growth profile of the intracellular zooxanthellae; which is known to depend not just on temperature, but also external (seawater) nutrient availability and zooxanthellae genotype. Here, I review this proposed bleaching linkage at a variety of observational scales, and find it to be parsimonious with the available evidence. Future experiments are suggested that can more formally test the linkage. If correct, the new cellular model delivers a valuable new perspective to consider the future prospects of the coral symbiosis in an era of rapid environmental change, including: (i) the underpinning mechanics (and biological significance) of observed changes in resident zooxanthellae genotypes, and (ii) the now crucial importance of reef water quality in co-determining thermal bleaching resistance.