Carbon dioxide concentrating mechanism in Chlamydomonas reinhardtii: inorganic carbon transport and CO2 recapture

Many microalgae are capable of acclimating to CO(2) limited environments by operating a CO(2) concentrating mechanism (CCM), which is driven by various energy-coupled inorganic carbon (Ci; CO(2) and HCO(3)(-)) uptake systems. Chlamydomonas reinhardtii (hereafter, Chlamydomonas), a versatile genetic model organism, has been used for several decades to exemplify the active Ci transport in eukaryotic algae, but only recently have many molecular details behind these Ci uptake systems emerged. Recent advances in genetic and molecular approaches, combined with the genome sequencing of Chlamydomonas and several other eukaryotic algae have unraveled some unique characteristics associated with the Ci uptake mechanism and the Ci-recapture system in eukaryotic microalgae. Several good candidate genes for Ci transporters in Chlamydomonas have been identified, and a few specific gene products have been linked with the Ci uptake systems associated with the different acclimation states. This review will focus on the latest studies on characterization of functional components involved in the Ci uptake and the Ci-recapture in Chlamydomonas.     

CO2 concentrating mechanisms in cyanobacteria: molecular components,their diversity and evolution

Cyanobacteria have evolved an extremely effective single-cell CO(2) concentrating mechanism (CCM). Recent molecular, biochemical and physiological studies have significantly extended current knowledge about the genes and protein components of this system and how they operate to elevate CO(2) around Rubisco during photosynthesis. The CCM components include at least four modes of active inorganic carbon uptake, including two bicarbonate transporters and two CO(2) uptake systems associated with the operation of specialized NDH-1 complexes. All these uptake systems serve to accumulate HCO(3)(-) in the cytosol of the cell, which is subsequently used by the Rubisco-containing carboxysome protein micro-compartment within the cell to elevate CO(2) around Rubisco. A specialized carbonic anhydrase is also generally present in this compartment. The recent availability of at least nine cyanobacterial genomes has made it possible to begin to undertake comparative genomics of the CCM in cyanobacteria. Analyses have revealed a number of surprising findings. Firstly, cyanobacteria have evolved two types of carboxysomes, correlated with the form of Rubisco present (Form 1A and 1B). Secondly, the two HCO(3)(-) and CO(2) transport systems are distributed variably, with some cyanobacteria (Prochlorococcus marinus species) appearing to lack CO(2) uptake systems entirely. Finally, there are multiple carbonic anhydrases in many cyanobacteria, but, surprisingly, several cyanobacterial genomes appear to lack any identifiable CA genes. A pathway for the evolution of CCM components is suggested.     

Sensing of inorganic carbon limitation in Synechococcus PCC7942 is correlated with the size of the internal inorganic carbon pool and involves oxygen

Freshwater cyanobacteria are subjected to large seasonal fluctuations in the availability of nutrients, including inorganic carbon (Ci). We are interested in the regulation of the CO2-concentrating mechanism (CCM) in the model freshwater cyanobacterium Synechococcus sp. strain PCC7942 in response to Ci limitation; however, the nature of Ci sensing is poorly understood. We monitored the expression of high-affinity Ci-transporter genes and the corresponding induction of a high-affinity CCM in Ci-limited wild-type cells and a number of CCM mutants. These genotypes were subjected to a variety of physiological and pharmacological treatments to assess whether Ci sensing might involve monitoring of fluctuations in the size of the internal Ci pool or, alternatively, the activity of the photorespiratory pathway. These modes of Ci sensing are congruent with previous results. We found that induction of a high-affinity CCM correlates most closely with a depletion of the internal Ci pool, but that full induction of this mechanism also requires some unresolved oxygen-dependent process.     

The evolution of inorganic carbon concentrating mechanisms in photosynthesis

Inorganic carbon concentrating mechanisms (CCMs) catalyse the accumulation of CO(2) around rubisco in all cyanobacteria, most algae and aquatic plants and in C(4) and crassulacean acid metabolism (CAM) vascular plants. CCMs are polyphyletic (more than one evolutionary origin) and involve active transport of HCO(3)(-), CO(2) and/or H(+), or an energized biochemical mechanism as in C(4) and CAM plants. While the CCM in almost all C(4) plants and many CAM plants is constitutive, many CCMs show acclimatory responses to variations in the supply of not only CO(2) but also photosynthetically active radiation, nitrogen, phosphorus and iron. The evolution of CCMs is generally considered in the context of decreased CO(2) availability, with only a secondary role for increasing O(2). However, the earliest CCMs may have evolved in oxygenic cyanobacteria before the atmosphere became oxygenated in stromatolites with diffusion barriers around the cells related to UV screening. This would decrease CO(2) availability to cells and increase the O(2) concentration within them, inhibiting rubisco and generating reactive oxygen species, including O(3).     

Inorganic carbon concentrating mechanisms in free-living and symbiotic dinoflagellates and chromerids

Photosynthetic dinoflagellates are ecologically and biogeochemically important in marine and freshwater environments. However, surprisingly little is known of how this group acquires inorganic carbon or how these diverse processes evolved. Consequently, how CO₂ availability ultimately influences the success of dinoflagellates over space and time remains poorly resolved compared to other microalgal groups. Here we review the evidence. Photosynthetic core dinoflagellates have a Form II RuBisCO (replaced by Form IB or Form ID in derived dinoflagellates). The in vitro kinetics of the Form II RuBisCO from dinoflagellates are largely unknown, but dinoflagellates with Form II (and other) RuBisCOs have inorganic carbon concentrating mechanisms (CCMs), as indicated by in vivo internal inorganic C accumulation and affinity for external inorganic C. However, the location of the membrane(s) at which the essential active transport component(s) of the CCM occur(s) is (are) unresolved; isolation and characterization of functionally competent chloroplasts would help in this respect. Endosymbiotic Symbiodiniaceae (in Foraminifera, Acantharia, Radiolaria, Ciliata, Porifera, Acoela, Cnidaria, and Mollusca) obtain inorganic C by transport from seawater through host tissue. In corals this transport apparently provides an inorganic C concentration around the photobiont that obviates the need for photobiont CCM. This is not the case for tridacnid bivalves, medusae, or, possibly, Foraminifera. Overcoming these long-standing knowledge gaps relies on technical advances (e.g., the in vitro kinetics of Form II RuBisCO) that can functionally track the fate of inorganic C forms.     

Algal and aquatic plant carbon concentrating mechanisms in relation to environmental change

Carbon dioxide concentrating mechanisms (also known as inorganic carbon concentrating mechanisms; both abbreviated as CCMs) presumably evolved under conditions of low CO(2) availability. However, the timing of their origin is unclear since there are no sound estimates from molecular clocks, and even if there were, there are no proxies for the functioning of CCMs. Accordingly, we cannot use previous episodes of high CO(2) (e.g. the Palaeocene-Eocene Thermal Maximum) to indicate how organisms with CCMs responded. Present and predicted environmental change in terms of increased CO(2) and temperature are leading to increased CO(2) and HCO(3)(-) and decreased CO(3)(2-) and pH in surface seawater, as well as decreasing the depth of the upper mixed layer and increasing the degree of isolation of this layer with respect to nutrient flux from deeper waters. The outcome of these forcing factors is to increase the availability of inorganic carbon, photosynthetic active radiation (PAR) and ultraviolet B radiation (UVB) to aquatic photolithotrophs and to decrease the supply of the nutrients (combined) nitrogen and phosphorus and of any non-aeolian iron. The influence of these variations on CCM expression has been examined to varying degrees as acclimation by extant organisms. Increased PAR increases CCM expression in terms of CO(2) affinity, whilst increased UVB has a range of effects in the organisms examined; little relevant information is available on increased temperature. Decreased combined nitrogen supply generally increases CO(2) affinity, decreased iron availability increases CO(2) affinity, and decreased phosphorus supply has varying effects on the organisms examined. There are few data sets showing interactions amongst the observed changes, and even less information on genetic (adaptation) changes in response to the forcing factors. In freshwaters, changes in phytoplankton species composition may alter with environmental change with consequences for frequency of species with or without CCMs. The information available permits less predictive power as to the effect of the forcing factors on CCM expression than for their overall effects on growth. CCMs are currently not part of models as to how global environmental change has altered, and is likely to further alter, algal and aquatic plant primary productivity.     

Role of carboxysomes in cyanobacterial CO2 assimilation: CO2 concentrating mechanisms and metabolon implications

Many carbon-fixing organisms have evolved CO₂ concentrating mechanisms (CCMs) to enhance the delivery of CO₂ to RuBisCO, while minimizing reactions with the competitive inhibitor, molecular O₂. These distinct types of CCMs have been extensively studied using genetics, biochemistry, cell imaging, mass spectrometry, and metabolic flux analysis. Highlighted in this paper, the cyanobacterial CCM features a bacterial microcompartment (BMC) called ‘carboxysome’ in which RuBisCO is co-encapsulated with the enzyme carbonic anhydrase (CA) within a semi-permeable protein shell. The cyanobacterial CCM is capable of increasing CO₂ around RuBisCO, leading to one of the most efficient processes known for fixing ambient CO₂. The carboxysome life cycle is dynamic and creates a unique subcellular environment that promotes activity of the Calvin–Benson (CB) cycle. The carboxysome may function within a larger cellular metabolon, physical association of functionally coupled proteins, to enhance metabolite channelling and carbon flux. In light of CCMs, synthetic biology approaches have been used to improve enzyme complex for CO₂ fixations. Research on CCM-associated metabolons has also inspired biologists to engineer multi-step pathways by providing anchoring points for enzyme cascades to channel intermediate metabolites towards valuable products.     

Inorganic carbon acquisition by eukaryotic algae: four current questions

The phylogenetically and morphologically diverse eukaryotic algae are typically oxygenic photolithotrophs. They have a diversity of incompletely understood mechanisms of inorganic carbon acquisition: this article reviews four areas where investigations continue. The first topic is diffusive CO₂ entry. Most eukaryotic algae, like all cyanobacteria, have inorganic carbon concentrating mechanisms (CCMs). The ancestral condition was presumably the absence of a CCM, i.e. diffusive CO₂ entry, as found in a small minority of eukaryotic algae today; however, it is likely that, as is found in several cases, this condition is due to a loss of a CCM. There are a number of algae which are in various respects intermediate between diffusive CO₂ entry and occurrence of a CCM: further study is needed on this aspect. A second topic is the nature of cyanelles and their role in inorganic carbon assimilation. The cyanelles (plastids) of the euglyphid amoeba Paulinella have been acquired relatively recently by endosymbiosis with genetic integration of an α-cyanobacterium with a Form 1A Rubisco. The α-carboxysomes in the cyanelles are presumably involved in a CCM, but further investigation is needed.Also called cyanelles are the plastids of glaucocystophycean algae, but is it now clear that these were derived from the β-cyanobacterial ancestor of all plastids other than that of Paulinella. The resemblances of the central body of the cyanelles of glaucocystophycean algae to carboxysomes may not reflect derivation from cyanobacterial β-carboxysomes; although it is clear that these algae have CCMs but these are now well characterized. The other two topics concern CCMs in other eukaryotic algae; these CCMs arose polyphyletically and independently of the cyanobacterial CCMs. It is generally believed that eukaryotic algal, like cyanobacterial, CCMs are based on active transport of an inorganic carbon species and/or protons, and they have C₃ biochemistry. This is the case for the organism considered as the third topic, i.e. Chlamydomonas reinhardtii, the eukaryotic alga with the best understood CCM. This CCM involves HCO₃ ^? conversion to CO₂ in the thylakoid lumen so the external inorganic carbon must cross four membranes in series with a final CO₂ effux from the thylakoid. More remains to be investigated about this CCM. The final topic is that of the occurrence of C₄-like metabolism in the CCMs of marine diatoms. Different conclusions have been reached depending on the organism investigated and the techniques used, and several aspects require further study.     

Algal evolution in relation to atmospheric CO2: carboxylases, carbon-concentrating mechanisms and carbon oxidation cycles

Oxygenic photosynthesis evolved at least 2.4 Ga; all oxygenic organisms use the ribulose bisphosphate carboxylase-oxygenase (Rubisco)-photosynthetic carbon reduction cycle (PCRC) rather than one of the five other known pathways of autotrophic CO(2) assimilation. The high CO(2) and (initially) O(2)-free conditions permitted the use of a Rubisco with a high maximum specific reaction rate. As CO(2) decreased and O(2) increased, Rubisco oxygenase activity increased and 2-phosphoglycolate was produced, with the evolution of pathways recycling this inhibitory product to sugar phosphates. Changed atmospheric composition also selected for Rubiscos with higher CO(2) affinity and CO(2)/O(2) selectivity correlated with decreased CO(2)-saturated catalytic capacity and/or for CO(2)-concentrating mechanisms (CCMs). These changes increase the energy, nitrogen, phosphorus, iron, zinc and manganese cost of producing and operating Rubisco-PCRC, while biosphere oxygenation decreased the availability of nitrogen, phosphorus and iron. The majority of algae today have CCMs; the timing of their origins is unclear. If CCMs evolved in a low-CO(2) episode followed by one or more lengthy high-CO(2) episodes, CCM retention could involve a combination of environmental factors known to favour CCM retention in extant organisms that also occur in a warmer high-CO(2) ocean. More investigations, including studies of genetic adaptation, are needed.     

Energy crosstalk between photosynthesis and the algal CO2-concentrating mechanisms

Microalgal photosynthesis is responsible for nearly half of the CO₂ annually captured by Earth’s ecosystems. In aquatic environments where the CO₂ availability is low, the CO₂-fixing efficiency of microalgae greatly relies on mechanisms – called CO_2-concentrating mechanisms (CCMs) – for concentrating CO₂ at the catalytic site of the CO₂-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). While the transport of inorganic carbon (C_i) across membrane bilayers against a concentration gradient consumes part of the chemical energy generated by photosynthesis, the bioenergetics and cellular mechanisms involved are only beginning to be elucidated. Here, we review the current knowledge relating to the energy requirement of CCMs in the light of recent advances in photosynthesis regulatory mechanisms and the spatial organization of CCM components.     

Introducing an algal carbon‐concentrating mechanism into higher plants: location and incorporation of key components

Many eukaryotic green algae possess biophysical carbon‐concentrating mechanisms (CCMs) that enhance photosynthetic efficiency and thus permit high growth rates at low CO₂ concentrations. They are thus an attractive option for improving productivity in higher plants. In this study, the intracellular locations of ten CCM components in the unicellular green alga Chlamydomonas reinhardtii were confirmed. When expressed in tobacco, all of these components except chloroplastic carbonic anhydrases CAH3 and CAH6 had the same intracellular locations as in Chlamydomonas. CAH6 could be directed to the chloroplast by fusion to an Arabidopsis chloroplast transit peptide. Similarly, the putative inorganic carbon (Ci) transporter LCI1 was directed to the chloroplast from its native location on the plasma membrane. CCP1 and CCP2 proteins, putative Ci transporters previously reported to be in the chloroplast envelope, localized to mitochondria in both Chlamydomonas and tobacco, suggesting that the algal CCM model requires expansion to include a role for mitochondria. For the Ci transporters LCIA and HLA3, membrane location and Ci transport capacity were confirmed by heterologous expression and H¹⁴CO₃^‐ uptake assays in Xenopus oocytes. Both were expressed in Arabidopsis resulting in growth comparable with that of wild‐type plants. We conclude that CCM components from Chlamydomonas can be expressed both transiently (in tobacco) and stably (in Arabidopsis) and retargeted to appropriate locations in higher plant cells. As expression of individual Ci transporters did not enhance Arabidopsis growth, stacking of further CCM components will probably be required to achieve a significant increase in photosynthetic efficiency in this species.     

Expression analysis of genes associated with the induction of the carbon-concentrating mechanism in Chlamydomonas reinhardtii

Acclimation to varying CO2 concentrations and light intensities is associated with the monitoring of environmental changes by controlling genetic and physiological responses through CO2 and light signal transduction. While CO2 and light signals are indispensable for photosynthesis, and these environmental factors have been proposed as strongly associated with each other, studies linking these components are largely limited to work on higher plants. In this study, we examined the physiological characteristics of a green alga, Chlamydomonas reinhardtii, exposed to various light intensities or CO2 concentrations. Acclimation to CO2-limiting conditions by Chlamydomonas requires the induction of a carbon-concentrating mechanism (CCM) to allow the uptake of inorganic carbon (Ci) and increase the affinity for Ci. We revealed that the induction of the CCM is not solely dependent on absolute environmental Ci concentrations but is also affected by light intensity. Using a cDNA array containing 10,368 expressed sequence tags, we also obtained global expression profiles related to the physiological responses. The induction of several CCM-associated genes was strongly affected by high light as well as CO2 concentrations. We identified novel candidates for Ci transporters and CO2-responsive regulatory factors whose expression levels were significantly increased during the induction of the CCM.     

The Chlamydomonas reinhardtii cia3 mutant lacking a thylakoid lumen-localized carbonic anhydrase is limited by CO2 supply to rubisco and not photosystem II function in vivo

The Chlamydomonas reinhardtii cia3 mutant has a phenotype indicating that it requires high-CO(2) levels for effective photosynthesis and growth. It was initially proposed that this mutant was defective in a carbonic anhydrase (CA) that was a key component of the photosynthetic CO(2)-concentrating mechanism (CCM). However, more recent identification of the genetic lesion as a defect in a lumenal CA associated with photosystem II (PSII) has raised questions about the role of this CA in either the CCM or PSII function. To resolve the role of this lumenal CA, we re-examined the physiology of the cia3 mutant. We confirmed and extended previous gas exchange analyses by using membrane-inlet mass spectrometry to monitor(16)O(2),(18)O(2), and CO(2) fluxes in vivo. The results demonstrate that PSII electron transport is not limited in the cia3 mutant at low inorganic carbon (Ci). We also measured metabolite pools sizes and showed that the RuBP pool does not fall to abnormally low levels at low Ci as might be expected by a photosynthetic electron transport or ATP generation limitation. Overall, the results demonstrate that under low Ci conditions, the mutant lacks the ability to supply Rubisco with adequate CO(2) for effective CO(2) fixation and is not limited directly by any aspect of PSII function. We conclude that the thylakoid CA is primarily required for the proper functioning of the CCM at low Ci by providing an ample supply of CO(2) for Rubisco.     

The molecular and biochemical analyses of CO2-concentrating mechanisms in cyanobacteria and microalgae

Abstract. The supply of dissolved inorganic carbon for photosynthesis in the aquatic environment is much more variable than that experienced by terrestrial plants. In response to this variability, cyanobacteria and many species of microalgae acclimate to rapid changes in the availability of dissolved inorganic carbon by the induction of high affinity/high capacity CO₂-concentrating systems (CCMs). Biochemical and molecular analyses of the acclimation response have recently identified several components that are required for efficient operation of the CCMs and CO₂ assimilation. This has been accomplished using in vivo labelling studies, and characterization of high inorganic carbon (C_i) requiring cyanobacterial and algal mutants. The identification and regulation of expression of polypeptides synthesized in response to limiting C_i concentrations, and the proposed role of the carboxysome and the pyrenoid in the functioning of the CCMs are examined.     

The environmental plasticity and ecological genomics of the cyanobacterial CO2 concentrating mechanism

Cyanobacteria probably exhibit the widest range of diversity in growth habitats of all photosynthetic organisms. They are found in cold and hot, alkaline and acidic, marine, freshwater, saline, terrestrial, and symbiotic environments. In addition to this, they originated on earth at least 2.5 billion years ago and have evolved through periods of dramatic O2 increases, CO2 declines, and temperature changes. One of the key problems they have faced through evolution and in their current environments is the capture of CO2 and its efficient use by Rubisco in photosynthesis. A central response to this challenge has been the development of a CO2 concentrating mechanism (CCM) that can be adapted to various environmental limitations. There are two primary functional elements of this CCM. Firstly, the containment of Rubisco in carboxysome protein microbodies within the cell (the sites of CO2) elevation), and, secondly, the presence of several inorganic carbon (Ci) transporters that deliver HCO3- intracellularly. Cyanobacteria show both species adaptation and acclimation of this mechanism. Between species, there are differences in the suites of Ci transporters in each genome, the nature of the carboxysome structures and the functional roles of carbonic anhydrases. Within a species, different CCM activities can be induced depending on the Ci availability in the environment. This acclimation is largely based on the induction of multiple Ci transporters with different affinities and specificities for either CO2 or HCO3- as substrates. These features are discussed in relation to our current knowledge of the genomic sequences of a diverse array of cyanobacteria and their ecological environments.     

Insights into the evolution of CCMs from comparisons with other resource acquisition and assimilation processes

Regarding inorganic carbon as 'just another' chemical resource used in the growth of aquatic photolithotrophs, we ask three questions and then attempt to answer them. (1) How common are catalysed chemical changes of the resource outside the cell, and accumulation of the resource inside the cell prior to assimilation, for the diverse chemical resources used? (2) Do acquisition and assimilation meet evolutionary optimality criteria with respect to the use of other resources? (3) Are there clues to the evolutionary origin of inorganic carbon concentrating mechanism (CCMs) in the mechanisms of acquisition of other resources and vice versa? Evidence considered includes molecular genetic similarities between CCM components and components of other resource acquisition mechanisms, and palaeogeochemical evidence on the timing of restrictions on the availability of the resources such that extracellular transformation of materials, and their accumulation within cells prior to assimilation, are needed. Provisional answers to the questions are as follows: (1) Many common chemical resources other than inorganic carbon are subject to extracellular chemical conversion and/or accumulation prior to assimilation, e.g. ammonium, nitrate, urea, amino acids, organic and inorganic phosphate and iron; (2) There is some evidence for optimality of CCMs and of less complex resource acquisition processes, exemplified by NH(4)(+) entry and assimilation, though many more data are needed and (3) There are molecular genetic similarities between CCM components and transporters for other solutes and components of respiratory NADH dehydrogenases that are consistent with their use in CCMs representing a derived evolutionary state. Palaeogeochemical evidence suggests that CCMs evolved later than did at least some of the extracellular chemical transformation and/or accumulation mechanisms for other resources.     

On-line stable isotope gas exchange reveals an inducible but leaky carbon concentrating mechanism in Nannochloropsis salina

Carbon concentrating mechanisms (CCMs) are common among microalgae, but their regulation and even existence in some of the most promising biofuel production strains is poorly understood. This is partly because screening for new strains does not commonly include assessment of CCM function or regulation despite its fundamental role in primary carbon metabolism. In addition, the inducible nature of many microalgal CCMs means that environmental conditions should be considered when assessing CCM function and its potential impact on biofuels. In this study, we address the effect of environmental conditions by combining novel, high frequency, on-line ¹³CO₂ gas exchange screen with microscope-based lipid characterization to assess CCM function in Nannochloropsis salina and its interaction with lipid production. Regulation of CCM function was explored by changing the concentration of CO₂ provided to continuous cultures in airlift bioreactors where cell density was kept constant across conditions by controlling the rate of media supply. Our isotopic gas exchange results were consistent with N. salina having an inducible “pump-leak” style CCM similar to that of Nannochloropsis gaditana. Though cells grew faster at high CO₂ and had higher rates of net CO₂ uptake, we did not observe significant differences in lipid content between conditions. Since the rate of CO₂ supply was much higher for the high CO₂ conditions, we calculated that growing cells bubbled with low CO₂ is about 40 % more efficient for carbon capture than bubbling with high CO₂. We attribute this higher efficiency to the activity of a CCM under low CO₂ conditions.