Carbon-concentrating Mechanism Activity Research Articles

The redox-sensitive R-loop of the carbon control protein SbtB contributes to the regulation of the cyanobacterial CCM

SbtB is a PII-like protein that regulates the carbon-concentrating mechanism (CCM) in cyanobacteria. SbtB proteins can bind many adenyl nucleotides and possess a characteristic C-terminal redox sensitive loop (R-loop) that forms a disulfide bridge in response to the diurnal state of the cell. SbtBs also possess an ATPase/ADPase activity that is modulated by the redox-state of the R-loop. To investigate the R-loop in the cyanobacterium Synechocystis sp. PCC 6803, site-specific mutants, unable to form the hairpin and permanently in the reduced state, and a R-loop truncation mutant, were characterized under different inorganic carbon (Ci) and light regimes. Growth under diurnal rhythm showed a role of the R-loop as sensor for acclimation to changing light conditions. The redox-state of the R-loop was found to impact the binding of the adenyl-nucleotides to SbtB, its membrane association and thereby the CCM regulation, while these phenotypes disappeared after truncation of the R-loop. Collectively, our data imply that the redox-sensitive R-loop provides an additional regulatory layer to SbtB, linking the CO2-related signaling activity of SbtB with the redox state of cells, mainly reporting the actual light conditions. This regulation not only coordinates CCM activity in the diurnal rhythm but also affects the primary carbon metabolism.

Temperature sensitivity of carbon concentrating mechanisms in the diatom Phaeodactylum tricornutum

Marine diatoms are key primary producers across diverse habitats in the global ocean. Diatoms rely on a biophysical carbon concentrating mechanism (CCM) to supply high concentrations of CO2 around their carboxylating enzyme, RuBisCO. The necessity and energetic cost of the CCM are likely to be highly sensitive to temperature, as temperature impacts CO2 concentration, diffusivity, and the kinetics of CCM components. Here, we used membrane inlet mass spectrometry (MIMS) and modeling to capture temperature regulation of the CCM in the diatom Phaeodactylum tricornutum (Pt). We found that enhanced carbon fixation rates by Pt at elevated temperatures were accompanied by increased CCM activity capable of maintaining RuBisCO close to CO2 saturation but that the mechanism varied. At 10 and 18 °C, diffusion of CO2 into the cell, driven by Pt’s ‘chloroplast pump’ was the major inorganic carbon source. However, at 18 °C, upregulation of the chloroplast pump enhanced (while retaining the proportion of) both diffusive CO2 and active HCO3− uptake into the cytosol, and significantly increased chloroplast HCO3− concentrations. In contrast, at 25 °C, compared to 18 °C, the chloroplast pump had only a slight increase in activity. While diffusive uptake of CO2 into the cell remained constant, active HCO3− uptake across the cell membrane increased resulting in Pt depending equally on both CO2 and HCO3− as inorganic carbon sources. Despite changes in the CCM, the overall rate of active carbon transport remained double that of carbon fixation across all temperatures tested. The implication of the energetic cost of the Pt CCM in response to increasing temperatures was discussed.

Rubisco and carbon-concentrating mechanism co-evolution across chlorophyte and streptophyte green algae.

Green algae expressing a carbon-concentrating mechanism (CCM) are usually associated with a Rubisco-containing micro-compartment, the pyrenoid. A link between the small subunit (SSU) of Rubisco and pyrenoid formation in Chlamydomonas reinhardtii has previously suggested that specific RbcS residues could explain pyrenoid occurrence in green algae. A phylogeny of RbcS was used to compare the protein sequence and CCM distribution across the green algae and positive selection in RbcS was estimated. For six streptophyte algae, Rubisco catalytic properties, affinity for CO2 uptake (K0.5 ), carbon isotope discrimination (δ13 C) and pyrenoid morphology were compared. The length of the βA-βB loop in RbcS provided a phylogenetic marker discriminating chlorophyte from streptophyte green algae. Rubisco kinetic properties in streptophyte algae have responded to the extent of inducible CCM activity, as indicated by changes in inorganic carbon uptake affinity, δ13 C and pyrenoid ultrastructure between high and low CO2 conditions for growth. We conclude that the Rubisco catalytic properties found in streptophyte algae have coevolved and reflect the strength of any CCM or degree of pyrenoid leakiness, and limitations to inorganic carbon in the aquatic habitat, whereas Rubisco in extant land plants reflects more recent selective pressures associated with improved diffusive supply of the terrestrial environment.

Pyrenoid loss in Chlamydomonas reinhardtii causes limitations in CO2 supply, but not thylakoid operating efficiency.

The pyrenoid of the unicellular green alga Chlamydomonas reinhardtii is a microcompartment situated in the centre of the cup-shaped chloroplast, containing up to 90% of cellular Rubisco. Traversed by a network of dense, knotted thylakoid tubules, the pyrenoid has been proposed to influence thylakoid biogenesis and ultrastructure. Mutants that are unable to assemble a pyrenoid matrix, due to expressing a vascular plant version of the Rubisco small subunit, exhibit severe growth and photosynthetic defects and have an ineffective carbon-concentrating mechanism (CCM). The present study set out to determine the cause of photosynthetic limitation in these pyrenoid-less lines. We tested whether electron transport and light use were compromised as a direct structural consequence of pyrenoid loss or as a metabolic effect downstream of lower CCM activity and resulting CO2 limitation. Thylakoid organization was unchanged in the mutants, including the retention of intrapyrenoid-type thylakoid tubules, and photosynthetic limitations associated with the absence of the pyrenoid were rescued by exposing cells to elevated CO2 levels. These results demonstrate that Rubisco aggregation in the pyrenoid functions as an essential element for CO2 delivery as part of the CCM, and does not play other roles in maintenance of photosynthetic membrane energetics.

Dynamics of carbon-concentrating mechanism induction and protein relocalization during the dark-to-light transition in synchronized Chlamydomonas reinhardtii.

In the model green alga Chlamydomonas reinhardtii, a carbon-concentrating mechanism (CCM) is induced under low CO2 in the light and comprises active inorganic carbon transport components, carbonic anhydrases, and aggregation of Rubisco in the chloroplast pyrenoid. Previous studies have focused predominantly on asynchronous cultures of cells grown under low versus high CO2. Here, we have investigated the dynamics of CCM activation in synchronized cells grown in dark/light cycles compared with induction under low CO2. The specific focus was to undertake detailed time course experiments comparing physiology and gene expression during the dark-to-light transition. First, the CCM could be fully induced 1 h before dawn, as measured by the photosynthetic affinity for inorganic carbon. This occurred in advance of maximum gene transcription and protein accumulation and contrasted with the coordinated induction observed under low CO2. Between 2 and 1 h before dawn, the proportion of Rubisco and the thylakoid lumen carbonic anhydrase in the pyrenoid rose substantially, coincident with increased CCM activity. Thus, other mechanisms are likely to activate the CCM before dawn, independent of gene transcription of known CCM components. Furthermore, this study highlights the value of using synchronized cells during the dark-to-light transition as an alternative means of investigating CCM induction.

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.

Effect of Temperature on Inorganic Carbon Acquisition of Chlamydomonas reinhardtii

ABSTRACT Carbon dioxide availability for microalgae in aquatic environments increases with decreasing water temperature, while photosynthetic activity generally decreases. Therefore, inorganic carbon acquisition by algal cells is greatly affected by temperature. We investigated half-saturation constants [Km(DIC), Km(CO2)] of inorganic carbon in photosynthesis under various temperatures for a strain of Chlamydomonas reinhadtii. C. reinhardtii showed an active carbon concentrating mechanism (CCM) at all temperature conditions investigated (5–25°C), implying that CCM activity is not diminished at low temperatures. The maximum photosynthetic rate was recorded at 15°C, while maximum CCM activity was detected at 20°C. A higher optimum temperature for CCM activity than for photosynthesis may compensate for lower photosynthetic rates above the optimum temperature. CCM may play a more significant role at higher temperatures in algal photosynthesis in aquatic environments.

EPR study on the photo-reduction of spin probe in the green alga Chlorella ellipsoidea

CO2 sensing system is believed to exist on the surface of plasmalemma of the green alga Chlorella ellipsoidea. It is well known that C. ellipsoidea can adjust the activity of carbon concentrating mechanism (CCM) in response to growth CO2 concentrations. We prepared two wild-type cells of C. ellipsoidea, i.e., High CO2 (5%)-grown cells and Low CO2-grown cells that have low and high CCM activities, respectively. CO2 insensitive mutants of C. ellipsoidea, which express the maximum level of CCM even grown in 5% CO2, were also cultured under low and high CO2-conditions. Cells were treated with 2 mM of 4-oxo-TEMPO(2,2,6,6-tetramethyl-4-oxopiperidinonxy) (TEMPONE), a spin probe, and 40 mM of K3Fe(CN)6, an oxidant. EPR signals from intracellular TEMPONE were observed under illumination or the dark in the presence of 150 mM of Na2MnEDTA, a signal quencher for external TEMPONE. The signal intensity of intracellular TEMPONE decreased gradually in a time dependent manner even under the dark, but the rate of reduction was stimulated as light intensity was increased. Two mM of DCMU, a specific inhibitor for the electron transport from QA to QB, halted the reduction of EPR signal. These data indicate that intracellular TEMPONE is reduced by photosynthetic electron flow primarily at PS II. Furthermore, the difference in effects of wavelength of light on the photoreduction of TEMPONE were also investigated.

Intra-specific variation in the photosynthetic responses of cyanobiont lichens from contrasting habitats.

The photosynthetic properties of cyanobiont lichens from contrasting habitats were measured to identify whether the increased assimilation rates which characterized Peltigera membranacea (Ach.) Nyl. from an exposed habitat were correlated with increased carbon-concentrating mechanism (CCM) activity. The results were contrasted with data obtained from two populations of Peltigera praetextata (Flörke ex Sommerf.) Zopf collected from dry and damp microhabitats within a shaded woodland and Peltigera leucophlebia (Nyl.) Gyelnik, which has been shown to lack a carbon-concentrating mechanism. The differences in assimilation rates between the cyanobiont lichens were not accounted for by differences in chlorophyll content. Peltigera membranacea from the exposed habitat which had the highest assimilation rates had the lowest Gamma; and K0·5 values and accumulated the greatest Ci -pool indicating that increased Ci accumulation contributed towards the higher assimilation rates shown by these species. The convexity of the light response curve for the cyanobiont lichens decreased with increasing assimilation rates. This might have indicated a diversion of electron transport to energize the carbon-concentrating mechanism. The apparent quantum efficiency of CO2 assimilation (ΦCO2 ) was correlated with the genus of lichen photobiont. All cyanobiont lichens had comparable values for ΦCO2 which were greater than that of the tripartite Peltigera leucophlebia. Light compensation points reflected the exposure of the habitats with higher compensation points characterizing the cyanobiont population from the exposed crag and the tri-partite population from the open grassland. Carbon isotope discrimination values for organic matter and measured instantaneously were the same for all cyanobiont lichens and were comparable with values recorded for species with a carbon-concentrating mechanism. Carbon isotope measurements for P. leucophlebia were typical of those recorded for species without a carbon-concentrating mechanism. Variation in source isotope signature and refixation of respiratory CO2 were considered to be significant factors in determining organic matter and instantaneous carbon-isotope discrimination. These factors might have masked any subtle variation in carbon-isotope discrimination which resulted from variable CCM activity. The functional significance of increased carbon-concentrating mechanism activity in cyanobiont lichens occupying exposed habitats is discussed.

Inorganic carbon pools in the bloom‐forming dinoflagellate Peridinium gatunense

Peridinum gatunense, the main bloom‐forming species in Lake Kinneret, acquires a carbon‐concentrating mechanism (CCM) during the progression of the annual winter–spring bloom. Previous indications for CCM activity in P. gatunense were based on decreased carbon isotope fractionation and higher activity of carbonic anhydrase (both external and internal). In the present study, CCM activity in low‐density, natural, and cultured P. gatunense populations was demonstrated by cells showing higher internal inorganic carbon (Ci) concentrations than those in the external medium. The presence, size, and dynamics of these Ci pools were measured using pulse‐chase experiments of 14C uptake into photosynthetic acid‐stable products under varying Ci, pH, CO2(aq), and light regimes. Internal Ci levels in P. gatunense were higher by a factor of ∼20–80 relative to the ambient Ci. The time required to reach isotopic equilibration of the pool ranged from 30 to 90 min, whereas the time required for the assimilation of the pool into cellular carbon ranged from 10 to 50 min. These long timescales are attributed to the larger vol: surface area ratio of this organism. Induction of the CCM was triggered by CO2(aq) rather than Ci and was evident at CO2(aq) concentrations <15 M. Preincubation in either the dark or with high CO2(aq) levels diminished the Ci pool. The data we report represent the steady‐state carbon metabolism of the species since the cells were not deprived of Ci prior to the measurements. The CCM in P. gatunense is an important adaptation mechanism contributing to the high primary production observed in the lake at the peak of the bloom under CO2‐limiting conditions [pH >9.5 and CO2(aq) <5 µM].