Affinity For Dissolved Inorganic Carbon Research Articles

Mitochondrial phosphoenolpyruvate carboxylase contributes to carbon fixation in the diatom Phaeodactylum tricornutum at low inorganic carbon concentrations.

Photosynthetic carbon fixation is often limited by CO2 availability, which led to the evolution of CO2 concentrating mechanisms (CCMs). Some diatoms possess CCMs that employ biochemical fixation of bicarbonate, similar to C4 plants, but whether biochemical CCMs are commonly found in diatoms is a subject of debate. In the diatom Phaeodactylum tricornutum, phosphoenolpyruvate carboxylase (PEPC) is present in two isoforms, PEPC1 in the plastids and PEPC2 in the mitochondria. We used real-time quantitative polymerase chain reaction, Western blots, and enzymatic assays to examine PEPC expression and PEPC activity, under low and high concentrations of dissolved inorganic carbon (DIC). We generated and analyzed individual knockout cell lines of PEPC1 and PEPC2, as well as a PEPC1/2 double-knockout strain. While we could not detect an altered phenotype in the PEPC1 knockout strains at ambient, low or high DIC concentrations, PEPC2 and the double-knockout strains grown under ambient air or lower DIC availability conditions showed reduced growth and photosynthetic affinity for DIC while behaving similarly to wild-type (WT) cells at high DIC concentrations. These mutants furthermore exhibited significantly lower 13 C/12 C ratios compared to the WT. Our data imply that in P. tricornutum at least parts of the CCM rely on biochemical bicarbonate fixation catalyzed by the mitochondrial PEPC2.

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.

Photophysiological responses of the marine macroalga Gracilariopsis lemaneiformis to ocean acidification and warming

To study the combined effects of ocean acidification (OA) and warming on the growth and photosynthetic performance of the economically important marine macroalga Gracilariopsis lemaneiformis, thalli were grown under ambient low CO2 (390 μatm, LC) and elevated high CO2 (1000 μatm, HC) conditions with culture temperatures of 20 °C and 24 °C. Based on the evaluation of growth and photosynthetic responses to light and dissolved inorganic carbon (DIC), HC decreased the growth rate and phycoerythrin (PE) and phycocyanin (PC) levels but increased contents of UV-absorbing compounds (UVACs) in G. lemaneiformis at 20 °C, and high temperature counteracted these effects. Photosynthetic responses such as chlorophyll fluorescence parameters (maximum relative electron transport rate, rETRmax; light use efficiency, α; saturation light intensity, Ik; maximum quantum yield, FV/FM; effective quantum yield, Y(II) and non-photochemical quenching, NPQ) were not different among the treatments. However, increased oxygen evolution (Pn) and dark respiration (Rd) rates were observed at 20 °C in the HC treatment. No significant effects of HC on apparent carboxylation efficiency (ACE), maximum oxygen evolution rate (Vmax) and DIC affinity for oxygen evolution (K1/2DIC) were found, and HC synergy with high temperature increased K1/2DIC. A lower C/N ratio with decreased tissue carbon but increased nitrogen was observed under HC and high-temperature treatment. Our results indicate that high temperature may counteract the negative effects of OA on the growth and pigment characteristics of G. lemaneiformis and improve food quality, as evidenced by enhanced N per biomass.

Carbon acquisition characteristics of six microalgal species isolated from a subtropical reservoir: potential implications for species succession.

CO2 levels in freshwater systems can fluctuate widely, potentially influencing photosynthetic rates and growth of phytoplankton. Given the right conditions, this can lead to bloom formation and affect water quality. This study investigated the acquisition of dissolved inorganic carbon (DIC) by six species of microalgae, a cyanobacterium Cylindrospermopsis raciborskii, the diatoms Cyclotella sp., Nitzschia sp., and the green algae Stichococcus sp., Staurastrum sp., and Monoraphidium sp., all isolated from a subtropical reservoir in Australia. Carbon acquisition characteristics, specifically the affinity for DIC, internal pH, and internal DIC concentrations were measured. Affinities for CO2 ( ) ranged between 0.7 and 6μM CO2 . This was considerably lower than air-equilibrated surface water CO2 concentrations, and below reported affinities for CO2 of RuBisCO suggesting operation of active carbon dioxide concentrating mechanisms (CCMs) in all species. Internal pH was lowest for Cyclotella sp. at 7.19, and highest for Staurastrum sp., at 7.71. At 180μM external DIC, ratios of internal:external CO2 ranged from 2.5 for Nitzschia sp. to 14 in C.raciborskii. Internal HCO3- concentration showed a linear relationship with surface area to biovolume ratio (SA:Vol). We hypothesized that species with a higher SA:Vol suffer more from diffusive escape of CO2 , thus storage of DIC as bicarbonate is favored in these strains. For C.raciborskii, under stratified summer conditions, its strong CCM, and resilient photosynthetic characteristics may contribute to its bloom forming capacity.

Characterization of a heat-tolerant Chlorella sp. GD mutant with enhanced photosynthetic CO2 fixation efficiency and its implication as lactic acid fermentation feedstock

BackgroundFermentative production of lactic acid from algae-based carbohydrates devoid of lignin has attracted great attention for its potential as a suitable alternative substrate compared to lignocellulosic biomass.ResultsA Chlorella sp. GD mutant with enhanced thermo-tolerance was obtained by mutagenesis using N-methyl-N′-nitro-N-nitrosoguanidine to overcome outdoor high-temperature inhibition and it was used as a feedstock for fermentative lactic acid production. The indoor experiments showed that biomass, reducing sugar content, photosynthetic O2 evolution rate, photosystem II activity (Fv/Fm and Fv′/Fm′), and chlorophyll content increased as temperature, light intensity, and CO2 concentration increased. The mutant showed similar DIC affinity and initial slope of photosynthetic light response curve (α) as that of the wild type but had higher dissolved inorganic carbon (DIC) utilization capacity and maximum photosynthesis rate (Pmax). Moreover, the PSII activity (Fv′/Fm′) in the mutant remained normal without acclimation process after being transferred to photobioreactor. This suggests that efficient utilization of incident high light and enhanced carbon fixation with its subsequent flux to carbohydrates accumulation in the mutant contributes to higher sugar and biomass productivity under enriched CO2 condition. The mutant was cultured outdoors in a photobioreactor with 6% CO2 aeration in hot summer season in southern Taiwan. The harvested biomass was subjected to separate hydrolysis and fermentation (SHF) for lactic acid production with carbohydrate concentration equivalent to 20 g/L glucose using the lactic acid-producing bacterium Lactobacillus plantarum 23. The conversion rate and yield of lactic acid were 80% and 0.43 g/g Chlorella biomass, respectively.ConclusionsThese results demonstrated that the thermo-tolerant Chlorella mutant with high photosynthetic efficiency and biomass productivity under hot outdoor condition is an efficient fermentative feedstock for large-scale lactic acid production.

Evolutionarily distinct strategies for the acquisition of inorganic carbon from seawater in marine diatoms.

The acquisition of dissolved inorganic carbon (DIC) in CO2-limited seawater is a central issue to understand in marine primary production. We previously demonstrated the occurrence of direct HCO3- uptake by solute carrier (SLC) 4 transporters in a diatom, a major marine primary producer. Homologs of SLC are found in both centric and pennate marine diatoms, suggesting that SLC transporters are generally conserved. Here, the generality of SLC-mediated DIC uptake in diatoms was examined using an SLC inhibitor, diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), and an inhibitor of external carbonic anhydrase, acetazolamide. DIDS suppressed high-DIC-affinity photosynthesis in the pennate diatom Phaeodactylum tricornutum and the centric diatom Chaetoceros muelleri, but there was no effect on either the pennate Cylindrotheca fusiformis or the centric Thalassiosira pseudonana. Interestingly, the DIC affinity of DIDS-insensitive strains was sensitive to treatment with up to 100 μM acetazolamide, displaying a 2-4-fold increase in K0.5[DIC]. In contrast, acetazolamide did not affect the DIDS-sensitive group. These results indicate the occurrence of two distinct strategies for DIC uptake-one primarily facilitated by SLC and the other being passive CO2 entry facilitated by external carbonic anhydrase. The phylogenetic independence of these strategies suggests that environmental demands drove the evolution of distinct DIC uptake mechanisms in diatoms.

Thylakoid luminal θ-carbonic anhydrase critical for growth and photosynthesis in the marine diatom Phaeodactylum tricornutum

The algal pyrenoid is a large plastid body, where the majority of the CO2-fixing enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) resides, and it is proposed to be the hub of the algal CO2-concentrating mechanism (CCM) and CO2 fixation. The thylakoid membrane is often in close proximity to or penetrates the pyrenoid itself, implying there is a functional cooperation between the pyrenoid and thylakoid. Here, GFP tagging and immunolocalization analyses revealed that a previously unidentified protein, Pt43233, is targeted to the lumen of the pyrenoid-penetrating thylakoid in the marine diatom Phaeodactylum tricornutum The recombinant Pt43233 produced in Escherichia coli cells had both carbonic anhydrase (CA) and esterase activities. Furthermore, a Pt43233:GFP-fusion protein immunoprecipitated from P. tricornutum cells displayed a greater specific CA activity than detected for the purified recombinant protein. In an RNAi-generated Pt43233 knockdown mutant grown in atmospheric CO2 levels, photosynthetic dissolved inorganic carbon (DIC) affinity was decreased and growth was constantly retarded; in contrast, overexpression of Pt43233:GFP yielded a slightly greater photosynthetic DIC affinity. The discovery of a θ-type CA localized to the thylakoid lumen, with an essential role in photosynthetic efficiency and growth, strongly suggests the existence of a common role for the thylakoid-luminal CA with respect to the function of diverse algal pyrenoids.

Photosynthesis in estuarine intertidal microphytobenthos is limited by inorganic carbon availability.

The effects of dissolved inorganic carbon (DIC) availability on photosynthesis were studied in two estuarine intertidal microphytobenthos (MPB) communities and in the model diatom species Phaeodactylum tricornutum. Kinetics of DIC acquisition, measured with a liquid-phase oxygen electrode, showed higher K(1/2)(DIC) (0.31 mM) and Vm (7.78 nmol min(-1) µg (Chl a)(-1)) for MPB suspensions than for P. tricornutum (K(1/2)(DIC) = 0.23 mM; Vm = 4.64 nmol min(-1) µg (Chl a)(-1)), suggesting the predominance of species with lower affinity for DIC and higher photosynthetic capacity in the MPB. The net photosynthetic rate of the MPB suspensions reached saturation at a DIC concentration of 1-1.5 mM. This range was lower than the concentrations found in the interstitial water of the top 5-mm sediment layer, suggesting no limitation of photosynthesis by DIC in the MPB communities. Accordingly, carbon isotope discrimination revealed a moderate activity of CO2-concentrating mechanisms in the MPB. However, addition of NaHCO3 to intact MPB biofilms caused a significant increase in the relative maximum photosynthetic electron transport rate (rETR max) measured by imaging pulse-amplitude modulated chlorophyll a fluorescence. These results suggest local depletion of DIC at the photic layer of the sediment (the first few hundred µm), where MPB cells accumulate during diurnal low tides. This work provides the first direct experimental evidence of DIC limitation of photosynthesis in highly productive intertidal MPB communities.

Transcriptional Response of the Sulfur Chemolithoautotroph Thiomicrospira crunogena to Dissolved Inorganic Carbon Limitation

The hydrothermal vent gammaproteobacterium Thiomicrospira crunogena inhabits an unstable environment and must endure dramatic changes in habitat chemistry. This sulfur chemolithoautotroph responds to changes in dissolved inorganic carbon (DIC) (DIC = CO(2) + HCO(3)(-) + CO(3)(-2)) availability with a carbon-concentrating mechanism (CCM) in which whole-cell affinity for DIC, as well as the intracellular DIC concentration, increases substantially under DIC limitation. To determine whether this CCM is regulated at the level of transcription, we resuspended cells that were cultivated under high-DIC conditions in chemostats in growth medium with low concentrations of DIC and tracked CCM development in the presence and absence of the RNA polymerase inhibitor rifampin. Induction of the CCM, as measured by silicone oil centrifugation, was hindered in the presence of rifampin. Similar results were observed for carboxysome gene transcription and assembly, as assayed by quantitative reverse transcription-PCR (qRT-PCR) and transmission electron microscopy, respectively. Genome-wide transcription patterns for cells grown under DIC limitation and those grown under ammonia limitation were assayed via microarrays and compared. In addition to carboxysome genes, two novel genes (Tcr_1019 and Tcr_1315) present in other organisms, including chemolithoautotrophs, but whose function(s) has not been elucidated in any organism were found to be upregulated under low-DIC conditions. Likewise, under ammonia limitation, in addition to the expected enhancement of ammonia transporter and P(II) gene transcription, the transcription of two novel genes (Tcr_0466 and Tcr_2018) was measurably enhanced. Upregulation of all four genes (Tcr_1019, 4-fold; Tcr_131, ∼7-fold; Tcr_0466, >200-fold; Tcr_2018, 7-fold), which suggests that novel components are part of the response to nutrient limitation by this organism, was verified via qRT-PCR.

Effects of dissolved inorganic carbon on competition of the bloom-forming cyanobacterium Microcystis aeruginosa with the green alga Chlamydomonas microsphaera

The availability of dissolved inorganic carbon (DIC) may have a considerable impact on species competition in phytoplankton communities. The growth, photosynthetic characteristics and competition of three strains, the toxic Microcystis aeruginosa FACHB 912, nontoxic M. aeruginosa FACHB 469 and Chlamydomonas microsphaera FACHB 52, were investigated under two different DIC concentrations (0.365 and 7.658 mmol l−1 KHCO3). In monoculture, DIC concentration did not affect the specific growth rates of any of the three strains. However, when grown in mixed culture with C. microsphaera, both toxic and nontoxic Microcystis strains showed increased percentages under low DIC concentration but decreased percentages under high DIC concentration. After 12 days’ mixed culture, the percentage of M. aeruginosa FACHB 912 or FACHB 469 decreased by 9−22% in high DIC medium, but increased by 6–11% in low DIC medium. Low DIC concentration decreased the cell size, cellular chlorophyll fluorescence and photosynthetic capacity of C. microsphaera, but it had little effect on those of M. aeruginosa FACHB 912 and FACHB 469. The apparent photosynthetic affinities for DIC were significantly higher in the two M. aeruginosa strains than in C. microsphaera. From the higher DIC affinity, more efficient photosynthetic capacity and increased percentages in competition experiments under low DIC concentration, it can be concluded that the bloom-forming M. aeruginosa possesses a competitive advantage in DIC-limited conditions.

Diversity of inorganic carbon acquisition mechanisms by intact microbial mats of Microcoleus chthonoplastes (Cyanobacteriae, Oscillatoriaceae)

The dissolved inorganic carbon (DIC) acquisition mechanisms were researched in intact microbial mats dominated by the cyanobacteria Microcoleus chthonoplastes Thuret, by determining the effect on photosynthesis of different inhibitors. The microbial mats exhibited high affinity for DIC at alkaline pH, with K(m(DIC)) values similar to the ones described for pure cultures of cyanobacteria and algae in which carbon concentrating mechanisms have been researched. Besides, the photosynthesis was non-sensitive to pH changes within the range of 5.6-9.6, indicating that HCO(3)(-) was the main DIC source used for photosynthesis. The M. chthonoplastes mats featured external and internal carbonic anhydrase (CA) activity as measured in intact cells and cell extracts, respectively. Acetazolamide (AZ, which slowly enters the cell and then inhibits mainly the external CA) and ethoxyzolamide (EZ, which inhibits both external and internal CA) reduced significantly the oxygen evolution rates, demonstrating that the CA was implied in the DIC acquisition. Vanadate inhibited photosynthesis by 60% although its application, when CA being inhibited (i.e. after applying AZ + EZ), did not produce any additional effect. It could indicate that ATPase-dependent HCO(3)(-) use occurred and also that this putative mechanism was coupled with CA-like activity at the plasma membrane. The involvement of Na(+)-dependent HCO(3)(-) transporters in DIC acquisition was also inferred as monensin and 4-4'-diisothiocyanatostibilene-2,2'-disulfonate (DIDS) reduced photosynthesis by 70%. DIDS produced a strong inhibitory effect even after application of AZ + EZ + vanadate, indicating that this mechanism was not related to CA activity. The microbial mats become subject to very unfavourable conditions for Rubisco carboxylation at their natural habitats (e.g. external pH of 10.5 and O(2) concentration doubled with respect to saturation concentration); therefore, this putative diversity of DIC acquisition mechanisms could ensure their growth under these extreme conditions.

CO2‐CONCENTRATING MECHANISMS OF THE POTENTIALLY TOXIC DINOFLAGELLATE PROTOCERATIUM RETICULATUM (DINOPHYCEAE, GONYAULACALES)1

The low CO2 concentration in seawater poses severe restrictions on photosynthesis, especially on those species with form II RUBISCO. We found that the potentially toxic dinoflagellate Protoceratium reticulatum Clap. et J. Lachm. possesses a form II RUBISCO. To cast some light on the mechanisms this organism undergoes to cope with low CO2 availability, we compared cells grown at atmospheric (370 ppm) and high (5000 ppm) CO2 concentrations, with respect to a number of physiological parameters related to dissolved inorganic carbon (DIC) acquisition and assimilation. The photosynthetic affinity for DIC was about one order of magnitude lower in cells cultivated at high [CO2]. End‐point pH‐drift experiments suggest that P. reticulatum was not able to efficiently use HCO3− under our growth conditions. Only internal carbonic anhydrase (CA) activity was detected, and its activity decreased by about 60% in cells cultured at high [CO2]. Antibodies raised against a variety of algal CAs were used for Western blot analysis: P. reticulatum extracts only cross‐reacted with anti‐ß‐CA sera, and the amount of immunoreactive protein decreased in cells grown at high [CO2]. No pyrenoids were observed under all growth conditions. Our data indicate that P. reticulatum has an inducible carbon‐concentrating mechanism (CCM) that operates in the absence of pyrenoids and with little intracellular CO2 accumulation. Calculations on the impact of the CA activity to photosynthetic growth [CO2] suggest that it is an essential component of the CCM of P. reticulatum and is necessary to sustain the photosynthetic rates observed at ambient CO2.

Competitive dominance of the cyanobacterium Microcystis aeruginosa in nutrient-rich culture conditions with special reference to dissolved inorganic carbon uptake

SUMMARY The aim of the present study was to determine what factors contribute to the competitive advantage of cyanobacteria in eutrophic conditions. Mixed species batch culture experiments were conducted at three pHs (8.2, 8.8, 10.2) and irradiances (30, 90, 180 μmol photons m−2 s_1) between Microcystis aeruginosa Kutzing and Staurastrum dorsidentiferum W. et West or Synedra ulna (Nitzsch) Ehrenberg, which always resulted in the dominance of M. aeruginosa. The final yields of competitors were often significantly lower than when cultured singly. The dominance of the surface-loving M. aeruginosa appears to be related to its advantageous C02 uptake. To clarify the importance of dissolved inorganic carbon (DIC) as a selective factor, the effect of aeration was examined with M. aeruginosa. The growth of M. aeruginosa quickly stopped in nonaerated conditions, while it continued growing throughout the culture when aerated. This result indicates that DIC limitation easily occurs in static conditions and the viewpoint of C02 exchange efficiency would be helpful in discussing the competitive advantage of M. aeruginosa. When the three species were cultured at various ratios of surface area to volume (s/v), postulating it as an index of the gas exchange efficiency, the increases in population densities strongly correlated with s/v, and the relationships between the specific growth rate and s/v corresponded well to a Monod type saturation function. We found that M. aeruginosa had the lowest half-saturation constant among the three, reflecting its high affinity for DIC.

The carbon-concentrating mechanism of the hydrothermal vent chemolithoautotroph Thiomicrospira crunogena.

Chemolithoautotrophic bacteria grow in habitats with a variety of dissolved inorganic carbon (DIC) concentrations and are likely to have transport-related adaptations to DIC scarcity. Carbon-concentrating mechanisms (CCMs) are present in many species of cyanobacteria, enabling them to grow in the presence of low concentrations of CO2 by utilizing bicarbonate transporters and CO2 traps to generate high intracellular concentrations of DIC. Similar CCMs may also be present in many other autotrophic bacteria. The sulfur-oxidizing γ-proteobacterial chemolithoautotroph Thiomicrospira crunogena experiences broad fluctuations in DIC availability at its hydrothermal vent habitat and may use a CCM to facilitate growth during periods of CO2 scarcity. T. crunogena was cultivated in chemostats under DIC limitation to determine whether it has a CCM. Its KDIC for growth was 0.22 mM, with a maximum growth rate of 0.44 h−1. In short-term incubations with [14C]DIC, DIC-limited cells had higher affinities for DIC (0.026 mM) than DIC-sufficient cells (0.66 mM). DIC-limited cells demonstrated an ability to use both extracellular CO2 and HCO3−, as assayed by isotopic disequilibrium incubations. These cells also accumulated intracellular DIC to concentrations 100× higher than extracellular, as determined using the silicone oil centrifugation technique. Cells that were not provided with an electron donor did not have elevated intracellular DIC concentrations. The inducible changes in whole-cell affinity for DIC, the ability to use both extracellular CO2 and HCO3−, and the energy-dependent generation of elevated intracellular concentrations of DIC are all consistent with the presence of a CCM in T. crunogena.

Modulation of photosynthesis and inorganic carbon acquisition in a marine microalga by nitrogen, iron, and light availability

The marine microalga Dunaliella tertiolecta Butcher expresses a high affinity for dissolved inorganic carbon (DIC) through a carbon-concentrating mechanism (CCM), known to be influenced by CO2 availability and instantaneous light supply. However, the regulation by light and nutrient supply during growth is less understood, although N and Fe limitation impose an energy limitation by compromising the photosynthetic apparatus. Dunaliella tertiolecta was grown under steady-state conditions of limited light, N, and Fe availability, and the affinity for DIC was measured under saturating light. High affinity DIC uptake capacity was maintained by D. tertiolecta under all growth-limiting conditions, but was modulated in response to the limiting resource. Affinity of photosynthesis for DIC(k0.5) was significantly reduced in cells grown under low light, both in turbidostats and in batch culture (p ≤ 0.03), although cell-normalized Pmax was not significantly affected. In contrast, N and Fe limitation resulted in a significant reduction in cell chlorophyll, Pmax, and maximum photosystem II quantum yield (Fv/Fm), but the affinity for DIC was enhanced with increasing N or Fe stress. While the affinity for DIC improved with increasing N stress (k0.5 < 17.8 µM at µ = 0.27 d–1 versus k0.5 > 26 µM at µ ≥ 0.77 d–1), light use efficiency (α) was impaired under N limitation, suggesting a trade-off between light harvesting capacity and active DIC uptake. Stable C isotope analysis of Fe-limited cells confirmed a lower fractionation by the most Fe-limited cells, consistent with the k0.5 data and more active DIC acquisition (δ13C = –19.56 at µ = 0.27 d–1 cf. δ13C = –26.28 at µ = 0.77 d–1). Assessment of affinity for DIC using k0.5 was supported by the close fit of P versus DIC curves to Michaelis–Menten kinetics; with the high DIC affinity of D. tertiolecta, there was poor resolution in the initial slope of the P versus DIC curve as a parameter of affinity for DIC. Enhanced DIC uptake efficiency under Fe and N limitation may relate to improved resource-use efficiency conferred by CCM activity.Key words: algae, carbon-concentrating mechanism, iron, light, nitrogen, nutrient limitation, photosynthesis.

Use of light and inorganic carbon acquisition by two morphotypes of Zostera noltii Hornem

The affinity for dissolved inorganic carbon (DIC) and the mechanisms to use HCO 3 − as a source of DIC for photosynthesis were investigated in two morphotypes of Zostera noltii Hornem. Both morphotypes were collected at Ria Formosa lagoon (Southern Portugal) at two different levels in the intertidal. Affinity for DIC at saturating photon fluence rate (PFR), estimated as photosynthetic conductance for CO 2 ( g p(CO 2) ), was reduced by 75% in the Z. noltii plants adapted to shade conditions (lower intertidal) in comparison to the sun morphotype (45×10 −6 and 182×10 −6 m s −1, respectively), indicating that the plants acclimated to sun conditions (higher intertidal) had a higher capacity to use HCO 3 − as DIC source for photosynthesis. Since external carbonic anhydrase activity was negligible and a large inhibitory effect was produced by Tris buffer addition, this HCO 3 − use was attributed to the operation of H + ATPases creating low pH zones in periplasmic space. The photosynthetic CO 2-flux supported for this mechanism was calculated to be 53 μmol O 2 m −2 s −1 in sun morphotype, about 80% out of maximum photosynthesis rate. In order to determine the possible photosynthetic energy cost of the HCO 3 − use, the effect of decreasing light on photosynthetic rates and g p(CO 2) was estimated. Photosynthetic conductance decreased in both morphotypes at non-saturating PFR. This dependence of g p(CO 2) on PFR indicated the existence of a positive interactive effect between DIC and PFR which was more pronounced in the shade morphotype since the ascending slope of O 2 evolution vs. PFR curves at limiting PFRs was reduced from 7.2 to 2.3 mmol O 2 mol photon −1 at 4 and 0.5 mol m −3 of DIC, respectively.

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The affinity for dissolved inorganic carbon (DIC) and the mechanisms to use HCO3− as a source of DIC for photosynthesis were investigated in two morphotypes of Zostera noltii Hornem. Both morphotypes were collected at Ria Formosa lagoon (Southern Portugal) at two different levels in the intertidal. Affinity for DIC at saturating photon fluence rate (PFR), estimated as photosynthetic conductance for CO2 (gp(CO2)), was reduced by 75% in the Z. noltii plants adapted to shade conditions (lower intertidal) in comparison to the sun morphotype (45×10−6 and 182×10−6 m s−1, respectively), indicating that the plants acclimated to sun conditions (higher intertidal) had a higher capacity to use HCO3− as DIC source for photosynthesis. Since external carbonic anhydrase activity was negligible and a large inhibitory effect was produced by Tris buffer addition, this HCO3− use was attributed to the operation of H+ ATPases creating low pH zones in periplasmic space. The photosynthetic CO2-flux supported for this mechanism was calculated to be 53 μmol O2 m−2 s−1 in sun morphotype, about 80% out of maximum photosynthesis rate. In order to determine the possible photosynthetic energy cost of the HCO3− use, the effect of decreasing light on photosynthetic rates and gp(CO2) was estimated. Photosynthetic conductance decreased in both morphotypes at non-saturating PFR. This dependence of gp(CO2) on PFR indicated the existence of a positive interactive effect between DIC and PFR which was more pronounced in the shade morphotype since the ascending slope of O2 evolution vs. PFR curves at limiting PFRs was reduced from 7.2 to 2.3 mmol O2 mol photon−1 at 4 and 0.5 mol m−3 of DIC, respectively.

Active transport of CO2 and bicarbonate is induced in response to external CO2 concentration in the green alga Chlorella kessleri

The time-course of induction of CO(2) and HCO(3)- transport has been investigated during the acclimation of high CO(2)-grown Chlorella kessleri cells to dissolved inorganic carbon (DIC)-limited conditions. The rate of photosynthesis of the cells in excess of the uncatalysed supply rate of CO(2) from HCO(3)- was taken as an indicator of HCO(3)- transport, while a stimulation of photosynthesis on the addition of bovine carbonic anhydrase was used as an indicator of CO(2) transport. The maximum rate of photosynthesis (Pmax) was similar for high CO(2)-grown and low CO(2)-grown cells, but the apparent whole cell affinity for DIC and CO(2) of high CO(2)-grown cells was found to be about 30-fold greater than in air-grown cells, which indicates a lower affinity for DIC and CO(2). It was found that HCO(3)- and CO(2) transport were induced in 5.5 h in cells acclimating to air in the light and in the presence and absence of 21% O(2), which indicates that a change in the CO(2)/O(2) ratio in the acclimating medium does not trigger induction of DIC transport. No active DIC transport was detected in high CO(2)-grown cells maintained on high CO(2) for 5.5 h in the presence of 5 mM aminooxyacetate, an aminotransferase inhibitor. These results indicate no involvement of photorespiration in triggering induction. Active DIC transport induction was inhibited in cells treated with 5 microgram ml(-1) cycloheximide, but was unaffected by chloramphenicol treatment, indicating that the induction process requires de novo cytoplasmic protein synthesis. The total DIC concentration eliciting the induction and repression of CO(2) and HCO(3)- transport was higher at pH 7.5 than at pH 6.6. The concentrations of external CO(2) required for the induction and repression of DIC transport were 0 and 120 microM, respectively, and was independent of the pH of the acclimation medium. Prolonged exposure to a critical external CO(2) concentration elicits the induction of DIC transport in C. kessleri.

A New Screening Method for Algal Photosynthetic Mutants (CO2-Insensitive Mutants of the Green Alga Chlorella ellipsoidea).

A new method has been developed for screening algal photosynthetic mutants. This method uses autoradiography to assess gross photosynthetic 14C fixation by green algal colonies on agar plates and allows the identification of clones that differ in photosynthetic characteristics from wild-type cells. Three wild-type cells, high-CO2-grown Chlorella ellipsoidea, air-grown C. ellipsoidea, and air-grown Chlorella saccharophila, had K0.5 values for dissolved inorganic carbon (DIC) of 1083, 250, and 50 [mu]M, respectively, and as plaques on agar plates at Chl densities greater than 25 [mu]g cm-2 exhibited relative amounts of 14C fixation of 15, 55, and 100%, respectively. Cells of C. ellipsoidea were mutagenized with x-rays and screened by this method. Growth of C. ellipsoidea in high CO2 represses DIC transport and thus lowers its affinity for DIC. Five of the mutants detected by this method showed high-affinity photosynthesis similar to air-grown wild-type cells even when grown in high CO2. Seven other mutants when grown in high CO2 showed affinities for DIC intermediate between air-grown and high-CO2-grown wild-type cells. The affinities of high-CO2-grown mutants were reflected precisely in their capacities to accumulate DIC intracellularly. These results indicate that the mutants are fully or partially insensitive to the repressive effect of ambient CO2 concentration on DIC transport.

Active uptake of inorganic carbon byChlorella saccharophilais not repressed by growth in high CO2

Regulation of transport of dissolved inorganic carbon (DIC) in response to CO 2 concentration in the external medium has been compared in two closely-related green algae, Chlorella ellipsoidea and Chlorella saccharophila. C. ellipsoidea, when grown in high CO 2 , had reduced activities of both CO 2 and HCO 3 - transport and DIC transport activities were increased after the cells had acclimated to air. However, high CO 2 -grown C. saccharophila had a comparable level of photosynthetic affinity for DIC to that of air-grown C. ellipsoidea and this was accompanied by a capacity to accumulate high internal concentrations of DIC. The high photosynthetic affinity and the high intracellular DIC accumulation did not change in cells grown in air except that the occurrence of external carbonic anhydrase (CA) in air-grown C. saccharophila stimulated the intracellular DIC accumulation in the absence of added CA. These data indicate that active DIC transport is constitutively expressed in C. saccharophila, presumably because this alga is insensitive to the repressive effect of high CO 2 on DIC transport. This strongly supports the existence of a direct sensing mechanism for external CO 2 in Chlorella species, but also indicates that external CA is regulated independently of DIC transport in Chlorella species.