Acclimation of wild-type cells and CO2-insensitive mutants of the green alga Chlorella ellipsoidea to elevated [CO2

CO(2)-insensitive mutants of the green alga Chlorella ellipsoidea were previously shown to be unable to repress an inorganic carbon-concentrating mechanism (CCM) when grown under 5% CO(2). When air-grown, wild-type (WT) cells were transferred to 5% CO(2), an abrupt drop of P(max) to 43% the original level of air-grown cells was observed within the initial 12 h. Photosynthetic affinities of WT cells to dissolved inorganic carbon (DIC) were maintained at high levels for the initial 4 d of acclimation, and then decreased gradually to lower levels over the next 6 d. In contrast to WT cells, the CO(2)-insensitive mutant, ENU16, exhibited a constant P(max) at maximum levels and a low K(1/2)[DIC] throughout the acclimation period. The rapid P(max) drop within 12 h of acclimation in WT cells was significantly reduced by treatment with 0.5 mm of 6-ethoxybenzothiazole-2-sulphonamide (EZA), a specific membrane-permeable inhibitor of carbonic anhydrase (CA), suggesting the participation of internal CAs in the temporary drop in P(max) in WT cells. WT and ENU16 cells were grown in controlled equilibrium [CO(2)], and the photosynthetic rate of each acclimated cell type was measured under equilibrated growth [DIC] conditions. In WT cells acclimated to 0.14-0.4% [CO(2)], K(1/2)[DIC] values increased as [CO(2)] increased, and the photosynthetic rates at growth DIC conditions were shown to decrease to about 70% the P(max) level in this intermediate [CO(2)] range. Such decreases in the net photosynthetic rates were not observed in ENU16. These results suggest that algal primary production could be depressed significantly under moderately enriched CO(2) conditions as a result of acquiring intermediate affinities for DIC because of their sensitive responses to changes in the ambient [CO(2)].     

A study of the control of glycolate excretion in chlorella

Chlorella pyrenoidosa cells grown on 5% CO(2) excreted glycolate when incubated in light with 10 mm bicarbonate, but excreted no glycolate under the same conditions when they were maintained on air for 7 hours prior to the assay. Incubation of 5% CO(2)-grown and air-grown cells with 10 mm isonicotinyl hydrazide or 10 mm alpha-hydroxypyridinemethane sulfonate during the assay stimulated the excretion of glycolate by CO(2)-grown cells severalfold that of air-grown cells.Adaptation of CO(2)-grown Chlorella to growth on air did not affect the levels of glycolate dehydrogenase in the cells and did not affect the rate of dark oxidation and metabolism of exogeneous (14)C-glycolate by the cells. These results indicate that the lack of glycolate excretion by air-grown or air-adapted cells of Chlorella cannot be explained by a concomitant change in the level of glycolate dehydrogenase.     

Induction of CO2 and Bicarbonate Transport in the Green Alga Chlorella ellipsoidea (I. Time Course of Induction of the Two Systems)

Changes in the physiological properties of the green alga Chlorella ellipsoidea (UTEX 20) were determined during adaptation from high CO2 to air. Cells of C. ellipsoidea, grown in high CO2, had an extremely low affinity for dissolved inorganic carbon (DIC). However, high-affinity DIC transport was induced rapidly after switching to air, which caused a massive decrease in the DIC concentration in the medium. Rates of O2 evolution without added carbonic anhydrase (CA) were compared with calculated rates of uncatalyzed CO2 formation in the medium as a measure of active HCO3-uptake. Cells were found to be able to use HCO3- after 5 h of adaptation and this capacity increased during the next 17 h. The stimulation of O2 evolution upon CA addition was used as a measurement of active CO2 transport: such stimulation occurred 2 h after transfer and increased during the next 5 h. Increases in O2 evolution rates were correlated closely with an increasing capacity to accumulate intracellular pools of acid-labile DIC and with decreases in K1/2(CO2) and CO2-compensation point of the cells. Treatment of cells with cycloheximide (5 [mu]g mL-1) during adaptation completely inhibited DIC transport induction, whereas treatment with chloramphenicol (400 [mu]g mL-1) had no effect, indicating the requirement for cytoplasmic protein synthesis in the induction. These results suggest that both CO2 and HCO3- transport are induced upon transfer of cells from high CO2 to air and that there is a temporal separation between the induction of the two systems.     

EXCRETION OF GLYCOLATE BY A SPECIES OF CHLORELLA (CHLOROPHYCEAE)1

The claim that Chlorella sp. (CCAP 211/8p), sometimes referred to as C. fusca, Shihira and Krauss, does not excrete glycolate has been reexamined. Chlorella sp. grown on 5% CO₂in air, excreted glycolate when incubated in light in 10 mM bicarbonate. Excretion ceased 30–60 min after transfer of the cells to air and no excretion could be detected with air-grown cells or with cells grown on 5% CO₂in media buffered at pH 8.0. Incubation with 10 mM isonicotinyl hydrazide, a glycolate pathway inhibitor, caused excretion in air-grown cells and stimulated excretion in CO₂-grown cells indicating that both the rate of glycolate synthesis and metabolism is higher in CO₂grown cells than in air-grown cells. Enhanced glycolate synthesis and excretion in CO₂-grown cells is correlated with law photosynthetic rate in 10 mM bicarbonate, and the photosynthetic rate of these cells doubles over a period of 2–2.5 h after initial transfer from high CO₂to bicarbonate. This correlation of photosynthetic induction with cessation of glycolate excretion is similar to that reported in a bluegreen alga and thought to occur in other green algae. These results indicate that glycolate excretion and its regulation in this species of Chlorella is not different from that in other algae.     

Cross-talk between photomixotrophic growth and CO(2) -concentrating mechanism in Synechocystis sp. strain PCC 6803

Simultaneous catabolic and anabolic glucose metabolism occurs in the same compartment during photomixotrophic growth of the model cyanobacterium Synechocystis sp. PCC 6803. The presence of glucose is stressful to the cells; it is reflected in the high frequency of suppression mutations in glucose-sensitive mutants. We show that glucose affects many cellular processes. It stimulates respiration and the rate of photosynthesis and quantum yield in low- but not high-CO(2) -grown cells. Fluorescence and thermoluminescence parameters of photosystem II are also affected but the results did not lend support to sustained glucose driven over reduction in the light. Glucose-sensitive mutants such as ΔpmgA (impaired in photomixotrophic growth) and Δhik31 (lacking histidine kinase 31) are far more susceptible under high than low air level of CO(2) . A glycine to tryptophan mutation in position 354 in NdhF3, involved in the high-affinity CO(2) uptake, rescued ΔpmgA. A rise in the apparent photosynthetic affinity to external inorganic carbon is observed in high-CO(2) -grown wild-type cells after the addition of glucose, but not in mutant ΔpmgA. This is attributed to upregulation of certain low-CO(2) -induced genes, involved in inorganic carbon uptake, in the wild type but not in ΔpmgA. These data uncovered a new level of interaction between CO(2) fixation (and the CO(2) -concentrating mechanism) and photomixotrophic growth in cyanobacteria.     

Active transport of CO(2) and bicarbonate is induced in response to external CO(2) 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.     

Induction of CO2 and Bicarbonate Transport in the Green Alga Chlorella ellipsoidea (II. Evidence for Induction in Response to External CO2 Concentration)

The critical species and concentrations of dissolved inorganic carbon (DIC) required for the induction of DIC transport during adaptation to low CO2 were determined for the green alga Chlorella ellipsoidea. The concentration of dissolved CO2 needed for the induction of both CO2 and HCO3- transport was independent of pH during adaptation, whereas the total DIC concentration required increased at alkaline pH. At pH 7.5, the minimum equilibrium DIC concentration at which high CO2 characteristics were maintained, i.e. transport was repressed, was 2100 [mu]M, whereas the maximum equilibrium DIC concentration below which DIC transport was fully induced (DICIND) was 500 [mu]M. Intracellular DIC concentration during adaptation to DICIND decreased temporarily after 2 h to 60% of the maximum level but recovered after 3 h of adaptation. After 3 h of adaptation to DICIND, cells exhibited maximum O2 evolution rate at DICIND. When cells partially adapted to DICIND were returned to high CO2, there was an immediate halt to the induction of transport and a gradual decrease in transport capacity over 23 h. The capacity for the induction of transport was unaffected by the absence of light. These results indicate that changes in the internal DIC pool during adaptation to low CO2 do not trigger the induction of DIC transport and that the induction is not light dependent. Induction of DIC transport in C. ellipsoidea appears to occur in response to the continuous exposure of cells to a critical CO2 concentration in the external medium.     

Active uptake of inorganic carbon by Chlorella saccharophila is not repressed by growth in high CO2

Regulation of transport of dissolved inorganic carbon (DIC)in response to CO₂ concentration in the external medium hasbeen compared in two closely-related green algae, Chlorellaellipsoidea and Chlorella saccharophila. C. ellipsoidea, whengrown in high CO₂, had reduced activities of both CO₂ and transport and DIC transport activitieswere increased after the cells had acclimated to air. However,high CO₂-grown C. saccharophila had a comparable level of photosyntheticaffinity for DIC to that of air-grown C. ellipsoidea and thiswas accompanied by a capacity to accumulate high internal concentrationsof DIC. The high photosynthetic affinity and the high intracellularDIC accumulation did not change in cells grown in air exceptthat the occurrence of external carbonic anhydrase (CA) in air-grownC. saccharophila stimulated the intracellular DIC accumulationin the absence of added CA. These data indicate that activeDIC transport is constitutively expressed in C. saccharophila,presumably because this alga is insensitive to the repressiveeffect of high CO₂ on DIC transport. This strongly supportsthe existence of a direct sensing mechanism for external CO₂in Chlorella species, but also indicates that external CA isregulated independently of DIC transport in Chlorella species. Key words: Carbonic anhydrase, Chlorella, CO₂-insensitive, DIC transport, wild type     

Effect of dissolved inorganic carbon on oxygen evolution and uptake by Chlamydomonas reinhardtii suspensions adapted to ambient and CO2-enriched air

Mass spectrometric measurements of ¹⁶O₂ and ¹⁸O₂ isotopes were used to compare the rates of gross O₂ evolution (E₀), O₂ uptake (U₀) and net O₂ evolution (NET) in relation to different concentrations of dissolved inorganic carbon (DIC) by Chlamydomonas reinhardtii cells grown in air (air-grown), in air enriched with 5% CO₂ (CO₂-grown) and by cells grown in 5% CO₂ and then adapted to air for 6h (air-adapted).At a photon fluence rate (PFR) saturating for photosynthesis (700 mol photons m^-2 s^-1), pH=7.0 and 28°C, U₀ equalled E₀ at the DIC compensation point which was 10M DIC for CO₂-grown and zero for air-grown cells. Both E₀ and U₀ were strongly dependent on DIC and reached DIC saturation at 480 M and 70 M for CO₂-grown and air-grown algae respectively. U₀ increased from DIC compensation to DIC saturation. The U₀ values were about 40 (CO₂-grown), 165 (air-adapted) and 60 mol O₂ mg Chl^-1 h^-1 (air-grown). Above DIC compensation the U₀/E₀ ratios of air-adapted and air-grown algae were always higher than those of CO₂-grown cells. These differences in O₂ exchange between CO₂- and air-grown algae seem to be inducable since air-adapted algae respond similarly to air-grown cells.For all algae, the rates of dark respiratory O₂ uptake measured 5 min after darkening were considerably lower than the rates of O₂ uptake just before darkening. The contribution of dark respiration, photorespiration and the Mehler reaction to U₀ is discussed and the energy requirement of the inducable CO₂/HCO₃ ^- concentrating mechanism present in air-adapted and air-grown C. reinhardtii cells is considered.Abbreviations DIC dissolved inorganic carbon - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - E₀ rate of photosynthetic gross O₂ evolution - PCO photosynthetic carbon oxidation - PFR photon fluence rate - PS I photosystem I - PS II photosystem II - U₀ rate of O₂ uptake in the light - MS mass spectrometer     

Inhibition of photosynthesis by intracellular carbonic anhydrase in microalgae under excess concentrations of CO2

When cells of Chlorococcum littorale that had been grown in air (air-grown cells) were transferred to extremely high CO₂ concentrations (>20%), active photosynthesis resumed after a lag period which lasted for 1–4 days. In contrast, C. littorale cells which had been grown in 5% CO₂ (5% CO₂-grown cells) could grow in 40% CO₂ without any lag period. When air-grown cells were transferred to 40% CO₂, the quantum efficiency of PS II (Φ_II) decreased greatly, while no decrease in Φ_II was apparent when the 5% CO₂-grown cells were transferred to 40% CO₂. In contrast to air-grown cells, 5% CO₂-grown cells showed neither extracellular nor intracellular carbonic anhydrase (CA) activity. Upon the acclimation of 5% CO₂-grown cells to air, photosynthetic susceptibility to 40% CO₂ was induced. This change was associated with the induction of CA. In addition, neither suppression of photosynthesis nor arrest of growth was apparent when ethoxyzolamide (EZA), a membrane-permeable inhibitor of CA, had been added before transferring air-grown cells of C. littorale to 40% CO₂. The intracellular pH value (pH_i) decreased from 7.0 to 6.4 when air-grown C. littorale cells were exposed to 40% CO₂ for 1–2 h, but no such decrease in pH_i was apparent in the presence of EZA. Both air- and 5% CO₂-grown cells of Chlorella sp. UK001, which was also resistant to extremely high CO₂ concentrations, grew in 40% CO₂ without any lag period. The activity of CA was much lower in air-grown cells of this alga than those in air-grown C. littorale cells. These results prompt us to conclude that intracellular CA caused intracellular acidification and hence inhibition of photosynthetic carbon fixation when air-grown C. littorale cells were exposed to excess concentrations of CO₂. No such harmful effect of intracellular CA was observed in Chlorella sp. UK001 cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

Regulation of energy balance in photosystems in response to changes in CO2 concentrations and light intensities during growth in extremely-high-CO2-tolerant green microalgae

Regulation of energy balance in photosystems in response to extremely-high-CO2 (40%) and low-CO2 (0.04%) stress was studied in extremely-high-CO2-tolerant green microalgae, Chlorococcum littorale and Chlorella sp. UK001. To investigate the energy input process, we assessed an F714/F685-ratio in a 77K fluorescence emission spectrum induced by 440-nm excitation in intact cells, which represents a ratio of fluorescence intensities derived from light-harvesting chlorophyll complexes in PSI and PSII. The F714/F685-ratio increased in several days after transferring C. littorale cells from air to 40% CO2, from 3% to 40% CO2 and from 3% to air. In all cases, the increase in the F714/F685-ratio was observed in high cell density culture, but no or a little increase was apparent in sparse cell density culture, when these cultures were illuminated at 250 micromol photon m-2 s-1. Even in the sparse culture, however, a similar increase in the F714/F685-ratio was observed when C. littorale cells were transferred from 3% to 40% CO2 at 20 micromol photon m-2 s-1. The cell density did not affect the F714/F685-ratio when CO2 concentration was kept at 3%. The activity of PSI electron (e-) transport was much higher in 40% CO2-grown cells than in 3% CO2-grown cells irrespective of the cell density during the culture, whereas the difference in PSII activity between them was small. The PSI activity at high cell density was higher also in air-grown cells than that in 3% CO2-grown cells. In both dense and sparse culture, 40% CO2-grown cells and air-grown cells showed higher relative quantum yield of PSI in the presence of DCMU than 3% CO2-grown cells, suggesting an increase in cyclic electron flow around PSI. Likewise, the increase in the F714/F685-ratio in response to the transfer to 40% CO2 was observed also in another extremely-high-CO2-tolerant alga, Chlorella sp. UK001. The possible role of the increases in the F714/F685-ratio, PSI/PSII activity ratio and cyclic e- transport activity in extremely-high-CO2 acclimation is discussed in comparison with low-CO2 acclimation.     

Evidence That an Internal Carbonic Anhydrase Is Present in 5% CO(2)-Grown and Air-Grown Chlamydomonas

Inorganic carbon (C_i) uptake was measured in wild-type cells of Chlamydomonas reinhardtii, and in cia-3, a mutant strain of C. reinhardtii that cannot grow with air levels of CO₂. Both air-grown cells, that have a CO₂ concentrating system, and 5% CO₂-grown cells that do not have this system, were used. When the external pH was 5.1 or 7.3, air-grown, wild-type cells accumulated inorganic carbon (C_i) and this accumulation was enhanced when the permeant carbonic anhydrase inhibitor, ethoxyzolamide, was added. When the external pH was 5.1, 5% CO₂-grown cells also accumulated some C_i, although not as much as air-grown cells and this accumulation was stimulated by the addition of ethoxyzolamide. At the same time, ethoxyzolamide inhibited CO₂ fixation by high CO₂-grown, wild-type cells at both pH 5.1 and 7.3. These observations imply that 5% CO₂-grown, wild-type cells, have a physiologically important internal carbonic anhydrase, although the major carbonic anhydrase located in the periplasmic space is only present in air-grown cells. Inorganic carbon uptake by cia-3 cells supported this conclusion. This mutant strain, which is thought to lack an internal carbonic anhydrase, was unaffected by ethoxyzolamide at pH 5.1. Other physiological characteristics of cia-3 resemble those of wild-type cells that have been treated with ethoxyzolamide. It is concluded that an internal carbonic anhydrase is under different regulatory control than the periplasmic carbonic anhydrase.     

Low Activation State of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Carboxysome-Defective Synechococcus Mutants

The high-CO2-requiring mutant of Synechococcus sp. PCC 7942, EK6, was obtained after extension of the C terminus of the small subunit of ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco). The carboxysomes in EK6 were much larger than in the wild type, but the cellular distribution of the large and small sub-units of Rubisco was not affected. The kinetic parameters of in vitro-activated Rubisco were similar in EK6 and in the wild type. On the other hand, Rubisco appeared to be in a low state of activation in situ in EK6 cells pretreated with an air level of CO2. This was deduced from the appearance of a lag phase when carboxylation was followed with time in cells permeabilized by detergent and subsequently supplied with saturating CO2 and RuBP. Pretreatment of the cells with high CO2 virtually abolished the lag. After low-CO2 treatment, the internal RuBP pool was much higher in mutant cells than in the wild-type cells; pretreatment with high CO2 reduced the pool in mutant cells. We suggest that the high-CO2-requiring phenotype in mutants that possess aberrant carboxysomes arises from the inactivated state of Rubisco when the cells are exposed to low CO2.     

Ribulose-1,5-bisphosphate carboxylase/oxygenase activase deficiency delays senescence of ribulose-1,5-bisphosphate carboxylase/oxygenase but progressively impairs its catalysis during tobacco leaf development.

Transgenic tobacco (Nicotiana tabacum L. cv W38) plants with an antisense gene directed against the mRNA of ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) activase grew more slowly than wild-type plants in a CO2-enriched atmosphere, but eventually attained the same height and number of leaves. Compared with the wild type, the anti-activase plants had reduced CO2 assimilation rates, normal contents of chlorophyll and soluble leaf protein, and much higher Rubisco contents, particularly in older leaves. Activase deficiency greatly delayed the usual developmental decline in Rubisco content seen in wild-type leaves. This effect was much less obvious in another transgenic tobacco with an antisense gene directed against chloroplast-located glyceraldehyde-3-phosphate dehydrogenase, which also had reduced photosynthetic rates and delayed development. Although Rubisco carbamylation was reduced in the anti-activase plants, the reduction was not sufficient to explain the reduced photosynthetic rate of older anti-activase leaves. Instead, up to a 10-fold reduction in the catalytic turnover rate of carbamylated Rubisco in vivo appeared to be the main cause. Slower catalytic turnover by carbamylated Rubisco was particularly obvious in high-CO2-grown leaves but was also detectable in air-grown leaves. Rubisco activity measured immediately after rapid extraction of anti-activase leaves was not much less than that predicted from its degree of carbamylation, ruling out slow release of an inhibitor from carbamylated sites as a major cause of the phenomenon. Nor could substrate scarcity or product inhibition account for the impairment. We conclude that activase must have a role in vivo, direct or indirect, in promoting the activity of carbamylated Rubisco in addition to its role in promoting carbamylation.     

Effect of Photon Fluence Rate on Oxygen Evolution and Uptake by Chlamydomonas reinhardtii Suspensions Grown in Ambient and CO(2)-Enriched Air

A closed system consisting of an assimilation chamber furnished with a membrane inlet from the liquid phase connected to a mass spectrometer was used to measure O₂ evolution and uptake by Chlamydomonas reinhardtii cells grown in ambient (0.034% CO₂) or CO₂-enriched (5% CO₂) air. At pH = 6.9, 28°C and concentrations of dissolved inorganic carbon (DIC) saturating for photosynthesis, O₂ uptake in the light (U_o) equaled O₂ production (E_o) at the light compensation point (15 micromoles photons per square meter per second). E_o and U_o increased with increasing photon fluence rate (PFR) but were not rate saturated at 600 micromoles photons per square meter per second, while net O₂ exchange reached a saturation level near 500 micromoles photons per square meter per second which was nearly the same for both, CO₂-grown and air-grown cells. Comparison of the U_o/E_o ratios between air-grown and CO₂-grown C. reinhardtii showed higher values for air-grown cells at light intensities higher than light compensation. For both, air-grown and CO₂-grown algae the rates of mitochondrial O₂ uptake in the dark measured immediately before and 5 minutes after illumination were much lower than U_o at PFR saturating for net photosynthesis. We conclude that noncyclic electron flow from water to NADP⁺ and pseudocyclic electron flow via photosystem I to O₂ both significantly contribute to O₂ exchange in the light. In contrast, mitochondrial respiration and photosynthetic carbon oxidation cycle are regarded as minor O₂ consuming reactions in the light in both, air-grown and CO₂-grown cells. It is suggested that the “extra” O₂ uptake by air-grown algae provides ATP required for the energy dependent CO₂/HCO₃⁻ concentrating mechanism known to be present in these cells.     

Photosynthetic characteristics of a multicellular green alga Volvox carteri in response to external CO2 levels possibly regulated by CCM1/CIA5 ortholog

When CO(2) supply is limited, aquatic photosynthetic organisms induce a CO(2)-concentrating mechanism (CCM) and acclimate to the CO(2)-limiting environment. Although the CCM is well studied in unicellular green algae such as Chlamydomonas reinhardtii, physiological aspects of the CCM and its associated genes in multicellular algae are poorly understood. In this study, by measuring photosynthetic affinity for CO(2), we present physiological data in support of a CCM in a multicellular green alga, Volvox carteri. The low-CO(2)-grown Volvox cells showed much higher affinity for inorganic carbon compared with high-CO(2)-grown cells. Addition of ethoxyzolamide, a membrane-permeable carbonic anhydrase inhibitor, to the culture remarkably reduced the photosynthetic affinity of low-CO(2) grown Volvox cells, indicating that an intracellular carbonic anhydrase contributed to the Volvox CCM. We also isolated a gene encoding a protein orthologous to CCM1/CIA5, a master regulator of the CCM in Chlamydomonas, from Volvox carteri. Volvox CCM1 encoded a protein with 701 amino acid residues showing 51.1% sequence identity with Chlamydomonas CCM1. Comparison of Volvox and Chlamydomonas CCM1 revealed a highly conserved N-terminal region containing zinc-binding amino acid residues, putative nuclear localization and export signals, and a C-terminal region containing a putative LXXLL protein-protein interaction motif. Based on these results, we discuss the physiological and genetic aspects of the CCM in Chlamydomonas and Volvox.     

Elevated CO2 Effects during Leaf Ontogeny (A New Perspective on Acclimation)

For many plants growth in elevated CO2 leads to reduced rates of photosynthesis. To examine the role that leaf ontogeny plays in the acclimation response, we monitored photosynthesis and some related parameters at short intervals throughout the ontogenetic development of tobacco (Nicotiana tabacum L.) leaves under ambient (350 [mu]L L-1)- and high (950 [mu]L L-1)-CO2 conditions. The pattern of photosynthetic rate over time was similar between the two treatments and consistent with the expected pattern for a typical dicot leaf. However, the photosynthesis pattern in high-CO2-grown tobacco was shifted temporally to an earlier maximum and subsequent senescent decline. Ribulose-1,5-biphosphate carboxylase/oxygenase activity appeared to be the main factor regulating photosynthetic rates in both treatments. Therefore, we propose a new model for interpreting the acclimation response. Lowered photosynthetic rates observed during acclimation appear to be the result of a shift in the timing of the normal photosynthetic stages of leaf ontogeny to an earlier onset of the natural decline in photosynthetic rates associated with senescence.     

Monensin Inhibition of Na+-Dependent HCO3- Transport Distinguishes It from Na+-Independent HCO3- Transport and Provides Evidence for Na+/HCO3- Symport in the Cyanobacterium Synechococcus UTEX 625

The effect of monensin, an ionophore that mediates Na+/H+ exchange, on the activity of the inorganic carbon transport systems of the cyanobacterium Synechococcus UTEX 625 was investigated using transport assays based on the measurement of chlorophyll a fluorescence emission or 14C uptake. In Synechococcus cells grown in standing culture at about 20 [mu]M CO2 + HCO3-, 50 [mu]M monensin transiently inhibited active CO2 and Na+-independent HCO3- transport, intracellular CO2 and HCO3- accumulation, and photosynthesis in the presence but not in the absence of 25 mM Na+. These activities returned to near-normal levels within 15 min. Transient inhibition was attributed to monensin-mediated intracellular alkalinization, whereas recovery may have been facilitated by cellular mechanisms involved in pH homeostasis or by monensin-mediated H+ uptake with concomitant K+ efflux. In air-grown cells grown at 200 [mu]M CO2 + HCO3- and standing culture cells, Na+-dependent HCO3- transport, intracellular HCO3- accumulation, and photosynthesis were also inhibited by monensin, but there was little recovery in activity over time. However, normal photosynthetic activity could be restored to air-grown cells by the addition of carbonic anhydrase, which increased the rate of CO2 supply to the cells. This observation indicated that of all the processes required to support photosynthesis only Na+-dependent HCO3- transport was significantly inhibited by monensin. Monensin-mediated dissipation of the Na+ chemical gradient between the medium and the cells largely accounted for the decline in the HCO3- accumulation ratio from 751 to 55. The two HCO3- transport systems were further distinguished in that Na+-dependent HCO3- transport was inhibited by Li+, whereas Na+-independent HCO3- transport was not. It is suggested that Na+-dependent HCO3- transport involves an Na+/HCO3- symport mechanism that is energized by the Na+ electrochemical potential.     

Growth at Low Temperature Mimics High-Light Acclimation in Chlorella vulgaris

Structural and functional alterations to the photosynthetic apparatus after growth at low temperature (5[deg]C) were investigated in the green alga Chlorella vulgaris Beijer. Cells grown at 5[deg]C had a 2-fold higher ratio of chlorophyll a/b, 5-fold lower chlorophyll content, and an increased xanthophyll content compared to cells grown at 27[deg]C even though growth irradiance was kept constant at 150 [mu]mol m-2 s-1. Concomitant with the increase in the chlorophyll a/b ratio was a lower abundance of light-harvesting polypeptides in 5[deg]C-grown cells as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and confirmed by western blotting.The differences in pigment composition were found to be alleviated within 12 h of transferring 5[deg]C-grown cells to 27[deg]C. Furthermore, exposure of 5[deg]C-grown cells to a 30-fold lower growth irradiance (5 [mu]mol m-2 s-1) resulted in pigment content and composition similar to that in cells grown at 27[deg]C and 150 [mu]mol m-2 s-1. Although both cell types exhibited similar measuring-temperature effects on CO2-saturated O2 evolution, 5[deg]C-grown cells exhibited light-saturated rates of O2 evolution that were 2.8-and 3.9-fold higher than 27[deg]C-grown cells measured at 27[deg]C and 5[deg]C, respectively. Steady-state chlorophyll a fluorescence indicated that the yield of photosystem II electron transport of 5[deg]C-grown cells was less temperature sensitive than that of 27[deg]C-grown cells. This appears to be due to an increased capacity to keep the primary, stable quinone electron acceptor of photosystem II (QA) oxidized at low temperature in 5[deg]C- compared with 27[deg]C-grown cells regardless of irradiance. We conclude that Chlorella acclimated to low temperature adjusts its photosynthetic apparatus in response to the excitation pressure on photosystem II and not to the absolute external irradiance. We suggest that the redox state of QA may act as a signal for this photosynthetic acclimation to low temperature in Chlorella.