A correlation between changes in luminescence decay kinetics and the appearance of a CO2-accumulating mechanism in Scenedesmus obliquus

In experiments with the unicellular green algae Scenedesmus obliquus a correlation was found between the presence of the CO₂-accumulating mechanism and the appearance of polyphasic luminescence decay kinetics. A potentiometric titration method was used to measure and calculate photosynthetic carbon uptake.Polyphasic luminescence decay kinetics was found when the algae showed photosynthetic characteristics typical of algae adapted to low-CO₂ conditions. When high-CO₂ grown algae were transferred to low-CO₂ conditions they gradually developed polyphasic decay kinetics during the first 25–30 minutes. When low-CO₂ grown algae were transferred to high-CO₂ conditions the polyphasic decay kinetics disappeared. To account for these results a working hypothesis is presented on the basis of the energy requirement for a CO₂-accumulating mechanism.     

Isolation of cDNA clones of genes induced upon transfer of Chlamydomonas reinhardtii cells to low CO2

Unicellular algae grow well under limiting CO₂ conditions, aided by a carbon concentrating mechanism (CCM). In C. reinhardtii, this mechanism is inducible and is present only in cells grown under low CO₂ conditions. We constructed a cDNA library from cells adapting to low CO₂, and screened the library for cDNAs specific to low CO₂-adapting cells. Six classes of low CO₂-inducible clones were identified. One class of clone, reported here, represents a novel gene associated with adaptation of cells to air. A second class of clones corresponds to the air-inducible periplasmic carbonic anhydrase I (CAH1). These clones represent genes that respond to the level of CO₂ in the environment.     

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     

Photosynthesis and apparent affinity for dissolved inorganic carbon by cells and chloroplasts of Chlamydomonas reinhardtii grown at high and low CO2 concentrations

Chloroplasts with high rates of photosynthetic O₂ evolution (up to 120 mol O₂· (mg Chl)^-1·h^-1 compared with 130 mol O₂· (mg Chl)^-1·h^-1 of whole cells) were isolated from Chlamydomonas reinhardtii cells grown in high and low CO₂ concentrations using autolysine-digitonin treatment. At 25° C and pH=7.8, no O₂ uptake could be observed in the dark by high- and low-CO₂ adapted chloroplasts. Light saturation of photosynthetic net oxygen evolution was reached at 800 mol photons·m^-2·s^-1 for high- and low-CO₂ adapted chloroplasts, a value which was almost identical to that observed for whole cells. Dissolved inorganic carbon (DIC) saturation of photosynthesis was reached between 200–300 M for low-CO₂ adapted chloroplasts, whereas high-CO₂ adapted chloroplasts were not saturated even at 700 M DIC. The concentrations of DIC required to reach half-saturated rates of net O₂ evolution (K_m(DIC)) was 31.1 and 156 M DIC for low- and high-CO₂ adapted chloroplasts, respectively. These results demonstrate that the CO₂ concentration provided during growth influenced the photosynthetic characteristics at the whole cell as well as at the chloroplast level.Abbreviations Chl chlorophyll - DIC dissolved inorganic carbon - K_m(DIC) coneentration of dissolved inorganic carbon required for the rate of half maximal net O₂ evolution - PFR photon fluence rate - SPGM silicasol-PVP-gradient medium     

Very high light resistant mutants of Chlamydomonas reinhardtii: Responses of Photosystem II,nonphotochemical quenching and xanthophyll pigments to light and CO2

We have isolated very high light resistant nuclear mutants (VHL ^R) in Chlamydomonas reinhardtii, that grow in 1500–2000 mol photons m^–2 s^–1 (VHL) lethal to wildtype. Four nonallelic mutants have been characterized in terms of Photosystem II (PS II) function, nonphotochemical quenching (NPQ) and xanthophyll pigments in relation to acclimation and survival under light stress. In one class of VHL ^R mutants isolated from wild type (S4 and S9), VHL resistance was accompanied by slower PS II electron transfer, reduced connectivity between PS II centers and decreased PS II efficiency. These lesions in PS II function were already present in the herbicide resistant D1 mutant A251L (L ^*) from which another class of VHL ^R mutants (L4 and L30) were isolated, confirming that optimal PS II function was not critical for survival in very high light. Survival of all four VHL ^R mutants was independent of CO₂ availability, whereas photoprotective processes were not. The de-epoxidation state (DPS) of the xanthophyll cycle pigments in high light (HL, 600 mol photons m^–2 s^–1) was strongly depressed when all genotypes were grown in 5% CO₂. In S4 and S9 grown in air under HL and VHL, high DPS was well correlated with high NPQ. However when the same genotypes were grown in 5% CO₂, high DPS did not result in high NPQ, probably because high photosynthetic rates decreased thylakoid pH. Although high NPQ lowered the reduction state of PS II in air compared to 5% CO₂ at HL in wildtype, S4 and S9, this did not occur during growth of S4 and S9 in VHL. L ^* and VHL ^R mutants L4 and L30, also showed high DPS with low NPQ when grown air or 5% CO₂, possibly because they were unable to maintain sufficiently high pH due to constitutively impaired PS II electron transport. Although dissipation of excess photon energy through NPQ may contribute to VHL resistance, there is little evidence that the different genes conferring the VHL ^R phenotype affect this form of photoprotection. Rather, the decline of chlorophyll per biomass in all VHL ^R mutants grown under VHL suggests these genes may be involved in regulating antenna components and photosystem stoichiometries.This revised version was published online in October 2005 with corrections to the Cover Date.     

Purification of highly active oxygen-evolving photosystem II from Chlamydomonas reinhardtii

A method is described for the isolation and purification of active oxygen-evolving photosystem II (PS II) membranes from the green alga Chlamydomonas reinhardtii. The isolation procedure is a modification of methods evolved for spinach (Berthold et al. 1981). The purity and integrity of the PS II preparations have been assesssed on the bases of the polypeptide pattern in SDS-PAGE, the rate of oxygen evolution, the EPR multiline signal of the S₂ state, the room temperature chlorophyll a fluorescence yield, the 77 K emission spectra, and the P700 EPR signal at 300 K. These data show that the PS II characteristics are increased by a factor of two in PS II preparations as compared to thylakoid samples, and the PS I concentration is reduced by approximately a factor ten compared to that in thylakoids.Abbreviations BSA bovine serum albumin - Chl chlorophyll - DCBQ 2,6-dichloro-p-benzoquinone - DCMU (diuron) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMQ 2,5-dimethyl-p-benzoquinone - EDTA ethylenediamine tetraacetic acid - EPR electron paramagnetic resonance - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MES 2-[N-Morpholino]ethanesulfonic acid - OEE oxygen evolving enhancer - PS II photosystem II - SDS-PAGE sodium dedocyl sulfate polyacrylamide gel electrophoresis     

Generation of expressed sequence tags from low-CO2 and high-CO2 adapted cells of Chlamydomonas reinhardtii.

To characterize genes whose expression is induced in carbon-stress conditions, 12,969 and 13,450 5'-end expressed sequence tags (ESTs) were generated from cells grown in low-CO2 and high-CO2 conditions of the unicellular green alga, Chlamydomonas reinhardtii. These ESTs were clustered into 4436 and 3566 non-redundant EST groups, respectively. Comparison of their sequences with those of 3433 non-redundant ESTs previously generated from the cells under the standard growth condition indicated that 2665 and 1879 EST groups occurred only in the low-CO2 and high-CO2 populations, respectively. It was also noted that 96.2% and 96.0% of the cDNA species respectively obtained from the low-CO2 and high-CO2 conditions had no similar EST sequence deposited in the public databases. The EST species identified only in the low-CO2 treated cells included genes previously reported to be expressed specifically in low-CO2 acclimatized cells, suggesting that the ESTs generated in this study will be a useful source for analysis of genes related to carbon-stress acclimatization. The sequence information and search results of each clone will appear at the web site: http://www.kazusa.or.jp/en/plant/chlamy/EST/.     

Time-resolved chlorophyll fluorescence studies on photosynthetic mutants of Chlamydomonas reinhardtii: origin of the kinetic decay components

The room temperature chlorophyll fluorescence decay kinetics of photosynthetic mutants of Chlamydomonas reinhardtii have been measured as a function of Photosystem 2 (PS2) trap closure, DNB-induced quenching at F_M, and time-resolved emission spectra. The overall decays have been analyzed in terms of three or four kinetic components where necessary. A comparison of the characteristics of the decay components exhibited by the mutants with the wild-type has been carried out to elucidate the precise origins of the different emissions in relation to the observed pigment-protein complexes. It is shown that a) charge recombination in PS2 is not necessary for the presence of long-lived decay components, b) there are two rapid PS1-associated emissions (=30 and 150–200 ps), c) a slow PS1 decay is observed (=1.73 ns) in the absence of PS1 reaction centres, d) the two variable components (=0.25–1.2 and 0.5–2.2 ns) observed in the wild-type arise from LHC2 and e) a rapid (=50–250 ps) decay is associated with the PS2 core antenna (CP3 and CP4). These results show that the intact thylakoid membrane system is too complex to distinguish all of the individual kinetic components.Abbreviations Aexp preexponential factor (Amplitude) - chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - DNB m, dinitrobenzene - F_M maximum chl fluorescence level - F₀ initial chl fluorescence level - F_v variable chl fluorescence (F_M–F₀) - LHC light harvesting chl a/b protein complex - PS photosystem - Q_A primary stable electron acceptor of PS2     

Effect of O2 and CO2 on net CO2 exchange in a high-CO2-requiring mutant of Chlamydomonas reinhardtii during dark-light-dark transitions

Net CO₂ exchange was monitored through a dark-light-dark transition, under 2% and 21% O₂ in the presence and absence of CO₂, in Chlamydomonas reinhardtii wild type and the high-CO₂-requiring mutant ca-1-12-1C. Upon illumination at 350 l/l CO₂, ca-1-12-1C cell exhibited a large decrease in net CO₂ uptake following an initial surge of CO₂ uptake. Net CO₂ uptake subsequently attained a steady-state rate substantially lower than the maximum. A large, O₂-enchanced post-illumination burst of CO₂ efflux was observed after a 10-min illumination period, corresponding to a minimum in the net CO₂ uptake rate. A smaller, but O₂-insensitive post-illumination burst was observed following a 30-min illumination period, when net CO₂ uptake was at a steady-state rate. These post-illumination bursts appeared to reflect the release of an intracellular pool of inorganic carbon, which was much larger following the initial surge of net CO₂ uptake than during the subsequent steady-state CO₂ uptake period.With the mutant in CO₂-free gas, O₂-stimulated, net CO₂ efflux was observed in the light, and a small, O₂-dependent post-illumination burst was observed. With wild-type cells no CO₂ efflux was observed in the light in CO₂-free gas under either 2% or 21% O₂, but a small, O₂-dependent post-illumination burst was observed. These results were interpreted as indicating that photorespiratory rates were similar in the mutant and wild-type cells in the absence of CO₂, but that the wild-type cells were better able to scavenge the photorespiratory CO₂.     

Secondary fluorescence kinetics of spinach leaves in relation to the onset of photosynthetic carbon assimilation

When spinach leaves are re-illuminated, after dark periods of 90 s or less, an initial fluorescence peak is observed which rapidly gives way to a much lower terminal value. After 2 min or more in the dark, however, there is a secondary rise, at about 50–70 s, which then gives way, more slowly, to approximately the same low terminal value as before. The secondary rise is eliminated or disguised by feeding D,L-glyceraldehyde (a specific inhibitor of photosynthetic carbon assimilation) and by manose, 2-deoxyglucose and glucosamine, all of which are believed to sequester cytoplasmic orthophosphate. This secondary rise in fluorescence is discussed in relation to photosynthetic induction and the manner in which these compounds may modulate fluorescence by their effect on the availability of orthophosphate and their consequent impact on the adenylate status of the stroma.Abbreviations DCMU 3(3,4-dichlorophenyl)-1,1-dimethylurea - CCCP carbonylcyanidchlorophenylhydrazon     

The impact of high CO2 concentrations on the structure and function of the photosynthetic apparatus and the role of polyamines

Here we examined the influence of high CO2 concentrations on the structure and functioning of the photosynthetic apparatus in the unicellular green alga Scenedesmus obliquus. Presented in this work are: chlorophyll (Chl) a fluorescence induction kinetics, measurements of photosynthetic and respiration rates, estimation of Chl a/Chl b ratios, isolation and quantitative assessment of the photosynthetic subcomplexes, quantitative analyses of thylakoid bound polyamines, and experiments with exogenously supplied polyamines with cultures grown in low- and high-CO2 concentrations. Together, they indicated that high-CO2 concentrations affect polyamines and, more specifically, increase the thylakoid bound putrescine (PUT) level that leads to an increase of the active reaction center density combined with a decrease in the LHCII-size and the ratio of LHCII-oligomeres/LHCII-monomeres. This reorganization of the photosynthetic apparatus leads to enhanced photosynthetic rates, which in combination with the high-CO2 concentrations, leads to an immense increase of biomass (800%). Further incubation for longer time periods under the same conditions produces, due to an increase in cell density, a self-shading effect and photoadaptation of the photosynthetic apparatus to low light conditions and therefore also results in reduction of the high-CO2 effect. The photoadaptation of the photosynthetic apparatus to high-light conditions (Kotzabasis et al. 1999) and the acclimation to high-CO2 concentrations (present work) lead to the same changes in the structure and function of the photosynthetic apparatus. These changes could be induced or inhibited through the manipulation of intracellular polyamines, especially through the putrescine/spermine ratio. The possibility that polyamines influence the photoadaptation of the photosynthetic apparatus and its acclimation to high-CO2 concentrations through a common mechanism is discussed.     

CO2 exchange characteristics during dark-light transitions in wild-type and mutant Chlamydomonas reinhardii cells

A burst of net CO₂ uptake was observed during the first 3–4 min after the onset of illumination in both wild-type Chlamydomonas reinhardii in which carbonic anhydrase was chemically inhibited with ethoxyzolamide and in a mutant of C. reinhardii (ca-1-12-1C) deficient in carbonic anhydrase activity. The burst was followed by a rapid decrease in the CO₂ uptake rate so that net evolution often occurred. After a 2–3 min period of CO₂ evolution, net CO₂ uptake again increased and ultimately reached a steady-state, positive rate. From [¹⁴CO₂]-tracer studies it was determined that CO₂ fixation proceeded at a nearly linear rate throughout the period of illumination. Thus, prior to reaching a steady state, there was a rapid accumulation of inorganic carbon inside the cells which apparently reached a supercritical concentration and the excess was excreted, causing a subsequent efflux of CO₂. A post illumination burst of net CO₂ efflux was also observed in ethoxyzolamide-inhibited wild type and ca-1 mutant cells, but not in the unihibited wild type. [¹⁴CO₂]-tracer experiments revealed that this burst was the result of a collapse of a large internal inorganic carbon pool at the onset of darkness rather than a photorespiratory post-illumination burst. These results indicate that upon illumination, chemical or genetic inhibition of carbonic anhydrase initially causes an accumulation of excess inroganic carbon in C. reinhardii cells, and that unknown regulatory mechanisms correct for this imbalance by first excreting the excess inorganic carbon and then, after several dampened oscillations, achieving an equilibrium between bicarbonate uptake, bicarbonate dehydration, and CO₂ fixation.     

CO2升高对枝角类群落结构影响的原位模拟

分别于春、夏两季在太湖梅梁湾进行原位试验,设置3个CO_2浓度梯度,270、380μL/L和750μL/L,以斜生栅藻作为枝角类的食物,研究了CO_2浓度升高对枝角类群落结构的影响。结果表明高CO_2浓度能促进斜生栅藻生长,显著提高枝角类的食物数量;此外CO_2浓度的变化能显著改变枝角类的群落结构,高CO_2浓度有利于象鼻蟤属、秀体蟤属和春季蟤属的生长,而不利于网纹蟤属的生长。这可能是由于CO_2浓度变化改变了枝角类的食物质量,浮游藻类的C∶P比值随CO_2浓度的升高而增加,从而有利于体内含磷量较低,高C∶P的枝角类生长。因此枝角类的群落结构主要受食物质量的影响而与食物的数量无关。研究为预测未来气候变化对太湖浮游动物的影响提供了一些理论依据。     

Effect of photon flux density on inorganic carbon accumulation and net CO2 exchange in a high-CO2-requiring mutant of Chlamydomonas reinhardtii

The effect of photon flux density on inorganic carbon accumulation and photosynthetic CO₂ assimilation was determined by CO₂ exchange studies at three, limiting CO₂ concentrations with a ca-1 mutant of Chlamydomonas reinhardiii. This mutant accumulates a large internal inorganic carbon pool in the light which apparently is unavailable for photosynthetic assimilation. Although steady-state photosynthetic CO₂ assimilation did not respond to the varying photon flux densities because of CO₂ limitation, components of inorganic-carbon accumulation were not clearly light saturated even at 1100 mol photons m^-2 s^-1, indicating a substantial energy requirement for inorganic carbon transport and accumulation. Steady-state photosynthetic CO₂ assimilation responded to external CO₂ concentrations but not to changing internal inorganic carbon concentrations, confirming that diffusion of CO₂ into the cells supplies most of the CO₂ for photosynthetic assimilation and that the internal inorganic carbon pool is essentially unavailable for photosynthetic assimilation. The estimated concentration of the internal inorganic carbon pool was found to be relatively insensitive to the external CO₂ concentration over the small range tested, as would be expected if the concentration of this pool is limited by the internal to external inorganic carbon gradient. An attempt to use this CO₂ exchange method to determine whether inorganic carbon accumulation and photosynthetic CO₂ assimilation compete for energy at low photon flux densities proved inconclusive.     

High CO2 partial pressure effects on dark and light CO2 fixation and metabolism in Vicia faba leaves

Stomatal opening on Vicia faba can be induced by high CO₂ partial pressures (10.2%) in dark as well as in light. Stomatal aperture was measured in both cases with a hydrogen porometer. The distribution of ¹⁴C among early products of photosynthesis was studied. Comparisons are made with carboxylations occurring when stomata were open in the dark with CO₂-free air and in light with 0.034% CO₂. Results showed that in high CO₂ partial pressure in light, less radioactivity was incorporated in Calvin cycle intermediates and more in sucrose. carboxylations and photorespiration seemed to be inhibited. In the dark in both CO₂ conditions, ¹⁴C incorporation was found in malate and aspartate but also in serine and glycerate in high CO₂ conditions. In light these changes in metabolic pathways may be related with the deleterious effects recorded on leaves after long-term expositions to high partial pressure of CO₂.Abbreviations DHAP dihydroxyacetone phosphate - PEP phosphonenolpyruvate - PEPCK phosphonenolpyruvatecarboxykinase - PGA 3-phosphoglyceric acid - RUBPc ribulose 1,5-bisphosphate carboxylase     

Regulation of the low-CO2-inducible polypeptides in Chlamydomonas reinhardtii

Polypeptides of 21, 36 and 37 kDa are induced in the unicellular green alga Chlamydomonas reinhardtii Dang. when cells are transferred from high (2%) to low (0.03%) CO₂ concentrations. The synthesis of these polypeptides is correlated with the induction of the CO₂-concentrating mechanism. In this work we studied the effect of the growth conditions on the synthesis of these polypeptides with the aim of clarifying whether the induction of all three of these low-CO₂-inducible polypeptides requires the same environmental factor. Our results showed that induction of the 21- and 36-kDa polypeptides under low-CO₂ conditions occurred only in the light, while the 37-kDa periplasmic carbonic anhydrase (EC 4.2.1.1) was induced in light, darkness, and in both synchronous and asynchronous cultures. In addition, induction of these polypeptides appeared to be determined more by the O₂/CO₂ ratio than by the CO₂ concentrations. None of these polypeptides could be induced in either of two different mutants of C. reinhardtii, one lacking ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) and the other with inactive enzyme. Our results indicate that the 21- and 36-kDa polypeptides are regulated by a mechanism different from that controlling the 37-kDa polypeptide.Abbreviations pCA (periplasmic) carbonic anhydrase - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - TAP Trisacetate phosphate medium The authors thank Prof. M. Spalding (Iowa State University, USA) for providing antisera to LIP-21 and LIP-36. We thank Prof. S. Bartlett and Dr. J. Moroney (Louisiana State University, USA) for providing antibodies to C. reinhardtii, Rubisco and 37-kDa pCA, respectively. This work was supported by the Instituto Tecnologico de Canarias.     

Fast and reversible response of thylakoid-associated polyamines during and after UV-B stress: a comparative study of the wild type and a mutant lacking chlorophyll b of unicellular green alga Scenedesmus obliquus

The functional and biochemical aspects of the photosynthetic apparatus in response to UV-B radiation were examined in unicellular oxygenic algae Scenedesmus obliquus. The wild type (Wt) and a chlorophyll b-less mutant (Wt-lhc) were used as a specific tool for the understanding of antenna role. Photosynthesis was monitored during and after UV-B stress by time resolved fluorescence spectroscopy and polarography. Carotenoids, such as neoxanthin, loroxanthin, lutein, violaxanthin, antheraxanthin, zeaxanthin, alpha- and beta-carotene, cellular and thylakoid-associated putrescine, spermidine, spermine and subcomplexes of light-harvesting complex (LHCII) of photosystem II (PSII) were investigated to assess their possible involvement in response to UV-B. Oxygen evolution depression by UV-B was higher in the Wt-lhc mutant than in the Wt. Photosynthesis recovery occurred in the Wt, but not in the mutant. The dissipation of excess excitation energy during UV-B stress was accompanied by changes in the thylakoid-associated polyamines which were much higher than changes in xanthophylls. We conclude that, at least in the unicellular green alga S. obliquus, mutants lacking chlorophyll b have significant lower capacity for recovery after UV-B stress. In addition, the comparison of xanthophylls and thylakoid-associated polyamines reveals that the latter are more responsive to UV-B stress and in a reversible manner.     

Treatment with NaHSO3 greatly enhances photobiological H2 production in the green alga Chlamydomonas reinhardtii

Treatment with NaHSO₃ induces a 10-fold increase in H₂ photoproduction in the filamentous N₂-fixing cyanobacterium Anabaena sp. strain PCC 7120. However, it is unclear whether this treatment also increases H₂ photoproduction in green alga. In this study, treatment with 13 mM NaHSO₃ resulted in about a 200-fold increase in H₂ production in Chlamydomonas reinhardtii, and this increase was most probably the result of reduced O₂ content and enhanced hydrogenase activity. Compared to the conventional strategy of sulfur deprivation, NaHSO₃ treatment results in a higher maximum rate of H₂ photoproduction, greater efficiency of conversion of light energy into H₂, shorter half-time to produce the maximum accumulated H₂ levels, and reduced costs because no centrifugation is involved. We therefore conclude that NaHSO₃ treatment is an efficient, rapid, and economic strategy for improving photobiological H₂ production in the green alga C. reinhardtii.     

The CO2 permeability of the plasma membrane of Chlamydomonas reinhardtii: mass-spectrometric 18O-exchange measurements from 13C18O2 in suspensions of carbonic anhydrase-loaded plasma-membrane vesicles

The unicellular green alga Chlamydomonas reinhardtii possesses a CO₂-concentrating mechanism. In order to measure the CO₂ permeability coefficients of the plasma membranes (PMs), carbonic anhydrase (CA) loaded vesicles were isolated from C. reinhardtii grown either in air enriched with 50 mL CO₂ · L?1} (high-C_i cells) or in ambient air (350 μL CO₂ · L?1}; low-C_i cells). Marker-enzyme measurements indicated less than 1% contamination with thylakoid and mitochondrial membranes, and that more than 90% of the PMs from high and low-C_i cells were orientated right-side-out. The PMs appeared to be sealed as judged from the ability of vesicles to accumulate [¹⁴C]acetate along a proton gradient for at least 10 min. Carbonic anhydrase-loaded PMs from high and low-C_i cells of C. reinhardtii were used to measure the exchange of ¹⁸O between doubly labelled CO₂ (¹³C¹⁸O₂) and H₂O in stirred suspensions by mass spectrometry. Analysis of the kinetics of the ¹⁸O depletion from ¹³C¹⁸O₂ in the external medium provides a powerful tool to study CO₂ diffusion across the PM to the active site of CA which catalyses ¹⁸O exchange only inside the vesicles but not in the external medium (Silverman et al., 1976, J Biol Chem 251: 4428–4435). The activity of CA within loaded PM vesicles was sufficient to speed-up the ¹⁸O loss to H₂O to 45360–128800 times the uncatalysed rate, depending on the efficiency of CA-loading and PM isolation. From the ¹⁸O-depletion kinetics performed at pH 7.3 and 7.8, CO₂ permeability coefficients of 0.76 and 1.49·10?3} cm·s?1}, respectively, were calculated for high C_i cells. The corresponding values for low-C_i cells were 1.21 and 1.8·10?3} cm·s?1}. The implications of the similar and rather high CO₂ permeability coefficients (low CO₂ resistance) in high and low-C_i cells for the CO_i-concentrating mechanism of C. reinhardtii are discussed.