High CO(2) Requiring Mutant of Anacystis nidulans R(2)

Some physiological characteristics of a mutant (E₁) of Anacystis nidulans R₂, incapable of growing at air level of CO₂, are described. E₁ is capable of accumulating inorganic carbon (C_i) internally as efficiently as the wild type (R₂). The apparent photosynthetic affinity for C_i in E₁, however, is some 1000 times lower than that of R₂. The kinetic parameters of ribulose 1,5-bisphosphate carboxylase/oxygenase from E₁ are similar to those observed in R₂. The mutant appears to be defective in its ability to utilize the intracellular C_i pool for photosynthesis and depends on extracellular supply of Ci in the form of CO₂. The very high apparent photosynthetic K_m (CO₂) of the mutant indicate a large diffusion resistance for CO₂. Data obtained here are used to calculate the permeability coefficient for CO₂ between the bulk medium and the carboxylation site of cyanobacteria.     

CO2 fixation and its regulation in Anacystis nidulans (Synechococcus)

Anacystis nidulans (Synechococcus) had a minimal doubling time of 5 hrs at 30 degrees C at saturating light intensity and carbon dioxide concentration. Half maximal growth rates in saturating CO2 occured at a light intensity of 0.54 mW per cm2, and there was an apparent threshold intensity of 0.13 mW per cm2 below which no growth occurred. Growth rate in saturating light was dependent on the concentration of CO2+H2CO3 in the medium, rather than on total dissolved CO2; half maximal rates were estimated at 0.1 mM CO2+H2CO3. Under saturating conditions of light and CO2, 14CO2 was fixed primarily into 3-PGA, and subsequently moved into sugar phosphates and amino acids. Incorporation into aspartate was relatively slow. CO2 fixation was strictly light-dependent. The changes in adenylate and pyridine nucleotide pools were followed in light/dark and dark/light transitions. Whereas adenylates relaxed slowly over 15-20 min to the concentrations characteristic of illuminated cells following the abrupt changes induced by darkening, the sharp drop in intracellular NADPH showed little dark recovery although rapid restoration occurred on reillumination. Other pyridine nucleotides showed no changes during these transitions. The nucleotide specificity and Km of partially purfied GAP dehydrogenase suggest a role for this enzyme in the regulation of CO2 fixation.     

Adaptation of the Photosynthetic Apparatus of Anacystis nidulans to Irradiance and CO2-Concentration

Homocontinuous cultures of the cyanobacterium Anacystis nidulans (syn. Synechococcus sp. PCC 6301) were grown at white light intensities of 2 and 20 W/m², and supplied with 0.03 and 3 % CO₂ enriched air. The mutual influence of these growth factors on the development of the photosynthetic apparatus was studied by analyses of the pigment content, by low temperature absorbance and fluorescence spectroscopy, by analyses of oxygen evolution light-saturation curves, and by SDS PAGE of isolated phycobilisomes. The two growth factors, light and CO₂, distinctly affect the absorption cross section of the photosynthetic apparatus, which is expressed by its pigment pattern, excitation energy distribution and capacity. In response to low CO₂ concentrations, the phycocyanin / allophycocyanin ratios were lower and one linker polypeptide L³⁰_R, of the phycobilisomes was no longer detectable in SDS PAGE. Apparently, low CO₂ adaptation results in shorter phycobilisome rods. Specifically, upon adaptation to low light intensities, the chlorophyll and the phycocyanin content on a per cell basis increase by about 50% suggesting a parallel increase in the amount of phycobilisomes and photosystem core-complexes. Low light adaptation and low CO₂ adaptation both cause a shift of the excitation energy distribution in favor of photosystem I. Variations in the content of the “anchor” polypeptides L⁶⁰_CM and L⁷⁵_CM are possibly related to changes in the excitation energy transfer from phycobilisomes to the photosystem II and photosystem I core-complexes.     

Adaptation to Low CO(2) Level in a Mutant of Anacystis nidulans R(2) which Requires High CO(2) for Growth

The mutant E₁ of Anacystis nidulans R₂ requires high CO₂ concentration for growth but was able to adapt to low CO₂ concentration. This was exhibited by the increased ability to accumulate inorganic carbon within the cells and the large increase in the amount of a 42-kilodalton polypeptide located in the cytoplasmic membrane. The adaptation occurred in E₁ cells at an extracellular CO₂ concentration as high as 0.3%, which was 8 times the concentration for maximal adaptation in R₂ cells. The ability of E₁ cells to exhibit low CO₂ characteristics at a higher CO₂ concentration was attributed to lower intracellular CO₂ concentration.     

Photochemical apparatus organization in Synechococcus 6301 (Anacystis nidulans). Effect of phycobilisome mutation

The photochemical apparatus organization in Synechococcus 6301 (Cyanophyceae) was investigated under various experimental conditions. Wild type (WT) Synechococcus produced phycobilisomes (PBSs) containing normal levels of phycocyanin (Phc) and allophycocyanin (Aphc). The ratio of reaction centers(RC) RCII/RCI of 0.4 was the same in WT and the mutant strain AN112, whereas RCH/PBS was 1.9:1 in WT and 1:1 in AN112. Excitation of WT cells with broad-band 620 nm light, which is absorbed primarily by Phc and Aphc and to a much lesser extent by chlorophyll (Chl), sensitized the RC of photosystem (PS) II at about 15 times the rate it sensitized RCI. This implies that PBSs are associated exclusively with PSII complexes and that PBS excitation is not transferred to PSI. The AN112 mutant of Synechococcus produced smaller PBSs consisting of the Aphc-containing core and of only six Phc-containing hexamers, respectively. It lacked about 65% of the Phccontaining rod substractures. Under our experimental conditions, the effective absorption cross section of the mutant PBS was only about half that of the WT. In agreement, the rate of RCII excitation by 620 nm light was also about half of that measured in the WT. Thus, the rate of light absorption by PSII depends directly on PBS size and composition. The low rate of RCI excitation with 620 nm light was the same in WT and AN112 cells, apparently independent of the PBS effective absorption cross section. We propose a strict structural-functional association between PBS and PSII complex. PSI is a structurally distinct entity and it receives excitation independently from its own Chl light-harvesting antenna.Abbreviations PBS phycobilisome - Phc phycocyanin - Aphc allophycocyanin - PS photosystem - RC reaction center - P700 reaction center of PSI - Q primary electron acceptor of PSII - Chl chlorophyll - MV methyl viologen - Tris Tris(hydroxymethyl)-aminomethane - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Biosynthesis of a 42-kD Polypeptide in the Cytoplasmic Membrane of the Cyanobacterium Anacystis nidulans Strain R2 during Adaptation to Low CO(2) Concentration

When cells of Anacystis nidulans strain R2 grown under high CO₂ conditions (3%) were transferred to low CO₂ conditions (0.05%), their ability to accumulate inorganic carbon (C_i) increased up to 8 times. Cytoplasmic membranes (plasmalemma) isolated at various stages of low CO₂ adaptation were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There was a marked increase of a 42-kilodalton polypeptide in the cytoplasmic membrane during adaptation; a linear relationship existed between the amount of this polypeptide and the C_i-accumulating capability of the cells. No significant changes were observed during this process in the amount of other polypeptides in the cytoplasmic membranes or in the polypeptide profiles of the thylakoid membranes, cell walls, and soluble fractions. Spectinomycin, an inhibitor of protein biosynthesis, inhibited both the increase of the 42-kilodalton polypeptide and the induction of high C_i-accumulating capability. The incorporation of [³⁵S]sulfate into membrane proteins was greatly reduced during low CO₂ adaptation. Radioautograms of the ³⁵S-labeled membrane proteins revealed that synthesis of the 42-kilodalton polypeptide in the cytoplasmic membrane was specifically activated during the adaptation, while that of most other proteins was greatly suppressed. These results suggested that the 42-kilodalton polypeptide in the cytoplasmic membrane is involved in the active C_i transport by A. nidulans strain R2 and its synthesis under low CO₂ conditions leads to high C_i-transporting activity.     

Enhanced Dark CO(2) Fixation by Preilluminated Chlorella pyrenoidosa and Anacystis nidulans

The products of short time photosynthesis and of enhanced dark ¹⁴CO₂ fixation (illumination in helium prior to addition of ¹⁴CO₂ in dark) by Chlorella pyrenoidosa and Anacystis nidulans were compared. Glycerate 3-phosphate, phosphoenolpyruvate, alanine, and aspartate accounted for the bulk of the ¹⁴C assimilated during enhanced dark fixation while hexose and pentose phosphates accounted for the largest fraction of isotope assimilated during photosynthesis. During the enhanced dark fixation period, glycerate 3-phosphate is carboxyl labeled and glucose 6-phosphate is predominantly labeled in carbon atom 4 with lesser amounts in the upper half of the C₆ chain and traces in carbon atoms 5 and 6. Tracer spread throughout all the carbon atoms of photosynthetically synthesized glycerate 3-phosphate and glucose 6-phosphate. During the enhanced dark fixation period, there was a slow formation of sugar phosphates which subsequently continued at 5 times the initial rate long after the cessation of ¹⁴CO₂ uptake. To explain the kinetics of changes in the labelling patterns and in the limited formation of the sugar phosphates during enhanced dark CO₂ fixation, the suggestion is made that most of the reductant mediating these effects did not have its origin in the preillumination phase.

It is concluded that a complete photosynthetic carbon reduction cycle operates to a limited extent, if at all, in the dark period subsequent to preillumination.

     

Effects of CO(2) Concentration during Growth on Fatty Acid Composition in Microalgae

The degree of unsaturation of fatty acids was higher in Chlorella vulgaris 11h cells grown with air (low-CO₂ cells) than in the cells grown with air enriched with 2% CO₂ (high-CO₂ cells). The change in the ratio of linoleic acid to α-linolenic acid was particularly significant. This change of the ratio was observed in four major lipids (monogalactosyldiacylglycerol, digalactosyldiacylglycerol, phosphatidylcholine, and phosphatidylethanolamine). The relative contents of lipid classes were essentially the same both in high-CO₂ and low-CO₂ cells. After high-CO₂ cells were transferred to low CO₂ condition, total amount of fatty acids remained constant but the relative content of α-linolenic acid increased during a 6-hour lag phase in growth with concomitant decreases in linoleic and oleic acids. When low-CO₂ cells were transferred to high CO₂ condition, total amount of fatty acids and relative content of oleic acid increased significantly. The amount of α-linolenic acid remained almost constant, while the amounts of palmitic, oleic, and linoleic acids increased. Similar, but smaller, changes in fatty acid compositions were observed in two species of green algae Chlamydomonas reinhardtii and Dunaliella tertiolecta. However, no difference was found in Euglena gracilis, Porphyridium cruentum, Anabaena variabilis, and Anacystis nidulans.     

Distinctive Light and CO(2)-Fixation Requirements of Nitrate and Ammonium Utilization by the Cyanobacterium Anacystis nidulans

The effect of light intensity on the rates of ammonium and nitrate uptake and of CO₂ fixation has been determined in intact Anacystis nidulans cells. Ammonium uptake became saturated at photon flux values of about 60 microeinsteins per square meter per second, whereas both nitrate uptake and CO₂ fixation reached saturation at about 250 microeinsteins per square meter per second, the rates of the two latter processes being tightly correlated at any light intensity assayed. Inhibition of ammonium assimilation resulted in the loss of correlation between CO₂ fixation and nitrate uptake, the latter process exhibiting then a reduced light requirement. The results establish a clear distinction between ammonium utilization and nitrate utilization with regard to their light requirement and to the nature of their dependence upon CO₂ fixation.     

Effect of CO(2) Concentration on Glycine and Serine Formation during Photorespiration

Amount and products of photosynthesis during 10 minutes were measured at different ¹⁴CO₂ concentrations in air. With tobacco (Nicotiana tabacum L. cv. Maryland Mammoth) leaves the percentage of ¹⁴C in glycine plus serine was highest (42%) at 0.005% CO₂, and decreased with increasing CO₂ concentration to 7% of the total at 1% CO₂ in air. However, above 0.03% CO₂ the total amount of ¹⁴C incorporated into the glycine and serine pool was about constant. At 0.005% or 0.03% CO₂ the percentage and amount of ¹⁴C in sucrose was small but increased greatly at higher CO₂ levels as sucrose accumulated as an end product. Relatively similar data were obtained with sugar beet (Beta vulgaris L. cv. US H20) leaves. The results suggest that photorespiration at high CO₂ concentration is not inhibited but that CO₂ loss from it becomes less significant.     

Inhibition of inorganic carbon transport by oxygen in a high CO2-requiring mutant (E1) of Anacystis nidulans R2

The effect of O₂ on inorganic carbon (C_i) transport was studied with a high CO₂-requiring mutant (E₁) of Anacystis nidulans R₂. Oxygen (above 2%) inhibited C_i transport by 15–35|X% at CO₂ concentrations above 200 μl/l, but had no apparent effect at low, limiting CO₂ concentration. The action spectra for C_i transport measured in the presence or absence of 20% O₂ showed two peaks around 684 and 625 nm, corresponding to chlorophyll a and phycocyanin absorption, respectively. The difference between these two spectra (anaerobic minus aerobic) showed one peak around 625 nm, which indicates that a linear electron transport from water to O₂ is involved in the O₂ inhibition of C_i transport. Dithiothreitol could overcome the inhibition by O₂. The results suggested that the O₂ inhibition is a result of inactivation of the C_i-transporting system.     

Separation and Characterization of Thylakoid and Cell Envelope of the Blue-green Alga (Cyanobacterium) Anacystis nidulans

The thylakoid and the cell envelope of the blue-green alga Anacystisnidulans were separated by mechanical disruption of lysozyme-treatedcells followed by differential and density gradient centrifugation.The prepared envelope was composed of an outer membrane, a peptidoglycanlayer and possibly a part of the cytoplasmic membrane. The preparedthylakoid retained the size and intricate structure typicalof the thylakoid membrane of this alga. Light absorption andfluorescence spectra revealed that the envelope contained carotenoids,a pigment with an absorption maximum at 748 nm (P750), and asmall amount of pheophytin-like pigment with an absorption maximumat 673 nm. The thylakoid contained chlorophyll a and carotenoidsbut no P750. The thylakoid contained five kinds of carotenoids,the major ones being rß-carotene and zeaxanthin, whereasthe cell envelope contained two kinds of carotenoids, zeaxanthinand nostoxanthin. Four kinds of lipids, abundant in the blue-greenalgae, were present in both the thylakoid and the cell envelope.However, the content of sulfolipid was very low in the cellenvelope. The polypeptide compositions differed between thethylakoid and the cell envelope. Similarities between blue-greenalgal cells and eukaryotic chloroplasts are discussed with respectto the spectrophotometric and biochemical characteristics ofthe thylakoid and the envelope. (Received March 7, 1981; Accepted May 22, 1981)     

Changes in the Polypeptide Composition of the Cytoplasmic Membrane in the Cyanobacterium Anacystis nidulans during Adaptation to Low CO2 Conditions

When cells of Anacystis nidulans grown under high CO₂ conditions(3%) were transferred to low CO₂ conditions (0.05%), their abilityto transport extracellular inorganic carbon (C_i) into the cellsincreased severalfold. There was a marked increase of 42-kDapolypeptide in the cytoplasmic membranes during the adaptationto low CO₂ conditions, while no changes were observed in thepolypeptide compositions of the thylakoid membranes and cellwalls. The results suggested that the increase of the 42-kDapolypeptide during adaptation is involved in the increased abilityto transport C_i (Received January 28, 1985; Accepted May 30, 1985)     

Entrapment of active ion-permeable cyanobacteria (Anacystis nidulans) in calcium alginate

Summary Cells of the unicellular cyanobacterium Anacystis nidulans were made permeable to ions by treating them with lysozyme and EDTA in a way that leaves the photosynthetic water-splitting function, the photoreduction of exogenous oxidants and the peptidoglycan exoskeleton of the cell virtually intact. The permeabilized cells (permeaplasts) were subsequently immobilized by entrapment in calcium alginate beads. The immobilized preparation exhibits remarkable stability both on storage and in action. On prolonged storage at room temperature in darkness, its photosynthetic activity deteriorates one-third as fast as the activity of immobilized intact cells. Illumination accelerates deactivation. Tested in prolonged runs, however, performed in an illuminated open reactor, alginate-immobilized Anacystis permeaplasts were capable of photoreducing ionic oxidants (ferricyanide) and of exporting ionic reductants (ferrocyanide) to the suspension medium continuously for more than 5 h before being totally inactivated. It is also shown that the major impediment to the photoreduction performance of immobilized permeaplasts arises from diffusion limitations, while the photonic limitation due to light reflection and scattering is approx. 7%.Abbreviations Chl chlorophyll - CSTR continuously stirred tank reactor - EDTA ethylenediaminetetraacetate - FeCN potassium ferricyanide - pBQ p-benzoquinone - PD p-phenylenediamine - PD_ox p-phenylenediamine in the presence of excess potassium ferricyanide - Hepes N-2-hydroxyethylpiperazine-N-2 ethane-sulphonic acid     

Action of Heavy Metals on Hill Activity and O(2) Evolution in Anacystis nidulans

Addition of 5 micromolar Cu²⁺, Cd²⁺, and Zn²⁺ was inhibitory to 10 micromolar H₂O₂-supported Hill activity (dichlorophenolindophenol reduction) and O₂ evolution in membrane preparation from Anacystis nidulans. The reversal of Cd²⁺ and Zn²⁺ inhibition, in contrast to Cu²⁺, by exogenously added catalase (EC 1.11.1.6) suggested that the former cations were inhibitory to H₂O₂ degradation. Ascorbic acid (20 micromolar) supported 27% of the Hill activity which was insensitive to DCMU (10 micromolar) and the remaining activity, attributable to the DCMU sensitive process, was sensitive to inhibition by Cu²⁺ only. It is suggestive that the action site of Cd²⁺ and Zn²⁺ is located between the electron donation sites of H₂O₂ and ascorbic acid, while that of Cu²⁺ is located beyond it. Electron donation by reduced glutathione was insensitive to DCMU and Cu²⁺, indicating that the action site of Cu²⁺ is prior to its electron donation site. Further, the phenanthroline (10 micromolar) reversal of Cu²⁺ inhibition of Hill activity suggested a tentative action site of Cu²⁺ at the level of cytochrome.     

Modulation of nitrate uptake in Anacystis nidulans by the balance between ammonium assimilation and CO2 fixation

Gradual inhibition of ammonium assimilation in Anacystis nidulans cells by increasing concentrations of 5-hydroxylysine resulted in a progressive enhancement of nitrate uptake. For 5-hydroxylysine-treated cells, the magnitude of the inhibition of nitrate uptake promoted by added ammonium was dependent on the ammonium assimilation capacity. In cells with a moderate ammonium assimilation activity, acceleration of CO2 fixation induced by bicarbonate addition antagonized the negative effect of ammonium, allowing full nitrate uptake activity. The results support the contention that nitrate utilization is under the feed-back control exerted by products of its own assimilation via ammonium, the inhibitory effect being potentiated by ammonium addition and alleviated by enhanced CO2 fixation. Results of amino acid analysis in cells exhibiting different capacities to utilize nitrate speak against these compounds as direct effectors of nitrate uptake.     

Response of Photosynthetic Apparatus of Spring Barley (Hordeum vulgare L.) to Combined Effect of Elevated CO2 Concentration and Different Growth Irradiance

The response of barley (Hordeum vulgare L. cv. Akcent) to various photosynthetic photon flux densities (PPFDs) and elevated [CO₂] [700 μmol (CO₂) mol⁻¹; EC] was studied by gas exchange, chlorophyll (Chl) a fluorescence, and pigment analysis. In comparison with barley grown under ambient [CO₂] [350 μmol (CO₂) mol⁻¹; AC] the EC acclimation resulted in a decrease in photosynthetic capacity, reduced stomatal conductance, and decreased total Chl content. The extent of acclimation depression of photosynthesis, the most pronounced for the plants grown at 730 μmol m⁻² s⁻¹ (PPFD₇₃₀), may be related to the degree of sink-limitation. The increased non-radiative dissipation of absorbed photon energy for all EC plants corresponded to the higher de-epoxidation state of xanthophylls only for PPFD₇₃₀ barley. Further, a pronounced decrease in photosystem 2 (PS2) photochemical efficiency (given as F_V/F_M) for EC plants grown at 730 and 1 200 μmol m⁻² s⁻¹ in comparison with AC barley was related to the reduced epoxidation of antheraxanthin and zeaxanthin back to violaxanthin in darkness. Thus the EC conditions sensitise the photosynthetic apparatus of high-irradiance acclimated barley plants (particularly PPFD₇₃₀) to the photoinactivation of PS2. This revised version was published online in August 2006 with corrections to the Cover Date.