Photosynthesis and Inorganic Carbon Accumulation in the Acidophilic Alga Cyanidioschyzon merolae

The intracellular pH and membrane potential were determined in the acidophilic algae Cyanidoschyzon merolae as a function of extracellular pH. The alga appear to be capable of maintaining the intracellular pH at the range of 6.35 to 7.1 over the extracellular pH range of 1.5 to 7.5. The membrane potential increase from −12 millivolts (negative inside) to −71 millivolts and thus ΔH⁺ decreased from −300 to −47 millivolts over the same range of extracellular pH. It is suggested that the ΔH⁺ may set the upper and lower limits of pH for growth. Photosynthetic performance was also determined as a function of pH. The cells appeared to utilize CO₂ from the medium as the apparent K_m(co2) was 2 to 3 micromolar CO₂ over the pH range of 1.5 to 7.5 C. merolae appear to possess a `CO₂ concentrating' mechanism.     

Acclimation of Photosynthetic Light Reactions during Induction of Inorganic Carbon Accumulation in the Green Alga Chlamydomonas reinhardtii

Cells of the unicellular green algae Chlamydomonas reinhardtii were grown in high dissolved inorganic carbon (DIC) concentrations (supplied with 50 milliliters per liter CO₂[g]) and transferred to low DIC concentrations (supplied with ≤ 100 microliters per liter CO₂[g]). Immediately after transfer from high to low DIC the emission of photosystem II related chlorophyll a fluorescence was substantially quenched. It is hypothesized that the suddenly induced inorganic carbon limitation of photosynthesis resulted in a phosphorylation of LHCII, leading to the subsequent state 1 to state 2 transition. After 2 hours of low-DIC acclimation, 77 K fluorescence measurements revealed an increase in the fluorescence emitted from photosystem I, due to direct excitation, suggesting a change in photosystem II/photosystem I stoichiometry or an increased light harvesting capacity of photosystem I. After 5 to 6 hours of acclimation a considerable increase in spillover from photosystem II to photosystem I was observed. These adjustments of the photosynthetic light reactions reached steady-state after about 12 hours of low DIC treatment. The quencher of fluorescence could be removed by 5 minutes of dark treatment followed by 5 minutes of weak light treatment, of any of four different light qualities. It is hypothesized that this restoration of fluorescence was due to a state 2 to state 1 transition in low-DIC acclimated cells. A decreased ratio of violaxanthin to zeaxanthin was also observed in 12 hour low DIC treated cells, compared with high DIC grown cells. This ratio was not coupled to the level of fluorescence quenching. The role of different processes during the induction of a DIC accumulating mechanism is discussed.     

Uptake and Accumulation of Inorganic Carbon by a Freshwater Diatom

Colman, B. and Rotatore, C. 1988. Uptake and accumulation ofinorganic carbon by a freshwater diatom.—J. exp Bot 39:1025–1032. The mechanism of uptake of inorganic carbon and its accumulationhas been studied in the freshwater diatom Navicula pelliculosa.No external carbonic anhydrase could be detected, although itwas detected in cell extracts. The rate of photosynthetic O₂evolution, in media in the range pH 7.5–8.5, exceededthe calculated rate of CO₂ supply 2- to 5-fold, indicating thatHCO^–₃ was taken up by the cells. At an external pH of7.5, the internal pH, measured by ¹⁴C-dimethyloxazolidine-2,4-dione distribution between the cells and the medium, was pH7.6 in the light and pH 7.4 in the dark. Accumulation of inorganiccarbon was determined by the silicone oil centrifugation methodand inorganic carbon pools of 23.5 mol m^–3 were found,a concentration 21.6-fold that in the external medium. The resultsindicate an active accumulation of inorganic carbon againstpH and concentration gradients in this diatom, probably by activeHCO^–₃ uptake. Key words: Bicarbonate transport, carbon dioxide, carbonic anhydrase, CO₂ affinity, CO₂ concentrating mechanism, internal pH, Navicula pelliculosa     

The Role of External Carbonic Anhydrase in Inorganic Carbon Acquisition by Chlamydomonas reinhardii at Alkaline pH

The role of external carbonic anhydrase in inorganic carbon acquisition and photosynthesis by Chlamydomonas reinhardii at alkaline pH (8.0) was studied. Acetazolamide (50 micromolar) completely inhibited external carbonic anhydrase (CA) activity as determined from isotopic disequilibrium experiments. Under these conditions, photosynthetic rates at low dissolved inorganic carbon (DIC) were far greater than could be maintained by CO₂ supplied from the spontaneous dehydration of HCO₃⁻ thereby showing that C. reinhardii has the ability to utilize exogenous HCO₃⁻. Acetazolamide increased the concentration of DIC required to half-saturate photosynthesis from 38 to 80 micromolar, while it did not affect the maximum photosynthetic rate. External CA activity was also removed from the cell-wall-less mutant (CW-15) by washing. This had no effect on the photosynthetic kinetics of the algae while the addition of acetazolamide to washed cells (CW-15) increased the K_½^DIC from 38 to 80 micromolar. Acetazolamide also caused a buildup of the inorganic carbon pool upon NaHCO₃ addition, indicating that this compound partially inhibited internal CA activity. The effects of acetazolamide on the photosynthetic kinetics of C. reinhardii are likely due to the inhibition of internal rather than a consequence of the inhibition of external CA. Further analysis of the isotopic disequilibrium experiments at saturating concentration of DIC provided evidence consistent with active CO₂ transport by C. reinhardii. The observation that C. reinhardii has the ability to take up both CO₂ and bicarbonate throws into question the role of external CA in the accumulation of DIC in this alga.     

Carbon Accumulation during Photosynthesis in Leaves of Nitrogen- and Phosphorus-Stressed Cotton

Leaves of cotton (Gossypium hirsutum L.) accumulate considerable dry mass per unit area during photosynthesis. The percentage of C in that accumulated dry mass was estimated as the regression coefficient (slope) of a linear regression relating C per unit area to total dry mass per unit area. Plants were grown on full nutrients or on N- or P-deficient nutrient solutions. In the fully nourished controls, the mass that accumulated over a 9-hour interval beginning at dawn contained 38.6% C. N and P stress increased the C concentration of accumulated mass to 49.7% and 45.1%, respectively. Nutrient stress also increased the starch concentration of accumulated mass, but starch alone could not account for the differences in C concentration. P stress decreased the estimated rate of C export from source leaves, calculated as the difference between C assimilation and C accumulation. The effect of P stress on apparent export was very sensitive to the C concentration used in the calculation, and would not have been revealed with an assumption of unchanged C concentration in the accumulated mass.     

Transport and Fixation of Inorganic Carbon during Photosynthesis of Anabaena Grown under Ordinary Air. I. Active Species of Inorganic Carbon Utilized for Photosynthesis

Inorganic carbon transport during photosynthesis of cyanobacteriumAnabaena variabilis grown under ordinary air was investigatedby supplying ¹⁴CO₂ or H¹⁴CO₃^– solution to three differentstrains. Both CO₂ and HCO₃^– were accumulated within thealgal cells. In the cell suspension from which dissolved inorganiccarbon had been depleted by pre-illumination, CO₂ was transportedand accumulated faster than HCO₃^–. When the concentrationof HCO₃^– injected into the cell suspension of A. variabilisM3 was 25 times as high as that of CO2 (the expected ratio atequilibrium at pH 7.8), the initial rates of fixation of bothinorganic carbon species were practically the same. On the otherhand, when ¹⁴CO₂ or H¹⁴CO₃^– was added under steady statephotosynthetic conditions, both carbon species were transportedat similar rates. The ratio of fixed to transported carbon measuredafter the initial 5 s was only 23–27% regardless of thecarbon species supplied. This percentage is much lower thanthat reported for Chlorella cells. ¹ To whom reprint requests should be addressed (Received June 30, 1986; Accepted December 16, 1986)     

Utilization of Exogenous Inorganic Carbon Species in Photosynthesis by Chlorella pyrenoidosa

The nature of the inorganic carbon utilized during photosynthesis by Chlorella pyrenoidosa was investigated using three experimental techniques (open gas analysis system with “artificial leaf” or “aqueous” chambers and O₂ electrode system) to measure carbon assimilation. Photosynthesis was studied as a function of pH and CO₂ concentration. The CO₂ concentration was inadequate to meet the requirements of photosynthesis only when HCO₃⁻ was added at high pH. Under all other conditions, the low and constant K_m (CO₂), in contrast to the highly variable K_m (HCO₃⁻), suggested that CO₂ was the major species utilized.     

Form of Inorganic Carbon Utilized for Photosynthesis in Chlamydomonas reinhardtii1

The effect of carbonic anhydrase (CA) on time courses of photosynthetic¹⁴C incorporation in the presence of ¹⁴CO₂ or NaH¹⁴CO₃ was studiedwith cells of Chlamydomonas reinhardtii which had been grownunder ordinary air (low-CO₂ cells) or air enriched with 4% CO₂(high-CO₂ cells). Experimental data obtained at 20°C andpH 8.0 suggested that the major form of inorganic carbon utilizedby high-CO₂ cells was CO₂, while that utilized by low-CO₂ cellswas HCO₃^–. The cell suspension showed CA activity which was comparableto that observed in the sonicate of cells. Both activities werehigher in low-CO₂ cells than in high-CO₂ cells. The mechanism by which HCO₃^– is utilized by low-CO₂ cellsof C. reinhardtii is discussed. ³Present address: Department of Biology, Faculty of Science,University of Niigata, Niigata 950-21, Japan. (Received August 4, 1982; Accepted January 19, 1983)     

The Interrelations of Cell Turgor Pressure, Gas-vacuolation, and Buoyancy in a Blue-green Alga

The increase in pressure required to collapse gas vacuoles onsuspending the cells of the blue-green alga Anabaena flos-aquaein hypertonic sucrose solutions shows the turgor pressure tovary over the range of 265 to 459 KN m^–2 under differentculture conditions. The cell turgor increased at a rate of upto KN m^–2 h^–1 on transferring the alga from lowto high light intensity. This rise appears to be a result ofthe accumulation of photosynthate, as it is dependent on thepresence of carbon dioxide in the gas phase and is inhibitedby DCMU. Experiments using ¹⁴CO² indicate that the increasedrate of photosynthesis during the high light exposure is easilysufficient to account for the observed turgor rise. The rise in turgor can bring about collapse of sufficient ofthe alga's gas vacuoles to destroy its buoyancy. Higher turgorpressures, and consequently a lower degree of gas vacuolationand buoyancy, were maintained when the alga was kept at highlight intensitives for a week and more. The significance ofthis behaviour is discussed in relation to stratification ofplanktonic blue-green algae in natural habitats.     

Utilization Modes of Inorganic Carbon for Photosynthesis in Various Species of Chlorella

Rates of CO₂ and HCC₃^– fixation in cells of various Chlorellaspecies in suspension were compared from the amounts of ¹⁴Cfixed during the 5 s after the injection of a solution containingonly ¹⁴CO₂ or H¹⁴CO₃^–. Results indicated that irrespectiveof the CO₂ concentration during growth, Chlorella vulgaris 11h and C. miniata mainly utilized CO₂, whereas C. vulgaris C-3,C. sp. K. and C. ellipsoidea took up HCO₃^– in additionto CO₂. Cells of C. pyrenoidosa that had been grown with 1.5%CO₂ (high-CO₂ cells) mainly utilized CO₂, whereas those grownwith air (low-CO₂ cells) utilized HCO₃^– in addition toCO₂. Cells that utilized HCO₃^– had carbonic anhydrase(CA) on their surfaces. The effects of Diamox and CA on the rates of CO₂ and HCO₃^–fixation are in accord with the inference that HCO₃^– wasutilized after conversion to CO₂ via the CA located on the cellsurface. CA was found in both the soluble and insoluble fractions;the CA on the cell surface was insoluble. Independent of the modes of utilization, the apparent K_m (NaHCO₃)for photosynthesis was much lower in low-CO₂ cells than in high-CO₂ones. The fact that the CA in the soluble fraction in C. vulgarisC-3 was closely correlated with the K_m(NaHCO₃) indicates thatsoluble CA lowers the K_m. ¹ Dedicated to the late Professor Joji Ashida, one of the foundersand first president of the Japanese Society of Plant Physiologists. ⁴ On leave from Research and Production Laboratory of Algology,Bulgarian Academy of Sciences, Sofia. (Received September 14, 1982; Accepted March 1, 1983)     

Some Observations on the Pattern of Sporulation in a Blue-green Alga, Anabaena doliolum

Sporulation in Anabaena doliolum begins in the middle of thetwo heterocysts and proceeds towards the heterocystous ends.Two inorganic nitrogen sources—potassium nitrate and ammoniumchloride inhibit sporulation, whereas glucose promotes it. Duringsporulation, the reductive ability of the heterocyst graduallydiminishes. It is concluded that spore differentiation in this alga is controlledby critical levels of nitrogen and of sugar in the cell. Thecritical levels are probably regulated by the heterocyst.     

Effect of pH on Inorganic Carbon Uptake in Algal Cultures

Biomass production by the green algae Scenedesmus obliquus and Chlorella vulgaris in intensive laboratory continuous cultures was considerably affected by the pH at which the cultures were maintained. Carbon photoassimilation experiments revealed that pH values in the range of 8 to 9 were important for determining the free CO₂ concentrations in the medium. With higher pH values, additional pH effects were observed involving a decrease in the relative high affinity of low CO₂-adapted algae to free CO₂. The carbon uptake rate by high CO₂-adapted algae after transfer to low free CO₂ medium was characterized by a lag period of about 30 min, after which the affinity of the algae to CO₂ increased considerably. Both continuous growth and carbon uptake experiments indicated that artificially maintained high free CO₂ concentrations are recommended for maximal production in intensive outdoor algal cultures.     

Oxygen Isotope Fractionation during Photosynthesis in a Blue-Green and a Green Alga

Oxygen isotope fractionation (¹⁸O/¹⁶O) at the natural abundance level has been measured during photosynthesis of a blue-green and a green alga. When sufficient attention is paid to removal of contaminating air O₂ before and during the experiments, then the photosynthetic O₂ evolved, as compared to the water O₂, had an average difference of −0.36% for a blue-green alga and −0.80% for a green alga. These experiments suggest that there is no reason to invoke an inverse isotope effect in photosynthesis as part of the explanation for the ¹⁸O enrichment in atmospheric O₂ relative to O₂ in oceanic waters. In addition, in an indirect way, the experiments also support the argument that the bulk of O₂ evolved during photosynthesis comes from water. A 10% contribution of O₂ arising from CO₂ would have been detectable in the present work.     

Photosynthesis and Inorganic Carbon Usage by the Marine Cyanobacterium, Synechococcus sp

The marine cyanobacterium, Synechococcus sp. Nägeli (strain RRIMP N1) changes its affinity for external inorganic carbon used in photosynthesis, depending on the concentration of CO₂ provided during growth. The high affinity for CO₂ + HCO₃⁻ of air-grown cells (K_½ < 80 nanomoles [pH 8.2]) would seem to be the result of the presence of an inducible mechanism which concentrates inorganic carbon (and thus CO₂) within the cells. Silicone-oil centrifugation experiments indicate that the inorganic carbon concentration inside suitably induced cells may be in excess of 1,000-fold greater than that in the surrounding medium, and that this accumulation is dependent upon light energy. The quantum requirements for O₂ evolution appear to be some 2-fold greater for low CO₂-grown cells, compared with high CO₂-grown cells. This presumably is due to the diversion of greater amounts of light energy into inorganic carbon transport in these cells.

A number of experimental approaches to the question of whether CO₂ or HCO₃⁻ is primarily utilized by the inorganic carbon transport system in these cells show that in fact both species are capable of acting as substrate. CO₂, however, is more readily taken up when provided at an equivalent concentration to HCO₃⁻. This discovery suggests that the mechanistic basis for the inorganic carbon concentrating system may not be a simple HCO₃⁻ pump as has been suggested. It is clear, however, that during steady-state photosynthesis in seawater equilibrated with air, HCO₃⁻ uptake into the cell is the primary source of internal inorganic carbon.

     

A Biochemical Study of Spore Germination in the Blue-green Alga Anabaena doliolum

The spores of Anabaena doliolum formed in light (light spores)and after transfer to darkness (dark spores) are biochemicallydifferent in that the light spores contain chlorophyll a andphycocyanin, while dark spores seem to lack them. The apparentbiosyntheses accompanying dark-spore germination seem to proceedin the following order: RNA, chlorophyll a, phycocyanin andDNA. Results of chloramphenicol treatment indicate that proteinsynthesis precedes RNA synthesis. The biosynthetic events followingRNA synthesis show a requirement for light.     

The Influence of Leaf Age on C(4) Photosynthesis and the Accumulation of Inorganic Carbon in Flaveria trinervia, a C(4) Dicot

Characteristics of C₄ photosynthesis were examined in young, mid-age, and mature leaves of Flaveria trinervia (an NADP-malic enzyme-type C₄ dicot). The turnover of [4-¹⁴C] (malate plus aspartate) following a pulse with ¹⁴CO₂ was similar in leaves of different ages (apparent half-time of 18-25 seconds). However, the rate of ¹⁴CO₂ incorporation in mid-age leaves was about 1.5-fold higher than in young leaves, and about 2.5-fold higher than in mature leaves. The rate of ¹⁴CO₂ fixation was proportional to the total active pool of malate plus aspartate but was not correlated with the total photosynthetically derived inorganic carbon pool. The leaf's ability to concentrate inorganic carbon photosynthetically declined during leaf expansion, from 29 down to 7 nanomoles per milligram chlorophyll. Similarly, the active aspartate pool also declined during leaf expansion, from about 123 down to 20 nanomoles per milligram chlorophyll. Enhanced metabolism of aspartate to CO₂ and pyruvate in young leaves is suggested to facilitate the maintenance of high CO₂ levels in bundle sheath cells which are thought to have a higher conductance to CO₂.     

Regeneration of NADPH by Frozen-thawed Cells of a Blue-green Alga,Synechococcus sp.

The photoreduction of NADP ⁺ and its associated reactions were studied in a blue-green algal preparation that was frozen in liquid nitrogen and thawed at room temperature. The preparation was capable of photoreducing exogeneous NADP⁺. Water was the ultimate electron donor for the reduction. The optimum pH was 7.5 ~ 8.0, and optimum temperature, around 50°C. Light saturation for NADP⁺ photoreduction was reached at 50 μEinstein/m²/sec. Factors limiting the stability of the preparation were examined and a possible application of this cofactor regeneration system is discussed.     

Uptake of Inorganic Carbon by Isolated Chloroplasts of the Unicellular Green Alga Chlorella ellipsoidea

Chloroplasts, isolated from protoplasts of the green alga, Chlorella ellipsoidea, were estimated to be 99% intact by the ferricyanide-reduction assay, and gave CO₂ and PGA-dependent rates of O₂ evolution of 64.5 to 150 micromoles per milligram of chlorophyll per hour, that is 30 to 70% of the photosynthetic activity of the parent cells. Intact chloroplasts showed no carbonic anhydrase activity, but it was detected in preparations of ruptured organelles. Rates of photosynthesis, measured in a closed system at pH 7.5, were twice the calculated rate of CO₂ supply from the uncatalyzed dehydration of HCO₃⁻ indicating a direct uptake of bicarbonate by the intact chloroplasts. Mass spectrometric measurements of CO₂ depletion from the medium on the illumination of chloroplasts indicate the lack of an active CO₂ transport across the chloroplast envelope.