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Changes in photosynthetic carbon metabolism during the glucose bleaching of Chlorella protothecoides cells were investigated using NaH14CO3 as tracer. Several hours after incubating the green algal cells in the glucose medium in the dark, the rates of 14C-incorporation into glucose polymers and sucrose decreased and the incorporation into the lipid fraction (fatty acids) greatly increased. At this stage, the rate of photosynthetic 14CO2 fixation and the chlorophyll content were practically the same as in the starting green cells. Afterwards, the photosynthetic capacity and chlorophyll content continued to decrease throughout the experimental period. In contrast, when photosynthetic 14CO2 fixation of green cells was carried out in the medium containing glucose, the rate of 14C-incorporation into glucose polymers increased, though there was no change in the incorporations into sucrose and the lipid fraction.

Illumination with blue light enhanced the production of ammonia by cells of C. vulgaris 11h, while no such effect was induced by red light illumination. Addition of ammonia caused increases in ATP levels and decreases in Pi and ADP levels. When 5 mm NH4Cl was added to phosphorylase and amylase isolated from the cells of C. vulgaris 11h, their activities increased about 5–15% and 40–100%, respectively.

Inorganic carbon transport during photosynthesis of cyanobacterium Anabaena variabilis grown under ordinary air was investigated by supplying 14CO2 or H14CO3− solution to three different strains. Both CO2 and HCO3− were accumulated within the algal cells. In the cell suspension from which dissolved inorganic carbon had been depleted by pre-illumination, CO2 was transported and accumulated faster than HCO3−. When the concentration of HCO3− injected into the cell suspension of A. variabilis M3 was 25 times as high as that of CO2 (the expected ratio at equilibrium at pH 7.8), the initial rates of fixation of both inorganic carbon species were practically the same. On the other hand, when 14CO2 or H14CO3− was added under steady state photosynthetic conditions, both carbon species were transported at similar rates. The ratio of fixed to transported carbon measured after the initial 5 s was only 23–27% regardless of the carbon species supplied. This percentage is much lower than that reported for Chlorella cells.

Antibody was raised against Porphyridium carbonic anhydrase (CA) which was electrophoretically recovered from the gel after SDS-polyacrylamide slab gel electrophoresis (SDS-PAGE) of the partially purified enzyme. The antiserum reacted with CA of Porphyridium, but not with that of Chlamydomonas reinhardtii. Even though the antiserum did not react with CA from P. cruentum R-l in Ouchterlony's double immunodiffusion, it blocked the enzyme activity in the presence of 1% Nonidet P-40 and 1% Triton X-100. After Western blotting and enzyme-linked immunostaining (ELIS), only one band which reacted with the antiserum was detected in the extract of low-CO2 cells (grown under ordinary air) of P cruentum, while no significant band was detected in that of high-CO2 cells (grown under air enriched with 1–5% CO2). Immunogold electron microscopy of low-CO2 cells of P. cruentum R-l using this antibody revealed that most of the CA was localized in the chloroplast, with some in the cytoplasm. No specific binding of gold particles was observed in the high-CO2 cells.

The requirement of sodium for growth of Anabaena variabilis M3 was investigated under low (0.04%) and high (1.5 or 5%) CO2 conditions. The growth rates under both conditions were strongly affected by NaCl concentrations up to 0.5 mm in the medium. In the presence of 40 μm NaCl, the cells were not able to grow under a low CO2 condition, but were able to grow under a high CO2 condition. The sodium requirement for growth was dependent on pH: in the Na+-deficient condition, cells could grow at pH6.8, while no growth occurred at pH 8.2, suggesting that the requirement of Na+ for growth observed in the low CO2 condition can be substituted for by a lower pH. In the presence of 20 mm NaCl at pH 7.8, 14CO2 as well as H14CO3− were actively transported into the cells which had been grown in air. In contrast, the transport of both of these inorganic carbon (IC) species was suppressed under the Na+-deficient condition. These results suggest that sodium is required for the stimulation of transport of IC during photosynthesis. This is one of the reasons why Na+ is required for the growth of Anabaena under ordinary air and alkaline conditions.

In cells of cyanobacterium Anabaena variabilis grown under ordinary air (low-CO2 cells), the transport of both CO2 and HCO3− was significantly enhanced by Na+. This effect was pronounced as the external pH increased. When low-CO2 cells were treated with an inhibitor of carbonic anhydrase (CA), only CO2 transport but not HCO3− transport, was inhibited. The initial rate of photosynthetic carbon fixation as a function of the concentration of internal inorganic carbon (IC) was practically the same irrespective of whether CO2 or HCO3− was externally supplied. These results suggest that IC is actively transported through the plasma membrane in a form of HCO3− probably by some transporter and that the transmembrane Na+ gradient is involved in this IC transport system. Free CO2 may be hydrated by CA to HCO3− and then transported to the cells by this transporter. On the other hand, CO2 is actively taken up by cells grown with air containing 5% CO2 (high-CO2 cells) though the enhancing effect of Na+ was much smaller in high- CO2 cells than in low-CO2 cells. The initial rate of fixation as a function of internal IC concentration indicated that the rate of the carboxylation reaction of accumulated IC is higher in I0W-CO2 cells than in high-CO2 cells. The studies with ethoxyzolamide indicated that even in low-CO2 cells, CA does not function inside Anabaena cells. These results suggest that inside the low-CO2 cells of Anabaena, some mediator(s) facilitates the transport of IC to RuBPCase.

When ammonia was added to cells of Chlorella which had fixed 14CO2 photo synthetically, 14C which had been incorporated into starch was greatly decreased. A similar effect was observed when potassium nitrate and sodium nitrite were added. The ammonia-induced decrease in 14C-starch was observed in all species of Chlorella tested. With cells of C. vulgaris 11h, most of the radioactivity in starch was recovered in sucrose, indicating that ammonia induces the conversion of starch into sucrose. The percent of 14C recovered in sucrose differed from species to species and practically no recovery in sucrose was observed in C. pyrenoidosa. In most species tested, the enhancing effects of blue light and ammonia on O2 uptake as well as the ammonia effect on starch degradation were greater in cells which had been starved in phosphate medium in the dark than in non-starved cells. In contrast, the enhancing effect of ammonia on dark CO2 fixation was much greater in non-starved cells. C. pyrenoidosa was unique in that blue light did not show any effect on its O2 uptake.

The activity and intracellular compartmentation of carbonic anhydrase was examined in mesophyll protoplasts of several C3 terrestrial species including wheat, since this enzyme may facilitate diffusion of inorganic carbon in solution by converting CO2 to bicarbonate. Carbonic anhydrase was located in the mesophyll chloroplast with little or no activity in the cytosolic fraction. In wheat, carbonic anhydrase was absent in etiolated leaves and increased in the light during greening. Thus the enzyme may have a role in photosynthesis in the chloroplast but not in the cytosol of mesophyll cells of higher C3 plants. The amount of CO2 required for half maximum rates of photosynthesis (under low O2) was about two-fold higher for isolated protoplasts than with isolated chloroplasts of wheat. The form of inorganic carbon taken up by protoplasts, like that of chloroplasts, is CO2. The results are discussed in relation to a possible resistance to CO2 transfer in the cytosol of mesophyll cells.

In Dunaliella tertiolecta, D. bioculata and D. viridis the activities of phosphoenolpyruvate carboxylase and carbonic anhydrase were higher in the cells grown in ordinary air (low-CO2 cells) than in those grown in air enriched with 1–5% CO2 (high-CO2 cells), whereas in Porphyridium cruentum R-1 there was no difference in phosphoenolpyruvate carboxylase activity between these two types of cells. Apparent Km(NaHCO3) values for photosynthesis in low-CO2 cells of all species tested were smaller than those in high-CO2 cells. Most of the 14C was incorporated into 3-phosphoglycerate, sugar mono- and di-phosphates during the initial periods of photosynthetic NaH14CO3 indicating that both types of cells in D. tertiolecta are C3 plants.

Cellular distribution and subunit constitution of carbonic anhydrase (CA) were compared between the CBSC strain of Chlamydomonas reinhardtii (purchased from Carolina Biological Suuply Co.) and C. reinhardtii C-9. CAs existed exclusively outside the plasma membrane inboth strains. Mol wts of the monomer and the holoenzyme from both strains were 35 kDa and 115–117 kDa, respectively. These results show that the CAs of both strains are identical or verysimilar.

Rotational streaming of the cytoplasm including chloroplasts was induced by L-histidine, as well as by light, on the anticlinal face of leaf cells of Egeria densa. In the case of treatment with L-histidine some of the chloroplasts remained stationary on the periclinal face of cells after rotational cytoplasmic streaming was initiated. However, these chloroplasts were easily dislodged and translocated to the centrifugal end of the histidine-treated cells by application of a centrifugal force that barely affected the location of chloroplasts in cells incubated in the dark without L-histidine. This result indicates that the anchoring of chloroplasts was weakened by L-histidine. Thus only the release of chloroplasts from anchoring was not enough for initiation of their streaming. The cytoplasmic pH (pHc) and vacuolar pH (pHv) were noninvasively monitored by in vivo 31P-nuclear magnetic resonance (NMR) spectroscopy. Compared with the dark control value, both illumination and treatment with L-histidine increased the pHc by 0.3 units. In contrast, pHv changed only a little with both illumination and treatment with L-histidine. Release of chloroplasts from anchoring and initiation of cytoplasmic streaming are discussed in relation to the increase in pHc induced by both light and L-histidine.

Treatment with trypsin of Chlamydomonas reinhardtii cells grown in ordinary air (low-CO2 cells) caused almost complete release of carbonic anhydrase (CA) into the suspending medium, but did not affect the shape and kinesis of the cells. These results indicate that most of the CA exists on the cell surface of low-CO2 cells. The released CA has the same molecular weight, specific activity and susceptibility to various CA inhibitors as that purified from non-treated low-CO2 cells.

Cells of Dunaliella tertiolecta which had been grown in ordinary air (low-CO2 cells) had high carbonic anhydrase (CA) activity on the cell surface and mainly utilized HCO3− for photosynthesis. When CA activity on the cell surface was inhibited by Diamox or subtilisin, the cells utilized CO2. When bovine CA was added, the subtilisin-treated low-CO2 cells utilized mainly HCO3−. When grown in air containing 2% CO2, the cells had low CA activity on the cell surface, and preferred CO2 to HCO3−. Kinetic analysis of these results indicated that low-CO2 cells of D. tertiolecta absorb CO2 which was converted from HCO3− via the CA located on the cell surface.

The pH changes in the blue-green alga (cyanobacterium) Anabaena cylindrica caused by addition of ammonia were investigated using 31P NMR spectroscopy. A pH shift of 0.9 or more was observed when 30 nM NH4OH was added to the cell suspension, but no significant cellular pH change was observed with 50 mM NH4CI, a concentration high enough to stimulate dark CO2 fixation of this alga. The change in cellular pH does not seem to cause ammonia-induced stimulation of dark CO2 fixation.

Affinity for inorganic carbon in photosynthesis of Euglena gracilis Z was higher in the cells grown in ordinary air than in those grown in 4% CO2. The cells grown in ordinary air accumulated inorganic carbon in the cells to the level far in excess of that expected from passive diffusion due to pH gradient across the cell surface membrane.

Blue light was specifically required for the induction of carbonic anhydrase (CA) activity in Chlamydomonas reinhardtii. The enhancing effect of blue light (460 nm) was saturated at energy fluence rate as low as 0.6-0.8 W/m2. The wavelength dependency curve had a peak at 460 nm with no effect at wavelengths above 510 nm, thus showing the strong similarities to other blue light responses in microalgae. CA induction was strongly inhibited by UV irradiation at 280 nm. Experiments with the flavin quencher, potassium iodide, suggested that flavin is somehow involved in CA induction.

The regulation of carbonic anhydrase (CA) activity by light was determined for Chlamydomonas reinhardtii. When high-CO2 (4-5% CO2 grown cells were transferred to low-CO2 condition (0.04% CO2, the induction of CA activity showed correlation with the energy fluence rate. Addition of 10 μM DCMU immediately after transferring to low-CO2 condition completely suppressed the induction of CA activity, while its addition one hour after air induction still caused an increase for a while. These results indicate that there are two light-requiring steps controlling CA induction in C. reinhardtii: a photosynthesis-dependent step followed by a photosynthesis-independent one. The latter step showed requirement for blue light but not for red light. Inhibitor experiments suggest that blue light exerted its regulatory effect on the post-transcriptional level of CA biosynthesis.

By 31P-in vivo nuclear magnetic resonance spectroscopy, two intracellular compartments were detected, and their pH values were estimated in intact cells of a halotolerant unicellular green alga Dunaliella tertiolecta. They were identified as the cytoplasm (pH 7.1) and vacuoles (pH 6.0). Vacuoles were also visualized with a fluorescence-differential interference microscope. During the adaptation to the salt stress (NaCl concentration from 0.17 to 1.0 M) where cells rapidly synthesize glycerol as osmoticum, both cytoplasmic and vacuolar pH showed transient increases to about 8 and 6.5, respectively. Subsequently, cytoplasmic inorganic phosphate level, as well as sugar phosphates and terminal phosphate group levels increased. These drastic changes in chemical environment in the cytoplasm including pH and inorganic phosphate concentration are discussed to be key factors for osmoregulation that activate the synthesis and inhibit the breakdown of glycerol.

The biosynthesis and degradation of carbonic anhydrase (CA; EC 4.3.1.1) was investigated during the course of synchronous culture of the unicellular green alga Chlamydomonas reinhardtii, carried out under a regime of 12 h of light and 12 h of darkness with bubbling of ordinary air. The enzymatic activity increased linearly during the light phase. A coordinate increase in the level of the 35-kDa mature CA polypeptide was demonstrated by immunostaining after poly-acrylamide gel electrophoresis and Western blotting. Pulse-labeling with [14C]arginine followed by immunoprecipitation showed that the biosynthesis of the CA polypeptide is very active in the early light phase and rapidly decreases after the middle of the light phase, indicating that the bio-synthetic activity does not reflect the quantity of enzyme protein or the level of enzymatic activity. The 42-kDa precursor but not the 35-kDa mature polypeptide was synthesized in the dark. The 35-kDa polypeptide, pulse-labeled at the beginning of the light phase, was gradually degraded throughout the light phase while it appeard to be stable in the dark. These results suggest that the messenger RNA coding for CA is present but its translation is limited in the dark. Normal translation of mRNA and processing of the precursor to yield the holoenzyme may both require light.

The levels of protochlorophyllide and protochlorophyll of pigment mutant C-2A′ of Scenedesmus obliquus grown in darkness depend upon the calcium concentration in the growth medium. In the presence of calcium both the protochlorophyllide and protochlorophyll levels decrease upon irradiation whereas the amount of photoreduced chlorophyllide increases. In contrast to light-dependent protochlorophyllide reduction, the activity of light-independent protochlorophyllide reduction is higher in calcium free cultures compared to those grown in the presence of calcium. It is discussed whether calcium acts directly on the activity of the protochlorophyllide oxidoreductase or stabilizes the newly formed chlorophyllide.

Most of the carbonic anhydrase (CA) activity was released into the growth medium, when a cell wall-deficient mutant strain of C. reinhardtii was cultured under low CO2 conditions. Treatment of wild-type cells carrying high CA activity with a gametic wall-lysing enzyme resulted in the release of the CA activity into the medium. Both data indicate that the majority of CA activity in C. reinhardtii is located outside the plasmalemma, either in the periplasmic space or attached to the cell wall.

Sakami's procedure for determination of intramolecular radio-tracer distribution in 3-phosphoglyceric acid or serine is modified. This new procedure uses ion exchange resin chromatography to separate formic acid and formaldehyde, minimizing cross contamination between C-2 and C-3 position. Specific activity was determined by initial weighing of Bal4CO3 on a fiberglass filter, followed by scintillation counting. The modified procedure is simpler and shorter than the original one, requiring much less starting material and it permits the parallel handling of multiple samples.

Temperature-sensitive (ts), high-CO2 requiring mutants of Anacystis nidulans R2 were isolated by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) mutagenesis and ampicillin enrichment. One of these mutants was able to grow under ordinary air enriched with 5% CO2, but not under ordinary air at 40�C. At 40�C, the concentration of CO2 at which the rate of oxygen evolution reached half the maximum velocity (apparent Km(CO2) in photosynthesis) was 1,000 times higher in mutant cells than in wild type cells, whereas there was no significant difference in the maximum rate of photosynthesis. When wild type and mutant cells were incubated with 55 μm NaHC03 under illumination at 40�C, the initial rate of inorganic carbon (IC) transport from the medium into the cells and the maximum internal IC accumulation were significantly higher in wild type cells than in mutant cells. These results indicate that the isolated high-CO2 requiring mutant lacks the ability in transporting IC into the cells at 40�C. Furthermore, the finding that the mutant cells which are defective in IC transport cannot grow under ordinary air suggests the importance of IC concentrating system(s) in photosynthesis of cyanobacteria.

Most of the radioactivity incorporated into malic acid during light-enhanced dark 14CO2-fixation was found in C-4, supporting our conclusion that phosphoenolpyruvic acid serves as a primary acceptor of 14CO2 to form the C4 acid.

1) With Chlorella ellipsoidea cells, in the presence of 5×10−6 M DSPD, or in its absence, the amounts of 14CO2 incorporated in P-esters, serine-plus-glycine and alanine were larger under red light than under blue light, whereas blue light specifically increased 14CO2-incorporation in aspartate, glutamate, malate and fumarate (blue light effect). The amount of total 14C fixed under blue or red light was greatly decreased by the addition of DSPD. When the concentration of DSPD was raised to 5×10−4 M, practically no radioactivity was found, under blue or red light, in aspartate, glutamate and fumarate. Radioactivity in alanine was greatly increased. Effects of higher concentration of DSPD are explained as due to the inhibition of PEP carboxylase activity in Chlorella cells. 2) The percentage incorporation of 14C into aspartate and the other compounds mentioned above, under near infra-red illumination was significantly smaller than that under blue light and was almost equal to that under red light. These results along with the effect of 5×10−6 M DSPD, exclude the possibility that cyclic photophosphorylation is involved in the “blue light effect” mechanism.