high-CO2 Cells Research Articles

Intracellular localization of a carbonic anhydrase in Porphyridium purpureum (Porphyridiales, Rhodophyta)

The intracellular localization of a carbonic anhydrase (CA) in the red alga, Porphyridium purpureum, was analysed by immunogold labelling experiments. A polyclonal antibody was raised against recombinant CA derived from cloned cDNA. Much higher CA activity was present in cells grown in ordinary air (low-CO2 cells) than in those grown in 5% CO2–enriched air (high-CO2 cells). An immunoblot analysis detected a single band in the low-CO2 cells, whereas no positive band was apparent in the high-CO2 cells. The antibody was used as a probe for immunogold labelling. The gold particles were predominantly localized along the plasma membrane of the low-CO2 cells, whereas no significant labelling was apparent in the high-CO2 cells. The exclusively intracellular localization of this CA activity and the absence of any membrane-spanning property of the enzyme suggests that the CA was associated with the cytoplasmic face of the plasma membrane. The results are discussed in relation to the possible role of CA.

Presence of the CO2-concentrating mechanism in some species of the pyrenoid-less free-living algal genus Chloromonas (Volvocales, Chlorophyta).

Physiological and morphological characteristics related to the CO2-concentrating mechanism (CCM) were examined in several species of the free-living, unicellular volvocalean genus Chloromonas (Chlorophyta), which differs morphologically from the genus Chlamydomonas only by lacking pyrenoids. The absence of pyrenoids in the chloroplasts of Chloromonas (Cr.) rosae UTEX 1337, Cr. serbinowii UTEX 492, Cr. clatharata UTEX 1970, Cr. rosae SAG 26.90, and Cr. palmelloides SAG 32.86 was confirmed by light and electron microscopy. In addition, immunogold electron microscopy demonstrated that ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) molecules were distributed almost evently throughout the chloroplasts in all five Chloromonas strains. However, Chloromonas exhibited two types of physiological characteristics related to the CCM depending on the species or strains examined. Chloromonas rosae UTEX 1337 and Cr. serbinowii had high photosynthetic affinities for CO2 in cells grown in culture medium bubbled with air (low-CO2 cells), compared with those grown in medium bubbled with 5% CO2 (high-CO2 cells), indicating the presence of the low-CO2-inducible CCM. In addition, these two Chloromonas strains exhibited low-CO2-inducible carbonic anhydrase (CA; EC 4.2.1.1) activity and seemed to have small intracellular inorganic carbon pools. Therefore, it appears that Cr. rosae UTEX 1337 and Cr. serbinowii possess the CCM as in pyrenoid-containing microalgae such as Chlamydomonas reinhardtii. By contrast, Cr. clatharata, Cr. rosae SAG 26.90 and Cr. palmelloides showed low photosynthetic affinities for CO2 when grown under both CO2 conditions. Moreover, these three strains exhibited an apparent absence of intracellular inorganic carbon pools and lacked low-CO2-inducible CA activity. Thus, Cr. clatharata, Cr. rosae SAG 26.90 and Cr. palmelloides, like other pyrenoid-less algae (lichen photobionts) reported previously, seem to lack the CCM. The present study is the first demonstration of the CCM in pyrenoid-less algae, indicating that pyrenoids or accumulation of Rubisco in the chloroplasts are not always essential for the CCM in algae. Focusing on this type of CCM in pyrenoid-less algae, the physiological and evolutionary significance of pyrenoid absence is discussed.

Polypeptide composition and enzyme activities of the pyrenoid and its regulation by CO2 concentration in unicellular green algae

Effects of environmental conditions on the pyrenoid were investigated in unicellular green algae. During adaptation to CO2 limitation, the pyrenoid and pyrenoid starch developed within several hours, while stroma starch was degraded, suggesting that metabolism around the pyrenoid is regulated independently from that in other stromal spaces. This pyrenoid development was light-dependent and inhibited by DCMU but was not affected by changes in nitrogen assimilation. Pyrenoids isolated from Chlamydomonas reinhardtii mainly consisted of the two subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), and several other polypeptides were detected. Phosphoribulokinase was not localized in the pyrenoid. Isolated chloroplasts were fractionated to separate intact pyrenoids from stroma and membranes. Rubisco activity was detected in both, and about 70% was distributed in the pyrenoid fraction in low-CO2 cells. The Rubisco content determined by SDS–PAGE, immunoblotting, and immunoelectron microscopy supported the above ratio. By fractionation of isolated chloroplasts under high ionic strength, a pyrenoid-like structure with high Rubisco activity was collected. It is suggested that the main carboxylation site in high-CO2 cells is the stroma, whereas Rubisco is concentrated in the pyrenoid and possibly functions in CO2 fixation under low-CO2 conditions. Key words: Chlamydomonas reinhardtii, chloroplast, CO2 concentration, photosynthetic CO2 fixation, pyrenoid, ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco).

Effects of CO2 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-CO(2) cells) than in the cells grown with air enriched with 2% CO(2) (high-CO(2) cells). The change in the ratio of linoleic acid to alpha-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-CO(2) and low-CO(2) cells. After high-CO(2) cells were transferred to low CO(2) condition, total amount of fatty acids remained constant but the relative content of alpha-linolenic acid increased during a 6-hour lag phase in growth with concomitant decreases in linoleic and oleic acids. When low-CO(2) cells were transferred to high CO(2) condition, total amount of fatty acids and relative content of oleic acid increased significantly. The amount of alpha-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.

Enzymatic Inactivation of Extracellular Carbonic Anhydrase and its Effect on <italic>K</italic><sub><italic>1/2</italic></sub> (CO<sup>2</sup>) for Photosynthesis in <italic>Chlorella ellipsoidea</italic> C-27

The ratio of the extracellular to the intracellular activity of carbonic anhydrase (CA) in cells of Chlorella ellipsoidea C-27, adapted to low levels of CO2 for 24 h (low-CO2 cells), was about one to one. Treatment of intact cells with Pronase P inactivated about one-half of the extracellular CA activity without affecting photosynthetic activity. The CA activity in cell homogenates and in cell-wall ghosts liberated during cell division was completely inactivated by the same treatment. Pretreatment with Glycosidase mix, Chitosanase and Macerozyme enhanced the inactivation of the CA activity in intact cells. These results suggest that extracellular CA is evenly distributed throughout the whole cell-wall region. The apparent K1/2 for dissolved inorganic carbon (DIC) in low-CO2 cells doubled when extracellular CA was inactivated by treatment with Pronase P, but the K1/2 obtained was still one-half of that in high-CO2 cells. Photosynthetic 14CO2-fixation in low-CO2 cells was enhanced by acetazolamide, whereas H14CO3−-fixation was suppressed. The results suggest that CO2 is a dominant substrate utilized by cells and that HCO3− is utilized after conversion to CO2. The present results show that both intracellular and extracellular CA contribute to the increase in affinity for DIC during photosynthesis in low-CO2 cells of Chlorella ellipsoidea C-27.

Mass Spectrometry Analysis of Oxygen Gas Exchange in High- and LOW-CO2 Cells of Chlorella vulgaris

Abstract Oxygen gas exchange was monitored in the unicellular green alga Chlorella vulgaris 211 - 11 h by means of a mass spectrometer equipped with a special membrane gas-inlet-system and a photosynthetic reaction vessel. CO2-dependent 18O2-uptake as well as 16O2-evolution were analyzed in both High- and Low-CO2 cells. In High-CO2 cells, the 18O2-ruptake in the light (UL) decreased by 65% upon addition of 3 mᴍ NaHCO3 , while 16O2-evolution (E) was increased approx. 1.8 times by the same treatment. 18O2-uptake in the dark (UD) was not affected by the addition of external inorganic carbon (Ci). The addition of 3.3 mᴍ NaHCO3 also affected UL and E in Low CO2-cells, however, to a minor extent. UL under CO2-saturating conditions was light intensity-independent up to 2 klux and 1.2 klux in High- and Low-CO2 cells, respectively. Above these light intensities UL increased approx. 4-fold in High- and approx. 6-fold in Low-CO2 cells. Under CO2-limiting conditions, however, UL increased in High-CO2 cells even under very low light intensities, showing that photorespiratory oxygen uptake occurred even in the near vicinity of the light compensation point. Under C02-saturating and strong light conditions UL represented almost half of E in Low-CO2 cells and about 30 % of E in High-CO2 cells. In Low-CO2 cells addition of ethoxyzolamide (EZA), an inhibitor of carbonic anhydrase, enhanced UL and suppressed E and NET under CO2-limiting conditions, whereas the compound had only a minor effect on High-CO2 cells. DCMU (3 μᴍ) strongly inhibited E and UL under CO2-saturating conditions, with the remaining UL being smaller than UD . KCN (1 mᴍ) and SHAM (1.5 mᴍ) added to DCMU-treated Low-CO2 cells suppressed UL by approx. 50 % . The resulting value corresponded to half of UD . KCN also inhibited E under CO2-saturating conditions, with UL being strongly enhanced showing a maximal uptake at 0.4 mᴍ KCN . Under these conditions NET was nearly zero. The effect seems to be due to an inhibition of RubisCO and an enhancement of Mehler reactions. At 0.7 mᴍ KCN , DCMU entirely inhibited UL , but oxygen uptake appeared increased after turning the light off. This uptake corresponded to approx. 60 % of UD . Whereas KCN and SHAM inhibited approx. 70 % of UD , only 16% of UL was suppressed. These results suggest that the contribution of mitochondrial respiration to UL was negligeable, since UL seemed to be suppressed in the light under CO2-saturated conditions. Iodoacetamide, which is an inhibitor of the Calvin cycle and thereby diverts carbon into the respiratory pathway, inhibited E and NET under CO2-saturating conditions, but did not affect UL . This result also shows that UL is not due to mitochondrial respiration. A hydroxylamine derivative [20, 21] which changes the ratio of the RuBP carboxylation to oxygenation activity in tobacco leaves did not affect this ratio in Chlorella.

Effect of pH on Glycolate and Ammonia Excretion in L-MSO Treated Chlorella Cells

Abstract The effect of pH changes on the excretion of ammonia and glycolate from algal cells into the medium was investigated in L-MSO (final concentration, 0.5 mм) -treated High-and Low CO2-cells of Chlorella vulgaris 211-11 h. The excretion was analyzed in the condition in which the cells were continuously gassed with air at 25 °C. At the values tested, generally more ammonia was excreted in L-MSO-treated Low CO2-cells than in L-MSO treated High CO2-cells. In both kinds of algal cells more ammonia was excreted at low pH-values and absolutely no ammonia was excreted at pH 8. In the dark, no or only slight ammonia excretion was observed in both L-MSO-treated High and Low CO2-cells. Under all these conditions no or only very low glycolate excretion was observed in both L-MSO treated High and Low CO2-cells. In High CO2-cells rates of photosynthesis were high at pH 6 and lower at higher pH values. On the other hand Low CO2-cells showed practically little dependence of photosynthetic rates on the pH. This result might indicate that the major part of the ammonia excretion observed, was not due to the inhibition of photosynthesis at acid pH values. It is known that ammonia excretion in L-MSO treated algal cells is due to the inhibition of the refixation of ammonia which originates from the glycine-serine aminotransferase reaction in the glycolate pathway. Our results demonstrate that glycolate production and glycolate metabolism are more intense at low pH values when compared to high pH values. This is valid for both High and Low CO2-cells. Low CO2-cells in Chlorella vulgaris 211-11 h exhibit a more active glycolate metabolism than High CO2-cells.

Carbonic anhydrase and CO2concentrating mechanisms in microalgae and cyanobacteria

Among the microbial phototrophs, those belonging to the cyanobacteria utilize CO2 and HCO−3 for photosynthesis. Some Chlorophyceae mainly take up CO2 in photosynthesis, and others, which have carbonic anhydrase (CA) on their cell surface can utilize HCO−3 as well as CO2. Kinetic studies revealed that most of the HCO−3 is utilized after this ion is converted to CO2 via CA located on the cell surface. Therefore, the actual molecular species which crosses the plasmalemma is mostly free CO2. There is apparent variation in the mode of utilization of dissolved inorganic carbon (DIC) for photosynthesis in microalgae in other classes. The apparent Km(CO2) values for photosynthesis in most microalgae grown in ordinary air (low-CO2 cells) are as low as in terrestrial C4 plants, although the algal cells fix CO2 via the C3 pathway. In contrast, the apparent Km(CO2) values in cells grown on CO2-enriched air (high-CO2 cells) are as high as those in the terrestrial C3 plants. Most low-CO2 cells show low photorespiration; a low CO2 compensation point, low rates of glycolate excretion and no or low O2 inhibition of photosynthesis. These results indicate that the efficiency of DIC utilization for photosynthesis in low-CO2 cells is very high. The activity of CA in low-CO2 cells is higher than that in high-CO2 cells, while no difference has been confirmed in the activities of other photosynthetic enzymes between low- and high-CO2 cells. In addition, low-CO2 cells can accumulate large amounts of DIC internally, indicating the existence of CO2-concentrating mechanisms in these cells. When CA activity or CO2 concentrating ability is reduced by inhibitors or by mutation, the apparent Km(CO2) values for photosynthesis and the rate of photorespiration increased notably. These results indicate that the high efficiency of DIC utilization in low-CO2 cells depends on both CA and a CO2-concentrating mechanism. It is concluded that CA facilitates the diffusion of DIC from outside the cells to the site(s) of the carboxylation reaction and the concentration of DIC is achieved via an active transporter.

Stimulation of Photorespiration by the Carbonic Anhydrase Inhibitor Ethoxyzolamide in Chlorella vulgaris

Ammonia was excreted at high rates in the presence of L-methionine sulfoximine (L-MSO) from Chlorella cells which have been grown and analyzed at normal CO2 partial pressure (330 ppm ). If these cells are analyzed at high CO2-concentration (3% CO2 in air) only little ammonia is excreted in the presence of L-MSO. In the absence of L-MSO no ammonia is excreted under either condition. In agreem ent with this observation Chlorella cells grown under high CO2 partial pressure (3% CO2 in air) but tested under normal CO2 partial pressure excreted only very little ammonia. Under these conditions neither “High CO2-cells” nor “Low CO2-cells” exhibited any glycolate excretion. However, glycolate excretion was observed in the presence of a-HPMS (a-hydroxy-2-pyridyl methanesulfonate) an inhibitor of glycolate dehydrogenase or INH (isonicotinyl hydrazide) an inhibitor of the glycine-serine am inotransferase, irrespective of the presence or absence of L-M SO. INH inhibited ammonia excretion. The above described high ammonia excretion in “Low CO2-cells” in the presence of L-MSO was suppressed or substantially reduced by 0.1 mм ethoxyzolamide an inhibitor of carbonic anhydrase which, however, at the same time caused a substantial excretion of glycolate into the medium. The same qualitative effect of ethoxyzolam ide was observed in “High CO2-cells” (tested under normal CO2 partial pressure) although the amount of glycolate excreted in this type of culture was very small. It was generally noted that glycolate excretion caused by ethoxyzolamide was stoichiometrically always more important than the rate of ammonia excretion which was inhibited. This shows that excretion and therefore most probably also the formation of glycolate are enhanced by ethoxyzolamide. The experiments seem to show that due to the inhibition of carbonic anhydrase the affinity of the ribulose-1,5-bisphosphate carboxylase/oxygenase system is increased towards oxygen, which leads to a stimulation of the photorespiratory carbon cycle.

Effect of the CO2-Concentration during Growth on the Oxygen Evolution Pattern under Flash Light in Chlorella

Photosynthetic O2-evolution patterns were determined in cells of Chlorella vulgaris 211 - 11 h, grown under air enriched with 2% CO2 (High CO2- cells) and under ordinary air (Low CO2-cells). Oxygen evolution in these algae was measured as consequence of short saturating light flashes with the three electrode system according to Schmid and Thibault, Z. Naturforsch. 34c, 414 (1979). It was shown that Low CO2-cells had the usual Joliot-Kok pattern of O2 evolution whereas High CO2-cells exhibited a more reduced pattern characterized by the fact that maximal flash yield was observed under the 4th flash and that damping due to misses was more important than in Low CO2-cells. Moreover, the amplitudes of the amperometric signals in High CO2-cells were consistently lower. The observations clearly speak in favor of a more reduced condition of the S-state system, when the cells are grown under high CO2. This was confirmed by the fact that higher concentrations of hydroxylamine had to be added to bring Low CO2-cells into the maximally reduced condition namely S-2, than was the case with High CO2-cells. Our observations suggest that the redox condition of the S-state system of photosystem II in Chlorella vulgaris is affected either by changes of CO2 concentrations during algal growth or that the S-state system is only maintained in the 4 state Kok condition when the enzyme carbonic anhydrase is present.

Factors Controlling Induction of Carbonic Anhydrase and Efficiency of Photosynthesis in Chlorella vulgaris llh Cells

When Chlorella vulgaris llh cells which had been grown in air enriched with 2–4% CO2 (high-CO2 cells) were bubbled with air containing ca. 400 ppm CO2, illumination at an intensity as low as the light compensation point (350 lux) was sufficient to increase the photosynthetic rate under limiting CO2 concentrations. The same treatment induced carbonic anhydrase (CA) activity. The induction of CA activity and increase in photosynthetic rate at limiting CO2 concentrations were observed in the presence of 10 μM DCMU which completely inhibits photosynthesis. These results indicate that photosynthetic electron transport is not involved in CA induction in Chlorella vulgaris llh cells. The parallelism between the changes in CA activity and the rate of photosynthesis under limiting CO2 concentrations agree with the previous conclusion that the transport of CO2 from outside to the site of CO2 fixation is facilitated by CA and hence lowers the apparent Km(CO2) for photosynthesis.

Effects of carbonic anhydrase on ribulose 1,5-biphosphate carboxylase and oxygenase

The apparent Km value for CO* in photosynthesis in algal cells grown with ordinary air (low-CO2 cells) is much lower than that in those grown with CO*enriched air (high-CO2 cells) [l-4]. Between the two types of cells there was practically no difference in activities as well as Km (COv (ii) HCO; formed from CO* in the stroma is converted again into CO* via CA and futed by RuBPCase (indirect use of COz). Under CO*-limiting conditions, photosynthetic CO2 fixation would be enhanced by the indirect use of COz, and CA is essential for this enhancement. COz-fixation by RuBPCase is actually enhanced by CA [7]: When [14C02] dissolved in the reaction medium containing RuBPCase waslow (in equilibrium with 400 ppm 14C02 gas), the addition of CA greatly

Operation of the reductive pentose phosphate cycle daring the induction period of photosynthesis in Chlorella1

When Chlorella oulgaris ll h cells grown in air containing 4% CO2 (high-CO2 cells) were given low concentrations of14CO2 (<150 ppm), the initial rate of photosynthetic 14CO2 fixation was very low and linear 14CO2 fixation was observed after an induction period which lasted for ca. 45 min. No such induction period was observed when high-CO2 cells were given high concentrations of 14CO2 (10,000 ppm) or when IOW-CO2 cells were given either low or high concentrations of 14CO2, supporting the observations by Briggs and Whittingham (l). However, irrespective of CO2 concentrations during growth and of 14CO2 concentrations during the experiments, most of the 14C was incorporated into phosphate esters during the initial periods of photosynthetic 14CO2 fixation. These results are in sharp contrast to the report by Graham and Whittingham (4).

Enhancement of ribulose 1,5-bisphosphate carboxylation reaction by carbonic anhydrase

It has been established that algal cells grown with ordinary air (low-CO2 cells) have a much lower Km value for COZ in photosynthesis than those grown with COz-enriched air (high-CO, cells) [l-4]. Carbonic anhydrase (EC 4.2.1 .I) is almost exclusively located in low-CO2 cells of Chlorella and its activity in high-CO2 cells is marginal, if any [S ,6]. COZ concentration during growth does not affect ribulose l$bisphosphate (Ru-P,) carboxylase (EC 4.1.1.39) activity [3,6,7]. The app. Km (CO*) in low-CO2 cells is greatly increased by Diamox, an inhibitor of CA [3,6]. These results indicate the possibility that the transport of CO? from outside the algal cells to the site of Ru-P2 carboxylase may be accelerated by CA and that the observed difference in Km (COZ) may be due to the difference in CA activity. We have found [8] that, irrespective of CO2 concentration during growth, the rate of photosynthetic CO* fixation in Chlorella vulgaris 11 h cells in the presence of low NaHCOa concentrations was greatly enhanced by the addition of CA, while the initial rate of COZ fixation immediately after bubbling of ordinary air was suppressed by this enzyme. Since CA catalyzes the dehydration of bicarbonate and the