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In the green alga Chlorella vulgaris UAM 101, a CO2-concentrating mechanism (CCM) is induced when cells are transferred from high (5%) to low (0.03%) CO2 concentrations. The induction of the CCM is correlated with de-novo synthesis of several polypeptides that remain to be identified. The internal carbonic anhydrase (CA; EC 4.2.1.1) activity increased 6- to 7-fold within 6 h of acclimation to air. When crude homogenates were further separated into soluble and insoluble fractions, nearly all of the CA activity was associated with the membrane fraction. Immunoblot analysis of cell homogenates probed with antibodies raised against the 37-kDa subunit of periplasmic CA of Chlamydomonas reinhardtii showed a cross-reaction with a single 38-kDa polypeptide in both high- and low-CO2-grown cells. The up-regulation of the expression of the 38-kDa polypeptide was closely correlated with the increase in internal CA activity. Furthermore, its subcellular location was also correlated with the distribution of the activity. Immunoblot analysis of pyrenoid fractions showed that the 38-kDa polypeptide was concentrated in the pyrenoids from low-CO2-grown cells but was not present in pyrenoids from high-CO2-grown cells. In addition, immunogold labeling experiments showed that the protein was mainly associated with membranes crossing the pyrenoid, while it was absent from the pyrenoid matrix. These studies have identified a putative intracellular CA polypeptide associated with the pyrenoid in Chlorella vulgaris, suggesting that this structure may play an important role in the operation of the CCM and the acclimation to low CO2 conditions. [ABSTRACT FROM AUTHOR]

The CO-concentrating mechanism (CCM) was induced in the green unicellular alga Chlorella when cells were transferred from high (5% CO) to low (0.03%) CO concentrations. The induction of the CCM correlated with the formation of a starch sheath specifically around the pyrenoid in the chloroplast. With the aim of clarifying whether the starch sheath was involved in the operation of the CCM, we isolated and physiologically characterized a starchless mutant of Chlorella pyrenoidosa, designated as IAA-36. The mutant strain grew as vigorously as the wild type under high and low CO concentrations, continuous light and a 12 h light/12 h dark photoperiod. The CO requirement for half-maximal rates of photosynthesis [K(CO)] decreased from 40 μM to 2-3 μM of CO when both wild type and mutant were switched from high to low CO. The high affinity for inorganic carbon indicates that the IAA-36 mutant is able to induce a fully active CCM. Since the mutant does not have the pyrenoid starch sheath, we conclude that the sheath is not involved in the operation of the CCM in Chlorella cells. [ABSTRACT FROM AUTHOR]

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%) CO concentrations. The synthesis of these polypeptides is correlated with the induction of the CO-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-CO-inducible polypeptides requires the same environmental factor. Our results showed that induction of the 21- and 36-kDa polypeptides under low-CO 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 O/CO ratio than by the CO 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. [ABSTRACT FROM AUTHOR]

Unicellular algae grown under low-CO conditions (0.03% CO) have developed a means of concentrating CO at the site of ribulose-1,5-bisphosphate carboxylase/oxygenase. Cells with the CO-concentrating mechanism (CCM) acquire the ability to accumulate inorganic carbon to a level higher than that obtained by simple diffusion. To identify proteins which are involved in the organization of the CCM, cells of Scenedesumus obliquus and Chlorella vulgaris grown in high CO (5% CO in air) were transferred to low-CO (0.03%) conditions in the presence of SOand, thereafter, polypeptides labeled with S were detected. Under low-CO conditions the inducton of 36-, 39-, 94- and 110- to 116kDa polypeptides were particularly observed in S. obliquus and 16-, 19-, 27-, 36-, 38- and 45-kDa polypeptides were induced in C. vulgaris. Western blots with antibodies raised against 37-kDa subunits of the periplasmic carbonic anhydrase (CA) of Chlamydomonas reinhardtii showed immunoreactive bands with the 39-kDa polypeptide in the whole-cell homogenates from S. obliquus and with 36 and 38-kDa polypeptides in both high- and low-COgrown cells of C. vulgaris. Anti-pea-chloroplast CA antibodies cross-reacted with a single polypeptide of 30 kDa in the whole-cell homogenates but not with thylakoid membranes. The CA activity was associated with soluble and membrane-bound fractions, except thylakoid membranes. [ABSTRACT FROM AUTHOR]

In the green marine alga Dunaliella tertiolecta, a CO-concentrating mechanism is induced when the cells are grown under low-CO conditions (0.03% CO). To identify proteins induced under low-CO conditions the cells were labelled with SO, and seven polypeptides with molecular weights of 45, 47, 49, 55, 60, 68 and 100 kDa were detected. The induction of these polypeptides was observed when cells grown in high CO (5% CO in air) were switched to low CO, but only while the cultures were growing in light. Immunoblot analysis of total cell protein against pea chloroplastic carbonic anhydrase polyclonal antibodies showed immunoreactive 30-kDa bands in both high- and low-CO-grown cells and an aditional 49-kDa band exclusively in low-CO-grown cells. The 30-kDa protein was shown to be located in the chloroplast. Western blot analysis of the plasmamembrane fraction against corn plasma-membrane AT-Pase polyclonal antibodies showed 60-kDa bands in both high- and low-CO cell types as well as an immunoreactive 100-kDa band occurring only in low-CO-grown cells. These results suggest that there are two distinct forms of both carbonic anhydrase and plasma-membrane ATPase, and that one form of each of them can be regulated by the CO concentration. [ABSTRACT FROM AUTHOR]

The pyrenoid is a prominent proteinaceous structure found in the stroma of the chloroplast in unicellular eukaryotic algae, most multicellular algae, and some hornworts. The pyrenoid contains the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase and is sometimes surrounded by a carbohydrate sheath. We have observed in the unicellular green alga Chlamydomonas reinhardtii Dangeard that the pyrenoid starch sheath is formed rapidly in response to a decrease in the CO concentration in the environment. This formation of the starch sheath occurs coincidentally with the induction of the CO-concentrating mechanism. Pyrenoid starch-sheath formation is partly inhibited by the presence of acetate in the growth medium under light and low-CO conditions. These growth conditions also partly inhibit the induction of the CO-concentrating mechanism. When cells are grown with acetate in the dark, the CO-concentrating mechanism is not induced and the pyrenoid starch sheath is not formed even though there is a large accumulation of starch in the chloroplast stroma. These observations indicate that pyrenoid starch-sheath formation correlates with induction of the CO-concentrating mechanism under low-CO conditions. We suggest that this ultrastructural reorganization under lowCO conditions plays a role in the CO-concentrating mechanism C. reinhardtii as well as in other eukaryotic algae. [ABSTRACT FROM AUTHOR]

We have assessed the effect of vanadate as an inhibitor of plasma-membrane ATPase on photosynthesis and the ATP/ADP ratio in Chlamydomonas reinhardtii CW-92 (a mutant strain lacking a cell wall). This effect was compared in low-CO-adapted cells grown in media bubbled with air containing 400 or 70 μL · L CO. Evidence is presented indicating that cells grown at 70 μL · L CO have a higher rate of photosynthetic O evolution than cells grown at 400 μL · L CO, at limiting carbon concentrations. Extracellular and intracellular carbonic-anhydrase activities were, however, similar in cells grown in both of the low-carbon conditions. Vanadate inhibited, to a different extent, the HCO-dependent O evolution in cells grown at 400 and 70 μL · L CO. At 400 μM vanadate, inhibition reached 70-75% in cells grown at 400 μL · L but only 50% in those grown at 70 μL · L CO. The ATP/ADP ratios determined with and without vanadate at limiting concentrations of dissolved inorganic carbon indicated that more ATP was hydrolysed in algae grown at 70 μL · L than in those grown at 400 μL · L CO. We conclude that the maximal capacity to accumulate dissolved inorganic carbon is inversely related to the CO concentration in the medium. Activation and - or synthesis of vanadate-sensitive ATPase may be the major explanation for the higher capacity for HCO-dependent O evolution in cells grown under limited CO concentrations. [ABSTRACT FROM AUTHOR]

It has been proposed that many marine macroalgae are able to utilize HCOfor photosynthesis and growth, and that energy-dependent ion pumping is involved in this process. We have therefore studied the light-dependent alkalization of the surrounding medium by two species of marine macroscopic brown algae, Fucus serratus L. and Laminaria saccharina (L.) Lamour. with the aim of investigating the role of extracellular carbonic anhydrase (EC 4.2.1.1.) in the assimilation of inorganic carbon from the seawater medium. In particular, the influence of membrane-impermeable or slowly permeable carbonic-anhydrase inhibitors on the rate of alkalization of the seawater has been investigated. Inhibition of the alkalization rate occurred in both species at an alkaline pH (pH 8.0) but no inhibition was observed at an acidic pH (pH 6.0). The alkalization was found to be light-dependent and inhibited by 3-(3′,4′-dichlorophenyl)-1, 1-dimethylurea and, thus, correlated with photosynthesis. Alkalization by macroalgae has previously been shown to be proportional to inorganiccarbon uptake. We suggest that alkalization of the medium at alkaline pH in both of the species examined is mainly the consequence of an extracellular reaction. The reaction is catalyzed by extracellular carbonic anhydrase which converts HCOto OH and CO; CO is then taken up through the plasmalemma. However, we do not exclude the involvement of other mechanisms of inorganic-carbon uptake. [ABSTRACT FROM AUTHOR]

It has been proposed that many marine macroalgae are able to utilize HCOfor photosynthesis and growth, and that energy-dependent ion pumping is involved in this process. We have therefore studied the light-dependent alkalization of the surrounding medium by two species of marine macroscopic brown algae, Fucus serratus L. and Laminaria saccharina (L.) Lamour. with the aim of investigating the role of extracellular carbonic anhydrase (EC 4.2.1.1.) in the assimilation of inorganic carbon from the seawater medium. In particular, the influence of membrane-impermeable or slowly permeable carbonic-anhydrase inhibitors on the rate of alkalization of the seawater has been investigated. Inhibition of the alkalization rate occurred in both species at an alkaline pH (pH 8.0) but no inhibition was observed at an acidic pH (pH 6.0). The alkalization was found to be light-dependent and inhibited by 3-(3′,4′-dichlorophenyl)-1, 1-dimethylurea and, thus, correlated with photosynthesis. Alkalization by macroalgae has previously been shown to be proportional to inorganiccarbon uptake. We suggest that alkalization of the medium at alkaline pH in both of the species examined is mainly the consequence of an extracellular reaction. The reaction is catalyzed by extracellular carbonic anhydrase which converts HCOto OH and CO; CO is then taken up through the plasmalemma. However, we do not exclude the involvement of other mechanisms of inorganic-carbon uptake. [ABSTRACT FROM AUTHOR]

The mechanism of inorganic-carbon (C) accumulation in the red seaweed Gracilaria tenuistipitata Zhang et Xia has been investigated. Extracellular and intracellular carbonic-anhydrase (CA) activities have been detected. Photosynthetic O evolution in thalli and protoplasts of G. tenuistipitata were higher at pH 6.5 than at pH 8.6, where HCOis the predominant form of C. Dextran-bound sulfonamide (DBS), a specific inhibitor of extracellular CA, reduced photosynthetic O evolution at pH 8.6 and did not have any effect at pH 6.5. After inhibition with DBS, O evolution was similar to the rate that could be supported by CO from spontaneous dehydration of HCO. The rate of photosynthetic alkalization of the surrounding medium by the algal thallus was dependent on the concentration of C and inhibited by DBS. We suggest that the general form of C that enters through the plasma membrane of G. tenuistipitata is CO. Bicarbonate is utilized mainly by an indirect mechanism after dehydration to CO, and this mechanism involves extracellular CA. [ABSTRACT FROM AUTHOR]

Mechanisms of carbon assimilation were investigated in thalli and protoplasts of Ulva rigida by measuring HCO-dependent O evolution at pH 6.5 and 8.6. In thalli, dextran-bound azetazolamide (DBAZ), a specific inhibitor of extracellular carbonic anhydrase (CA), inhibited the rate of O evolution at pH 8.6 when HCOwas the only available form of inorganic carbon (C) in the medium. At pH 6.5 when CO is accessible, DBAZ did not affect photosynthetic O evolution. Inhibition of total CA activity (extracellular and intracellular) by ethoxyzolamide (EZ) inhibited photosynthesis at pH 6.5 and 8.6. During illumination of thalli the medium was alkalized at a rate which increased with increasing light. This alkalization decreased during inhibition of extracellular CA by DBAZ. Protoplasts at pH 6.5 exhibited a higher rate of O evolution than in pH 8.6. Addition of CA to protoplasts at pH 8.6 increased the rate of O evolution, whereas EZ was inhibitory at both pH 6.5 and 8.6, and DBAZ did not affect photosynthesis at either pH. We suggest that both extracellular and intracellular CA are present and that Ulva rigida assimilates HCOby an indirect mechanism. A theoretical scheme for carbon utilization is suggested. [ABSTRACT FROM AUTHOR]

In Chlamydomonas reinhardtii the formation of a starch sheath surrounding the pyrenoid is observed when cells grown under high CO2 (5% CO2 in air) are transferred to low CO2 (0.03%) conditions. Formation of the starch sheath occurs coincidentally with induction of the CO2 concentrating mechanism and with de novo synthesis of 5 polypeptides with molecular masses of 21, 36, 37, 42-44 kDa. We studied the effect of CO2 concentrations on photosynthesis, ultrastructure and protein synthesis in Chlamydomonas reinhardtii cw-15 (wild phenotype for photosynthesis) and in the starch-less mutant BAFJ-6, with the aim to clarify the role of the pyrenoid starch sheath in the operation of the CO2 concentrating mechanism and whether these low CO2-inducible polypeptides are involved in the formation of starch sheath. When wild type and starch-less mutant cells were transferred from high to low CO2 the CO2 requirement for half-maximal rates of photosynthesis decreased from 40 μM to 2 μM CO2. 35SO42- labeling analyses showed that the starch-less mutant induced the 5 low CO2-inducible polypeptides. These observations suggest that the starch-less mutant was able to induce a fully active CO2 concentrating mechanism. Since the starch-less mutant did not form a pyrenoid starch sheath, we suggest that the starch sheath is not involved in the operation of the CO2 concentrating mechanism and that none of these 5 low CO2-inducible proteins is involved in the formation of the starch sheath in Chlamydomonas.

The effect of CO2 concentration on the rate of photorespiratory ammonium excretion and on glutamine synthetase (GS) and carbonic anhydrase (CA) isoenzymes activities has been studied in Chlamydomonas reinhardtii cw‐15 mutant (lacking cell wall) and in the high CO2‐requiring double mutant cia‐3/cw‐15 (lacking cell wall and chloroplastic carbonic anhydrase). In cw‐15 cells, both the extracellular (CAext) and chloroplastic (CAchl) CA activities increased after transferring cells from media bubbled with 5% CO2 in air (v/v, high‐Ci cells) to 0.03% CO2 (low‐Ci cells), whereas in cia‐3/cw‐15 cells only the CAext was induced after adaptation to low‐Ci conditions and the CAchl activity was negligible. During adaptation to low‐Ci conditions in the presence of 1 mM of l‐methionine‐D,L‐sulfoximine (MSX), a specific inhibitor of GS activity, both mutant strains excreted photorespiratory ammonium into nitrogen free medium. In addition, the ammonium excretion rate by cw‐15 in the presence of MSX was lower in cells grown and kept at 5% CO2 than in high‐Ci cells adapted to 0.03% CO2. The double mutant cia‐3/cw‐15 excreted photorespiratory ammonium at a higher rate than did cw‐15. Total GS activity (GS‐1 plus GS‐2) increased during adaptation to 0.03% CO2 in both strains of C. reinhardtii. However, only the activity GS‐2, which is located in the chloroplast, increased during the adaptation to low CO2, whereas the cytosolic GS‐1 levels remained similar in high and low‐Ci cells. We conclude that: (1) cia‐3/cw‐15 cells lack chloroplastic CA activity; (2) in C. reinhardtii photorespiratory ammonium is refixed in the chloroplasts through the GS‐2/GOGAT cycle; and (3) chloroplastic GS‐2 concentration changes in response to the variation of environmental CO2 concentration. [ABSTRACT FROM AUTHOR]

Thalli of Ulva reticulata Forskaal, Ulva rigida C. Ag., and Ulva pulchra Jaasund were incubated at different concentrations of dissolved CO[SUB2]. Incubation at a high CO[SUB2]2 concentration resulted in decreased oxygen evolution rate and lower affinity for inorganic carbon at high pH conditions, i.e. the ability to use HCO[SUP-][SUB3] as a carbon source was reduced. This effect was reversible, and plants regained this HCO[SUP-][SUB3] uptake capacity when transferred to air concentrations of CO[SUB2] The phytosynthetic oxygen evolution rate of plants grown at high C02 concentration was reduced by high O[SUB2] concentrations, whereas thalli and protoplasts from cultures grown at air concentration were not affected. This is interpreted as a deactivation of the carbon-concentrating mechanism during conditions of high CO[SUB2] resulting in high photorespiration when plants are exposed to high O[SUB2] concentrations. Protoplasts were not affected by high O[SUB2] to the same extent and were not able to utilize HCO[SUP-][SUB3] from the medium. The algae were able to grow at very low CO[SUB2] concentrations, but growth was suppressed when ah inhibitor of external carbonic anhydrase was present. Assay of carbonic anhydrase activities showed that external and internal CA activities were lower in plants grown at a high CO[SUB2] concentration compared to plants grown at a low concentration of CO[SUB2] possible mechanisms for HCO[SUP-][SUB3] utilization in these Ulva species are discussed. [ABSTRACT FROM AUTHOR]