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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]

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]

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.

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]