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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]

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.