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In the green marine alga Dunaliella tertiolecta, a CO2-concentrating mechanism is induced when the cells are grown under low-CO2 conditions (0.03% CO2). To identify proteins induced under low-CO2 conditions the cells were labelled with 35SO42-, 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 CO2 (5% CO2 in air) were switched to low CO2, 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-CO2-grown cells and an aditional 49-kDa band exclusively in low-CO2-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-CO2 cell types as well as an immunoreactive 100-kDa band occurring only in low-CO2-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 CO2 concentration.

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 CO2 concentration in the environment. This formation of the starch sheath occurs coincidentally with the induction of the CO2-concentrating mechanism. Pyrenoid starch-sheath formation is partly inhibited by the presence of acetate in the growth medium under light and low-CO2 conditions. These growth conditions also partly inhibit the induction of the CO2-concentrating mechanism. When cells are grown with acetate in the dark, the CO2-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 CO2-concentrating mechanism under low-CO2 conditions. We suggest that this ultrastructural reorganization under lowCO2 conditions plays a role in the CO2-concentrating mechanism C. reinhardtii as well as in other eukaryotic algae.

Abstract.: 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.

The CO2-concentrating mechanism (CCM) was induced in the green unicellular alga Chlorella when cells were transferred from high (5% CO2) to low (0.03%) CO2 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 Chlorellapyrenoidosa, designated as IAA-36. The mutant strain grew as vigorously as the wild type under high and low CO2 concentrations, continuous light and a 12 h light/12 h dark photoperiod. The CO2 requirement for half-maximal rates of photosynthesis [K0.5(CO2)] decreased from 40 µM to 2–3 µM of CO2 when both wild type and mutant were switched from high to low CO2. 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.

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%) CO2 concentrations. The synthesis of these polypeptides is correlated with the induction of the CO2-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-CO2-inducible polypeptides requires the same environmental factor. Our results showed that induction of the 21- and 36-kDa polypeptides under low-CO2 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 O2/CO2 ratio than by the CO2 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.

Unicellular algae grown under low-CO2 conditions (0.03% CO2) have developed a means of concentrating CO2 at the site of ribulose-1,5-bisphosphate carboxylase/oxygenase. Cells with the CO2-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 CO2 (5% CO2 in air) were transferred to low-CO2 (0.03%) conditions in the presence of 35SOinf4sup2-and, thereafter, polypeptides labeled with 35S were detected. Under low-CO2 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-CO2grown 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.

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-CO2-adapted cells grown in media bubbled with air containing 400 or 70 µL · L-1 CO2. Evidence is presented indicating that cells grown at 70 µL · L-1 CO2 have a higher rate of photosynthetic O2 evolution than cells grown at 400 µL · L-1 CO2, 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 HCO3--dependent O2 evolution in cells grown at 400 and 70 µL · L-1 CO2. At 400 µM vanadate, inhibition reached 70–75% in cells grown at 400 µL · L-1 but only 50% in those grown at 70 µL · L-1 CO2. 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-1 than in those grown at 400 µL · L-1 CO2. We conclude that the maximal capacity to accumulate dissolved inorganic carbon is inversely related to the CO2 concentration in the medium. Activation and — or synthesis of vanadate-sensitive ATPase may be the major explanation for the higher capacity for HCO3--dependent O2 evolution in cells grown under limited CO2 concentrations.