Uptake of Inorganic Carbon by Isolated Chloroplasts from Air-Adapted Dunaliella

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Abstract
Neither Dunaliella cells grown with 5% CO2 nor their isolated chloroplasts had a CO2 concentrating mechanism. These cells primarily utilized CO2 from the medium because the K(0.5) (HCO3-) increase from 57 micromolar at pH 7.0 to 1489 micromolar at pH 8.5, where as the K(0.5) CO2 was about 12 micromolar over the pH range. After air adaptation for 24 hours in light, a CO2 concentrating mechanism was present that decreased the K0.5 (CO2) to about 0.5 micromolar and K0.5 (HCO3-) to 11 micromolar at pH 8. These K0.5 values suggest that air-adapted cells preferentially concentrated CO2 but could also use HCO3- from the medium. Chloroplasts isolated from air-adapted cells had a K(0.5) for total inorganic carbon of less than 10 micromolar compared to 130 micromolar for chloroplasts from cell grown on high CO2. Chloroplasts from air-adapted cells, but not CO2-grown cells, concentrate inorganic carbon internally to 1 millimolar in 60 seconds from 240 micromolar in the medium. Maximum uptake rates occurred after preillumination of 45 seconds to 3 minutes. The CO2 concentrating mechanism by chloroplasts from air-adapted cells was light dependent and inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) or flurocarbonyl-cyamidephenylhydrazone (FCCP). Phenazine-methosulfate at 10 micromolar to provide cyclic phosphorylation partially reversed the inhibition by DCMU but not by FCCP. One to 0.1 millimolar vanadate, an inhibitor of plasma membrane ATPase, inhibited inorganic carbon accumulation by isolated chloroplasts. Vanadate had no effect on CO2 concentration by whole cells, as it did not readily cross the cell plasmalemma. Addition of external ATP to the isolate chloroplast only slightly stimulated inorganic carbon uptake and did not reverse vanadate inhibition by more than 25%. These results are consistent with a CO2 concentrating mechanism in Dunaliella cells which consists in part of an inorgaic carbon transporter at the chloroplast envelope that is energized by ATP from photosynthetic electron transport.