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

Abstract

Neither Dunaliella cells grown with 5% CO(2) nor their isolated chloroplasts had a CO(2) concentrating mechanism. These cells primarily utilized CO(2) from the medium because the K((0.5)) (HCO(3) (-)) increase from 57 micromolar at pH 7.0 to 1489 micromolar at pH 8.5, where as the K((0.5)) CO(2) was about 12 micromolar over the pH range. After air adaptation for 24 hours in light, a CO(2) concentrating mechanism was present that decreased the K(0.5) (CO(2)) to about 0.5 micromolar and K(0.5) (HCO(3) (-)) to 11 micromolar at pH 8. These K(0.5) values suggest that air-adapted cells preferentially concentrated CO(2) but could also use HCO(3) (-) 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 cells grown on high CO(2). Chloroplasts from air-adapted cells, but not CO(2)-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 CO(2) 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 CO(2) concentration by whole cells, as it did not readily cross the cell plasmalemma. Addition of external ATP to the isolated chloroplast only slightly stimulated inorganic carbon uptake and did not reverse vanadate inhibition by more than 25%. These results are consistent with a CO(2) concentrating mechanism in Dunaliella cells which consists in part of an inorganic carbon transporter at the chloroplast envelope that is energized by ATP from photosynthetic electron transport.