ATP‐dependent carbon transport in perfused Chara cells

Abstract

ABSTRACTCarbon transport across the plasma membrane, and carbon fixation were measured in perfused Chara internodal cells. These parameters were measured in external media of pH 5·5 and pH 8·5, where CO2 and HCO3‐ are, respectively, the predominant carbon species in both light and dark conditions. Cells perfused with medium containing ATP could utilize both CO2 and HCO3‐ from the external medium in the light. Photosynthetic carbon fixation activity was always higher at pH 5·5 than at pH 8·5. When cells were perfused either with medium containing hexokinase and 2‐deoxyglucose to deplete ATP from the cytosol (HK medium) or with medium containing vanadate, a specific inhibitor of the plasma membrane H+‐ATPase (V medium), photosynthetic carbon fixation was strongly inhibited at both pH 5·5 and 8·5. Perfusion of cells with medium containing pyruvate kinase and phosphoenolpyruvate (PEP) to maximally activate the H+‐ATPase (PK medium), stimulated the photosynthetic carbon fixation activities. Oxygen evolution of isolated chloroplasts and the carbon fixation of cells supplied 14C intracellularly were not inhibited by perfusion media containing either hexokinase and 2‐deoxyglucose or vanadate. The results indicate that Chara cells possess CO2 and HCO3‐ transport systems energized by ATP and sensitive to vanadate in the light. In the dark, intact cells also fix carbon. By contrast, in cells perfused with medium containing ATP, no carbon fixation was detected in 1 mol m ‐3 total dissolved inorganic carbon (TDIC) at pH 8·5. By increasing TDIC to 10 mol m‐3, dark fixation became detectable, although it was still lower than that of intact cells at 1mol m‐3 TDIC. Addition of PEP or PEP and PEP carboxylase to the perfusion media significantly increased the dark‐carbon fixation. Perfusion with vanadate had no effect on the dark‐carbon fixation.