Inorganic Phosphate Concentration in the Stroma of Isolated Chloroplasts and Its Influence on Photosynthesis

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The concentration of inorganic orthophosphate (Pi) was determined in the stroma of isolated chloroplasts during photosynthesis under Pi-saturated and Pi-limited conditions. Pi was determined calorimetrically or by high performance liquid chromatography of extracts of chloroplasts labelled with 32Pi. When chloroplasts were illuminated in the absence of added Pi, photosynthesis soon declined due to Pi-depletion. After 5 min in the light, photosynthesis had declined to 2% of the maximum rate. At this point, stromal Pi was estimated to be 1.4 mM by the colorimetric method and 0.2 mM by 32P chromatography. Using the colorimetric method, Pi equivalent to approximately 1 mM in the stroma was found to be associated with thylakoid membranes isolated from chloroplasts, irrespective of the Pi content of the intact chloroplasts. This was considered to be a non-metabolic pool of Pi. During steady- state photosynthesis with optimal concentrations of Pi added to the reaction medium, the stromal Pi concentration was estimated to be 2.6 mM and 1.6 mM with the colorimetric and 32P methods, respectively. Measurement of stromal 32Pi in chloroplasts illuminated with varying concentrations of 32Pi in the reaction medium suggested that photosynthesis was saturated at stromal Pi concentrations above 2.0-2.5 mM. Photophosphorylation by thylakoid membranes was saturated at Pi concentrations above 1.2-1.5 mM. It is concluded that, during photosynthesis in isolated chloroplasts in the presence of an optimal supply of Pi from the reaction medium, the stromal Pi concentration is just above that required to saturate photophosphorylation. Any decrease in the supply of Pi from the medium results in a rapid decrease in stromal Pi to the point where photophosphorylation may become Pi-limited, decreasing the rate of CO2 fixation.