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

Abstract

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