Regulation of photosynthesis in isolated spinach chloroplasts during orthophosphate limitation

The stromal concentration of orthophosphate in intact spinach chloroplasts (prepared in the absence of orthophosphate or pyrophosphate but supplied with both in the reaction medium) fell from a value of approx. 20 mM in the dark to a steady-state concentration of approx. 8 mM in the light. Chloroplasts illuminated in the absence of orthophosphate or pyrophosphate showed a similar trend. However, in this situation the stromal inorganic phosphate (P_i) concentration rapidly decreased from approx. 10 mM in the dark to a constant steady-state concentration of between 1.5 and 2.5 mM in the light. This P_i concentration was not further diminished (even though CO₂-dependent O₂ evolution had ceased) and was therefore considered to be stromal orthophosphate not freely available to metabolism. In the P_i-deficient chloroplasts the rate of photosynthesis declined rapidly after 1–2 min in the light such that CO₂-dependent O₂ evolution ceased with 5 min of the onset of illumination. The decline in O₂ evolution was accompanied by an increase in the transthylakoid ΔpH (as measured by 9-aminoacridine fluorescence quenching) and in the high-energy state, non-photochemical component of chlorophyll fluorescence quenching (q_E). Measurements of stromal metabolite concentrations showed that the ATP/ADP ratio was decreased in the P_i-deficient chloroplasts relative to chloroplasts illuminated in the presence of P_i. The stromal concentration of glycerate 3-phosphate was comparable in the P_i-deficient chloroplasts and those to which P_i had been supplied. Chloroplasts which were illuminated in P_i-free media showed a large accumulation of ribulose-1,5-bisphosphate relative to those supplied with P_i, suggesting inhibition of ribulose-1,5-bisphosphate carboxylase under these conditions. When P_i was added to chloroplasts illuminated in the absence of P_i, both non-photochemical quenching (q_E), photochemical quenching (q_Q) and ΔpH increased. This suggests that electron transport was not limited by inability to discharge transthylakoid ΔpH. These observation are consistent with the hypothesis that P_i limitation results in decreased ATP production by the thylakoid ATP synthase. The data presented here show that there are multiple sites of flux control exerted by low stromal P_i in the chloroplast. At least three factors contribute to the inhibition of photosynthesis under phosphate limitation: (1) there appears to be a direct effect of P_i on the energy-transducing system; (2) there is direct inhibition of the Calvin cycle decreasing the ability of the pathway to act as a sink for ATP and NADPH; and (3) feedback inhibition of primary processes occurs either via ΔpH or the redox state of electron carriers. However, ΔpH does not appear to be a limiting factor, but rather an inability to regenerate NADP as electron acceptor is suggested. The addition of DCMU to chloroplasts during illumination in the absence of P_i for periods of up to 10 min showed that there was very little loss of variable fluorescence despite a 60% reduction in the capacity for O₂ evolution. This would suggest that photoinhibitory damage to Photosystem II was not the major cause of the inhibition of photosynthesis observed with low P_i.