Coregulation of electron transport and Benson-Calvin cycle activity in isolated spinach chloroplasts: Studies on glycerate 3-phosphate reduction

Abstract

Glycerate 3-phosphate-dependent O 2 evolution was measured in intact chloroplasts in the absence of CO 2. At all concentrations of added glycerate 3-phosphate oxygen evolution ceased before stoichiometric amounts of oxygen were evolved. The inhibition of glycerate 3-phosphate-dependent-O 2 evolution increased with increasing concentrations of substrate added. A similar response was observed in chloroplasts treated with KCN which inhibits ribulose-1,5-bisphosphate carboxylase-oxygenase. Oxygen uptake via the oxygenase activity of this enzyme is therefore not the cause of the discrepancy in stoichiometry of oxygen release in this system. The addition of NaHCO 3 to chloroplasts in which oxygen evolution was inhibited by glycerate 3-phosphate caused an immediate sustained rate of oxygen evolution in the absence of KCN but not with KCN present. Simultaneous measurements of chlorophyll a fluorescence showed that q Q remained oxidized, although net O 2 evolution had ceased. As O 2 evolution decreased, q E and ΔpH increased. Upon the addition of NaHCO 3, Q A became more oxidized while ΔpH and q E were decreased, suggesting that the inhibition of electron transport at high glycerate 3-phosphate concentrations was mediated by photosynthetic control via ΔpH. However, the levels of ATP, ADP, ribulose 1,5-bisphosphate, and P i in the stroma remained constant during the period when O 2 evolution was inhibited. The addition of NaHCO 3 caused a decrease in the stromal ribulose 1,5-bisphosphate and P i concentrations and ATP/ ADP ratio. The stromal glycerate 3-phosphate content declined upon illumination until O 2 evolution ceased. At this time a constant stromal glycerate 3-phosphate concentration of 8–10 m m was maintained while net import of glycerate 3-phosphate into the stroma had virtually ceased. The stromal triosephosphate content remained at a constant low level throughout but the glycerate 3-phosphate level increased slightly after addition of NaHCO 3. The data provided by the measurements of thylakoid reactions and stromal metabolites suggest that photosynthetic electron transport is tightly coupled to the requirements of the stroma for ATP and NADPH. Glycerate 3-phosphate reduction requires much less ATP than the operation of the complete Benson-Calvin cycle since the stoichiometry of ATP and NADPH utilization is reduced to 1:1. We conclude that thylakoid electron flow is not sufficiently flexible to maintain NADPH and ATP production in the ratio of 1:1. This situation will favor overenergization of the thylakoid membrane, increased leakiness of protons, increased electron drainage to O 2, and result in progressive inhibition of noncyclic electron flow.