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Low concentrations of oligomycin, which strongly inhibit mitochondrial oxidative phosphorylation but do not affect chloroplast photophosphorylation, caused an inhibition of photosynthesis by 30 to 40% in barley (Hordeum vulgare L.) leaf protoplasts. This inhibition is reversed and the full rate of photosynthesis is regained when the protoplasts are ruptured so as to leave the chloroplasts intact. Oligomycin fed into barley leaves by the transpiration stream inhibited photosynthesis in these leaves by up to 60%. The measurement of metabolites in protoplast and leaf extracts showed that oligomycin caused a decrease in the ATP/ADP ratio and an increase in the content of glucose-and fructose 6-phosphate. Subcellular analysis of protoplasts revealed that the decrease in ATP/ADP ratio in the cytosol was larger than in the stroma and that the increase in hexose monophosphates was restricted to the cytosol, whereas the stromal hexosemonophosphates decreased upon the addition of oligomycin. Moreover, oligomycin caused an increase in the triosephosphate-3-phosphoglycerate ratio. It is concluded from these results that during photosynthesis of a plant leaf cell mitochondrial oxidative phosphorylation contributes to the ATP supply of the cell and prevents overreduction of the chloroplast redox carriers by oxidizing reductive equivalents generated by photosynthetic electron transport.It has been a matter of debate if in a plant leaf cell mitochondrial oxidative phosphorylation contributes to the supply of ATP during photosynthesis metabolism. In a previous publication (1 1), we investigated how to inhibit mitochondrial ATP synthesis in plant protoplasts without affecting photophosphorylation. A suitable inhibitor appeared to be oligomycin. It strongly inhibits mitochondrial oxidative phosphorylation (16) and has only a weak effect on photophosphorylation (10).Very low concentrations of oligomycin inhibiting mitochondrial oxidative phosphorylation caused a partial inhibition of protoplast photosynthesis that was observed only when the protoplasts were intact (1 1). When protoplasts that had been inhibited by oligomycin were ruptured by forcing them through a 5 ,um net, a procedure leaving chloroplasts and Supported by the Deutsche Forschungsgemeinschaft. mitochondria intact, photosynthesis of the released chloroplasts was recovered to the rate obtained in the absence of oligomycin.In a chloroplast suspension the volume of the external medium is about 1000 times larger than the stromal space of the chloroplasts. The phosphate in the medium functions as a reservoir for chloroplast photosynthesis and the products DHAP and PGA are diluted in the medium to such an extent that for the duration of the measurements photosynthesis is not affected by an accumulation of external products. In protoplasts, on the other hand, the volume of the cytosolic space is similar to the stromal one. Therefore, in the intact protoplasts photosynthesis can operate only if the products DHAP2 and PGA are further metabolized in the cytosol and th...

Oligomycin was used for the selective inhibition of mitochondrial oxidative phosphorylation in barley leaf protoplasts.The addition of oligomycin inhibited the photosynthesis of intact protoplasts by 4&6Oq6, this being accompanied by a pronounced decrease in cellular ATP content. When the protoplasts were ruptured in a way leaving the chloroplasts intact, the photosynthesis of the released chloroplasts was not inhibited by oligomycin. It is therefore concluded that in illuminated protoplasts, ATP formation by oxidative phosphorylation is required for utilization of the photosynthate by the cell. Thus, mitochondrial oxidative phosphorylation appears to serve an essential function for supplying the cytosol with ATP during photosynthesis.

An oligomycin concentration that specifically inhibits oxidative phosphorylation was added to isolated barley (Hordeum vurgare 1.) leaf protoplasts at various irradiances and carbon dioxide concentrations. At saturating as well as low light intensities, photosynthetic oxygen evolution was decreased as a result of the oligomycin treatment, whereas no effect was observed at intermediate light intensities. This was the same for photorespiratory and nonphotorespiratory conditions. These results were confirmed by measurements of fluorescence quenching under the same conditions. Metabolite analysis in the presence of oligomycin revealed a drastic decrease in the mitochondrial and cytosolic ATP/ADP ratios, whereas there was little or no effect on the chloroplastic ratio. Concomitantly, sucrose phosphate synthase activity was reduced. Under high irradiances, this inhibition of sucrose synthesis by oligomycin apparently caused a feedback inhibition on the Calvin cycle and the photosynthetic activity. Under low irradiances, a feedback regulation compensated, indicating that light was more limiting than the activity of regulative enzymes. Thus, the importance of mitochondrial respiratory activity might be different in different metabolic situations. At saturating light, the oxidation of excess photosynthetic redox equivalents is required to sustain a high rate of photosynthesis. At low light, the supply of ATP to the cytosol might be required to support biosynthetic reactions.