Tricarboxylic Acid Cycle Activity in Mitochondria from Soybean Nodules and Cotyledons

Infected cells of soybean (Glycine max) nodules require NADH, ATP, and 2-oxoglutarate for ammonia assimilation. The role of mitochondria in nodule metabolism was investigated by determining their respiratory properties and comparing them with cotyledon mitochondria. Nodule mitochondria oxidized malate at a rate twice that of any other NAD-linked substrate although their malic enzyme activity was very low, accounting for only 12% of malate oxidation at pH 6.4 compared to 56% for cotyledon mitochondria. The reduction of NAD+ in mitochondria of nodules on adding malate (determined by fluorescence) was rapid and reached a stable level, whereas in cotyledon mitochondria the NADH level declined rapidly as oxaloacetate accumulated. An oxaloacetate scavenging system in the mitochondrial reaction medium increased malate oxidation by cotyledon mitochondria 4-fold, but increased that of nodule mitochondria by less than 50%. This demonstrates that the efflux of oxaloacetate by the oxaloacetate carrier is highly regulated by the extra-mitochondrial oxaloacetate concentration in cotyledon mitochondria compared to nodule mitochondria. The activity of TCA cycle enzymes, except malate and succinate dehydrogenases, was low in nodule mitochondria. Their oxaloacetate export during malate oxidation was rapid. The aspartate amino transferase activity associated with nodule mitochondria was sufficient to account for significant formation of 2-oxoglutarate from oxaloacetate and glutamate. These results suggest that nodule mitochondria operate a truncated form of the TCA cycle and primarily oxidize malate to provide oxaloacetate and ATP for NH3 assimilation.