The Site of Oxygen Limitation in Soybean Nodules

In legume nodules the [O-2] in the infected cells limits respiration and nitrogenase activity, becoming more severe if nodules are exposed to subambient O-2 levels. To identify the site of O-2 limitation, adenylate pools were measured in soybean (Glycine max) nodules that were frozen in liquid N-2 before being ground, lyophilized, sonicated, and separated on density gradients of nonaqueous solvents (heptane/tetrachloroethylene) to yield fractions enriched in bacteroid or plant components. In nodules maintained in air, the adenylate energy charge (AEC = [ATP + 0.5 ADP]/[ATP + ADP + AMP]) was lower in the plant compartment (0.65 +/- 0.04) than in the bacteroids (0.76 +/- 0.095), but did not change when the nodulated root system was exposed to 10% O-2. In contrast, 10% O-2 decreased the bacteroid AEC to 0.56 +/- 0.06, leading to the conclusion that they are the primary site of O-2 limitation in nodules. To account for the low but unchanged AEC in the plant compartment and for the evidence that mitochondria are localized in O-2-enriched microenvironments adjacent to intercellular spaces, we propose that steep adenylate gradients may exist between the site of ATP synthesis (and ADP use) in the mitochondria and the extramitochondrial sites of ATP use (and ADP production) throughout the large, infected cells.