The effect of excision on O2 diffusion and metabolism in soybean nodules

Abstract

Nitrogen‐fixing nodules of soybean [Glycine max (L.) Merr. cv. Maple Arrow inoculated with Bradyrhizobium japonicum USDA 16] were studied before and after excision from the root to determine the role the O2 regulation plays in the inhibition of nodule activity and the potential for using excised nodules nodules in studies of nodule metabolism. Relative nitrogenase (EC 1.7.99.2) activity (H2 evolution in N2:O2) and nodule respiration (CO2 evolution) were monitored first in intact nodulated roots and then in freshly excised nodules of the same plant to determine the time course of the decline in nodule metabolism. Folowing excision, nitrogenase activity and respiration declined rapidly in the first minute and then more gradually. After 40 min the rate of H2 evolution was only 14–28% of that in the intact plant. In some nodules activity declined steadily, and in others there was a partial recovery in activity ca 10 min after detachment. Infected cell O2 concentration (Oi), measured by a spectro‐photometric technique, also declined after nodule detachment with a time course similar to the declines in nitrogenase activity and respiration. Following excision, Oi levels declined rapidly from ca 21 nM in attached nodules to 8–12 nM at 4–10 min after excision and then more gradually to 2–3 nM O2 at 30–40 min after excision. These results show that the nodules' permeability to gas diffusion continued to be regulated for up to 40 min after detachement. At 40 min after detachment, when excised nodules displayed steady‐state rates of gas exchange, linear increases in pO2 from 20 to 100% at 4% min−1 resulted in recoveries of H2 and CO2 evolution, indicating that Oi limited nitrogenase activity durig this period, and that energy reserves were greatly in excess of the O2 available for respiration. When detached nodules were equilibrated for 12 h at 20, 30 and 50% O2, Oi values measured at supra‐ambient pO2 were greater than those at 20% O2 and were linked with a more rapid decline in nitrogenase activity. Also, increases in external pO2 (Oc) failed to stimulate nodule metabolism, suggesting that the nodules' energy reserves were no longer greatly in excess of their respiratory demands. It was concluded that soybean nodules could provide useful material for steady‐state studies of nodule metabolism between 40 and 240 min after detachment, but to attain metabolic rates equivalent to in vivo rates the nodules must be exposed to above‐ambient pO2.