The argon-induced decline in nitrogenase activity commences before the beginning of a decline in nodule oxygen uptake

Replacement of N2 by argon in the air around nodules directs nitrogenase electron flow in its total onto H+ resulting in increased nodule H2 evolution (total nitrogenase activity (TNA)). However, argon application induces a so-called argon-induced decline in nitrogenase activity (Ar-ID) connected with decreased nodule oxygen permeability. Consequently, TNA measurements tend to underestimate total nitrogenase activity. It is unclear whether the decline in oxygen diffusion into nodules induces the Ar-ID, or whether a decline in nitrogenase activity is followed by lower nodule O2 uptake. The objective of the present work was to examine the time sequence of the decline in nodule H2 evolution and O2 uptake after argon application. In addition, the reliability of TNA values, taken as quickly as possible after the switch to Ar/O2, was tested through comparative measurement of 15N2 uptake of the same plants. Short-term TNA measurements in an optimized gas exchange measurement system yielded reliable results, verified by parallel determination of 15N2 uptake. A five min application of Ar/O2 was without effect on the subsequent H2 evolution in ambient air. A parallel experiment on control plants revealed that a decrease in nodule oxygen uptake began several minutes after the onset of the decline in H2 evolution. We conclude that the primary effect of the replacement of N2 by argon differs from oxygen diffusion control. A gas exchange system allowing an immediate taking of TNA yields reliable results and does not disturb nodule activity. Gas exchange measurements provide a powerful tool for studying nodule physiology and should be combined with material from molecular studies.