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

Replacement of N(2) by argon in the air around nodules directs nitrogenase electron flow in its total onto H(+) resulting in increased nodule H(2) 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 O(2) uptake. The objective of the present work was to examine the time sequence of the decline in nodule H(2) evolution and O(2) uptake after argon application. In addition, the reliability of TNA values, taken as quickly as possible after the switch to Ar/O(2), was tested through comparative measurement of (15)N(2) uptake of the same plants. Short-term TNA measurements in an optimized gas exchange measurement system yielded reliable results, verified by parallel determination of (15)N(2) uptake. A five min application of Ar/O(2) was without effect on the subsequent H(2) 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 H(2) evolution. We conclude that the primary effect of the replacement of N(2) 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.