Current Nitrogen Fixation Is Involved in the Regulation of Nitrogenase Activity in White Clover (Trifolium repens L.)

Previous studies have shown that nitrogenase activity decreases dramatically after defoliation, presumably because of an increase in the O2 diffusion resistance in the infected nodules. It is not known how this O2 diffusion resistance is regulated. The aim of this study was to test the hypothesis that current N2 fixation (ongoing flux of N2 through nitrogenase) is involved in the regulation of nitrogenase activity in white clover (Trifolium repens L. cv Ladino) nodules. We compared the nitrogenase activity of plants that were prevented from fixing N2 (by continuous exposure of their nodulated root system to an Ar:O2 [80:20] atmosphere) with that of plants allowed to fix N2 (those exposed to N2:O2, 80:20). Nitrogenase activity was determined as the amount of H2 evolved under Ar:O2. An open flow system was used. In experiment I, 6 h after complete defoliation and the continuous prevention of N2 fixation, nitrogenase activity was higher by a factor of 2 compared with that in plants allowed to fix N2 after leaf removal. This higher nitrogenase activity was associated with a lower O2 limitation (measured as the partial pressure of O2 required for highest nitrogenase activity). In experiment II, the nitrogenase activity of plants prevented from fixing N2 for 2 h before leaf removal showed no response to defoliation. The extent to which nitrogenase activity responded to defoliation was different in plants allowed to fix N2 and those that were prevented from doing so in both experiments. This leads to the conclusion that current N2 fixation is directly involved in the regulation of nitrogenase activity. It is suggested that an N feedback mechanism triggers such a response as a result of the loss of the plant's N sink strength after defoliation. This concept offers an alternative to other hypotheses (e.g. interruption of current photosynthesis, carbohydrate deprivation) that have been proposed to explain the immediate decrease in nitrogenase activity after defoliation.