Nitrogen Fixation by Subterranean Clover at Varying Stages of Nodule Dehydration

Changes in nitrogenase activity (C2H2 reduction and H2 production), nodulated root respiration and the efficiency of nitrogenase functioning were measured in response to progressive dehydration of nodules on intact well-watered plants of subterranean clover (Trifolium subterraneum L.) cv. Seaton Park. The nodulated roots of vegetative plants grown to the 14-leaf stage were incubated in a gas exchange system through which a continuous dry airstream was passed over an 8 d period. The root tips were immersed in an N-free nutrient solution during this time so that water and ion uptake was unimpeded. The decline in nodulated root respiration resulting from nodule drying was associated with a continual reduction in respiration coupled to nitrogenase activity. As nodule water potential (ψnod) decreased, the proportion of total nodulated root respiration which was nitrogenase-linked declined from 50% (day 1) to 33% (day 8). This was accompanied by a 79% reduction in specific nitrogenase activity (from 3.79 to 0.81 umol C2H4 g−1 nodule dry weight min−1). Nodule dehydration also induced a decline in hydrogen (H2) production in air. The relative decline in hydrogen production exceeded that of acetylene reduction activity and this resulted in an increase in the relative efficiency of nitrogenase functioning. However, the carbon cost of nitrogenase activity progressively increased above 2.0 mol CO2 respired per mol C2H4 reduced as rood decreased below −0.4 to −0.5 MPa. Consecutive measurements of the rates of hydrogen evolution, 15N2 fixation and acetylene reduction activity on intact unstressed plants resulted in a C2H4/N2 conversion factor of 4.08 and an electron balance of 1.08. These results indicated that the pre-decline rate of acetylene reduction activity measured in a flow-through system provided a valid measure of the total electron flux through nitrogenase.