The entry of nitrate into the infected region of soybean nodules and the possibility of a subsequent nitrite accumulation was studied. Nitrate was observed to gain access to the infected region in the short-term and significant amounts could be measured within 2 d of nitrate supply. The availability of nitrate in the bacteroid-containing region did not cause free-nitrite accumulation for at least 8 d. However, to avoid the artefactual production of nitrite during extraction it was necessary to disrupt nodules in the presence of zinc acetate and ethanol, to prevent bacteroid nitrate reductase activity. Nitrite rapidly accumulated if nodules were extracted without prior enzyme-inactivation, or if bacteroids were allowed access to nitrate, or, more significantly, if nodules were not extracted immediately following detachment. Nitrate accumulation in detached nodules was mediated by oxygen concentration within the nodule; in the presence of pure N2 gas, nitrite accumulation was three times greater than in air and, conversely, it was prevented by exposure to pure O2. Furthermore, nitrite produced in detached nodules under atmospheric conditions was scavenged by transfering these nodules into 100% oxygen. However, measurements of apparent functional leghaemoglobin, using a nodule oximeter, suggested that after 8 d nitrate exposure up to 83% of Lb activity was lost, possibly due to interactions with nitrite produced in the nodule interior leading to the formation of nitrosylleghaemoglobin.
Bibliographical noteFunding Information:
We thank CL James and EL Fox for their excellent technical assistance. CAI greatfully acknowledges the grant from the DGICYT of Spain (Programa Sectorial de Formación de Profesorado y Personal Investigador) and RFD was supported in part by a travel fellowship from the OECD Project on Biological Resource Management. The MAC236 antibody was kindly provided by Dr N Brewin IPSR, Norwich, UK.
- Glycine max
- Infected region