Submitted to: Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/17/2003
Publication Date: 10/1/2003
Citation: Moran, J.F., James, E.K., Rubio, M.C., Sarath, G., Klucas, R.V., Becana, M. 2003. Functional characterization, expression, and subcellular localization of a novel iron-superoxide dismutase from cowpea (vigna unguiculata) root nodules. Plant Physiology, 133(2):773-782 Interpretive Summary: Root nodules are symbiotic structures that are the site for the fixation of atmospheric nitrogen in many land plants. This process is a critical component of the global nitrogen cycle and has profound implications in land management and cropping systems. A fundamental event in all aerobic life forms is the removal of toxic compounds produced from oxygen. These mechanisms are both enzymatic and non-enzymatic. One of the enzymes needed for maintaining cellular health is superoxide dismutase. In this study, we have purified, cloned and studied the properties of a novel iron-containing superoxide dismutase from cowpea root nodules. The biochemical properties indicate this is an active enzyme that could play a role in senescent root nodules. We have also demonstrated for the first time that this enzyme is present in the cytosolic-compartment of cells, unlike other iron containing superoxide dismutases that are present in cell organelles. The implications of our findings are (1) that enzymes of this class could be involved in cellular protection in aging tissues and (2) even though these enzymes are predominantly found in organelles, the root nodule form is strictly cytosolic in localization
Technical Abstract: A novel iron-superoxide dismutase, VuFeSOD, has been purified from cowpea (Vigna unguiculata) nodules and leaves. The enzyme has two identical subunits of 27 kD that are not covalently bound. Comparison of its N-terminal sequence (NVAGINLL) with the cDNA-derived amino acid sequence showed that VuFeSOD is synthesized as a precursor with seven additional amino acids. The mature protein was overexpressed in Escherichia coli and the recombinant enzyme used to generate a polyclonal monospecific antibody. Phylogenetic and immunological data demonstrate that there are at least two types of FeSODs in plants. An enzyme homologous to VuFeSOD is present in soybean and common bean nodules, but not in alfalfa and pea nodules. The latter two species also contain FeSODs in the leaves and nodules, but the enzymes are presumably localized to the chloroplasts and plastids. In contrast, immunoblots of the soluble nodule fraction and immunoelectron microscopy of crio-processed nodule sections demonstrate that VuFeSOD is localized to the cytosol. Immunoblot analysis showed that the content of VuFeSOD protein increases in senescent green nodules, undergoing leghemoglobin degradation, which suggests a protective role of VuFeSOD against iron toxicity. Therefore, contrary to the widely held view, FeSODs in plants are not restricted to the chloroplast stroma and may become an important defensive mechanism against the oxidative stress associated with senescence.