Submitted to: Molecular Plant-Microbe Interactions
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/27/2004
Publication Date: 12/7/2004
Citation: Rubio, M.C., James, E.K., Clemente, M.R., Bucciarelli, B., Fedorova, M., Vance, C.P., Becana, M. 2004. Localization of superoxide dismutases and hydrogen peroxide in legume root nodules. Molecular Plant-Microbe Interactions. 17(12):1294-1305. Interpretive Summary: Legume plants such as pea and alfalfa form a symbiotic association with soil bacteria known as rhizobia. The plant provides nutrients for the bacteria and the bacteria give the host plant nitrogen fertilizer. This process, known as symbiotic nitrogen fixation (SNF), occurs in small wart-like structures called root nodules. For SNF to occur, the plant and bacteria must be compatible and the bacteria must be protected from highly reactive forms of oxygen known as reactive oxygen. In order to define where and how reactive oxygen molecules occur in root nodules, we investigated two enzymes, protein catalysts, involved in the synthesis of reactive oxygen. We also developed a stain for identifying where reactive oxygen occurs in nodules. The results show that the enzyme super oxide dismutase along with an oxidase enzyme are required for production of reactive oxygen. Moreover, as the bacteria residing in root nodules age, reactive oxygen is produced. These findings are important because the identification of enzymes involved in production of reactive oxygen can lead to new strategies to control and reduce the formation of reactive oxygen in root nodules. Reducing the production of reactive oxygen can potentially increase the time span over which root nodules and SNF remain functional.
Technical Abstract: Superoxide dismutases (SODs) catalyze the dismutation of superoxide radicals to O2 and H2O2, and thus represent a primary line of antioxidant defense in all aerobic organisms. H2O2 is a signal molecule involved in the plant's response to pathogen attack and other stress conditions as well as in nodulation. In this work, we have tested the hypothesis that SOD is a source of H2O2 in indeterminate alfalfa (Medicago sativa) and pea (Pisum sativum) nodules. The transcripts and proteins of the major SODs of nodules were localized, respectively, by in situ RNA hybridization and immunogold electron microscopy, whereas H2O2 was localized cytochemically by electron microscopy of cerium-perfused nodule tissue. The transcript and protein of cytosolic CuZnSOD are most abundant in the meristem (I) and invasion (II) zones, interzone II-III, and distal part of the N2-fixing zone (III), and those of MnSOD in zone III, especially in the infected cells. At the subcellular level, CuZnSOD was found in the infection threads, cytosol adjacent to cell walls, and apoplast, whereas MnSOD was in the bacteroids, bacteria within infection threads, and mitochondria. The distinct expression pattern of CuZnSOD and MnSOD suggests specific roles of the enzymes in nodules. Large amounts of H2O2 were found at the same three nodule sites as CuZnSOD but not in association with MnSOD. This colocalization led us to postulate that cytosolic CuZnSOD is a source of H2O2 in the plant's response to rhizobia. Furthermore, the absence of H2O2 staining in nodule tissue preincubated with enzyme inhibitors (cyanide, azide, diphenyleneiodonium, diethyldithiocarbamate) provides strong support to the hypothesis that, at least in infection threads, H2O2 originates by the sequential operation of an NADPH oxidase-like enzyme and CuZnSOD. Results also show that there is abundant H2O2 associated with degrading bacteroids in the senescent zone (IV), which reflects the oxidative stress ensued during nodule senescence.