Localization of superoxide dismutases and hydrogen peroxide in legume root nodules

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. H ₂O₂ 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 SODs are a source of H ₂O₂ in indeterminate alfalfa (Medicago sativa) and pea (Pisum sativum) nodules. The transcripts and proteins of the major SODs of nodules were localized by in situ RNA hybridization and immunogold electron microscopy, respectively, 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 N ₂-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 H ₂O₂ in nodules. Furthermore, the absence or large reduction of H₂O₂ in nodule tissue preincubated with enzyme inhibitors (cyanide, azide, diphenyleneiodonium, diethyldithiocarbamate) provides strong support to the hypothesis that at least some of the H ₂O₂ originates by the sequential operation of an NADPH oxidase-like enzyme and CuZnSOD. Results also show that there is abundant H ₂O₂ associated with degrading bacteroids in the senescent zone (IV), which reflects the oxidative stress ensued during nodule senescence.