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Localization of Superoxide Dismutases and Hydrogen Peroxide in Legume Root Nodules

¹Departamento de Nutrición Vegetal, Estación Experimental de Aula Dei, Consejo Superior de Investigaciones Científicas, Apdo 202, 50080 Zaragoza, Spain; ²Centre for High Resolution Imaging and Processing, MSI/WTB Complex, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K.; ³Department of Agronomy and Plant Genetics, University of Minnesota, 411 Borlaug Hall, 1991 Upper Buford Circle, St Paul, Minnesota 55108, U.S.A.

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 SODs are 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 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 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 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 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.