Localization of Superoxide Dismutases and Hydrogen Peroxide in Legume Root Nodules

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 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.

The response of the Rhizobium leguminosarum bv. trifolii wild-type and exopolysaccharide-deficient mutants to oxidative stress

“…H 2 O 2 is a signal molecule involved in plant response to pathogen attack and other stress conditions as well as nodulation (Rubio et al 2004). It is also known that the proper level of H 2 O 2 is required for optimal establishment of symbiosis.…”

Metal Transport in the Rhizobium-Legume Symbiosis

“…In the nodule, ROS are created by respiration in mitochondria and bacteroids, the direct reduction of O 2 by nitrogenase, hydrogenase and ferredoxin, and the autooxidation of leghemoglobin (Puppo et al 1981, Dalton et al 1991. Manganese and ferric-SOD are expressed in the nitrogen fi xing areas of the nodule, where they seem to be controlling ROS (Rubio et al 2004(Rubio et al , 2007. The relative levels of Mn-SOD or of Fe-SOD appear to be controlled by iron availability in the nodule, in such a way that as the nodule senesces and heminic iron from the leghemoglobin is released, the relative levels of Fe-SOD increase, as a means to both protect against ROS, and to sequester free iron (Rubio et al 2007).…”

Expression and Localization of a Rhizobium-Derived Cambialistic Superoxide Dismutase in Pea (Pisum sativum) Nodules Subjected to Oxidative Stress

“…MnSODs are typical bacterial and mitochondrial SODs, and in nodules are localized on bacteroids and on mitochondria within both infected and uninfected cells (Rubio et al 2004(Rubio et al , 2009). …”

“…Cytosolic and plastidial CuZnSOD isozymes are common to all plant cells, and are found in determinate and indeterminate nodules (Moran et al 2003;Rubio et al 2004;. As well as being present in the cytoplasm of cells from various nodule types (Rubio et al 2004; cytosolic CuZnSODs are also localized on chromatin in the nuclei of infected and uninfected cells in root nodules, stem nodules and leaves of the semiaquatic tropical legume Sesbania rostrata (Rubio et al 2009). Plastidial CuZnSOD isoenzymes are less abundant in nodules, as they are strongly linked to photosynthesis, but an exception are the photosynthetic stem nodules on S.…”