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Functional characterization of glutathione peroxidases from legume root nodules

AuthorsSaiz Rodríguez, Ana ; Matamoros Galindo, Manuel Ángel ; Peñuelas Hortelano, Maria ; Bustos-Sanmamed, Pilar ; James, Euan Kevin; Becana Ausejo, Manuel
Issue Date7-Sep-2014
Citation11th European Nitrogen Fixation Conference (Tenerife, España. 07 - 10.09.2014)
AbstractNitrogen fixation requires a robust antioxidant system to maintain reactive oxygen/nitrogen species (RONS) in adequate levels [1]. Glutathione peroxidases (Gpxs) catalyze the reduction of H2O2 or organic peroxides to water or the corresponding alcohols. They protect cell membranes from peroxidative damage and participate in RONS homeostasis and redox signaling. In plants, Gpxs are encoded by a multigene family of five to eight members that are distributed in different tissues with distinct subcellular locations [2]. In a previous study, we characterized six Gpx genes in the model legume Lotus japonicus. In this work, we investigated in detail two isoforms (LjGpx1 and LjGpx3) that are highly expressed in the nodules. Biochemical characterization of the purified recombinant proteins showed that both enzymes catalyze the Trx-dependent (but not glutathione-dependent) reduction of inorganic and organic peroxides. Enzymatic constants indicated a high affinity and catalytic efficiency of both proteins for phospholipid hydroperoxides. Expression analyses showed that LjGpx1 and LjGpx3 are differentially expressed in response to abiotic stress (cadmium) and signaling compounds (hormones). In situ RNA hybridization revealed that the LjGpx1 and LjGpx3 mRNAs are particularly abundant in the infected zone of Lotus nodules. Besides, significant amounts of LjGpx3 mRNA could be detected in the nodule cortex and in the vascular bundles. The localization of the respective proteins was examined using immunogold electron microscopy. LjGpx1 labeling was mainly observed in the plastids, whereas LjGpx3 was localized in the cytosol. The biotin switch assay [3] and streptetavidin-agarose affinity chromatography allowed us to detect the presence of nitrosylated Gpxs in nodules from plants treated with the nitric oxide (NO) donor S-nitrosoglutathione (GSNO). In addition, the His-tag switch method coupled to mass spectrometry was used to identify the position of the nitrosylated Cys residues. The experiments suggest a higher susceptibility to nitrosylation of LjGpx3 with respect to LjGpx1. Accordingly, treatment of purified proteins with 1mM GSNO caused a 40% reduction of LjGpx3 activity but had no effect on LjGpx1 activity. 1) Becana et al. 2010. New Phytol. 188: 960-976. 2) Margis et al. 2008. FEBS J. 275: 3959¿3970. 3) Jaffrey et al. 2001. Nat. Cell Biol. 3: 193¿197.
Appears in Collections:(EEAD) Comunicaciones congresos
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