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Nitrogen 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.

1 copia .pdf (a3) de póster original presentado por los autores. 4 Figs., The thiol tripeptide GSH (yGIu-Cys-GIy) is a major antioxidant and redox buffer in plants, where it also performs critical functions in ccli cycle regulation, development, sulfur transport and storage, stress response, and heavy metal detoxification. In legumes, hGSH (yGlu-Cys-bAla) may partially or completely replace GSH with presumably the same functions. The synthesis of GSH is accomplished in two sequential reactions catalyzed by yECS and GSHS, whereas the synthesis of hGSH shares the same first enzyme and then requires a specific hGSHS. A better understanding of the regulation of GSH and hGSH biosynthesis in legumes during the stress response requires a precise determination of the subcellular localization of the enzymes and a quantitative expression analysis of the genes involved. For this purpose, two types of experiments were performed. First, these proteins were immunolocalized in legumes using electron microscopy. Second, the expression pattern of the three genes was determined in the model legume Lotus japonicus following treatment with several hormones that are crucial for plant development and stress signaling., This work was funded by grant AGL2008-01298 from MICINN-FEDER.

2 Pag., 1 Tabl., We have identified nine genes encoding peroxiredoxins (Prxs) in the model legume Lotus japonicus. The genes belong to the typical four classes of Prxs found in plants: 1Cys (1C-Prx), 2Cys (C2-PrxA and C2-PrxB), PrxQ (PrxQ1, PrxQ2, and PrxQ3), and type II (PrxIIB/C, PrxIIE, and PrxIIF). All genes were found to be transcribed, except PrxQ3. The expression pattern among the various plant organs and tissues is gene specific, with some overlapping in genes of the same class. Additionally, we have assessed the response of Prx genes to treatments with classical hormones and related stress and signaling compounds in roots of hydroponically-grown plants. Most Prx genes, especially 1C-Prx, were responsive to indole-3-acetic acid (IAA) or cytokinins (CKs). There were limited and specific changes of Prx expression after plant treatment with abscisic acid (ABA), 1- aminocyclopropane-1-carboxylic acid (ACC), gibberellic acid (GA), or jasmonic acid (JA). The unique expression pattern for each Prx gene within the plant and the specific response of Prx expression to hormones support a non-redundant role for most of these genes., AT-M is the recipient of a postdoctoral contract (“JAEPostdoc”) from CSIC and PB-S is the recipient of a predoctoral fellowship (‘‘Formación de Personal Investigador’’) from the Ministerio de Ciencia e Innovación (MICINN). This work was funded by MICINN (AGL2008-01298, co-financed by FEDER) and Gobierno de Aragón (group A53).

143 Pag., 15 Tabl., 27 Fig., Objetivos: 1. Estudio fisiológico, bioquímico y molecular de la respuesta antioxidante de Lotus japonicus al estrés salino. 2. Determinación de los mecanismos de regulación de la ruta de biosíntesis de tioles en Lotus japonicus en respuesta a óxido nítrico y hormonas. 3. Caracterización estructural y funcional de los genes de las hemoglobinas no simbióticas de Lotus japonicus. Este estudio incluye el análisis de la expresión génica en respuesta a óxido nítrico, estrés abiótico y hormonas, así como la localización de los transcritos y de la actividad de los promotores en los nódulos.

16 Pags., 7 Figs. The definitive version is available at: http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1365-3040, Boron (B) is an essential nutrient for N(2)-fixing legume-rhizobia symbioses, and the capacity of borate ions to bind and stabilize biomolecules is the basis of any B function. We used a borate-binding-specific resin and immunostaining techniques to identify B ligands important for the development of Pisum sativum- Rhizobium leguminosarum 3841 symbiotic nodules. arabinogalactan-extensin (AGPE), recognized by MAC 265 antibody, appeared heavily bound to the resin in extracts derived from B-sufficient, but not from Bdeficient nodules. MAC 265 stained the infection threads and the extracellular matrix of cortical cells involved in the oxygen diffusion barrier. In B-deprived nodules, immunolocalization of MAC 265 antigens was significantly reduced. Leghaemoglobin (Lb) concentration largely decreased in B-deficient nodules. The absence of MAC 203 antigens in B-deficient nodules suggests a high internal oxygen concentration, as this antibody detects an epitope on the lipopolysaccharide (LPS) of bacteroids typically expressed in microaerobically grown R. leguminosarum 3841. However, B-deprived nodules did not accumulate oxidized lipids and proteins, and revealed a decrease in the activity of the major antioxidant enzyme ascorbate peroxidase (APX). Therefore, B deficiency reduced the stability of nodule macromolecules important for rhizobial infection, and for regulation of oxygen concentration, resulting in non-functional nodules, but did not appear to induce oxidative damage in low-B nodules., This work was supported by Ministerio de Educación y Ciencia, BIO2008-05736-CO2-01 and by MICROAMBIENTECM Program from Comunidad de Madrid. M.R. is the recipient of a contract from Comunidad de Madrid.

2 Pag., 2 Fig., In plants, three distinct types of hemoglobins (Hbs) are known: symbiotic, nonsymbiotic, and truncated. Nonsymbiotic Hbs are further divided into class-1 and class-2 based on primary sequences and oxygen-binding properties. Symbiotic Hbs are well-characterized, whereas the functions of the other two types of Hbs are not fully understood. We have identified five genes encoding two class-1 (GLB1-1, GLB1-2), one class-2 (GLB2), and two truncated (GLB3-1, GLB3-2) Hbs in Lotus japonicus. GLB1-2 and GLB3-2 are expressed in all plant organs, whereas GLB1-1, GLB2, and GLB3-1 are expressed abundantly only in nodules. We have assessed also the response of GLB genes in nodules to hormone treatments. GLB1-1 was up-regulated by abscisic acid (ABA), 1-aminocyclopropane-1-carboxylic acid (ACC), and polyamines (PAs), whereas GLB2 and GLB3-1 were drastically down-regulated by cytokinins (CKs). In situ RNA hybridization showed that the GLB1-1 and GLB1-2 mRNAs are mainly localized in the cortex, vascular bundles, and fixation zone, whereas GLB2 and GLB3 mRNAs are mainly confined to the cortex and slightly to the vascular bundles. GLB3-2 promoter activity was localized by GUS staining in the vascular and endodermis/sclerenchyma cells in the cortex of mature nodules., PB-S is the recipient of a predoctoral fellowship (‘‘Formación de Personal Investigador’’) from MICINN and AT-M is the recipient of a postdoctoral contract (“JAE-Postdoc”) from