The MicroRNA390/TAS3 Pathway Mediates Symbiotic Nodulation and Lateral Root Growth

Legume roots form two types of postembryonic organs, lateral roots and symbiotic nodules. Nodule formation is the result of the interaction of legumes with rhizobia and requires the mitotic activation and differentiation of root cells as well as an independent, but coordinated, program that allows infection by rhizobia. MicroRNA390 (miR390) is an evolutionarily conserved microRNA that targets the Trans-Acting Short Interference RNA3 (TAS3) transcript. Cleavage of TAS3 by ARGONAUTE7 results in the production of trans-acting small interference RNAs, which target mRNAs encoding AUXIN RESPONSE FACTOR2 (ARF2), ARF3, and ARF4. Here, we show that activation of the miR390/TAS3 regulatory module by overexpression of miR390 in Medicago truncatula promotes lateral root growth but prevents nodule organogenesis, rhizobial infection, and the induction of two key nodulation genes, Nodulation Signaling Pathway1 (NSP1) and NSP2. Accordingly, inactivation of the miR390/TAS3 module, either by expression of a miR390 target mimicry construct or mutations in ARGONAUTE7, enhances nodulation and rhizobial infection, alters the spatial distribution of the nodules, and increases the percentage of nodules with multiple meristems. Our results revealed a key role of the miR390/TAS3 pathway in legumes as a modulator of lateral root organs, playing opposite roles in lateral root and nodule development.Facultad de Ciencias Exacta

Thiol synthetases of legumes: immunogold localization and differential gene regulation by phytohormones

In plants and other organisms, glutathione (GSH) biosynthesis is catalysed sequentially by γ-glutamylcysteine synthetase (γECS) and glutathione synthetase (GSHS). In legumes, homoglutathione (hGSH) can replace GSH and is synthesized by γECS and a specific homoglutathione synthetase (hGSHS). The subcellular localization of the enzymes was examined by electron microscopy in several legumes and gene expression was analysed in Lotus japonicus plants treated for 1–48 h with 50 μM of hormones. Immunogold localization studies revealed that γECS is confined to chloroplasts and plastids, whereas hGSHS is also in the cytosol. Addition of hormones caused differential expression of thiol synthetases in roots. After 24–48 h, abscisic and salicylic acids downregulated GSHS whereas jasmonic acid upregulated it. Cytokinins and polyamines activated GSHS but not γECS or hGSHS. Jasmonic acid elicited a coordinated response of the three genes and auxin induced both hGSHS expression and activity. Results show that the thiol biosynthetic pathway is compartmentalized in legumes. Moreover, the similar response profiles of the GSH and hGSH contents in roots of non-nodulated and nodulated plants to the various hormonal treatments indicate that thiol homeostasis is independent of the nitrogen source of the plants. The differential regulation of the three mRNA levels, hGSHS activity, and thiol contents by hormones indicates a fine control of thiol biosynthesis at multiple levels and strongly suggests that GSH and hGSH play distinct roles in plant development and stress responses

Regulation of heterogenous lexA expression in staphylococcus aureus by an antisense RNA originating from transcriptional read-through upon natural mispairings in the sbrB intrinsic terminator

Bacterial genomes are pervasively transcribed, generating a wide variety of antisense RNAs (asRNAs). Many of them originate from transcriptional read-through events (TREs) during the transcription termination process. Previous transcriptome analyses revealed that the lexA gene from Staphylococcus aureus, which encodes the main SOS response regulator, is affected by the presence of an asRNA. Here, we show that the lexA antisense RNA (lexA-asRNA) is generated by a TRE on the intrinsic terminator (TTsbrB) of the sbrB gene, which is located downstream of lexA, in the opposite strand. Transcriptional read-through occurs by a natural mutation that destabilizes the TTsbrB structure and modifies the efficiency of the intrinsic terminator. Restoring the mispairing mutation in the hairpin of TTsbrB prevented lexA-asRNA transcription. The level of lexA-asRNA directly correlated with cellular stress since the expressions of sbrB and lexA-asRNA depend on the stress transcription factor SigB. Comparative analyses revealed strain-specific nucleotide polymorphisms within TTsbrB, suggesting that this TT could be prone to accumulating natural mutations. A genome-wide analysis of TREs suggested that mispairings in TT hairpins might provide wider transcriptional connections with downstream genes and, ultimately, transcriptomic variability among S. aureus strains.This work was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant no. 646869 to A.T.-A.) and by the Spanish Ministry of Science and Innovation grants (BIO2017-83035-R to I.L. and PID2019-105216GB-I00 to A.T.-A.). Funding for open access charge was provided by the CSIC Open Access Publication Support Initiative, Unit of Information Resources for Research (URICI)

The MicroRNA390/TAS3 Pathway Mediates Symbiotic Nodulation and Lateral Root Growth

Legume roots form two types of postembryonic organs, lateral roots and symbiotic nodules. Nodule formation is the result of the interaction of legumes with rhizobia and requires the mitotic activation and differentiation of root cells as well as an independent, but coordinated, program that allows infection by rhizobia. MicroRNA390 (miR390) is an evolutionarily conserved microRNA that targets the Trans-Acting Short Interference RNA3 (TAS3) transcript. Cleavage of TAS3 by ARGONAUTE7 results in the production of trans-acting small interference RNAs, which target mRNAs encoding AUXIN RESPONSE FACTOR2 (ARF2), ARF3, and ARF4. Here, we show that activation of the miR390/TAS3 regulatory module by overexpression of miR390 in Medicago truncatula promotes lateral root growth but prevents nodule organogenesis, rhizobial infection, and the induction of two key nodulation genes, Nodulation Signaling Pathway1 (NSP1) and NSP2. Accordingly, inactivation of the miR390/TAS3 module, either by expression of a miR390 target mimicry construct or mutations in ARGONAUTE7, enhances nodulation and rhizobial infection, alters the spatial distribution of the nodules, and increases the percentage of nodules with multiple meristems. Our results revealed a key role of the miR390/TAS3 pathway in legumes as a modulator of lateral root organs, playing opposite roles in lateral root and nodule development.Facultad de Ciencias Exacta

NERNST: a genetically-encoded ratiometric non-destructive sensing tool to estimate NADP(H) redox status in bacterial, plant and animal systems

NADP(H) is a central metabolic hub providing reducing equivalents to multiple biosynthetic, regulatory and antioxidative pathways in all living organisms. While biosensors are available to determine NADP+ or NADPH levels in vivo, no probe exists to estimate the NADP(H) redox status, a determinant of the cell energy availability. We describe herein the design and characterization of a genetically-encoded ratiometric biosensor, termed NERNST, able to interact with NADP(H) and estimate E NADP(H). NERNST consists of a redox-sensitive green fluorescent protein (roGFP2) fused to an NADPH-thioredoxin reductase C module which selectively monitors NADP(H) redox states via oxido-reduction of the roGFP2 moiety. NERNST is functional in bacterial, plant and animal cells, and organelles such as chloroplasts and mitochondria. Using NERNST, we monitor NADP(H) dynamics during bacterial growth, environmental stresses in plants, metabolic challenges to mammalian cells, and wounding in zebrafish. NERNST estimates the NADP(H) redox poise in living organisms, with various potential applications in biochemical, biotechnological and biomedical research

Function of glutathione peroxidases in legume root nodules

© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.[EN] Glutathione peroxidases (Gpxs) are antioxidant enzymes not studied so far in legume nodules, despite the fact that reactive oxygen species are produced at different steps of the symbiosis. The function of two Gpxs that are highly expressed in nodules of the model legume Lotus japonicus was examined. Gene expression analysis, enzymatic and nitrosylation assays, yeast cell complementation, in situ mRNA hybridization, immunoelectron microscopy, and LjGpx-green fluorescent protein (GFP) fusions were used to characterize the enzymes and to localize each transcript and isoform in nodules. The LjGpx1 and LjGpx3 genes encode thioredoxin-dependent phospholipid hydroperoxidases and are differentially regulated in response to nitric oxide (NO) and hormones. LjGpx1 and LjGpx3 are nitrosylated in vitro or in plants treated with S-nitrosoglutathione (GSNO). Consistent with the modification of the peroxidatic cysteine of LjGpx3, in vitro assays demonstrated that this modification results in enzyme inhibition. The enzymes are highly expressed in the infected zone, but the LjGpx3 mRNA is also detected in the cortex and vascular bundles. LjGpx1 is localized to the plastids and nuclei, and LjGpx3 to the cytosol and endoplasmic reticulum. Based on yeast complementation experiments, both enzymes protect against oxidative stress, salt stress, and membrane damage. It is concluded that both LjGpxs perform major antioxidative functions in nodules, preventing lipid peroxidation and other oxidative processes at different subcellular sites of vascular and infected cells. The enzymes are probably involved in hormone and NO signalling, and may be regulated through nitrosylation of the peroxidatic cysteine essential for catalytic function.AS and PBS were the recipients of predoctoral (Formacion de Personal Investigador) and postdoctoral (Marie Curie) contracts, respectively. We thank Martin Crespi for help with in situ RNA hybridization and Simon Avery for sharing the yeast mutant and for helpful advice. This work was supported by Ministerio de Economia y Competitividad-Fondo Europeo de Desarrollo Regional (AGL2011-24524 and AGL2014-53717-R). The UMR1136 is supported by a grant overseen by the French National Research Agency (ANR) as part of the 'Investissements d'Avenir' programme (ANR-11-LABX-0002-01, Lab of Excellence ARBRE). MM and KJD acknowledge support within SPP1710. The proteomic analysis was performed in the CSIC/UAB Proteomics Facility of IIBB-CSIC that belongs to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001.Matamoros, MA.; Saiz Andres, A.; Peñuelas, M.; Bustos-Sanmamed, P.; Mulet Salort, JM.; Barja, MV.; Rouhier, N.... (2015). Function of glutathione peroxidases in legume root nodules. Journal of Experimental Botany. 66(10):2979-2990. https://doi.org/10.1093/jxb/erv066S297929906610Astier, J., Kulik, A., Koen, E., Besson-Bard, A., Bourque, S., Jeandroz, S., … Wendehenne, D. (2012). Protein S-nitrosylation: What’s going on in plants? Free Radical Biology and Medicine, 53(5), 1101-1110. doi:10.1016/j.freeradbiomed.2012.06.032Avery, A. M., & Avery, S. V. (2001). Saccharomyces cerevisiaeExpresses Three Phospholipid Hydroperoxide Glutathione Peroxidases. Journal of Biological Chemistry, 276(36), 33730-33735. doi:10.1074/jbc.m105672200Avsian-Kretchmer, O., Gueta-Dahan, Y., Lev-Yadun, S., Gollop, R., & Ben-Hayyim, G. (2004). The Salt-Stress Signal Transduction Pathway That Activates the gpx1 Promoter Is Mediated by Intracellular H2O2, Different from the Pathway Induced by Extracellular H2O2. Plant Physiology, 135(3), 1685-1696. doi:10.1104/pp.104.041921Balmer, Y., Koller, A., del Val, G., Manieri, W., Schurmann, P., & Buchanan, B. B. (2002). Proteomics gives insight into the regulatory function of chloroplast thioredoxins. Proceedings of the National Academy of Sciences, 100(1), 370-375. doi:10.1073/pnas.232703799Becana, M., Matamoros, M. A., Udvardi, M., & Dalton, D. A. (2010). Recent insights into antioxidant defenses of legume root nodules. New Phytologist, 188(4), 960-976. doi:10.1111/j.1469-8137.2010.03512.xBrigelius-Flohé, R., & Maiorino, M. (2013). Glutathione peroxidases. Biochimica et Biophysica Acta (BBA) - General Subjects, 1830(5), 3289-3303. doi:10.1016/j.bbagen.2012.11.020Bright, J., Desikan, R., Hancock, J. T., Weir, I. S., & Neill, S. J. (2005). ABA-induced NO generation and stomatal closure in Arabidopsis are dependent on H2 O2 synthesis. The Plant Journal, 45(1), 113-122. doi:10.1111/j.1365-313x.2005.02615.xBroughton, W. J., & Dilworth, M. J. (1971). Control of leghaemoglobin synthesis in snake beans. Biochemical Journal, 125(4), 1075-1080. doi:10.1042/bj1251075Camerini, S., Polci, M. L., Restuccia, U., Usuelli, V., Malgaroli, A., & Bachi, A. (2007). A Novel Approach to Identify Proteins Modified by Nitric Oxide:  the HIS-TAG Switch Method. Journal of Proteome Research, 6(8), 3224-3231. doi:10.1021/pr0701456Chang, C. C. C., Ślesak, I., Jordá, L., Sotnikov, A., Melzer, M., Miszalski, Z., … Karpiński, S. (2009). Arabidopsis Chloroplastic Glutathione Peroxidases Play a Role in Cross Talk between Photooxidative Stress and Immune Responses. Plant Physiology, 150(2), 670-683. doi:10.1104/pp.109.135566Colebatch, G., Kloska, S., Trevaskis, B., Freund, S., Altmann, T., & Udvardi, M. K. (2002). Novel Aspects of Symbiotic Nitrogen Fixation Uncovered by Transcript Profiling with cDNA Arrays. Molecular Plant-Microbe Interactions, 15(5), 411-420. doi:10.1094/mpmi.2002.15.5.411Dalton, D. A. (1995). Antioxidant Defenses of Plants and Fungi. Oxidative Stress and Antioxidant Defenses in Biology, 298-355. doi:10.1007/978-1-4615-9689-9_9FOYER, C. H., & NOCTOR, G. (2005). Oxidant and antioxidant signalling in plants: a re-evaluation of the concept of oxidative stress in a physiological context. Plant, Cell and Environment, 28(8), 1056-1071. doi:10.1111/j.1365-3040.2005.01327.xFu, L.-H., Wang, X.-F., Eyal, Y., She, Y.-M., Donald, L. J., Standing, K. G., & Ben-Hayyim, G. (2002). A Selenoprotein in the Plant Kingdom. Journal of Biological Chemistry, 277(29), 25983-25991. doi:10.1074/jbc.m202912200Gaber, A., Ogata, T., Maruta, T., Yoshimura, K., Tamoi, M., & Shigeoka, S. (2012). The Involvement of Arabidopsis Glutathione Peroxidase 8 in the Suppression of Oxidative Damage in the Nucleus and Cytosol. Plant and Cell Physiology, 53(9), 1596-1606. doi:10.1093/pcp/pcs100Daniel Gietz, R., & Woods, R. A. (2002). Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods in Enzymology, 87-96. doi:10.1016/s0076-6879(02)50957-5Gueta-Dahan, Y., Yaniv, Z., Zilinskas, B. A., & Ben-Hayyim, G. (1997). Salt and oxidative stress: similar and specific responses and their relation to salt tolerance in Citrus. Planta, 203(4), 460-469. doi:10.1007/s004250050215Herbette, S., Lenne, C., Leblanc, N., Julien, J.-L., Drevet, J. R., & Roeckel-Drevet, P. (2002). Two GPX-like proteins fromLycopersicon esculentumandHelianthus annuusare antioxidant enzymes with phospholipid hydroperoxide glutathione peroxidase and thioredoxin peroxidase activities. European Journal of Biochemistry, 269(9), 2414-2420. doi:10.1046/j.1432-1033.2002.02905.xHerbette, S., Roeckel-Drevet, P., & Drevet, J. R. (2007). Seleno-independent glutathione peroxidases. FEBS Journal, 274(9), 2163-2180. doi:10.1111/j.1742-4658.2007.05774.xJaffrey, S. R., Erdjument-Bromage, H., Ferris, C. D., Tempst, P., & Snyder, S. H. (2001). Protein S-nitrosylation: a physiological signal for neuronal nitric oxide. Nature Cell Biology, 3(2), 193-197. doi:10.1038/35055104Jung, B. G., Lee, K. O., Lee, S. S., Chi, Y. H., Jang, H. H., Kang, S. S., … Lee, S. Y. (2002). A Chinese Cabbage cDNA with High Sequence Identity to Phospholipid Hydroperoxide Glutathione Peroxidases Encodes a Novel Isoform of Thioredoxin-dependent Peroxidase. Journal of Biological Chemistry, 277(15), 12572-12578. doi:10.1074/jbc.m110791200Koh, C. S., Didierjean, C., Navrot, N., Panjikar, S., Mulliert, G., Rouhier, N., … Corbier, C. (2007). Crystal Structures of a Poplar Thioredoxin Peroxidase that Exhibits the Structure of Glutathione Peroxidases: Insights into Redox-driven Conformational Changes. Journal of Molecular Biology, 370(3), 512-529. doi:10.1016/j.jmb.2007.04.031Kuranda, K., Leberre, V., Sokol, S., Palamarczyk, G., & Francois, J. (2006). Investigating the caffeine effects in the yeast Saccharomyces cerevisiae brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways. Molecular Microbiology, 61(5), 1147-1166. doi:10.1111/j.1365-2958.2006.05300.xLivak, K. J., & Schmittgen, T. D. (2001). Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method. Methods, 25(4), 402-408. doi:10.1006/meth.2001.1262Maiorino, M., Gregolin, C., & Ursini, F. (1990). [47] Phospholipid hydroperoxide glutathione peroxidase. Methods in Enzymology, 448-457. doi:10.1016/0076-6879(90)86139-mMargis, R., Dunand, C., Teixeira, F. K., & Margis-Pinheiro, M. (2008). Glutathione peroxidase family - an evolutionary overview. FEBS Journal, 275(15), 3959-3970. doi:10.1111/j.1742-4658.2008.06542.xMiao, Y., Lv, D., Wang, P., Wang, X.-C., Chen, J., Miao, C., & Song, C.-P. (2006). An Arabidopsis Glutathione Peroxidase Functions as Both a Redox Transducer and a Scavenger in Abscisic Acid and Drought Stress Responses. The Plant Cell, 18(10), 2749-2766. doi:10.1105/tpc.106.044230Mullineaux, P. M., Karpinski, S., Jimenez, A., Cleary, S. P., Robinson, C., & Creissen, G. P. (1998). Identification of cDNAS encoding plastid-targeted glutathione peroxidase. The Plant Journal, 13(3), 375-379. doi:10.1046/j.1365-313x.1998.00052.xNakagawa, T., Kurose, T., Hino, T., Tanaka, K., Kawamukai, M., Niwa, Y., … Kimura, T. (2007). Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation. Journal of Bioscience and Bioengineering, 104(1), 34-41. doi:10.1263/jbb.104.34Navrot, N., Collin, V., Gualberto, J., Gelhaye, E., Hirasawa, M., Rey, P., … Rouhier, N. (2006). Plant Glutathione Peroxidases Are Functional Peroxiredoxins Distributed in Several Subcellular Compartments and Regulated during Biotic and Abiotic Stresses. Plant Physiology, 142(4), 1364-1379. doi:10.1104/pp.106.089458Passaia, G., Queval, G., Bai, J., Margis-Pinheiro, M., & Foyer, C. H. (2014). The effects of redox controls mediated by glutathione peroxidases on root architecture in Arabidopsis thaliana. Journal of Experimental Botany, 65(5), 1403-1413. doi:10.1093/jxb/ert486Perazzolli, M., Dominici, P., Romero-Puertas, M. C., Zago, E., Zeier, J., Sonoda, M., … Delledonne, M. (2004). Arabidopsis Nonsymbiotic Hemoglobin AHb1 Modulates Nitric Oxide Bioactivity. The Plant Cell, 16(10), 2785-2794. doi:10.1105/tpc.104.025379Puppo, A., Herrada, G., & Rigaud, J. (1991). Lipid Peroxidation in Peribacteroid Membranes from French-Bean Nodules. Plant Physiology, 96(3), 826-830. doi:10.1104/pp.96.3.826Puppo, A., Pauly, N., Boscari, A., Mandon, K., & Brouquisse, R. (2013). Hydrogen Peroxide and Nitric Oxide: Key Regulators of the Legume—Rhizobium and Mycorrhizal Symbioses. Antioxidants & Redox Signaling, 18(16), 2202-2219. doi:10.1089/ars.2012.5136Ramos, J., Matamoros, M. A., Naya, L., James, E. K., Rouhier, N., Sato, S., … Becana, M. (2008). The glutathione peroxidase gene family of Lotus japonicus : characterization of genomic clones, expression analyses and immunolocalization in legumes. New Phytologist, 181(1), 103-114. doi:10.1111/j.1469-8137.2008.02629.xMilla, M. A. R., Maurer, A., Huete, A. R., & Gustafson, J. P. (2003). Glutathione peroxidase genes in Arabidopsis are ubiquitous and regulated by abiotic stresses through diverse signaling pathways. The Plant Journal, 36(5), 602-615. doi:10.1046/j.1365-313x.2003.01901.xROMERO-PUERTAS, M. C., RODRIGUEZ-SERRANO, M., CORPAS, F. J., GOMEZ, M., DEL RIO, L. A., & SANDALIO, L. M. (2004). Cadmium-induced subcellular accumulation of O2.- and H2O2 in pea leaves. Plant, Cell and Environment, 27(9), 1122-1134. doi:10.1111/j.1365-3040.2004.01217.xRubio, M. C., Becana, M., Kanematsu, S., Ushimaru, T., & James, E. K. (2009). Immunolocalization of antioxidant enzymes in high-pressure frozen root and stem nodules of Sesbania rostrata. New Phytologist, 183(2), 395-407. doi:10.1111/j.1469-8137.2009.02866.xSainz, M., Pérez-Rontomé, C., Ramos, J., Mulet, J. M., James, E. K., Bhattacharjee, U., … Becana, M. (2013). Plant hemoglobins may be maintained in functional form by reduced flavins in the nuclei, and confer differential tolerance to nitro-oxidative stress. The Plant Journal, 76(5), 875-887. doi:10.1111/tpj.12340Seidel, T., Kluge, C., Hanitzsch, M., Roß, J., Sauer, M., Dietz, K.-J., & Golldack, D. (2004). Colocalization and FRET-analysis of subunits c and a of the vacuolar H+-ATPase in living plant cells. Journal of Biotechnology, 112(1-2), 165-175. doi:10.1016/j.jbiotec.2004.04.027Serrano, R., Mulet, J. M., Rios, G., Marquez, J. A., Larrinoa, I. igo F. de, Leube, M. P., … Montesinos, C. (1999). A glimpse of the mechanisms of ion homeostasis during salt stress. Journal of Experimental Botany, 50(Special_Issue), 1023-1036. doi:10.1093/jxb/50.special_issue.1023Tovar-Méndez, A., Matamoros, M. A., Bustos-Sanmamed, P., Dietz, K.-J., Cejudo, F. J., Rouhier, N., … Becana, M. (2011). Peroxiredoxins and NADPH-Dependent Thioredoxin Systems in the Model Legume Lotus japonicus. Plant Physiology, 156(3), 1535-1547. doi:10.1104/pp.111.177196Wolff, S. P. (1994). [18] Ferrous ion oxidation in presence of ferric ion indicator xylenol orange for measurement of hydroperoxides. Oxygen Radicals in Biological Systems Part C, 182-189. doi:10.1016/s0076-6879(94)33021-

Regulación de la expresión de antioxidantes y hemoglobinas de Lotus japonicus en respuesta a estrés y hormonas

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.Peer reviewe

The fission yeast Map4 protein is a novel adhesin required for mating

AbstractCell adhesion is required for many cellular processes. In fungi, cell–cell contact during mating, flocculation or virulence is mediated by adhesins, which typically are glycosyl phosphatidyl inositol (GPI)-modified cell wall glycoproteins. Proteins with internal repeats (PIR) are surface proteins involved in the response to stress. In Schizosaccharomyces pombe no adhesins or PIR proteins have been described. Here we study the S. pombe Map4p, which defines a new class of surface protein that is not GPI-modified and has a serine/threonine rich domain and internal repeats that differ from those present in PIR proteins. Map4p is a mating type-specific adhesin required for mating in h+ cells and enhances cell adhesion when overexpressed

Ligands of boron in Pisum sativum nodules are involved in regulation of oxygen concentration and rhizobial infection

16 Pags., 7 Figs. The definitive version is available at: http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1365-3040Boron (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.Peer reviewe