MtCOPT1 mediates copper transport to Medicago truncatula nodules

Copper is an essential oligonutrient. Its redox properties allow it to be a suitable cofactor for many proteins, such as cytochromes or superoxide dismutases. Copper is key for Symbiotic Nitrogen Fixation (SNF); For instance, bacteroids contain copper-dependent cytochrome oxidases that provide energy in the microaerobic environment within the nodule. Once copper is in the plant, it is delivered by the vasculature to the apoplast of zone II. From there, a plasma membrane transporter introduces this nutrient into the cell for copper-protein assembly. COPT family transporters mediate high-affinity copper transport towards the cytosol. Therefore, they are good candidates to introduce copper in nodule cells. From the 8 COPT family genes present in M. truncatula genome, MtCOPT1 is the only one induced specifically in nodule. MtCOPT1 can restore Saccharomyces cerevisae ?ctr1 capacity to uptake copper. Inmunolocalization and GUS fusion studies localize MtCOPT1 in the nodule. Moreover, a Tnt-1-derived knockdown mutant line for MtCOPT1 shows decreased nitrogenase activity when compared with the wild-type line. This activity is, at least, partially rescued when a wild-type copy of MtCOPT1 gene is reintroduced. Taken together, all this data suggest an important role of MtCOPT1 copper-mediated transport for SNF

MtMTP2-Facilitated Zinc Transport Into Intracellular Compartments Is Essential for Nodule Development in Medicago truncatula

Zinc (Zn) is an essential nutrient for plants that is involved in almost every biological process. This includes symbiotic nitrogen fixation, a process carried out by endosymbiotic bacteria (rhizobia) living within differentiated plant cells of legume root nodules. Zn transport in nodules involves delivery from the root, via the vasculature, release into the apoplast and uptake into nodule cells. Once in the cytosol, Zn can be used directly by cytosolic proteins or delivered into organelles, including symbiosomes of infected cells, by Zn efflux transporters. Medicago truncatula MtMTP2 (Medtr4g064893) is a nodule-induced Zn-efflux protein that was localized to an intracellular compartment in root epidermal and endodermal cells, as well as in nodule cells. Although the MtMTP2 gene is expressed in roots, shoots, and nodules, mtp2 mutants exhibited growth defects only under symbiotic, nitrogen-fixing conditions. Loss of MtMTP2 function resulted in altered nodule development, defects in bacteroid differentiation, and severe reduction of nitrogenase activity. The results presented here support a role of MtMTP2 in intracellular compartmentation of Zn, which is required for effective symbiotic nitrogen fixation in M. truncatula

Papel de COPT1, COPT2, MTP2 y ZIP6 en el transporte y la homeostasis de metales en Medicago truncatula

El cobre y el zinc son cofactores esenciales de enzimas involucradas en múltiples procesos fisiológicos (Finkelstein, 2009). En plantas participan, entre otros procesos, en la fotosíntesis (Yruela, 2013), en la respiración oxidativa (Ravet & Pilon, 2013), la floración (Takahashi et al., 2003), y, en el caso de las leguminosas, en la fijación simbiótica de nitrógeno (Brear et al., 2013; González-Guerrero et al., 2016). Dada su importancia como nutrientes, se ha realizado un importante esfuerzo para entender cómo las plantas distribuyen y usan estos oligonutrientes metálicos. Sin embargo, este trabajo ha sido principalmente realizado en la planta modelo Arabidopsis thaliana (Peñarrubia et al., 2015; Brumbarova et al., 2015) y en cereales (Nozoye et al., 2011; von Wirén et al., 1994). Por ello, mucho menos se conoce sobre cómo estos metales son transferidos por las leguminosas a sus nódulos radicales. En la presente Tesis Doctoral, hemos estudiado dos transportadores involucrados en el suministro de zinc para la fijación simbiótica de nitrógeno, y uno involucrado en la incorporación de cobre. Además, trabajando en este último, se ha identificado a un homólogo cercano que podría estar involucrado en la captación de cobre de hongos formadores de micorrizas arbusculares. ----------ABSTRACT---------- Copper and zinc are essential cofactors of enzymes involved in multiple physiological processes (Finkelstein, 2009). In plants, they participate in photosynthesis (Yruela, 2013), oxidative respiration (Ravet & Pilon, 2013), flowering (Takahashi et al., 2003), and, in the case of legumes, in symbiotic nitrogen fixation (Brear et al., 2013; González-Guerrero et al., 2016), among others. Given their importance as nutrients, a significant effort has been invested in understanding how plants distribute and used these essential metallic oligonutrients. However, this work has been mainly done in model plant Arabidopsis thaliana (Peñarrubia et al., 2015; Brumbarova et al., 2015) and in cereals (Nozoye et al., 2011; von Wirén et al., 1994). Consequently, much less is known on how these metals are delivered by the legumes to their root nodules. In this PhD thesis, we have studied two transporters involved in zinc delivery for symbiotic nitrogen fixation, and one involved in copper uptake. In addition, while working on the later, we have also identified a close homologue putatively involved in copper uptake form arbuscular mycorrhizal fungi

MtCOPT2 is a Cu+ transporter specifically expressed in Medicago truncatula mycorrhizal roots

Arbuscular mycorrhizal fungi are critical participants in plant nutrition in natural ecosystems and in sustainable agriculture. A large proportion of the phosphorus, nitrogen, sulfur, and transition metal elements that the host plant requires are obtained from the soil by the fungal mycelium and released at the arbuscules in exchange for photosynthates. While many of the plant transporters responsible for obtaining macronutrients at the periarbuscular space have been characterized, the identities of those mediating transition metal uptake remain unknown. In this work, MtCOPT2 has been identified as the only member of the copper transporter family COPT in the model legume Medicago truncatula to be specifically expressed in mycorrhizal roots. Fusing a C-terminal GFP tag to MtCOPT2 expressed under its own promoter showed a distribution pattern that corresponds with arbuscule distribution in the roots. When expressed in tobacco leaves, MtCOPT2-GFP co-localizes with a plasma membrane marker. MtCOPT2 is intimately related to the rhizobial nodule-specific MtCOPT1, which is suggestive of a shared evolutionary lineage that links transition metal nutrition in the two main root endosymbioses in legumes.This research was funded by a European Research Council Starting Grant (ERC-2013-StG-335284) to MGG. IA is the recipient of a Juan de la Cierva -Formación- postdoctoral fellowship from Ministerio de Ciencia, Innovación y Universidades (FJCI-2017-33222). VE was partially funded by the Severo Ochoa Programme for Centres of Excellence in R&D from Agencia Estatal de Investigación of Spain (grant SEV-2016-0672) received by Centro de Biotecnología y Genómica de Plantas (UPM-INIA)

The Pseudomonas syringae pv. Tomato DC3000 PSPTO_0820 multidrug transporter is involved in resistance to plant antimicrobials and bacterial survival during tomato plant infection

Multidrug resistance efflux pumps protect bacterial cells against a wide spectrum of antimicrobial compounds. PSPTO_0820 is a predicted multidrug transporter from the phytopatho-genic bacterium Pseudomonas syringae pv. tomato DC3000. Orthologs of this protein are conserved within many Pseudomonas species that interact with plants. To study the potential role of PSPTO_0820 in plant-bacteria interaction, a mutant in this gene was isolated and characterized. In addition, with the aim to find the outer membrane channel for this efflux system, a mutant in PSPTO_4977, a TolC-like gene, was also analyzed. Both mutants were more susceptible to trans-cinnamic and chlorogenic acids and to the flavonoid (+)-catechin, when added to the culture medium. The expression level of both genes increased in the presence of (+)-catechin and, in the case of PSPTO_0820, also in response to trans-cinnamic acid. PSPTO_0820 and PSPTO_4977 mutants were unable to colonize tomato at high population levels. This work evidences the involvement of these two proteins in the resistance to plant antimicrobials, supporting also the importance of chlorogenic acid, trans-cinnamic acid, and (+)-catechin in the tomato plant defense response against P. syringae pv. tomato DC3000 infection.This research has been supported by the Spanish Plan Nacional I+D+i grants AGL-2012- 32516 and AGL 2015-63851-R. JJRH was funded by the Ramo´n y Cajal program from the Spanish Ministry of Science and Innovation (RYC-2007- 01045). PMMG was supported by the Campus de Excelencia Internacional Andalucı´a TECH

Medicago truncatula Zinc-Iron Permease6 provides zinc to rhizobia-infected nodule cells

International audienceZinc is a micronutrient required for symbiotic nitrogen fixation. It has been proposed that in model legume Medicago truncatula, zinc is delivered by the root vasculature into the nodule and released in the infection/differentiation zone. There, transporters must introduce this element into rhizobia-infected cells to metallate the apoproteins that use zinc as a cofactor. MtZIP6 (Medtr4g083570) is an M. truncatula Zinc-Iron Permease (ZIP) that is expressed only in roots and nodules, with the highest expression levels in the infection/differentiation zone. Immunolocalization studies indicate that it is located in the plasma membrane of nodule rhizobia-infected cells. Down-regulating MtZIP6 expression levels with RNAi does not result in any strong phenotype when plants are fed mineral nitrogen. However, these plants displayed severe growth defects when they depended on nitrogen fixed by their nodules, losing of 80% of their nitrogenase activity. The reduction of this activity was likely an indirect effect of zinc being retained in the infection/differentiation zone and not reaching the cytosol of rhizobia-infected cells. These data are consistent with a model in which MtZIP6 would be responsible for zinc uptake by rhizobia-infected nodule cells in the infection/differentiation zone

Talleres en la escuela : medio didáctico para la integración de niños con problemas de adaptación social (II parte)

Proyecto de creación de talleres, iniciado el curso pasado, que se centra en la elaboración de diferentes estrategias encaminadas a la socialización y al desarrollo de valores socio-emocionales que faciliten la adaptación e integración social de un alumnado, principalmente de raza gitana, que se desenvuelve en un entorno marginal (Barrio del Pozo del Huevo). Los objetivos son: promover la asunción de responsabilidades y toma de decisiones; fomentar alternativas para un empleo adecuado del ocio y tiempo libre; interiorizar hábitos elementales de convivencia y respeto hacia los demás y hacia el entorno; adquirir habilidades útiles para su posterior inserción profesional en la sociedad; y desarrollar sus capacidades motrices. Para ello, a través de los cuatro talleres creados (huerto, madera, experiencias y educación física), se trabajan de forma lúdica los objetivos y contenidos de las distintas áreas curriculares. Así, en el taller de experiencias, dirigido al ciclo inicial de EGB, se realizan actividades de carácter global, mediante la creación de rincones de trabajo (lectura, composición, expresión matemática, plástica y dinámica, y huerto), como juegos de prelectura, preescritura, técnicas de manipulación, juegos lógicos, expresión corporal y psicomotricidad. En el taller de huerto, dirigido a todo el alumnado del centro, se continúa la labor iniciada cursos anteriores centrada en el mantenimiento del huerto y con la preparación de nuevos terrenos y semilleros. El taller de madera, dirigido al ciclo superior, que se organiza en torno a agrupamientos flexibles y al trabajo en equipo, consiste en la aplicación práctica de lo trabajado en el resto de las áreas (carácter técnico-manual). Y, por último, en el taller de educación física, dirigido a todo el alumnado, se realiza una adaptación de los programas a las necesidades del alumno, haciendo hincapié en los objetivos de carácter social y primando el desarrollo psicomotor y el aprovechamiento del tiempo de ocio. La evaluación de la experiencia se considera muy positiva, aunque señala la necesidad de seguir trabajando para alcanzar los objetivos planteados ya que su propuesta es a largo plazo. Se presentan memorias individuales por talleres..Madrid (Comunidad Autónoma). Consejería de Educación y CulturaMadridMadrid (Comunidad Autónoma). Subdirección General de Formación del Profesorado. CRIF Las Acacias; General Ricardos 179 - 28025 Madrid; Tel. + 34915250893ES

Image_1_MtMTP2-Facilitated Zinc Transport Into Intracellular Compartments Is Essential for Nodule Development in Medicago truncatula.PDF

<p>Zinc (Zn) is an essential nutrient for plants that is involved in almost every biological process. This includes symbiotic nitrogen fixation, a process carried out by endosymbiotic bacteria (rhizobia) living within differentiated plant cells of legume root nodules. Zn transport in nodules involves delivery from the root, via the vasculature, release into the apoplast and uptake into nodule cells. Once in the cytosol, Zn can be used directly by cytosolic proteins or delivered into organelles, including symbiosomes of infected cells, by Zn efflux transporters. Medicago truncatula MtMTP2 (Medtr4g064893) is a nodule-induced Zn-efflux protein that was localized to an intracellular compartment in root epidermal and endodermal cells, as well as in nodule cells. Although the MtMTP2 gene is expressed in roots, shoots, and nodules, mtp2 mutants exhibited growth defects only under symbiotic, nitrogen-fixing conditions. Loss of MtMTP2 function resulted in altered nodule development, defects in bacteroid differentiation, and severe reduction of nitrogenase activity. The results presented here support a role of MtMTP2 in intracellular compartmentation of Zn, which is required for effective symbiotic nitrogen fixation in M. truncatula.</p