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14 Pags.- 7 Figs., Mercury (Hg) is one of the most hazardous pollutants released by humans and is of global environmental concern. Mercury causes oxidative stress and strong cellular damages in plants, which can be attenuated by the biosynthesis of thiol-rich peptides (biothiols), including glutathione (GSH) and phytochelatins (PCs). We analysed Hg tolerance and speciation in five Arabidopsis thaliana genotypes, the wild-type Col-0, three knockdown γ-glutamylcysteine synthetase (γECS) mutants and a knockout PC synthase (PCS) mutant. Mercury-PC complexes were detected in roots by HPLC-ESI-TOFMS, with its abundance being limited in γECS mutants. Analysis of Hg-biothiol complexes in the xylem sap revealed that HgPC2 occurs in wild-type Col-0 Arabidopsis, suggesting that Hg could be translocated associated with thiol-rich metabolites. Twenty genes involved in sulphur assimilation, GSH and PCs synthesis were differentially expressed in roots and shoots, implying a complex regulation, possibly involving post-translational mechanisms independent of GSH cellular levels. In summary, the present study describes the importance of biothiol metabolism and adequate GSH levels in Hg tolerance and identifies for the first time Hg-PC complexes in the xylem sap. This finding supports the notion that Hg-biothiol complexes could contribute to Hg mobilisation within plants., Work supported by the Spanish State Research Agency (AEI) co-financed with the European Regional Development Fund (ERDF) (projects AGL2014-53771-R and AGL2017-87591-R to LEH and AGL2016-75226-R to AAF and JA; AEI/ERDF, UE).

Main conclusion The ratio of nicotianamine to deoxymugenic acid controls tissue-specific metal homeostasis in rice and regulates metal delivery to the endosperm. Abstract The metal-chelating phytosiderophores nicotianamine (NA) and 2′deoxymugenic acid (DMA) are significant factors for the control of metal homeostasis in graminaceous plants. These compounds are thought to influence metal homeostasis, but their individual roles and the effect of altering the NA:DMA ratio are unknown. We purposely generated rice lines with high and low NA:DMA ratios (HND and LND lines, respectively). The HND lines accumulated more iron (Fe), zinc (Zn), manganese (Mn) and copper (Cu) in the endosperm through the mobilization of Fe, Zn and Mn from the seed husk to the endosperm. In contrast, Fe, Zn and Mn were mobilized to the husk in the LND lines, whereas Cu accumulated in the endosperm. Different groups of metals are, therefore, taken up, transported and sequestered in vegetative tissues in the HND and LND lines to achieve this metal distribution pattern in the seeds. We found that different sets of endogenous metal homeostasis genes were modulated in the HND and LND lines to achieve differences in metal homeostasis. Our findings demonstrate that the NA:DMA ratio is a key factor regulating metal homeostasis in graminaceous plants. These findings can help formulate refined strategies to improve nutrient composition and nutrient use efficiency in crop plants., We acknowledge support from the European Research Council IDEAS Advanced Grant Program (BIOFORCE) to P.C., Generalitat de Catalunya Grant 2017 SGR 828 to ABBU (BIO2014-54426-P) and the Spanish Ministry of Economy and Competitivity (MINECO; projects AGL2016-75226-R, co-financed with FEDER) and the Aragón Government (Group A09_17R) to J.A. R.B was supported by a PhD fellowship from the University of Lleida, Spain.

17 Pags.- 7 Figs.- 1 Tabl. Copyright © 2018 Díaz-Benito, Banakar, Rodríguez-Menéndez, Capell, Pereiro, Christou, Abadía, Fernández and Álvarez-Fernández. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms., Iron and Zn deficiencies are worldwide nutritional disorders that can be alleviated by increasing the metal concentration of rice (Oryza sativa L.) grains via bio-fortification approaches. The overproduction of the metal chelator nicotianamine (NA) is among the most effective ones, but it is still unclear whether this is due to the enrichment in NA itself and/or the concomitant enrichment in the NA derivative 2′-deoxymugineic acid (DMA). The endosperm is the most commonly consumed portion of the rice grain and mediates the transfer of nutrients from vegetative tissues to the metal rich embryo. The impact of contrasting levels of DMA and NA on the metal distribution in the embryo and endosperm of rice seeds has been assessed using wild-type rice and six different transgenic lines overexpressing nicotianamine synthase (OsNAS1) and/or barley nicotianamine amino transferase (HvNAATb). These transgenic lines outlined three different DMA/NA scenarios: (i) in a first scenario, an enhanced NA level (via overexpression of OsNAS1) would not be fully depleted because of a limited capacity to use NA for DMA synthesis (lack of -or low- expression of HvNAATb), and results in consistent enrichments in NA, DMA, Fe and Zn in the endosperm and NA, DMA and Fe in the embryo; (ii) in a second scenario, an enhanced NA level (via overexpression of OsNAS1) would be depleted by an enhanced capacity to use NA for DMA synthesis (via expression of HvNAATb), and results in enrichments only for DMA and Fe, both in the endosperm and embryo, and (iii) in a third scenario, the lack of sufficient NA replenishment would limit DMA synthesis, in spite of the enhanced capacity to use NA for this purpose (via expression of HvNAATb), and results in decreases in NA, variable changes in DMA and moderate decreases in Fe in the embryo and endosperm. Also, quantitative LA-ICP-MS metal map images of the embryo structures show that the first and second scenarios altered local distributions of Fe, and to a lesser extent of Zn. The roles of DMA/NA levels in the transport of Fe and Zn within the embryo are thoroughly discussed., This work was supported by the grants of the Spanish Ministry of Science, Innovation and Universities (AGL2016-75226-R, BIO2014-54426-P and AGL2017-85377-R, all co-financed with FEDER), Aragón Government (Group A09_17R) and Generalitat de Catalunya (Grant 2017 SGR 828). PD-B was supported by a MINECO-FPI contract. RB was supported by a Ph.D. fellowship from the University of Lleida. SR-M was supported by a research contract from the Fundación Universidad de Oviedo (FUO-069-17). BF was supported by a MINECO research contract (RYC-2014-14985; “Ramón y Cajal Program”).

14 Pags.- 6 Figs.- 1 Tabl. Copyright © 2020 The Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms., Symbiotic nitrogen fixation carried out by the interaction between legumes and diazotrophic bacteria known as rhizobia requires relatively large levels of transition metals. These elements are cofactors of many key enzymes involved in this process. Metallic micronutrients are obtained from soil by the roots and directed to sink organs by the vasculature, in a process mediated by a number of metal transporters and small organic molecules that facilitate metal delivery in the plant fluids. Among the later, nicotianamine is one of the most important. Synthesized by nicotianamine synthases (NAS), this molecule forms metal complexes participating in intracellular metal homeostasis and long-distance metal trafficking. Here we characterized the NAS2 gene from model legume Medicago truncatula. MtNAS2 is located in the root vasculature and in all nodule tissues in the infection and fixation zones. Symbiotic nitrogen fixation requires of MtNAS2 function, as indicated by the loss of nitrogenase activity in the insertional mutant nas2-1, phenotype reverted by reintroduction of a wild-type copy of MtNAS2. This would result from the altered iron distribution in nas2-1 nodules shown with X-ray fluorescence. Moreover, iron speciation is also affected in these nodules. These data suggest a role of nicotianamine in iron delivery for symbiotic nitrogen fixation., This research was funded by a European Research Council Starting Grant (ERC-2013-StG-335284) and a Ministerio de Economía y Competitividad (MINECO) grant (AGL2015-65866-P), to MG-G, and a MINECO grant (AGL2016-75226-R) to JA and AÁ-F. 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) to CBGP. IA is recipient of a Juan de la Cierva- Formación postodoctoral fellowship from Ministerio de Ciencia, Innovación y Universidades (FJCI-2017-33222). Development of M. truncatula Tnt1 mutant population was, in part, funded by the National Science Foundation, USA (DBI-0703285) to KM.

195 Pag., [ES] Los objetivos generales de esta tesis doctoral son: a) desarrollar y aplicar nuevas metodologías analíticas que permitan la especiación química del Fe en los fluidos vegetales involucrados en el transporte a larga distancia de este elemento en plantas y b) estudiar el perfil de metabolitos de plantas cultivadas en condiciones de deficiencia de Fe. Para alcanzar estos objetivos generales se plantean los siguientes objetivos específicos: 1. Estudiar el efecto del pH y las reacciones de intercambio del metal o el ligando sobre la formación de complejos metálicos con nicotianamina mediante espectrometría de masas (MS) con analizador de tiempo de vuelo (TOF) y fuente de electrospray (ESI). 2. Desarrollar un método que permita la determinación de complejos naturales de Fe en plantas involucrados en el transporte de este elemento vía xilema mediante cromatografía líquida de alta resolución (HPLC) acoplada a ESI-TOFMS y espectrometría de masas con fuente de plasma de acoplamiento inductivo (ICP-MS). 3. Desarrollar un método que permita la determinación de ácidos orgánicos en tejidos vegetales mediante HPLC-ES-TOFMS. 4. Caracterizar los cambios en el perfil de metabolitos que se producen en condiciones de deficiencia y reaporte de Fe en las raíces de las plantas mediante cromatografía de gases acoplada a espectrometría de masas (GC-MS). 5. Caracterizar los cambios en el perfil de metabolitos que se producen en condiciones de deficiencia y reaporte de Fe en la savia de xilema y las hojas de las plantas mediante GC-MS., [EN] The general objectives of this Thesis are: i) to develop and apply new analytical methodologies for the determination of the Fe forms involved in long-distance Fe transport in plants, and ii) to study the metabolite profile of plants grown under Fe-deficiency conditions. In order to achieve these general objectives the specific objectives are: 1. To study the formation of metal complexes with nicotianamine as affected by pH, ligand and metal exchange by means of electrospray time-of-flight mass spectrometry (ESI-TOFMS). 2. To develop a method for the determination of naturally occurring Fe complexes in xylem sap, using high performance liquid chromatography coupled to ESI-TOFMS and inductively coupled plasma mass spectrometry (HPLC-ESI-TOFMS and HPLC-ICP-MS, respectively). 3. To develop an HPLC-ESI-TOFMS method for the determination of organic acids in plant tissues. 4. To characterize the changes induced in the metabolite profile of plant roots in response to Fe deficiency and resupply, using gas chromatography coupled to mass spectrometry (GC-MS). 5. To characterize the changes induced in the metabolite profile of xylem sap and leaves of plants in response to Fe deficiency and resupply, using GC-MS., This Thesis was supported by the Spanish MICINN (projects AGL2004- 00194 and AGL2007-61948, co-financed with FEDER), the European Commission (Thematic Priority 5- Food Quality and Safety, 6th Framework RTD Programme, Contract no. FP6- FOOD-CT-2006-016279) and the Aragón Government (group A03). Rubén Rellán Álvarez was supported by a FPI fellowship from the Spanish Ministry of Science and Innovation (MICINN).

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. fecha de lectura: 11/2/2011

231 Pag., Tabl., Fig. Esta Tesis Doctoral ha sido escrita en la modalidad de compendio de trabajos previamente publicados o aceptados para su publicación y consta de las siguientes publicaciones: 1. Orera Irene, Abadía Javier, Abadía Anunciación, Álvarez-Fernández Ana. Analytical technologies to study the biological and environmental implications of iron-fertilisation using synthetic ferric chelates: the case of Fe(III)-EDDHA – a review. Journal of Horticultural Science and Biotechnology (2009) 84(1): 7-12. 2. Álvarez-Fernández Ana*, Orera Irene*, Abadía Javier, Abadía Anunciación. Determination of synthetic ferric chelates used as fertilizers by liquid Chromatography- Electrospray/Mass Spectrometry in agricultural matrices. Journal of the American Society for Mass Spectrometry (2007) 18: 37-47. 3. Orera Irene, Abadía Anuciación, Abadía Javier, Álvarez-Fernández Ana. Determination of o,oEDDHA -a xenobiotic chelating agent used in Fe-fertilizers- in plant tissues by liquid chromatography-electrospray mass spectrometry: overcoming matrix effects. Rapid Communications in Mass Spectrometry (2009) 23:1694-1702. DOI:10.1002/rcm.4056. 4. Orera Irene, Orduna Jesús, Abadía Javier, Álvarez-Fernández Ana. Electrospraycollision- induced dissociation mass spectrometry: a tool to characterize synthetic polyaminocarboxylate ferric chelates used as fertilizers. Rapid Communications in Mass Spectrometry (2010) 24(1):109-119. DOI: 10.1002/rcm.4361. 5. Orera Irene, Rodríguez-Castrillón Jose Ángel, Moldovan Mariella, García-Alonso Jose Ignacio, Abadía Anunciación, Abadía Javier, Álvarez-Fernández Ana. Using a dualstable isotope tracer method to study the uptake, xylem transport and distribution of Fe and its chelating agent from stereoisomers of a xenobiotic Fe(III)-chelate used as fertilizer in Fe-deficient Strategy I plants. Metallomics (enviado)., Los fertilizantes a base de quelatos férricos sintéticos son xenobióticos de elevado precio comúnmente utilizados para corregir o prevenir uno de los desórdenes nutricionales más habituales de las plantas, la deficiencia de Fe. De todos los quelatos férricos sintéticos que se comercializan en el mercado, aquellos basados en el quelato Fe(III)-o,oEDDHA son los más utilizados, sobre todo en cultivos de alto valor añadido crecidos sobre suelos calizos como los de Aragón. El uso de quelatos sintéticos con fines agrícolas o industriales está siendo cuestionado en la actualidad, ya que son difícilmente degradables, muy persistentes en el medio ambiente y pueden alterar el equilibrio natural de los metales (nutrientes o tóxicos) en el suelo. Los estudios sobre el efecto de los quelatos férricos sintéticos en el medio ambiente y su mecanismo de acción son aún escasos, debido a la falta de metodologías analíticas capaces de determinar concentraciones muy bajas de estos compuestos en matrices tan complejas como el sistema suelo-planta. Los equipos de espectrometría de masas ofrecen alta sensibilidad, selectividad y versatilidad en el análisis de compuestos químicos tanto a nivel atómico como molecular. Por este motivo, en esta Tesis Doctoral hemos apostado por esta técnica para desarrollar y validar un método analítico que permita determinar de manera directa y simultánea los principales quelatos férricos sintéticos usados como fertilizantes. Este método además permite cuantificar los quelatos en fertilizantes comerciales y matrices agrícolas líquidas como la solución del suelo, solución nutritiva, agua de riego y savia de xilema. Igualmente, se ha desarrollado otro método analítico de espectrometría de masas para determinar dos agentes quelantes de Fe(III), racémico y meso o,oEDDHA, en distintos tejidos (raíces, hojas, savia de xilema y fruto) y especies vegetales (tomate, remolacha y melocotonero) con muy alta selectividad y sensibilidad. La elección del estándar interno ha sido un factor crítico en el desarrollo de este método. Los quelatos marcados con isótopos estables de Fe no han resultado ser adecuados como estándares internos para esta aplicación, sin embargo, un análogo estructural, el compuesto metilado del o,oEDDHA, ha resultado satisfactorio., Los patrones de fragmentación de la mayoría de los quelatos férricos sintéticos utilizados como fertilizantes han sido descritos en esta Tesis Doctoral utilizando la técnica de espectrometría de masas en tándem. En general, los iones producto más intensos correspondieron a descarboxilaciones, a excepción de los quelatos Fe(III)-CDTA, Fe(III)-o,pEDDHA, Fe(III)-EDDCHA y Fe(III)-EDDHSA, en los que correspondieron a pérdidas en el anillo. Cuando los espectros se obtuvieron con un analizador de masas QTOF, se consiguió diferenciar entre isómeros posicionales como el Fe(III)-o,oEDDHA y el Fe(III)-o,pEDDHA. La espectrometría de masas en tándem ha sido utilizada también para proponer las identidades de dos compuestos de Fe desconocidos presentes en un fertilizante comercial a base del quelato férrico Fe(III)-EDDHA. Estos compuestos han resultado ser también quelatos férricos con una estructura similar a la del Fe(III)-EDDHA, derivados, probablemente, de impurezas de los reactivos de partida en la síntesis del agente quelante. Por último el marcaje diferencial con dos isótopos estables de Fe (54Fe y 57Fe) de los dos fertilizantes, racémico y meso Fe(III)-o,oEDDHA, ha resultado ser una herramienta de gran utilidad para estudios de nutrición de hierro en plantas, incluyendo absorción, transporte y distribución del Fe aportado. Los resultados de este trabajo muestran que el fertilizante meso Fe(III)-o,oEDDHA es dos veces más eficiente suministrando Fe que el racémico Fe(III)-o,oEDDHA en plantas de remolacha deficientes en Fe crecidas en hidroponía. Este hecho puede ser explicado por la mayor eficiencia de la enzima quelato reductasa en la reducción del meso Fe(III)-o,oEDDHA, que fue dos veces superior a la mostrada con el racémico Fe(III)-o,oEDDHA. Los agentes quelantes racémico y meso o,oEDDHA son rápidamente absorbidos por las plantas de remolacha deficientes en Fe, encontrándose cantidades significativas en todos los tejidos después de sólo 3 horas de tratamiento. Además, ambos agentes quelantes son absorbidos y distribuidos en una relación 1:1 en plantas de remolacha deficientes en Fe, siendo la cantidad de los mismos un orden de magnitud menor que la de Fe en todos los tejidos analizados. Este hecho indica que, aunque la mayoría del Fe se absorbe a través de un mecanismo que implica la disociación del quelato férrico, una pequeña parte puede ser tomado mediante mecanismos que no incluyen disociación., Esta tesis ha sido financiada por los proyectos de investigación AGL2006-1416 y AGL2007-61948 del Ministerio de Ciencia en Innovación (MICINN) y el proyecto ISAFRUIT No.FP6-FOOD-CT-2006-016279 de la Comunidad Europea. Irene Orera Utrilla ha sido financiada por una beca predoctoral CONAID-DGA del Gobierno de Aragón. Las estancias en centros de investigación fuera de Aragón han sido financiadas por el Programa Europa de la Caja de Ahorros de la Inmaculada.