plant stress physiology | plant stress physiology · no presentable página 13 de 19. 3.9...

Resumen de la documentación de la solicitud de ProyectoIndividual

Plan Nacional de I + D + I (2008-2011)

El investigador :Javier Abadía Bayona

Ha presentado la solicitud de código (Identificador de solicitud - PIN):361516533-16533-4-10

En el Programa : INVESTIGACIÓN FUNDAMENTAL

En el Subprograma: Proyecto de Investigación Fundamental no orientada

Con los siguientes anexos :

Memoria técnica

Memoria técnica: Sumario_Ejecutivo_METALPLANT.pdfMemoria técnica en inglés: Technical_Annex_METALPLANT.pdf

MINISTERIO DE CIENCIA E INNOVACIÓNDirección General de Programas y Transferencia de Conocimiento

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Currículos

Investigador Principal :Javier Abadía Bayona

Currículo:cv_JA.pdf

Otros Investigadores :

Nombre Investigador Currículo

Ana Flor López Millán cv_AFLM.pdf

Ana María Álvarez Fernández cv_AAF.pdf

Rubén Rellán Álvarez cv_RR.pdf

Giuseppe Lattanzio cv_GL.pdf

Oliver Fiehn cv_OF.pdf

Jian Feng Ma cv_JFM.pdf

Jorge Rodríguez Celma cv_JRC.pdf

Ferenc Fodor cv_FF.pdf

Fermin Morales Iribas cv_FM.pdf

Michael Grusak cv_MG.pdf

Otros documentos

Autorización para participar el investigador Michael Grusak : Aut_MG.pdfAutorización para participar el investigador Jian Feng Ma : Aut_JFM.pdfDocumento acreditativo de E.P.O: EPO_Timac-Agro_Bruker.pdfAutorización para participar el investigador Ferenc Fodor : Aut_FF.pdfAutorización para participar el investigador Oliver Fiehn : Aut_OF.pdf


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Plan Nacional de I + D + I (2008-2011)

Solicitud de ayuda para

Proyectos de Investigación Fundamental no orientada

PROGRAMA INVESTIGACIÓN FUNDAMENTAL

AREA TEMÁTICA GESTIÓN :Agricultura (AGL-AGR)

AREA PRINCIPAL ANEP :Agricultura

AREAS SECUNDARIAS ANEP :

Biología Vegetal, Animal y Ecología


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1. SOLICITUD

1.1. DATOS DEL PROYECTO

Título:Metalómica vegetal: una aproximación a la homeostasis de metales en plantas mediante espectrometría de masas integrada

Acrónimo:METALPLANT

Area de Gestión Científico-Técnica:Agricultura (AGL-AGR)

Código NABS:Agricultura

Clasificación UNESCO:2417:BIOLOGIA VEGETAL (BOTANICA), 3101:AGROQUIMICA, 3103:AGRONOMIA

Tipo:B-Tradicional

¿Estima que el proyecto de investigación que presenta puede ser susceptible de generar resultados en los que haya queproteger la propiedad intelectual (patentes)?:Sí

¿Considera usted que su proyecto puede clasificarse en una de las Áreas Estratégicas del Plan Nacional?No

¿Contempla el proyecto el desarrollo o la aplicación de herramientas de análisis masivo (genómica, proteónica u otras"ómicas")?:Sí

Duración (en años):3


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1.2. PALABRAS CLAVE PARA LA IDENTIFICACIÓN DEL PROYECTO

deficiencia de hierro, toxicidad de zinc, toxicidad de cadmio, metabolómica, proteómica, complejos metálicos

1.3. DATOS DE LA ENTIDAD SOLICITANTE

Entidad:CONSEJO SUPERIOR DE INVESTIGACIONES CIENTIFICAS

C.I.F.:Q2818002D

Centro:ESTACION EXPERIMENTAL DE AULA DEI (EEAD-CSIC)

Nombre del representante legal:JOSE LUIS ARRUE UGARTE

Correo electrónico:[email protected]

Dirección postal completa del departamento:

AVDA. DE MONTAÑANA, 1005Zaragoza50059 - ZARAGOZA

1.4. DATOS DEL INVESTIGADOR PRINCIPAL

Nombre:Javier Apellidos:Abadía Bayona

Nacionalidad:ESPAÑA

Dirección postal completa:c/ Monasterio de Siresa 15 6BZaragoza50002 - ZARAGOZA

Correo electrónico:[email protected] Teléfono:976497779(Ext.)

¿Es doctor?SI

¿Ha sido IP de un proyecto aprobado de más de un año?SI

Vinculación con el Centro:Funcionario

1.5. RÉGIMEN DE SUBVENCIÓN (ver apartados 5º.6 y 5º.7 de la Convocatoria):

Costes Marginales

Costes Totales


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1.6. SOLICITUD DE INCLUSIÓN EN EL PROGRAMA DE FORMACIÓN DE INVESTIGADORES(FPI)

¿Solicita que este subproyecto sea incluido en el Programa de Formación de Investigadores? SI NO

Número de investigadores que solicita: 1

1.7. SOLICITUD DE INCLUSIÓN DE UN TÉCNICO DE APOYO:

¿Solicita la inclusión de un Técnico de Apoyo? SI NO

1.8. ¿Existe alguna EPO, española o extranjera, interesada en los resultados del Proyecto deInvestigación?(La presencia de EPO es obligatoria para ser incluido en el Programa de Formación deTécnicos y recomendable en el Programa de Formación de Personal Investigador)

SI NO

Timac Agro España S.A. (CIPAV) CIF: A31007644 Tipo Vinculación: Seguimiento del proyecto

Bruker CIF: A84206051 Tipo Vinculación: Seguimiento del proyecto

1.9. ANTÁRTIDA

¿Su proyecto se va a desarrollar en las Bases Antárticas? SI NO


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2. RELACIÓN DE PERSONAL INVESTIGADOR

2.1.GRUPO INVESTIGADOR DE LA ENTIDAD SOLICITANTE

Personal titulado superior vinculado estatutaria o contractualmente a la Entidad.

Investigador Principal:

Resto de Investigadores:

Apellidos: Abadía Bayona Nombre: Javier

NIF: 17850563X Sexo: V M Año de nacimiento: 02/04/1954

Titulación académica: Doctor en Ciencias Grado: Doctor Licenciado/Ingeniero/Arquitecto

Categoría profesional: Profesor de Investigación

Dedicación al proyecto:Única (EDP = 1)Compartida en 2 proyectos (EDP = 0.5)

Apellidos: López Millán Nombre: Ana Flor

NIF: 29101691K Sexo: V M Año de nacimiento: 14/04/1970

Titulación académica: Doctor Ciencias Químicas Grado: Doctor Licenciado/Ingeniero/Arquitecto

Categoría profesional: Cientifíco Titular


Apellidos: Álvarez Fernández Nombre: Ana María

NIF: 09392534R Sexo: V M Año de nacimiento: 08/10/1968

Titulación académica: Doctor Ciencias Químicas Grado: Doctor Licenciado/Ingeniero/Arquitecto

Categoría profesional: Cientifíco Titular


Apellidos: Morales Iribas Nombre: Fermin

NIF: 18204666M Sexo: V M Año de nacimiento: 31/01/1965


Categoría profesional: Investigador Científico (CSIC)


EDP DEL GRUPO INVESTIGADOR DE LA ENTIDAD SOLICITANTE, SIN CONTAR OTROS MIEMBROS: P1 = 3.0


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2.2. OTROS MIEMBROS DEL GRUPO INVESTIGADOR DE LA ENTIDAD SOLICITANTE

(Profesores eméritos, doctores ad honorem, académicos numerarios y titulados superiores ligados al centro mediante contrato por obra o servicio o que

disfruten de una beca de formación. Ver aparatado 5 del Anexo 1 de la Convocatoria)

Apellidos: Rellán Álvarez Nombre: Rubén

NIF: 71644347Z Sexo: V M Año de nacimiento: 13/01/1980

Titulación académica: Ldo.en CC. Ambientales Grado: Doctor Licenciado/Ingeniero/Arquitecto

Categoría profesional: Investigador Contratado


Apellidos: Lattanzio Nombre: Giuseppe

NIE: X9168479N Sexo: V M Año de nacimiento: 21/04/1976

Titulación académica: Licenciado en Farmacia Grado: Doctor Licenciado/Ingeniero/Arquitecto

Categoría profesional: Becario Predoctoral


Apellidos: Rodríguez Celma Nombre: Jorge

NIF: 72986569W Sexo: V M Año de nacimiento: 11/06/1983

Titulación académica: Licenciado en Bioquímica Grado: Doctor Licenciado/Ingeniero/Arquitecto

Categoría profesional: Becario Predoctoral


EDP DE OTROS MIEMBROS DEL GRUPO INVESTIGADOR DE LA ENTIDAD SOLICITANTE: P2 = 3.0


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2.3. INVESTIGADORES DE OTRAS ENTIDADES (INCLUIDOS CENTROS EXTRANJEROS) (Deberán tener autorización de sus entidades de origen).

Apellidos: Fiehn Nombre: Oliver

Pasaporte: 249858032 Sexo: V M Año de nacimiento: 02/01/1967


Categoría profesional: Profesor Titular Universidad

Apellidos: Ma Nombre: Jian Feng

Pasaporte: TG7146756 Sexo: V M Año de nacimiento: 20/07/1963


Categoría profesional: Catedrático de Universidad

Apellidos: Fodor Nombre: Ferenc

Pasaporte: ZE599425 Sexo: V M Año de nacimiento: 28/01/1966


Categoría profesional: Profesor Titular Universidad

Apellidos: Grusak Nombre: Michael

Pasaporte: 820307839 Sexo: V M Año de nacimiento: 26/10/1957


Categoría profesional: Investigador

EDP DE INVESTIGADORES DE OTRAS ENTIDADES: P3 = 0.0

EDP DEL GRUPO INVESTIGADOR COMPLETO DE LA ENTIDAD SOLICITANTE: P = P1 + P2 = 6.0

EDP TOTAL DEL EQUIPO DEL PROYECTO: Q = P1 + P2 + P3 = 6.0


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3.9 PRESUPUESTO DE COSTES MARGINALES3.9.1. COSTES DEL PERSONAL CONTRATADO CON CARGO AL PROYECTO

Para la evaluación de este apartado se tendrá en cuenta la dedicación del grupo investigador al proyecto.

Perfil Coste imputable Justificación de necesidad y tareas que realizará

Formación Profesional de GradoSuperior 75.376 Apoyo técnico cultivo plantas, muestreo xilema y floema, técnicas de espectrofotometría, HPLC

y MS (contrato de Técnico Superior, G.P. 3).

Complementos Salariales 37.800

TOTAL 113.176


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3.9 PRESUPUESTO DE COSTES MARGINALES3.9.2 GASTOS DE EJECUCIÓN: Pequeño equipamiento científico-técnico y material bibliográfico

Descripción Coste ImputableEURO

Justificación de uso

Equipos accesorios de HPLC-espectrómetros de masas:-Segundo nebulizador Electro-spray para calibraciones: 4.000euros-Bomba de HPLC (controlada por software) para aplicacióncontinua de calibrante en espectrómetro de masas: 6.000 euros-Agitador refrigerado para preparación de muestras: 2.500 euros-Fuente de electroforesis: 2.500 euros

15.000 Necesarios para la implantación de nuevas metodologías noexistentes en el Instituto.

TOTAL 15.000


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3.9 PRESUPUESTO DE COSTES MARGINALES3.9.3 GASTOS DE EJECUCIÓN: Material Fungible



Material hidroponía y cámaras de cultivo de plantas (12.000euros); columnas y accesorios de HPLC y nHPLC (18.000 euros);isótopos estables para MS (10.000 euros); material diverso devidrio (cubetas espectrofotómetro, etc.; 2.000 euros), reactivospara ensayos (7.000 euros); N2 gas y líquido (2.000 euros);elementos de equipos de espectrometría de masas (9.000 euros);fungibles proteómica, electroforesis 2-D, HPLC(MS)-TOF y nHPLC-MS-MS (25.000 euros); estándares MS (10.000 euros); fungiblepara trabajos de metabolómica en UC Davis, USA (10.000 euros);fungible para trabajos en Univ. Okayama, Japón (5.000 euros);fungible para trabajos en USDA-ARS (5.000 euros), fungible paratrabajos en Budapest (5.000 euros), fungible informático (1.500euros).

121.500 Diversos fungibles para experimentación.

TOTAL 121.500


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3.9 PRESUPUESTO DE COSTES MARGINALES3.9.4 GASTOS DE EJECUCIÓN: Viajes y dietas



Viaje a Congreso Internacional de Fe (ISINIP) 2012 (1; 1.500euros), Viaje Congreso Tree Nutrition ISHS Tailandia 2012 (1;1.500 euros), Viaje Congreso ICPN Turquía 2013 (1; 1,000 euros),Viaje Oliver Fiehn a Zaragoza (1; 1.500 euros), Viaje Jian FengMa a Zaragoza (1; 1.500 euros), viaje Michael Grusak a Zaragoza(1; 1.500 euros), viajes Ferenc Fodor a Zaragoza (2; 2x1.000euros).

10.500 Viajes a Congresos especializados para presentación deresultados, y viajes de colaboradores dentro del proyecto aZaragoza.

TOTAL 10.500


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3.9 PRESUPUESTO DE COSTES MARGINALES3.9.5 GASTOS DE EJECUCIÓN: Varios



a) Publicaciones, gastos de edición y presentación (2.000 euros);b) Gastos de mantenimiento y actualización de la página web(1.800 euros); c) Cuotas de Sociedades Científicas (ASPB, SEFV,SeProt) (1.000 euros); d) Gastos de mensajería (2.000 euros), y e)Utilización de Servicios Generales de Apoyo a la Investigación,colaboraciones externas (6.000 euros)

12.800 a) Separatas, paneles, etc.; b) para la difusión de resultados delproyecto; c) hacen más económica la asistencia a Congresos ysuponen el acceso a Newsletters; d) envío de muestras entrelaboratorios participantes, y e) análisis de muestras en servicios ylaboratorios externos

TOTAL 12.800


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3.9 PRESUPUESTO DE COSTES MARGINALES3.9.6 RESUMEN DEL PRESUPUESTO DESGLOSADO POR CONCEPTOS

CONCEPTOAyuda que se solicita % total

EURO

COSTE DEPERSONAL

CONCEPTO COSTE IMPUTABLE

Gastos de personal 75376.0

Complementos Salariales 37800.0

TOTAL GASTOS DE PERSONAL CON CARGO AL PROYECTO 113.176

COSTE DEEJECUCIÓN

CONCEPTO COSTE IMPUTABLE

Pequeño equipamiento y material bibliográfico (a) 15000.0

Material Fungible (b) 121500.0

Viajes y Dietas (c) 10500.0

Varios (d) 12800.0

TOTAL GASTOS DE EJECUCIÓN(a+b+c+d) 159.800

TOTAL COSTES DIRECTOS 272.976 100,00%

TOTAL COSTES INDIRECTOS 57.325

TOTAL COSTES DIRECTOS (Directos + Indirectos)... 330.301 100,00%


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DOCUMENTACIÓN ADICIONAL A ADJUNTAR.

Documento acreditativo del poder que ostenta el representante legal del Organismo solicitante (original o copia compulsada).


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4. IMPLICACIONES ÉTICAS O DE BIOSEGURIDAD DE LA INVESTIGACIÓN PROPUESTA

- Este documento deberá remitirse,debidamente firmado, junto con el impreso de solicitud del proyecto.- El escrito de autorización del correspondiente Comité de Ética o Bioseguridad deberá remitirse preferentemente junto con la solicitud del proyecto,

para que pueda ser considerado en su evaluación. Si no dispone del mismo en el momento de presentar la solicitud, podrá enviarlo con posterioridad,peroserá un requisito para la aprobación del proyecto. En caso de que el Organismo solicitante no disponga de Comité de Ética o Bioseguridad, laautorización corresponderá al Responsable legal del Organismo o persona en quién delegue.

- En aquellos proyectos en los que las muestras o tejidos humanos procedan de un Hospital o de un Banco de Tejidos, se deberá aportar la certificacióndel Comité de Ética de dicha institución, en la que se autorice su utilización en el proyecto solicitado. Indicar si la propuesta contempla alguno de lossiguientes aspectos que puedan tener implicaciones éticas o relativas a la bioseguridad:

1. Indicar si la propuesta contempla alguno de los siguientes aspectos que puedan tener implicaciones éticas o relativas a la

bioseguridad:

2. En el caso de que haya contestado afirmativamente en algunos de los supuestos A, B, C o D y para complementar la información de

la Memoria del proyecto, debe detallar a continuación los siguientes aspectos referidos a la investigación propuesta:

- Número de pacientes, selección y protocolos previstos. Diseño estadístico de la experimentación.

- Tipo y características de los tejidos o muestras que se proponen utilizar.

- Datos personales o información genética que se van a utilizar.

- Procedencia y protocolos previstos para su utilización en investigación.

- Procedimientos previstos para salvaguardar la confidencialidad de los datos.

- Otra información que considere oportuna.

- En todos estos supuestos deberá remitir la preceptiva autorización del Comité Ético de Investigación del Organismo solicitante.- En el caso de experimentación con células troncales embrionarias humanas o líneas derivadas de ellas, deberá remitir la preceptiva

autorización de la Comisión de seguimiento y control de la donación y utilización de células y tejidos humanos.

Se le recuerda que la investigación propuesta deberá cumplir los principios éticos de respeto a la dignidad humana, confidencialidad, nodiscriminación y proporcionalidad entre los riesgos y los beneficios esperados, y, si procede, deberá disponer del consentimiento informado y escritode la personas implicadas o de sus representantes legales.

SI NOA. Experimentación clínica con seres humanos

B. Utilización de células troncales embrionarias humanas, o líneas derivadas de ellas, procedentes depreembriones sobrantesC. Utilización de tejidos o muestras biológicas de origen humano

D. Uso de datos personales, información genética, etc.

E. Experimentación animal.

F. Utilización de agentes biológicos de riesgo para la salud humana, animal o para las plantas.

G. Uso de organismos modificados genéticamente (OMGs)

H. Liberación de OMGs


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3. En el caso de Experimentación Animal y para complementar la información de la Memoria del proyecto, debe detallar acontinuación los siguientes aspectos referidos a la investigación propuesta:

- Número y tipo de animales que se proponen utilizar. Diseño estadístico de la experimentación.

- Tipo de ensayos a realizar, especialmente cuando impliquen dolor, estrés o lesión; así como los métodos paliativos previstos.

- Razones para no utilizar métodos alternativos, si se dispone de ellos.

- En el caso de modificación genética de animales, justificar su necesidad y los beneficios esperados.

- Método de sacrificio previsto.

- Deberá remitir la autorización del Comité de Ética del Organismo solicitante (o Comité de Ética de Experimentación Animal cuando

se haya constituido)

4. En el caso de utilización de agentes biológicos de riesgo para la salud humana, animal o para las plantas, debe precisar los

siguientes aspectos referidos a la investigación propuesta:

- Tipo de agente biológico y nivel de contención necesario.

- Medidas e instalaciones de contención de las que se dispone.

- Precisiones de bioseguridad que se han considerado.

- Deberá remitir una certificación del Comité de Bioseguridad o, en su caso, del Organismo solicitante que acredite que se dispone

de las instalaciones bioseguridad adecuadas para la experimentación propuesta.

5. En el caso de utilización de OMGs, precise a continuación los siguientes aspectos referidos a la investigación propuesta:

- Tipo de organismo y nivel de contención necesario en relación con el posible riesgo.

- Medidas e instalaciones de contención de las que se dispone.

- Previsiones sobre bioseguridad que se han considerado.

- Deberá remitir una certificación del Comité de Bioseguridad o, en su caso, del Organismo solicitante que acredite que se dispone

de las instalaciones bioseguridad adecuadas para la experimentación propuesta.

6. Si se tiene previsto liberar OMGs al medio ambiente, precise a continuación el siguiente aspecto referido a la investigación

propuesta:

- Detallar sus previsiones sobre bioseguridad y control de posibles riesgos en relación con la experimentación propuesta.

- Deberá remitir la preceptiva autorización de la autoridad competente para los ensayos previstos en el proyecto.

7. Indicar y valorar las posibles implicaciones éticas de la investigación propuesta o de los resultados científicos esperados.

El abajo firmante, en calidad de investigador principal de este proyecto informa que:

No hay implicaciones éticas.


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- La investigación propuesta respeta los principios fundamentales de la Declaración de Helsinki, del Convenio del Consejo de Europa relativo a los

derechos humanos y la biomedicina, de la Declaración Universal de la UNESCO sobre el genoma humano y los derechos humanos, y del Conveniopara la protección de los derechos humanos y la dignidad del ser humano con respecto a las aplicaciones de la Biología y la Medicina (Convenio deOviedo relativo a los derechos humanos y la biomedicina).

- Conoce y cumplirá la legislación vigente y otras normas reguladoras, pertinentes al proyecto, en materia de ética, experimentación animal obioseguridad.

D./D.ªJavier Abadía Bayona


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Convocatoria de ayudas de Proyectos de Investigación Fundamental no orientada

TECHNICAL ANNEX FOR TYPE A or B PROJECTS

1. SUMMARY OF THE PROPOSAL (the summary must be also filled in Spanish)

PROJECT TITLE: PLANT METALLOMICS: AN INTEGRATED MASS SPECTROMETRY

APPROACH TO STUDY METAL HOMEOSTASIS IN PLANTS PRINCIPAL INVESTIGATOR: Javier Abadía SUMMARY (brief and precise, outlining only the most relevant topics and the proposed objectives) Metals such as Zn and Fe are essential microelements required by crops. On the other hand, plants also acquire toxic metals such as Cd when they are present in the growth media. When nutrient metals are scarce or when metals are accumulated in excess, alterations in different biological processes essential for plant development occur. Thus, plants must carefully regulate metal acquisition, transport and partitioning within different organ and cell compartments in order to prevent excess accumulation while obtaining an adequate intake. The tendency toward a relatively stable equilibrium between those interdependent mechanisms, as maintained by physiological processes, is called homeostasis. Despite recent advances, we only have a very limited knowledge of metal homeostasis in plants. The objectives of the project are: i) to study metal speciation in plant fluids, carrying out detection and identification of metal-complexes and progressing towards metal bio-complexes quantification; ii) to develop novel methodologies to study crucial steps in metal homeostasis using mass spectrometry, including Fe(III)-reductase assays, assays for metal fluxes and studies on root accumulation and excretion of metabolites; iii) to study the metabolism of metal imbalances with a combined metabolomics/proteomics approach, and to exploit the main differences found by using targeted analysis; and iv) to carry out the micro-localization of metals and metal homeostasis-related components (proteins and metabolites) in plants. A better understanding of processes involved in the acquisition, transport, chelation and storage of metals within plants would help us not only to control agricultural problems such as metal deficiencies and toxicities, but also to improve the metal ion content of plant foods, as well as to promote phytoremediation of metal contaminated soils. This proposal is a continuation of the project currently in course, AGL2007-61948, which ends in October 2010, and complements the efforts of the group to study metal homeostasis in plants. The proposal includes four staff CSIC scientists, one scientist more on contract and two graduate students. Four foreign scientists, two from the USA, one from Hungary and another from Japan, will collaborate on a part time basis in some tasks of the project.

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TÍTULO DEL PROYECTO: Metalómica vegetal: una aproximación a la homeostasis de metales en plantas mediante espectrometría de masas integrada

RESUMEN (breve y preciso, exponiendo sólo los aspectos más relevantes y los objetivos propuestos) Algunos metales como Zn y Fe son microelementos esenciales necesarios para los cultivos. Además, las plantas pueden adquirir metales tóxicos como el Cd, si éstos están presentes en los medios de crecimiento. Cuando los metales esenciales son escasos o cuando los metales se acumulan en exceso se presentan alteraciones en diferentes procesos biológicos fundamentales para el desarrollo de la planta. Así, las plantas deben regular cuidadosamente la adquisición de los metales, su transporte y la distribución entre los diferentes órganos y compartimentos celulares, a fin de evitar su acumulación en exceso, manteniendo un suministro adecuado de aquellos que son esenciales. Se denomina homeostasis a la tendencia hacia un equilibrio relativamente estable entre mecanismos interdependientes sustentados por procesos fisiológicos. A pesar de los avances recientes, aún tenemos un conocimiento muy limitado de la homeostasis de metales en plantas. Los objetivos del proyecto son: i) estudiar la especiación de metales en fluidos vegetales, llevando a cabo la detección e identificación de complejos metálicos y avanzando hacia la cuantificación de los mismos; ii) desarrollar nuevas metodologías para estudiar algunos pasos cruciales en la homeostasis de metales por espectrometría de masas: ensayos de Fe(III)-reductasa, ensayos para medir flujos de metales y estudios sobre la acumulación en la raíz y excreción por la misma de metabolitos al medio de crecimiento; iii) estudiar el metabolismo de los desequilibrios causados por la deficiencia y toxicidad de metales con un enfoque combinado metabolómica/proteómica, investigando las principales diferencias encontradas mediante un análisis dirigido; y iv) estudiar la micro-localización de metales y componentes relacionados con la homeostasis de metales (proteínas y metabolitos) en plantas. Una mejor comprensión de los procesos implicados en la adquisición, transporte, quelación y almacenamiento de los metales en las plantas nos ayudarán a controlar no sólo problemas agrícolas, tales como deficiencias y toxicidades de metales, sino también a mejorar el contenido de metales en los alimentos de origen vegetal, así como promover la fitorremediación de suelos contaminados con metales. Esta propuesta es una continuación del proyecto actualmente en curso, AGL2007-61948, que acaba en octubre de 2010, y complementa los esfuerzos actuales del grupo en el estudio de la homeostasis de metales en plantas. La propuesta incluye cuatro científicos de plantilla del CSIC, uno más contratado, y dos estudiantes de postgrado. Cuatro científicos extranjeros, dos de EE.UU., uno de Hungría y otro de Japón, colaborarán a tiempo parcial en algunas tareas del proyecto.

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2. INTRODUCTION (max. 5 pages)

• The introduction should include: the aims of the project; the background and the state of the art of the

scientific knowledge, including the essential references; the most relevant national and international groups working in the same or related topics.

Aim The aim of this proposal is to improve the current state of knowledge of metal homeostasis in plants, with the goals of revealing metal species and the role of metabolites and proteins participating in these processes. The project is a continuation of previous ones and proposes four objectives, based on the findings obtained so far, which are supported by the advanced technologies available in our research group and in those of collaborating laboratories, and also by the expertise of the group personnel. The major focus of the group in previous years has been Fe deficiency, but some of the objectives of this proposal also involve other metals such as Zn and Cd, in which the group has also experience. The proposal includes the work of four EEAD-CSIC staff scientists, a scientist on contract and two graduate students. Four foreign scientists (two from the US, one from Hungary and another from Japan) will collaborate in specific tasks of the project.

Background

Metals such as Zn and Fe are essential microelements required by crops. On the other hand, plants also acquire toxic metals such as Cd when they are present in the growth media. When metals accumulate in excess, they cause alterations in different biological processes basic for plant development, including transpiration, photosynthesis, chlorophyll biosynthesis and maintenance of biological membranes (see Sagardoy et al., 2009, López-Millán et al., 2009, and references therein). Therefore, plants must carefully regulate metal acquisition and partitioning, in order to prevent excess accumulation while obtaining an adequate intake in the case of nutrients. Metal uptake pathways include transport proteins localized to various membranes, as well as long-distance systems (xylem and phloem) crucial to move water and nutrients throughout the plant. Thus, a complex, integrated system of tissues and membrane processes is required to move the metals from the root-soil interface to cells throughout the plant. Iron (after reduction by a plasma membrane Fe(III)-chelate reductase) and Zn are primarily taken up by roots as divalent cations, and subsequently they are thought to be transported to the shoot through the xylem, either as free divalent ions or complexed with small molecules such as organic acids or aminoacids; a tri-Fe, tri-citrate complex has been recently found in the xylem sap of tomato by our group (Rellán-Álvarez et al., 2010). Once in the leaf apoplast, these metals are taken up by mesophyll cells (Larbi et al., 2001), and then utilized in leaf biochemical processes, stored in the vacuoles (González et al., 1999) and/or remobilized to other organs via the phloem pathway (Grusak, 1994). Toxic metals such as Cd share some of the uptake and transport pathways used by metal nutrients.

Metal movement within the plant has been mainly studied by molecular biology approaches, which have provided in the last decade a breadth of information (see reviews by Puig and Peñarrubia, 2009, Palmer and Guerinot, 2009, and Morrisey and Guerinot, 2009). Different families of metal transporters, including ZIP and YSL, are involved not only in root uptake from the soil but also in transport to other plant organs and/or in subcellular membrane transport within the plant (Guerinot, 2000; Curie et al., 2001; Hall and Guerinot, 2006; Bauer and Hell, 2006; Morrissey and Guerinot, 2009). Other gene families involved in membrane metal transport are OPT, VIT, PIC, CDF and ATM; members of these families are localized in different subcellular compartments such as vacuoles, mitochondria and chloroplasts and/or are responsible for metal loading in different tissues, including xylem, phloem and seeds. Most of these transporters have a broad substrate range (generally Fe(II), Cd(II), Zn(II) and Mn(II); Korshunova et al., 1999), are localized in different membranes, and are transcriptionally regulated by metal concentrations (Guerinot, 2000). Also, the study of unique mutants that exhibit altered metal concentrations in different plant organs is promising, not only for gene discovery, but also to understand the regulation and interactions of these genes. These mutants include man-1 (Arabidopsis), raz (Medicago truncatula), chloronerva (tomato) and brz and dgl (pea) (Grusak et al., 1990; Delhaize, 1996; Ellis et al., 2003). Less information is available regarding chelators acting in long and short distance metal transport. For instance, the organic acid citrate has been described to chelate Fe in xylem sap (Rellán-Álvarez et al., 2010), whereas in phloem sap the non-proteinogenic amino acid nicotianamine (NA) and small proteins such as ITP are candidates for Fe transport (Kruger et al., 2002). Within the cell, NA is thought to be a short distance transporter, and phytate and ferritin have been described to store Fe.

A better understanding of the processes involved in the acquisition, transport, chelation and storage of metal ions within plants would help us not only to control agricultural problems such as metal deficiencies and toxicities in crops, but also to improve the metal ion content of plant foods, as well as to progress in the phytoremediation of metal contaminated soils.

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State-of-the-art of the scientific knowledge (according to the proposed objectives) Objective 1. To study metal speciation in plant fluids. The detection, identification and quantification of metal species in plant fluids and/or sub-cellular compartments are still some of the biggest challenges in plant metal transport (Hider et al. 2004; Spuznar, 2005; Callahan et al., 2006). Plant metal homeostasis relies on bioligands synthesized by the plant, which are used for metal transport (to specific organs and/or compartments) and/or metal inactivation and storage. Until now, proofs for the occurrence of specific metal species in plant fluids are scarce, since analytical problems faced are of major importance. They include: i) the very low concentrations of metal species in plant materials, with the additional challenge of maintaining metal species in the same chemical form(s) found in vivo; ii) the complexity of the matrix; iii) the difficulties to distinguish among metal complexes; and iv) the challenges in identifying the metal species involved, because of the lack of standards and the still limited development of standard-free, metal species-specific methodologies (Schaumlöffel and Lobinski 2005). These problems could be successfully tackled by hyphenated techniques, based on the combination of chromatographic separation with dual-detection methods, including metal-specific (inductively coupled plasma mass spectrometry; ICP-MS) and molecule-specific (electro-spray mass spectrometry; ESI-MS-MS) technologies. This combined approach, referred to as "integrated mass spectrometry" (Meija et al., 2006), has been recently reviewed by Mounicou et al. (2009). Plant fluids are considered as good materials to study metal coordination in plants, since the use of hyphenated techniques requires the presence of the metal species in liquid phase. Using whole plant tissues, metal species must be determined after tissue homogenization and extraction, leading to the possibility of the formation of metal-bioligand complexes that do not exist in vivo. Even when working with plant fluids, the maintenance of the original forms of metal species occurring in vivo is a critical issue, since many metal complexes could be very labile.

In spite of these difficulties, the potential of hyphenated techniques in bio-inorganic speciation analysis has been demonstrated successfully in some cases. Aluminum-citrate and Al-aconitate were found to be predominant Al-complexes in the xylem sap of Sempervivum tectorum and Sansevieria trifasciata (Bantan et al., 1999). In a water extract of the latex of a hyper-accumulator tree, Sebertia acuminata, two major Ni-complexes were found to occur in vivo: 99.4% of the Ni was complexed by citrate (as Ni(II)Cit2), whereas 0.3% was bound to NA (Schaumlöffel et al., 2003). More recently, a methylated aldaric acid and other organic acids have been identified as Ni(II) bioligands in the same material (Callahan et al., 2008). In the xylem sap of the metal hyper-accumulator plant Thlaspi caerulescens part of the Ni was also found as a stable complex with NA (Mari et al., 2006), and the same complex was found in the xylem sap of Arabidopsis grown in Ni-containing nutrient solution (Xuan et al., 2007). However, Cd was found by NMR to be transported as free ion forms in the xylem of Arabidopsis halleri (Ueno et al., 2008). Identification of a tri-Fe(III), tri-citrate complex in the xylem sap of Fe-deficient tomato resupplied with Fe was recently reported by our group, using HPLC-ESI-MS and HPLC-ICP-MS (Rellán-Álvarez et al., 2010). Many of the studies published so far have focused on metal hyper-accumulating plants and toxic metals, whereas less information is available for crops or model plants and essential micronutrients. In this project we propose to use hyphenated techniques to further study the metal (Fe, Zn and Cd) species transported in plant fluids.

Objective 2. To develop novel methodologies to study crucial steps in plant metal homeostasis using mass spectrometry Methodologies for studying some crucial steps in metal homeostasis have undergone few changes in recent years. Three good examples are methodologies used to measure: i) Fe(III)-reductase activities; 2) metal fluxes in and out of cells and organelles; and iii) the root accumulation and excretion of metabolites to the growth media in cases of Fe deficiency.

Iron is reduced at the root plasma membrane by an Fe(III)-reductase enzyme, and the activity is always assayed by using a strong Fe(II) chelator (e.g., BPDS), and then measuring spectrophotometrically the Fe(II)-BPDS chelate formed (Chang et al., 2003; Gogorcena et al., 2004). This assay procedure is questionable, because the strong affinity of the chelator for Fe(II) fully displaces the equilibrium (Pierre et al., 2002), leading to activities that are much higher than those occurring physiologically, since they reflect the reduction capacity during the assay but not the in vivo real activity. In this project we propose to use the new MS-based methodologies developed in the last years (Álvarez-Fernández et al., 2007; Orera et al., 2009, 2010; Rellán-Álvarez et al., 2010) to design assays based in monitoring the decreases in the concentrations of the enzyme substrates (Fe(III)-chelates or Fe(III)-complexes).

Metal fluxes are usually measured in cells and organelles by exposing them to metal-containing assay media (radioactive or not), and then measuring metal fluxes by traditional analysis techniques such as AAS or radioactivity. Recently, new techniques such as multi-elemental ICP-MS have been applied with the same purpose (Pedas et al., 2008). In this project we propose to use stable isotopes and isotope dilution analysis (IDA) techniques (Schaumlöffel and Lobinski 2005) to

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design methods to study the metal (Fe and others) fluxes in cells (protoplasts and oocites) and isolated organelles (chloroplasts).

When submitted to metal deprivation, plants accumulate and excrete a number of substances, including organic acids, flavins, phenolics and others (Walker et al., 2003). The accumulation and excretion of metabolites has been mainly studied by traditional techniques, generally involving the use of target analysis of specific metabolites (e.g., excretion of flavins by sugar beet roots with Fe deficiency; Susín et al., 1994) or even less specific techniques, just assaying broad types of compounds (e.g., phenolics excretion by clover roots; Jin et al., 2007). In this project we propose to study the root accumulation and excretion of metabolites to the growth media when plants are grown under Fe deficiency, using targeted (flavins, organic acids, phenolic compounds by HPLC-MS(MS)) and untargeted analysis (GC-MS).

Objective 3. To study the metabolism of metal imbalances in plants with a combined metabolomics/proteomics approach Plant metabolomics and proteomics have gained strength in the last years, with the improvement of technologies such as GC-MS and HPLC-MS(-MS) and the development of protein and metabolite databases (see Jorrín-Novo et al., 2009, and Hall, 2006, for recent reviews on plant proteomics and metabolomics, respectively). These high-throughput techniques can provide a breadth of information about metabolite and protein changes in plants under different metal imbalances, complementing previous studies and facilitating the elaboration of new hypothesis. Examples of proteomic and/or metabolomic studies in plants with different metal stresses are those on B (Roessner et al., 2006) and Cd toxicity (Sarry et al., 2006) and Fe deficiency (Li et al. 2008; Brumbarova et al. 2008). Reviews on the subject have been published recently (Shulaev et al., 2008; Ahsan et al. 2009). However, most of the works published include either metabolomics or proteomics data alone, and very few attempts to combine both types of studies have been made (Sarry et al., 2006). The comprehensive combination of metabolomics and proteomics data is one of the main issues that need to be faced in this field (Weckwerth, 2008). Other limitation of the works carried out so far is that most of them have focused on the effects of metal imbalances in leaves, and to a lesser extent on the effects in roots. Furthermore, plant compartments such as those related to plant fluids, including xylem (Alvarez et al., 2006), apoplastic fluid (Haslam et al., 2003) and phloem (Waltz et al., 2004) have been even less studied. Our group has studied the changes in thylakoid protein profiles with Fe deficiency (Andaluz et al., 2006), and we have started work on the changes in the metabolomic and proteomic profiles of sugar beet root tips grown under Fe deficiency, and the proteomic profiles of tomato roots grown under Cd toxicity. In this project we propose to use the combined metabolomics/proteomics approach to study changes in plants caused by metal deficiency (Fe) and metal toxicity (Zn and Cd) in different plant materials.

Objective 4. To study the micro-localization of metals and metal homeostasis-related components in plants Most plant studies, including biochemical, photosynthetic and metabolomic ones, consider plant organs as a unit. However, leaves, stems and roots are composed of different cell types. The localization of metals in different plant tissues and cells is essential to understand the plant processes involved in metal homeostasis and will open new ways to study mineral nutrition in plants (Ma et al., 2007a). In the last years, several image techniques have been applied to improve the knowledge on tissue-specific metal location associated to different nutritional status (metal toxicities and deficiencies). For instance, 2-dimensional, synchrotron radiation-induced, X-ray fluorescence (µ-SRXF) has been used to study the distribution of Fe in peach leaves by our group (Jiménez et al., 2009). However, µ-SRXF data correspond to the full leaf depth (thickness), and no information about the distribution of Fe across the transversal section of Fe-deficient leaves is still available. Microscopy-based techniques coupled to microprobes, such as scanning electron microscopy coupled to energy-dispersive x-ray microanalysis (SEM-EDX), have been successfully used to obtain transversal foliar metal distribution in some hyper-accumulator (Frey et al., 2000; Küpper et al., 2001; Fernando et al., 2008) and monocot species (Karley et al., 2000). However, in non-hyper-accumulator species difficulties based on the inadequate detection limits for metals have been reported (Ralle and Lutsenko, 2009). To cope with these analytical restrictions, more sensitive microanalysis techniques such as particle induced, X-ray emission (PIXE) are being used (Vogel-Mikus et al., 2008). For metal distribution at a deeper scale (sub-cellular distribution) the use of high-resolution transmission electron microscopy (TEM-EDX) is necessary. Using this technique, some promising data have been obtained for several plant species (Hirsch et al., 2006). Another approach to micro-localize metals is the use of fluorophores with high affinity to metals, using different isolated plant compartments (protoplasts, chloroplasts, etc.; Maruyama et al., 2002). Finally, image analysis techniques can be used to study the distribution of other components related to the metal homeostasis processes, such as proteins by immuno-localization (Dell’Orto et al., 2002) and flavins by fluorescence microscopy; in the last years we have acquired experience in both types of techniques. In this project we propose to use image and metal detection technologies to study the distribution of metals (Fe, Zn and Cd) and metal-homeostasis

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related components (metabolites and proteins) in plant tissues.

Most relevant groups dealing with the same or similar aspects to the ones presented in this project Spanish groups. We are in contact with Spanish groups dealing with similar topics, including Fe uptake (U. Córdoba, E. Alcántara and J. Romera), chemical equilibria in hydroponics and fertilizer research (U.A. Madrid, J.J. Lucena; Timac-Agro/U. Navarra, J. García-Mina; U. Córdoba, M.C. del Campillo). Other Spanish groups working in the subject metals and plants, under a molecular point of view, are those of L. Peñarrubia (U. Valencia; Cu and Fe), L. Romero (IBVF-CSIC Seville; Cd) and J. Barceló/Ch. Poschenrieder (U.A. Barcelona; heavy metals). Finally, groups studying the subject under different points of view are those of R. Carpena (U.A. Madrid; heavy metals) and R. Millán (CIEMAT Madrid; Hg). We maintain contacts with many of these groups within the Network for Abiotic Stress in Plants funded by the Spanish MICINN (REAP). We also have active mass spectrometry-related collaborations with the groups of J.I. García Alonso (U. Oviedo; ICP-MS) and J. Orduna (ICM-CSIC Zaragoza; HPLC-MS/MS).

International groups. There are many international groups relevant in metal homeostasis in plants. We maintain contacts and collaborations with a number of them. Four of them are included in specific tasks in this proposal (O. Fiehn, U.C. Davis, US; M. Grusak, USDA-ARS Houston, US; J.F. Ma, U. Okayama, Japan; F. Fodor, U. Budapest, Hungary). We also collaborate currently in another project with other European groups in a trilateral project (J.F. Briat, U. Montpellier, France; N. von Wirén, IPK-Gatersleben, Germany; K. Philippar, U. Munchen, Germany), with many agronomical groups within the EU project Isafruit, and with other groups in bilateral actions (G. Zocchi, U. Milano, Italy; J. Spuznar, U. Pau, France). We are also in touch with participants in the International Fe Nutrition and Interaction in Plants Symposia, which are held every 2 years (the IP belongs to the Steering Committee). We are also in contact and exchange information with participants in mineral nutrition symposia (ICPN) and the tree nutrition ISHS meetings, and participate actively in two COST actions (603 and 905).

References (This list contains all papers quoted in the proposal; in grey, papers of the proponent group, including partners abroad) -Ahsan N et al., 2009. Recent developments in the application of proteomics to the analysis of plant responses

to heavy metals. Proteomics 9:2602. -Alvarez et al., 2006. Characterization of the maize xylem sap proteome. J Proteome Res 5:963. -Álvarez-Fernández et al., 2007. Determination of synthetic ferric chelates used as fertilizers by liquid

chromatography-electrospray mass spectrometry in agricultural matrices. J Am Soc Mass Spectrom 18:37. -Andaluz, 2005. PhD Thesis, University of Zaragoza. -Andaluz et al., 2006. Proteomic profiles of thylakoid membranes and changes in response to iron deficiency.

Photosynth Res 89:141. -Bantan et al., 1999. Combination of various analytical techniques for speciation of low molecular weight

aluminium complexes in plant sap. Fresenius J Anal Chem 365:545. -Bauer and Hell, 2006. In: Iron Nutrition in Plants and Rhizospheric Microorganisms. Barton LL and Abadía J

Eds. Springer. p:279. -Brumbarova et al., 2008. A proteomic study showing differential regulation of stress, redox regulation and

peroxidase proteins by iron supply and the transcription factor FER. Plant J 54:321. -Callahan et al., 2006. Metal ion ligands in hyperaccumulating plants. J Biol Inorg Chem 11:2. -Callahan et al., 2008. LC-MS and GC-MS metabolite profiling of nickel(II) complexes in the latex of the nickel-

hyperaccumulating tree Sebertia acuminata and identification of methylated aldaric acid as a new nickel(II) ligand. Phytochemistry 69:240.

-Chang et al., 2003. Effects of cadmium and lead on ferric chelate reductase activities in sugar beet roots. Plant Physiol Biochem 41:999.

-Curie et al., 2001. Maize yellow stripe 1 encodes a membrane protein directly involved in Fe(III) uptake. Nature 409:346.

-Delhaize, 1996. A metal-accumulator mutant of Arabidopsis thaliana. Plant Physiol 111:849. -Dell’Orto et al., 2002. Localization of the membrane H+-ATPase in Fe-deficient cucumber roots by

immunodetection. Plant Soil 241:11-17. -Ellis et al., 2003. Metal physiology and accumulation in a Medicago truncatula mutant exhibiting an elevated

requirement for zinc. New Phytol 158:207. -Fernando et al., 2008. Novel pattern of foliar metal distribution in a manganese hyperaccumulator. Funct Plant

Biol 35:193. -Fiehn et al., 2008. Quality control for plant metabolomics: reporting MSI-compliant studies. Plant J. 53:691. -Frey et al., 2000. Distribution of Zn in functionally different leaf epidermal cells of the hyperaccumulator

Thlaspi caerulescens. Plant Cell Environ 23:675. -Gogorcena et al., 2004. New technique for screening iron-efficient genotypes in peach rootstoocks: Elicitation

of root ferric chelate reductase by manipulation of external iron concentrations. J Plant Nutr 27:1701. -González et al., 1999. A comparison of Zn, Mn, Cd and Ca transport mechanisms in oat root tonoplast vesicles.

Physiol Plant 106:203. -Grusak et al., 1990. Physiological characterization of a single-gene mutant of Pisum sativum exhibiting excess

iron accumulation. I. Root iron reduction and iron uptake. Plant Physiol 93:976. -Grusak, 1994. Iron transport to developing ovules of Pisum sativum I. Seed import characteristics and phloem

iron-loading capacity of source regions. Plant Physiol 104:649. -Guerinot, 2000. The Zip family of metal transporters. Biochim Biophys Acta 1465:190. -Hall, 2006. Plant metabolomics: from holistic hope, to hype, to hot topic. New Phytol 169:453. -Hall and Guerinot, 2006. In: Iron Nutrition in Plants and Rhizospheric Microorganisms. Barton LL and Abadía J

Eds. Springer. p:311. -Haslam et al., 2003. The assessment of enriched apoplastic extracts using proteomic approaches. Ann Appl

Biol 143:81. -Hider et al., 2004. Competition or complementation: the iron-chelating abilities of nicotianamine and

phytosiderophores. New Phytol 164:204.

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-Hirsch et al., 2006 Phosphate deficiency promotes modification of iron distribution in Arabidopsis plants. Biochimie 88:1767.

-Igartua et al., 2000. Prognosis of iron chlorosis from the mineral composition of flowers in peach. J Hort Sci 75:111.

-Jiménez et al., 2009. Elemental 2-D mapping and changes in leaf iron and chlorophyll in response to iron re-supply in iron-deficient GF 677 peach-almond hybrid. Plant Soil 315:93.

-Jin et al., 2007. Iron deficiency-induced secretion of phenolics facilitates the reutilization of root apoplastic iron in red clover. Plant Physiol 144:278.

-Jorrín-Novo et al., 2009. Plant proteomics update (2007-2008): Second-generation proteomic techniques, an appropriate experimental design, and data analysis to fulfill MIAPE standards, increase plant proteome coverage and expand biological knowledge. J Proteomics 72:285.

-Karley, et al., 2000. Where do all the ions go? The cellular basis of differential ion accumulation in leaf cells. Trends Plant Sci 5:465.

-Korshunova et al., 1999. The IRT1 protein from Arabidopsis thaliana is a metal transporter with a broad substrate range. Plant Mol Biol 40:37.

-Krüger et al., 2002. A metal-binding member of the late embryogenesis abundant protein family transports iron in the phloem of Ricinus communis L. J Biol Chem 277:25062.

-Küpper et al., 2001. Cellular compartmentation of nickel in the hyperaccumulator Alyssum berolonii and Thlaspi caerulescens. J Exp Bot 52:2291.

-Larbi et al., 2001. Technical advance: Reduction of Fe(III)-chelates by mesophyll leaf disks of sugar beet. Multi-component origin and effects of Fe deficiency. Plant Cell Physiol 42:94.

-Larbi et al., 2010. Changes in organic acid and iron concentrations in xylem sap and apoplastic fluid of Beta vulgaris in response to iron deficiency and resupply. J Plant Physiol, in press.

-Li et al., 2008. Proteomic response to iron deficiency in tomato root. Proteomics 397:2299. -López-Millán et al., 2000. Effects of iron deficiency on the composition of the leaf apoplastic fluid and xylem

sap in sugar beet. Implications for iron and carbon transport. Plant Physiol 124:873. -López-Millán et al., 2001. Iron deficiency-associated changes in the composition of the leaf apoplastic fluid

from field-grown pear (Pyrus communis L.) trees. J Exp Bot 52:1489. -López-Millán et al., 2009. Cadmium toxicity in tomato (Lycopersicon esculentum) plants grown in hydroponics.

Environ Exp Bot 65:376. -Ma et al., 2007a. Subcellular localisation of Cd and Zn in the leaves of a Cd-hyperaccumulating ecotype of

Thlaspi caerulescens. Planta 220:731. -Ma et al., 2007b. An efflux transporter of silicon in rice. Nature 448:209. -Marentes and Grusak, 1998. Mass determination of low-molecular-weight proteins in phloem sap using matrix-

assisted laser desorption/ionization time-of-flight mass spectrometry. J Exp Bot 49:903. -Mari et al., 2006. Root-to-shoot long-distance circulation of nicotianamine and nicotianamine-nickel chelates in

the metal hyperaccumulator Thlaspi caerulescens. J Exp Bot 57:4111. -Maruyama et al., 2002. A novel, cell-permeable, fluorescent probe for ratiometric imaging of Zinc ion. J Am

Chem Soc 124:10650. -Meija et al., 2006. Integrated mass spectrometry in (semi-) metal speciation and its potential in

phytochemistry. Trends Anal Chem 25:44. -Morrissey and Guerinot, 2009. Iron uptake and transport in plants: the good, the bad, and the ionome. Chem

Rev 109:4553. -Mounicou et al., 2009. Metallomics: the concept and methodology. Chem Soc Rev 38:1119. -Orera et al., 2009. 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 Commun Mass Sp 23:1694.

-Orera et al., 2010. Electrospray-collision-induced dissociation mass spectrometry: a tool to characterize synthetic polyaminocarboxilate ferric chelates used as fertilizers. Rapid Commun Mass Sp 24:109

-Palmer and Guerinot, 2009. Facing the challenges of Cu, Fe and Zn homeostasis in plants. Nat Chem Biol 5:333.

-Pedas et al., 2008. Manganese efficiency in barley: Identification and characterization of the metal transporter HvIRT1. Plant Physiol 148:455.

-Pierre et al., 2002. Chemistry for an essential biological process: the reduction of ferric iron. Biometals 15:341. -Puig and Peñarrubia, 2009. Placing metal micronutrients in context: transport and distribution in plants. Curr

Opin Plant Biol 12:299. -Ralle and Lutsenko, 2009. Quantitative imaging of metals in tissues. Biometals 22:197–205. -Rellán-Álvarez et al., 2006 Direct and simultaneous determination of reduced and oxidized glutathione and

homoglutathione by liquid chromatography-electrospray/mass spectrometry in plant tissue extracts. Anal Biochem 356:254.

-Rellán-Álvarez et al., 2010. Identification of a tri-iron(III), tri-citrate complex in the xylem sap of iron-deficient tomato resupplied with iron: new insights into plant iron long-distance transport. Plant Cell Physiol 51:91.

-Roessner et al., 2006. An investigation of boron toxicity in barley using metabolomics Plant Physiol 142:1087. -Sagardoy et al., 2009. Effects of Zinc toxicity in sugar beet (Beta vulgaris L.) plants grown in hydroponics.

Plant Biol 11:339. -Sarry et al., 2006. The early responses of Arabidopsis thaliana cells to cadmium exposure explored by protein

and metabolite profiling analyses. Proteomics 6:2180. -Schaumlöffel et al., 2003. Speciation analysis of nickel in the latex of a hyperaccumulating tree Sebertia

acuminata by HPLC and CZE with ICP MS and electrospray MS-MS detection. J Anal Atom Spectrom 18:120. -Schaumlöffel and Lobinski, 2005. Isotope dilution technique for quantitative analysis of endogenous trace

element species in biological systems. Int J Mass Spectrom 242:217. -Shulaev et al., 2008. Metabolomics for plant stress response. Physiol Plant 132: 199. -Susín et al., 1994. Flavin excretion from roots of iron-deficient sugar beet (Beta vulgaris L.). Planta 193:514. -Szpunar, 2005. Advances in analytical methodology for bioinorganic speciation analysis: metallomics,

metalloproteomics and heteroatom-tagged proteomics and metabolomics. Analyst 130:442. -Ueno et al., 2008 Characterization of Cd Translocation and identification of the Cd form in xylem sap of the Cd-

hyperaccumulator Arabidopsis halleri. Plant Cell Physiol 49:540. -Vogel-Mikus et al., 2008. Comparison of essential and non-essential element distribution in leaves of the

Cd/Zn hyperaccumulator Thlaspi praecox as revealed by micro-PIXE. Plant Cell Environ 31:1484. -Walker et al., 2003. Root exudation and rhizosphere biology. Plant Physiol 132:44. -Waltz et al., 2004. Proteomics of curcurbit phloem exudate reveals a network of defence proteins.

Phytochemistry 65:1795. -Weckwerth, 2008. Integration of metabolomics and proteomics in molecular plant physiology – coping with the

complexity by data-dimensionality reduction. Physiol Plant 132: 176. -Xuan et al., 2007. CE of phytosiderophores and related metal species in plants. Electrophoresis 28:3507. -Zouari et al., 2001. Iron is required for the induction of root ferric chelate reductase activity in iron-deficient

tomato. J Plant Nutr 24:383.

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3. OBJECTIVES (max. 2 pages)

3.1 Reasons to present this proposal and the initial hypotheses which support its objectives

(maximum 20 lines) This project is a follow-up of the research on metals in plants developed by the proponent group (project AGL2007-00194, finishing December 2010, among others). The proposal is based on the results obtained and takes advantage of the state-of-the-art analytical methodologies available and the expertise of the group personnel. Four foreign researchers (2 in the US, 1 in Hungary and 1 in Japan) will cooperate in specific tasks. The rationale of the proposal is that new knowledge of metal homeostasis in plants, which is still fragmentary in many aspects, can be improved by using advanced mass spectrometry techniques. Several hypotheses will be tested as follows: -Metals are present in plant fluids as metal-ligand complexes. -Crucial steps in metal homeostasis, such as Fe(III)-reductase activity, metal fluxes and root metabolite accumulation and excretion, can be better studied using mass spectrometry techniques. -Metal deficiencies and toxicities in plants result in changes in the proteome and metabolome, either as adaptive strategies of as a result of the disruption of plant metabolism. -Metals and other components involved in metal homeostasis are distributed non-homogenously within plant tissues. The project proposes studies on metabolites (target analysis, metabolite/xenomic profiling, metabolomics), proteins (proteomics, localization by image techniques) and metals (including stable isotopes, isotope dilution analysis and image techniques), to investigate changes in different plant tissues and compartments in metal stresses such as Fe deficiency and Zn and Cd toxicities. All these studies would allow for a progress towards achieving a more rational approach to avoid metal deficiencies and toxicities in plants, and to improve metal nutrient content in foods. 3.2. Background and previous results of the group or the results of other groups that support the

initial hypothesis

This proposal is based on the results obtained in previous projects of the group and other teams developing research in the field of metal homeostasis in plants. These data have outlined the need to develop new research lines aimed to progress in the knowledge of this issue, which is still far from being understood. Objectives are proposed taking advantage of the state-of-the-art analytical technologies available in our laboratory and the laboratories of collaborating partners, and the expertise of the participating personnel. This proposal has a broader approach than that used in previous projects, since studies on metabolomics/proteomics, stable isotopes and image techniques are included. The background information on all these issues, including a literature review, is included in the Introduction (Section 2) of this proposal.

The proponent research group has devoted, since its creation in 1988, a large part of its time and efforts to study plant mineral nutrition, and specially Fe deficiency, both in model plant species and in fruit trees, and is currently a reference team in this issue at international level. Because of this reason, many of the previous results of the group have been already quoted in the current state of knowledge (see Section 2; other studies have been submitted for publication).

Both the proponent research group and other teams have obtained data indicating that:

-The speciation of metals in plant fluids is still poorly known: Metal-ligand species have been detected in only a few cases. New MS techniques have permitted the finding of Ni species in plant exudates (Callahan et al., 2008) and Fe in tomato xylem (Rellán-Álvarez et al., 2010).

-Methodologies to study some crucial steps in metal homeostasis can benefit from developments in mass spectrometry techniques: Current developments have allowed for the accurate determination of Fe(III)-chelates in solution (Álvarez-Fernández et al., 2007). Also, the use of ICP-MS (Pedas et al., 2008) and isotope dilution analysis techniques (Schaumlöffel and Lobinski 2005) can potentially be used for determining and measuring metal fluxes (in and out) in cells and isolated organelles. Finally, both targeted (HPLC-MS(-MS)) and untargeted (GC-MS) techniques can shed light on the role of root accumulation and excretion of metabolites in metal acquisition processes. -Metal imbalances in plants lead to changes at the proteome and metabolome level: Both metal deficiencies and toxicities lead to changes in the metabolome and proteome of plants. The combined use of metabolomics and proteomics techniques to delve into the overall metal effects in plants could give exciting new results.

-The distribution of metals and metal-homeostasis components in plants is not homogeneous. There is ample evidence that both metals and other components are localized in a specific manner in plant compartments. For instance, metals in hyper-accumulators can be sequestered in locations that do not interfere with normal metabolism. Also, proteins and metabolites associated to metal uptake in cases of metal deficiency are localized in specific membranes or cells; in some cases proteins have a polar localization in one or the other side of the membrane (Ma et al., 2007b).

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3.3. Objectives

Objective 1. To study metal speciation in plant fluids This objective focuses on unraveling the chemical species of metals that occur in plant fluids, including metal nutrients and toxic ones. We will look for natural and synthetic metal complexes involved in metal transport, using stable metal isotopes. Metabolite target analysis and profiling studies will be carried out using an integrated mass spectrometry approach, including both molecular-specific ((nano)HPLC-MS(-MS), available in our laboratory) and metal-specific techniques (HPLC-ICP-MS, available in collaborating laboratories). The materials to be studied include plant xylem sap, apoplastic fluid and phloem sap. Metals to be studied will be Fe (as nutrient) and Cd and Zn (as toxic metals). The plant species that will be used include sugar beet, tomato (including mutants), Lupinus texensis (in collaboration with Grusak's lab in USA), and barley (in collaboration with Ma's lab in Japan), as well as the field species pear.

Objective 2. To develop novel methodologies to study crucial steps in plant metal homeostasis using mass spectrometry This objective includes studies on three crucial steps in metal homeostasis: Fe(III) reduction activities, metal fluxes in different plant boundaries, and root accumulation and excretion of metabolites to the growth medium. New HPLC-MS-based methods will be designed for assaying Fe(III)-reductase activity in whole roots, excised root tips, protoplasts and chloroplasts. Also, new stable isotope, IDA-based methods will be designed for assessing metal fluxes in cells (protoplasts and oocites) and organelles (chloroplasts). The role of root accumulation and excretion of metabolites to the growth media in the case of Fe deficiency will be explored using (nano)HPLC-MS(-MS) and GC-MS (the latter technique in Fiehn's lab in US). Species to be used will be sugar beet and cucumber.

Objective 3. To study the metabolism of metal imbalances in plants with a combined metabolomics/proteomics approach This objective includes studies on the changes in plant proteomics and metabolomics associated to different levels of metals in the growth medium. Studies will be focused on the metabolome/proteome of roots, xylem sap, leaf apoplastic fluid and phloem, using different plant species grown with Fe deficiency or with toxicities of Zn or Cd. Techniques used will be (nano)HPLC-MS(-MS) and GC-MS.

Objective 4. To study the micro-localization of metals and metal homeostasis-related components in plants This objective includes studies on the localization of metals (Fe, Zn and Cd) by LT-SEM-EDX, TEM-EDX and PIXE. Fluorophores will be also used to study the localization of Zn and Cd in isolated protoplasts. Also, the localization of metal homeostasis-related proteins and metabolites such as flavins will be studied by using antibodies and fluorescence image analysis techniques. The materials to be used in this objective will be sugar beet and cucumber.

Novelty and scientific relevance of the objectives The novelty of the proposal is based on: i) the holistic approach to assess overall changes in plant metabolites and proteins related to metal homeostasis; ii) the use of state-of-the-art, advanced technologies, such as (nano)HPLC-MS(-MS), GC-MS and metal detection microscopy techniques; and iii) the aim to tackle metal homeostasis issues little explored so far.

The proposal is a follow-up of previous projects in metal homeostasis developed by our research group, and is coherent with work recently published and in progress. Funding of the proposal would also facilitate the use of advanced state-of-the-art analytical techniques in the Agricultural Sciences area. The proposal further expands the application of the techniques used in the current project, which finishes at the end of 2010.

The scientific relevance of the proposal is supported by the previous results and publications of the proponent research group (45 SCI papers in the last 5 years), as well as by the related invited talks in specialized Symposia delivered by members of the group. The relevance is strengthened by the importance of these issues under both agronomical and environmental viewpoints, since both metal deficiencies and toxicities are common in the world. The group is in close touch with other groups working in this area, both within Spain and abroad, working either with model plants or with field crops, and also with other groups working with targeted and untargeted MS analytical techniques (see Part 2).

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4. METHODOLOGY AND WORKING PLAN Detail and justify precisely the methodology and the working plan. Describe the working chronogram. ♦ The working plan should contain tasks, milestones and deliverables. ♦ If personnel costs are requested, the tasks to be developed by the personnel to be hired must be detailed

and justified. Remember that personnel costs are eligible only when personnel is contracted, fellowships are not eligible as personnel costs.

It should be taken into consideration that this proposal was written in February 2010 and that in case of success, the project would probably begin to be financed in January 2011. This time lag could imply that changes and/or improvements concerning the methodologies employed, and changes in the staff available, may take place throughout the year 2010.

All personnel indicated in the application form official list (EEAD-CSIC: staff JA, AAF, AFLM and FM, hired scientist RR, graduate students JRC and GL) will participate in objectives and tasks as indicated below. Concerning international collaborators, staff U.C. Davis OF, U. Okayama JFM, U. Budapest FF and USDA-ARS Houston MG will also participate in some objectives and tasks as indicated below. The tasks where a graduate student (Bap) ascribed to the project will work (if approved) are indicated just for reviewers’ information.

Scientists not included in the participant list but participating in the project Other staff of the "Abiotic Plant Stress Physiology" group in the EEAD-CSIC (A. Abadía and S. Vázquez) will participate in the project, but currently (February 2010) do not comply with the administrative requirements of the proposal and are not included in the application form. Despite studies may take place within different research projects, the investigations developed within the whole group are always complementary.

Financial support for a research fellow (graduate student) is requested in the proposal. Should the position will be granted, the fellow would be included automatically as project participant.

Foreign scientists participating in the project

Four researchers working in foreign institutions are involved in this proposal:

-Oliver Fiehn (U.C. Davis, US): One of the world leader experts in plant metabolomics. He already participated in the previous project, and two members of our group have worked in short stages in his lab. He is included in several tasks related to metabolomics studies using GC-MS.

-Jian Feng Ma (U. Okayama, Japan): A world leader in plant nutrition research. He already participated in the previous project and also in a Japan-Spain program with our group, and two members of our team have worked in short stages in his lab. He is included in tasks related to metal transport in the xylem, metal fluxes and protein immuno-localization.

-Ferenc Fodor (U. Budapest, Hungary): With ample experience in thylakoid composition and function and heavy metals effects in plants. He joined the previous project, and has also participated in two Hungary-Spain programs with our group. Two members of our group have worked in his lab in short periods, and members of his group work frequently in our lab in short stages. He is included in tasks related to metal fluxes and proteomics.

-Michael Grusak (USDA-ARS Houston, US). A leading expert in the field of nutrient distribution in plants. One member of our group has worked in his lab for several years. He is included in tasks related to Fe uptake and metal fluxes.

Methodology

Methodology common to all Objectives (cites are included in the literature list of Part 2)

The majority of tasks will be carried out in the EEAD-CSIC, although some of the proposed experiments will be performed in U.C. Davis, U. Okayama, U. Budapest and USDA-ARS Houston. Some analysis will be carried out in external laboratories or services, following our current collaboration policies.

Plant material: The use of several model plants is necessary for the development of the project, because it is not possible to reach all the objectives proposed with only one or two plant species. Sugar beet has been the model plant used by the proponent group in several projects, and will be used because a good deal of information is available on the physiology and biochemistry of this species, and also because it is possible to isolate plant fluids and purified membrane preparations. However, the information available on the genomics and proteomics of this species is limited. Tomato and cucumber are two species where more genomic and proteomic data are available, and in the case of tomato there are mutants with altered metal homeostasis. Phloem can be obtained in sufficient quantities for analysis from some lupine species such as Lupinus texensis. Barley is a model plant used by the Japanese group, and poplar is used routinely as a model species by the

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Hungarian group. Finally, some fruit tree species grown in the field such as peach and pear will be used in some project tasks.

The species to be used are as follows: 1) Sugar beet (Beta vulgaris L.); 2) tomato (Lycopersicon esculentum Mill.), including the semi lethal mutant 'Chloronerva', that shows high concentrations of Fe, interveinal chlorosis and other symptoms of Fe deficiency (Zouari et al., 2001); 3) cucumber (Cucumis sativus L.); 4) Lupinus texensis; 5) peach (Prunus persica L. Batsch) and 6) pear (Pyrus communis L.) trees grown in field conditions. Barley and poplar plants will be used by the partner groups in Japan and Hungary, respectively.

Plant culture: In the EEAD-CSIC plants will be grown at 25°C in growth chambers with controlled light (PPFD 400 µE m-2 s-1), humidity (80% RH) and photoperiod conditions (16 h light/8 h darkness) (Sagardoy et al., 2009; López-Millán et al., 2009; Rellán-Álvarez et al., 2010). Plants will be pre-grown in hydroponics for approximately 7-10 days in standard solution, and after this period metal deficiency (Fe) and toxicity treatments (Zn and Cd) will be imposed. In the case of peach and pear, leaves will be sampled from trees grown under field conditions with or without Fe deficiency. In the case of our partners abroad, growth conditions will be as described in their papers.

Xylem sap and apoplastic fluid collection: Xylem sap by centrifugation in sugar beet (López-Millán et al., 2000), detopping in tomato (López-Millán et al., 2009), lupine and barley, and using a Schölander chamber in pear (Larbi et al., 2010); leaf apoplastic fluid by centrifugation in sugar beet (López-Millán et al., 2000) and pear (López-Millán et al., 2001).

Phloem sap collection: Using Lupinus texensis, according to Marentes and Grusak (1998).

Nutrient status of plants: The nutrient status of plants will be assessed by measuring: i) shoot and root dry mass; ii) mineral concentrations in roots, shoots and leaves; and iii) photosynthetic pigment concentrations in leaves. Plant tissue mineral concentrations (N, P, K, Ca, Mg, Fe, Mn, Cu and Zn) will be determined by the techniques available in the Analysis Service of the Plant Nutrition Department at the EEAD-CSIC, by Dumas, FAAS and FES (Igartua et al., 2000). Low concentrations of Fe and other metals will be determined by graphite furnace AAS in the Analysis Service of the U. Barcelona (López-Millán et al., 2000).

Methodology for Objective 1 (To study metal speciation in plant fluids; Responsible AAF).

This objective is aimed to reveal metabolites that could transport metals in the xylem sap (in sugar beet, tomato, lupine and pear in Spain; in barley in Japan), leaf apoplastic fluid (sugar beet and pear) and phloem sap (lupine). The in vivo detection of metal species will be carried out by means of metal-specific and metal species-specific target analysis (including natural and xenobiotic compounds). This will be done in fluids of plants grown under different metal status (Fe deficiency, Zn -only in sugar beet- and Cd -only in tomato and sugar beet- toxicities), and in some cases with metal stable isotopes. In the case of Fe a special effort will be made to lower the detection limit of Fe-complexes, using nanoHPLC. Metabolite profile and/or metabolomic analysis may allow for the identification of other possible ligands different from those proposed in the literature (see Objective 3). Objective 1 is structured in two tasks (Tasks 1 and 2), one involving detection and identification of metal complexes and a second one involving quantification.

Metal-specific and metal species-specific target analysis: For the detection, identification and quantification of metal-ligand complexes in plant fluids we will use hyphenated techniques, following an integrated MS approach. This includes the combination of a liquid chromatographic separation (HPLC or nanoHPLC) coupled to high resolution, metal specific (ICP-MS) and molecule specific detections (by ESI-TOFMS and ESI-CID-MS-MS, respectively). Most of the instrumentation is available in the EEAD-CSIC: two HPLC devices (Waters 2795 HPLC and Agilent 1200 nanoHPLC) coupled either to an ESI-TOFMS (micrOTOF, Bruker Daltoniks) or to an ESI-CID-MS-MS ion trap (HCT Ultra, Bruker Daltoniks) detector. Other instrumentation, including HPLC-ICP-MS, will be used in external laboratories or services following our current collaboration policies.

The determination of xenobiotic Fe-complexes in plant fluids will carried out applying the MS-based methods recently developed and validated by our group (Álvarez-Fernández et al., 2007; Orera et al., 2009, 2010). These HPLC-ESI-TOFMS methods are capable of determining very small quantities of the major synthetic (xenomic) Fe chelates used in agriculture (with EDTA, DTPA, EDDHA and EDDHMA).

The determination of natural metal-complexes in plant fluids will be carried out following a similar approach to that used successfully by our group to achieve the first direct and unequivocal identification of a natural Fe complex in plant xylem sap (Rellán-Álvarez et al., 2010). This approach includes: i) the development of integrated MS-based methods for the determination of pure metal complexes (obtained with standards of bio-molecules putatively involved in metal transport in plants), and ii) the application of the methods developed to plant fluid (labeled or not with stable isotopes) studies. This includes the detection of metal-containing ions by using metal

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isotopic signatures, and the identification of such ions through determination of empirical formulae and collision induced dissociation fragmentation patterns (ESI-CID-MS-MS). As in the work of Rellán-Álvarez et al. (2010), all analyses will use (i) pH values similar to those of the plant fluid studied throughout the analysis and sample preparation, to preserve the integrity of the metal-complexes, (ii) an hydrophilic interaction chromatography separation that allows for the retention of polar compounds, thus reducing ion suppression in ESI-ionization, and (iii) stable Fe isotopes for identification and quantification purposes. The quantification of metal-biocomplexes will be tackled using metal-specific and metal species-specific isotope dilution (IDA) techniques (Schaumlöffel and Lobinski 2005).

Methodology for Objective 2 (To develop novel methodologies to study crucial steps in plant metal homeostasis using mass spectrometry; Responsible JA). This objective is structured in three different tasks (Tasks 3, 4 and 5), aimed to: i) develop MS-based methods for assaying Fe(III)-reductase activity in whole roots, excised root tips, protoplasts and chloroplasts (sugar beet), and whole roots and excised root tips of cucumber; ii) develop stable isotope, isotope dilution analysis (IDA)-based methods for assessing metal fluxes in cells (sugar beet protoplasts and transformed oocites) and organelles (sugar beet and poplar chloroplasts); and iii) study root accumulation and excretion of metabolites to the growth media in sugar beet and cucumber plants grown under Fe-deficiency.

Reductase assays: Iron(III)-reductase enzyme activities will be assayed by using a strong Fe(II) chelator (e.g., BPDS), and measuring spectrophotometrically the Fe(II)-BPDS chelate formed (Chang et al., 2003; Gogorcena et al., 2004). Also, we will use the new MS methodologies developed in the last years for synthetic Fe(III)-chelates (Álvarez-Fernández et al., 2007) and natural Fe(III)-complexes (Rellán-Álvarez et al., 2010) to design assays based in monitoring the decreases in the concentrations of the enzyme substrates. In this task we will also collaborate with Grusak's team.

Metal fluxes assays: Metal fluxes are usually assessed in cells and organelles by exposing them to metal-containing assay media (radioactive or not), and then measuring metal fluxes by traditional analysis techniques such as AAS, ICP-MS or radioactivity (Ellis et al, 2003; Pedas et al., 2008). In this project we propose to use stable isotopes and isotope dilution analysis (IDA) techniques (Schaumlöffel and Lobinski 2005; in collaboration with J.I. García-Alonso at U. Oviedo) to design methods to study the fluxes of metals in cells (protoplasts and transformed oocites) and isolated organelles (chloroplasts). In this task we will also collaborate with the US-Houston, Japanese and Hungarian laboratories, which have extensive experience in work with metal fluxes, protoplasts and oocites, and intact chloroplasts, respectively.

Metabolite accumulation and excretion: The accumulation and excretion of metabolites will be studied in sugar beet roots by targeted analysis of flavins, organic acids and phenolic compounds by HPLC-MS-MS. Also, untargeted, metabolomic analysis (GC-MS) will be used to obtain a perspective of the metabolites being released by roots to the medium. In the latter analysis we will collaborate with Fiehn's lab with the methods indicated below.

Methodology for Objective 3 (To study the metabolism of metal imbalances in plants with a combined metabolomics/proteomics approach; Responsible AFLM).

This objective is aimed to study the changes in the plant metabolome and proteome associated to different metal imbalances. Studies will be focused on: i) root tips of sugar beet (Fe deficiency, Zn and Cd toxicity) and tomato (Fe deficiency and Cd toxicity); ii) xylem of sugar beet (Fe deficiency, Zn and Cd toxicity), tomato (Fe deficiency and Cd toxicity), lupine (Fe deficiency) and peach (Fe deficiency); iii) leaf apoplastic fluid of sugar beet (Fe deficiency, Zn and Cd toxicity); and iv) phloem of lupine (Fe deficiency). Key steps in this objective will be the optimization of the sampling process of these plant compartments and the adequate combination of proteomics and metabolomics data. The objective is structured in two tasks (Tasks 6 and 7), the first devoted to study the metabolomics/proteomics changes and the second to exploit in depth the major differences found, by using targeted analysis.

Metabolomics: For metabolomic analysis we will use methods developed in Fiehn’s laboratory (Fiehn et al., 2008). All information related to the experiment design will be introduced into SetupX, an XML based system that enables entering biological metadata for steering laboratory workflows, by generating "classes" that reflect experimental designs. Samples will be extracted using protocols developed in Fiehn's lab (Fiehn et al., 2008), in which we already have experience from previous collaborations. Samples will be analyzed in a randomized way with a GC-TOFMS apparatus (Pegasus III, Leco). Quality control standards at five different concentrations will be injected with each sample set. Initial peak detection and mass spectra deconvolution will be performed with Leco Chroma-TOF software v.2.25. GC-MS chromatograms will be processed as described previously. Different statistical tools will be used, such as ANOVA, principal component analysis (PCA) and partial least squares (PLS). These tools can help us find relevant metabolites

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that can explain differences between different classes or relationships between metabolites in the same class. The samples would be obtained and processed in the EEAD-CSIC. Metabolomic analysis will be carried out by personnel from our group in Fiehn's lab.

Proteomics: Proteins will be separated by two-dimensional (2-D SDS-PAGE) electrophoresis (Protean IEF system, Biorad) according to Andaluz (2005) and Andaluz et al. (2006). Following 2-D SDS-PAGE, gels will be stained with silver, Coomasie Blue or Sypro, and individual protein gel spots will be excised and collected with a spot gel cutter (ExQuest, BioRad). Gels could also be stained with specific staining for metal containing proteins, such as Ferene for Fe (Krüger et al., 2002). Protein spots will be identified using MALDI-TOF mass fingerprinting in external service laboratories. In previous studies it has been shown (Andaluz 2005) that when using plant materials little characterized such as phloem and root tips a significant number of spots separated by 2-D electrophoresis techniques could not be identified by MALDI-TOF mass fingerprinting, making necessary de novo sequencing. Protein spots will be digested enzymatically, and peptides will be separated in micro-columns with the nanoHPLC-MS-MS system indicated above, that permits to carry out de novo sequencing of moderately large proteins. Exact molecular mass can also be obtained by using the ESI-TOFMS system indicated above, available in our laboratory. Databases will be used to find homologies with known proteins, EST and/or genomic sequences using Mascot (v. 9) as a search engine. Statistical analyses will be similar to those used for metabolomics data.

Combination of the proteomics and metabolomics data: Combined metabolomics and proteomic studies will be carried out in the same plant material. Plant material sampling, conservation and extraction processes will be done in parallel for both types of analysis to avoid any kind of analysis-specific deviation. After acquisition of metabolite and protein profiles, both sets of data will be normalized for an adequate integration between metabolites and proteins (Weckwerth, 2008). Multivariate statistics will be used to examine sample pattern recognition based on data-dimensionality reduction techniques such as PLS. We will also investigate possible correlations between proteins, metabolites and protein-metabolites.

Methodology for Objective 4. (To study the micro localization of metals and metal homeostasis-related components in plants; Responsible FM). This objective is structured in two tasks (Tasks 8 and 9), aimed to study: i) the micro-localization of metals both by using microanalysis-coupled electron microscopy (EM) techniques (LT-SEM-EDX, TEM-EDX, micro-PIXE; in roots and leaves of sugar beet with Fe deficiency, Zn and Cd toxicity, and in roots and leaves of cucumber with Fe deficiency) and by using fluorophores (leaf protoplasts of sugar beet with Zn or Cd toxicity); and ii) the distribution of important molecules related to the metal homeostasis in roots and leaves such as proteins by immuno-localization and flavins by fluorescence microscopy (roots and leaves of sugar beet and cucumber, with Fe deficiency only).

Electron microscopy: Scanning electron microscopy (SEM-EDX) will be carried out in the nearby ICB-CSIC Institute, where we have an established collaboration (Hitachi S3400N, with a Röntek EDX detector for elemental analysis). The image resolution of the SEM is 10-15 nm (in low vacuum) and the elemental analysis resolution is approximately 4 µm. Sample analysis by low-temperature-scanning electron microscopy (LT-SEM-EDX; Zeiss 960, with an Oxford Link Isis EDX microanalysis system) and transmission electron microscopy (TEM; LEO 910, 120 Kv) will be conducted in the CCMA-CSIC in Madrid.

Collection of fresh sections: Fresh plant material will be embedded in agar, and 50 µm sections will be obtained using a vibratome (Leica VT 1000S). Cross- and longitudinal sections will be obtained from leaves and roots.

Localization of proteins and metabolites in fresh sections: Two main types of determinations will be carried out in fresh sections:

-Immuno-localization of Fe-related proteins by using specific antibodies for PEPCase, ATPase, IRT1 and ferritin in roots (Dell’Orto et al., 2002) and leaves.

-Flavin localization in Fe-deficient root sections will be carried out by using an inverted microscope (Leica DMIL LED) with a fluorescence kit, equipped with a CCD camera (Leica).

Protoplast isolation: Intact protoplasts will be isolated from sugar beet leaves under different metal status following optimized protocols (Sagardoy et al., paper in preparation).

Fluorophores application in intact protoplasts: Highly metal-specific fluorophores (Zn and Cd) will be used to determine the metal distribution in leaf protoplasts. Suitable fluorophores will be selected in order to use ratio image techniques (Maruyama et al., 2002).

Other image-related analysis tools: Laser capture micro-dissection (LCM, Corpas et al., 2006), image mass spectrometry (MALDI-TOFMS), laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and micro-proton-induced X-ray emission (micro-PIXE) microanalysis may be used in some works if needed. LCM can be done at the U. Jaén (J.B. Barroso), image mass spectrometry in Bruker’s laboratories in Bremen, LA-ICP-MS at the University of Oviedo (J.I.

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García-Alonso) and micro-PIXE in the Jožef Stefan Institute Microanalytical Center (Ljubljana, Slovenia).

Other collaborations within the project We have already established collaborations for specific subjects within the project with:

-U. Oviedo (J.I. García Alonso) for stable isotopes studies, ICP-MS and IDA analysis. -ICMA-CSIC (J. Orduna, Zaragoza, Spain) for ESI-QTOFMS-MS. -ICB-CSIC (J.M. Andrés, Zaragoza, Spain) for SEM-EDX. -U. Milano (G. Zocchi) for protein localization and proteomics.

In Objective 4 some external services will be involved:

-CCMA-CSIC (Madrid, Spain) for LT-SEM-EDX (freeze fracture) and TEM. -PCB (Barcelona, Spain) for high pressure freezing and freeze-substitution.

Workplan This is just a tentative workplan, which can change considerably depending on the knowledge at the start of the project and also on the findings obtained during the three years (underlined are the initials of the task responsible; Bap: pre-doctoral fellow associated to the project; in brackets [], partners abroad):

First year (2011)

Task 1 (Obj. 1) (AAF, RR, JA, Bap, [JFM]). Detection and identification of metal-complexes. This includes integrated MS-analysis of: a) standard solutions containing metals (e.g. Fe, Zn, Cd) and bio-molecules putatively involved in transport in plants, at different metal:biomolecule ratios and at the typical pH values found in plant fluids; and b) xylem sap of Fe-, Zn- and Cd-stressed plants. Standard solution analyses would involve i) the optimization of ESI ionization conditions for the best ESI-MS detection, preserving the integrity of the metal-complex species, ii) optimization of HPLC separation conditions that allow for the retention, separation and ICP-MS/ESI-MS detection of putative metal-bioligand complexes; and iii) characterization of CID fragmentation patterns of the metal-biocomplexes detected, which will add authority to their identification in complex matrices such as plant tissues, where isobaric interferences can appear. Plant fluid analyses (xylem sap in the first year and other fluids in the following years) would involve: i) the application and/or modification of the HPLC separation and ESI ionization conditions found optimal for putative metal-biocomplexes; ii) the detection of metals by HPLC-ICP-MS; iii) HPLC-ESI-TOFMS detection of metal-biocomplex species by using specific metal isotopic signatures; iv) determination of the elemental formulae for metal-containing ions; and v) CID fragmentation of metal-containing ions. Metal-biocomplexes that cannot be identified will be isolated for characterization by other means. Deliverable (SCI paper) X.

Task 3 (Obj. 2) (JA, RR, AFLM, AAF, [MG]). Iron reductase assays. This includes: i) characterization of Fe(III)-reductase affinity for the Fe2Cit2 and Fe3Cit3 complexes described in Rellán-Álvarez et al. (2010), using roots, leaf protoplasts and chloroplasts, and comparing the results with those of the well-characterized Fe(III)-EDTA reduction; and ii) measurement of Fe(III)-reductase activity in the same materials by quantification of substrate concentration, using the MS-method described by Orera et al. (2009).

Task 5 (Obj. 2) (JA, JRC, RR, AAF, [OF]). Root accumulation and excretion studies. This includes: i) the development of efficient methods to sample metabolites excreted by Fe-deficient roots; and ii) the optimization of extraction processes for a good detection by MS-techniques, including LC-MS(-MS) and GC-MS. Deliverable (SCI paper) X.

Task 6 (Obj. 3) (AFLM, RR, GL, JRC, AAF, JA, [OF, FF]). Metabolomics/proteomics studies. This includes: i) optimization of plant material sampling and metabolite/protein extraction procedures compatible with metabolomic and proteomic analyses; and ii) metabolomic/proteomic analyses of sugar beet tissues and fluids under Fe deficiency and Zn and Cd toxicities. Deliverable (SCI paper) X.

Task 8 (Obj. 4) (FM, JA). Micro-localization of metals using microanalysis-coupled, electron microscopy techniques (SEM-EDX). Micro-localization of Zn and Cd in intact sugar beet protoplasts, using highly specific fluorophores (ratio image technique). Deliverable (SCI paper) X.

Task 9 (Obj. 4) (FM, JA, [JFM]). Localization of metabolites. Micro-localization of flavins by fluorescence microscopy in sugar beet root sections.

Task 10 (Publications, Dissemination) (JA, all project members). Writing of communications to Symposia, papers to Scientific SCI journals and dissemination papers. Milestone O: Annual Report.

Second year (2012)

15

Task 1 (Obj. 1) (AAF, RR, JA, Bap, [JFM]). Detection and identification of metal-complexes. This includes analyses of leaf apoplast of Fe-, Cd- and Zn-stressed plants. These analyses will be carried out as indicated in the first year.

Task 3 (Obj. 2) (JA, RR, AFLM, AAF, [MG]). Iron reductase assays. Continuing assays of Fe(III)-reductase activity by substrate (Fe(III)-EDTA, Fe(III)-EDDHA, Fe(III)-Cit complexes) quantification using the method described by Orera et al. (2009). Methodology setup for quantification using species-specific IDA. Deliverable (SCI paper) X.

Task 4 (Obj. 2) (JA, AFLM, AAF, Bap, [MG, JFM, FF]). Assays for metal fluxes. Methods to assess metal fluxes will be designed using stable isotopes and IDA to be used with protoplasts and chloroplasts.

Task 5 (Obj. 2) (JA, RR, AAF, [OF]). Root accumulation and excretion studies. After optimizing sampling and extraction, we will analyze the metabolomics of roots and excreted compounds using plants grown under Fe deficiency, to identify the main changes occurring in this system. Organic acids excreted will be studied by HPLC-MS(MS).

Task 6 (Obj. 3) (AFLM, RR, GL, AAF, JA, [OF, FF]). Metabolomics/proteomics studies. This includes: i) continuing the sugar beet experiments, and ii) experiments with tomato. Deliverable (SCI paper) X.

Task 8 (Obj. 4) (FM, JA, Bap). Micro-localization of metals using microanalysis-coupled, electron microscopy techniques (TEM-EDX). Continuing micro-localization of Zn and Cd in intact sugar beet protoplasts, using highly specific fluorophores (ratio image technique). Deliverable (SCI paper) X.

Task 9 (Obj. 4) (FM, JA, Bap, [JFM]). Localization of proteins and metabolites. Immunolocalization of Fe-related proteins in sugar beet roots and leaves with specific antibodies (PEPCase, ATPase, IRT1, ferritin and other available in JFM lab). Continuing the micro-localization of flavins by fluorescence microscopy in sugar beet root sections.


Third year (2013)

Task 1 (Obj. 1) (AAF, JA, Bap, [JFM]). Detection and identification of metal-complexes. This includes the follow-up of studies with leaf apoplast and new studies on phloem sap. These analyses will be carried out as indicated in the first year. Deliverable (SCI paper) X.

Task 2 (Obj. 1) AAF, JA, Bap). Towards metal bio-complexes quantification in plant fluids (those used in Task 1) includes, besides the typical quantification methods used in MS analysis, two different isotopic dilution (IDA) techniques: species-unspecific IDA for quantification of metal elements bound to unknown biomolecules and species-specific IDA for quantification of well-defined compounds.

Task 3 (Obj. 2) (JA, AFLM, AAF, [MG]). Iron reductase assays. Continuing assays of Fe(III)-reductase activity by substrate (Fe(III)-EDTA, Fe(III)-EDDHA, Fe(III)-Cit complexes), and quantification using species-specific IDA.

Task 4 (Obj. 2) (JA, AFLM, AAF, Bap, [MG, JFM]). Assays for metal fluxes. The development of methods to assess metal fluxes will continue in this year, including work with oocites expressing specific metal transporters in the Japanese lab. Deliverable (SCI paper) X.

Task 5 (Obj. 2) (JA, AAF). Root accumulation and excretion studies. We will continue analysis of the metabolomics of roots and excreted compounds with Fe deficiency. Phenolic compounds excreted will be studied by HPLC-MS(-MS). Deliverable (SCI paper) X.

Task 6 (Obj. 3) (AFLM, AAF, JA, [OF, FF]). Metabolomics/proteomics studies. This includes i) continuing the experiments with tomato and ii) experiments with peach and lupine.

Task 7 (Obj. 3) (AFLM, AAF, Bap, JA, Bap). Exploitation of metabolomics/proteomic studies by targeted analysis. Compounds identified as having marked changes in metabolomics/proteomic studies will be analyzed in depth using targeted analysis by HPLC-MS(-MS).

Task 8 (Obj. 4) (FM, JA, Bap). Micro-localization of metals using microanalysis-coupled, electron microscopy techniques (micro-PIXE).

Task 9 (Obj. 4) (FM, JA, Bap, [JFM]). Localization of proteins and metabolites. Immunolocalization of Fe-related proteins with specific antibodies (PEPCase, ATPase, IRT1, ferritin, and other available in JFM lab) in cucumber roots and leaves and micro-localization of flavins by fluorescence microscopy in cucumber root sections. Deliverable (SCI paper) X.

16


Justification of Personnel to be on Contract

The Laboratory Technician to be contracted will work in growing plants in the different metal treatments, sampling different tissues and fluids and in the following analytical tasks of the project:

• Task 5: Accumulation and excretion studies. • Task 6: Metabolomics/proteomics studies. • Task 7: Exploitation by targeted analysis.

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4.1 CHRONOGRAM The name of the person supervising each task is underlined.

Activities/Tasks Center

Responsible person and others involved

First year (*) 2011

Second year (*) 2012

Third year (*) 2013

Objective 1. Metal speciation in plant fluids

Task 1. Detection and identification of metal-complexes.

EEAD-CSIC, [U Okayama]

AAF, RR, JA, Bap, [JFM]

X|X|X|X|X|X|X|X|X|X|X| X |X|X X|X|X|X|X|X|X|X|X|X|X |X|X X| X |X|X|X|X|X|X|X|X|X|O |X|X

Task 2. Towards metal bio-complexes quantification

EEAD-CSIC AAF, JA, Bap X|X|X|X|X|X|X|X|X|X|X|O

Objective 2. Novel methodologies to study crucial steps in metal homeostasis using mass spectrometry

Task 3. Iron reductase assays EEAD-CSIC, [USDA-ARS]

JA, RR, AFLM, AAF, [MG]

X|X|X|X|X|X|X|X|X|X|X|X |X|X X|X|X|X|X|X|X|X|X|X|X| X |X|X X|X|X|X|X|X|X|X|X|X|X|O |X|X

Task 4. Assays for metal fluxes EEAD-CSIC, [USDA-ARS, U Okayama, U. Budapest]

JA, AFLM, AAF, Bap, [MG, JFM, FF]

X|X|X|X|X|X|X|X|X|X|X|X |X|X X|X|X|X|X|X|X|X|X|X| X|O |X|X

Task 5. Accumulation and excretion studies

EEAD-CSIC, [UC Davis]

JA, JRC, RR, AAF, [OF] X|X|X|X|X|X|X|X|X|X|X| X |X|X X|X|X|X|X|X|X|X|X|X|X|X |X|X X|X|X| X |X|X|X|X|X|X|X|O |X|X

Objective 3. Metabolism of metal imbalances with a combined metabolomics/proteomics approach

Task 6. Metabolomics/proteomics studies

EEAD-CSIC, [UC Davis, U Budapest]

AFLM, RR, GL, JRC, AAF, JA, [OF, FF]

X|X|X|X|X|X|X|X|X|X|X| X |X|X X|X|X|X|X|X|X|X|X|X|X| X |X|X X|X|X|X|X|X|X|X|X|X|X|O |X|X

Task 7. Exploitation by targeted analysis

EEAD-CSIC AFLM, AAF, JA, Bap X|X|X|X|X|X|X|X|X|X|X|O

Objective 4. Micro localization of metals and metal homeostasis-related components in plants

Task 8. Localization of metals EEAD-CSIC FM, JA, Bap X|X|X|X|X|X|X|X|X|X|X| X |X|X X|X|X|X|X|X|X|X|X|X|X| X |X|X X|X|X|X|X|X|X|X|X|X|X|O |X|X

Task 9. Localization of proteins and metabolites

EEAD-CSIC, [U. Okayama]

FM, JA, Bap [JFM]

X|X|X|X|X|X|X|X|X|X|X|X |X|X X|X||X|X|X|X|X|X|X|X|X|X X |X|X X| X |X|X|X|X|X|X|X|X|X|O |X|X

Dissemination

Task 10. Publications, presentations, Dissemination, etc.

EEAD-CSIC JA, all members X|X|X|X|X|X|X|X|X|X|X|O X|X|X|X|X|X|X|X|X|X|X|O X|X|X|X|X|X|X|X|X|X|X|O

*JRC until March 2011 Deliverable (SCI paper) X *RR until Feb 2012 Milestone O *GL until October 2012 *Bap = Fellow associated to the project *In brackets [], partners abroad.

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5. BENEFITS DERIVED FROM THE PROJECT, DIFUSION AND EXPLOTATION OF RESULTS (maximum 1 page) ♦ Scientific and technical contributions expected from the project, potential application or transfer of the expected

results in the short, medium or large term, benefits derived from the increase of knowledge and technology. ♦ Diffusion plan and, if appropriate, exploitation plan of the results.

Scientific and technical contributions expected from the project We expect that, alike in previous projects developed by our research group, the project shall result in a number of papers in SCI Scientific Journals, as well as in papers in Technical and Dissemination Journals and presentations in International and National Symposia. We will send our findings to Scientific Journals in the first SCI quartile of the disciplines Plant Science, Analytical Chemistry, Agriculture-Multidisciplinary, Agronomy and Forestry. In the past five years we have published papers in New Phytol, Plant Cell Physiol, Photosynth Res, J Plant Physiol, Plant Physiol Biochem, Environ Exp Bot, Plant Sci and Plant Biol (Plant Science), J Am Soc Mass Spectrom, Anal Biochem and Rapid Commun Mass Sp (Analytical Chemistry), J Agric Food Chem (Agriculture-Multidisciplinary), Plant Soil (Agronomy) and Tree Physiol (Forestry), among others. We also aim at disseminating results for a broader community of readers, by publishing in Technical and Dissemination Journals such as Acta Hortic, Vida Rural and Italus Hortus, as well as in local newspapers. We have published in all these media types in the last few years. The most significant Symposia in which we will present our work (as we have done on a regular basis) are the next ones in the following Series: International Symposium on Iron Nutrition and Interactions in Plants (ISINIP; 2012), International Symposia on Mineral Nutrition of Fruit Plants of the ISHS (2012), International Congress on Plant Nutrition (ICPN; 2013) and Spanish-Portuguese Symposia on Plant Mineral Nutrition (2012) and Plant Physiology (2011, 2013).

Potential transfer of expected results to specialized sectors Prior projects led to a number of R+D Contracts with Companies related to the agricultural sector, including Spanish, German, Swiss and Japanese companies. Cooperation with companies will ensure knowledge transfer within the sector (largely in the scenario of fruit production and fertilizer technology). Should the project give rise to a patent, the corresponding OTT-CSIC Unit would process and manage it as it has been done previously.

Expected benefits derived from the increase in knowledge Granting this proposal will give an impulse to the knowledge of metal homeostasis processes in plants. Knowing the form of transport of metals in plant fluids is crucial to understand metal homeostasis. Studies focusing in crucial steps in the plant response to Fe deficiency (reductase activities, metal fluxes and metabolite accumulation and excretion) are expected to change the way researchers approach this issue. Also, new knowledge on changes in plant metabolism as a result of metal deficiency or toxicity would be gained from the proteomics/metabolomics studies. Localization studies will be also very helpful, for instance to optimize fertilization practices. The benefits obtained from the project could potentially be of application to fields ranging from agricultural fertilization management in metal-deficient crops to phyto-remediation of metal-contaminated areas. The topic is of global interest at a worldwide scale. Moreover, the proposed aims will enable advanced applications of state-of-the-art technologies to Agricultural Sciences, including the use of mass spectrometry ((nano)ESI-MS(-MS) and GC-MS for metabolomic, xenomic and proteomic applications.

Proposals in European Frameworks in relation to this project We are currently participating in the EU-6th FP project Isafruit (2006-2010) and in a Germany/France/Spain Trilateral Project (2009-2012). We also participate in COST actions 603 and 905. In the past, our research group coordinated a EU AIR Project (1995-1999), and participated in two more (1 STD, 1990-1994; 1 INCO 1998-2001).

Knowledge Dissemination plan As indicated above, should the proposal be financed results will be published in Scientific and Dissemination Journals, and presented in relevant scientific Congresses and Meetings. A distinctive feature of our group is the web page, which is updated at least monthly. According to our experience, making results available to the public in such way facilitates direct dissemination to both technical and scientific sectors of interest and the society in general. The members of our group also participate in different local dissemination activities focused on the general public, especially young people. These activities include open door days, stands in science fairs and publication of science dissemination articles in local newspapers.

http://www.stressphysiology.com

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6. BACKGROUND OF THE GROUP (max. 2 pages) ♦ Previous activities and achievements of the group in the field of the project:

In this section we summarize the research activities of our group in issues related with the proposal (only in the period 2005-2010). This includes Projects and R+D contracts1, Publications2, PhD Theses3 and Keynote talks in Symposia4. We also include additional information5 on results related to the objectives of the last Spanish R+D Plan project (AGL2007-61948, that will end in October 2010), although at this moment (February 2010) many of the studies proposed are still in progress and some publications are in preparation at different stages. Finally, we include information6 on our experience in the techniques necessary for the objectives of the current proposal (metabolomics, proteomics, etc).

Further additional information can be found in the research group web page, which is frequently updated. 1Projects and R+D Contracts, 2005-2010 The PI of this proposal has been leader in 7 previous projects of the Spanish National R+D Plan, from 1988 to 2010. In the period 2005-2010, the research group has worked in the following Research Projects and R+D Contracts in the field "metals and plants" (those underlined are still in progress in February 2010):

-National R+D Plan 2005-2010: AGL2003-01999 (2003-2006), AGL2004-00194 (2004-2007), AGL2006-01416 (2006-2009), AGL2007-61948 (2007-2010), EUI2008-03618 (2009-2012) and AGL2009-09018 (2010-2013). -National R+D Plan, other Programs 2005-2009: PTR1995-0580-OP (2002-2005). -Local Government (DGA) Programs 2001-2006: Support for Consolidated Group A03 (2003-2010), Projects PM014-2004 (2004-2006) and PM003-2006 (2006-2008). -EU FP projects 2005-2010: Isafruit (6th FP 2006-2010, 016279 Food). -R+D Contracts with companies 2001-2009: 1 with Agromillora Catalana (2002-2005), and 6 with foreign companies. -International cooperation 2005-2010. Bilateral projects: Italy (1), Tunisia (4+1), Hungary (1+1) and Japan (1). 2Publications, 2005-2009 Many of the 45 SCI publications of the group in the period 2005-2009 are on metals in plants or related subjects. Publications include (see web page for full titles) 9 SCI papers in 2005, 8 in 2006, 6 in 2007, 9 in 2008 and 13 in 2009 (6 more in 2010). In the same period, 7 dissemination articles in Technical Journals, 6 invited Book chapters and 2 Books have been published. The PI has currently an "h index" of 27 (27 papers quoted 27 or more times). The thematic of some of the works is as follows (publications underlined in the list at the end were supported by the 2 previous grants of the Spanish National R+D Plan (AGL2004-00194 and AGL2007-61948)(note that not all SCI papers are quoted because of lack of space): Heavy metal effects in plants. On Cd (2, 25), Zn (28), Zn and Mn (3), and Cd and Hg (13, 16). Iron nutrition in plants. On Fe acquisition and transport (21, 23, 24), photoinhibition and photoprotection (10, 11), diagnostic methods and nutritional status in fruit trees (4, 19), interactions between plant nutrients (5), treatments for Fe deficiency (1, 8, 17, 18, 22), and Fe deficiency and fruit quality (6). Metabolomics and xenomics. On glutathione (13), synthetic Fe chelates (14, 26, 27, 31), natural Fe complexes (20, 29), and capsicinoids (9, 15). Proteomics. On thylakoids and Fe deficiency (7). 3PhD Theses, 2005-2009 -Studies on changes in the plant proteome induced by iron deficiency (Sofía Andaluz, October 2005). 4Invited and keynote talks in Symposia, 2005-2009 2005 V International Symposium on Mineral Nutrition of Fruit Crops ISHS, Talca, Chile (J. Abadía) 2006 XIII International Symposium on Iron Nutrition and Interactions in Plants, Montpellier, France (A. Álvarez-Fernández); XI Simposio Ibérico sobre Nutrición Mineral de las Plantas, Pamplona, Spain (J. Abadía) 2007 XVII Reunión de la Sociedad Española de Fisiología Vegetal-X Congreso Hispano-Luso de Fisiología Vegetal, Alcalá de Henares, España (J. Abadía) 2008 VI International Symposium on Mineral Nutrition of Fruit Crops ISHS, Faro, Portugal (V. Fernández) 2009 XVI International Plant Nutrition Colloquium, Sacramento, CA, USA (R. Rellán-Álvarez)



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5Published results within the objectives of the last Spanish R+D Plan project, AGL2007-61948 (many works are still in progress, since this project will end in October 2010). See papers below for full references.

Objective 1 (To study metal homeostasis-related changes in plant metabolites). We have published a paper on the determination of metal complexes with nicotianamine (20). We have also just published a paper on the citrate-Fe complexes existing in tomato xylem (29). We have used HPLC-ESI-TOFMS techniques to determine metabolites such as glutathione (12) and xenobiotics (Fe chelates) in plant materials (14, 26, 27, 31). In other papers we have progressed in the characterization of the effects of Fe deficiency in tomato (24), Medicago truncatula (21) and sugar beet (30), Cd toxicity in tomato (25) and Zn toxicity in sugar beet (28).

Objective 2 (To study metal homeostasis-related changes in plant proteins). We have progressed in the works proposed, that include studies with protoplasts, vacuoles, tonoplast, xylem and thylakoids with Fe deficiency and Cd toxicity. We are studying the changes in the root proteome of sugar beet with Fe deficiency and of tomato with Cd toxicity.

Objective 3. (To study metal homeostasis-related gradients in plant leaves and roots). We have published one paper on the changes in the structure of peach leaves with Fe deficiency (18) and sent one manuscript including changes in the structure of sugar beet leaves with Zn toxicity. 6Experience on the techniques needed for the present proposal Objective 1 (To study metal speciation in plant fluids). In the last years we have studied metabolite target analysis and xenomics profiling using HPLC-MS(-MS) techniques. Methods developed so far (see full references below) are for synthetic Fe chelates (14, 26, 27, 31), natural metal complexes with citrate (29) and nicotianamine (20), glutathione and ascorbate (12) and capsicinoids (9, 15). Methods for carboxylates and flavins have manuscript drafts already in preparation. We have published one paper including HPLC-ICP-MS techniques (29). Two members of the team (RR, AAF) have worked in stages of several months using GC-MS techniques in Fiehn's lab in the Genome Center, U.C. Davis, USA.

Objective 2 (To develop a new perspective on crucial steps in metal homeostasis using mass spectrometry and ICP-MS techniques). The techniques to be used are HPLC-MS(-MS) and HPLC-ICP-MS. We have ample experience in HPLC-MS and have collaborated in HPLC-ICP-MS with the University of Oviedo (29).

Objective 3 (To study the metabolism of metal imbalances with a combined metabolomics/proteomics approach). Two PhD Theses including plant 2-D studies were carried out in our laboratory (defended in 2000 and 2005). In the last years we have developed work in proteomics by using for protein identification MALDI-TOFMS and MALDI-MS-MS techniques available in external laboratories (7). The automated spot cutter and nanoHPLC-MS-MS ion trap system installed in our laboratory will allow carrying out de novo sequencing of proteins obtained by 2-D or other techniques.

Objective 4 (To study the micro localization of metals and metal homeostasis-related components in plants). Our group has acquired experience in image analysis and metal detection techniques in the past two years (18; some papers also in preparation), in collaboration with several external laboratories. Papers quoted (all of them with co-authors from the proposing group; underlined those published within the project AGL2007-61948).

(1) Álvarez-Fernández et al. (2005) Eur J Agron 22:119-130; (2) Fodor et al. (2005) Tree Physiol 25:1173-1180; (3) López-Millán et al. (2005) Plant Sci 168:1015-1022; (4) Pestana et al. (2005) Sci Hortic 104:25-36; (5) Rombolà et al. (2005) Plant Soil 271:39-45; (6) Álvarez-Fernández et al. (Book Chapter, 2006); (7) Andaluz et al. Photosynth Res (2006) 89:141-155; (8) Fernández et al. (2006) Plant Soil 289:239-252; (9) Garcés-Claver et al. (2006) J Agric Food Chem 54:9303-9311; (10) Larbi et al. (2006) Photosynth Res 89:113-12; (11) Morales et al. (Book Chapter, 2006), (12) Rellán-Álvarez et al. (2006) Anal Biochem 356:254-264; (13) Rellán-Álvarez et al. (2006) Plant Soil 279:41-50; (14) Álvarez-Fernández et al. (2007) J Am Soc Mass Spectrom 18:37-47; (15) Garcés-Claver et al. (2007) J Agric Food Chem 55:6951-6957; (16) Ortega-Villasante et al. (2007) New Phytol 176:96-107; (17) Fernández et al. (2008) Sci Hortic 117:241-24; (18) Fernández et al. (2008) Plant Soil 331:161-17; (19) Jiménez et al. (2008) HortScience 43:304-309; (20) Rellán-Álvarez et al. (2008) Rapid Commun Mass Sp 22, 1553-1562; (21) Andaluz et al. (2009) Plant Physiol Biochem 47:1082-1088; (22) Fernández et al. (2009) J Hortic Sci Biotechnol 84:1-6; (23) Jiménez et al. (2008) Plant Soil 315:93-106; (24) López-Millán et al. (2009) J Plant Physiol 166:375-384; (25) López-Millán et al. (2009) Environ Exp Bot 65:376-385; (26) Orera et al. (2009) Rapid Commun Mass Sp 23:1694-1702; (27) Orera et al. (2009) J Hortic Sci Biotech 84:7-12; (28) Sagardoy et al. (2009) Plant Biol 11:339-350; (29) Rellán-Álvarez et al. (2010) Plant Cell Physiol 51:91-102; (30) Larbi et al. (2010) J Plant Physiol, in press; (31) Orera et al. (2010) Rapid Commun Mass Sp 24:109-119.

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6.2 PUBLIC AND PRIVATE GRANTED PROJECTS AND CONTRACTS OF THE RESEARCH GROUP Project and contract grants during the last 5 years (2005-2009) (national, regional or international)

Project or contract title

Relation with the current

proposal(1)

Principal researcher (PI) Funding (in €) Funding entity and Project Reference

Validity period or date of application of the proposal(2)

Selection of fruit rootstocks tolerant to iron chlorosis 2 Mª Angeles Moreno 53.057 MCYT PTR1995-0580-OP

May 02-May 05 G

Support for Consolidated Research Group DGA-A03 2 Javier Abadía ca.

12.000/year DGA (Aragón Autonomic Government)

2005-2010 G

Iron nutrition in fruit trees: strategies for the control of iron chlorosis

2 Anunciación Abadía 171.550 PN AGL2003-1999

Dec 03-Dec 06 G

Acquisition and transport of metals in plants 1 Javier Abadía 160.050 PN AGL2004-00194

Dec 04-Dec 07 G

Cyanobacteria as a source for plant fertilizers 2 Ana-Flor López-Millán 25.000 DGA (Aragón Autonom. Govmnt.) PM014-2004

Dec 04-Nov 06 G

7 Iron fertilizer testing Contracts (with different companies, confidential in part, see PI's c.v.). 3 Javier Abadía 43.000 2 Foreign Companies 05-09 G

Increasing fruit consumption through a trans-disciplinary approach delivering high quality produce from environmentally friendly, sustainable production methods (ISAFRUIT)

2 Ole Callesen

(Javier Abadía, RI) 80.811

EU-6th Framework Program Isafruit 016279 Food

Jan 06-Jun 10 G

Scientific basis for the optimization of foliar fertilization 2 Javier Abadía 25.000 DGA (Aragón Autonom. Govmnt.) PM003-2006

Dec 06-Nov 08 G

Basis for a rational use of micronutrient fertilizers in fruit species


Dec 06-Dec 09 G

Studies on metal homeostasis in plants 1 Javier Abadía 196.000 PN AGL2007-61948

Oct 07-Oct 10 G

Homeostasis and Transport of Iron – improving Plant Productivity

2 Javier Abadía 205.000

(+177.000 for a company)

PN EUI2008-03618

Mar 09-Apr 12 G

New approaches to study Fe availability, trafficking and localization in the context of fruit tree fertilization


Jan 10-Feb 13 G

(1) (1) 0, 1, 2 o 3 correspond to: 0, similar project; 1, very related project; 2, related project; 3 unrelated project (2) (2) G means “granted” project. (3) Rows with grey background are active projects in February 2010.

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7. TRAINING CAPACITY OF THE PROJECT AND THE GROUP Granting a 4 year-FPI graduate student fellowship associated to the project is essential for an appropriate development of the proposal.

Research training capacity of the project The graduate student associated to the project shall have good training opportunities, ranging from applied to basic research techniques, always within the field of Agricultural Sciences. Techniques available include very advanced ones, such as HPLC-TOFMS, 2-D electrophoresis and nanoHPLC-MS-MS (ion trap). Since 1992, the project PI has supervised 9 PhD and 5 MSc Theses, and three more PhD Thesis are in final elaboration stages and will be defended in 2010. Most graduates that have received research fellowships to undergo training in our group have been able to complete their degrees, and 2 out of 9 PhD Theses received Extraordinary University Doctoral Awards.

Research training capacity of the group This can be assessed from the positions hold currently by the trainees, from the number of PhD and MSc theses, from the number of people that have come to our group for training, as well as from the scientific publications and projects in which technicians and graduate students have participated (see PI's c.v.).

Current positions hold by Doctors and Technicians trained in our Group Eight out of 9 PhDs trained in our group are working in R+D, 2 as staff Spanish CSIC scientists1, 2 as staff scientists abroad (1 in the French CNRS2 and 1 in the Tunisian Ministry of Agriculture3), 3 in laboratories of Spanish private companies4 and 1 as Coordinator of Postgraduate Courses in an International Institute (CIHEAM-IAMZ)5. Five out of 6 technicians trained in our group are currently working as staff or contracted technicians in Agriculture/Research Units (2 in the Local DGA Government and 4 in the CSIC).

Ph.D. Theses 1. Changes mediated by environmental stresses in the photosynthetic apparatus of higher plants (U.

Zaragoza, F. Morales1, 1992). 2. Responses induced by Fe deficiency in the root system of Beta vulgaris L. (U. Zaragoza, S. Susín2,

1994). 3. Changes induced by Fe deficiency and LCM mutation in the organisation of photosynthetic apparatus

of Beta vulgaris L. (U. Zaragoza, R. Quílez4, 1994). 4. Environmental stress tolerance evaluation by chlorophyll fluorescence techniques, pigment analysis

and mineral content in agronomic plants (U. Lleida, R. Belkhodja5, 1998). Extraordinary Doctoral Award.

5. Study of the Fe-deficiency response mechanisms in plants (U. Zaragoza, E. González-Vallejo4, 1999). 6. Dissipation of the energy excess gathered by the photosynthetic apparatus in conditions of

environmental stress: oxygen related mechanisms (U. Zaragoza, D. Tobías, 1999). 7. Transport and acquisition of Fe in plants (U. Zaragoza, A.F. López-Millán1, 2000). Extraordinary

Doctoral Award. 8. Iron chlorosis: plant responses and correction methods (U. Lleida, A. Larbi3, 2003). 9. Studies on changes in the plant proteome induced by iron deficiency (U. Zaragoza, S. Andaluz4,

2005).

M.Sc. Theses 1) La fluorescence de la chlorophylle sur l'orge (Hordeum vulgare L.): une possible voie pour le criblage de variétés

tolérantes à la salinité et à la sécheresse (R Belkhodja, 1993). MSc CIHEAM-IAMZ. Mark 9.0/10.0. 2) Réponses radiculaires face à la deficience en fer chez differents genotypes de tomate et de betterave (M. Zouari,

1996). MSc CIHEAM-IAMZ. Mark 9.5/10.0. 3) Quelques changements de la composition chimique de la sève du xylème sous deficience en Fe chez la tomate, le

pêcher et l'amandier (J Chatti, 1997). MSc CIHEAM-IAMZ. Mark 8.5/10.0. 4) Effet de la chlorose férrique sur la réduction de fer par le mesophyle de feuilles de la betterave à sucre (Beta

vulgaris L.) et du pêcher (Prunus persica L. Batsch.). MSc CIHEAM-IAMZ (A. Larbi, 1999). Mark 9.0/10.0. 5) Necesidades de nutrientes minerales en melocotonero (Prunus persica L. Bastch). MSc CIHEAM-IAMZ (Hamdi El-

Jendoubi, 2009). Mark 7.8/10.

Postgraduate Training A total of 117 pre- and post-doctoral students from 14 different countries have carried out short training stages in our group.

Several group members usually participate in Ph.D. and M.Sc. courses organized by the U. Zaragoza, U.A. Madrid and the IAMZ-CIHEAM.

Convocatoria de ayudas de Proyectos de Investigación Fundamental no orientada

Sumario Ejecutivo de la Propuesta (la propuesta completa está en el documento en inglés)

TÍTULO DEL PROYECTO: Metalómica vegetal: una aproximación a la homeostasis de metales en plantas mediante espectrometría de masas integrada

INVESTIGADOR PRINCIPAL: Javier Abadía

Objetivos específicos del proyecto

Objetivo 1. Estudiar la especiación de metales en fluidos vegetales Este objetivo se centra en desentrañar las especies químicas de metales existentes en los fluidos de plantas, tanto de los que son nutrientes esenciales como de los que son tóxicos. Se buscarán complejos metálicos que participen en el transporte de metales, ya sea con ligandos naturales o bien sintéticos, utilizando isótopos estables. Se harán estudios de perfiles de metabolitos y se utilizará un enfoque integrado por espectrometría de masas, utilizando técnicas específicas tanto para moléculas ((nano) HPLC-MS(-MS), disponible en nuestro laboratorio) como para metales (HPLC-ICP-MS, disponible en los laboratorios colaboradores). Los materiales a estudiar incluyen savia de xilema, fluido apoplástico y savia de floema. Los metales que se estudiarán serán Fe (como nutriente) y Cd y Zn (como metales tóxicos). Las especies modelo que se utilizarán serán remolacha, tomate (incluyendo mutantes), Lupinus texensis (en colaboración con el laboratorio de Grusak en EE.UU.) y cebada (en colaboración con el laboratorio de Ma en Japón), así como la

RESUMEN:

Algunos metales como Zn y Fe son microelementos esenciales necesarios para los cultivos. Además, las plantas pueden adquirir metales tóxicos como Cd, si los mismos están presentes en los medios de crecimiento. Cuando los metales esenciales son escasos o cuando los metales se acumulan en exceso se presentan alteraciones en diferentes procesos biológicos fundamentales para el desarrollo de las plantas. Así, las plantas deben regular cuidadosamente la adquisición de los metales, su transporte y la distribución entre los diferentes órganos y compartimentos celulares, a fin de evitar su acumulación en exceso, al mismo tiempo manteniendo un suministro adecuado de aquellos que son esenciales. Se denomina homeostasis a la tendencia hacia un equilibrio relativamente estable entre mecanismos interdependientes sustentados por procesos fisiológicos. A pesar de los avances recientes, aún tenemos un conocimiento muy limitado de la homeostasis de metales en plantas. Los objetivos del proyecto son: i) estudiar la especiación de metales en fluidos vegetales, llevando a cabo la detección e identificación de complejos metálicos y avanzando hacia la cuantificación de los mismos; ii) desarrollar nuevas metodologías para estudiar algunos pasos cruciales en la homeostasis de metales por espectrometría de masas: ensayos de Fe(III)-reductasa, ensayos para medir flujos de metales y estudios sobre la acumulación en la raíz y excreción por la misma de metabolitos al medio de crecimiento; iii) estudiar el metabolismo de los desequilibrios causados por la deficiencia y toxicidad de metales mediante un enfoque combinado metabolómica/proteómica, investigando las principales diferencias encontradas mediante un análisis dirigido; y iv) estudiar la micro-localización de metales y componentes relacionados con la homeostasis de metales (proteínas y metabolitos) en plantas. Una mejor comprensión de los procesos implicados en la adquisición, transporte, quelación y almacenamiento de los metales en las plantas nos ayudarán a controlar no sólo problemas agrícolas, tales como las deficiencias y toxicidades de metales, sino también a mejorar el contenido de metales en los alimentos de origen vegetal, así como a promover la fitorremediación de suelos contaminados con metales. Esta propuesta es una continuación del proyecto actualmente en curso, AGL2007-61948, que acaba en octubre de 2010, y complementa los esfuerzos actuales del grupo en el estudio de la homeostasis de metales en plantas. La propuesta incluye cuatro científicos de plantilla del CSIC, uno más contratado y dos estudiantes de postgrado. Cuatro científicos extranjeros, dos de EE.UU. (O. Fiehn, U.C. Davis; M. Grusak, USDA-ARS Houston), uno de Hungría (F. Fodor, U. Budapest) y otro de Japón (J.F. Ma, U. Okayama), colaborarán a tiempo parcial en algunas tareas del proyecto.

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Objetivo 2. Desarrollar nuevas metodologías para estudiar pasos cruciales en la homeostasis de metales en plantas utilizando espectrometría de masas Este objetivo incluye estudios sobre tres pasos cruciales en la homeostasis de metales en plantas: las actividades de reducción de Fe(III), los flujos de metales en diversas fronteras de la planta, y la acumulación en la raíz y excreción por la misma de metabolitos al medio de crecimiento. Se diseñarán nuevos métodos de HPLC-MS para medir la actividad Fe(III)-reductasa en raíces, protoplastos y cloroplastos. Además, se utilizarán métodos basados en la utilización de isótopos estables y discriminación isotópica para medir flujos de metales en células (protoplastos y oocitos) y orgánulos vegetales (cloroplastos). El papel de la acumulación en la raíz y excreción por la misma de metabolitos a los medios de crecimiento se estudiará, en el caso de la deficiencia de Fe, mediante (nano)HPLC-MS(-MS) y GC-MS (la última técnica en colaboración con Fiehn en EE.UU.). Las especies que se utilizarán serán remolacha y pepino.

Objetivo 3. Estudiar el metabolismo de los desequilibrios metálicos en plantas con un enfoque combinado metabolómica/proteómica Este objetivo incluye estudios sobre los cambios en el proteoma y metaboloma de las plantas asociados con la deficiencia y toxicidad de metales en el medio de cultivo. Los estudios se centrarán en el metaboloma/proteoma de raíces, savia de xilema, fluido apoplástico y floema, utilizando distintos materiales vegetales, en los casos de deficiencia de Fe y toxicidad de Zn y Cd. Las técnicas utilizadas serán (nano)HPLC-MS(-MS) y GC-MS.

Objetivo 4. Estudiar la micro-localización de metales y otros componentes relacionados con la homeostasis de metales en plantas Este objetivo incluye estudios sobre la localización de metales (Fe y Cd) mediante LT-SEM-EDX, TEM-EDX y PIXE. Se utilizarán también fluoróforos para estudiar la localización de Zn y Cd en protoplastos aislados. Además, se estudiará la localización de proteínas y metabolitos (flavinas, etc.) relacionados con la homeostasis de metales, mediante el uso de anticuerpos y de técnicas de análisis de imagen (fluorescencia, etc.). Los materiales que se utilizarán en este objetivo serán remolacha y pepino.

Novedad y relevancia científica de los objetivos La novedad de la propuesta se basa en: i) el enfoque holístico para evaluar los cambios globales en proteínas y metabolitos relacionados con la homeostasis de metales en plantas; ii) el uso de tecnologías avanzadas, tales como (nano)HPLC-MS(-MS), GC-MS y técnicas de análisis de imagen; y iii) el abordaje de aspectos de homeostasis de metales en plantas muy poco explorados hasta la fecha.

La propuesta es la continuación de proyectos anteriores sobre homeostasis de metales en plantas desarrollados por nuestro grupo de investigación, y es coherente con el trabajo en proceso y las publicaciones recientes. La financiación de la propuesta facilitaría el uso de técnicas de análisis avanzadas en el área de Ciencias Agrarias. La propuesta amplía la aplicación de las técnicas ya utilizadas en el proyecto actual, que concluye a finales de 2010.

La importancia científica de la propuesta se apoya en los resultados y publicaciones anteriores del grupo de investigación autor de la propuesta, así como en las presentaciones invitadas en simposios especializados a cargo de los miembros del grupo. Su relevancia se ve reforzada por la importancia de la homeostasis de metales en plantas bajo puntos de vista tanto agronómicos como medioambientales, ya que tanto las deficiencias de metales como las toxicidades son muy comunes. El grupo está en estrecho contacto con otros grupos españoles y extranjeros que trabajan en este ámbito, ya sea con plantas modelo o con cultivos de campo, así como con especialistas en diversas técnicas de espectrometría de masas (véase Parte 2 del proyecto en inglés).

Fortalezas del equipo de investigación

El líder del grupo proponente ha sido IP en 7 proyectos anteriores del PN de I+D y coordinador de un proyecto Europeo, y su “índice h” es de 27 (27 trabajos SCI citados 27 o más veces). El grupo español proponente ha publicado 45 trabajos en revistas SCI en el período 2005-2009 (6 más en 2010), así como diversos artículos de divulgación, capítulos de libro y libros (ver página web del grupo. El grupo trabaja actualmente con financiación de la comunidad autónoma de Aragón, del PN y del 6º PM de la UE. Aproximadamente un 5% de su financiación procede de proyectos con empresas. De un total de 9 Doctores formados en el grupo, 8 han seguido trabajando en tareas de I+D en España, Francia y Túnez. Los cuatro investigadores extranjeros son líderes en sus campos y han publicado en las mejores revistas del área.

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