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Convocatoria de ayudas de Proyectos de Investigación Fundamental no orientada

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

PROJECT TITLE: New approaches to study Fe availability, trafficking and localization in the context of fruit tree fertilization PRINCIPAL INVESTIGATOR: Anunciación Abadía Bayona SUMMARY (brief and precise, outlining only the most relevant topics and the proposed objectives):

Iron-deficiency is a nutritional disorder that limits crop yields in many agricultural areas. Fruit tree growers need to fertilize orchards to improve fruit quality and yield. The aim of this proposal is to improve the current state of knowledge of Fe nutrition in fruit tree species -peach, pear, olive and citrus-, by studying Fe availability, trafficking and localization in the context of Fe-fertilization. Analytical technologies for fertilizers used in cases of Fe chlorosis will be developed, focusing on the determination of Fe-containing compounds and the characterization of interactions between formulation components. Three main objectives are proposed: i) to study Fe-trafficking in plants after Fe fertilization, by using innovative experimental approaches that involve the application of one or more Fe stable isotopes to the same plant. This includes studies on Fe uptake from different fertilizers, distribution of the Fe applied inside the plant, changes in nutrient balance in leaves, and changes in specific metabolites and proteins related to Fe nutrition in different plant materials, including apoplast and xylem sap; ii) to localize nutrients and compounds related to Fe nutrition after fertilization. This includes elemental micro-localization, using specific stains for Fe in leaves and roots and other techniques, and studies on the leaf gradients of Fe, Fe compounds and related metabolites and proteins; and iii) to characterize micronutrient fertilizer solutions used in Fe fertilization, especially in the case of foliar fertilization, by developing new analytical approaches. This project is a follow-up of the research on fruit tree mineral nutrition carried out in the past years by the proponent research group. The proposal is based on the results obtained in previous projects and opens some new research lines, taking advantage of the expertise of the new personnel in our group, the new analytical methodologies available, and the work in collaboration with other teams that complement our activities.

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TITULO DEL PROYECTO: Nuevos enfoques para el estudio de la disponibilidad, movimiento y localización del Fe en la fertilización de árboles frutales RESUMEN (breve y preciso, exponiendo sólo los aspectos más relevantes y los objetivos propuestos): La deficiencia de Fe es un desorden nutricional que limita la producción en muchas áreas agrícolas en todo el mundo. Debido a ello, los agricultores necesitan fertilizar las plantaciones frutales para mejorar tanto la calidad de los frutos como la producción. El propósito de la propuesta es aumentar el conocimiento actual de la nutrición férrica en frutales -melocotonero, peral, olivo y cítricos- estudiando para ello la localización y el movimiento del Fe tras la fertilización. También se propone el desarrollo de una tecnología analítica para la identificación de compuestos de Fe usados en casos de clorosis férrica y para la caracterización de interacciones entre componentes de diferentes formulaciones. Se proponen tres objetivos concretos: i) estudiar el movimiento del Fe en la planta tras la fertilización, utilizando nuevos enfoques que implican la aplicación de uno o más isótopos estables a la misma planta. Este objetivo incluye estudios de toma de Fe a partir de diferentes productos fertilizantes, de distribución del Fe aplicado dentro de la planta, cambios en el balance de nutrientes en hoja y cambios específicos en metabolitos y proteínas en diferentes materiales como apoplasto y savia de xilema; ii) localizar nutrientes y compuestos relacionados con la nutrición mineral tras la fertilización. Incluye la localización elemental y tinciones específicas de Fe en hojas y raíces, y estudios de concentración de Fe, compuestos de Fe y proteínas y otros metabolitos en gradientes en hoja; y iii) caracterizar las soluciones de fertilizantes de micronutrientes, especialmente en el caso de aspersión foliar, mediante nuevos desarrollos analíticos. Este proyecto es continuación de la investigación sobre nutrición mineral en frutales desarrollada por el grupo proponente en los últimos años. Por ello, se basa en los resultados obtenidos, pero también abre nuevas vías de investigación, debido a la especialización de los nuevos miembros del equipo, las nuevas tecnologías analíticas disponibles y el trabajo en colaboración con otros equipos que complementan nuestras actividades.

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2. INTRODUCTION (maximum 5 pages)

Aim

The aim of this proposal is to improve the current state of knowledge of mineral nutrition in fruit tree species, with the objective of decreasing costs associated with the correction of mineral deficiencies and minimizing the potential side effects deriving from such amendments. The project is a follow-up of previous ones and proposes three objectives, based on the findings obtained so far, which are also supported by the expertise of new personnel and the implementation of new technologies that have been recently introduced in our research group.

Background

Importance of iron chlorosis in agricultural areas

In spite of the relatively low Fe requirement of plants and the high abundance of Fe in soils, Fe-deficiency is a nutritional disorder that limits crop yields in many agricultural areas of the World, specially under high pH, calcareous soil conditions, such as those prevailing in many Mediterranean climate agricultural areas. The cause of Fe-deficiency is generally a combination of limited bioavailability of Fe in the soil and the use of susceptible plant genotypes that have insufficient activation of one or more Fe-deficiency defense mechanisms. Fruit tree crops such as peach, pear, kiwifruit, apricot, plum, cherry, olive trees, citrus and avocado are sensitive to shortages of Fe. Typical Fe chlorosis symptoms include leaf interveinal chlorosis starting from the shoot apex, development of leaf necrotic spots and shoot defoliation during the growing season (Rombolà and Tagliavini, 2006). Apart from leaf chlorophyll and carotenoid concentration decreases, reductions in leaf size, fresh and dry weight have been found associated with lime-induced chlorosis (Hutchinson, 1970; Anderson, 1984; Morales et al., 1998; Larbi et al., 2006). Also, Fe chlorosis has deleterious effects on fruit production, reducing the number of fruits per tree, fruit size and total yield, and affecting fruit quality parameters such as color, firmness and acidity (Álvarez-Fernández et al., 2003; Álvarez-Fernández et al., 2006). In addition to these effects in fruit production, Fe deficiency has an important economic impact on the fruit sector because of the need to add fertilizers to correct the deficiency, which is usually carried out with expensive products (the cost of treatment is 200 – 400 € ha–1 year–1; Rombolà and Tagliavini, 2006).

Physiological pathways

Iron deficiency alters markedly the morphology and physiology of plants (Briat, 2007) and maintaining Fe homeostasis in plants is crucial for an adequate plant metabolism activity. The long-distance allocation of Fe between organs and tissues, as well as its subcellular compartmentalization and remobilization, involve various chelation and oxidation/reduction steps, protein transporters and small metabolites and proteins that buffer and store this metal (Briat et al., 2007). The first step is the root uptake of Fe (either native soil Fe or Fe coming from fertilizers), which could be mediated or not by a reduction step (Strategies I and II, respectively). Then, Fe is transported via xylem to the leaf apoplast mainly as -so far uncharacterized- Fe citrate complexes (Tiffin 1966, Brown et al., 1971, White et al., 1981, Cataldo et al., 1988, Schmidt 1999, Lopez-Millán et al., 2000). The immobilization and accumulation of Fe in inactive forms somewhere in the leaf is suggested to be associated with the appearance of chlorosis (Abadía et al., 2002). Concerning the Fe supply to leaves by foliar fertilizers, there is controversial information about the mechanisms of penetration, which could occur via the cuticle, cuticular cracks or stomata. Changes in cuticle structure and composition due to Fe deficiency have been recently reported in fruit tree leaves (Fernández et al, 2008a).

Correction of iron chlorosis

Strategies to alleviate Fe chlorosis in fruit crops include: (i) the use of rootstocks tolerant to soil conditions that induce development of the disorder and with improved Fe uptake mechanisms; (ii) modifying soil characteristics; and/or (iii) treatment with Fe-containing substances via root, trunk, or canopy application(s) (Abadía et al., 2004b; Lucena, 2006). Iron-fertilization is the best and most commonly used technique to correct Fe-deficiency in established fruit tree orchards. There is scientific evidence that Fe fertilization increases fruit quality and yield in many crops (Álvarez-Fernández et al., 2006). The active ingredients can be either inorganic or organic Fe-containing compounds, which in many cases are still not well characterized. Trunk injection with liquid Fe fertilizers or solid branch implants of Fe compounds are not often used, in spite of the long-lasting efficacy that can be obtained with only one application every several years (Abadía et al., 2004b).

Root Fe fertilization is the most reliable and widely-used technique to control Fe deficiency, and commercial Fe(III)-EDDHA-based products are the most effective fertilizers to correct Fe chlorosis under severe soil conditions (Lucena, 2006). The most used synthetic Fe(III)-chelates for Fe supply to soils are Fe(III)-o,o-EDDHA, Fe(III)-o,p-EDDHA, Fe(III)-o,o-EDDHMA, Fe(III)-o,p-EDDHMA, or Fe(III)-EDDSHA. The efficiency of synthetic Fe(III)-chelates depends on the stability and persistence in the soil and nutrient solutions, as well as on the ability to supply Fe to plants (Lucena, 2006). However, these synthetic chemicals are expensive and may perform differently according to the particular Fe(III)-EDDHA formulation (Cerdán et al., 2007). On the other hand, the use of chelates has environmental

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consequences not well studied yet. Persistence in the media, phytotoxicity or toxicological effects are some aspects to be considered for future studies (Orera et al., 2009).

Foliar Fe fertilization with inorganic Fe compounds (e.g., FeSO4) and some organic Fe complexes, including natural (e.g., citrate) and synthetic ligands (such as Fe(III)-EDTA, Fe(III)-HEEDTA, and Fe(III)-DTPA) could alleviate Fe-deficiency, and these treatments could be a cheaper and more targeted strategy to correct plant Fe deficiency (Abadía et al., 2002; Álvarez-Fernández et al., 2004; Fernández et al., 2008a). However, the response to Fe sprays has been shown to vary according to many fertilizer-and plant-related, environmental and physico-chemical factors, and problems of reproducibility and interpretation of results from foliar and cuticular Fe-application studies have been described (Fernández and Ebert, 2005). Our current limited understanding of both i) the chemical characterization (determination of active ingredients) of micronutrient spray formulations and ii) the factors involved on the penetration, translocation, and bio-availability of leaf-applied Fe fertilizers makes it difficult to develop effective spray formulations for agricultural purposes. At present, foliar nutrition is considered to be only a valuable complement to the application of nutrients via the root system (Weinbaum, 1996).

Current state of knowledge (according to the proposed objectives)

Objective 1. To study Fe trafficking in the plant after Fe fertilization

Many of the studies on Fe trafficking after Fe fertilization have used so far non-labeled Fe fertilizers. However, important data could be obtained by using isotopically-labeled Fe fertilizers (isotopic tracers). Iron has four different stable isotopes: 54Fe, 56Fe, 57Fe and 58Fe. The utilization of Fe isotopic tracers permits to follow the traffic of the Fe applied inside the plant during Fe acquisition, translocation and distribution. For instance, it is possible to test if plants have preference for a specific Fe-compound or application way, by feeding a given plant with more than one different stable isotope Fe tracers. In the past, the application of Fe radioactive isotopes (55Fe or 59Fe) to label Fe fertilizers gave some insights on (see Álvarez-Fernández, 2006 for review): i) the absorption and breakdown of synthetic Fe(III)-chelates by different plant species; ii) the shoot translocation of the Fe supplied from Fe(III) complexes of siderophores, phytosiderophores and synthetic chelates; iii) the existence of two different higher plant strategies for the solubilization and uptake of Fe in response to Fe deficiency; iv) the first evidence for a specific uptake system for Fe(III)-phytosiderophores in roots of grasses, recently confirmed by the identification of a Fe(III)-MA protein transporter (Murata et al., 2006); and v) the comparison of the uptake rates of Fe from different external sources, including phytosiderophores, microbial siderophores and synthetic chelates. In the last two decades, the use of stable isotope tracers to study important issues in mineral nutrition (including Fe nutrition) has expanded rapidly, particularly in human (Turnlund, 2006) and animal nutrition studies (Suzuki et al., 2006), but the use in plant studies is scarce so far (Álvarez-Fernández et al., 2007). Stable isotopes have some advantages over radioisotopes: there is no exposure to radiation, and multiple stable isotopes of one element and isotopes of multiple elements can be fed to plants, simultaneously or sequentially, without interfering with one another. In plant Fe nutrition, the application of Fe stable isotope tracers has just started and it has been used in fertilizer studies to obtain more conclusive data on the relative efficiency of two ferric synthetic chelate fertilizers, Fe(III)-o,oEDDHA and Fe(III)-o,pEDDHA. For instance, 57Fe(III)-o,o-EDDHA and 57Fe(III)-o,p-EDDHA were used to compare the efficacy to supply Fe i) to cucumber plants grown in nutrient solution (Rodríguez-Castrillón et al., 2007) and ii) to peach trees grown in nutrient solution and in a calcareous soil (Rojas et al., 2008). The results obtained showed that Fe(III)-o,o-EDDHA was capable of providing sufficient Fe to plants in both nutrient solution and calcareous soils. However, Fe(III)-o,p-EDDHA was capable of providing sufficient Fe to plants in nutrient solution but not in calcareous soils, where this kind of fertilizers are normally used. Simultaneous multiple labeling and feeding of Fe stable isotope fertilizers to a plant followed by simultaneous detection has not been applied so far (Álvarez-Fernández, 2007). This experimental approach would make possible to evaluate biological data such as absorption, distribution and losses accurately and sensitively (Suzuki et al., 2006).

Changes in metabolites and proteins induced by Fe fertilization have not been studied extensively so far. The effects of Fe-fertilization in proteomic and metabolomic patterns are not known, although some studies to characterize the organic acid composition in xylem sap after branch solid implants have been carried out (Larbi et al., 2003b).

Concerning nutrient balance, foliar treatments with Fe-containing solutions induced significant changes in the concentration of several nutrients as compared to Fe-deficient peach leaves, even with the treatment did not cause increases in leaf chlorophyll concentrations (Fernández et al., 2008b). This indicates that some leaf mineral composition changes typical of chlorotic leaves would depend on leaf Fe concentration rather than on leaf Chl levels. It is still unknown if this is a general behavior in all species after a foliar treatment.

Objective 2. To localize nutrients and compounds related to mineral nutrition after fertilization

Most studies on plant leaves, including biochemical, photosynthetic and metabolomic studies, consider the leaf as a whole. However, leaves are composed of different cell layers, including epidermal, and mesophyll palisade and spongy cells. During the last years, gradients across leaves have been studied, and it has been found that carbon fixation gradients differ from light gradients, and that they are

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correlated with Rubisco levels (Nishio et al., 1993). Differences in leaf epidermal and internal structure in Fe-sufficient and Fe-deficient Mexican lime, pear and peach have been reported (Maldonado-Torres et al., 2006; Fernández et al., 2008a). There is no information about changes in proteins or metabolites in gradient leaves after Fe-fertilization, although we are currently carrying out preliminary research work to characterize of gradients in control and deficient nutritional status.

In order to improve the knowledge about the tissue-specific Fe location associated to chlorosis phenomenon, two-dimensional synchrotron radiation-induced X-ray fluorescence (µ-SRXF) imaging of Fe distribution has been carried out in Fe-sufficient and Fe-deficient peach leaves (Jiménez et al., 2009). In this study we showed that in chlorotic leaves Fe is preferentially located in midribs and veins. These results are in accordance to the idea of Fe immobilization in leaf as a possible cause of Fe-deficiency chlorosis. However, µ-SRXF data corresponded to the full depth (thickness) of the leaf and no information about the distribution of Fe across transversal sections of Fe-deficient leaves is available so far. The metal localization among different leaf tissues (epidermis, mesophyll, xylem, etc) is essential to understand plant processes involved in metal homeostasis, and will provide new insights in the study of plant mineral nutrition. In the last years, scanning electron microscopy and energy-dispersive x-ray microanalysis (SEM/EDX) has been successfully used to obtain transversal foliar metal distribution in some hyper-accumulator species (Küpper et al., 2001; Frey et al., 2000; Fernando et al., 2008). Most of these studies have indicated that the epidermis is the principal site of heavy metal accumulation. Differential accumulation of nutrients between epidermis and mesophyll cell layers has been also reported in monocot species (Karley et al., 2000). Other techniques, such as particle induced x-ray emission (PIXE) (Vogel-Mikus et al., 2008), laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) (Punshon et al., 2004), and 3D-nuclear magnetic resonance imaging (MRI) (Lambert et al., 2006) have been used in the last years to determine the localization of elements in plant tissue.

Objective 3. To characterize micronutrient fertilizer solutions used in Fe fertilization

To characterize micronutrient-containing solutions used in Fe fertilization, Fe and other micronutrients such as Cu, Zn and Mn are usually added to fertilizers in chelate forms. Micronutrient fertilizers are frequently dissolved and/or mixed with other compounds (i.e. co-adjuvants) to prepare fertilizer solutions, and interactions between compounds can affect considerably the stability of micronutrient fertilizers and therefore its expected efficacy. Metal chelate stability is very dependent on pH and the presence of other anions and cations in the solution (Lucena, 2006). Decomposition - recomposition reactions of Fe(III)-o,oEDDHA and Fe(III)-o,oEDDHMA was found to occur, both under acidic conditions and/or in presence of Cu, Ni, Zn and P, in fertilizer tanks of concentrated solutions used in fertirrigation (Juárez et al., 2001; Bermúdez et al., 2002). Ions derived from the synthesis of Fe(III)-EDTA have been shown to cause ionisation of non-ionic surfactants, and this can seriously affect their performance as adjuvants in foliar sprays (Fernández et al., 2009). These studies have been limited by the availability of analytical methods permitting the determination of micronutrient-chelates and/or surfactants both in the original product (fertilizer) and in fertilizer solutions. In Europe there is an increasing regulatory interest in the chelated micronutrient contents of fertilizers (specially those containing synthetic chelates), but at present the Official Methods for quantifying the authorized synthetic chelates (4 different methods to determine 10 compounds) do not allow to distinguish between the free and metal-bound forms of the chelating agents. The lack of methods for the simultaneous determination of as many as possible micronutrient-chelates is due to the low specificity of the detection technique (UV/VIS) generally used, that makes it mandatory to have very good chromatographic separations, specially for compounds which have a similar molecular structure. The utilization of high resolution mass spectrometry (MS) techniques, along with electrospray ionization (ESI), allows for a highly selective identification of metal-complexes, based on the determinations of the exact mass and isotopic signatures (the ratios of isotopes of a given metal, which are also preserved in the metal complexes). These techniques have been used to determine different metal-EDTA chelates in a single analysis, either in standard solutions by direct injection (Baron and Hering, 1998) or in soil solution and plant xylem samples by HPLC-ESI/MS (Collins et al., 2001). Also, all synthetic Fe(III)-chelates authorized in Europe to be used as fertilizers have been successfully determined in agricultural matrices by HPLC-ESI/MS (Álvarez-Fernández et al., 2007). External calibrations were always used in these studies to quantify Fe-chelates. However, the high resolution capability of some current MS instruments (i.e. time of flight devices) could yield accurate determination of isotopic signatures and provide, in combination with isotope dilution analysis methods (see Rodríguez-González et al., 2005 for review), a most precise, rapid and less standard-depending approach to quantify metal-complexes in fertilizers. This approach has not been pursued yet.

Groups dealing with the same or similar aspects to the ones presented in this project

Spanish groups. We are in contact with groups dealing with similar topics: Fe uptake, Drs. Alcántara and Romera (Universidad de Córdoba); Chemical equilibria in hydroponics, fertilizer and nutrient deficiency correction investigations, Drs. Lucena (Universidad Autónoma de Madrid), García-Mina (Roullier Group-Universidad de Navarra, and del Campillo (Universidad de Córdoba).

International groups. Since 2006 we take part in the EU project ISAFRUIT concerning the nutrition of fruit species, in which we collaborate with 61 research groups and companies. In 2009 an ERANET KBBA Project will begin with Drs. Philippar (University of München), Briat (University of Montpellier), von

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Wirén (University of Hohenheim) and García-Mina (Roullier Group), to study regulatory processes linking Fe homeostasis in the chloroplast and the cytosol of plant cells with mechanisms of Fe acquisition and transport. We are also in touch with many of the participants in International Fe Nutrition and Interactions in Plants Symposia Series, which are held every 2 years. One of the researchers of our team belongs to the Steering Committee of these Symposia. We are also in contact and exchange information with participants in plant mineral nutrition (ICPN) and tree nutrition (ISHS) Congresses. In relation to nutrient demands and deficiency correction, we have collaborations with the University of Bolzano (Dr. Tagliavini), the Institut de l’Olivier-Tunis (Drs. Msallem and Larbi) and the University of Faro (Dr. Pestana). Also, in 2006 a member of the team (HE-J) worked for one month in the University of Bologna for the development of the nutrient demand model, and during 2009 this same graduate student will carry out a 2-month stage with Dr. Neilsen in the Pacific Agri-Food Research Centre, British Columbia (Canada) to study some aspects of mineral nutrition in trees.

References (This list contains all papers quoted in the proposal; in black, papers of the proponent research group, in grey those from other groups)

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Álvarez-Fernández A. et al., 2003. Effects of Fe deficiency-chlorosis on yield and fruit quality in peach (Prunus persica L. Batsch). J Agric Food Chem, 51:5738.

Álvarez-Fernández A. et al., 2004. Foliar fertilization to control iron chlorosis in pear (Pyrus communis L.) trees. Plant Soil, 263:5.

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Álvarez-Fernández A. et al., 2006. Iron deficiency, fruit yield and fruit quality. In: Iron Nutrition in Plants and Rhizospheric Microorganisms. (Barton, L. L. andAbadía, J., Eds.). Springer, Dordrecht,The Netherlands. 85–101.

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Hutchinson T.C. 1970. Lime chlorosis as a factor in seedling establishment on calcareous soils. II. The development of leaf water deficits in plants showing lime-chlorosis. New Phytol, 69:143.

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Pestana M. et al., 2004. Floral analysis as a tool to diagnose iron chlorosis in orange trees. Plant Soil, 259:287. Punshon T. et al., 2004. Mass loading of nickel and uranium on plant surfaces: application of laser ablation-ICP-MS. J Environ Monit,

6:153.

Rellán-Álvarez R. et al., 2008. Metal-nicotianamine complexes formation as affected by pH, ligand exchange with citrate and metal exchange. A study by electrospray-time of flight mass spectrometry. Rapid Commun Mass Spectrom, 22:1553.

Rodríguez-Castrillón J. A. et al., 2007. Isotope pattern deconvolution as a tool to study iron metabolism in plants. Anal Bianal Chem, 390:579.

Rodríguez-González P. et al., 2005. Isotope dilution analysis for elemental speciation: A tutorial review. Spectrochimica Acta Part B, 60:151.

Rojas C. L. et al., 2008. Efficacy of Fe(o,o-EDDHA) and Fe(o,p-EDDHA) isomers in supplying Fe to Strategy I plants differs in nutrient solution and calcareous soil. J Agric Food Chem, 56:10774.

Rombolá A and Tagliavini M. 2006. Iron nutrition of fruit tree crops. In: Iron Nutrition in Plants and Rhizospheric Microorganisms. (Barton, L. L. and Abadía, J., Eds.). Springer, Dordrecht,The Netherlands. 61–83.

Rombolà A. et al., 2005. Iron deficiency-induced changes in carbon fixation and leaf elemental composition of sugar beet (Beta vulgaris) plants. Plant Soil, 271:39.

Schmidt W. 1999. Review: Mechanisms and regulation of reduction-based iron uptake in plants. New Phytol, 141:1.

Suzuki K.T. et al., 2006. Absolute labeling and simultaneous speciation in tracer experiments with multiple isotopes. J Health Sci, 52:590.

Tiffin L.O. 1966. Iron translocation. II. Citrate:iron ratios in plant stem exudates. Plant Physiol, 41:515.

Turnlund J.R. 2006. Mineral bioavailability and metabolism determined by using stable isotope tracers. J Anim Sci, 84(E. Suppl.):E73-E78.

Vogel-Mikus K. 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.

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White M.C. et al. 1981. Metal complexation in xylem fluid. II. Theoretical equilibrium model and computational computer program. Plant Physiol, 67:301.

Zaharieva T. et al., 2004. Dynamics of metabolic responses in sugar beet roots to iron deficiency. Plant Sci, 166:1045.

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3. OBJETIVES (maximum 2 pages)

3.1 Describe the reasons to present this proposal and the initial hypothesis which support its

objectives (maximum 20 lanes)

The project proposes to study the Fe availability, trafficking and localization in the context of Fe-fertilization in fruit trees (peach, pear, olive and citrus). Analytical technologies for fertilizers used in cases of Fe chlorosis will be developed, focusing on the determination of Fe-containing compounds and the characterization of interactions between formulation components. This project is a follow-up of the research on fruit tree mineral nutrition developed in the past years by the proponent research group. The proposal is based on the results obtained in previous projects and opens some new research lines, taking advantage of the expertise of the new personnel in our group, the new analytical methodologies available, and the work in collaboration with other teams that complement our activities. The project is aimed to tackle some major needs in this field: a) to increase the knowledge on the strategies to optimize Fe-fertilization, both in terms of decreasing fertilizer costs and in terms of avoiding nutritional imbalances associated to fertilization; b) to study Fe trafficking after Fe resupply to achieve an appropriate fertilizer use; and c) to minimize the environmental problems caused by an excess of micronutrient fertilizers, including the possible deleterious effects on the quality of agricultural products as well as the possible contamination of soil and waters.

3.2. Indicate the background and previous results of your group or the results of other groups

that support the initial hypothesis

This proposal is based in the results obtained in previous projects of the group. These previous projects have outlined the need to develop new research lines aimed to progress in the knowledge of fruit tree nutrition needs, which are still not fully known. Furthermore, new objectives have been proposed taking advantage of the expertise of the new personnel and the new analytical technologies available in our laboratory. Also, this project aims a goal that has not been tackled so far, the study of mineral nutrition by using imaging techniques, and in particular the direct elemental analysis in tissue sections. The background information on all these issues is included in the Introduction Section 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 plants and in fruit tree species. The proponent group is a reference group in this issue, both at national and international levels. Because of this reason, many of the previous results of the group have been already quoted in the current state of knowledge (see Introduction Section). Many of the results obtained so far have been already published (see References in Part 2) or are in preparation for publication (see Part 6).

The proponent research group has already carried out work in the specific objectives of the present proposal as follows:

On micronutrient fertilization. We have already developed studies on i) the effects of Fe supply on the changes in photosynthesis, chlorophyll fluorescence, photosynthetic pigments, xylem sap and apoplastic fluid composition in sugar beet; ii) the effects of different Fe fertilization techniques (foliar sprays, solid implants, soil chelates) on the changes in photosynthesis, chlorophyll fluorescence, and photosynthetic pigments in peach and pear trees; iii) the use of foliar sprays for Fe chlorosis correction (including the differences in cuticle surfaces induced by Fe-deficiency and the effect of the formulations in the effectiveness of the Fe-compounds); and iv) on analytical HPLC-MS methodologies for the separation of metal chelates, both of natural and synthetic origin.

Concerning the elemental microlocalization, i) we have showed the preferential localization of Fe in midribs and veins in chlorotic leaves and ii) we have preliminary results on the differences in gradient concentrations of elements and other compounds in control and chlorotic fruit tree leaves (publication in preparation). Some authors have described gradients within the leaf (i.e. Evans and Vogelman, 2006), and in the case of hyper-accumulator species the transversal foliar distribution of metals has been documented (i.e. Fernando et al. 2008). So far, there is no information in these issues in leaves of fruit tree species.

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3.3. Describe briefly the objectives of the project.

1. To study Fe-trafficking in the plant after Fe fertilization. This includes studies on Fe uptake from different fertilizers, distribution of the Fe applied once inside the plant, changes in nutrient balance in leaves, and changes in specific metabolites and proteins related to Fe nutrition in different plant materials, including apoplast and xylem sap. This objective will be based on the use of stable Fe isotopes.

2. To localize nutrients and compounds related to mineral nutrition after Fe fertilization. This includes elemental micro-localization, using specific stains for Fe in leaves and roots and other techniques, and studies on the leaf gradients of Fe, Fe compounds and related metabolites and proteins.

3. To characterize micronutrient fertilizer solutions used in Fe fertilization. This objective is aimed to the development of analytical technologies to i) identify and quantify fertilizers used to correct micronutrient deficiencies and ii) study interactions between surfactants and Fe-compounds. In this objective, mass spectrometry (time-of-flight) and isotope dilution analysis techniques will be used.

Novelty and scientific relevance of the objectives

The novelty of the proposal is based on: i) the use of advanced technologies available in our laboratory (HPLC-MS(TOF) and nHPLC-MS-MS) and other collaborating laboratories (GC-MS and SEM-EDX with High Pressure Freezing) to study metabolites, proteins and elemental micro-localization after Fe fertilization; ii) the use of stable Fe isotopes and ICP-MS analysis to follow the movement of Fe once it is taken up by the plant; and iii) the aim to tackle the so far little explored gradient changes within leaves and roots after Fe-fertilization, in an attempt to provide new insights on plant mineral nutrition.

The objectives proposed are the logical follow-up of the Fe-fertilization research line in our group and are coherent with the previous work. In addition, the proposal is moving in with some goals that have not been addressed in previous projects, such as the study of mineral nutrition through elemental characterization profiling in leaves and roots.

The scientific relevance of the proposal is supported by the previous results and publications of the proponent research group, as well as the related invited talks in specialized Symposia delivered by members of the group. The relevance is strengthened by the repercussion of these issues, since Fe deficiency chlorosis affects a large percentage of the existing fruit tree orchards in the Mediterranean area and represents one of the most significant constraints for fruit production. Furthermore, the group is in close contact with other groups working in this area, both within Spain and abroad (see Part 2).

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4. METHODOLOGY AND WORKING PLAN The proposal was written in January 2009 and if the application is successful the project would start in approximately December 2009. This time lag could imply that changes and/or improvements concerning the methods employed and the staff available (specially because of the currently under evaluation pre- or post-doctoral JAE-CSIC calls) may take place throughout this period. Only the staff already confirmed to participate in the project has been taken into account in the proposed chronogram (AA, AA-F, SV, HE-J and a Technician to be contracted-Tc).

Staff not included in the research group but participating in the project

* As it has been the case in previous projects, we count on the collaboration of the remaining personnel from the Plant Stress Physiology group (EEAD), which is not participating on the proposal in administrative terms (Drs. J. Abadía, F. Morales and A.F. López-Millán). It must be noted that although studies may be allocated in different research projects, the investigations developed by the whole group are always complementary. * This project seeks to be included in the training program, and therefore we request a research pre-doctoral fellowship. If this is approved, the fellow would be included as project participant.

Foreign personnel participating in the project

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

-Dr. Maribela Pestana (MP) from CDCTPV, University of Algarve (UA), Faro, Portugal. She is working on citrus mineral nutrition. Her main research topic focuses on studies on Fe chlorosis (chelate treatments and influence on fruit quality). The IP of this proposal (AA) is advisor in the last project financed by Fundaçao para a Ciência e a Tecnologia FCT (Portugal) led by MP. The relationship with this group is stable from 1993 (we worked together in an EU Project).

-Dr. Ajmi Larbi (AL) from The Institut de l’Olivier (IO), Tunisia. He is working on mineral nutrition in olive trees. He is interested in topics such as tree fertilization (foliar or soil applied). He obtained his PhD Thesis in our group and has continued to work –he already has a staff permanent position- with us through Tunisia-Spain bilateral projects.

Both of them provide the possibility of having very different plant material from those used in earlier projects: olive and citrus. These fruit trees have perennial leaves and different leaf cuticles than those studied by our group, and the behavior could be different to those of species studied so far. Another important advantage of both collaborating teams is that they have tightly controlled orchards, so there are no external factors (apart from the environmental conditions) that can alter the results.

Methodology (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 analyses will be performed in external laboratories or services, following our normal collaboration policy (See Part 2).

Plant material.

The fruit tree species used in the different studies will be peach (Prunus persica L. Batsch) and pear (Pyrus communis L.) grown on calcareous soils, growing either in controlled conditions in growth chambers, in large pots or in field orchards located on the Ebro River basin in Spain. Also, in this proposal we will use olive trees (Olea europaea) in collaboration with Dr. Larbi in Tunis and Citrus trees in collaboration with Dr. Pestana in Algarve, Portugal. In both cases, only materials that can be transported (dried, freeze-dried or dry-ice conserved material) would be analysed. In some objectives, the use of model plants, such as sugar beet (Beta vulgaris L.) or tomato (Lycopersicon esculentum L.) will be necessary. As a common feature to all tasks, studies developed in growth chambers will use hydroponic cultures in growth chambers and under controlled environmental conditions as follows: light intensity of 400 µE m-2 s-1, 16 h light / 8 h dark photoperiod, 80% RH and 22°C (Zouari et al., 2001). Plants will be grown in Hoagland type solutions as described in previous works.

Methodology applied in all objectives.

Mineral analysis of plant material (leaves and roots, and flowers, wood and fruits if necessary) will be carried out as described by Igartua et al. (2000) and Alvarez-Fernández et al. (2003). Mineral elements (N, P, K, Ca, Mg, Fe, Mn, Cu and Zn) will be determined in the Analysis Service of the Plant Nutrition Department of the EEAD-CSIC (Dumas, FAAS, FES). Except for the quantification of small Fe concentrations, to be performed by AAS with graphite furnace or ICP-MS in the Analysis Service, PCB-Barcelona, all required apparatus and techniques are available in the EEAD-CSIC.

Leaf chlorophyll content will be estimated with SPAD-Minolta devices. The SPAD meters will be calibrated against spectrophotometric methods, as described in Abadía and Abadía (1993).

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Concerning Fe-treatments, in some tasks both foliar and soil applied Fe-products will be used (Tasks 1, 2, 3, 5 and 6). In task 4 only foliar treatments will be carried out. Iron products that will be used in foliar sprays include Fe(II)-sulfate, Fe(III)-DTPA and Fe(III)-EDTA. Surfactants such as Break-Thru and others will be used for foliar sprays. For soil Fe treatments, products will include Fe(III)-EDDHA, Fe(III)-EDDHMA and some natural Fe compounds. Doses of compounds to be used will depend on the specific experiments: in field experiments the doses recommended by manufacturers will be used, and when using stable isotopes we will try to minimize the amounts and treatment time because of the high cost of products.

Objective 1. To study Fe-trafficking in the plant after Fe fertilization

Tasks 1. Iron-uptake from different fertilizers, and Task 2. Distribution of Fe applied inside the plant

Experiments with one or two stable isotope-labeled Fe-fertilizers will be carried out by treating tomato, sugar beet and peach plants grown in hydroponics. Experiments involving the use of multiple stable isotope tracers will include the simultaneous treatment of each plant either by foliar application of two different Fe-compounds, by nutrient solution application of two different Fe-compounds or by using one Fe-compound each in foliar and nutrient solution applications. Iron stable isotopes in different plant materials (tissues and fluids), nutrient solutions and/or foliar sprays will be quantified by inductively coupled plasma mass spectrometry (ICP-MS) and isotope dilution analysis, using the procedure developed by Rodríguez-Castrillón et al. (2007). The ICP-MS analysis will be carried out in collaboration with the group of José I. García Alonso (Department of Physical and Analytical Chemistry, University of Oviedo). When treatments involve the use of synthetic chelates, the analysis of i) chelating agents in plant tissues and ii) Fe-chelates (natural and synthetic) in plant fluids and nutrient and foliar spray solutions will be carried out by ESI/MS(TOF) mass spectrometry (Bruker MicrOTOF) in the EEAD-CSIC. ESI/MS(TOF) analysis will be done either by direct injection or by coupling to a liquid chromatograph (Waters Alliance 2795 HPLC) also in the EEAD-CSIC. Materials enriched in the four Fe stable isotopes are available from the Oak Ridge National Laboratory (ORNL).

Task 3. Xylem sap and apoplast fluid characterization after Fe-fertilization

Xylem sap and apoplastic fluid isolation will be carried out as described by Larbi (2003). Xylem will be obtained from tree branches with a Model 600 Pressure Chamber (PMS Instruments).

For protein characterization, fractions will be separated by two-dimensional (2-D SDS-PAGE) electrophoresis (Protean IEF system, Biorad) according to González-Vallejo (1999, PhD Thesis), Andaluz (2005, PhD Thesis), and Andaluz et al. (2006). Following electrophoretic separation (2-D SDS-PAGE), gels will be stained with silver, Coomasie Blue or Sypro, and individual protein spots will be excised from gels and collected with a spot cutter (ExQuest, BioRad). In previous studies it has been shown (Andaluz, 2005) that when using plant materials little characterized such as root tips and xylem of fruit trees a large number of spots separated by 2-D electrophoresis techniques could not be identified by MALDI-TOF mass fingerprinting, making necessary de novo sequencing. Separated proteins will be digested enzymatically, and peptides will be separated in micro-columns with a nHPLC-MS-MS system in the EEAD-CSIC. A nHPLC (1200 Agilent) device coupled to a MS-MS ion trap (Bruker HCT Ultra) will be used. The exact molecular mass can also be obtained by using ESI-MS(TOF) (Bruker MicrOTOF) mass spectrometry.

For metabolite characterization, analysis will be carried out by using gas chromatography coupled to mass spectrometry (GC-MS), following the recommendations of the Metabolomics Standards Initiative (Fiehn et al., 2008). Metabolomic analyses will be done in collaboration with the group of Dr. Fiehn (University of California-Davis). Targeted analysis of nutrients and metabolites (i.e. organic acids) will also be performed by ICP-MS and LC-ESI-MS(TOF), respectively.

Task 4. Nutrient balance in leaves

Some foliar treatments will be applied in peach and other species to test changes in nutrient balance (Fernández et al., 2008b). The effects of exogenous Fe (applied as Fe(II) sulfate or Fe(III) DTPA with a surfactant) will be estimated after treating a leaf zone and measuring chlorophyll on an area basis and nutrients on a dry matter basis, in treated and untreated leaf parts.

Objective 2. To localize nutrients and compounds related to mineral nutrition after fertilization

Task 5. Micro-localization of mineral elements

This study will be carried out by scanning electron microscopy with an energy dispersive X-ray microprobe (SEM-EDX). Spot analysis will be carried out in the nearby ICB-CSIC Institute (Hitachi S3400N, with a Röntek EDX detector for elemental analysis) or in the CCMA-CSIC Institute in Madrid. The image resolution of the SEM is 10-15 nm (in low vacuum) and the elemental analysis resolution is approximately 4 µm. For mapping analysis, a High Pressure Freezing device is required to preserve

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the anatomy of the plant tissue and we will use the service of the PCB (Univ. Barcelona, Spain). Iron staining techniques will be performed according to established methods for plant material described by Pearse (1972) and Green and Rogers (2004). For cross-sections, after staining with K ferrocyanide solution, plant tissue will be embedded in 5% (w/v) low-melt agarose. Approximately 100 µm-thick sections will be cut with a vibratome for observation by light microscopy.

Tasks 6. Leaf gradient studies

Collection of sections: Fine paradermal sections (around 40 µm thick) will be obtained from leaves and also from roots with a cryo-sectioning method (Evans and Vogelmann, 2003) in a Leica cryotome. The technique is already set up in our lab.

Section analysis: Gradients across leaf and root sections will be analyzed using target analysis, metabolite/xenomic profiling, metabolomics and proteomics techniques described above.

Objective 3. To characterize micronutrient fertilizer solutions used in Fe fertilization

Task 7. Development of analytical technologies for the identification and quantification of fertilizers used to correct micronutrient deficiencies.

Standard solutions of stable isotope-labeled and non-labeled micronutrients (mainly Fe, Cu and Zn) complexes with different ligands (mainly the CE-authorized synthetic chelates and aminoacids) will be obtained as described by Lucena et al. (1996). Combined and non-combined standard solutions of the non-labeled compounds, commercial fertilizers (mainly those containing different micronutrients) and foliar spray solutions will be analyzed by ESI-MS(TOF) (Bruker MicrOTOF, EEAD-CSCI) and ESI-MS/MS(Q-TOF) (Bruker MicroTof-Q, ICMA-CSIC). The ESI-MS and ESI-MS/MS analysis will be carried out by direct injection and also by coupling to liquid chromatography, adding just prior the ESI process a known spike of a given stable isotope-labeled micronutrient complex solution. The spike will be directly introduced into the ESI chamber with a dual spray, in order to avoid possible stable isotope exchange reactions between the labeled and unlabeled molecules. Isotopic dilution analysis will be carried out to quantify each micronutrient metal-complex in every sample (including standards, fertilizers and foliar spray solutions). To validate the viability of the utilization of ESI/MS(TOF) analysis together with isotopic dilution analysis in the identification and quantification of metal-complexes, all samples will also be analyzed i) by ICP-MS (Agilent Technologies Q-ICP-MS 7500ce, University of Oviedo), using isotopic dilution analysis for quantification and liquid chromatography to obtain molecular specificity and ii) by ESI-MS(TOF) or LC-ESI-MS(TOF), using external calibration for quantification.

Task 8. Study of interactions between surfactants and Fe-products

The products (Fe-containing compounds and surfactants) to be used are those described in Fernández et al. (2008b). Interactions will be studied by ESI-MS(TOF) (Bruker MicrOTOF, EEAD-CSIC) as described in Fernández et al. (2009) and also by ESI-MS/MS(Q-TOF) (Bruker MicroTof-Q, ICMA-CSIC) to obtain fragmentation spectra.

Knowledge dissemination

Task 9 All members participating on the proposal will be engaged in this task. As specified in Section 6, this task will be developed by means of publications in specialized and dissemination journals and presentations in Congresses. Moreover, research results of the group will be available in the group webpage (http://www.eead.csic.es/stressphysiology).

Collaborations within the project

We have already collaborations for specific subjects within the project with: -ICB-CSIC (Dr. Andrés, Zaragoza, Spain) for SEM-EDX. -Metabolomics lab in the Genome Center (Dr. Fiehn), UC Davis, USA. -Research Institute for Bioresources (Dr.Ma), Okayama University, Japan, for microscopy techniques. -ICMA-CSIC (Dr. Orduna, Zaragoza, Spain) for ESI-MS/MS (Q-TOF). -Oviedo University (Dr. García Alonso) for stable isotopes studies and ICP-MS analysis.

In Objective 2 some external services will be involved: -CCMA-CSIC (Madrid-Spain) for SEM-EDX (freeze fracture). -PCB (Barcelona, Spain) for high pressure freezing and freeze-substitution.

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Working Plan

The contract of SV will end in March 2011, thereby his participation in the project beyond this date has not been considered. From this date, in case SV is no longer in our group, his tasks will be under the responsibility of AA. However, it is expected that SV could obtain financial support in the form of a postdoctoral research contract well before the mentioned deadline. Hamdi El-Jendoubi (HE-J), holding a FPI grant corresponding to the previous AGL project, will finish his grant in July 2011. The work of HE-J is focused on mineral nutrition studies, with an active participation in Tasks 2, 4 and 8.

Since fruit species will be investigated under field conditions, there are tasks that must be adjusted to fixed dates. Treatment applications and sampling must be done at specific dates. In contrast, growth chamber trials do not depend upon the season. The proposed chronogram has been devised with the aim that the beginning of the project is coincident with the commencement of the year 2010.

First year (2010)

Tasks 1 & 2 (Obj. 1) (AA-F, AA, HE-J, Tc) Tissues and fluids from tomato and sugar beet plants, treated with stable isotope labeled Fe-fertilizers, will be obtained. Plant materials will be analyzed for Fe stable isotopes by ICP-MS and for chelating agents and Fe-complexes by LC-ESI-MS.

Task 3 (Obj. 1) (AA, AA-F, HE-J, Tc) The characterization of xylem sap and apoplast fluid in control and deficient material will be completed. The first attempts to characterize both tissues after fertilization will be carried out.

Task 4 (Obj. 1) (AA, HE-J, MP, AL, Tc) Iron treatments will be carried out in model plants growing in growth chambers. Some experiments will be carried out in olive (Tunis) and citrus (Portugal) and sampled material will be analyzed.

Tasks 5 (Obj. 2) (SV, AA, MP, AL, Tc) Studies with plants grown in hydroponics and first year of sampling in field experiments after Fe-treatment.

Task 6 (Obj. 2) (SV, AA, Tc) Studies with plants grown in hydroponics and first year of sampling in field experiments after Fe-treatment.

Task 7 (Obj. 3) (AA-F, AA) Development of ESI-MS(TOF) and isotopic dilution methodologies to determine micronutrient-complexes used as fertilizers in standard solutions. Validation of the method using i) ICP-MS and isotopic dilution and ii) ESI-MS(TOF) and external calibrations.

Task 8 (Obj. 3) (AA-F, HE-J) Analysis by ESI-MS(TOF) and ESI-MS/MS(Q-TOF) of surfactants and Fe-fertilizers. The optimum conditions for surfactant analysis will be studied.

Task 9 (Dissemination) The task will be carried out throughout the complete project duration, as described in section 5.

Second year (2011)

Task 1 & 2 (Obj. 1) (AA-F, AA, HE-J, Tc) Tissues and fluids from peach trees treated with stable isotope labeled Fe-fertilizers will be obtained. Analysis of Fe stable isotopes, and chelating agents and Fe-complexes will be carried out by ICP-MS and LC-ESI-MS, respectively.

Task 3 (Obj. 1) (AA, AA-F, HE-J, MP, AL, Tc) Iron treatments and sampling of xylem sap and apoplast fluid will be carried out. Proteomic and metabolic analysis will be performed. Some experiments will be carried out in Portugal and Tunis to obtain xylem sap.

Task 4 (Obj. 1) (AA, HE-J, MP, AL, Tc) Parallel experiments in Spain (peach and pear), Tunis (olive) and Portugal (citrus) will be done to study mineral balance after Fe-treatments. Experiments with Fe localization in roots.

Tasks 5 (Obj. 2) (SV, AA, MP, AL, Tc) Second year of sampling in field experiments after Fe-treatments.

Task 6 (Obj. 2) (SV, AA, Tc) Field experiments and analysis of leaf sections.

Task 7 (Obj. 3) (AA-F, AA, MP, AL) Application of the developed ESI-MS(TOF) methods to the analysis of micronutrient fertilizers containing synthetic chelates commercialized in Spain, Portugal, and Tunis. ESI-MS/MS(TOF) analysis of standard solutions and micronutrient fertilizers containing Fe complexed by different ligands of those present in major synthetic chelates.

Task 8 (Obj. 3) (AA-F, HE-J) Analysis by ESI-MS(TOF) and ESI-MS/MS(Q-TOF) of combined solutions of well-characterized surfactants and synthetic Fe-fertilizers. Evaluation of possible interactions between products.

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Task 9 (Dissemination) This task will be carried out throughout the complete project duration, as described in section 5.

Third year (2012)

Task 1 & 2 (Obj. 1) (AA-F, Tc) Tissues and fluids from peach trees treated with stable isotope labeled Fe-fertilizers will be obtained. Analysis of Fe stable isotopes, and chelating agents and Fe-complexes will be carried out by ICP-MS and LC-ESI-MS, respectively.

Task 3 (Obj. 1) (AA, AA-F, MP, AL, Tc) Metabolic and proteomic analysis of xylem and apoplast in different plant species after Fe treatments.

Task 4 (Obj. 1) (AA, MP, AL, Tc) Parallel experiments in Spain (peach and pear), Tunis (olive) and Portugal (citrus) will be done to study mineral balance after Fe-treatments.

Task 6 (Obj. 2) (AA-FAA, Tc) Study of proteins and metabolites in leaf sections.

Task 8 (Obj. 3) (AA-F, AA) Analysis by ESI-MS(TOF) and ESI-MS/MS(Q-TOF) of combined solutions of well-characterized surfactants and non synthetic Fe-fertilizers. Evaluation of possible interactions between products.

Task 9 (Dissemination) This task will be carried out throughout the complete project duration, as described in section 5.

Justification of the research training fellowship requested

The postgraduate fellow will work on Fe-trafficking, plant material analysis, and MS analysis of fertilizers. We believe that the group has the capacity to provide an appropriate training to the research fellow, as reflected on the description of the group’s history and training capacity (Sections 6 and 7).

Justification of economic costs

Personnel costs. The agricultural technician to be on contract within the project will be engaged in all tasks related to plant treatment and sampling. Therefore, this person will work in Objectives 1 and 2 (Tasks 1, 2, 3, 4, 5 and 6). In more detail, the person holding such technical position will apply treatments and collect samples (leaves, xylem and apoplast) before and after treatments, and maintain hydroponic cultures. In the last years, a similar position has been funded via projects, in a discontinuous way, and the current post is only supported until September 2009. The group does not have any permanent technical staff to carry out field work. Thereby, financial support of a field technician contract is essential for the development of this proposal.

Durable equipment. A microtome equipped with a vibrating blade –vibratome- and a binocular loupe is required for Objective 2 (Task 5). The use of this device is mandatory to preserve the cellular structure when fresh tissue cuts are required for Fe localization.

Consumables. The costs of consumables for plant material analysis (carried out in our Department, but by an independent analysis service), chemicals for laboratory work, mass spectrometry, HPLC, hydroponic cultures and other small materials are considered in common for all the objectives. Stable isotopes will be used in Objectives 1 and 3 and are the most expensive chemicals used in the project: the isotope cost required for a single plant grown with 54Fe in hydroponics is approximately 37 euros and the cost of a 1-day resupply is approximately 12 euros. Costs related to microscopy studies will be specific for Objective 2.

Travel and subsistence. The travel budget associated with sampling (2.500 €) is justified, since the majority of field tasks corresponding to all objectives have to be carried out far away from the Institute. Other travel and stages (3,500 €) corresponding to collaborations and external analysis must be done (Univ. of Oviedo, Madrid and Barcelona). Concerning Symposia, we estimated a budget (3.000 €) for two persons to attend the Iron Nutrition Congress (ISINIP) to be held in 2010 in Budapest. Should any of the group members be invited to the Congress (e.g. as a Invited Speaker or Committee Member), funds would be used to participate in other Congresses (i.e. SEFV Iberic Plant Nutrition and FESPB Plant Physiology Symposia in 2010). A part of the budget (3.000 €) is devoted to finance two trips to Zaragoza of foreign personnel participating in the project (MP and AL, one trip each).

Other costs. Under this heading, general costs associated with external analysis, plant materials, web page maintenance, computer materials and dissemination have been included.

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

Tasks Centre Persons

First Year (*) 2010

Second Year (*) 2011

Third Year (*) 2012

OBJ 1. To study Fe-trafficking in the plant after Fe fertilization

Task 1. Fe uptake from different fertilizers EEAD-CSIC AA-F, AA, Tc 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

Task 2. Distribution of applied Fe inside the plant EEAD-CSIC AA-F, HE-J, Tc 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

Task 3. Xylem sap and apoplast fluid characterization after Fe-fertilization

EEAD-CSIC, IO, AU

AA, AA-F, HE-J, Tc, MP, AL

......|X|X|X|X|X|X| ......|X|X|X|X|X|X| ......|X|X|X|X|X|X|

Task 4. Nutrient balance in leaves EEAD-CSIC, IO, AU

AA, HE-J, Tc, MP, AL

X|X|X|X|X|X|X|X|X|X|X|X ......|X|X|X|X|X|X| ......|X|X|X|X|X|X|

OBJ 2. To localize nutrients and compounds related to mineral nutrition after fertilization

Task 5. Micro-localization of mineral elements EEAD-CSIC, IO, AU

SV, AA, Tc, MP, AL 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

Tasks 6. Leaf gradient studies EEAD-CSIC SV, AA, AA-F, Tc X|X|X|X|X|X|X|X|X|X|X|X X|X|X|X|

OBJ 3. To characterize micronutrient fertilizers solutions used in Fe fertilization

Task 7. Development of analytical technologies for the identification and quantification of fertilizers

EEAD-CSIC, IO, AU

AA-F, AA, MP, AL X|X|X|X|X|X|X|X|X|X|X|X X|X|X|X|X|X|X|X|X|X|X|X

Task 8. Study of interactions between surfactant and Fe-products

EEAD-CSIC AA, AA-F, HE-J X|X|X|X|X|X X|X|X|X|X|X|X|X|X|X|X|X X|X|X|X|

Task 9. Dissemination EEAD-CSIC IO, UA

AA, AA-F, SV, MP, AL

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

SV until March 2011 HE-J until July 2011 Tc Technician to be on contract

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5. BENEFITS DERIVED FROM THE PROJECT, DIFFUSION AND EXPLOITATION OF RESULTS

Expected benefits

Granting financial support for this proposal will help to improve the current state of knowledge concerning mineral nutrition of fruit species, with especial regard to correcting Fe deficiency status in fruit trees grown in calcareous soils. This would improve the rational use of fertilizers, with an appropriate use of inputs, a decrease of production costs and a reduction of the environmental impact of fertilization. The benefits derived from the project are applicable to other environments where Fe deficiency could occur (i.e. areas of low organic matter, high pH). The results obtained can be extrapolated to the whole of the Mediterranean region, and since approximately one third of the earth’s crust soils are calcareous, the topic is of global interest at a worldwide scale. Moreover, some of the proposed aims will enable advances in new technologies applied to Agricultural Sciences, including the use of Mass Spectrometry (ESI-MS TOF and MS-MS) for proteomic, metabolomic and xenomic applications, the use of stable isotopes and ICP-MS for the study of nutrient trafficking in fruit trees and the use of electron microscopy techniques for element localization in fruit tree leaves.

Scientific-technical contributions

Alike in previous projects developed by our research group, spreading of results will be carried out via publications in specialized scientific journals and presentations in Symposia (some group members belong to national and international Scientific Committees). Relevant journals in which we regularly publish are (Impact Factor 2007, in parentheses): Plant Soil (1.82), Rapid Commun Mass Sp (2.97), J Plant Nutr (0.59), J Plant Physiol (2.24), Physiologia Plantarum (2.19), Functional Plant Biology (2.38), Tree Physiol (2.14), J Hort Sci Biotech (0.66) and others (see http://www.eead.csic.es/stressphysiology). We also aim at disseminating results for a broader community of readers, by publishing in technical magazines and journals such as Phytoma, Nutri-Fitos, Vida Rural and others. The most significant Symposia in which we present our work on a regular basis are of the series: International Symposium on Iron Nutrition and Interactions in Plants (ISINIP); International Congress on Plant Nutrition (ICPN); Simposia Ibéricos sobre Nutrición Mineral de las Plantas or Fisiología Vegetal; and International Symposium on Mineral Nutrition of Deciduous Fruit Plants (ISHS).

European RTD projects

Our research group was granted an AIR EC Project (95-99) concerning plant nutrition, which was coordinated by our team. Financial support was obtained later for the development of a large EC Project within the 6th EU Framework (ISAFRUIT, 2006- Sep 2010). An ERANET project (PLANT-KBBE) will be developed in 2009-2012.

Technology transfer and knowledge transfer to specialised sectors

The results of prior projects led to 10 contracts with companies related to the agricultural sector, mainly on fertilizer issues: three with Spanish companies (2 with Caffaro and 1 with Agromillora), and seven contracts with foreign companies subjected to confidentiality (2 contracts with a Swiss company, 2 with a German and 3 with a Japanese companies). Cooperation with companies ensures knowledge transfer within the sector (largely in the scene of fruit production and fertilizer technology). Should the project give rise to a patent, the corresponding CSIC-OTT unit would process and manage it as it was done with the existing patent in 2004.

Objectives of the Spanish R&D&I Plan (2008-2011)

The project is included in Spanish R&D&I Plan within Innovation Projects in an Unguided Fundamental Research. The area of the research is Agriculture. The proposal covers general objectives O1.1 and O1.3 of Area 1.

Knowledge Dissemination plan

Should the proposed project be financed, results will be published in scientific and dissemination journals, will be presented in relevant scientific congresses and meetings and will be available at a specific site in the group webpage (http://www.eead.csic.es/stressphysiology). The webpage will be updated at least once every three months.

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6. BACKGROUND OF THE GROUP In this section the history of our research group concerning the proposed research is summarized (only since 2004). This includes Projects and RTD contracts1, Publications2, Patents3, PhD Theses4, Invited presentations in Symposia5, Results of the last project6 and Experience needed for this proposal7. The researcher responsible for this proposal has been leader in 5 previous projects from 1994 to 2009. Those publications underlined were supported by the 2 previous grants of the Spanish National R&D Plan (Agro-Food Technologies) (AGL2003-01999 and AGL2006-1416). The project AGL2006-1416 will finish in October 2009 and at this point of time (January 2009) many of the studies proposed are still in progress and some publications are in preparation. The work of the group is shown in our webpage (http://www.eead.csic.es/stressphysiology). 1Projects and RTD Contracts (2004-2008)

✔ National R&D&I for Agro-Food Sciences: AGL 2003-01999 (2003-2006), AGL2004-00194 (2004-2007), AGL2006-1416 (2006-2009) and AGL2007-61948 (2007-2010). ✔ PGC (General Knowledge Program): BOS2001-2343 (2001-04). ✔ VI EU Framework program: ISAFRUIT (2006-2010) Ref FP6-FOOD-CT-2006-016279. ✔ ERANET: KBBE (2009-2011). ✔ Bilateral projects with Italy, Hungary, Japan, Tunisia and Portugal ✔ Testing of new fertilizers for the correction of Fe deficiency chlorosis has led to 7 contracts with different foreign companies (Spanish, Swiss, German and Japanese companies). 2Publications

Twenty publications included in SCI, of which 15 were financed by the two last projects (AGL2003-01999 and AGL2006-1416). These include (see http://www.eead.csic.es/stressphysiology/publication_list.html for full titles) 6 in 2004, 2 in 2005, 3 in 2006, 1 in 2007, and 8 in 2008-2009. In the same period, 5 dissemination articles in grower’s journals, 3 invited book chapters and a Book have been published. The PI has currently a “h index” of 25 (25 papers quoted 25 or more times). A very short description of some of the works is as follows (publications underlined were supported by the 2 previous grants of the Spanish National R&D&I Plan):

General studies on Fe-deficiency Several studies have been developed to assess the diagnosis and nutritional status of fruit trees: Pestana et al. (2004) Plant and Soil, 259:287-295; Abadía et al. (2004) Vida Rural, 186:54-57; and Grasa et al. (2006) Acta Horticulturae, 721:99-102. About nutrient interactions in mineral nutrition: Rombolà et al. (2005) Plant and Soil, 271:39-45; and Fodor et al. (2005) Tree Physiology, 25:1173-1180. About Fe deficiency and physiological changes: Fernández et al. (2008a) Plant and Soil, 311:161-172. A book chapter about the effects of deficiency on fruit quality has been written (Álvarez-Fernández et al. 2006).

Fe acquisition and transport About tolerance responses: Gogorcena et al. (2004) Journal of Plant Nutrition, 27: 1701-1715; Zaharieva et al. (2004) Plant Science, 166:1045-1050; Jiménez et al. (2008) HortScience, 43:304-309; Jiménez et al. (2009) Plant and Soil, 315:93-106; López-Millán et al. (2009) Journal of Plant Physiology, in press. A book chapter on the use of isotopes to investigate Fe transport has been written (Álvarez-Fernández, 2006).

Studies related to the leaf A book chapter about photoinhibition and photoprotection has been written (Morales et al., 2006). Mechanisms of dissipation in Fe-deficient and Fe-resupply plants: Larbi et al. (2004) Photosynthesis Research, 79:59-69; and Larbi et al. (2006) Photosynthesis Research, 89:113-126. Proteomics studies on thylakoid membrane: Andaluz et al. (2006) Photosynthesis Research, 89:141-155.

Treatments to correct Fe deficiency Concerning foliar sprays and solid trunk implants for the correction of Fe deficiency: Álvarez-Fernández et al. (2004) Plant and Soil, 263:5-15; Fernández et al. (2006) Plant and Soil, 289:239-252; Fernández et al. (2008b) Science Horticulturae, 117:241-248; Fernández et al. (2009) Journal of Horticultural Science and Biotechnology, 84:1-6. Reviewing Fe deficiency correction methods: Abadía et al. (2004) Soil Science and Plant Nutrition, 50:965-971.

Specific publications on MS technology in agricultural studies Ferric chelate studies: Álvarez-Fernández et al. (2007) Journal of the American Society of Mass Spectrometry, 18:37-47; Orera et al. (2009) Journal of Horticultural Science and Biotechnology, 84:7-12. Nicotianamine complexes: Rellán et al. (2008) Rapid Communication in Mass Spectrometry, 22:1553-1562.

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3Patents

The screening rootstocks method published in 2002 (Gogorcena et al. 2000) gave origin to a patent (ref 200002827) accepted in 2004. 4PhD Theses (2003-2009)

✔ Iron chlorosis: plant responses and correction methods (Ajmi Larbi, February 2003). ✔ Studies on changes in the plant proteome induced by iron deficiency (Sofía Andaluz, October 2005). ✔ Novel analytical technologies for the study of Fe-fertilizers (Irene Orera, in prep 2009). ✔ Iron long-distance transport and metabolomics of Fe-deficiency in plants (Rubén Rellán, in prep 2009). 5Invited presentations (only titles) in Simposia (2006-2008). Six of a total of 27 contributions correspond to invited presentations. Also, some group members belong to Organizational or Scientific Committees of some periodic Congresses.

2006 XIII International Symposium on Iron Nutrition and Interactions in Plants. Montpellier (Francia). New analytical technologies to tackle the biological and environmental implications of iron fertilization. XI Simposio Ibérico de Nutrición Mineral de Plantas. Pamplona (España) Absorción, transporte y uso de hierro en plantas. Estudios de biología vegetal vs. Prácticas agronómicas de fertilización. 14 Tagung Arbeitskreis Blattdüngung. Grundlagen, Beratung und Praxis, Wurzburg, Germany. Regreening of chlorotic peach leaves after foliar treatment. 2007 XVII Reunión de la Sociedad Española de Fisiología Vegetal-X Congreso Hispano-Luso de Fisiología Vegetal. Alcalá de Henares, España. Long-distance metal transport in plants. Isafruit Meeting. Bologna, Italia. Recent developments in fruit tree Fe-fertilization: foliar spray formulations and Fe-chelate analysis. 2008 VI International ISHS Symposium on Mineral Nutrition of Fruit Crops, Faro, Portugal. Foliar fertilisation: a reliable strategy to control plant nutrient deficiencies? 6Results within the objectives of the last Spanish R&D&I Plan project, AGL2006-01416 (many works are still in progress, since this project will end in October 2009).

Objective 1. (To assess micronutrient budgets in peach tree orchards) A Master Thesis will be defended in January 2009 with two years of data. A preliminary model for nutrient demand will be included in this Thesis. Uptake, transport and Fe accumulation in a peach-almond hybrid has been reported (Plant Soil (2009), 315:93-106).

Objectives 2 and 3. (To study the action mechanisms of fertilizers and to assess the persistence of the micronutrient fertilizers in the water/soil/plant system) A method to study the formation of metal-nicotianamine complexes has been published (Rapid Commun Mass Spectrom 2008, 1553-1562). The organic acid metabolism in deficient plants has been characterized (J Plant Physiol 2009, in press). Analytical technologies to tackle biological and environmental implicatios of Fe-fertilizers have been reviewed (J Hortic Sci Biotech 2009, 84:7-12). One PhD Thesis on this subject will be presented during the first semester of 2009 and another PhD Thesis related to these objectives will be presented after summer, also in 2009. Both Theses are co-directed by one of the researchers participating in this proposal (AA-F).

Objective 4. (To develop and optimize foliar spray methodologies for correcting iron chlorosis) Three studies about Fe formulations (Sci Hort 2008, 117:241-248; J Hortic Sci Biotech 2009, 84:1-6) and effects of Fe-deficiency on leaf surface (Plant Soil 2008, 331:161-172) have been published. 7Experience on the techniques needed for the present proposal

In the last few years we have developed work in metabolite target analysis and xenomics profiling using HPLC-MS(TOF) techniques. There are some publications of the group that developed methods based on this technology: Fe-chelates, glutathion and ascorbate, capsicinoids and metal-nicotianamine complexes. Work with stable isotopes is also underway in collaboration with Dr. J. I. García-Alonso, Univ. of Oviedo. Preliminary results of xylem characterization in fruit trees peach) has been achieved. For metabolic analysis we collaborate with the Metabolomics Fiehn lab in the Genome Center, UC Davis, USA.

With regard to the protein analysis, an automated spot cutter and a nHPLC-MS-MS ion trap system is now installed and working in our lab and it shall permit to carry out sequencing de novo proteins obtained by 2D techniques. Our group has experience on electrophoretic separation of proteins: BN-PAGE, SDS-PAGE, 2D-PAGE, and some specific stains.

The technique to perform the analysis of leaf gradient by cutting adaxial sections has been developed in the EEAD by one of the researchers included in the proposal (SV). For elemental micro-localization studies we have collaborations with some services in SEM-EDX (CCMA-CSIC and ICB-CSIC), freeze fracture (CCMA-CSIC) and freeze-substitution (PCB-UAB) techniques. Some expertise in some other microscopy techniques has been acquired by SV in the Dr. Ma Laboratory at Okayama University, Japan.

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6.2 PUBLIC AND PRIVATE GRANTED PROJECTS AND CONTRACTS OF THE RESEARCH GROUP

Budget

Title of the project or contract

Relationship with this

proposal (1)

Principal Investigator

EUROS

Funding agency and

project reference

Project period

(2) Physiology and biochemistry of iron deficiency in the model plant Beta vulgaris

2 Javier Abadía 92.435,38 DGCYT-MCYT (PGC) BOS 2001-2343

Dic 01-Dic 04 C

Selection of fruit rootstock tolerant to iron chlorosis 3 Mª Angeles Moreno 53.057 MCYT PTR1995-0580-OP May 02-May 05 C

Iron nutrition of fruit-trees: strategies for the control of iron deficiency

1 Anunciación Abadía 171.550 DGI-MCYT (PN de Agricultura) AGL2003-1999

Dic 03- Dic 06 C

Support for Consolidated Research Group DGA-A01 2 Javier Abadía ca.12.000/year DGA (Aragón Autonomic Govmnt.)

03/04/05/06/07/08G

Acquisition and transport of iron in plants 1 Javier Abadía 160.050 DGCYT-MEC (PN de Agricultura) AGL 2004-00194

Dic 04-Dic 07 C

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

1 Ole Callesen PI (Javier Abadía Task Leader)

80.691 EU- Sixth Framework Program

Jan 06-Jan 09 C

Scientific basis for the optimization of foliar fertilization

1 Javier Abadía 25.000 DGA (Aragón Auton. Govmnt.) PM003-2006

Dec 06-Nov 08 C

Basis for a rational use of micronutrient fertilizers in fruit species

1 Anunciación Abadía 199.650 DGI-MICINN (Plan Nacional) AGL 2006-01416

Oct 06- Sep 09 C

Studies on metal homeostasis in plants 2 Javier Abadía 237.000 DGI-MICINN (Plan Nacional) AGL 2007-61948

Oct 07-Sep 10

Homeostasis and Transport of Iron improving Plant Productivity and Growth (HOT IRON: PLANT PROGROW)

1 Katrin Philippar Approved, funding in evaluation

ERANET, PLANT-KBBE Call 2008

(1) Write 0, 1, 2 or 3 according to: 0 = Similar project; 1 = Very related; 2 = Low related; 3 = Unrelated. (2) Write C or S if the project has been funded or it is under evaluation, respectively.

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7. TRAINING CAPACITY OF THE PROJECT AND THE GROUP Granting of 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 and of the group

The candidates have good training opportunities, ranging from applied to basic research and always within the field of Agricultural Sciences. On one hand, the internationalization of the group (with active projects with Italy, Japan, Hungary, Portugal, Tunisia, and France/Germany), and on the other hand, the facilities of the group, as much in apparatus as in analytical developments, allow for the students to have a great opportunity for a complete training. In the past, 90% of the graduates that received research fellowships within the group were able to complete their PhDs. Since 1992, the responsible of the proposal has co-directed 8 doctoral theses, 5 MSc theses and 2 “final year” graduate research projects. Two PhD theses directed by one of the participant investigators (AA-F) will be defended in 2009. It is important to remark that 2 PhD theses of the group have received Extraordinary Doctoral Awards.

Current positions hold by the PhDs and Technicians trained in our Group

Eight out of 9 PhDs trained in our group are working in R&D&I, 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. Four out of 5 technicians trained in our group are currently working as staff or contracted technicians in agriculture/research Units (2 in the Local DGA Government and 2 in the CSIC).

PhD Theses

1. Changes mediated by environmental stresses in the photosynthetic apparatus of higher plants (Zaragoza, F Morales1, 1992).

2. Responses induced by Fe deficiency in the root system of Beta vulgaris L. (Zaragoza, S Susín2, 1994).

3. Changes induced by Fe deficiency and LCM mutation in the organization of photosynthetic apparatus of Beta vulgaris L. (Zaragoza, R Quílez4, 1994).

4. Environmental stress tolerance evaluation by chlorophyll fluorescence techniques, pigment analysis and mineral content in agronomic plants (Lleida, R Belkhodja5, 1998). Extraordinary Doctoral Award.

5. Study of the Fe-deficiency response mechanisms in plants (Zaragoza, E González-Vallejo4, 1999).

6. Dissipation of the energy excess gathered by the photosynthetic apparatus in conditions of environmental stress: oxygen related mechanisms (Zaragoza, D Tobías, 1999).

7. Transport and acquisition of Fe in plants (Zaragoza, AF López-Millán1, 2000). Extraordinary Doctoral Award.

8. Iron chlorosis: plant responses and correction methods (Lleida, A Larbi3, 2003).

9. Studies on changes in the plant proteome induced by iron deficiency (Zaragoza, S Andaluz4, 2005).

Postgraduate Training

A total of 105 pre- and post-doctoral students from 14 different countries have carried out short training stages in our laboratories. Several group members usually participate in doctoral and MS courses organized by the University of Zaragoza (Spain), the IAMZ-CIHEAM, the UAM (Spain) and the University of Chihuahua (Mexico).

Master Theses IAMZ-CIHEAM and “final year” research projects (ETSIA Huesca) ✔ 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). Mark 9.0/10.0. ✔ Réponses radiculaires face à la deficience en fer chez differents genotypes de tomate et de betterave (M. Zouari, 1996). Mark 9.5/10.0. ✔ 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) Mark: 8.5/10.0. ✔ 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.) (A. Larbi, 1999) Mark: 9.0/10.0. ✔ Necesidades de nutrientes minerales en melocotonero (H. El-Jendoubi, to be defended in January 2009). ✔ Tratamientos foliares para la corrección de la clorosis férrica. (P García Laviña, 1998). ✔ Efecto de distintos factores en la corrección de clorosis férrica en peral. (C Fidalgo Pinilla, 1998).