Mineral Improved Crop Production for Healthy Food and Feed

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Cost Action FA 0905 First Annual Meeting

Mineral Improved Crop Production for Healthy Food and Feed

Belek – Antalya TURKEY

1-5 November 2010

Local Organizing Committee:

Ismail Cakmak Levent Ozturk U. Baris Kutman Bahar Yildiz S. Bahar Aciksoz

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First Annual Meeting of the COST Action FA 0905

Mineral Improved Crop Production for Healthy Food and Feed Antalya, Turkey, 1-5 November 2010

PROGRAM

Monday, 1November 2010

14:00-24:00 Arrival of participants and check-in 14:00-19:00 Registration

19:00-20:30 Welcome Cocktail

Tuesday, 2November 2010 (Conference Room: Ottoman-I) 08:00-09:00 Registration

09:10-09:30 Opening Speeches (Bal Ram Singh, Ismail Cakmak)

09:30-12.20 Session I: Soil-Plant Interactions & Physiology (Working Group I) Chair: Bal Ram Singh, Norway

09:30-10:10 Keynote Lecture:

Philip J. White, UK

Contribution of Plant Mineral Nutrition to Sustainable Development and Global Health

10:10-10:30 Micronutrient Management Strategies - How to Utilize Local and Site-Specific Resources to Produce High Quality Products?

Ingrid Öborn, Sweden 10:30-11:00 Coffee Break

11:00-11:20 Roles of Nitrogen and Zinc Nutrition in Biofortification of Wheat Grain

Ümit Barış Kutman, Türkiye

11:20-11:40 Zn Uptake Kinetics of two Wheat Cultivars Differing in Zn Efficiency and Development of the Permeation Liquid Membrane Technique (PLM) for Free Zn Measurements in Solutions

Anja Gramlich, Switzerland

11:40-12:00 Integrated Experimental and Modeling Approach to Identify Processes Controlling Micronutrient Bioavailability in the Rhizosphere

Ellis Hoffland, Netherlands

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12:00-12.20 Effectiveness of Iodine Biofortification and Mineral Composition of Spinach Depending on Iodine Form and Method of its Application Wlodzimierz Sady, Poland

12:20-13:30 Lunch

13:30-15:00 Poster Session

15:00-17.30 Session II: Soil-Plant Interactions & Physiology (Working Group I) Chair: Rainer Schulin, Switzerland

Steve McGrath, UK

Agronomic and molecular approaches to reduce accumulation of non-nutritive elements (e.g., cadmium and arsenic) in food crops

15:40-16:00 HarvestPlus-Global Zinc Fertilizer Project Ismail Cakmak, Türkiye

16:00-16:30 Coffee Break

16:30-16:50 Copper Uptake and Phenotypic Plasticity of Sunflower Mutant Lines

Michel Mench, France

16:50-17:10 Speciation and Si Influence on As in Lettuce Maria Greger, Sweden

17:10-17:30 Exponential Decrease in the Concentration of Essential (Cu, Zn, Fe, Mn) and Toxic (As, Cd, Pb) Trace Elements in Shoots during the Development of Field-Grown Maize and its Correlation with the Concentration in the Grain

Christophe Nguyen, France 17:30-18:00 General Discussions

Wednesday, 3November 2010

09:00-12:30 Session III:

(Working Group II)

Molecular Biology, Genetics and Breeding Chair: Jan K. Schjoerring, Denmark

Elsbeth L. Walker, USA

Molecular Biological Approaches to Improving Iron Concentration of Food Crops

09.40-10.00 Quantitative Localization of Iron in Arabidopsis thaliana Seeds Magali Schnell Ramos, France

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10.00-10.20 Identification of Zinc-Binding Proteins in Rice Endosperm James Stangoulis, Australia

10.20-10.40 Modification of the Plant Zinc Deficiency Response towards Increased Zinc Biofortification

Mark Aarts, The Netherlands

10.40-11.10 Coffee Break

11.10-11.30 Learning from Extremes: Elevated Levels of Nicotianamine are Important for Zn Accumulation in Arabidopsis halleri

Stephan Clemens, Germany

11.30-11.50 Cadmium efflux from the root appears to be a major determinant controlling cadmium accumulation in lettuce leaves

Pierre Berthomieu, France

11.50-12.10 Tolerance and Detoxification Mechanisms of Arsenic in Plants:

Role of Antioxidant Enzymes Annabelle Austruy, France

12.10-12.30 Insights into the Ionomic and Transcriptomic Responses of Plants to Magnesium Availability

Christian Hermans, Belgium 12:30-13:00 General Discussions

13:00-14:00 Lunch

14:15-17:45 Excursion (Ancient Pamphylian Cities: Aspendos and Perge; attendance fee:

20 EURO including the entrance fees) 19:30-22:30 Conference Dinner

Thursday, 4 November 2010

09:00-12:10 Session IV:

Working Groups 3&4

Plant Product Processing and Human Nutrition Chair: Martin Broadley, UK

Jan K. Schjoerring, Denmark

Enhanced Content and Bioavailability of Iron and Zinc in the Endosperm of Rice Seeds by Activation-Tagging of Nicotianamine Synthase

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09:40-10:00 Biofortification in Common Bean: Identification and Characterization of a LPA (Low Phytic Acid) Mutant

Francesca Sparvoli, Italy

10:00-10:20 In vitro Digestion Model of Food Components by Using Human Gastrointestinal Enzymes

Gerd Elisabeth Vegarud, Norway 10:20-10:50 Coffee Break

10:50-11:10 Evaluation of Iron Bioavailability from Potato Cultivars for Human Consumption

Lisa Miranda, Luxembourg

11:10-11:30 Identification and Quantification of Nutritionally Relevant Forms of Iron within Cereal Endosperm to Assess Potential Bioavailability, Using Size Exclusion Chromatography Coupled with Inductively Coupled Plasma Mass Spectrometry

Tristan Eagling, UK

11:30-11:50 A Bread Wheat Zinc Biofortification Project: Chelating Action of Food Additives in Fortified Flour and Technological Development of Alternatives to Potentiate Zinc Bioavailability

Maria Manuela Abreu da Silva, Portugal 11:50-13:30 Lunch

12:30-14:00 MC Meeting 13:30-15:00 Poster Session 15:00-16:20 Session V:

Working Groups 3&4

Plant Product Processing and Human Nutrition Chair: Elsbeth L. Walker, USA

Martin Broadley, UK

Agronomic Biofortification of Foods with Macro- and Micro-Mineral Elements

15:40-16:00 Chromium Uptake by Trigonella Foenum-Graecum, a Medicinal Herb with Anti-Diabetic Activity

Charlotte Poschenrieder, Spain

16:00-16:20 Practical Approaches to Increase Selenium as Essential Metalloid in Human Diet

Hassan Azaizeh, Israel 16:20-16:50 General Discussions

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16:50-17:20 Coffee Break

17:20-18:20 Conference Panel (Panelists: Stephan Clemens, Francesca Sparvoli, Steve McGrath, Michel Mench, Lisa Miranda, Bal Ram Singh)

18:20-18:40 Poster Prizes and Closing Speeches (e.g., next meetings/activities) Peter Schröder and Bal Ram Singh

Friday, 5 November 2010

……-12:30 Check-out / Departures

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POSTER PRESENTATIONS

1. Immobilization of Cadmium in Rhizosphere of Vegetables by Application of Compost

Stefan SHILEV, Todor BABRIKOV, Enrique D. SANCHO

2. Effects of Irrigation with Metal-Contaminated Water on Soil Solution Speciation and Metal Soil-Plant Transfer

Fayçal KERBOUA, Laurence DENAIX, Nancy ZAAROUR, Valérie SAPPIN- DIDIER

3. Effective Utilization of Crop Production at Low Contaminated Site in Czech Republic

Šárka PETROVÁ, Mikuláš MADARAS, Petr SOUDEK, Jan LIPAVSKÝ, Petr HUTLA, Tomáš VANĚK

4. Cd and Zn Speciation and Mobility in Contaminated Soil: Physical Micro- Characterization, Chemical Extraction and Isotopic Exchange Kinetics Methods Valérie SAPPIN-DIDIER, Yann SIVRY, Marguerite MUNOZ, Jean RIOTTE, Laurence DENAIX, Bernard DUPRÉ

5. Uptake and Localization of Cadmium and Lead in Typha latifolia Lyudmila LYUBENOVA, Paula PONGRAC, Peter SCHRÖDER

6. Quantification of Trace Element Fluxes (As, Cd, Cu, Pb, Zn) to Agricultural Fields Amended with Pig Slurry

Laurence DENAIX, Christophe NGUYEN, Julien HÉROULT, Corinne PARAT, Gaetane LESPES, Martine POTIN-GAUTIER, Regis COUDURE, Sylvie DAUGUET 7. Evaluation of the Soil Toxicity before and after Culture of Cannabis sativa L.

Using Standardized Ecotoxicological Bioassays

A. AUSTRUY, Q. SICARD, P. VERNAY, C. GAUTHIER-MOUSSARD, A. HITMI 8. Associations between Soil P Dynamics and Buckwheat Biomass Yields

Saulius MARCINKONIS

9. Zinc Nutrition Effect on Antioxidative Responses to Cadmium of three Wheat Genotypes Differing in Zinc Efficiency

A. SANAEIOSTOVAR, A.H. KHOSHGOFTARMANESH, H. SHARIATMADARI, M. AFYUNI, R. SCHULIN

10. Elevated Concentration of Nitrogen Oxides in atmosphere as cause of nutrient leaching from vegetation

Edita BALTRĖNAITĖ, Pranas BALTRĖNAS

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11. Microelements and Cadmium in Grain of Wheat Grown in Poland Grzegorz SIEBIELEC, Rafal GALAZKA, Tomasz STUCZYNSKI

12. Selenium in the Soil-Plant System from South-Eastern Part of Romania

Radu LĂCĂTUŞU, Anca-Rovena LĂCĂTUŞU, Mihaela Monica ALDEA, Mihaela LUNGU, Mihaela Venera STROE

13. The Plant Cell Wall in Plant Protection

D. RICHTEROVÁ, Z. VATEHOVÁ, K. KOLLÁROVÁ, A. MALOVÍKOVÁ, I.

ZELKO, D. LIŠKOVÁ

14. Beneficial Effects of Nickel Nutrition: Nickel Ameliorates Toxicity Symptoms Caused by Foliar Urea Application in Soybean

Bahar YILDIZ, U. Baris KUTMAN, Ismail CAKMAK

15. Nitrogen Nutrition Increased Release of Phytosiderophores and Root Uptake of Fe(III)-Phytosiderophore Complex in Fe-Deficient Wheat

Seher Bahar ACIKSOZ, Levent OZTURK, Ozay Ozgur GOKMEN, Volker ROMHELD, Ismail CAKMAK

16. Influence of Fertilizers on Bioavailability of Lead and Persistent Organic Pollutants

Lidiya MOKLYACHUK, Nataliya MAKARENKO, Volodymyr MAKARENKO, Olexandr NIKITJUK

17. The Possible Interactions of Copper in Vineyards: Soil and Plant, Physiology and Biochemistry

Tjasa JUG, Denis Rusjan

18. Reaction of Root Tissues to Cadmium Treatment Alexander LUX, Michal MARTINKA, Marek VACULÍK

19. Spatial Variability of Cadmium Absorption in Intact Roots of Sunflower (Helianthus annuus L.)

Marie-Aline LAPORTE, Laurence DENAIX, Sylvie DAUGUET, Francis FLENET, Stéphane THUNOT, Christophe NGUYEN

20. Agricultural Use Impact of Urban Waster; Chemical Study of Trace Metals Dynamics in Soil-Plant System

Senda ZARROUK, Naïma KOLSI BENZINA, Alain BERMOND

21. Relationship between Available Phosphorus and Total Cadmium under Different Land Uses

Vladimir IVEZIĆ, Bal Ram SINGH, Zdenko LONČARIĆ 22. Phytoremediation of Agricultural Soils

Tommy LANDBERG and Maria GREGER

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23. Photosynthetic Performance in Barley – An Evaluating Tool of Cadmium Tolerance

Fernando Cebola LIDON, Andon VASSILEV, Benvindo MAÇÃS, José Prates COUTINHO, Ana Sofia ALMEIDA, Ana Luisa FERNANDO, Maria Paula DUARTE, Maria Manuela ABREU DA SILVA, António Eduardo LEITÃO, Ana Isabel RIBEIRO, José Cochicho RAMALHO

24. A Bread Wheat Zinc Biofortification Project – Genotypes Screening to Obtain a Prototype

Ana Sofia ALMEIDA, Benvindo MAÇÃS, José Prates COUTINHO, Ana Luisa FERNANDO, José Cochicho RAMALHO, António Eduardo LEITÃO, Ana Isabel RIBEIRO, Maria Paula DUARTE, Maria Manuela ABREU DA SILVA, Fernando Cebola LIDON

25. A Bread Wheat Zinc Biofortification Project – Nutritiotnal and Technological Flour Evaluation

António Eduardo LEITÃO, Maria Paula DUARTE, Maria Manuela ABREU DA SILVA, Benvindo MAÇÃS, José Prates COUTINHO, Ana Sofia ALMEIDA, Ana Luisa FERNANDO, José Cochicho RAMALHO, Ana Isabel RIBEIRO, Fernando Cebola LIDON

26. A Bread Wheat Zinc Biofortification Project – Physiological and Molecular Characterization of Grain Filling

José Cochicho RAMALHO, Ana Isabel RIBEIRO, Benvindo MAÇÃS, José Prates COUTINHO, Ana Sofia ALMEIDA, Ana Luisa FERNANDO, Maria Paula DUARTE, Maria Manuela ABREU DA SILVA, António Eduardo LEITÃO, Fernando Cebola LIDON

27. A Bread Wheat Zinc Biofortification Project – Assessment of Low-Technology Processing Methods to Reduce the Phytate Content of Cereals

Ana Luisa FERNANDO, Maria Paula DUARTE, António Eduardo LEITÃO, José Cochicho RAMALHO, Ana Sofia ALMEIDA, Benvindo MAÇÃS, José Prates COUTINHO, Maria Manuela ABREU DA SILVA, Ana Isabel RIBEIRO, Fernando Cebola LIDON

28. Is AtHMA4 a Good Candidate for Biofortification Strategies?

Justyna RUDZKA, Anna BARABASZ, Lorraine WILLIAMS, Danuta M.

ANTOSIEWICZ

29. Evaluation of Andean Potatoes as Sources of Mineral Micronutrients; Focus on Iron

Danièle EVERS, Sylvain LEGAY, Isabelle LEFÈVRE, Cédric GUIGNARD, Joanna ZIEBEL, Torsten BOHN

30. Threats Concerning Consumption of Self-Provisioning Crops Produced in Areas with Elevated Concentrations of Pb and Cd in Soil

Jadwiga GZYL

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31. Differences in Cadmium Concentration in Barley Grains Depending on Cultivars and Sites

Marta POGRZEBA, Aleksandra SAS-NOWOSIELSKA, Jacek KRZYŻAK 32. In vitro Conversion of Selenium Species by Human Intestinal Microbiota

R.V. Srikanth. LAVU, Tom van de Wiele, Katrijn Van den Broeck, Filip Tack, Gijs Du Laing

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ABSTRACTS OF

ORAL PRESENTATIONS

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*S1.1.CONTRIBUTIONSOFPLANTMINERALNUTRITION TOSUSTAINABLEDEVELOPMENTANDGLOBALHEALTH

Philip J. White

Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK Keywords: Biofortification, Fertiliser, Genetics, Ionomics, Mineral

This lecture reflects on the definition of food security as having sufficient, safe and nutritious food to meet the dietary needs of an active and healthy life. It provides an introduction to plant mineral nutrition and explains how mineral elements are acquired by roots and distributed within plants. It observes that crop production is often limited by low phytoavailability of essential mineral elements and/or the presence of excessive concentrations of potentially toxic mineral elements. It describes the strategies by which plants strive to overcome these environmental limitations. It introduces the concept of the ionome (the elemental composition of a subcellular structure, cell, tissue or organism), and describes how the ionome is affected by environmental and genetic factors. It observes that the activities of key transport proteins can determine tissue and cellular ionomes. It reviews how current research in agronomy and plant breeding is addressing the problems of mineral toxicities in agricultural soils to improve food security and the optimization of fertilizer applications for economic and environmental sustainability. It concludes with a perspective on how agriculture can produce edible crops that contribute sufficient mineral elements for adequate animal and human nutrition through a combination of appropriate agronomic and genetic strategies.

* S1.1: Session 1; 1. presentation

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S1.2. MICRONUTRIENT MANAGEMENT STRATEGIES - HOW TO UTILIZE LOCAL AND SITE SPECIFIC RESOURCES TO PRODUCE HIGH QUALITY

PRODUCTS?

Ingrid Öborn¹

Robin Walker⁴, Christine A Watson⁴, Maria Wivstad¹

, Jan Bertilsson¹, Colin D Campbell², A Sigrun Dahlin¹, Anthony C Edwards³, Jan Eriksson¹, Bodil E Frankow-Lindberg¹, Stephen Hillier², Bodil Lindström¹, Lisbeth

Linse¹, David Lumsdon², Atefeh Ramezanian¹, Charlie Shand^2, Alex H Sinclair⁴,

1Swedish University of Agricultural Sciences (SLU), SE-750 07 Uppsala, Sweden, [email protected],²Macaulay Institute, Aberdeen AB15 8QH, UK; ³Nether Backhill, Aberdeenshire, UK; ⁴Scottish Agricultural College (SAC), Aberdeen, UK.

Keywords: forage crops, geochemistry, plant uptake, soil amender, soil mineralogy

There has been a significant increase in demand for organic products in recent years. In addition, climate change conscious consumers show preference for local products with low food miles. A greater emphasis on farming systems that rely upon local sources while being environmentally friendly, may increase the longer-term risk of issues related to micronutrient imbalances. How can these trends be combined with an effort to optimise food and feed quality, e.g. in relation to micronutrients? This research is designed to gain knowledge on how soils can be managed and plant species selected to supply the optimum micronutrient (e.g. B, Co, Cu, Fe, Mo, Mn, Ni, Se and Zn) profile of forage crops and to contribute to the development of sustainable farm management strategies based on local resources. The potential of utilizing site specific properties, common and novel herbage species is explored together with options for local micronutrient (re)cycling. A suite of complementary approaches are used including national soil, plant and veterinary survey and monitoring data, and long-term experiments from Sweden and Scotland. Soils of contrasting geochemistry and mineralogy are used in experiments testing a range of forage species and varieties, and soil amenders. Plant performance and quality, and soil-microbe interactions are examples of aspects included in the studies.

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S1.3. ROLES OF NITROGEN AND ZINC NUTRITION IN BIOFORTIFICATION OF WHEAT GRAIN

Umit Baris Kutman¹

Emin Bulent Erenoglu², Ismail Cakmak¹

, Bahar Yildiz¹, Yasemin Ceylan¹,

1Sabanci University, Faculty of Engineering and Natural Sciences, 34956, Istanbul, Turkey

2Cukurova University, Department of Soil Science and Plant Nutrition, 01330, Adana, Turkey

Keywords: biofortification, wheat, zinc, nitrogen, iron

Deficiencies of zinc (Zn) and iron (Fe) are widespread nutritional problems, caused mainly by low dietary intake. Biofortification of cereal grains with Zn and Fe in order to alleviate the health problems associated with these deficiencies is a global challenge. In this project, the potential of N fertilization in biofortification of wheat grain was investigated. For this purpose, wheat plants were grown with different N and Zn treatments under greenhouse or growth chamber conditions. Increasing N application improved the grain Zn and Fe concentrations by up to 100%. This impact of N on grain Zn concentration disappeared at low Zn supply, whereas the combination of high N and Zn treatments gave rise to synergistic results. Improving the N nutrition enhanced the Zn and Fe concentrations not only in the whole grain but also the endosperm, the most widely consumed part of wheat grain. In both short-term and long-term experiments, some of which utilized the radioisotope ⁶⁵Zn, it was clearly demonstrated that an improved N nutritional status is associated with enhanced uptake, root-to-shoot translocation and remobilization of Zn. As an agronomic biofortification tool, optimized N applications may rapidly and effectively contribute to the mitigation of Zn and Fe deficiency problems in developing countries.

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S1.4. ZINC UPTAKE KINETICS OF TWO WHEAT CULTIVARS DIFFERING IN ZINC EFFICIENCY AND DEVELOPMENT OF THE

PERMEATION LIQUID MEMBRANE TECHNIQUE (PLM) FOR FREE ZINC MEASUREMENTS IN SOLUTIONS

Anja Gramlich¹, Susan Tandy¹, Emmanuel Frossard², Rainer Schulin¹

1Institute of Terrestrial Ecosystems, ETH Zurich, Switzerland

2Institute for Plant, Animal and Agroecosystems Sciences, ETH Zurich, Switzerland

Keywords: Free Zn, Organo-Zn-Complexes, Permeation Liquid Membrane, Uptake Kinetics, Wheat

Organic ligands can increase the mobility of metals in soils. Little is known about whether organo-zinc-complexes in the soil solution can also play a direct role in the uptake of zinc (Zn) by plants. In a preliminary experiment for studying the role of soil organic ligands in Zn uptake by wheat roots we studied the kinetics of Zn influx into seedlings of two Iranian wheat genotypes: the Zn-inefficient cultivar Kavir and the Zn-efficient cultivar Back Cross Rushan.

Zinc influxes into roots and shoots were determined at free Zn concentrations between 0.005- 20 µM in short-term hydroponic uptake experiments using 65-Zn labeling and EDTA for buffering free Zn concentrations. Michaelis-Menten constants (K_m) were between 1-2 µM Zn²⁺ in both cultivars. When comparing the effect of ligands on Zn-uptake, the free Zn in solution is an important parameter. Free Zn concentrations were therefore modeled and the predictions were verified experimentally using the Permeation Liquid Membrane (PLM) technique, a method described in the literature for Cd, Cu and Pb speciation in aquatic chemistry. However, the method has never been used for Zn speciation. At free Zn concentrations, the method was stable and gave reproducible results, giving a pre- concentration factor of 639±39 after 2h contact time at pH 7.2. We will now use the determined Km values and the PLM method in subsequent experiments in which we are comparing the influence of different organic ligands on Zn uptake by wheat.

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S1.5. INTEGRATED EXPERIMENTAL AND MODELING APPROACH TO IDENTIFY PROCESSES CONTROLLING

MICRONUTRIENT BIOAVAILABILITY IN THE RHIZOSPHERE

Ellis Hoffland, Xiaopeng Gao, Andreas Duffner and Erwin Temminghoff Wageningen University, Dept. Soil Quality

Our work should contribute to alleviation of micronutrient deficiency problems and aims at identifying soil and plant factors affecting Zn and Fe bioavailability. In a project on Zn we identified Zn mobilization mechanisms by rice. Genotypes with a higher Zn uptake responded to Zn deficiency with increased root exudation of malate, but the increase was too small to explain the higher Zn uptake. In a new project we aim to integrate rhizosphere chemistry, physics and biology. Our methodology will be generalized such that it can be used to evaluate rhizosphere metal bioavailability in a broader context, including contaminated soils.

Genotypes of rice and rapeseed will be grown in rhizoboxes for in situ sampling of rhizosphere and bulk soil solution with micro-suction cups. High resolution analyses of metals and low molecular weight organic acids and other relevant parameters (pH, DOC) will be done. These rhizosphere parameters will be used to simulate rhizosphere conditions in batch experiments to understand the chemical speciation of trace metals using the Donnan Membrane Technique. A model will be developed for numerical studies on speciation and transport processes in the rhizosphere and to connect the different scales of observation and of controlling processes.

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S1.6. EFFECTIVENESS OF IODINE BIOFORTIFICATION AND MINERAL COMPOSITION OF SPINACH DEPENDING ON IODINE FORM AND

METHOD OF ITS APPLICATION

Włodzimierz Sady, Sylwester Smoleń

Department of Soil Cultivation and Fertilization of Horticultural Plants Faculty of Horticulture, University of Agriculture in Kraków, Poland

Keywords: biofortification, biological quality, iodine, mineral composition, spinach

Objectives: Biofortification of vegetables with iodine can become an alternative (in reference to salt

iodization) method of introducing this element to human diet. Iodine is not an essential nutrient for plants and its effect on plant growth and development has not yet been sufficiently examined.

Methods: In the present research, pot cultivation of spinach ‘Olbrzym zimowy’ cv. was carried on

heavy soil and differentiated iodine forms and ways of application were studied: 1- control (without iodine application), 2- pre-sowing KI fertilization, 3- pre-sowing KIO3 fertilization, 4- fertigation with KI, 5- fertigation with KIO3. In combinations no.2 and 3 iodine was applied in a dose of 1 mg I per 1 dm³ of soil. In combinations no.4 and 5, water solution of iodine in the concentration of 0.0004% was used – total amount of ≈ 1 mg I∙dm ^-3was introduced to soil throughout spinach cultivation. The content of I, P, K, Mg, Ca, S, Na, B, Cu, Fe, Mn, Zn, Mo, Cd and Pb was determined in spinach plants using ICP-OES technique, N-total by Kiejdahl method, while nitrates – with FIA technique.

Results: Introduction of iodine through fertigation proved to be much more effective in respect to

iodine biofortification of spinach than pre-sowing fertilization with this element (by 339% - 498%

with higher results obtained for KIO3 than KI). Soil fertilization with KI contributed to increased concentration of nitrates in spinach. Lowered content of N-total and Ca as well as higher accumulation of Fe were found in plants from combinations no. 2, 4 and 5. Generally, application of iodine in spinach cultivation (combinations 2-5) reduced Na and Zn content in plants. Pre-sowing fertilization with KI and KIO3 decreased Pb accumulation in spinach. No influence of iodine fertilization and fertigation was found in reference to the content of P, K, Mg, S, B, Cu, Mn, Mo and Cd in spinach plants.

Conclusions: Fertigation with iodine (particularly in the form of KIO3) turned out more effective for biofortification of spinach with this element than pre-sowing fertilization. Higher iodine contents were determined in plants treated with KIO3 than KI. Introduction of iodine to soil variously influenced mineral composition of spinach plants.

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S2.1. AGRONOMIC AND MOLECULAR APPROACHES TO REDUCE ACCUMULATION OF NON-NUTRITIVE ELEMENTS

(e.g. CADMIUM, ARSENIC etc.) IN FOOD CROPS

Steve P. McGrath and Fang-Jie Zhao Rothamsted Research, Harpenden, Herts AL5 2JQ, United Kingdom Keywords: accumulation, arsenic, cadmium, food, remediation

Cadmium and arsenic are non-essential for plants and harmful to human health. The European Food Standard Authority has recently called for reductions in the intake of these elements by humans from food. It is therefore important to understand the soil, agronomic and plant factors that influence the accumulation of Cd and As in the edible parts of food crops. Both elements may be present in agricultural soils at levels that are not phytotoxic but may lead to accumulation exceeding the current limits. Plant species vary widely in the accumulation of Cd and As. Some vegetables are accumulators of Cd whilst rice is particularly efficient in As accumulation. The bioavailability of both As and Cd is strongly influenced by the redox conditions of paddy soil; aerobic cultivation can greatly decrease As accumulation by rice but increase Cd accumulation. Recently, significant progress has been made in understanding the molecular mechanisms of As and Cd accumulation in rice. Arsenite, the predominant chemical species of As in anaerobic soil, is taken up by rice mainly through the silicon pathway; two Si transporters which are strongly expressed in rice roots are able to transport arsenite (Ma et al. 2008). Adding Si fertilizer can substantially decrease As accumulation by rice. A P1B-type ATPase, HMA3, has been identified as a Cd transporter localised to the tonoplast of root cells of rice (Ueno et al. 2010). Over-expression of this gene in rice enhances Cd sequestration in roots and reduces its translocation to shoots. Some indica cultivars of rice possess a non-functional allele of this gene and consequently a highly efficient Cd translocation from roots to shoots, a character that may be exploited for the phytoremediation purposes. These new findings open the way for developing strategies to decrease As and Cd transfer in the food chain.

References:

Ma JF, Yamaji N, Mitani N, Xu XY, Su YH, McGrath SP, Zhao FJ (2008) Transporters of arsenite in rice and their role in arsenic accumulation in rice grain. Proc Nat Acad Sci USA 105: 9931–9935 Ueno D, Yamajia N, Kono I, Huang CF, Ando T, Yano M, Ma JF (2010) Gene limiting cadmium accumulation in rice. Proc Nat Acad Sci USA 107: 16500-16505

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S2.2. HARVESTPLUS: GLOBAL ZINC FERTILIZER PROJECT

Ismail Cakmak

Sabanci University, Faculty of Engineering and Natural Sciences, Istanbul, Turkey Keywords: agronomic biofortification, HarvestPlus, nitrogen, zinc, wheat

Zinc deficiency is a global nutritional problem in crop plants and human populations. In general regions with Zn deficient soils coincide with the regions where Zn deficiency in humans is widespread. This highlights the importance of soil Zn deficiency in widespread occurrence of Zn deficiency in human populations. Since cereal grains have inherently low Zn concentrations, growing them on micronutrient deficient soils further decreases grain Zn and thus the daily intake of Zn through diets. Application of Zn-containing fertilizers (agronomic biofortification) offers a rapid solution to the problem, and represents an important complementary approach to genetic solutions. HarvestPlus Zinc Fertilizer Project, initiated on April 2008, evaluates the potential of various Zn-containing fertilizers for increasing Zn concentration of cereal grains and improving crop production in different target countries.

Based on the results to-date, grain Zn concentrations were significantly increased by foliar Zn applications while soil Zn applications was less effective. Wheat has been found to be the most promising cereal crop for increasing Zn in grains through foliar Zn fertilization. The trials also showed that N-nutritional status of plants appears to be a very critical factor in enrichment of grain with Zn. The project represents an excellent social responsibility project and a successful global partnership that seeks a short-term solution to the well-known Zn deficiency problem in human nutrition and also in crop production.

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S2.3. COPPER UPTAKE AND PHENOTYPIC PLASTICITY OF SUNFLOWER MUTANT LINES

Mench M.¹,Kolbas A.^1,2, Herzig R.³, Nehnevajova E.⁴

1UMR BIOGECO INRA 1202, Ecology of Communities, University of Bordeaux 1, Bât B8, RdC Est, avenue des Facultés, F-33405 Talence, France²Brest State University named after A.S. Pushkin, Boulevard of Cosmonauts, 21, 224016, Brest, Belarus; ³Phytotech Foundation, Quartiergasse 12, CH - 3013 Berne, Switzerland; ⁴Institute of Biology/Applied Genetics, Free University of Berlin, Albrecht-Thaer-Weg 6, DE - 14195 Berlin, Germany

Keywords: copper, phenotypic trait, soil solution, sunflower, uptake

Sunflower mutant lines have been selected for metal uptake using a fast-track breeding method (Nehnevajova et al. 2009). The purpose was to assess their Cu uptake and phenotypic plasticity vs.

total soil Cu and Cu concentration in soil pore water.

Sunflower cultivars and mutant lines were cultivated in 2008 and 2009 in field plots (n=31, 21-1170 Cu kg^-1,Gironde, SW France). Concurrently, pot experiments were carried out on soils from the plots and a soil series obtained by the fading technique (21-832 mg Cu kg^-1). Soil pore waters were collected using Rhizon MOM moister samplers.

(1) Shoot FW and DW, capitulum and seed yields, foliar and shoot elemental concentrations were determined. At high Cu exposure, Cu removal peaked in mutant lines and one commercial cultivar.

(2) (1 month-old) mutant line 1 [6^th generation, 1/67-35-190-04]: chlorophyll and carotenoid densities, stem length, water content, leaf, stem and root biomasses, leaf area and asymmetry were determined.

Cu uptake indicators were computed: TF (translocation factor: shoot Cu conc. vs. root Cu conc.) and BCF (bioconcentration factor: shoot Cu conc. vs. total soil Cu conc.). Phenotypic plasticity of sunflower mutant lines, and leaf and shoot Cu concentrations are discussed in relation to Cu exposure.

Reference:

Nehnevajova, E., Herzig, R., Bourigault, C., Bangerter, S., Schwitzguébel, J.P. 2009a. Stability of enhanced yield and metal uptake by sunflower mutants for improved phytoremediation. Int. J.

Phytorem. 4, 329–346.

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S2.4. SPECIATION AND SI INFLUENCE ON AS IN LETTUCE

Maria Greger, Arifin Sandhi and Tommy Landberg Department of Botany, Stockholm university, 106 91 Stockholm, SWEDEN

Among vegetables, lettuce has a high As accumulation. To humans, organic As like monomethylarsonate (MMA) and dimethylarsinate (DMA) is more harmful than the inorganic form, arsenite and arsenate. Arsenic speciation in lettuce was therefore focused on in this work. Furthermore, since silicon (Si) influences accumulation of toxic elements in plants the effect by Si on uptake and speciation of As in lettuce was investigated. Different experiments on uptake and effect of As in lettuce were performed under controlled conditions. Plants were treated with arsenite and arsenate with or without Si. The content of total As as well as As speciation in root and shoot was analysed. Arsenic was accumulated, more in roots than in shoots, especially when arsenite was added, and 48% was bound to cell walls. Silicon increased the shoot:root ratio of As when arsenite was added. In short term studies, Si decreased the uptake of As. Independently of how As was added, there was always higher concentration of arsenate than arsenite in the plant. Only when arsenate was added lettuce contained organic As, MMA but no DMA. Silicon decreased the content of MMA. Silicon addition decreased the As toxicity to the plant when arsenate was added.

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S2.5. EXPONENTIAL DECREASE IN THE CONCENTRATION OF ESSENTIAL (Cu, Zn, Fe, Mn) AND TOXIC (As, Cd, Pb) TRACE ELEMENTS IN SHOOTS

DURING THE DEVELOPMENT OF FIELD -GROWN MAIZE AND ITS CORRELATION WITH THE CONCENTRATION IN THE GRAIN

NGUYEN¹ Christophe, DENAIX¹ Laurence, BUSSIÈRE¹ Sylvie, COUDURE² Régis, HÉROULT³ Julien, PARAT³ Corinne, LESPES³ Gaëtane, POTIN-GAUTIER³ Martine,

DAUGUET⁴ Sylvie

1UMR 1220 INRA, TCEM, BP 71, F33883 Villenave d'Ornon, France, [email protected]; ²Maïzeurop - 21 chemin de Pau - F64121 Montardon France; ³Université de Pau et Pays de l'Adour/CNRS, LCABIE, UMR 5254 IPREM, 2 av. P.

Angot F64053 Pau cedex 9, France; ⁴CETIOM rue Monge, Parc industriel F33600, Pessac, France

Keyword: Crop quality, Maize, Model, Trace element accumulation

It is important to understand the dynamics of accumulation of essential and contaminant trace elements by food crops in order to improve both their quality and their safety. The same maize cultivar was planted in 4 different farms having contrasted soil conditions. The concentrations of essential (Cu, Zn, Fe, Mn) and toxic (As, Cd, Pb) elements in shoots and in grain were determined at 6 growth stages covering the entire cycle.

Whatever the trace element, the concentration in shoots decreased with growth indicating a dilution in biomass. The decrease was more important for toxic trace elements than for essential ones. The concentration in shoots was also correlated with the soil concentration of total metals in soil, with soil pH, soil carbon and soil Cation Exchange Capacity, the nature of the variable depending on the element. Hence, the shoot concentration in trace element can be described by the multiplicative exponential model Conc=α(Shoot biomass)^β(Soil variable)^γ with β<0.

The concentration of trace element in grain was not correlated with that of shoot except for As. The results suggest that trace element accumulation in grain significantly derived from the further root absorption and subsequent translocation in addition to possible remobilisation from shoots.

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S3.1. MOLECULAR BIOLOGICAL APPROACHES TO IMPROVING IRON CONCENTRATION OF FOOD CROPS

Elsbeth L. Walker¹

University of Massachusetts¹; University of Nebraska²; Dartmouth College³ , Brian M. Waters², and Heng-Hsuan Chu³

Keywords: Iron, Zinc, Copper, Yellow Stripe-Like, Nicotianamine

Objectives: An understanding of the biochemical and molecular mechanisms underlying plant nutrient transport and storage is necessary before safe and effective biofortification strategies can be implemented. To contribute to this understanding, we are characterizing the Yellow Stripe-Like (YSL) family of metal-nicotianamine (NA) transporters.

Methods: A double mutant that has null lesions in AtYSL1 and AtYSL3 displays strong chlorosis and iron deficiency. Reproduction is also severely affected in double mutant plants.

We have analyzed the metal content of double mutant plants, and furthermore have performed grafting experiments to understand the roles of YSL1 and YSL3 in seed production and seed metal content.

Results and Conclusions: The double mutants failed to mobilize zinc and copper from leaves. Seeds produced by the double mutant plants contained reduced levels of zinc and copper. This demonstrates a role for YSLs in the re-distribution of metals from shoots to seeds. We used inflorescence grafting of ysl1ysl3 scions to WT rosettes, predicting that seed development would occur normally, and that levels of metals would be restored to normal in seeds. However, while seed weight was restored by grafting, the levels of Fe, Zn and Cu in seeds remained low, indicating complex roles for YSLs in reproduction.

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S3.2. QUANTITATIVE LOCALIZATION OF IRON IN ARABIDOPSIS THALIANA SEEDS

Magali Schnell Ramos¹, Hicham Khodja², Sébastien Thomine¹

1Institut des Sciences du Végétal, CNRS, Gif-sur-Yvette, France; ²Laboratoire d'Etudes des Eléments Légers, UMR 3299 CEA/CNRS SIS2M, Gif-sur-Yvette, France

Keywords: elemental imaging, Fe, seed, µPIXE

Since plant seeds feed a large part of the global human population, the understanding of seed storage and remobilization processes of essential micronutrients, such as iron (Fe), manganese (Mn) or zinc (Zn) is crucial to improve seed nutritional quality. In particular, Fe deficiency is a major problem for agriculture as well as for human health. AtNRAMP3 and AtNRAMP4 are tonoplastic transporters involved in Fe and Mn vacuolar remobilization^1,2. The nramp3nramp4 double knockout mutant fails to remobilize vacuolar Fe stores and thus exhibits strong hypersensitivity to Fe deficiency during seed germination. To explore the mechanisms controlling Fe storage and remobilization in seeds, we have i) initiated a genetic screen for suppressors based on our knowledge of the mechanisms of Fe mobilization and ii) developed a quantitative elemental imaging method for seeds of the model plant Arabidopsis thaliana.

Previous imaging techniques of Arabidopsis seeds revealed that Fe localizes around provascular tissues but provided either non-quantitative data or approximate quantification of Fe concentrations in the different seed tissues^1,3,4. At the meeting, we will present the µPIXE (Particle-Induced X-ray emission with the use of focused ion beams) technique we used to localize and quantify Fe in Arabidopsis WT and nramp3nramp4 mutant seeds.

References:

1 Lanquar et al. (2005) EMBO J. 24, 4041-51

2 Lanquar et al. (2010) Plant Physiol 152, 1986-1999

3 Kim et al. (2006) Science 314, 1295-98

4 Roschzttardtz et al. (2009) Plant Physiol. 151, 1329-38

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S3.3. IDENTIFICATION OF ZINC-BINDING PROTEINS IN RICE ENDOSPERM

James Stangoulis, Georgia Guild and Eun Young Choi

School of Biological Sciences, Flinders University, Sturt Rd, Bedford Park, South Australia 5042, Australia

Keywords: proteomics, rice, zinc, zinc-binding protein

Unlike most cereals, rice is able to accumulate significant amounts of zinc (Zn) in the endosperm of its seed. The mechanism associated with this retention is still not known. The aim of this study was to identify Zn-binding proteins in the endosperm of rice which would contribute toward a physiological dissection of the mechanism of seed endosperm, Zn accumulation. The concentration of Zn present in the endosperm of rice was correlated (r = 0.65) to the concentration of protein bound sulfhydryl groups. Zn-containing protein fractions in rice endosperm were identified by size-exclusion (SE) - Fast Protein Liquid Chromatography (FPLC), anion-exchange (AE)-FPLC, and ICP-OES (Inductively Coupled Plasma-Optical Emission Spectrometry). After the fractions were separated by SDS-PAGE, a proteomics approach identified eighteen putative Zn-binding proteins. FPLC analysis was also conducted on grains with high and low Zn concentration to enable a comparative analysis. The absorbance at 254 nm (metal-thiolate charge transfer spectra) of the Zn- containing peak was 2-fold greater in the Zn-dense genotype. Results suggest that an increase in Zn-thiolate formation within protein structures may be associated with a higher density of Zn in rice grains.

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S3.5. LEARNING FROM EXTREMES: ELEVATED LEVELS OF NICOTIANAMINE ARE IMPORTANT FOR ZINC ACCUMULATION IN Arabidopsis halleri

Ulrich Deinlein¹, Michael Weber¹, Ina Talke², Ute Krämer³, Holger Schmidt¹, Stefan Rensch¹, Aleksandra Trampczynska¹, Stephan Clemens¹

1Department of Plant Physiology, University of Bayreuth, Germany

2Max-Planck Institute for Molecular Plant Physiology, Golm, Germany

3Department of Plant Physiology, Ruhr University Bochum, Germany

A promising approach to dissect Zn distribution and Zn accumulation pathways in plants focuses on the ability of certain plant species to hyperaccumulate Zn. One such plant is Arabidopsis halleri, a close relative of A. thaliana, and growing, for instance, in old mining areas in Central Europe. Comparative transcriptome studies revealed that key homeostatic processes such as Zn transport and Zn chelation are regulated differently in metal hyperaccumulators. Among these metal hyperaccumulation candidate genes are nicotianamine synthase (NAS) genes. Their higher expression in A. halleri roots translates into elevated levels of nicotianamine (NA), a chelator of transition metal ions. We confirmed this for several A. halleri populations from both metallicolous and non-metallicolous sites. Recently, we found evidence for in vivo formation of NA-Zn complexes. In order to determine the role of NAS genes for metal hyperaccumulation we generated A. halleri RNAi lines. They show strongly reduced nicotianamine levels in their roots. A. halleri lines with strong suppression of NAS2 transcript levels translocate significantly less Zn into the shoots. This finding supports the hypothesis that nicotianamine keeps Zn ions mobile in the root for loading into the xylem and plays a key role in governing Zn accumulation rates in plants.

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S3.6. CADMIUM EFFLUX FROM THE ROOT APPEARS TO BE A MAJOR DETERMINANT CONTROLLING CADMIUM ACCUMULATION

IN LETTUCE LEAVES

Walid ZORRIG^1;2, Catherine SARROBERT³, Aïda ROUACHED¹, Brigitte MAISONNEUVE⁴, Jean-Claude DAVIDIAN¹, Chedly ABDELLY², Pierre BERTHOMIEU¹

1Biochimie & Physiologie Moléculaire des Plantes, UMR CNRS-INRA-Université Montpellier II-Montpellier SupAgro, Place Viala, 34060 Montpellier Cédex 1, France;

2Laboratoire des Plantes Extrêmophiles, Centre de Biotechnologie, BP 901, Hammam-Lif 2050, Tunisie; ³Groupe de Recherches Appliquées en Phytotechnologie, DEVM, CEN Cadarache, 13108 St Paul les Durance, France; ⁴Génétique et Amélioration des Fruits et Légumes, UR INRA, 1052 Domaine St Maurice, BP 94, 84143 Montfavet Cédex, France Keywords: cadmium accumulation, cadmium transport, food security, lettuce

We aim at characterizing mechanisms controlling cadmium accumulation in lettuce, which is a food crop showing one of the highest capacities to accumulate this toxic compound.

Starting from a phenotypic and transcriptomic analysis of 18 lettuce genotypes representing the genetic diversity of the species, we identified trends linking cadmium and zinc accumulation in roots an shoots, cadmium translocation from roots to shoot, cadmium tolerance, and expression of a dozen of candidate genes proposed to be involved in cadmium transport. A positive correlation linked cadmium accumulation and the mRNA accumulation of an heavy metal efflux transporter.

Interestingly, measurements of ¹⁰⁹Cd influx in roots and ¹⁰⁹Cd efflux from roots revealed that the genotype displaying the least cadmium accumulation could be discriminated from the genotype displaying the highest one by its markedly increased ability to efflux cadmium from the roots to the culture medium. Progeny analysis from crosses between the genotypes displaying extreme performances for cadmium accumulation or cadmium translocation from roots to shoots revealed that these traits could be supported by single genetic determinism.

In conclusion, starting from the analysis of cadmium accumulation in a large set of lettuce genotypes, a major determinant limiting cadmium accumulation in this species was revealed, that is the ability to efflux cadmium from the root to the culture medium. A candidate gene has been identified for this function, which paves the way towards the breeding for lettuce with reduced cadmium accumulation in leaves.

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S3.7. TOLERANCE AND DETOXIFICATION MECHANISMS OF ARSENIC IN PLANTS: ROLE OF ANTIOXIDANT ENZYMES

Austruy A.^1,2, Gauthier Moussard C.¹, Vernay P.¹, Goupil P.², Ledoigt G.², Hitmi A.¹

1Clermont Universités, IUT Clermont Ferrand, Laboratoire de Physiologie et Biotechnologies Végétales, 100 rue de l’Egalité, F-15000 Aurillac Cedex, France

2PIAF UMR 547 Université-INRA, Physiologie et Génétique Végétales, Université Blaise-Pascal, 24 Avenue des Landais, 63177 Aubière Cedex, France

[email protected]

The soil is an essential resource for human societies and ecosystems. In the context, the preservation of soils and the rehabilitation of polluted sites represent one of the major challenges of sustainable development policy. The human activity, responsible for an increasing contamination of water and soils, leads to structural and functional perturbations of ecosystems. The plants, because of their immobility, are particularly exposed. It is interesting to look into the toxicity of heavy metals and metalloids on vegetation and the tolerance mechanisms to their adaptation and development on contaminated soil. This study focuses on the impact of arsenic in plants and defence mechanisms brought into play by them to grow and maintain in presence of the pollutant. Indeed, despite all the interest in arsenic by the scientific community, the action mechanisms of this metalloid, as well as those that determine its penetration into the plant or cells, are little known. Previous works have shown an impact of arsenic on the development and physiological activity of plants characterizing the toxicity from this metal in them. This work concentrates about tolerance mechanisms of sentinel plant species, Vicia faba, sensible of arsenic, cultivated in hydroponic solution in presence of inorganic arsenic (arsenite and arsenate). The early response of plants to increasing exposure to inorganic As was used to determine the effects of this metal to short time of exposure with a special:

- the uptake mechanisms of arsenic and its translocation to aerial parts (genes expression coding for metallothionein and phytochelatins involved in the detoxification and sequestration of metalloid, enzyme activities of phytochelatin synthase and arsenate reductase);

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- the evolution of three stress bioindicators, the content of ascorbic acid, the lipid peroxidation and the gaz exchange activities;

- the response of the plant to this exposure with the study of the activity of various antioxidant enzymes (SOD, catalase, glutathione reductase), and the expression of genes coding for these enzymes.

This study focuses therefore to highlight the role of antioxidant metabolism in the detoxification and adaptation of plants to arsenic. In the sensible specie, the high production of ROS causes extensive damage (lipid peroxidation, DNA breaks, inhibition of physiological activity). Parallel to these perturbations, modulations of antioxidant enzyme activities were observed, notably an increase of catalase and SOD activity. All alterations of enzyme activities studied are discussed in relation with the gene expression coding for these enzymes and the perturbations observed at the physiological level.

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S3.8. INSIGHTS INTO THE IONOMIC AND TRANSCRIPTOMIC RESPONSES OF PLANTS TO MAGNESIUM AVAILABILITY

Hermans C and Verbruggen N

Lab of Plant Physiology and Molecular Genetics, Université Libre de Bruxelles, Belgium [email protected]

Mg is one of the nine essential macronutrients that plants utilize in large quantities for their growth. In addition, Mg is the 4^th most common cation in the human body and half of its dietary intake is from plant origin. Mg deficiency is a widespread problem, affecting productivity and quality in agriculture [1]. In many plants, the earliest symptoms observed within days to weeks consist of an impairment in sugar partitioning and an enhancement of antioxidative mechanisms [2,3]. We are currently using two approaches to expand knowledge on the mechanisms underlying Mg homeostasis in Arabidopsis thaliana, a domain relatively unexplored up to date [4]. Our experimental outlines are (i) the exploitation of the Mg content variation and (ii) the identification of transcriptome changes related to Mg availability. First, we studied the natural variation in accessions as a source of biodiversity to find new genes and new alleles controlling Mg content in tissues. Second, in a thorough description of the early transcriptomic responses to Mg deprivation, we recently documented the perturbation of the central oscillator of the circadian clock, and the triggering of the ethylene biosynthetic pathway and the photoprotection of the photosynthetic apparatus [5,6]. Through the understanding of Mg homeostasis mechanisms, this work may provide new tools to biofortify Mg concentration and to improve the tolerance to Mg deficiency in Brassica crops.

References:

[1] Bennett WF (1997) Nutrients Deficiencies & Toxicities in Crop Plants. APS Press, The American Phythopathological Society.

[2] Çakmak I. and Kirkby E. (2008) Physiol. Plant. 133: 692-704.

[3] Hermans C. and Verbruggen N. (2005) J. Exp. Bot. 56, 2153-2161.

[4] Hermans C. and Verbruggen N. (2008) Dietary Magnesium: New Research. ISBN 978-1- 60692-109-8, pg 159-175.

[5] Hermans C. et al. (2010) New Phytol. 187, 119-131.

[6] Hermans C. et al. (2010) New Phytol. 187, 132-144.

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S4.1. ENHANCED CONTENT AND BIOAVAILABILITY OF IRON AND ZINC IN THE ENDOSPERM OF RICE SEEDS BY ACTIVATION-TAGGING OF

NICOTIANAMINE SYNTHASE

Jan K. Schjoerring¹

Un Sil Jeon², Yoon-Keun Kim² and Gynheung An²

, Sichul Lee², Daniel P. Persson¹, Søren Husted¹, Thomas H. Hansen¹,

1University of Copenhagen, Faculty of Plant Sciences, Plant and Soil Science Laboratory, Thorvaldsensvej 40, DK-1871 Frederiksberg C; ²Department of Life Science, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Republic of Korea.

e-mail: [email protected]

Iron (Fe) and zinc (Zn) deficiency in human populations is a widespread problem, particularly in the developing world. This has stressed the need for development of new strategies aiming at improving the content and bio-availability of these elements in the edible part of the cereal grain; i.e. the endosperm. We have used activation tagging and ectopic over-expression of nicotianamine synthase genes NAS2 and NAS3 to generate rice lines with increased content of nicotianamine which is a key ligand involved in metal transport and homeostasis in plants (Lee et al. 2009). Our results demonstrate for the first time that not only the content but also the bioavailability of Zn in rice grain can be improved by the use of biotechnology. A further novel aspect is that Zn bio-fortification can be achieved without an accompanying increase in cadmium content and bioavailability.

Rice seeds harvested from the activation-tagged rice plants had elevated amounts of both Fe (2.9-fold) and Zn (2.2-fold), as well as NA (9.6 fold). The plants also showed an increased tolerance to Fe and Zn deficiencies as well as to Zn, Cu and Ni toxicities.

Furthermore, when fed to anemic mice, Fe and Zn deficiencies were ameliorated within two weeks, whereas the mice fed with WT seeds remained anemic.

In order to elucidate the chemical speciation of Fe and Zn, the rice seeds were milled, and the remaining endosperm was extracted in an aqueous buffer prior to analysis by size exclusion chromatography hyphenated to an inducively coupled plasma mass spectrometer (SEC-ICP-MS; Persson et al. 2009). Compared to the WT, the seeds of the activation-

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tagged lines contained 7-fold more Fe and 16-fold more Zn bound to a low molecular weight complex. The elemental peaks were collected, lyophilized and re-injected onto a hydrophilic interaction liquid chromatography (HILIC) column, coupled to ESI-TOF-MS.

The mass spectrum confirmed that the main compound found in the collected Fe and Zn peaks indeed was nicotianamine. In addition, deoxymugineic acid (DMA), a compound traditionally regarded as a phytosiderophore in grasses, was present. Since NA and DMA follow the same biosynthetic pathway, elevated content of DMA is not surprising. The content of Cu, and Ni associated with the NA/DMA pool was increased 9.7 and 2.4-fold relative to the WT. In contrast, this pool did not contain an increased level of cadmium.

It is concluded that over-expression of rice NAS genes can increase the content and bioavailability of Fe and Zn in the endosperm of rice grain. This approach may prove to be a successful strategy in the battle against global Fe and Zn malnutrition.

References:

Lee S, Jeon US, Lee SJ, Kim YK, Persson DP, Husted S, Schjorring JK, Kakei Y, Masuda H, Nishizawa NK, An G 2009 Iron fortification of rice through activation of the nicotianamine synthase gene. Proceedings of the National Academy of Sciences of the United States of America 106, 22014-22019.

Persson DP, Hansen TH, Laursen KH, Schjoerring JK, Husted S 2009 Simultaneous iron, zinc, sulphur and phosphorus analysis of barley grain tissues using SEC-ICP-MS and IP- ICP-MS. Metallomics 1, 418-426.

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S4.2. BIOFORTIFICATION IN COMMON BEAN: IDENTIFICATION AND CHARACTERISATION OF A LPA (LOW PHYTIC ACID) MUTANT

Panzeri D¹, Cassani E², Tagliabue G¹, Daminati MG¹, Doria E³, Bollini R¹, Nielsen E³, Pilu R², Sparvoli F¹

1Istituto di Biologia e Biotecnologia Agraria, CNR, Milano, Italy;

2Dipartimento di Produzione Vegetale, Università degli Studi di Milano, Milan, Italy;

3Dipartimento di Genetica e Microbiologia, Università di Pavia, Pavia, Italy;

Keywords: myo-inositol, Phaseolus vulgaris, phytic acid transporter, seed

Phytic acid (InsP₆) represents the main phosphorous fraction of plant seeds, which is undigested by monogastric animals ad thus largely found in manure. It’s reduction in feed would be beneficial for both productivity and environmental aspect. InsP₆ is also the main responsible for poor micronutrient bioavailability to humans, in fact, it chelates various mineral cations (i.e., calcium, iron, zinc), resulting in micronutrient deficiencies. In common bean, InsP₆ content is a serious problem for human nutrition, particularly in developing countries.

Screening and EMS mutagenised population, we have identified a lpa (low phytic acid) 280- 10 line that carries a monogenic recessive mutation conferring a 90% reduction of InsP6

content. Differently from other lpa mutants, the bean lpa-280-10 apparently does not display negative pleiotropic effects associated to the mutation.

Using SSRs markers, the mutation was mapped on the bean chromosome 1, in a region that resulted syntenic to soybean chromosomes 19 and 3, where the lpa mutant CX1834 was previously mapped and found to be in an ATP-binding cassette (ABC).

Our transcriptional and metabolic analysis of InsP6 pathway demonstrate the existence of a negative feedback mechanism of InsP6 acting on genes coding for key enzymes of phytic acid pathway and leading to a 30% reduction of myo-inositol. We show that the reduced content of myo-inositol is responsible of the ABA hypersensitive response found in lpa-280-10 seed germination, indicating a key role of this metabolite in the many pleiotropic effects often reported for lpa mutants.

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S4.3. IN VITRO DIGESTION MODEL OF FOOD COMPONENTS BY USING HUMAN GASTRIOINTESTINAL ENZYMES

Gerd E. Vegarud

Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, P. Box 1432-Ås, Norway

In order to mimic human digestion, an in vitro model to degrade raw food, processed food or single food components using human gastrointestinal enzymes has been developed. The digestion is performed in the two following steps; Step 1 by human gastric juice (HGJ) at pH 2.0 (or pH 4 or pH 6) and Step 2. using human duodenal juice (HDJ) at pH 7-8/370C. The human juices aspirated from 20 individuals have been charcterised with the following parameters HGJ: pH, volume and pepsin activity (U) and HDJ: pH, total proteolytic (U), lipase (U), amylase (U) activities, bile salts (mg/ml), bilirubin and total protein content.

A starting food sample food of is solubilized then HGJ is added and pH is adjusted to the gastic pH of 2 or 4 or to simulated adults or infants stomach at 370C under continues stirring. At different time 5- 10-20-30-60 min samples are taken out and used for further analysis. Futher digestion in step 2 as comparable to the upper human duodenal tract is done by djusting pH to 7-8 and adding HDJ for 60min. more. By protein, mineral, fat or polysaccharide analysis during and after digestion it is possible to understand the fate of degradation of components as protein degradation (SDS-PAGE) or peptide formation/identification (Lc-MS, Maldi-MS etc), stability of fatty acids (GC-MS, Lc-MS etc), mineral Fe release etc. and degradation of polysaccharides. This model digestion may be useful in selecting new food/feed varieties or in designing spesific components in food products.

References:

Govasmark E., A. B.Kjelsen, J. Szpunar, K. Bierla, G. Vegarud and B. Salbu (2010). Bioaccessibilty of Selenium from Se-enriched wheat and chicken meat. Pure and Applied Chemistry Vol 82, No 2;

461-471

Eriksen E, Halvor H, Jensen E and G. Vegarud (2010) Different digestion of caprine whey proteins by human and porcine gastrointestinal enzymes. Brit J Nutr 104 (3), 374-381

H. Almås, H. Holm, Ragnar Flengsrud,Thor Langsrud and G. Vegarud (2006) . Model Digestion of whey proteins by human gastric and duodenal juice. British Journal of Nutrition, 96 (3) 562-569