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Ascorbate Efflux as a New Strategy for Iron Reduction and Transport in Plants*

  • Louis Grillet
    Footnotes
    Affiliations
    Laboratoire de Biochimie et Physiologie Moléculaire des Plantes, Institut de Biologie Intégrative des Plantes, Centre National de la Recherche Scientifique (UMR5004), Institut National de la Recherche Agronomique, Université Montpellier II, Ecole Nationale Supérieure d'Agronomie, 34060 Montpellier Cedex 2, France and
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  • Laurent Ouerdane
    Footnotes
    Affiliations
    the Laboratoire de Chimie Analytique Bio-Inorganique et Environnement, Institut Pluridisciplinaire de Recherche sur l'Environnement et les Matériaux, Centre National de la Recherche Scientifique (UMR5254), Université de Pau et des Pays de l'Adour, 64063 Pau Cedex 9, France
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  • Paulina Flis
    Affiliations
    the Laboratoire de Chimie Analytique Bio-Inorganique et Environnement, Institut Pluridisciplinaire de Recherche sur l'Environnement et les Matériaux, Centre National de la Recherche Scientifique (UMR5254), Université de Pau et des Pays de l'Adour, 64063 Pau Cedex 9, France
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  • Minh Thi Thanh Hoang
    Affiliations
    Laboratoire de Biochimie et Physiologie Moléculaire des Plantes, Institut de Biologie Intégrative des Plantes, Centre National de la Recherche Scientifique (UMR5004), Institut National de la Recherche Agronomique, Université Montpellier II, Ecole Nationale Supérieure d'Agronomie, 34060 Montpellier Cedex 2, France and
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  • Marie-Pierre Isaure
    Affiliations
    the Laboratoire de Chimie Analytique Bio-Inorganique et Environnement, Institut Pluridisciplinaire de Recherche sur l'Environnement et les Matériaux, Centre National de la Recherche Scientifique (UMR5254), Université de Pau et des Pays de l'Adour, 64063 Pau Cedex 9, France
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  • Ryszard Lobinski
    Affiliations
    the Laboratoire de Chimie Analytique Bio-Inorganique et Environnement, Institut Pluridisciplinaire de Recherche sur l'Environnement et les Matériaux, Centre National de la Recherche Scientifique (UMR5254), Université de Pau et des Pays de l'Adour, 64063 Pau Cedex 9, France
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  • Catherine Curie
    Affiliations
    Laboratoire de Biochimie et Physiologie Moléculaire des Plantes, Institut de Biologie Intégrative des Plantes, Centre National de la Recherche Scientifique (UMR5004), Institut National de la Recherche Agronomique, Université Montpellier II, Ecole Nationale Supérieure d'Agronomie, 34060 Montpellier Cedex 2, France and
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  • Stéphane Mari
    Correspondence
    To whom correspondence should be addressed: Laboratoire de Biochimie et Physiologie Moléculaire des Plantes, Institut de Biologie Intégrative des Plantes, Centre National de la Recherche Scientifique (UMR5004), Institut National de la Recherche Agronomique, Université Montpellier II, Ecole Nationale Supérieure d'Agronomie, 34060 Montpellier Cedex 2, France. Tel.: 33-4-99-61-25-72; Fax: 33-4-67-52-57-37
    Affiliations
    Laboratoire de Biochimie et Physiologie Moléculaire des Plantes, Institut de Biologie Intégrative des Plantes, Centre National de la Recherche Scientifique (UMR5004), Institut National de la Recherche Agronomique, Université Montpellier II, Ecole Nationale Supérieure d'Agronomie, 34060 Montpellier Cedex 2, France and
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  • Author Footnotes
    * This work was supported by the Centre National de la Recherche Scientifique, by l'Institut National de la Recherche Agronomique, and by Agence Nationale pour la Recherche Grants 23643 (CIDS) and 07-3-18-8-87 (DISTRIMET).
    1 Both authors contributed equally to this work.
    2 Supported by a Ph.D. fellowship from the Plant Biology Department of the Institut National de la Recherche Agronomique.
Open AccessPublished:December 17, 2013DOI:https://doi.org/10.1074/jbc.M113.514828
      Iron (Fe) is essential for virtually all living organisms. The identification of the chemical forms of iron (the speciation) circulating in and between cells is crucial to further understand the mechanisms of iron delivery to its final targets. Here we analyzed how iron is transported to the seeds by the chemical identification of iron complexes that are delivered to embryos, followed by the biochemical characterization of the transport of these complexes by the embryo, using the pea (Pisum sativum) as a model species. We have found that iron circulates as ferric complexes with citrate and malate (Fe(III)3Cit2Mal2, Fe(III)3Cit3Mal1, Fe(III)Cit2). Because dicotyledonous plants only transport ferrous iron, we checked whether embryos were capable of reducing iron of these complexes. Indeed, embryos did express a constitutively high ferric reduction activity. Surprisingly, iron(III) reduction is not catalyzed by the expected membrane-bound ferric reductase. Instead, embryos efflux high amounts of ascorbate that chemically reduce iron(III) from citrate-malate complexes. In vitro transport experiments on isolated embryos using radiolabeled 55Fe demonstrated that this ascorbate-mediated reduction is an obligatory step for the uptake of iron(II). Moreover, the ascorbate efflux activity was also measured in Arabidopsis embryos, suggesting that this new iron transport system may be generic to dicotyledonous plants. Finally, in embryos of the ascorbate-deficient mutants vtc2-4, vtc5-1, and vtc5-2, the reducing activity and the iron concentration were reduced significantly. Taken together, our results identified a new iron transport mechanism in plants that could play a major role to control iron loading in seeds.

      Introduction

      Iron is an essential micronutrient for plants. It is used as an enzymatic cofactor in a wide range of metabolic processes, such as photosynthetic and respiratory electron transfer reactions, reduction of nitrate and sulfate, synthesis of fatty acids, and branched amino acids. Iron was selected on the basis of the redox capacity of the Fe2+/Fe3+ couple that can exchange one electron. This chemical property is also responsible for the potential toxicity of iron in excess conditions because ferrous ions can promote, in the presence of oxygen, the generation of reactive oxygen species and oxidative stress. Plants, as sessile organisms, must maintain a strict iron balance to cope with deficiency or excess conditions. At the whole plant level, this is achieved by the fine regulation of root absorption, allocation to the aerial parts, storage, and remobilization.
      Although very abundant in soils, iron is often poorly available to plants because the main iron form, iron(III), has a very limited solubility and is prone to precipitate in alkaline and neutral conditions. Therefore, plants have developed strategies to efficiently acquire iron. In roots of dicotyledonous species, such as the model plant Arabidopsis thaliana, the uptake of iron requires the acidification of the rhizosphere by the proton pumping activity of the ATPase AHA2 (
      • Santi S.
      • Schmidt W.
      Dissecting iron deficiency-induced proton extrusion in Arabidopsis roots.
      ) to release Fe3+ bound to humic substances and mineral phases, the reduction of ferric ions by the ferric chelate reductase encoded by FRO2 (
      • Robinson N.J.
      • Procter C.M.
      • Connolly E.L.
      • Guerinot M.L.
      A ferric-chelate reductase for iron uptake from soils.
      ),
      The abbreviations used are: FRO
      ferric reductase oxidase
      NA
      nicotianamine
      YSL
      yellow stripe 1-like
      ESL
      embryo sac liquid
      XANES
      x-ray absorption near edge structure
      ESI
      electrospray ionization
      BPDS
      bathophenanthroline disulfonate
      AOX
      ascorbate oxidase
      Cit
      citrate
      Mal
      malate
      VTC
      vitamin C
      HILIC
      hydrophilic interaction liquid chromatography
      ICP
      inductively coupled plasma.
      and the subsequent uptake of Fe2+ by the metal transporter IRT1 (
      • Eide D.
      • Broderius M.
      • Fett J.
      • Guerinot M.L.
      A novel iron-regulated metal transporter from plants identified by functional expression in yeast.
      ,
      • Henriques R.
      • Jásik J.
      • Klein M.
      • Martinoia E.
      • Feller U.
      • Schell J.
      • Pais M.S.
      • Koncz C.
      Knock-out of Arabidopsis metal transporter gene IRT1 results in iron deficiency accompanied by cell differentiation defects.
      ,
      • Varotto C.
      • Maiwald D.
      • Pesaresi P.
      • Jahns P.
      • Salamini F.
      • Leister D.
      The metal ion transporter IRT1 is necessary for iron homeostasis and efficient photosynthesis in Arabidopsis thaliana.
      ,
      • Vert G.
      • Grotz N.
      • Dédaldéchamp F.
      • Gaymard F.
      • Guerinot M.L.
      • Briat J.F.
      • Curie C.
      IRT1, an Arabidopsis Transporter essential for iron uptake from the soil and for plant growth.
      ).
      When in root cells, iron atoms must reach the vascular system and enter the xylem vessels to be transported to the aerial parts. Xylem loading is controlled, at least in part, by the FRD3 protein that belongs to the multidrug and toxic compound extrusion family of transporters (
      • Green L.S.
      • Rogers E.E.
      FRD3 controls iron localization in Arabidopsis.
      ). FRD3 is a citrate effluxer expressed in the root pericycle, whose activity is required to solubilize iron in the xylem sap (
      • Durrett T.P.
      • Gassmann W.
      • Rogers E.E.
      The FRD3-mediated efflux of citrate into the root vasculature is necessary for efficient iron translocation.
      ), where citrate was identified as the main iron ligand (
      • Rellán-Alvarez R.
      • Giner-Martínez-Sierra J.
      • Orduna J.
      • Orera I.
      • Rodríguez-Castrillón J.A.
      • García-Alonso J.I.
      • Abadía J.
      • Alvarez-Fernández A.
      Identification of a tri-iron(III), tri-citrate complex in the xylem sap of iron-deficient tomato resupplied with iron. New insights into plant iron long-distance transport.
      ), hence promoting iron movement into the xylem. In leaves, unloading of iron from xylem vessels and distribution in the surrounding cells also requires FRD3 (
      • Roschzttardtz H.
      • Séguéla-Arnaud M.
      • Briat J.F.
      • Vert G.
      • Curie C.
      The FRD3 citrate effluxer promotes iron nutrition between symplastically disconnected tissues throughout Arabidopsis development.
      ). Likewise, the oligopeptide transporter OPT3 (
      • Stacey M.G.
      • Patel A.
      • McClain W.E.
      • Mathieu M.
      • Remley M.
      • Rogers E.E.
      • Gassmann W.
      • Blevins D.G.
      • Stacey G.
      The Arabidopsis AtOPT3 protein functions in metal homeostasis and movement of iron to developing seeds.
      ) contributes to iron movement in leaves, but its substrate, whether it is an iron ligand or an iron complex, is still unknown (
      • Stacey M.G.
      • Patel A.
      • McClain W.E.
      • Mathieu M.
      • Remley M.
      • Rogers E.E.
      • Gassmann W.
      • Blevins D.G.
      • Stacey G.
      The Arabidopsis AtOPT3 protein functions in metal homeostasis and movement of iron to developing seeds.
      ). To date, the number of organic molecules shown to form complexes with iron in vivo is extremely limited. Beside citrate, most of the information concerns nicotianamine (NA). This aminopropyl polymer, enzymatically synthesized from S-adenosylmethionine, has a high affinity for iron, copper, and zinc. Several studies have shown that the chemical properties and the binding capacity of NA make it an ideal ligand for iron in neutral, cytosolic conditions, whereas in acidic, apoplastic conditions, citrate will prevail (
      • von Wiren N.
      • Klair S.
      • Bansal S.
      • Briat J.F.
      • Khodr H.
      • Shioiri T.
      • Leigh R.A.
      • Hider R.C.
      Nicotianamine chelates both FeIII and FeII. Implications for metal transport in plants.
      ,
      • Rellán-Alvarez R.
      • Abadía J.
      • Alvarez-Fernández A.
      Formation of metal-nicotianamine complexes as affected by pH, ligand exchange with citrate and metal exchange. A study by electrospray ionization time-of-flight mass spectrometry.
      ). In planta, NA is involved in the root-to-shoot transport of copper in tomato (
      • Pich A.
      • Scholz G.
      Translocation of copper and other micronutrients in tomato plants (Lycopersicon esculentum Mill). Nicotianamine-stimulated copper transport in the xylem.
      ) and nickel and zinc in the hyperaccumulator species Thlaspi caerulescens (
      • Mari S.
      • Gendre D.
      • Pianelli K.
      • Ouerdane L.
      • Lobinski R.
      • Briat J.F.
      • Lebrun M.
      • Czernic P.
      Root-to-shoot long-distance circulation of nicotianamine and nicotianamine-nickel chelates in the metal hyperaccumulator Thlaspi caerulescens.
      ,
      • Trampczynska A.
      • Küpper H.
      • Meyer-Klaucke W.
      • Schmidt H.
      • Clemens S.
      Nicotianamine forms complexes with Zn(II) in vivo.
      ). For nickel, the formation of a complex between nicotianamine and nickel in xylem exudates has been demonstrated unambiguously by the coupling of chromatography and mass spectrometry (
      • Mari S.
      • Gendre D.
      • Pianelli K.
      • Ouerdane L.
      • Lobinski R.
      • Briat J.F.
      • Lebrun M.
      • Czernic P.
      Root-to-shoot long-distance circulation of nicotianamine and nicotianamine-nickel chelates in the metal hyperaccumulator Thlaspi caerulescens.
      ,
      • Ouerdane L.
      • Mari S.
      • Czernic P.
      • Lebrun M.
      • Lobinski R.
      Speciation of non-covalent nickel species in plant tissue extracts by electrospray Q-TOF MS/MS after their isolation by 2D size-exclusion-hydrophilic interaction LC (SEC-HILIC) monitored by ICP MS.
      ).
      In A. thaliana, several genetic approaches have allowed to pinpoint the role of NA in the transport of iron. The impairment of NA production, through the inactivation of all four NA synthase-encoding genes, provokes the expected phenotype of interveinal chlorosis described for the NA-free tomato mutant chloronerva and, more interestingly, a reduced accumulation of iron in seeds (
      • Klatte M.
      • Schuler M.
      • Wirtz M.
      • Fink-Straube C.
      • Hell R.
      • Bauer P.
      The analysis of Arabidopsis nicotianamine synthase mutants reveals functions for nicotianamine in seed iron loading and iron deficiency responses.
      ). Such an impact on seeds has also been reported in mutants of the yellow stripe 1-like (YSL) family of NA-metal transporters. The mutation in AtYSL1 results in decreased iron and NA accumulation in seeds (
      • Le Jean M.
      • Schikora A.
      • Mari S.
      • Briat J.F.
      • Curie C.
      A loss-of-function mutation in AtYSL1 reveals its role in iron and nicotianamine seed loading.
      ), most likely caused by a reduced remobilization from old leaves, as shown for the Arabidopsis ysl1ysl3 double mutant (
      • Le Jean M.
      • Schikora A.
      • Mari S.
      • Briat J.F.
      • Curie C.
      A loss-of-function mutation in AtYSL1 reveals its role in iron and nicotianamine seed loading.
      ,
      • Waters B.M.
      • Chu H.H.
      • Didonato R.J.
      • Roberts L.A.
      • Eisley R.B.
      • Lahner B.
      • Salt D.E.
      • Walker E.L.
      Mutations in Arabidopsis yellow stripe-like1 and yellow stripe-like3 reveal their roles in metal ion homeostasis and loading of metal ions in seeds.
      ). Taken together, these results indicate that disturbing the long distance transport of iron has a major impact on the process of seed loading (reviewed in Ref.
      • Walker E.L.
      • Waters B.M.
      The role of transition metal homeostasis in plant seed development.
      ). Seeds represent the most important sink for plants and the end point of long distance circulation of nutrients (
      • Patrick J.W.
      Phloem unloading. Sieve element unloading and post-sieve element transport.
      ,
      • Patrick J.W.
      • Offler C.E.
      Compartmentation of transport and transfer events in developing seeds.
      ). Virtually nothing is known about the transport pathway that leads to iron loading in the seeds and on the chemical forms (i.e. the speciation) of iron during this process. The speciation of a metal ion in a specific biological compartment is crucial information to further understand how the metal is kept soluble, transported across membranes, and delivered to its final target.
      In this work, the pea (Pisum sativum) was chosen as a biochemical model to study the mechanisms of iron acquisition by the embryo. Grain legumes are particularly suited to study the processes of transport in seeds. One specific feature of these seeds is that the bulk flow of nutrients delivered by the seed coat accumulates in large amounts, forming a liquid endosperm also termed embryo sac liquid (ESL). This particular liquid is of high biological importance because it serves directly to feed the embryo (
      • Van Dongen J.T.
      • Ammerlaan A.M.
      • Wouterlood M.
      • Van Aelst A.C.
      • Borstlap A.C.
      Structure of the developing pea seed coat and the post-phloem transport pathway of nutrients.
      ). We first analyzed the speciation of iron in isolated ESL using chromatography coupled to mass spectrometry and synchrotron radiation x-ray absorption spectroscopy. This information was crucial to further characterize the transport machinery that is expressed at the embryo surface to acquire iron from maternal tissues.

      EXPERIMENTAL PROCEDURES

      Plant Material

      Pea (P. sativum L., Dippes Gelbe Viktoria, DGV, cultivar) and A. thaliana (cultivar Col-0) were used in this study. Plants were grown in a greenhouse at 23 °C, in 3-liter pots containing 0.5 liter of quartz sand and 2.25 liters of Humin substrate N2 Neuhaus (Klasmann-Deilmann, Geeste, Germany) irrigated with tap water. Plants used for pea root ferric-chelate reductase measurement were grown hydroponically for 15 days in a climate chamber at 20 °C, 70% hygrometry, 16 h light/8 h dark, and 250 μmol photons/m2/s of photosynthetically active radiation. The nutrient solution contained 1.25 mm KNO3, 1.5 mm Ca(NO3)2, 0.75 mm MgSO4, 0.5 mm KH2PO4, 25 μm H3Bo3, 2 μm MnSO4, 2 μm ZnSO4, CuSO4, Na2MoO4, and 0.1 μm NiCl2, buffered pH 5 with 1 mm 2-(N-morpholino)ethanesulfonic acid. For iron-deficient plants, no iron was added to this solution, and iron-sufficient plants were fed with 50 μm iron(III)-EDTA.
      For Arabidopsis root ferric chelate reductase experiments, plants were cultivated in vitro on half strength Murashige/Skoog medium supplemented with 50 μm iron-EDTA. After 10 days, plantlets were transferred in an MS medium without iron and containing 30 μm FerroZine for 3 days.

      Liquid Endosperm Sampling

      Developing pods were dissected with a surgical blade. Two holes were pierced into each seed using a glass capillary tube. The liquid endosperm was then extracted quickly with another glass capillary using a peristaltic pump and blown gently at the bottom of an Eppendorf tube kept on ice. Pumping was carried out carefully to avoid bubbling. Samples were then maintained frozen in liquid nitrogen and kept at −80 °C until further analysis.

      Iron Speciation by X-ray Absorption Spectroscopy

      Iron K-edge x-ray absorption near edge structure (XANES) measurements were performed on LUCIA Beamline at Soleil Synchrotron, Gif sur Yvette, France, operating at 2.75 GeV (
      • Flank A.M.
      • Cauchon G.
      • Lagarde P.
      • Bac S.
      • Janousch M.
      • Wetter R.
      • Dubuisson J.M.
      • Idir M.
      • Langlois F.
      • Moreno T.
      • Vantelon D.
      LUCIA, a microfocus soft XAS beamline.
      ). The x-ray beam was monochromatized using a Si(111) two-crystal monochromator, and spectra were collected in fluorescence mode using a Bruker Silicon Drift diode and a non-focused beam. All measurements were done in a vacuum and in cryo conditions with a He cryostat providing a temperature of 110 K on the sample holder. A few drops of glycerol were added to liquid references to minimize the formation of ice crystals. No glycerol was added to the ESL because it already contains high concentrations of sugars. Energy was calibrated with metallic iron. The XANES spectra were corrected from background absorption by subtracting a linear function before the edge and normalizing using a linear or to-degree polynome. A fingerprint approach was then used to simulate the experimental spectra by a linear combination of iron reference compound spectra. The quality of the fits was estimated by the normalized sum-squares residuals NSS = Σ (Xanesexperimental − Xanesfit)2 / Σ (Xanesexperimental)2 × 100 in the 7090-7380 eV range. The iron valence was also estimated by the examination of the pre-edge structure (
      • Wilke M.
      • Farges F.
      • Petit P.E.
      • Brown G.E.
      • Martin F.
      Oxidation state and coordination of Fe in minerals. An FeK-XANES spectroscopic study.
      ).
      Iron reference compounds were prepared for comparison with ESL samples. Aqueous 50 mm iron(II) and iron(III) was prepared by dissolving FeCl2 and FeCl3 in deionized water at pH 2. Iron(II) preparation was performed in an argon atmosphere just before analysis to prevent iron oxidation. 25 mm iron(III)-citrate, iron(II)-citrate, and iron(III)-malate were prepared with metal/ligand (M/L) = 1/10 at pH 5.6 and 5.2, respectively. On the basis of the Visual MINTEQ software for chemical speciation (
      • Gustafsson J.P.
      ), under these conditions, more than 99% of iron is found as organic complexes. Iron(III)-histidine was synthesized at pH 7.2 with a 1/10 ratio and a total iron concentration of 3.1 mm.

      Iron Complexes by Chemical Analysis

      Instrumentation

      Chromatographic separations were performed using a model 1100 HPLC pump (Agilent, Wilmington, DE) as the delivery system. The HILIC column was a TSK gel amide 80 (250 × 1 mm inner diameter) (Tosoh Bioscience, Germany). All connections were made of fused silica/PEEK tubing (0.050 mm inner diameter). The ICP MS instrument was Agilent 7500cs (Agilent Technologies, Japan) equipped with a collision cell with hydrogen as collision gas. The interface between HPLC and the Agilent 7500cs consisted of a glass Cinnabar cyclonic spray chamber (Glass Expansion, Australia), a Micromist U-series nebulizer (Glass Expansion), a 1-mm inner diameter quartz torch (Agilent Technologies), and a set of platinum cones (Agilent Technologies). The ESI MS instrument was an LTQ Orbitrap Velos mass spectrometer (Thermo Scientific, San Jose, CA).

      LC MS Experimental Conditions

      The mobile phase was 5 mmol/liter ammonium acetate buffer (pH adjusted to 5.5 by addition of acetic acid) (A) and acetonitrile (B). Analytical reagent-grade chemicals were used for buffer preparation and were purchased from Sigma-Aldrich (Saint Quentin, Fallavier, France). Water (18.2 mΩ/cm) was obtained with a Milli-Q system (Millipore, Bedford, MA). The optimized HILIC gradient started with 10% of A and was increasing to 50% of A during 25 min. The samples (diluted in acetonitrile 1/2, v/v, and 7-μl injection) were eluted at a flow rate of 50 μl/min. Iron-containing species were detected by direct online coupling of the HILIC column to the ICP MS instrument (following signals for 54Fe and 56Fe isotopes) and to the ESI MS instrument. The settings and parameters of ICP MS and ESI MS instruments were optimized daily for highest intensities and lowest interferences using a built-in software procedure (ICP) and a standard mixture recommended by the constructor (ESI). ES MS spectra were acquired with a maximum injection time of 400 ms in the range of 230–1000 mass units. The electrospray voltage was set at 3 kV in positive ion mode and −3 kV in negative ion mode. The ions were fragmented by higher-energy collisional dissociation at various energy levels.

      Ferric Reduction Assays

      Ferric chelate reductase activity was estimated as the quantity of iron-bathophenanthroline disulfonate (BPDS) complexes formed in the assay solution calculated from the optical density measured at 535 nm with a Hitachi U-2800 spectrophotometer. A535 was measured after incubation in the dark at 22–25 °C with 250 rpm shaking in an assay solution that contained 5 mm MES buffer (pH 5.5), 300 μm (BPDS), and 100 μm iron(III)-EDTA unless stated otherwise. Pea embryos were dissected with a surgical blade as fast as possible and kept in 5 mm MES buffer (pH 5.5) for 30 min, representing the time to dissect and weigh around 15 embryos. Embryos were then incubated in the assay solution.
      To measure root ferric chelate reductase activity, whole root systems from individual pea plants were excised, rinsed three times with demineralized water, and incubated in 50 ml of assay solution following the same protocol. The comparison of embryo ferric chelate reductase activities was repeated five times independently, and each experiment consisted of the incubation of five embryos in 5 ml of assay solution with the usual protocol. Final results are the mean ± S.D. of the five values obtained. Arabidopsis root ferric chelate reductase activity was measured from individual root systems of iron-deficient seedlings in 1 ml of assay solution.

      Ferric Reduction Activity Measurement of Exudates

      Exudates consisted of 5 ml Milli-Q water in which five pea embryos were incubated, representing a total fresh weight of 0.7–1 g. For the efflux kinetics, embryos were incubated 10, 20, and 30 min or immersed and removed immediately for the 0-min time point. 100 μm iron(III)-EDTA and 300 μm BPDS were then added to exudates and allowed to react for 1 h in the dark prior to A535 determination. For the comparison of exudate activity with embryo activity, exudates were collected in Milli-Q water after 30 min of incubation and allowed to react with 100 μm iron(III)-EDTA and 300 μm BPDS for 1 h in the dark. In the case of ascorbate oxidase (AOX) treatment, 1.5 unit of AOX/ml of exudate was added 2 min before iron(III)-EDTA and BPDS addition. In this experiment, embryo ferrireduction activity was quantified following 30 min of embryo incubation in Milli-Q water containing 100 μm iron(III)-EDTA and 300 μm BPDS and 1 h incubation in the dark without embryos. All final values are the mean ± S.D. of three independent experiments.

      Time Course of Embryo Fe Reduction

      In this experiment, individual embryos were incubated in 2 ml of assay solution containing 0, 0.15, or 1.5 AOX units/ml. A535 was determined after 0, 10, 20, 30, 40, and 50 min of incubation from 2.5 μl of solution using a Nanodrop 1000 (Thermo Scientific). The experiment was carried out with five individual embryos per treatment and repeated three times. Values are the mean of the three experiments ± S.D.

      55Fe Accumulation in Pea Embryos

      Embryos weighting 20–50 mg were isolated and weighed as fast as possible after pod harvesting. They were individually disposed in 24-well plates containing 2 ml of the following solution: 100 mm KCl, 5 mm CaSO4, and 5 mm MES (pH 5.5). Influx was initiated by replacing this solution with influx buffer, which consisted of the same solution supplemented with 100 μm iron(III)-EDTA and 0.2 μCi/ml 55Fe. In the BPDS- and AOX-treated samples, 4 mm BPDS or 1.5 AOX units/ml were also provided in the uptake buffer. Embryos were incubated for 40 min in the dark at either 30 or 4 °C. The reaction was stopped by rinsing embryos three times with an ice-cold solution containing 1 mm KCl, 10 mm EDTA, 1 mm FeCl3, 10 mm CaCl2, 5 mm MgSO4, and 5 mm MES (pH = 5.5).
      55Fe was quantified following PerkinElmer Life Sciences instructions. Embryos were transferred to glass vials and dried for 48 h at 50 °C. They were then dissolved in 100 μl of perchloric acid at 50 °C for 24 h. Sample were cleared by adding 200 μl of hydrogen peroxide and incubated at 50 °C until complete bleaching. 3 ml of HionicFluor scintillating mixture (PerkinElmer Life Sciences) was added to the samples. Counts per minute were measured with a Tri-carb liquid scintillation counter (PerkinElmer Life Sciences) and converted to degradations per minute by QuantaSmart software (PerkinElmer Life Sciences) using the tSIE/AEC quenching correction. The iron quantity was deduced from the activity of a standard vial containing 10 μl of uptake buffer and 3 ml of HionicFluor. Net influx values were obtained by subtracting the quantity of 55Fe accumulated at 4 °C. The final Fe accumulation corresponds to the mean ± S.E. (n = 3) of data obtained with 12 individual embryos from three independent experiments.

      DISCUSSION

      In this work, we studied the mechanism of iron entry into the seed through two approaches: an iron speciation analysis at the interphase between the seed coat and embryo in the pea, associated with a biochemical dissection of iron transport on isolated embryos of the pea and Arabidopsis. We established that iron is delivered by the maternal tissues as ferric complexes with citrate and malate, that the embryos are capable of reducing the ferric ions of these complexes through the efflux of ascorbate, and that this reducing activity is an obligatory step for iron transport as iron(II). The analysis of iron speciation is by far the least documented aspect of iron homeostasis, both in animals and plants. This is principally due to the difficulties of obtaining biologically reliable samples (i.e. without mixing cellular compartments and creating artifact complexes) and the low concentration of iron in living tissues. To our knowledge, the iron speciation analysis reported in this study is one of the few unambiguous reports on the identification of iron complexes in vivo. Ferric-citrate complexes have long been proposed to occur in apoplastic compartments, but only recently a has a speciation approach led to the molecular identification of a tri-iron(III) tricitrate complex as the major iron-circulating form in the xylem of tomato plants (
      • Rellán-Alvarez R.
      • Giner-Martínez-Sierra J.
      • Orduna J.
      • Orera I.
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      Identification of a tri-iron(III), tri-citrate complex in the xylem sap of iron-deficient tomato resupplied with iron. New insights into plant iron long-distance transport.
      ). The ESL of pea seeds, although highly concentrated in nutrients (sugars, amino acids, etc.) from symplastic origin (
      • Melkus G.
      • Rolletschek H.
      • Radchuk R.
      • Fuchs J.
      • Rutten T.
      • Wobus U.
      • Altmann T.
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      • Borisjuk L.
      The metabolic role of the legume endosperm. A noninvasive imaging study.
      ), is characterized by an acidic pH (5.4) that is similar to the pH of an apoplastic compartment. Thus, our findings reinforce previous in silico (
      • von Wiren N.
      • Klair S.
      • Bansal S.
      • Briat J.F.
      • Khodr H.
      • Shioiri T.
      • Leigh R.A.
      • Hider R.C.
      Nicotianamine chelates both FeIII and FeII. Implications for metal transport in plants.
      ) and in vitro studies (
      • Rellán-Alvarez R.
      • Abadía J.
      • Alvarez-Fernández A.
      Formation of metal-nicotianamine complexes as affected by pH, ligand exchange with citrate and metal exchange. A study by electrospray ionization time-of-flight mass spectrometry.
      ) that demonstrated that, under mildly acidic conditions, ferric citrate would be the most stable and abundant type of complex, despite the presence of other ligands like nicotianamine. Moreover, our data identified malate as a new player in iron speciation in plants. Although one of the metabolic responses to iron deficiency is the accumulation of organic acids, including malate (
      • López-Millán A.F.
      • Morales F.
      • Abadía A.
      • Abadía J.
      Iron deficiency-associated changes in the composition of the leaf apoplastic fluid from field-grown pear (Pyrus communis L.) trees.
      ), a direct role of malic acid has never been clearly established. Instead, malate has been involved in the hyperaccumulation of nickel in T. caerulescens leaf extract (
      • Ouerdane L.
      • Mari S.
      • Czernic P.
      • Lebrun M.
      • Lobinski R.
      Speciation of non-covalent nickel species in plant tissue extracts by electrospray Q-TOF MS/MS after their isolation by 2D size-exclusion-hydrophilic interaction LC (SEC-HILIC) monitored by ICP MS.
      ) and Alyssum serpyllifolium with the formation of complexes with nickel in the xylem sap (
      • Alves S.
      • Nabais C.
      • Simões Gonçalves Mde L.
      • Correia Dos Santos M.M.
      Nickel speciation in the xylem sap of the hyperaccumulator Alyssum serpyllifolium ssp. lusitanicum growing on serpentine soils of northeast Portugal.
      ). Malate was also found to be involved in the storage of zinc in the leaves of the hyperaccumulator Arabidopsis halleri (
      • Sarret G.
      • Willems G.
      • Isaure M.P.
      • Marcus M.A.
      • Fakra S.C.
      • Frérot H.
      • Pairis S.
      • Geoffroy N.
      • Manceau A.
      • Saumitou-Laprade P.
      Zinc distribution and speciation in Arabidopsis halleri × Arabidopsis lyrata progenies presenting various zinc accumulation capacities.
      ). Malate is also a key molecule for the tolerance to aluminium because it is excreted from roots by aluminium-activated malate transporters to chelate aluminium in the rhizosphere (
      • Delhaize E.
      • Ryan P.R.
      • Hebb D.M.
      • Yamamoto Y.
      • Sasaki T.
      • Matsumoto H.
      Engineering high-level aluminum tolerance in barley with the ALMT1 gene.
      ,
      • Sasaki T.
      • Yamamoto Y.
      • Ezaki B.
      • Katsuhara M.
      • Ahn S.J.
      • Ryan P.R.
      • Delhaize E.
      • Matsumoto H.
      A wheat gene encoding an aluminum-activated malate transporter.
      ,
      • Hoekenga O.A.
      • Maron L.G.
      • Piñeros M.A.
      • Cançado G.M.
      • Shaff J.
      • Kobayashi Y.
      • Ryan P.R.
      • Dong B.
      • Delhaize E.
      • Sasaki T.
      • Matsumoto H.
      • Yamamoto Y.
      • Koyama H.
      • Kochian L.V.
      AtALMT1, which encodes a malate transporter, is identified as one of several genes critical for aluminum tolerance in Arabidopsis.
      ). Aluminium-activated malate transporter family members, therefore, represent putative candidate transporters whose function could be to load malate in the embryo sac liquid. Belonging to the same multidrug and toxic compound extrusion family of transporters as aluminium-activated malate transporters, FRD3 is a citrate efflux transporter that has been proposed recently to play a critical role in the movement of iron between tissues that are not symplastically connected. A detailed analysis of the expression pattern of FRD3 and a phenotypical analysis of frd3 knockout mutants have demonstrated the importance of FRD3-mediated citrate efflux in iron unloading in the leaf (from the xylem to adjacent cells) and in the anther (from the tapetum to pollen grains) (
      • Roschzttardtz H.
      • Séguéla-Arnaud M.
      • Briat J.F.
      • Vert G.
      • Curie C.
      The FRD3 citrate effluxer promotes iron nutrition between symplastically disconnected tissues throughout Arabidopsis development.
      ). Interestingly, the seed coat and the embryo protodermis are also disconnected completely, and FRD3 is highly expressed in both tissues in A. thaliana (
      • Roschzttardtz H.
      • Séguéla-Arnaud M.
      • Briat J.F.
      • Vert G.
      • Curie C.
      The FRD3 citrate effluxer promotes iron nutrition between symplastically disconnected tissues throughout Arabidopsis development.
      ). Thus, citrate efflux by FRD3 could be important as well to load the ESL with citrate and, subsequently, induce the formation of iron-citrate complexes. More generally, our results strengthen the importance of citrate in the transport of iron between cells and organs. Citrate may, in fact, be a ubiquitous and important intercellular iron ligand. Indeed, in mammals citrate is the main ligand of the non-transferrin-bound iron species (
      • Evans R.W.
      • Rafique R.
      • Zarea A.
      • Rapisarda C.
      • Cammack R.
      • Evans P.J.
      • Porter J.B.
      • Hider R.C.
      Nature of non-transferrin-bound iron. Studies on iron citrate complexes and thalassemic sera.
      ,
      • Hider R.C.
      • Silva A.M.N.
      • Podinovskaia M.
      • Ma Y.M.
      ), which appear to be the most abundant chemical form of iron circulating in the extracellular medium of the brain (
      • Bradbury M.W.
      Transport of iron in the blood-brain-cerebrospinal fluid system.
      ,
      • Moos T.
      • Rosengren Nielsen T.
      • Skjørringe T.
      • Morgan E.H.
      Iron trafficking inside the brain.
      ), used as a source of iron for brain cells (
      • Bishop G.M.
      • Dang T.N.
      • Dringen R.
      • Robinson S.R.
      Accumulation of non-transferrin-bound iron by neurons, astrocytes, and microglia.
      ). Most of the iron present in the liquid endosperm was found to be Fe3+ bound to a mixture of citrate and malate. It is the first observation in vivo of such mixed complexes. Compounds 1 and 2 correspond to two iron complexes that most likely participate in binding iron(III) to form a soluble, stable (contrary to the iron(III)Cit2 complex, for example), but low-reactive species. Indeed, iron(III) alone would precipitate, leading to iron deficiency, whereas its presence in a weakly complexed form would probably lead to increased stress for the plant because of expected frequent random exchanges of the Fe3+ ion with other nonspecific binding sites in surrounding molecules. Therefore, these mixed complexes with organic acids probably participate in making iron(III) available in the liquid endosperm for transport/reaction, such as reduction by ascorbate in the vicinity of the embryo, without inducing significant metal or oxidative stress.
      The identification of iron(II)-nicotianamine in the ESL represents the definitive proof of the in vivo role of nicotianamine in the movement of iron, although, in our case, the proportion of this complex is several orders of magnitude lower than the iron bound to organic acids. Nevertheless, the presence of iron-NA in the ESL could be interpreted as the signature of the activity of YSL transporters. The fact that iron translocation to the seed is controlled, in part, by YSL transporter activity has been shown in Arabidopsis. The closely related members AtYSL1 and AtYSL3 have been involved in the loading of iron, zinc, and copper to the seeds (
      • Le Jean M.
      • Schikora A.
      • Mari S.
      • Briat J.F.
      • Curie C.
      A loss-of-function mutation in AtYSL1 reveals its role in iron and nicotianamine seed loading.
      ,
      • Waters B.M.
      • Chu H.H.
      • Didonato R.J.
      • Roberts L.A.
      • Eisley R.B.
      • Lahner B.
      • Salt D.E.
      • Walker E.L.
      Mutations in Arabidopsis yellow stripe-like1 and yellow stripe-like3 reveal their roles in metal ion homeostasis and loading of metal ions in seeds.
      ). In particular, the mutation in AtYSL1, which is expressed in the chalazal endosperm of the seed, provokes a reduced accumulation of iron and NA in mature seeds (
      • Le Jean M.
      • Schikora A.
      • Mari S.
      • Briat J.F.
      • Curie C.
      A loss-of-function mutation in AtYSL1 reveals its role in iron and nicotianamine seed loading.
      ). Thus, the presence of iron-NA complexes, the bona fide substrate of YSL transporters, in the ESL leads to the assumption that iron is delivered from the seed coat to the ESL by YSL proteins.
      Up to now, there has been no direct functional link between the metabolism of ascorbate and iron homeostasis. Instead, several reports had described the induction of ascorbate accumulation in response to iron deficiency and proposed that it could represent a mechanism to cope with potential reactive oxygen species production (
      • Zaharieva T.B.
      • Abadía J.
      Iron deficiency enhances the levels of ascorbate, glutathione, and related enzymes in sugar beet roots.
      ,
      • Urzica E.I.
      • Casero D.
      • Yamasaki H.
      • Hsieh S.I.
      • Adler L.N.
      • Karpowicz S.J.
      • Blaby-Haas C.E.
      • Clarke S.G.
      • Loo J.A.
      • Pellegrini M.
      • Merchant S.S.
      Systems and trans-system level analysis identifies conserved iron deficiency responses in the plant lineage.
      ). The most unexpected finding of this study was the discovery of ascorbate efflux as the mechanism to reduce and acquire iron from the ferric complexes. Although other organic molecules, such as phenolics, can be excreted by roots of strategy I plants in response to iron deficiency, these molecules are not capable of reducing iron(III) in the medium (
      • Jin C.W.
      • You G.Y.
      • He Y.F.
      • Tang C.
      • Wu P.
      • Zheng S.J.
      Iron deficiency-induced secretion of phenolics facilitates the reutilization of root apoplastic iron in red clover.
      ). Furthermore, the iron reduction activity has, so far, only been attributed to membrane proteins of the FRO family, such as the plasma membrane proteins FRO2, FRO4, and FRO5 (
      • Robinson N.J.
      • Procter C.M.
      • Connolly E.L.
      • Guerinot M.L.
      A ferric-chelate reductase for iron uptake from soils.
      ,
      • Bernal M.
      • Casero D.
      • Singh V.
      • Wilson G.T.
      • Grande A.
      • Yang H.
      • Dodani S.C.
      • Pellegrini M.
      • Huijser P.
      • Connolly E.L.
      • Merchant S.S.
      • Krämer U.
      Transcriptome sequencing identifies SPL7-regulated copper acquisition genes FRO4/FRO5 and the copper dependence of iron homeostasis in Arabidopsis.
      ) and the chloroplastic protein FRO7 (
      • Jeong J.
      • Cohu C.
      • Kerkeb L.
      • Pilon M.
      • Connolly E.L.
      • Guerinot M.L.
      Chloroplast Fe(III) chelate reductase activity is essential for seedling viability under iron limiting conditions.
      ). In contrast, this report describes a new strategy of iron acquisition on the basis of a reduction activity that is independent of the FRO proteins but, instead, relies on the property of ascorbate as a powerful reductant. Unlike the root ferric reductase activity, the ascorbate-mediated reduction was not induced by iron deficiency or in the dgl mutant that constitutively expresses the iron deficiency responses, irrespective of the iron nutritional status, strengthening the idea that this new mechanism is molecularly distinct from the FRO-based iron transport and not regulated by the iron deficiency signaling pathway. Furthermore, we have shown that the ascorbate efflux activity is also expressed in A. thaliana embryos. To further confirm the link between ascorbate metabolism and iron acquisition, we used mutants affected in ascorbate synthesis. The VTC2 and VTC5 genes encode two isoforms of GDP-l-galactose phosphorylase, one of the last enzymes of the ascorbate biosynthetic pathway (
      • Dowdle J.
      • Ishikawa T.
      • Gatzek S.
      • Rolinski S.
      • Smirnoff N.
      Two genes in Arabidopsis thaliana encoding GDP-l-galactose phosphorylase are required for ascorbate biosynthesis and seedling viability.
      ,
      • Laing W.A.
      • Wright M.A.
      • Cooney J.
      • Bulley S.M.
      The missing step of the l-galactose pathway of ascorbate biosynthesis in plants, an l-galactose guanyltransferase, increases leaf ascorbate content.
      ). In terms of expression, VTC2 is predominant, and disruption of this gene provokes a 70% decrease in ascorbate accumulation in leaves, whereas mutations in VTC5 barely affect ascorbate accumulation (
      • Dowdle J.
      • Ishikawa T.
      • Gatzek S.
      • Rolinski S.
      • Smirnoff N.
      Two genes in Arabidopsis thaliana encoding GDP-l-galactose phosphorylase are required for ascorbate biosynthesis and seedling viability.
      ). Both genes are expressed in A. thaliana embryos, and we show here that both mutations greatly impact the ascorbate efflux, iron accumulation in seeds and mature embryos. These data confirm the biochemical characterization performed in pea embryos and establish a direct and genetic link between ascorbate metabolism and iron transport in the plant.
      Interestingly, a comparable iron uptake mechanism has been proposed in mammals. Erythroleukemia K562 cells and astrocytes are capable of reducing iron(III) using ascorbate efflux, generating iron(II) and dehydroascorbate that is reabsorbed to regenerate an ascorbate pool in the cytosol (
      • Lane D.J.
      • Lawen A.
      Non-transferrin iron reduction and uptake are regulated by transmembrane ascorbate cycling in K562 cells.
      ,
      • Lane D.J.
      • Robinson S.R.
      • Czerwinska H.
      • Bishop G.M.
      • Lawen A.
      Two routes of iron accumulation in astrocytes. Ascorbate-dependent ferrous iron uptake via the divalent metal transporter (DMT1) plus an independent route for ferric iron.
      ). Transmembrane cycling of ascorbate coupled to ferrous transport by the divalent transporter DMT1 appears as one possible route for iron accumulation in these cell types. Yet, in contrast to the constitutively high ascorbate efflux activity measured in embryos, ascorbate efflux was only triggered when cells were supplied with elevated dehydroascorbate levels. The molecular identity of the efflux transport system remains unknown in both plants and mammals, but the biochemical features of ascorbate efflux in mammalian K562 cells were compatible with the activity of an anion channel (
      • Lane D.J.
      • Lawen A.
      Non-transferrin iron reduction and uptake are regulated by transmembrane ascorbate cycling in K562 cells.
      ). Ascorbate efflux by pea embryos was insensitive to anion channel inhibitors (data not shown) but required a pH gradient, suggesting that it could be mediated by an H+/ascorbate antiporter. The importance of the proton motive force has already been reported for the transport of nutrients in pea embryos on the basis of the specific expression in the outer cell layer of the cotyledons of genes encoding H+/sucrose and H+/amino acid cotransporters (
      • Tegeder M.
      • Offler C.E.
      • Frommer W.B.
      • Patrick J.W.
      Amino acid transporters are localized to transfer cells of developing pea seeds.
      ,
      • Tegeder M.
      • Wang X.D.
      • Frommer W.B.
      • Offler C.E.
      • Patrick J.W.
      Sucrose transport into developing seeds of Pisum sativum L.
      ). Future work will be aimed at molecularly identifying this ascorbate efflux system.

      Acknowledgments

      We thank the Soleil Synchrotron (Gif sur Yvette, France) for the provision of beamtime (Project 20110430) and Nicolas Trcera from the LUCIA Beamline for help and advice during data collection. We also thank Nicholas Smirnoff for discussions regarding the vtc mutants.

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