Stéphane Mari

Christophe Bailly

Sébastien Thomine
email: sebastien.thomine@i2bc.paris-saclay.fr

Handing off iron to the next generation: how does it get into seeds and what for?

To ensure the success of the new generation in annual species, the mother plant transfers a large proportion of the nutrients it has accumulated during its vegetative life to the next generation through its seeds. Iron (Fe) is required in large amounts to provide the energy and redox power to sustain seedling growth. However, free Fe is highly toxic as it leads to the generation of reactive oxygen species. Fe must therefore be tightly bound to chelating molecules to allow seed survival for long periods of time without oxidative damage.

Nevertheless, when conditions are favorable, the seed Fe stores have to be readily remobilized to achieve the transition towards active photosynthesis before the seedling becomes able to take up Fe from the environment. This is likely critical for the vigor of the young plant. Seeds constitute an important dietary source of Fe, which is essential for human health.

Understanding the mechanisms of Fe storage in seeds is key to improve their Fe content and availability in order to fight the Fe deficiency. Seed longevity, germination efficiency and seedling vigor are also important traits that may be affected by the chemical form under which Fe is stored. In this review, we summarize the current knowledge on seed Fe loading during development, long-term storage and remobilization upon germination. We highlight how this knowledge may help seed Fe biofortification and discuss how Fe storage may affect seed quality and germination efficiency.

Introduction

Seed is a very special stage in the life of a plant. It is the basis for the next generation and the survival of the species. To ensure the success of the new generation, the mother plant transfers a large proportion of the nutrients it has accumulated during its vegetative life to its seeds.

Most work on seed nutrient storage focuses on carbohydrate, lipids and proteins, which provide the bulk energy, carbon and nitrogen required to initiate development. Depending on species, 30 to 90% of plant nitrogen is allocated to seeds at the end of the plant life cycle [START_REF] Have | Nitrogen remobilization during leaf senescence: lessons from Arabidopsis to crops[END_REF] .

However, mineral stores are also very important to sustain seedling growth. Especially, iron (Fe) is required in large amount in mitochondria, which provide the energy and redox power during the initial stage of seedling development. For example, in the case of Arabidopsis (Arabidopsis thaliana), 50 to 55% of the iron (Fe) is allocated to seeds at the end of the life cycle [START_REF] Pottier | Autophagy is essential for optimal translocation of iron to seeds in Arabidopsis[END_REF][START_REF] Waters | Whole-plant mineral partitioning throughout the life cycle in Arabidopsis thaliana ecotypes Columbia, Landsberg erecta, Cape Verde Islands, and the mutant line ysl1ysl3[END_REF] . Moreover, the nutrients need to be stored in a compact and stable form. Seed storage proteins form pseudo crystalline aggregates in protein bodies within seed embryo cells. Free Fe is highly toxic as it leads to the generation of reactive oxygen species through the Fenton reaction. In cells, Fe must therefore be tightly bound to chelating molecules to prevent the formation of ROS. This is probably even more important in seeds than in other organs, as seeds represent resistance forms designed to survive for long periods of time, often several years, and through stresses. Nevertheless, when conditions are favorable, the nutrients stored in seeds have to be readily remobilized to sustain the early development of the seedling before it acquires photosynthetic capacity and becomes able to take up nutrients from the environment. Iron is a major component of photosystem I and its availability to the young seedling is necessary to achieve the transition towards active photosynthesis. This is likely critical for the vigor of the young plant.

From the point of view of animals and especially human beings, seeds are a major food resource. They provide carbohydrates, proteins and fat but also micronutrients such as Fe and zinc, which are essential for human health. About 2 billion human beings suffer from Fe deficiency, which leads to asthenia, anemia and in extreme cases to death. The prevalence of Fe deficiency is highest in populations that rely mostly on a plant based diet because plants and seeds in particular do not provide sufficient Fe [START_REF] Murgia | Biofortification for combating 'hidden hunger' for iron[END_REF] . Understanding the mechanisms of Fe storage in seeds is key to improve their Fe content and availability in order to fight Fe deficiency using biofortification. From the agricultural perspective, seed longevity, germination efficiency and seedling vigor are also very important traits that breeders and farmers seek to improve. Here also, seed nutrient content and especially the content and chemical form of Fe may be critical. In this review, we will summarize the current knowledge on seed Fe loading during development, long-term storage and remobilization upon germination, updating the last review on the topic [START_REF] Grillet | Iron in seeds -loading pathways and subcellular localization[END_REF] . We will highlight how this knowledge may help seed Fe biofortification, longevity, germination efficiency and stand establishment.

Iron uptake and translocation to the shoots

Plants take up iron using distinct strategies according to species [START_REF] Connorton | Iron homeostasis in plants -a brief overview[END_REF][START_REF] Kobayashi | Iron uptake, translocation, and regulation in higher plants[END_REF] . Most plant species use strategy I, which is based on the reduction of Fe in the rhizosphere followed by its uptake by a transporter for Fe(II). In contrast, graminaceous species, which include the major grain crops such as rice, wheat and barley, have evolved strategy II. Strategy II is based on the release of small Fe(III) chelating molecules called phytosiderophores in the rhizosphere and subsequent uptake of siderophore-Fe(III) complexes. Under Fe deficiency, strategy I plants activate the expression of the genes involved in Fe solubilization and uptake. The molecular players in this response have been identified in Arabidopsis but are essentially conserved in other species. The proton pump Arabidopsis plasma membrane H + -ATPase 2 (AHA2) drives rhizosphere acidification which is important to solubilize Fe 8 . The coumarin transporter Pleiotropic Drug Resistance 9 (PDR9) allows the secretion of coumarin with iron binding and reducing properties in the rhizosphere. Enzymes of the coumarin biosynthetic pathway are induced along with PDR9 [START_REF] Rajniak | Biosynthesis of redox-active metabolites in response to iron deficiency in plants[END_REF][START_REF] Tsai | Mobilization of Iron by Plant-Borne Coumarins[END_REF] . The membrane bound Ferric chelate Reductase FRO2 reduces chelate-bound Fe(III) to Fe(II), which is taken up into root cells by the divalent metal Iron Regulated Transporter 1 (IRT1) [START_REF] Robinson | A ferric-chelate reductase for iron uptake from soils[END_REF][START_REF] Vert | IRT1, an Arabidopsis transporter essential for iron uptake from the soil and for plant growth[END_REF] . IRT1 is not specific for Fe(II) and drives the uptake of a range of other divalent metal cations including Zn, Mn, Co, Cd, Ni [START_REF] Connolly | Expression of the IRT1 metal transporter is controlled by metals at the levels of transcript and protein accumulation[END_REF][START_REF] Eide | A novel iron-regulated metal transporter from plants identified by functional expression in yeast[END_REF][START_REF] Korshunova | The IRT1 protein from Arabidopsis thaliana is a metal transporter with a broad substrate range[END_REF] . A sophisticated mechanism enables the removal of IRT1 from the plasma membrane when intracellular concentrations of non-Fe metals raise [START_REF] Dubeaux | Metal Sensing by the IRT1 Transporter-Receptor Orchestrates Its Own Degradation and Plant Metal Nutrition[END_REF] . In addition, several genes involved in the sequestration of these metal cations are activated under Fe deficiency [START_REF] Thomine | Iron transport in plants: better be safe than sorry[END_REF] . In graminaceous species such as rice, maize and barley, the components that are upregulated for Fe acquisition under Fe deficiency include the phytosiderophore efflux and Fe-sirerophore influx transporters as well as the biosynthetic pathway of phytosiderophores of the mugineic acid family [START_REF] Kobayashi | Iron uptake, translocation, and regulation in higher plants[END_REF] . The S-adenosyl methionine cycle is activated to produce methionine, which is used by the nicotianamine synthase (NAS) to produce nicotianamine (NA) the precursor of mugineic acids. Whereas NA is also synthetized in strategy I species, mugineic acids are unique to graminaceous species. NA is converted to mugineic acid by the nicotianamine aminotransferase (NAAT) and to deoxymugineic acid by the deoxymugineic acid synthase (DMAS). MA and DMA are secreted to the rhizosphere by an efflux transporter, Transporter Of Mugineic acid 1 (TOM1) in rice (Oryza sativa), Yellow Stripe 3 (YS3) in maize (Zea mais) [START_REF] Chan-Rodriguez | Analysis of Yellow Striped Mutants of Zea mays Reveals Novel Loci Contributing to Iron Deficiency Chlorosis[END_REF][START_REF] Nozoye | The Phytosiderophore Efflux Transporter TOM2 Is Involved in Metal Transport in Rice[END_REF] . Fe-siderophore complexes are taken up by specific influx transporters, Yellow Stripe 1 (YS1) in maize and YS1-Like 15 (YSL15) in rice [START_REF] Curie | Maize yellow stripe1 encodes a membrane protein directly involved in Fe(III) uptake[END_REF][START_REF] Inoue | Rice OsYSL15 is an iron-regulated iron(III)-deoxymugineic acid transporter expressed in the roots and is essential for iron uptake in early growth of the seedlings[END_REF][START_REF] Lee | Disruption of OsYSL15 leads to iron inefficiency in rice plants[END_REF] . Despite the difference in their uptake strategies, the control of Fe deficiency responses involves a similar network of negatively and positively regulating bHLH (basic Helix Loop Helix) in graminaceous plants and the other species [START_REF] Gao | The Transcriptional Control of Iron Homeostasis in Plants: A Tale of bHLH Transcription Factors?[END_REF][START_REF] Kobayashi | Iron transport and its regulation in plants[END_REF] . In addition, Hemerythrin Domain containing RING proteins that act as negative regulators of Fe deficiency responses by mediating the Fe dependent degradation of specific bHLH, are also conserved among strategy I and strategy II plants [START_REF] Kobayashi | Iron-binding haemerythrin RING ubiquitin ligases regulate plant iron responses and accumulation[END_REF][START_REF] Long | The bHLH transcription factor POPEYE regulates response to iron deficiency in Arabidopsis roots[END_REF][START_REF] Rodriguez-Celma | Arabidopsis BRUTUS-LIKE E3 ligases negatively regulate iron uptake by targeting transcription factor FIT for recycling[END_REF] .

To be translocated to the shoots, Fe has to go through the endodermis cell layer and to be released into the xylem sap together with ligands that maintain it in soluble form. Recently, suberin deposition around the endodermis has been shown to provide a control point for Fe translocation. Under Fe deficiency, the suberin layer is degraded allowing the transfer of Fe from the root cortex to the central cylinder where it can be loaded into the xylem for translocation to the aerial parts 28,[START_REF] Barberon | Adaptation of Root Function by Nutrient-Induced Plasticity of Endodermal Differentiation[END_REF] . Based on its expression pattern and plasma membrane localization, the Fe efflux transporter Ferroportin (FPN1)/Iron REGulated 1 (IREG1) has been proposed to mediate Fe loading in the xylem in Arabidopsis [START_REF] Morrissey | The ferroportin metal efflux proteins function in iron and cobalt homeostasis in Arabidopsis[END_REF] . The MATE family citrate efflux transporters, Ferric Reductase Deficient 3 (FRD3) in Arabidopsis and FRD3-Like 1(FRDL1) in rice, play an important role in the translocation of Fe and other metals, by loading citrate which chelates Fe and prevent its precipitation in the xylem sap [START_REF] Durrett | The FRD3-mediated efflux of citrate into the root vasculature is necessary for efficient iron translocation[END_REF][START_REF] Flis | Inventory of metal complexes circulating in plant fluids: a reliable method based on HPLC coupled with dual elemental and high-resolution molecular mass spectrometric detection[END_REF][START_REF] Rogers | FRD3, a member of the multidrug and toxin efflux family, controls iron deficiency responses in Arabidopsis[END_REF][START_REF] Roschzttardtz | The FRD3 citrate effluxer promotes iron nutrition between symplastically disconnected tissues throughout Arabidopsis development[END_REF][START_REF] Yokosho | OsFRDL1 is a citrate transporter required for efficient translocation of iron in rice[END_REF] .

Remobilization from other organs to the seeds

At the vegetative stage, most of the iron translocated to aerial parts is used for photosynthesis in chloroplasts in mesophyll cells. Photosystem I and Ferredoxin, which are very abundant in chloroplasts, contain numerous FeS clusters. This assumption based on biochemical needs is supported by cell fractionation experiments and Fe imaging in leaves [START_REF] Lanquar | Export of vacuolar manganese by AtNRAMP3 and AtNRAMP4 is required for optimal photosynthesis and growth under manganese deficiency[END_REF][START_REF] Roschzttardtz | New insights into Fe localization in plant tissues[END_REF][START_REF] Shingles | Ferrous ion transport across chloroplast inner envelope membranes[END_REF] . Upon transition to the reproductive stage, a large part of the Fe present in vegetative tissues is transferred to the seeds. In Arabidopsis, 50 to 55% of total plant Fe ends up in the seeds at the end of the life cycle (the Iron Harvest Index, Figure 1) [START_REF] Pottier | Autophagy is essential for optimal translocation of iron to seeds in Arabidopsis[END_REF][START_REF] Waters | Whole-plant mineral partitioning throughout the life cycle in Arabidopsis thaliana ecotypes Columbia, Landsberg erecta, Cape Verde Islands, and the mutant line ysl1ysl3[END_REF] . A recent study based on Fe isotope labeling indicated that most of the Fe stored in seeds originates from vegetative tissue, implying a major remobilization of Fe 2 .

Prior to its movement from the vegetative tissues to the seeds, Fe has to be made available in the senescing tissues. Autophagy was proposed to play a key role in nutrient remobilization from vegetative tissues to seeds [START_REF] Pottier | Autophagy as a possible mechanism for micronutrient remobilization from leaves to seeds[END_REF] . This was based on the finding that autophagy is involved in nitrogen remobilization to seeds [START_REF] Guiboileau | Autophagy machinery controls nitrogen remobilization at the whole-plant level under both limiting and ample nitrate conditions in Arabidopsis[END_REF] . Recently, the finding that a mutation in Autophagy 5 (ATG5), which strongly impairs autophagy, decreases the proportion of Fe allocated to seeds from 50-55% to 15-20% highlighted the importance of autophagy for Fe remobilization 2 .

Similar results were obtained for other micronutrients such as Zn and Mn. The role of autophagy is thus clearly not limited to Fe. Recently, several pathways have been implicated in the degradation of chloroplasts, which contain most of the cellular Fe, during leaf senescence [START_REF] Otegui | Vacuolar degradation of chloroplast components: autophagy and beyond[END_REF] . They include (i) the senescence-associated vacuoles (SAV) that are important for the degradation of stromal proteins such as RuBisCo and glutamine synthase, (ii) chlorophagy in which whole chloroplast are encapsulated in autophagosomes [START_REF] Izumi | Entire Photodamaged Chloroplasts Are Transported to the Central Vacuole by Autophagy[END_REF] (iii) a vesicular pathway depending on the ATG8 Interacting protein (ATI) which degrades envelope and thylakoid membranes in addition to stromal proteins [START_REF] Michaeli | Arabidopsis ATG8-INTERACTING PROTEIN1 is involved in autophagydependent vesicular trafficking of plastid proteins to the vacuole[END_REF] and (iv) CV-Containing vesicles (CCVs) that contain the CV protein (Chloroplast Vesiculation) responsible for chloroplast destabilization [START_REF] Wang | Stress-induced chloroplast degradation in Arabidopsis is regulated via a process independent of autophagy and senescence-associated vacuoles[END_REF] . In contrast to the others, the CCV pathway does not require autophagy. It will be interesting to determine the respective importance of these pathways for Fe remobilization during vegetative organ senescence, as they could allow to target some nutrients more specifically than autophagy.

In addition to autophagy, the timing of senescence is also an important factor controlling remobilization. If senescence occurs too fast, the time window for remobilization is reduced and less nutrients are allocated to seeds. Hence, NAC transcription factors, which control the onset of senescence, also play a major role in the control of nutrient remobilization from leaves to seeds [START_REF] Qiu | EIN3 and ORE1 Accelerate Degreening during Ethylene-Mediated Leaf Senescence by Directly Activating Chlorophyll Catabolic Genes in Arabidopsis[END_REF][START_REF] Ricachenevsky | kNACking on heaven's door: how important are NAC transcription factors for leaf senescence and Fe/Zn remobilization to seeds?[END_REF][START_REF] Uauy | A NAC Gene regulating senescence improves grain protein, zinc, and iron content in wheat[END_REF] . More work will be required to understand how the interplay between autophagy and senescence controls Fe allocation to seeds in different species.

After it has been made available in vegetative tissue, Fe has to be transported to developing seeds through the phloem. Many lines of evidence have pointed to a key role of NA in this process. Complete loss of NA synthesis leads to sterility [START_REF] Klatte | The analysis of Arabidopsis nicotianamine synthase mutants reveals functions for nicotianamine in seed iron loading and iron deficiency responses[END_REF][START_REF] Ling | Map-based cloning of chloronerva, a gene involved in iron uptake of higher plants encoding nicotianamine synthase[END_REF][START_REF] Takahashi | Role of nicotianamine in the intracellular delivery of metals and plant reproductive development[END_REF] . This has initially prevented a direct analysis of the role of NA in Fe transport to seeds, as the mutants did not set seeds. The first evidence that NA is important for Fe transport to the seeds came from the study of mutants impaired in YS1-like genes (YSL). The strong expression of OsYSL2 in phloem tissues of rice aerial parts and developing seeds and the ability of this transporter to take up NA-Fe and NA-Mn complexes when expressed in Xenopus oocytes provided the first indication that NA is important for Fe transport through the phloem to the seeds [START_REF] Koike | OsYSL2 is a rice metal-nicotianamine transporter that is regulated by iron and expressed in the phloem[END_REF] . Analysis of rice lines in which OsYSL2 expression was silenced further supported this hypothesis [START_REF] Ishimaru | Rice metal-nicotianamine transporter, OsYSL2, is required for the long-distance transport of iron and manganese[END_REF] . In these lines, Fe transport to the shoots and the seeds was strongly decreased. Conversely, increased OsYSL2 expression under a phloem specific promoter led to strong increase in seed endosperm Fe concentration [START_REF] Ishimaru | Rice metal-nicotianamine transporter, OsYSL2, is required for the long-distance transport of iron and manganese[END_REF] . More recently, another YSL transporter from rice, OsYSL9, was implicated in Fe delivery to developing rice grain. OsYSL9 expression is upregulated by Fe deficiency in roots but down regulated in leaves under the same conditions [START_REF] Senoura | The iron-chelate transporter OsYSL9 plays a role in iron distribution in developing rice grains[END_REF] . The role of YSL in metal transport to seeds is not restricted to rice. Similar conclusions were obtained from the analysis of Arabidopsis knockout mutants targeting AtYSL1 gene or double mutants affecting AtYSL1 and AtYSL3 [START_REF] Chu | Successful reproduction requires the function of Arabidopsis Yellow Stripe-Like1 and Yellow Stripe-Like3 metal-nicotianamine transporters in both vegetative and reproductive structures[END_REF][START_REF] Jean | A loss-of-function mutation in AtYSL1 reveals its role in iron and nicotianamine seed loading[END_REF][START_REF] Waters | Mutations in Arabidopsis yellow stripe-like1 and yellow stripelike3 reveal their roles in metal ion homeostasis and loading of metal ions in seeds[END_REF] . Like OsYSL2, AtYSL1 is expressed in phloem tissues of leaves as well as in developing siliques. Similar to OsYSL9, it is down regulated under Fe deficiency. Loss of function mutants in AtYSL1 accumulated higher levels of NA in shoots but strongly decreased NA and Fe concentrations in mature seeds [START_REF] Jean | A loss-of-function mutation in AtYSL1 reveals its role in iron and nicotianamine seed loading[END_REF] . Combining a mutation in AtYSL1 with a mutation in another Arabidopsis YSL strongly expressed in phloem, AtYSL3, leads to more severe phenotypes: the ysl1ysl3 mutants display symptoms of strong Fe deficiency with interveinal chlorosis as well as decreased fertility. Like ysl1, they also accumulate lower concentrations of Fe in their seeds when provided with a high Fe concentration in the medium, which alleviates the defect in fertility and up-regulate YSL1 expression [START_REF] Jean | A loss-of-function mutation in AtYSL1 reveals its role in iron and nicotianamine seed loading[END_REF][START_REF] Waters | Mutations in Arabidopsis yellow stripe-like1 and yellow stripelike3 reveal their roles in metal ion homeostasis and loading of metal ions in seeds[END_REF] . Further inflorescence grafting experiments showed that expression of AtYSL1 and AtYSL3 in the rosette was sufficient to restore fertility but not seed Fe content [START_REF] Chu | Successful reproduction requires the function of Arabidopsis Yellow Stripe-Like1 and Yellow Stripe-Like3 metal-nicotianamine transporters in both vegetative and reproductive structures[END_REF] . Furthermore, the identification of a quadruple mutant combining knockout in 3 out of the 4 genes encoding NAS in Arabidopsis and a knockdown in the fourth one allowed to obtain fertile plants with drastically decreased levels of NA [START_REF] Klatte | The analysis of Arabidopsis nicotianamine synthase mutants reveals functions for nicotianamine in seed iron loading and iron deficiency responses[END_REF] . In this quadruple nas mutant, Fe and NA levels are lower in seeds, similar to the phenotype observed in ysl1. Interestingly, in NA biosynthesis mutants, Fe accumulates in phloem cells indicating that NA is required to retrieve Fe from the phloem and provide it to sink tissues, such as seeds [START_REF] Schuler | Nicotianamine functions in the Phloem-based transport of iron to sink organs, in pollen development and pollen tube growth in Arabidopsis[END_REF] . This role may be shared between NA and mugineic acids in graminaceous plants. On the other hand, all YSL transporters characterized so far using expression in yeast or in Xenopus oocytes mediate the influx of NA-Fe or MA-Fe complexes into cells. The finding that NA and YSL are important for phloem transport of Fe does not imply that Fe is transported as NA-Fe complex in the phloem.

Another important player in Fe transport from vegetative organs to the seeds is the oligopeptide transporter OligoPeptide Transporter 3 (OPT3). Full loss of function of OPT3 leads to embryo lethality suggesting that the substrate of OPT3 is an essential cellular metabolite [START_REF] Stacey | AtOPT3, a member of the oligopeptide transporter family, is essential for embryo development in Arabidopsis[END_REF] . Even though the closest homologue of OPT3, BjGT1, is a glutathione transporter [START_REF] Bogs | Functional characterization and expression analysis of a glutathione transporter, BjGT1, from Brassica juncea: evidence for regulation by heavy metal exposure[END_REF] , transport assays in yeast failed to show OPT3 ability to transport GSH [START_REF] Zhai | OPT3 Is a Phloem-Specific Iron Transporter That Is Essential for Systemic Iron Signaling and Redistribution of Iron and Cadmium in Arabidopsis[END_REF] .

Instead, expression of OPT3 could complement the yeast fet3fet4 mutant impaired in Fe uptake, suggesting that it transports Fe complexes. The exact substrate of OPT3 remains to be identified. The identification of a weak allele of opt3, opt3-2 allowed investigating its function at the reproductive stage [START_REF] Stacey | The Arabidopsis AtOPT3 protein functions in metal homeostasis and movement of iron to developing seeds[END_REF] . In opt3-2 mutants, Fe concentration is increased in vegetative organs but decreased in seeds, indicating a role in Fe transfer from vegetative organs to seeds. More recently, OPT3 was shown to be targeted to the plasma membrane and mainly expressed in phloem cells [START_REF] Zhai | OPT3 Is a Phloem-Specific Iron Transporter That Is Essential for Systemic Iron Signaling and Redistribution of Iron and Cadmium in Arabidopsis[END_REF][START_REF] Mendoza-Cozatl | OPT3 is a component of the iron-signaling network between leaves and roots and misregulation of OPT3 leads to an over-accumulation of cadmium in seeds[END_REF] . In opt3-2 mutants, Fe concentration was strongly increased in the xylem sap and decreased in the phloem sap, pointing to role of OPT3 in Fe transfer from the xylem to the phloem [START_REF] Zhai | OPT3 Is a Phloem-Specific Iron Transporter That Is Essential for Systemic Iron Signaling and Redistribution of Iron and Cadmium in Arabidopsis[END_REF] . Combined with its expression in phloem cells, this suggests that OPT3 is involved in Fe loading into the phloem. Hence, both OPT3 and NA play critical roles in long distance transport of Fe in the phloem to sink organs and especially seeds: OPT3 would be necessary for loading Fe into the phloem, while NA would be required for Fe retrieval from the phloem [START_REF] Schuler | Nicotianamine functions in the Phloem-based transport of iron to sink organs, in pollen development and pollen tube growth in Arabidopsis[END_REF][START_REF] Zhai | OPT3 Is a Phloem-Specific Iron Transporter That Is Essential for Systemic Iron Signaling and Redistribution of Iron and Cadmium in Arabidopsis[END_REF] . Interestingly, OPT3 is also important for Fe deficiency signaling and opt3-2 up-regulated Fe-deficiency responsive gene expression even under Fe sufficient conditions [START_REF] Zhai | OPT3 Is a Phloem-Specific Iron Transporter That Is Essential for Systemic Iron Signaling and Redistribution of Iron and Cadmium in Arabidopsis[END_REF][START_REF] Mendoza-Cozatl | OPT3 is a component of the iron-signaling network between leaves and roots and misregulation of OPT3 leads to an over-accumulation of cadmium in seeds[END_REF] . In rice, OsOPT7, a homologue of OPT3, was characterized [START_REF] Bashir | Iron deficiency regulated OsOPT7 is essential for iron homeostasis in rice[END_REF] .

OsOPT7 could not transport GSH or Fe when expressed in Xenopus oocytes and yeast. Like AtOPT3, OsOPT7 is induced under Fe deficiency but its expression is more widespread than that of OPT3 [START_REF] Stacey | The Arabidopsis AtOPT3 protein functions in metal homeostasis and movement of iron to developing seeds[END_REF][START_REF] Bashir | Iron deficiency regulated OsOPT7 is essential for iron homeostasis in rice[END_REF] . Moreover, the phenotype of opt7 mutant does not resemble that of opt3-2.

Whether graminaceous plant genomes carry orthologues of OPT3 remains to be determined.

Iron loading into seed tissues

There is no continuity between the vasculature of the mother plant and the pro-vasculature of the embryo. Therefore, Fe, as other nutrients, has to be released from the phloem and re absorbed by the embryo. In Arabidopsis and other dicotyledonous plants, nutrients are released at the level of the chalaze and the nucellus into the embryo sac fluid from which the embryo takes up nutrients (Figure 2). Accordingly, AtYSL1 expression is detected in the funiculus and the chalazal endosperm [START_REF] Jean | A loss-of-function mutation in AtYSL1 reveals its role in iron and nicotianamine seed loading[END_REF] . Analyses of Fe speciation in the embryo sac fluid of developing pea seeds revealed that Fe is present as ferric Fe bound to citrate and malate in this extracellular compartment [START_REF] Grillet | Ascorbate efflux as a new strategy for iron reduction and transport in plants[END_REF] . In agreement with this finding, the citrate efflux transporter FRD3 is expressed in the peripheral cell layer of the embryo and the cell layer of the tegument facing the embryo sac during seed development (Figure 2) [START_REF] Roschzttardtz | The FRD3 citrate effluxer promotes iron nutrition between symplastically disconnected tissues throughout Arabidopsis development[END_REF] . This suggest that FRD3 secretes citrate in the embryo sac to maintain Fe solubility and availability for uptake by the embryo.

In dicotyledonous plants, Fe is taken up as ferrous Fe. This implies that Fe must be reduced prior to uptake by the embryo. However, genetic analyses in Arabidopsis failed to identify a membrane bound ferric chelate reductase that is important for Fe acquisition by the embryo among FRO2 homologues. Instead, further analysis of the embryo sac fluid in pea showed the presence of a high concentration of ascorbate, sufficient to reduce Fe(III) to Fe(II) prior to its uptake by the embryo [START_REF] Grillet | Ascorbate efflux as a new strategy for iron reduction and transport in plants[END_REF] . Consistently, in Arabidopsis vtc (vitamin c) mutants deficient in ascorbate biosynthesis, the seed Fe content is decreased. The speciation of Fe in the embryo sac is thus similar to that encountered in other extracellular compartments of the plant, such as the xylem sap. In contrast, it is striking that ascorbate is used for Fe reduction, whereas membrane bound ferric chelate reductases are used for uptake in roots and leaves as well as in intracellular organelles such as mitochondria and plastids [START_REF] Robinson | A ferric-chelate reductase for iron uptake from soils[END_REF][START_REF] Jain | The diverse roles of FRO family metalloreductases in iron and copper homeostasis[END_REF][START_REF] Jeong | Chloroplast Fe(III) chelate reductase activity is essential for seedling viability under iron limiting conditions[END_REF] . The transporters responsible for secreting ascorbate and Fe in the embryo sac remain to be identified. In the case of Zn, the Heavy Metal pumping P-type ATPases HMA2 and HMA4 release Zn from the mother tissues for subsequent uptake by the embryo [START_REF] Olsen | Mother-plant-mediated pumping of zinc into the developing seed[END_REF] . In the case of Fe, the plasma membrane Fe efflux transporter IREG1/FPN1 may be involved in this process but so far no defect in Fe supply to the embryo has been reported for ireg1/fpn1 mutant [START_REF] Morrissey | The ferroportin metal efflux proteins function in iron and cobalt homeostasis in Arabidopsis[END_REF] .

In wheat and barley, the nutrients, including Fe, are provided to the grain by a single vascular strand along the ventral crease [START_REF] Borg | Iron Transport, deposition and bioavailability in the wheat and barley grain[END_REF] . Fe is mostly provided by the phloem and moves through several specialized cell layers: the crease vascular parenchyma, the pigment strand and the nucellar projection. Fe ends up in the transfer cells that are facing the embryo. From there, Fe has to be released in the extracellular space separating the mother plant from the embryo (Figure 3). The transfer cells, as the modified aleurone cells facing them on the side of the embryo, have highly invaginated plasma membranes favoring nutrient release and reabsorption [START_REF] Borg | Iron Transport, deposition and bioavailability in the wheat and barley grain[END_REF] . Fe accumulation and speciation in these structures show marked contrasts in the mature wheat grain. Fe is highly concentrated in the nucellar projection and co-localizes with sulfur. Fe accumulates to a lesser extend in the modified aleurone, from where it is probably distributed to the other aleurone cells, the embryo and the endosperm [START_REF] De Brier | Element distribution and iron speciation in mature wheat grains (Triticum aestivum L.) using synchrotron X-ray fluorescence microscopy mapping and X-ray absorption near-edge structure (XANES) imaging[END_REF] . X-ray absorption spectra indicate that in the nucellar projection, Fe is mostly associated with NA, whereas in the modified aleurone it is associated with phytate [START_REF] De Brier | Element distribution and iron speciation in mature wheat grains (Triticum aestivum L.) using synchrotron X-ray fluorescence microscopy mapping and X-ray absorption near-edge structure (XANES) imaging[END_REF] . In agreement with the speciation, barley homologues of AtVIT1, which drives Fe influx into the vacuole where phytate is localized, are strongly expressed in the aleurone [START_REF] Borg | Iron Transport, deposition and bioavailability in the wheat and barley grain[END_REF][START_REF] Connorton | Wheat Vacuolar Iron Transporter TaVIT2 Transports Fe and Mn and Is Effective for Biofortification[END_REF][START_REF] Kim | Localization of iron in Arabidopsis seed requires the vacuolar membrane transporter VIT1[END_REF][START_REF] Lott | X-ray Analysis Studies of Elements Stored in Protein Body Globoid Crystals of Triticum Grains[END_REF] . In contrast, the genes encoding NAAT, NAS and YSL, that favor Fe mobility, are expressed at high levels in transfer cells [START_REF] Borg | Iron Transport, deposition and bioavailability in the wheat and barley grain[END_REF][START_REF] Curie | Metal movement within the plant: contribution of nicotianamine and yellow stripe 1-like transporters[END_REF] (Figure 3). In the future, it will be important to determine the specific expression pattern and the role of each of the genes involved in Fe transport in this complex structure. This should allow the identification of the key transporters that release Fe to the extracellular space from the transfer cells as well as those responsible for taking it up into the embryo in the modified aleurone cells.

Iron storage in seeds

The localization of Fe in seeds differs according to species and seed developmental stage. The localization and subcellular localization are strongly associated with Fe speciation, i.e. the nature of the ligand that binds Fe and determines it bioavailability [START_REF] Clemens | Zn and Fe biofortification: the right chemical environment for human bioavailability[END_REF] . For example, Fe phytate complexes that are stored in vacuoles are notoriously poorly bioavailable [START_REF] Hallberg | Perspective on nutritional iron deficiency[END_REF] . In contrast, ferritin Fe stored in plastids constitutes a highly bioavailable source of Fe [START_REF] Briat | Plant ferritin and human iron deficiency[END_REF] .

Iron and other metals are not distributed evenly in seed tissues. In contrast, Fe distribution follows striking patterns. For example, in Arabidopsis mature embryo, Fe is highly concentrated around the vascular tissues [START_REF] Kim | Localization of iron in Arabidopsis seed requires the vacuolar membrane transporter VIT1[END_REF][START_REF] Chu | The Arabidopsis MTP8 transporter determines the localization of manganese and iron in seeds[END_REF][START_REF] Eroglu | Metal Tolerance Protein 8 Mediates Manganese Homeostasis and Iron Reallocation during Seed Development and Germination[END_REF][START_REF] Mary | Bypassing Iron Storage in Endodermal Vacuoles Rescues the Iron Mobilization Defect in the natural resistance associated-macrophage protein3natural resistance associated-macrophage protein4 Double Mutant[END_REF][START_REF] Roschzttardtz | Identification of the endodermal vacuole as the iron storage compartment in the Arabidopsis embryo[END_REF][START_REF] Schnell Ramos | Using muPIXE for quantitative mapping of metal concentration in Arabidopsis thaliana seeds[END_REF] . Interestingly, the pattern of Fe distribution is distinct from that of other metals: Mn is concentrated in the sub-epidermal cell layers of Arabidopsis cotyledons in mature seeds while Zn is evenly distributed in Arabidopsis embryo 71, 77, 78, 81 . The patterns of metal localization were initially discovered using synchrotron X-ray Fluorescence imaging of intact seeds and were then confirmed and refined using additional approaches. The use of Perls staining intensified by Diaminobenzidine (Perls DAB) allowed identification of the precise cell type accumulating Fe as the proto endodermis of the embryo [START_REF] Roschzttardtz | Identification of the endodermal vacuole as the iron storage compartment in the Arabidopsis embryo[END_REF] . The use of micro Particle Induced X-ray Emission (PIXE) allowed quantification of the pattern: even though it is concentrated in proto-endodermal cells, Fe is present in other cells, albeit at much lower concentrations, and the Fe concentrated around pro-vascular tissues accounts for 50% of total seed Fe [START_REF] Schnell Ramos | Using muPIXE for quantitative mapping of metal concentration in Arabidopsis thaliana seeds[END_REF] . Finally, the use of Electron Dispersive X-ray imaging coupled to Transmission Electron Microscopy (TEM EDX) provided higher spatial resolution allowing the determination of the subcellular localization of Fe in the globoids of vacuoles of proto-endodermal cells (Figure 4) [START_REF] Mary | Bypassing Iron Storage in Endodermal Vacuoles Rescues the Iron Mobilization Defect in the natural resistance associated-macrophage protein3natural resistance associated-macrophage protein4 Double Mutant[END_REF][START_REF] Lanquar | Mobilization of vacuolar iron by AtNRAMP3 and AtNRAMP4 is essential for seed germination on low iron[END_REF] . This subcellular localization is in agreement with the finding that mutations in AtVIT1 (Vacuolar Iron Transporter 1) disrupt the pattern of Fe distribution [START_REF] Kim | Localization of iron in Arabidopsis seed requires the vacuolar membrane transporter VIT1[END_REF][START_REF] Mary | Bypassing Iron Storage in Endodermal Vacuoles Rescues the Iron Mobilization Defect in the natural resistance associated-macrophage protein3natural resistance associated-macrophage protein4 Double Mutant[END_REF] . In vit1 mutants, Fe is not concentrated around vascular tissues but instead co-localizes with Mn in sub-epidermal cells [START_REF] Kim | Localization of iron in Arabidopsis seed requires the vacuolar membrane transporter VIT1[END_REF][START_REF] Mary | Bypassing Iron Storage in Endodermal Vacuoles Rescues the Iron Mobilization Defect in the natural resistance associated-macrophage protein3natural resistance associated-macrophage protein4 Double Mutant[END_REF] . Nevertheless, Fe is still localized in vacuoles in vit1 mutants [START_REF] Mary | Bypassing Iron Storage in Endodermal Vacuoles Rescues the Iron Mobilization Defect in the natural resistance associated-macrophage protein3natural resistance associated-macrophage protein4 Double Mutant[END_REF] . Recently, the vacuolar Mn transporter AtMTP8 (Metal Tolerance Protein 8) was shown to be responsible for concentrating Mn in sub-epidermal cells in wild type seeds and Fe localization in these cells in the vit1 mutant background [START_REF] Chu | The Arabidopsis MTP8 transporter determines the localization of manganese and iron in seeds[END_REF][START_REF] Eroglu | Metal Tolerance Protein 8 Mediates Manganese Homeostasis and Iron Reallocation during Seed Development and Germination[END_REF] . In mtp8 knockout mutant seeds, Mn is concentrated around the vascular tissues together with Fe. In a double mutant combining vit1 and mtp8 mutations, Mn and Fe are evenly distributed in Arabidopsis embryo [START_REF] Eroglu | Metal Tolerance Protein 8 Mediates Manganese Homeostasis and Iron Reallocation during Seed Development and Germination[END_REF] . Therefore, by creating strong sinks for Fe or Mn, vacuolar metal transporters determine the pattern of metal distribution in mature Arabidopsis embryo. The phenotype of vit1 mutant, which survives poorly when germinated under Fe-deficient conditions, indicates that adequate tissue localization in the embryo is crucial [START_REF] Kim | Localization of iron in Arabidopsis seed requires the vacuolar membrane transporter VIT1[END_REF][START_REF] Mary | Bypassing Iron Storage in Endodermal Vacuoles Rescues the Iron Mobilization Defect in the natural resistance associated-macrophage protein3natural resistance associated-macrophage protein4 Double Mutant[END_REF] . Little information is available on how the Fe distribution pattern is generated during embryo development. Consistent with a strong expression of VIT1 during seed development, Fe patterning is already apparent at the torpedo stage at the center of the cotyledons where the provascular tissue will differentiate [START_REF] Kim | Localization of iron in Arabidopsis seed requires the vacuolar membrane transporter VIT1[END_REF][START_REF] Roschzttardtz | Identification of the endodermal vacuole as the iron storage compartment in the Arabidopsis embryo[END_REF] . However, in Brassica napus, high resolution Perls DAB imaging detects Fe in the nuclei of all embryo cells at the torpedo stage [START_REF] Ibeas | Dynamic Subcellular Localization of Iron during Embryo Development in Brassicaceae Seeds[END_REF] . Further during development, at the bent cotyledon stage, Fe is detected in cytoplasmic vesicles and finally ends up in the vacuoles of the cells surrounding the vasculature in mature embryo [START_REF] Ibeas | Dynamic Subcellular Localization of Iron during Embryo Development in Brassicaceae Seeds[END_REF] .

The observation of Fe in the nucleus at the torpedo stage in B. napus is consistent with the detection of high Fe concentrations in the nucleus of embryo cells in developing pea (pisum sativum) seeds [START_REF] Roschzttardtz | Plant cell nucleolus as a hot spot for iron[END_REF] . Therefore, Fe subcellular localization evolves during embryo development.

These changes in localization are most likely paralleled by changes in speciation and therefore of Fe bioavailability. The analysis of Fe bioavailability in pea seeds at different developmental stages show that Fe bioavailability in immature peas is higher than in mature pea seeds [START_REF] Moore | The stage of seed development influences iron bioavailability in pea (Pisum sativum L.)[END_REF] .

Whether the pattern of Fe distribution in the vacuoles of cells surrounding the vasculature described in Arabidopsis is conserved among angiosperm has been investigated. In beans (Phaseolus vulgaris), Fe stores also concentrates around vascular tissues [START_REF] Cvitanich | Iron and ferritin accumulate in separate cellular locations in Phaseolus seeds[END_REF] . Recently, this observation was extended to Brassicales and even Rosids, which represent a wide group including a third of the angiosperms [START_REF] Eroglu | The Conservation of VIT1-Dependent Iron Distribution in Seeds[END_REF][START_REF] Ibeas | The Diverse Iron Distribution in Eudicotyledoneae Seeds: From Arabidopsis to Quinoa[END_REF] . However, in many cases the number of cell layers that accumulate Fe is increased, to 2, the endodermis and one cortical cell layer in Brassicales, or even more cortical cell layers in other Rosids, compared to endodermis only in Arabidopsis [START_REF] Ibeas | Dynamic Subcellular Localization of Iron during Embryo Development in Brassicaceae Seeds[END_REF][START_REF] Eroglu | The Conservation of VIT1-Dependent Iron Distribution in Seeds[END_REF][START_REF] Ibeas | The Diverse Iron Distribution in Eudicotyledoneae Seeds: From Arabidopsis to Quinoa[END_REF] . While screening representatives of the different groups of angiosperm, interesting exceptions were identified in Chenopodium quinoa and Carica papaya [START_REF] Eroglu | The Conservation of VIT1-Dependent Iron Distribution in Seeds[END_REF][START_REF] Ibeas | The Diverse Iron Distribution in Eudicotyledoneae Seeds: From Arabidopsis to Quinoa[END_REF] . There are certainly more diverse patterns to be discovered.

In grains, the seeds of graminaceous species, the distribution of Fe is very different from that observed in Brassicales. In grains, in contrast to Arabidopsis, the embryo represents only a small volume of the seed, while the major part is constituted by the starchy endosperm. The endosperm is surrounded by the so called aleurone cell layer. During germination, the aleurone layer is activated by gibberellins, releases enzymes that digest the carbohydrates stored in the endosperm and undergoes programmed cell death [START_REF] Fath | Barley aleurone cell death is not apoptotic: characterization of nuclease activities and DNA degradation[END_REF][START_REF] Jones | Gibberellic acid and the fine structure of barley aleurone cells : II. Changes during the synthesis and secretion of alpha-amylase[END_REF] . The embryo is equipped with an absorptive structure called the scutellum, somewhat equivalent to human placenta, which take up the nutrients from the endosperm. In grains, a large part of the Fe is concentrated in the aleurone layer and another pool is found in the embryo [START_REF] De Brier | Element distribution and iron speciation in mature wheat grains (Triticum aestivum L.) using synchrotron X-ray fluorescence microscopy mapping and X-ray absorption near-edge structure (XANES) imaging[END_REF][START_REF] Iwai | Dynamic changes in the distribution of minerals in relation to phytic acid accumulation during rice seed development[END_REF][START_REF] Singh | Pattern of iron distribution in maternal and filial tissues in wheat grains with contrasting levels of iron[END_REF] (Figure 3).

In wheat (Triticum durum), the highest concentrations of Fe are found in the aleurone layer and in the scutellum [START_REF] Singh | Pattern of iron distribution in maternal and filial tissues in wheat grains with contrasting levels of iron[END_REF] . Separate measurements of wheat flour and bran indicated that almost 60% of grain Fe is in the bran which includes the aleurone layer and therefore represents the major Fe pool in grains [START_REF] De Brier | Distribution of Minerals in Wheat Grains (Triticum aestivum L.) and in Roller Milling Fractions Affected by Pearling[END_REF] . Within the aleurone layer, Fe is concentrated together with phosphorus and other minerals in globoids inside the protein storage vacuole, similar to the subcellular localization observed in Arabidopsis [START_REF] Lott | X-ray Analysis Studies of Elements Stored in Protein Body Globoid Crystals of Triticum Grains[END_REF] . In situ X-ray absorption spectroscopy analyses have indicated that in the aleurone layer, most Fe is bound to phytate, either as Fe(II) or Fe(III) in good agreement with its subcellular localization [START_REF] De Brier | Element distribution and iron speciation in mature wheat grains (Triticum aestivum L.) using synchrotron X-ray fluorescence microscopy mapping and X-ray absorption near-edge structure (XANES) imaging[END_REF][START_REF] Singh | Pattern of iron distribution in maternal and filial tissues in wheat grains with contrasting levels of iron[END_REF] . Another pool is bound to citrate, which may be involved in the transport from modified aleurone cells present in the crease to aleurone cells [START_REF] De Brier | Element distribution and iron speciation in mature wheat grains (Triticum aestivum L.) using synchrotron X-ray fluorescence microscopy mapping and X-ray absorption near-edge structure (XANES) imaging[END_REF] . In rice, Fe accumulates in the aleurone layer together with phytate and in the scutellum, as observed in wheat [START_REF] Iwai | Dynamic changes in the distribution of minerals in relation to phytic acid accumulation during rice seed development[END_REF][START_REF] Takahashi | In vivo analysis of metal distribution and expression of metal transporters in rice seed during germination process by microarray and X-ray Fluorescence Imaging of Fe, Zn, Mn, and Cu[END_REF] . The exact speciation of Fe in the embryo and in the endosperm has not yet been determined in these tissues that contain lower Fe concentration, probably because of limitations in the sensitivity of X-ray absorption spectroscopy. Based on co-occurrence of elements, it has been proposed that Fe is bound to phosphate (possibly as phytate) in the scutellum in wheat and to other ligands in the other parts of the embryo and in the endosperm [START_REF] De Brier | Element distribution and iron speciation in mature wheat grains (Triticum aestivum L.) using synchrotron X-ray fluorescence microscopy mapping and X-ray absorption near-edge structure (XANES) imaging[END_REF][START_REF] Singh | Pattern of iron distribution in maternal and filial tissues in wheat grains with contrasting levels of iron[END_REF] . These ligands could include proteins or smaller molecules such as NA, which is present in grains as in seeds of non graminaceous species [START_REF] Jean | A loss-of-function mutation in AtYSL1 reveals its role in iron and nicotianamine seed loading[END_REF][START_REF] Lee | Iron fortification of rice seeds through activation of the nicotianamine synthase gene[END_REF] . In wheat flour which corresponds to the starchy endosperm, NA is the main ligand for Fe [START_REF] Eagling | Distribution and speciation of iron and zinc in grain of two wheat genotypes[END_REF] .

Little is known on the mechanisms that control the pattern of Fe localization in grains. A developmental analysis showed that throughout rice grain development Fe co-localizes with phosphorus in the aleurone cell layer, indicating that Fe is most likely bound to phytate as soon as it is stored [START_REF] Iwai | Dynamic changes in the distribution of minerals in relation to phytic acid accumulation during rice seed development[END_REF] . Mutations in OsVIT1, OsVIT2 and Mitochondrial Iron Transporter (MIT) have been shown to perturb iron localization within the embryo [START_REF] Bashir | The knockdown of OsVIT2 and MIT affects iron localization in rice seed[END_REF][START_REF] Zhang | Vacuolar membrane transporters OsVIT1 and OsVIT2 modulate iron translocation between flag leaves and seeds in rice[END_REF] . OsYSL9 is strongly expressed in the scutellum and participates to Fe storage in the embryo [START_REF] Senoura | The iron-chelate transporter OsYSL9 plays a role in iron distribution in developing rice grains[END_REF] . The phytosiderophore efflux transporter TOM2 is expressed in the dorsal vascular bundle, epithelium and the scutellum of the embryo and may also participate in the distribution of Fe [START_REF] Nozoye | The Phytosiderophore Efflux Transporter TOM2 Is Involved in Metal Transport in Rice[END_REF] . Silencing TOM2 gene expression did not alter seed total Fe content. Analysis of Fe distribution using sXRF, Perls staining or seed dissection could provide more insights in its function in developing rice grains.

Biofortification

The abundance, the localization and the speciation of Fe are equally important parameters to be taken into account in attempts to increase Fe content and bioavailability in grains, which are two of the major targets of biofortification projects. Having the major Fe pool stored as Fe phytate in the aleurone layer is probably the worst combination for human nutrition, as phytate has extremely high affinity for metals, which drastically limits Fe absorption in the intestine [START_REF] Hallberg | Perspective on nutritional iron deficiency[END_REF] . Moreover, the aleurone layer is most of the time discarded during grain processing, such polishing for rice or preparation of white flour for wheat [START_REF] De Brier | Distribution of Minerals in Wheat Grains (Triticum aestivum L.) and in Roller Milling Fractions Affected by Pearling[END_REF][START_REF] Eagling | Distribution and speciation of iron and zinc in grain of two wheat genotypes[END_REF][START_REF] Kawakami | Molecular processes in iron and zinc homeostasis and their modulation for biofortification in rice[END_REF] . Hence, biofortification strategies using targeted molecular approaches have aimed at changing both the localization and the speciation of Fe in grains. Several reviews have been recently published on the attempts made to biofortify wheat and rice grain [START_REF] Kawakami | Molecular processes in iron and zinc homeostasis and their modulation for biofortification in rice[END_REF][100][101] . This review will therefore not extensively cover the topic of biofortification and rather just highlight a few examples. The initial attempt to biofortify rice grains through biotechnology used the gene encoding ferritin, which provides Fe under a highly bioavailable form, driven by an endosperm specific promoter 102 . Although the work provided a critical proof of concept, the increase in Fe content was modest and variable 103 . Probably the most efficient strategy for biofortification by manipulating a single gene has been the activation or the over expression of NA synthase in rice grains [START_REF] Lee | Iron fortification of rice seeds through activation of the nicotianamine synthase gene[END_REF] . This resulted not only in a strong increase in grain Fe concentration but also in Fe bioavailability, as NA appears to favor Fe intestinal absorption [START_REF] Lee | Iron fortification of rice seeds through activation of the nicotianamine synthase gene[END_REF] .

More recently, expression of a Fe vacuolar transporter TaVIT2 in wheat endosperm was also shown to be a relevant approach to increase Fe content and bioavailability [START_REF] Connorton | Wheat Vacuolar Iron Transporter TaVIT2 Transports Fe and Mn and Is Effective for Biofortification[END_REF] . Probably, the expression of TaVIT2 in the endosperm vacuoles creates a strong Fe sink during development. Subsequently, Fe becomes available when endosperm cells die and lose their internal structure. Current efforts use pyramiding of several constructs to combine the benefits of multiple transgenes. This way, by combining the expression of transporters that limit vacuolar storage with elevated ferritin and NA synthesis, an increase in Fe concentration in grains by up to 6-fold has been obtained 104 .

A role for iron in the regulation of seed germination?

Seed germination starts with water uptake by the dry seed and ends with radicle protrusion and initiation of cell division 105 . This is the critical phase of plant emergence because it is tightly regulated by water availability, temperature, oxygen, and light conditions. Germination is also controlled by endogenous factors such as the plant hormones abscisic acid (ABA), gibberellins (GA) and ethylene that play a major role in regulating early seed germination. These endogenous signals regulate germination through the process of dormancy, which is an endogenous block to the completion of germination of a mature seed 106 . Germination is followed by seedling growth during which mobilization of reserves and nutrient stored within the seed sustain active cell metabolism till the acquisition of autotrophy. Thus, stand establishment, the first critical component of crop yield, results from the successful and fast completion of both germination and early seedling growth.

Although the role of iron in post-germinative events is quite well documented (see below) its possible involvement in the regulation of germination, i.e. the sum of molecular events that allow radicle protrusion, is rather unknown. The effect of iron on seed germination has mostly been documented in the context of soil toxicity, since high Fe concentrations are toxic and inhibit germination 107,108 , but this is a common feature to many metals. Increase in phytatedegrading enzyme activity has been reported to occur during seed germination, with a concomitant decline in phytate [109][110][111] thus increasing iron bioavailability 109 . Müller et al. (2009) showed that hydroxyl radicals were likely to play a role in cell wall loosening during radicle elongation and weakening of the endosperm of cress seeds 112 . Although Fenton reaction is a natural candidate mechanism for explaining hydroxyl radical production they did not provide any evidence about its possible involvement in vivo. Thus, whether iron availability participates to the numerous biochemical and molecular processes involved in the regulation of radicle protrusion is actually not known.

Murgia and Morandini (2017) demonstrated that, when seeds developed on the mother plant under Fe deficiency, they were more dormant at harvest, thus suggesting that seed Fe availability can regulate sensu stricto germination 113 . It is highly likely that Fe could regulate germination by controlling ROS homeostasis, through its involvement in the Fenton reaction.

ROS indeed have been proposed to be key players in seed germination and dormancy [114][115][116] .

Seed dormancy alleviation requires controlled ROS generation 117 but excessive levels of ROS trigger oxidative stress and prevent germination. This is occurring when seeds are germinated in unappropriated environmental conditions or when they have aged 115 . With regards to the relationship between Fe and ROS metabolism, we can thus hypothesize that Fe homeostasis, as controlled by sequestration and release by vacuoles, is likely to participate in the ability of seeds to germinate and to play a role in the regulation of dormancy, by buffering ROS homeostasis. However whether Fe speciation (and localization) is important for seed longevity and control of dormancy and germination remains to be addressed.

Iron remobilization after germination

Besides human nutrition, the main role of seed Fe stores is to sustain the early development of seedlings after germination. In dicots in which most of the Fe is probably stored as Fe phytate in vacuoles, retrieving Fe stores likely requires first to break down the phytate, then to transport Fe out of the vacuole and out of the cells. In Arabidopsis, Perls DAB staining allowed monitoring the fate of Fe stores after germination: the high Fe concentration around vascular tissues observed in dry seeds disappeared within 4 days, indicating that Fe was rapidly redistributed the growing organs of the seedling [START_REF] Mary | Bypassing Iron Storage in Endodermal Vacuoles Rescues the Iron Mobilization Defect in the natural resistance associated-macrophage protein3natural resistance associated-macrophage protein4 Double Mutant[END_REF][START_REF] Roschzttardtz | Identification of the endodermal vacuole as the iron storage compartment in the Arabidopsis embryo[END_REF] . In Arabidopsis, this process requires two redundant vacuolar transporters AtNRAMP3 and AtNRAMP4 (Natural Resistance Associated Macrophage Protein) [START_REF] Lanquar | Mobilization of vacuolar iron by AtNRAMP3 and AtNRAMP4 is essential for seed germination on low iron[END_REF] . AtNRAMP3 and AtNRAMP4 are highly expressed after radicle protrusion. In loss of AtNRAMP3 and AtNRAMP4 function mutants, seedling growth is arrested before the onset of photosynthesis [START_REF] Lanquar | Mobilization of vacuolar iron by AtNRAMP3 and AtNRAMP4 is essential for seed germination on low iron[END_REF] . In the nramp3nramp4 double mutant, Fe remains blocked inside the endodermal vacuoles after germination [START_REF] Mary | Bypassing Iron Storage in Endodermal Vacuoles Rescues the Iron Mobilization Defect in the natural resistance associated-macrophage protein3natural resistance associated-macrophage protein4 Double Mutant[END_REF][START_REF] Roschzttardtz | Identification of the endodermal vacuole as the iron storage compartment in the Arabidopsis embryo[END_REF][START_REF] Lanquar | Mobilization of vacuolar iron by AtNRAMP3 and AtNRAMP4 is essential for seed germination on low iron[END_REF] .

The phenotype of nramp3nramp4 is partially suppressed by mutations in AtVIT1 by redirecting Fe storage in vacuoles of cortical cells [START_REF] Mary | Bypassing Iron Storage in Endodermal Vacuoles Rescues the Iron Mobilization Defect in the natural resistance associated-macrophage protein3natural resistance associated-macrophage protein4 Double Mutant[END_REF] . The phenotype of nramp3nramp4 is also rescued by Fe supplementation. However, even in the presence of Fe in the medium, the mutant activates Fe deficiency responses 118 . Interestingly, chloroplast functions and especially Fe-requiring plastidial enzymes are repressed in the mutant while mitochondrial function is maintained 118 . This suggests that Fe is prioritized to mitochondria or that these organelles rely on an independent pool of Fe. AtNRAMP3 and AtNRAMP4 encode divalent cation transporters. As Fe is most likely stored as Fe(III) in the vacuole, it means that Fe reduction is probably required prior to Fe export by AtNRAMP3 and AtNRAMP4. However, the Fe reduction system active in vacuoles of germinating cells remains to be identified. The phytase allowing Fe release prior to its reduction and export also remains to be identified. The importance of such activity is highlighted by the finding that expression of the bacterial phytase US417 or mutation in the phytate biosynthetic enzyme IPK1 (Inositol Phosphate Kinase 1) in Arabidopsis significantly accelerate Fe remobilization 119 . After its export from the vacuole, Fe needs to be exported from storage cells (the endodermis in Arabidopsis).

However, the transporters involved in Fe efflux from cells during germination have not been identified. One candidate could be IREG1/FPN1, the plant homologue of the transporter involved in the release of Fe into the blood flow from intestine epithelial cells [START_REF] Morrissey | The ferroportin metal efflux proteins function in iron and cobalt homeostasis in Arabidopsis[END_REF] . Once it is exported from the cells, Fe needs to remain in a soluble form to diffuse to other cells. The citrate efflux transporter FRD3 is expressed in germinating seeds and could be involved in the release of citrate to form soluble Fe complexes in the apoplast. This hypothesis is supported by the finding that frd3 mutants are chlorotic and exhibit slow root growth after germination [START_REF] Roschzttardtz | The FRD3 citrate effluxer promotes iron nutrition between symplastically disconnected tissues throughout Arabidopsis development[END_REF] . This phenotype can be rescued by supplementation of Fe or citrate in the cytosol.

In relation with their distinct organization, Fe remobilization mechanisms in seeds of graminaceous species are likely very different. In grains, most of the Fe is in the aleurone layer, outside of the embryo. During germination, under the positive control of gibberellins, aleurone cells secrete amylases that digest the starch in the endosperm and eventually undergo programmed cell death (PCD) [START_REF] Fath | Barley aleurone cell death is not apoptotic: characterization of nuclease activities and DNA degradation[END_REF] . Membranes are destroyed in this process and it is likely that the content of globoids including Fe bound to phytate is released in the endosperm. Iron efflux from vacuoles is probably not a necessary step for mobilization. Accordingly, no orthologues of AtNRAMP3 or AtNRAMP4 have been reported in graminaceous plants. In the endosperm, phytate is probably hydrolyzed by phytase and the released Fe bound to NA. As in dicotyledonous species, the precise phytase involved have not yet been identified. Then, the main question is how is Fe taken up from the endosperm into the embryo and further distributed to growing organs within the embryo. Time course analysis of gene expression and Fe localization during rice germination has highlighted several candidates [START_REF] Takahashi | In vivo analysis of metal distribution and expression of metal transporters in rice seed during germination process by microarray and X-ray Fluorescence Imaging of Fe, Zn, Mn, and Cu[END_REF]120 . X-ray fluorescence imaging revealed that after 24h of germination, Fe accumulates in the epithelium and scutellum, the Fe level decreases in the scutellum and increases in the coleoptile. Iron is already visible in the root tip after 36 hours [START_REF] Takahashi | In vivo analysis of metal distribution and expression of metal transporters in rice seed during germination process by microarray and X-ray Fluorescence Imaging of Fe, Zn, Mn, and Cu[END_REF] . Genes encoding NA and phytosiderophore biosynthesis were upregulated during germination 120 . Interestingly, NA synthase expression was prominent in the endosperm while NA amino transferase (NAAT) was expressed in the embryo. Accordingly, several members of the YSL NA and phytosiderophore transporters, YSL2, 6, 10, 12 and 14 are upregulated 120 . The expression of TOM2, a NA and phytosiderophore efflux transporter and of OsYSL2 increased markedly in the scutellum during germination [START_REF] Nozoye | The Phytosiderophore Efflux Transporter TOM2 Is Involved in Metal Transport in Rice[END_REF]120 . These data indicate that Fe is transported as a complex with NA into the embryo or phytosiderophores within the embryo. Several transporters for free divalent Fe were also upregulated during germination: OsIRT1, several OsNramp genes and a Ferroportin/IREG [START_REF] Takahashi | In vivo analysis of metal distribution and expression of metal transporters in rice seed during germination process by microarray and X-ray Fluorescence Imaging of Fe, Zn, Mn, and Cu[END_REF]120 . As in dicotyledonous species, the citrate efflux transporter OsFRDL1 is upregulated indicating that citrate is also secreted to maintain Fe solubility during cell-tocell transport [START_REF] Roschzttardtz | The FRD3 citrate effluxer promotes iron nutrition between symplastically disconnected tissues throughout Arabidopsis development[END_REF][START_REF] Yokosho | OsFRDL1 is a citrate transporter required for efficient translocation of iron in rice[END_REF] . Finally, the rice homologue of the chloroplast Fe influx system in Arabidopsis PIC1 is upregulated after 3 days probably to sustain the differentiation of plastids into chloroplast during early seedling development 121 . Many transporters of Fe 2+ or Fe complexes with NA or siderophores are apparently involved in Fe remobilization after grain germination. However, the individual contribution of each of these transporters remains to be analyzed to identify the key steps in Fe remobilization in graminaceous species.

With its uptake into chloroplasts of the developing seedling, Fe completes its journey from the senescing leaves of the mother plant to the expanding photosynthetic apparatus of its progeny, via the seed.

Conclusions and perspectives

Many transporters and ligands for Fe have been characterized and this knowledge has been used to design targeted biofortification strategies. However, many key steps remain to be elucidated at the molecular level. The mechanisms that make iron available for reallocation to the seeds during leaf senescence need to be explored in more details. It would be important to determine which of the different pathways that contribute to chloroplast degradation are most important for Fe mobilization. Important questions also still need to be answered concerning phloem transport: the ligand for Fe in the phloem is still not unequivocally identified.

Moreover, whereas OPT3 plays a key role in Fe transport to seeds, the specific substrate of this transporter is still unknown. The transfer of Fe from the mother plant to the embryo has been under investigated so far and probably deserves more attention (Figure 2). The pioneering discovery that ascorbic acid is responsible for Fe reduction in the extracellular space separating the mother tissues from the embryo raises important questions. How general this mechanism is remains to be determined. For example, the relevance of this finding for grain staples needs to be addressed. There is also a need to identify the molecular players involved in ascorbate secretion and regeneration. Moreover, even though FRD3 has been proposed to be responsible for citrate efflux in the intracellular space separating the maternal tissues and the embryo, this hypothesis would need to be substantiated and other transporters potentially involved in citrate and malate efflux need to be identified. Last but not least, the transport mechanisms responsible for Fe efflux from mother tissues and reuptake into the embryo remain completely unknown. Tissue specific transcriptomic analyses have provided candidates for mediating these steps in barley [START_REF] Borg | Iron Transport, deposition and bioavailability in the wheat and barley grain[END_REF] When considering Fe remobilization after germination, many questions are still open. In Arabidopsis and in dicots in general, several steps upstream and downstream of the action of vacuolar efflux by NRAMP3/4 remain to be investigated. The molecular identity of the phytases that make Fe available for transport has to be determined. Whether Fe needs to be reduced prior to transport by vacuolar NRAMP and by which mechanism are still open questions. Downstream of vacuolar efflux the plasma membrane transporters that allow Fe efflux from endodermal cells to other cell types, as well as the mechanisms that allow Fe distribution to plastids, are still to be identified. Similar questions arise concerning the use of Fe stored in aleurone cells after the germination of grains of monocots. Finally, the master regulators that orchestrate Fe remobilization in response to hormonal cues, such as gibberellins, also need to be defined.

In conclusion, even though many players involved in Fe storage and remobilization from seeds have been discovered in the last decades, we still lack a molecular understanding of several key steps. In addition to improving our basic knowledge of how plants transfer nutrients from one generation to the next, unraveling these steps holds great promises for seed Fe biofortification and possibly for improving seed quality and stand establishment.

  . The role of each candidate has now to be analyzed in detail and similar analyses are lacking in Arabidopsis or other dicots. Manipulating these steps would certainly open new perspectives for biofortification. Besides the effectors of Fe transport and complexation during Fe loading into seeds, it would also be important to get insights into the master regulators that control Fe storage in seeds. How Fe storage is connected to the signaling pathways and transcription factors that regulate seed development, such as Leafy Cotyledon 2 (LEC2) or WRINCKLED, needs to be unraveled 122 .

Ackowledgements

The work on seed iron storage in ST, SM and CB laboratories is supported by the CNRS, the INRA and Paris Sorbonne University, as well as by the collaborative ANR grant ISISTOR (ANR-16-CE20-0019-02). References

Fe is transported to the maternal tissues of the developing seed but has to cross an extracelluar space between the teguments and the embryo. B. Fe is bound to malate and citrate in the extracellular space separating the maternal tissues from the embryo. In Arabidopsis and pea, Fe is reduced by ascorbate prior to its uptake into the embryo. The transporters responsible for Fe, malate and ascorbate secretion to the extracellular space as well as the transporter responsible for Fe uptake into the embryo remain to be identified.


Fanny Peigneguy

Charles Cougnon

Raphaëlle Barry-Martinet

Christine Bressy

Frédéric Gohier

Antifouling activities of immobilized ferrocenyl glucose on glassy carbon surface

Keywords: Antifouling, ferrocene, electroactivity, amine oxidation

In this article a new coating for antifouling applications has been developed. This coating has two distinct parts, one being a carbohydrate to render the surface polar and the other being a ferrocene which is electrically activatable to produce charges periodically. This material has been grafted onto carbon surfaces by oxidation of an amine group and has shown anti-bacterial activities.

Introduction.

Submerged surfaces in the marine environment are prone to microorganisms that quickly allow colonization by larger marine species responsible for the deterioration of the surface. 1 Prevention is generally done by coatings that can release active ingredients that are toxic to the

microorganisms. Among others copper-based, 2,3 zinc-based biocides, [START_REF] Omae | General Aspects of Tin-Free Antifouling Paints[END_REF][START_REF] Martins | Review: ecotoxicity of organic and organo-metallic antifouling co-biocides and implications for environmental hazard and risk assessments in aquatic ecosystems[END_REF] and boosters [START_REF] Konstantinou | Worldwide occurrence and effects of antifouling paint booster biocides in the aquatic environment: a review[END_REF] can be caged and released gradually to avoid any colonization. In order to last in time, regular applications of the coating are necessary. They represent 90% of the coating used. The released products accumulate in the marine environment; they are not necessarily degraded and has side-effects on flora and fauna. [START_REF] Amara | Antifouling processes and toxicity effects of antifouling paints on marine environment. A review[END_REF] It exists non-release coatings that can act either by preventing the approach of microorganisms or coatings that allow the release of microorganisms which attempt to bind on the surface. [START_REF] Lejars | Fouling Release Coatings: A Nontoxic Alternative to Biocidal Antifouling Coatings[END_REF][START_REF] Hu | Silicone-Based Fouling-Release Coatings for Marine Antifouling[END_REF] Surfaces can be highly hydrophobic or highly hydrophilic, both presenting advantages and inconvenience faces to fouling. For example, super hydrophilic surfaces will see a watery layer forming like a shield to prevent the adhesion of proteins. This is the case of materials with poly(ethyleneglycol) (PEG) [START_REF] Jeon | Protein-surface interactions in the presence of polyethylene oxide: I. Simplified theory[END_REF] or zwitterionic materials. [START_REF] Chen | Surface hydration: Principles and applications toward low-fouling/nonfouling biomaterials[END_REF] Carbohydrates also have a high polarity making them good candidates as antifouling materials. Colavita et al. [START_REF] Zen | Modulation of Protein Fouling and Interfacial Properties at Carbon Surfaces via Immobilization of Glycans Using Aryldiazonium Chemistry[END_REF][START_REF] Angione | Enhanced Antifouling Properties of Carbohydrate Coated Poly(ether sulfone) Membranes[END_REF] have grafted on polyethersulfone membranes or simply on carbon, glucose, galactose and lactose with good antifouling properties. Cao et al. [START_REF] Cao | Resistance of Polysaccharide Coatings to Proteins, Hematopoietic Cells, and Marine Organisms[END_REF] showed with alginic acid, hyaluronic acid and pectic acid immobilized on the surface a good activity against adhesion of Ulva spores and barnacle larvae.

However, Kerchove et al. [START_REF] De Kerchove | Calcium and Magnesium Cations Enhance the Adhesion of Motile and Nonmotile Pseudomonas aeruginosa on Alginate Films[END_REF] demonstrated the development of a bacterial biofilm on alginic acid which is hydrophilic. On the other hand, Matsunaga [START_REF] Okochi | Electrochemical sterilization of bacteria using a graphite electrode modified with adsorbed ferrocene[END_REF] by grafting ferrocene onto a carbon surface showed an antibacterial activity by scanning a small range of electrical potential. Bressy et al. [START_REF] Nguema Edzang | Bacterial anti-adhesion activity based on the electrochemical properties of polymethacrylates bearing ferrocenyl pendant groups[END_REF] also confirmed this result by immobilizing ferrocene on polymethacrylate. This presents an alternative to the classical weapons against fouling. With this in mind, the association of ferrocene with a carbohydrate targeting antifouling properties has never been demonstrated. The association of two entities could be cooperative since one has very polar interactions that can preserve a protective aqueous layer while ferrocene is electrically switchable and can generate charges when the latter is solicited. This system would be active as protein-repellent on one hand and on the other hand it would be possible to change the wettability, making the surface not very propitious to bacteria. Synthesis, electrochemical properties, grafting on surfaces and biological methodologies on ferrocene bound to glucose are described.

Experimental section

Material:

Ferrocene 6 was synthesized in 8 steps from glucose (see supp. info.). Compounds 5 and 6 are described below and 1, 2, 3 and 4 are described in the SI.

6-Deoxy-6-(4-ferrocenyl-1H-1,2,3-triazol-1-yl)-1-(4-nitrophenyl)-β-D-glucopyranose 5

To a solution of 4-nitrophenyl 6-azido-6-deoxy-β-D-glucopyranose (3.8 mmol, 1 equiv) in a mixture of DMF:H2O (20 mL, 3:1), ethynylferrocene (4.18 mmol, 1.1eq), sodium ascorbate (3 mmol, 0.8 equiv,) and CuSO4.5H2O (1.52 mmol, 0.4eq,) were added at room temperature. The reaction mixture was stirred at room temperature overnight. The mixture was poured into saturated NH4Cl solution, and then extracted with EtOAc and washed with brine. The organic phase was concentrated under reduced pressure. The crude product was purified by flash column chromatography (EtOAc/EtOH, 100:0 to 95:5) to give an orange-brown powder in 69 % yield. Spectral data were consistent with the results reported in the literature.

6-Deoxy-6-(4-ferrocenyl-1H-1,2,3-triazol-1-yl)-1-(4-aminophenyl)-β-D-glucopyranose 6

SnCl2.2H2O (1.89 g, 5eq, 8.39 mmol) was added to a solution of 5 (900 mg, 1.67 mmol) in ethyl acetate (40 ml) at room temperature. The reaction mixture was refluxed overnight. Then, the mixture was cooled to room temperature and poured into a saturated aqueous NaHCO3. A white precipitate was formed and filtered. Then, the reaction mixture was extracted with ethyl acetate, washed with water and brine. The organic phase was dried over anhydrous MgSO4 and concentrated in vacuo. The residue was purified by column chromatography on silica gel (EtOAct/EtOH, 100:0 to 90:10) to afford the final compound in 40 % yield as an orange solid. Spectral data were consistent with the results reported in the literature.

For the different immobilizations of compound 6, 1mM solutions were prepared. In aqueous medium, it was dissolved in a solution of artificial seawater (36 g Sea-salt (Aldrich) in 100 ml ultrapure water). For electrodeposition, a 96-well microplate with 3 electrodes (ref DRP-96X110) associated with a connector (ref CONNECTOR96X) connected to a potentiostat (ref DRP-STAT8000P) was purchased from DROPSENS. The 3 electrodes at the bottom of the wells are in carbon (C) for the working electrode (3 mm) and the counter-electrode and in silver for the reference electrode. Marine bacteria (TC8, Pseudoalteromonas lipolytica) were isolated in Toulon Bay (Mediterranean Sea, February 2008) from an artificial surface. This bacterial strain is Gramnegative and classified as hydrophilic bacteria. It was cultured on Vaatanen nine salts solution (VSSN) under agitation (120 rpm) at 20°C and harvested in the stationary phase for bacterial adhesion tests. [START_REF] Camps | Antifouling activity of commercial biocides vs. natural and natural-derived products assessed by marine bacteria adhesion bioassay[END_REF] Experimental procedures: Electrodeposition of compound 6 in wells.

A solution of compound 6 at 1mM in ASW (36 g/L) was prepared and 20 equivalents of 1M NaOH solution was added. This solution was sonicated to ultrasounds to make compound 6 completely soluble. 200 µL of the solution were introduced in each well. 6 was grafted by cyclic voltammetry (5 cycles) from -0.3 V to + 0.8 V at 100 mV/s. Then, the solution was removed from each well. All the wells were rinsed by ASW (three times). Fresh ASW was then placed back into each well to study the grafting responses by cyclic voltammetry (10 cycles) from -0.3 V to + 0.6 V, 100 mV/s. ASW was removed again to do the antiadhesion assay.

Antifouling test

Bacterial cultures were performed in VNSS, starting with an optical density (OD600nm) equal to 0.1. This bacterial suspension was incubated at 20°C at 120 rpm. The growing was controlled every hour for 6 hours until reaching the stationary phase to enhance their adhesion on substrates.

The bacterial suspension was centrifuged for 15 min. The VNSS was removed and replaced by ASW (Seasalt, Sigma Aldrich), with the same volume. This new suspension (bacteria+ASW) was diluted in ASW in order to reach an optical density (OD600nm) equal to 0.3. 200 µL of bacterial suspension (at OD600nm = 0.3) were introduced in each wells of columns 8 and 9 noted WB, and free bacteria ASW solution in the well of columns 8 and 9 noted WoB. The plate was connected to the potentiostat and was incubated at 20°C at 120 rpm for 16 hours. Potential current scans were applied from -0.3 V to 0.6 V at 100 mV/s, for 16 hours, on one column. After 16 hours, wells were rinsed 3 times by NaCl (36 g/L). A 1 µM Syto ® 61 solution (λexc = 620 nm, λem = 650 nm) diluted in NaCl (36 g/L) was prepared without light. 200 µL of Syto ® 61 solution were introduced in each well (WB and WoB) and were incubated in the dark at 20°C at 120 rpm for 20 min. Then, the fluorescence intensity (FI) was measured by the Infinit 200pro microplate fluorescence reader (TECAN). In parallel to the adhesion test done on coatings, a polystyrene (PS) plate was prepared with the same procedure and was used as a reference. The percentage of adhesion for the PS reference was assumed to be 100%. The percentage of adhesion of TC8 on coatings was calculated as follows:

% 𝑎𝑎𝑎𝑎ℎ𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 = (𝐹𝐹𝐹𝐹 𝑤𝑤𝑒𝑒𝑤𝑤ℎ 𝑏𝑏𝑎𝑎𝑏𝑏𝑤𝑤𝑒𝑒𝑏𝑏𝑒𝑒𝑎𝑎 -𝐹𝐹𝐹𝐹 𝑤𝑤𝑒𝑒𝑤𝑤ℎ𝑒𝑒𝑜𝑜𝑤𝑤 𝑏𝑏𝑎𝑎𝑏𝑏𝑤𝑤𝑒𝑒𝑏𝑏𝑒𝑒𝑎𝑎)𝑏𝑏𝑒𝑒𝑎𝑎𝑤𝑤𝑒𝑒𝑒𝑒𝑐𝑐 (𝐹𝐹𝐹𝐹 𝑤𝑤𝑒𝑒𝑤𝑤ℎ 𝑏𝑏𝑎𝑎𝑏𝑏𝑤𝑤𝑒𝑒𝑏𝑏𝑒𝑒𝑎𝑎 -𝐹𝐹𝐹𝐹 𝑤𝑤𝑒𝑒𝑤𝑤ℎ𝑒𝑒𝑜𝑜𝑤𝑤 𝑏𝑏𝑎𝑎𝑏𝑏𝑤𝑤𝑒𝑒𝑏𝑏𝑒𝑒𝑎𝑎) 𝑃𝑃𝑃𝑃

Toxicity assay

After 16h of incubation, 100 µL of solution from WB and WoB wells were kept and moved to a PS plate. In additional PS wells, 100 µL of a bacterial suspension at an OD600nm equal to 0.6 were introduced. In these later, ethanol at different concentrations (100 µL; 0%, 5%, 10%, 20%, 40%, 70%, 100%) was introduced to lead to a bacterial suspension at OD = 0.3. The PS plate was incubated at 20 °C, 120 rpm for 1 hour. Then, 100 µL of a mixture Syto ® 9 nucleic acid stain (λexc = 470nm, λem = 515nm) and propidium iodide (PI) (λexc = 538nm, λem = 617nm) in NaCl (36 g/L)

were introduced in each well and the plate was incubated for 20 min at 20°C at 120rpm in dark.

The PI shows the dead bacteria whereas the Syto ® 9 shows the living and the dead bacteria. Their FI were measured by the Infinit 200pro microplate fluorescence reader TECAN. The ratio Syto ® 9/PI allows to estimate the toxicity of the coating and to compare it to the toxicity of various % of ethanol. If this ratio is close to zero, the coating is considered as toxic.

Results

The immobilization of the targeted antifouling ferrocenyl glucose derivative by a covalent bond can be achieved in different ways, all using the amino group as surface anchoring functionality, such as the reduction of diazonium salts, [START_REF] Pinson | Attachment of organic layers to conductive or semiconductive surfaces by reduction of diazonium salts[END_REF] or the spontaneous reaction of the amine derivatives with the surface [START_REF] Gallardo | Spontaneous Attachment of Amines to Carbon and Metallic Surfaces[END_REF] or by electrochemical oxidation of amine functions. [START_REF] Barbier | Electrochemical Bonding of Amines to Carbon Fiber Surfaces Toward Improved Carbon-Epoxy Composites[END_REF][START_REF] Bélanger | Electrografting: a powerful method for surface modification[END_REF] The latter route is the most compatible with the ferrocene entity since the diazonium salt is not stable in the presence of ferrocene. [START_REF] Berlin | Polymers with conjugated double bonds and heteroatoms in the conjugated chain-XXVI. Polyazophenyleneferrocenes[END_REF][START_REF] Rosenblum | Synthesis of ferrocenylacetylenes[END_REF] Ferrocene is bound to glucose by 1,3-dipolar cycloaddition, the glucose azide having been prepared in 5 steps from glucose as shown in scheme 1.

Scheme 1: Synthesis of glucose clicked ferrocene.

The covalent immobilization of the ferrocenyl aminophenyl glucose derivative 6 was achieved by electrochemical oxidation in artificial sea water (ASW) which has more practical significance for antifouling applications than organic medium commonly used for surface modification. Indeed, acetonitrile/nBu4NPF6, usually used in electrochemistry could lead to entrapment of these derivatives in the coating. We have already shown that acetonitrile and nBu4NPF6 can have some toxicity to bacteria (unpublished results). In order to really attribute the anti-fouling effect to the grafted material, it was better to put ourselves in the conditions closest to biological tests. Another reason to use ASW medium is technical, as the plates with the microwells do not support organic media. In this respect, it should be noted that electrografting process by oxidation of amines in aqueous medium are rarely reported in literature, especially in the natural seawater electrolyte for which no published data exist. Geneste et al. [START_REF] Geneste | Electrochemically linking TEMPO to carbon via amine bridges[END_REF] showed the possibility of grafting TEMPO and nitrobenzylamine in both acidic and basic media. Other derivatives such as aminobenzylphosphonic acid, [START_REF] Yang | Covalent modification of glassy carbon electrode during electrochemical oxidation process of 4-aminobenzylphosphonic acid in aqueous solution[END_REF] aminobenzoic acid, [START_REF] Yang | Copper hexacyanoferrate multilayer films on glassy carbon electrode modified with 4-aminobenzoic acid in aqueous solution[END_REF] PAMAM dendrimers [START_REF] Kim | Modification of a glassy carbon surface with amine-terminated dendrimers and its application to electrocatalytic hydrazine oxidation[END_REF] and cyclodextrine derivatives [START_REF] Silva | Covalent Immobilization of Amino-β-Cyclodextrins on Glassy Carbon Electrode in Aqueous Media[END_REF] could be immobilized in glassy carbon electrode using aqueous medium.

Cyclic voltammograms (CVs) presented in Figure 1 show that molecules essentially retain the redox characteristics of both ferrocene and amine groups, with features of a reversible system centered at 0.23 V vs. ECS and an irreversible one-electron anodic wave located at 0.58 V vs.

ECS, proving that in the time scale of the cyclic voltammetry, the amine cation radical is unstable and underwent an overall deprotonation/dimerization sequence. Importantly, the fate of the oxidation products of amine is pH dependent. [START_REF] Cauquis | Les propriétés électrochimiques des diphénylamines et de leurs produits d'oxydation en milieu organique-II. Propriétés des diaryl-5,10 dihydro-5,10 phénazines et des tétraarylhydrazines[END_REF] In ASW (pH 8.3), the irreversible oxidation of the amine is accompanied by a new reversible system appearing at around 0.03 V vs. ECS, attributable to an electroactive head-to-tail dimer as demonstrated by Bacon and Adams in aqueous media. [START_REF] Bacon | Anodic oxidations of aromatic amines. III. Substituted anilines in aqueous media[END_REF] When ASW was adjusted at pH 12 with NaOH 2M, a huge decrease in the current peak intensity of the oxidation of amine is obtained after the first cycle. A cathodic shift of amine signal is also observed, the amine oxidation seems to be facilitated in very basic medium, and the reversible system oxidation has become difficult to see.

These changes in the shape of the CVs as the pH increases demonstrate that it was possible to minimize the formation of the dimer to make grafting favourable. Indeed, at pH 12, the reversible oxidation peak at 0.03 V is less and less visible and the oxidation peak of the amine loses its intensity with each cycle indicating a progressive passivation of the electrode. [START_REF] Peigneguy | Preparation and study of a carbohydrate-functionalized carbon surface by electrochemical oxidation of 4-aminophenyl-β-D-glucopyranoside[END_REF] The cyclic voltammetry study in pure ASW of glassy carbon (GC) electrodes previously treated as in Figures 1a and1b is also very instructive about the reactivity of the amino cation radical toward the GC electrode surface. At pH 8.3, the modified electrode shows two weak reversible systems with decreasing intensities upon the first CVs, which can be identified as a coupling product physisorbed onto the electrode surface. On the contrary, at pH 12, both the electrode passivation in ASW (figure 1c) containing 1 mM of 6 and the stable pair of reversible peaks at the same potential location than the one of the ferrocene/ferrocenium couple in pure ASW (figure 1d) are evidence for the robust grafting of the electrode surface. It should be noted that the recovery rate at pH 8.3 is 6.2 10 -11 mol/cm 2 and increases to 2.25 10 -10 mol/cm 2 at pH 12.2. Comparing these results with the literature, it would appear that the layer formed on the surface of the electrode is fairly compact and is close to a monolayer (4.4 10 -10 mol/cm 2 for a compact layer of ferrocene derivatives). [START_REF] Leroux | Efficient Covalent Modification of a Carbon Surface: Use of a Silyl Protecting Group To Form an Active Monolayer[END_REF] To make the covalent grafting possible, the carbon surface should be attacked by an aminyl radical produced by deprotonation of the amino cation radical. [START_REF] Adenier | Electrochemical Oxidation of Aliphatic Amines and Their Attachment to Carbon and Metal Surfaces[END_REF] With reference to the well-established reaction mechanism of the electrografting of amines, the stability of the cation radical governs the grafting efficiency of amines and a base is sometimes added in solution when the basic strength of the neutral amine is not sufficient to make the deprotonation of the exergonic amino cation radical. [START_REF] Anex | Base-Assisted Electrografting of Aromatic Amines[END_REF] This applies especially when electron-donating groups are present on the phenyl ring of aniline derivatives, the amino cation radical of which suffers from relative low acidity. As examples, Amatore et al. demonstrate that, in DMF, neither the lutidine nor the collidine significantly deprotonates the cation radical of the p-anisidine, [START_REF] Simon | Mechanistic investigation of the oxidation of panisidine in unbuffered DMF using fast scan rates at ultramicroelectrodes[END_REF] while the neutral pethoxyphenazyl free radical was detected by ESR in water only at pH 11-12. [START_REF] Fischer | Direct electron spin resonance detection of free radical intermediates during the peroxidase catalyzed oxidation of phenacetin metabolites[END_REF] This is the reason why the pH of the amine solution needs to be adjusted at 12 to make the grafting possible. At pH 7.5-8, the anodic behavior of the amino-containing compound mostly involves cation radicals, yielding a dimerization product. To test the antifouling properties of this compound, a protocol that we have already tested on polymer materials was used. [START_REF] Nguema Edzang | Bacterial anti-adhesion activity based on the electrochemical properties of polymethacrylates bearing ferrocenyl pendant groups[END_REF][START_REF] Brisset | 96X Screen-Printed Gold Electrode Platform to Evaluate Electroactive Polymers as Marine Antifouling Coatings[END_REF] The equipment consists of a microplate with 96 wells at the bottom of which are three electrodes (WE, CE, Ref; see figure 2 for illustration), a configuration identical to what can be done in a conventional electrochemical cell. This microplate is placed on a connector which is connected to a potentiostat. The potentiostat allows to apply the same potential per column (as shown in yellow in figure 2), however not all columns can be solicited at the same time. The working electrodes of the same column can be functionalized under the conditions described. The different voltamperograms recorded (corresponding to wells a to h) A live/dead test was also performed on columns 8 and 9 to establish the toxicity of the coatings.

The degree of toxicity of coatings is evaluated by comparison with the toxicity of ethanol (see the table SI-2 for the test results). Syto ® 9 and Propidium Iodide (PI) dyes were used to stain all the cells and dead cells, respectively, in each well, after 1 hour of incubation. The ratio of the two fluorescent dyes intensities indicated that the toxicity of the coatings is comparable to the toxicity of 5 % ethanol solution, toxicity that cannot be detected.

An ageing test via ferrocene signal monitoring in electrochemistry allowed us to estimate the condition of the layers in each well (figure 3c). There is a decrease in the signal of the ferrocene, so the recovery rate is lower but the layer obtained seems to be stabilized till 7 hours of ageing.

The coatings were oxidized and reduced during 16 h of incubation. Then, the polarity of the layer was controlled by measuring the water contact angle at time t = 0 and after 16 hours of solicitation. The measured polarity remains unchanged after the stress and angles of 30 to 40° could be measured indicating a rather polar coating. The contact angle of the bare carbon was measured around 140 °. Thus we can show that the carbon has been modified and that ageing only slightly alters the layer on the C electrode.

Conclusion

The method we have developed makes it possible to covalently immobilize the substrate on the surface and avoid any release of active substances into the environment, as is the case with most coatings used on ship hulls, for example. On the other hand, amine immobilization for antibacterial effects is new since the most relevant work is that of P. Colavita showing an anti-protein effect after immobilization by reduction of diazonium salts. Thus, it has been shown that a glucosebound ferrocene can be grafted by oxidation of an amine on the carbon surface in aqueous medium such as sea water. Functionalization was confirmed by cyclovoltamperometry and contact angle measurement and an ageing test ensured the perenity of the Fc-grafted layer on the electrode surface. The biological tests were carried out using a device linking a potentiostat to a microplate with 96 wells in which electrochemistry can be carried out. The coatings alone tend to have a marine antibacterial effect on the TC8 strain, an effect that becomes more pronounced if ferrocene is oxidized. Indeed, the developed system allows an electric cycling with a potential that allows to extract an electron from the ferrocene and transform it into ferrocenium. This charge will disappear in the return cycle. The to-and-fro of this charge has an additional effect on the layer deposited material on the prevention of bacteria adhesion.

Figure 1 .

 1 Figure 1. a) Grafting by cyclic voltammetry (10 CVs) from -0.3 to + 0.8 V of 6 (1 mM in ASW);

Figure 2 . 3 (

 23 Figure 2. a) Illustration of ferrocene derivatives grafted in a column of wells; CE: counter

Figure 3 .

 3 Figure 3. a) Columns 8 and 9 of a microplate; in blue: non solicited; in orange: solicited; WB:

We thank the Direction Générale de l'Armement (DGA) and the Région Pays de la Loire for the thesis of Fanny Peigneguy. We thank Dr Jean-François Briand for his help in performing bioassays.

ASSOCIATED CONTENT Supporting Information. The following files are available free of charge.

Syntheses of 1 to 4, Adhesion tests (table SI-1), Toxicity tests (table SI-2) and Voltamperograms of wells a9-h9.

AUTHOR INFORMATION

Corresponding Author

*Email: frederic.gohier@univ-angers.fr ACKNOWLEDGMENT