NRAMP6 and NRAMP1 cooperatively regulate root growth and manganese translocation under manganese deficiency in Arabidopsis.

The essential micronutrient manganese (Mn) in plants regulates multiple biological processes including photosynthesis and oxidative stress. Some Natural Resistance-Associated Macrophage Proteins (NRAMPs) have been reported to play critical roles in Mn uptake and reutilization in low Mn conditions. NRAMP6 was demonstrated to regulate cadmium tolerance and iron utilization in Arabidopsis. Nevertheless, it is unclear whether NRAMP6 plays a role in Mn nutrition. Here, we report that NRAMP6 cooperates with NRAMP1 in Mn utilization. Mutation of NRAMP6 in nramp1 but not in a wild-type background reduces root growth and Mn translocation from the roots to shoots under Mn deficient conditions. Grafting experiments revealed that NRAMP6 expression in both the roots and shoots is required for root growth and Mn translocation under Mn deficiency. We also showed that NRAMP1 could replace NRAMP6 to sustain root growth under Mn deficiency, but not vice versa. Mn deficiency does not affect the transcript level of NRAMP6, but is able to increase and decrease the protein accumulation of NRAMP6 in roots and shoots, respectively. Furthermore, NRAMP6 can be localized to both the plasma membrane and endomembranes including the endoplasmic reticulum, and Mn deficiency enhances the localization of NRAMP6 to the plasma membrane in Arabidopsis plants. NRAMP6 could rescue the defective growth of the yeast mutant Δsmf2, which is deficient in endomembrane Mn transport. Our results reveal the important role of NRAMP6 in Mn nutrition and in the long-distance signaling between the roots and shoots under Mn deficient conditions.

Golgi in and out: multifaceted role and journey of manganese.

Manganese (Mn) is pivotal for plant growth and development but little is known about the processes that control its homeostasis in the cell. A spotlight on the pools of intracellular manganese and their cellular function has recently been gained through the characterization of new Mn transporters. In particular, transporters catalyzing the ins and outs of Mn at the various Golgi membranes have revealed the central role of the Golgi pool of Mn in the synthesis of the cell wall and as a reservoir for the numerous cellular Mn-dependent pathways whose calibration relies on a set of Golgi-resident transporters of the BICAT and NRAMP families.

NICOTIANAMINE SYNTHASE activity affects nucleolar iron accumulation and impacts rDNA silencing and RNA methylation in Arabidopsis.

In plant cells, a large pool of iron (Fe) is contained in the nucleolus, as well as in chloroplasts and mitochondria. A central determinant for intracellular distribution of Fe is nicotianamine (NA) generated by NICOTIANAMINE SYNTHASE (NAS). Here, we used Arabidopsis thaliana plants with disrupted NAS genes to study the accumulation of nucleolar iron and understand its role in nucleolar functions and more specifically in rRNA gene expression. We found that nas124 triple mutant plants, which contained lower quantities of the iron ligand NA, also contained less iron in the nucleolus. This was concurrent with the expression of normally silenced rRNA genes from nucleolar organizer regions 2 (NOR2). Notably, in nas234 triple mutant plants, which also contained lower quantities of NA, nucleolar iron and rDNA expression were not affected. In contrast, in both nas124 and nas234, specific RNA modifications were differentially regulated in a genotype dependent manner. Taken together, our results highlight the impact of specific NAS activities in RNA gene expression. We discuss the interplay between NA and nucleolar iron with rDNA functional organization and RNA methylation.

Manganese triggers phosphorylation-mediated endocytosis of the Arabidopsis metal transporter NRAMP1.

The NATURAL RESISTANCE-ASSOCIATED MACROPHAGE PROTEIN 1 (NRAMP1) transporter guarantees plant survival of manganese (Mn) deficiency by mediating Mn entry into root cells. Unlike other high-affinity metal transporters, NRAMP1 is only slightly regulated at the transcriptional level. We show here that adequate Mn content in tissues is safeguarded through a tight control of the quantity of NRAMP1 present at the surface of root cells. Depending on Mn availability, an NRAMP1-GFP fusion protein cycles dynamically between the plasma membrane (PM) and endosomal compartments. This involves a clathrin-mediated endocytosis pathway, as disrupting this pathway in auxilin-overexpressor lines prevents NRAMP1 internalization. Mutation of the phosphorylated serine residues 20, 22 and 24 in the cytosol-exposed N terminus of NRAMP1 alters its membrane distribution. Indeed, a phospho-dead mutation stabilizes NRAMP1 at the PM, regardless of the Mn regime, and dramatically reduces plant tolerance to Mn toxicity. Conversely a phosphomimetic mutant is constitutively internalized into endosomes. Together, these data establish that phosphorylation of NRAMP1 is the trigger for its Mn-induced endocytosis and represents the main level of regulation of this transporter. Furthermore, the extent of Mn toxicity observed when interrupting NRAMP1 membrane cycling undermines the dogma that Mn is only marginally toxic to plants.

AtDTX25, a member of the multidrug and toxic compound extrusion family, is a vacuolar ascorbate transporter that controls intracellular iron cycling in Arabidopsis.

Iron (Fe) is an essential element, its transport is regulated by the cell redox balance. In seeds, Fe enters the embryo as Fe2+ and is stored in vacuoles as Fe3+ . Through its ferric reduction activity, ascorbate plays a major role in Fe redox state and therefore Fe transport within the seed. We searched for ascorbate membrane transporters responsible for controlling Fe reduction by screening the yeast ferric reductase-deficient fre1 strain and isolated AtDTX25, a member of the Multidrug And Toxic compound Extrusion (MATE) family. AtDTX25 was shown to mediate ascorbate efflux when expressed in yeast and Xenopus oocytes, in a pH-dependent manner. In planta, AtDTX25 is highly expressed during germination and encodes a vacuolar membrane protein. Isolated vacuoles from AtDTX25-1 knockout mutant contained less ascorbate and more Fe than wild-type (WT), and mutant seedlings were highly sensitive to Fe deficiency. Iron imaging further showed that the remobilisation of Fe from vacuoles was highly impaired in mutant seedlings. Taken together, our results established AtDTX25 as a vacuolar ascorbate transporter, required during germination to promote the reduction of the pool of stored Fe3+ and its remobilisation to feed the developing seedling.

Intracellular Distribution of Manganese by the Trans-Golgi Network Transporter NRAMP2 Is Critical for Photosynthesis and Cellular Redox Homeostasis.

Plants require trace levels of manganese (Mn) for survival, as it is an essential cofactor in oxygen metabolism, especially O2 production via photosynthesis and the disposal of superoxide radicals. These processes occur in specialized organelles, requiring membrane-bound intracellular transporters to partition Mn between cell compartments. We identified an Arabidopsis thaliana member of the NRAMP family of divalent metal transporters, NRAMP2, which functions in the intracellular distribution of Mn. Two knockdown alleles of NRAMP2 showed decreased activity of photosystem II and increased oxidative stress under Mn-deficient conditions, yet total Mn content remained unchanged. At the subcellular level, these phenotypes were associated with a loss of Mn content in vacuoles and chloroplasts. NRAMP2 was able to rescue the mitochondrial yeast mutant mtm1∆ In plants, NRAMP2 is a resident protein of the trans-Golgi network. NRAMP2 may act indirectly on downstream organelles by building up a cytosolic pool that is used to feed target compartments. Moreover, not only does the nramp2 mutant accumulate superoxide ions, but NRAMP2 can functionally replace cytosolic superoxide dismutase in yeast, indicating that the pool of Mn displaced by NRAMP2 is required for the detoxification of reactive oxygen species.

Phosphatidylinositol 3-phosphate-binding protein AtPH1 controls the localization of the metal transporter NRAMP1 in Arabidopsis.

"Too much of a good thing" perfectly describes the dilemma that living organisms face with metals. The tight control of metal homeostasis in cells depends on the trafficking of metal transporters between membranes of different compartments. However, the mechanisms regulating the location of transport proteins are still largely unknown. Developing Arabidopsis thaliana seedlings require the natural resistance-associated macrophage proteins (NRAMP3 and NRAMP4) transporters to remobilize iron from seed vacuolar stores and thereby acquire photosynthetic competence. Here, we report that mutations in the pleckstrin homology (PH) domain-containing protein AtPH1 rescue the iron-deficient phenotype of nramp3nramp4 Our results indicate that AtPH1 binds phosphatidylinositol 3-phosphate (PI3P) in vivo and acts in the late endosome compartment. We further show that loss of AtPH1 function leads to the mislocalization of the metal uptake transporter NRAMP1 to the vacuole, providing a rationale for the reversion of nramp3nramp4 phenotypes. This work identifies a PH domain protein as a regulator of plant metal transporter localization, providing evidence that PH domain proteins may be effectors of PI3P for protein sorting.

New routes for plant iron mining.

Contents 521 I. 521 II. 522 III. 523 IV. 524 525 References 525 SUMMARY: Plant iron (Fe) uptake relies to a large extent on the capacity of cells to control and extract Fe pools safely conserved in extracytoplasmic environments such as the apoplast and vacuoles, at least as much as on the transport machinery nested in plasma membranes. Recent studies on root and embryo Fe nutrition support this assertion and show that the root Fe-deficiency response also includes the dynamic use of a large Fe reservoir bound to cell wall components in the root apoplast, secretion in the apoplast of phenolic compounds of the coumarin family, which solubilize Fe in calcareous soils, and inhibition of suberization of endodermal cells in order to allow apoplastic and transcellular radial transport of Fe. All of these responses are regulated by the stress hormones ethylene and abscisic acid (ABA), suggesting an integrated strategy within the root to adapt to Fe shortage. For its nutrition, the embryo has developed both an original uptake mechanism, in which ascorbate is effluxed to chemically reduce Fe3+ to the transport-competent Fe2+ form, and an efficient strategy to control utilization of a large Fe pool in vacuoles. This review will attempt to summarize exciting new insights into the diverse routes that Fe takes to feed plant tissues.

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.

Description of metal species in plant fluids such as xylem, phloem or related saps remains a complex challenge usually addressed either by liquid chromatography-mass spectrometry, X-ray analysis or computational prediction. To date, none of these techniques has achieved a complete and true picture of metal-containing species in plant fluids, especially for the least concentrated complexes. Here, we present a generic analytical methodology for a large-scale (> 10 metals, > 50 metal complexes) detection, identification and semiquantitative determination of metal complexes in the xylem and embryo sac liquid of the green pea, Pisum sativum. The procedure is based on direct injection using hydrophilic interaction chromatography with dual detection by elemental (inductively coupled plasma mass spectrometry) and molecular (high-resolution electrospray mass spectrometry) mass spectrometric detection. Numerous and novel complexes of iron(II), iron(III), copper(II), zinc, manganese, cobalt(II), cobalt(III), magnesium, calcium, nickel and molybdenum(IV) with several ligands including nicotianamine, citrate, malate, histidine, glutamine, aspartic acid, asparagine, phenylalanine and others are observed in pea fluids and discussed. This methodology provides a large inventory of various types of metal complexes, which is a significant asset for future biochemical and genetic studies into metal transport/homeostasis.

The Arabidopsis YELLOW STRIPE LIKE4 and 6 transporters control iron release from the chloroplast.

In most plant cell types, the chloroplast represents the largest sink for iron, which is both essential for chloroplast metabolism and prone to cause oxidative damage. Here, we show that to buffer the potentially harmful effects of iron, besides ferritins for storage, the chloroplast is equipped with specific iron transporters that respond to iron toxicity by removing iron from the chloroplast. We describe two transporters of the YELLOW STRIPE1-LIKE family from Arabidopsis thaliana, YSL4 and YSL6, which are likely to fulfill this function. Knocking out both YSL4 and YSL6 greatly reduces the plant's ability to cope with excess iron. Biochemical and immunolocalization analyses showed that YSL6 resides in the chloroplast envelope. Elemental analysis and histochemical staining indicate that iron is trapped in the chloroplasts of the ysl4 ysl6 double mutants, which also accumulate ferritins. Also, vacuolar iron remobilization and NRAMP3/4 expression are inhibited. Furthermore, ubiquitous expression of YSL4 or YSL6 dramatically reduces plant tolerance to iron deficiency and decreases chloroplastic iron content. These data demonstrate a fundamental role for YSL4 and YSL6 in managing chloroplastic iron. YSL4 and YSL6 expression patterns support their physiological role in detoxifying iron during plastid dedifferentiation occurring in embryogenesis and senescence.

Ascorbate efflux as a new strategy for iron reduction and transport in plants.

Iron (Fe) is essential for virtually all living organisms. The identification of the chemical forms of iron (the speciation) circulating in and between cells is crucial to further understand the mechanisms of iron delivery to its final targets. Here we analyzed how iron is transported to the seeds by the chemical identification of iron complexes that are delivered to embryos, followed by the biochemical characterization of the transport of these complexes by the embryo, using the pea (Pisum sativum) as a model species. We have found that iron circulates as ferric complexes with citrate and malate (Fe(III)3Cit2Mal2, Fe(III)3Cit3Mal1, Fe(III)Cit2). Because dicotyledonous plants only transport ferrous iron, we checked whether embryos were capable of reducing iron of these complexes. Indeed, embryos did express a constitutively high ferric reduction activity. Surprisingly, iron(III) reduction is not catalyzed by the expected membrane-bound ferric reductase. Instead, embryos efflux high amounts of ascorbate that chemically reduce iron(III) from citrate-malate complexes. In vitro transport experiments on isolated embryos using radiolabeled (55)Fe demonstrated that this ascorbate-mediated reduction is an obligatory step for the uptake of iron(II). Moreover, the ascorbate efflux activity was also measured in Arabidopsis embryos, suggesting that this new iron transport system may be generic to dicotyledonous plants. Finally, in embryos of the ascorbate-deficient mutants vtc2-4, vtc5-1, and vtc5-2, the reducing activity and the iron concentration were reduced significantly. Taken together, our results identified a new iron transport mechanism in plants that could play a major role to control iron loading in seeds.

Over-expression of the bacterial phytase US417 in Arabidopsis reduces the concentration of phytic acid and reveals its involvement in the regulation of sulfate and phosphate homeostasis and signaling.

Phytic acid (PA) is the main phosphorus storage form in plant seeds. It is recognized as an anti-nutrient for humans and non-ruminant animals, as well as one of the major sources of phosphorus that contributes to eutrophication. Therefore, engineering plants with low PA content without affecting plant growth capacity has become a major focus in plant breeding. Nevertheless, lack of knowledge on the role of PA seed reserves in regulating plant growth and in maintaining ion homeostasis hinders such an agronomical application. In this context, we report here that the over-expression of the bacterial phytase PHY-US417 in Arabidopsis leads to a significant decrease in seed PA, without any effect on the seed germination potential. Interestingly, this over-expression also induced a higher remobilization of free iron during germination. Moreover, the PHY-over-expressor lines show an increase in inorganic phosphate and sulfate contents, and a higher biomass production after phosphate starvation. Finally, phosphate sensing was altered because of the changes in the expression of genes induced by phosphate starvation or involved in phosphate or sulfate transport. Together, these results show that the over-expression of PHY-US417 reduces PA concentration, and provide the first evidence for the involvement of PA in the regulation of sulfate and phosphate homeostasis and signaling.

The FRD3 citrate effluxer promotes iron nutrition between symplastically disconnected tissues throughout Arabidopsis development.

We present data supporting a general role for FERRIC REDICTASE DEFECTIVE3 (FRD3), an efflux transporter of the efficient iron chelator citrate, in maintaining iron homeostasis throughout plant development. In addition to its well-known expression in root, we show that FRD3 is strongly expressed in Arabidopsis thaliana seed and flower. Consistently, frd3 loss-of-function mutants are defective in early germination and are almost completely sterile, both defects being rescued by iron and/or citrate supply. The frd3 fertility defect is caused by pollen abortion and is associated with the male gametophytic expression of FRD3. Iron imaging shows the presence of important deposits of iron on the surface of aborted pollen grains. This points to a role for FRD3 and citrate in proper iron nutrition of embryo and pollen. Based on the findings that iron acquisition in embryo, leaf, and pollen depends on FRD3, we propose that FRD3 mediated-citrate release in the apoplastic space represents an important process by which efficient iron nutrition is achieved between adjacent tissues lacking symplastic connections. These results reveal a physiological role for citrate in the apoplastic transport of iron throughout development, and provide a general model for multicellular organisms in the cell-to-cell transport of iron involving extracellular circulation.

High-affinity manganese uptake by the metal transporter NRAMP1 is essential for Arabidopsis growth in low manganese conditions.

In contrast with many other essential metals, the mechanisms of Mn acquisition in higher eukaryotes are seldom studied and poorly understood. We show here that Arabidopsis thaliana relies on a high-affinity uptake system to acquire Mn from the soil in conditions of low Mn availability and that this activity is catalyzed by the divalent metal transporter NRAMP1 (for Natural Resistance Associated Macrophage Protein 1). The nramp1-1 loss-of-function mutant grows poorly, contains less Mn than the wild type, and fails to take up Mn in conditions of Mn limitation, thus demonstrating that NRAMP1 is the major high-affinity Mn transporter in Arabidopsis. Based on confocal microscopy observation of an NRAMP1-green fluorescent protein fusion, we established that NRAMP1 is localized to the plasma membrane. Consistent with its function in Mn acquisition from the soil, NRAMP1 expression is restricted to the root and stimulated by Mn deficiency. Finally, we show that NRAMP1 restores the capacity of the iron-regulated transporter1 mutant to take up iron and cobalt, indicating that NRAMP1 has a broad selectivity in vivo. The role of transporters of the NRAMP family is well established in higher eukaryotes for iron but has been controversial for Mn. This study demonstrates that NRAMP1 is a physiological manganese transporter in Arabidopsis.

Phosphorylation by CIPK23 regulates the high-affinity Mn transporter NRAMP1 in Arabidopsis.

Manganese (Mn) is essential for plants but is toxic when taken up in excess. To maintain Mn homeostasis, the root Mn transporter natural resistance associated macrophage protein 1 (NRAMP1) cycles from the plasma membrane to endosomes upon phosphorylation. To identify the kinase involved, a split-luciferase screening was carried out between NRAMP1 and kinases of the CIPK family and identified CIPK23 as a partner of NRAMP1. The interaction was confirmed by split-mCitrine bimolecular fluorescence complementation and co-immunoprecipitation assays. In vitro phosphorylation assays pinpointed two CIPK23 target residues in NRAMP1, among which serine 20, important for endocytosis. Interestingly, Mn-induced internalization of NRAMP1 was unaffected by cipk23 mutation suggesting a potential redundancy between CIPK23 and other kinase(s). How CIPK23 could regulate NRAMP1 in response to Mn availability is discussed.

Study of the Stability, Uptake and Transformations of Zero Valent Iron Nanoparticles in a Model Plant by Means of an Optimised Single Particle ICP-MS/MS Method.

In the context of the widespread distribution of zero valent iron nanoparticles (nZVI) in the environment and its possible exposure to many aquatic and terrestrial organisms, this study investigates the effects, uptake, bioaccumulation, localisation and possible transformations of nZVI in two different forms (aqueous dispersion-Nanofer 25S and air-stable powder-Nanofer STAR) in a model plant-Arabidopsis thaliana. Seedlings exposed to Nanofer STAR displayed symptoms of toxicity, including chlorosis and reduced growth. At the tissue and cellular level, the exposure to Nanofer STAR induced a strong accumulation of Fe in the root intercellular spaces and in Fe-rich granules in pollen grains. Nanofer STAR did not undergo any transformations during 7 days of incubation, while in Nanofer 25S, three different behaviours were observed: (i) stability, (ii) partial dissolution and (iii) the agglomeration process. The size distributions obtained by SP-ICP-MS/MS demonstrated that regardless of the type of nZVI used, iron was taken up and accumulated in the plant, mainly in the form of intact nanoparticles. The agglomerates created in the growth medium in the case of Nanofer 25S were not taken up by the plant. Taken together, the results indicate that Arabidopsis plants do take up, transport and accumulate nZVI in all parts of the plants, including the seeds, which will provide a better understanding of the behaviour and transformations of nZVI once released into the environment, a critical issue from the point of view of food safety.

Plant cell nucleolus as a hot spot for iron.

Many central metabolic processes require iron as a cofactor and take place in specific subcellular compartments such as the mitochondrion or the chloroplast. Proper iron allocation in the different organelles is thus critical to maintain cell function and integrity. To study the dynamics of iron distribution in plant cells, we have sought to identify the different intracellular iron pools by combining three complementary imaging approaches, histochemistry, micro particle-induced x-ray emission, and synchrotron radiation micro X-ray fluorescence. Pea (Pisum sativum) embryo was used as a model in this study because of its large cell size and high iron content. Histochemical staining with ferrocyanide and diaminobenzidine (Perls/diaminobenzidine) strongly labeled a unique structure in each cell, which co-labeled with the DNA fluorescent stain DAPI, thus corresponding to the nucleus. The unexpected presence of iron in the nucleus was confirmed by elemental imaging using micro particle-induced x-ray emission. X-ray fluorescence on cryo-sectioned embryos further established that, quantitatively, the iron concentration found in the nucleus was higher than in the expected iron-rich organelles such as plastids or vacuoles. Moreover, within the nucleus, iron was particularly accumulated in a subcompartment that was identified as the nucleolus as it was shown to transiently disassemble during cell division. Taken together, our data uncover an as yet unidentified although abundant iron pool in the cell, which is located in the nuclei of healthy, actively dividing plant tissues. This result paves the way for the discovery of a novel cellular function for iron related to nucleus/nucleolus-associated processes.

Proteasome-mediated turnover of the transcriptional activator FIT is required for plant iron-deficiency responses.

Plants display a number of responses to low iron availability in order to increase iron uptake from the soil. In the model plant Arabidopsis thaliana, the ferric-chelate reductase FRO2 and the ferrous iron transporter IRT1 control iron entry from the soil into the root epidermis. To maintain iron homeostasis, the expression of FRO2 and IRT1 is tightly controlled by iron deficiency at the transcriptional level. The basic helix-loop-helix (bHLH) transcription factor FIT represents the most upstream actor known in the iron-deficiency signaling pathway, and directly regulates the expression of the root iron uptake machinery genes FRO2 and IRT1. However, how FIT is controlled by iron and acts to activate transcription of its targets remains obscure. Here we show that FIT mRNA and endogenous FIT protein accumulate in Arabidopsis roots upon iron deficiency. However, using plants constitutively expressing FIT, we observed that FIT protein accumulation is reduced in iron-limited conditions. This post-transcriptional regulation of FIT is perfectly synchronized with the accumulation of endogenous FIT and IRT1 proteins, and therefore is part of the early responses to low iron. We demonstrated that such regulation affects FIT protein stability under iron deficiency as a result of 26S proteasome-dependent degradation. In addition, we showed that FIT post-translational regulation by iron is required for FRO2 and IRT1 gene expression. Taken together our results indicate that FIT transcriptional and post-translational regulations are integrated in plant roots to ensure that the positive regulator FIT accumulates as a short-lived protein following iron shortage, and to allow proper iron-deficiency responses.

MCO1 and MCO3, two putative ascorbate oxidases with ferroxidase activity, new candidates for the regulation of apoplastic iron excess in Arabidopsis.

Iron (Fe) is an essential metal ion that plays a major role as a cofactor in many biological processes. The balance between the Fe2+ and Fe3+ forms is central for cellular Fe homeostasis because it regulates its transport, utilization, and storage. Contrary to Fe3+ reduction that is crucial for Fe uptake by roots in deficiency conditions, ferroxidation has been much less studied. In this work, we have focused on the molecular characterization of two members of the MultiCopper Oxidase family (MCO1 and MCO3) that share high identity with the Saccharomyces cerevisiae ferroxidase Fet3. The heterologous expression of MCO1 and MCO3 restored the growth of the yeast fet3fet4 mutant, impaired in high and low affinity Fe uptake and otherwise unable to grow in Fe deficient media, suggesting that MCO1 and MCO3 were functional ferroxidases. The ferroxidase enzymatic activity of MCO3 was further confirmed by the measurement of Fe2+-dependent oxygen consumption, because ferroxidases use oxygen as electron acceptor to generate water molecules. In planta, the expression of MCO1 and MCO3 was induced by increasing Fe concentrations in the medium. Promoter-GUS reporter lines showed that MCO1 and MCO3 were mostly expressed in shoots and histochemical analyses further showed that both promoters were highly active in mesophyll cells. Transient expression of MCO1-RFP and MCO3-RFP in tobacco leaves revealed that both proteins were localized in the apoplast. Moreover, cell plasmolysis experiments showed that MCO1 remained closely associated to the plasma membrane whereas MCO3 filled the entire apoplast compartment. Although the four knock out mutant lines isolated (mco1-1, mco1-2, mco3-1, and mco3-2) did not display any macroscopic phenotype, histochemical staining of Fe with the Perls/DAB procedure revealed that mesophyll cells of all four mutants overaccumulated Fe inside the cells in Fe-rich structures in the chloroplasts, compared with wild-type. These results suggested that the regulation of Fe transport in mesophyll cells had been disturbed in the mutants, in both standard condition and Fe excess. Taken together, our findings strongly suggest that MCO1 and MCO3 participate in the control of Fe transport in the mesophyll cells, most likely by displacing the Fe2+/Fe3+ balance toward Fe3+ in the apoplast and therefore limiting the accumulation of Fe2+, which is more mobile and prone to be transported across the plasma membrane.