Since the identification of the genes controlling the root acquisition of iron (Fe), the control of inter- and intracellular distribution has become an important challenge in understanding metal homeostasis. The identification of the yellow stripe-like (YSL) transporter family has paved the way to decipher the mechanisms of long-distance transport of Fe. Once in the plant, Fe will systematically react with organic ligands whose identity is poorly known so far. Among potential ligands, nicotianamine has been identified as an important molecule for the circulation and delivery of metals since it participates in the loading of copper (Cu) and nickel in xylem and prevents Fe precipitation in leaves. Nicotianamine is a precursor of phytosiderophores, which are high-affinity Fe ligands exclusively synthesized by Poaceae species and excreted by roots for the chelation and acquisition of Fe. Maize YS1 is the founding member of a family of membrane transporters called YS1-like (YSL), which functions in root Fe–phytosiderophore uptake from the soil. Next to this well-known Fe acquisition role, most of the other YSL family members are likely to function in plant-wide distribution of metals since (a) they are produced in vascular tissues throughout the plant and (b) they are found in non-Poaceae species that do not synthesize phytosiderophores. The hypothesized activity as Fe–nicotianamine transporters of several YSL members has been demonstrated experimentally by heterologous expression in yeast or by electrophysiology in Xenopus oocytes but, despite numerous attempts, proof of the arabidopsis YSL substrate specificity is still lacking. Reverse genetics, however, has revealed a role for AtYSL members in the remobilization of Cu and zinc from senescing leaves, in the formation of pollen and in the Fe, zinc and Cu loading of seeds. Preliminary data on the YSL family of transporters clearly argues in favour of its role in the long-distance transport of metals through and between vascular tissues to eventually support gametogenesis and embryo development.

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.

NRAMP (natural resistance-associated macrophage protein) homologues are evolutionarily conserved bivalent metal transporters. In Arabidopsis, AtNRAMP3 and AtNRAMP4 play a key role in iron nutrition of the germinating plantlet by remobilizing vacuolar iron stores. In the present paper we describe the molecular and physiological characterization of AtNRAMP6. AtNRAMP6 is predominantly expressed in the dry seed embryo and to a lesser extent in aerial parts. Its promoter activity is found diffusely distributed in cotyledons and hypocotyl, as well as in the vascular tissue region of leaf and flower. We show that the AtNRAMP6 transcript coexists with a partially spliced isoform in all shoot cell types tested. When expressed in yeast, AtNRAMP6, but not its misspliced derivative, increased sensitivity to cadmium without affecting cadmium content in the cell. Likewise, Arabidopsis transgenic plants overexpressing AtNRAMP6 were hypersensitive to cadmium, although plant cadmium content remained unchanged. Consistently, a null allele of AtNRAMP6, named nramp6-1, was more tolerant to cadmium toxicity, a phenotype that was reverted by expressing AtNRAMP6 in the mutant background. We used an AtNRAMP6::HA (where HA is haemagglutinin) fusion, shown to be functional in yeast, to demonstrate through immunoblot analysis of membrane fractions and immunofluorescence localization that, in yeast cells, AtNRAMP6 is targeted to a vesicular-shaped endomembrane compartment distinct from the vacuole or mitochondria. We therefore propose that AtNRAMP6 functions as an intracellular metal transporter, whose presence, when modified, is likely to affect distribution/availability of cadmium within the cell.

Summary: Plants display a number of biochemical and developmental responses to low iron availability in order to increase iron uptake from the soil. The ferric‐chelate reductase FRO2 and the ferrous iron transporter IRT1 control iron entry from the soil into the root epidermis. In Arabidopsis, expression of IRT1 and FRO2 is tightly controlled to maintain iron homeostasis, and involves local and long‐distance signals, as well as transcriptional and post‐transcriptional events. FIT encodes a putative basic helix‐loop‐helix (bHLH) transcription factor that regulates iron uptake responses in Arabidopsis. Here, we uncover a new regulation of the root iron uptake genes. We show that IRT1, FRO2 and FIT are repressed by the exogenous addition of cytokinins (CKs), and that this repression acts at the level of transcript accumulation, and depends on the AHK3 and CRE1 CK receptors. The CKs and iron‐deficiency signals act through distinct pathways to regulate the soil iron uptake genes, as (i) CK repression is independent of the iron status, (ii) IRT1 and FRO2 downregulation is unchanged in a fit loss‐of‐function mutant, indicating that FIT does not mediate CK repression, and (iii) the iron‐regulated genes AtNRAMP3 and AtNRAMP4 are not downregulated by CKs. We show that root growth‐inhibitory conditions, such as abiotic stresses (mannitol, NaCl) and hormonal treatments (auxin, abscissic acid), repress the iron starvation response genes. We propose that CKs control the root iron uptake machinery through a root growth dependent pathway in order to adapt nutrient uptake to the demand of the plant.

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.