Dual regulation of the Arabidopsis high-affinity root iron uptake system by local and long-distance signals

Regulation of the root high-affinity iron uptake system by whole-plant signals was investigated at the molecular level in Arabidopsis, through monitoring FRO2 and IRT1 gene expression. These two genes encode the root ferric-chelate reductase and the high-affinity iron transporter, respectively, involved in the iron deficiency-induced uptake system. Recovery from iron-deficient conditions and modulation of apoplastic iron pools indicate that iron itself plays a major role in the regulation of root iron deficiency responses at the mRNA and protein levels. Split-root experiments show that the expression of IRT1 and FRO2 is controlled both by a local induction from the root iron pool and through a systemic pathway involving a shoot-borne signal, both signals being integrated to tightly control production of the root iron uptake proteins. We also show that IRT1 and FRO2 are expressed during the day and down-regulated at night and that this additional control is overruled by iron starvation, indicating that the nutritional status prevails on the diurnal regulation. Our work suggests, for the first time to our knowledge, that like in grasses, the root iron acquisition in strategy I plants may also be under diurnal regulation. On the basis of the new molecular insights provided in this study and given the strict coregulation of IRT1 and FRO2 observed, we present a model of local and long-distance regulation of the root iron uptake system in Arabidopsis.     

IRT1, an Arabidopsis transporter essential for iron uptake from the soil and for plant growth

Plants are the principal source of iron in most diets, yet iron availability often limits plant growth. In response to iron deficiency, Arabidopsis roots induce the expression of the divalent cation transporter IRT1. Here, we present genetic evidence that IRT1 is essential for the uptake of iron from the soil. An Arabidopsis knockout mutant in IRT1 is chlorotic and has a severe growth defect in soil, leading to death. This defect is rescued by the exogenous application of iron. The mutant plants do not take up iron and fail to accumulate other divalent cations in low-iron conditions. IRT1-green fluorescent protein fusion, transiently expressed in culture cells, localized to the plasma membrane. We also show, through promoter::beta-glucuronidase analysis and in situ hybridization, that IRT1 is expressed in the external cell layers of the root, specifically in response to iron starvation. These results clearly demonstrate that IRT1 is the major transporter responsible for high-affinity metal uptake under iron deficiency.     

Characterization of FRO1, a pea ferric-chelate reductase involved in root iron acquisition

To acquire iron, many plant species reduce soil Fe(III) to Fe(II) by Fe(III)-chelate reductases embedded in the plasma membrane of root epidermal cells. The reduced product is then taken up by Fe(II) transporter proteins. These activities are induced under Fe deficiency. We describe here the FRO1 gene from pea (Pisum sativum), which encodes an Fe(III)-chelate reductase. Consistent with this proposed role, FRO1 shows similarity to other oxidoreductase proteins, and expression of FRO1 in yeast conferred increased Fe(III)-chelate reductase activity. Furthermore, FRO1 mRNA levels in plants correlated with Fe(III)-chelate reductase activity. Sites of FRO1 expression in roots, leaves, and nodules were determined. FRO1 mRNA was detected throughout the root, but was most abundant in the outer epidermal cells. Expression was detected in mesophyll cells in leaves. In root nodules, mRNA was detected in the infection zone and nitrogen-fixing region. These results indicate that FRO1 acts in root Fe uptake and they suggest a role in Fe distribution throughout the plant. Characterization of FRO1 has also provided new insights into the regulation of Fe uptake. FRO1 expression and reductase activity was detected only in Fe-deficient roots of Sparkle, whereas both were constitutive in brz and dgl, two mutants with incorrectly regulated Fe accumulation. In contrast, FRO1 expression was responsive to Fe status in shoots of all three plant lines. These results indicate differential regulation of FRO1 in roots and shoots, and improper FRO1 regulation in response to a shoot-derived signal of iron status in the roots of the brz and dgl mutants.     

The metal ion transporter IRT1 is necessary for iron homeostasis and efficient photosynthesis in Arabidopsis thaliana

The mutants irt1-1 and irt1-2 of Arabidopsis thaliana were identified among a collection of T-DNA-tagged lines on the basis of a decrease in the effective quantum yield of photosystem II. The mutations responsible interfere with expression of IRT1, a nuclear gene that encodes the metal ion transporter IRT1. In irt1 mutants, photosensitivity and chlorophyll fluorescence parameters, as well as abundance and composition of the photosynthetic apparatus, are significantly altered. Additional effects of the mutation under greenhouse conditions, including chlorosis and a drastic reduction in growth rate and fertility, are compatible with a deficiency in iron transport. Propagation of irt1 plants on media supplemented with additional quantities of iron salts restores almost all aspects of wild-type behaviour. The irt2-1 mutant, which carries an En insertion in the highly homologous IRT2 gene of Arabidopsis thaliana, was identified by reverse genetics and shows no symptoms of iron deficiency. This, together with the finding that irt1-1 can be complemented by 35S::IRT1 but not by 35S::IRT2, demonstrates that, although the products of the two genes are closely related, only AtIRT1 is required for iron homeostasis under physiological conditions.     

Expression profiling of the Arabidopsis ferric chelate reductase (FRO) gene family reveals differential regulation by iron and copper

The Arabidopsis FRO2 gene encodes the iron deficiency-inducible ferric chelate reductase responsible for reduction of iron at the root surface; subsequent transport of iron across the plasma membrane is carried out by a ferrous iron transporter (IRT1). Genome annotation has identified seven additional FRO family members in the Arabidopsis genome. We used real-time RT-PCR to examine the expression of each FRO gene in different tissues and in response to iron and copper limitation. FRO2 and FRO5 are primarily expressed in roots while FRO8 is primarily expressed in shoots. FRO6 and FRO7 show high expression in all the green parts of the plant. FRO3 is expressed at high levels in roots and shoots, and expression of FRO3 is elevated in roots and shoots of iron-deficient plants. Interestingly, when plants are Cu-limited, the expression of FRO6 in shoot tissues is reduced. Expression of FRO3 is induced in roots and shoots by Cu-limitation. While it is known that FRO2 is expressed at high levels in the outer layers of iron-deficient roots, histochemical staining of FRO3-GUS plants revealed that FRO3 is predominantly expressed in the vascular cylinder of roots. Together our results suggest that FRO family members function in metal ion homeostasis in a variety of locations in the plant.     

Arabidopsis IRT2 gene encodes a root-periphery iron transporter

Iron uptake from the soil is a tightly controlled process in plant roots, involving specialized transporters. One such transporter, IRT1, was identified in Arabidopsis thaliana and shown to function as a broad-range metal ion transporter in yeast. Here we report the cloning and characterization of the IRT2 cDNA, a member of the ZIP family of metal transporters, highly similar to IRT1 at the amino-acid level. IRT2 expression in yeast suppresses the growth defect of iron and zinc transport yeast mutants and enhances iron uptake and accumulation. However, unlike IRT1, IRT2 does not transport manganese or cadmium in yeast. IRT2 expression is detected only in roots of A. thaliana plants, and is upregulated by iron deficiency. By fusing the IRT2 promoter to the uidA reporter gene, we show that the IRT2 promoter is mainly active in the external cell layers of the root subapical zone, and therefore provide the first tissue localization of a plant metal transporter. Altogether, these data support a role for the IRT2 transporter in iron and zinc uptake from the soil in response to iron-limited conditions.     

Differential regulation of nramp and irt metal transporter genes in wild type and iron uptake mutants of tomato

Metal transporters regulated by iron can transport a variety of divalent metals, suggesting that iron regulation is important for specificity of iron transport. In plants, the iron-regulated broad-range metal transporter IRT1 is required for uptake of iron into the root epidermis. Functions of other iron-regulated plant metal transporters are not yet established. To deduce novel plant iron transport functions we studied the regulation of four tomato metal transporter genes belonging to the nramp and irt families with respect to environmental and genetic factors influencing iron uptake. We isolated Lenramp1 and Lenramp3 from tomato and demonstrate that these genes encode functional NRAMP metal transporters in yeast, where they were iron-regulated and localized mainly to intracellular vesicles. Lenramp1 and Leirt1 revealed both root-specific expression and up-regulation by iron deficiency, respectively, in contrast to Leirt2 and Lenramp3. Lenramp1 and Leirt1, but not Lenramp3 and Leirt2, were down-regulated in the roots of fer mutant plants deficient in a bHLH gene regulating iron uptake. In chloronerva mutant plants lacking the functional enzyme for synthesis of the plant-specific metal chelator nicotianamine Leirt1 and Lenramp1 were up-regulated despite sufficient iron supply independent of a functional fer gene. Lenramp1 was expressed in the vascular root parenchyma in a similar cellular pattern as the fer gene. However, the fer gene was not sufficient for inducing Lenramp1 and Leirt1 when ectopically expressed. Based on our results, we suggest a novel function for NRAMP1 in mobilizing iron in the vascular parenchyma upon iron deficiency in plants. We discuss fer/nicotianamine synthase-dependent and -independent regulatory pathways for metal transporter gene regulation.     

The effect of sodium bicarbonate on plant performance and iron acquisition system of FA-5 (Forner-Alcaide 5) citrus seedlings

This work studies the effect of bicarbonate on plant performance and the iron acquisition system of Forner-Alcaide 5 (FA-5) seedlings, a citrus genotype known for its tolerance to calcareous soils. Plants were irrigated for 6 weeks with or without 10 mM NaHCO₃. Treatment significantly decreased shoot growth, photosynthetic levels and iron concentration in shoots and roots. o,o-⁵⁷FeEDDHA experiments indicated that ⁵⁷Fe uptake by roots was inhibited in treated plants. Moreover, those seedlings accumulated more ⁵⁷Fe in roots, and enhanced mRNA accumulation of ferric reductase genes FRO1 and FRO2 and FC-R activity in roots. H⁺-ATPase activity and HA1 gene expression were also increased, while HA2 was not affected. In addition, expression of the iron transporter gene IRT1 was increased, while IRT2 was not significantly affected. Finally, according to PEPC enzymatic activity, PEPC1 gene expression was higher in treated roots. In conclusion, it appears that bicarbonate prevents medium acidification by roots, thus reducing Fe²⁺ uptake. Accordingly, Fe deficiency enhanced the expression of some genes related with the Fe acquisition system (IRT1, FRO1, FRO2, HA1 and PEPC1) and the activity of the corresponding enzymes, which appear to constitute an adaptive mechanism of FA-5 in these soils.     

The IRT1 protein from Arabidopsis thaliana is a metal transporter with a broad substrate range

The molecular basis for the transport of manganese across membranes in plant cells is poorly understood. We have found that IRT1, an Arabidopsis thaliana metal ion transporter, can complement a mutant Saccharomyces cerevisiae strain defective in high-affinity manganese uptake (smf1). The IRT1 protein has previously been identified as an iron transporter. The current studies demonstrated that IRT1, when expressed in yeast, can transport manganese as well. This manganese uptake activity was inhibited by cadmium, iron(II) and zinc, suggesting that IRT1 can transport these metals. The IRT1 cDNA also complements a zinc uptake-deficient yeast mutant strain (zrt1zrt2), and IRT1-dependent zinc transport in yeast cells is inhibited by cadmium, copper, cobalt and iron(III). However, IRT1 did not complement a copper uptake-deficient yeast mutant (ctr1), implying that this transporter is not involved in the uptake of copper in plant cells. The expression of IRT1 is enhanced in A. thaliana plants grown under iron deficiency. Under these conditions, there were increased levels of root-associated manganese, zinc and cobalt, suggesting that, in addition to iron, IRT1 mediates uptake of these metals into plant cells. Taken together, these data indicate that the IRT1 protein is a broad-range metal ion transporter in plants.     

Arabidopsis cpFtsY mutants exhibit pleiotropic defects including an inability to increase iron deficiency-inducible root Fe(III) chelate reductase activity

All plants, except for the grasses, must reduce Fe(III) to Fe(II) in order to acquire iron. In Arabidopsis, the enzyme responsible for this reductase activity in the roots is encoded by FRO2. Two Arabidopsis mutants, frd4-1 and frd4-2, were isolated in a screen for plants that do not induce Fe(III) chelate reductase activity in their roots in response to iron deficiency. frd4 mutant plants are chlorotic and grow more slowly than wild-type Col-0 plants. Additionally, frd4 chloroplasts are smaller in size and possess dramatically fewer thylakoid membranes and grana stacks when compared with wild-type chloroplasts. frd4 mutant plants express both FRO2 and IRT1 mRNA normally in their roots under iron deficiency, arguing against any defects in systemic iron-deficiency signaling. Further, transgenic frd4 plants accumulate FRO2-dHA fusion protein under iron-deficient conditions, suggesting that the frd4 mutation acts post-translationally in reducing Fe(III) chelate reductase activity. FRO2-dHA appears to localize to the plasma membrane of root epidermal cells in both Col-0 and frd4-1 transgenic plants when grown under iron-deficient conditions. Map-based cloning revealed that the frd4 mutations reside in cpFtsY, which encodes a component of one of the pathways responsible for the insertion of proteins into the thylakoid membranes of the chloroplast. The presence of cpFtsY mRNA and protein in the roots of wild-type plants suggests additional roles for this protein, in addition to its known function in targeting proteins to the thylakoid membrane in chloroplasts.     

Kinetic properties of a micronutrient transporter from<Emphasis Type="Italic"> Pisum sativum</Emphasis> indicate a primary function in Fe uptake from the soil

Fe uptake in dicotyledonous plants is mediated by a root plasma membrane-bound ferric reductase that reduces extracellular Fe(III)-chelates, releasing Fe²⁺ ions, which are then absorbed via a metal ion transporter. We previously showed that Fe deficiency induces an increased capacity to absorb Fe and other micronutrient and heavy metals such as Zn²⁺ and Cd²⁺ into pea (Pisum sativum L.) roots [Cohen et al. (1998) Plant Physiol 116:1063–1072). To investigate the molecular basis for this phenomenon, an Fe-regulated transporter that is a homologue of the Arabidopsis IRT1 micronutrient transporter was isolated from pea seedlings. This cDNA clone, designated RIT1 for root iron transporter, encodes a 348 amino acid polypeptide with eight putative membrane-spanning domains that is induced under Fe deficiency and can functionally complement yeast mutants defective in high- and low-affinity Fe transport. Chelate buffer techniques were used to control Fe²⁺ in the uptake solution at nanomolar activities representative of those found in the rhizosphere, and radiotracer methodologies were employed to show that RIT1 is a very high-affinity ⁵⁹Fe²⁺ uptake system (K _m =54–93 nM). Additionally, radiotracer (⁶⁵Zn, ¹⁰⁹Cd) flux techniques were used to show that RIT can also mediate a lower affinity Zn and Cd influx (K _m of 4 and 100 M, for Zn²⁺ and Cd²⁺, respectively). These findings suggest that, in typical agricultural soils, RIT1 functions primarily as a high-affinity Fe²⁺ transporter that mediates root Fe acquisition. This is consistent with recent findings with Arabidopsis IRT1 knockout mutants that strongly suggest that this transporter plays a key role in root Fe uptake and nutrition. However, the ability of RIT1 to facilitate Zn and Cd uptake when these metals are present at elevated concentrations suggests that RIT1 may be one pathway for the entry of toxic metals into the food chain. Furthermore, the finding that plant Fe deficiency status may promote heavy metal uptake via increased expression of this transporter could have implications both for human nutrition and also for phytoremediation, the use of terrestrial plants to sequester toxic metals from contaminated soil.