Copper and iron homeostasis in Arabidopsis: responses to metal deficiencies, interactions and biotechnological applications

Plants have developed sophisticated mechanisms to tightly control the acquisition and distribution of copper and iron in response to environmental fluctuations. Recent studies with Arabidopsis thaliana are allowing the characterization of the diverse families and components involved in metal uptake, such as metal-chelate reductases and plasma membrane transporters. In parallel, emerging data on both intra- and intercellular metal distribution, as well as on long-distance transport, are contributing to the understanding of metal homeostatic networks in plants. Furthermore, gene expression analyses are deciphering coordinated mechanisms of regulation and response to copper and iron limitation. Prioritizing the use of metals in essential versus dispensable processes, and substituting specific metalloproteins by other metal counterparts, are examples of plant strategies to optimize copper and iron utilization. The metabolic links between copper and iron homeostasis are well documented in yeast, algae and mammals. In contrast, interactions between both metals in vascular plants remain controversial, mainly owing to the absence of copper-dependent iron acquisition. This review describes putative interactions between both metals at different levels in plants. The characterization of plant copper and iron homeostasis should lead to biotechnological applications aimed at the alleviation of iron deficiency and copper contamination and, thus, have a beneficial impact on agricultural and human health problems.     

Electron spin echo envelope modulation evidence for carbonate binding to iron(III) and copper(II) transferrin and lactoferrin

Iron binding to transferrin and lactoferrin requires a synergistic anion, which is carbonate in vivo. The anion is thought to play a key role in iron binding and release. To understand better the iron-carbonate interaction, experiments were performed with iron(III) and copper(II) complexes of human milk lactoferrin and serum transferrin with carbon-13-labeled carbonate. Modulation frequencies were present in the Fourier transforms of two-pulse and three-pulse electron spin echo envelope modulation data for the Fe(III) and Cu(II) complexes, consistent with binding of carbonate to both metals. The metal-13C interaction was similar for the lactoferrin and transferrin complexes. Spin coupling to the nitrogen of a coordinated histidine imidazole was observed for both metals. Both the metal-nitrogen and the metal-carbon spin coupling constants were about a factor of 5 smaller for the iron complexes than for the copper complexes, which indicated substantial similarity in the metal-carbonate and metal-imidazole binding for the two metals.     

A proteomic approach to iron and copper homeostasis in cyanobacteria.

Cyanobacteria, which are considered to be the chloroplast precursors, are significant contributors to global photosynthetic productivity. The ample variety of membrane and soluble proteins containing different metals (mainly, iron and copper) has made these organisms develop a complex homeostasis with different mechanisms and tight regulation processes to fulfil their metal requirements in a changing environment. Cell metabolism is so adapted as to synthesize alternative proteins depending on the relative metal availabilities. In particular, plastocyanin, a copper protein, and cytochrome c(6), a haem protein, can replace each other to play the same physiological role as electron carriers in photosynthesis and respiration, with the synthesis of one protein or another being regulated by copper concentration in the medium. The unicellular cyanobacterium Synechocystis sp. PCC 6803 has been widely used as a model system because of completion of its genome sequence and the ease of its genetic manipulation, with a lot of proteomic work being done. In this review article, we focus on the functional characterization of knockout Synechocystis mutants for plastocyanin and cytochrome c(6), and discuss the ongoing proteomic analyses performed at varying copper concentrations to investigate the cyanobacterial metal homeostasis and cell response to changing environmental conditions.     

Iron and copper homeostasis and intestinal absorption using the Caco2 cell model

Whole body homeostasis can be viewed as the balance between absorption and excretion, which can be regulated independently. Present evidence suggests that for iron, intestinal absorption is the main site for homeostatic regulation, while for copper it is biliary excretion. There are connections between iron and copper in intestinal absorption and transport. The blue copper plasma protein, ceruloplasmin, and its intracellular homologue, hephaestin, play a role in cellular iron release. The studies reviewed here compare effects of Fe(II) and Cu(II) on their uptake and overall transport by monolayers of polarized Caco2 cells, which model intestinal mucosa. In the physiological range of concentrations, depletion of cellular iron or copper (by half) increased uptake of both metal ions. Depletion of iron or copper also enhanced overall transport of iron from the apical to the basal chamber. Copper depletion enhanced overall copper transport, but iron depletion did not. Pretreatment with excess copper also stimulated copper absorption. Plasma ceruloplasmin (added to the basal chamber) failed to enhance basolateral iron release, and Zn(II) failed to compete with Cu(II) for uptake. Neither copper nor iron deficiency altered expression of IREG1 or DMT1 (-IRE form) at the mRNA level. Thus, in the low-normal range of iron and copper availability, intestinal absorption of both metals appears to be positively related to the need for these elements by the whole organism. The two metal ions also influenced each other's transport; but with copper excess, other mechanisms come into play.     

Targeting of the mouse Slc39a2 (Zip2) gene reveals highly cell-specific patterns of expression, and unique functions in zinc, iron, and calcium homeostasis

Fourteen members of the Slc39a superfamily of metal ion uptake transporters have been identified in mice and humans, but the physiological functions of most remain obscure. Herein, we created mice with Zip2 (Slc39a2) genes in which the open reading frame was replaced with that of the enhanced green fluorescent protein (EGFP), to study temporal and spatial patterns of Zip2 gene expression and examine the physiological roles of this transporter. Expression of this gene was remarkably cell-type specific and developmentally regulated in pericentral hepatocytes, developing keratinocytes, and a subset of immature dendritic cells in the immune system. In addition, the Zip2 gene was transiently expressed in giant trophoblast cells in the placenta. Although the Zip2 gene was not essential under conditions of normal dietary zinc, it played an important role in adapting to dietary zinc deficiency during pregnancy, and in the homeostasis of iron in the liver as well as iron and calcium in developing embryos. These studies suggest that active expression of the Zip2 gene in these few specific cell types, aforementioned, plays a particularly important role during zinc deficiency. These studies further reveal novel interactions between zinc transporter function and the homeostasis of other essential metals.     

Globus pallidus: a target brain region for divalent metal accumulation associated with dietary iron deficiency

Recently, iron deficiency has been connected with a heterogeneous accumulation of manganese in the rat brain. The striatum is particularly vulnerable, for there is a significant negative correlation between accumulated manganese and gamma-aminobutyric acid levels. The effect of dietary iron deficiency on the distribution of zinc and copper, two other divalent metals with essential neurobiological roles, is relatively unexplored. Thus, the primary goal of this study was to examine the effect of manipulating dietary iron and manganese levels on the concentrations of copper, iron, manganese and zinc in five rat brain regions as determined with inductively coupled plasma mass spectrometry analysis. Because divalent metal transporter has been implicated as a transporter of brain iron, manganese, and to a lesser extent zinc and copper, another goal of the study was to measure brain regional changes in transporter levels using Western blot analysis. As expected, there was a significant effect of iron deficiency (P < 0.05) on decreasing iron concentrations in the cerebellum and caudate putamen; and increasing manganese concentrations in caudate putamen, globus pallidus and substantia nigra. Furthermore, there was a significant effect of iron deficiency (P < 0.05) on increasing zinc concentration and a statistical trend (P = 0.08) toward iron deficiency-induced copper accumulation in the globus pallidus. Transporter protein in all five regions increased due to iron deficiency compared to control levels (P < 0.05); however, the globus pallidus and substantia nigra revealed the greatest increase. Therefore, the globus pallidus appears to be a target for divalent metal accumulation that is associated with dietary iron deficiency, potentially caused by increased transporter protein levels.     

Copper Deficiency Leads to Anemia,Duodenal Hypoxia,Upregulation of HIF-2α and Altered Expression of Iron Absorption Genes in Mice

Iron and copper are essential trace metals, actively absorbed from the proximal gut in a regulated fashion. Depletion of either metal can lead to anemia. In the gut, copper deficiency can affect iron absorption through modulating the activity of hephaestin - a multi-copper oxidase required for optimal iron export from enterocytes. How systemic copper status regulates iron absorption is unknown. Mice were subjected to a nutritional copper deficiency-induced anemia regime from birth and injected with copper sulphate intraperitoneally to correct the anemia. Copper deficiency resulted in anemia, increased duodenal hypoxia and Hypoxia inducible factor 2α (HIF-2α) levels, a regulator of iron absorption. HIF-2α upregulation in copper deficiency appeared to be independent of duodenal iron or copper levels and correlated with the expression of iron transporters (Ferroportin - Fpn, Divalent Metal transporter – Dmt1) and ferric reductase – Dcytb. Alleviation of copper-dependent anemia with intraperitoneal copper injection resulted in down regulation of HIF-2α-regulated iron absorption genes in the gut. Our work identifies HIF-2α as an important regulator of iron transport machinery in copper deficiency.     

Genotoxicity and mutagenicity of iron and copper in mice

The toxicity of trace metals is still incompletely understood. We have previously shown that a single oral dose of iron or copper induces genotoxic effects in mice in vivo, as detected by single cell gel electrophoresis (comet assay). Here, we report the effect of these metals on subchronic exposure. Mice were gavaged for six consecutive days with either water, 33.2 mg/kg iron, or 8.5 mg/kg copper. On the 7th day, the neutral and alkaline comet assays in whole blood and the bone marrow micronucleus (MN) test were used as genotoxicity and mutagenicity endpoints, respectively. Particle induced X-ray emission was used to determine liver levels of the metals. Females showed a slightly lower DNA damage background, but there was no significant difference between genders for any endpoint. Iron and copper were genotoxic and mutagenic. While copper was more genotoxic in the neutral version, iron was more genotoxic in the alkaline version of the comet assay. Copper induced the highest mutagenicity as evaluated by the MN test. Iron was not mutagenic to male mice. Iron is thought to induce more oxidative lesions than copper, which are primarily detected in the alkaline comet assay. Treatment with iron, but not with copper, induced a significant increase in the hepatic level of the respective metal, reflecting different excretion strategies.     

Differential effects of basolateral and apical iron supply on iron transport in Caco-2 cells

Iron homeostasis in the human body is maintained primarily through regulation of iron absorption in the duodenum. The liver peptide hepcidin plays a central role in this regulation. Additionally, expression and functional control of certain components of the cellular iron transport machinery can be influenced directly by the iron status of enterocytes. The significance of this modulation, relative to the effects of hepcidin, and the comparative effects of iron obtained directly from the diet and/or via the bloodstream are not clear. The studies described here were performed using Caco-2 cell monolayers as a model of intestinal epithelium, to compare the effects of iron supplied in physiologically relevant forms to either the apical or basolateral surfaces of the cells. Both sources of iron provoked increased cellular ferritin content, indicating iron uptake from both sides of the cells. Supply of basolateral transferrin-bound iron did not affect subsequent iron transport across the apical surface, but reduced iron transport across the basolateral membrane. In contrast, the apical iron supply led to subsequent reduction in iron transport across the apical cell membrane without altering iron export across the basolateral membrane. The apical and basolateral iron supplies also elicited distinct effects on the expression and subcellular distribution of iron transporters. These data suggest that, in addition to the effects of cellular iron status on the expression of iron transporter genes, different modes and direction of iron supply to enterocytes can elicit distinct functional effects on iron transport.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-015-0463-5) contains supplementary material, which is available to authorized users.     

The interplay between siderophore secretion and coupled iron and copper transport in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120

Iron uptake is essential for Gram-negative bacteria including cyanobacteria. In cyanobacteria, however, the iron demand is higher than in proteobacteria due to the function of iron as a cofactor in photosynthesis and nitrogen fixation, but our understanding of iron uptake by cyanobacteria stands behind the knowledge in proteobacteria. Here, two genes involved in this process in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 were identified. ORF all4025 encodes SchE, a putative cytoplasmic membrane-localized transporter involved in TolC-dependent siderophore secretion. Inactivation of schE resulted in an enhanced sensitivity to high metal concentrations and decreased secretion of hydroxamate-type siderophores. ORF all4026 encodes a predicted outer membrane-localized TonB-dependent iron transporter, IacT. Inactivation of iacT resulted in decreased sensitivity to elevated iron and copper levels. Expression of iacT from the artificial trc promoter (P_trc) resulted in sensitization against tested metals. Further analysis showed that iron and copper effects are synergistic because a decreased supply of iron induced a significant decrease of copper levels in the iacT insertion mutant but an increase of those levels in the strain carrying P_trc-iacT. Our results unravel a link between iron and copper homeostasis in Anabaena sp. PCC 7120.     

Iron and the heart: A paradigm shift from systemic to cardiomyocyte abnormalities

Iron is a key micronutrient for the human body and participates in biological processes, such as oxygen transport, storage, and utilization. Iron homeostasis plays a crucial role in the function of the heart and both iron deficiency and iron overload are harmful to the heart, which is partly mediated by increased oxidative stress. Iron enters the cardiomyocyte through the classic pathway, by binding to the transferrin 1 receptor (TfR1), but also through other routes: T-type calcium channel (TTCC), divalent metal transporter 1 (DMT1), L-type calcium channel (LTCC), Zrt-, Irt-like Proteins (ZIP) 8 and 14. Only one protein, ferroportin (FPN), extrudes iron from cardiomyocytes. Intracellular iron is utilized, stored bound to cytoplasmic ferritin or imported by mitochondria. This cardiomyocyte iron homeostasis is controlled by iron regulatory proteins (IRP). When the cellular iron level is low, expression of IRPs increases and they reduce expression of FPN, inhibiting iron efflux, reduce ferritin expression, inhibiting iron storage and augment expression of TfR1, increasing cellular iron availability. Such cellular iron homeostasis explains why the heart is very susceptible to iron overload: while cardiomyocytes possess redundant iron importing mechanisms, they are equipped with only one iron exporting protein, ferroportin. Furthermore, abnormalities of iron homeostasis have been found in heart failure and coronary artery disease, however, no clear picture is emerging yet in this area. If we better understand iron homeostasis in the cardiomyocyte, we may be able to develop better therapies for a variety of heart diseases to which abnormalities of iron homeostasis may contribute.     

Iron deficiency and its epigenetic effects on iron homeostasis

Iron deficiency is a common micronutrient deficiency associated with metabolic changes in the levels of iron regulatory proteins, hepcidin and ferroportin. Studies have associated dysregulation of iron homeostasis to other secondary and life-threatening diseases including anaemia, neurodegeneration and metabolic diseases. Iron deficiency plays a critical role in epigenetic regulation by affecting the Fe²⁺/α-ketoglutarate-dependent demethylating enzymes, Ten Eleven Translocase 1–3 (TET 1–3) and Jumonji-C (JmjC) histone demethylase, which are involved in epigenetic erasure of the methylation marks on both DNA and histone tails, respectively. In this review, studies involving epigenetic effects of iron deficiency associated with dysregulation of TET 1–3 and JmjC histone demethylase enzyme activities on hepcidin/ferroportin axis are discussed.     

A novel role for thioredoxin reductase in the iron metabolism of S. cerevisiae

Intracellular levels of iron are tightly regulated. Saccharomyces cerevisiae uses well-defined pathways to extract iron molecules from the environment. Once inside the cell, the iron molecules must be transferred to target sites via an intracellular iron transporter. Although analogous carriers have been described for other metals, such as copper, an iron transporter has yet to be identified. We used two-dimensional gel electrophoresis and mass spectrometry techniques to attempt to identify the iron transporter from cytosolic fraction of S. cerevisiae. In this study, we identified the iron-binding activity of thioredoxin reductase, and our data suggest a potential role for this enzyme in intracellular iron transport.     

综述:金属离子代谢平衡失调与阿尔茨海默病早期发病机制

研究表明,脑内金属离子代谢失衡与阿尔茨海默病(AD)有关,但其机理尚需深入探讨．结合本实验室研究结果,作者对金属离子代谢紊乱与氧化应激,金属离子代谢紊乱与β-淀粉样蛋白、转铁蛋白和转铁蛋白受体、铁调节蛋白、二价金属离子转运体以及天然抗氧化剂通过调节金属离子代谢平衡缓解β-淀粉样蛋白的毒性和保护细胞的作用进行探讨．提出：铁、铜等金属离子缺乏可能主要与AD早期关系密切,而铁、铜等金属离子过载可能主要与AD后期损伤关系密切的学术观点．     

Decreased hephaestin expression and activity leads to decreased iron efflux from differentiated Caco2 cells

Iron is transported across intestinal brush border cells into the circulation in at least two distinct steps. Iron can enter the enterocyte via the apical surface through several paths. However, iron egress from the basolateral side of enterocytes converges on a single export pathway requiring the iron transporter, ferroportin1, and hephaestin, a ferroxidase. Copper deficiency leads to reduced hephaestin protein expression and activity in mouse enterocytes and intestinal cell lines. We tested the effect of copper deficiency on differentiated Caco2 cells grown in transwells and found decreased hephaestin protein expression and activity as well as reduced ferroportin1 protein levels. Furthermore, the decrease in hephaestin levels correlates with a decrease of ⁵⁵Fe release from the basolateral side of Caco2 cells. Presence of ceruloplasmin, apo‐transferrin or holo‐transferrin did not significantly alter the results observed. Repletion of copper in Caco2 cells leads to reconstitution of hephaestin protein expression, activity, and transepithelial iron transport. J. Cell. Biochem. 107: 803–808, 2009. © 2009 Wiley‐Liss, Inc.     

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.     

The Ferroxidase Hephaestin But Not Amyloid Precursor Protein is Required for Ferroportin-Supported Iron Efflux in Primary Hippocampal Neurons

Iron efflux in mammalian cells is mediated by the ferrous iron exporter ferroportin (Fpn); Fpn plasma membrane localization and function are supported by a multicopper ferroxidase and/or the soluble amyloid precursor protein (sAPP). Fpn and APP are ubiquitously expressed in all cell types in the central nervous system including neurons. In contrast, neuronal ferroxidase(s) expression has not been well characterized. Using primary cultures of hippocampal neurons, we examined the molecular mechanism of neuronal Fe efflux in detail. Developmental increases of Fpn, APP, and the ferroxidase hephaestin (Hp) were observed in hippocampal neurons. Iron efflux in these neurons depended on the level of Fpn localized at the cell surface; as noted, Fpn stability is supported by ferroxidase activity, an enzymatic activity that is required for Fe efflux. Iron accumulation increases and iron efflux decreases in Hp knockout neurons. In contrast, suppression of endogenous APP by RNAi knockdown does not affect surface Fpn stability or Fe efflux. These data support the model that the neuronal ferroxidase Hp plays a unique role in support of Fpn-mediated Fe efflux in primary hippocampal neurons. Our data also demonstrate that Hp ferroxidase activity relies on copper bioavailability, which suggests neuronal iron homeostasis will be modulated by cellular copper status.     

Regulation of iron uptake in Saccharomyces cerevisiae. The ferrireductase and Fe(II) transporter are regulated independently.

Iron is required for the growth of Saccharomyces cerevisiae. High concentrations of iron, however, are toxic, forcing this yeast to tightly regulate its concentration of intracellular free iron. We demonstrate that S. cerevisiae accumulates iron through the combined action of a plasma membrane ferrireductase and an Fe(II) transporter. This transporter is highly selective for Fe(II). Several other transition metals did not inhibit iron uptake when these metals were present at a concentration 100-fold higher than the Km (0.15 microM) for iron transport. Pt(II) inhibited ferrireductase activity but not the ability of cells to transport iron that was chemically reduced to Fe(II). Incubation of cells in a synthetic iron-limited media resulted in the induction of both ferrireductase and Fe(II) transporter activities. In complex media, Fe(II) transport activity was regulated in response to media iron concentration, while the activity of the ferrireductase was not. When stationary phase cells were inoculated into fresh media, ferrireductase activity increased independent of the iron content of the media; in contrast, transporter activity varied inversely with iron levels. These results demonstrate that the ferrireductase and Fe(II) transporter are separately regulated and that iron accumulation may be limited by changes in either activity.     

Yeast, a model organism for iron and copper metabolism studies

Virtually all organisms on earth depend on transition metals for survival. Iron and copper are particularly important because they participate in vital electron transfer reactions, and are thus cofactors of many metabolic enzymes. Their ability to transfer electrons also render them toxic when present in excess. Disturbances of iron and copper steady-state levels can have profound effects on cellular metabolism, growth and development. It is critical to maintain these metals in a narrow range between utility and toxicity. Organisms ranging from bacteria and plants to mammals have developed sophisticated mechanisms to control metal homeostasis. In this review, we will present an overview of the current understanding of iron and copper metabolism in yeast, and the utility of yeast as a model organism to investigate iron and copper metabolism in mammals and plants.     

A role for copper in protozoan grazing – two billion years selecting for bacterial copper resistance

The Great Oxidation Event resulted in integration of soft metals in a wide range of biochemical processes including, in our opinion, killing of bacteria by protozoa. Compared to pressure from anthropologic copper contamination, little is known on impacts of protozoan predation on maintenance of copper resistance determinants in bacteria. To evaluate the role of copper and other soft metals in predatory mechanisms of protozoa, we examined survival of bacteria mutated in different transition metal efflux or uptake systems in the social amoeba Dictyostelium discoideum. Our data demonstrated a strong correlation between the presence of copper/zinc efflux as well as iron/manganese uptake, and bacterial survival in amoebae. The growth of protozoa, in turn, was dependent on bacterial copper sensitivity. The phagocytosis of bacteria induced upregulation of Dictyostelium genes encoding the copper uptake transporter p80 and a triad of Cu(I)‐translocating P_IB‐type ATPases. Accumulated Cu(I) in Dictyostelium was monitored using a copper biosensor bacterial strain. Altogether, our data demonstrate that Cu(I) is ultimately involved in protozoan predation of bacteria, supporting our hypothesis that protozoan grazing selected for the presence of copper resistance determinants for about two billion years.