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.     

Metal Acquisition and Homeostasis in Fungi

Transition metals, particularly iron, zinc and copper, have multiple biological roles and are essential elements in biological processes. Among other micronutrients, these metals are frequently available to cells in only limited amounts, thus organisms have evolved highly regulated mechanisms to cope and to compete with their scarcity. The homeostasis of such metals within the animal hosts requires the integration of multiple signals producing depleted environments that restrict the growth of microorganisms, acting as a barrier to infection. As the hosts sequester the necessary transition metals from invading pathogens, some, as is the case of fungi, have evolved elaborate mechanisms to allow their survival and development to establish infection. Metalloregulatory factors allow fungal cells to sense and to adapt to the scarce metal availability in the environment, such as in host tissues. Here we review recent advances in the identification and function of molecules that drive the acquisition and homeostasis of iron, copper and zinc in pathogenic fungi.     

Transporters of ligands for essential metal ions in plants

Essential metals are required for healthy plant growth but can be toxic when present in excess. Therefore plants have mechanisms of metal homeostasis which involve coordination of metal ion transporters for uptake, translocation and compartmentalization. However, very little metal in plants is thought to exist as free ions. A number of small, organic molecules have been implicated in metal ion homeostasis as metal ion ligands to facilitate uptake and transport of metal ions with low solubility and also as chelators implicated in sequestration for metal tolerance and storage. Ligands for a number of essential metals have been identified and proteins involved in the transport of these ligands and of metal-ligand complexes have been characterized. Here we review recent advances in understanding the role of mugineic acid, nicotianamine, organic acids (citrate and malate), histidine and phytate as ligands for iron (Fe), zinc (Zn), copper (Cu), manganese (Mn) and nickel (Ni) in plants, and the proteins identified as their transporters.     

Nicotianamine: mediator of transport of iron and heavy metals in the phloem?

Recent work has demonstrated that minerals in plants are circulated between root and shoot. This occurs during the whole life time and renders possible response to changing environmental conditions. This mineral circulation occurs through intensive solute exchange between xylem and phloem in roots, stems, and leaves. The transport form of heavy metals such as iron, manganes, zinc and copper in the phloem, whether ionic or chelated, is unclear in most cases.
The unusual amino acid nicotianamine (NA) is ubiquitous throughout the plant kingdom. It is a chelator of several divalent transition metals. Its physiological role was investigated with the tomato mutant chloronerva, the only known NA-free multicellular plant. The mutant also exhibits disturbances of its iron metabolism and that of other heavy metals. This leads, among others, to a typical intercostal chlorosis and progressive iron accumulation in the leaves. From the heavy metal chelating properties of NA and from the phenotype of the mutant chloronerva it is concluded that NA is needed for normal distribution of heavy metals in young growing tissues fed via the phloem. This function could be fulfilled by mediating phloem loading or unloading of heavy metals as well as by preventing their precipitation in the alkaline phloem sap. An attempt is made to explain the chloronerva phenotype in the light of the phloem transport hypothesis of chelated iron.     

The chloroplast permease PIC1 regulates plant growth and development by directing homeostasis and transport of iron

The membrane-spanning protein PIC1 (for permease in chloroplasts 1) in Arabidopsis (Arabidopsis thaliana) was previously described to mediate iron transport across the inner envelope membrane of chloroplasts. The albino phenotype of pic1 knockout mutants was reminiscent of iron-deficiency symptoms and characterized by severely impaired plastid development and plant growth. In addition, plants lacking PIC1 showed a striking increase in chloroplast ferritin clusters, which function in protection from oxidative stress by sequestering highly reactive free iron in their spherical protein shell. In contrast, PIC1-overexpressing lines (PIC1ox) in this study rather resembled ferritin loss-of-function plants. PIC1ox plants suffered from oxidative stress and leaf chlorosis, most likely originating from iron overload in chloroplasts. Later during growth, plants were characterized by reduced biomass as well as severely defective flower and seed development. As a result of PIC1 protein increase in the inner envelope membrane of plastids, flower tissue showed elevated levels of iron, while the content of other transition metals (copper, zinc, manganese) remained unchanged. Seeds, however, specifically revealed iron deficiency, suggesting that PIC1 overexpression sequestered iron in flower plastids, thereby becoming unavailable for seed iron loading. In addition, expression of genes associated with metal transport and homeostasis as well as photosynthesis was deregulated in PIC1ox plants. Thus, PIC1 function in plastid iron transport is closely linked to ferritin and plastid iron homeostasis. In consequence, PIC1 is crucial for balancing plant iron metabolism in general, thereby regulating plant growth and in particular fruit development.     

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.     

Role of ROS and auxin in plant response to metal-mediated stress

Being unable to move away from their places of germination, in order to avoid excess metal-induced damages, plants have to evolve different strategies and complex regulatory mechanisms to survive harsh conditions. While both ROS and auxin are documented to be important in plant response to metal stress, the mechanisms underlying the crosstalk between ROS and auxin in metal stress are poorly understood. In this review, we provide an update on the regulation of plant responses to metal-stress by ROS and auxin signaling pathways, primarily, with a focus on the copper, aluminum and cadmium stress. We aim at surveying the mechanisms underlying how metal stress modulates the changes in auxin distribution and the network of ROS and auxin in plant response to metal stress based on recent studies.     

Recent developments in the application of proteomics to the analysis of plant responses to heavy metals

Pollution of soils by heavy metals is an ever‐growing problem throughout the world, and is the result of human activities as well as geochemical weathering of rocks and other environmental causes such as volcanic eruptions, acid rain and continental dusts. Plants everywhere are continuously exposed to metal‐contaminated soils. The uptake of heavy metals not only constrains crop yields, but can also be a major hazard to the health of humans and to the entire ecosystem. Although analysis of gene expression at the mRNA level has enhanced our understanding of the response of plants to heavy metals, many questions regarding the functional translated portions of plant genomes under metal stress remain unanswered. Proteomics offers a new platform for studying complex biological functions involving large numbers and networks of proteins, and can serve as a key tool for revealing the molecular mechanisms that are involved in interactions between toxic metals and plant species. This review focuses on recent developments in the applications of proteomics to the analysis of the responses of plants to heavy metals; such studies provide a deeper understanding of protein responses and the interactions among the possible pathways that are involved in detoxification of toxic metals in plant cells. In addition, the challenges faced by proteomics in understanding the responses of plants to toxic metal are discussed, and some possible future strategies for meeting these challenges are proposed.     

Homeostasis of metals in the progression of Alzheimer’s disease

In order to study the involvement of metals in the progression of Alzheimer’s disease, serum samples from patients with Alzheimer and mild cognitive impairment were investigated. For this purpose, metal content was analyzed after size-fractionation of species and then, inter-element and inter-fraction ratios were computed. In this way, the analysis allowed discovering changes that could be used as markers of disease, but also provided a new insight into the interactions in the homeostasis of elements in neurodegeneration and its progression. Aluminum and labile forms of iron and copper were increased in demented patients, while manganese, zinc and selenium were reduced. Interestingly, levels of different elements, principally iron, aluminum and manganese, were closely inter-related, which could evidence a complex interdependency between the homeostasis of the different metals in this disorder. On the other hand, imbalances in metabolism of copper, zinc and selenium could be associated to abnormal redox status. Therefore, this study may contribute to our understanding of the pathological mechanisms related to metals in Alzheimer’s disease.     

PIC1, an ancient permease in Arabidopsis chloroplasts, mediates iron transport

In chloroplasts, the transition metals iron and copper play an essential role in photosynthetic electron transport and act as cofactors for superoxide dismutases. Iron is essential for chlorophyll biosynthesis, and ferritin clusters in plastids store iron during germination, development, and iron stress. Thus, plastidic homeostasis of transition metals, in particular of iron, is crucial for chloroplast as well as plant development. However, very little is known about iron uptake by chloroplasts. Arabidopsis thaliana PERMEASE IN CHLOROPLASTS1 (PIC1), identified in a screen for metal transporters in plastids, contains four predicted alpha-helices, is targeted to the inner envelope, and displays homology with cyanobacterial permease-like proteins. Knockout mutants of PIC1 grew only heterotrophically and were characterized by a chlorotic and dwarfish phenotype reminiscent of iron-deficient plants. Ultrastructural analysis of plastids revealed severely impaired chloroplast development and a striking increase in ferritin clusters. Besides upregulation of ferritin, pic1 mutants showed differential regulation of genes and proteins related to iron stress or transport, photosynthesis, and Fe-S cluster biogenesis. Furthermore, PIC1 and its cyanobacterial homolog mediated iron accumulation in an iron uptake-defective yeast mutant. These observations suggest that PIC1 functions in iron transport across the inner envelope of chloroplasts and hence in cellular metal homeostasis.     

Molecular aspects of Cu, Fe and Zn homeostasis in plants

Proper metal transport and homeostasis are critical for the growth and development of plants. In order to potentially fortify plants pre-harvest with essential metals in aid of human nutrition, we must understand not only how metals enter the plant but also how metals are then delivered to the edible portions of the plant such as the seed. In this review, we focus on three metals required by both plants and humans: Cu, Fe and Zn. In particular, we present the current understanding of the molecular mechanisms of Cu, Fe and Zn transport, including aspects of uptake, distribution, chelation and/or sequestration.     

Molecular mechanisms of plant metal tolerance and homeostasis

Transition metals such as copper are essential for many physiological processes yet can be toxic at elevated levels. Other metals (e.g. lead) are nonessential and potentially highly toxic. Plants – like all other organisms – possess homeostatic mechanisms to maintain the correct concentrations of essential metal ions in different cellular compartments and to minimize the damage from exposure to nonessential metal ions. A regulated network of metal transport, chelation, trafficking and sequestration activities functions to provide the uptake, distribution and detoxification of metal ions. Some of the components of this network have now been identified: a number of uptake transporters have been cloned as well as candidate transporters for the vacuolar sequestration of metals. Chelators and chaperones are known, and evidence for intracellular metal trafficking is emerging. This recent progress in the molecular understanding of plant metal homeostasis and tolerance is reviewed. Received: 14 July 2000 / Accepted: 22 September 2000     

Dissecting plant iron homeostasis under short and long-term iron fluctuations

A wealth of information on the different aspects of iron homeostasis in plants has been obtained during the last decade. However, there is no clear road-map integrating the relationships between the various components. The principal aim of the current review is to fill this gap. In this context we discuss the lack of low affinity iron uptake mechanisms in plants, the utilization of a different uptake mechanism by graminaceous plants compared to the others, as well as the roles of riboflavin, ferritin isoforms, nitric oxide, nitrosylation, heme, aconitase, and vacuolar pH. Cross-homeostasis between elements is also considered, with a specific emphasis on the relationship between iron homeostasis and phosphorus and copper deficiencies. As the environment is a crucial parameter for modulating plant responses, we also highlight how diurnal fluctuations govern iron metabolism. Evolutionary aspects of iron homeostasis have so far attracted little attention. Looking into the past can inform us on how long-term oxygen and iron-availability fluctuations have influenced the evolution of iron uptake mechanisms. Finally, we evaluate to what extent this homeostastic road map can be used for the development of novel biofortification strategies in order to alleviate iron deficiency in human.     

Influence of Soil Chemistry and Plant Physiology in the Phytoremediation of Cu,Mn, and Zn

Different anthropogenic sources of metals can result from agricultural, industrial, military, mining and urban activities that contribute to environmental pollution. Plants can be grown for phytoremediation to remove or stabilize contaminants in water and soil. Copper (Cu), manganese (Mn) and zinc (Zn) are trace essential metals for plants, although their role in homeostasis in plants must be strictly regulated to avoid toxicity. In this review, we summarize the processes involved in the bioavailability, uptake, transport and storage of Cu, Mn and Zn in plants. The efficiency of phytoremediation depends on several factors including metal bioavailability and plant uptake, translocation and tolerance mechanisms. Soil parameters, such as clay fraction, organic matter content, oxidation state, pH, redox potential, aeration, and the presence of specific organisms, play fundamental roles in the uptake of trace essential metals. Key processes in the metal homeostasis network in plants have been identified. Membrane transporters involved in the acquisition, transport and storage of trace essential metals are reviewed. Recent advances in understanding the biochemical and molecular mechanisms of Cu, Mn and Zn hyperaccumulation are described. The use of plant-bacteria associations, plant-fungi associations and genetic engineering has opened a new range of opportunities to improve the efficiency of phytoremediation. The main directions for future research are proposed from the investigation of published results.     

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.