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Homing in on iron homeostasis in plants
Review
Homing in on iron homeostasis in plants Jeeyon Jeong and Mary Lou Guerinot Department of Biological Sciences, Dartmouth College, Hanover, NH 03755, USA
Iron is essential for plants but is not readily accessible and is also potentially toxic. As plants are a major dietary source of iron worldwide, understanding plant iron homeostasis is pivotal for improving not only crop yields but also human nutrition. Although iron acquisition from the environment is well characterized, the transporters and reductases involved in plant organellar iron transport and some of the transcription factors that regulate iron uptake have only recently been discovered. Here, we discuss newly characterized molecular players, focusing on Arabidopsis. Localization of iron to the right compartment and accessibility of iron stores are proving crucial for maintaining proper iron homeostasis and will need to be considered in biofortification efforts currently underway. The significance of iron homeostasis in plants For most organisms, iron serves as a cofactor in vital metabolic pathways such as the electron transport chain of respiration. Plants have an additional need for iron because photosynthesis and chlorophyll biosynthesis both require iron. However, excess iron generates cytotoxic hydroxyl radicals via the Fenton reaction, and iron is not readily bioavailable because it forms insoluble complexes under aerobic conditions at neutral or alkaline pH [1]. Therefore, how plants maintain iron homeostasis is a biologically relevant question. Here, we outline recent progress made in our understanding of how the uptake of iron is regulated and then transported within plants. We also discuss the use of ionomics (Box 1) as a novel approach to studying iron homeostasis and highlight recent attempts to develop iron-fortified crops (Box 2). Iron transport from soil to roots Plants obtain iron from the environment using mechanisms based on reduction or chelation. Here, we discuss iron acquisition strategies, focusing on recent studies with Arabidopsis thaliana. The reduction strategy Non-graminaceous plants use the Reduction Strategy, or Strategy I, to obtain iron. Upon iron deficiency, protons are released to increase the solubility of iron, via H+-ATPases of the root plasma membrane [2,3]. Several Arabidopsis H+-ATPase (AHA) family members are induced in irondeficient roots [4,5], suggesting possible roles for these enzymes in iron-deficiency responses. Corresponding author: Guerinot, M.L. ([email protected]).
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After acidification, Fe3+ is reduced to Fe2+ by a membrane-bound ferric reductase oxidase (FRO), FRO2 [6], one of eight members of the FRO family. FRO genes are expressed differentially at the tissue level; for example, FRO2 is specific to roots, whereas FRO6 and FRO7 are specific to shoots [7,8]. FRO proteins are also predicted and/or experimentally shown to localize to different subcellular compartments [7,9,10]. Therefore, each FRO family member has a specific role in different organs or subcellular compartments, signifying that reduction-based iron transport is not limited to the root plasma membrane. Once Fe3+ is reduced, Fe2+ is transported into the root by iron-regulated transporter 1 (IRT1), a member of the zincregulated transporter (ZRT)-, IRT-like protein (ZIP) family [11]. Analysis of plants overexpressing IRT1 from the cauliflower mosaic virus 35S promoter shows that IRT1 is present only in iron-deficient roots, suggesting that it is controlled post-transcriptionally [12]. A recent follow-up study showed that iron-induced turnover of IRT1 requires two lysine residues located in the intracellular loop of IRT1 between transmembrane domains III and IV [13]. This is consistent with the turnover of ZRT1, a yeast zinc transporter that also belongs to the ZIP family [14]. In its variable loop region, ZRT1 has a lysine residue that is ubiquitinated to target it for protein degradation under zinc-sufficient conditions. When either IRT1 lysine residue was substituted with arginine and the variant was overexpressed, the plants accumulated higher levels of iron than did wild-type plants; by contrast, plants overexpressing wild-type IRT1 contained iron levels similar to those of wild-type plants [13]. However, the accumulation of iron was not associated with increased ferric chelate reductase activity [13], implying that this activity is not rate limiting, at least under the conditions tested. The chelation strategy To acquire iron, grasses use a mechanism based on chelation, known as Strategy II, in which phytosiderophores (PSs), such as mugineic acids (MAs), are released to chelate Fe3+. The resulting Fe(III)–PS complexes are then transported into the roots via transporters belonging to the Yellow Stripe (YS) family, named for the YS1 PS transporter of maize (Zea mays) [15]. Grasses can also take up Fe2+ in addition to Fe(III)–PS [16,17]. Rice (Oryza sativa) plants that cannot synthesize PS, owing to a mutation in the NICOTIANAMINE AMINOTRANSFERASE (NAAT) gene, do not show growth defects if Fe2+ is supplied [16]. However, unlike Strategy I plants, neither H+-ATPase nor Fe3+–chelate reductase activity is induced
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Review Box 1. Interaction of iron with other mineral elements and ionomics Changes in iron homeostasis influence other elements and vice versa. It has long been proposed that multiple mineral nutrients have roles in plants facing deficiency or toxicity caused by a specific nutrient [65]. Iron can be substituted for copper, under copper deficiency, as seen in the examples of cytochrome oxidase and diiron oxidase or Cu- or Zn-superoxide dismutase (SOD) and Fe-SOD [66]. Non-metal minerals, such as phosphate, also interact with iron. Iron homeostasis genes were affected in phosphate-deficient plants; expression of FER1 was increased, whereas IRT1 expression was decreased [67]. It seems that phosphate deficiency makes iron more available to plants, and inhibition of the primary root elongation, a typical symptom of phosphate deficiency, can be alleviated by reducing the amount of iron in the medium [68]. The issue of interactions among minerals can be tackled by applying the concept of the ‘ionome’, which refers to the mineral nutrients and trace elements, both essential and non-essential, found in an organism [69]. Advances in inductively coupled plasma (ICP) mass spectroscopy (MS) techniques, along with the development of bioinformatics and large-scale genetic tools, provides a potentially powerful strategy to understand genes involved in maintaining the ionome and how they affect physiological and developmental processes in plants [69]. This approach, in turn, will facilitate efforts to understand the roles of individual trace elements, including iron. The Purdue Ionomics Information Management System (PiiMS) provides an open-access web-based tool for large datasets generated by a high-throughput ICP-MS phenotyping platform [70]. Concentrations of 16 elements (phosphate, calcium, potassium, magnesium, copper, iron, zinc, manganese, cobalt, nickel, boron, selenium, molybdenum, sodium, arsenic and cadmium) in >60 000 samples of Arabidopsis shoots from >800 experiments can be found in PiiMS (http://www.ionomicshub.org/home/PiiMS). Using the ionomics approach, it was shown that the Arabidopsis leaf ionome contains signatures that reflect the physiological status and elemental composition of the plant, and statistical models were established to detect changes in iron homeostasis [71]. The study reveals that iron content itself is not a good indicator of the iron status of the plant because shoot iron concentration is tightly regulated. However, even under sustained shoot iron levels, increases in manganese, cobalt, zinc and cadmium concentrations and a decrease in molybdenum concentration are observed, and these changes are good predictors of the iron status of a plant [71]. When the authors screened previously collected ionomics data, known iron mutants such as frd1 and frd3 were identified, validating their model.
under iron deficiency. This probably reflects an adaptation to flooded rice paddies, where Fe2+ is more abundant than Fe3+ owing to reduced levels of oxygen [17]. Regulation of iron-deficiency responses in Strategy II plants was recently reviewed [18] so will not be covered further here. Regulation of the reduction strategy In Arabidopsis, Fe-regulated (Fer)-like iron-deficiencyinduced transcription factor (FIT), a basic helix–loop–helix (bHLH) transcription factor orthologous to the tomato (Solanum lycopersicum) FER protein [19], is required to regulate iron-deficiency responses [4,20,21]. FIT is highly induced in the epidermis of iron-deficient roots, and fit mutants are chlorotic and seedling lethal unless watered with supplemental iron [4], showing phenotypes similar to those of irt1 plants [22]. FRO2 is transcriptionally regulated by FIT, and FRO2 transcripts and root ferric chelate reductase activity are both absent in fit mutants [4,20]. Meanwhile, IRT1 is post-transcriptionally regulated by FIT [4]. fit mutants do not accumulate IRT1 protein, despite the induction of IRT1 mRNA by iron deficiency.
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Box 2. Resolving ‘hidden hunger’ with iron-fortified crops Understanding iron homeostasis in plants is crucial not only for improving plant growth and crop yields but also for improving human nutrition. According to the World Health Organization, iron deficiency is the most prevalent nutritional disorder in the world today, affecting an estimated 2.7 billion people in both developed and developing countries (http://www.who.int/nutrition/topics/ida/en/index.html). Along with deficiencies of vitamin A and iodine, iron deficiency is often referred to as ‘hidden hunger’. Because plants are the primary source of dietary iron worldwide, this calls for the need to enrich crops with bioavailable iron to help solve the issue of iron malnutrition. Biofortification refers to the strategy of enhancing the nutritional value of crops to solve malnutrition worldwide sustainably [72]. Because mineral nutrients, such as iron, are acquired from the environment, iron-fortification strategies should be based on increasing iron transport to, or storage in, the edible portions of plants. There have been multiple promising reports of iron-fortified crops. For example, iron-fortified rice was developed by overexpressing the iron storage protein ferritin [73]. It was also shown that seeds of transgenic maize expressing ferritin, in combination with phytase, which digests phytic acid that inhibits iron absorption, contained more iron [74]. In this study, it was also found that the expression level of phytase correlated with increased cellular iron absorption, as detected by in vitro digestion assays with Caco-2, a human intestinal cell line. Ferric chelate reductases have also been expressed in crop plants. FRO2 was expressed in soybean (Glycine max) [75], and a modified version of yeast ferric reductase 1 (FRE1), selected for enhanced activity at alkaline pH, was expressed in rice [76]. Although these attempts did not result in fortifying the crops with iron, in both cases, expression of ferric chelate reductase improved growth under iron-limiting conditions. Moreover, in the transgenic rice, the grain yield was increased nearly eightfold [76]. Although conventional breeding could be another approach for biofortification, it might not be feasible depending on the crop species or the mineral nutrient of interest [72,77]. Therefore, it is necessary to understand the molecular mechanisms involved in plant mineral nutrition because this will provide insights to develop better strategies for biofortification.
Because overexpressing FIT does not affect FRO2 and IRT1 expression, FIT was postulated to function as a heterodimer [4]. Quantitative reverse transcriptase polymerase chain reaction (qRT–PCR) results showed that genes encoding four additional bHLH transcription factors (AtbHLH38, AtbHLH39, AtbHLH100 and AtbHLH101) were induced by iron deficiency in roots and leaves [23]. Because FIT is not expressed in leaves, the partially overlapping expression pattern suggests the four bHLH proteins also have other roles besides interacting with FIT. Biomolecular fluorescence complementation showed that AtbHLH38 and AtbHLH39 interact with FIT [24]. FIT– AtbHLH38 or FIT–AtbHLH39 complexes activated transcription driven by the FRO2 and IRT1 promoters in yeast, suggesting that FRO2 and IRT1 are direct targets of these complexes [24]. AtbHLH38 and AtbHLH39 are seemingly functionally redundant because their single null mutants lack phenotypes [24]. When either AtbHLH38 or AtbHLH39 was overexpressed with FIT, the plants accumulated more iron in their shoots than did wild-type plants [24], consistent with the idea that FIT functions as a heterodimer. The fact that all these genes are themselves iron-regulated indicates there must be an upstream iron sensor. Iron signaling and sensing in plants Hormones have roles in iron-deficiency signaling. Physiological studies showed that ferric chelate reductase activity 281
Review decreased upon ethylene inhibitor treatment but increased upon addition of ethylene precursors [25]. Similarly, treatment with ethylene inhibitors repressed expression of IRT1, FRO2 and FIT, whereas expression of these genes was enhanced when ethylene precursors were added [26,27]. Cytokinins negatively regulated IRT1, FRO2 and FIT expression at the transcript level, independent of the iron status of the plants [28]. This repression required cytokinin receptors but not FIT, and conditions that inhibit root growth, such as osmotic stress induced by mannitol or NaCl and hormonal treatments with auxin or abscissic acid, repressed iron-deficiency response genes [28]. Because cytokinins inhibit root growth [29], the results imply that cytokinin treatment restricted nutrient uptake via a growth-dependent pathway by transiently arresting root elongation to reduce nutrient demand. Another example of the close link between root growth and nutrient uptake is provided by phosphate uptake. Phosphate starvation responses require continuous root growth, whereas inhibiting cell-cycle activity represses phosphate uptake in response to decreased phosphate demand [30]. It will be interesting to test whether this is also the case for iron. Nitric oxide (NO) is proposed to transmit iron-deficiency signals [31–33]. NO reverted the chlorotic phenotypes of maize mutants defective in iron uptake [31] and stimulated accumulation of Arabidopsis ferritin transcripts and protein by acting downstream of iron [32]. In tomato, NO was rapidly produced in roots as an early response to irondeficiency; this helped to facilitate iron uptake, presumably by regulating root hair growth and by enhancing expression of iron-uptake-related genes, because treatment with NO enhanced FER, LeFRO1 and LeIRT1 mRNA levels [33]. NO, plant hormones and other molecules could also act in concert or downstream or upstream of each other. With more evidence accumulating for different molecules involved in iron signaling, a necessary task will be to determine how they integrate into a bigger pathway.
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Iron transport within the plant For proper storage and use of iron, it must be safely translocated to multiple parts of the plant and compartmentalized into specific organelles in the cell. Indeed, it is essential to maintain iron homeostasis both at the intercellular and intracellular levels.
loading citrate into the xylem [36]. frd3 xylem exudate contained less citrate and iron than did exudate from wildtype plants, and frd3 mutant phenotypes were rescued by supplementing with citrate [36], consistent with the role of FRD3 as a citrate transporter. Heterologous studies in Xenopus oocytes confirmed that FRD3 does transport citrate. After citrate loaded into the xylem chelates iron, Fe(III)–citrate complexes are either taken up at different locations via as yet unidentified transporters or the complexes might be reduced by FROs and Fe2+ would then be transported into various cells of the plant. NA is a non-proteogenic amino acid that chelates both Fe2+ and Fe3+, in addition to other divalent metals such as copper, zinc, manganese, cobalt and nickel [34]. NA is synthesized and used in all plants, regardless of their iron uptake strategy, and is a precursor of MA, a PS that is only found in graminaceous plants [15,34]. NA is structurally similar to PSs and chelates iron for intercellular transport in the phloem. A characteristic phenotype of plants lacking NA is interveinal chlorosis in young growing leaves, as seen in the tomato chloronerva (chln) mutant, which is defective in NA synthase [37]. YS-like (YSL) family members are thought to transport metal–NA complexes [15,34]. There are eight YSLs in Arabidopsis, and their proposed functions have been recently reviewed [15]. YSL1 and YSL3 are suggested to be involved in mobilizing metals, including iron, from leaves for use in developing seeds [38]. ysl1 and ysl3 are functionally redundant because the single mutants lack visible phenotypes, whereas ysl1 ysl3 double mutants show severe interveinal chlorosis, lower iron content in roots, leaves and seeds, decreased fertility, arrested pollen and embryo development and defects in mobilizing metals from leaves during senescence [38]. YSLs represent a subfamily of the Arabidopsis oligopeptide transporter (OPT) family, and AtOPT3 was also reported to be involved in supplying iron for seed development [39]. OPT3 is expressed in the vasculature, pollen and developing embryos [40,41]. In opt3-2 mutant plants, where OPT3 expression is reduced, both the yield and iron content of opt3-2 seeds decreased [39]. Although the mutant roots exhibited constitutive irondeficiency responses, their leaves were necrotic and accumulated high levels of iron. These results imply that OPT3 is also involved in regulating iron at the wholeplant level.
Intercellular iron transport To avoid handling toxic, free cellular iron during translocation throughout the plant, chelates such as citrate and nicotianamine (NA) are used [15,34]. Depending on the iron–chelate complex formed, different transport systems are involved in distributing iron throughout the plant. Fe(III)–citrate is the major form of iron present in xylem exudates, and citrate is thought to be involved in longdistance iron transport from roots to shoots [1]. Ferric reductase defective 3 (FRD3), a multidrug and toxin efflux (MATE) family member, is localized to the plasma membrane of cells in the pericycle and vasculature [35] and functions in iron translocation from roots to shoots by
Subcellular iron transport Organelles such as chloroplasts and mitochondria require iron to carry out various metabolic processes and serve as reservoirs to keep iron for later use. This is essential to regulate iron not only at the cellular level but also at the organismal level. For example, defects in organellar iron homeostasis can cause a lethal phenotype, as seen in the Arabidopsis frataxin mutant, which is defective in mitochondrial iron homeostasis [42]. Although our understanding of subcellular iron transport remains limited, studies are gradually providing insights into subcellular trafficking of iron in plants (Figure 1). Vacuoles. Vacuoles are crucial compartments for iron storage and sequestration within plant cells. In particular,
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Figure 1. The molecular components involved in subcellular iron trafficking in plant cells. At the plasma membrane, Fe3+–chelate reductases and transporters of the ZIP and Nramp families are involved in iron uptake [7,45]. The three major compartments that have key roles in iron homeostasis are the chloroplasts, mitochondria and vacuoles. Recent reports suggest that PIC1 imports iron into chloroplasts [50], and an Fe3+–chelate reductase, FRO7, is involved in chloroplast iron acquisition [9]. Although the mitochondrial iron importer remains to be identified, Fe-S clusters are exported from the mitochondria via STA1 [57], and Fe3+–chelate reductases, such as FRO8 [10] and FRO3 (M.L. Guerinot, unpublished data), have been localized to the mitochondria [10]. In the vacuole, iron is taken up by VIT1 [44] and effluxed by Nramp3 and Nramp4 [43]. Dotted lines indicate the porous outer membrane of chloroplasts and mitochondria. The colored circles represent Fe3+–chelate reductases (pink); transporters that import iron into the organelle (green); and transporters that export iron from an organelle (blue).
studies have shown that the vacuole is an essential compartment for iron storage in seeds [43,44]. Vacuolar iron transporter 1 (VIT1) imports iron into the vacuole [44]. Heterologous expression of VIT1 in yeast complemented the high iron-sensitive phenotype of the Ca2+ cross complementer 1 (ccc1) mutant by increasing its vacuolar iron content, indicating that VIT1 transports iron into the vacuole [44]. In Arabidopsis, VIT1 is highly expressed in developing seeds, and vit1 mutants are unable to survive when germinated in alkaline soil, where iron availability is greatly limited [44]. Synchrotron X-ray fluorescence microtomography revealed that iron is concentrated in the provasculature of developing embryos [44]. Most strikingly, such distribution of iron was abolished in vit1-1 mutant seeds, even though the seed iron content was not altered. These results highlight the relevance of proper localization of vacuolar iron stores in the embryo for seedling development. Two metal transporters of the natural resistanceassociated macrophage protein (NRAMP) family, NRAMP3 and NRAMP4, both export iron from vacuoles [43]. NRAMP3 and NRAMP4 genes are induced under iron deficiency and, although the single mutants lack phenotypes owing to presumed functional redundancy, germination of nramp3 nramp4 double mutants was arrested under iron-limiting conditions [43]. Mutant seeds contain wild-type levels of iron; however, electron microscopy showed the disappearance of iron from wildtype vacuoles during germination, whereas vacuoles of
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the nramp3 nramp4 mutant remained unaltered, supporting a role for these transporters in iron mobilization from the vacuole. Taken together with the vit1 phenotypes, these data suggest that vacuoles are an important site of iron storage and that iron remobilization during germination is crucial for seedling development when iron supply is low. Chloroplasts. Iron is required for photosynthetic electron transport, chlorophyll biosynthesis, Fe-S cluster assembly, heme biosynthesis and other essential metabolic processes that occur in chloroplasts [45]. It is also quantitatively significant in plastids, which contain 80% of the iron found in a leaf cell [46]. Because the photosynthetic electron transport chain produces reactive oxygen species (ROS), iron should be tightly regulated in chloroplasts to avoid oxidative damage via the Fenton reaction. In plants, the iron storage protein ferritin, which stores up to 4500 iron atoms, is found in plastids [47]. There are four FER genes in Arabidopsis. FER1 is proposed to be involved in senescence [48]. Age-dependent senescence was accelerated in fer1 loss-of-function mutants owing to iron toxicity under excessive ROS accumulation. A recent study with mutants that lack seed ( fer2) or leaf ferritins ( fer1 fer3 fer4), showed that ferritins are essential for protection against oxidative damage but are not the major iron pool for either seedling development or proper functioning of the photosynthetic apparatus [49]. Recently, a chloroplast ferric chelate reductase, FRO7, was characterized that was required for seedling survival under iron-limiting conditions [9]. Chloroplasts isolated from fro7 mutants had 75% less ferric chelate reductase activity and contained 33% less iron than did wild-type chloroplasts, demonstrating that FRO7 has a role in chloroplast iron acquisition. To our knowledge, no ZIP transporters are localized to chloroplasts. However, a cyanobacterial permease-like protein, permease in chloroplasts 1 (PIC1), was reported as a chloroplast iron transporter [50]. Although PIC1 was also identified as part of the chloroplast inner envelope protein-conducting channel [51], its expression complemented the yeast fet3 fet4 mutant defective in iron uptake; furthermore, pic1 mutant plants show phenotypes consistent with a defect in iron transport, such as severe chlorosis, heterotrophic growth and accumulation of ferritins [50]. Whether or not PIC1 transports Fe2+ or Fe3+ and whether or not a reductase is associated with the process are still unknown. Mitochondria. Mitochondria, similar to chloroplasts, are organelles with a high iron demand [45]. Iron is used as a cofactor in the respiratory electron transport chain and FeS clusters are assembled in mitochondria in addition to chloroplasts. In animals, mitochondrial ferritins have been identified [52,53], and proteomics and electron microscopy suggest that mitochondrial ferritins are also present in Arabidopsis [54]. As in chloroplasts, mitochondria must deal with ROS generated from the electron transport chain and must strictly maintain iron homeostasis. Although plant mitochondrial iron importer(s) have not yet been identified, three orthologs of the yeast (Saccharomyces cerevisiae) ABC transporter of the mitochondrion 1 (ATM1) are found in Arabidopsis [55,56]. ScATM1 exports 283
Review Fe-S clusters from the mitochondrial matrix, and the yeast atm1 mutants exhibit slow growth, respiration defects, lack cytochromes and constitutively accumulate high levels of mitochondrial iron [56]. AtATM1, AtATM2 and AtATM3 are localized to mitochondria. Among the three ATMs in Arabidopsis, AtATM3, also known as STARIK (STA1), is the most similar to ScATM1 and complements the yeast atm1 phenotypes [57]. AtATM3 is thought to export Fe-S clusters in planta, and sta1 mutant plants are dwarf and chlorotic. AtATM1 (STA2) partially suppressed the Arabidopsis sta1 and the yeast atm1 mutant phenotypes [57], whereas the function of AtATM2 remains uncharacterized [55]. FRO8 was detected in a mitochondrial proteomics study [10]. This implies that ferric chelate reductase(s) might also be involved in mitochondrial iron transport, as seen for chloroplast iron transport [9]. Perspectives The recent studies discussed here have considerably expanded our knowledge of iron homeostasis in plants. However, crucial tasks remain, such as identifying the iron sensor. The hypothetical iron sensor must be upstream of FIT, because FIT itself is iron regulated [4]. Mechanisms for sensing cellular iron levels have been characterized in bacteria, animals and yeast. The bacterial ferric uptake regulator (Fur) protein transcriptionally represses promoters of iron-regulated genes in an Fe2+-dependent manner [58]. In yeast, the iron-regulated transcription factor activator of ferrous transport 1 (Aft1) senses Fe-S clusters exported from the mitochondria [59–61]. The iron regulon does not directly respond to cytosolic iron levels, but inactivation of Aft1 under iron-replete conditions requires mitochondrial Fe-S cluster export. Animals post-transcriptionally regulate iron metabolism genes using iron regulatory proteins 1 and 2 (IRP1 and IRP2), aconitase family members that are adapted to sense cytosolic iron levels [62]. In plants, aconitase is not a significant component in iron homeostasis [63], and whether Fe-S clusters are involved or whether a transcription factor itself responds to cytosolic iron concentrations remains to be determined. Because many of the players regulating iron are controlled by iron themselves, their expression profiles from publicly available datasets have not been that informative. However, high resolution expression profiling [5], which reports how gene expression responds to iron-deficiency in each cell layer of the root, will be a significant resource for studying iron homeostasis. This dataset is consistent with the expression patterns based on b-glucoronidase (GUS) assays or mRNA in situ studies previously reported for important genes, including IRT1, FRO2 and FRD3 [22,35,64]. Despite the questions that remain, we are beginning to unravel the mechanisms of iron homeostasis in plants. A thorough understanding of the ‘big picture’ of iron homeostasis will substantially contribute to plant biology, agriculture and human nutrition. Acknowledgements This work was supported by a grant to M.L.G. from the National Science Foundation (IBN 0344305).
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Review 26 Lucena, C. et al. (2006) Ethylene could influence ferric reductase, iron transporter, and H+-ATPase gene expression by affecting FER (or FER-like) gene activity. J. Exp. Bot. 57, 4145–4154 27 Waters, B.M. et al. (2007) Ethylene involvement in the regulation of the H+-ATPase CsHA1 gene and of the new isolated ferric reductase CsFRO1 and iron transporter CsIRT1 genes in cucumber plants. Plant Physiol. Biochem. 45, 293–301 28 Seguela, M. et al. (2008) Cytokinins negatively regulate the root iron uptake machinery in Arabidopsis through a growth-dependent pathway. Plant J. 55, 289–300 29 Dello Ioio, R. et al. (2007) Cytokinins determine Arabidopsis rootmeristem size by controlling cell differentiation. Curr. Biol. 17, 678–682 30 Lai, F. et al. (2007) Cell division activity determines the magnitude of phosphate starvation responses in Arabidopsis. Plant J. 50, 545–556 31 Graziano, M. and Lamattina, L. (2005) Nitric oxide and iron in plants: an emerging and converging story. Trends Plant Sci. 10, 4–8 32 Arnaud, N. et al. (2006) An iron-induced nitric oxide burst precedes ubiquitin-dependent protein degradation for Arabidopsis AtFer1 ferritin gene expression. J. Biol. Chem. 281, 23579–23588 33 Graziano, M. and Lamattina, L. (2007) Nitric oxide accumulation is required for molecular and physiological responses to iron deficiency in tomato roots. Plant J. 52, 949–960 34 Haydon, M.J. and Cobbett, C.S. (2007) Transporters of ligands for essential metal ions in plants. New Phytol. 174, 499–506 35 Green, L.S. and Rogers, E.E. (2004) FRD3 controls iron localization in Arabidopsis thaliana. Plant Physiol. 136, 2523–2531 36 Durrett, T.P. et al. (2007) The FRD3-mediated efflux of citrate into the root vasculature is necessary for efficient iron translocation. Plant Physiol. 144, 197–205 37 Ling, H.Q. et al. (1999) Map-based cloning of chloronerva, a gene involved in iron uptake of higher plants encoding nicotianamine synthase. Proc. Natl. Acad. Sci. U. S. A. 96, 7098–7103 38 Waters, B.M. et al. (2006) Mutations in Arabidopsis Yellow StripeLike1 and Yellow Stripe-Like3 reveal their roles in metal ion homeostasis and loading of metal ions in seeds. Plant Physiol. 141, 1446–1458 39 Stacey, M.G. et al. (2008) The Arabidopsis AtOPT3 protein functions in metal homeostasis and movement of iron to developing seeds. Plant Physiol. 146, 589–601 40 Stacey, M.G. et al. (2002) AtOPT3, a member of the oligopeptide transporter family, is essential for embryo development in Arabidopsis. Plant Cell 14, 2799–2811 41 Stacey, M.G. et al. (2006) Expression analyses of Arabidopsis oligopeptide transporters during seed germination, vegetative growth and reproduction. Planta 223, 291–305 42 Vazzola, V. et al. (2007) Knockout of frataxin gene causes embryo lethality in Arabidopsis. FEBS Lett. 581, 667–672 43 Lanquar, V. et al. (2005) Mobilization of vacuolar iron by AtNRAMP3 and AtNRAMP4 is essential for seed germination on low iron. EMBO J. 24, 4041–4051 44 Kim, S.A. et al. (2006) Localization of iron in Arabidopsis seed requires the vacuolar membrane transporter VIT1. Science 314, 1295–1298 45 Briat, J.F. et al. (2007) Iron utilization and metabolism in plants. Curr. Opin. Plant Biol. 10, 276–282 46 Shikanai, T. et al. (2003) PAA1, a P-type ATPase of Arabidopsis, functions in copper transport in chloroplasts. Plant Cell 15, 1333–1346 47 Hintze, K.J. and Theil, E.C. (2006) Cellular regulation and molecular interactions of the ferritins. Cell. Mol. Life Sci. 63, 591–600 48 Murgia, I. et al. (2007) Knock-out of ferritin AtFer1 causes earlier onset of age-dependent leaf senescence in Arabidopsis. Plant Physiol. Biochem. 45, 898–907 49 Ravet, K. et al. (2008) Ferritins control interaction between iron homeostasis and oxidative stress in Arabidopsis. Plant J. 57, 400–412 50 Duy, D. et al. (2007) PIC1, an ancient permease in Arabidopsis chloroplasts, mediates iron transport. Plant Cell 19, 986–1006 51 Teng, Y.S. et al. (2006) Tic21 is an essential translocon component for protein translocation across the chloroplast inner envelope membrane. Plant Cell 18, 2247–2257 52 Levi, S. and Arosio, P. (2004) Mitochondrial ferritin. Int. J. Biochem. Cell Biol. 36, 1887
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53 Missirlis, F. et al. (2006) Characterization of mitochondrial ferritin in Drosophila. Proc. Natl. Acad. Sci. U. S. A. 103, 5893–5898 54 Zancani, M. et al. (2004) Evidence for the presence of ferritin in plant mitochondria. Eur. J. Biochem. 271, 3657–3664 55 Chen, S. et al. (2007) Functional characterization of AtATM1, AtATM2 and AtATM3, a subfamily of Arabidopsis half-molecule ABC transporters implicated in iron homeostasis. J. Biol. Chem. 282, 21561–21571 56 Rouault, T.A. and Tong, W-H. (2005) Iron-sulphur cluster biogenesis and mitochondrial iron homeostasis. Nat. Rev. Mol. Cell Biol. 6, 345 57 Kushnir, S. et al. (2001) A mutation of the mitochondrial ABC transporter Sta1 leads to dwarfism and chlorosis in the Arabidopsis mutant starik. Plant Cell 13, 89–100 58 Lee, J.W. and Helmann, J.D. (2007) Functional specialization within the Fur family of metalloregulators. Biometals 20, 485–499 59 Chen, O.S. et al. (2004) Transcription of the yeast iron regulon does not respond directly to iron but rather to iron-sulfur cluster biosynthesis. J. Biol. Chem. 279, 29513–29518 60 Kumanovics, A. et al. (2008) Identification of FRA1 and FRA2 as genes involved in regulating the yeast iron regulon in response to decreased mitochondrial iron-sulfur cluster synthesis. J. Biol. Chem. 283, 10276– 10286 61 Rutherford, J.C. et al. (2005) Activation of the iron regulon by yeast Aft1/Aft2 transcription factors depends on mitochondrial but not cytosolic iron-sulfur protein biogenesis. J. Biol. Chem. 280, 10135– 10140 62 Rouault, T.A. (2006) The role of iron regulatory proteins in mammalian iron homeostasis and disease. Nat. Chem. Biol. 2, 406–414 63 Arnaud, N. et al. (2007) The iron responsive element (IRE)/iron regulatory protein1 (IRP1)-cytosolic aconitase iron-regulatory switch does not operate in plants. Biochem. J. 405, 523–531 64 Connolly, E.L. et al. (2003) Overexpression of the FRO2 iron reductase confers tolerance to growth on low iron and uncovers posttranscriptional control. Plant Physiol. 133, 1102–1110 65 Marschner, H. (1995) Mineral Nutrition of Higher Plants. Academic Press 66 Puig, S. et al. (2007) Copper and iron homeostasis in Arabidopsis: responses to metal deficiencies, interactions and biotechnological applications. Plant Cell Environ. 30, 271–290 67 Misson, J. et al. (2005) A genome-wide transcriptional analysis using Arabidopsis thaliana Affymetrix gene chips determined plant responses to phosphate deprivation. Proc. Natl. Acad. Sci. U. S. A. 102, 11934–11939 68 Ward, J.T. et al. (2008) The effect of iron on the primary root elongation of Arabidopsis during phosphate deficiency. Plant Physiol. 147, 1181– 1191 69 Salt, D.E. et al. (2008) Ionomics and the study of the plant ionome. Annu. Rev. Plant Biol. 59, 709–733 70 Baxter, I. et al. (2007) Purdue Ionomics Information Management System. An integrated functional genomics platform. Plant Physiol. 143, 600–611 71 Baxter, I.R. et al. (2008) The leaf ionome as a multivariable system to detect a plant’s physiological status. Proc. Natl. Acad. Sci. U. S. A. 105, 12081–12086 72 Zhu, C. et al. (2007) Transgenic strategies for the nutritional enhancement of plants. Trends Plant Sci. 12, 548–555 73 Goto, F. et al. (1999) Iron fortification of rice seed by the soybean ferritin gene. Nat. Biotechnol. 17, 282–286 74 Drakakaki, G. et al. (2005) Endosperm-specific co-expression of recombinant soybean ferritin and Aspergillus phytase in maize results in significant increases in the levels of bioavailable iron. Plant Mol. Biol. 59, 869–880 75 Vasconcelos, M. et al. (2006) Molecular and phenotypic characterization of transgenic soybean expressing the Arabidopsis ferric chelate reductase gene, FRO2. Planta 224, 1116–1128 76 Ishimaru, Y. et al. (2007) Mutational reconstructed ferric chelate reductase confers enhanced tolerance in rice to iron deficiency in calcareous soil. Proc. Natl. Acad. Sci. U. S. A. 104, 7373–7378 77 White, P.J. and Broadley, M.R. (2005) Biofortifying crops with essential mineral elements. Trends Plant Sci. 10, 586–593
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Homing in on iron homeostasis in plants Jeeyon Jeong and Mary Lou Guerinot Department of Biological Sciences, Dartmouth College, Hanover, NH 03755, USA
Iron is essential for plants but is not readily accessible and is also potentially toxic. As plants are a major dietary source of iron worldwide, understanding plant iron homeostasis is pivotal for improving not only crop yields but also human nutrition. Although iron acquisition from the environment is well characterized, the transporters and reductases involved in plant organellar iron transport and some of the transcription factors that regulate iron uptake have only recently been discovered. Here, we discuss newly characterized molecular players, focusing on Arabidopsis. Localization of iron to the right compartment and accessibility of iron stores are proving crucial for maintaining proper iron homeostasis and will need to be considered in biofortification efforts currently underway. The significance of iron homeostasis in plants For most organisms, iron serves as a cofactor in vital metabolic pathways such as the electron transport chain of respiration. Plants have an additional need for iron because photosynthesis and chlorophyll biosynthesis both require iron. However, excess iron generates cytotoxic hydroxyl radicals via the Fenton reaction, and iron is not readily bioavailable because it forms insoluble complexes under aerobic conditions at neutral or alkaline pH [1]. Therefore, how plants maintain iron homeostasis is a biologically relevant question. Here, we outline recent progress made in our understanding of how the uptake of iron is regulated and then transported within plants. We also discuss the use of ionomics (Box 1) as a novel approach to studying iron homeostasis and highlight recent attempts to develop iron-fortified crops (Box 2). Iron transport from soil to roots Plants obtain iron from the environment using mechanisms based on reduction or chelation. Here, we discuss iron acquisition strategies, focusing on recent studies with Arabidopsis thaliana. The reduction strategy Non-graminaceous plants use the Reduction Strategy, or Strategy I, to obtain iron. Upon iron deficiency, protons are released to increase the solubility of iron, via H+-ATPases of the root plasma membrane [2,3]. Several Arabidopsis H+-ATPase (AHA) family members are induced in irondeficient roots [4,5], suggesting possible roles for these enzymes in iron-deficiency responses. Corresponding author: Guerinot, M.L. ([email protected]).
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After acidification, Fe3+ is reduced to Fe2+ by a membrane-bound ferric reductase oxidase (FRO), FRO2 [6], one of eight members of the FRO family. FRO genes are expressed differentially at the tissue level; for example, FRO2 is specific to roots, whereas FRO6 and FRO7 are specific to shoots [7,8]. FRO proteins are also predicted and/or experimentally shown to localize to different subcellular compartments [7,9,10]. Therefore, each FRO family member has a specific role in different organs or subcellular compartments, signifying that reduction-based iron transport is not limited to the root plasma membrane. Once Fe3+ is reduced, Fe2+ is transported into the root by iron-regulated transporter 1 (IRT1), a member of the zincregulated transporter (ZRT)-, IRT-like protein (ZIP) family [11]. Analysis of plants overexpressing IRT1 from the cauliflower mosaic virus 35S promoter shows that IRT1 is present only in iron-deficient roots, suggesting that it is controlled post-transcriptionally [12]. A recent follow-up study showed that iron-induced turnover of IRT1 requires two lysine residues located in the intracellular loop of IRT1 between transmembrane domains III and IV [13]. This is consistent with the turnover of ZRT1, a yeast zinc transporter that also belongs to the ZIP family [14]. In its variable loop region, ZRT1 has a lysine residue that is ubiquitinated to target it for protein degradation under zinc-sufficient conditions. When either IRT1 lysine residue was substituted with arginine and the variant was overexpressed, the plants accumulated higher levels of iron than did wild-type plants; by contrast, plants overexpressing wild-type IRT1 contained iron levels similar to those of wild-type plants [13]. However, the accumulation of iron was not associated with increased ferric chelate reductase activity [13], implying that this activity is not rate limiting, at least under the conditions tested. The chelation strategy To acquire iron, grasses use a mechanism based on chelation, known as Strategy II, in which phytosiderophores (PSs), such as mugineic acids (MAs), are released to chelate Fe3+. The resulting Fe(III)–PS complexes are then transported into the roots via transporters belonging to the Yellow Stripe (YS) family, named for the YS1 PS transporter of maize (Zea mays) [15]. Grasses can also take up Fe2+ in addition to Fe(III)–PS [16,17]. Rice (Oryza sativa) plants that cannot synthesize PS, owing to a mutation in the NICOTIANAMINE AMINOTRANSFERASE (NAAT) gene, do not show growth defects if Fe2+ is supplied [16]. However, unlike Strategy I plants, neither H+-ATPase nor Fe3+–chelate reductase activity is induced
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Review Box 1. Interaction of iron with other mineral elements and ionomics Changes in iron homeostasis influence other elements and vice versa. It has long been proposed that multiple mineral nutrients have roles in plants facing deficiency or toxicity caused by a specific nutrient [65]. Iron can be substituted for copper, under copper deficiency, as seen in the examples of cytochrome oxidase and diiron oxidase or Cu- or Zn-superoxide dismutase (SOD) and Fe-SOD [66]. Non-metal minerals, such as phosphate, also interact with iron. Iron homeostasis genes were affected in phosphate-deficient plants; expression of FER1 was increased, whereas IRT1 expression was decreased [67]. It seems that phosphate deficiency makes iron more available to plants, and inhibition of the primary root elongation, a typical symptom of phosphate deficiency, can be alleviated by reducing the amount of iron in the medium [68]. The issue of interactions among minerals can be tackled by applying the concept of the ‘ionome’, which refers to the mineral nutrients and trace elements, both essential and non-essential, found in an organism [69]. Advances in inductively coupled plasma (ICP) mass spectroscopy (MS) techniques, along with the development of bioinformatics and large-scale genetic tools, provides a potentially powerful strategy to understand genes involved in maintaining the ionome and how they affect physiological and developmental processes in plants [69]. This approach, in turn, will facilitate efforts to understand the roles of individual trace elements, including iron. The Purdue Ionomics Information Management System (PiiMS) provides an open-access web-based tool for large datasets generated by a high-throughput ICP-MS phenotyping platform [70]. Concentrations of 16 elements (phosphate, calcium, potassium, magnesium, copper, iron, zinc, manganese, cobalt, nickel, boron, selenium, molybdenum, sodium, arsenic and cadmium) in >60 000 samples of Arabidopsis shoots from >800 experiments can be found in PiiMS (http://www.ionomicshub.org/home/PiiMS). Using the ionomics approach, it was shown that the Arabidopsis leaf ionome contains signatures that reflect the physiological status and elemental composition of the plant, and statistical models were established to detect changes in iron homeostasis [71]. The study reveals that iron content itself is not a good indicator of the iron status of the plant because shoot iron concentration is tightly regulated. However, even under sustained shoot iron levels, increases in manganese, cobalt, zinc and cadmium concentrations and a decrease in molybdenum concentration are observed, and these changes are good predictors of the iron status of a plant [71]. When the authors screened previously collected ionomics data, known iron mutants such as frd1 and frd3 were identified, validating their model.
under iron deficiency. This probably reflects an adaptation to flooded rice paddies, where Fe2+ is more abundant than Fe3+ owing to reduced levels of oxygen [17]. Regulation of iron-deficiency responses in Strategy II plants was recently reviewed [18] so will not be covered further here. Regulation of the reduction strategy In Arabidopsis, Fe-regulated (Fer)-like iron-deficiencyinduced transcription factor (FIT), a basic helix–loop–helix (bHLH) transcription factor orthologous to the tomato (Solanum lycopersicum) FER protein [19], is required to regulate iron-deficiency responses [4,20,21]. FIT is highly induced in the epidermis of iron-deficient roots, and fit mutants are chlorotic and seedling lethal unless watered with supplemental iron [4], showing phenotypes similar to those of irt1 plants [22]. FRO2 is transcriptionally regulated by FIT, and FRO2 transcripts and root ferric chelate reductase activity are both absent in fit mutants [4,20]. Meanwhile, IRT1 is post-transcriptionally regulated by FIT [4]. fit mutants do not accumulate IRT1 protein, despite the induction of IRT1 mRNA by iron deficiency.
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Box 2. Resolving ‘hidden hunger’ with iron-fortified crops Understanding iron homeostasis in plants is crucial not only for improving plant growth and crop yields but also for improving human nutrition. According to the World Health Organization, iron deficiency is the most prevalent nutritional disorder in the world today, affecting an estimated 2.7 billion people in both developed and developing countries (http://www.who.int/nutrition/topics/ida/en/index.html). Along with deficiencies of vitamin A and iodine, iron deficiency is often referred to as ‘hidden hunger’. Because plants are the primary source of dietary iron worldwide, this calls for the need to enrich crops with bioavailable iron to help solve the issue of iron malnutrition. Biofortification refers to the strategy of enhancing the nutritional value of crops to solve malnutrition worldwide sustainably [72]. Because mineral nutrients, such as iron, are acquired from the environment, iron-fortification strategies should be based on increasing iron transport to, or storage in, the edible portions of plants. There have been multiple promising reports of iron-fortified crops. For example, iron-fortified rice was developed by overexpressing the iron storage protein ferritin [73]. It was also shown that seeds of transgenic maize expressing ferritin, in combination with phytase, which digests phytic acid that inhibits iron absorption, contained more iron [74]. In this study, it was also found that the expression level of phytase correlated with increased cellular iron absorption, as detected by in vitro digestion assays with Caco-2, a human intestinal cell line. Ferric chelate reductases have also been expressed in crop plants. FRO2 was expressed in soybean (Glycine max) [75], and a modified version of yeast ferric reductase 1 (FRE1), selected for enhanced activity at alkaline pH, was expressed in rice [76]. Although these attempts did not result in fortifying the crops with iron, in both cases, expression of ferric chelate reductase improved growth under iron-limiting conditions. Moreover, in the transgenic rice, the grain yield was increased nearly eightfold [76]. Although conventional breeding could be another approach for biofortification, it might not be feasible depending on the crop species or the mineral nutrient of interest [72,77]. Therefore, it is necessary to understand the molecular mechanisms involved in plant mineral nutrition because this will provide insights to develop better strategies for biofortification.
Because overexpressing FIT does not affect FRO2 and IRT1 expression, FIT was postulated to function as a heterodimer [4]. Quantitative reverse transcriptase polymerase chain reaction (qRT–PCR) results showed that genes encoding four additional bHLH transcription factors (AtbHLH38, AtbHLH39, AtbHLH100 and AtbHLH101) were induced by iron deficiency in roots and leaves [23]. Because FIT is not expressed in leaves, the partially overlapping expression pattern suggests the four bHLH proteins also have other roles besides interacting with FIT. Biomolecular fluorescence complementation showed that AtbHLH38 and AtbHLH39 interact with FIT [24]. FIT– AtbHLH38 or FIT–AtbHLH39 complexes activated transcription driven by the FRO2 and IRT1 promoters in yeast, suggesting that FRO2 and IRT1 are direct targets of these complexes [24]. AtbHLH38 and AtbHLH39 are seemingly functionally redundant because their single null mutants lack phenotypes [24]. When either AtbHLH38 or AtbHLH39 was overexpressed with FIT, the plants accumulated more iron in their shoots than did wild-type plants [24], consistent with the idea that FIT functions as a heterodimer. The fact that all these genes are themselves iron-regulated indicates there must be an upstream iron sensor. Iron signaling and sensing in plants Hormones have roles in iron-deficiency signaling. Physiological studies showed that ferric chelate reductase activity 281
Review decreased upon ethylene inhibitor treatment but increased upon addition of ethylene precursors [25]. Similarly, treatment with ethylene inhibitors repressed expression of IRT1, FRO2 and FIT, whereas expression of these genes was enhanced when ethylene precursors were added [26,27]. Cytokinins negatively regulated IRT1, FRO2 and FIT expression at the transcript level, independent of the iron status of the plants [28]. This repression required cytokinin receptors but not FIT, and conditions that inhibit root growth, such as osmotic stress induced by mannitol or NaCl and hormonal treatments with auxin or abscissic acid, repressed iron-deficiency response genes [28]. Because cytokinins inhibit root growth [29], the results imply that cytokinin treatment restricted nutrient uptake via a growth-dependent pathway by transiently arresting root elongation to reduce nutrient demand. Another example of the close link between root growth and nutrient uptake is provided by phosphate uptake. Phosphate starvation responses require continuous root growth, whereas inhibiting cell-cycle activity represses phosphate uptake in response to decreased phosphate demand [30]. It will be interesting to test whether this is also the case for iron. Nitric oxide (NO) is proposed to transmit iron-deficiency signals [31–33]. NO reverted the chlorotic phenotypes of maize mutants defective in iron uptake [31] and stimulated accumulation of Arabidopsis ferritin transcripts and protein by acting downstream of iron [32]. In tomato, NO was rapidly produced in roots as an early response to irondeficiency; this helped to facilitate iron uptake, presumably by regulating root hair growth and by enhancing expression of iron-uptake-related genes, because treatment with NO enhanced FER, LeFRO1 and LeIRT1 mRNA levels [33]. NO, plant hormones and other molecules could also act in concert or downstream or upstream of each other. With more evidence accumulating for different molecules involved in iron signaling, a necessary task will be to determine how they integrate into a bigger pathway.
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Iron transport within the plant For proper storage and use of iron, it must be safely translocated to multiple parts of the plant and compartmentalized into specific organelles in the cell. Indeed, it is essential to maintain iron homeostasis both at the intercellular and intracellular levels.
loading citrate into the xylem [36]. frd3 xylem exudate contained less citrate and iron than did exudate from wildtype plants, and frd3 mutant phenotypes were rescued by supplementing with citrate [36], consistent with the role of FRD3 as a citrate transporter. Heterologous studies in Xenopus oocytes confirmed that FRD3 does transport citrate. After citrate loaded into the xylem chelates iron, Fe(III)–citrate complexes are either taken up at different locations via as yet unidentified transporters or the complexes might be reduced by FROs and Fe2+ would then be transported into various cells of the plant. NA is a non-proteogenic amino acid that chelates both Fe2+ and Fe3+, in addition to other divalent metals such as copper, zinc, manganese, cobalt and nickel [34]. NA is synthesized and used in all plants, regardless of their iron uptake strategy, and is a precursor of MA, a PS that is only found in graminaceous plants [15,34]. NA is structurally similar to PSs and chelates iron for intercellular transport in the phloem. A characteristic phenotype of plants lacking NA is interveinal chlorosis in young growing leaves, as seen in the tomato chloronerva (chln) mutant, which is defective in NA synthase [37]. YS-like (YSL) family members are thought to transport metal–NA complexes [15,34]. There are eight YSLs in Arabidopsis, and their proposed functions have been recently reviewed [15]. YSL1 and YSL3 are suggested to be involved in mobilizing metals, including iron, from leaves for use in developing seeds [38]. ysl1 and ysl3 are functionally redundant because the single mutants lack visible phenotypes, whereas ysl1 ysl3 double mutants show severe interveinal chlorosis, lower iron content in roots, leaves and seeds, decreased fertility, arrested pollen and embryo development and defects in mobilizing metals from leaves during senescence [38]. YSLs represent a subfamily of the Arabidopsis oligopeptide transporter (OPT) family, and AtOPT3 was also reported to be involved in supplying iron for seed development [39]. OPT3 is expressed in the vasculature, pollen and developing embryos [40,41]. In opt3-2 mutant plants, where OPT3 expression is reduced, both the yield and iron content of opt3-2 seeds decreased [39]. Although the mutant roots exhibited constitutive irondeficiency responses, their leaves were necrotic and accumulated high levels of iron. These results imply that OPT3 is also involved in regulating iron at the wholeplant level.
Intercellular iron transport To avoid handling toxic, free cellular iron during translocation throughout the plant, chelates such as citrate and nicotianamine (NA) are used [15,34]. Depending on the iron–chelate complex formed, different transport systems are involved in distributing iron throughout the plant. Fe(III)–citrate is the major form of iron present in xylem exudates, and citrate is thought to be involved in longdistance iron transport from roots to shoots [1]. Ferric reductase defective 3 (FRD3), a multidrug and toxin efflux (MATE) family member, is localized to the plasma membrane of cells in the pericycle and vasculature [35] and functions in iron translocation from roots to shoots by
Subcellular iron transport Organelles such as chloroplasts and mitochondria require iron to carry out various metabolic processes and serve as reservoirs to keep iron for later use. This is essential to regulate iron not only at the cellular level but also at the organismal level. For example, defects in organellar iron homeostasis can cause a lethal phenotype, as seen in the Arabidopsis frataxin mutant, which is defective in mitochondrial iron homeostasis [42]. Although our understanding of subcellular iron transport remains limited, studies are gradually providing insights into subcellular trafficking of iron in plants (Figure 1). Vacuoles. Vacuoles are crucial compartments for iron storage and sequestration within plant cells. In particular,
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Figure 1. The molecular components involved in subcellular iron trafficking in plant cells. At the plasma membrane, Fe3+–chelate reductases and transporters of the ZIP and Nramp families are involved in iron uptake [7,45]. The three major compartments that have key roles in iron homeostasis are the chloroplasts, mitochondria and vacuoles. Recent reports suggest that PIC1 imports iron into chloroplasts [50], and an Fe3+–chelate reductase, FRO7, is involved in chloroplast iron acquisition [9]. Although the mitochondrial iron importer remains to be identified, Fe-S clusters are exported from the mitochondria via STA1 [57], and Fe3+–chelate reductases, such as FRO8 [10] and FRO3 (M.L. Guerinot, unpublished data), have been localized to the mitochondria [10]. In the vacuole, iron is taken up by VIT1 [44] and effluxed by Nramp3 and Nramp4 [43]. Dotted lines indicate the porous outer membrane of chloroplasts and mitochondria. The colored circles represent Fe3+–chelate reductases (pink); transporters that import iron into the organelle (green); and transporters that export iron from an organelle (blue).
studies have shown that the vacuole is an essential compartment for iron storage in seeds [43,44]. Vacuolar iron transporter 1 (VIT1) imports iron into the vacuole [44]. Heterologous expression of VIT1 in yeast complemented the high iron-sensitive phenotype of the Ca2+ cross complementer 1 (ccc1) mutant by increasing its vacuolar iron content, indicating that VIT1 transports iron into the vacuole [44]. In Arabidopsis, VIT1 is highly expressed in developing seeds, and vit1 mutants are unable to survive when germinated in alkaline soil, where iron availability is greatly limited [44]. Synchrotron X-ray fluorescence microtomography revealed that iron is concentrated in the provasculature of developing embryos [44]. Most strikingly, such distribution of iron was abolished in vit1-1 mutant seeds, even though the seed iron content was not altered. These results highlight the relevance of proper localization of vacuolar iron stores in the embryo for seedling development. Two metal transporters of the natural resistanceassociated macrophage protein (NRAMP) family, NRAMP3 and NRAMP4, both export iron from vacuoles [43]. NRAMP3 and NRAMP4 genes are induced under iron deficiency and, although the single mutants lack phenotypes owing to presumed functional redundancy, germination of nramp3 nramp4 double mutants was arrested under iron-limiting conditions [43]. Mutant seeds contain wild-type levels of iron; however, electron microscopy showed the disappearance of iron from wildtype vacuoles during germination, whereas vacuoles of
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the nramp3 nramp4 mutant remained unaltered, supporting a role for these transporters in iron mobilization from the vacuole. Taken together with the vit1 phenotypes, these data suggest that vacuoles are an important site of iron storage and that iron remobilization during germination is crucial for seedling development when iron supply is low. Chloroplasts. Iron is required for photosynthetic electron transport, chlorophyll biosynthesis, Fe-S cluster assembly, heme biosynthesis and other essential metabolic processes that occur in chloroplasts [45]. It is also quantitatively significant in plastids, which contain 80% of the iron found in a leaf cell [46]. Because the photosynthetic electron transport chain produces reactive oxygen species (ROS), iron should be tightly regulated in chloroplasts to avoid oxidative damage via the Fenton reaction. In plants, the iron storage protein ferritin, which stores up to 4500 iron atoms, is found in plastids [47]. There are four FER genes in Arabidopsis. FER1 is proposed to be involved in senescence [48]. Age-dependent senescence was accelerated in fer1 loss-of-function mutants owing to iron toxicity under excessive ROS accumulation. A recent study with mutants that lack seed ( fer2) or leaf ferritins ( fer1 fer3 fer4), showed that ferritins are essential for protection against oxidative damage but are not the major iron pool for either seedling development or proper functioning of the photosynthetic apparatus [49]. Recently, a chloroplast ferric chelate reductase, FRO7, was characterized that was required for seedling survival under iron-limiting conditions [9]. Chloroplasts isolated from fro7 mutants had 75% less ferric chelate reductase activity and contained 33% less iron than did wild-type chloroplasts, demonstrating that FRO7 has a role in chloroplast iron acquisition. To our knowledge, no ZIP transporters are localized to chloroplasts. However, a cyanobacterial permease-like protein, permease in chloroplasts 1 (PIC1), was reported as a chloroplast iron transporter [50]. Although PIC1 was also identified as part of the chloroplast inner envelope protein-conducting channel [51], its expression complemented the yeast fet3 fet4 mutant defective in iron uptake; furthermore, pic1 mutant plants show phenotypes consistent with a defect in iron transport, such as severe chlorosis, heterotrophic growth and accumulation of ferritins [50]. Whether or not PIC1 transports Fe2+ or Fe3+ and whether or not a reductase is associated with the process are still unknown. Mitochondria. Mitochondria, similar to chloroplasts, are organelles with a high iron demand [45]. Iron is used as a cofactor in the respiratory electron transport chain and FeS clusters are assembled in mitochondria in addition to chloroplasts. In animals, mitochondrial ferritins have been identified [52,53], and proteomics and electron microscopy suggest that mitochondrial ferritins are also present in Arabidopsis [54]. As in chloroplasts, mitochondria must deal with ROS generated from the electron transport chain and must strictly maintain iron homeostasis. Although plant mitochondrial iron importer(s) have not yet been identified, three orthologs of the yeast (Saccharomyces cerevisiae) ABC transporter of the mitochondrion 1 (ATM1) are found in Arabidopsis [55,56]. ScATM1 exports 283
Review Fe-S clusters from the mitochondrial matrix, and the yeast atm1 mutants exhibit slow growth, respiration defects, lack cytochromes and constitutively accumulate high levels of mitochondrial iron [56]. AtATM1, AtATM2 and AtATM3 are localized to mitochondria. Among the three ATMs in Arabidopsis, AtATM3, also known as STARIK (STA1), is the most similar to ScATM1 and complements the yeast atm1 phenotypes [57]. AtATM3 is thought to export Fe-S clusters in planta, and sta1 mutant plants are dwarf and chlorotic. AtATM1 (STA2) partially suppressed the Arabidopsis sta1 and the yeast atm1 mutant phenotypes [57], whereas the function of AtATM2 remains uncharacterized [55]. FRO8 was detected in a mitochondrial proteomics study [10]. This implies that ferric chelate reductase(s) might also be involved in mitochondrial iron transport, as seen for chloroplast iron transport [9]. Perspectives The recent studies discussed here have considerably expanded our knowledge of iron homeostasis in plants. However, crucial tasks remain, such as identifying the iron sensor. The hypothetical iron sensor must be upstream of FIT, because FIT itself is iron regulated [4]. Mechanisms for sensing cellular iron levels have been characterized in bacteria, animals and yeast. The bacterial ferric uptake regulator (Fur) protein transcriptionally represses promoters of iron-regulated genes in an Fe2+-dependent manner [58]. In yeast, the iron-regulated transcription factor activator of ferrous transport 1 (Aft1) senses Fe-S clusters exported from the mitochondria [59–61]. The iron regulon does not directly respond to cytosolic iron levels, but inactivation of Aft1 under iron-replete conditions requires mitochondrial Fe-S cluster export. Animals post-transcriptionally regulate iron metabolism genes using iron regulatory proteins 1 and 2 (IRP1 and IRP2), aconitase family members that are adapted to sense cytosolic iron levels [62]. In plants, aconitase is not a significant component in iron homeostasis [63], and whether Fe-S clusters are involved or whether a transcription factor itself responds to cytosolic iron concentrations remains to be determined. Because many of the players regulating iron are controlled by iron themselves, their expression profiles from publicly available datasets have not been that informative. However, high resolution expression profiling [5], which reports how gene expression responds to iron-deficiency in each cell layer of the root, will be a significant resource for studying iron homeostasis. This dataset is consistent with the expression patterns based on b-glucoronidase (GUS) assays or mRNA in situ studies previously reported for important genes, including IRT1, FRO2 and FRD3 [22,35,64]. Despite the questions that remain, we are beginning to unravel the mechanisms of iron homeostasis in plants. A thorough understanding of the ‘big picture’ of iron homeostasis will substantially contribute to plant biology, agriculture and human nutrition. Acknowledgements This work was supported by a grant to M.L.G. from the National Science Foundation (IBN 0344305).
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