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

Authors

  • SERGI PUIG,

    1. Departament de Bioquímica i Biologia Molecular. Universitat de València. Av. Doctor Moliner, 50 E-46100 Burjassot, Valencia, Spain
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  • NURIA ANDRÉS-COLÁS,

    1. Departament de Bioquímica i Biologia Molecular. Universitat de València. Av. Doctor Moliner, 50 E-46100 Burjassot, Valencia, Spain
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  • ANTONI GARCÍA-MOLINA,

    1. Departament de Bioquímica i Biologia Molecular. Universitat de València. Av. Doctor Moliner, 50 E-46100 Burjassot, Valencia, Spain
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  • LOLA PEÑARRUBIA

    1. Departament de Bioquímica i Biologia Molecular. Universitat de València. Av. Doctor Moliner, 50 E-46100 Burjassot, Valencia, Spain
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Lola Peñarrubia. Fax: 34 963544635; e-mail: penarrub@uv.es

ABSTRACT

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.

INTRODUCTION

Copper (Cu) and iron (Fe) are essential micronutrients for virtually all living organisms. Both metals exhibit redox properties that allow them to participate as catalytic cofactors in multiple metabolic pathways. In higher plants, both metals play key roles in photosynthetic and respiratory electron-transport chains at chloroplasts and mitochondria, respectively. Cu is also involved in crucial processes including ethylene perception, cell wall metabolism and oxidative stress protection. More recently, a role for Cu in molybdenum cofactor biosynthesis has been reported (Kuper et al. 2004). Fe is mainly required for photosynthesis, respiration, sulphate assimilation, hormone synthesis, nitrogen fixation, as well as DNA synthesis and repair. The essentiality of Cu and Fe is evidenced by the symptoms that their deficiencies provoke in plants. Cu deficiency induces plant chlorosis, mostly affecting young leaves and reproductive organs. Fe deficiency induces severe chlorosis, and affects both the yield and the nutritional value of crops (Märschner 2002).

Fe deficiency is one of the most widespread nutrient imbalances in agriculture. Despite its abundance, Fe bioavailability is very low because of the extreme insolubility of Fe3+ in alkaline calcareous soils, which represent one third of world's cultivated lands. The decrease in Fe bioavailability is a consequence of the appearance of oxygen in the atmosphere, whereas concomitantly, Cu becomes available as soluble Cu2+ (Crichton & Pierre 2001). The best-adapted organisms develop new strategies to solubilize and acquire Fe3+, but they also incorporate Cu in multiple processes requiring higher redox potentials. Cu proteins are, therefore, a more recent biochemical achievement that coincides with the appearance of multicellular organisms. Depending on metal bioavailability, multiple organisms coordinately regulate the alternative use of Cu- versus Fe-containing enzymes to catalyse the same biochemical reaction with completely different apoproteins. Key examples in plants include cytochrome oxidase versus diiron oxidase, Cu versus haem nitrite reductases and Cu/Zinc-superoxide dismutase (Cu/ZnSOD) versus FeSOD.

The first evidence of a connection between Cu and Fe homeostasis is based on nutritional studies with rats, which show that Cu fortification can overcome anaemia (Hart et al. 1928, reviewed by Fox 2003). Studies with Saccharomyces cerevisiae have demonstrated that this connection relies on the multicopper ferroxidase Fet3, responsible for yeast high-affinity Fe uptake (Askwith et al. 1994; Dancis et al. 1994b). In mammals, Fet3 homologs include ceruloplasmin and hephaestin, which are essential for Fe distribution and acquisition, respectively (Harris et al. 1999;Vulpe et al. 1999). The photosynthetic algae Chlamydomonas reinhardtii possesses both Cu-dependent (orthologue to Fet3) and Cu-independent pathways for Fe acquisition (La Fontaine et al. 2002). However, multiple evidences indicate that Fe acquisition by roots does not require Cu, but depends instead on Cu-independent transporters. In any case, further studies are necessary to determine whether Cu directly participates in Fe transport or in other steps of Fe homeostasis.

When present at elevated concentrations, the same redox properties that make Cu and Fe essential elements trigger the formation of reactive oxygen radicals that damage cells at the level of membranes, proteins and nucleic acids (Halliwell & Gutteridge 1984). Moreover, under metal excess, unavoidable ectopic binding to mistargeted proteins can also inactivate and disturb protein structure (Koch, Pena & Thiele 1997; Yang et al. 2006). This essential versus toxic duality has driven the development of finely tuned homeostatic networks devoted to acquiring appropriate amounts of Cu and Fe in diverse environmental conditions, and precisely delivering them to specific compartments and target metalloproteins, while avoiding their deleterious effects.

The yeast S. cerevisiae has tremendously contributed to deciphering the basic cellular components of Cu and Fe homeostasis in eukaryotic organisms. Several families of proteins involved in plant metal homeostasis have primarily been identified by sequence homology to a yeast counterpart. Furthermore, yeast mutant strains defective in specific steps of metal transport and distribution have allowed the development of screening strategies to identify and functionally characterize both conserved and plant-specific components of metal homeostasis.

The growing interest in Fe and Cu homeostasis is reflected in a number of excellent reviews that focus on higher plants (Curie & Briat 2003; Colangelo & Guerinot 2006; Grotz & Guerinot 2006; Pilon et al. 2006) as well as other organisms (Puig & Thiele 2002; Kosman 2003; Balamurugan & Schaffner 2006; Kaplan et al. 2006). The present review highlights recent progress made on the transport, distribution and regulation of Cu and Fe in response to metal deficiencies in Arabidopsis thaliana, with special attention on Cu and Fe homeostasis coordination and interaction. We also discuss the potential contribution of these research advances to the design of new biotechnological strategies to alleviate Cu toxicity and Fe deficiency, two global agricultural and human nutrition concerns.

IRON AND COPPER UPTAKE COMPONENTS

Higher plants are traditionally divided into two types depending on their strategy for high-affinity Fe acquisition from Fe-deficient soils: Strategy I plants (dicotyledonous and non-graminaceous) acidify the soil and reduce Fe3+ before transport, while Strategy II plants (graminaceous) synthesize and transport Fe3+-chelating agents named phytosiderophores. The Strategy I plant A. thaliana induces three enzymatic activities within the roots in response to Fe deficiency: firstly, a rhizosphere acidification proton-ATPase, probably attributable to one of the 12 members of the Arabidopsis H+-ATPase (AHA) gene family; secondly, a ferric-chelate reductase that reduces Fe3+ to soluble Fe2+ at the root surface; and thirdly, a high-affinity Fe2+ transporter (Curie & Briat 2003). On the other hand, Cu acquisition by plant roots depends on specific high-affinity Cu+ uptake transporters at the plasma membrane that belong to the conserved copper transporter (CTR) family (Sancenon et al. 2003, 2004). These high-affinity Cu transporters together with the ferric-chelate reductases and the Fe2+ transporters of Strategy I plants are described subsequently.

Figure 1 summarizes the subcellular location of multiple Cu and Fe transport components in a generic Arabidopsis cell. For a more complete picture, Tables 1 and 2 show different families of Arabidopsis metal transport components, together with their members, subcellular localization and function. Table 3 shows tissue and organ-specific expression levels for Cu and Fe homeostasis factors.

Figure 1.

Arabidopsis Fe and Cu transport components. A speculative diagram representing Fe and Cu transport components in a generic plant cell is shown. Fe transporters and Fe proteins are in red, while Cu transporters and Cu proteins are blue. Positive and negative charges represent electrochemical gradient across membranes created by H+-ATPases. Arrows indicate the proposed direction for metal transport. Question marks indicate unclear components or steps. ITP, iron transport protein; NA, nicotianamine; FRO, ferric reductase oxidase; COPT, copper transporter; ZIP2, ZRT, IRT-like protein 2; IRT1, iron-regulated transporter 1; AHA, Arabidopsis H+-ATPase; HMA, heavy metal P-type ATPase; RAN1, responsive-to-antagonist 1; NRAMP, natural resistance-associated macrophage proteins; PAA, P-type ATPase of Arabidopsis; SOD, superoxide dismutase; PC, plastocyanin; YSL, yellow stripe-like.

Table 1.  Iron homeostasis factors in Arabidopsis thaliana
FamilyNameMIPS IDSubcellular localizationDescriptionReferences
  1. Putative subcellular localization and function is described for multiple Arabidopsis Fe homeostasis components. Question marks indicate putative, but not demonstrated, subcellular localizations.

  2. bHLH, basic helix-loop-helix; FRO, ferric reductase oxidase; ZIP, ZRT, IRT-like protein; IRT, iron-regulated transporter; NRAMP, natural resistance-associated macrophage proteins; MATE, multidrug and toxin efflux; OPT, oligopeptide transporter; YSL, yellow stripe-like; FIT1, Fe-deficiency-induced transcription factor 1.

FROFRO2At1g01580Plasma membraneFe3+-chelate reductase Robinson et al. (1999)
FRO3At1g23020Plasma membraneFe3+-chelate reductase Mukherjee et al. (2006); Wu et al. (2005)
FRO6At5g49730Chloroplast envelope?Fe3+-chelate reductase Feng et al. (2006); Mukherjee et al. (2006)
ZIPIRT1At4g19690Plasma membraneFe2+ transporter Eide et al. (1996); Connolly, Fett & Guerinot (2002); Henriques et al. (2002); Varotto et al. (2002); Vert et al. (2002)
IRT2At4g19680Plasma membrane?Fe2+ transporter Vert, Briat & Curie (2001); Varotto et al. (2002); Vert et al. (2002)
NRAMPNRAMP1At1g80830Chloroplast envelopeFe2+ transporter Curie et al. (2000)
NRAMP3At2g23150Vacuolar membraneFe2+ transporter Thomine et al. (2003); Lanquar et al. (2005)
NRAMP4At5g67330Vacuolar membraneFe2+ transporter Lanquar et al. (2005)
MATEFRD3At3g08040Plasma membraneCitrate efflux protein Rogers & Guerinot (2002); Green & Rogers (2004)
OPTYSL1At4g24120Plasma membraneFe-phytosiderophore/Fe-NA transporter Le Jean et al. (2005); Waters et al. (2006)
YSL2At5g24380Plasma membraneFe-phytosiderophore/Fe-NA transporter DiDonato et al. (2004); Schaaf et al. (2005)
bHLHFIT1At2g28160NucleusFe-regulated transcription factor Colangelo & Guerinot (2004); Jakoby et al. (2004)
Table 2.  Copper homeostasis factors in Arabidopsis thaliana
FamilyNameMIPS IDSubcellular localizationDescriptionReferences
  1. Putative subcellular localization and function is described for multiple Arabidopsis Cu homeostasis components. Question marks indicate putative, but not demonstrated, subcellular localizations.

  2. ZIP, ZRT, IRT-like protein; COPT, copper transporter; HMA, heavy metal P-type ATPase; PAA1, P-type ATPase of Arabidopsis 1; RAN1, responsive-to-antagonist 1; COX, cytochrome c oxidase; SOD, superoxide dismutase; ATX, antioxidant; CCS, Cu chaperone for Cu/ZnSOD; CCH, copper chaperone.

ZIPZIP2At5g59520Plasma membrane?Divalent cation uptake transporter Grotz et al. (1998); Wintz et al. (2003)
ZIP4At1g10970Plasma membrane?Divalent cation uptake transporter 
COPTCOPT1At5g59030Plasma membrane?High-affinity copper transporter Kampfenkel et al. (1995); Sancenon et al. (2004)
COPT2At3g46900Plasma membrane?High-affinity copper transporter Sancenon et al. (2003)
COPT3At5g59040Internal localization?High-affinity copper transporter 
COPT5At5g20650Internal localization?High-affinity copper transporter 
COPT6At2g26975Plasma membrane?High-affinity copper transporter 
HMAHMA1At4g37270Chloroplast envelopeCu2+-transporting P-type ATPase Seigneurin-Berny et al. (2006)
HMA5At1g63440Secretory pathway?Cu+-transporting P-type ATPase Andres-Colas et al. (2006)
PAA1 (HMA6)At4g33520Chloroplast envelopeCu+-transporting P-type ATPase Shikanai et al. (2003); Abdel-Ghany et al. (2005b);
RAN1 (HMA7)At5g44790Trans-Golgi Network?Cu+-transporting P-type ATPase Hirayama et al. (1999); Woeste & Kieber (2000); Chen et al. (2002)
PAA2 (HMA8)At5g21930Thylakoid membraneCu+-transporting P-type ATPase Abdel-Ghany et al. (2005b)
ATXCCHAt3g56240CytosolATX1-like CCH Himelblau et al. (1998)
ATX1At1g66240CytosolATX1-like CCHOur unpublished results
CCSCCSAt1g12520Cytosol and chloroplastCCH for Cu/ZnSOD Abdel-Ghany et al. (2005a); Chu et al. (2005)
COXCOX17-1At3g15352UnknownCOX17-like CCH Balandin & Castresana (2002); Wintz & Vulpe (2002)
COX17-2At1g53030UnknownCOX17-like CCH Balandin & Castresana (2002); Wintz & Vulpe (2002)
Table 3.  Tissue and organ-specific expression levels for copper and iron homeostasis factors
  1. The signal intensity values from ATH1/Col0 chip integrated into the Gene Atlas tool of Genevestigator are depicted for the genes included in Tables 1 and 2 except unavailable or misleading data (FRO2, FRO6, COPT6 and PAA2). n denotes the number of chip experiments available by September 2006. The Arabidopsis image is courtesy of Genevestigator (https://www.genevestigator.ethz.ch/at/). Expression levels significantly higher than the average are highlighted blue for Cu and red for Fe homeostasis factors. Lower values are in dotted boxes.

  2. FRO, ferric reductase oxidase; IRT, iron-regulated transporter; NRAMP, natural resistance-associated macrophage proteins; YSL, yellow stripe-like; FIT1, Fe-deficiency-induced transcription factor 1; ZIP, ZRT, IRT-like protein; COPT, copper transporter; RAN1, responsive-to-antagonist 1; HMA, heavy metal P-type ATPase; PAA1, P-type ATPase of Arabidopsis 1; CCH, copper chaperone; COX, cytochrome c oxidase; ATX1, antioxidant 1; CCS, Cu chaperone for Cu/ZnSOD.

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Ferric-chelate reductases

In Strategy I plants, Fe3+ solubilization is achieved by extracellular reduction to Fe2+, which is catalysed by ferric-chelate reductases that belong to a large family of electron transport flavocytochromes. Although Arabidopsis high-affinity Cu+ acquisition probably requires extracellular Cu2+ reduction (Sancenon et al. 2003, 2004), no root Cu reductase gene responsible for this activity has been identified yet (Fig. 1). Interestingly, evidence suggests that plasma membrane Cu- and Fe-chelate reductase activities are inextricably linked. In pea plants, root plasma membrane Cu and Fe reductase activities are specifically induced upon both Cu and Fe depletion, but not upon other nutrient deficiencies (Cohen, Norvell & Kochian 1997). Biochemical characterization of this response suggests that both Cu and Fe activities depend on the same reductase (Cohen et al. 1997). In Arabidopsis, induction of ferric-reductase activity by simultaneous Fe and Cu deficiency is synergistic rather than additive, which is also consistent with a single gene responding to both Fe and Cu deficiency (Romera, Frejo & Alcantara 2003).

The preliminary characterization of the eight-member family of Arabidopsis metal reductases points to ferric reductase oxidase 2 (FRO2) and FRO3 as the main components responsible for Fe acquisition and metabolism in Arabidopsis roots (Wu et al. 2005; Mukherjee et al. 2006) (Fig. 1, and Tables 1 and 3). FRO2, which is induced in Arabidopsis roots by iron deficiency, was previously identified by sequence homology and complementation of Arabidopsis frd1 (defective in FRO2) mutants, which exhibit reduced Fe uptake and growth impairment when Fe is limiting (Yi & Guerinot 1996; Robinson et al. 1999). Arabidopsis FRO2 also exhibits Cu reductase activity under Fe deficiency, which is absent in frd1 mutants (Yi & Guerinot 1996; Robinson et al. 1999). Experiments addressing Cu accumulation in frd1 mutants (Yi & Guerinot 1996) and Cu sensitivity in FRO2-overexpressing plants (Connolly et al. 2003) have not been conclusive regarding an in vivofunction for FRO2 in root Cu reduction. FRO3 expression is increased in Arabidopsis roots upon both Fe and Cu limitation suggesting a role for FRO3 in Fe and Cu acquisition from soil (Wu et al. 2005; Mukherjee et al. 2006). The expression of FRO genes at different plant tissues accounts for the requirement of metal-chelate reductase activity not only in roots but at diverse plant locations (Wu et al. 2005; Mukherjee et al. 2006). Several data are consistent with FRO6 functioning in Fe uptake into chloroplasts. Firstly, FRO6 is specifically expressed in green tissues (Feng et al. 2006; Mukherjee et al. 2006) (Table 3). Secondly, light-responsive elements within the FRO6 promoter region mediate gene activation by exposure to light (Feng et al. 2006). And thirdly, FRO6 expression is decreased in shoot tissues depleted for Cu, which is also essential for photosynthesis (Mukherjee et al. 2006).

The ZIP family of divalent metal transporters

Fe acquisition by Arabidopsis roots under Fe-deficient conditions mostly depends on iron-regulated transporter 1 (IRT1), a member of the ZRT, IRT-like proteins (ZIP) divalent metal transporter family (Connolly, Fett & Guerinot 2002; Henriques et al. 2002; Varotto et al. 2002; Vert et al. 2002) (Fig. 1 and Table 1). IRT1 is identified by its ability to complement the growth defect on Fe-limited conditions of a fet3Δfet4Δ yeast strain defective in both high- and low-affinity Fe acquisition (Eide et al. 1996). Arabidopsis irt1-1 knockout mutants exhibit chlorosis and severe growth defects, which are rescued by exogenous Fe application (Vert et al. 2002). Metal uptake experiments and growth assays in yeast suggest that IRT1 is a broad-range divalent metal ion transporter able to transport Fe, Zn, Mn and Cd, but not Cu (Eide et al. 1996; Korshunova et al. 1999). Interestingly, the replacement of specific residues has been shown to alter IRT1 selectivity (Rogers, Eide & Guerinot 2000). In agreement with a wide range of metal specificity, a tomato IRT homolog, but not Arabidopsis IRT1, complements yeast mutants defective in Cu transport (Eckhardt, Mas Marques & Buckhout 2001). The ZIP family of divalent metal transporters contains 14 additional members in Arabidopsis (Maser et al. 2001). IRT2, which is the closest IRT1 homolog, is expressed in root epidermal cells under Fe deficiency, and it reverts fet3Δfet4Δ yeast mutant growth defect (Vert, Briat & Curie 2001). However, IRT2 does not seem to play a relevant role in root Fe acquisition because its overexpression cannot substitute for the loss of IRT1 (Vert et al. 2002), and irt2-1 insertion mutants show no symptoms of Fe deficiency (Varotto et al. 2002).

Despite being a Strategy II plant, rice plants contain two IRT1 homologs, OsIRT1 and OsIRT2, which have been suggested to function in Fe acquisition (Bughio et al. 2002; Ishimaru et al. 2006). Visualization of Fe translocation by positron-emitting tracer imaging system has demonstrated that rice plants directly absorb Fe2+ (Ishimaru et al. 2006). These authors suggest that Fe2+ is the major form of Fe acquired by rice plants, which do not secrete sufficient Fe3+ phytosiderophores. The higher abundance of Fe2+ over Fe3+ on paddy soils would compensate for the lack of effective Fe-chelate reductases in rice roots (Ishimaru et al. 2006). This Fe acquisition strategy can be especially advantageous for plants adapted to submerged conditions.

Arabidopsis ZIP2 and ZIP4 family members seem not to be involved in Fe transport, but instead, they complement growth defects of yeast Zn and Cu transport mutants (Grotz et al. 1998; Wintz et al. 2003). Furthermore, expression of both genes is up-regulated in Arabidopsis by deficiency in Zn and Cu, but not in Fe (Grotz et al. 1998; Wintz et al. 2003). Interestingly, in addition to its role in zinc homeostasis (van de Mortel et al. 2006), ZIP2 tissue expression pattern would be consistent with a function in Cu acquisition by Arabidopsis roots (Table 3). Although the role of these proteins in plant Cu transport still requires further characterization, the preference that ZIP family members show for divalent metals suggests that ZIP2 and ZIP4 proteins may transport Cu2+ ions (Fig. 1 and Table 2).

The COPT family of high-affinity copper transporters

The conserved CTR family of Cu transport proteins mediates high-affinity Cu acquisition from the exterior into the cytoplasm of eukaryotic cells ranging from yeast to mammalian organisms (Dancis et al. 1994a,b; Kampfenkel et al. 1995; Knight et al. 1996; Zhou & Gitschier 1997; Lee, Prohaska & Thiele 2001). CTR proteins are small integral membrane proteins with three putative transmembrane domains (TMDs) (Dancis et al. 1994b; Lee et al. 2002). Biochemical studies have shown that the CTR amino terminus is located in the extracellular space, whereas the carboxyl terminus and the loop between TMD1 and TMD2 face the cytosol (Eisses & Kaplan 2002; Puig et al. 2002; Klomp et al. 2003) (Fig. 2). The amino-terminal domain contains conserved methionine-rich motifs, which are important for transport when Cu is scarce (Puig et al. 2002) (Fig. 2). Furthermore, genetic data and in vivo Cu uptake experiments have demonstrated that an extracellular methionine residue, located approximately 20 amino acids before TMD1, and an MxxxM motif within TMD2, are essential for Cu acquisition, and probably mediate metal coordination during transport (Puig et al. 2002). Genetic and biochemical evidence has suggested that CTR proteins assemble and function as homotrimers (Dancis et al. 1994b; Pena, Puig & Thiele 2000; Lee et al. 2002; Klomp et al. 2003). The recently reported projection structure of human Ctr1 in a phospholipid bilayer has revealed a compact and symmetrical trimer with a novel channel-like architecture where a conserved GxxxG motif within TMD3 is essential for trimerization (Aller et al. 2004; Aller & Unger 2006) (Fig. 2). In vivo Cu transport experiments have shown that the CTR family is highly specific for Cu+ ion with a Km in the lower micromolar range (Eisses & Kaplan 2002; Lee et al. 2002). CTR proteins do not use ATP for Cu import, but their transport ability is stimulated by extracellular K+ (Lee et al. 2002). Cu uptake is probably facilitated by the extremely low cytosolic concentration of free Cu ions (Rae et al. 1999). In addition to the plasma membrane, other CTR members have been localized to the tonoplast, where they function in Cu mobilization from vacuoles (Bellemare et al. 2002; Rees, Lee & Thiele 2004).

Figure 2.

Predicted membrane topology for COPT and heavy metal P-type ATPase (HMA) families of copper transporters. Transmembrane domains (TMDs) are numbered starting at the amino-terminal end. COPT proteins contain three TMDs, and assemble as trimers. HMA proteins contain eight TMDs, with most of the protein facing the cytoplasm. Conserved residues in putative functional domains are represented in one-letter amino acid code and the ATP-binding motif is shown as a blue box. Arrows indicate the probable direction of Cu transport. Black N and C letters indicate N-terminal and C-terminal residues. COPT, copper transporter.

In A. thaliana, the CTR family is known as copper transporter (COPT). The first member, COPT1, is isolated by its ability to rescue yeast ctr1Δ mutant respiratory defects (Kampfenkel et al. 1995). Subsequent members have been identified by sequence homology to COPT1 and yeast complementation (Sancenon et al. 2003). While COPT1 and COPT2 family members fully rescue yeast ctr1Δ growth defect, COPT3 and COPT5 only partially complement defective mutants in Cu transport (Sancenon et al. 2003). These results suggest that COPT1 and COPT2 can be plasma membrane proteins, whereas COPT3 and COPT5 proteins can function in intracellular Cu transport (Fig. 1 and Table 2). COPT4 does not contain methionine residues essential for Cu transport such as the MxxxM motif, and its function in Cu homeostasis is currently questioned. The best characterized member of this family is COPT1 (Sancenon et al. 2004). Several observations indicate that COPT1 functions in root Cu acquisition by Arabidopsis. Firstly, COPT1 antisense plants result in increased root length, growth defects on Cu-deficient media and up-regulation of genes that respond to Cu limitation, such as COPT2 and CCH Cu chaperone (see Copper Chaperones). Secondly, COPT1-defective plants exhibit a 50% decrease in both in vivo plant Cu uptake and Cu accumulation in leaves. Thirdly, GUS expression driven by the COPT1 promoter is restricted to the tips of primary and secondary roots, although this pattern of expression does not agree with the Genevestigator microarray data for COPT1 (Table 3). And fourthly, COPT1 mRNA levels increase when Cu is limiting. Furthermore, COPT1 expression in pollen grains, as well as pollen defects observed in COPT1-deficient plants, also suggest a crucial role for Cu transport in pollen development. Finally, COPT1 expression in cells probably lacking Cu-transporting competent plasmodesmata, such as embryos, trichomes, pollen and stomata, suggests a function in apoplastic Cu transport (Oparka & Roberts 2001; Sancenon et al. 2004) (Table 3). An additional member of the COPT family, designated as COPT6, has been identified in a more recent annotation of the Arabidopsis genome (our unpublished results and Table 2). Further characterization of the COPT family members will improve our understanding of Cu function in higher plants.

INTRACELLULAR AND INTERCELLULAR IRON AND COPPER DISTRIBUTION

The NRAMP family of divalent metal transporters

The natural resistance associated macrophage proteins (NRAMP) family of divalent metal transporters participates in plant intracellular Fe distribution. The presence of NRAMP transporters in both Strategy I and II plants suggests a common function in metal homeostasis. In Arabidopsis, three NRAMP proteins (NRAMP1, NRAMP3 and NRAMP4) revert yeast fet3Δfet4Δ growth in Fe-limited conditions, and exhibit increase mRNA levels upon Fe starvation (Curie et al. 2000; Thomine et al. 2000). Increased resistance to toxic Fe levels of NRAMP1 overexpressing plants and a preferential root localization suggest that Arabidopsis NRAMP1 functions in intracellular root Fe distribution (Curie et al. 2000). Studies on single and double knockout mutants demonstrate that NRAMP3 and NRAMP4 play a redundant role in Fe mobilization from vacuoles in Arabidopsis seeds (Lanquar et al. 2005; Thomine et al. 2003) (Fig. 1 and Table 1). The nramp3nramp4 double mutant exhibits germination arrest under low Fe nutrition, which is fully rescued by exogenous Fe supply (Lanquar et al. 2005). The in vivo imaging of individual metals in plant organelles has shown that nramp3nramp4 mutant seeds fail to retrieve Fe from vacuolar globoids during germination, which is crucial to support Arabidopsis early development previous to Fe acquisition from the soil (Lanquar et al. 2005). Finally, increased Cd hypersensitivity when overexpressed and suppression of growth defects associated with yeast cells deleted for the Mn transporter suggest that Arabidopsis NRAMP proteins also participate in Mn and Cd transport (Thomine et al. 2000).

More recently, a new vacuolar iron transporter 1 (VIT1), which does not belong to the NRAMP family, has been characterized in Arabidopsis plants (Kim et al. 2006). VIT1 complements the sensitivity of ccc1Δ yeast mutants to elevated levels of Fe, which strongly suggests that VIT1 functions in vacuolar Fe storage (Li et al. 2001; Kim et al. 2006). VIT1 localizes to the Arabidopsis vacuolar membrane, and it is highly expressed in the developing embryo and seed. Synchrotron X-ray fluorescence microtomography data have demonstrated that Fe localizes to the provascular strands of wild-type seeds, a distribution completely abolished in vit1-1 mutants (Kim et al. 2006). Furthermore, vit1-1 plants grow poorly in Fe-limiting soils, which is indicative of a VIT1 function in seedling development (Kim et al. 2006).

Copper-transporting P-type ATPases

The heavy metal P-type ATPase (HMA) family, also know as P1B-ATPases, utilizes ATP hydrolysis to efflux positively charged metals from the cytoplasm (Fig. 2). The Arabidopsis genome encodes eight predicted HMA proteins that are classified in two groups: HMA1 to HMA4, which have been implicated in divalent cation transport, including Zn2+, Cd2+ and Cu2+; and HMA5 to HMA8, which presumably function in transport of monovalent Cu+ ions (Fig. 1 and Table 2). HMA5 to HMA8 exhibit two particular characteristics: a CPC ion transduction motif in the sixth predicted TMD, and MxCxxC Cu+-binding motifs at the amino-terminal domain of the protein (Axelsen & Palmgren 2001; Williams & Mills 2005) (Fig. 2).

Arabidopsis responsive-to-antagonist 1 (RAN1, or HMA7) was isolated in a genetic screen for seedlings that evoked the ethylene triple response in the presence of an ethylene antagonist (Hirayama et al. 1999). Given that Cu coordination is required for ethylene binding to the ETR1 ethylene receptor (Rodriguez et al. 1999), RAN1 has been proposed to participate in the biogenesis of functional ethylene receptors by supplying its metal cofactor at the endoplasmic reticulum (Hirayama et al. 1999; Woeste & Kieber 2000; Chen et al. 2002) (Fig. 1). Loss-of-function alleles of RAN1 affect multiple processes including cell elongation, which are partially alleviated by Cu supply (Woeste & Kieber 2000). Therefore, RAN1 will function in Cu delivery to the ethylene receptor, and presumably to other Cu proteins within the secretory pathway. On the contrary, several lines of evidence suggest that Arabidopsis HMA5, the closest RAN1 relative in the P-type ATPase family, functions in root Cu detoxification (Andres-Colas et al. 2006). Firstly, HMA5 is primarily expressed in roots, and is strongly and specifically induced by Cu treatment. Secondly, T-DNA insertion mutants are more sensitive to excess Cu, but not to increases in other metals, as shown by growth defect and root growth arrest. And thirdly, when grown under Cu excess, HMA5-defective plants accumulate Cu in roots to a greater extent than wild-type plants (Andres-Colas et al. 2006). While further studies on cellular and tissue HMA5 expression are needed, these data suggest that HMA5 can be involved in Cu efflux at specific root cells (Fig. 1). If this is the case, HMA5 overexpression in plants can be a strategy for improving Cu detoxification under Cu excess (see Copper phytoremediation).

Cu transport into the chloroplasts depends on the HMA proteins P-type ATPase of Arabidopsis 1 (PAA1, or HMA6) and PAA2 (or HMA8). Both genes were isolated in a screen for mutants that exhibited a high-chlorophyll-fluorescence phenotype, which is a consequence of reduced photosynthetic electron transport activity (Shikanai et al. 2003; Abdel-Ghany et al. 2005b). Both PAA1 and PAA2 proteins contain amino-terminal cleavable transit sequences that, when fused to GFP, target this reporter protein to the chloroplast (Abdel-Ghany et al. 2005b). Interestingly, while full-length fusion of PAA1 to GFP localizes to the chloroplast periphery, an amino-terminal portion of PAA2 protein including the first four TMDs localizes to the thylakoid membranes (Abdel-Ghany et al. 2005b). Analyses of chloroplast Cu proteins in wild-type paa1 and paa2 mutants demonstrate that both ATPases are required for Cu delivery to the thylakoidal protein plastocyanin (PC), while only the paa1 mutant shows a decrease in Cu delivery to the stromal Cu/ZnSOD (Abdel-Ghany et al. 2005b) (Fig. 1). Consistent with these data, PAA1 is expressed in both roots and shoots, while PAA2 is only detected in shoots (Abdel-Ghany et al. 2005b). A double paa1paa2 mutant results in seedling lethality, a more severe phenotype than that observed for plants defective for both PC genes (Weigel et al. 2003; Abdel-Ghany et al. 2005b), suggesting that PAA proteins play additional roles such as Cu delivery to Cu/ZnSOD.

Cu import into chloroplasts is not completely abolished in paa1 mutants. An additional HMA family member, HMA1, localized in the chloroplast envelope, has been recently implicated in Cu transport into the chloroplasts (Seigneurin-Berny et al. 2006). hma1 mutants exhibit diminished chloroplast Cu/ZnSOD activity, but normal PC content (Seigneurin-Berny et al. 2006), suggesting that Cu from HMA1 would be preferentially delivered to Cu/ZnSOD (Fig. 1). Furthermore, HMA1 may have specific functions in plants grown under adverse light conditions (Seigneurin-Berny et al. 2006). It should be stressed that the HMA1 protein does not contain the canonical MxCxxM amino-terminal Cu+-binding motifs, but instead, it is histidine rich at the amino terminus, suggesting that, rather than Cu+, it may transport Cu2+.

Copper chaperones

Once inside the cell, the limited solubility and high reactivity of Cu+ requires the participation of specialized cellular factors named Cu chaperones. These factors are Cu+-binding soluble proteins that mediate intracellular Cu delivery to specific target apoproteins to form biological active Cu proteins (O'Halloran & Culotta 2000; Huffman & O'Halloran, 2001). Several independent Cu chaperones have been described in S. cerevisiae: Antioxidant 1 (Atx1) shuttles Cu to a P-type ATPase located within a post-Golgi compartment for translocation into the secretory pathway (Pufahl et al. 1997); Cu chaperone for Cu/ZnSOD (Ccs1) is required for Cu insertion into the active site of cytosolic Cu/ZnSOD (reviewed by Culotta, Yang & O'Halloran T 2006), and Cox17 is involved in Cu trafficking towards mitochondrial cytochrome c oxidase (COX) (Cobine, Pierrel & Winge 2006). Cu chaperones seem to be conserved in most eukaryotes, but unique metallochaperone characteristics have emerged in plants, probably aimed to fit plant-specific requirements in their Cu delivery pathways (Fig. 3).

Figure 3.

Subcellular distribution of copper by Arabidopsis metallochaperones. A speculative diagram showing subcellular distribution of copper in a generic plant cell is represented. Cu transporters and Cu proteins are represented in blue, while Cu chaperones are in orange, green and pink. Dotted lines indicate putative Cu delivery pathways; continuous lines represent interactions demonstrated by yeast two-hybrid, and discontinuous lines mean the lack of interaction unless the protein is processed. Question marks indicate unclear chaperones or steps. COPT, copper transporter; COX, cytochrome c oxidase; CCH, copper chaperone; ATX1, antioxidant 1; CCS, Cu chaperone for Cu/ZnSOD; SOD, superoxide dismutase; HMA, heavy metal P-type ATPase; RAN1, responsive-to-antagonist 1; PAA, P-type ATPase of Arabidopsis; PC, plastocyanin.

The Arabidopsis CCH protein was the first Cu chaperone described in plants (Himelblau et al. 1998). Although constitutively expressed, CCH is up-regulated in leaves undergoing senescence (Himelblau et al. 1998). At its amino-terminal domain, CCH exhibits the conserved features of the ATX1-like metallochaperone family including a predicted overall βαββαβ fold structure and an MxCxxC Cu+-binding motif. However, CCH presents a plant-exclusive carboxy-terminal domain with special structural characteristics (Mira, Martinez-Garcia & Penarrubia 2001a; Mira et al. 2001b; Mira et al. 2004). This domain adopts an extended conformation in solution and forms well-ordered amyloid-like fibrils. Moreover, in the presence of anionic detergents, the carboxy-terminal domain displays altered electrophoretic mobility (Mira et al. 2004). Because CCH is present in phloem-enucleated sieve elements, a plant-specific role in Cu symplasmic transport through the plasmodesmata during senescence-associated nutrient mobilization has been proposed for this extra carboxy-terminal domain of CCH (Mira et al. 2001a) (Fig. 3). Low CCH expression in cells lacking functional plasmodesmata, such as pollen, is consistent with this putative function (Table 3). The Arabidopsis genome contains a gene encoding an additional Atx1 homolog (MIPS code: At1g66240), which lacks the CCH plant-specific carboxy-terminal extension. Recent yeast two-hybrid studies show interaction between Arabidopsis ATX1 metallochaperone, and both RAN1 and HMA5 P-type ATPases metal-binding domains (Andres-Colas et al. 2006 and our unpublished results) (Fig. 3). Interestingly, deletion of the CCH carboxy-terminal domain is required for the interaction between the metallochaperone and P-type ATPases metal binding domains (Andres-Colas et al. 2006, and our unpublished results). These results suggest either a regulatory role for the CCH carboxy-terminal extension, the processing of this domain prior to interaction with ATPases or the requirement for an intermediate regulatory protein. Copper delivery to other P-type ATPases, such as chloroplastic PAA1 and PAA2, has not been described so far in higher plants. Because Cu delivery to Cu-transporting ATPases for thylakoid import in chloroplast-related cyanobacteria involves Atx1-like metallochaperones (Banci et al. 2006), candidates for higher plant chloroplast Cu chaperones include CCH and ATX1, as well as other proteins with ATX1-like Cu+-binding motifs encoded by the Arabidopsis genome (Wintz & Vulpe 2002) (Fig. 3).

Yeast Ccs1 contains three conserved and functionally distinct domains: the amino-terminal domain bears striking homology to the Atx1 metallochaperone; the central domain with sequence homology to its target protein Cu/ZnSOD, which physically interacts with SOD; and the carboxy-terminal domain that carries a conserved CxC motif that plays a crucial role in copper transfer to Cu/ZnSOD (Casareno, Waggoner & Gitlin 1998; Schmidt et al. 1999). Furthermore, Ccs1 oxidizes an intrasubunit disulphide in Cu/ZnSOD in an oxygen-dependent process necessary for dimerization into the catalytically active form (reviewed by Culotta et al. 2006). The Arabidopsis genome contains only one gene with significant homology to CCS metallochaperone (Table 2), but three Cu/ZnSOD isoforms that localize to cytosol, chloroplast and peroxisome. This raises the question of how plants specifically deliver Cu to three different subcellularly located SODs with just one chaperone. Recent results show that Arabidopsis CCS rescues the growth defects associated with yeast ccs1Δ mutants, and localizes to chloroplasts in plant cells (Abdel-Ghany et al. 2005a). However, ccs T-DNA insertion lines show a decrease in all three Cu/ZnSOD activities (Chu et al. 2005). Furthermore, a CCS truncated form lacking the 66 amino-terminal amino acids, which target the protein to the chloroplasts, only rescues cytosolic and peroxisomal Cu/ZnSOD activities (Chu et al. 2005). These results suggest that CCS dual targeting to the chloroplasts and the cytosol would allow proper Cu delivery to different SODs (Fig. 3). It has been demonstrated that mammalian and Caenorhabditis elegans Cu/ZnSOD can acquire Cu by a not yet completely understood CCS-independent mechanism (Carroll et al. 2004; Jensen & Culotta 2005). This pathway for Cu delivery would explain the basal Cu/ZnSOD activities and the lack of a more severe phenotype associated with Arabidopsis ccs knockout mutants.

Studies in yeast postulated that Cox17 is the Cu chaperone implicated in Cu trafficking to the mitochondrial COX (reviewed by Cobine et al. 2006). However, a Cox17 protein tethered to the mitochondrial inner membrane resulted in complementation of the respiratory defect of yeast cox17Δ mutants (Maxfield, Heaton & Winge 2004). Most mitochondrial Cu is located within the matrix in a soluble and accessible non-proteinaceous form, suggesting that this pool can be the source of Cu for Cox17 (Cobine et al. 2004). Within the inner membrane space, Cox17 delivers Cu to two mitochondrial inner membrane proteins, Sco1 and Cox11, which are thought to be Cu donors for the COX CuA and CuB sites, respectively (reviewed by Cobine et al. 2006). Although two Cox17 homologs have been identified in Arabidopsis by complementation of the respiratory deficiency of yeast cox17Δ mutants (Balandin & Castresana 2002; Wintz & Vulpe 2002) (Fig. 3 and Table 2), further studies are necessary to determine their role within the plant.

The presence of putative Cu2+ transporters in plants (e.g. HMA1) suggests that additional mechanisms may exist for proper Cu delivery. In this sense, the Arabidopsis CUTA protein, which binds Cu2+ and localizes to the intermembrane chloroplastic space, has been proposed as a candidate for a Cu2+ metallochaperone (Burkhead et al. 2003).

Long-distance transport

Once incorporated into the root, Fe and Cu are loaded into the xylem sap by a not yet characterized system. It has been proposed that organic acids, especially citrate, are the main metal-chelators in xylem (Rauser 1999; Curie & Briat 2003; Hell & Stephan 2003) (Fig. 1). A key component of Fe xylem loading is FRD3, a member of the multidrug and toxin efflux (MATE) family of small molecule transporters. Arabidopsis plants defective in FRD3 exhibit constitutive Fe deficiency responses independently of Fe supply and are chlorotic, in spite of accumulating elevated Fe levels (Rogers & Guerinot 2002). The explanation for these apparently contradictory results is that in frd3 mutants, Fe accumulates within the xylem, while the Fe content in leaf protoplasts is half the level of wild type (Green & Rogers 2004). Interestingly, it has been recently shown that FRD3 is capable of efflux citrate when expressed in Xenopus oocytes (E.E. Rogers, personal communication) (Fig. 1).

The enhanced demand of phloem-mediated transport of metals during reproductive phases, especially under mineral deficiency, poses the question of the identity of the associated ligands. Studies with the castor bean Ricinus communis implicate iron transport protein (ITP), a 96-amino acid protein with high similarity to the stress-related family of late embryogenesis abundant proteins, and the non-proteinogenic amino acid nicotianamine (NA) in phloem Fe and maybe Cu distribution (Kruger et al. 2002; Hell & Stephan 2003). ITP binds Fe3+in vivo in the phloem, but it also complexes Cu2+ and other metals in vitro (Kruger et al. 2002) (Fig. 1). NA is a ubiquitous metal-chelator in all plants, which is synthesized by nicotianamine synthase (NAS) from S-adenosyl-L-methionine. The first evidence for a role of NA in internal metal transport came from Cu and Fe-related phenotypes associated with the NA synthesis-defective chloronerva tomato mutant, which shows interveinal chlorosis (Ling et al. 1996; Mori 1999). In Strategy II plants, NA is a substrate for nicotianamine aminotransferase (NAAT), acting as intermediate in the phytosiderophore biosynthesis pathway. Given that NAAT is absent in Strategy I plants, NA has been greatly reduced in tobacco plants by expressing a barley NAAT (Takahashi et al. 2003). NA-defective tobacco plants exhibit phenotypes that point to the essentiality of NA for metal transport in veins and interveinal areas, and for reproductive growth and fertility (Takahashi et al. 2003) (Fig. 1). Because NA is mostly complexed with Fe2+ in the phloem (von Wiren et al. 1999), while transported as an Fe3+-ITP complex, it has been proposed that NA can act as a shuttle by chelating Fe2+ from ITP-bound Fe3+ during loading and unloading of Fe in the phloem (Kruger et al. 2002). If this is the case, a reduction system, perhaps including an FRO family member, would also be necessary, as well as the participation of specific Fe2+-NA transporters.

YSL transporters

The primary uptake of Fe3+-phytosiderophore complexes in Strategy II plants is mediated by a member of the oligopeptide transporter (OPT) superfamily known in maize as yellow stripe 1 (YS1) (Curie et al. 2001). Yeast complementation assays and transport studies with Xenopus oocytes demonstrate that YS1 encodes a proton-coupled transporter for phytosiderophores and NA broad-range metal chelates (Schaaf et al. 2004). Despite the fact that Strategy I plants do not synthesize and secrete phytosiderophores, they express multiple yellow stripe-like (YSL) genes (Table 1), which have been proposed to function in transport of Fe-NA complexes. Arabidopsis YSL1 loss-of-function mutants contain lower levels of Fe-NA in their seeds and display a transient defect in germination that can be rescued by Fe supply (Le Jean et al. 2005). An exacerbated phenotype is observed for ysl1ysl3 double knockout mutants, which exhibit interveinal chlorosis in leaves caused by decreased Fe levels, and reduced fertility as a consequence of defective anther and embryo development (Waters et al. 2006). These phenotypes are at least partially rescued by addition of exogenous Fe and ectopic expression of YSL3. Interestingly, ysl1ysl3 double mutants are less efficient in mobilizing metals, especially Cu, from senescent leaves. These results, and YSL1/YSL3 expression in the vasculature of shoots and reproductive organs, suggest a function in metal delivery from vascular tissues, as well as in Fe-NA delivery to seeds (Waters et al. 2006) (Fig. 1). Localization of Arabidopsis YSL2 to root endodermis and pericycle cells facing the meta-xylem tubes has suggested that it also participates in lateral movement of Fe and/or Cu within the veins (DiDonato et al. 2004; Schaaf et al. 2005). However, further studies are necessary to elucidate YSL2 substrate specificity, because contradictory results have been reported for YSL2-mediated transport in yeast (DiDonato et al. 2004; Schaaf et al. 2005).

The assumption that the members of the OPT family transport only peptides is being challenged by the surprising discovery that some OPTs may also be capable of divalent metal ion transport. AtOPT2 and AtOPT3 are differentially expressed under metal deficiencies, and heterologous expression of AtOPT3 in yeast suggests that it can transport Cu2+, Mn2+ and Fe2+ (Wintz et al. 2003).

REGULATION OF IRON AND COPPER DEFICIENCY RESPONSES

Living organisms have developed multiple regulatory mechanisms to respond to environmental stresses. Because of their sessile nature, plants probably are the organisms that explore a widest variety of responses to environmental nutrient deficiencies. At least three different molecular strategies can be distinguished in response to Fe and Cu starvation. The first strategy is addressed to improve metal acquisition and includes increased expression of metal reductases and high-affinity transporters. The second mechanism consists in prioritizing the use of metals in essential versus dispensable pathways. Finally, if metalloproteins with different metallic ligands perform similar or overlapping functions, a specific metalloprotein can be substituted by another when its metal is scarce. In this section, we describe our current knowledge of how A. thaliana regulates gene expression in response to Fe and Cu deficiencies.

Regulation of gene expression in response to iron deficiency

The primary response of plants to Fe deficiency is controlled through coordinated transcriptional activation. Studies with the chlorotic fer tomato mutant have led to the identification of the LeFER basic helix-loop-helix (bHLH) transcription factor, which controls root Fe3+ reductase and LeIRT1 induction upon Fe limitation (Ling et al. 2002). The Arabidopsis LeFER orthologue is the Fe-deficiency-induced transcription factor 1 (FIT1), also known as bHLH29/FRU (Colangelo & Guerinot 2004; Jakoby et al. 2004; Yuan et al. 2005). FIT1 expression in the root epidermis of the differentiation zone upon Fe deficiency overlaps with the expression pattern of FRO2 and IRT1, which are controlled at different levels by FIT1 (Vert et al. 2002; Connolly et al. 2003; Colangelo & Guerinot 2004; Jakoby et al. 2004). Thus, in fit1 mutants, no increase in FRO2 mRNA levels and Fe3+-chelate reductase activity is observed upon Fe depletion (Colangelo & Guerinot 2004; Jakoby et al. 2004). However, whereas IRT1 mRNA up-regulation is not significantly affected in fit1 mutants, the IRT1 protein is undetectable (Colangelo & Guerinot 2004; Jakoby et al. 2004). These results indicate that FRO2 is a direct target for transcriptional regulation by FIT1 (Fig. 4), while an FIT1-dependent mechanism of post-transcriptional regulation controls IRT1 protein levels. It has been proposed that an FIT1 target gene can inhibit IRT1 protein turnover upon Fe starvation (Colangelo & Guerinot 2004). A post-transcriptional mechanism may also regulate FRO2 in Arabidopsis, because FRO2 overexpressing plants only result in increased FRO2 mRNA levels and elevated Fe-chelate reductase activity when plants are starved for Fe (Connolly et al. 2003). Furthermore, overexpression of FIT1 does not alter FRO2 and IRT1 expression patterns, suggesting that an additional protein is required for FIT1-mediated transcriptional activation (Colangelo & Guerinot 2004) (Fig. 4). In this sense, it has been recently shown that FIT1 interacts with other proteins, probably forming heterodimers that would mediate Fe-dependent changes in gene expression (H.Q. Ling, personal communication). Besides FRO2, FIT1 regulates 71 out of the 179 genes, whose expression is modified in roots after a 3 d period of Fe deficiency (Colangelo & Guerinot 2004). This group of genes includes the Fe-transporters IRT2 and NRAMP1, the COPT2 Cu transporter (Fig. 4), the plasma membrane H+-ATPase AHA7 and NAS1. The cis regulatory elements bound by FIT1 remain unclear because the bHLH recognition E-box sequence, 5′-CANNTG-3′, is not significantly over-represented in the promoter region of the greatest deregulated 20 genes in fit1 mutants (Colangelo & Guerinot 2004). Further experiments, such as gene-expression microarrays on specific groups of cells (Birnbaum et al. 2005), or chromatin immunoprecipitation, may help characterize Arabidopsis FIT1 target genes and their regulation mechanism.

Figure 4.

Regulation of gene expression under Fe and Cu deficiency in Arabidopsis. Scheme of the promoter region of several genes regulated by Fe and Cu. Fe transcription factors and repressors (Rep) are represented as red circles, while Cu factors are blue. Putative cis elements responsive to low Fe or low Cu are indicated. Green arrows indicate transcriptional activation and black lines indicate transcriptional repression. FIT1, Fe-deficiency-induced transcription factor 1; FRO2, ferric reductase oxidase 2; COPT, copper transporter; IRT1, iron-regulated transporter 1; IDE, iron-deficiency-responsive element; IDRS, iron-dependent regulatory sequence; SPL, squamosa protein-like; CuRE, Cu-responsive element.

The fact that many important genes induced upon Fe starvation, including IRT1, are independent of FIT1, suggests additional regulatory mechanisms (Fig. 4). In Strategy II plants, evidence obtained in both barley promoter-reporter fusion experiments and rice genome-wide microarrays under Fe deficiency points to two iron-deficiency-responsive elements (IDE) 1 and IDE2, as responsible for transcriptional induction during Fe deprivation (Kobayashi et al. 2003, 2005). Interestingly, sequences homologous to IDE1 are also found in Arabidopsis IRT1 and FRO2 promoter regions (Kobayashi et al. 2003).

Additional genome-wide approaches have addressed Arabidopsis gene expression changes upon Fe limitation. An expression analysis of 53 metal-related genes in response to Fe deficiency shows that FRO2 and FRO3 metal-chelate reductases, IRT2 transporter (IRT1 was not present in the microarray), and NAS1 and NAS3 are induced upon Fe deficiency in roots (Wintz et al. 2003). Furthermore, a microarray analysis (containing approximately 6000 genes) in both roots and shoots has revealed a profound metabolic rearrangement upon Fe deficiency, which affects 25% of expressed genes (Thimm et al. 2001). These changes in gene expression profiles suggest that the elevated root energy demand in response to Fe deficiency induces the mobilization of carbohydrates from shoots to roots and an increase in both aerobic and anaerobic root respiration. The induction of both CO2 fixation and O2-dependent respiration in plants indicates that the stimulated root metabolism under Fe deficiency may result in O2 deficiency (Lopez-Millan et al. 2000). Interestingly, responses to Cu deficiency are also related to hypoxia in Chlamydomonas (Quinn et al. 2000). Enhanced activity in the citric acid cycle and the total nucleotide, mitochondrial quinone and ATP pools are also observed in response to Fe deficiency, which points to an increase in root mitochondrial activity (Thimm et al. 2001). However, in unicellular model organisms such as bacteria and yeast, Fe-dependent pathways including mitochondrial respiration are post-transcriptionally down-regulated in response to Fe deprivation (Masse & Arguin 2005; Puig, Askeland & Thiele 2005). In S. cerevisiae, a protein induced by Fe deficiency named Cth2 binds mRNAs that encode proteins involved in Fe-dependent processes, accelerating their degradation (Puig et al. 2005). This coordinated control of Fe-dependent pathways may prioritize the delivery of limited Fe to essential versus dispensable Fe proteins. Mechanisms for the optimized use of Fe, when scarce, may also be present in plants.

Iron-storage proteins such as ferritin, encoded by the FER gene, are repressed during Fe starvation (Lescure et al. 1991). Site-directed mutagenesis analyses and gel shift assays have allowed the identification of a 15 bp iron-dependent regulatory sequence (IDRS) within maize and Arabidopsis FER1 promoter region, which is responsible for ferritin transcriptional repression under Fe deficiency (Petit et al. 2001) (Fig. 4). Upon Fe supply, FER1 mRNA rapidly accumulates, allowing ferritin synthesis to protect plants from Fe-induced oxidative stress. Recent studies with Arabidopsis cultured cells have shown that treatment with a specific 26S proteasome inhibitor completely abolishes FER1 induction upon Fe loading, strongly suggesting that the ferritin repressor is ubiquitinated and degraded after Fe treatment in a process where nitric oxide plays a key role (Murgia, Delledonne & Soave 2002; Arnaud et al. 2006) (Fig. 4).

Molecular responses to copper deficiency

The mechanisms that regulate plant gene expression responses to Cu deficiency in Arabidopsis are currently unknown. Elegant genetic and biochemical studies have established that transcriptional regulation mediates the primary response to Cu deprivation in the photosynthetic algae C. reinhardtii. Mutational analyses and promoter fusions to reporter genes have allowed the identification of Cu-responsive elements (CuREs) and hypoxia-responsive elements (HyRE) with a critical GTAC core sequence, as responsible for Cu deficiency and hypoxia transcription activation of two Chlamydomonas genes, cytochrome c6 (CYC6) and coproporphyrinogen III oxidase 1 (CPX1) (Quinn et al. 2000). More recently, a genetic screen has revealed copper response regulator (CRR1) as the locus responsible for CYC6 and CPX1 activation upon Cu deprivation (Eriksson et al. 2004). The Crr1 protein contains a plant DNA-binding domain named squamosa binding protein (SBP), ankyrin repeats and a carboxy-terminal cysteine-rich region with similarity to Drosophila metallothionein (MT) (Kropat et al. 2005). In vitro electrophoretic mobility assays have shown that the Crr1-SBP domain specifically binds CuREs within CYC6 and CPX1 promoter regions (Kropat et al. 2005). Interestingly, the Arabidopsis genome contains 16 proteins with SBP domains denoted squamosa protein-like (SPL) proteins, some of them involved in flower development (Birkenbihl et al. 2005). A subset of the Arabidopsis SPL proteins contain additional motifs also found in Crr1 that include an amino-terminus aromatic and hydrophobic motif embedded in an acidic context similar to heat stress transcription factors, a region with SBP homology linked to the SBP domain and three ankyrin repeats. The existence of several SPL members suggests a pattern of Cu regulation maybe specifically modulated in different tissues and developmental stages. Further studies will elucidate whether any of the Crr1-type SPL proteins are responsible for gene expression regulation under Cu deficiency in Arabidopsis (Fig. 4).

Although no genome-wide microarray data are currently available, several Arabidopsis genes with increased expression in response to low Cu availability have been identified, including COPT1, COPT2 and ZIP2 transporters, FRO3 metal reductase, CCH Cu chaperone and chloroplastic FeSOD (Himelblau et al. 1998; Sancenon et al. 2003; Wintz et al. 2003; Abdel-Ghany et al. 2005b; Mukherjee et al. 2006). As mentioned previously, COPT2 is also induced upon Fe deficiency in a FIT1-dependent manner (Colangelo & Guerinot 2004). The theoretical analysis of the COPT2 promoter sequence shows putative cis elements responsive to both low Fe and low Cu, suggesting that this promoter can integrate signalling pathways of deficiencies in both metals (Fig. 4). The characterization of the genome-wide response of Arabidopsis to Cu deficiency will tremendously contribute to elucidate the coordination between Cu and Fe responses in higher plants.

Cu-protein substitution by functionally equivalent Fe proteins under low Cu is well-documented in Chlamydomonas (Merchant et al. 2006). In fact, upon Cu deficiency, cells replace PC within the photosynthetic chain by the Fe-containing CYC6, a Cu-independent/haem protein (Quinn & Merchant 1995). Because PC is essential and represents around 50% of Cu in a photosynthetic cell, this Cu versus Fe substitution is tightly regulated by proteolysis of apoplastocyanin and transcriptional activation of CYC6 upon Cu deficiency (Li & Merchant 1995; Quinn & Merchant 1995). Higher plants do not substitute PC, but instead, the abundant chloroplastic Cu/ZnSOD is replaced by the Fe counterpart upon Cu limitation. This coordinated substitution allows plants to economize Cu, when scarce, for essential chloroplastic PC. Arabidopsis plants grown on Cu-replete conditions express cytosolic, peroxisomal and chloroplastic Cu/ZnSOD, but not chloroplastic FeSOD (Fig. 5). Under low Cu conditions, chloroplastic FeSOD mRNA, protein and activity levels dramatically increase while chloroplastic and cytosolic Cu/ZnSOD levels are barely detectable (Abdel-Ghany et al. 2005b) (Fig. 5). A concomitant decrease in the expression of the corresponding metallochaperone CCS has also been observed under Cu deficiency (Abdel-Ghany et al. 2005b). This coordinated regulation of nuclear encoded genes is probably directed to the optimal use of chloroplastic available metal ions in essential versus dispensable proteins and suggest that stromal Cu levels regulate nuclear expression through a still unknown signalling pathway (Pilon et al. 2006).

Figure 5.

Substitution of chloroplastic superoxide dismutase (SOD) enzymes depending on Cu bioavailability in Arabidopsis. Cu/ZnSOD is the predominant SOD within the chloroplast during Cu-replete conditions, whereas it is substituted by FeSOD upon Cu limitation. Cu proteins are represented in blue, while Fe proteins are in red. Thick and thin arrows represent, respectively, the main and minor Cu delivery pathways under Cu deficiency. PC, plastocyanin; SOD, superoxide dismutase.

PLANT BIOTECHNOLOGY APPLICATIONS IN AGRICULTURE AND NUTRITION

The concentration of both nutrient and toxic elements in soils and waters directly influences plant growth and development. For elements such as Cu and Fe, soil deficiencies are found worldwide. Although naturally occurring metal contamination is relatively uncommon, human activities have contributed to the increased levels of Cu and other trace metals on the upper layer of soils used for agricultural applications (He, Yang & Stoffella 2005). In this section, we describe how plant biotechnology strategies can contribute to alleviate two important agricultural problems such as Fe deficiency and Cu excess in soils.

Copper phytoremediation

Cu is one of the elements that most often contaminates cultivating soils and irrigating waters. The frequent use of fungicides, pesticides and herbicides with high Cu concentrations, the excessive application of metal-containing fertilizers to correct soil nutrient deficiencies and the use of organic manures from animals fed with metal-based additives, as well as dry and wet deposits, industrial wastewaters and mining activities, have tremendously contributed to this Cu contamination (He et al. 2005). Effects of Cu toxicity in plants include reduction in plant size, inhibition of root growth, decrease in the number and size of leaves, chlorosis, defects in flowering and decrease in germination index (Märschner 2002). Cultivation practices can increase Cu concentration in the fields by 10–20 times, and among the most affected cultures are citrus, vineyards and other fruit groves (He et al. 2005).

Over the past two decades, the utilization of plants for environmental decontamination of soils and waters (a process termed phytoremediation) has emerged as an attractive low-cost alternative technology (Kramer 2005). An approach for decontamination of toxic metals consists of phytoextraction by metal hyperaccumulating plants, which are metal-tolerant plants able to transport and accumulate up to 100–1000 times soil metal concentrations in above-ground tissues for a subsequent easy harvest and processing. Elsholtzia splendens, a Chinese native herb of the Labiata family, is a Cu-tolerant and accumulating plant with phytoremediation potential (Jiang, Yang & He 2004; Weng et al. 2005). Also known as the ‘copper flower’, E. splendens grows predominantly on Cu mining deposits and has been used as a Cu indicator for metal prospecting. Although more than 25 Cu hyperaccumulating plant species have been identified, the underlying molecular mechanisms leading to this Cu accumulation are largely unknown.

The development of efficient Cu phytoremediation strategies requires the identification of relevant traits and responsible genes in natural Cu-accumulating plants, the characterization of their functional mechanisms in model organisms and the assay of biotechnological strategies in Arabidopsis plants and eventually in crops. Putative candidates to improve Cu phytoremediation include root Cu reductases and transporters, NA synthases and two Cu detoxification proteins: P-type ATPases and MTs. Regarding P-type ATPases, the Cu-tolerant plant Silene vulgaris displays enhanced ATP-dependent Cu efflux across the root cell plasma membrane (Van Hoof et al. 2001). Furthermore, the inactivation of the ActP gene, which encodes a P-type ATPase, provokes Cu hypersensitivity in Rhizobium leguminosarum and Sinorhizobium meliloti (Reeve et al. 2002). In Arabidopsis, an interesting candidate for overexpression in order to improve Cu detoxification is HMA5 (Andres-Colas et al. 2006). MTs are a group of low molecular weight and cysteine-rich proteins that chelate metallic cations avoiding binding to unspecific ligands. In Arabidopsis, MTs are involved in metal detoxification, binding to released ions during protein degradation in senescent organs, and in metal secretion through foliar trichomes (Cobbett & Goldsbrough 2002). Overexpression of the yeast Cu-binding MT CUP1 in tobacco produces a two- to threefold increase in Cu accumulation when these plants are grown on contaminated soils (Thomas et al. 2003). A predominant Cu-homeostasis function has also been attributed to the plant metal hyperaccumulator Thlaspi caerulescens MT 3 (TcMT3) (Roosens et al. 2004). TcMT3 exhibits important differences from its Arabidopis homolog that favour Cu binding, probably to ensure adequate Cu homeostasis (Roosens et al. 2004).

Different studies, such as the molecular and genetic analyses carried out with the Zn hyperaccumulators Arabidopsis halleri and T. caerulescens, and genetic engineering-based phytoremediation strategies assayed for mercury and arsenic, suggest that multiple genes are implicated in the mechanisms of metal hyperaccumulation in plants (Kramer 2005; Meagher & Heaton 2005). The optimal design of biotechnological applications will require the molecular characterization of plant Cu homeostasis mechanisms.

Iron biofortification

The frequently low Fe bioavailability in cultivated soils limits plant growth and nutritional values, and it represents a serious agricultural and human health problem. Fe deficiency anaemia is the primary nutritional disorder in the world as it affects over two billion people, specially infants and pregnant women. Main consequences include mental retardation and decreased immune function. Plant biofortification through biotechnology has emerged as animportant approach to fight Fe deficiency anaemia (Grotz & Guerinot 2002; Zimmermann & Hurrell 2002).

Multiple strategies have been assayed to improve acquisition and accumulation of Fe in the edible parts of plants. One of the pioneering attempts in Strategy I plants is the overexpression of S. cerevisiae plasma membrane Fe reductases (Samuelsen et al. 1998). Transgenic tobacco lines overexpressing yeast FRE1 and FRE2 reductases exhibit an increased tolerance to chlorosis induced by Fe deprivation, and a 50% increase in leaf Fe content under both Fe-replete and Fe-deficient conditions (Samuelsen et al. 1998). Similarly, overexpression of Arabidopsis FRO2 results in an improved growth under Fe-limiting conditions, although no significant variation in plant Fe content is observed (Connolly et al. 2003). This is probably a consequence of the post-transcriptional mechanisms that regulate FRO2 in Arabidopsis (Connolly et al. 2003). Interestingly, Arabidopsis FRO2 is not subjected to post-transcriptional control in soybean plants, where its overexpression leads to a threefold increase in Fe accumulation under Fe-replete conditions (Vasconcelos et al. 2006). However, the optimal approach would be combining Fe reductase overexpression with enhanced Fe-transport capacity. The Arabidopsis root Fe transporter IRT1 does not seem to be the best candidate for this approach because of post-transcriptional regulation mechanisms (Connolly et al. 2002) and its broad cation range that allows toxic metals, such as Cd, to accumulate (Connolly et al. 2002). The replacement of specific residues has been shown to alter IRT1 selectivity by eliminating Zn or Cd transport without affecting Fe transport (Rogers et al. 2000; Guerinot 2006). The modification of metal transporters affecting their selectivity represents a new strategy to design transporters that specifically improve essential metal uptake while avoiding the entrance of toxic metals.

The Fe3+-chelating strategy used by Strategy II has been tested to improve Fe acquisition. Thus, tobacco plants overexpressing NAS exhibit an improved Fe utilization under Fe limitation (Douchkov et al. 2005). Furthermore, expression of two barley NAAT genes in rice, which is more sensitive to Fe deficiency, improves phytosiderophore secretion, and it significantly enhances rice growth and grain yield in alkaline soils with low Fe bioavailability (Takahashi et al. 2001). Fe acquisition can be further improved by combining phytosiderophore synthesis, which would require both NAS and NAAT, with increased expression of Fe-siderophore transporters such as maize YS1.

Fe-storage proteins have also provided interesting results in plant Fe accumulation strategies. Ferritins are multimeric proteins that store up to 4500 Fe atoms in a soluble, non-toxic and bioavailable form. Plant ferritins have both ferroxidase and Fe nucleation activity. Arabidopsis possesses four ferritin family members that are differentially expressed. Fer1 and Fer3 accumulate in response to high Fe and oxidative stress, and Fer2 has been postulated to play a role in seed Fe storage (Petit et al. 2001). Specific overexpression of soybean ferritin in rice endosperm results in a two- to threefold increase in seed Fe content (Goto et al. 1999). Among other divalent metals tested, only Zn, but not the toxic Cd, showed a significant increase in ferritin-overexpressing rice seeds (Qu le et al. 2005). It will be interesting to explore the possibilities of the recently characterized VIT1 vacuolar transporter in seed Fe accumulation (Kim et al. 2006). Important efforts have been made to improve Fe bioavailability in the intestine. Phytic acid is one of the anti-nutrient compounds that most contributes to Fe deficiency anaemia because it binds to Fe and other cations in the intestine preventing uptake while enhancing Fe excretion. Plants deficient in phytic acid biosynthesis obtained from breeding strategies and overexpression of heat-resistant phytases have been used to decrease phytic acid content (White & Broadley 2005). Cysteine-rich peptides have also been considered to potentiate Fe absorption. A combined expression of a ferritin, a thermotolerant phytase and a cysteine-rich MT-like protein in rice endorsperm has been checked in order to improve both Fe accumulation in rice seeds and Fe bioavailability. A twofold increase in rice seed Fe content has been obtained, and cysteine content and phytase activity have increased seven times in transgenic plants. However, Fe bioavailability still needs to be carefully evaluated, because only residual phytase activity was detected after rice cooking (Lucca, Hurrell & Potrykus 2002).

The elucidation of the mechanisms that govern Cu and Fe homeostasis and their interactions with other metals in higher plants should help the development of efficient biotechnological strategies for Cu phytoremediation and Fe biofortification while avoiding deleterious side effects. Benefits range from increased yield and nutritional value of crops to a safer and improved human health.

CONCLUDING REMARKS

The use of S. cerevisiae, both as a model eukaryotic cell to characterize basic components of Cu and Fe homeostasis, and as a tool to identify orthologues in other organisms, has fuelled our current understanding of subcellular metal transport and distribution in plants. Although far from being complete, the emerging picture of metal homeostatic networks defines many plant peculiarities. Compared with other organisms, plants dedicate a higher percentage of their genomes to transport issues. Plants have multiple families of Cu and Fe homeostasis factors that function in different tissues or subcellular locations, are differentially expressed and regulated, or have diverged their substrate specificity. The challenge now is to elucidate the specific contribution of each family member to plant metal homeostasis. In the case of the Arabidopsis HMA family of Cu transporters, the different members exhibit diverse subcellular locations providing Cu to the chloroplasts (PAA1 and HMA1), the thylakoids (PAA2), the secretory pathway (RAN1) or performing a detoxification function (HMA5). However, the ZIP family of transporters seems to have wide and divergent substrate specificity in plants. In this respect, the Arabidopsis IRT1 Fe transporter also mediates Mn, Zn and Cd uptake, and other members of the same divalent cation transporter family, such as ZIP2 and ZIP4, seem to transport Cu and Zn.

Metallochaperones also constitute a widespread family covering divergent roles in plants. CCH is one of the ATX-like chaperones in Arabidopsis and contains a plant-specific domain that can regulate its interaction with ATPases and/or function in Cu mobilization during senescence. A particular case is Cu delivery to Cu/ZnSOD, where a single CCS chaperone is responsible for Cu trafficking to three differentially localized enzymes. Plants seem to have developed a CCS dual targeting to the cytosol and the chloroplasts, although a CCS-independent Cu delivery pathway cannot be discarded. Additional unidentified chaperones would be necessary in plants to guarantee Cu delivery to the chloroplast.

Although the regulation of plant responses to Fe and Cu deficiencies is just starting to be deciphered, tightly controlled transcriptional and post-transcriptional mechanisms can be anticipated. Activation of gene expression upon Fe starvation in Arabidopsis seems not to be exclusively controlled by the FIT1 transcription factor, but to include other transcription factors (IRT1 transcriptional regulation) and repressors (FER1 transcriptional repression), as well as unknown post-transcriptional mechanisms that can involve mRNA stability, translational control and protein turnover. Plants also need to coordinate other metabolic processes to optimize the utilization of the scarce metal. A striking example for this coordinated regulation is the alternative use of Cu/ZnSOD versus FeSOD in Arabidopsis chloroplasts depending on Cu availability. A further characterization of plant responses to Cu and Fe deficiencies will contribute to understanding how plants coordinate metal homeostasis, and to developing strategies for efficient Cu and Fe phytoextraction from contaminated or deficient soils to meet agricultural and nutritional demands.

ACKNOWLEDGMENTS

We thank our colleagues M.J. Cornejo, E. Dorcey and J. Moreno for help and stimulating discussions. N. Andrés-Colás and A. García-Molina are recipients of predoctoral fellowships from the Spanish Ministerio de Educación y Ciencia. We are grateful to the Spanish Ministerio de Educación y Ciencia grant BIO2005-07120 and a Ramón y Cajal contract to S. Puig.

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