Available online at www.sciencedirect.
com
Essential transition metal homeostasis in plants
Marinus Pilon1, Christopher M Cohu1, Karl Ravet2, Salah E Abdel-Ghany3
and Frederic Gaymard2
The homeostasis of the essential transition metals copper, iron,
manganese and zinc requires balanced activities of
transporters that mediate import into the cell, distribution to
organelles and export from the cell. Transcriptional control is
important for the regulation of cellular homeostasis. In the case
of Fe and Cu much progress has been made in uncovering the
regulatory networks that mediate homeostasis, and key
transcription factors have now been described. A master
regulator of Cu homeostasis in Arabidopsis thaliana, AtSPL7, is
related to the Chlamydomonas master regulator CCR1,
suggesting that the key switch is conserved between the two
systems even though different sets of targets are regulated in
the two systems.
Addresses
1
Biology Department and Program in Molecular Plant Biology, Colorado
State University, Fort Collins, CO 80523-1878, USA
2
Laboratoire de Biochimie et Physiologie Moléculaire des Plantes, UMR
5004 Supagro/CNRS/INRA/UMII, Bat. 7, 2 Place Viala, 34060
Montpellier Cedex 1, France
3
Botany Department, Faculty of Science, Zagazig University, Zagazig
44519, Egypt
Corresponding author: Pilon, Marinus (pilon@lamar.colostate.edu),
Cohu, Christopher M (ccohu@lamar.colostate.edu), Ravet, Karl
(ravet@supagro.inra.fr), Abdel-Ghany, Salah E (salahghany@gmail.com)
and Gaymard, Frederic (gaymard@supagro.inra.fr)
Current Opinion in Plant Biology 2009, 12:347–357
This review comes from a themed issue on
Physiology and metabolism
Edited by David Salt and Lorraine Williams
Available online 27th May 2009
1369-5266/$ – see front matter
# 2009 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.pbi.2009.04.011
Introduction
The transition metals copper (Cu), iron (Fe), manganese
(Mn) and zinc (Zn) are essential trace elements for plants
as cofactors of various proteins [1]. In cells, zinc exists
only in its stable Zn2+ form; by contrast, the redox active
metals Cu, Fe and Mn occur in variable redox states in
cells and therefore can participate in electron transfer
reactions [2]. Owing to their redox activity, Cu, Fe and
Mn can catalyze the formation of undesired radicals. The
redox potential of Cu and Fe is such that these elements
would be especially prone to radical formation in the cells.
The Irving-Williams series indicates that the relative
binding affinities of the divalent forms of these metals
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for ligands is Mn < Fe < Zn < Cu. Thus relative to Mn
and Fe, the potential for Zn and especially Cu to displace
other metals in binding sites of essential proteins is
relatively high [2]. To balance need and avoid potential
toxic excess, the cellular concentrations of Mn, Zn, Fe
and Cu are tightly controlled. For most natural soils,
deficiency for Cu, Fe and Zn seems to be a far larger
problem than potential excess, and much insight has
been gained by comparing the physiology of plants grown
under replete versus limiting conditions. Nevertheless,
there are several reports in the literature where very high
metal ion concentrations have been used in such excess
that cellular damage is observed. While such studies may
potentially give insight into general (oxidative) stress
recovery mechanisms, we think that this type of approach
has yielded little useful information on metal ion homeo-
stasis, and such approaches are not further considered
here. We aim to summarize key findings in the homeo-
stasis of essential metal ions with emphasis on the past
two years. We discuss these metals in the order of what
seems to be increased tightness of their control mech-
anisms: Mn, Zn, Fe and finally Cu. This same order
corresponds to how well we presently understand each
system.
Manganese
Cellular Mn homeostasis in A. thaliana seems to be
maintained by the activities of transporters. ZIP family
and NRAMP family transporters regulate transport
towards the cytosol [4]. The AtMTP11 (CDF) transporter
allows transport of excess Mn into a prevacuolar compart-
ment [5,6]. Some members of the CAX family of trans-
porters, which typically serve as Ca2+/H+ antiporters, may
also contribute to Mn homeostasis [64]. A Golgi or endo-
some localized P-type ATPase AtECA3, expressed highly
in root tips and the vasculature, can transport both Mn and
Ca into an endomembrane system compartment as
demonstrated by complementation studies in yeast
[7,8]. Mutants for eca3 in A. thaliana show severe growth
phenotypes and chlorosis on low Mn (and to a lesser
extent low Ca), a phenotype that is rescued on moderate
Mn (or Ca) supply [8]. However, the eca3 mutant is also
extra sensitive to excess Mn. The expression of AtECA3 is
not affected by metal ions. AtECA3 may be required to
supply Mn to an essential endomembrane system enzyme
but also to help cells avoid Mn excess in the cytosol.
Plants have a functional homologue of yeast MTM1, a
mitochondrial carrier family member and a putative metal
ion chaperone that is needed to activate manganese-
superoxide dismutase in the mitochondria (MnSOD)
Current Opinion in Plant Biology 2009, 12:347–357
348 Physiology and metabolism
[9]. Expression of AtMTM1 was reported to be induced
in plants by paraquat, which promotes superoxide for-
mation [9]. Plants that were silenced for MnSOD had
mild-growth phenotypes, reduced activities of TCA cycle
enzymes and were affected in their antioxidant responses
in the mitochondria [10]. However, the phenotypes of a
plant mtm1 mutant, possibly defective in MnSOD
activity, have not been reported yet.
Zinc
Even though Zn is not redox active, too high levels of Zn
are toxic because Zn can displace other metals in the cell.
Unlike what is reported for Fe, Cu and perhaps Mn, there
are no specialized assembly systems known for Zn cofac-
tors. Thus, proteins seem to acquire their Zn cofactors by
spontaneous assembly. To allow this a bio-available Zn
pool should be maintained in cells. Interestingly, low
basal cellular Zn levels may also be required for cell
survival as it was found that Zn levels mediate apoptosis
in Norway Spruce embryos [11]. Cellular Zn homeostasis
seems to be maintained by the activities of transporters in
the cell and vacuolar membranes [4]. In A. thaliana and
other plants ZIP family transporters allow uptake into the
cell [4]. The CDF (cation diffusion family) transporters
AtMTP1 (root and shoot) and AtMTP3 (root) move Zn
into vacuoles [12,13]. AtZIF1, a major facilitator protein
in the tonoplast, may transport Zn bound to an organic
ligand into vacuoles [14]. AtMTP1, AtMTP3 and AtZIF1
all may help to buffer cellular Zn levels. The AtHMA2
and AtHMA4 transporters mediate Zn export from the
cell [15–17]. AtHMA4, which is active around the vascu-
lature in roots, is particularly important in regulating root
to shoot transport by allowing Zn to be exported from the
root symplast into the xylem [15]. Zn can be bound by
nicotianamine, and YSL family transporters could con-
tribute to Zn (and Fe and Cu) re-allocation from vege-
tative tissue [18].
Still much is to be learned about how any of the plant
metal transporters are regulated by substrate availability.
Work on AtHMA2 provided insights in possible regulation
of this and related P1B-type-ATPase transporters. The
Zn-specific ATP-dependent HMA transporters have an
N-terminal and a C-terminal Zn-binding domain. The
transporter without the N-terminal metal-binding
domain still transports metal, indicating that the metal
is directly bound by the transmembrane translocation site
[19]. Structural work on related bacterial Cu-transporters
lends support to the hypothesis that the N-terminal
regions inhibit ATPase and transport activity in the
absence of the cognate metal ion [20]. In this fashion,
the metal export activity from the cell may be regulated
by the affinity of the metal binding domains.
The AtMTP1 transporter contains a histidine-rich cyto-
plasmic loop that is not essential for Zn2+ transport into
the vacuole but may serve to increase the affinity of the
Current Opinion in Plant Biology 2009, 12:347–357
transporter for Zn2+ and perhaps binds Zn ions. As such,
this loop may help to buffer and control the cytosolic free
Zn2+ levels [65].
Transcriptional control contributes to Zn homeostasis.
The ZIP transporters involved in uptake are upregulated
by Zn and Fe deficiency in A. thaliana [21]. Thus Fe-
deficiency, which leads to upregulation of the ZIP family
members IRT1 and IRT2, can cause excess Zn uptake.
The vacuolar transporter ZIF1 is induced by Zn excess
[14]. Genome wide transcript profiling is a powerful
method to find candidate genes that respond to changes
in metal availability. However, because both deficiency
and excess can lead to secondary phenotypes it is always
challenging to distinguish which components are directly
involved in homeostasis of the metal in question. By
comparing RNA expression levels at different Zn levels
in the non-accumulator A. thaliana and the related Zn/Cd
hyperaccumulator Arabidopsis halleri genes that respond
directly to Zn status could be identified [21]. The
regulation of transporters is altered in A. halleri where
the roots seem to constitutively express homologous
components that mediate responses to Zn deficiency in
A. thaliana such as transporters involved in uptake [21].
At the same time, the transcript of AhHMA4 is upregu-
lated in A. halleri, both by increased gene copy number
and more active promoter sequences, and as a con-
sequence Zn transport to the shoot is increased [22].
The vacuolar Zn transporter TgMTP1 in the Zn hyper-
accumulator Thlaspi goesingense allows this plant to tolerate
excess Zn. By removing Zn from the cytosol TgMTP1
induces a systemic Zn-deficiency response that in turn
leads to upregulation of Zn uptake [66].
Iron
Transport and use of iron
An overview of Fe transport in plants will be given
elsewhere in this issue [4]. Iron is utilized as a cofactor
in three major forms: iron–sulfur clusters (with five sub-
types in plants), in the porphyrin ring of heme and
siroheme, and as non-heme iron [1]. Specialized assembly
machineries exist for Fe cofactors. The major steps in
heme and siroheme biosynthesis that branch off from the
chlorophyll biosynthetic pathway are localized in plastids,
and regulation of these pathways and their coordination
with chlorophyll synthesis have been described [1].
Regulation of Fe–S clusters biogenesis
Iron–sulfur (Fe–S) clusters are ancient and important
cofactors with roles in catalysis and electron transport
[1]. Both plastids and mitochondria harbour a Fe–S clus-
ter assembly machinery. Fe–S cluster biogenesis can be
divided into iron and sulfur mobilization, their pre-assem-
bly on scaffolds and finally transfer to apoproteins. In the
past years several potential scaffolds as well as systems
that mobilize S have been identified mainly in A. thaliana.
In the mitochondria, frataxin functions to provide Fe to
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the Fe–S cluster biosynthesis machinery [23]. In plastids
a similar function had been postulated for ferritin, how-
ever a recent report has clearly shown that ferritin does
not serve this role [24]. Sulfur is mobilized from
cysteine by cysteine desulfurases. Mitochondrial and
plastidial cysteine desulfurases are encoded by the essen-
tial nuclear genes AtNFS1 and AtNFS2, respectively
[25,26]. The cysteine desulfurase activity of the plastid
and possibly also the mitochondrial cysteine desulfurase
is regulated by SufE proteins. The Arabidopsis genome
contains three AtSufE genes. The three corresponding
proteins are chloroplast-localized and were shown to
enhance AtNFS2 activity [27,28,31]. AtSufE1 is also
targeted to the mitochondria where it may activate
AtNFS1 [28]. AtSufE1 has been shown to interact, in
vitro, with both AtNFS1 and AtNFS2 [27,28]. A sufe1
mutant is embryo lethal and can be rescued only when
AtSufE1 is targeted to both plastids and mitochondria,
revealing the essential function of this protein in both
compartments [28]. Interestingly, two of the SufE
proteins exhibit additional domains that could integrate
other metabolic signals. AtSufE1 contains a BolA-like
domain. In yeast, the cytosolic BolA has been shown to
interact with monothiolglutaredoxins and to regulate the
iron-deficiency responsive genes [29]. The two plastidial
monothiolglutaredoxins GrxS14 and GrxS16 from poplar
have been shown to act as potential scaffold proteins for
the assembly of Fe–S clusters [30]. It is tempting to
hypothesize that glutaredoxins participate in Fe–S cluster
biogenesis by interacting with the BolA domain of SufE1
proteins. AtSufE3 contains a NadA domain that provides
quinolinate synthase activity to the protein required for
NAD/NADP synthesis [31]. Quinolinate synthase con-
tains a highly oxygen sensitive Fe–S cluster, and its link
to a SufE domain in AtSufE3 may provide a necessary
direct link to the core of the Fe–S assembly system that
could ensure synthesis and repair of the cluster in the
oxygen producing chloroplast.
In yeast, cytosolic Fe–S cluster proteins depend on the
mitochondrial cysteine desulfurase and its associated
machinery but the form in which Fe–S clusters or cluster
intermediates are exported from the mitochondria is as
yet unknown (for review see [32]). In mammals, convin-
cing evidence for a cytosolic cysteine desulfurase and
assembly scaffold that are required for Fe–S assembly has
been reported [33]. Thus, in plants three origins for
cytosolic Fe–S clusters would be possible. In A. thaliana,
two mitochondrial ABC transporters (AtATM1 and
AtATM3) that are homologous to ATM1 of S. cerevisae
have been shown to complement the yeast atm1 mutant.
Therefore, these proteins could participate in the assem-
bly of Fe–S clusters in the cytosol [34]. AtNPB35, a P-
loop NTPase homologous to that described in the yeast
and human CIA (cytosolic iron–sulfur cluster assembly)
machinery, could act as a scaffold protein for cytosolic Fe–
S cluster assembly [35]. The disruption of the A. thaliana
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Essential transition metal homeostasis in plants Pilon et al. 349
AtNBP35 gene caused an arrest of embryo development,
which points to an essential role of this cytosolic machin-
ery [35].
Regulation of gene expression in response to iron
deficiency
Fe uptake is highly regulated in plants. Non-graminac-
eous (strategy I) plants use the ferric-chelate reductase
FRO2 and Fe transporter IRT1 [4]. These proteins are
regulated in response to Fe deficiency via the bHLH
transcription factor FIT1 [36]. Recently, two other bHLH
transcription factors, AtbHLH38 and AtbHLH39, have
been shown to interact with AtFIT1 and to regulate
AtFRO2 and AtIRT1 expression in A. thaliana [37]. More-
over, AtFRO2 and AtIRT1 expression is also regulated via
thus far uncharacterized post-transcriptional mechanisms
since overexpression of AtFRO2 or AtIRT1 results in
accumulation of the corresponding mRNA in both shoots
and roots regardless of the iron status of the plant, but
ferric reductase activity and AtIRT1 protein are only
detected in iron-deficient roots. Lysine residues present
in an intracellular loop of AtIRT1 have been shown to be
involved in AtIRT1 turnover [38]. Graminaceous
(strategy II) plants secrete mugineic acid family phyto-
siderophores (PS) to chelate iron followed by Fe-MA
chelate uptake. A large part of the regulatory network
that mediates responses to Fe deficiency has been uncov-
ered. The first cis-elements of the ‘iron-deficiency-
responsive’ pathway, named IDE1 and IDE2, have been
identified in the promoter of the IDS2 gene in barley.
Two trans-acting factors, OsIDEF1 and OsIDEF2, which
bind to respectively the IDE1 and IDE2 elements have
been isolated in rice [39,40]. Transcriptomic analysis in
rice allowed the identification of another trans factor,
named OsIRO2 [41]. OsIRO2 was shown to be an essen-
tial regulator of genes involved in PS synthesis and iron
uptake [42]. Interestingly, putative IDE1 and IDE2
elements are also present in the OsIRO2 promoter,
suggesting that the expression of OsIRO2 is under the
control of the IDEFs [42]. IRO2 binding sites have also
been localized in the promoter regions of other transcrip-
tion factors [42] suggesting that additional activation
steps occur downstream of IRO2 (see Figure 1). Combi-
natorial control that utilizes different cis-elements and trans
acting factors in the iron-deficiency response pathway
may be needed for adjustment to various possible
environmental conditions.
Alteration of iron homeostasis: consequence for
metabolism and oxidative stress
Iron homeostasis is maintained by the coordinated regu-
lation of its transport between the different cellular
compartments, its utilization and its storage. Because
electron transport chains and primary carbon metabolism
require Fe, both the chloroplasts and mitochondria are
major sites for Fe utilization in the cell. The mechanisms
that allow Fe fluxes into and out of these organelles are
Current Opinion in Plant Biology 2009, 12:347–357
350 Physiology and metabolism

Figure 1

Model of the regulatory network that mediates response to Fe deficiency in monocots. In response to Fe-deficiency signals, IDEF1 and IDEF2 may
bind IDE1 and IDE2 motifs present in the promoter of the OsIRO2 gene and activate OsIRO2 expression. Then, IRO2 may activate expression of two
IRO2 binding sequence-containing transcription factors, TF1 (AK073848) and TF2 (AK109390), suggesting that other trans-activation steps may occur
downstream of IRO2. Interestingly, both IDE1, IDE2 and IRO2-binding sequences are present in the promoter region of a subset of genes involved in
the plant Fe-deficiency responses including Fe uptake and homeostasis. Some genes also contain IDEs and IRO2-binding sequences, which suggests
that the different transcription factors that appear to act sequentially in the transduction pathway could also act coordinately in the regulation of the
genes involved in the plant acclimation to low Fe. Solid red lines represent interactions of regulatory proteins with upstream regulatory sequences
based on experimental evidence. Dashed red lines are for proposed interactions.

largely unknown. Two proteins are reported to be
involved in plastidial Fe transport in A. thaliana. The
plastid-localized ferric reductase AtFRO7 may participate
in chloroplast Fe acquisition, and the corresponding
mutant exhibits Fe-deficiency symptoms especially in
the seedling stage [43]. AtPIC1 is targeted to the plas-
tidial inner envelope and complemented iron acquisition
defective yeast mutants [44]. The redox state of the iron
transported by AtPIC1 is unknown. Compared to the fro7
mutant, the pic1 mutant exhibited severe growth defects.
Moreover, unlike pic1, fro7 can be rescued by supplying
Fe. Perhaps there are redundant systems for iron uptake
into the chloroplast that can bypass AtFRO7. Notably,
the observed overaccumulation of ferritins in the pic1
mutant [44] suggests a plastidial Fe content increase,
which would contrast with the expected phenotype of the
mutant. The deregulation of iron transport over chloro-
plast envelopes could lead to strong alteration in iron
homeostasis. In plants ferritins are upregulated on mild
Fe excess and are central components involved in the
maintenance of Fe homeostasis, by preventing free Fe-
induced ROS production in the plastids [24]. In mito-

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 Essential transition metal homeostasis in plants Pilon et al. 351

Figure 2

Model for SPL7 and Cu-microRNA-mediated responses to low Cu availability in A. thaliana. Low cellular Cu activates SPL7-mediated transcription of
genes involved in copper uptake and assimilation as well as the four Cu-microRNAs (miR397, miR398, miR408 and miR857). The Cu-microRNAs in
turn mediate the RISC (RNA induced silencing complex)-dependent cleavage of transcripts that encode non-essential Cu proteins. As a consequence,
essential Cu-proteins such as plastocyanin can obtain their Cu-cofactor when plants are grown in soils with either high and with low Cu availability.

chondria, the frataxin protein may serve a related function
in protection against oxidative stress. In Chlamydomonas,
ferritin is upregulated not by Fe excess but under
deficiency [45,46]. In this condition Chlamydomonas re-
models PSI, a major iron sink and ferritin seems to be
required as a temporary safe storage site in this condition
[46]. Thus, despite the seemingly opposite regulation by
Fe, ferritins in plants and Chlamydomonas have related
functions. Clearly, iron storage within cellular organelles
contributes to the protection against iron reactivity with
oxygen to avoid oxidative stress. The factors involved in
Fe homeostasis that are discussed in this paper are listed
in Table 1.
Copper
Regulation of Cu-protein expression via the
Cu-microRNAs
The delivery of Cu to cellular compartments in A. thaliana
involves AtCOPT transporters for import into the cytosol
and export to organellar compartments or the apoplast by
AtHMA5-8. The response to impending Cu deficiency in
roots and vegetative tissue is well described especially in
A. thaliana (Figure 2). AtCOPT1 expression in roots is
upregulated by Cu limitation. On limited Cu, cytosolic
and plastid Cu/ZnSODs are downregulated in A. thaliana
[47] and a number of other plants in non-stress conditions
[48]. At the same time a FeSOD is upregulated [47,48]. In
Cu-supplemented conditions the reciprocal expression
pattern was observed for the SODs. A microRNA,
miR398 targets mRNAs that encode the Cu/Zn super-
oxide dismutases AtCSD1 and AtCSD2 [49]. Strong
oxidative stress was shown to reduce expression of mature
miR398 levels, which in turn led to increased AtCSD1 and
AtCSD2 mRNA accumulation and thus more CSD1 and
CSD2 activity [49]. However, Cu availability also
directly affects miR398 expression; on low Cu miR398
accumulates while AtCSD1 and AtCSD2 mRNA abun-
dance decreased. The reverse was found on high Cu
[50]. Cu-availability was a much more important factor
for miR398 regulation than oxidative stress [50]. The
presence of sucrose in plant tissue culture media was
found to be another important regulator of miR398 and
Cu/ZnSOD activity independently of Cu [51]. The
biological significance of Cu/ZnSOD regulation by
sucrose is so far not clear. Three additional microRNAs,
miR408, miR397 and miR857 are also Cu regulated
[52]. These microRNAs also target Cu proteins (lac-
cases and plantacyanin). Additional Cu-regulated micro-
RNAs may regulate further laccases, although not all
laccases were found to be Cu regulated [52]. Thus at
least four microRNAs, now called the Cu-microRNAs, are
regulated by Cu availability. We hypothesize that the
induction of the Cu-microRNAs and, therefore, a
reduction in Cu-protein expression during Cu-limited
conditions would allow for preferential allocation of lim-
ited Cu to the most essential Cu proteins such as plas-

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352 Physiology and metabolism

Table 1

Proteins involved in Fe homeostasis discussed in this paper.

Name Description Gene identifier Other names Cellular l Reference(s)

ocalization
Fe homeostasis (Arabidopsis)
AtFH Frataxin homologue At4g03240 – Mitochondria [23]
AtFer1 Ferritin 1 At5g01600 – Plastid [24]
AtFer2 Ferritin 2 At3g11050 – Plastid [24]
AtFer3 Ferritin 3 At3g56090 – Plastid [24]
AtFer4 Ferritin 4 At2g40300 – Plastid [24]
AtFRO7 Ferric Reduction Oxidase 7 At5g49740 – Plastid [43]
membrane
AtPIC1 Permease in Chloroplast 1 At2g15290 AtTIC21 (Translocon at Plastid [44]
Inner Membrane) membrane
AtCIA5 (Chloroplast Import
Apparatus 5)
Fe–S cluster biosynthesis (Arabidopsis)
AtNFS1 Cystein desulfurase At5g65720 AtNIFS1 Mitochondria [25]
AtNFS2 Cystein desulfurase At1g08490 AtCpNIFS Plastid [26]
AtSUFS
AtSufE1 Cystein desulfurase activator At4g26500 AtSULFURE1 Plastid, [27,28]
mitochondria
AtCpSUFE
AtSufE2 Cystein desulfurase activator At1g67810 AtSULFURE2 Plastid [31]
AtSuFE3 Cystein desulfurase activator At5g50210 AtSULFURE3 Plastid [31]
AtQS (Quinolate Synthase)
AtOLD5 (Onset of Leaf
Death 5)
GrxS14 Monothiolglutaredoxin At3g54900 AtCIPX1 (Cax Interacting Plastid [30]
Protein 1)
AtGRXcp
AGrxS16 Monothiolglutaredoxin At2g38270 AtCIPX2 (Cax Interacting Plastid [30]
Protein 2)
AtGRX2
AtNBP35 Nucleotide binding protein 35 At5g50960 – Cytosol [35]
Fe-deficiency signalling (Arabidopsis and rice)
AtIRT1 Iron-regulated transporter 1 At4g19690 – Plasma [4]
membrane
AtFRO2 Ferric reduction oxidase 2 At1g01580 – Plasma [4]
membrane
AtFIT1 Fe-deficiency induced At2g28160 AtBHLH029 Nucleus [36]
transcription factor 1
AtFRU
FER-like
AtBHLH038 Transcription factor At3g56070 AtORG2 (OBP3-responsive Nucleus [37]
gene 2)
AtBHLH039 Transcription factor At3g56980 AtORG3 (OBP3-responsive Nucleus [37]
gene 3)
OsIDEF1 IDE1 (Iron deficiency-responsive AK107456 – Nucleus [39]
element 1)-binding transcription factor
OsIDEF2 IDE2 (Iron deficiency-responsive AK072874 – Nucleus [40]
element 2)-binding transcription factor
OsIRO2 bHLH transcription factor AK073385 – Nucleus [41,42]
OsAP2 AP2-domain transcription factor AK073848 – Nucleus [42]
OsNAC4 NAC-domain transcription factor AK109390 – Nucleus [42]

tocyanin, which is absolutely required for photoauto-
trophic growth in plants.
The master Cu homeostasis regulator SPL7
Whereas plastocyanin is essential in higher plants, Chla-
mydomonas switches between cytochrome c6 (a heme
protein) and plastocyanin (Cu-protein) depending on Cu
availability [53]. This switch is mediated by a transcription
factor called copper response regulator (CRR1), which
binds to a GTAC-containing core motif in the promoters
of Cu-regulated genes [53]. CRR1 expression is not
regulated by Cu, which suggests that the presence or
absence of Cu causes a change in protein activity. CRR1
belongs to the family SPL (SQUAMOSA promoter-binding

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like) transcription factors, of which there are over a dozen
members in Arabidopsis. The closest homologue in A.
thaliana is AtSPL7 [54]. Interestingly, AtSPL7 activates
miR398 transcription by binding at a GTAC core motif
present in miR398b and c during Cu-limitation [54]. In an
spl7 T-DNA mutant miR397, miR398, miR408 and
miR857 were not detected even when Cu was limited.
spl7 mutants grown on low Cu demonstrated severe growth
phenotypes, which indicates that AtSPL7 is an important
regulator during Cu-limitation [54]. The analysis of a
moss CRR1 and SPL7 homologue called PpSBP2 indicated
that it binds to a GTACT core motif of the FeSOD
promoter regulating the expression of this gene in response
to Cu [55]. In A. thaliana, spl7 mutants did not increase
FeSOD expression during Cu-limitation [54].
AtSPL7 seems to be a master regulator for the response to
low Cu. Wild-type A. thaliana plants increase mRNA
expression for the transporters COPT1, COPT2, ZIP2,
FRO3 and YSL2 and the Cu-chaperone CCH when plants
are Cu-limited [54,56,57], but in the spl7 mutant the
mRNA of these transporters did not increase [54].
Surprisingly, AtSPL7 is expressed highly in the roots
although expression is detected throughout the plant
[54]. The Cu-microRNAs are found throughout the
plant [52,54]. The high expression of AtSPL7 in
the roots could suggest a role in detecting Cu availability
at the site where Cu enters the plant, after which it
orchestrates whole plant Cu delivery. miR398 and
miR408 were found in the phloem of Brassica napus,
which suggests that Cu homeostasis signals could move
systemically throughout the plant [58]. This method of
signal delivery could be very important for young devel-
oping leaves during Cu-limitation so that proper Cu
delivery to essential Cu-proteins is maintained during
initial development. Some other transporters such as
AtHMA5 and AtFRO6 are also regulated by Cu, but
AtSPL7 activity does not seem to influence these
[54]. AtHMA5 is induced by high Cu and believed to
be involved in removing excess Cu from the cytosol to the
apoplast [56] similar to the role of AtHMA2 and AtHMA4
in Zn homeostasis. The Cu-chaperone AtATX1 that
interacts with AtHMA5 is constitutively expressed and
not under AtSPL7 control [54]. In vegetative tissue the
expression of AtFRO6, a possible metal reductase, is
decreased during Cu-limitation [57] but the mechanism
for this suppression has not been determined. It is
possible that yet another Cu sensitive regulatory mech-
anism for these transporters exists. In this context it may
be noted that next to AtSPL7, two additional SPL tran-
scription factors, AtSPL1 and AtSPL12, are related in
sequence to CRR1 albeit more distantly [53].
Cu-protein accumulation requires Cu supply. Cu is deliv-
ered to Cu/ZnSOD by the chaperone CCS [59]. Chlor-
oplastic Cu/ZnSOD levels are drastically reduced in A.
thaliana plants that are deficient in AtCCS or in the
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Essential transition metal homeostasis in plants Pilon et al. 353
AtPAA1 Cu-transporter [47,59]. Arabidopsis and most
other higher plants have two plastocyanins, PC1 and
PC2. PC1 is expressed at low levels relative to PC2.
The two plastocyanin proteins seem to have a similar
function with respect to electron transport and mRNAs
levels of both genes are not affected by Cu [60]. However,
PC2 protein accumulates strongly as Cu levels increase.
Interestingly, this PC2 accumulation did not positively
correlate with increased photosynthetic capacity,
suggesting that PC2 may serve an additional function
to sequester excess Cu [60]. PC1 seemed more tolerant to
reduced Cu levels and may serve to provide a basal level
of electron transport capacity even when Cu levels
become limiting.
SPL7 could affect other metals besides Cu. SPL7-
mediated and miR398-mediated downregulation of
CuZnSODs reduces the sink size for both Cu and Zn
but is also accompanied by an increase in FeSOD expres-
sion, increasing the pool size for Fe. The AtYSL2 pro-
moter contains 5 GTAC core motifs and is regulated by
AtSPL7 in response to Cu [54]; this transporter could
transport Fe, Zn or Cu. It is striking that the microRNA-
mediated downregulation is only seen for Cu proteins.
Perhaps only Cu binds its target sites so tightly that the
absence of these sites is required to allow the remaining
Cu to be distributed to other essential targets. It is
possible that Cu-protein accumulation, which is partly
regulated by the Cu-microRNAs and partly by protein
stability, allows plants to store Cu when it is present in
excess of physiological needs. Such buffering capacity
could be meaningful in times that Cu needs to be re-
allocated to new tissue or it could serve an ecological role,
allowing plants to sequester Cu that otherwise could be
used by competitors.
Concluding remarks and outlook
Whereas much is still to be learned about Mn and Zn use,
transport and homeostasis, we seem to be much closer to a
full understanding of Fe and especially Cu homeostasis.
It is evident that the regulation of metal ion homeostasis
is largely mediated by membrane transporters. It is clear
that in many cases transcriptional control of these trans-
porters contributes to homeostasis, but too little is known
about post-transcriptional and post-translational control
of transporter activity. Compared to yeast and Chlamydo-
monas, where there is clear evidence for interaction be-
tween Fe and Cu homeostasis, there is limited evidence
for coordination of the regulatory networks that control
Mn, Zn, Fe and Cu homeostasis in plants. However, there
is some crosstalk. Because Fe, Mn and Zn are all taken up
by the IRT1 transporter, these metals affect each other’s
uptake. For example, low Fe leads to IRT upregulation,
which in turn allows more Zn uptake. Zn toxicity could
therefore be a secondary effect of Fe deficiency. Further-
more, Fe and Cu both need to be reduced before import,
which requires FRO2 (for details see [4]). Therefore Cu
Current Opinion in Plant Biology 2009, 12:347–357
354 Physiology and metabolism
and Fe affect each other’s uptake. Finally FeSOD and
CuZnSOD expression is reciprocal in plastids.
A more detailed understanding of cellular metal ion
homeostasis will allow us to see how these systems are
integrated to allow homeostasis at a whole plant level. For
agriculture it will be especially important to understand
the regulation of seed loading of these essential transition
elements. In this respect it was interesting that a single
NAC transcription factor (NAM-B1) affects senescence
and both Zn and Fe content of wheat grains as well as
protein content [61]. It will be interesting to see how the
activity of this NAC transcription factor and other more
global regulators can be connected to known homeostasis
models. As can be expected, there is evidence for this
type of whole plant control, at least for Fe. A defect in the
biogenesis of chloroplast thylakoids, a major Fe sink,
affects Fe uptake [62] and the hormone cytokinin affects
both growth and Fe uptake [63]. Therefore, Fe uptake
seems to be integrated with utilization and growth.
Acknowledgements
We apologize to authors whose work could not be cited owing to space
limitations. Work in Marinus Pilon’s lab was supported by NSF grant
#NSF-IBN-0418993. Marinus Pilon’s sabbatical stay in the group of Dr
Gaymard and Dr Briat at the Laboratoire de Biochimie et Physiologie
Moléculaire des Plantes, UMR 5004 Supagro/CNRS/INRA/UMII was made
possible by funds from the Centre National de la Recherche Scientifique
(CNRS) and the Agropolis Foundation.
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Current Opinion in Plant Biology 2009, 12:347–357
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Essential transition metal homeostasis in plants Pilon et al. 357
A yeast expression system was used to study the kinetics of Zn transport
of wild-type AtMTP1 as well as a mutant that lacks a cytoplasmic
histidine-rich loop.
66. Gustin JL, Loureiro ME, Kim D, Na G, Tikhonova M, Salt DE:
 MTP1-dependent Zn sequestration into shoot vacuoles
suggests dual roles in Zn tolerance and accumulation in
Zn-hyperaccumulating plants. Plant J 2009, 57:1116-1127.
MTP1 from the Zn hyperaccumulator Thlaspi goesingense was localized
to the tonoplast and not the plasmalemma. Expression in A. thaliana
indicates that the protein mediates Zn sequestration in vacuoles and
tolerance. Furthermore, high MTP1 activity induces a systemic Zn defi-
ciency response characteristic of hyperaccumulators.
Current Opinion in Plant Biology 2009, 12:347–357