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Copper homeostasis
Author for correspondence:
Marinus Pilon
Tel: +1 970 491 0803
Email: [email protected]
Jason L. Burkhead, Kathryn A. Gogolin Reynolds, Salah E. Abdel-Ghany,
Christopher M. Cohu and Marinus Pilon
Biology Deparment, Colorado State University, Fort Collins, CO 80523-1878, USA
Received: 16 December 2008
Accepted: 2 March 2009
Contents
Summary
799
VII. Uptake in the root and distribution to aerial tissues
804
I.
Introduction
800
VIII. Uptake in the shoot symplast, redistribution of Cu
during flowering, seed set and senescence
806
II.
The origins of Cu homeostasis
800
IX.
Cu delivery inside the cell
806
III.
Copper homeostasis in unicellular photosynthetic
model organisms
801
X.
Regulation of Cu homeostasis
809
IV.
Functions of Cu in plants
802
XI.
Conclusions and outlook
811
V.
Typical levels of Cu in plants, deficiency and toxicity
802
Acknowledgements
811
VI.
Copper abundance in soils and appropriate Cu
concentrations in media
References
811
804
Summary
New Phytologist (2009) 182: 799–816
doi: 10.1111/j.1469-8137.2009.02846.x
Key words: copper (Cu), deficiency,
homeostasis, metallochaperone,
photosynthesis, regulation, small RNA,
transport.
© The Authors (2009)
Journal compilation © New Phytologist (2009)
Copper (Cu) is a cofactor in proteins that are involved in electron transfer reactions
and is an essential micronutrient for plants. Copper delivery is accomplished by the
concerted action of a set of evolutionarily conserved transporters and metallochaperones. As a result of regulation of transporters in the root and the rarity of natural
soils with high Cu levels, very few plants in nature will experience Cu in toxic excess
in their tissues. However, low Cu bioavailability can limit plant productivity and plants
have an interesting response to impending Cu deficiency, which is regulated by an
evolutionarily conserved master switch. When Cu supply is insufficient, systems to
increase uptake are activated and the available Cu is utilized with economy. A number
of Cu-regulated small RNA molecules, the Cu-microRNAs, are used to downregulate
Cu proteins that are seemingly not essential. On low Cu, the Cu-microRNAs are upregulated by the master Cu-responsive transcription factor SPL7, which also activates
expression of genes involved in Cu assimilation. This regulation allows the most important proteins, which are required for photo-autotrophic growth, to remain active over
a wide range of Cu concentrations and this should broaden the range where plants
can thrive.
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Fig. 1 Models for copper (Cu) homeostasis in two heterotrophs, Enterococcus hirae (a) and Saccharomyces cerevisiae (b). (a) The cop operon
of E. hirae is required for Cu tolerance and encodes the two Cu-transporting P-type ATPases CopA and CopB, the Cu-chaperone CopZ which
provides Cu to CopB and to the Cu-regulated repressor, CopY. CopB and CopZ function as an extrusion system. The biological function of CopA
is not completely clear. It might provide Cu to activate CopZ and affect gene expression via CopY. The most likely directions for transport are
indicated in the figure but direct evidence for Cu transport by CopA into the cell is still lacking. For details see Magnani & Solioz (2007).
(b) Copper homeostasis in yeast. Major targets for Cu delivery in yeast are the cytosolic Cu/ZnSOD (Sod1), a cell surface ferric reductase (Fet3),
and cytochrome c oxidase (Cco) in the mitochondria. Copper is acquired by the high-affinity transporters Ctr1 and Ctr3. Lys7/Ccs is a Cu
chaperone for the cytosolic Cu/Zn superoxide dismutase Sod1. The Cu-chaperone for superoxide dismutase (SOD), CCS, has an N-terminal
Atx-like heavy-metal binding domain, a central region with similarity to its target Cu/ZnSOD and a C-terminal domain with two additional
cysteine residues. Copper is delivered to the Golgi-localized Cu-transporting P-type ATPase Ccc2 by the Atx1 Cu chaperone. Ccc2p supplies Cu
for Fet3p, the plasma membrane multicopper oxidase that is in turn required for high-affinity Fe assimilation. The Cu chaperones Cox17, Sco1
and Cox11 act in the mitochondrial inter-membrane space and deliver Cu ions that originate from a Cu pool in the mitochondrial matrix to
mitochondrial cytochrome c oxidase. The Cu-binding metallothionein-like proteins Cup1 and Crs5 bind excess Cu. All of these yeast proteins have
homologues in mammals and plants. Intracellular levels of Cu in yeast are largely controlled by regulation of the high-affinity transporter Ctr1
and its homologues with responses to Cu levels transcriptionally regulated by Mac1 and Ace1. Mac1 induces transcriptional activation of Ctr1,
Ctr3 and the Cu reductase Fre1 under Cu-limiting conditions. A switch to high Cu levels induces internalization and degradation of Ctr1, as well as
Ace1-stimulated transcription of Cu/ZnSOD and the yeast metallothioneins. White rectangles, Cu reductases; gray rectangles, copper transporters;
white hexagons, metallothioneins; white circles, Cu targets. Solid lines depict interactions demonstrated through experimental data. For details
see: Gralla et al. (1991); Dancis et al. (1994); Culotta et al. (1997); Pena et al. (1998, 2000); Harrison et al. (1999); Carr & Winge (2003).
I. Introduction
The transition metal copper (Cu) is essential to life on earth as
a cofactor in numerous proteins (Linder & Goode, 1991). The
utility of Cu in biochemical reactions results from its ability
to cycle between oxidized Cu(II) and reduced Cu(I) states in
enzymes and electron carriers (Lippard & Berg, 1994). The
majority of Cu proteins are active in electron transport or
function as enzymes that catalyse redox reactions that involve
oxygen (Linder & Goode, 1991). However, Cu can also be toxic
when in excess. Redox cycling between Cu1+ and Cu2+ can also
catalyse the production of highly toxic hydroxyl radicals, with
subsequent damage to macromolecules (Halliwell & Gutteridge,
1984). In addition, Cu is highly reactive to thiols and can possibly
displace other essential metals in proteins (Lippard & Berg,
1994). Because of this, Cu homeostasis is tightly controlled in
all organisms.
II. The origins of Cu homeostasis
Many of the key components that function in Cu homeostasis
in plants are evolutionarily conserved. It is likely that the oldest
Cu homeostasis factors functioned in detoxification and origi-
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nated before Cu had an essential role in metabolism. Early in
the history of life, iron (Fe) must have been readily available in
the anoxic biosphere and would have had roles in catalysis and
redox reactions, including those now filled by Cu enzymes.
Copper probably did not become available to living organisms
in significant amounts before the appearance of cyanobacteria
and dioxygen-generating photosynthesis (Harrison & Hoare,
1980; Chapman & Schopf, 1983). Indeed, a bioinformatic
comparison of sequenced genomes reveals a strong link between
the utilization of Cu in biochemistry and oxygen (Ridge et al.,
2008). The oxidizing atmosphere could liberate Cu from insoluble sulfide salts, yielding the soluble Cu(II) form; at the same
time Fe became less available because of the formation of
insoluble iron oxides (Chapman & Schopf, 1983). It can be
hypothesized that organisms in the early oxidizing atmosphere
would initially require Cu detoxification systems, probably
similar to the systems utilized by present-day bacteria (Fig. 1a).
The analysis of Cu homeostasis in all of the main branches
of the tree of life has made it apparent that much of the
machinery which functions to provide Cu tolerance in
bacteria is conserved in mammals, yeast (Fig. 1b) and higher
plants (see later) where it serves roles in delivery to specific
targets. Most likely, the ancient sequestration and extrusion
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Fig. 2 Copper (Cu) homeostasis in the cyanobacterial model
Synechocystis. Solid lines, interactions demonstrated through
experimental data; gray rectangles, copper transporters of the P-type
ATPase family; white circles, copper targets. Atx1 is a Cu chaperone;
CO, cytochrome c oxidase; PC, plastocyanin. See text for details.
systems were linked with new uptake systems to form a
cellular Cu homeostasis and delivery system for the efficient
incorporation of Cu into biomolecules. This allowed Cu
to participate in directed chemical reactions, often replacing
Fe-containing enzymes. Interestingly, many Cu proteins have
a functional counterpart that uses Fe (see Merchant et al.,
2006) and sometimes one can serve as backup for the other
during deficiency.
III. Copper homeostasis in unicellular
photosynthetic model organisms
Because much of the Cu homeostasis machinery found in cyanobacteria and green algae is conserved in plants, it is useful to
give a brief overview of these systems. The cyanobacteria, which
share a common ancestor with plant chloroplasts, are responsible
for creating the oxidizing atmosphere in which most of life must
operate. Cyanobacteria acquire Cu for cytochrome c oxidase and
the thylakoid lumen photosynthetic electron carrier plastocyanin
(Fig. 2). However, some cyanobacteria such as Synechocystis and
Anabaena can replace plastocyanin with cytochrome c6 under
Cu deficiency (Bovy et al., 1992; Zhang et al., 1992). Copper is
delivered by two P-type ATPases: CtaA in the plasma membrane
(Phung et al., 1994) and PacS in the thylakoid membrane
(Kanamura et al., 1994; Tottey et al., 2001). Mutants in CtaA
are slightly less sensitive to excess Cu, whereas mutants in PacS
are more sensitive to excess Cu. These findings indicate that these
transporters also have a role in Cu homeostasis. In this respect,
PacS is similar to Enterococcus hirae CopB, which exports Cu
from the cytosol (Magnani & Solioz, 2007). In Synechocystis
PCC 6803 the Atx1 Cu chaperone specifically interacts with
CtaA and PacS and functions to supply Cu for both plastocyanin
and cytochrome c oxidase (Tottey et al., 2002). Work in Synecho-
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cystis PCC 6803 has shown that the location of protein folding
may affect metal occupancy (Tottey et al., 2008). In vivo, the
periplasmic proteins CucA and MncA bind Cu2+ and manganese
(Mn2+) respectively. The proteins have similar folds and
metal chelation sites, which raises the question of how the
specificity in metal binding can be explained. When expressed
and folded in vitro MncA prefers to bind Cu2+ over Mn2+. In
vivo, however, McnA folds in the cytoplasm, where most Cu
is chelated but where a pool of free Mn exists, and McnA
is subsequently exported by the Tat pathway to the periplasm
as a folded protein with bound Mn. Once in the periplasm Cu
can no longer replace Mn in the folded protein. CucA is
exported in an unfolded state via the Sec pathway and picks
up Cu2+ in the periplasm because of the higher binding
affinity of this metal (Tottey et al., 2008).
The main targets for Cu in the eukaryotic green alga Chlamydomonas rheinhardtii are plastocyanin, cytochrome c oxidase
and FOX1, a homolog of the yeast cell surface ferric reductase
(for a review see Merchant et al., 2006). Chlamydomonas can
adjust to low Cu availability because it has backup systems for
plastocyanin and FOX1, and a regulatory switch. In Chlamydomonas, plastocyanin can be functionally replaced by a cytochrome c6 when Cu levels are insufficient and Cu deficiency
induces proteolysis of plastocyanin (Li & Merchant, 1995).
However, a mutation that causes a loss of plastocyanin function
results in a complete loss of photosynthetic growth when cells
are grown with sufficient Cu to suppress the expression of cytochrome c6 (reviewed in Merchant et al., 2006). The Chlamydomonas genome encodes homologs of Ctr transporters and three
Cu-transporting P-type ATPases (Hanikenne et al., 2005). Two
of these may be functional homologs of cyanobacterial CtaA
and PacS and higher plant PAA1 and PAA2, which function to
provide Cu to plastocyanin (Hanikenne et al., 2005). Homologs
of the yeast Cu-chaperone Atx1 and the transporter Ccc2 have
been identified in Chlamydomonas (La Fontaine et al., 2002),
and these components probably function to provide Cu to the
FOX1 ferric reductase for Fe acquisition.
The Chlamydomonas protein CRR1 (Cu Response Regulator)
is a transcription factor that regulates the response to Cu deficiency (Kropat et al., 2005). The targets of CRR1 (e.g. the CYC6
gene which encodes cytochrome c6) contain a Cu-responsive
regulatory element (CuRe) that includes a GTAC core motif.
This cis-acting element is required for activation by CRR1 under
low Cu. The CRR1 protein has similarity to higher plant SPL
(SQUAMOSA PROMOTER BINDING PROTEIN-LIKE)
transcription factors (Cardon et al., 1999; Kropat et al., 2005).
It contains a possible zinc-binding SPL DNA binding motif
and a C-terminal cysteine-rich region. These domains of CRR1
may be required for the regulation by Cu (Kropat et al., 2005)
but the exact mechanism by which CRR1 regulates gene expression has not yet been reported. It is likely that CRR1 is a metalsensing transcription factor. CRR1 and its plant homologs are
of interest as very few metal sensors have been reported in
higher eukaryotes.
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Fig. 3 Copper (Cu) transport pathways in
Arabidopsis thaliana. Solid lines, interactions
demonstrated through experimental data;
dashed lines, hypothetical pathways; gray
rectangles, copper transporters; white circles,
copper targets. Metallothionein (MT) proteins
may help buffer intracellular Cu levels. See
text for details.
IV. Functions of Cu in plants
1. Symptoms of deficiency
Experiments with tomato established Cu as an essential nutrient
for plants (Arnon & Stout, 1939). Copper plays roles in photosynthesis, respiration, perception of ethylene, reactive oxygen
metabolism and cell wall remodeling. A listing of the main Cu
proteins found in plants is given in Table 1 and the major targets
are also indicated in Fig. 3. In addition to its role as a stable
cofactor Cu may also play a role in thylakoid grana stacking
(see for example Bernal et al., 2006). There is a possible indirect
role for Cu in nitrogen assimilation and abscisic acid (ABA) biosynthesis, which are functions of molybdenum (Mo) cofactorrequiring enzymes. While determining the structure of CNX1,
an enzyme that functions in Mo cofactor synthesis, it was found
that a Cu ion temporarily occupies the site for Mo insertion in
the bound molybdopterin substrate (Kuper et al., 2004). Unlike
what has been found for yeast and Chlamydomonas, there is no
direct requirement for Cu in Fe acquisition in plants.
Symptoms of Cu deficiency include decreased growth rate,
distortion or whitening (chlorosis) of young leaves, curling of
leaf margins and damage to the apical meristem, as well as a
decrease in fruit formation (for reviews see: Marschner, 1995;
Epstein & Bloom, 2005). Copper deficiency in forests severely
affects wood production (Ruiter, 1969). A secondary effect of
Cu deficiency can be insufficient water transport caused by a
decrease in cell wall formation and lignification in several tissues,
including xylem tissue (Marschner, 1995). Because of the
elevated levels of Cu found in reproductive tissue, deficiency has
a severe effect on pollen development and viability, fruit and
seed production, in addition to embryo development and seed
viability (see also below).
V. Typical levels of Cu in plants, deficiency and
toxicity
Epstein & Bloom (2005) report Cu levels in plants ranging
from 2 to 50 µg g−1 DW (ppm) with 6 µg g−1 considered as
adequate in the shoots. The amount of Cu contained in healthy
plants varies considerably within this range and depends both
on the species and the Cu-feeding status (Cohu & Pilon, 2007).
Typically, symptoms of deficiency start when Cu decreases below
5 µg g−1 DW in vegetative tissues, while toxicity levels are
observed above 20 µg g−1 DW or higher in the same tissue
(Marschner, 1995).
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2. Symptoms of toxic excess
Copper toxicity thresholds vary greatly between species of plants
and affect tissues differently depending on metabolic requirements. Excess Cu concentrations in the soil tend to decrease root
growth before shoot growth because of preferential Cu accumulation in that organ (Alaoui-Sossé et al., 2004; Navari-Izzo et al.,
2006). The most common general symptom of toxicity is
chlorosis of vegetative tissue. Copper toxicity can also reduce Fe
uptake, even to the point of deficiency, depending on the form
of Fe available in the soil (Marschner, 1995). In the shoot, the
thylakoid membrane in the chloroplast, especially photosystem
II (PSII), is a primary target of Cu toxicity (Yruela et al., 1996,
Bernal et al., 2004). Both PSII and chlorophyll are major targets
for Cu toxicity in aquatic algae (Küpper et al., 2003).
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Table 1 Functions and cellular locations major copper (Cu) proteins in plants
Protein
Cellular location
Function
No. of Cu
Remarks
References
Plastocyanin
Thylakoid lumen
Electron transport
1
Electron transport, proton
pumping, terminal oxidase
Dismutation of superoxide:
2O2– + 2H+ → H2O2 + O2
Hormone perception
Cell wall modeling
Metabolism of phenolic
compounds (anthocyanins
and lignin )
Suggested roles in cell
expansion, salt tolerance
Cell wall differentiation,
wound healing; response
to pathogens
3 per unit
Two genes in most plants
(loss of both copies is lethal)
Three core Cu-binding subunits
encoded in MT genome
Active as dimer
Katoh (1960); Weigel et al. (2003);
Pesaresi et al. (2009); Abdel-Ghany (2009)
Carr and Winge (2003)a;
Welchen et al. (2004)
Bowler et al. (1992)a;
Kliebenstein et al. (1998)
Rodriguez et al. (1999); Chen et al. (2002)
Gavnholt & Larsen (2002); McCaig et al.
(2005); Pourcel et al. (2005); Nakamura
& Go (2005); Cai et al. (2006)
Cytochrome c
oxidase
Cu/ZnSOD
Mitochondrial
inner membrane
cytosol (CSD1) stroma (CSD2)
peroxisome (CSD3)
Ethylene receptors Endoplasmic reticulum
Laccase
Apoplast (secretory pathway)
Ascorbate oxidase Apoplast
Amine oxidase
Apoplast
Phytocyanin
(plantacyanin)
Polyphenol
oxidase
Apoplast
Thylakoid lumen
1 per
mono-mer
1 per unit Five genes in Arabidopsis
4
Multigene family in plants (17 members
in. Arabidopsis thaliana) Multicopper
oxidase related to ascorbate
oxidase, ceruloplasmin + ferroxidase
4
Multicopper oxidase
Pignocchi et al. (2003);
Yamamoto et al. (2005)
Kumar et al. (1996); Frebort et al. (2000);
Rea et al. (2002); Paschalidis et al. (2005)
An et al. (2008); Angelini et al. (2008);
Marina et al. (2008)
Kim et al. (2003); Dong et al. (2005)
1
Contain a special topaquinone
group formed from tyrosine.
Abundant in pea cell walls
Possible roles in reproduction
1
Structurally similar to plastocyanin
Conversion of monophenols to
diphenols; conversion of dihydroxy
phenols to orthoquinones
2
Proposed role in wounding and pathogen Arnon (1949); Thipyapong et al. (1997);
Mayer (2006)a; Schubert et al. (2002)
responses. Seven members in tomato;
absent in Arabidopsis
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SOD, superoxide dismutase. aReviews.
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VI. Copper abundance in soils and appropriate Cu
concentrations in media
Copper concentrations range from 3 to 110 ppm with an average
abundance of 55 ppm in the earth’s crust (Misra, 2000). Copper
is comparable in average abundance to zinc (Zn, 70 ppm) but
is rare when compared with Fe (50 000 ppm), aluminum (Al,
81 000 ppm) and Mn (950 ppm). The bioavailability of Cu
to plants is dependent on the soil type. Copper, especially as
Cu(II), has a high affinity to bind to organic matter and therefore, organic soils are more likely to be Cu deficient (Marschner,
1995). Even though Cu tends to bind to agar, it is much more
bioavailable in agar media when compared with soils. Copper
is even more bioavailable in hydroponic growth media. For
example, Arabidopsis plants grown on agar medium with Cu
below 0.05 µm (0.0032 ppm) start to become deficient (AbdelGhany et al., 2005a; Cohu & Pilon, 2007) whereas toxicity
occurs above 20 µm (1.27 ppm) in agar media (Murphy & Taiz,
1995). Standard Murashige and Skoog (MS; Murashige &
Skoog, 1962) agar medium has 0.1 µm Cu and is slightly
deficient in Cu. For Arabidopsis in hydroponic media, Cu is
only deficient below 5 nm while 100 nm or higher is already
toxic (Abdel-Ghany & Pilon, 2008).
VII. Uptake in the root and distribution to aerial
tissues
1. Cu uptake
Copper most likely enters the cytosol of root cells via a cell
surface COPT-family transporter (Kampfenkel et al., 1995;
Sancenon et al., 2003, 2004). The COPT transporters (Fig. 4a)
belong to the conserved Ctr family (Dancis et al., 1994). There
are four expressed members of the COPT family in Arabidopsis
(Sancenon et al., 2003). Some of the COPT proteins may be
active at the plasma membrane whereas others may be active
in internal membranes such as the vacuoles, facilitating release
from intracellular stores. Perhaps COPT1, which is highly
expressed in roots (Sancenon et al., 2004), and COPT2, which
is highly expressed in shoots (Sancenon et al., 2003; Wintz et al.,
2003) are the key Cu-regulated cell surface localized uptake
systems, while COPT3 and COPT5 may function in mobilization of Cu from intracellular stores. However, for plant COPT
proteins, the complete expression pattern and subcellular localization for all of the gene products has not yet been reported.
The COPT/Ctr-like proteins transport Cu in a reduced form
(Eisses & Kaplan 2005) but the most bioavailable form of Cu
in soils is Cu(II). Therefore, reduction of Cu will likely facilitate
uptake into the root cells. Arabidopsis and other dicots use a
root cell surface ferric reductase, FRO2, for Fe uptake (Robinson et al., 1999). This ferric reductase activity may also facilitate
the uptake of Cu (Welch et al., 1993). Plants have reduced Fe
uptake activity and reduced Fe content when given excess Cu,
and vice versa (Welch et al., 1993; Chen et al., 2004). There are
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Fig. 4 Schematic diagrams of the likely topologies of the Ctr/CopT
family of transporters (a) and the heavy metal P1B-type ATPase
transporters (b). (a) Ctr transporters have three transmembrane
domains and a high methionine content, which is thought to play a
role in copper (Cu) translocation. The Ctr proteins likely act as trimers
and transport Cu in the form of Cu(I). These transporters are not ATP
dependent and the high chelating capacity of the cytosol may provide
the driving force for Cu(I) transport by the Ctr transporters.
(b) Cu-transporting P-type ATPases utilize the energy of ATP
hydrolysis to transport Cu(I), in most cases, out of the cytosol.
Ion specificity is determined by a sequence motif (CPx) in the sixth
transmembrane region, together with sequences in transmembrane
regions 7 and 8. Most prokaryotic Cu transporting P-type ATPases
have a single Atx-like N-terminal heavy-metal binding domain that
may be regulatory. Eukaryotic transporters can have two or
sometimes six of these domains. The actuator (A) domain and the
ATPase domain together mediate phosphorylation state-driven
conformational changes, which allow the protein to function as an
ATP-dependent pump.
eight genes in the FRO family in Arabidopsis. In addition to
FRO2, FRO3 may be involved in Cu uptake. FRO3 is highly
expressed in roots, especially in lateral roots and in the vasculature, and FRO3 is induced by Cu deficiency (Mukherjee
et al., 2006).
The COPT1 transporter is likely to be active in the plasma
membrane and its expression is negatively regulated by Cu
(Sancenon et al., 2003, 2004). The gene is highly expressed in
root tips, stomata, trichomes, pollen and embryos; all these cell
types lack functional plasmodesmata, which would block nutrient diffusion via a symplastic route (Sancenon et al., 2004).
Antisense silenced COPT1 plants had lower Cu levels as a result
of decreased Cu uptake and these plants were sensitive to Cu
chelators (Sancenon et al., 2004). Plants with reduced COPT1
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expression also show a pollen development defect and a root
elongation phenotype, both of which are reversed by Cu feeding
(Sancenon et al., 2004). The genes for the COPT family members, COPT1, COPT2, COPT3 and COPT5 as well as ZIP2
and ZIP4 complement a yeast ctr1 deletion mutant defective in
high-affinity Cu uptake (Sancenon et al., 2003, Wintz et al.,
2003). Therefore, ZIP2 and ZIP4 (members of the ATPindependent ZIP family of transporters) may function in addition to COPT transporters for Cu uptake in plant cells (Wintz
et al., 2003). A link with Cu for ZIP2 and ZIP4 was found by
expression profiling of plants grown on low Cu (Wintz et al.,
2003). The same study reports that ZIP2 expression was relatively higher in roots, while ZIP4 was expressed more in the
leaves (Wintz et al., 2003).
2. Responses to subtoxic Cu excess
Non-tolerant species, such as Arabidopsis, can use chelation and
export as mechanisms to avoid Cu-induced damage in the
cytosol during moderate Cu excess. The cysteine-rich metallothionein (MT) proteins, which are upregulated by Cu stress
(Zhou & Goldsbrough, 1994), and possibly the glutathionederived phytochelatins may buffer cytosolic Cu concentrations
(for a review see Cobbett & Goldsbrough, 2002). There are
four types of MT in Arabidopsis and each type of plant MT
could have a role in binding Cu and Zn, based on overexpression
studies in yeast (Guo et al., 2003, 2008). Arabidopsis plants
that lack individual MT1a and MT2b accumulate 30% less
Cu in the roots when treated with high concentrations of
Cu. These plants show very mild phenotypes compared with
the wild type even with elevated concentrations of Cu and
Zn (Guo et al., 2008). Plants that lack both MT1a/MT2b
and phytochelatin showed more exacerbated phenotypes on
elevated Cu (Guo et al., 2008). A full understanding of the
physiological functions of MTs may require the analysis of
plants in which multiple MTs are disrupted (Guo et al., 2008).
The regulation of MT genes by metal ions may be complex.
In pea plants, MT mRNA levels were found to be drastically
upregulated on low Fe whereas mild upregulation was observed
on low Cu (Fordham-Skelton et al., 1997). In addition to the
MTs, the HMA5 transporter, which exports Cu from the cell
(see below) contributes to reducing the possible toxic effects
of Cu overload.
It would be interesting to know how plants sense Cu excess
and transmit this signal to affect physiology and gene expression. In wheat seedlings, increased phospholipase D activity
(Navari-Izzo et al., 2006) and a spike in the production of reactive oxygen species (Sgherri et al., 2007) have been reported as
possible early signals during Cu excess in roots. However, the
CuSO4 concentration that was used in these studies was 100 µm
(Navari-Izzo et al., 2006; Sgherri et al., 2007) which is so high
that we suspect that these data likely have bearing on a general
oxidative stress response and perhaps have less relevance for
Cu homeostasis.
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3. Copper-tolerant plants
There are a number of plant species that hyperaccumulate
specific metal ions such as Zn, Ni, arsenic (As), or selenium (Se)
in their shoots to levels that are orders of magnitude above toxic
levels for normal plants. These hyperaccumulators thrive when
the metal contents are high and it is likely that such toxic metal
levels provide protection against herbivory or microbial attack
(Boyd, 2007). There are very few reports of possible Cu accumulators, but such plants have been reported in Zaire in central
Africa and in China (for a review see Reeves & Baker, 2000).
The reported Cu contents of these possible Cu accumulators
(sometimes above of 6000 mg kg−1 in shoots) are highly variable
(Reeves & Baker, 2000). In several cases, the high levels of Cu
in plants from central Africa could be attributed to dust that
could be removed by washing (Faucon et al., 2007). It would
be interesting to see how the putative Cu hyperaccumulators
plants perform in glasshouse conditions with controlled Cu
levels. Plants that survive on old Cu mine wastes such as Elsholtzia
splendens in central China have increased Cu tolerance and have
two- to three-fold fold elevated Cu contents relative to nontolerant species (Tang et al., 1999). Nevertheless, these plants still
seem to be mainly excluders that avoid excessive Cu levels in
their tissues and even these plants suffer from high Cu levels
(Wang et al., 2008).
4. Root to shoot transport
Copper must be exported from the root symplast before entering
the xylem. Transpiration would take Cu through the xylem to
mature leaves where it could be loaded into the phloem in order
to reach sink tissues such as newly developing leaves, flowers and
seeds. Most likely, export from the root symplast involves the
action of the Cu-transporting P-type ATPase HMA5 (AndrésColás et al., 2006). A schematic structure of Cu-transporting
P-type ATPases is given in Fig. 4b. The accumulation of Cu in
roots of a hma5 mutant is consistent with a role of this transporter
in export from the cell (Andrés-Colás et al., 2006). The HMA5
gene is expressed mainly in roots and flowers (Andrés-Colás et al.,
2006). The Cu sensitive phenotype of hma5 is the opposite of
the phenotype observed for a COPT1 antisense line, which
supports the idea that COPT1 and HMA5 transport Cu in
opposite directions (Sancenon et al., 2004; Andrés-Colás et al.,
2006). Typically, plants that are given excess Cu have elevated
Cu in the roots and several studies report that excess Cu does
not reach the shoot, at least in the first days of Cu exposure
(Alaoui-Sossé et al., 2004; Navari-Izzo et al., 2006). Thus, export
from root cells and long-distance transport to the shoot may
be limiting in the translocation of excess Cu. The P-type ATPases
such as HMA5 most likely export Cu as Cu(I). However, it is
not known for certain in which oxidation state Cu travels in
the xylem.
Long-distance transport may involve chelators such as the
methionine-derived compound nicotianamine. Nicotianamine
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is a metal-chelating compound derived from methionine that
is implicated in the transport of Fe and possibly other metal
ions in plants (see Briat et al., 2007). The tomato mutant chloronerva, which lacks nicotianamine, overaccumulates Fe, Zn,
Mn and Cu, with Cu accumulation restricted to the roots
(Scholtz et al., 1987; Stephan & Grun, 1989). Nicotianamine
has a high affinity for Cu and Cu transport in the xylem is greatly
reduced in the chloronerva mutant. Xylem Cu levels returned
to wild type levels when the mutant was given exogenous nicotianamine (Pich & Scholz, 1996). Tobacco plants deficient in
nicotianamine are deficient in leaf Fe, Zn and Cu and display
reproductive deficiency (Takahashi et al., 2003). These observations all support a role for nicotianamine in Cu transport in
the xylem.
VIII. Uptake in the shoot symplast, redistribution
of Cu during flowering, seed set and senescence
If COPT-family transporters are again responsible for Cu uptake
in the symplast of leaves and other shoot organs then a reduction
may be necessary at the cell surface. The FRO6 and FRO7
ferric chelate reductase family genes are highly expressed in green
parts. FRO7 plays a role in Fe uptake in plastids (Jeong et al.,
2008). The function of FRO6 is not clear yet but its expression
is reduced when plants are Cu limited (Mukherjee et al., 2006).
Copper is not efficiently redistributed from older leaves to
young leaves and meristems. Therefore, younger leaves and stems
are generally more affected by deficiency than mature leaves
(Marschner, 1995). Flowering and efficient seed set require Cu
(Marschner, 1995; Epstein & Bloom, 2005). In Arabidopsis, the
majority of the Fe, Mn and Cu that ends up in reproductive
tissues seems to take a direct route from the roots via the vasculature (Waters & Grusak, 2008). However, some of the minerals,
including Cu, that accumulate in reproductive tissues are mobilized from vegetative tissue vasculature (Waters & Grusak, 2008),
possibly via the phloem.
Nicotianamine is a precursor of the phytosiderophore that
functions in high affinity Fe uptake from the soil via the Yellow
Stripe-Like (YSL) transporters in monocots with strategy-II Fe
uptake (Briat et al., 2007). Arabidopsis and most other dicots
acquire Fe via a strategy-I mechanism involving the root surface
ferric reductase FRO2 and the high-affinity Fe transporter IRT1
of the ZIP family (Briat et al., 2007). Nevertheless, the Arabidopsis genome encodes for eight YSL transporters (Briat et al.,
2007). The YSL transporters most likely function in the cellular
uptake of metal ion–chelate (nicotianamine) complexes (Schaaf
et al., 2004; DiDonato et al., 2004). In Arabidopsis, the function
of these YSL transporters is not yet fully understood but one
possible function is in redistribution of minerals between tissues
(Briat et al., 2007). High nicotianamine content in the phloem
is consistent with this role. Interestingly, plants that are mutated
in YSL2 and YSL3 have lower Cu levels in their flowers and seeds
and maintain higher levels of Cu in rosette and cauline leaves
(Waters et al., 2006). Furthermore, plants deficient in YSL2 and
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YSL3 did not remobilize Cu and Fe as efficiently as the wild type
(Waters & Grusak 2008). We note that nicotianamine not only
binds metal ions but also has high nitrogen content. It would
be beneficial for growing, biosynthetically active sink tissue to
receive both nitrogen and essential metal ions at the same time.
During senescence, expression of the Atx1-like Cu chaperone
CCH (Himelblau et al., 1998) is upregulated in Arabidopsis
(Mira et al., 2002). Therefore, the CCH Cu chaperone may
function in mobilization during senescence. It was suggested
that the C-terminal domain of CCH could facilitate transport
through plasmodesmata and therefore CCH may be present in
the phloem. Finally, phloem transport of Cu may also involve
the metallothionein family members MT1a and MT2b (Guo
et al., 2003). MT1 is upregulated during senescence (Mira et al.,
2002).
IX. Cu delivery inside the cell
Figure 3 depicts our present knowledge about the cellular Cu
delivery machinery in plants.
1. Delivery to ethylene receptors in the endomembrane
system and to the apoplast
The RAN1/HMA7 gene product is needed to deliver Cu to the
ethylene receptors. RAN1 (RESPONSIVE TO ANTAGONIST
1) is a functional homolog of the yeast and human genes encoding Cu transporting P-type ATPases functional in the endomembrane system (Hirayama et al., 1999). In Arabidopsis, RAN1 was
the first functionally characterized heavy metal ATPase (HMA)
of which there are eight family members, called HMA1–8, in
Arabidopsis (for review see Williams & Mills, 2005). Homologs
of RAN1 in yeast and mammalian cells function in the removal
of Cu from the cytosol into a secretory compartment or the extra
cellular space (Lutsenko et al., 2007). Mild alleles of ran1 affect
only ethylene signaling (Hirayama et al., 1999). Stronger loss
of function alleles, in addition, affect ethylene independent
processes including cell elongation (Woeste & Kieber, 2000).
Mutants in RAN1 are not Cu sensitive; instead the phenotypes
are suppressed by Cu feeding. The closest homolog of RAN1
in Arabidopsis is HMA5 (Williams & Mills, 2005). The HMA5
gene is expressed mainly in roots and flowers (Andrés-Colás et al.,
2006). The phenotypes of T-DNA insertion mutants in HMA5
are Cu sensitivity, a wave-like root growth, growth arrest and
an elevated Cu content. The sensitivity to excess Cu and the
increased Cu content of the plants are consistent with a role
of HMA5 in Cu export from the cell. The hma5 mutants do
not display the ethylene-signaling related defects noted for ran1
(Andrés-Colás et al., 2006). However, both ran1 and hma5
loss of function mutants have phenotypes associated with cell
expansion (Woeste & Kieber, 2000; Andrés-Colás et al., 2006).
It is possible that Cu cofactor delivery to Cu-requiring apoplastic
oxidases and laccases, which could all have roles in cell wall
modeling and thus cell expansion, is affected in the ran1 and
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hma5 mutants. Recent reports indicate roles for apoplastic
ascorbate oxidase in cell expansion and salt tolerance (Pignocchi
et al., 2003, Yamamoto et al., 2005). However, the activities of
these Cu enzymes have not yet been reported for the ran1 and
hma5 mutants. Another Cu protein in the apoplast is plantacyanin, which plays a role in pollen tube guidance (Dong et al.,
2005). The available data indicate that it could be of interest
to further explore the link between Cu and cell expansion and
to consider the roles of COPT members and HMA5 or RAN1.
The specific roles of RAN1 and HMA5 are probably related
to their intracellular location, which has not been determined
for either protein. However, based on the phenotypes of the
mutants it seems likely that RAN1 is active in an endomembrane
system compartment, perhaps the endoplasmic reticulum, while
HMA5 likely is in the plasma membrane or a late secretory
compartment. This situation is indicated in Fig. 3.
Review
activity in plants may result from the use of an alternative translational start site that skips the chloroplast-targeting peptide
(Chu et al., 2005). Expression in the T-DNA mutant of the CCS
protein without its transit sequence rescues only cytosolic and
peroxisomal Cu/ZnSOD activities. Although yeast Cu/ZnSOD
has an absolute requirement for CCS, it was found that in the
worm Caenorhabditis elegans and to a lesser extent in mammals
Cu/ZnSOD can also be activated via a CCS-independent pathway that uses glutathione (Carroll et al., 2004). It is not clear
if this CCS-independent delivery pathway operates in plants,
but plants without CCS have very low if any CSD1 and CSD2
activity (Chu et al., 2005). The requirement for CCS may be
correlated with the presence of two proline residues in the motif
PRP (residues 142–144) in the Cu/ZnSOD sequences of fungi
(Carroll et al., 2004). In Arabidopsis Cu/ZnSODs the amino
acid residues at the corresponding positions are GRV (for CSD1)
and GRL (for CSD2).
2. Cytosolic metallochaperones
Arabidopsis has two homologs of yeast Atx1, CCH (Himelblau
et al., 1998) and ATX1 (Andrés-Colás et al., 2006, Puig et al.,
2007). CCH has a C-terminal extension relative to yeast Atx1
and Arabidopsis ATX1 proteins (Mira et al., 2001a; Puig et al.,
2007). Both CCH and ATX1 proteins complement the yeast
atx1 mutant and interact with the N-terminus of HMA5, as
shown by a yeast two-hybrid assay (Puig et al., 2007). The Cterminus of CCH has a negative effect on interaction with
HMA5 (Andrés-Colás et al., 2006, Puig et al., 2007). The gene
is expressed in areas around the vascular tissue and the protein
was detected in phloem (Mira et al., 2001b). It is possible that
the C-terminal domain of CCH is involved in the transport
of the protein through plasmodesmata to nonnucleated cells,
such as sieve elements, thus providing a symplastic pathway
for intercellular Cu transport. However, this notion has not yet
been tested and the phenotypes for loss of function mutants in
CCH are not yet known. Copper deficiency, senescence, oxidative stress as well as mechanical stress, and jasmonic acid treatment are factors that lead to upregulation of CCH and ATX1
in either Arabidopsis (Himelblau et al., 1998; Mira et al., 2002;
Puig et al., 2007) or poplar (Lee et al., 2005).
Plant homologs of the Cu chaperone for superoxide dismutase
(SOD) (CCS) have been reported for tomato (Zhu et al., 2000),
potato (Trindade et al., 2003), maize (Ruzsa & Scandalios,
2003) and Arabidopsis (Wintz & Vulpe, 2002). Arabidopsis has
just one functional homolog of the yeast CCS, (Wintz & Vulpe,
2002; Chu et al., 2005). When the full-length Arabidopsis protein is fused to green fluorescent protein (GFP) it localizes to
chloroplasts only (Abdel-Ghany et al., 2005b). However, a TDNA KO in CCS affects all three CuZnSOD activities (Chu
et al., 2005). Therefore CCS is active both in plastids, delivering
Cu to CSD2, and in the cytosol delivering Cu to both CSD1
and CSD3. CSD3 has a peroxisomal targeting sequence and may
be imported by the peroxisomes – which can import proteins
in a folded state – with its cofactor bound. The cytosolic CCS
© The Authors (2009)
Journal compilation © New Phytologist (2009)
3. Copper delivery to mitochondria
Genetic screens in yeast have identified the assembly factors
required for the formation of functional cytochrome c oxidase
(Carr & Winge, 2003). A functional plant homolog of the yeast
Cox19 protein has been described (Attallah et al., 2007). There
are two Cox17 genes in Arabidopsis, which both complement
the yeast mutant (Balandin & Castresana, 2002). The other
mitochondrial Cu chaperones required for cytochrome c oxidase
activation, Cox11 and Sco1, are conserved (Carr & Winge,
2003) and most likely functional homologs exist in plants.
The mitochondrial matrix seems to be a storage site for metals
including Cu (for review see Pierrel et al., 2007).
4. Delivery of Cu to chloroplasts
Targets for Cu delivery in plant chloroplasts are plastocyanin
in the thylakoid lumen and the Cu/ZnSOD CSD2 in the stroma,
both are nuclear encoded. Plastocyanin is indispensable in plants
(Molina-Heredia et al., 2003; Weigel et al., 2003) and therefore
a priority for Cu delivery. The apoprotein (Garrett et al., 1984)
has a structure that is highly similar to holoplastocyanin (Colman
et al., 1978) and therefore cofactor insertion could be spontaneous after Cu is delivered to the lumen where the protein
acquires its cofactor (Li et al., 1990). PAA1 (Tabata et al., 1997;
Shikanai et al., 2003) and PAA2 encode Cu-transporting P-type
ATPases located in the chloroplast envelope inner membrane
and thylakoids respectively (Abdel-Ghany et al., 2005a; Bernal
et al., 2007). They are similar in sequence to bacterial Cu transporters such as E. hirae CopB and cyanobacterial CtaA and PacS.
Characterization of paa1 and paa2 mutants has indicated that
the two transporters have distinct functions, whereas both transporters are required for Cu delivery to plastocyanin and efficient
electron transport, Cu delivery to the stroma and CSD2 is only
inhibited in paa1 but not in paa2 mutants. Therefore, PAA1
and PAA2 can be considered functional homologs of CtaA and
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PacS respectively (Abdel-Ghany et al., 2005a). A paa1 paa2
double mutant was seedling lethal (Abdel-Ghany et al., 2005a)
underscoring the importance of Cu to photosynthesis, and the
essential role of plastocyanin for photoautotrophic growth in
Arabidopsis (Weigel et al., 2003). Interestingly, the phenotypes
of the paa1 and, to a lesser extent, paa2 mutants (but not the
paa1 paa2 double mutant) were alleviated by Cu-feeding, suggesting that an alternative lower-affinity pathway for Cu delivery
exists (Shikanai et al., 2003; Abdel-Ghany et al., 2005a). A
candidate for the alternative transporter in the envelope may
be HMA1, which was localized in envelopes and affects Zn and
Cu uptake activity when expressed in yeast (Seigneurin-Berny
et al., 2006). A T-DNA insertion mutant in hma1 shows no
defect in plastocyanin but was reported to be defective in total
plastid SOD activity and to have reduced Cu levels in plastids
(Seigneurin-Berny et al., 2006). HMA1 does not have a canonical N-terminal heavy metal binding domain and the sequence
in TM domain six is SPC instead of CPC. A possibility is that
HMA1 transports Cu(II) or other divalent ions. Energy independent Cu(II) transport into isolated pea thylakoids was measured
in an assay that utilized a Cu(II) sensitive fluorescent dye (Shingles
et al., 2004). This Cu transport activity, which was inhibited by
Zn and by a low internal pH (Shingles et al., 2004), perhaps
explains the low levels of plastocyanin activity in the PAA2
mutants. Mutants in PAA1 were slightly more sensitive to Cu
excess while mutants in PAA2 were about as sensitive as the wild
type (Abdel-Ghany et al., 2005a) and this observation is consistent with the view that the thylakoid membrane is a primary target
for Cu toxicity (Yruela et al., 1996; Bernal et al., 2006).
5. The mechanism of action of the chloroplast Cu
transporters PAA1 and PAA2
Many of the Cu-transporting P-Type ATPases function together
with a Cu chaperone that is thought to interact with the Nterminal HMB domain (Arnesano et al., 2002). HMA5 and
RAN1 interact with CCH and ATX1. For PAA1 and PAA2
such a chaperone has not yet been reported, and it may not exist.
Arabidopsis has a homolog of the conserved CutA protein (Burkhead et al., 2003) and CutA has some structural resemblance
to ATX (Arnesano et al., 2003). CutA binds Cu(II) in vitro and
was detected in chloroplasts of overexpressing plants (Burkhead
et al., 2003), but its location has not yet been verified by GFP
fusions and a T-DNA knockout does not affect plastocyanin
function. Research in Synechocystis PCC 6803 on Atx1 has shed
light on the role of metallochaperones (Borrely et al., 2004).
Three P-Type ATPases are present in the cell membrane that
function in the uptake of Cu (CtaA), the extrusion of Zn (ZiaA)
or cobalt (Co, CoaA) respectively. The fourth P-Type ATPase
heavy metal transporter, PacS, delivers Cu to the thylakoids.
Atx1 functions to deliver Cu to PacS but there are no metallochaperones for delivery to ZiaA or CoaA. The N-terminal domains
of PacS and ZiaA bind their cognate metal ion (Cu and Zn
respectively), but Cu binds to the ZiaA N-terminal domain more
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strongly than Zn. Perhaps this would block Zn extrusion when
both Zn and Cu are in excess. However, in the cell the ZiaA Nterminus cannot accept Cu from cyanobacterial Atx1 because
the proteins do not have matching complementary surfaces
(Borrelly et al., 2004). Therefore Atx1 acts as a true chaperone
in cyanobacteria; it prevents an undesired interaction while facilitating delivery to the correct partner. As a side note: a comparable
role may be taken in cyanobacteria by the periplasmic Fe(III)binding protein called FutA2 (Badarau et al., 2008). FutA2 functions primarily in Fe uptake in cyanobacteria. But when FutA2
is mutated, Cu delivery to cytochrome c oxidase and plastocyanin becomes impaired, presumably because in the absence
of FutA2 Fe now competes with Cu for binding to Cu import
systems in the relatively oxidizing environment of the periplasm
(Waldron et al., 2007). It unclear whether FutA homologs
operate in the plastid envelope in plants. Perhaps because a Zn
transporter such as the cyanobacterial ZiaA (Borrelly et al.,
2004) is absent from chloroplasts there is no need for a soluble
Cu chaperone in the stroma of plants. It is possible that the
single N-terminal HMB domains of PAA1 and PAA2 interact
if the topology of the transporters allows it; thus PAA1 could
possibly serve a Cu chaperone function for PAA2 and vice versa.
The mechanism of bacterial P-type ATPases has been studied
in some detail. The major domains of these proteins are indicated in Fig. 4b. ATP hydrolysis-driven conformational shifts in
the A (actuator) domain and ATPase domain are thought to
drive transmembrane movement of ions in the P-type pumps
(for reviews see Williams & Mills, 2005; Arguello et al., 2007).
Ion transduction specificity is determined by a sequence motif
(CPC) in the sixth TM region together with sequences in TM 7
and TM 8 (Mandal et al., 2004). Several functions have been
proposed for the ATX-like HMB domains in Cu P-type
ATPases. These HMB domains may be an entry point for Cu,
accepting the ion from a Cu-chaperone and donating it to the
TM region for transport. This view is supported in the literature
by metallochaperone–HMB interactions (Huffman & O’Halloran, 2000). However, when HMB regions are removed the
transporters still display Cu transport and Cu-driven ATP
hydrolysis. Furthermore, evidence exists for direct donation of
Cu by a metallochaperone to the TM transport sites (GonzálezGuerrero & Argüello, 2008). Structural evidence (Sazinsky et al.,
2006; Wu et al., 2008) suggests that the HMB domain without Cu interacts with the A domain, restricting its motion and
thus autoinhibiting the transporter. This regulatory ‘braking’
model is appealing. A third possible role especially emphasized
in mammalian systems is a function in Cu-dependent intracellular membrane trafficking (for a review see Lutsenko et al.,
2007). Perhaps we should consider that PAA1 and PAA2 regulate each other’s location and activities by protein contact.
6. Central role of the vacuole?
Both the mitochondria and plastids are important Cu sinks
(most Cu in the green cell is in the chloroplast) but there seem
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to be no delivery systems (Cu chaperones) targeted to the surface
of these organelles (or at least they have not been discovered
yet). It is thought (at least in yeast) that the chelating capacity
of the cytosol allows for fewer than one free Cu ion per cell
(Rae et al., 1999). How then is Cu delivery to these organelles
possible? Schematic images of cells such as Fig. 3 are static and
do not do justice to the true volume and dynamics of the
vacuole. In reality, the organelles of a plant cell could often be
close to the tonoplast. Therefore, the vacuole could really be
a delivery pathway within the cell and following transport over
the tonoplast to the cytosol a metal ion may need to diffuse only
a very short distance before encountering a transporter such as
PAA1. Work with cultured soybean cell cultures has indicated
that the chloroplast, the vacuole and the cell wall are the major
sites of Cu accumulation (Bernal et al., 2006). We should learn
much more if mutants in possible vacuolar exporters such as
COPT3, COPT5 and ZIP proteins are analysed for defects in
specific Cu enzymes.
X. Regulation of Cu homeostasis
1. Regulation of transporters
Plants will require mechanisms to regulate Cu uptake and distribution because of the variable availability of Cu in the environment. Copper demand in tissues will also depend on the
growth environment, light conditions and developmental stage.
There is still much to be learned about the mechanistic aspects
of Cu transport by Ctr and P-type ATPase transporters, especially in plants. We do, however, have some knowledge about
regulation at the transcript abundance level and at the protein
level. The COPT1 and COPT2 transporters were found to be
downregulated in response to high Cu; COPT3–5 were not
affected by Cu (Sancenon et al., 2003). The transcript of
HMA5 was upregulated by high Cu (Andrés-Colás et al., 2006).
In roots, the downregulation of the importer COPT1 and
upregulation of the exporter HMA5 by high Cu suggests the
existence of a feedback mechanism that regulates Cu loading
and export from roots. We now understand the mechanism of
COPT1 and COPT2 regulation via the transcription factor
SPL7 (Yamasaki et al., 2009; see below). A transcriptomic study
that investigated 8300 genes of the Arabidopsis genome has
provided the first evidence that a number of genes that are
possibly involved in Cu homeostasis are coregulated by Cu
deficiency (Wintz et al., 2003). The ZIP2 and ZIP4 transporters
were upregulated on low Cu, but were even more influenced
by Zn (Wintz et al., 2003). The same study found COPT2 to
be upregulated in leaves by both Cu and Zn deficiency. Furthermore, AtOPT3 and three nicotianamine synthase genes were
upregulated by Cu deficiency in roots (Wintz et al., 2003). A
knockout of AtOPT3 is embryo lethal (Stacey et al., 2002);
however, further analysis of a milder knock-down allele showed
a number of Fe-related phenotypes: constitutive expression
of genes involved in Fe uptake in the roots, overload of Fe in
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Review
vegetative tissues and decreased Fe accumulation in seeds.
Nicotianamine–metal complexes are possible substrates for YSL
family transporters and these have been mainly linked to Fe
homeostasis. Two YSL transporters (YSL2 and YSL3) were
implicated in redistribution of Cu in senescence (see above;
Waters et al., 2006).
2. Copper economy and the Cu-microRNAs
Cu availability is a major factor in the expression of Cu/ZnSOD
genes in plants (Kurepa et al., 1997; Wintz et al., 2003; Cohu
& Pilon, 2007). Wintz et al. (2003) first noted the coregulation
in response to Cu of the Cu/ZnSOD genes (CSD1 and CSD2)
and the Cu chaperone CCS, all of which are downregulated
on low Cu (Abdel-Ghany et al., 2005a,b). In addition to Cu/
ZnSOD, plants have a FeSOD in the stroma of the chloroplast.
Interestingly, Cu availability regulates the activity of stromal
and cytosolic SOD isoforms (Abdel-Ghany et al., 2005a). At
low Cu levels, the FeSOD, FSD1, is active and Cu/ZnSOD
expression is shut off, so Cu may be preferentially targeted to
plastocyanin in the thylakoid lumen. At higher Cu levels, FSD1
expression is shut off, possibly saving Fe for other uses, and
Cu/ZnSOD becomes a major sink for Cu in the stroma (AbdelGhany et al., 2005a,b; Cohu & Pilon, 2007). The reciprocal
regulation of Cu/ZnSOD and FeSOD in response to Cu is not
only seen for Arabidopsis grown on agar plates but is also observed
for Arabidopsis along with other plants when grown on hydroponics (Cohu & Pilon, 2007). Copper limitation does not affect
plastocyanin mRNA accumulation (Abdel-Ghany & Pilon,
2008). The regulation of CSD2 by Cu did not involve its
promoter elements, instead it involves a microRNA, miR398
(Yamasaki et al., 2007; Dugas & Bartel, 2008). MiR398 was
predicted to target the mRNA of CSD1, CSD2 and COX5b
(Jones-Rhoades & Bartel, 2004; Sunkar & Zhu, 2004; Sunkar
et al., 2005; see also the small RNA database at http://asrp.cgrb.
oregonstate.edu/). These transcripts all encode Cu proteins. The
conserved miR398 family consists of three genes, miR398a,
miR398b and miR398c. The sequences of mature miR398b
an d miR398c are identical, whereas the 3′ end nucleotide is
different in miR398a. The function of COX5b in cytochrome
c oxidase and the meaning of its mild regulation by miR398
is not yet clear (Yamasaki et al., 2007). Direct evidence for the
strong regulation of CSD1 and CSD2 by miR398 was shown
by the analysis of plants with altered miR398 and plants with
altered miRNA binding sites in the targets (Sunkar et al. 2006;
Yamasaki et al., 2007; Dugas & Bartel 2008). Three other
conserved families of miRNA (miR397, 408, 857) are predicted
to target transcripts for laccases and plantacyanin. Because
they target genes that encode Cu proteins, we now propose to
call miR397, miR398, miR408 and miR857 collectively the
Cu-microRNAs. All of these Cu-microRNAs accumulate
at low Cu concentrations and disappear at sufficiently high
Cu concentrations (Yamasaki et al., 2007; Abdel-Ghany &
Pilon, 2008). The range of Cu concentrations in which
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miR398-mediated Cu/ZnSOD regulation occurs are not stressful
for the plants (Cohu & Pilon, 2007). miR398 expression is
turned on by low Cu before deficiency symptoms are apparent,
while the amount of Cu required to turn miR398 expression
off is not in the toxic range (Yamasaki et al., 2007; Abdel-Ghany
& Pilon, 2008). The targets for miR397, miR408 and miR857
were confirmed by cleavage site analyses via 5′-rapid amplification of cDNA ends (RACE; Abdel-Ghany & Pilon, 2008).
The targets of Cu-microRNAs are also Cu responsive, but in
a reciprocal fashion compared to the miRNAs (Yamasaki
et al., 2007; Abdel-Ghany & Pilon, 2008). Sunkar et al. (2006)
presented evidence for miR398 regulation in the context of
oxidative stress protection. However, Cu, much more than
oxidative stress is the primary factor that determines the
expression of miR398 (Yamasaki et al., 2007). We hypothesize
that the Cu-microRNAs are used to downregulate nonessential
Cu proteins and to save Cu for essential functions such as
plastocyanin under impending Cu deficiency. In addition
to Cu, miR398 is also regulated by sucrose (Dugas & Bartel
2008) but the biological role of the sucrose regulation is
not yet evident. Regulation by Cu and sucrose seem to be
independent of each other (Dugas & Bartel 2008).
3. Possible regulatory factors
In Chlamydomonas, the protein CRR1 (Cu response regulator)
is a transcription factor that upregulates specific genes in Cu
deficiency (Kropat et al., 2005). The gene targets of CRR1 (e.g.
CYC6 and CPX1) contain a GTAC core in the promoter region.
Interestingly, in plants, a GTAC sequence can be found in high
frequency in the promoter regions of the Cu-microRNAs and
in the promoter of FeSOD, whereas this motif is not found
as frequently in other miRNA promoters. The regulation of
FeSOD by Cu is conserved in the moss Barbula unguiculata, and
there is evidence that this regulation involves a homolog of CRR1
(Nagae et al., 2008). While it was suggested that the response
of FeSOD to Cu in B. unguiculata involves negative regulation
(switching off when Cu is high) the same data can also be
interpreted as positive regulation when Cu is low (Nagae et al.,
2008). In Arabidopsis the protein SPL7, a member of the SPL
family of transcription factors (Cardon et al., 1999), has good
sequence similarity to the CRR1 protein. Indeed, SPL7 seems
to be a conserved key regulator of Cu-responsive genes in plants.
In an spl7 mutant the Cu-microRNAs are not expressed on
low Cu. In addition, the transporters COPT1, COPT2, ZIP2,
YSL2, the reductase FRO3, the Cu-chaperones CCH and CCS
and the FeSOD FSD1, plus a set of novel proteins including
some transcription factors are misregulated (Yamasaki et al.,
2008). Many of the genes that are upregulated on low Cu, such
as several Cu-miRNAs and FSD1, carry GTAC motifs in their
promoter regions. For miRNA398c, a set of these GTAC motifs
was indeed shown to be essential for Cu regulation (Yamasaki
et al., 2009). It should be noted that the T-DNA insertion in
the spl7 line may not result in full loss of function but that
New Phytologist (2009) 182: 799–816
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instead this mutation may cause the expression of a truncated
protein (Yamasaki et al., 2009) that may still bind to DNA. Thus
the results obtained with this spl7 mutant should be interpreted
with care. Nevertheless, this spl7 mutant shows strong growth
phenotypes on low Cu indicating that this master switch is
important in Cu homeostasis (Yamasaki et al., 2009).
The A351 mutant of maize has reduced Cu/ZnSOD expression relative to the wild type, possibly because a MT gene is overexpressed and chelates Cu. Cytochrome c oxidase and ascorbate
oxidase activities are also reduced (Rusza &Scandalios, 2003).
The expression level of these Cu proteins responds to Cu. Interestingly, a sequence element very similar to the cis-acting region
of yeast genes such as CUP1, CRS5 and SOD1 that respond
to the transcription factor Ace1 (Gralla et al., 1991; Pena et al.,
1998) is found in the promoters of three Cu/ZnSOD genes
in maize (Rusza & Scandalios, 2003). If an Ace1-like transcription factor affects SOD expression in maize in response to Cu,
this mechanism is not conserved in Arabidopsis because CSD2
regulation by Cu is not via the promoter region (Sunkar et al.,
2006; Yamasaki et al., 2007; Dugas & Bartel, 2008).
4. Why do plants downregulate Cu proteins using the
Cu-microRNAs?
The model of downregulation of Cu-binding proteins in favor
of plastocyanin (Fig. 5) is appealing because plastocyanin is
essential. However, a few questions remain. Are the other Cu
enzymes abundant enough to compete with plastocyanin for Cu?
Probably this is the case. For example, proteomic data suggests
that in chloroplasts the Cu/ZnSODs can be highly abundant
(Zybailov et al., 2008). We may also wonder if other Cu proteins
such as Cu/ZnSOD enzymes are really dispensable. Although
plants that overaccumulate CSD2 are reported to be more
tolerant to oxidative stresses (Sunkar et al., 2006) these effects
were quite small and very extreme treatments were needed to
reveal differences between CSD2 overaccumulators and WT
plants (Sunkar et al., 2006). Furthermore, Dugas & Bartel
(2008) reported that plants with altered levels of miR398 and
thus altered levels of CSD1 and CSD2 showed no noticeable
phenotypes. The mild phenotype of paa1 mutants, which lack
CSD2 activity, supports the same notion (Shikanai et al., 2003).
Thus, in some of the present literature the importance of
Cu/ZnSOD might be exaggerated, and perhaps a previously
described CSD2-knockdown phenotype (Rizhsky et al., 2003)
resulted from an unrelated genetic change.
The prioritized delivery of Cu to plastocyanin and other
essential Cu proteins by downregulation of nonessential Cucontaining proteins could be an essential part of the Cu homeostasis mechanism allowing plants to adjust to a broad range of
Cu supply. In addition to the Cu-microRNAs, further miRNAs
have been described whose expression is regulated by nutrient
availability. miR395 may regulate sulfate assimilation and allocation by targeting APS1 and APS4 transcripts for the APS (ATP
sulfurylase) enzyme and a sulfate transporter, SULTR2;1
© The Authors (2009)
Journal compilation © New Phytologist (2009)
Tansley review
Review
and Mn. Copper is at the top of the Irving–Williams series and
is known to bind very tightly to its target polypeptides (Lippard
& Berg, 1994). Therefore, the Cu sinks must be eliminated, if
possible, when Cu becomes limiting in order to allow Cu delivery
to essential targets. Metabolism of Cu evolved relatively late in
the history of life (Ridge et al., 2008); therefore, perhaps many
Cu enzymes are less essential and can be downregulated during
deficiency. In addition to their enzymatic function many Cu proteins may function to sequester or buffer Cu. Such a secondary
role may even be postulated for plastocyanin on the basis of the
observation that plants have an excess of plastocyanin over what
they need for maximum electron transport under most conditions (Shikanai et al., 2003; Abdel-Ghany, 2009). Indeed, plastocyanin accumulates when excess Cu is provided (Bernal et al.,
2006; Abdel-Ghany & Pilon, 2008; Abdel-Ghany, 2009).
XI. Conclusions and outlook
Fig. 5 Model for copper (Cu)-microRNA mediated regulation of Cu
protein expression. PolII, RNA polymerase II; DCL, dicer-like
dependent microRNA processing machinery; RISC, RNA-induced
silencing complex; SPL7, squamosa promoter binding-like
transcription factor similar to Chlamydomonas CRR1.
(Bonnet et al., 2004; Joanes-Rhoades et al., 2006). miR399 is
induced in response to phosphate starvation. In this condition,
miR399 downregulates its target transcript, which encodes a
ubiquitin-conjugating E2 enzyme that is required for the turnover of a phosphate transporter (Chiou et al., 2006; Fujii et al.,
2005). In grafting experiments, miR399 was shown to be a
phloem-mobile long-distance signal for the regulation of Pi
homeostasis, which suggests a role of miRNAs in systemic nutrient homeostasis (Pant et al., 2008).
The observations on microRNA-mediated regulation of sulfur
(S) and phosphorus (P) homeostasis raise additional questions
when we consider the function of the Cu-microRNAs. The
presence of miR399 in the phloem of P-starved plants suggests
that machinery exists for systemic regulation via microRNAs.
Therefore, Cu homeostasis could also have a systemic component. In this context it is highly significant that several CumicroRNAs were detected in the phloem of Cu-starved Brassica
napus plants (Buhtz et al., 2008). The types of proteins that
are targeted by the Cu-microRNAs and the miRNAs involved
in S and P homeostasis are different. The miRNAs involved in
S and P homeostasis target the machinery responsible for assimilation; by contrast, in the case of Cu homeostasis the miRNAs
target Cu proteins. It is striking that this mechanism only seems
to be used for Cu and not for other trace elements such as Fe
© The Authors (2009)
Journal compilation © New Phytologist (2009)
Copper cofactor delivery and assembly is a highly regulated process that involves specific protein interactions and a sophisticated
regulatory system that in plants involves small RNA and a
regulatory protein that is conserved from Chlamydomonas to
mosses and plants. In plants, we seem closer to a complete understanding of homeostasis for Cu than for most other metal ions.
Perhaps much of this progress resulted from the analysis of
mutants in genes such as RAN1, PAA1, PAA2 and CCS that
proved to be involved in Cu delivery to specific targets with
important physiological roles. The evolutionary conservation
of Cu homeostasis mechanisms and the perhaps fortuitous
discovery of the Cu-microRNAs have also helped our progress.
Exactly how the Cu-microRNAs are regulated and the biological
significance of this regulation deserves further attention. Furthermore, we still have a limited understanding of the molecular
mechanisms by which Cu is distinguished from other ions.
While we have a relatively good understanding of Cu homeostasis, we are unsure of the biological function of many Cu
enzymes in plants. In this respect, plastocyanin, cytochrome c
oxidase and the ethylene receptors, for which a clear biological
function is evident, are the exceptions. Further research on Cu
homeostasis should also enhance our understanding of the role
of Cu enzymes for which we have not yet identified a clear
biological function.
Acknowledgements
Work in the authors’ laboratory was supported by a grant from
the US National Science Foundation to M.P. (NSF-IBN0418993). We apologize to colleagues whose work could not
be cited because of the limitations of space.
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