ANTIOXIDANTS & REDOX SIGNALING

Volume 19, Number 9, 2013

ª Mary Ann Liebert, Inc.
DOI: 10.1089/ars.2012.5084

FORUM REVIEW ARTICLE

Copper and Iron Homeostasis in Plants:

The Challenges of Oxidative Stress

Karl Ravet1,2 and Marinus Pilon1

Abstract

Significance: Photosynthesis, the process that drives life on earth, relies on transition metal (e.g., Fe and Cu)
containing proteins that participate in electron transfer in the chloroplast. However, the light reactions also
generate high levels of reactive oxygen species (ROS), which makes metal use in plants a challenge. Recent
Advances: Sophisticated regulatory networks govern Fe and Cu homeostasis in response to metal ion availability
according to cellular needs and priorities. Molecular remodeling in response to Fe or Cu limitation leads to its
economy to benefit photosynthesis. Fe toxicity is prevented by ferritin, a chloroplastic Fe-storage protein in plants.
Recent studies on ferritin function and regulation revealed the interplay between iron homeostasis and the redox
balance in the chloroplast. Critical Issues: Although the connections between metal excess and ROS in the
chloroplast are established at the molecular level, the mechanistic details and physiological significance remain to
be defined. The causality/effect relationship between transition metals, redox signals, and responses is difficult to
establish. Future Directions: Integrated approaches have led to a comprehensive understanding of Cu homeo-
stasis in plants. However, the biological functions of several major families of Cu proteins remain unclear. The
cellular priorities for Fe use under deficiency remain largely to be determined. A number of transcription factors
that function to regulate Cu and Fe homeostasis under deficiency have been characterized, but we have not
identified regulators that mediate responses to excess. Importantly, details of metal sensing mechanisms and cross
talk to ROS-sensing mechanisms are so far poorly documented in plants. Antioxid. Redox Signal. 19, 919–932.

Introduction als are essential for most prokaryotic and eukaryotic cells.
However, the same redox properties of Fe and Cu that are

T he metals iron (Fe), copper (Cu), manganese (Mn), and

zinc (Zn) are employed by organisms to perform a re-
markable array of functions that are critical for life. Proteins
utilized by metalloproteins can be harmful to aerobic cells
because the uncontrolled production of ROS is deleterious for
the cell. Thus, living organisms have developed various and
that carry these metal ions as cofactors mediate diverse bio- sophisticated cellular and systemic homeostatic processes that
chemical processes, including energy conversion, synthesis, allow an efficient use of the metals, despite the unavoidable
and regulation of nucleic acids, biosynthesis of complex danger they constitute for the cell.
molecules, reactive oxygen species (ROS) detoxification, as Among the different living organisms, plants had to face
well as signaling events that trigger molecular, cellular, and specific challenges related to metal homeostasis. Plants and
systemic responses. algae have an additional intracellular organelle where carbon
During the evolution of life, the metal-based biochemical fixation takes place, the chloroplast. Similar to respiration in
reactions have been selected and conserved because the che- the mitochondria, the chloroplast relies on a complex protein
mical properties of these metals make them ideal structural machinery to mediate electron transport. However, chloro-
and/or active cofactors in enzymes. In particular, the transi- plasts use photosystems to capture sun light in order to drive
tion metals Fe and Cu take a central place in redox control and the electron transport chain and reduce NADP + , thereby
electron transport in the cell. Therefore, these transition met- converting solar energy into chemical energy. The processes

1
Biology Department, Colorado State University, Fort Collins, Colorado.
2
Biochemistry and Plant Molecular Physiology, CNRS/INRA/UM2, Montpellier, France.

919
920 RAVET AND PILON

of photosynthesis are strictly dependent on metals (57, 71). It

is now clear that most of the cellular Fe and Cu pools in plants
are found in the chloroplast (78). However, photosynthetic
electron transport is also a potential ROS generative process.
When light is in excess, or when the electron transport ca-
pacity in the chloroplast is limited, electrons can directly re-
duce molecular oxygen (29). Plants are sessile organisms and
are therefore extra sensitive to biotic and abiotic stresses.
Stress promotes ROS fluctuations in plants, particularly in the
chloroplast (9). Therefore, a fine-tuned regulation of homeo-
static processes has evolved in plants in order to maintain a
tight equilibrium between cellular ‘‘labile’’ pools of metals,
ROS detoxification capacity, and photosynthesis efficiency.
In this review we will focus on the interplay between metal
use and ROS metabolism in plants, with an emphasis on Cu
and Fe in the chloroplast. We will address how plants cope
with low Cu availability and the consequences of this adap-
tation for the photosynthetic apparatus and chloroplast lo-
calized redox processes. Another part of this review will
emphasize the dark side of Fe, and focuses on ferritin function
FIG. 1. Major metal-dependent cellular processes in plant
and regulation. These storage proteins are unique to Fe in chloroplast. The main pathways and proteins that utilize
metal homeostasis and constitute a molecular tool to identify metals in plant chloroplasts are listed. PPO, polyphenol ox-
Fe-specific signaling pathways. In this context, we will discuss idase; TIC55, translocon inner membrane complex 55; NFU,
the tight interconnection between Fe excess and oxidative ‘‘nitrogen fixing bacteria’’ (Nif) U-like proteins; SUF, ‘‘sulfur
stress, both at the physiological and molecular levels in plants. limitation’’ proteins; APR, adenosine 5¢-phosphosulfate
(APS) reductase; SIR, sulfite reductase; NiR, nitrite reductase;
GOGAT, glutamine oxoglutarate aminotransferase ( = glu-
The Use of Metals in Plant Cell Biology tamate synthase); HCAR, 7-hydroxymethyl chlorophyll a
Metals are important bio-components because they can reductase; CHL27, chlorophyll deficient 27 ( = CRD1: copper
response defect 1); LLS1, pheophorbide a oxygenase; SiRB,
interact with macromolecules and affect protein structure and
sirohydrochlorin ferrochelatase B; FC, ferrochelatase; SOD,
activity. Among the transition metals important for biology superoxide dismutase; APX, ascorbate peroxidase; FER,
are Fe, Cu, Mn, Mo, and Ni (53). These metal ions exist in more ferritin.
than one oxidation state when bound to protein active sites.
Therefore, they readily accept or donate electrons from their
external orbitals to other molecules. Mo and Ni only occur in a
chineries and metal-binding structures into proteins, their
limited set of enzymes active in the cytosol and peroxisomes
presence in specific kingdoms, and the evolution of the
(35). The much more abundant Fe, as well as Cu and Mn ions,
chemistry of the different trace elements in primitive oceans
serve as critical cofactors for electron transport chain in the
(27). It is now established that the geochemistry of the trace
mitochondria and in the plastids, the two power generators in
elements has been a driving factor in the evolution of cellular
plant cells. These three transition metals are also found in
metal ion use. For their redox chemistry, primitive prokary-
active centers of diverse proteins and act as electron acceptors
otic cells mainly used Fe, the most available metal, together
and donors in numerous cellular processes, particularly in the
with Mn in the anoxic and reducing Archean ocean 4.5 billion
chloroplast in the green lineage (Fig. 1) (56, 94). Among the
years ago. The increase of oxygen levels as a consequence of
different transition metals, Fe is the most widely utilized. Fe is
the invention of photosynthesis roughly 2.4 billion years ago
used as cofactor in a variety of chemical structures: directly
decreased Fe availability in the Proterozoic ocean and some
bound to the polypeptide (nonheme iron), incorporated into
metallo-proteins evolved toward the use of Mn. Over the past
heme structures or into iron-sulfur (Fe-S) clusters. These
0.8 billion years, the emergence of an aerobic and oxidizing
various types of Fe-cofactor allow Fe-proteins to cover a very
environment led to a dramatic decline of Fe and Mn avail-
large spectrum of redox potentials (Fig. 2). Therefore,
abilities (21, 80, 91). Concomitantly, Cu, which was scarcely
Fe-proteins are ubiquitously utilized in diverse biochemical
available to this point, became bio-available and appeared in
environments. Transition metals can also have structural
biological systems (75) together with Zn.
functions where the binding of the metal cofactor allows
specific protein folding, stability, and functionality. The ab-
Metal Toxicity in Plants
sence of redox activity for zinc (Zn) explains why Zn is the
metal of choice as a safe structural cofactor in many enzymes The chemical properties that make the transition metals key
and transcription factors [reviewed in (17), for plants see (79)]. components for beneficial cellular redox reactions can also
The use of the different transition metals in modern plants lead to undesired reactivity that is deleterious for the cell.
results from a long evolutionary process, involving dramatic Partially reduced oxygen species are produced when molec-
changes in cell complexity and environmental fluctuations. ular oxygen accepts electrons from other molecules. Many
Recent advances in large-scale genome and proteome analytic metal-dependent and metal-independent cellular processes
technologies allowed the systematic analysis of the relation- reduce oxygen to superoxide (O2 - ) or hydrogen peroxide
ship between the temporal emergence of metal-specific ma- (H2O2) (8). Although these molecules have by themselves a
METAL HOMEOSTASIS IN PLANTS AND OXIDATIVE STRESS 921

FIG. 2. Metals are crucial for the electron transport in the photosynthetic apparatus in plants. The electron transfer from
water to the final electron acceptor in the plant photosynthetic complexes is presented (black arrow). The oxygen evolving
complex (OEC), photosystem II, the cytochrome b6/f complex, photosystem I, and ferredoxin (FD) are depicted with respect
to their functional arrangement in the electron transport chain and orientation in the chloroplast thylakoids and stroma
compartments. Metalloproteins are depicted with black lined symbols. Electron carriers unrelated to metals are presented in
gray-lined symbols. Proteins involved in electron transport are depicted according to their standard redox potential (mV). Fe
complex, nonheme Fe protein of the photosystem II; Pheo, pheophytin; Q, quinone; PQ, plastoquinone; Cyt, cytochrome; PC,
plastocyanin; FX/FA/FB, electron acceptor X/A/B of photosystem I.

limited cytotoxicity, they are the substrates for the production

of the very harmful hydroxyl radical (OH$) which cannot be
removed enzymatically and therefore is responsible for most
of the oxidative damage in biological systems (34). The pro-
duction of hydroxyl radicals is catalyzed by Fe ions and, to a
lesser extent by Cu ions, in what is known as the Haber-Weiss
cycle (Fig. 3):

Mn þ 1 þ O2 )/Mn þ O2 ðaÞ

Mn þ H2 O2 )/Mn þ 1 þ OH þ OH  ðbÞ
O2 þ H2 O2 )/O2 þ OH  þ OH ðcÞ

The first reaction (reaction a) is due to the cycling of the

transition metal (M) between its reduced Mn and oxidized
Mn + 1 states, which can result in the reduction of molecular
oxygen. The single-electron reduction of oxygen is thermo-
dynamically unfavorable because of the electron configura-
tion, but transition metals (M) such as Fe and Cu can harbor
unpaired electrons and therefore can be catalysts of oxygen
reduction (32, 34). The second reaction (reaction b) consists of
the metal-mediated reduction of H2O2 leading to the pro- FIG. 3. Photosynthesis and stresses in plant chloroplast
duction of the hydroxyl radicals (reaction b). This reaction is promote the Haber-Weiss cycle. Under high light condi-
known as the Fenton reaction when Fe is the catalyst (33, 34). tions, electrons can escape from the photosynthetic machin-
The net reaction (c) illustrates how the presence of superoxide ery (Fig. 2) and can directly reduce molecular oxygen (O2)
and H2O2 can lead to the production of hydroxyl radicals in present in the chloroplast. The resulting superoxide ion
(O2 - ) reacts with hydrogen peroxide (H2O2) in the presence
the presence of transition metals (Fig. 3). The hydroxyl radi-
of catalytic transition metals such as Fe and Cu to produce
cals produced can oxidize biomolecules leading to major the highly toxic hydroxyl radical (OH ). In plants, stress
cellular alterations and ultimately to cell death. The hydroxyl $
promotes the accumulation of O2 - and H2O2 in the chloro-
radical can be responsible for mutations at very high rates. plast. In that particular context, redox cycling of the metal
The presence of ROS can also lead to oxidative modifications constitutes a major issue for the plants. (a) and (b) refer to the
of proteins and release of metal cofactors. Protein oxidation or equations presented in the text.
922 RAVET AND PILON

loss of the cofactor leads to protein misfolding, aggregation, Comparative genomics and phylogenetic analyses of vari-
and/or degradation (25, 32). Another deleterious effect ous classes of metalloproteins suggest that the use of the dif-
of metal reactivity is the peroxidation of lipids (34). Thus, ferent metals by living organisms has evolved under the
intracellular membrane systems and cell integrity can be pressure of aerobic conditions on earth (94). Catalase, which is
altered. considered as an ancient metalloprotein, is found ubiqui-
tously in all aerobic organisms. Catalase is a hemoprotein and
therefore uses Fe as redox-active metal ion. Primitive SOD
How Do Plants Tackle the Challenge Posed
also used Fe as a cofactor (11, 54). When Fe availability de-
by the Redox-Active Metals?
creased on earth, the use of Fe in SOD enzymes progressively
Plants are sessile organisms and therefore face major fluc- evolved toward the alternative use of Mn. Phylogenetic data
tuations in environmental conditions such as temperature, support the idea that the MnSODs originate from the FeSOD
moisture (drought), and nutrient availability in addition to (4). However, the later emergence of a third category of SOD
biotic stress from pathogens and herbivores. It is interesting using both Cu and Zn is a biological novelty since the amino
to note that ROS are important signaling molecules involved acid sequences of the Cu/ZnSODs are unrelated to the ones of
in the plant’s response to stress and development, and the the FeSODs and MnSODs (11, 54). The use of Cu as the redox-
chloroplast is emerging as a pivotal organelle for the cellular active metal in these novel SODs probably emerged in re-
redox balance. On the other hand, the chemistry of the redox- sponse to the rise in oxygen levels on earth and the consequent
active metals such as Fe and Cu in the chloroplast has to be decline of bio-available Fe and Mn (75).
tightly tuned to avoid the deleterious effect of the hydroxyl
radical production (Fig. 3), as well as the alteration of the ROS-
Iron and Copper Homeostasis in Plant Chloroplasts
mediated signaling events. Therefore, both biotic and abiotic
challenges have driven the evolution of defense mechanisms Over the last decade, metal homeostasis research has fo-
in plants allowing them to cope with adverse conditions. In cused on the identification of molecular factors involved in
addition, photosynthesis itself is a potentially dangerous metal transport, distribution, chelation, and utilization in the
process that combines molecular oxygen production, electron model plant Arabidopsis thaliana, and more recently in crops
transport, and the utilization of metal cofactors (Figs. 2 and 3) and trees. This has been extensively reviewed elsewhere
(29). The presence in chloroplasts of nature’s strongest oxi- (14, 19, 44, 64, 67). In comparison to Fe, the cellular need for
dizer (photosystem II [PSII]) and nature’s strongest reducer Cu is lower and Cu cofactor use is restricted to a relatively
(photosystem I [PSI]) poses a challenge (56). In particular, at small set of proteins (19). The concentration of each metal
high light intensities the saturation of the electron transport in plant leaves illustrates these differential requirements (53).
capacity of the photosynthetic apparatus leads to the unde- Under optimal growth conditions, Fe accumulates at around
sired reduction of molecular oxygen in the chloroplast. Such 50–100 lg$g - 1 dry weight (DW) when Cu accumulates at
electron congestion in the photosystems can originate not 5–20 lg$g - 1 DW in leaves of different plant species. At least
only from excessive light irradiance, but also from diverse half of the Cu is found in the chloroplast in green cells of A.
environmental and nutritional events that alter the molecular thaliana (1, 78, 82). The major copper proteins are plastocyanin
integrity or the proper redox cycling of the photosynthetic and cytochrome-c oxidase involved in the electron transport
apparatus (29). Plants have developed fine-tuned homeostatic in the chloroplast and the mitochondria, respectively, and the
mechanisms allowing the delivery of the specific metal to Cu/ZnSODs that are found in the cytosol and the chloroplast
metalloproteins while limiting uncontrolled metal reactivity in plants (Fig. 5). Other cuproproteins found in plants such as
[reviewed in (64)]. Due to their extremely high reactivity, the the chloroplastic polyphenol oxidases (PPO) and the extra-
concentrations of metals as free ions must be very low inside cellular laccases are also redox-active enzymes (Fig. 5) (19).
cells. Metal toxicity avoidance is possible by the chelation of Fe is more widely utilized in plant redox biology and many
the metal to ligands, metallochaperones, or storage proteins. of the Fe-dependent proteins still remain to be discovered.
The control of the subcellular distribution of metals is another With the exception of the Mn-dependent oxygen evolving
strategy that allows plants to cope with metal toxicity. In this complex that provides the electrons from the water-splitting
context, the accumulation of metals in the vacuole is emerging complex to PSII and Cu-dependent plastocyanin that carries
as an important component of metal homeostasis in plants. the electrons from the cytochrome b6/f complex and PSI, the
For instance, tonoplast-located metal transporters and metal photosynthetic electron transport machinery relies on Fe-
chelating molecules such as nicotianamine play pivotal based cofactors (57, 71). Therefore, Fe is indeed qualitatively
functions for metal storage and mobilization in the vacuole and quantitatively the most important metal for photosyn-
(36, 42, 43, 45). thesis (Fig. 2). Up to 80% of the cellular Fe is in the chloroplast
To further tackle the toxicity of the redox-active metals, (35, 78, 84). The PSII complex contains a polypeptide to which
another strategy used by plants is the strict control on the Fe is directly coordinated as well as a heme protein subunit.
accumulation of superoxide and H2O2 (5), the two important The cytochrome b6/f complex contains two hemoproteins and
players in the undesired Haber-Weiss cycle. Antioxidant en- a Rieske-type [2Fe-2S] cluster protein. The subunits PsaA,
zymes are involved in the breakdown of the reduced forms of PsaB, and PsaC of the plant PSI are all [4Fe-4S] cluster pro-
oxygen in plant cells: superoxide dismutases (SODs) catalyze teins. The ultimate electron acceptor ferredoxin, which has
the dismutation of O2 - $into O2 and H2O2, followed by the diverse and crucial redox functions in the chloroplast, is also a
catalase and peroxidase mediated decomposition of H2O2 to [2Fe-2S] cluster protein (10). In total, 22 Fe atoms can be in-
produce H2O and O2 [reviewed in (5, 63)]. Strikingly, many of volved in a single electron transfer throughout a photosyn-
these proteins require Fe or Cu and Zn as cofactors in the thetic chain (71). Apart from this, Fe is ubiquitously used
chloroplast and the cytosol (Fig. 4), or Mn in the mitochondria. in oxidation–reduction processes (Fig. 1). For instance, the
METAL HOMEOSTASIS IN PLANTS AND OXIDATIVE STRESS 923

FIG. 4. The enzymatic reactive oxygen species (ROS)-detoxifying mechanisms in plant chloroplasts are dependent on
metal-based reactions. In plant chloroplasts, ROS homeostasis mainly relies on a core machinery in which metalloenzymes are
pivotal (triangle). Fe- and Cu-dependent enzymes are depicted with black-lined symbols. The superoxide ion produced through
the photosynthetic activity is converted into molecular oxygen and H2O2 by SODs. SODs found in plant chloroplasts are all
dependent on Fe or Cu cofactors. H2O2 is then subsequently reduced to water (H2O). The enzyme APX requires AsA and generates
monodehydroascorbate (MDA). The enzyme glutathione peroxidase (GPX) requires GSH and generates glutathione disulphide
(GSSG). The Fe-dependent APX is thought to be the major pathway used by plants for H2O2 removal. The catabolism of H2O2
through these cycles is described as the ascorbate-glutathione cycle. The regeneration of the reducing power (AsA and GSH) is
allowed by the cyclic transfer of reducing equivalents (peripheric cycles). Within the chloroplast, MDA can be directly reduced to
AsA by the MDA reductase (MDAR). MDA (two molecules) can also be spontaneously converted into AsA and dehydroascorbate
(DHA). DHA is then reduced to AsA by the action of the DHA reductase (DHAR), using GSH as the source of reducing
equivalents. The GSSG produced by both pathways is further reduced back to GSH using the NADPH as an ultimate electron
donor, a reaction catalyzed by the glutathione reductase (GR). Oxidized and reduced forms for each molecule are indicated.

FIG. 5. Cu transport, delivery, utilization, and chelation in plant cell. The different proteins involved in Cu homeostasis
are presented with respect to their membrane and organelle location. Rectangles indicate Cu-transporters and arrows Cu
transport orientation. White circles represent Cu-chaperones, black circles Cu-utilizing proteins, and gray circles Cu-chelating
proteins. COPT, copper transporter; HMA, heavy metal associated transporter; RAN, response to antagonist ( = HMA7);
PAA, P-type ATPase (PAA1 = HMA6, PAA2 = HMA8), ATX, antioxidant; CCH, copper chaperone; CCS, copper chaperone
for superoxide dismutase; CSD, Cu/ZnSOD; ETR, ethylene receptor; COX, cytochrome c oxydase; LAC, laccase; AO,
ascorbate oxidase; AAO, amine oxidase; ARPN, plantacyanin.
924 RAVET AND PILON

synthesis of chlorophyll, heme, and siroheme occurs in the through soil acidification, reduction of the metal, or excretion
chloroplast and is dependent on several Fe-proteins (55, 58, of phytosiderophores [reviewed in (69)]. A common feature of
60, 87). Nitrate and sulfur assimilation also rely on the use of plant responses to metal limitation is the upregulation of high-
Fe as redox cofactor in essential enzymes such as the nitrate affinity transporters in concert with subsequent distribution
reductase (heme) in the cytosol and nitrite reductase ([4Fe- mechanisms. Under metal excess, plants limit their metal
4S]), glutamate synthetase ([3Fe-4S]), adenosine phospho- uptake, induce the synthesis of metal-chelating molecules and
sulfate reductase ([2Fe-2S]), and sulfite reductase ([4Fe-4S]) in proteins, and activate ROS detoxifying mechanisms (67).
the chloroplast (10).
With such a critical demand for Fe and Cu in the oxygen- How Plants Deal with Cu Scarcity: A Prioritization
producing chloroplast, photosynthetic organisms have of Cu Toward Plastocyanin and a Metal-Switch
evolved regulatory mechanisms that serve to constantly ad- to Maintain SOD Activity in the Chloroplast
just the organism’s physiology in order to optimize metal use.
The progressive discovery of almost all Cu-proteins in
In addition, while metal toxicity is a constant challenge at the
plants is important for our understanding of Cu homeostasis
cellular and molecular levels, plants have to face low metal
(Fig. 5) (19). In addition, we identified what probably is the
bioavailability in the rhizosphere (38). Plants therefore have
majority of all Cu transporters and Cu chaperones. Further
systems that allow the increase of metal bioavailability
understanding of Cu homeostasis has been made possible
by integration of genetic and biochemical data obtained
for individual components into larger models via systems
approaches, thus linking regulatory mechanisms and physi-
ological responses. The identification and analyses of regu-
latory transcription factors from algae and plants indicate that
copper homeostasis relies on highly conserved mechanisms
and that the intracellular Cu levels are the main regulators of
its distribution and use [reviewed in (19, 56)].
Plastocyanin, a copper-containing protein in the lumen of
the thylakoids, is essential for photosynthesis in plants (92).
Any environmental or genetic limitation of the delivery of Cu
to the chloroplast decreases the plastocyanin content likely
because the protein is less stable in the absence of Cu (1, 78).
As a consequence, the electron transport rate throughout the
photosynthetic apparatus is decreased under Cu deficiency.
Under these conditions, ROS accumulation may occur in the

FIG. 6. Plant response to Cu deficiency: the ‘‘economy’’

model. In plants, Cu limitation triggers a large molecular
remodeling that allows Cu to be preferentially allocated to
PC in the chloroplast lumen. Central to this response is the
conserved transcription factor SPL7 (for squamosa promoter
binding protein-like 7) which is active under Cu limitation
and which regulates almost all Cu responses in Arabidopsis
(lower panel). SPL7 is required for the up-regulation of the
expression of the plasma membrane COPT1 Cu transporter
involved in the primary Cu uptake from the rhyzosphere.
SPL7 induces also the plasma membrane COPT6 Cu trans-
porter in the shoot, likely involved in the Cu loading into the
photosynthetic cells. Concomitantly, SPL7 induces the ex-
pression of the so-called Cu microRNAs, which in turn
down-regulate transcripts encoding for most of the Cu pro-
teins in various subcellular compartments (see text for de-
tails). This regulatory mechanism allows the prioritization of
Cu use toward PC, which is not a target of the Cu micro-
RNAs. Cu transport into the chloroplast lumen is also facil-
itated by the stabilization of the P-type ATPase PAA2 at the
thylakoids under low Cu condition. This regulation is not
dependent on SPL7. Therefore, this additional layer of reg-
ulation allows the chloroplast to control its own Cu alloca-
tion, independently to the other subcellular compartments.
One major consequence of this Cu-microRNA-dependent
economy model is the depletion of Cu/ZnSOD activity in
both the chloroplast and the cytosol. Interestingly, an FeSOD
is up-regulated by SPL7 and accumulates in the chloroplast
to compensate for the lack of the Cu-requiring functional
counterpart. FSD, FeSOD.
METAL HOMEOSTASIS IN PLANTS AND OXIDATIVE STRESS 925

light because of photo-reduction of oxygen at PSII in the ab- magnitude of Cu-microRNA-mediated Cu pool savings.
sence of downstream electron acceptors. In addition, photo- However, given the fact that Cu/ZnSOD and PPO are abun-
synthetic electron transport is required for the maintenance of dant proteins in the chloroplast (73, 95) at least under Cu
high NADPH/NADP + and GSH/GSSG ratios and such a sufficiency, it is likely that the absence of competition for Cu in
reduced state of the stroma is important for ROS scavenging this organelle, and potentially also at the cellular and systemic
systems such as glutathione dependent peroxidases. In the levels, gives an advantage to maturation of plastocyanin when
green alga Chlamydomonas, a heme-containing cytochrome c6 Cu is deficient (Fig. 6). In this context, a recent study in poplar
is expressed under Cu deficiency and serves to functionally revealed that plastocyanin is the first protein to be recovered
replace plastocyanin while the latter is actively degraded (56). when Cu is re-supplied to previously Cu-deficient plants (73).
However, there is no evidence for compensation by a cyto- Strikingly, the photosynthetic efficiency was quickly recovered
chrome c6-homolog in higher plants (59, 78). Instead, plants after Cu-resupply, while the restoration of other Cu-depen-
prioritize Cu distribution toward plastocyanin (Fig. 6) (73, 82). dent enzymatic activities, such as the Cu/ZnSOD or the PPO
In plants, Cu homeostasis is regulated by the SPL7 (squa- activities, was delayed for several days (73). These data lend
mosa promoter binding protein-like) transcription factor that strong support to the idea that Cu delivery to plastocyanin is
is active under Cu starvation (Fig. 6) (12, 93). In Arabidopsis indeed a priority and that the Cu-microRNAs serve to allow
important targets of SPL7 are the COPT1, COPT5, and COPT6 Cu economy, the optimal use of Cu when it is scarce.
genes. COPT1 encodes for the high-affinity Cu transporter of While this economy strategy may be efficient for continued
the roots that is responsible for the primary uptake of Cu (76). biomass production through photosynthesis, the depletion of
COPT6 is expressed in shoots and is located at the plasma the Cu/ZnSODs would at the same time have the potential to
membrane (40). Both are upregulated in Cu-deficient plants in impact the cellular redox balance. Cu/ZnSODs are by far the
order to increase the absorption capacity at a systemic level most abundant both in the cytosol and the chloroplast under
(COPT1) and more specifically in photosynthetic cells Cu-replete growth conditions. Other SOD enzymes in plants
(COPT6) (40, 76). COPT5 is also induced by Cu deficiency and are the mitochondrial MnSOD and the plastidial FeSOD [re-
is responsible for Cu efflux from the vacuole, suggesting its viewed in (68)]. In the chloroplast of mature plants, Cu/
function in Cu remobilization (43). At the top of the Irving- ZnSOD was the only SOD active at detectable level in native
Williams series, Cu binds very tightly to its targets (50). gel-assays in Cu-sufficient conditions (24). Interestingly,
Therefore, any competing Cu sinks (i.e., Cu-utilizing proteins) FeSOD accumulates in the chloroplast under Cu deficiency
must be eliminated when Cu becomes limiting in order to conditions (Fig. 6). The transcription of the corresponding
allow the preferential Cu delivery to plastocyanin. In plants, gene is upregulated by SPL7 and therefore its expression is
this mechanism of ‘‘copper economy’’ involves the post- tightly coordinated with the loss of the Cu/ZnSODs (19).
transcriptional regulation of dispensable Cu-enzymes by Likewise the metal-based switch between the plastocyanin
various microRNAs, which are themselves under the control and cytochrome c6 in Chlamydomonas (56), the synthesis of the
of SPL7 (2, 93). Interestingly, transcripts encoding the essen- FeSOD likely serves to compensate for the loss of the Cu/
tial Cu-proteins such as plastocyanin are not targeted for ZnSOD; however, there is so far little insight into the physi-
degradation by the microRNAs (2), suggesting that these ological relevance of the Cu-regulated SOD enzyme switch,
proteins are priority targets for scarcely available Cu. because both Cu/ZnSOD and FeSOD seem redundant under
To date, five microRNA families have been shown to various laboratory growth conditions [reviewed in (68)].
down-regulate dispensable Cu-proteins in plants: miR397, In addition to the preferential allocation of Cu to plastocy-
miR398, miR408, miR857, and miR1444 (2, 52, 73). These anin facilitated by the removal of competitive sinks, copper
microRNAs, which are up-regulated by Cu limitation, are translocation into the thylakoids is also activated in response to
known as the copper microRNAs [Cu-microRNAs; see (19)]. Cu deficiency (Fig. 6) (82). Plastocyanin is a nuclear-encoded
MiR398 targets transcripts of the Cu/ZnSOD proteins as well protein that needs to be translocated into the thylakoid lumen,
as the transcript of copper chaperone for superoxide dis- prior to acquisition of its metal cofactor for maturation. Similar
mutase, the chaperone responsible for Cu delivery to Cu/ to the peptide, Cu has to be transported across two membrane
ZnSOD [reviewed in (19)]. MiR398 also targets one of two systems in the chloroplast: the inner envelope of the chloro-
isoforms of COX5b, a subunit of cytochrome c oxidase in plast and the thylakoids (19). Two P-type ATPases (PAA) act
the mitochondria, but the relevance of this mild regulation as Cu transporters. PAA1/HMA6 and PAA2/HMA8 (HMA
by miR398 is not understood (93). Three other families of for Heavy-Metal Associated protein) are responsible for Cu
miRNAs, miR397, miR408, and miR857, target transcripts of transport through the inner envelope of the chloroplast and the
extracellular Cu-proteins such as laccases and a secreted thylakoids, respectively (1, 78). Upon Cu deficiency, the PAA2
plastocyanin-like protein, plantacyanin (2). The miR1444 transporter accumulates in the thylakoids. PAA2 half-live is
family targets the chloroplastic PPO (51, 52, 73). These Cu- increased by 2-fold when plants are grown on very low Cu
microRNAs accumulate at extremely low Cu concentrations concentrations similar to those that activate the SPL7 pathway
and disappear at higher Cu concentrations. This observation (82). However, this PAA2 stabilization is not dependent on
suggests that the Cu economy strategy is employed when the SPL7 since the regulation is still observed in the SPL7-deleted
use of the few available Cu atoms has to be optimized (19). Arabidopsis. Instead, low Cu levels in the chloroplast could be
Both laccase and PPO are present in many copies in plants. the signal for PAA2 stabilization (82). The modulation of PAA2
Either 4 or 2 Cu atoms per monomer, respectively, are nec- accumulation, simultaneously with the SPL7-mediated re-
essary for their activities. Cu/ZnSOD functions as a dimer sponses, likely controls the flow of available Cu ions in the
and requires 1 Cu atom per monomeric subunit. The relative chloroplast stroma to the thylakoid lumen.
abundance of each Cu-protein in a plant cell remains to be The available data support a model where regulatory
determined, which limits any firm conclusion about the mechanisms have evolved to largely respond to Cu limitation.
926 RAVET AND PILON

All the known Cu-specific regulatory mechanisms are acti- homeostasis, the phytohormone ethylene as well as the free
vated at very low Cu concentrations ( < 1 lM CuSO4 in agar radical nitric oxide (NO) and derived nitroso- and glutathione
media or < 50 nM CuSO4 in hydroponics), which is far below complexes are receiving particular attention (30, 70). Ethylene
the reported toxicity levels (2, 19). It is tempting to consider and nitric oxide have the potential to signal over longer dis-
that Cu deficiency is more prevalent than Cu toxicity in na- tances and might therefore help to communicate the need for
ture. Most of the Cu-proteins are abundant under nonlimiting iron in distant plant parts.
Cu conditions, but their activities are seemingly dispensable. Fe is abundant in plant cells and is widely implicated in
These Cu-proteins may themselves act as Cu buffers when Cu redox machineries. However, Fe is a relatively ‘‘labile’’ co-
is present at levels above the optimum. Interestingly, with factor when compared to Zn and Cu and free iron is highly
respect to electron transport function plastocyanin accumu- reactive and toxic. Thus, iron homeostasis has to be tightly
lates at supra-optimal levels in plants grown under Cu- controlled. Among the transition metals, Fe is the only one
repleted conditions (3, 73). This suggests that plastocyanin that required the evolution of a dedicated storage protein
might buffer excess Cu when Cu levels in the thylakoid lumen called ferritin. Ferritin is almost ubiquitously found in all
exceed the needs for electron transfer. Abundance of plasto- kingdoms of life with the exception of some fungi, including
cyanin transcripts is not affected by high Cu concentration in yeast. Yeast tackles Fe toxicity using the vacuole as a storage
the media (2). Therefore, a significant fraction of plastocyanin compartment (48). In plants also, Fe storage in the vacuole is
may be in the apo form and could readily bind excess Cu emerging as an important component of Fe homeostasis as
within the thylakoid lumen. Interestingly, the stabilization of shown by the involvement of the tonoplast-located VIT1,
PAA2 observed in response to low copper levels is abolished NRAMP3, and NRAMP4 transporters in Fe storage and mo-
in Arabidopsis by plastocyanin depletion (82). This indicates bilization during the development of Arabidopsis (42, 45).
that the presence of apo- and holo-plastocyanin, as an electron However, ferritin is found from bacteria to humans and
carrier or as a chelating protein, may be a prerequisite for higher plants and has important physiological functions (16,
loading of Cu into the thylakoid lumen in order to avoid 85). The recent studies on ferritin function and regulation in
nonspecific reactions of Cu. plants have revealed a tight interplay between these proteins,
the efficient use of Fe, and the protection against Fe-mediated
oxidative stress (Figs. 7 and 8) (6, 72, 74).
How Plants Deal with Fe Toxicity: Ferritin’s Function
and Regulation and Interplay with ROS
The Fe proteome (ironome) is much larger than the Cu
proteome. The identification of the complete ironome in
plants is essential in order to get a much better view of Fe
homeostasis. Another challenge for studies of Fe homeostasis
is the lack of conservation of regulatory mechanisms in dif-
ferent kingdoms. In yeast and mammals, Fe homeostasis
signaling involves Fe-S clusters because disruption of Fe-S
cluster assembly systems disrupts the regulation of cellular Fe
uptake and storage. In yeast, the master transcriptional reg-
ulator called activator of ferrous transport (AFT) shuttles from
the cytosol to the nucleus, where it activates Fe-responsive
genes. AFT activation is negatively regulated by Fe-S cluster-
containing glutaredoxin complexes in the cytosol. In this
fashion iron uptake in yeast is regulated in response to cyto-
solic Fe-S cluster availability, which in turn relies on the mi-
tochondrial Fe-S assembly system (49). In animals, the iron
responsive element-iron regulatory protein (IRE-IRP) system
regulates at the post-transcriptional level Fe acquisition and FIG. 7. Ferritin is important for the redox balance in the
chloroplast and allows plants to benefit from increased Fe
storage. Mammalian IRP is a cytosolic form of aconitase, a
bioavailability. Fe is considered as a limiting factor in most
4Fe-4S cluster-binding protein (37). These IRE-IRP systems soils for plant growth. The supply of a bioavailable source of
are not conserved in plants and the involvement of Fe-S Fe to the soil increases the growth of Arabidopsis plants. The
cluster proteins in Fe status sensing is not yet demonstrated. absence of ferritin does not lead to any macroscopic pheno-
Therefore, molecular studies in plants have first focused on type when grown in soil containing low Fe ( - Fe), yet the lack
the identification and biochemical characterization of indi- of Fe chelation leads to an increase of ROS levels. The mutant
vidual components of Fe homeostasis, and the analysis of can circumvent oxidative damage by increasing ROS detoxi-
their regulation. Because of its major consequences for the fying enzyme activities. By contrast, under high Fe condition
world’s feedstock quality and quantity, plant responses to Fe ( + Fe), while the wild-type takes advantage of the available Fe,
deficiency have received most attention. Major progress has the ferritin mutant undergoes severe oxidative stress. The
ROS detoxifying mechanisms are overwhelmed and are not
been made in the identification of the high affinity transport
capable anymore of preventing ROS toxicity. Consequently,
systems responsible for Fe uptake from the soil as well as in pleiotropic defects both in vegetative and reproductive organs
the identification of the signaling pathways responsible for are observed in the mutant, including a reduced carbon as-
their induction upon Fe deficiency [for a recent review, see similation that ultimately results in a decreased biomass.
(44)]. Long distance signaling events are involved in this Thus, the beneficial effect of the high Fe availability fully relies
control. Among the potential signal molecules involved in Fe on the presence of ferritin in the chloroplast.
METAL HOMEOSTASIS IN PLANTS AND OXIDATIVE STRESS 927

activities are catalase and ascorbate peroxidase (APX), both

Fe-dependent activities. Strikingly, while control plants grew
faster when the soil was supplemented with Fe, suggesting
that Fe is limiting, the growth of the ferritin-less mutant was
reduced (Fig. 7) (72). This result illustrates that ferritins are
required to take advantage of Fe bioavailability, while
avoiding its toxicity. The absence of ferritin had a strong im-
pact on the carbon fixation rate, but had no effect on the
photosynthetic electron transport itself (72). Therefore, the
observed reduction of biomass in the ferritin-less mutant
likely results from the effects of the Fe-mediated oxidative
stress on the subsequent carbon fixation reactions, and not
from a direct oxidation of the photosystem components.
Other Fe toxicity-related phenotypes are observed in the seed
and the flower of the ferritin mutants (72), suggesting that
ferritins not only are important for photosynthesis in green
FIG. 8. A model for ferritin regulation: the interconnec- tissues but also for versatile functions in different cell types in
tions between Fe and ROS signals. Ferritin is regulated at plants.
the transcriptional level by iron (Fe) through a cis-element
When ferritins are experimentally overexpressed in plant
iron dependent regulatory sequence (IDRS), which is neces-
sary for the repression of the plant ferritin gene under low Fe cells, two effects are observed. First is the induction of the Fe
condition. This pathway involves the accumulation of nitric acquisition system because the chelation of Fe at higher than
oxide (NO) in the chloroplast. The ferritin gene expression is desired levels likely alters the proper delivery of Fe to
also controlled by H2O2 at the transcriptional level, but this the metalloproteins (89). Second is the activation of the ROS-
regulation is independent on the IDRS. This suggests that detoxifying mechanism. Illegitimate Fe accumulation result-
two different pathways lead to the accumulation of ferritin ing from the overexpression of ferritin in the chloroplast leads
transcripts in response to Fe and H2O2. Ferritin expression is to an increase of various peroxidase, catalase, and glutathione
also controlled at the post-transcriptional level by Fe and reductase activities (13). These plants show phenotypic signs
H2O2. Fe and H2O2 promote the ferritin mRNA decay few of Fe excess and chloroplast structure alterations (13). There-
hours after the accumulation of the transcripts. This addi-
fore, both the absence and the overaccumulation of ferritins
tional layer of regulation likely constitutes a feedback control
allowing the plants to prevent overproduction of the ferritin impair the redox balance (13, 89). Because the proper syn-
transcript. Ultimately, the storage of Fe in the newly syn- thesis of ferritin is a key feature of the control of Fe homeo-
thesized ferritin decreases the free Fe present in the chloro- stasis, the mechanism controlling ferritin expression has been
plast, leading to the arrest of the transcription and the re- extensively studied. In animals, ferritin synthesis, together
stabilization of the transcript. This multilayered regulatory with the Fe acquisition systems, is regulated at the post-
network allows a tight control of ferritin synthesis, with re- transcriptional level in response to the cellular Fe levels and
spect with the presence of Fe and the production of ROS. oxidative stress. This regulation involves the IRP/IRE system
(37). In plants, this regulatory mechanism is not present (7)
and ferritin accumulation in response to Fe is primarily con-
Ferritins are macromolecular complexes made of 24 sub- trolled at the transcriptional level through a cis-element,
units that form a cage-like structure in which up to 4500 Fe called iron-dependent regulatory sequence (IDRS) present in
atoms can be stored in a nonreactive form. The Fe storage the promoter region of some ferritin genes (Fig. 8). This se-
mechanism is based on two intrinsic catalytic activities of the quence is necessary for the repression of the ferritin synthesis
peptide, (i) the ferroxidase activity that first oxidizes Fe2 + in under low Fe conditions (65). Thus, ferritin accumulation in
Fe3 + , and then (ii) the nucleation of the Fe3 + in a mineral core response to Fe addition to the media results from the de-
in which Fe is associated with phosphate (90). In mammals, repression of the transcription of the ferritin gene. Interestingly,
ferritins are located in the cytosol and constitute a pool of Fe an accumulation of NO in the chloroplast is detected already
required for early development (86). They are also expressed a few minutes after Fe treatment and this NO burst is nec-
in the mitochondria of specific cell types where they protect essary for the de-repression of the ferritin synthesis (6). The
against oxidative stress (47). Recent studies in both algae and NO -mediated de-repression is dose dependent and active
plants have revealed that, in these species, ferritin does not even when NO is artificially produced in plant cell in absence
constitute a major iron pool, but is solely involved in the of Fe, suggesting that NO acts as a downstream signaling
protection against Fe-mediated oxidative stress (20, 72). Fer- molecule in the pathway (Fig. 8) (6, 61). Oxidative signals are
ritins are thought to localize mainly to the chloroplast in definitively in the signal transduction pathway since extra-
plants, but low levels are detected in the mitochondria as well cellular treatments with antioxidant such as N-acetylcysteine
(83). Ferritins accumulate when plants are grown on high Fe and glutathione abolish ferritin accumulation (31, 77). While
concentrations [reviewed in (15, 16)]. Their involvement in Fe the source of the NO production in the chloroplast is still
chelation to prevent its reactivity with oxygen has been pro- under investigation, it seems dependent on the glutathione
posed (13). Indeed, recent characterization of Arabidopsis levels (88).
possessing single, double, triple, and quadruple disruptions H2O2 also triggers the increase of ferritin transcripts in
of ferritin genes showed that the levels of ROS and the activity plants (66, 77). The pathway by which H2O2 activates ferritin
of the ROS-detoxifying enzymes are elevated in the absence of transcription is independent of the IDRS-mediated Fe-
ferritin (Fig. 7) (72). Notably, among the increased enzyme induced pathway described above (6). Co-treatment with Fe
928 RAVET AND PILON

and H2O2 revealed an additive accumulation of ferritin tran- anism preventing the deleterious effects of the excessive ac-
scripts, relative to the accumulation observed for individual cumulation of the protein in plant cell. At the molecular level,
treatments (6). Therefore, two synergistic pathways involving this mRNA degradation process relies on the ribo-nucleolytic
specific oxidative signals lead to ferritin transcript accumu- activity of the previously identified mRNA destabilization
lation (Fig. 8). However, H2O2 only resulted in a mild ferritin (DST) machinery (62). Indeed, two mutants affected in
accumulation at the protein level, while Fe triggered a strong the mRNA degradation machinery (39) showed an over-
ferritin accumulation. Fe incorporation into ferritin is also an accumulation of ferritin mRNA and other target transcripts
important determinant of its stability (46). Therefore, the lack and as a consequence, the plant response to oxidant and pro-
of Fe may prevent ferritin protein accumulation in response of oxidant treatment such as Fe and high light was impaired, and
H2O2. In the absence of free iron ions, H2O2 is not nearly as photosynthesis and plant growth was decreased (74).
dangerous for the cell when compared to the presence of free Therefore, ferritins are at the heart of the Fe-oxygen
Fe that can promote the Haber-Weiss cycle (5). Therefore, it is chemistry in the chloroplast. They act as fine regulators of the
tempting to hypothesize that the H2O2-induced transcription free Fe in the chloroplast, providing the most advantageous
of ferritin genes serves as a mechanism to prepare cells so that balance between Fe use and sequestration. Both the protein
they have increased Fe sequestering capacity in an oxidizing and its mode of regulation are intimately interconnected with
environment. oxygen-derived molecules (Figs. 7 and 8). In this context,
Fe regulates ferritins along with other ROS-detoxifying ferritins definitively function as homeostatic proteins. Loss of
systems. In some plants, a Cu/ZnSOD transcript accumula- ferritin regulation or function results in the activation of oxi-
tion in response to Fe has been reported (81). This also occurs dative stress responses. The next challenge is to understand
in response to high light, other metals, and various oxidative the significance of these oxidative stress responses. What is
stresses [reviewed in (68)]. However, due to the very large really metal toxicity: what is specific to a metal and what is
effect of Cu on their expression, each of these conditions due to a general oxidative stress response? The interplay be-
previously shown to affect Cu/ZnSOD expression should be tween metals and ROS make it challenging to tease apart
revised under controlled Cu nutrition conditions. An unam- cause and effect. In vitro studies are also hard to interpret
biguous Fe-responsive protein is the cytosolic enzyme ascor- because of the difficulty to reproduce the cellular redox en-
bate peroxidase APX1 (28). Similar to SOD, APX1 is also vironment. A major barrier to tackle is the identification of the
regulated by high light (41). The effect of Fe on APX1 pro- molecular actors responsible for ferritin responses, this will
moter activity has been clearly documented at the molecular require the identification of transcription factors, for instance
level (28). Interestingly, this APX member is located in the through yeast one-hybrid experiments, as well as identifica-
cytosol and the expression of the chloroplastic counterparts is tion of the sensors that signal ferritin transcript turn-over,
not affected by Fe (28). It is now accepted that ROS detoxifi- which might require a genetic approach. Physiological and
cation relies on the concerted action of different cellular pharmacological experiments (6, 61, 72) as well as the recent
compartments especially in the case of H2O2 which is known progress in cellular imaging of ROS using specific probes (6,
to diffuse through the biological membranes (34). Interest- 72) can provide information concerning the nature of the ROS
ingly, the expression of APX in response to Fe and high light is involved. However, genetic approaches are now required to
also dependent on the glutathione level (28, 41). Ferritins are further investigate the primary signals responsible for the
also transcriptionally regulated by the circadian clock, and sensing of Fe. The identification of the source of NO pro-
this regulation is important for Fe homeostasis in Arabidopsis duction in response to Fe in the chloroplast, as well as the
(26). Given the dramatic effect of the daily light oscillations identification of the protein involved in Fe-induced decay of
on chloroplast metabolism and on the abundance of the Fe- ferritin mRNA, would be an excellent entry point. This would
proteins in the plastid, it is likely that the clock-dependent lead to further researches toward the discovery of the Fe
ferritin regulation serves to anticipate the potential Fe reac- sensor and signaling pathway(s). The integration of these Fe-
tivity during transitions from dark to light. signals in the oxidative stress response network should ulti-
Ferritins are also regulated together with other Fe-respon- mately allow a better understanding on the interplay between
sive proteins at the post-transcriptional level (Fig. 8) (74). Fe and oxygen.
Surprisingly, this regulatory mechanism decreases the mRNA
stability of some early Fe-induced genes. This apparent par-
Conclusion and Outlook
adox was explained by kinetic studies: this additional level of
regulation by mRNA turnover, together with the activation of Photosynthetic organisms frequently grow faster when Fe
transcription, allows some proteins to accumulate only tran- is supplied to their growth site, indicating that this micro-
siently in response to Fe or H2O2. Both Fe and H2O2 signaling nutrient is limiting in their native habitat (18, 72). Cu defi-
involve an oxidative step, since treatments with antioxidants ciency is rare in nature, but in agricultural systems Cu
abolished both the Fe- and the H2O2-mediated ferritin mRNA supplementation can improve crop yields (19). An explana-
destabilization (74). Thus, the existence of two independent tion for the beneficial effect of Fe feeding is that the high
pathways is not yet established. Interestingly, a trans- requirement for Fe is thwarted by its poor bioavailability in
criptomic study showed that, next to ferritin, most of the other most of the arable soils. Classic symptoms of Fe and Cu de-
genes affected by this regulation are transcription factors. ficiencies include reduction in vegetative biomass, chlorosis,
Among these Fe-induced transcription factors, some are im- decreased photosynthetic activity, defects in plant morphol-
portant for protection against oxidative stress in plants (22, ogy and seed production, or in the case of Cu deficiency
23). Transient expression of these transcription factors is lodging of cereals. Deficient plants are also, directly or indi-
pivotal for their proper function in gene regulation. In the case rectly, more susceptible to various stresses (drought, diseases,
of ferritin, this regulation likely constitutes a feedback mech- pathogens). Metal toxicity is not as prevalent in nature.
METAL HOMEOSTASIS IN PLANTS AND OXIDATIVE STRESS 929

However, plants that are grown in flooded fields such as rice, 3. Abdel-Ghany SE. Contribution of plastocyanin isoforms to
which represents an important part of the staple food photosynthesis and copper homeostasis in Arabidopsis thali-
worldwide, are particularly prone to accumulate Fe in excess, ana grown at different copper regimes. Planta 229: 767–779,
as well as toxic metals such as cadmium. 2009.
The importance of Fe in plant biology makes it a chal- 4. Abreu IA and Cabelli DE. Superoxide dismutases—a review
lenging ion to study. Constant progress is made with inputs of the metal-associated mechanistic variations. Biochim Bio-
from molecular, biochemical and phenotypic analyses yet we phys Acta 1804: 263–274, 2010.
still have a very elusive picture of Fe homeostasis. A partic- 5. Apel K and Hirt H. Reactive oxygen species: metabolism,
ularly understudied field is Fe utilization by plant cells. In a oxidative stress, and signal transduction. Annu Rev Plant Biol
55: 373–399, 2004.
context of Fe-limiting conditions, prioritization of the use of
6. Arnaud N, Murgia I, Boucherez J, Briat JF, Cellier F, and
the metal has to be orchestrated. A minimal activity of es-
Gaymard F. An iron-induced nitric oxide burst precedes
sential metabolisms such as respiration and photosynthesis is
ubiquitin-dependent protein degradation for Arabidopsis
required for plant survival. The depletion of the Fe-dependent AtFer1 ferritin gene expression. J Biol Chem 281: 23579–
ROS detoxifying mechanisms, simultaneously with the pro- 23588, 2006.
gressive imbalance of the metabolism that leads to the pro- 7. Arnaud N, Ravet K, Borlotti A, Touraine B, Boucherez J,
duction of ROS, must be limited to avoid cell death. Therefore, Fizames C, Briat JF, Cellier F, and Gaymard F. The iron-
the integrated approaches that led to a nearly complete un- responsive element (IRE)/iron-regulatory protein 1 (IRP1)-
derstanding of Cu homeostasis in plants, if applied to Fe, cytosolic aconitase iron-regulatory switch does not operate
should provide new insights into Fe utilization by plants. in plants. Biochem J 405: 523–531, 2007.
An important remaining challenge for Cu homeostasis is 8. Asada K. The water-water cycle in chloroplasts: scavenging
the discovery of the biological function of several Cu-utilizing of active oxygens and dissipation of excess photons. Annu
proteins. Among them, SOD should deserve most of the at- Rev Plant Physiol Plant Mol Biol 50: 601–639, 1999.
tention. Despite the numerous functions attributed to this 9. Asada K. Production and scavenging of reactive oxygen
protein, we have to admit that the phenotypic and molecular species in chloroplasts and their functions. Plant Physiol 141:
characterization of mutants virtually depleted of SOD activity 391–396, 2006.
in the chloroplast and the cytosol belies all biology textbooks 10. Balk J and Lobreaux S. Biogenesis of iron-sulfur proteins in
because such mutants do not show dramatic phenotypes even plants. Trends Plant Sci 10: 324–331, 2005.
under stress conditions. These SOD have been conserved in 11. Bannister WH, Bannister JV, Barra D, Bond J, and Bossa F.
plants during evolution and the switch between the Fe and Cu Evolutionary aspects of superoxide dismutase: the copper/
dependent SOD in plants gives rise to a strict requirement of zinc enzyme. Free Radic Res Commun 12–13: 349–361, 1991.
12. Bernal M, Casero D, Singh V, Wilson GT, Grande A, Yang H,
SOD activity maintenance in the chloroplast. The extracellular
Dodani SC, Pellegrini M, Huijser P, Connolly EL, Merchant
laccases form an important Cu-protein family with poorly
SS, and Krämer U. Transcriptome sequencing identifies
understood biological function. Some laccase family members
SPL7-regulated copper acquisition genes FRO4/FRO5 and
(3 out of 17 in Arabidopsis) have been implicated in cell wall the copper dependence of iron homeostasis in Arabidopsis.
formation and remodeling, but the majority of the laccases Plant Cell 24: 738–761, 2012.
have not been characterized functionally. Homeostatic pro- 13. Briat JF, Lobréaux S, Grignon N, and Vansuyt G. Regulation
cesses allow immobile plants to respond and acclimate to of plant ferritin synthesis: how and why. Cell Mol Life Sci 56:
environmental stresses. Molecular remodeling triggers phys- 155–166, 1999.
iological adaptation in order to cope with challenging condi- 14. Briat JF, Curie C, and Gaymard F. Iron utilization and me-
tions. While molecular studies can elucidate the regulatory tabolism in plants. Curr Opin Plant Biol 10: 276–282, 2007.
mechanisms that control these changes in response to metal 15. Briat JF, Ravet K, Arnaud N, Duc C, Boucherez J, Touraine
availability, pivotal will be the identification of metal sensing B, Cellier F, and Gaymard F. New insights into ferritin
mechanisms that are so far poorly defined in plants. synthesis and function highlight a link between iron
homeostasis and oxidative stress in plants. Ann Bot 105: 811–
Acknowledgments 822, 2010.
Work in the authors’ laboratory is supported by the US 16. Briat JF, Duc C, Ravet K, and Gaymard F. Ferritins and iron
National Science Foundation (Grant number MCB 0950726) storage in plants. Biochim Biophys Acta 1800: 806–814, 2010.
17. Broadley MR, White PJ, Hammond JP, Zelko I, and Lux A.
and USDA-NIFA (Award Number: 2012-67013-19416). KR
Zinc in plants. New Phytol 173: 677–702, 2007.
has received funding from the European Union’s Seventh
18. Buesseler KO, Andrews JE, Pike SM, and Charette MA. The
Framework Programme (FP7/2007–2013) under Grant
effects of iron fertilization on carbon sequestration in the
agreement no. 273586.
southern ocean. Science 304: 414–417, 2004.
19. Burkhead JL, Reynolds KA, Abdel-Ghany SE, Cohu CM,
References
and Pilon M. Copper homeostasis. New Phytol 182: 799–816,
1. Abdel-Ghany SE, Müller-Moulé P, Niyogi KK, Pilon M, and 2009.
Shikanai T. Two P-type ATPases are required for copper 20. Busch A, Rimbauld B, Naumann B, Rensch S, and Hippler
delivery in Arabidopsis thaliana chloroplasts. Plant Cell 17: M. Ferritin is required for rapid remodeling of the photo-
1233–1251, 2005. synthetic apparatus and minimizes photo-oxidative stress in
2. Abdel-Ghany SE and Pilon M. MicroRNA-mediated sys- response to iron availability in Chlamydomonas reinhardtii.
temic down-regulation of copper protein expression in re- Plant J 55: 201–211, 2008.
sponse to low copper availability in Arabidopsis. J Biol Chem 21. Chapman DJ and Schopf JW. Biological and biochemical
283: 15932–15945, 2008. effects of the development of an aerobic environment. In:
930 RAVET AND PILON

Earth’s Earliest Biosphere: Its Origin and Evolution, edited by 38. Ivanov R, Brumbarova T, and Bauer P. Fitting into the harsh
Schopf JW. Princeton, NJ: Princeton University Press, 1983, reality: regulation of iron-deficiency responses in dicotyle-
pp. 302–320. donous plants. Mol Plant 5: 27–42, 2012.
22. Chen H, Lai Z, Shi J, Xiao Y, Chen Z, and Xu X. Roles of 39. Johnson MA, Perez-Amador MA, Lidder P, and Green PJ.
Arabidopsis WRKY18, WRKY40 and WRKY60 transcription Mutants of Arabidopsis defective in a sequence-specific
factors in plant responses to abscisic acid and abiotic stress. mRNA degradation pathway. Proc Natl Acad Sci U S A 97:
BMC Plant Biol 10: 281, 2010. 13991–13996, 2000.
23. Christianson JA, Wilson IW, Llewellyn DJ, and Dennis 40. Jung HI, Gayomba SR, Rutzke MA, Craft E, Kochian LV,
ES. The low-oxygen-induced NAC domain transcription and Vatamaniuk OK. COPT6 is a plasma membrane trans-
factor ANAC102 affects viability of Arabidopsis seeds porter that functions in copper homeostasis in arabidopsis
following low-oxygen treatment. Plant Physiol 149: 1724– and is a novel target of SQUAMOSA promoter binding
1738, 2009. protein-like 7. J Biol Chem 287: 33252–33267, 2012.
24. Cohu CM, Abdel-Ghany SE, Gogolin-Reynolds KA, Onofrio 41. Karpinski S, Escobar C, Karpinska B, Creissen G, and Mulli-
AM, Bodecker JR, Kimbrel JA, Niyogi KK, and Pilon M. neaux PM. Photosynthetic electron transport regulates the
Copper delivery by the copper chaperone for chloroplast expression of cytosolic ascorbate peroxidase genes in Arabi-
and cytosolic copper/zinc-superoxide dismutases: regula- dopsis during excess light stress. Plant Cell 9: 627–640, 1997.
tion and unexpected phenotypes in an Arabidopsis mutant. 42. Kim SA, Punshon T, Lanzirotti A, Li L, Alonso JM, Ecker JR,
Mol Plant 2: 1336–1350, 2009 Kaplan J, and Guerinot ML. Localization of iron in Arabi-
25. Davies KJA, Lin SW, and Pacifici RE. Protein damage and dopsis seed requires the vacuolar membrane transporter
degradation by oxygen radicals. IV. Degradation of dena- VIT1. Science 314: 1295–1298, 2006.
tured protein. J Biol Chem 262: 9914–9920, 1987. 43. Klaumann S, Nickolaus SD, Fürst SH, Starck S, Schneider S,
26. Duc C, Cellier F, Lobréaux S, Briat JF, and Gaymard F. Ekkehard Neuhaus H, and Trentmann O. The tonoplast
Regulation of iron homeostasis in Arabidopsis thaliana by the copper transporter COPT5 acts as an exporter and is re-
clock regulator time for coffee. J Biol Chem 284: 36271–36281, quired for interorgan allocation of copper in Arabidopsis
2009. thaliana. New Phytol 192: 393–404, 2011.
27. Dupont CL, Butcher A, Valas RE, Bourne PE, and Caetano- 44. Kobayashi T and Nishizawa NK. Iron uptake, translocation,
Anollés G. History of biological metal utilization inferred and regulation in higher plants. Annu Rev Plant Biol 63: 131–
through phylogenomic analysis of protein structures Proc 152, 2012.
Natl Acad Sci U S A 107: 10567–10572, 2010. 45. Lanquar V, Lelievre F, Bolte S, Hames C, Alcon C, Neumann
28. Fourcroy P, Vansuyt G, Kushnir S, Inzé D, and Briat JF. Iron- D, Vansuyt G, Curie C, Schroder A, Kramer U, Barbier-
regulated expression of a cytosolic ascorbate peroxidase Brygoo H, and Thomine S. Mobilization of vacuolar iron by
encoded by the APX1 gene in Arabidopsis seedlings. Plant AtNRAMP3 and AtNRAMP4 is essential for seed germina-
Physiol 134: 605–613, 2004. tion on low iron. EMBO J 24: 4041–4051, 2005.
29. Foyer CH and Noctor G. Redox regulation in photosynthetic 46. Laulhère JP, Labouré AM, and Briat JF. Mechanism of the
organisms: signaling, acclimation, and practical implica- transition from plant ferritin to phytosiderin. J Biol Chem 264:
tions. Antioxid Redox Signal 11: 861–905, 2009. 3629–3635, 1989.
30. Garcia MJ, Suarez V, Romera FJ, Alcantara E, and Perez- 47. Levi S and Arosio P. Mitochondrial ferritin. Int J Biochem Cell
Vicente R. A new model involving ethylene, nitric oxide and Biol 36: 1887–1889, 2004.
Fe to explain the regulation of Fe-acquisition genes in 48. Li L, Chen OS, McVey Ward D, and Kaplan J. CCC1 is a
strategy I plants. Plant Physiol Biochem 49: 537–554, 2011. transporter that mediates vacuolar iron storage in yeast. J
31. Gaymard F, Boucherez J, and Briat JF. Characterization of a Biol Chem 276: 29515–29519, 2001.
ferritin mRNA from Arabidopsis thaliana accumulated in re- 49. Lill R. Function and biogenesis of iron-sulphur proteins.
sponse to iron through an oxidative pathway independent Nature 460: 831–838, 2009.
of abscisic acid. Biochem J 318: 67–73, 1996. 50. Lippard SJ and Berg JM, eds. Principles of Bioinorganic
32. Goldstein S and Czapski G. The role and mechanism of Chemistry. New York, NY: University Science Books, 1994.
metal ions and their complexes in enhancing damage in bi- 51. Lu S, Sun YH, and Chiang VL. Stress-responsive microRNAs
ological systems or in protecting these systems from the in Populus. Plant J 55: 131–151, 2008.
toxicity of superoxide. Free Radic Biol Med 2: 3–11, 1986. 52. Lu S, Yang C, and Chiang VL. Conservation and diversity of
33. Halliwell B and Gutteridge JM. Oxygen toxicity, oxygen microRNA-associated copper-regulatory networks in Popu-
radicals, transition metals and disease. Biochem J 219: 1–14, lus trichocarpa. J Integr Plant Biol 53: 879–891, 2011.
1984. 53. Marschner H, eds. Mineral Nutrition of Higher Plants. Lon-
34. Halliwell B and Gutteridge JM. Role of iron in oxygen rad- don, UK: Academic Press, 1995.
ical reactions. Methods Enzymol 105: 47–56, 1984. 54. McCord JM and Fridovich I. Superoxide dismutase. An en-
35. Hänsch R and Mendel RR. Physiological functions of min- zymatic function for erthyrocuprein (hemocuprein). J Biol
eral micronutrients (Cu, Zn, Mn, Fe, Ni, Mo, B, Cl). Curr Chem 244: 6049–6055, 1969.
Opin Plant Biol 12: 259–266, 2009. 55. Meguro M, Ito H, Takabayashi A, Tanaka R, and Tanaka A.
36. Haydon MJ, Kawachi M, Wirtz M, Hillmer S, Hell R, and Identification of the 7-hydroxymethyl chlorophyll a reduc-
Kramer U. Vacuolar nicotianamine has critical and distinct tase of the chlorophyll cycle in Arabidopsis. Plant Cell 23:
roles under iron deficiency and for zinc sequestration in 3442–3453, 2011.
Arabidopsis. Plant Cell 24: 724–737, 2012. 56. Merchant SS and M Sawaya. The light reactions: a guide to
37. Hentze MW and Kuhn LC. Molecular control of vertebrate recent acquisitions for the picture gallery. Plant Cell 17: 648–
iron metabolism: mRNA-based regulatory circuits operated 663, 2005.
by iron, nitric oxide, and oxidative stress. Proc Natl Acad Sci 57. Merchant SS. Trace metal utilization in chloroplasts. In:
U S A 93: 8175–8182, 1996. The Structure and Function of Plastids, edited by Wise RR
METAL HOMEOSTASIS IN PLANTS AND OXIDATIVE STRESS 931

and Hoober JK. The Netherlands: Springer, 2006, pp. 199– 75. Ridge PG, Zhang Y, and Gladyshev VN. Comparative ge-
218. nomic analyses of copper transporters and cuproproteomes
58. Mochizuki N, Tanaka R, Grimm B, Masuda T, Moulin M, reveal evolutionary dynamics of copper utilization and its
Smith AG, Tanaka A, and Terry MJ. The cell biology of link to oxygen. PLoS One 3: e1378, 2008.
tetrapyrroles: a life and death struggle. Trends Plant Sci 15: 76. Sancenon V, Puig S, Mateu-Andres I, Dorcey E, Thiele DJ,
488–498, 2010. and Penarrubia L. The Arabidopsis copper transporter
59. Molina-Heredia FP, Wastl J, Navarro JA, Bendall DS, Hervas COPT1 functions in root elongation and pollen develop-
M, Howe CJ, and De La Rosa MA. Photosynthesis: a new ment. J Biol Chem 279: 15348–15355, 2004.
function for an old cytochrome? Nature 424: 33–34, 2003. 77. Savino G, Briat JF, and Lobréaux S. Inhibition of the iron-
60. Moseley J, Quinn J, Eriksson M, and Merchant SS. The Crd1 induced ZmFer1 maize ferritin gene expression by antioxi-
gene encodes a putative di-iron enzyme required for pho- dants and serine/threonine phosphatase inhibitors. J Biol
tosystem I accumulation in copper deficiency and hypoxia in Chem 272: 33319–33326, 1997.
Chlamydomonas reinhardtii. EMBO J 19: 2139–2151, 2000. 78. Shikanai T, Muller-Moule P, Munekage Y, Nyogi KK, and
61. Murgia I, Delledonne M, and Soave C. Nitric oxide mediates Pilon M. PAA1, a P-type ATPase of Arabidopsis, functions
iron-induced ferritin accumulation in Arabidopsis. Plant J 30: in copper transport in chloroplasts. Plant Cell 15: 1333–1346,
521–528, 2002. 2003.
62. Newman TC, Ohme-Takagi M, Taylor CB, and Green PJ. 79. Sinclair SA and Krämer U. The zinc homeostasis network of
DST sequences, highly conserved among plant SAUR genes, land plants. Biochim Biophys Acta 1823: 1553–1567, 2012.
target reporter transcripts for rapid decay in tobacco. Plant 80. Stevenson J. The nature of the Earth prior to the oldest
Cell 5: 701–714, 1993. known rock record: the Hadean Earth. In: Earth’s Earliest
63. Noctor G and Foyer CH. Ascorbate and glutathione: Keep- Biosphere: Its Origin and Evolution, edited by Schopf JW.
ing active oxygen under control. Annu Rev Plant Physiol Princeton, NJ: Princeton University Press, 1983, pp. 32–40.
Plant Mol Biol 49: 249–279, 1998. 81. Sunkar R, Kapoor A, and Zhu JK. Posttranscriptional
64. Palmer CM and Guerinot ML. Facing the challenges of Cu, induction of two Cu/Zn superoxide dismutase genes in
Fe and Zn homeostasis in plants. Nat Chem Biol 5: 333–340, Arabidopsis is mediated by downregulation of miR398 and
2009. important for oxidative stress tolerance. Plant Cell 18: 2051–
65. Petit JM, van Wuytswinkel O, Briat JF, and Lobréaux S. 2065, 2006.
Characterization of an iron-dependent regulatory sequence 82. Tapken W, Ravet K, and Pilon M. Plastocyanin controls the
involved in the transcriptional control of AtFer1 and stabilization of the thylakoid Cu-transporting P-type ATPase
ZmFer1 plant ferritin genes by iron. J Biol Chem 276: 5584– PAA2/HMA8 in response to low copper in Arabidopsis.
5590, 2001. J Biol Chem 287: 18544–18550, 2012.
66. Petit JM, Briat JF, and Lobréaux S. Structure and differential 83. Tarantino D, Casagrande F, Soave C, and Murgia I.
expression of the four members of the Arabidopsis thaliana Knocking out of the mitochondrial AtFer4 ferritin does not
ferritin gene family. Biochem J 359: 575–782, 2001. alter response of Arabidopsis plants to abiotic stresses.
67. Pilon M, Cohu CM, Ravet K, Abdel-Ghany SE, and Gay- J Plant Physiol 167: 453–460, 2010.
mard F. Essential transition metal homeostasis in plants. 84. Terry N and Abadia J. Function of iron in chloroplasts.
Curr Opin Plant Biol 12: 347–357, 2009. J Plant Nutr 9: 609–646, 1986.
68. Pilon M, Ravet K, and Tapken W. The biogenesis and 85. Theil EC. Iron, ferritin, and nutrition. Annu Rev Nutr 24: 327–
physiological function of chloroplast superoxide dismutases. 343, 2004.
Biochim Biophys Acta 1807: 989–998, 2011. 86. Thompson K, Menzies S, Muckenthaler M, Torti FM,
69. Puig S and Peñarrubia L. Placing metal micronutrients in Wood T, Torti SV, Hentze MW, Beard J, and Connor J.
context: transport and distribution in plants. Curr Opin Plant Mouse brains deficient in H-ferritin have normal iron
Biol 12: 299–306, 2009. concentration but a protein profile of iron deficiency and
70. Ramirez L, Simontacchi M, Murgia I, Zabaleta E, and increased evidence of oxidative stress. J Neurosci Res 71:
Lamattina L. Nitric oxide, nitrosyl iron complexes, ferritin 46–63, 2003.
and frataxin: a well equipped team to preserve plant iron 87. Tottey S, Block MA, Allen M, Westergren T, Albrieux C,
homeostasis. Plant Sci 181: 582–592, 2011. Scheller HV, Merchant SS, and Jensen PE. Arabidopsis
71. Raven JA, Evans MCW, and Korb RE. The role of trace CHL27, located in both envelope and thylakoid membranes,
metals in photosynthetic electron transport in O2-evolving is required for the synthesis of protochlorophyllide. Proc Natl
organisms. Photosynthesis Research 60: 111–149, 1999. Acad Sci U S A 100: 16119–16124, 2003.
72. Ravet K, Touraine B, Boucherez J, Briat JF, Gaymard F, and 88. Touraine B, Briat JF, and Gaymard F. GSH threshold re-
Cellier F. Ferritins control interaction between iron homeo- quirement for NO-mediated expression of the Arabidopsis
stasis and oxidative stress in Arabidopsis. Plant J 57: 400– AtFer1 ferritin gene in response to iron. FEBS Lett 586: 880–
412, 2009. 883, 2012.
73. Ravet K, Danford FL, Dihle A, Pittarello M, and Pilon M. 89. Van Wuytswinkel O, Vansuyt G, Grignon N, Fourcroy P,
Spatiotemporal analysis of copper homeostasis in Populus and Briat JF. Iron homeostasis alteration in transgenic to-
trichocarpa reveals an integrated molecular remodeling for bacco overexpressing ferritin. Plant J 17: 93–97, 1999.
a preferential allocation of copper to plastocyanin in the 90. Wade VJ, Treffry A, Laulhère JP, Bauminger ER, Cleton MI,
chloroplasts of developing leaves. Plant Physiol 157: 1300– Mann S, Briat JF, and Harrison PM. Structure and compo-
1312, 2011. sition of ferritin cores from pea seed (Pisum sativum). Bio-
74. Ravet K, Reyt G, Arnaud N, Krouk G, Djouani el-B, Bou- chim Biophys Acta 1161: 91–96, 1993.
cherez J, Briat JF, and Gaymard F. Iron and ROS control of 91. Walker JCG, Cornelis K, Schidlowski M, Schopf JW,
the DownSTream mRNA decay pathway is essential for Stevenson J, and Walter MR. Environmental evolution
plant fitness. EMBO J 31: 175–186, 2011. of the Archean-Early Proterozoic Earth. In: Earth’s Earliest
932 RAVET AND PILON

Biosphere: Its Origin and Evolution, edited by Schopf JW.

Princeton, NJ: Princeton University Press, 1983, pp. 260–290. DHA ¼ dehydroascorbate
92. Weigel M, Varotto C, Pesaresi P, Finazzi G, Rappaport F, DHAR ¼ DHA reductase
Salamini F, and Leister D. Plastocyanin is indispensable for DST ¼ destabilization element
photosynthetic electron flow in Arabidopsis thaliana. J Biol DW ¼ dry weight
Chem 278: 31286–31289, 2003. ETR ¼ ethylene receptor
93. Yamasaki H, Abdel-Ghany SE, Cohu CM, Kobayashi Y, FC ¼ ferrochelatase
Shikanai T, and Pilon M. Regulation of copper homeostasis FD ¼ ferredoxin
by micro-RNA in Arabidopsis. J Biol Chem 282: 16369–16378, FER ¼ ferritin
2007. FX /FA /FB ¼ electron acceptor X/A/B of photosystem I
94. Zhang Y and Gladyshev VN. Comparative genomics of trace GOGAT ¼ glutamine oxoglutarate aminotransferase
elements: emerging dynamic view of trace element utiliza- (=glutamate synthase)
tion and function. Chem Rev 109: 4828–4861, 2009. GPX ¼ glutathione peroxidase
95. Zybailov B, Rutschow H, Friso G, Rudella A, Emanuelsson GR ¼ glutathione reductase
O, Sun Q, and van Wijk KJ. Sorting signals, N-terminal GSH ¼ glutathione (reduced form)
modifications and abundance of the chloroplast proteome. GSSG ¼ glutathione disulphide
PLoS One 3: e1994, 2008. HCAR ¼ 7-hydroxymethyl chlorophyll a reductase
HMA ¼ heavy metal associated transporter
Address correspondence to: IDRS ¼ iron dependent regulatory sequence
Dr. Karl Ravet IRE ¼ iron responsive element
Biology Department IRP ¼ iron regulatory protein
Colorado State University LAC ¼ laccase
Fort Collins, CO 80523-1878 LLS1 ¼ pheophorbide a oxygenase
M ¼ metal
E-mail: [email protected] MDA ¼ monodehydroascorbate
MDAR ¼ MDA reductase
Date of first submission to ARS Central, November 12, 2012; NFU ¼ ‘‘nitrogen fixing bacteria’’ (Nif)
date of acceptance, December 1, 2012. U-like proteins
NiR ¼ nitrite reductase
NO ¼ nitric oxide
Abbreviations Used NRAMP ¼ natural resistance-associated
macrophage protein
AAO ¼ amine oxidase
PAA ¼ P-type ATPase
AFT ¼ activator of ferrous transport
PC ¼ plastocyanin
AO ¼ ascorbate oxidase
Pheo ¼ pheophytin
APR ¼ adenosine 5¢-phosphosulfate (APS)
PPO ¼ polyphenol oxidase
reductase
PQ ¼ plastoquinone
APX ¼ ascorbate peroxidase
PSI ¼ photosystem I
ARPN ¼ plantacyanin
PSII ¼ photosystem II
AsA ¼ ascorbate (reduced form)
Q ¼ quinone
ATX ¼ antioxidant
RAN ¼ response to antagonist (=HMA7)
CCH ¼ copper chaperone
ROS ¼ reactive oxygen species
CCS ¼ copper chaperone for superoxide dismutase
SIR ¼ sulfite reductase
CHL27 ¼ chlorophyll deficient 27 (=CRD1:
SiRB ¼ sirohydrochlorin ferrochelatase B
copper response defect 1)
SOD ¼ superoxide dismutase
COPT ¼ copper transporter
SPL7 ¼ squamosa promoter binding protein-like 7
COX ¼ cytochrome c oxidase
SUF ¼ sulfur limitation proteins
CSD ¼ Cu/ZnSOD
TIC55 ¼ translocon inner membrane complex 55
Cyt ¼ cytochrome
VIT1 ¼ vacuolar iron transporter 1