Available online at www.sciencedirect.
com
Homeostasis of the micronutrients Ni, Mo and Cl with specific
biochemical functions
Manuel Tejada-Jiménez, Aurora Galván, Emilio Fernández and
Ángel Llamas
Homeostasis of three elements nickel, molybdenum and
chloride is analysed. These micronutrients, at amounts varying
in orders of magnitude, fulfil important cell functions. In general
terms, cells use similar strategies to ensure that the elements
are within physiological ranges avoiding high toxic
concentrations. These strategies correspond to specific
carriers, channels and pumps, intermediate steps (chelating/
sequestration/binding/metabolic conversion/storage), final
steps related to specific enzyme functionality and putative
sensing proteins. Single cell homeostasis, coordinated with an
efficient redistribution by xylem loading, ensures in turn
homeostasis at the whole plant level. Recent advances are
based on the molecular identification of some key components.
Address
Departamento de Bioquı́mica y Biologı́a Molecular, Facultad de
Ciencias, Universidad de Córdoba. Campus de Rabanales, Edif. Severo
Ochoa, 14071 Córdoba, Spain
Corresponding author: Fernández, Emilio (bb1feree@uco.es)
Current Opinion in Plant Biology 2009, 12:358–363
This review comes from a themed issue on
Physiology and metabolism
Edited by David Salt and Lorraine Williams
Available online 30th May 2009
1369-5266/$ – see front matter
# 2009 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.pbi.2009.04.012
Introduction
Living organisms are open systems in a continuous
exchange with the environmental medium. Ion homeo-
stasis refers to the ability of an organism to regulate
internal ion concentrations within ranges compatible with
its physiological role also preventing the toxic effects.
Nickel (Ni) is present in essentially all soils. Plants only
require trace amounts of nickel (between 2 and 4 ng g 1
dry weight, DW), showing toxicity at 10–50 mg g 1 DW
[1]. The activation of urease, which catalyses the hydroly-
sis of urea to carbon dioxide and ammonia, appears to be
the only function of Ni in plants [2].
Molybdenum (Mo) is essential for almost all livings
beings. Mo is present in soils at average concentration
Current Opinion in Plant Biology 2009, 12:358–363
around 1–5 mg kg 1 [3] and can be found in several
oxidation states between zero and VI. The availability
of molybdate for plants strongly depends on soil&s pH and
humidity. Molybdate, the acquirable form of Mo, is the
most abundant Mo soluble form at pH higher than 4.2; the
solubility/availability of molybdate increases when the
pH increases [4] and when humidity of soils is high. Mo is
one of the less abundant micronutrients of plants, about
100 000 Mo atoms per cell in Chlamydomonas reinhardtii
[5] and at the range of mg g 1 DW in Arabidopsis tissues
[6]. In eukaryotes Mo is biologically active in form of a
pterin-cofactor called Mo cofactor (Moco) [7] that in
plants participates as a prosthetic group in the active
centre of four Mo-enzymes: nitrate reductase (NR), alde-
hyde oxidase, xanthine dehydrogenase and sulfite oxidase
[8]. A shortage of Mo availability causes a block in Moco
biosynthesis and thus a pleiotropic failure of all the above
enzyme activities that leads to the loss of crucial meta-
bolic functions and, in most cases, to plant death [7,8].
Soil contains sufficient chloride (Cl ) for optimal plant
growth (1–5 mM). However, Cl toxicity is common in
non-halotolerants plants cultured in saline soils. Reliable
measurements of intracellular Cl concentrations and its
distribution into different organelles are difficult, since
they depend not only on the plant type but also on the
different plant tissues. In general terms, internal Cl
concentrations (5–133 mM, for different non-halophyte
plants) mainly represent Cl stored in vacuoles [9,10].
Chloride is an essential nutrient for plants and a major
osmotically active solute in the vacuole, participating in
key processes such as enzyme activity regulation, photo-
synthetic O2 evolution, stabilisation of membrane poten-
tial and intracellular pH regulation [9,10].
Plant cell homeostasis for these micronutrients involves
strategies for obtaining them from soils, intracellular
compartmentalisation into organelles, coordinating trans-
port mechanisms through different plant tissues and the
sensing mechanisms providing a link between environ-
mental changes and cellular responses. We focus herein
on recent advances on the homeostasis of these three
micronutrients by considering a single cell model.
Nickel homeostasis
Members from three families of transporters, ZIP (ZRT/
IRT-like Protein; zinc regulated transporters/iron
regulated transporters), NRAMP (Natural resistance-
associated macrophage protein) and YSL (Yellow Stripe
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 Homeostasis of the micronutrients Ni, Mo and Cl with specific biochemical functions Tejada-Jiménez et al. 359

Figure 1

Scheme of the Ni homeostasis process in a single plant cell. Nickel (Ni) refers to the Ni2+. Ni or Ni–NA enters the cell by transporters belonging to ZIP or
YSL families. Inside the cell Ni can be incorporated by the enzyme urease, may be by binding to UreG, or it can be stored in the vacuole or in the
cytosol complexed by organic compounds (Ni-X). The mechanism implicated in the Ni efflux from cell is unknown.

Like), participate in Ni transport and homeostasis [11]
(Figure 1). Yeast transformation with the Thlapsi japoni-
cum ZNT1 or ZNT2 conferred Ni tolerance, partially
correlated with Zn influx, which inhibits Ni uptake [12].
In contrast, NRAMP4 transformation confers Ni sensi-
tivity in yeast explained by Ni efflux from the vacuole
[11–13].
However, to date there are no experimental data about
the real roles of ZIP and NRAMP in plants. In addition, it
is often recognized that over expression of plant genes in
yeast results in miss location of protein in unexpected
organelles, and thus different characteristics from the
native proteins might appear. To clarify the participation
and roles of these transporters in plant cells, further
functional analyses using transgenic plants expressing
these transporters genes are needed.
In Arabidopsis the AtIREG2, which encodes a IRT-like
membrane protein sharing homology with Fe2+ exporters,
had been proposed to have a role in vacuole loading of
nickel under iron deficiency. This role is supported by the
localisation in Arabidopsis tonoplast of AtIREG2-GFP
fusion proteins and by the increased tolerance to elevated
concentrations of nickel in transgenic plants over expres-
sing AtIREG2. Therefore the physiological function of
AtIREG2 could be the deposition of excess nickel into
the vacuole to counterbalance the low substrate speci-
ficity of AtIRT1 and other iron transport systems [14].
Nicotianamine synthase (NAS) whose expression is
restricted to the leaves is involved in cellular tolerance
to Ni [15]. It is proposed that in response to Ni,
nicotianamine (NA) is translocated to the roots where
it chelates the absorbed Ni and facilitates its transport to
the shoot. NAS overexpression conferred Ni tolerance in
Arabidopsis [16]. TcYSL3 is able to transport both Ni–NA
and Fe–NA complexes [17]. In addition, plant tolerance
to Ni also derives from chelation by histidine [18] or
organic acids as citrate [19]. Increased salicylic acid levels
in Arabidopsis plants are linked to elevated glutathione
amounts that prevent Ni toxicity by buffering its oxidiz-
ing effect [20].
Plant urease contains two Ni ions in its catalytic centre.
Plant orthologues of UreD, UreF and UreG bacterial genes
for urease activation have been identified but not UreE
[21]. The precise role of the accessory proteins is not yet
clearly understood. Apo-urease and UreD, UreF, and
UreG form a complex that is competent to incorporate
nickel upon GTP hydrolysis carried out by UreG. It was
hypothesized that the function of the bacterial metallo-
chaperone UreE may have been taken over in plants by
the UreG [22]. The cis elements and trans factors
involved in transcriptional responses of plants to their
Ni status are unknown. Ni deficiency causes urea
accumulation [23], affects amino acid metabolism [24]
and induces metabolism nitrogen deficiency [2].
Molybdenum homeostasis
A precise molybdate transport process is crucial for plants
owing to the relatively low availability of molybdate in soils
together with the biological importance of a proper supply
of Mo (Figure 2). However until recently, it was thought

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360 Physiology and metabolism
Figure 2
Scheme of the Mo homeostasis process in a single plant cell. Mo, as molybdate, enters in the cell by specific transporters, as MOT1. Inside the cell Mo
is transferred to MPT to form Moco or it is complexed by anthocyanin or malic acid. MOT1 has been proposed to be located in mitochondrial
membrane. The process of Mo efflux from the cell is unknown. Moco is used by Mo-enzymes to carry out its biological activity or is stored and
protected against oxidation by MCP.
that molybdate transport was carried out by anion transport
systems able to transport molybdate unspecifically. Phys-
iological evidence for the presence of specific molybdate
transporters in plants [25] was confirmed by the identifi-
cation of MOT1 in Chlamydomonas [26] and Arabidopsis
[27]. MOT1 is a high-affinity molybdate transporter (Km
at nM range) distantly related to plant sulfate transporter
SULTR, with homologous proteins from bacteria to plants,
with the exception of animals [26]. In Arabidopsis MOT1
appears to be located at the mitochondrial membrane [6],
is found throughout the plant body and seems to be crucial
in ensuring an efficient uptake of molybdate from soil and
in the maintenance of its intracellular level [6,27]. By
contrast, in Chlamydomonas the absence of MOT1 is coun-
terbalanced by the presence of a second molybdate trans-
porter not related to MOT1 and molecularly unknown that
maintains the normal Mo supply [26]. After MOT1
isolation it has been described that the plant sulfate trans-
porter SHST1 from Stylosanthes hamata is able to transport
molybdate in addition to sulfate when this protein is
expressed in Saccharomyces cerevisiae [28]. The physiologi-
cal meaning of this molybdate transport activity in the plant
is still unknown.
AtMOT1 transcription is activated in the presence of
molybdate in shoots but not in roots [27]. CrMOT1
transcription and CrMOT1 activity are insensitive to
Mo availability but strongly activated when nitrate is
the only nitrogen source [26]. While the role of
AtMOT1 seems to be related to the maintenance of
Mo concentration in the plant, CrMOT1 is directly linked
Current Opinion in Plant Biology 2009, 12:358–363
to the requirement of the Mo-enzyme NR to reduce
nitrate to nitrite in the first step of nitrate assimilation.
This is a strong evidence of a cross-linking between Mo
and N metabolism to ensure an optimal coordination of
both pathways.
Once inside the cells Mo is incorporated to molybdop-
terin (MPT) in the last step of Moco biosynthesis cata-
lysed by CNX1 through an intermediate of adenylated-
molybdate [29]. CNX1 from Arabidopsis binds to cytos-
keleton [30]. This could be an adequate localisation to
interact with an integral membrane protein like a mol-
ybdate transporter facilitating a coordinated Moco bio-
synthesis [30]. However, there are no experimental
evidences to confirm this hypothesis yet.
Synthesized Moco is stored or transferred to Mo-
enzymes. Moco is very labile and oxygen sensitive
[31], thus it is not free in the cells. A Moco-Carrier-Protein
(MCP), identified in Chlamydomonas [32,33], binds Moco,
protects it against oxidation and transfers it to apoNR
[34]. To date, no homologues to MCP have been found
in higher plants, however Arabidopsis has a family of nine
proteins classified as lysine decarboxylase-like proteins
that show high structural similarity to Chlamydomonas
MCP. Taking into account that Mo export mechanisms
are unknown, the sequestration of Mo whether in form of
Moco or chelated as organic complex with anthocyanin or
malic acid [35,36] would explain why plants having
extremely low requirements for this element are fairly
tolerant to Mo.
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 Homeostasis of the micronutrients Ni, Mo and Cl with specific biochemical functions Tejada-Jiménez et al. 361
Chloride homeostasis
A view of elements involved in chloride homeostasis is
given considering data from Arabidopsis. Different
protein families could contribute to chloride homeosta-
sis in plants as CLC (Chloride Channel), CCC (cation
chloride cotransporter), CLIL (Chloride intracellular
channel), ICln (nucleotide-sensitive chloride conduc-
tance regulator protein) and ABC (ATP-Binding Cas-
sette) transporter. CLC proteins, widely distributed in
eukaryotes and prokaryotes, function as anion channel/
transporters permeable among others to chloride and
nitrate [37]. This characteristic has physiological
importance in plants where CLCs control nitrate
accumulation and ion homeostasis. Arabidopsis has
seven CLC members (AtCLC-a to AtCLC-g) with
different localisation, regulation and physiological role.
AtCLC-a resides in the vacuolar membrane, is nitrate
upregulated and mediates 2NO3 :1H+ exchanger and is
responsible for nitrate storage into this organellum [38].
AtCLCd and AtCLCf are Golgi membrane proteins
required for optimal acidification in the trans-Golgi
network during endocytic and secretory processes
[37]. AtCLCe is in thylakoid membranes of chloroplasts
so that AtCLCe mutants show a phenotype related to
photosynthetic activity [39].
AtCCC is a plasma membrane protein catalysing a
Na+,K+:Cl cotransport. At high salt CCC-defective
mutants accumulate high chloride concentration in shoots
and low in roots suggesting that this protein is involved in
long distance Cl transport [40] and probably in phloem
loading.
Arabidopsis has a family of proteins related to glutathione
S-transferase, four of which (At-DHAR 1-4) are homolo-
gous to animal CLICs. AtDHAR1 can exist in both
soluble and membrane form and generates ion conduc-
tance in heterologous systems explained by a CLIC-like
channel activity or by regulation of endogenous protein
channels. In addition, AtDHAR1 appears upregulated in
osmotic and salt stresses [41].
ICln is a multifunctional protein essential for cell volume
regulation. Arabidopsis contains uncharacterized ICln
proteins that in animal cells can also be in soluble and
membrane-associated forms and generate ion currents to
regulate cell swelling [42].
Arabidopsis genome shows genes related to the chloride
channel from animals CFTR (cystic fibrosis transmem-
brane regulator). In fact, the related ABC protein
AtMRP5 is a central regulator of anion and calcium
channels in guard cells in response to abscic acid and
Ca2+ signals [43].
The sensing mechanisms to regulate precisely chloride
homeostasis and thus of these chloride transporters are
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unknown. In yeast, a formate-nitrite transporter (FNT)
might be part of a chloride-sensing mechanism that
activates high affinity chloride transport [44]. However,
plants lack FNT proteins so that other proteins such as
the multifunctional CLIC, ICln or MRP proteins could
play this role.
Some open questions
The compartmentation in organelle of plant cells and the
specialisation of different plant tissues require a huge
number of channels/transporters for precise homeostasis.
Genomic and proteomic data are currently allowing the
identification of putative elements involved. New
insights on the role and function of these transporters
could come from analysis of ions/metabolites pools in the
different compartments and saps in the light on the cross-
relationships among them.
Since AtMOT1 seems to control the Mo plant content, it
would be expected that it is localized in plasma mem-
brane, so what is the physiological meaning of a mito-
chondrial localisation of AtMOT1? Arabidopsis has two
members of the MOT1 family, even though they share a
high similarity the second member appeared not to trans-
port molybdate when expressed in yeast; is it really a
molybdate transporter? In Chlamydomonas the lack of
MOT1 activity is counterbalanced by a second molybdate
transporter not related to MOT1 family, what type of
protein is this transporter? Plant requirement for Mo is
very low however the correct intracellular concentration
of this metal is vital since activity of Mo-enzymes
involved in abscisic acid biosynthesis and nitrate assim-
ilation have to be present in different cells; what trans-
porters are present in specific cell types? Is there any
mechanism to sense intracellular Mo? Since CNX1 has a
molybdate-binding site and could be in direct contact
with molybdate transporters, could it be a Mo sensor of
plant cells?
In general, sensing mechanisms for homeostasis of ana-
lysed elements are poorly understood and require further
insights.
Acknowledgements
Authors thank funding from ‘Ministerio de Educación y Ciencia’, Spain
(BFU 2008-01798) and Junta de Andalucı́a, Spain (PAI, BIO-0128 and CVI-
1609, CVI04157).
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associated to non-soluble microsomal membranes. The expressed protein
increased conductance is mostly due to the chloride channel activity.
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as a plasma membrane protein. The mrp5 mutant is impaired in abscisic
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regulatory role of this protein for signal transduction is proposed.
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Current Opinion in Plant Biology 2009, 12:358–363