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com
Physiological functions of mineral micronutrients (Cu, Zn, Mn, Fe,
Ni, Mo, B, Cl)
Robert Hänsch and Ralf R Mendel
Micronutrients are involved in all metabolic and cellular
functions. Plants differ in their need for micronutrients, and we
will focus here only on those elements that are generally
accepted as essential for all higher plants: boron (B), chloride
(Cl), copper (Cu), iron (Fe), manganese (Mn), molybdenum (Mo),
nickel (Ni), and zinc (Zn). Several of these elements are redox-
active that makes them essential as catalytically active
cofactors in enzymes, others have enzyme-activating
functions, and yet others fulfill a structural role in stabilizing
proteins. In this review, we focus on the major functions of
mineral micronutrients, mostly in cases where they were shown
as constituents of proteins, making a selection and highlighting
some functions in more detail.
Address
Institut für Pflanzenbiologie, Technische Universität Braunschweig,
Humboldtstraße 1, 38106 Braunschweig, Germany
Corresponding author: Mendel, Ralf R (r.mendel@tu-bs.de)
Current Opinion in Plant Biology 2009, 12:259–266
This review comes from a themed issue on
Physiology and metabolism
Edited by David Salt and Lorraine Williams
1369-5266/$ – see front matter
# 2009 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.pbi.2009.05.006
Introduction
Micronutrients are elements that are essential for plant
growth but are required in much smaller amounts (Table 1)
than those of the primary nutrients such as nitrogen,
phosphorus, sulfur, and potassium. Plants show different
needs for certain micronutrients, but the elements that are
generally accepted as essential for all higher plants are:
boron (B), chloride (Cl), copper (Cu), iron (Fe), manganese
(Mn), molybdenum (Mo), nickel (Ni), and zinc (Zn). This
list may grow as more protein structures are elucidated. All
organisms have to acquire appropriate amounts of each
micronutrient that requires a metal homeostasis network
involving mobilization, uptake and distribution within the
plant, intracellular trafficking, and storage. Several essen-
tial metal ions are redox-active that is the basis for their
occurrence as catalytically active cofactors in many metal-
loenzymes. Other metals (like zinc) fulfill in addition to
their catalytic role a structural role in stabilizing proteins.
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However, when present in elevated concentrations, the
same redox properties that make metal ions essential
elements lead to the formation of reactive oxygen species
with detrimental consequences for the cell. Moreover,
metal excess can lead to ectopic binding of metals to
proteins thus disturbing protein structures [1]. This
beneficial versus detrimental duality caused the develop-
ment of precisely tuned homeostatic cellular networks
involving metal chaperones. One can expect that most
metal ions do not exist as free ion in the cell [2]. Micro-
nutrients are involved in virtually all metabolic and cellular
functions, like energy metabolism, primary and secondary
metabolism, cell protection, gene regulation, hormone
perception, signal transduction, and reproduction among
others. Historically, their physiological role was first
described on the basis of deficiency symptoms. In this
review, we focus on the major functions of mineral micro-
nutrients, concentrating on cases where the micronutrient
is a constituent of a particular protein. The list of proteins
given in Table 1 is for some microelements only a short
selection (e.g. zinc where we show eight proteins out of
over one thousand), for others the list is nearly compre-
hensive (e.g. molybdenum) and again for others vague and
elusive (e.g. boron). The reader will find many more details
in Marschner’s handbook [3] that still is a basic reference
for plant nutrients.
Boron
The unusual nature of boron chemistry suggests the
possibility of a wide variety of biological functions for
the micronutrient; however, the exact metabolic func-
tions are not finally understood. Boron is involved in
numerous important processes, including protein syn-
thesis, transport of sugars, respiration, RNA and carbo-
hydrate metabolism, and the metabolism of plant
hormones (indole acetic acid). Moreover, functions of
boron are related to cell wall synthesis, lignification,
and cell wall structure by cross-linking of cell wall poly-
saccharides as well as the structural integrity of biomem-
branes. It increases the transport of chlorine and
phosphorus as a result of plasmalemma ATPase induc-
tion. Other investigations have shown that boron can
stimulate proton pumping that causes hyperpolarization
of the membrane potential (for review see [4]). More than
90% of the boron in plants is found in cell walls, and
rhamnogalacturonan II was shown to bind boron [3].
Because the wall-associated kinase in the plasmamem-
brane has an extracellular matrix connection with the
pectin molecule [5], the membrane cell wall connection
is finally also boron-dependent. Boron was found to
Current Opinion in Plant Biology 2009, 12:259–266
260 Physiology and metabolism

Table 1

Micronutrients in plants.

Element Symbol Absorbed Concentration Protein complexed Literature

by plant in plant [mg g 1 with the micronutrient
dry weight] a (or other effects)
Boron (B) H3BO3 3–100 Rhamnogalacturonan II [5]
b
Chlorine (Cl) Cl 20 000 Oxygen evolving complex [9]
Seismonastic movement [11]
Copper (Cu) Cu2+ 1–20 Ascorbate oxidase [49]
Polyphenol oxidase [50]
Cu–Zn superoxide dismutase [51]
Cytochrome c oxidase [52]
Plastocyanin [53]
Cu-metallothionein [54]
Ethlyene receptor [18]
Mo-cofactor biosynthesis [19]
Iron (Fe) Fe3+, Fe2+ 50–150
Fe–S-cluster Aconitase [55]
Succinate dehydrogenase [56]
NADH-Q oxidoreductase [57]
Thioredoxin reductase [58]
Xanthine dehydrogenase [59]
Aldehyde oxidase [60]
Ferredoxin [61]
Heme Cytochromes [3]
Catalase, Peroxidase [62]
Cytochrome c oxidase [63]
Nitrate reductase [64]
Nitrite reductase [65]
Cytochrome P450 [66]
Leg hemoglobin [67]
Non-heme Fe-superoxide dismutase [68]
Lipoxygenase [69]
Alternative oxidase [70]
Ferritin [23]
Manganese (Mn) Mn2+ 10–100 Mn-superoxide dismutase [51]
PEP-carboxykinase [3]
Allantoate amidohydrolase [71]
Malic enzyme [3]
Isocitrate lyase [72]
PEP carboxylase [73]
Molybdenum (Mo) MoO42 0.1–1 Nitrate reductase [27]
Sulfite oxidase [74]
Aldehyde oxidase [27,60]
Xanthine dehydrogenase [59]
Nickel (Ni) Ni+ 15–22 Urease [33]
Ni-chaperone [36]
Zinc (Zn) Zn2+ 15–50 SPP [39]
Carbonic anhydrase [75]
Cu–Zn superoxide dismutase [51]
Alcohol dehydrogenase [76]
Peptide deformylase [44]
a-Mannosidase [46]
Matrix metalloproteinase [47]
a
The concentration of micronutrients in plants can vary widely depending on the species, genotype, organ, tissue, and growth condition. Therefore
ranges are given.
b
Requirement for optimal growth: 200–400 mg g 1 dry weight.

promote the structural integrity of biomembranes and the shoot and root tips so that the whole plant may be stunted
formation of lipid rafts [3]. Since all these functions are (rosetting). Flower retention, pollen formation, pollen
fundamental to meristematic tissues, boron deficiency is tube growth or germination, nitrogen fixation, and nitrate
predominantly damaging actively growing organs such as assimilation are also affected by boron [6].

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 Physiological functions of mineral micronutrients (Cu, Zn, Mn, Fe, Ni, Mo, B, Cl) Hänsch and Mendel 261
Chlorine
Chlorine is known to exist in more than 130 organic
compounds in plants [7]. Most soils contain sufficient
levels of chlorine. However, chlorine deficiencies have
been described in sandy soils in high rainfall areas or
could be created artificially in experiments to prove its
requirement as a micronutrient for higher plants [8].
Because chloride is a mobile anion in plants, most of
its functions are related to electrical charge balance. In
the chloroplast, chloride is a structural constituent of
photosystem II in the oxygen evolving complex as one
of the three important cofactors [9]. Proton-pumping
ATPase at the tonoplast is specifically stimulated by
chloride [10]. The overall chlorine concentration in the
whole plant is too low to be an effective osmo-regulator;
however, chlorine is accumulated in certain tissues or
single cells (e.g. guard cells). Opening and closing of the
guard cells is mediated by the flux of potassium and
anions such as malate and chloride. Therefore, chlorine
indirectly affects plant growth by stomatal regulation.
Reduction of leaf surface area, wilting of the plant, and
restricted, highly branched root systems are the main
chlorine-deficiency symptoms. On the contrary, seismo-
nastic leaf movement of Mimosa pudica is directly
chlorine-dependent. The ‘osmotic motor’ for the leaf
movement is powered by a plasma membrane proton
ATPase, which drives KCl and water fluxes. The move-
ment results from different volume and turgor changes in
the two oppositely positioned parts in the specialized
motor leaf organs called pulvinus (for details see [11]).
Copper
Copper is of utmost importance for life. Copper is essen-
tial for photosynthesis and mitochondrial respiration,
for carbon and nitrogen metabolism, for oxidative stress
protection, and is required for cell wall synthesis, to name
only a few of its cellular tasks. Under physiological
conditions, copper exists in the two oxidation states
Cu1+ and Cu2+ and can interchange between these forms
(monovalent copper is unstable). This allows copper to
function as a reducing or oxidizing agent in biochemical
reactions. But at the same time, this property makes
copper also potentially toxic as copper ions can catalyze
the production of free radicals, in particular through
Fenton chemistry, thus leading to the damage of proteins,
DNA, and other biomolecules. Therefore, immediately
after uptake the vast majority of copper ions is bound by
scavenging proteins like metallothioneins to prevent
copper from accumulating in a toxic form. However, part
of the imported copper bypasses this system and becomes
captured by small binding proteins, so called copper
chaperones [2,12] that spare copper from the detoxifica-
tion systems and guide it to the target sites in the cell. If
the target is a cytosolic protein, the copper chaperone also
directly inserts the metal in the cognate site of the target
protein. While in yeast and mammals only a handful of
copper chaperones are known, the genome of Arabidopsis
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thaliana possesses over 30 sequence homologs that might
encode for copper chaperones; however, only very few of
these proteins are already characterized [13].
In total, more than 100 Arabidopsis proteins are predicted
to be complexed with copper [14], comprising two
groups: copper binding proteins/chaperones and
enzymes. Copper has a particularly high affinity to dioxy-
gen molecules that explains why copper is the catalytic
metal in many oxidases. The most prominent member of
this group is mitochondrial cytochrome c oxidase as prin-
cipal catalyst of the terminal oxidation. Copper is also
found in electron carrier proteins like plastocyanin that
accounts for about 50% of the plastidic copper [15]. More
than half of the copper in plants is found in chloroplasts
and participates in photosynthetic reactions. Hence, cop-
per deficiency becomes first visible in young leaves and
reproductive organs, later consequences are stunted
growth of the whole plant and pale green leaves that
wither easily. Interestingly, copper metabolism is inti-
mately linked to iron metabolism. Depending on the
bioavailability of copper and iron, plants possess enzymes
for the alternative use of copper versus iron thus catalyz-
ing the same biochemical reaction with completely differ-
ent apoproteins [16], a process that involves regulation by
miRNAs [17]. Examples include Cu-nitrite versus
heme-nitrite reductase, Cu/Zn-superoxide dismutase ver-
sus Fe-superoxide dismutase, and cytochrome oxidase
versus diiron oxidase. Finally, copper is also part of the
ethylene receptor [18] and is involved in molybdenum
cofactor biosynthesis [19].
Iron
Like copper, iron is also of great importance for life. As
redox-active metal it is involved in photosynthesis, mito-
chondrial respiration, nitrogen assimilation, hormone bio-
synthesis (ethylene, gibberellic acid, jasmonic acid),
production and scavenging of reactive oxygen species,
osmoprotection, and pathogen defense. Up to 80% of the
cellular iron is found in the chloroplasts that is consistent
with its major function in photosynthesis. Depending on
the type of iron ligand, three groups of iron-containing
proteins can be defined: (1) proteins with iron–sulfur
clusters (Fe–S), (2) heme-containing proteins, and (3)
other iron proteins.
Fe–S proteins: Fe–S clusters have pivotal functions in
electron transfer, they constitute part of substrate binding
sites in enzymes, they form iron storage moieties, they are
involved in transcriptional or translational regulation,
they can control protein structure in the vicinity of the
cluster, and finally they have been shown to be involved
in disulfide reduction and sulfur donation (e.g. thioredox-
ins). Hence, Fe–S proteins serve functions as enzymes, as
electron carriers (e.g. ferredoxin), and as regulator
proteins (e.g. aconitase). Fe–S cluster formation occurred
very early in evolution and was strictly conserved later on.
Current Opinion in Plant Biology 2009, 12:259–266
262 Physiology and metabolism
Fe–S clusters are built from inorganic iron and sulfide.
While in vitro they can form spontaneously under
anaerobic conditions, the low concentrations of both
components in the cell preclude a spontaneous assembly.
Therefore, a complex assembly system has been evolved
in bacteria that have counterparts in mitochondria and
chloroplasts [20]. The cytoplasm receives its Fe–S clus-
ters from the mitochondria.
Heme proteins: The well-known hemoproteins are the
photosynthetic and respiratory cytochromes, involved
in electron transfer, and the globins that bind oxygen.
Other examples include the oxidative enzymes catalase,
peroxidase, and NADPH oxidase, involved in the pro-
duction and/or scavenging of free radicals, and the very
large group of cytochrome P450 enzymes. In plants and
microbes, these latter catalyze mono-oxygenation reac-
tions in biosynthetic pathways, such as for sterols and
many secondary metabolites, whereas in animals their
major role is in the detoxification of xenobiotics. Further,
globins like leg-hemoglobin are involved in oxygen bind-
ing and transport. Nitrite reductase and sulfite reductase
harbor both a siroheme and an Fe–S cluster in the
enzyme. Little is known about the coordination between
apoprotein and heme synthesis or the assembly into a
functional protein. In plants, hemoproteins are distribu-
ted in all subcellular locations, but the situation is made
more complex by the fact that heme is synthesized in
both mitochondria and chloroplasts [21], and it is not
known which organelle supplies heme to other users in
the cell. For example, cytochrome P450 localizes to the
endoplasmic reticulum, catalase to the peroxisomes and
other enzymes to the cytoplasm.
Other iron proteins: These proteins (that are sometime also
grouped as non-heme proteins) bind iron ions directly, i.e.
neither as heme nor in the Fe–S form. Among these
proteins, ferritins are most prominent. Ferritins are plas-
tidic iron storage proteins and control the interaction
between iron homeostasis and oxidative stress in Arabi-
dopsis [22]. They are high molecular weight 24-mer
proteins that can store up to 4500 ion atoms in a soluble
and bioavailable form [23]. Ferritins occur mostly in non-
green plastids like etioplasts and amyloplasts but not in
mature chloroplasts [24].
Manganese
Manganese is essential for plant metabolism and devel-
opment and occurs in oxidation states II, III, and IV in
approximately 35 enzymes of a plant cell [25]. Manganese
can fulfill two functions in proteins: (1) it serves as
catalytically active metal, or (2) it exerts an activating
role on enzymes. Examples for the catalytic role are
manganese-containing superoxide dismutase protecting
the cell from damaging effects of free radicals, the oxalate
oxidase, and the manganese-containing water splitting
system of photosystem II [26]. Examples for the manga-
Current Opinion in Plant Biology 2009, 12:259–266
nese-activated enzymes are malic enzyme, isocitrate
dehydrogenase, PEP carboxykinase, and phenylalanin
ammonia lyase. Among the rather large group of manga-
nese-activated enzymes, the role of manganese is less
specific as in many cases it can be replaced by magnesium
[3]. Proteins belonging to this group are involved in the
shikimic acid pathway and subsequent pathways leading
to the formation of aromatic amino acids, lignins, flavo-
noids, and the phytohormone indole acetic acid. Manga-
nese activation was seen in enzymes of nitrogen
metabolism (glutamin synthetase, arginase), gibberellic
acid biosynthesis, RNA polymerase activation, and fatty
acid biosynthesis.
Molybdenum
Only a handful of plant proteins are known to contain
molybdenum. These proteins, however, are very import-
ant as they are involved in nitrogen assimilation, sulfur
metabolism, phytohormone biosynthesis, and stress reac-
tions [27]. Nitrate reductase is the key-enzyme for nitrate
assimilation while nitrogenase is found in nitrogen fixing
bacteria inside nodules of symbiotically growing species.
The last step of abscisic acid biosynthesis is catalyzed by
the molybdenum-enzyme aldehyde oxidase, and sulfite
oxidase protects the plant against toxic levels of sulfite
(acid rain!). Hence a defect in molybdenum-metbolism
leads to the pleiotropic loss of these enzyme activities
with lethal consequences for the organism. Recently, a
novel molybdenum-enzyme (‘mitochondrial amidoxime
reducing component’ mARC) was found on the envelope
of mammalian mitochondria [28] catalyzing the reduction
of N-hydroxylated amidines in concert with cytochrome
b5 and cytochrome b5 reductase, a reaction that may be
associated with detoxification tasks. This new enzyme is
wide spread in nature as homologs were found among
plants and eubacteria [29]. In all organisms, molybdenum
has to be complexed by a pterin compound thereby
forming the molybdenum cofactor in order to gain bio-
logical activity. This pterin compound is a unique pterin
named molybdopterin or metal-containing pterin. With
the exception of nitrogen-fixing nitrogenase all molyb-
denum-containing enzymes characterized to this end
contain the pterin-type cofactor [30]. Interestingly, mol-
ybdenum enzymes produce reactive oxygen species (sul-
fite oxidase, aldehyde oxidase, xanthine dehydrogenase)
or NO (nitrate reductase) as side reaction products. Mol-
ybdenum metabolism is intimately linked to iron and
copper metabolism at several crosspoints (Figure 1). In all
organisms, enzymes participating in the first step of
molybdenum cofactor biosynthesis were found to contain
Fe–S clusters. Further down the molybdenum pathway,
nitrogenase, aldehyde oxidase, and xanthine dehydrogen-
ase bind Fe–S clusters as well. And also nitrate reductase
contains iron in the form of the heme group. Another
crosstalk was discovered between molybdenum and cop-
per metabolism as copper was found to be essential for the
formation of a molybdenum cofactor intermediate [19].
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 Physiological functions of mineral micronutrients (Cu, Zn, Mn, Fe, Ni, Mo, B, Cl) Hänsch and Mendel 263

Figure 1

Schematic representation of the metabolic link between the three micronutrients molybdenum, iron, and copper as it is found in molybdenum
metabolism. In order to become biologically active, molybdenum has to be complexed by a pterin compound thereby forming the molybdenum
cofactor (Moco). The first step of Moco biosynthesis taking place inside mitochondria is dependent on Fe–S clusters that form part of the enzymes
involved in this step. Mitochondria are the site of Fe–S cluster synthesis and support the biogenesis of extra-mitochondrial Fe–S proteins by exporting
Fe–S cluster equivalents through a specific transporter into the cytosol. There are indications that this transporter is also involved in exporting a Moco
intermediate. Mitochondria synthesize and export heme groups as well. With the exception of sulfite oxidase and mARC, molybdenum-containing
enzymes also need iron, either in the form of Fe–S clusters or as heme-iron. Finally, copper is involved in Moco biosynthesis by facilitating
molybdenum insertion into the cofactor precursor. In summary, molybdenum metabolism is fully dependent on a functional iron and copper
metabolism.

Nickel substrate to carbon dioxide and ammonia. Moreover,

Nickel is essential in numerous prokaryotic enzymes
like dehydrogenases, hydrogenases, and methyl-
reductases but is barely used as cofactor in eukaryotes.
Among plants, it occurs not only in oxidation states II,
but also in states I and III. A deficiency symptom in
plants is the accumulation of toxic urea [31–33] that
could be explained with the complete loss of urease
activity within the cell. Plant urease hydrolyzes its
recent findings of Follmer suggest that plant ureases
have a protective role against phytopathogens, unrelated
to the release of ammonia [34]. The bacterial homo-
trimeric or homotetrameric enzyme contains two Ni2+-
ions per subunit in its catalytic center [35]. An additional
Ni2+-binding protein could be identified in soybean
that acts as Ni-metallochaperone essential for urease
activity [36]. It seems to be possible that a few more

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264 Physiology and metabolism
Ni-dependent enzymes will be identified in plants in
the future.
Zinc
Zinc is important as a component of enzymes for protein
synthesis and energy production and maintains the struc-
tural integrity of biomembranes. More than 1200 proteins
are predicted to contain, bind, or transport Zn2+, including
– among others – a large numbers of zinc-finger contain-
ing proteins and transcription factors, oxidoreductases,
and hydrolytic enzymes such as metalloproteases [14].
Zinc plays also an important role in seed development,
and zinc-deficient plants show a delayed maturity.
Most of the zinc enzymes are involved in regulation of
DNA-transcription, RNA-processing, and translation. Up
to 4% of the predicted proteins in Arabidopsis contain the
zinc-finger motif [37] that is well known in transcription
factors and is important for protein–protein interactions.
Beside transcription factors, several enzymes involved in
DNA or RNA synthesis and maintenance are zinc-de-
pendent: all types of DNA-polymerases and RNA-poly-
merases, histone deacetylases, splicing factors, and
enzymes involved in RNA-editing in mitochondria and
chloroplasts [14]. Furthermore, zinc was found in a
number of tRNA synthetases and in the translation
initiation factor elF-5 from maize that is required for
the formation of a functional 80S initiation complex [38].
In chloroplasts, zinc-dependent enzymes fulfill several
major functions. The stromal processing peptidase SPP
[39] is zinc-dependent in analogy to the mitochondrial
system [40]. Additional zinc metalloproteases in these
organelles have to destroy the cleaved signal-peptide
[41]. Moreover inside the chloroplasts proteolytic activi-
ties are dependent on zinc, for example, the repair pro-
cesses of photosystem II by turning over photo-damaged
D1 protein [42]. Zinc deficiency also reduces net photo-
synthesis in plants by disturbing the activity of carbonic
anhydrase, which is the limiting enzyme for CO2 fixation
in C4-plants, Cu–Zn superoxide dismutase, and D-ribu-
lose-5-phosphate 3-epimerase, an enzyme of the Calvin
cycle and oxidative pentose phosphate pathway [43].
In addition to chloroplasts and mitochondria, also the
cytoplasm, lysosome, and the apoplastic space are com-
partments with zinc-dependent hydrolytic activities:
different nucleases and aminopeptidases, peptide defor-
mylases [44], the 26S-proteasome [45], the a-mannosi-
dase [46], and matrix-metalloproteinases associated with
the extracellular matrix [47]. Further, Zinc was found to
be involved in signal transduction via mitogen-activated
protein kinases [48].
Conclusion
Essential micronutrients were found as constituents in
over 1500 proteins where they fulfill catalytic, (co-)acti-
Current Opinion in Plant Biology 2009, 12:259–266
vating, and/or structural functions. The largest group
(>1200) is formed by zinc-proteins (with transcription
factors as major subgroup). Proteins containing iron,
copper, or manganese make up groups in the range of
50–150 members each, while molybdenum and nickel
proteins can be counted on one hand each. Boron and
chlorine are very important, but proteins or compounds
that were unambiguously shown to contain these micro-
nutrients are very rare and mostly elusive.
References and recommended reading
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 of special interest
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 Physiological functions of mineral micronutrients (Cu, Zn, Mn, Fe, Ni, Mo, B, Cl) Hänsch and Mendel 265
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