Future Perspectives in Plant Biology
The Elements of Plant Micronutrients1
Sabeeha S. Merchant*
Institute for Genomics and Proteomics and Department of Chemistry and Biochemistry, University of
California, Los Angeles, California 90095–1569
Amino acid R groups in proteins provide a limited
repertoire of functional groups for catalyzing biochemical transformations. The use of inorganic elements, particularly the first row transition metals,
expands greatly the range of chemistry that can be
catalyzed in a cell. Zinc ions are key for enzymatic
catalysis of reactions that require an electrophile, while
iron, manganese, copper, nickel, and molybdenum are
brokers of redox transformations. These elements are
therefore essential nutrients for plants. They are referred to as micronutrients because they are less abundant (by 1–4 orders of magnitude) compared to the
macronutrients like sulfur and phosphorus. Even
among the micronutrients, the amount of individual
transition metals in plant tissues varies over several
orders of magnitude, with iron being the most abundant (approximately 100 mg/g) and molybdenum the
least (Fig. 1).
Each metal has unique chemical properties including ligand preferences, coordination geometries, and
redox potentials, which are exploited for diverse, yet
highly specific, chemistry. In the photosynthetic electron transfer chain, the midpoint potentials of the
metal centers span nearly 1.5 V. The import of metals
in biology is evident from the association of approximately 30% to 40% of proteins with a metal (Waldron
et al., 2009). Transition metal-protein associations are
highly specific, at least in vivo, because mismetallation
can block activity or yield undesirable chemistry. In
vitro, the associations occur according to thermodynamic preferences described by the Irving-Williams
series: For divalent ions, copper and zinc ions bind
most tightly relative to manganese and iron ions
(Waldron and Robinson, 2009).
The in vivo specificity is achieved by kinetic control
of metal ion assimilation, distribution, and storage and
of metalloprotein assembly, or in other words, metal
metabolism. For nickel and copper proteins in bacteria, specificity can be achieved by direct protein to
protein transfer via metallochaperones coupled with
structural reorganization and stabilization of the resulting holoprotein so that the metal is kinetically
1
This work was supported by the Department of Energy (grant
no. DE–FG02–04ER15529) and the National Institutes of Health
(grant no. GM42143).
* E-mail [email protected].
www.plantphysiol.org/cgi/doi/10.1104/pp.110.161810
512
trapped. For a periplasmic manganese protein in bacteria, one study showed that correct metallation is
achieved by restricting holoprotein formation to the
cytoplasm where the concentration of another competing metal ion is reduced by sequestration in binding protein (Tottey et al., 2008). Compartmentation of
metal ions is, therefore, a key consideration in metalloprotein biogenesis pathways.
Nonessential metals like cadmium, mercury, and
silver can compete with the essential transition metals
for uptake and metalloprotein assembly pathways
because protein flexibility can reduce the selectivity
of metal-binding sites. A consequence of the redox
reactivity and the promiscuous binding of transition
metals to thiol, thioether, imidazole, and carboxylate
ligands is that inappropriate accumulation of metals
is harmful in biology. Therefore, metal metabolism
is under homeostatic regulation (plant homeostasis
pathways reviewed in Palmer and Guerinot, 2009).
Studies of metalloregulation in bacteria, involving
nutrient acquisition pathways as well as metal resistance pathways, indicate that regulators for each metal
have coevolved in a single organism as a set of regulators with ranked relative affinities for various metals
(Waldron and Robinson, 2009). These regulators determine the ranges (between deficiency and excess) of
the number of atoms of metal available in a bacterial
cell to match the number of metal-binding sites. The
problem is more complex in a eukaryotic cell where
subcellular compartmentation of metals and metalloproteins is another consideration. In multicellular organisms, there is the question of how regulation at the
cellular level is integrated into the context of the whole
organism.
The subject of metal-protein interactions in plants
needs more attention at all levels of study, from
molecular to cellular to whole plant. From a practical
perspective, this is important for effective cross-species
transfer of metalloenzymes and metal-binding domains to ensure that they are populated with the
correct metals. Plants are a dietary source of minerals
for a large fraction of the human population and zinc
and iron deficiency are pressing problems in human
nutrition. The understanding of metalloprotein biochemistry and plant mineral nutrition, therefore, has
global impact. In the coming years, a greater understanding of human nutrient metabolism will have an
impact on the design of micronutrient stores in crop
plants.
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Micronutrients
of plants that are found in diverse (with respect to
mineral content) soil types to laboratory-created variants with enhanced substrate selectivity.
Interactions and Networks
Figure 1. Mineral nutrient composition of Chlamydomonas. Elemental
analysis was by inductively coupled plasma mass spectrometry.
LOGISTICS
Transport and Distribution
Decades ago, the focus in studying metals in plants
was on establishing micronutrient requirements and
describing symptoms of individual deficiencies. One
important outcome of these studies was an appreciation for the characteristic phenotype resulting from
individual metal deficiencies. These studies underscored the unique biochemical functions of each metal
micronutrient. More recent research, using classical
and molecular genetics, has emphasized the discovery
of metal transporters and processes that facilitate
transport, including mobilization by redox chemistry
(particularly important for copper and iron), chelation
(relevant for iron and nickel), and extracellular acidification (for review, see Palmer and Guerinot, 2009).
Assimilatory pathways are now quite well described,
and continued investigation in the near term will
distinguish transporters involved in intercellular
transport—processes for metal loading into the xylem
for root to shoot delivery or for recovery of metal
nutrients prior to leaf senescence—and intracellular distribution to the metal utilizing versus storing organelles.
Each type of transporter, ZIP, NRAMP, CTR, CDF/
MTP, is found as a family of genes encoding variant
proteins (Hanikenne et al., 2009). The distinct functions of individual members are just starting to be
explored. It is possible that sequence variants are
exploited to handle variation in environmental supply
of the nutrient or to handle the presence of competing metals, whether essential or nonessential. In the
coming decade, we can expect increased functional
understanding of the many metal transporters in
mediating selective metal uptake and intracellular
distribution. Genome surveys of ecotypes isolated
from diverse soils or of species adapted to contaminated or marginal soils are promising routes for
distinguishing the contributions of individual assimilation and distribution pathways. It will be interesting
to compare natural sequence variations in transporters
The ion complement of cells should be viewed as a
complex networked structure. The impact of metal ions
on biochemistry depends upon their intracellular abundance, speciation (i.e. association with ligands), redox
state, the abundance and speciation of other essential or
nonessential metals (i.e. ratios), compartmentation, and
source-sink relationships. The study of one element at a
time served us well for discovery of individual metalhandling proteins, but systems-level understanding
demands systems-level experimental design.
The application of high-throughput quantitative
elemental analyses (to describe the ionome) for classical genetic screens or for surveying ecotypes in combination with genome association studies is a powerful
approach for probing the interactions of metals with
each other and with other metabolic pathways (Baxter
et al., 2008; Baxter, 2009; Buescher et al., 2010). These
interactions are described presently only at a phenomenological level. Cross talk between iron and manganese, iron and zinc, manganese and phosphate, and
copper and zinc, are among the many documented
interactions (Baxter, 2009). There is speculation that the
links might be reactive oxygen species (for iron and
manganese), various transporters (for iron and manganese or iron and zinc or manganese and phosphate),
or metallothioneins (for copper and zinc), but at present there is very little mechanistic insight. A genetic
approach, especially if applied with spatial and temporal resolution ionomics, has the potential to uncover
the molecular bases of these connections. Will the
technology have practical application in the field?
Can we titrate micronutrient amendments by elemental profiling?
Instrument development is giving us the capability
for ultrahigh resolution (to avoid isobaric interferences
of conventional inductively coupled plasma mass
spectrometry) and simultaneous global detection and
quantitation of elements. We will also be able to combine
proteome with ionome analyses as sample processing
improvements preserve structures and interactions.
Nano-secondary ion mass spectrometry combines microscopy with mass spectrometry to distinguish
where elements are located in cells with 50 nm resolution. As these technologies become more accessible
to plant biologists, they will drive our understanding of
individual metal dynamics and metal-metal interactions at a whole organism level rather than in a test tube.
ECONOMY
Supply and Demand
Trace metal distribution is particularly relevant in a
situation of deficiency, since particular developmental
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Merchant
processes and biochemical pathways will be prioritized
as a function of growth, development, metabolic status,
and environment. In Chlamydomonas (Chlamydomonas
reinhardtii), copper distribution to plastocyanin is deprioritized because another (copper-independent) protein, cytochrome c6, can serve as a backup. However, in
Arabidopsis (Arabidopsis thaliana) chloroplasts, plastocyanin is prioritized over copper-zinc superoxide dismutase because there is no backup for plastocyanin in
most land plants, but an iron-containing superoxide
dismutase can cover for the loss of the copper-zinc
enzyme. In Chlamydomonas, plastocyanin is degraded
by an inducible protease whereas in Arabidopsis the
mRNAs for dispensable copper proteins are degraded
via a miRNA-dependent mechanism. Despite the billion years of evolutionary separation and the distinct
molecular events involved in copper sparing in the
two organisms, both responses are controlled by a
conserved plant-specific transcription factor (CRR1 in
Chlamydomonas, SPL7 in Arabidopsis), which emphasizes the fundamental importance of metal homeostasis
pathways (for review, see Merchant et al., 2006; Pilon
et al., 2009).
Is the prioritizing of copper to plastocyanin in
Arabidopsis restricted to photosynthetic organs? The
question of copper allocation to individual enzymes in
other compartments or other cell types has not been
given as much attention. It is likely that there are cellspecific and developmental-stage specific regulators
that feed into the metal-responsive circuits, and these
will be revealed in the near future through the application of “omic” approaches for the study of transition
metal metabolism. The individualistic response of
copper-deficient Chlamydomonas versus Arabidopsis
(cited above) or even within a single organism in two
different metabolic states, as in iron-deficient Chlamydomonas grown on CO2 versus acetate (Terauchi et al.,
2010), suggests that the hierarchy of micronutrient
utilization might show species- or niche-specific variations.
In the case of zinc proteins, which have expanded in
eukaryotes (Dupont et al., 2010), the affinity of zinc for
protein metal-binding sites (Waldron et al., 2009), suggests that specific mechanisms for selective loading
might not be necessary. However, in a zinc-deficient
cell, other metals might compete more effectively. Zincsparing mechanisms and zinc protein assembly are
presently underinvestigated in plants and ripe for
exploitation. Proteins carrying a COG0523 domain,
which is suggested to function in zinc homeostasis
in all kingdoms of life (Haas et al., 2009), might be
involved in plant zinc homeostasis.
Sparing and Recycling
Metal-sparing phenomena are well documented in
diverse microbes, including cyanobacteria, algae, and
diatoms, but are just beginning to be appreciated in
land plants. Transcriptome and proteome profiling will
reveal more examples of micronutrient-dependent utilization of enzyme isoforms, and metabolomics might
tell us why cofactor flexibility is maintained. What is
the advantage of plastocyanin over cytochrome c6 or
ferredoxin over flavodoxin or copper-zinc superoxide
dismutase over iron-containing superoxide dismutase?
One possibility is that the nutrient-replete isoform is
better and this is established, for example, for ferredoxin, which has a greater range of substrates, over
flavodoxin. Another possibility is that the nutrientreplete isoform serves as a metal reservoir to supply
metal for essential pathways when deficiency is encountered. This model is more difficult to establish because
conventional metabolic tracer techniques, which establish precursor-product relationships, are not easily applied to transition metal ions. They exchange rapidly
with their ligands, they are noncovalently associated
with proteins (which precludes precipitation steps for
enrichment), and radioisotopes of many metals can be
impractical because of issues of half-life or isotopic
purity for conventional pulse-chase experiments.
Therefore, the mechanisms underlying metal cofactor
recycling are unexplored. Nevertheless, the advent of
metal-selective sensors (chemical as well as genetically
encoded) opens the door to monitoring intra- and
intercellular metal movement (Fig. 2; Domaille et al.,
2008; Dittmer et al., 2009; Vinkenborg et al., 2009). In
combination with optical and fluorescence spectroscopy, we can watch the location and movement of
metal ion pools within and between cells in real time.
And by combining probes it might be possible to
Figure 2. Staining of Chlamydomonas cells with
a fluorescent sensor. A, C, E, and G, Bright field.
B, D, F, and H, A cuprous sensitive fluorescent
dye (courtesy of Chris Chang, University of California, Berkeley) was used to visualize Cu(I).
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Micronutrients
monitor interactions between metals. In vivo imaging
will illuminate delivery processes during growth and
recovery pathways during senescence or deficiency
situations. The technology also expands the experimental repertoire for studying mechanisms of longdistance sensing—that is, how does the root sense the
metal status of the shoot? Is there a systemic signal?
This question is of fundamental interest and, most
likely, involves interactions between multiple nutrients.
Real-time imaging with metal-selective sensors also
provides dynamic information for visualizing transient fluxes. Transition metals have not been evaluated
for their potential as messengers in signal transduction
pathways (analogous to calcium, but with a built-in
redox switch). This is completely unexplored in plants,
but the path to discovery is now open.
ACKNOWLEDGMENTS
Janette Kropat and Anne Hong-Hermesdorf are gratefully acknowledged
for contributing figures and Profs. Nigel Robinson and Marinus Pilon for
advice.
Received June 24, 2010; accepted July 8, 2010; published October 6, 2010.
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