INVITED REVIEW
Metal movement within the plant: contribution of nicotianamine and yellow
stripe 1-like transporters
Catherine Curie
1,
*, Gae¨lle Cassin
1
, Daniel Couch
1
, Fanchon Divol
1
, Kyoko Higuchi
2
, Marie Le Jean
1
,
Julie Misson
1
, Adam Schikora
1
, Pierre Czernic
1
and Ste´phane Mari
1
1
Laboratoire de Biochimie et Physiologie Mole´culaire des Plantes CNRS UMR5004/SupAgro/INRA/Universite´ Montpellier2, 1
place Viala, F-34060 Montpellier cedex1, France and
2
Laboratory of Plant Production Chemistry, Department of Applied
Biology and Chemistry, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo, 156-8502, Japan
Received: 6 June 2008 Returned for revision: 5 September 2008 Accepted: 30 September 2008 Published electronically: 31 October 2008
† Background Since the identification of the genes controlling the root acquisition of iron (Fe), the control
of inter- and intracellular distribution has become an important challenge in understanding metal homeostasis.
The identification of the yellow stripe-like (YSL) transporter family has paved the way to decipher the
mechanisms of long-distance transport of Fe.
† Scope Once in the plant, Fe will systematically react with organic ligands whose identity is poorly known so far.
Among potential ligands, nicotianamine has been identified as an important molecule for the circulation and
delivery of metals since it participates in the loading of copper (Cu) and nickel in xylem and prevents Fe pre-
cipitation in leaves. Nicotianamine is a precursor of phytosiderophores, which are high-affinity Fe ligands exclu-
sively synthesized by Poaceae species and excreted by roots for the chelation and acquisition of Fe. Maize YS1 is
the founding member of a family of membrane transporters called YS1-like (YSL), which functions in root Fe–
phytosiderophore uptake from the soil. Next to this well-known Fe acquisition role, most of the other YSL family
members are likely to function in plant-wide distribution of metals since (a) they are produced in vascular tissues
throughout the plant and (b) they are found in non-Poaceae species that do not synthesize phytosiderophores. The
hypothesized activity as Fe–nicotianamine transporters of several YSL members has been demonstrated exper-
imentally by heterologous expression in yeast or by electrophysiology in Xenopus oocytes but, despite numerous
attempts, proof of the arabidopsis YSL substrate specificity is still lacking. Reverse genetics, however, has
revealed a role for AtYSL members in the remobilization of Cu and zinc from senescing leaves, in the formation
of pollen and in the Fe, zinc and Cu loading of seeds.
† Conclusions Preliminary data on the YSL family of transporters clearly argues in favour of its role in the long-
distance transport of metals through and between vascular tissues to eventually support gametogenesis and
embryo development.
Key words: Metals, iron, nicotianamine, yellow stripe-like, YS1-like, circulation, transport, phytosiderophore,
xylem, phloem.
INTRODUCTION
Cell life is reliant on iron (Fe) for its unique property of being
able to catalyse oxidation/reduction reactions. Fe serves as a
prosthetic group in proteins to which it is associated either
directly or through a haem or an iron-sulfur cluster. Since in
solution it exists under two redox states, reduced ferrous
and oxidized ferric, Fe can lose or gain an electron within
metalloproteins. A wide range of metabolic pathways rely on
Fe redox enzymes, including the electron transfer chains of
respiration and photosynthesis (cytochromes), the biosynthesis
of DNA (ribonucleotide reductase), lipids (lipoxygenase) and
hormones [1-aminocyclopropane 1-carboxylic acid (ACC)
oxidase], the detoxification of reactive oxygen species (ROS)
(peroxidase, catalase) and the nitrogen assimilation (nitrite
and nitrate reductase). These cellular processes take place in
distinct intracellular compartments, which therefore need to
be provided with an adequate amount of Fe.
Fe is relatively insoluble in its inorganic state, and therefore
is scarcely available to plant roots. As a result, plants have
developed specialized systems to optimize Fe uptake in case
of Fe shortage (Curie and Briat, 2003). To increase soil Fe
solubility, Poaceae, that include the major crop plants, rely
on chelation of Fe by small metabolites generically termed
phytosiderophores (PS) that they synthesize and secrete.
Dicotyledonous and non-grass monocotyledonous plants,
however, improve Fe solubility by lowering rhizospheric pH
through the extrusion of organic acids and protons. While
the first class of plants acquires Fe as Fe(III)–PS complexes,
the latter reduces solubilized Fe(III) through activation of a
ferric-chelate reductase and take Fe up in its reduced state
by the ferrous Fe transporter IRT1 (Eide et al., 1996; Vert
et al., 2002; Vacchina et al., 2003). The dogma of the classi-
fication of plants in two distinct uptake strategies has been
recently disturbed by the finding that rice, a Poaceae, can
induce both Fe(II) uptake by OsIRT1 and Fe(III)–PS uptake
in response to Fe starvation (Ishimaru et al., 2006). These
sophisticated physiological mechanisms, as well as root
* For correspondence. E-mail curie@supagro.inra.fr
# The Author 2008. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
For Permissions, please email: journals.permissions@oxfordjournals.org
Annals of Botany 103: 1–11, 2009
doi:10.1093/aob/mcn207, available online at www.aob.oxfordjournals.org

morphological changes necessary to increase foraging, enable
plants to adapt to an ever-changing environment for Fe nutri-
tion. Severe Fe deficiency limits plant growth and provokes the
development of a characteristic interveinal chlorosis.
Conversely, an excess of Fe in the organism can be detrimen-
tal. Indeed, the same chemical properties that make Fe a
central player in many essential enzymatic reactions, can
lead to harmful effects. Iron, specially Fe(II), catalyses the pro-
duction of reactive oxygen species, including the nasty
hydroxyl radical OH
†
, through the Fenton reaction. Because
free radicals can react with many cell components and gener-
ate some so-called oxidative stress, cells prevent noxious
effects of excessive Fe in the cell by storing it either associated
with organic acids in the vacuole or engulfed in ferritin, a ubi-
quitous Fe storage protein, which in plants is located in plas-
tids (Briat et al., 2006).
Iron chelation too is a central process in Fe homeostasis
because it can be viewed both as a way to enhance its avail-
ability by helping its solubilization, or as a way to scavenge
Fe in a non-Fenton active form. Indeed, Fe reactivity is such
that Fe exists mainly in cells as highly stable complexes
with organic ligands or inorganic phosphate. Yet very little
is known about the nature of the Fe species in the different
cell compartments. Furthermore, circulation of Fe throughout
the plant and distribution to tissues and organelles relies on
transmembrane transporters whose nature depends on the
type of Fe substrate. Contrary to the transporters responsible
for the entry of Fe from the soil solution into the root, candi-
date transporters that mediate long-distance transport within
the plant have only been discovered in recent years. This
review focuses on one such candidate family of proteins, the
yellow stripe-like (YSL) transporters, and present data support-
ing the view that YSLs mediate long-distance transport of
specific Fe species, along with other metals, within the
plant. Because internal transport of Fe is tightly integrated
with Fe speciation, recent advances are also highlighted and
old data on the nature and physiological role of a plant-specific
Fe ligand, nicotianamine (NA), revisited.
THE NA MOLECULE: BIOCHEMICAL
PROPERTIES
The metabolite NA, ubiquitous in the plant kingdom, is indis-
pensable for a plant to complete its life cycle. NA (molecular
weight 303) results from the enzymatic condensation of three
amino-carboxylpropyl groups of three S-adenosyl-methionine
molecules, by NA synthase. During the enzymatic reaction,
three covalent bonds are broken to release three amino-
carboxypropyl groups from S-adenosyl-methionine and three
new covalent bonds are formed, including an internal cycliza-
tion, leading to the formation of an azetidin ring. The presence
of three amino and three carboxy groups in the molecule
allows the formation of an hexadentate co-ordination that
drives the formation of very stable octahedral chelates with a
central metal ion (Fig. 1A). In vitro, NA is able to form
stable complexes with manganese (Mn), Fe(II), cobalt (Co),
zinc (Zn), nickel (Ni) and copper (Cu), in increasing order
of affinity (Benes et al., 1983; Anderegg and Ripperger,
1989). It has been shown only recently that NA can complex
Fe(III) in vitro with a high affinity (Fig. 1B; von Wiren
et al., 1999). In that study, based on potentiometric and spec-
trophotometric measurements of the chelation capabilities of
NA, computer analyses were performed to simulate the
pH-dependent stability of different metal–NA complexes.
Several important conclusions were drawn: (a) for all the
metals considered, the stability of the complexes was
maximal at pH values above 6
.
5, indicating that NA would
be more likely a ‘symplastic’ chelator of metals; (b) among
essential metals Cu is the exception as the Cu–NA complex
is very stable in mild acidic conditions, which is a strong argu-
ment in favour of the possible occurrence of a Cu–NA
complex in an ‘apoplastic’ environment such as the xylem
sap; (c) in vitro, NA can chelate Fe(III) with a high affinity,
although the kinetic stability of this complex is much lower
than Fe(II)–NA; (d ) for pH values around 5
.
5 organic acids
like citrate would be the main chelators of metal ions
whereas in conditions of neutrality NA would be almost the
exclusive chelator; (e) when mixing NA and deoxymugineic
acid (DMA; a PS derived from NA) with Fe(III), DMA
would complex Fe in the pH range 3
.
5–5
.
5 whereas above
this value NA would prevail, which illustrates that ligand
exchanges can occur when switching from an apoplastic to a
symplastic environment (von Wiren et al., 1999). These obser-
vations are based on computer simulations and it is only very
recently that the metal–NA complexes have been studied in
vitro with mass spectrometry techniques, namely electrospray
ionization time-of-flight mass spectrometry (Rellan-Alvarez
et al., 2008). In that work, the formation of different metal–
NA complexes has been analysed by measuring free NA
and metal–NA complexes at different pH conditions. The
authors have confirmed biochemically, without ambiguity,
the computer simulations proposed above concerning the
pH-dependent stability of the several metal–NA complexes,
the high stability of Fe(II)–NA and finally the possibility of
ligand exchange of Fe from NA to citrate at pH 5
.
5. In
summary, NA has ideal structural features for a stable metal
chelator, through the formation of hexadentate octahedral
complexes. The different complexes studied either through
computational prediction (modellization) and/or in vitro are
more stable in neutral conditions, which implies that NA is
likely to be a symplastic metal chelator. The capacity of NA
to chelate Fe(III), somehow controversial, has been actually
reported and demonstrated in different ways (von Wiren,
NH
COO
OOC
N
COO
Mn(II)
B
A
log K 8·8 12·1 14·7 14·8 16·1 18·6 20·6
Fe(II) Zn(II) Co(II) Ni(II) Cu(II) Fe(III)
NH
2
Me
FIG. 1. Biochemical properties of nicotianamine: (A) proposed chemical
structure of the nicotianamine–metal complex; (B) in vitro affinity constants
of complexes of nicotianamine with various metals. Me, Metal.
Curie et al. — YSL transporters and metal circulation2