Download Cloning an iron-regulated metal transporter from rice

Journal of Experimental Botany, Vol. 53, No. 374, pp. 1677±1682, July 2002
DOI: 10.1093/jxb/erf004
Cloning an iron-regulated metal transporter from rice
Naimatullah Bughio1, Hirotaka Yamaguchi1,3, Naoko K. Nishizawa2,3, Hiromi Nakanishi1 and
Satoshi Mori1,3,4
1
Laboratory of Plant Molecular Physiology, Department of Applied Biological Chemistry, The University of Tokyo,
1-1 Yayoi, Bunkyo-ku, 113-8657 Tokyo, Japan
2
Laboratory of Plant Biotechnology, Department of Global Agricultural Sciences, The University of Tokyo,
1-1 Yayoi, Bunkyo-ku, 113-8657 Tokyo, Japan
3
Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation,
4-1-8 Honcho, Kawaguchi-shi, 332-0012, Saitama, Japan
Received 21 September 2001; Accepted 20 March 2002
Abstract
Rice cDNA and genomic libraries were screened in
order to clone an Fe(II) transporter gene. A cDNA
clone highly homologous to the Arabidopsis Fe(II)
transporter gene IRT1 was isolated from Fe-de®cient
rice roots. The cDNA clone was named OsIRT1. A
genomic clone corresponding to the cDNA was also
obtained, sequenced and analysed. When expressed
in yeast cells, OsIRT1 cDNA reversed the growth
defects of the yeast iron-uptake mutant. Northern
blot analysis revealed that OsIRT1 mRNA was predominantly expressed in roots and was induced by
Fe- and Cu-de®ciency. This suggests that OsIRT1 is
a functional metal transporter for iron, and is actively
engaged in Fe uptake from soils, especially under
limiting conditions.
Key words: Copper, Fe(II) transporter, iron, IRT1, rice.
Introduction
Decades of research on plants have established that there
are two distinct iron-uptake systems based on the response
of plants to Fe-de®ciency. These are known as Strategy I
and Strategy II (RoÈmheld, 1987; RoÈmheld and Marschner,
1986). Strategy I plants include all dicots and nongraminaceous monocots. These plants respond to Fede®ciency by decreasing rhizosphere pH and reducing
sparingly soluble ferric iron. These mechanisms employ
releasing protons and reductants, and morphological
changes in the roots. The most important step of the
4
mechanism is the reduction of ferric iron at the root surface
by membrane-resident ferric chelate reductase (Chaney
et al., 1972). In Arabidopsis, this reductase is encoded by
FRO2 (Robinson et al., 1999). Reduced ferrous iron is
absorbed into root cells by the Fe(II) transporter. IRT1 in
Arabidopsis, a member of the ZIP metal transporter family,
was the ®rst metal transporter involved in Fe(II) transport
to be identi®ed in higher plants (Eide et al., 1996). IRT1
was also the ®rst member of the ZRT, IRT-like protein
(ZIP) family to be described (Guerinot, 2000). To date,
IRT1-like Fe(II) transporters have been isolated from
several dicotyledonous species (Eckhardt et al., 2001; Vert
et al., 2001).
Strategy II iron-uptake systems are limited to graminaceous monocots. These plants release mugineic acid-family
phytosiderophores to the rhizosphere, where they solubilize sparingly soluble iron by chelation. The chelated
complex is then absorbed into the roots. Genes for the
synthesis of phytosiderophores have been isolated from
barley (Higuchi et al., 1999; Kobayashi et al., 2001;
Okumura et al., 1994; Takahashi et al., 1999). The ®rst
step of phytosiderophore synthesis is also crucial in
Strategy I plants (Ling et al., 1999), which is conversion
of three molecules of S-adenosyl methionine to nicotianamine. Recently, a strong candidate for the transporter of
the iron±phytosiderophore complex was cloned from
maize (Curie et al., 2001).
It is generally believed that plants depend on a single
system (either Strategy I or II) for iron acquisition. In
graminaceous plants, such as barley and maize, the
inducible Fe-de®ciency Fe(II) transport system is either
absent or is expressed only at very low levels (Zaharieva
To whom correspondence should be addressed. Fax: +81 3 5841 8009. E-mail: [email protected]
ã Society for Experimental Biology 2002
1678 Bughio et al.
32
P-
and RoÈmheld, 2001). It is possible that some plants have
both uptake systems. Rice is a strategy II plant and releases
phytosiderophores under Fe-de®ciency. In fact, rice is the
®rst plant in which the root-secreted iron-solubilizing
substance, the phytosiderophore, was observed (Takagi,
1976). Under submerged conditions, such as in a paddy
®eld where rice is normally grown, however, ferrous iron is
more abundant than ferric iron. Up until now, quite limited
information has been available as to the existence of an
Fe(II) transport system for iron uptake in rice (Eide et al.,
1996).
This paper reports the isolation of cDNA and a genomic
clone for a functional Fe(II) transporter from rice. This is
the ®rst report of the isolation of a functional Fe(II)
transporter from a Strategy II plant. It is proposed that the
Fe(II) transport system participates in iron uptake in rice in
the reductive environment in which rice is normally
grown.
onto nylon membranes. The membranes were hybridized with
labelled probes speci®c for the cDNA fragments for OsIRT1.
Materials and methods
Functional expression in yeast
A yeast expression vector (pYH23) was constructed as follows. The
plasmid YEplac181 (Gietz and Sugino, 1988) was cleaved with XbaI
and EcoRI, blunt ended with T4 DNA polymerase, and re-ligated in
order to remove most of the multiple cloning sites. The HindIII site
was similarly deleted. The plasmid was digested with SphI and the
728 bp fragment from pVT100U (Vernet et al., 1987) containing the
ADH1 expression cassette was inserted. To insert a NotI site in the
multicloning site of the cassette, the plasmid was cleaved with
BamHI and blunt ended with T4 DNA polymerase and the
phosphorylated linker AGCGGCCGCT (TaKaRa Shuzo, Japan)
was inserted. The new plasmid pYH23 has HindIII, PvuII, PstI,
XhoI, SstI, XbaI, and NotI sites in the ADH1 expression cassette. For
constitutive expression of OsIRT1 in yeast cells, OsIRT1 cDNA was
inserted into the XhoI±NotI site in pYH23 to form pYH23-OsIRT1.
Yeast transformation was carried out using the Li-acetate transformation method (Gietz and Schiestl, 1995).
Plant material
Two varieties of rice plants, Oryza sativa cv. Notohikari or
Nihonbare, were used for cDNA library construction or Northern
analysis, respectively. Seeds were germinated and seedlings were
hydroponically grown as previously described (Mori and Nishizawa,
1987). For micronutrient de®ciency treatment, 3-week-old plants
were transferred to nutrient solution without Cu, Fe, Mn, or Zn, and
grown for three more weeks.
cDNA cloning
The OsIRT1 cDNA clone, identical to a rice expression sequence tag
(EST) (accession no. D49213) homologous to IRT1, was isolated as
follows. RNA was extracted from the roots of rice plants grown
under Fe-de®cient conditions for 2 weeks. A cDNA library was
constructed according to the SUPER SCRIPT Plasmid System for
cDNA Synthesis and Plasmid Cloning instruction manual (GibcoBRL, USA). The OsIRT1 cDNA clone was isolated using a PCRbased strategy. Approximately 105 independent colonies of
Escherichia coli from the library were divided into 96 sublibraries.
Ninety-six pools of plasmid were prepared, and each sublibrary
was screened for the EST clone by PCR using speci®c primers
(5¢-CGAGCTGCAGGATCCGAATTCTCGGCGGCTGCATCCTGC
and 5¢-GGGCAACGTCCGTCTTGAAC). Any sub-library in which a
fragment was ampli®ed was further analysed. Repeated subdivision
and screening allowed an OsIRT1 cDNA clone to be isolated.
Cloning a genomic sequence
A commercial genomic library of rice Oryza sativa L. cv. IR36 was
used for screening (CLONTECH Inc., USA) by plaque hybridization. A DNA fragment used as a probe was ampli®ed using the above
primer set from a cDNA pool prepared from RNA isolated from 3month-old rice leaves.
DNA and RNA procedures
Plasmid puri®cation, subcloning, electrophoresis, blotting, and
hybridization were carried out according to standard protocols
(Sambrook et al., 1989). For Northern analysis, total RNA was
extracted from plant materials by the SDS-phenol method (Naito
et al., 1988). Total RNA (20 mg) was electrophoresed and blotted
Yeast strains and growth media
The following strains of the yeast, Saccharomyces cerevisiae, were
used in this study: CM3260 (parent strain) MATalpha trp1-63 leu23,112 gcn4-101 his3-609 ura3-52, YH001 MATalpha trp1-63 leu23,112 gcn4-101 his3-609 ura3-52 Dftr1::URA3, YH003 MATalpha
trp1-63 leu2-3,112 gcn4-101 his3-609 ura3-52 Dftr1::URA3
Dfre1::HIS3 Dfet4::TRP1, M3 MATalpha trp1-63 leu2-3,112 gcn4101 his3-609 ura3-52 FRE1-HIS3::URA3 ctr1-3, FTRUNB1 MATa
ino1-13 leu2-3,112 gcn4-101 his3-609 ura3-52 Dctr1::URA3. Yeast
disruption mutant strains, YH001 and YH003, were made by
homologous recombination.
Yeast cells were grown in 1% yeast extract, 2% peptone, and 2%
glucose (YPD), 1% yeast extract, 2% peptone, and 2% glycerol
(YPG), and synthetic de®ned medium (SD) supplied with appropriate amino acids. Two per cent agar was added for solid plate media
(Sherman, 1991). For iron- or copper-depleted media, bathophenanthroline disulphonic acid disodium salt (BPDS) or bathocuproine
disulphonic acid disodium salt (BPCS) (Wako Pure Chemical
Industries, Ltd., Japan) was added, respectively.
Results
Cloning the cDNA and genomic sequences of OsIRT1
The database contains expression sequence tag (EST) from
rice (accession no. D49213) that is highly homologous to
the metal transporter IRT1 from Arabidopsis (Eide et al.,
1996). Speci®c PCR primers were designed based on the
EST sequence in order to isolate a corresponding cDNA
clone. These primers were used to amplify a fragment (280
bp) clone from cDNA prepared from leaves of rice with
91% identity to the EST. The PCR-based strategy
successfully allowed the isolation of a cDNA clone
identical to the ampli®ed fragment from a cDNA library
made from Fe-de®cient rice roots. This cDNA clone had
high sequence similarity to IRT1. The cDNA was designated as OsIRT1. A rice genomic library was also screened
by hybridization using the 280 bp ampli®ed fragment as a
probe. After several rounds of screening, a clone containing the genomic sequence of OsIRT1 cDNA was isolated.
Cloning rice Fe(II) transporter 1679
Fig. 2. Growth of the Dftr1 Dfet4 Dfre1 yeast mutants on irondepleted media containing 50 mM BPDS, transformed with vector only
(left) or pYH23-OsIRT1 (right).
Fig. 1. Alignment of the deduced OsIRT1 protein and ZIP proteins
from higher plants. Residues conserved in all sequences are shaded in
gray. Slashes indicate a gap. Transmembrane domains and a variable
region are indicated. The histidine-rich metal-binding domain is
underlined. An arrowhead indicates the corresponding position of an
intron in OsIRT1 gene.
The ®rst (699 bp) and second (679 bp) exons of OsIRT1
cDNA are separated by an intron (4.3 kb).
Sequence of OsIRT1 cDNA and the deduced amino
acid sequence of OsIRT1 protein
The OsIRT1 cDNA is 1395 bp long and contains an open
reading frame of 374 amino acids (accession no.
AB070226). OsIRT1 protein has a strong similarity to
the metal transport protein IRT1 from Arabidopsis (53%
amino acid identity, accession no. U27590). IRT1 and
related metal transporters comprise the ZIP metal transporter family (Guerinot, 2000). Within the ZIP family,
Rit1, an iron transport protein from pea, has the highest
amino acid identity to OsIRT1 (55%, AF065444). LeIRT1
and LeIRT2 from tomato have a strong similarity as well
(55% and 55%, AF2(6266 and AF2(6266, respectively)
(Eckhardt et al., 2001). The amino acid alignment of these
proteins is shown in Fig. 1. The hydrophobicity pro®le
reveals that OsIRT1 has eight transmembrane domains
with a `variable region' between domains 3 and 4. These
are typical features found in the ZIP metal transporter
family (Guerinot, 2000). The variable region was less
similar among these proteins. The region of OsIRT1
contains a potential metal-binding domain rich in histidine
residues, which was also found in IRT1. OsIRT1 has only
one intron, while IRT1 has two. However, the position of
the ®rst intron of IRT1 is same as OsIRT1 (Fig. 1).
OsIRT1 reversed growth defect of ferrous uptake
mutants of yeast
The yeast strain YH003 (Dftr1 Dfet4 Dfre1) has disrupted
null mutations in the genes for high- and low-af®nity iron
transporters and ferric reductase (Dancis et al., 1992; Dix
et al., 1994; Stearman et al., 1996). YH003 could not be
grown on SD media containing 50 mM of BPDS, a strong
chelator of the ferrous iron. Heterologous expression of
OsIRT1, however, reversed the growth defect of the mutant
on iron-depleted media (Fig. 2). The effect of OsIRT1
expression in copper uptake mutant ctr1 was also checked.
Two strains, M3 (ctr1-3) and FTRUNB1 (Dctr1) (Dancis
et al., 1994) were tested. However, no signi®cant effect on
the growth defect of these strains could be found on media
containing non-fermentable carbon source (YPG) or SD
media containing 50 mM or 100 mM BPCS, a strong
chelator of the copper ion (data not shown).
Expression of OsIRT1 under trace metal nutrient
de®ciency
Induction of OsIRT1 expression in rice roots and leaves
under trace metal-limiting conditions was examined by
Northern hybridization (Fig. 3). Transcripts of OsIRT1
from plants grown under nutrient-suf®cient conditions
were very faint in roots, and not visible in shoots. In roots,
Fe-de®ciency signi®cantly increased the level of OsIRT1
transcripts. Cu-de®ciency also increased the transcript
level. Under Mn- and Zn-de®ciency, induction was not
observed. In shoots, however, no increase in transcript
level was found under any metal de®ciency conditions.
1680 Bughio et al.
Fig. 3. Transcript levels of the OsIRT1 gene in the roots and leaves of
rice plants grown under trace metal de®ciency conditions. Ten mg of
total RNA were used. `C' indicates plants grown in normal nutrient
solution.
Discussion
In this paper, the isolation of a cDNA and genomic clone of
OsIRT1, an IRT1-like metal transport protein from rice is
reported. OsIRT1 has features typical of the ZIP metal
transporter family, such as eight transmembrane domains
and a variable region with a histidine-rich metal binding
domain (Guerinot, 2000). So far, several members of this
family have been isolated from dicotyledonous species
(Eckhardt et al., 2001; Eide et al., 1996; Vert et al., 2001).
OsIRT1 is the ®rst member of the ZIP metal transporter
family to be isolated from a graminaceous plant.
It was also shown that OsIRT1 cDNA reversed the
growth defect of the yeast iron-uptake mutant ftr1 fet4 fre1
on iron-depleted media when expressed in the yeast cells.
This suggests that OsIRT1 encodes a functional iron
transporter. OsIRT1 transcripts were predominantly expressed in the roots, and were induced by Fe-de®ciency.
The fact that expression of OsIRT1 is limited to the roots
and is induced by Fe-de®ciency indicates the direct
involvement of OsIRT1 in uptake of iron from soil.
It has been reported that IRT1 is a metal transporter with
a broad substrate speci®city (Eide et al., 1996; Korshunova
et al., 1999; Rogers et al., 2000). In addition to ferrous
iron, IRT1 can transport manganese, zinc, and probably
cadmium and cobalt, when expressed in yeast cells.
LeIRT1 and LeIRT2 from tomato can also complement
yeast mutants defective in the uptake of iron, zinc,
manganese, and copper (Eckhardt et al., 2001). Although
IRT1 has a broad substrate speci®city, the transcript level
was not increased by any of these metal de®ciency
conditions other than iron (Eide et al., 1996; Korshunova
et al., 1999). Therefore, it is interesting that expression of
OsIRT1 was also induced by Cu-de®ciency in addition to
Fe-de®ciency. It was reported that LeIRT1 and LeIRT2
from tomato could reverse the growth defects of the yeast
high-af®nity copper uptake mutant, ctr1 (Dancis et al.,
1994), while IRT1 from Arabidopsis could not (Eckhardt
et al., 2001). Although no effects of OsIRT1 expression on
the growth defects of ctr1 could be found in these
experiments, OsIRT1 may participate in copper uptake
when it is Cu-limited. The effects of OsIRT1 expression on
manganese or zinc uptake yeast mutants were not tested.
Although OsIRT1 was not induced by these metal
de®ciency conditions, it may have a broad substrate
speci®city for these metals similar to IRT1, LeIRT1, and
LeIRT2.
To date, it has been generally accepted that
graminaceous plants can utilize only ferric iron in the
rhizosphere in a phytosiderophore-dependent manner
(RoÈmheld, 1987; RoÈmheld and Marschner, 1986).
Compared with dicotyledonous plants, Fe-de®ciency
inducible Fe(II) transport systems are absent or
expressed at lower levels in the roots of barley and
maize (Zaharieva and RoÈmheld, 2001). This report on
the isolation of a root-speci®c IRT1-like Fe(II) transporter clearly demonstrates that rice possesses an iron
uptake system for Fe(II), in addition to a phytosiderophore-mediated Fe(III) transport system. This is a new
mechanism of iron uptake that combines both Strategy
I and Strategy II. Rice is grown under submerged
conditions in which the dominant form of soil iron is
ferrous iron (Yoshida, 1981). Therefore, it seems
reasonable that rice may have an iron uptake system
for Fe(II) as well as Fe(III) in its roots. Rice also
transports oxygen from the shoots to the roots, and
secretes oxygen from roots to oxidize the rhizosphere.
Therefore, rice plants may induce one or the other of
these iron transport systems by sensing the concentration of ferrous iron or the redox potential around their
rhizoplane. Perhaps rice is unique among graminaceous
plants in possessing a transport system for Fe(II).
There is another class of metal transporters, the Nramp
family, involved in Fe(II) transport in higher plants. Nramp
genes are widely distributed in mammals, yeasts, and
higher plants, and are known to be involved in a variety of
processes, including metal transport and resistance to
microbial infection (Cellier et al., 1996; Cohen et al.,
2000; Gunshin et al., 1997). AtNramp1 and OsNramp1 can
complement the fet3 fet4 yeast mutant defective both in
low- and high-af®nity iron transporters. The expression of
AtNramp1, however, did not enhance iron uptake in yeast
mutant cells. These proteins were speculated to be
localized to intracellular compartments, such as chloroplasts, and to facilitate intracellular iron transport (Curie
et al., 2000). In another paper, it was reported that
AtNramp3 and AtNramp4 could complement the yeast
metal uptake-de®cient strain smf1, in addition to the fet3
fet4 mutant. The authors suggested the involvement of
Arabidopsis Nramp proteins in the transport of iron,
manganese, and cadmium, including Fe-de®ciency inducible iron transport, especially AtNramp3 (Thomine et al.,
2000). There are at least three isologues of Nramp proteins
in rice (Belouchi et al., 1997). However, the function of
these proteins remains unclear. Recently, yellow stripe 1
(YS1), a strong candidate for the gene for the transport
Cloning rice Fe(II) transporter 1681
protein for the ferric phytosiderophore complex, was
isolated from maize (Curie et al., 2001). Several other rice
YS1 homologues were found in the database. Further study
might elucidate the function of each of these three classes
of metal transport proteins (IRT, Nramp, and YS1), and
how the expression of these proteins is regulated developmentally and spatially. Analysis of these genes will
allow an understanding of iron nutrition from the viewpoint not only of iron uptake from soils, but iron transport
in planta, as well as organellar iron transport.
Acknowledgements
The authors thank Dr Andrew Dancis for the gift of the yeast strains
used for the complementation analysis. We also thank Dr Kyoko
Higuchi for her gift of a cDNA library and help interpreting data.
This work was supported by the Japan Science and Technology
Corporation (JST).
References
Belouchi A, Kwan T, Gros P. 1997. Cloning and characterization
of the OsNramp family from Oryza sativa, a new family of
membrane proteins possibly implicated in the transport of metal
ions. Plant Molecular Biology 33, 1085±1092.
Chaney RL, Brown JC, Tif®n LO. 1972. Obligatory reduction of
ferric chelates in iron uptake by soybeans. Plant Physiology 50,
208±213.
Cellier M, Belouchi A, Gros P. 1996. Resistance to intracellular
infections: comparative genomic analysis of Nramp. Trends in
Genetics 12, 201±204.
Cohen A, Nelson H, Nelson N. 2000. The family of SMF metal ion
transporters in yeast cells. Journal of Biological Chemistry 275,
33388±33394.
Curie C, Alonso JM, Jean MLE, Ecker JR, Briat JF. 2000.
Involvement of NRAMP1 from Arabidopsis thaliana in iron
transport. Biochemical Journal 347, 749±755.
Curie C, Panaviene Z, Loulergue C, Dellaporta SL, Briat JF,
Walker EL. 2001. Maize yellow stripe1 encodes a membrane
protein directly involved in Fe(III) uptake. Nature 409, 346±
349.
Dancis A, Roman DG, Anderson GJ, Hinnebusch AG, Klausner
RD. 1992. Ferric reductase of Saccharomyces cerevisiaeÐ
molecular characterization, role in iron uptake, and
transcriptional control by iron. Proceedings of the National
Academy of Sciences, USA 89, 3869±3873.
Dancis A, Yuan DS, Haile D, Askwith C, Eide D, Moehle C,
Kaplan J, Klausner RD. 1994. Molecular characterization of a
copper transport protein in S. cerevisiae: an unexpected role for
copper in iron transport. Cell 76, 393±402.
Dix DR, Bridgam JT, Broderius MA, Byersdorfer CA, Eide DJ.
1994. The FET4 gene encodes the low-af®nity Fe(II) transport
protein of Saccharomyces cerevisiae. Journal of Biological
Chemistry 269, 26092±26099.
Eckhardt U, Marques AM, Buckhout TJ. 2001. Two ironregulated cation transporters from tomato complement metal
uptake-de®cient yeast mutants. Plant Molecular Biology 45, 437±
448.
Eide D, Broderius M, Fett J, Guerinot ML. 1996. A novel ironregulated metal transporter from plants identi®ed by functional
expression in yeast. Proceedings of the National Academy of
Sciences, USA 93, 5624±5628.
Gietz RD, Schiestl RH. 1995. Transforming yeast with DNA.
Methods in Molecular and Cellular Biology 5, 255±269.
Gietz RD, Sugino A. 1988. New yeast and Escherichia coli shuttle
vectors constructed with in vitro mutagenized yeast genes lacking
six-base pair restriction sites. Gene 72, 527±534.
Guerinot ML. 2000. The ZIP family of metal transporters.
Biochimica et Biophysica Acta 1465, 190±198.
Gunshin H, Mackenzie B, Berger UV, Gunshin Y, Romero MF,
Boron WF, Nussberger S, Gollan JL, Hediger MA. 1997.
Cloning and characterization of a mammalian proton-coupled
metal-ion transporter. Nature 388, 482±488.
Higuchi K, Suzuki K, Nakanishi H, Yamaguchi H, Nishizawa
NK, Mori S. 1999. Cloning of nicotianamine synthase genes,
novel genes involved in the biosynthesis of phytosiderophores.
Plant Physiology 119, 471±479.
Kobayashi T, Nakanishi H, Takahashi M, Kawasaki S,
Nishizawa NK, Mori S. 2001. In vivo evidence that Ids3 from
Hordeum vulgare encodes a dioxygenase that converts 2Âdeoxymugineic acid to mugineic acid in transgenic rice. Planta
212, 864±871.
Korshunova YO, Eide D, Clark WG, Guerinot ML, Pakrasi HB.
1999. The IRT1 protein from Arabidopsis thaliana is a metal
transporter with a broad substrate range. Plant Molecular Biology
40, 37±44.
Ling HQ, Koch G, Baumlein H, Ganal MW. 1999. Map-based
cloning of chloronerva, a gene involved in iron uptake of higher
plants encoding nicotianamine synthase. Proceedings of the
National Academy of Sciences, USA 96, 7098±7103.
Mori S, Nishizawa NK. 1987. Methionine as a dominant precursor
of phytosiderophores in Gramineae plants. Plant and Cell
Physiology 28, 1081±1092.
Naito S, Duke PH, Beachy RN. 1988. Differential expression of
conglycinin alpha and beta subunit genes in transgenic plants.
Plant Molecular Biology 11, 109±124.
Okumura N, Nishizawa NK, Umehara Y, Ohata T, Nakanishi H,
Yamaguchi H, Chino M, Mori S. 1994. A dioxygenase gene
(Ids2) expressed under iron de®ciency condition in the roots of
Hordeum vulgare. Plant Molecular Biology 25, 705±719.
Robinson NJ, Procter CM, Connolly EL, Guerinot ML. 1999. A
ferric-chelate reductase for iron uptake from soil. Nature 397,
694±697.
Rogers EE, Eide DJ, Guerinot ML. 2000. Altered selectivity in an
Arabidopsis metal transporter. Proceedings of the National
Academy of Sciences, USA 97, 12356±1260.
RoÈmheld V. 1987. Different strategies for iron acquistion in hgher
plants. Physiologia Plantarum 70, 321±234.
RoÈmheld V, Marschner H. 1986. Evidence for a speci®c system
for iron phytosiderophores in roots of grasses. Plant Physiology
80, 175±180.
Sambrook J, Fritsch E, Maniatis T. 1989. Molecular cloning: a
laboratory manual. New York: Cold Spring Harbor Laboratory
Press.
Sherman F. 1991. Getting started with yeast. Methods in
Enzymology 194, 3±21.
Stearman R, Yuan DS, Yamaguchi-Iwai Y, Klausner RD,
Dancis A. 1996. A permease-oxidase complex involved in highaf®nity iron uptake in yeast. Science 271, 1552±1557.
Takagi S. 1976. Naturally occurring iron-chelating compounds in
oat- and rice-root washing. I. Activity measurement and
preliminary characterization. Soil Science and Plant Nutrition
22, 423±433.
Takahashi M, Yamaguchi H, Nakanishi H, Nishizawa NK, Mori
S. 1999. Cloning two genes for nicotianamine aminotransferase, a
critical enzyme in iron acquisition (Strategy II) in graminaceous
plants. Plant Physiology 121, 947±956.
Thomine S, Wang R, Ward JM, Crawford NM, Schroeder JI.
1682 Bughio et al.
2000. Cadmium and iron transport by members of a plant metal
transporter family in Arabidopsis with homology to Nramp genes.
Proceedings of the National Academy of Sciences, USA 97, 4991±
4996.
Vernet T, Dignard D, Thomas DV. 1987. A family of yeast
expression vectors containing the phage f1 intergenic region.
Gene 52, 225±233.
Vert G, Briat JF, Curie C. 2001. Arabidopsis IRT2 gene encodes a
root-periphery iron transporter. The Plant Journal 26, 181±189.
Yoshida S. 1981. Fundamentals of rice crop science. Laguna:
International Rice Research Institute.
Zaharieva T, RoÈmheld V. 2001. Speci®c Fe2+ uptake system in
Strategy I plants inducible under Fe de®ciency. Journal of Plant
Nutrition 23, 1733±1744.