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Plant Physiology 132:1989-1997 (2003) © 2003 American Society of Plant Biologists Three Nicotianamine Synthase Genes Isolated from Maize Are Differentially Regulated by Iron Nutritional StatusDepartments of Applied Biological Chemistry (D.M., K.H., H.N., S.M.) and Global Agricultural Sciences (T.S., N.K.N.), Graduate School of Agricultural and Life Science, University of Tokyo, 1–1–1 Yayoi, Bunkyoku, Tokyo 113–8657, Japan; and Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, 2–1–6 Sengen, Tsukuba 305–0047, Japan (K.H., S.M., N.K.N.)
Nicotianamine synthase (NAS) is an enzyme that is critical for the biosynthesis of the mugineic acid family of phytosiderophores in graminaceous plants, and for the homeostasis of metal ions in nongraminaceous plants. We isolated one genomic NAS clone, ZmNAS3, and two cDNA NAS clones, ZmNAS1 and ZmNAS2, from maize (Zea mays cv Alice). In agreement with the increased secretion of phytosiderophores with Fe deficiency, ZmNAS1 and ZmNAS2 were positively expressed only in Fe-deficient roots. In contrast, ZmNAS3 was expressed under Fe-sufficient conditions, and was negatively regulated by Fe deficiency. This is the first report describing down-regulation of NAS gene expression in response to Fe deficiency in plants, shedding light on the role of nicotianamine in graminaceous plants, other than as a precursor in phytosiderophore production. ZmNAS1-green fluorescent protein (sGFP) and ZmNAS2-sGFP were localized at spots in the cytoplasm of onion (Allium cepa) epidermal cells, whereas ZmNAS3-sGFP was distributed throughout the cytoplasm of these cells. ZmNAS1 and ZmNAS3 showed NAS activity in vitro, whereas ZmNAS2 showed none. Due to its duplicated structure, ZmNAS2 was much larger (65.8 kD) than ZmNAS1, ZmNAS3, and previously characterized NAS proteins (30–38 kD) from other plant species. We reveal that maize has two types of NAS proteins based on their expression pattern and subcellular localization.
To acquire Fe, graminaceous plants secrete Fe chelators, known as mugineic-acid family phytosiderophores (MAs). MAs dissolve Fe in the rhizosphere, followed by reabsorption of the Fe(III)-MA complexes through YS1 transporters in the plasma membrane (Takagi, 1976  ;
Curie et al., 2001). Only
graminaceous plants use the MA mechanism of acquiring Fe(III), classified as
the Strategy II mechanism (Marschner et
al., 1986). Fe deficiency is a problem in crop production
worldwide, especially in calcareous soils, where Fe is sparingly soluble due
to the high soil pH. The ability of graminaceous plants to tolerate Fe
deficiency is thought to depend on the quantity of MAs secreted during Fe
deficiency (Takagi, 1976; Mori
et al., 1987,
1988;
Römheld, 1987;
Kawai et al., 1988;
Mihashi and Mori, 1989;
Singh et al., 1993). The
biosynthetic pathways of MAs (Fig.
1A) have been determined (Mori
and Nishizawa, 1987; Kawai et
al., 1988; Shojima et al.,
1990; Ma et al.,
1999), and almost all the genes involved have been isolated in our
laboratory (Higuchi et al.,
1999b; Takahashi et al.,
1999; Nakanishi et al.,
2000; Kobayashi et al.,
2001). NAS is a key enzyme in MA biosynthesis, catalyzing the
trimerization of SAM into one molecule of NA
(Higuchi et al., 1999b). NAS
activity in graminaceous plants is well correlated with tolerance to Fe
deficiency. In maize (Zea mays), a plant susceptible to Fe
deficiency, NAS activity is very low
(Higuchi et al., 1996a), and
maize secretes lower amounts of MAs than barley or oat (Avena
sativa), cereals that are tolerant of low Fe supply
(Römheld, 1987;
Lytle and Jolley, 1991). NAS
is also important for growth in nongraminaceous plants, which do not
synthesize MAs (Higuchi et al.,
1996a). In these plants, NA has been implicated in the internal
transport of metal ions (Scholz et al.,
1992; Stephan and Scholz,
1993; Pich et al.,
1994; Stephan et al.,
1994). Since Higuchi et al.
(1999b) isolated the first
NAS gene from barley, NAS genes have been isolated from
barley again (Herbik et al.,
1999), and from tomato (Lycopersicon esculentum;
Ling et al., 1999),
Arabidopsis (Suzuki et al.,
1999), and rice (Oryza sativa;
Higuchi et al., 2001). In this
study, we focus on maize, an economically important cereal that is susceptible
to Fe deficiency. We isolated and characterized three NAS genes in
maize (ZmNAS), and demonstrated that there are two types of
NAS genes with different expression patterns; ZmNAS1 and
ZmNAS2 are expressed only in roots under Fe-deficient conditions, and
ZmNAS3 is expressed under Fe-sufficient conditions. We discuss the
role of NA in Fe-deficient roots and in Fe-sufficient leaves.
Isolation of a Genomic Clone Containing a Full-Length NAS-Like Sequence
We screened a maize genomic library (Clontech, Palo Alto, CA) for
NAS clones using the rice NAS gene (OsNAS1), and
found one genomic clone with an NAS-like sequence. This clone, named
ZmNAS3 (accession no. AB042551), contains a full-length
NAS-like sequence with a putative open reading frame of 1,080 bp,
with a calculated molecular mass for ZmNAS3 of 38.8 kD and a pI value of 5.43.
Like the NAS genomic clones from barley and rice
(Higuchi et al., 2001
We prepared a cDNA library from Fe-deficient maize roots and screened this library for NAS clones using ZmNAS3 as a probe. DNA sequence analysis of the isolated cDNA clones revealed that two had NAS-like sequences. The NAS-like insert of one clone, ZmNAS1, was 1,250 bp long; the other insert, ZmNAS2, was much longer (2,154 bp) than previously characterized NAS genes from other plant species.
The ZmNAS1 and ZmNAS2 cDNAs have open reading frames encoding 327 and 601 amino acids, respectively. We calculated molecular mass and pI values of 35.6 kD and 6.11 for ZmNAS1, and 64.1 kD and 6.08 for ZmNAS2. Deduced amino acid sequences for ZmNAS1 and ZmNAS2 are shown with those of other plant NASs in Figure 2. Both proteins are highly homologous to other NASs. ZmNAS1 has nearly the same molecular mass as the others, whereas ZmNAS2 is approximately twice as large. Interestingly, the ZmNAS2 sequence partially duplicates that of ZmNAS1. The first and second NAS domains, comprising amino acids 1 to 281 and 282 to 601, are very similar to ZmNAS1 (with 93% and 85% amino acid identity, respectively; Fig. 3).
We confirmed the existence of longer NAS transcripts by northern-blot analysis. The ZmNAS2 probe detected transcripts of approximately 1.3 and 2.0 kb (data not shown), corresponding to ZmNAS1 and ZmNAS2, respectively, in Fe-deficient roots, but not in Fe-deficient or Fe-sufficient leaves. The amount of each transcript in Fe-deficient roots increased gradually until d 5, plateaued until d 9 with ZmNAS1 and ZmNAS2, and decreased 5 d after Fe was resupplied. Transcripts of both genes were present in low concentrations in Fe-sufficient roots (Fig. 4A). In contrast, ZmNAS3 was expressed in Fe-sufficient roots and leaves. Its expression was decreased by Fe deficiency and was induced by Fe resupply. The results of quantitative reverse transcription-PCR using specific primers for ZmNAS3 were consistent with the results of northern-blot analysis using specific probes for ZmNAS3 (data not shown). A supply of excess Fe suppressed the expression of ZmNAS1 and ZmNAS2, whereas the expression of ZmNAS3 was not changed by the Fe excess treatment (Fig. 4B).
At least four protein spots were detected in Fe-deficient maize roots by two-dimensional-PAGE western-blot analysis (Fig. 5). We identified two spots corresponding to proteins derived from ZmNAS1 and ZmNAS2 from their calculated molecular mass and pI values (35.6 kD and 6.11 for ZmNAS1, and 64.1 kD and 6.08 for ZmNAS2). However, no spot corresponding to ZmNAS3 (38.8 kD and a pI value of 5.43) was detected in Fe-deficient roots. We designated the other spots ZmNAS4 and ZmNAS5. ZmNAS4 corresponds to the protein derived from an expressed sequence tag clone (GenBank accession no. Q619504) judging from its predicted molecular mass and pI value. Spots corresponding to ZmNAS1 and ZmNAS2 were larger than those of ZmNAS4 and ZmNAS5; therefore, we concluded that ZmNAS1 and ZmNAS2 are the most abundant known NAS isoforms in Fe-deficient maize roots.
To confirm the enzymatic function of their gene products, ZmNAS genes were fused to the maltose-binding protein (MBP) gene and the resulting fusion proteins were produced in Escherichia coli. MBP-NASs were purified using amylose resin affinity columns, and 1 µg of each fusion protein was used for each enzyme assay. MBP-ZmNAS1 and MBP-ZmNAS3 displayed NAS activity (Fig. 6). In contrast, MBP-ZmNAS2 showed no NAS activity, even though ZmNAS2 has two NAS-like domains.
The ZmNAS protein fused to the N terminus of sGFP was transiently expressed under the control of the cauliflower mosaic virus (CaMV) 35S promoter in onion epidermal cells. The green fluorescence of sGFP was viewed with a confocal microscopy. When sGFP alone was expressed (Fig. 7A), the protein was localized in the cytoplasm and nucleus. The fluorescence of ZmNAS1-sGFP and ZmNAS2-sGFP was observed as spots in the cytoplasm of onion epidermal cells (Fig. 7, B and C). On the other hand, the fluorescence of ZmNAS3-sGFP was distributed throughout the cytoplasm of onion epidermal cells (Fig. 7D).
Two Different Expression Patterns with Different Localization The ZmNAS3 gene was isolated by screening a genomic library using a heterologous probe. Subsequently, using ZmNAS3 as a probe, two genes that encode NAS-like proteins, ZmNAS1 and ZmNAS2, were isolated from a cDNA library prepared from Fe-deficient maize roots. The molecular masses of ZmNAS1 and ZmNAS3 were similar to previously characterized NAS isoforms (Fig. 2); however, ZmNAS2 had a higher molecular mass and contained two NAS-like sequence domains (Fig. 3). ZmNAS1 and ZmNAS3 showed NAS activity in vitro, but ZmNAS2 did not (Fig. 6).
Although ZmNAS2 did not exhibit NAS activity, the expression of
ZmNAS1 and ZmNAS2 was coordinated, with both genes being
up-regulated by Fe deficiency and suppressed by a resupply of Fe
(Fig. 4). Transcripts of both
genes were detected in Fe-deficient roots, but not in Fe-deficient leaves, not
even in chlorotic leaves. This is similar to the case in barley, in which
there is no HvNAS gene expression in leaves
(Higuchi et al., 2001
The localization of ZmNAS1-sGFP fusion protein and ZmNAS2-sGFP fusion
protein was different from that of ZmNAS3-sGFP fusion protein. ZmNAS1-sGFP and
ZmNAS2-sGFP were localized as small spots in the cytoplasm, whereas
ZmNAS3-sGFP was distributed throughout the cytoplasm. PSORT
(http://psort.nibb.ac.jp/)
predictions were determined using full-length predicted protein sequences. All
of the ZmNAS proteins were predicted to be localized at the membrane of the
endoplasmic reticulum. Therefore, the pattern of ZmNAS1-sGFP and ZmNAS2-sGFP
localization is interpreted as vesicles derived from the endoplasmic
reticulum. The prediction of the ZmNAS3 localization did not match the result
from the ZmNAS3-sGFP fusion, which indicated cytosolic localization. We have
previously shown that "particular vesicles" in the Fe-deficient
barley root cell is related to the secretion of MAs
(Nishizawa and Mori, 1987
NA is essential not only for Strategy II plants, but also for Strategy I
plants (nongraminaceous plants). The chloronerva mutant of tomato
(Lycopersicon esculentum), which lacks NAS activity
(Higuchi et al., 1996b
NA is also thought to play an important role in the detoxification of
excess intracellular Fe (von Wirén
et al., 1999
The unrooted phylogenic tree (Fig.
8) suggests that NASs in graminaceous plants are also classified
into two groups as are ZmNAS proteins. OsNAS3 and NASHOR2 have a longer
amino-terminal region and are closely related to ZmNAS3 in the unrooted
phylogenic tree. In fact, OsNAS3 was expressed in leaves under
Fe-sufficient conditions and was suppressed by Fe deficiency, as is
ZmNAS3 (H. Inoue, personal communication). Presumably,
NASHOR2 has similar characteristics to ZmNAS3 and
OsNAS3 and may show a similar expression pattern in barley. Seven of
nine HvNAS genes were isolated from an Fe-deficient root cDNA library
(Higuchi et al., 1999a
HvNAS1 showed NAS activity in vitro as a MBP fusion (MBP-HvNAS1;
Fig. 6). Three MBP fusions of
Arabidopsis NAS proteins (AtNAS1, AtNAS2, and AtNAS3) were also active in
vitro (Suzuki et al., 1999
As described earlier, NAS is responsible for tolerance to low Fe
availability in graminaceous plants. Many genes encoding NAS proteins have
been isolated from graminaceous plants: nine from barley
(Herbik et al., 1999 It is possible that ZmNAS2 has some biological function. It is conceivable that a proteolytic cleavage takes place, producing an active enzyme when needed. We detected an uncloned NAS isoform (ZmNAS5) in Fe-deficient maize roots, possibly arising from the cleavage of ZmNAS2. In the absence of this cleavage, it is possible that the long polypeptide manages to titrate the substrate and lead to some kind of regulation.
A recent study showed that transgenic rice containing NAAT genes
from barley is more tolerant than nontransgenic individuals to low Fe
availability in calcareous soil (Takahashi
et al., 2001
Plant and Culture Conditions Seeds of maize (Zea mays cv Alice) were germinated on paper towels soaked with distilled water in a tray covered with a sheet of aluminum foil for 5 d at 27°C. After germination, plants were transferred to a polythene net floating on standard culture solution (pH 5.5) and were placed in a growth chamber under conditions of 30°C/16 h of light and 25°C/8 h of dark. The standard culture solution consisted of 2 x 10–3 M Ca(NO3)2, 7 x 10–4 M K2SO4, 1 x 10–4 M KCl, 1 x 10–4 M KH2PO4, 5 x 10–4 M MgSO4, 1 x 10–5 M H3BO3, 5 x 10–7 M MnSO4, 5 x 10–7 M ZnSO4, 2 x 10–8 M CuSO4, 1 x 10–8 M (NH4)6Mo7O24, and 1.5 x 10–4 M Fe-EDTA. After 4 d, plants were transplanted into 20-liter plastic boxes and were cultured for 5 d in standard culture solution with adequate amounts of Fe. Plants were then kept in standard culture solution without Fe for 9 d. Finally, Fe was added to the culture solution in the form of 6.0 x 10–4 M Fe-EDTA. For preparing the Fe-excess plants, 1.0 x 10–3 M Fe-EDTA was added to the culture solution and plants were harvested after 10 d. Culture solutions were prepared with distilled water, adjusted to pH 5.5 every day using 1.0 M HCl, and renewed every 5 d. Roots and leaves were harvested at various stages, frozen in liquid nitrogen, and stored at –80°C until use.
To isolate an NAS homolog, a genomic library (Corn Genomic
Library, variety B73; CLONTECH) was screened using plaque hybridization
techniques and a probe consisting of the open reading frame of the rice
(Oryza sativa) NAS gene (OsNAS1; accession no.
AB021746). The probe was labeled with [
Total RNA was extracted from Fe-deficient maize roots using the SDS-phenol
method. A cDNA library was constructed using the cloning vector pSPORT1
(Invitrogen, Carlsbad, CA) as described previously
(Higuchi et al., 2001
Northern-blot analyses were conducted using specific probes for
ZmNAS1, ZmNAS2, and ZmNAS3 labeled with [32P-]dATP.
Specific primers for each gene were designed as follows and were used for
preparing specific probes: ZmNAS1,
5'-GAGGAGATGGCGACCACGACAG-3' and
5'-GAAGTGCATGAGAAATTCAGCA-3'; ZmNAS2,
5'-ATCGACGCCCATGCAAACAC-3' and
5'-ATCCTCTGCGCGTCCGTGCC-3'; and ZmNAS3,
5'-GCCATGGCCGTCATGGGCAA-3' and
5'-ATCTTGGCACCAGCGCCGACT-3'. Total RNA was extracted from maize
roots and leaves using the SDS-phenol method. Ten micrograms of total RNA was
separated, blotted, and hybridized with the probes at 42°C as described
previously (Higuchi et al.,
1999b
Two mice were immunized with a total of 100 µg of NAS peptides prepared from Fe-deficient barley (Hordeum vulgare) roots. For the first injection, the immunogen was emulsified in complete Freund's adjuvant. For the second and subsequent infections, incomplete Freund's adjuvant was used. After the fourth induction, whole blood was collected and the antiserum was stored at –80°C until use.
Proteins were extracted from Fe-deficient maize roots, separated using
two-dimensional PAGE, and blotted as described previously
(Higuchi et al., 1999a
To subclone ZmNAS1 into pMAL-c2 (New England Biolabs, Beverly,
MA), an annealed oligomer (5'-CCATGCGGAATTCCG-3') was inserted
into the NcoI site (including the first ATG) of ZmNAS1
cloned in pSPORT. This ZmNAS1 plasmid was then digested with
EcoRI and HindIII, and the excised fragment containing the
ZmNAS1 coding sequence was subcloned into pMAL-c2. To subclone
ZmNAS2 into pMAL-c2, an EcoRI site was introduced close to
the first ATG using PCR mutagenesis, using two primers:
5'-GAGACTCTGAATTCGCCATGGAGGCCCAGAACGTGGA-3' and
5'-GAGACTCTAAGCTTCATATGAGTTCCATGCATCAGATGGACA-3'. The amplified
fragments were digested with EcoRI and HindIII, and the
excised fragment was cloned into pBluescriptII (SK-). This clone was
designated ZmNAS2B. We exchanged the region between EcoRI
and SmaI of ZmNAS2 in pSPORT with the
EcoRI/SmaI ZmNAS2B fragment. The resulting
ZmNAS2 construct was digested with EcoRI and
HindIII, and the excised fragment containing the ZmNAS2
coding sequence was subcloned into pMAL-c2. To subclone ZmNAS3 into
pMAL-c2, an EcoRI site was introduced close to the first ATG using
PCR mutagenesis, using two primers:
5'-GAGACTCTGAATTCCATATGGCCGTCATGGGCAAGGAG-3'and
5'-GAGACTCTGGATCCCACTTAATACAATCACGGTGAC-3'. The amplified
fragments were digested with EcoRI and BamHI, and the
excised fragment was cloned into pBluescriptII (SK-). The resulting
ZmNAS3 construct was digested with EcoRI and XbaI,
and the excised fragment containing the ZmNAS3 coding sequence was
subcloned into pMAL-c2. These pMAL-c2 plasmids, containing the coding
sequences of ZmNAS, were introduced into E. coli
XL1-Blue, which was induced to produce the recombinant fusion proteins. These
proteins were purified as described previously
(Higuchi et al., 1999b
NAS enzyme activity was assayed using the cell-free system modified by
Higuchi et al. (1994
Plasmid pUC18 containing the construct, CaMV 35S promoter-sGFP (S65T)-NOS3', was kindly provided by Dr. Yasuo Niwa (University of Shizuoka, Shizuoka, Japan). The construct had SalI and NcoI sites on the 3' side of the CaMV 35S promoter. The NcoI site "CCATGG" included the initiation codon for sGFP. An annealed oligomer (5'-TCGAGGGCCC-3') was inserted into the SalI site of CaMV35S-sGFP (S65T)-NOS3' to produce ApaI site "GGGCCC." This modified plasmid was designated CaMV35S-ApaI-sGFP (S65T)-NOS3'. The open reading frame of ZmNAS1 was amplified using two primers: 5'-GAGACTCTGGGCCCATGGAGGCCCAGAACGTGGA-3' and 5'-GAGACTCTGAATTCATGAAGATGGACAGCTCTGTCGTGG-3'. The amplified fragments were digested with ApaI and EcoRI, and the excised fragment was cloned into pBluescriptII (SK-). The resulting ZmNAS1 construct was digested with ApaI and BspHI, and the excised fragment containing the ZmNAS1 coding sequence was subcloned into CaMV35S-ApaI-sGFP (S65T)-NOS3' digested with ApaI and NcoI. The open reading frame of ZmNAS2 was amplified using two primers: 5'-GAGACTCTGGGCCCATGGAGGCCCAGAACGTGG A-3' and 5'-GAGACTCTGAATTCATGAACGCCGTTGCCATCTCCTCCCT-3'. The amplified fragments were subcloned into CaMV35S-ApaI-sGFP (S65T)-NOS3' in the same way. Two annealed oligomers, 5'-TCGAACCATGGAGCAGCTGCCGTCCGG-3' and 5'-CATGCCGGACGGCAGCTGCTCCATGGT-3', which encoded five N-terminal amino acid residues of ZmNAS3, were inserted into the SalI/NcoI site of CaMV35S-sGFP(S65T)-NOS3'. The NcoI fragment excised from ZmNAS3 was inserted in to the NcoI site of the resulting plasmid.
Transformation of onion epidermal cells was carried out by the Biolistic PDS-1000/He Particle Delivery System (Bio-Rad, Hercules, CA). White onion bulbs were purchased locally and were stored in the dark until they were used. Inner epidermal layers were peeled and placed inside up on a Murashige and Skoog plate solidified with 2% (w/v) Gellan Gum. Gold particles with a diameter of 1.0 µm were coated with each plasmid DNA containing the different constructs and were prepared for bombardment according to the manufacturer's protocol. Plated onion epidermal layers were placed under the stopping screen at a distance of 8 cm and were bombarded in a vacuum of 28 inches of mercury using a helium pressure of 1,350 psi to accelerate the macrocarrier. Bombarded cells were kept in the dark at 28°C for 20 h. Bombarded onion cells were mounted on a slide glass, and fluorescence viewed with a laser-scanning confocal microscope (LSM510; Karl Zeiss, Jena, Germany) equipped with an argon laser and a GFP filter set.
We thank Dr. E. Yoshimura, Dr. P. Blamey for assistance with English expression, and an anonymous reviewer for a suggestion regarding the possible function of ZmNAS2. Received December 29, 2002; returned for revision February 25, 2003; accepted May 12, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.102.019869. * Corresponding author; e-mail annaoko{at}mail.ecc.u-tokyo.ac.jp; fax 813–5841–5337.
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ABSTRACT
RESULTS
-32P]dATP using a
Random Primer Labeling kit (version 2; TaKaRa, Kusatsu, Japan) and was
purified using a microcolumn (ProbeQuant G-50; Pharmacia, Uppsala). Of
approximately 400,000 plaques, one positive clone was plaque purified and its
insert was subcloned into the pBluescriptII (SK-) vector. This clone was
designated ZmNAS3 (accession no. AB042551). 
