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Plant Physiol. (1999) 119: 471-480
Cloning of Nicotianamine Synthase Genes, Novel Genes Involved in
the Biosynthesis of Phytosiderophores
Kyoko Higuchi,
Kazuya Suzuki,
Hiromi Nakanishi,
Hirotaka Yamaguchi,
Naoko-Kishi Nishizawa, and
Satoshi Mori*
Laboratory of Plant Molecular Physiology, Department of Applied
Biological Chemistry, The University of Tokyo, 1-1 Yayoi, Bunkyo-ku,
113-8657 Tokyo, Japan (K.H., K.S., H.N., H.Y., N.-K.N., S.M.); and Core Research for Evolutional Science and Technology, Japan Science and
Technology Corporation, 4-1-8 Honcho, Kawaguchi-shi, 332-0012,
Saitama, Japan (K.H., K.S., H.Y., S.M.)
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ABSTRACT |
Nicotianamine synthase (NAS), the key
enzyme in the biosynthetic pathway for the mugineic acid family of
phytosiderophores, catalyzes the trimerization of
S-adenosylmethionine to form one molecule of
nicotianamine. We purified NAS protein and isolated the genes
nas1, nas2, nas3,
nas4, nas5-1, nas5-2, and
nas6, which encode NAS and NAS-like proteins from
Fe-deficient barley (Hordeum vulgare L. cv Ehimehadaka
no. 1) roots. Escherichia coli expressing nas1 showed NAS activity, confirming that this gene
encodes a functional NAS. Expression of nas genes as
determined by northern-blot analysis was induced by Fe deficiency and
was root specific. The NAS genes form a multigene family in the barley
and rice genomes.
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INTRODUCTION |
Graminaceous plants that adopt the Strategy II mechanism of Fe
acquisition (Römheld, 1987 ) secrete Fe chelators, called
phytosiderophores, from their roots to solubilize sparingly soluble Fe
in the rhizosphere. The amount of phytosiderophore secreted increases
dramatically under Fe-deficiency stress. To our knowledge, the MA is
the only class of phytosiderophore so far identified in plants (Takagi, 1976). Tolerance to Fe deficiency in graminaceous plants is thought to
depend on the quantity of MAs secreted by plants under Fe-deficiency stress (Takagi et al., 1984; Römheld and Marschner, 1986;
Marschner et al., 1987; Mori et al., 1987, 1988; Kawai et al., 1988;
Mihashi and Mori, 1989; Singh et al., 1993). Of the graminaceous
plants, rice secretes the least MAs and is the species most susceptible to Fe deficiency in calcareous soils. Transgenic rice overexpressing the genes of MA biosynthesis under Fe deficiency should secrete greater
amounts of MAs and tolerate Fe deficiency.
The biosynthetic pathway for MAs has been determined (Fig. 1). SAM is
synthesized from Met by SAM synthetase. Subsequently, three molecules
of SAM are combined to form one molecule of NA by NAS. NA is then
converted to [3"-keto acid] by NAAT, and deoxymugineic acid is
synthesized by the subsequent action of a reductase. A further series
of hydroxylation steps produces the other MA members from deoxymugineic
acid (Fig. 1; Mori and Nishizawa, 1987; Shojima et al., 1989, 1990; Ma
and Nomoto, 1993). Three cDNAs encoding SAM synthetase from barley
(Hordeum vulgare L.) roots have been cloned (Takizawa et
al., 1996; accession nos. D63835, D85273, and D85238), but these genes
are not induced by Fe deficiency. Recently, NAAT was purified and two
NAAT cDNAs (naat-A and naat-B) were cloned from
Fe-deficient barley roots (Takahashi et al., 1997). naat-A
expression was shown to be specifically induced in Fe-deficient roots.
A clone encoding the putative mugineic acid synthase Ids3,
which converts deoxymugineic acid to mugineic acid and is strongly
induced by Fe deficiency, was cloned from the barley genome using
differential hybridization (Nakanishi et al., 1993; accession no.
D37796).
The synthesis of NA from SAM is similar to polyamine synthesis from
decarboxy-SAM. In contrast to spermidine synthase (Pajula et al.,
1979), however, NAS catalyzes the polymerization of three SAM molecules
with the release of adenine and the azetidine ring formation at the
same time (Fig. 2). Therefore, NAS is a novel type of enzyme.
Previously, we reported the partial purification of NAS from the roots
of Fe-deficient barley and showed that NAS activity was induced under
Fe-deficiency stress (Higuchi et al., 1994, 1995; Kanazawa et al.,
1995). Since NAS is vulnerable to degradation by proteases using the
purification method previously described, it has been difficult to
purify sufficient quantities to determine its partial amino acid
sequence. In this paper we describe an improved purification procedure
for NAS that allowed us to obtain sufficient quantities for amino acid
sequencing. Subsequently, we cloned and characterized several genes
encoding NAS or NAS-like proteins.
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| Figure 2.
NAS catalyzes trimerization of SAM and ring
formation to synthesize NA. Bold lines indicate the unit incorporated
in NA. Hatched lines indicate newly formed N---C bonds.
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MATERIALS AND METHODS |
Preparation of Plant Material
Barley (Hordeum vulgare L. cv Ehimehadaka no. 1) was
grown in hydroponic culture as previously described (Higuchi et al., 1994). Two weeks after Fe-deficiency treatment, when severe Fe chlorosis appeared on the fourth or fifth leaf, the roots were harvested, crushed in liquid N2, and stored at
 80°C until use.
Assay of NAS Activity
Enzyme solutions were equilibrated with reaction buffer (50 mM Tris, 1 mM EDTA, 3 mM DTT, 10 µM p-APMSF, and 10 µM E-64, pH 8.7) and
concentrated by ultrafiltration using Ultrafree C3LGC NMWL10000
(Millipore). The details of the method used for detecting NAS activity
were described by Higuchi et al. (1996a).
Purification of NAS Proteins
The roots were crushed into a fine powder in liquid
N2 and then homogenized in a household juicer
with 200 mL of extraction buffer (0.2 M Tris, 10 mM EDTA, 5% [v/v] glycerol, 10 mM DTT, 0.1 mM E-64, 0.1 mM p-APMSF, and 5% [w/v]
insoluble PVP, pH 8.0) per 100 g fresh weight. The homogenate was
centrifuged for 30 min at 22,500g. Ammonium sulfate was
added to the supernatant to yield a final concentration of 400 mM. After 1 h on ice, the precipitate was
removed by centrifugation for 30 min at 22,500g.
The supernatant was loaded onto a TSK gel Butyl Toyopearl 650M column
(10-mL bed volume for every 100 g fresh weight roots; Fractogel
TSK Butyl-650M, Merck, Darmstadt, Germany) that had been equilibrated
with a loading buffer (20 mM Tris, 1 mM EDTA, 3 mM DTT, 400 mM
(NH4)2SO4,
and 0.1 mM p-APMSF, pH 8.0). NAS was eluted with elution
buffer (10 mM Tris, 1 mM EDTA, 3 mM
DTT, 0.1 mM p-APMSF, 5% glycerol, and 0.05% Chaps, pH
8.0).
KCl was added to the active fraction to give a final concentration of
0.4 M, and 1 M potassium phosphate buffer (pH
8.0) was added to a final concentration of 1 mM. The NAS
fraction was loaded onto a hydroxyapatite column (100 to
approximately 350 mesh; Nacalai Tesque, Kyoto, Japan; 10-mL bed
volume for each 100 mg of protein in the NAS fraction) equilibrated
with loading buffer (1 mM potassium phosphate buffer, 10 mM KCl, 3 mM DTT, and 0.1 mM
p-APMSF, pH 8.0). NAS activity was eluted in the void volume.
The resulting NAS fraction was then loaded onto another TSK gel Butyl
Toyopearl 650M column (1-mL bed volume for each 10 mg of
protein in the NAS fraction) and eluted in the manner described above.
The active fraction was loaded onto a DEAE-Sepharose fast-flow column
(5-mL bed volume for each 25 mg of protein in the NAS fraction,
Pharmacia) equilibrated with the loading buffer (20 mM
Tris, 1 mM EDTA, 3 mM DTT, and 0.1 mM p-APMSF, pH 8.0). The column was eluted using a stepwise
gradient with increasing KCl concentration in the elution buffer (20 mM Tris, 1 mM EDTA, 3 mM DTT, 0.1 mM p-APMSF, and 0.05% Chaps, pH 8.0, with 50, 100, 150, or
200 mM KCl). The fraction with NAS activity was eluted with
the buffer that contained 150 mM KCl.
The active fraction was loaded onto an Ether Toyopearl 650M
column (0.5-mL bed volume; Fractogel TSK Butyl-650M, Merck)
equilibrated with loading buffer (20 mM Tris, 1 mM EDTA, 3 mM DTT, 0.1 mM p-APMSF, and 1.2 M
[NH4]2SO4,
pH 8.0). NAS did not bind to the column and was eluted in the void
volume. This unbound fraction was subsequently loaded onto a Butyl
Toyopearl 650M column (bed volume, 0.3 mL) equilibrated
with loading buffer (20 mM Tris, 1 mM EDTA, 3 mM DTT, 0.1 mM p-APMSF, and 1.2 M
[NH4]2SO4,
pH 8.0). The column was washed with buffer (20 mM Tris, 1 mM EDTA, 3 mM DTT, 0.4 M [NH4]2SO4,
and 0.1 mM p-APMSF, pH 8.0) and the NAS activity was then
eluted with the elution buffer (10 mM Tris, 1 mM EDTA, 3 mM DTT, 0.1 mM p-APMSF,
5% glycerol, and 0.05% Chaps, pH 8.0).
The proteins in the active fraction were separated by SDS-PAGE at 4°C
using 11% acrylamide slab gels. After SDS-PAGE the gel was stained
with 0.3 M CuCl2 (Dzandu et al.,
1988), and then the stained bands were cut out. The gel fragments were
destained with 0.25 M EDTA/0.25 M Tris (pH 9.0)
and then homogenized with 1% SDS, 25 mM Tris, and 192 mM Gly. Each homogenate was placed in an elution cup and
polypeptides were electroeluted with SDS-free buffer containing 25 mM Tris and 192 mM Gly. The NAS fractions from
all the steps described above were stored at 4°C with 10 µM E-64.
Gel Filtration
The active NAS fraction after hydroxyapatite chromatography was
applied to a Sephacryl S300HR column (1.5 × 71 cm, 125 mL; Pharmacia) equilibrated with buffer (50 mM Tris, 1 mM EDTA, 100 mM KCl, 0.05% Chaps, 0.1 mM p-APMSF, and 3 mM DTT, pH 8.0). The column
was calibrated with standard proteins: thyroglobulin
(Mr 670,000),  -globulin
(Mr 158,000), ovalbumin
(Mr 44,000), and myoglobin
(Mr 17,000). Linear flow was 10 cm
h 1.
Determination of Partial Amino Acid Sequence
Proteins were digested with CNBr using the method of Gross (1967)
with the following modifications. NAS peptide isolated on SDS-PAGE gels
was eluted from the gel by homogenizing in a 10-fold volume of 70%
(v/v) formic acid containing 1% (w/v) CNBr in a 1.5-mL tube and by
subsequent overnight incubation at 4°C. The supernatant was
collected, dried under a partial vacuum, resuspended in the SDS-PAGE
sample buffer, and incubated overnight at room temperature. After the
proteins were digested, the small peptides were separated by
electrophoresis using Tricine SDS-PAGE (Schägger and Jagow, 1987)
in 16.5% (w/v) acrylamide gels. The peptides were transferred onto a
PVDF membrane by electroblotting (Towbin et al., 1979) and stained with
amido black. Each band on the PVDF membrane was cut out and the amino
acid sequence was determined by automated Edman degradation in a
gas-phase sequencer (model 492A protein sequencer, model 785A
programmable absorbance detector, and model 140C microgradient system,
Applied Biosystems).
Cloning of nas Genes
A pYH23 cDNA library prepared from the poly(A+) RNA
of Fe-deficient barley roots was screened with a PCR product
corresponding to the sequence of the NAS homolog of rice, expressed
sequence tag cDNA clone RICR2562A (D24790), and RICR0168A (D23792). The
primers used
were:5 -ATGGAGGCTCAGAACCAAGAGGTCGC-3(N-terminal forward primer) and 5-GGATGAGCTCCTCCCGCATCGCCT-3
(N-terminal reverse primer) from D24790, and
5-CAACCTGAGCAAGCTGGAGTACGACC-3 (internal forward primer)
and 5-TTCTTCTCGGCCGCCATGCCCACGA-3 (internal reverse
primer) from D23792 (Fig. 5). The probe obtained by PCR amplification
was labeled with [ -32P]dATP using the random
primer-labeling kit version 2 (TaKaRa, Shiga, Japan). The labeled DNA
was purified in a ProbeQuant G-50 microcolumn (Pharmacia). The isolated
cDNA clones were sequenced by the Thermo Sequenase Cycle Sequencing Kit
(Shimadzu, Tokyo, Japan), following the protocol of the manufacturer,
using a DNA sequencer (model DSQ-1000L, Shimadzu).
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| Figure 5.
Elution pattern of NAS activity from the
gel-filtration column. The black circles indicate NAS activity. The
solid line indicates protein concentration monitored at 280 nm. The
active NAS fraction after hydroxyapatite chromatography was applied to
a Sephacryl S300HR column (1.5 × 71 cm, 125 mL). The column was
calibrated with standard proteins: thyroglobulin
(Mr 670,000), -globulin
(Mr 158,000), ovalbumin
(Mr 44,000), and myoglobin
(Mr 17,000). The linear flow was 10 cm
h1.
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Expression in Escherichia coli
An EcoRI site was introduced near the first ATG
of the nas1 cDNA, and a PstI site was introduced
near the first stop codon of the nas1 cDNA by PCR
mutagenesis. The primers used were
5-GAGAGAGAGAATTCGCCATGGATGCCCAGAACAAGGAG-3 and
5-GAGAGAGAGGATCCCTGCAGCTTCAATCAAAAGGCCAGCTC-3
(EcoRI and PstI sites are underlined). An
EcoRI-PstI fragment containing the
nas1-coding sequence was excised from the PCR product
and cloned into pMAL-c2 (New England Biolabs) to give pMAL-NAS1, which was introduced into E. coli XL1-Blue, and the recombinant
bacteria were cultured in Luria-Bertani medium containing 100 µg mL1 ampicilllin and 20 µg
mL1 tetracyclin at 37°C until the
A600 of the culture reached 0.5. At this
time isopropyl  -D-thiogalactopyranoside was
added to a final concentration of 0.3 mM. After
4 h a crude extract from the cells was prepared as described
by the manufacturer of the pMAL kit.
Northern-Blot Analysis
The hybridization probe used for northern blots was a
HindIII-NotI restriction fragment containing the
full-length nas1 cDNA labeled with
[-32P]dATP. The labeled DNA was purified in
a ProbeQuant G-50 microcolumn (Pharmacia). Total RNA was isolated from
the roots or leaves of barley according to the procedure of Naito et
al. (1988). RNA (5 µg per lane) was separated on 1.4% (w/v) agarose
gels containing 5% (v/v) formaldehyde and blotted onto membranes
(Hybond-N+, Amersham). The membrane was hybridized with the
labeled probe in 0.5 M Church phosphate buffer
(Church and Gilbert, 1984), 1 mM EDTA, and 7%
(w/v) SDS with 100 µg mL1 salmon sperm DNA at
65°C overnight. After hybridization the blot was washed twice with 40 mM Church phosphate buffer and 1% (w/v) SDS at
65°C for 10 min and at high stringency with 2× SSPE and 0.1% (w/v)
SDS at 65°C for 10 min, and radioactivity was then detected using a
BAS-2000 image analyzer (Fuji, Tokyo, Japan).
Southern-Blot Analysis
Genomic DNA prepared from leaves of barley and rice was digested
with BamHI, EcoRI, or HindIII,
separated on a 0.8% (w/v) agarose gel (10 µg per lane), and alkali
transferred onto a Hybond-N+ membrane. The
membrane was hybridized with the same probes under the same conditions
as described above.
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RESULTS |
Improvement of the Extraction and Column Chromatography Procedure
Used to Purify NAS from Fe-Deficient Barley Roots
We previously reported the partial purification of NAS (Higuchi et
al., 1994), but we were unable to obtain sufficient amounts of protein
to determine its partial amino acid sequence. Subsequently, we
discovered that E-64, a thiol protease inhibitor, was very effective in
protecting NAS from degradation (Higuchi et al., 1996a). In this study
frozen roots were crushed to a fine powder in liquid
N2 and then rapidly homogenized with buffer
containing 0.1 mM E-64 to avoid degradation of NAS. This
improvement increased the recovery of NAS about 20-fold.
In the previous work NAS activity was detected in the 30- to 35-kD
protein fraction recovered from the SDS-polyacrylamide gel after the
removal of SDS (Higuchi et al., 1994), but the rate of recovery was
very low. Therefore, we further improved the column chromatography
procedures. NAS is relatively hydrophobic and a buffer containing Chaps
effectively increased the rate of recovery and the resolution of the
column chromatography. Several ion-exchange chromatography media were
tested, and DEAE-Sepharose fast-flow and DEAE Sephacel were found to
be the most effective. Both Butyl Toyopearl and another
hydrophobic chromatography medium, Ether Toyopearl,
effectively removed impurities from the 30- to 35-kD fraction.
Comparison of the Peptides on SDS-PAGE in the Purified NAS Fraction
from Fe-Deficient Barley Roots and Control Barley Roots
In general, NAS activity was detected as a broad peak on the
SDS-polyacrylamide gel from 30 to 35 kD. To identify the NAS protein,
we compared the peptides in the NAS fraction obtained from Fe-deficient
barley roots with those from Fe-sufficient (control) barley roots. NAS
was purified from 200 g of Fe-deficient and control roots. The NAS
activity of the control roots was about one-quarter of the Fe-deficient
roots at each purification step. The active NAS fraction from each
purification step was analyzed by SDS-PAGE (Fig.
3). An almost identical pattern was
observed in both Fe-deficient and control roots before the
DEAE-Sepharose step (Fig. 3A). After the DEAE-Sepharose step it became
clear that the 30- and 31-kD peptides were induced by Fe deficiency (Fig. 3B). After the Ether Toyopearl step, the 31-kD peptide was eliminated from the active NAS fraction. Two more
Fe-deficiency-inducible peptides that were 32 and 33 kD in size were
also detected in the active NAS fraction (Fig. 3C). Both the 32- and
33-kD peptides had NAS activity, but the 30-kD peptide was inactive
(Fig. 4).
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| Figure 3.
Comparison of NAS purification from Fe-deficient
() and Fe-sufficient (control, +) barley roots. SDS-PAGE was carried
out using 12.5% acrylamide slab gels (Laemmli, 1970). Gels were
stained with Coomassie brilliant blue. A, top, From Fe-deficient barley
roots; bottom, from control barley roots. Lanes 1, Crude extract, 200 µg of protein. Lanes 2, After Butyl Toyopearl 650M, 100 µg of protein. Lanes 3, After hydroxyapatite, 20 µg of protein.
Lanes 4, After Butyl Toyopearl 650M, 15 µg of protein. B,
After DEAE-Sepharose fast flow, 25 µg of protein. C, After Ether
Toyopearl 650M, one-quarter of each fraction.
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| Figure 4.
Preparative SDS-PAGE was carried out using 11%
acrylamide slab gels. A portion of the gel in this figure was stained
with Coomassie brilliant blue and the rest of the gel was stained with
Cu. The gel containing proteins between 30 and 35 kD in size was cut
into seven fragments (indicated by the short lines). The recovery was
low but NAS activity was reproducibly detected by elimination of SDS
and Cu during electroelution of peptide from the gels. The thick bars
indicate relative NAS activity of peptides from each gel fragment. The
activity of the most active fraction was set at 100%.
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Estimation of the Mr of NAS Using Gel
Filtration
According to previous data, the Mr of
NAS is between 40,000 and 50,000 (Higuchi et al., 1994), but this did
not correspond with the value estimated by SDS-PAGE. In this study the
buffer containing Chaps effectively increased the recovery of active enzyme and the resolution of the peak pattern of the column.
Consequently, the Mr of NAS was more
accurately determined to be 35,000 (Fig. 5) and corresponds well to the value
estimated by SDS-PAGE.
Determination of the Partial Amino Acid Sequence of NAS
The active NAS fraction was purified from 1 kg of Fe-deficient
barley roots. Because 32- and 33-kD peptides from the preparative SDS-PAGE gel could not be completely separated from each other, they
were digested together with CNBr, whereas the 30-kD peptide was
digested separately. The partial amino acid sequences of the fragments
obtained from the 32- plus 33-kD peptides and the 30-kD peptide were
homologous to each other (Fig. 6). The
Mr of the 33- plus 32-kD-1
fragment (Fig. 6) was almost the same as the
original; thus, we speculated that this sequence corresponded to the
N-terminal region of NAS. A search of the database revealed that these
putative NAS amino acid sequences were similar to the translation
products of rice expressed sequence tag cDNA clones of unknown function (accession nos. D23792 and D24790), with 80.0% identity in a 33-amino
acid overlap in the former and 68.4% identity in a 19-amino acid
overlap in the latter (Fig. 6).
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| Figure 6.
Partial amino acid sequence of combined 32- plus
33-kD peptides and that of the 30-kD peptide. The amino acid sequences
of two rice homologs are also shown. The arrows indicate PCR primers
(for the DNA sequences of primers, see ``Materials and Methods'').
IF, Internal forward primer; IR, internal reverse primer; NF,
N-terminal forward primer; NR, N-terminal reverse primer.
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Cloning and Nucleotide Sequences of cDNA Clones Encoding NAS
PCR amplification of total cDNA prepared from Fe-deficient barley
roots using degenerate primers designed from the partial amino acid
sequence of barley NAS was not successful. We then used partial
sequences from the rice expressed sequence tag clones (Fig. 6, arrows)
as the primers. The resulting 205-bp amplification product using
N-terminal forward and N-terminal reverse primers and a 274-bp
amplification product using internal forward and internal reverse
primers were used as probes for hybridization. A cDNA library prepared
using poly(A+) RNA from Fe-deficient barley roots was screened
and 19 positive clones using the 205-bp probe and 88 positive clones
using the 274-bp probe were obtained.
Nucleotide sequence analysis of one of these positive clones,
designated nas1, revealed an open reading frame of 985 bp
encoding a 328-amino acid polypeptide with a predicted
Mr of 35,144. This corresponded well with
the size of the NAS polypeptide estimated by SDS-PAGE. The partial
amino acid sequences of the 32- plus 33-kD peptide matched the portions
of the deduced amino acid sequence of nas1 (Fig.
7, underlined). The predicted pI of 5.2 matched the value estimated by native IEF electrophoresis well (data
not shown). Another six NAS-like clones, nas2,
nas3, nas4, nas5-1, nas5-2,
and nas6, were also obtained (Table
I; Fig. 8).
The nucleotide sequences for the NAS genes from barley have been
deposited in the database and given the following accession numbers:
AB010086 (nas1), AB011265 (nas2), AB011264
(nas3), AB011266 (nas4), AB011267
(nas5-1), AB011268 (nas5-2), and AB011269
(nas6). The partial amino acid sequences of the 30-kD
peptide matched portions of the deduced amino acid sequence of
nas5-1 and nas5-2 (Fig. 8, underlined). The 5
and 3 noncoding region of these seven clones differed from each other,
except nas5-1 and nas5-2. Sequences of
nas5-1 and nas 5-2 were identical, with the
exception of the 122 to 140 amino acid residues of nas5-2.
nas5-1 may be a deletion clone of nas5-2, although we
did not determine whether this deletion clone is an artifact.
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| Figure 7.
Nucleotide and deduced amino acid sequence of the
barley nas1 cDNA clone. The underlined sequences
indicate four partial amino acid sequences of fragments from combined
32- plus 33-kD peptides. The nucleotide sequence is numbered from the
5 end of the cDNA clone and indicated to the right of each row. Amino
acid numbers are indicated on the left of each row.
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Table I.
Properties of deduced peptides from nas clones
pI values were predicted by the DNASIS program (Hitachi, Tokyo, Japan).
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| Figure 8.
Comparison of the deduced amino acid sequences of
barley proteins NAS 1 to NAS 6. Asterisks indicate identical
amino acid residues in all sequences. Asterisks in parentheses indicate
identical amino acid residues in all sequences except NAS 5-1 and 5-2. The underlined sequences indicate the partial amino acid sequences of
fragments from 30-kD peptide.
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Expression of NAS1 in E. coli
To confirm the enzymatic function of the gene product,
nas1 was expressed as a maltose-binding protein fusion in
E. coli. The bacterial strains containing the NAS1
expression vector or the empty vector were induced with isopropyl
-D-thiogalactopyranoside, and the crude
extracts were analyzed for NAS activity by TLC (Fig. 9). The crude extract from the strain
transformed with pMAL-NAS1 had NAS activity, whereas the crude extract
from the strain transformed with the vector had no NAS activity.
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| Figure 9.
TLC analysis of NAS activity assay mixture from
E. coli expressing NAS1. Lane 1, Standard NA; lane 2, pMAL only; lane 3, pMAL-NAS1.
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Northern-Blot Analysis
Northern hybridization analysis revealed that nas1-mRNA
was not detected in either the leaves or roots of the control plants. In contrast, nas1 was highly expressed in the roots but not
in the leaves of Fe-deficient plants (Fig.
10). This result corresponds well with
the previously described expression pattern of NAS activity (Higuchi et
al., 1994).
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| Figure 10.
Northern-blot analysis of nas1.
RNA was extracted from Fe-deficient () and control (+) roots and
leaves. Each lane was loaded with 5 µg of RNA. Total RNA was
extracted after 1 week of Fe-deficiency treatment.
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Southern-Blot Analysis
The number of nas and nas-like genes in the
barley and rice genomes was assessed by Southern-blot analysis (Fig.
11). With the barley genome DNA,
cutting with BamHI and EcoRI each produced 7 bands, which were detected on Southern blots, whereas cutting with
HindIII produced 10 bands. None of the seven clones obtained in this work have BamHI or EcoRI sites, and only
nas2 and nas4 have HindIII sites. This
result corresponds well with the existence of at least seven
nas cDNA clones.
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| Figure 11.
Southern-blot analysis of nas-like
genes. Genomic DNA from barley and rice were digested with
BamHI (lanes B), EcoRI (lanes E), and
HindIII (lanes H) and probed with nas1
under high-stringency conditions.
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DISCUSSION |
In this paper we describe the partial purification of several NAS
proteins induced by Fe deficiency (Fig. 4) and their corresponding genes (Fig. 8). The enzymatic function of the nas1 product
was confirmed (Fig. 9). In addition, a 30-kD peptide and its gene, nas5-1 or nas5-2, which are homologous to NAS but
lack NAS activity, were also found (Fig. 4). Several NAS proteins and
their corresponding genes were found, but it remains unclear whether
each NAS protein functions differently. It is possible that a
heteropolymer composed of these homologous 30-, 32-, and 33-kD peptides
forms the native enzyme. However, the Mr of
NAS estimated by gel filtration was 35,000 (Fig. 5), and we did not
observe increased NAS activity by combining the 30-, 32-, and 33-kD
peptides in vitro (data not shown). Therefore, we tentatively conclude
that native NAS exists as a monomer.
Another possibility is that native NAS exists as a complex of 30-, 32-, and 33-kD peptides, because NAS proteins are relatively hydrophobic,
indicating that they may be membrane bound and localized in some
organelle (Fig. 12). This may allow the
efficient catalysis of trimerization of SAM. We have already shown that
radioactivity from [1-14C]Met, the precursor of
MAs, is localized in "particular vesicles" in the Fe-deficient
barley root cell (Nishizawa and Mori, 1987; Nishizawa et al., 1990).
This suggests that biosynthesis of MAs occurs inside particular
vesicles.
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| Figure 12.
Hydropathy profile of NAS1. Hydrophobicity was
analyzed by the DNASIS program, as described by Kyte and Doolittle
(1982), with a window size of 10 amino acid residues. Hydrophilic
domains are below the zero line. Bold lines indicate hydrophobic
regions.
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The catalytic mechanism of NA synthesis from SAM by NAS may be similar
to that of methyl transferase using SAM as a methyl donor or those of
spermidine synthase and spermine synthase using decarboxy-SAM as a
substrate (Pajula et al., 1979). The common catalytic domain of these
enzymes has been discussed in relation to amino acids occupying similar
positions in the secondary and tertiary structures (Schluckebier et
al., 1995; Hashimoto et al., 1998). If the 30-kD peptide lacking NAS
activity has SAM-binding activity, comparison of NAS protein and the
inactive NAS homolog may reveal the catalytic or the binding domain
structure of NAS. Because NA is contained in all plants surveyed so
far, NAS may be widespread among plant species. The translation
products of the putative rice NAS homologs, D23792 and D24790, were very similar to NAS1 protein, with 80.2% identity in a 111-amino acid
overlap in the former and 78.9% identity in a 71-amino acid overlap in
the latter. Putative Arabidopsis NAS homologs, AC003114 (function
unknown) and AB005245 (function unknown), were also found with 45%
identity in the former and 46% identity in the latter to NAS1.
Therefore, comparison of NAS protein homology among many species may
also reveal information about its catalytic domain.
Since full-length nas1 used as a probe could also detect
other nas genes, a 1.3-kb band smear was seen on northern
blots (Fig. 10), and many bands were detected on Southern blots (Fig.
11). Therefore, we conclude that nas belongs to a multigene
family in the barley genome and probably also in the rice genome. The
number of copies should be confirmed by Southern-blot analysis using
conserved sequences of nas as the probes.
On northern blots a smeared band produced the strongest signal, but
weak and short bands were also detected. These may represent degradation products of nas1 mRNA (Fig. 10). Since NAS
proteins are vulnerable to degradation, it is quite possible that the
expression of the nas gene in barley roots is strictly
controlled to avoid overproduction of MAs. For instance, resupplying Fe
to Fe-deficient barley decreased NAS activity within 24 h (Higuchi
et al., 1996a).
We could tentatively categorize the seven nas clones into
several types based on homology of amino acid sequences: type 1, nas1; type 2, nas2, 3, 4,
and 6; and type 3, nas5 (Table I). Nineteen
independent nas1 clones were obtained from a cDNA library prepared using poly(A+) RNA from Fe-deficient barley roots,
but only one nas4 clone was obtained. The numbers of clones
of nas2, nas3, nas5-1,
nas5-2, and nas6 were two, four, five, three, and
three, respectively. These results suggest that the expression of
nas genes under Fe deficiency differs from one
nas gene family to the next. Northern-blot analysis using a
specific probe for each nas gene will reveal the precise
expression pattern of each gene and suggest the function of each
nas gene under different stress conditions.
Until now, NA, the reaction product of NAS, has been thought to
function only as an intermediate substrate for MAs synthesis in
graminaceous plants (Fig. 1). Since the nas gene belongs to a multigene family, NA has a function in the survival of graminaceous plants other than as a precursor of MAs. In Strategy I plants, which
lack the ability to produce MAs, it has been proposed that NA plays a
key role as an endogenous chelator of divalent metal cations, such as
Fe2+, Cu2+,
Zn2+, and Mn2+, in the
xylem and phloem and that it contributes to the homeostasis of those
metals in plants (Stephan et al., 1994; Higuchi et al., 1996b). NA may
also play a role in the metabolism of Fe or other metals in Strategy II
plants. The fact that NAS activity is not enhanced by Fe deficiency in
Strategy I plants (Higuchi et al., 1995, 1996b) suggests that
expression of the nas gene is not regulated by Fe in these
plants. Transgenic plants with suppressed NAS expression will help
clarify the role of NA in both Strategy I and Strategy II plants.
| |
FOOTNOTES |
*
Corresponding author; e-mail
asmori{at}hongo.ecc.u-tokyo.ac.jp; fax 81-3-3812-0544.
Received June 18, 1998;
accepted October 20, 1998.
| |
ABBREVIATIONS |
Abbreviations:
Chaps, 3-[(3-cholamidopropyl)dimethyl-ammonio]propanesulfonic
acid.
CNBr, cyanogen bromide.
E-64, trans-epoxysuccinyl-leucylamido-(4-guanidino)butane.
MAs, mugineic acid
family.
NA, nicotianamine.
NAAT, nicotianamine aminotransferase.
NAS, nicotianamine synthase.
p-APMSF, (p-amidinophenyl)methanesulfonyl fluoride.
SAM, S-adenosylmethionine.
| |
ACKNOWLEDGMENTS |
We thank Dr. Naimatullah Bughio, Mr. Nobuyuki Sato, and Dr.
Michiko Takahashi for the preparation of Fe-deficient plant material. We are thankful to Dr. Emmanuel Delhaize, Commonwealth Scientific and
Industrial Research Organization, Australia, for editing.
| |
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Top
Abstract
Introduction
