Nitrite Reductase Gene Enrichment Improves
Assimilation of NO2 in Arabidopsis1
Misa Takahashi, Yukari Sasaki, Shoji Ida, and Hiromichi Morikawa*
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1–3–1
Kagamiyama, Higashi-Hiroshima, 739–8526 Japan (M.T., Y.S., H.M.); and Research Institute for Food Science,
Kyoto University, Uji, Kyoto, 611–0011 Japan (S.I.)
Transgenic plants of Arabidopsis bearing the spinach (Spinacia oleracea) nitrite reductase (NiR, EC 1.7.7.1) gene that catalyzes
the six-electron reduction of nitrite to ammonium in the second step of the nitrate assimilation pathway were produced by
use of the cauliflower mosaic virus 35S promoter and nopaline synthase terminator. Integration of the gene was confirmed
by a genomic polymerase chain reaction (PCR) and Southern-blot analysis; its expression by a reverse transcriptase-PCR and
two-dimensional polyacrylamide gel electrophoresis western-blot analysis; total (spinach ⫹ Arabidopsis) NiR mRNA
content by a competitive reverse transcriptase-PCR; localization of NiR activity (NiRA) in the chloroplast by fractionation
analysis; and NO2 assimilation by analysis of the reduced nitrogen derived from NO2 (NO2-RN). Twelve independent
transgenic plant lines were characterized in depth. Three positive correlations were found for NiR gene expression; between
the total NiR mRNA and total NiR protein contents (r ⫽ 0.74), between the total NiR protein and NiRA (r ⫽ 0.71), and
between NiRA and NO2-RN (r ⫽ 0.65). Of these twelve lines, four had significantly higher NiRA than the wild-type control
(P ⬍ 0.01), and three had significantly higher NO2-RN (P ⬍ 0.01). Each of the latter three had one to two copies of spinach
NiR cDNA per haploid genome. The NiR flux control coefficient for NO2 assimilation was estimated to be about 0.4. A
similar value was obtained for an NiR antisense tobacco (Nicotiana tabacum cv Xanthi XHFD8). The flux control coefficients
of nitrate reductase and glutamine synthetase were much smaller than this value. Together, these findings indicate that NiR
is a controlling enzyme in NO2 assimilation by plants.
NO2, a major air pollutant that causes acid rain,
reacts with volatile organic compounds in the atmosphere, producing photooxidants, including ozone. A
1980 estimate put the total natural and anthropogenic
emissions of nitrogen oxides (NOx) at 150 million
tons per year, more than one-half of which were from
natural sources (Yunus et al., 1996). Road transport,
the major anthropogenic source of NOx in most developed countries, in 1984 accounted for as much as
75% of the NOx in some metropolitan cities, and the
percentage has continued to rise with increases in
road traffic volume. In many developing countries as
well, petro-fueled motor vehicles are the principal
source of NOx (Yunus et al., 1996).
Plants take up NO2 (Hill, 1971) and assimilate its
nitrogen through a primary nitrate assimilation pathway (Zeevaart, 1976; Yoneyama and Sasakawa,
1979a; Kaji et al., 1980; Rowland et al., 1985; Wellburn, 1990; Morikawa et al., 1998; Ramge et al., 1993).
Moreover, nitrate reductase (NR, EC 1.6.6.1) and nitrite reductase (NiR, EC 1.7.7.1) activities in leaves
1
This work was supported in part by the program Research for
the Future from the Japanese Society for the Promotion of Science
(no. JSPS–RFTF96L00604) and by a Grant-in-Aid for Scientific Research on Priority Areas (no. 05266213) from the Ministry of Education, Science, Culture and Sports, Japan. We also gratefully
acknowledge financial support from the Naito Foundation and
Electric Technology Research Foundation of Chugoku.
* Corresponding author; e-mail [email protected].
ac.jp; fax 81– 824 –24 – 0749.
frequently are enhanced by exposure to NO2 (Zeevaart, 1974; Yoneyama and Sasakawa, 1979a; Wellburn et al., 1981; Murray and Wellburn, 1985; Takeuchi et al., 1985; Wingsle et al., 1987; Yu et al., 1988), as
when nitrate is supplied. Apart from the study by
Rowland-Bamford et al. (1989), which showed that
the rate of NO2 uptake in NR-deficient barley mutants was similar to that in non-mutated plants, little
is known about the role of these enzymes of the
primary nitrate assimilation pathway in NO2
assimilation.
Nitrite ions, considered toxic to plant cells
(Shimazaki et al., 1992; Vaucheret et al., 1992; Duncanson et al., 1993; Lea, 1993), are increased in leaf cells of
a variety of plant species after fumigation with NO2
(Zeevaart, 1976; Yoneyama and Sasakawa, 1979a;
Takeuchi et al., 1985; Yu et al., 1988; Shimazaki et al.,
1992; Morikawa et al., 1998), sometimes resulting in
visible leaf injury (Yoneyama et al., 1979b; Shimazaki
et al., 1992). Plants that have high NiR activity (NiRA)
are considered to have a high tolerance for NO2 (Yoneyama et al., 1979b; Shimazaki et al., 1992). We speculated that NiR is a controlling enzyme in NO2 assimilation and that overexpression of the NiR gene and an
increase in the amount of NiR enzyme produced by
genetic engineering would improve the ability of
plants to assimilate NO2.
Ferredoxin-dependent NiR, which catalyzes the
six-electron reduction of nitrite (oxidation state ⫹3)
to ammonium (oxidation state ⫺3) in the second step
of the nitrate assimilation pathway, is localized in the
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Takahashi et al.
chloroplast and its gene is nuclear encoded. NiR
cDNAs of various higher plant species have been
cloned: spinach (Back et al., 1988), maize (Lahners et
al., 1988), birch (Friemann et al., 1992), tobacco (Nicotiana tabacum cv Xanthi XHFD8; Vaucheret et al.,
1992; Kronenberger et al., 1993), Arabidopsis (Tanaka
et al., 1994), rice (Terada et al., 1995), kidney bean
(Sander et al., 1995), and beet (Schneider et al., 1999).
With an eventual aim to produce “NO2-philic”
plants that can grow with NO2 as sole nitrogen
source, we have been studying the metabolism of
NO2 in plants (Morikawa et al., 1998). As far as we
are aware, only one study by Crété et al. (1997) on
NiR overexpressors has been reported, but no reports
on the analysis of NO2 assimilation by transgenic
plants have been published. Here we produced transgenic plants of Arabidopsis bearing chimeric spinach
NiR gene, characterized the integration of the transgene in the genome and its expression in those plants
by various methods such as Southern blot, quantitative reverse transcriptase (RT)-PCR and twodimensional PAGE western-blot analyses, and analyzed NO2 assimilation in transgenic plants by
fumigation with 15N-labled NO2 followed by Kjeldahl digestion and mass spectrometry. Moreover,
results were compared with those of transformants in
which NR and Gln synthetase (GS) genes are
overexpressed.
RESULTS
Plasmid pSNIRH bearing spinach NiR cDNA controlled by the cauliflower mosaic virus (CaMV) 35S
promoter, nopaline synthase (NOS) terminator, and
the chimeric hpt expression cassette (Fig. 1) was introduced to root sections of Arabidopsis by particle
bombardment. Putative transgenic calli were selected
Figure 1. Diagram of plasmid pSNIRH. CaMV 35S, CaMV 35S
promoter; NOS ter, NOS terminator; hpt, hygromycin phosphotransferase.
732
on hygromycin-containing medium. Thirty-seven
shoots, which developed from these independent
hygromycin-resistant calli and which had a 352-bp
band in a PCR with primers specific to spinach NiR
cDNA, were allowed to mature and set seeds. The T2
to T4 plants obtained by self-pollination were randomly chosen. About 20 seedlings per each line were
first tested for segregation of the 325-band specific to
spinach NiR cDNA. Homozygous lines for the transgene as determined by the presence of the 325-bp
PCR band were called “hm” lines and heterozygous
ones that segregated the band were called “ht” ones.
Those plants bearing the introduced spinach NiR
cDNA were analyzed further.
Typical RT-PCR analysis results for expressions of
the introduced spinach NiR cDNA and endogenous
NiR gene in the 12 transgenic lines of Arabidopsis are
shown in Figure 2A. Their mRNA extracts were analyzed by the use of primers specific to spinach NiR
and Arabidopsis NiR (see “Materials and Methods”).
All the transgenic plants had the 352-bp band specific
to spinach NiR cDNA (lanes 1–12), whereas no wildtype ones had it (lane WT). The fact that both bands
specific to spinach and Arabidopsis NiR are present
in all the transgenic plants indicates that both NiR
genes are expressed in all of them. Total (spinach ⫹
Arabidopsis) NiR mRNAs were quantified by the
competitive RT-PCR (Fig. 2B; see also Table I), in
which a 1,668-bp intron-containing DNA fragment of
NiR gene was used as the competitor (see “Materials
and Methods”). In Figure 2B, PCR signals of the
competitor that showed were closest to those of the
target cDNA are shown. Quantitative results, based
on the competitive RT-PCR analysis, of the total NiR
mRNA content of each transgenic line will be discussed below.
Figure 3 shows typical results of two-dimensional
PAGE western-blot analysis with anti-spinach NiR
antibody of the protein extract from a transgenic line
(line 121, which had the highest NiRA among the 12,
see Table I), together with results for wild-type Arabidopsis and spinach. The transgenic plants gave six
spots at about the molecular mass of 60 kD and pIs
ranging from 5 to 6. In contrast, wild-type Arabidopsis gave four (acidic pIs) and spinach gave three
(basic pIs) spots that had similar molecular masses.
Three to four of the acidic pI spots of the transgenic
plants corresponded to the spots of wild-type Arabidopsis, and the remaining two to three basic pI spots
to those of spinach, evidence that the introduced
spinach NiR cDNA was successfully expressed and
translated in the transgenic plants. Arabidopsis NiR
protein has an estimated molecular mass of 63 kD
and a pI of 5.6, based on cDNA sequence data
(Tanaka et al., 1994), and spinach NiR protein had a
molecular mass of 63 kD and a pI of 5.9, based on its
NiR cDNA sequence data (Back et al., 1988) obtained
with DNASIS software (Hitachi Software Engineering Co., Yokohama, Japan). Ida and Mikami (1986)
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Plant Physiol. Vol. 126, 2001
NO2 Assimilation by Transgenic Plants
Figure 2. RT-PCR analysis of NiR mRNA from transgenic (lanes 111–10) and wild-type (lane WT) Arabidopsis plants. A,
Without competitor DNAs. Product sizes are 352 bp for the introduced spinach NiR cDNA (top), 219 bp for Arabidopsis NiR
gene (middle), and 1,563 bp for the ␤-tubulin of Arabidopsis (bottom). B, Typical results of competitive RT-PCR for
quantification of total (spinach ⫹ Arabidopsis) NiR mRNAs. Product sizes are 1,314 bp for total NiR cDNAs and 1,668 bp
for competitor. The amount of the competitor varied from 2.5 ⫻ 105 to 5 ⫻ 106 copies per the PCR reaction mixture (see
“Materials and Methods”).
previously reported the molecular mass of spinach
NiR polypeptide as 63 kD. These values all are consistent with ours found by western-blot analysis.
Based on quantification of the NiR protein spots on
the two-dimensional gels as shown in Figure 3, the
contribution of the transgene in the total NiR proteins in the line 121 was estimated to be about 50%.
The introduced spinach NiR cDNA had the 5⬘ transit peptide for chloroplastic targeting. Whether this
peptide functions in transgenic Arabidopsis cells
needed to be clarified. Protoplasts were isolated from
transgenic and wild-type Arabidopsis leaves and
were ruptured by osmotic treatment to isolate chloroplasts. Western-blot analysis with anti-lettuce GS
antibody (provided by Dr. Go Takeba, Kyoto Prefectural University) showed that the chloroplast fraction
had only chloroplastic, and no cytosolic GS—evidence that this fraction is free of cytosol contamination (data not shown). NiRA in the chloroplast fraction was determined next and the value was
compared with the total NiRA of the intact leaf.
Results are shown in Table II. Based on recovery of
the RBCS from the chloroplast fraction, chloroplast
yields (intensity of the RBCS, band of the chloroplast
fraction/intensity of RBCS, band in the intact leaf)
were estimated as 34.7% for the transgenic and 39.2%
for the wild-type plants (Table II). The NiRA in the
chloroplast fraction (NiRA in isolated chloroplasts/
chloroplast yield) was 157.2 for the transgenic and
81.9 (nmol NO2⫺ min⫺1 mg⫺1 chlorophyll) for the
wild-type plants. The former value was 97.5% of the
Plant Physiol. Vol. 126, 2001
total NiR leaf activity in the transgenic and 90.3% in
the wild-type Arabidopsis plants, a clear indication
that the chloroplast transit peptide derived from
spinach functions in the cells of Arabidopsis in the
transport of ectopically expressed spinach NiR translates to the chloroplast.
Table I shows the copy number of the introduced
spinach NiR cDNA per haploid genome of Arabidopsis, total (spinach ⫹ Arabidopsis) NiR mRNA content
as determined by the competitive RT-PCR, total NiR
protein content as determined by western-blot analysis, NiRA, and NO2-RN of the 12 transgenic lines.
Six “hm” lines (693, 116, 10, 117, 121, and 191) did not
segregate the 325-bp band specific to spinach NiR
cDNA when tested as described above. Each of six
“ht” lines (147, 571, 146, 703, 111, and 221) segregated
the band. The copy number of spinach NiR cDNA
per haploid genome varied from one to six or more.
The segregation ratio varied from 16:4 to 17:3, which
is very close to a Mendelian 3:1 ratio, suggesting that
the transgenes are located in a single locus in these
transgenic lines. In general, lines with a low copy
number had higher total NiR mRNA content. The
relative total NiR mRNA content of the transgenic
lines varied markedly from 80.6% to 967.7%, where
the average signal intensity of the competitive PCR
band corresponding to the total NiR mRNA of the
control wild-type plants being taken as 100. Lines
693, 10, 121, and 147, all of which had a low copy
number of the transgene, had high total mRNA
(more than or close to 500% of the control). On the
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733
Takahashi et al.
Table I. Copy number of spinach NiR cDNA, total NiR mRNA, total NiR protein, NiRA, and NO2-RN in transgenic Arabidopsis plants carrying spinach NiR cDNA under control of the CaMV 35S promoter and NOS terminator
hm Lines did not segregate the PCR band specific to spinach NiR cDNA, whereas ht lines segregated this band (see text for details). Values
are means ⫾ SD of more than two independent experiments. One experiment consisted of a sample prepared from a 5- to 6-week-old plant of
transgenic or wild-type Arabidopsis. Nos. in parentheses are NiRA- or NO2-derived reduced nitrogen relative to the value for wild-type plants.
Nos. in brackets are the no. of experiments. Values with asterisks (P ⬍ 0.01) are significantly different as determined by ANOVA with Dunnett’s
test. Copy no. of spinach NiR cDNA per haploid genome was determined by Southern-blot analysis of genomic DNA with spinach NiR cDNA
as the probe. Total mRNA was determined by competitive RT-PCR with primers specific to spinach NiR and Arabidopsis NiR. Total NiR protein
content was determined by western-blot analysis with anti-spinach NiR antibody. NO2-RN was determined after fumigation with 4 ␮L L⫺1 NO2
for 8 h followed by Kjeldahl digestion of the leaves, as described in “Materials and Methods.”
Line No.
Wild type
693 hm
116 hm
10 hm
117 hm
121 hm
191 hm
147 ht
571 ht
146 ht
703 ht
111 ht
221 ht
Copy
No.
1
⭌5
1
⭌4
2
⭌6
1
⭌5
⭌2
⭌3
⭌1.5
⭌4.5
Total NiR mRNA
100.0 ⫾ 75.1 [3]
967.7 ⫾ 0 [3]
145.2 ⫾ 68.4 [3]
709.7 ⫾ 365.0 [3]
145.2 ⫾ 68.4 [3]
483.9 ⫾ 0 [3]
80.6 ⫾ 22.8 [2]
806.5 ⫾ 228.1 [3]
129.0 ⫾ 45.6 [3]
80.6 ⫾ 22.8 [3]
129.0 ⫾ 45.6 [3]
80.6 ⫾ 22.8 [3]
80.6 ⫾ 22.8 [2]
Total NiR Protein
100.0 ⫾ 0 [3]
142.2 ⫾ 11.8 [3]
119.1 ⫾ 22.8 [3]
150.0 ⫾ 12.1 [3]
108.9 ⫾ 43.1 [3]
176.6 ⫾ 4.1 [3]
82.1 ⫾ 4.0 [3]
132.4 ⫾ 22.3 [3]
82.8 ⫾ 26.8 [3]
123.9 ⫾ 48.9 [3]
116.8 ⫾ 4.7 [3]
91.7 ⫾ 9.3 [3]
75.9 ⫾ 34.5 [3]
other hand, those lines having multiple copies of the
transgene such as lines 191 and 221 had low total NiR
mRNA. These results suggest that cosuppression of
NiR expression may have occurred at least in some
plants of these transgenic lines. The total NiR protein
content of the transgenic plants was also not always
greater than that of the wild-type control, where the
average signal intensity of the two-dimensional western band corresponding to the NiR protein of the
control wild-type plants being taken as 100. This
value varied in a rather narrow range (from 75.9%–
176.6% of the controls) even though the total NiR
mRNA of the transgenic plants varied by a factor of
more than 10 times. This suggests that posttranscriptional regulation is active in NiR gene expression, which is consistent with previous results
(Crété et al., 1997). A positive correlation, however,
was obtained for the total NiR mRNA and total NiR
protein contents (r ⫽ 0.75).
Another positive correlation existed between the
total NiR protein content and NiRA (r ⫽ 0.71). The
NiRA of the transgenic plants ranged from 82% to
181% of the values for the wild-type controls. A
Dunnett’s test for two samples of different sizes
showed that four of the transgenic lines (121, 10, 117,
and 693) had significantly higher NiRA than the controls (P ⬍ 0.01). Except for line 117, all had high NiR
protein contents. Based on quantification of the NiR
protein spots on the two-dimensional gels (Fig. 3),
the contribution of the transgene in the total NiR
proteins was estimated to be about 50% (see above).
It is conceivable that the transgene may make a sim734
NiRA
Reduced Nitrogen Derived
from NO2 (NO2-RN)
nmol NO2⫺ min⫺1 mg⫺1
protein
mg nitrogen g⫺1 dry wt
124.7 ⫾ 19.0 (100) [3]
192.1 ⫾ 2.2* (154) [3]
134.2 ⫾ 13.8 (108) [3]
197.0 ⫾ 2.6* (158) [2]
196.7 ⫾ 7.4* (158) [3]
226.2 ⫾ 9.9* (181) [3]
102.6 ⫾ 9.4 (82) [3]
143.0 ⫾ 9.8 (115) [5]
112.6 ⫾ 18.0 (90) [5]
121.9 ⫾ 15.1 (98) [5]
103.3 ⫾ 11.2 (83) [3]
162.4 ⫾ 26.3 (130) [5]
108.4 ⫾ 13.9 (87) [3]
1.18 ⫾ 0.08 (100) [3]
1.65 ⫾ 0.14* (140) [3]
1.68 ⫾ 0.33 (142) [2]
1.55 ⫾ 0.04* (131) [3]
1.39 ⫾ 0.22 (118) [2]
1.47 ⫾ 0.06* (125) [3]
1.20 ⫾ 0.10 (102) [3]
0.92 ⫾ 0.08 (78) [2]
0.98 ⫾ 0.11 (83) [3]
1.06 ⫾ 0.05 (90) [2]
1.18 ⫾ 0.33 (100) [2]
1.31 ⫾ 0.04 (111) [3]
1.02 ⫾ 0.15 (86) [3]
ilar contribution to the observed NiRA in line 121.
Further study is needed to address whether this estimation is applicable to other transgenic lines.
A third positive correlation was found between
NiRA and NO2-RN (r ⫽ 0.65). The NO2-RN of the
transgenic plants was 78% to 142% the values for the
controls. A similar Dunnett’s test as described above
showed that except for 117, the lines (121, 10, and
693) had higher NO2-RN values than the controls
(P ⬍ 0.01). Each of the three had one to two copies of
spinach NiR cDNA per haploid genome.
Four lines bearing low copy number of the transgene (lines 693, 10, 121, and 147) had high levels in
four parameters; total NiR mRNA content, total NiR
protein content, NiRA (except for 147), and NO2-RN
(except for 147). As described above, NiRA and
NO2-RN values of the three lines 693, 10, and 121
were statistically significant over the wild-type control. It should also be note that all of these three lines
are homozygous in the transgene, as described
above. Although the reason(s) why line 147 having a
high NiR protein content showed a low NiRA and
NO2-RN values is not clear, it is likely that homozygous transgenic plants bearing low copy number of
NiR transgene have a high level of NiR enzyme
activity and high ability to assimilate NO2. Lines 116
and 117 are somewhat unique because both appeared
to have multiple copies of the transgene, but had
increased levels of total NiR mRNA, total NiR protein, NiRA, and NO2-RN, although their NO2-RN
values were statistically not significant over the wildtype control. Line 111 is also unique because al-
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Plant Physiol. Vol. 126, 2001
NO2 Assimilation by Transgenic Plants
Figure 3. Western-blot analysis of NiR proteins
from transgenic and wild-type Arabidopsis and
spinach plants. Two-dimensional PAGE was
done with a polyclonal antibody raised against
spinach NiR.
though it is not low in the copy in the transgene or
high in the level of total NiR mRNA, it had increased
levels of NiRA and NO2-RN (but statistically not
significant). Line 146 had a high NiR protein content,
but had low NiRA and NO2-RN values. Taken together, in addition to the copy number of the transgene and homozygosity of the transgene locus, other
factors also control NiRA and thereby the ability of
plants to assimilate NO2.
The flux control coefficient, a measure of the effect
of change in a single enzyme activity on the flux
(Kacser and Porteous, 1987; Stitt and Sonnewald,
1995), of NiR for NO2 assimilation, estimated as reported elsewhere (Runquist and Kruger, 1999) from
the data for the 12 lines (Table I) was about 0.4. We
presume that the conversion of nitrite ions to ammonia is a controlling step in the NO2 assimilation
pathway.
To determine whether this is so we analyzed the
assimilation of NO2 in a transgenic tobacco line
(clone 271) in which NiR antisense mRNA is expressed, NiRA therefore being greatly reduced
(Vaucheret et al., 1992) (see “Materials and Methods”). Results are shown in Table III. The NiRA of the
transgenic tobacco leaf was 3.5% and the corresponding NO2-RN value was 62.1% that of the wild-type
control. The estimated flux control coefficient of NiR
was 0.4, a value very close to that obtained with the
NiR sense transformants as described above.
Using particle bombardment we produced transgenic Arabidopsis plants bearing chimeric tobacco
NR cDNA (Vincentz and Caboche, 1991) and plants
bearing chimeric cDNAs for the GSs (GS1 and GS2)
from Arabidopsis (M. Takahashi, Y. Sasaki, S. Ida,
and H. Morikawa, unpublished data). All these
cDNAs were under the control of the CaMV 35S proPlant Physiol. Vol. 126, 2001
moter and NOS terminator. GS1 and GS2 cDNAs had
a sense or antisense orientation in their respective
expression vectors. Ten transgenic plant lines were
analyzed for enzyme activity by the NR method of
Wray and Fido (1990) and 25 were analyzed by the
GS transferase assay (Rhodes et al., 1975). NR transformants had NR activity that was, at the most, about
twice that of the non-transformed control. Similar
results were obtained for the GS transformants.
These findings are very similar to those for the NiR
transformants given earlier (Table I). NO2-RN values
were determined for the NR and GS transformants,
as described above. No significant increase in the
ability to assimilate NO2 was found for these transformants; the percentage of NO2-RN of the nontransformed control (mean of three plants ⫾ sd) at
the most was 111.8 ⫾ 6.0 for the NR- and 99.0 ⫾ 5.8
for the GS transformants. Based on the analysis of
five NR- and eight GS-transformants, the respective
flux control coefficients of NR and GS for NO2 assimilation were ⫺0.01 and ⫺0.1. These values are
much smaller than those for NiR, another indication
that among the genes for primary nitrogen metabolism, NiR controls the assimilation of NO2 by plants.
DISCUSSION
Plants convert the nitrogen of NO2 taken from the
atmosphere to organic nitrogen (Hill, 1971; Zeevaart,
1976; Yoneyama and Sasakawa, 1979a; Kaji et al.,
1980; Rowland et al., 1985; Wellburn, 1990; Morikawa
et al., 1998; Ramge et al., 1993). The function of acting
as a sink for this air pollutant is very important.
Molecular physiological studies on the controlling
steps in the NO2 assimilation pathway of plants are
required to improve the ability of plants to incorpo-
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735
Takahashi et al.
Table II. NiR activity in chloroplast fractions from transgenic and
wild-type Arabidopsis plants
Values in parentheses are for chloroplast NiRA relative to the total
NiRA in intact leaves. Protoplasts were isolated from wild-type and
transgenic plants (line 121) of Arabidopsis. Chloroplasts were isolated as described in “Materials and Methods.” NiRAs were measured in extracts from intact leaves and from isolated chloroplasts.
Chloroplast yield was estimated by a comparison of the intensity of
the RBCS band in the SDS-PAGE gel after silver staining of the intact
leaf sample with that of the corresponding chloroplast fraction band.
NiRA (calc.) in the chloroplast fraction ⫽ [NiRA of chloroplast
fraction/(RBCS band intensity in the chloroplast fraction/RBCS band
intensity in RBCS in the intact leaf)] ⫻ 100 was estimated from these
two values. Experiments were repeated twice. The deviation of NiRA
(calc.) was less than 10%. One experiment used all leaves from 10
plants of a transgenic line or non-transformed plant.
NiRA
Whole
leaf
Chloroplast
fraction
Relative Intensity of
Small Rubisco
Subunit (RBCS) Band
Whole
leaf
Chloroplast
fraction
nmol NO2⫺ ⫻
min⫺1 mg⫺1
chlorophyll
Wild type
121
90.7
161.3
28.4
61.6
NiRA (calc.) in
Chloroplasts
nmol NO2⫺ ⫻
min⫺1 mg⫺1
chlorophyll
100
100
34.7
39.2
81.9 (90.3)
157.2 (97.5)
gave NR and GS flux control coefficients of ⫺0.01
and ⫺0.1.
Elsewhere, we showed that among the 217 taxa of
naturally occurring plants, there is more than a 600fold variation in the ability to assimilate NO2
(Morikawa et al., 1998). The molecular biological
causes of this variation, however, have yet to be
determined. Species that show the highest assimilation may have genes for the efficient incorporation of
NO2 and/or for metabolizing it in such physiological
processes as stomatal conductance, cell wall and
membrane transport, or nitrogen and carbon metabolite sensing in their primary and secondary metabolisms. We are currently investigating which regulatory genes (“NO2-philic genes”) are responsible for
high NO2 assimilation using differential analysis of
genes of species with the highest and lowest abilities.
The availability of roadside trees transformed with
such genes and enriched with the NiR gene will be
central to improving the ability of roadside vegetation in developed and developing countries to clean
up air pollution in situ.
MATERIALS AND METHODS
Construction of the NiR cDNA Expression Vector
rate NO2. The primary nitrate assimilation pathway
is considered to have a key role in the assimilation of
NO2 (Zeevaart, 1976; Yoneyama and Sasakawa,
1979a; Kaji et al., 1980; Rowland et al., 1985; Wellburn, 1990; Morikawa et al., 1998; Ramge et al., 1993).
We, therefore, produced transgenic plants that bear
chimeric expression vectors for NR, NiR, and GS
cDNA and analyzed gene expression and NO2 assimilation in them. Of these three genes, NiR is the
controlling gene in NO2 assimilation for the following reasons.
In transgenic Arabidopsis plants bearing chimeric
spinach NiR cDNA, four parameters, total NiR
mRNA content, total NiR protein content, NiRA, and
NO2-RN, were positively correlated. An increase in
NiR gene transcription therefore increases NO2
assimilation.
Of the 12 NiR-transformant lines studied, four had
significantly higher NiRA than the wild-type control
(P ⬍ 0.01), and three had significantly higher
NO2-RN than that control (P ⬍ 0.01). Each of the
latter three had one to two copies of spinach NiR
cDNA per haploid genome. Two lines had 140%
NO2-RN, higher than the control value. NR transformants had NR activity that was, at most, about twice
that of the non-transformed control. Similar results
were obtained with the GS transformants. The NR
and GS transformants did not show a significant
increase in NO2-RN.
The flux control coefficient of NiR for NO2 assimilation was estimated as about 0.4, based on analyses
of the NiR sense and antisense transformants. Estimations based on the results for the transgenic plants
736
The chimeric gene construct pCIB400 (Back et al., 1988)
that carries the expression cassette of spinach (Spinacia
oleracea) NiR cDNA was a gift from Dr. S. Rothstein (University of Guelph, Ontario, Canada). The 2.0-kb EcoRI fragment bearing spinach NiR cDNA was excised from
pCIB400 and was ligated into the deletion plasmid derived
from pBI221 in which the 1.9-kb SstI/BamHI fragment of
the ␤-glucuronidase gene had been deleted and end-filled
to produce plasmid pSNIR. To obtain plasmid pCaMVH,
the 1.0-kb BamHI fragment carrying the neomycin phosphotransferase gene in plasmid pCaMVNEO (Fromm et al.,
1986) was replaced by a 1.3-kb BamHI fragment that had
the hygromycin phosphotransferase (hpt) gene excised
from pCH (Goto et al., 1993). The 1.8-kb HindIII fragment
of the hpt expression cassette was excised from plasmid
pCaMVH, end-filled, and ligated into the filled SphI sites of
pSNIR to produce plasmid pSNIRH (Fig. 1).
Transgenic Arabidopsis Plants
Root sections of Arabidopsis ecotype C24 were bombarded with pSNIRH by means of a pneumatic particle gun
Table III. NiRA and NO2-RN in transgenic (clone 271) and wildtype tobacco plant leaves fumigated with 4 ␮L L⫺1 15N-labeled
NO2 for 8 h
Values are mean ⫾ SD of three independent experiments. One
experiment consisted of a sample prepared from a 10-week-old plant
of transgenic or wild-type tobacco.
NiRA
NO2⫺-RN
␮mol NO2
Wild type
271
102 ⫾ 7
3.6 ⫾ 2.4
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2001 American Society of Plant Biologists. All rights reserved.
⫺
h
⫺1
g
⫺1
fresh wt
0.63 ⫾ 0.07
0.39 ⫾ 0.05
Plant Physiol. Vol. 126, 2001
NO2 Assimilation by Transgenic Plants
device. Transformed shoots were selected for hygromycin
resistance as reported elsewhere (Takahashi and
Morikawa, 1996). Shoots that developed from independent
hygromycin-resistant calli and that had a band at 352 bp in
the PCR with primers specific to spinach NiR cDNA (see
below) were transferred to rock fiber cubes (Nittoboseki
Co., Tokyo) and were allowed to mature (T0 plants) and set
seeds (T1 seeds).
Seeds of the progenies of the transgenic Arabidopsis
plants were sown in vermiculite and perlite (1:1, v/v) in
plastic pots and grown at 22°C for 5 to 6 weeks under
continuous fluorescent light (70 ␮mol m⫺2 s⫺1) and 70%
humidity in a growth chamber (model ER-20-A; Nippon
Medical and Chemical Instruments, Osaka). Plants were
irrigated every 4 d with a one-half-strength solution of the
inorganic salts used in Murashige-Skoog medium (Murashige and Skoog, 1962) containing 10.3 mm NH4NO3 and 9.4
mm KNO3 (Cheng et al., 1991). A tiny piece of a leaf was
taken from plants of each transgenic line and was analyzed
by a PCR (Takahashi and Morikawa, 1996) with primers that
define a 352-bp fragment in the 5⬘ region of spinach NiR
cDNA. The primer sequences used in the analysis of spinach
NiR cDNA were 5⬘-AGCCGAGAGTGGAGGAGAGA-3⬘
and 5⬘-TACATCCGCACATCCATCTTTTCC-3⬘, which define a 352-bp fragment in the 5⬘ region of this cDNA (Back et
al., 1988). The 5- to 6-week-old plants that showed this band
were used for further analyses.
Transgenic Tobacco (Nicotiana tabacum cv Xanthi
XHFD8) Plants
Seeds of wild-type plants (provided by Dr. Michel Caboch, Laboratoire de Biologie Cellulaire, Institut National de
la Recherche Agronomique, Versailles, France) and transgenic plant clone 271, which expressed the NiR antisense
mRNA from tobacco under the control of the CaMV 35S
promoter, which lacks NiRA (Vaucheret et al., 1992), were
surface sterilized and sown in vitro on B-medium adjusted
to pH 5.6, which contained 20 mm KNO3 as the sole nitrogen source (Vaucheret et al., 1992). Two weeks after sowing, plants were transferred to B-medium containing 10
mm ammonium succinate as the sole nitrogen source and
were grown for 4 weeks. After transfer to soil in a growth
chamber and growth at 22°C and 70% humidity for 4 weeks
under continuous fluorescent light (100 ␮mol m⫺2 s⫺1) in
B-medium containing 10 mm ammonium succinate as the
sole nitrogen source, the plants were analyzed for their
NiRA and ability to assimilate NO2. For determination of
NiRA, a sample of crude enzyme extract was prepared
from the youngest fully expanded leaf (500 mg) taken from
the 10-week-old plant. For determination of NO2-RN, a
sample powder was prepared from the youngest and second youngest fully expanded leaves (total of about 1 g)
taken from the 10-week-old plants before and after fumigation was used.
Southern Hybridization Analysis
Total DNAs from the whole shoots of a 5- to 6-week-old
plant (approximately 100 mg) of transgenic line or wildPlant Physiol. Vol. 126, 2001
type control plant were isolated according to Murray and
Thompson (1980) as modified by Rogers and Bendich
(1985). The isolated DNA (2 ␮g) was digested with EcoRI/
PstI (which produces a 1.8-kb fragment of the spinach NiR
cDNA from pSNIRH), SacI (which has a unique site in the
NiR cDNA of pSNIRH), SalI (which has no site in pSNIRH),
or KasI/ScaI (which produces a 3.1-kb fragment of the
spinach NiR cDNA expression cassette). Digests were electrophoresed in a 1% (w/v) agarose gel and were then
transferred to a Hybond-N⫹ membrane (Amersham, Buckinghamshire, UK) and hybridized at 65°C with a gene
probe labeled with [␣-32P]dCTP and a T7QuickPrime Kit
(Pharmacia Biotech, Piscataway, NJ) in a solution (Sambrook et al., 1989) containing 5⫻ sodium chloride/sodium
phosphate/EDTA, 0.5% [w/v] SDS, 200 mg L⫺1 denatured
salmon sperm DNA, and 5⫻ Denhardt’s solution (50⫻
Denhardt’s solution containing 1% [w/v] Ficoll, 1% [w/v]
polyvinylpyrrolidone [PVP], and 1% [w/v] bovine serum
albumin). The 1.8-kb fragment of spinach NiR cDNA excised from pSNIRH by EcoRI/PstI digestion was the gene
probe. Final washes were done at 65°C in 0.5⫻ sodium
chloride/sodium phosphate/EDTA and 0.1% (w/v) SDS.
Hybridization signals were made visible by autoradiography with an intensifying screen at ⫺80°C for 1 to 2 d.
Total RNA and Reverse Transcription
Total RNAs from the whole shoots of a 5- to 6-week-old
plant (approximately 100 mg) of transgenic line or those of
wild-type control plants were extracted according to Verwoerd et al. (1989). Reverse transcription of the total RNA
was done with oligo-dT as the primer: Total Arabidopsis
RNA (1 ␮g) was mixed with the oligo-dT, incubated at 70°C
for 10 min, then cooled on the ice, after which 10 mm
dithiothreitol, 0.5 m dNTPs, RT buffer (Gibco-BRL, Cleveland), and Superscript II reverse transcriptase (Gibco-BRL)
were added. This reaction mixture (20 ␮L) first was incubated at 42°C for 50 min and then at 70°C for 15 min. After
being cooled, RNase H was added, and the mixture incubated at 37°C for 20 min. This RT reaction mixture was used
for RT-PCR analysis.
RT-PCR Analysis of Spinach, Arabidopsis, and Total
NiR mRNAs
For the analysis of mRNAs of transgene spinach NiR and
endogenous Arabidopsis NiR genes, the PCR mixture contained 20 ␮m dNTPs, PCR buffer (Takara Shuzo, Shiga,
Japan), rTaq (Takara Shuzo), the primers, and 0.5 ␮L of the
RT reaction mixture for a total volume of 20 ␮L. This
mixture was heated to 95°C for 3 min, amplified for 30
cycles at 95°C for 1 min, then at 63°C for 2 min, and 72°C
for 2 min. The primer sequences were 5⬘-AGCCGAGAGTGGAGGAGAGA-3⬘ (forward) and 5⬘-TACATCCGCACATCCATCTTTTCC-3⬘ (reverse) for amplification of the NiR
cDNA from the introduced spinach NiR cDNA (Back et al.,
1988), 5⬘-TGCTTGTGGGAGGATTCTTTAGTC-3⬘ (forward)
and 5⬘-TTGGCATTCTCTTCTCTACCTCAG-3⬘ (reverse) for
amplification of the Arabidopsis NiR cDNA (Tanaka et al.,
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Copyright © 2001 American Society of Plant Biologists. All rights reserved.
737
Takahashi et al.
1994), and 5⬘-TTCATATCCAAGGCGGTCAATGTG-3⬘ (forward) and 5⬘-CCATGCCTTCTCCTGTGTACCAA-3⬘ (reverse) for amplification of the Arabidopsis ␤-tubulin cDNA
(Marks et al., 1987). The PCR products were electrophoresed
in 1.8% (w/v) agarose gels that then were stained with
ethidium bromide. The resulting bands were quantified by a
Gel Documentation System (Gel Doc 2000, Bio-Rad, Hercules, CA) and software (Quantity One, PDI, Inc., New
York).
For the quantitative analysis of total (spinach ⫹ Arabidopsis) NiR mRNAs, the PCR mixture contained 20 ␮m
dNTPs, PCR buffer (Takara Shuzo), rTaq (Takara Shuzo),
the primers, 1 ␮L of competitors (dilution series of 2.5 ⫻
105 to 5 ⫻ 106 copies/␮L, see below), and 0.5 ␮L of the RT
reaction mixture for a total volume of 20 ␮L. The competitor (1,668 bp) consisted of a partial Arabidopsis NiR
genomic clones containing three (the first to third) introns
(Tanaka et al., 1994) that were prepared by PCR using 500
ng of genomic DNA of Arabidopsis as the template and 0.5
pmol of primers for total NiR cDNA listed below, as described elsewhere (Takahashi and Morikawa, 1996) after
which competitor DNAs were purified by the Suprec-02
column according to the manufacturer’s instruction
(Takara Shuzo). The mixture was then heated to 95°C for 5
min, amplified for 30 cycles at 94°C for 1 min, then at 63°C
for 2 min, and 72°C for 1 min. The primers used for total
NiR cDNAs were 5⬘-GTTAGACTCAAGTGGC-3⬘ (forward)
and 5⬘-ATGCGAGTCACTTCCT-3⬘ (reverse). The PCR
products were electrophoresed, stained with ethidium bromide, and the resulting bands were quantified as described
above. Different concentrations of the competitor were
tested so that total NiR mRNA contents were determined
from the intersection of curves depicting the levels of product from the competitor and levels of product from the
target cDNAs.
Protein Extraction and Western-Blot Analysis
Whole shoots from a 5- to 6-week-old plant (approximately 100 mg) of a transgenic line or wild-type control
plant were frozen in liquid nitrogen and then ground in a
mortar with a pestle to which 0.3 mL of extraction buffer
(per 100 mg of tissue) containing 50 mm potassium phosphate buffer (pH 7.5), 1 mm EDTA, 10 mm 2-mercaptoethanol, 100 ␮m phenylmethylsulfonyl fluoride (PMSF),
and 5 mg PVP had been added. The homogenate was
centrifuged at 12,000g and 4°C for 5 min, and the resulting
supernatant (protein extract) used for western-blot analysis. The protein content of the extract was measured by the
method of Bradford (1976) with bovine serum albumin as
the standard.
An extract sample containing 10 ␮g of protein was layered on a 12% (w/v) acrylamide slab gel (Laemmli, 1970)
and was electrophoresed for 3 h at 35 mA. NiR bands were
detected with a polyclonal antibody raised against spinach
NiR (Ida, 1987) and were quantified by an enhanced chemiluminescence western-blot analysis system and a detection
kit (Renaissance, NEN Life Science Products, Boston).
738
Two-Dimensional PAGE
Two-dimensional PAGE was done as described by Görg
et al. (1985). An approximate 200 ␮g of extracted protein
was loaded on the first-dimension gel (Immobiline Dry
Plate, pH 4–7, Pharmacia Biotech). Isoelectric focusing was
done horizontally with a Multiphor II apparatus (Pharmacia Biotech) for 4 h at 300 V and 18 h at 3,500 V. The
isoelectric focusing gel was equilibrated for 30 min in SDS
sample buffer containing 50 mm Tris-HCl (pH 6.8), 30%
(w/v) glycerol, and 1% (w/v) SDS and was then mounted
on a 12% (w/v) polyacrylamide-SDS slab gel and was
electrophoresed for 3 h at 35 mA. Proteins that separated in
the two-dimensional gels were transferred to a membrane
(Immobilon-P, Millipore, Bedford, MA) by an electroblotter
(Trans-Blot SD, Bio-Rad). Immunodetection was done with
polyclonal antibody raised against spinach NiR (Ida, 1987),
as described above.
Chloroplast Isolation
The 5- to 6-week-old Arabidopsis plants were kept in the
dark for 24 h before chloroplast isolation, as described by
Somerville et al. (1981). Leaves of 10 such plants (approximately 1,000 mg) of a transgenic line or wild-type control
plant were used for protoplast isolation. The lower leaf
epidermis was scratched on ice with a razor. The scratched
leaves were floated on 0.5 m sorbitol, 10 mm MES [2-(Nmorpholino)-ethanesulfonic acid, pH 5.5], 1 mm CaCl2,
1.6% (w/v) Macerozyme R-10 (Yakult Honsha, Tokyo), and
1.6% (w/v) Cellulase Onozuka R-10 (Yakult Honsha) in a
Petri dish and shaken at 40 rpm for 1.5 h. After removal of
the medium, the protoplasts on the bottom of the Petri dish
were washed several times with cold washing medium
containing 0.5 m sorbitol, 10 mm MES (pH 6.0), 1 mm CaCl2,
and 1 mm PMSF and were then transferred to a new tube
containing washing medium. After centrifugation at 100g
at 4°C for 4 min, the pellet was resuspended in the cold
washing medium. The protoplast suspension obtained was
layered on a base of 0.42 m sorbitol, 60% (v/v) Percoll, 10
mm MES (pH 6.0), 1 mm CaCl2, and 1 mm PMSF and was
then centrifuged at 100g at 4°C for 4 min. The protoplasts
were collected and ruptured by resuspension in 0.3 m
sorbitol, 50 mm HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid , pH 7.5], 1 mm MgCl2, 1 mm MnCl2, 2
mm EDTA, 30 mm KCl, 0.25 mm KH2PO4, and 1 mm PMSF.
The chloroplasts released were recovered by centrifugation
at 270g for 35 s at 4°C and were ruptured by resuspension
in 50 mm phosphate buffer, 1 mm EDTA, 0.07% (w/v)
2-mercaptoethanol, and 1 mm PMSF. This ruptured chloroplast preparation, which contained 10 ␮g of chlorophyll
as determined by the method of Mackinney (1941), was
layered on a 12% (w/v) polyacrylamide-SDS gel and electrophoresed as described above. The intact leaf homogenate (see below) was similarly analyzed by SDS-PAGE. The
gels were silver-stained with a detection kit (Daiichi Pure
Chemicals, Tokyo). RBCS band intensities in the chloroplast and intact leaf gels were quantified as described
above. The chloroplast yield (intensity of RBCS band of
chloroplast fraction/intensity of RBCS band of intact leaf)
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Copyright © 2001 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 126, 2001
NO2 Assimilation by Transgenic Plants
was estimated from these two values. The chloroplast and
intact leaf NiRAs were measured as described below, and
the NiRA in the chloroplast fraction (NiRA in isolated
chloroplasts/chloroplast yield) was calculated.
spectrometer (Delta C; Finnigan MAT, Bremen, Germany)
connected directly to an elemental analyzer (EA/NA;
Fisons Instrument, Milano, Italy) to determine the amount
of NO2-RN in the fumigated leaf sample.
NiR Enzyme Activity Analysis
Statistical Analysis
Whole shoots of a 5- to 6-week-old plant (approximately
100 mg) of transgenic line or wild-type control plant were
frozen in liquid nitrogen then ground in a mortar with a
pestle. The powdered tissues were added to 0.3 mL of
extraction buffer (per 100 mg of tissue) containing 50 mm
potassium phosphate buffer (pH 7.5), 1 mm EDTA, 10 mm
2-mercaptoethanol, 100 ␮m PMSF, and 5 mg PVP and were
then homogenized. The homogenate was centrifuged and
the supernatant (crude enzyme solution) was used for the
NiRA analysis. In an alternate manner, the chloroplasts
were ruptured and centrifuged, as described above, and
the supernatant was used for the NiRA analysis.
NiRA was assayed as reported by Wray and Fido (1990),
with modification, to measure the decrease of nitrite ion in
the assay mixture. A 45-␮L sample of the crude enzyme
solution was transferred to a 1.5-mL centrifuge tube, and
195 ␮L of assay solution containing 50 mm potassium
phosphate buffer (pH 7.5), 1 mm NaNO2, and 1 mm methyl
viologen was added. The reaction was started by the addition of 60 ␮L of 57.4 mm Na2S2O4 in 290 mm NaHCO3
(final Na2S2O4 concentration in the assay solution was 11.5
mm), and the reaction was run for 5 min at 30°C. A 20-␮L
sample was transferred to a new tube containing 480 ␮L of
water, and the whole was vigorously mixed to stop the reaction, after which 500 ␮L of 1% (w/v) sulfanilamide in 3 n HCl
and 500 ␮L of 0.02% (w/v) N-1-naphthylethylenediameine
dihydrochloride was added. The absorbance of this mixture
at 540 nm was measured.
All results for Arabidopsis are, except for Table II, reported in the form of means ⫾ sd of at least two independent experiments. Each experiment consisted of the result
of the whole shoots isolated from a 5- to 6-week-old Arabidopsis plant (approximately 100 mg) of transgenic line or
wild-type control plant, except that in chloroplasts isolation experiment (Table II), 10 identical plants were used.
For results for tobacco in Table III, details are described in
“Materials and Methods.” A linear regression to calculate
the correlation coefficient (r) and Dunnett’s test for two
samples of different sizes were made using Excel (Microsoft, Redmond, WA). Significant differences over the
control non-transformed wild-type plants were estimated.
NO2 Assimilation Analysis
The 5- to 6-week-old plants of a transgenic line or wildtype control plant, grown as described above, were fumigated with 15N-labeled NO2 (4.0 ⫾ 0.1 ␮L L⫺1, 51.9 atom
percentage of 15N) in a fumigation chamber (model
NC1000-SC, Nippon Medical and Chemical Instruments)
for 8 h during the day (9 am–5 pm) in light (70 ␮mol m⫺2
s⫺1). The chamber was maintained at 22.0°C ⫾ 0.3°C, with
a relative humidity of 70% ⫾ 4% and an atmospheric level
CO2 concentration (0.03%–0.04%), as described elsewhere
(Morikawa et al., 1998). Whole shoots were harvested from
the 15NO2-fumigated plants, washed with distilled water,
lyophilized for 12 h after which they were ground into fine
powder in a mortar with a pestle, and the powder digested
by the Kjeldahl method. (A sample for the NO2 assimilation analysis experiment was prepared from whole shoots
harvested from one fumigated plant of transgenic line or
wild-type one.) Preparation of the reduced nitrogen fraction was essentially as described elsewhere (Morikawa et
al., 1998), except that the reducing agents (CuSO4 and
K2SO4) were omitted from the digestion mixture. The 15N
in the reduced nitrogen fraction was measured with a mass
Plant Physiol. Vol. 126, 2001
ACKNOWLEDGMENTS
We thank Junko Uchiyama and Orie Mori for their technical assistance.
Received January 17, 2001; accepted January 19, 2001.
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