全 文 ：Received 10 Jun. 2003 Accepted 5 Oct. 2003
Supported by the National Natural Science Foundation of China (30390080) and the State Key Basic Research and Development Plan of China
(G1998010208, G1998010205).
* Author for correspondence. Tel: +86 (0)10 64889381; E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (3): 347-354
Isolation and Expression Analysis of a High-Affinity Nitrate
Transporter TaNRT2.3 from Roots of Wheat
ZHAO Xue-Qiang1, 2, LI Yu-Jing1, LIU Jian-Zhong1, LI Bin1, LIU Quan-You3,
TONG Yi-Ping 3*, LI Ji-Yun3, LI Zhen-Sheng1
(1. State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental
Biology, The Chinese Academy of Sciences, Beijing 100101, China;
2. Graduate School of The Chinese Academy of Sciences, Beijing 100039, China;
3. Research Center for Eco-environmental Sciences, The Chinese Academy of Sciences, Beijing 100085, China)
Abstract: This paper reports the cloning and expression analysis of a high-affinity nitrate transporter in
wheat (Triticum aestivum L.). A full-length cDNA, TaNRT2.3 (accession number AY053452), was isolated
from NO3–-induced roots of wheat. The cDNA encodes a polypeptide with 507 amino acids and 12
transmembrane domains, belongs to nitrate/nitrite porter (NNP) family within the major facilitator superfamily
(MFS), and is closely related to other NRT2 proteins from plants. The expressions of TaNRT2 genes in
wheat tissues were analyzed using Northern blot, results indicated that TaNRT2 were induced specifically
in roots but not in shoots in response to both low (5-200 mmol/L) and high (2.0 mmol/L) concentrations
of NO3–. TaNRT2 transcripts were undetectable in N-deprived or NH4+-grown plant roots. The significant
correlation between the time course of TaNRT2 transcription accumulation in the roots of wheat plants
grown in 0.2 mmol/L NO3– and the time course of the nitrate uptake rates by wheat plants grown under the
same conditions suggested that TaNRT2 played an important role in high-affinity NO3– uptake. Using the
split root system, we found that supplying NO3– to one part of the roots induced the expression of TaNRT2
in the other part not supplied with NO3– or supplied with NH4+, which implied that N cycling within plants
acted as a regulatory signal for N uptake.
Key words: Triticum aestivum ; high-affinity nitrate transporter; nitrate induction; high-affinity nitrate
uptake rate
Worldwide nitrogen use efficiency for cereal produc-
tion is approximately 33%. The unaccounted 67% repre-
sents a $15.9 billion annual loss of nitrogen fertilizer (Raun
and Johnson, 1999). Loss of N fertilizer resulted in serious
environmental problems, such as greenhouse warming and
pollution of drinking water resources (Tilman, 1999). Wheat
is one of the most important food crops in the world, and as
high as 20% of the global N fertilizer is applied to raise
wheat yields (Harris, 1998). Improving nitrogen use effi-
ciency in wheat production is important in sustainable
agriculture.
Nitrate is one of the most important macronutrients and
also acts as a signal for plant growth. It is the major inor-
ganic nitrogen form in aerobic agricultural soils, and the
main nitrogen source taken up by wheat. Understanding
the molecular mechanisms of how wheat absorbs NO3– and
assimilates it is a critical step to improve nitrogen use effi-
ciency and reduce the nitrogen loss.
Nitrate uptake is a key step in NO3– assimilation. Kinetic
studies of NO3– uptake indicate that roots of higher plants
have two kinetically distinct NO3– uptake systems, one is
the high-affinity transport system (HATS) and the low-af-
finity transport system (LATS). According to the differ-
ences in response to NO3– induction, the HATS can be
further divided into inducible HATS (iHATS) and constitu-
tive HATS (cHATS) components. The cHATS has low Km
(typically 6-20 mmol/L), while the iHATS has higher Km
(typically 20-100 mmol/L), and appears to play a major role
in NO3– uptake when NO3– concentrations in the soil are
very low (< 250 mmol/L) (Crawford and Glass, 1998). The
LATS generally has a larger capacity than the HATS does,
and shows linear kinetics above 0.5 mmol/L and constitu-
tive expression in plants (Siddiqi et al., 1990; Glass and
Siddiqi, 1995; Crawford and Glass, 1998). It has long been
thought that the LATS has only a constitutive component
(Glass and Siddiqi, 1995). However, more recent studies
show that the LATS also has two components analogous
to iHATS and cHATS in Arabidopsis (Huang et al., 1996;
Acta Botanica Sinica 植物学报 Vol.46 No.3 2004348
1999).
Genes involved in low and high-affinity NO3– uptake
have been identified in fungi, algae and, more recently, in
plants. These genes fall into two families: NRT1 for LATS
and NRT2 for HATS. The first NRT2 gene crnA was iso-
lated from Aspergillus nidulans (Johnstone et al., 1990;
Unkles et al., 1991). The crnA mutants can not survive on
growth media using NO3– as the sole N source at conid-
iospore and young mycelia stages, for they are defective in
NO3– uptake (Brownlee and Arst, 1983). The crnA gene,
which encodes a protein with 507 amino acids and 12 mem-
brane spanning domains, is able to restore NO3– uptake in
the crnA mutants (Johnstone et al., 1990; Unkles et al.,
1991). After the crnA was isolated, a number of crnA ho-
mologous genes have been cloned from other eukaryotes,
including Chlamydomonas reihardtii (Quesada et al., 1994),
Hansenula polymorpha (Perez et al., 1997), Barley (Trueman
et al., 1996b; Vidmar et al., 2000a), tobacco (Quesada et al.,
1997), soybean (Amarasinghe et al., 1998) and Arabidopsis
(Zhuo et al., 1999). The transcription levels for these genes
increase dramatically within hours exposure to NO3-, the
expression patterns correlate well with NO3– influx, provid-
ing indirect evidence that NRT2 encodes inducible high-
affinity transporters (Quesada et al., 1997; Zhuo et al., 1999;
Vidmar et al., 2000a). Recently direct experimental evidence
demonstrates that NRT2 gene product does contribute to
high-affinity NO3– uptake (Tong and Miller, unpublished
data), but it requires another gene, NAR2, to co-produce a
functional HATS in Xenopus oocytes.
iHATS is subject to regulation by both positive and
negative effects. NO3- is the signal for the induction of
NRT2 expression, while NH4+ and N assimilation
intermediate(s) are responsible for the down-regulation of
NO3- influx and NRT2 transcript (Crawford and Glass, 1998;
Forde, 2000). In barley, NH4+ and amino acids (Asp, Asn,
Glu and Gln) make high-affinity NO3- influx and HvNRT2
transcript abundance in roots decrease, NO3– itself is not
responsible for down-regulating HvNRT2 transcript levels,
but it may act post-transcriptionally (Vidmar et al., 2000b).
Here we report the cloning and characterization of two
crnA homolog, TaNRT2.1 and TaNRT2.3 from wheat.
1 Materials and Methods
1.1 Plant materials
Wheat (Triticum aestivum L. cv. Chinese Spring) seeds
were surface-sterilized with 1% NaOCl for 10 min, rinsed
with deionized water, and germinated in sterilized moist
sands in the dark. The seeds were placed on nylon mesh
fitted to 150 mm glass petri dishes, and covered with 5 mm
of moist sands. After 3 d of germination in the dark, the
dishes were transferred into incubator with 12 h light/12 h
dark cycle at 25 ± 2 ℃. Four days later, the seedlings were
transferred into 2 L hydroponic pots with 12 plants per pot.
All the nutrient solution was prepared with deionized water
and reagent-grade chemicals. The solution contained (mol/L)
2× 10–3 (NH4)2SO4, 2× 10–4 KH2PO4, 5× 10–4
MgSO4·7H2O, 1.5×10–3 KCl, 1.5×10–3 CaCl2, 1×10–4
Fe-EDTA, 1×10–6 H3BO3, 1×10–6 ZnSO4·7H2O, 5×10–7
CuSO4·5H2O, 3× 10–7 Na2MoO4·2H2O, 1× 10–6
MnSO4·H2O. The solution was refreshed every two days,
and the pH was maintained at 6.0 ± 0.2 by adding excess
CaCO3 powder. The hydroponic pots were placed in a
greenhouse with average 20 ℃ day/10 ℃ night. The plants
were grown in hydroponic pots for two weeks prior to analy-
sis of net NO3- uptake and TaNRT2 expression in plant
tissues.
1.2 Expression analysis
1.2.1 Nitrogen forms and concentrations Three-week-
old wheat plants, which were grown in full strength nutri-
ent solution as described above, were deprived of nitrogen
for 48 h. Then the plants were transferred to the nutrient
solution containing 0 mmol/L KNO3, 1 mmol/L (NH4)2SO4,
0.2 mmol/L KNO3 and 2.0 mmol/L KNO3 as sole nitrogen
sources for the times indicated, and to the solution con-
taining 0, 5, 25, 50, 100, and 200 mmol/L KNO3 for 4 h. The
solution was refreshed daily and the pH was maintained at
6.0 ± 0.2 by adding excess CaCO3 powder. The roots were
harvested and frozen in liquid nitrogen for RNA extraction.
1.2.2 Split-root experiments For the split-root
experiments, seedlings were prepared as described above.
After pretreatment in N-free nutrient solution for 48 h, the
roots were separated into two parts and placed into sepa-
rated containers containing nutrient solution with different
concentrations or forms of nitrogen. In NO3–-0 split-root
system, one part of roots was treated with 2 mmol/L NO3–
as KNO3, whereas the other part was placed in N-free nutri-
ent solution. In NO3–-NH4+ split-root system, one part of
roots was treated with 2 mmol/L NO3– as KNO3, the other
part was treated with 2.0 mmol/L NH4+ as (NH4)2SO4. The
wheat plants were grown in the split-root system for 1 to 8
h before being harvested. Roots of the two parts were
harvested separately and were frozen in liquid nitrogen for
RNA extraction or kept at -80 ℃ until use.
1.3 Isolation of TaNRT2.3 cDNA
1.3.1 RT-PCR and cDNA cloning The first strand of
cDNA was synthesized by reverse transcription (RT) and
amplified through polymerase chain reaction (PCR) using
Superscript® Preamplification System kit (Gibco, BRL)
ZHAO Xue-Qiang et al.: Isolation and Expression Analysis of a High-Affinity Nitrate Transporter TaNRT2.3 from Roots of Wheat 349
according to manufacturer’s protocol. Five microgrammes
of total RNA was isolated according to Wei (1999) from the
roots of 21-day-old plants, which induced in culture solu-
tion containing 0.2 mmol/L KNO3 for 24 h was used in each
RT-PCR reaction. The degenerative primers used for the
RT-PCR were P1 (5 -GA(T/C)AA(T/C)GT(A/T/C/G)AT(A/
T/C)GC(A/T/C/G)GA(A/G)TA(T/C)-3 ) and P2 (5-(A/T/C/
G)(C/G)(T/A)(A/T/C/G)CC CCA(T/C)TG(A/T/C/G)GG (A/
G)AA(A/G)TG-3 ) which were designed according to the
conserved sequences of known plant NRT2 proteins. The
PCR reaction was carried out with the following program:
94 ℃ for 2 min followed by 5 cycles of 94 ℃ for 1 min, 46 ℃
for 2 min and 72 ℃ for 2 min, which then continued with 35
cycles of 94 ℃ for 1 min, 55 ℃ for 2 min and 72 ℃ for 2 min
and ended with a final extension of 72 ℃ for 10 min. The RT-
PCR products were gel-purified by Agrose Gel DNA Extrac-
tion kit (Roche Diagnostics GmbH) and subsequently cloned
into pGEM-T easy vector (Promega). The cDNA clones
were sequenced using the ABI PRISM™ Big Dye™ Termi-
nator Cycle (Perkin-Elmer, San Jose, CA). The DNA se-
quence was compared with published gene sequences in
the GenBank database using BLAST programs from Na-
tional Center for Biotechnology Information (NCBI).
1.3.2 cDNA library construction and screening To
construct a cDNA library from NO3--induced wheat roots,
total RNA was isolated as previously described (Wei, 1999)
from the roots of 21-day-old plants, which had been in-
duced in culture solution containing 0.2 mmol/L KNO3 for
24 h. The polyadenylated RNA was isolated from total RNA
by oligo-dT cellulose chromatography. Using the
polyadenylated RNA as a template, first and second strand
cDNAs were synthes ized wi th the Unive rsa l
RiboClone®cDNA Synthesis System (Promega). After add-
ing an EcoRⅠ adaptor in both ends, the cDNAs were li-
gated into EcoRⅠ-digested lgt10 vector (Promega), and
packaged into bacteriophage using Packagene® Lambda
DNA Packaging System (Promega) to yield a primary li-
brary stock. The stock was amplified and stored at – 80 ℃
until use.
The library of a size 5×105 plaque-forming unit (pfu)
was plated out on a lawn of Escherichia Coli C600Hfr strain.
The membrane lifts of this library was screened with the [a-
32P]dCTP-labeled (Prime-a-Gene Labeling System, Promega)
TaNRT2 RT-PCR fragment. Titering, plaque lifting and filter
hybridization and washing were performed according to
the manufacturer’s protocol. Phage DNAs of positive
clones were prepared using Wizard® Lambda Preps DNA
Purification System (Promega).
1.3.3 5 rapid amplification of cDNA ends (5 RACE-
PCR) Since the positive clone screened from cDNA li-
brary contained only 3 end of TaNRT2. The missing part of
TaNRT2 at 5 end was rescued by rapid amplification of 5
cDNA ends (5-RACE) using SMARTTM-RACE cDNA Am-
plification kit (Clontech, USA). 5 RACE was performed ac-
cording to manufacturer’s protocol. The gene specific prim-
e r s o f Ta N R T 2 u s e d i n 5 R AC E wa s : 5 -
GTACAGAGCAGGAACGCAAAA-3, corresponding to
nucleotides of 1 724-1 744 of TaNRT2.3 . The 5 RACE PCR
products were cloned into pGEM-T easy vector and se-
quenced using the ABI PRISM™ Big Dye™ Terminator
Cycle (Perkin-Elmer, San Jose, CA).
1.4 RNA isolation and Northern blotting analysis Total
RNA from roots was phenol/chloroform-extracted and pu-
rified according to Wei (1999). Total RNA of 20 mg isolated
from wheat roots was separated in 1.2% agarose gel con-
taining 1×MOPS buffer with 2.2 mol/L formaldehyde. The
RNA was then transferred by capillary to nylon membrane
(Hybond N+, Amersham Pharmacia Biotech.). The membrane
was baked for 2 h at 80 ℃ to cross-link RNA. The pre-
hybridization and hybridization were preformed by stan-
dard procedures (Sambrook et al., 1989). The 504 bp frag-
ment cloned by RT-PCR was randomly labeled (Prime-A-
Gene, Promega) and used to probe the RNA. The mem-
brane was washed at medium stringency (0.5×SSC, 0.1%
SDS) and the autoradiograph of the RNA blot was obtained
by exposing the blot to X-ray film (Fuji, Japan) at – 80 ℃ for
7 d. As TaNRT2.3 shares high homology with TaNRT2.1
and TaNRT2.2 (>90% at nucleotide level), Northern blot in
this study represents the expressions of TaNRT2 genes in
wheat roots. Ribosomal RNA was stained with methylene
blue to ensure equal RNA loading (Sambrook et al., 1989).
1.5 Net NO3– uptake analysis
Wheat plants pretreated with 0.2 mmol/L NO3- for 0, 1,
2, 4, 8, 24, 48, 96 h (detailed in 1.2.1) were used in net NO3–
uptake analysis. Six plants were shifted to a flask with 100
mL uptake solution containing 0.2 mmol/L KNO3 at pH 6.0.
The flasks were then wrapped with black plastic cloth and
incubated for 30 min at 25 ℃. After incubation, 1 mL of
the residual uptake solution was sampled and the amount
of depleted NO3– was determined by nitration of salicylic
acid as described by Cataldo et al. (1975) with minor
modifications. Briefly, 50 mL sampled solution was trans-
ferred to a 1.5 mL eppendorf tube and concentrated to 10
mL using Concentrator 5301 (Eppendorf, Germany) and then
mixed thoroughly with 40 mL of 5% (W/V) salicylic acid in
concentrated H2SO4. After cooling for 20 min, 950 mL of 2
mol/L NaOH was added to terminate the reaction and again
let it cool to room temperature. A series of solutions of
Acta Botanica Sinica 植物学报 Vol.46 No.3 2004350
known NO3- concentrations were used to obtain calibra-
tion graph. The absorbance of the solution was determined
at 410 nm and NO3- level was determined from the calibra-
tion graph. Each of the treatments was repeated four times.
Net NO3- uptake rates were calculated according to the
amount of NO3- depleted and root fresh weights
(mmol.L-1.g-1 FW.h-1).
2 Results
2.1 Isolation and sequence analysis of full length
TaNRT2.3 cDNA
In an attempt to isolate TaNRT2 genes, a 504 bp frag-
ment was amplified from total RNA isolated from NO3–-
induced wheat roots by RT-PCR using a pair of degenera-
tive primers designed based on the conserved sequences
of known plant NRT2 proteins. Using this 504 bp fragment
as a probe, a cDNA library, which was constructed in lgt10
vector (Promega) from NO3–-induced wheat root, was
screened. After two rounds of screening, the putative cDNA
clone was isolated and sequenced. The positive clone
(designated as TaNRT2.3) was not a full-length cDNA, but
the BLAST result showed that it was indeed a TaNRT2
homolog. So 5 RACE approach was used to amplify 5 end
of TaNRT2.3. To create the maximum overlap with 5 trun-
cated cDNA, a TaNRT2.3 gene-specific primer (GSP) was
designed at the far 3 end of 5 truncated cDNA
(corresponding to nucleotides of 1 724-1 744 of
TaNRT2.3) and used in the RACE reactions. As a result,
the full-length of TaNRT2.3 cDNA sequence was obtained
(accession number AY053452). The nucleotide sequence
of TaNRT2.3 contains an open reading frame from nucle-
otide of 63 to 1 583 and encodes a protein of 507 amino
acids (Fig.1). Hydropathy analysis of the deduced protein
of TaNRT2.3 indicated that it contains 12 putative trans-
membrane domains with a short central loop (24 aa) and a
long, hydrophilic C-terminal domain (69 aa), like high-affin-
ity NO3– transporters in other higher plants (Trueman et
al., 1996a; Forde, 2000).
Comparison of the deduced amino acid sequence of
TaNRT2.3 with other NRT2 homologs from both eukary-
otes and prokaryotes revealed that all species shared a
high degree of sequence similarity (Fig.2).
Based on BLAST of the ammonium acid sequence of
TaNRT2.3, it contains the A-G-W/L-G-N-M-G consensus
motif (Fig.1, in shaded box) within transmembrane domain
5, which has been proposed as a signature motif for NNP
(nitrate/nitrite porter) family (Trueman et al., 1996a), imply-
ing that TaNRT2.3 belongs to the NNP family of NO3– and
NO2– transporters, one of Major Facilitator superfamily
(MFS). We analyzed the TaNRT2.3 protein with NetPhos 2.0
program (Blom et al., 1999) and found a number of possible
phosphorylation sites. Three of them are conserved pro-
tein kinase C recognition motif (S/T-X-R/K) (Fig.1, boxed).
Ser-28 is part of a S/T-X-R motif found in all HvNRT2 se-
quences and in OsNRT2; Ser-381 is part of S-X-R motif
found in all the plant and algal sequences; and Ser-484 is
part of a S-X-R motif found in the algal and the majority of
the higher plant sequences (Forde, 2000).
2.2 Effects of nitrogen sources on the expression of
TaNRT2
The effects of different forms of nitrogen (NO3– and
NH4+) on the expression of TaNRT2 were investigated by
Northern blotting analysis using the 504 bp RT-PCR prod-
uct as a probe. As the 504 bp fragment of TaNRT2.3 shares
high homology with the same segments both in TaNRT2.1
and TaNRT2.2, Northern blots in this study actually reflect
Fig.1. The deduced amino acid sequences of TaNRT2.3 (AY053452). Using HMMTOP 2.0 (Tusnady and Simon, 2001), 12
transmembrane spanning domains (underlined) and cytoplasmically orientated N- and C-terminal were predicted. Signature motif for
NNP (nitrate/nitrite porter) is boxed and shaded. Conserved protein kinase C recognition motifs (S/T-X-R/K) are boxed. The
TaNRT2.3 residues underlined were used for designing degenerative primers for RT-PCR.
ZHAO Xue-Qiang et al.: Isolation and Expression Analysis of a High-Affinity Nitrate Transporter TaNRT2.3 from Roots of Wheat 351
the expressions of TaNRT2 genes in wheat.
Figure 3A showed that the expression of TaNRT2 was
only induced in the roots by 0.2 mmol/L or 2 mmol/L of
NO3– but not by NH4+ and H2O, and not in the upper parts
of wheat plants (Fig.3A), suggesting that the induction of
TaNRT2 was NO3– -dependent and root-specific.
No accumulation of TaNRT2 transcripts was seen in the
roots at 0 h after the plants were deprived of NO3– for 48 h.
The TaNRT2 transcripts accumulated rapidly within 1 h,
peaked at 8 h and kept at a significant level till 24 h after the
plants were shifted to a solution containing both 0.2 mmol/L
or 2.0 mmol/L NO3-, then dropped drastically to a barely
detectable level after 48 h. The expression pattern of
TaNRT2 in 2.0 mmol/L NO3– was similar to that in 0.2
mmol/L NO3–.
2.3 Minimal NO3– concentration for the induction of
TaNRT2 transcript accumulation
To determine the minimal NO3– concentration for the
induction of TaNRT2 transcripts, wheat roots were treated
with a series of NO3– concentrations from 5 to 200 mmol/L
for 4 h. As shown in Fig.4, as low as 5 mmol/L NO3– could
induce the accumulation of TaNRT2 transcripts, but the
transcript level was quite low. The transcript levels in-
creased as the NO3– concentration increased, and dramati-
cally increase was seen when the NO3– concentration
shifted from 100 mmol/L to 200 mmol/L. Thus we concluded
that 0.2 mmol/L NO3– was enough to induce a high level of
TaNRT2 mRNA in the roots (Figs.3A, 4).
Fig.2. Phylogenetic relationship of NRT2 proteins. The homol-
ogy tree was created using DNAMAN, and the NRT2 homologs
used in this sequence alignment are Triticum aestivum TaNRT2.1
(AF288688), TaNRT2.2 (AF332214), TaNRT2.3 (AY053452),
Hordeum vulgare HvNRT2.1 (U34198) and HvNRT2.2
(U34290), Oryza sativa OsNRT2.1 (AB008519), Zea mays
ZmNRT2.1 (AY129953), Glycine max GmNRT2.1 (AF047718),
Arabidopsis thaliana AtNRT2.1 (AF019748), AtNRT2.2
(AF019749), Lycopersicon esculentum LeNRT2.1 (AF092655),
Nicotiana plumbaginifolia NpNRT2.1 (Y08210), Lotus japonicus
LjNRT2.1 (AJ292342), Aspergillus nidulans crnA (M61125), and
Chlamydomonas reinhanltii CrNRT2.1 (Z25438). The bar indi-
cates branch length.
Fig.3. Northern analysis of TaNRT2 expression in roots and
leaves and the effects of 200 mmol/L NO3- pretreatment time on
NO3- uptake rate. A. TaNRT2.3 expressions in the roots and
leaves treated with different N sources. Three-week-old wheat
plants were thoroughly rinsed with deionized distilled water be-
fore transferred to N-deprived nutrient solution. After 48 h of N
starvation treatment, the plants were shifted to the nutrient solu-
tion contained 0.0, 0.2, 2.0 mmol/L KNO3, and 1 mmol/L
(NH4)2SO4, pH 6.0, for the times indicated. The nutrient solution
was refreshed daily. For the Northern blot, 20 mg total RNA was
introduced into each lane and washed at medium stringency. B.
Net NO3- uptake rates of plants pretreated with 0.2 mmol/L
KNO3 for the times indicated. The 0.2 mmol/L NO3- pretreated
plants (for the procedure, see A) were shifted to 100 mL of 0.2
mmol/L KNO3 solution at pH 6.0. After 30 min at 25 ℃, the
amount of depleted NO3- was determined by nitration of sali-
cylic acid as described in Materials and Methods. Each of the
treatments consisted of four replicates.
Acta Botanica Sinica 植物学报 Vol.46 No.3 2004352
2.4 Correlation between the TaNRT2 transcript levels
and NO3– uptake rates
To test the hypothesis that TaNRT2 is involved in high-
affinity NO3– uptake, the net NO3– uptake rates were mea-
sured for wheat roots pretreated with 200 mmol/L NO3– for
different period of time (0-96 h) (Fig.3B). Before NO3–
induction, the NO 3– uptake ra te was only 0 .46
mmol·g–1FW·h–1. After feeding wheat roots with 200 mmol/
L NO3–, it increased dramatically and reached the peak at 8
h (3.33 mmol· g–1 FW· h–1). The NO3– uptake rate gradually
declined after 8 h but still remained at relative high level till
96 h (1.61 mmol· g–1 FW· h–1). The significant correlation
between the time course of TaNRT2 transcription accumu-
lation and the time course of the nitrate uptake rates for the
period of 0-24 h suggested that TaNRT2 played a very
important role in high-affinity NO3– uptake.
2.5 The expressions of TaNRT2 in split root systems
A root-splitting system was used to test the N signal
transduction in wheat plant. Insupplying NO3– to one part
of roots induced the expression of TaNRT2 in the other
part not supplied with NO3– (H2O-treated roots , and NH4+
-treated roots), the induction appeared at 2 h, and became
apparent at 8 h (Fig.5A, B).
3 Discussion
In this study, a new member (TaNRT2.3) of the TaNRT2
nitrate transporter gene family was cloned using RT-PCR,
cDNA library screening and 5-RACE approaches. Like other
TaNRT2 gene cloned, TaNRT2.3 contains all the conserved
motifs of NNP family (Trueman et al., 1996a). Homology
analysis indicated that TaNRT2.3 was closerly clustered to
its counterparts from monocots than to these from dicots
(Fig.2).
The expression of TaNRT2 genes is NO3–-inducible and
root-specific. These results, together with the sequence
analysis, provide the support for the participation of
TaNRT2.3 in HATS. We found that as low as 5 mmol/L NO3–
could induce the expression of TaNRT2, but the transcripts
did not increase significantly until the NO3– concentration
was at 200 mmol/L (Fig.4).
Nitrate induction can increase the activity of HATS up
to 30 folds in many plant species which vary depending on
plant species and genotypes (Quesada et al., 1997;
Amarashinghe et al., 1998; Lejay et al., 1999; Zhuo et al.,
1999; Vidmar et al., 2000a). In this study, the net NO3– up-
take rates in wheat roots was about 7 folds higher when
induced by 0.2 mmol/L NO3– (3.33 mmol·g–1FW·h–1) than
that in unduced roots (0.46 mmol·g–1FW·h–1) (Fig.3B), which
showed significant correlation between the time course of
TaNRT2 transcription accumulation and the time course of
the nitrate uptake rates for the period of 0 -24 h. Moreover,
the induced high-affinity NO3– uptake rates at 0.2 mmol/L
NO3– (Fig. 3B) were close to the Vmax values (3.17-4.91
mmol·g–1FW·h–1) of six Chinese wheat varieties which were
measured at 2 mmol/L NO3– (Tong et al., 1999), suggesting
that high-affinity uptake systems also play important roles
in the NO3– uptake when the NO3– concentrations in the
soils are high.
N cycling between the shoot and the roots was pro-
posed as the signal regulating N uptake (Cooper and
Clarkson, 1989; Marschner et al., 1996; Forde, 1999), which
was supported by this study. It has been reported that
over 50% of the N in wheat plants is cycling between shoots
and roots (Simpson et al., 1982; Cooper and Clarkson, 1989).
Fig.4. Effects of NO3- concentrations on TaNRT2 transcript
accumulation in roots. Three-week-old wheat plants were de-
prived of N for 48 h after grown in full strength nutrient solution
as described in Materials and Methods. The plants were then
shifted to the nutrient solution containing 0, 5, 25, 50, 100, and
200 mmol/L KNO3 for 4 h.
Fig.5. TaNRT2 transcript accumulation in split root systems
unevenly supplied with nitrogen. The root systems of three-
week-old wheat plants were divided into two parts after deprived
of N for 48 h. Then, one part was supplied with 2.0 mmol/L
NO3- as KNO3, the other part was supplied with nil nitrogen
(H2O-treated), or 2.0 mmol/L NH4+ as (NH4)2SO4 (NH4+-treated).
A. mRNA levels of TaNRT2 in H2O-treated roots. B. mRNA
levels of TaNRT2 in NH4+-treated roots.
ZHAO Xue-Qiang et al.: Isolation and Expression Analysis of a High-Affinity Nitrate Transporter TaNRT2.3 from Roots of Wheat 353
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(Managing editor: ZHAO Li-Hui)