Journal of Experimental Botany, Vol. 53, No. 375, pp.

17111721, August 2002

DOI: 10.1093/jxb/erf023

Effects of nitrate pulses on BnNRT1 and BnNRT2 genes:

mRNA levels and nitrate inux rates in relation to the
duration of N deprivation in Brassica napus L.
Sandrine Faure-Rabasse1, Erwan Le Deunff1, Philippe Laine1, James H. Macduff2 and Alain Ourry1,3
1

Institut de Recherche en Biologie Appliquee, UMR INRA/UCBN de Physiologie et Biochimie Vegetales,

Esplanade de la Paix, Universite de Caen, F-14032 Caen Cedex, France
2

Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Ceredigion SY23 3EB, UK

Received 17 October 2001; Accepted 18 April 2002

Abstract
A de-repression mechanism based on the disappearance of `signals' down-regulating N transporter activity has been proposed in the literature to explain the
transient increase of NO3 uptake by the roots following N deprivation in higher plants. This hypothesis
was investigated at the physiological and molecular
levels by measuring NO3 inux into roots of
Brassica napus L. grown under low or high external
concentrations of KNO3 following N deprivation.
Parallel measurements were made of endogenous
NO3, amino acid concentrations and abundance of
mRNA for BnNRT1 and BnNRT2, genes encoding
nitrate-inducible transport proteins. The effect of
NO3 pulsing on NO3 transport components in Ndeprived plants was also investigated by measuring
inux of high- and low-afnity transport system
(HATS and LATS) and assaying mRNA levels. Inux
of NO3 via HATS and LATS, and transcript levels of
BnNRT2 and BnNRT1 decreased with the duration of
N deprivation. The results suggested that the
absence of de-repression of NO3 inux and BnNRT2
gene expression following N starvation was related
to a high amino acid status. Pulsing with NO3
induced a large increase in BnNRT2 mRNA level, but
a comparatively small increase in NO3 inux via
HATS. The level of BnNRT1 mRNA also increased,
but there was no effect on LATS uptake activity. The
absence of a strict correlation between the NO3
transport activity and the mRNA BnNRT1 and
BnNRT2 levels is discussed in terms of possible
post-transcriptional regulation by the amino acids.
3

Key words: Brassica napus L., high-afnity nitrate

transporter, low-afnity nitrate transporter, nitrate inux,
nitrate transporter genes.

Introduction
The availability of N for uptake by roots is one of the main
factors limiting plant productivity in agricultural systems.
Although the roots of higher plants can absorb simple
organic compounds such as amino acids (Bush, 1993;
Chapin et al., 1993), most crop species depend on mineral
N forms, particularly NO3. Until relatively recently the
initial reduction of NO3 by nitrate reductase (NR) was
considered to be the main `growth limiting step' in the N
assimilation pathway. However, molecular studies of NR
(Hoff et al., 1994) and the use of transgenic plants, with
increased or decreased NR expression, have indicated little
relationship between plant growth and NR level or activity.
Consequently, attention has switched to the mechanisms
regulating the uptake of mineral N into the roots, at both
physiological and molecular levels (for review, see
Crawford and Glass, 1998; Forde, 2000; Touraine et al.,
2001).
It has been shown for a number of plant species that
inux of NO3 involves several different carrier systems.
At low external NO3 concentrations (< 1 mM), a
constitutive high-afnity transport system (CHATS),
operating at a low rate and displaying MichaelisMenten
kinetics, is regarded as genetically distinct and independent from an inducible high-afnity transport system
(IHATS) that is substrate saturable and inducible
(Siddiqi et al., 1990). Several full-length cDNAs with

To whom correspondence should be addressed. Fax: +33 2 31 56 53 60. E-mail: ourry@ibba.unicaen.fr

Society for Experimental Biology 2002

1712 Faure-Rabasse et al.

sequence homology to the crnA gene of Aspergillus

nidulans (Unkles et al., 1991, 1995) encoding a highafnity NO3 transporter, have been characterized from
Chlamydomonas reinhardtii (Quesada et al., 1994; Galvan
et al., 1996), Hordeum vulgare (Trueman et al., 1996;
Vidmar et al., 2000), Glycine max (Amarasinghe et al.,
1998), Nicotiana plumbaginifolia (Quesada et al., 1997;
Krapp et al., 1998), and Arabidopsis thaliana (Filleur and
Daniel-Vedele, 1999; Zhuo et al., 1999). In N. plumbaginifolia, G. max, A. thaliana, and H. vulgare similar
patterns for the expression of NRT2 and NO3 inux rates
of HATS have recently been reported (Krapp et al., 1998;
Amarasinghe et al., 1998; Zhuo et al., 1999; Vidmar et al.,
2000).
At higher external concentrations of NO3 (i.e. in the
mM range) the rate of NO3 uptake increases linearly with
increasing substrate concentration (Siddiqi et al., 1990).
This low-afnity transport system (LATS) has been
distinguished from the active high-afnity systems by its
lower sensitivity to cold temperatures and to several
metabolic inhibitors (Glass et al., 1990). At the molecular
level, a putative substrate inducible NO3 transporter has
been identied in Arabidopsis thaliana by using transgenic
plants with an insertional mutagen further selected for
resistance to chlorate-herbicide (Tsay et al., 1993). The
functional translation in Xenopus oocytes of AtNRT1:1
mRNA has been demonstrated successfully (Tsay et al.,
1993). Moreover it has been found that the corresponding
mRNA was synthesized predominantly in roots of plants
previously induced by NO3 supply (Tsay et al., 1993).
Lauter et al. (1996) and Zhou et al. (1998) have also
characterized two tomato genes and one rape gene,
respectively, for NRT1 (LeNRT1:1, LeNRT1:2 and
BnNRT1:2), which are inducible by NO3. Huang et al.
(1996) and Liu et al. (1999) proposed a two-gene model
for the low-afnity NO3 uptake system that may explain
the discrepancy between the inducibility of NRT1 and the
apparent constitutive expression of the low-afnity transport system of NO3. Recently, another NRT1 gene
(AtNRT1:2) has been characterized from Arabidopsis that
encodes a constitutive low-afnity nitrate transporter
(Huang et al., 1999). Consequently, the low-afnity
transport system seems to have an inducible (ILATS)
and a constitutive (CLATS) component, somewhat
analagous to that for the high-afnity nitrate uptake
system.
Nitrogen uptake is a highly regulated process, the rate of
uptake matching the rate at which N is required for the
synthesis and expansion of new tissues (Touraine et al.,
1994). One of the most commonly cited mechanisms for
co-ordinating NO3 uptake by the roots and NO3 assimilation in the shoot is the model proposed by Clarkson
(1986). It postulates that nitrogen uptake is normally
down-regulated if N is freely available to the plant. The
transient increase in uptake rate occurring during the rst

2448 h after N starvation is interpreted as de-repression

resulting from the progressive disappearance of `signals'
down-regulating N-transporter activity. This model was
conrmed at the molecular level in Arabidospsis N-starved
plants and revealed at least two discrete processes: an
initial AtNRT2:1 de-repression observed 2448 h after
starvation, followed by a down-regulation corresponding
to a de-induction of the gene AtNRT2:1 under more
prolonged starvation (Lejay et al., 1999). AtNRT1 was not
subjected to this de-repression process or affected by the
N-status of the plant (Lejay et al., 1999).
The mechanism by which shoot demand for N, following N deprivation, regulates N uptake could rely on a
common amino-N transport pool between shoot and root
(Cooper and Clarkson, 1989). Using specic inhibitors of
GS, GOGAT or amino transferases and externally supplied
amino acids, Lee et al. (1992) showed under N starvation
that treatments raising intracellular concentrations of
glutamine and/or asparagine led to the suppression of net
uptake of NH4+ and NO3 by maize seedlings. Conversely,
conditions, which lowered root glutamine and/or asparagine, stimulated the net uptake of NO3. Using a GS
specic inhibitor (azaserine) in Hordeum vulgare plants,
Vidmar et al. (2000) suggested that glutamine (but not
glutamate) is responsible for the down-regulation of
HvNRT2 expression. Nevertheless, the model of N uptake
regulation by phloem-translocated amino acids remains
controversial (Laine et al., 1995; Tillard et al., 1998).
Split-root experiments have shown that N deprivation to
half of the root system can be entirely and rapidly
compensated for by an increase in NO3 inux into the
other half of the root system supplied with NO3, whilst
levels of amino acids in the roots were unaffected or only
slightly increased.
The aims of this study were (i) to investigate the
occurrence of the de-repression mechanism postulated by
Clarkson (1986) for NO3 uptake by Brassica napus L., at
the inux and transcript levels, following N deprivation,
and (ii) to measure the concurrent changes in the main N
substrate pool in root and shoot tissues. The experimental
approach included exposure of NO3-deprived plants to a
brief NO3 pulse in order to alleviate the de-induction
mechanism of NO3 during starvation, so that the effect of
NO3 induction could be discriminated from other putative
regulation processes. As a preliminary, the kinetics of
NO3 inux were also established in induced and noninduced plants.
Materials and methods
Plant culture
Experiment 1 (Culture conducted in Caen, France; results in Fig. 1):
Brassica napus L. cv. Capitol were germinated and grown
hydroponically (50 seedlings per 1 dm3 plastic tank) in a greenhouse.
The aerated nutrient solution contained 0.4 mM KH2PO4, 0.15 mM

N deprivation in Brassica napus L. 1713

K2HPO4, 1.0 mM K2SO4, 0.50 mM MgSO4, 3.0 mM CaCl2, 0.20 mM
FeNa EDTA, 14 mM H3BO3, 5.0 mM MnSO4, 3.0 mM ZnSO4,
0.7 mM CuSO4, 0.7 mM (NH4)6Mo7O24, and 0.1 mM CoCl2, and was
renewed every 3 d. Natural light was supplemented with highpressure sodium lamps (170 mmol m2 s1 of photosynthetically
active radiation at the height of canopy) for 16 h d1. The
thermoperiod was 24 C (day) and 18 C (night).
Experiment 2 (Culture conducted in Aberystwyth, UK; results in
Figs 27): Seeds of Brassica napus L. cv. Capitol were imbibed for
48 h on tissue paper saturated with 10 mM CaSO4 and then sown into
six culture units of a owing solution culture (FSC) system
incorporating automatic control of concentrations of NO3, K+ and
H+ in solution (Clement et al., 1974; Hatch et al., 1986). Each
culture unit contained 200 dm3 of recirculating nutrient solution and
24 culture vessels, each containing three plants. The FSC system was
located in a greenhouse, solution temperature was maintained at
2060.5 C and air temperature at 2062/1561 C day/night (09.00
21.00 h) throughout the experiment. The plants were established
under natural illumination until day 17 after sowing. Supplementary
light (09.0021.00 h) of 200 mmol m2 s1 PAR was provided
between days 1824 by a single 400 W SON-T lamp (Philips
Lighting Ltd, Croydon, Surrey, UK) suspended 1.5 m above the
surface of each culture unit. On day 24 after sowing, natural light
was excluded and thereafter illumination was provided by paired 400
W SON-T and HPI/T lamps (Philips Lighting Ltd, Croydon, Surrey,
UK) giving 500 mmol m2 s1 PAR at canopy height, over 12 h.
Nutrient concentrations in each culture unit were initially (mM):
NO3, 250; K+, 250; H2PO4, 50; Mg2+, 100; SO42, 325; Fe2+, 5.4;
with micronutrients as previously described by Clement et al.
(1978). All culture units were drained and relled with fresh nutrient
solution of the same composition on day 18 after sowing. Nutrient
concentrations were allowed to deplete by plant uptake until day 24
when automatic monitoring (every 27 min) and resupply of nutrients
was introduced. Thereafter, the concentration of K+ was maintained
at 2062 mM in each culture unit by automatic resupply at a rate
equal to the depletion of K+ by plant uptake. All other nutrients
except NO3 were supplied automatically in xed ratios to the net
uptake of K+, for 1 mol of K, 0.645, 0.057, 0.045, 0.00075, and 0.522
mol of S, Mg, P, Fe, and Ca, respectively, were supplied, with
micronutrients as described by Clement et al. (1978). Solution pH
was maintained at 6.060.1 by automatic delivery of H2SO4 or
Ca(OH)2 to each culture unit throughout the experiment. A
concentration of 2062 mM NO3 was maintained automatically in
each culture unit from day 24 until the start of the N deprivation
period (day 26). Net uptake of K+ and NO3 was calculated on an
hourly basis from the amounts required to maintain the `set point'
concentrations in the owing solutions.
Experimental treatments
Experiment 1 (results in Fig. 1): On day 15 after sowing, half of the
total number of plants previously grown without N was supplied
with 1 mM KNO3 for 24 h to induce the NO3 uptake system. Nitrate
inux rates were measured on day 16 with induced and non-induced
plants.
Experiment 2 (results in Figs 27): On day 26 after sowing the
automatic supply of NO3 was terminated to culture units and the
concentration of NO3 in these units allowed to deplete by plant
uptake to <1 mM over 2 h. This point was taken as time zero (t0) for
the N-deprivation treatment.
Nitrate inux rates and mRNA abundance were measured on Ndeprived plants (N plants) at intervals during the period of N
deprivation (0, 24, 48, 72, and 96 h). On each occasion additional
batches of plants were exposed to a NO3 pulse 412 h prior to these

measurements, for comparison with N plants. All measurements

were made on three culture vessels (nine plants) for each combination treatment and time. The NO3 pulse exposure was performed
by transferring plants for a period of 30 min into separate FSC units
containing 200 dm3 of full (N) nutrient solution with the addition of
100 mM NO3. Afterwards the roots were rinsed twice in 1 mM
CaSO4 solution for 1 min before Returning the plants to their
original N culture units until required for measurement of inux or
mRNA. For one batch of plants, the NO3 pulse application was
performed 12 h prior to the measurement of NO3 inux, and for
another batch of plants the NO3 pulse exposure was performed 4 h
prior to their harvest for mRNA analysis.
Measurement of NO3 inux and plant harvesting
Experiment 1 (results in Fig. 1): Inux of 15NO3 was measured on
three batches of 50 seedlings on each occasion. The roots of each
batch were rinsed twice for 1 min in 1 mM CaSO4 solution at 20 C
before being immersed for 5 min in 250 ml of nutrient solution
(described above for Experiment 1) containing different concentrations (10, 25, 50, 75, 100, 135, 250, 1000, 2500, 5000, 7500 mM) of
K15NO3 (99.9% atom% 15N). The extent of NO3 depletion from
these solutions during the inux assays was less than 4% in each
case. The roots were then rinsed twice for 1 min in 1 mM CaSO4
solution at 4 C before harvesting. Shoots and roots were separated,
weighed and frozen in liquid nitrogen prior to freeze-drying. The
freeze-dried tissues were weighed and ground to a ne powder and
stored at 80 C for subsequent analysis.
Experiment 2 (results in Figs 27): NO3 inux was measured 2 h
prior to the end of the photoperiod, on each occasion using six
culture vessels per treatment. The vessels were removed from the N
culture units and their roots immersed for 5 min in nutrient solution
of the same composition (1 dm3 per vessel) with the addition of
either 100 mM (HATS activity) or 5 mM (LATS activity) of K15NO3
(labelled at 99.9 and 30 atom% 15N, respectively). Roots were rinsed
twice for 1 min in 1 mM CaSO4 solution at 4 C and harvested
immediately. Shoots and roots were separated and treated as
described previously for subsequent analysis. Three additional
vessels per treatment were harvested at the same time for RNA
analysis.
Nitrogen, nitrate, isotope, and amino acid analysis
Nitrogen and 15N contents of plant freeze-dried samples were
measured in continuous ow using a C/N analyser linked to an
isotope ratio mass spectrometer (Roboprep CN and 2020 mass
spectrometer, Europa PDZ, Crewe, UK). Inux of NO3 was
calculated from the 15N contents of the roots and shoots. Nitrate and
amino acids were extracted from freeze-dried tissue (100 mg) with
10 ml of methanol:dichloromethane:water (60:25:15, by vol.) for 1
h. After centrifugation (10 000 g, 20 min), the pellet was re-extracted
under the same conditions. The supernatants were mixed, 5 ml of
dichloromethane and 5 ml of water were added, and stored overnight
at 4 C. The dichloromethane phase was discarded and the remaining
upper phase containing amino acids, sugars and NO3 was collected
and evaporated to dryness under vacuum at 30 C. The residue was
resuspended in 2 ml of water and ltered through a 0.45 mm
membrane. 1 ml was used for the quantication of nitrate by high
performance anionic chromatography (DX 100 with an Ionpac AS9
analytical column, Dionex Corporation, Sunnyvale, USA) and the
other 1 ml was used for amino acid analysis. After a dilution by 10,
aliquots of 15 ml each were analysed by high performance liquid
chromatography (HPLC) as ophthaldialdehyde derivatives on a C-18
column using Gold System 8.0 (Beckman Instruments, San Ramon,
CA, USA) as previously described by Murray et al. (1996) and

1714 Faure-Rabasse et al.

Fig. 1. Changes in 15NO3 inux into roots of intact Brassica napus L. seedlings as a function of low (A) or high nitrate (B) concentrations in the
uptake solution. Plants were either non-induced (i.e. grown without N supply, open squares) or induced prior to measurements (1 mM KNO3
supplied during previous 24 h, lled squares). Inux ascribed to a constitutive high-afnity transport system (CHATS) and a constitutive lowafnity transport system (CLATS) was calculated from measurements on non-induced plants. Inux through an inducible high-afnity transport
system (IHATS) and both IHATS+ILATS was calculated by subtracting the rates measured with non-induced plants from those with induced
plants. The inset in (B) represents the `putative ILATS' and CLATS activities after subtraction of theoretical calculated IHATS and CHATS inux
values from the inux at 5 mM (HATS+ILATS). Equations for MichaelisMenten and linear components were calculated using Sigma plot
software (Sigma Co, USA). Vertical bars indicate 6SD for n=3 when larger than the symbol.

specic amino acids were quantied using the a-aminobutyric acid

as an internal standard.
Cloning of BnNRT2 and BnNRT1
BnNRT2 and BnNRT1 cDNA were obtained by the conjunction of
RT-PCR, 3 and 5 RACE with the aid of the Marathon cDNA
amplication kit (Clontech Laboratories, Palo Alto, USA). One
specic pair of oligonucleotide primers was designed for BnNRT1
from the sequence of BnNRT1 gene (Muldin and Ingemarsson, 1995)
and AtNRT1 gene (Tsay et al., 1993). For BnNRT2, the primers were
obtained from Hordeum vulgare (Trueman et al., 1996). These two
pairs of oligonucleotide primers: NRT1F (5-TAC CGG GAC TGA
GAC CAC CAA GAT-3) NRT1R (5-GGA CTG CGC GAC CGA
TAA TGT-3) and NRT2F (5-GGT TGC ACA TCA TCA TGG
GAG TC-3) NRT2R (5-GCA ACG TGC AGG CAA CTA TCA
TCA CTC CC-3), were used in an RT-PCR reaction to amplify a
736 bp and 643 bp fragments. Therefore, these resulting probes were
gel puried (Quiagen Gmbh, Hilden, Germany) and cloned in a
pGEM-T vector (Promega Corporation, Madison, USA). Plasmids
DNA were extracted and sequenced to check the sequence
similarity.
To obtain the full-length sequence the method of 3 and 5 RACE
was used. 1 mg Poly (A)+ RNA was used for the rst and secondstrands cDNA synthesis before the ligation of double strand cDNA
adaptator according to the manufacturer's instructions (Clontech
laboratories, Palo Alto, USA). 5 and 3 Rapid Amplication of
cDNA ends (RACE) cloning of the BnNRT2 and BnNRT1 cDNA
were conducted by using two pairs of designed primers: RACENRT2F
(5-GCT TCA CAC TGC CGG AAT CAT CGC AGC-3)
RACENRT2R (5-GTG GCT CCA CAA GCT GCT TGT GCT CCT3) and RACENRT1F (5-GGC TAT GGC ATT TGC GCG TTG GCA
ATC G-3) RACENRT1R (5-GAC GGG TCC GAT GGC AAC
TCG AGC CGC-3) that produce overlapping of 5 and 3-RACE
products. Touchdown Polymerase Chain Reaction was performed
with Advantage 2 Polymerase Mix (Clontech laboratories, Palo Alto,
USA) for 35 cycles: 5 cycles at 94 C for 30 s, 72 C for 4 min; 5
cycles at 94 C for 30 s, 70 C for 4 min and 25 cycles at 94 C for
30 s, 68 C for 4 min. Amplied products were then gel puried with

QIAquick Gel Extraction Kit (Quiagen Gmbh, Hilden, Germany)

and cloned directly into the pGEM-Teasy cloning vector following
the manufacturer's instructions (Promega Corporation, Madison,
USA). For nucleotide sequence analysis, plasmids were isolated with
exiprep kit (Amersham, Buckinghamshire, UK), sequenced with
ABI PRISM dRhodamine terminator of Perkin Elmer and run on an
ABI PRISM 377 automated sequencer (Perkin Elmer Applied
Biosystem). Analysis of the sequenced fragment by Blast algorithm
showed that the sequence of BnNRT1 (EMBL access. AJ278966)
had 96% of similarity with the clone BnNRT1 (Muldin and
Ingemarsson, 1995) and that BnNRT2 (EMBL accession no.
AJ293028) had 89% of similarity with AtNRT2 clone (Zhuo et al.,
1999).
Synthesis of cDNA selective RT-PCR probes
The cDNA selective probes were obtained by RT-PCR with a
specic pair of primers for the BnNRT1 gene (NRT1FNRT1R) and
for the BnNRT2 gene (NRT2FNRT2R). 1 mg of Poly (A)+ RNA were
used for reverse transcription with the M-MLV ReverseTranscriptase (Life Technologies, Paisley, UK) and primed with
each specic reverse primer according to the manufacturer's
instructions. Then PCRs were performed with 2.5 U Taq DNA
polymerase (Life Technologies, Paisley, UK) in 50 ml reaction
containing 1 ng of cDNA, 50 pmol of each primer, 1.5 mM
magnesium chloride, and 0.2 mM dNTPs. The reactions were
performed for 35 cycles at 95 C for 1 min, 50 C for 1 min, and
72 C for 1 min followed by a nal extension step at 72 C for 5 min.
Amplied products of BnNRT1 (736 bp) and BnNRT2 (643 bp) were
puried and cloned as described in previous section. After plasmid
digestion, cDNA fragments were labelled with a32P dCTP (3000 Ci
mmol1) by using random priming NEBlot kit (New Englands
Biolabs, Beverley, USA).
Isolation of RNA and northern blot analysis
20 mg of total RNA previously extracted from root tissue with TriReagent according to the manufacturer's instructions (MCR
Euromedex, Cincinnati, USA), were fractionated on 1.2% agarose
gel containing formaldehyde and transferred to Hybond-N+ blotting

N deprivation in Brassica napus L. 1715

Fig. 2. Effect of N deprivation on the growth of Brassica napus L.

plants: total dry weight of plants supplied continuously with 20 mM
NO3 (lled squares) and plants deprived of NO3 (open squares).
Vertical bars indicate 6SD for n=3 when larger than the symbol.

membranes (Amersham, Buckinghamshire, UK) using 103 SSC

(1.5 M NaCl, 0.15 M sodium citrate, pH 7), and xed onto the
membranes by backing at 80 C for 2 h. After blotting, the blots were
prehybridized for 2 h at 62 C in the Church buffer (Church and
Gilbert, 1984). After addition of the probes, membranes were
hybridized overnight at 62 C in buffer containing: SDS 7%,
Na2HPO4 0.25 M, EDTA 2 mM, heparin 0.2 mg ml1, and calf
thymus DNA 0.1 mg ml1 (Church and Gilbert, 1984). Then, the
membranes were washed successively with: (1) 23 SSC, 0.1% SDS
20 min at 50 C, (2) 13 SSC, 0.1% SDS 20 min at 60 C, (3) 0.23
SSC, 0.1% SDS 20 min at 60 C before being analysed.
Analysis of NRT1 and NRT2 transcript levels
The blots were exposed to radiographic Kodak BioMax MS lm for
35 d at 80 C and developed as described by the manufacturer
(Eastman Kodak Company, New York, USA). The signal intensities
have been quantied by image analyser (Wilbert Lourmat, France).
In order to correct RNA loading differences, the ribosomic RNA 28S
and 18S stained with ethidium bromide were quantied and used for
the determination of NRT genes transcript levels (Fig. 4A, B).

Fig. 3. Changes in 15NO3 inux into roots of intact Brassica napus L.

seedlings from solutions containing (A) 100 mM or (B) 5 mM KNO3,
after depriving plants of NO3 (open squares). Also shown are the
effects of a 30 min pulse exposure with 100 mM NO3 supplied 12 h
prior to measurement of inux (lled squares). The insert shows the
differences in inux between plants with and without a NO3 pulse,
measured at 100 mM (open circles) and 5 mM KNO3 (lled circles).
Vertical bars indicates 6SD for n=3 when larger than the symbol.

Results
Kinetics of NO3 inux in induced and non-induced
plants

At least three different systems for NO3 inux were

distinguished in Brassica napus L. on the basis of the
kinetic characterization (Fig. 1). Two of them were
constitutive systems in non-induced seedlings. At concentrations of NO3 below 200 mM, inux rates approximated
MichaelisMenten kinetics (Fig. 1A, CHATS), with an
estimated Vmax of 26.3 mmol h1 g1 DW and a Km of
15.9 mM. A second, low afnity system (Fig. 1B, CLATS)
exhibited non-saturable kinetics between 17.5 mM NO3.
When seedlings were induced by exposure to 1 mM NO3
for 24 h prior to assaying inux, NO3 inux increased

across the entire range of concentrations. The concentration effects for the inducible high-afnity (Fig. 1A,
IHATS) and the putative inducible low-afnity (Fig. 1B,
ILATS) systems were calculated by subtracting inux
measured in non-induced plants from the rates measured
with induced plants. The inducible high-afnity system
approximated MichaelisMenten kinetics at substrate
concentrations lower than 1 mM (Fig. 1A, IHATS), with
a Vmax of 135 mmol h1 g1 DW and a Km of 85 mM. Inux
attributable to the IHATS was 5-fold higher than the one
associated with the CHATS. The kinetics of NO3 uptake
determined at high concentrations (17.5 mM NO3)
seems to show that the LATS system is devoid of an

1716 Faure-Rabasse et al.

Fig. 4. Changes in relative abundance of BnNRT1 (white bars), BnNRT2 (black bars) mRNA in roots of Brassica napus L. over 4 d following
withdrawal of the N supply to plants. Plants either received (B, D) or did not receive (A, C) a NO3 pulse (30 min, 100 mM NO3) 4 h prior to the
harvest of the roots for mRNA extraction and analysis by Northern hybridization (C, D). The relative transcript level of NRT genes was corrected
after quantication of RNAr 28S and 18S loading differences under UV by image analyser (A, B). Data for only one repetition of three
experiments is presented. The EMBL nucleotide sequence database accession numbers for BnNRT1 and BnNRT2 are AJ278966 and AJ293028,
respectively.

inducible component (Fig. 1B, inset). Indeed, the values of

`ILATS activity' calculated by subtraction of the theoretical IHATS (Vmax of 135 mmol h1 g1 DW and a Km of
85 mM) from both IHATS+ILATS activity (Fig. 1B) do not
show a signicant difference with the values calculated for
CLATS activity (Fig. 1B, inset). Taken as a whole these
results conrm that 100 mM and 5 mM external concentrations of NO3 were appropriate for assessing inux
mediated, respectively, by the high- and the low-afnity
systems.
Effects of N deprivation and NO3 pulses on inux
rates and gene expression
Plant growth (i.e. dry matter production) was not
signicantly affected during the rst 4 d of NO3
deprivation (Fig. 2). Consequently, effects of N
deprivation on NO3 inux and gene expression during
this period were unrelated to changes in growth rate or
senescence. Nitrate inux through the high-afnity

systems (Fig. 3A, 100 mM) decreased progressively

over the four days of N deprivation (from 125 to 30
mmol h1 g1 DW). Seventy per cent of this decline
was observed during the rst 48 h of N starvation.
Pulsing the plants with NO3 for 30 min, 12 h before
inux was measured, reversed the trend for the rst
24 h and lowered the subsequent rate of decline in
inux (Fig. 3A). The NO3 pulse application increased
HATS activity during the rst 24 h and resulted in a
higher inux during the next three days, (77 mmol h1
g1 DW after 4 d) compared with N plants, the
difference being at least 2-fold throughout.
The activity of the low-afnity system was determined
by measuring inux from 5 mM NO3. Uptake from this
higher NO3 concentration resulted from LATS and HATS
activities (Fig. 1B). Inux decreased with increasing
duration of N deprivation (Fig. 3B), declining by 60%
over four days. The overall trend was similar to that
observed for the high-afnity system. Pulsing plants with

N deprivation in Brassica napus L. 1717

Fig. 5. Changes in endogenous NO3 and total amino acid concentrations in the shoots (A, C) and roots (B, D) of Brassica napus L. plants as a
function of the time of NO3 deprivation. Plants either received (lled squares) or did not receive (open squares) a NO3 pulse (30 min, 100 mM
NO3) applied 12 h prior to harvest. Vertical bars indicate 6SD of the mean for n=3 when larger than the symbol.

NO3 for 30 min, 12 h before inux was measured,

increased inux through LATS+HATS by about 40%, and
delayed the decline in inux by 24 h (Fig. 3B). Comparison
of the differences in inux at 100 mM and 5 mM
attributable to NO3 pulsing (Fig. 3B inset) suggested
that the main effect of the NO3 pulse was to increase
IHATS.
The abundance of mRNA encoding for the BnNRT1 and
BnNRT2 NO3 transporters, showing respectively 96% of
homology with BnNRT1 clone (Muldin and Ingemarsson,
1995) and 89% of homology with AtNRT2:1 (Zhuo et al.,
1999), were followed in N plants with and without
exposure to the NO3 pulse. Southern analysis (data not
shown) suggested that these probes BnNRT1 and BnNRT2
recognized at least two genes of each family. Therefore,
the probes used in Northern studies (described below)
reveal the expression of several BnNRT1 or BnNRT2
genes. In N plants, transcripts levels of BnNRT1 and
BnNRT2 (Fig. 4A, C) decreased as a function of the
duration of NO3 deprivation. In comparison with the
plants (t0), pulsing plants with NO3 for 30 min, 4 h prior to
harvest, increased by 4 the relative abundance of BnNRT1
mRNA and by 14 the relative abundance of BnNRT2
mRNA (Fig. 4B, D).

Effects of N deprivation and NO3 pulsing on

endogenous NO3 and amino acid concentrations in
shoots and roots
The effect of exposing plants to a NO3 pulse during N
deprivation on endogenous NO3 and amino acid concentrations in shoots and roots was investigated to establish
whether there was a relationship between variation in these
compounds (Fig. 5) and nitrate uptake (Figs 3, 4).
Generally, the effect of the NO3 pulse was more
pronounced in shoots (Fig. 5A, C) than in roots (Fig. 5B,
D), and particularly so for amino acid concentrations
(Fig. 5C).
The concentration of NO3 was initially higher in the
shoots than in the roots (Fig. 5A, B), but following
termination of the N supply it decreased more rapidly in
the roots (Fig. 5B), reaching negligible levels after 2 d,
compared with 4 d in the shoots. Pulsing the plants with
NO3, 12 h prior to harvest, accelerated the depletion of
NO3 in the shoots between 2448 h of N deprivation, but
had no other effect on endogenous NO3 concentrations in
shoots or roots.
Surprisingly, total amino acid concentrations increased
in both shoots (Fig. 5C) and roots (Fig. 5D) of N plants
during the rst 48 h of N deprivation, but decreased

1718 Faure-Rabasse et al.

The dynamics of the physiological attributes and

molecular components of NO3 transport as affected by
N deprivation and by N deprivation + NO3 pulsing are
summarized on a relative basis in Fig. 7. In the case of N
deprivation, the effects are relative to plants at t0, the start
of the N deprivation period (Fig. 7A, B), whilst the NO3
pulsing effect is relative to the corresponding plants
without NO3 pulsing (Fig. 7C, D). Expression of the
results in this form highlights the positive effect of NO3
pulsing on mRNA BnNRT2 levels (Fig. 7D).
Discussion
Nitrate uptake systems in Brassica napus L.
Nitrate inux in Brassica napus L. appears to involve at
least three kinetically different transport systems, including constitutive low-afnity, inducible and constitutive
high-afnity systems (Fig. 1A, B). At high nitrate
concentrations, this kinetic study does not reveal the
existence of an inducible low-afnity transporter system as
previously reported by Touraine and Glass (1997) in
Arabidopsis thaliana. Wang et al. (1998) and Liu et al.
(1999) had suggested that the protein carrier AtNRT1.1
may also be an important component of both the highafnity and the low-afnity nitrate uptake systems and that
AtNRT1.1 may be a dual-afnity nitrate transporter in
Arabidopsis thaliana. In addition, unexpected expression
of this transporter in rapidly dividing cells prompted Guo
et al. (2001) to re-examine AtNRT1.1 functions. They
suggested that this nitrate transporter supports the growth
of nascent organs in roots and shoots and that it could also
play a role in the induction of the owering.

Fig. 6. Comparison of specic amino acid concentrations in the roots

of Brassica napus L. plants either receiving (black bars) or not
receiving (white bars) a NO3 pulse (30 min, 100 mM NO3) after 0,
48 and 120 h of N deprivation. Vertical bars indicate 6SD of the
mean for n=3 when larger than the symbol.

thereafter. Pulsing the plants with NO3 did not affect the
trend in amino acid concentration in the roots, although the
absolute values were invariably slightly lower compared
with those in N plants (Fig. 5D). By contrast, NO3
pulsing markedly accelerated the decrease in total amino
acid levels in the shoots (Fig. 5C). In terms of specic
amino acids in the roots, the largest decrease attributable to
NO3 pulsing was in glutamate followed by glutamine,
after 48 h of N starvation (Fig. 6A, B), whereas the sum of
the other amino acids increased slightly (Fig. 6C).

Effect of N deprivation
The concept that N uptake is regulated at the whole plant
level to match the N demand associated with growth is
widely accepted (Touraine et al., 1994). Although the
mechanism of regulation at this level is not fully understood, several authors have suggested that the size,
composition or rate of internal recycling of the free
amino acid pool within the plant might be involved
(Cooper and Clarkson, 1989; Marschner et al., 1996). One
of the simplest experimental approaches for altering the
internal availability of N in roots and shoots is the
termination of the external N supply to the plant. In the
short-term (up to 4 d), it has been hypothesized that N
deprivation might progressively up-regulate the capacity
of the N uptake system and its transporters (Clarkson,
1986). This up-regulation would be the result of depletion
of the internal NO3 and amino acid pools through
continued translocation, assimilation and protein synthesis
in growing tissues (Cooper and Clarkson, 1989).
In the present study with Brassica napus L. growth was
reduced only after 5 d of NO3 deprivation although

N deprivation in Brassica napus L. 1719

Fig. 7. Relative effects of N deprivation (A, B) and NO3 pulsing (30 min, 100 mM NO3) of N-deprived plants (C, D) on different physiological
attributes (A, C) and molecular (B, D) components of NO3 transport. For N deprivation, the values are expressed relative to control plants (t0).
For NO3 pulsing the values are expressed relative to corresponding plants without pulsing: root amino acid concentration (lled circles), root
nitrate concentration (open circles), HATS inux (open triangles), LATS inux (inverted open triangles), BnNRT1 mRNA level (inverted lled
triangles), BnNRT2 mRNA level (lled triangles), and photosynthesis (open squares).

photosynthesis was affected sooner (Figs 2, 7). A comparison between the concentrations of N compounds
occurring in B. napus plants grown under eld conditions
(Colnenne et al., 1998) and those measured in the present
study (total N higher than 6% DW, high NO3 and aminoacid concentrations), suggests that the latter had optimum
N status at the start of the period of N starvation.
Consequently, it is likely that N uptake systems were in
a down-regulated state at t0, and hence the effects of N
deprivation on their activity were expected to be signicant. However, at the carrier level and at the levels of
putative gene expression the results showed that NO3
uptake activity was not up-regulated shortly after the
withdrawal of the external supply. Indeed, contrary to the
results obtained by Lejay et al. (1999) in Arabidopsis, no
correlative de-repression of both activity of high-afnity
NO3 uptake systems and BnNRT2 expression was
observed during the entire period of N deprivation in
Brassica napus. N starvation resulted in decreased activity
of both low- and high-afnity NO3 uptake systems and the
expression of BnNRT1 and BnNRT2.
The absence of up-regulation induced by N starvation in
the short-term has also been reported by Siddiqi et al.

(1989) in Hordeum vulgare, where N deprivation of plants

previously fed with NO3 caused a decrease in NO3 inux
during the rst 24 h to levels similar to those in plants
which had not been exposed to NO3. Under these
experimental conditions, it is suggested that the unexpected repression of NO3 inux in Brassica napus was
due to a rapid assimilation of nitrate pools during the rst
48 h of N starvation, resulting in increased levels of free
amino acids. This phenomenon may be an adaptive
mechanism enabling fast-growing species like B. napus
to maintain rapid growth during a short period of N
starvation. Beyond 48 h of N deprivation it is probable that,
in the absence of an external NO3 signal, the de-induction
mechanism suggested by Clarkson (1986), more than
counterbalanced the effect of decreasing levels of amino
acids, leading to a decline in nitrate inux and expression
of BnNRT genes.
The effect of a NO3 pulse during N deprivation:
maintaining NO3 induction?

Because several components of the NO3 uptake and

assimilation processes are known to be substrate-inducible
(Crawford, 1995; Stitt, 1999), the application of a NO3

1720 Faure-Rabasse et al.

pulse during N deprivation would be expected to increase

the potential activity of the NO3 transport systems, via
increased transcription and translation of genes encoding
for transporters (Siddiqi et al., 1990; Redinbaugh and
Campbell, 1991). The results showed that a NO3 pulse
during N deprivation increased the activity of HATS and
the mRNA BnNRT2 level compared with untreated (N)
plants. This component of NO3 pulse-induced up-regulation appears to be inversely correlated with endogenous
nitrate levels. By contrast, it was found that the LATS
activity was unaffected by NO3 pulsing, despite the
increase in mRNA BnNRT1 level. This suggests that these
two families of NO3 transporters are differentially regulated with respect to NO3 per se.
Relationships between inux rates and gene
expressions

The responses measured in mRNA NRT2 levels and NO3

inux to N deprivation were generally similar in trend.
However, they differed in magnitude (Fig. 7). For
example, the relative increase in BnNRT2 mRNA level
associated with exposure to a NO3 pulse was greater than
the corresponding increase in NO3 inux mediated by the
HATS. As reported in Nicotiana plumbaginifolia by
Fraisier et al. (2000), the present results suggest that the
HATS activity may be subject to a post-transcriptional
regulation. The observation that the concentration of free
amino acids (notably Gln) was high during the rst 48 h of
N deprivation suggests that the amino acid pool could be
involved in this post-transcriptional regulation. However,
although this interpretation would appear to support the
ndings of Fraisier et al. (2000), it is possible that mRNAs
for other genes encoding for components of the HATS
were not picked up by the probe used in the current study.
The increase in endogenous free amino acids levels
coinciding with a decrease in NO3 inux measured in
present work, together with the results obtained with an
external supply of glutamine in Nicotiana plumbaginifolia
(Krapp et al., 1998) and in Hordeum vulgare (Vidmar et al.,
2000) support the hypothesis that amino acids are involved
in the transcriptional and/or post-transcriptional regulation
of inux during the rst 48 h of N deprivation. However,
the case for their involvement after 48 h is far less
convincing. In the plants exposed to a NO3 pulse, the
decrease in amino acid concentrations (notably Gln and
Glu) between 48 h and 120 h of N deprivation coincided
with a decline in HATS activity. This suggests that
`signals' other than amino acids are implicated in the
regulation of NO3 uptake at this time. A detailed
quantication of the translocatory ux and cellular
compartmentation of amino acids in parallel with measurements of inux and NRT2 mRNA abundance is required
before the speculative hypothesis for the transcriptional or/
and post-transcriptional role of endogenous amino acids in

the regulation of NO3 uptake by Brassica napus can be

conrmed or refuted.
In conclusion, these results indicate that regulation of
NO3 inux activity in Brassica napus during N starvation
involves several components acting differentially over
time. These include the putative negative feedback regulation by amino acids at transcriptional and/or posttranscriptional levels, linkage to the endogenous nitrate
pool and the inductive effect of exogenous NO3 on the
expression of NO3 transporter genes.
Acknowledgements
This research was supported in part by an INRA/BBSRC grant. The
French authors would like to acknowledge the contribution of staff
from the Institute of Grassland and Environmental Research,
Aberystwyth, for their collaboration in this work. We thank Dr B
Forde (Department of Biological Sciences, University of Lancaster,
UK) for his generous gift of the primers BnNRT2:1 of Hordeum
vulgare L. and Christelle Le Dantec (Research Institut of Applied
Biology, Caen University) for her kind assistance with the
sequencing.

References
Amarasinghe BHR, De Bruxelles GL, Braddon M, Onyeocha I,
Forde BG, Udvardi MK. 1998. Regulation of GmNRT2
expression and nitrate transport activity in roots of soybean
(Glycine max). Planta 206, 4452.
Bush DR. 1993. Proton-coupled sugar and amino acid transporters
in plants. Annual Review of Plant Physiology and Plant
Molecular Biology 44, 513542.
Chapin III FS, Moilanen L, Kielland K. 1993. Preferential use of
organic nitrogen for growth by a non-mycorrhizal artic sedge.
Nature 361, 150153.
Church GM, Gilbert W. 1984. Genomic sequencing. Proceedings
of the National Academy of Sciences, USA 81, 1991.
Clarkson DT. 1986. Regulation of absorption and release of nitrate
by plant cells: a review of current ideas and methodology. In:
Lambers H, Neeteson JJ, Stulen I, eds. Developments in plant and
soil sciences: fundamental, ecological and agricultural aspect of
nitrogen metabolism in higher plants. Dordrecht: Marttinus
Nijhoff Publishers, 327.
Clement CR, Hopper MJ, Canaway RJ, Jones LPH. 1974. A
system for measuring the uptake of ions by plants from solutions
of controlled composition. Journal of Experimental Botany 25,
8199.
Clement CR, Hopper MJ, Jones LPH. 1978. The uptake of nitrate
by Lolium perenne from owing nutrient solution. Journal of
Experimental Botany 109, 453464.
Colnenne C, Meynard JM, Reau R, Justes E, Merrien A. 1998.
Determination of a critical dilution curve for winter oilseed rape.
Annals of Botany 81, 311317.
Cooper HD, Clarkson DT. 1989. Cycling of amino-nitrogen and
other nutrients between shoots and roots in cerealsa possible
mechanism integrating shoot and root in the regulation of nutrient
uptake. Journal of Experimental Botany 40, 753762.
Crawford NM. 1995. Nitrate: nutrient and signal for plant growth.
The Plant Cell 7, 859868.
Crawford NM, Glass ADM. 1998. Molecular and physiological
aspects of nitrate uptake in plants. Trends in Plant Sciences 3,
389395.

N deprivation in Brassica napus L. 1721

Filleur S, Daniel-Vedele F. 1999. Expression analysis of a highafnity nitrate transporter isolated from Arabidopsis thaliana by
differential display. Planta 207, 461469.
Fraisier V, Gojon A, Tillard P, Daniel-Vedele F. 2000.
Constitutive expression of a putative high-afnity nitrate
transporter in Nicotiana plumbaginifolia: evidence for posttranscriptional regulation by reduced nitrogen source. The Plant
Journal 23, 489449.
Forde BG. 2000. Nitrate transporters in plants: structure, function
and regulation. Biochimica et Biophysica Acta 1465, 219218.
Galvan A, Quesada A, Fernandez E. 1996. Nitrate and nitrite are
transported by different specic transport systems and by a
bispecic transporter in Chlamydomonas reinhardtii. Journal of
Biological Chemistry 271, 20882092.
Glass ADM, Ruth TJ, Rufty TW. 1990. Studies of the uptake of
nitrate in barley. II. Energetics. Plant Physiology 93, 15851589.
Guo FQ, Wang R, Chen M, Crawford NM. 2001. The
Arabidopsis dual-afnity nitrate transporter gene AtNRT1.1
(CHL1) is activated and functions in nascent organ
development during vegetative and reproductive growth. The
Plant Cell 13, 17611777.
Hatch DJ, Hopper MJH, Dhanoa MS. 1986. Measurement of
ammonium ions in owing solution culture and diurnal variation
in uptake in Lolium perenne. Journal of Experimental Botany 37,
589596.
Hoff T, Truong HN, Caboche M. 1994. The use of mutants and
transgenic plants to study nitrate assimilation. Plant, Cell and
Environment 17, 489506.
Huang NC, Chiang CS, Crawford NM, Tsay YF. 1996. CHL1
encodes a component of the low-afnity nitrate uptake system in
Arabidopsis and shows cell type-specic expression in roots. The
Plant Cell 8, 21832191.
Huang NC, Liu KH, Lo HJ, Tsay YF. 1999. Cloning and
functional characterization of an Arabidopsis nitrate transporter
gene that encodes a constitutive component of low-afnity
uptake. The Plant Cell 11, 13811392.
Krapp A, Fraisier V, Scheible WR, Quesada A, Gojon A, Stitt
M, Caboche M, Daniel-Vedele F. 1998. Expression studies of
NRT2:1Np, a putative high afnity nitrate transporter: evidence
for its role in nitrate uptake. The Plant Journal 14, 723731.
Laine P, Ourry A, Boucaud J. 1995. Shoot control of nitrate
uptake rates by roots of Brassica napus L.: effects of localized
nitrate supply. Planta 196, 7783.
Lauter FR, Ninnemann O, Bucher M, Reismeier JW, Frommer
WB. 1996. Preferential expression of an ammonium transporter
and of two putative nitrate transporters in roots hairs of tomato.
Proceedings of the National Academy of Sciences, USA 93, 8139
8144.
Lee RB, Purves JV, Ratcliffe RG, Saker LR. 1992. Nitrogen
assimilation and the control of ammonium and nitrate absorption
by maize roots. Journal of Experimental Botany 43, 13851396.
Lejay L, Tillard P, Lepetit M, Olive FD, Filleur S, DanielVedele F, Gojon A. 1999. Molecular and functional regulation of
two NO3 uptake systems by N- and C-status of Arabidopsis
plants. The Plant Journal 18, 509519.
Liu KH, Huang CY, Tsay YF. 1999. CHLI is a dual-afnity nitrate
transpoter of Arabidopsis involved in multiple phases of nitrate
uptake. The Plant Cell 11, 865874.
Marschner H, Kirkby EA, Cakmak I. 1996. Effect of mineral
nutritional status on shootroot partioning of photoassimilates
and cycling of mineral nutrients. Journal of Experimental Botany
47, 12551263.
Muldin I, Ingemarsson B. 1995. A cDNA from Brassica napus L.
encoding a putative nitrate transporter. Plant Physiology 108,
1341.
Murray PJ, Hatch DJ, Cliquet JB. 1996. Impact of insect

herbivory on the growth, nitrogen and carbon contents of white

clover (Trifolium repens L.). Canadian Journal of Botany 74,
15921595.
Quesada A, Galvan A, Fernandez E. 1994. Identication of
nitrate transporter genes in Chlamydomonas reinhardtii. The
Plant Journal 5, 407419.
Quesada A, Krapp A, Trueman LJ, Daniel-vedele F, Fernandez
E, Forde BG, Caboche M. 1997. PCR identication of a
Nicotiana plumbaginifolia cDNA homologous to the high-afnity
nitrate transpoters of the crnA family. Plant Molecular Biology
34, 265274.
Redinbaugh MG, Campbell WH. 1991. Higher plant responses to
environmental nitrate. Physiologia Plantarum 82, 640650.
Siddiqi MY, Glass ADM, Ruth TJ, Fernando M. 1989. Studies of
the regulation of nitrate inux by barley seedlings using 13NO3.
Plant Physiology 90, 806813.
Siddiqi MY, Glass ADM, Ruth TJ, Rufty TW. 1990. Studies of
the uptake of nitrate in barley. I. Kinetics of 13NO3 inux. Plant
Physiology 93, 14261432.
Stitt M. 1999. Nitrate regulation of metabolism and groth. Current
Opinion in Plant Biology 2, 178186.
Tillard P, Passama L, Gojon A. 1998. Are phloem amino acids
involved in the shoot to root control of NO3 uptake in Ricinus
communis plants. Journal of Experimental Botany 49, 1371
1379.
Touraine B, Clarkson DT, Muller B. 1994. Regulation of nitrate
uptake at the whole plant level. In: Roy J, Garnier E, eds. A whole
plant perspective on carbonnitrogen interactions. The Hague:
SPB Academic Publishers, 1130.
Touraine B, Daniel-Vedele F, Forde B. 2001. Nitrate uptake and
its regulation. In: Peter JL, Morot-Gaudry JF, eds. Plant nitrogen.
Berlin: Springer-Verlag Publishers, 136.
Touraine B, Glass ADM. 1997. NO3 and ClO3 uxes in the chl15 mutant of Arabidopsis thaliana. Plant Physiology 114, 137
144.
Trueman LJ, Richardson A, Forde BG. 1996. Molecular cloning
of higher plant homologues of the high-afnity nitrate
transporters of Chlamydomonas reinhardtii and Aspergillus
nidulans. Gene 175, 223231.
Tsay YF, Schroeder JI, Feldmann KA, Crawford NM. 1993. The
herbicide sensitivity gene CHL1 of Arabidopsis encodes a nitrateinducible nitrate transporter. Cell 72, 705713.
Unkles SE, Hawker KL, Grieve C, Campbell EI, Montague P,
Kinghorn JR. 1991. crnA encodes a nitrate transporter in
Aspergillus nidulans. Proceedings of the National Academy of
Sciences, USA 88, 204208.
Unkles SE, Hawker KL, Grieve C, Campbell EI, Montague P,
Kinghorn JR. 1995. crnA encodes a nitrate transporter in
Aspergillus nidulans. (Correction) Proceedings of the National
Academy of Sciences, USA 92, 3076.
Vidmar JJ, Zhuo D, Siddiqui MY, Schjoerring JK, Touraine B,
Glass ADM. 2000. Regulation of high-afnity nitrate transporter
genes and high-afnity nitrate inux by nitrogen pools in roots of
barley. Plant Physiology 123, 307318.
Wang R, Liu D, Crawford NM. 1998. The Arabidopsis CHL1
protein plays a major role in high-afnity nitrate uptake.
Proceedings of the National Academy of Sciences, USA 95,
1513415139.
Zhou JJ, Theodoulou FL, Muldin I, Ingemarsson B, Miller AJ.
1998. Cloning and functional characterization of Brassica napus
transporter that is able to transport nitrate and histidine. Journal
of Biological Chemistry 273, 1201712023.
Zhuo D, Okamoto M, Vidmar JJ, Glass ADM. 1999. Regulation
of a putative high-afnity nitrate transporter (NRT2;1At) in roots
of Arabidopsis thaliana. The Plant Journal 17, 563568.