Putative high-affinity nitrate (NO3–) transporter genes, designatedNrt2;1AtandNrt2;2At, were isolated fromArabidopsis thalianaby RT–PCR using degenerate primers. The genes shared 86% and 89% identity at the amino acid and nucleotide levels, respectively, while their proteins shared 30–73% identities with other eukaryotic highaffinity NO3– transporters. Both genes were induced by NO3–, butNrt2;1Atgene expression was not apparent in 2- and 5-day-old plants. By 10 days, and thereafter,Nrt2;1Atgene expression in roots was substantially higher than for theNrt2;2Atgene. RootNrt2;1Atexpression levels were strongly correlated with inducible high-affinity 13NO3– influx into intact roots under several treatment conditions. The use of inhibitors of N assimilation indicated that downregulation ofNrt2;1Atexpression was mediated by NH4+, gln and other amino acids.
Three transport systems participate in nitrate (NO3–) absorption by plant roots, namely a low-capacity constitutively expressed system (CHATS), a high-capacity NO3–inducible system (IHATS), and a low-affinity constitutively expressed system, designated LATS ( Crawford & Glass 1998; Glass & Siddiqi 1995). Absorbed NO3– may be assimilated into amino acids by the enzymes nitrate (NR) and nitrite (NiR) reductases, respectively, and glutamine synthetase (GS) and glutamate synthase (GOGAT). In addition to root assimilation, significant quantities of NO3– are stored in root vacuoles, or translocated to the shoot for assimilation or storage (see Crawford & Glass 1998 for a review).
High-affinity nitrate transporter genes belonging to the highly conserved crnA family have been cloned from Aspergillus nidulans ( Johnstone et al. 1990; Unkles et al. 1991), the yeast Hansenula polymorpha ( Perez et al. 1997), Chlamydomonas ( Quesada et al. 1994), Hordeum vulgare ( Trueman et al. 1996) and Nicotiana plumbaginifolia ( Quesada et al. 1997). However, little is known concerning the regulation of these genes. Krapp et al. (1998) demonstrated that expression levels of the N. plumbaginifolia homologue (Nrt2:1Np) were downregulated by exposing plants to 5 m m NH4+ or 5 m m gln. However, it is unclear whether these effects resulted from NH4+ or gln themselves or from their assimilation products.
We used RT–PCR to isolate Arabidopsis thaliana Nrt2;1At and Nrt2;2At genes, belonging to the crnA gene family, and characterized the Nrt2;1At gene and 13NO3– influx by IHATS, with respect to induction and regulation by different cellular N sources.
Results and discussion
Characterization of the Nrt2;1At and Nrt2;2At genes
The Nrt2;1At gene was isolated using PCR. The complete cDNA consisted of 1751 bp, and showed 75% identity with barley HvNRT2 genes ( Trueman et al. 1996 ). The genomic DNA sequences downstream from Nrt2;1At, generated by PCR and inverse PCR ( Ochman et al. 1990 ), revealed the presence of a homologous gene ( Fig. 1a). Southern blot analysis under low stringency conditions confirmed this finding ( Fig. 1b). Each gene was present as a single copy, separated by 1.5 kb, in a tail to tail configuration. Located at the same position in each were two introns; the crnA gene of A. nidulans contains three introns, while the barley HvNRT2 genes contain none (data not shown).
The coding regions of Nrt2;1At and Nrt2;2At genes shared about 89% nucleotide identity and encoded two putative transmembrane polypeptides of 530 and 522 amino acids, respectively. These shared 87% identity and 96% similarity. The predicted Arabidopsis NRT2 proteins shared 30–77% identities with high-affinity nitrate transporters from other eukaryotes ( Perez et al. 1997 ; Quesada et al. 1994 , 1997; Trueman et al. 1996 ; Unkles et al. 1991 ). Regions of high hydrophobicity, consistent with 12 putative membrane spanning regions, corresponded exactly for the NRT2;1At and Nrt2;2At proteins, and closely for all members of the crna family.
Nrt2;1At and Nrt2;2At genes are differentially expressed
cDNA cloning experiments suggested that the Nrt2;1At and Nrt2;2At genes might be differentially expressed. RT–PCR products (40 cycles), using RNA isolated from whole plants and from roots of plants at different ages revealed that Nrt2;1At gene RT–PCR products were absent from 2- and 5-day-old plants, but present in 10-day-old root material ( Fig. 1c). Nrt2;1At RT–PCR products from root material increased substantially by days 15 and 20. Touraine & Glass (1997) reported that Arabidopsis seedlings failed to significantly deplete medium NO3– until > 10 days after germination. This may reflect a greater dependence on seed reserves of N at early stages of development. Expression levels of the Nrt2;1At gene in leaves were typically less than 1% of root levels (data not shown); however, given that xylem sap typically contains 5–20 m m NO3– ( Glass & Siddiqi 1995), low-affinity NO3– transporters related to the Nrt1 family ( Tsay et al. 1993) may have greater relevance to leaf NO3– uptake.
Compared to Nrt2;1At gene expression, root levels of Nrt2;2At transcript were substantially lower at all ages examined ( Fig. 1c), and expression levels in shoots were < 1% of those detected in roots. The functional significance of the Nrt2;2At gene is presently unknown.
Induction of Nrt2;1At gene expression by NO3–
Nrt2;1At expression in roots of 3-week-old plants, previously grown on 1 m m ammonium citrate, was induced by exposing roots of these plants to 1 m m KNO3. Transcript levels increased within 30 min of exposure to NO3– ( Fig. 2a), peaked by 3 h, and then declined to a steady level. In parallel experiments, 13NO3– influx followed an almost identical pattern ( Fig. 2c). The relatively high initial value of 13NO3– influx was probably due to CHATS activity.
Nrt2;1At gene expression under steady-state conditions
In barley roots, 13NO3– influx via IHATS was inversely correlated with levels of NO3– pretreatment ( Siddiqi et al. 1989 ). In roots of Arabidopsis plants, Nrt2;1At gene expression was absent in ammonium-grown roots, highest in roots of 100 μm NO3––grown plants and lowest in 15 m m NO3––grown plants. ( Fig. 3a). The pattern of inducible high-affinity 13NO3– influx corresponded exactly to that of Nrt2;1At gene expression ( Fig. 3c), although plants grown in 1 m m ammonium citrate had relatively high values of constitutive 13NO3– influx, probably due to CHATS activity.
Nrt2;1At gene expression in leaf tissue was less than 1% of that of roots and, in contrast to the root pattern, it increased as the [NO3–] of media increased (data not shown). This may reflect the increasing proportion of absorbed NO3– that is transported to the shoot as external [NO3–] is increased ( Andrews 1986). Nevertheless, the result is perplexing, given that a role for high-affinity NO3– uptake in leaf tissue (as stated above) is unlikely.
To address this question, plants grown for 3 weeks in 1 m m ammonium citrate were exposed to various N treatments and/or inhibitors of N assimilation for 3 h. Using the 1 m m KNO3 treatment as a control (set at 100%), 3 h exposure to 2 m m ammonium citrate, 2 m m KNO3 or 1 m m NH4NO3 resulted in Nrt2;1At expression levels of 9%, 74% or 50%, respectively ( Fig. 4a, lanes 1–4). These results confirm the dependence on NO3– for induction of Nrt2;1At expression and the downregulation associated with increasing external [NO3–]. l-methionine sulphoximine (MSX), which inhibits the assimilation of NH4+ to gln by GS, reduced Nrt2;1At gene expression to 10% and 22%, respectively, when administered together with 1 m m NH4NO3 or 2 m m KNO3 ( Fig. 4a, lanes 5 and 6, respectively). In maize roots, MSX treatment increased cytosolic [NH4+] 10-fold, to 80 m m ( Lee et al. 1992 ), and in barley roots reduced 13NO3– incorporation into amino acids to < 1% of control values ( King et al. 1993 ). Thus, the present observations strongly suggest that NH4+ itself is a potent regulator of Nrt2;1At gene expression. The small effect of tungstate inhibition of NR activity on Nrt2;1At expression levels ( Fig. 4a, lane 7) suggests that NO3– is not important in downregulating this gene. Similar conclusions were obtained by Krapp et al. (1998 ) using NR mutants of N. plumbaginifolia.
The inhibitors aminooxyacetate (AOA) and azaserine (AZA) increase tissue concentrations of glu and gln, respectively, by inhibiting aspartate aminotransferase and GOGAT. These inhibitors reduced Nrt2;1At expression levels to 36% and 13%, of controls, respectively ( Fig. 4a, lanes 8 and 9), indicating that glu and gln are potent regulators of Nrt2;1At expression. Exogenous application of 1 m m KNO3 together with 1 m m arg, asn or gln reduced Nrt2;1At expression levels to 18%, 38%, and 77%, respectively, of the 1 m m KNO3 control ( Fig. 4a, lanes 10–12). The lack of effect of gln was surprising, given the result of the AZA application. However, gln was found to be relatively ineffective in blocking NO3– transport when applied exogenously to Arabidopsis, wheat and soybean, while asp, glu or arg were strong inhibitors ( Doddema & Otten 1979; Muller & Touraine 1992; Rodgers & Barneix 1993). Results based on exogenous applications of amino acids must be interpreted with caution because of differences in rates of amino acid uptake and subsequent assimilation. In contrast to observations based on physiological induction of IHATS NO3– influx by NO2– pretreatment in barley ( Aslam et al. 1996a ; King et al. 1993 ), a 3-h exposure to 1 m m NO2– failed to induce a detectable level of Nrt2;1At expression ( Fig. 4a, lane 13). Nrt2;1At gene expression was compared to IHATS activity by measuring 13NO3– influx in selected treatments, using 100 μm K13NO3. In uninduced plants, pretreated with ammonium citrate for 3 h, 13NO3– influx was 1.4 μmol g–1 fresh weight (FW) h–1. This high value, corresponding to the CHATS influx, was subtracted from all flux values to determine the treatment effects upon IHATS activity. Pretreatment with 1 m m KNO3– increased 13NO3– influx (due to IHATS induction) by 100%, while 1 m m KNO3– plus 1 m m MSX, 1 m m NH4NO3, and 1 m m NH4NO3 plus 1 m m MSX completely abolished the IHATS component of 13NO3– influx in all three treatments. Exogenous application of 1 m m arg, asn and gln reduced the IHATS component of influx by 62%, 72% and 76%, respectively. Given that exogenous application of gln failed to strongly reduce Nrt2;1At expression ( Fig. 4a, lane 12), the observed pronounced effect on 13NO3– influx may have resulted from direct effects of gln on the NO3– transporter.
In summary, we have demonstrated strong correlations between Nrt2;1At expression levels and IHATS activity in A. thaliana under a wide range of conditions. The use of various inhibitors of N assimilation provides strong evidence that several forms of N, particularly NH4+ and the amino acids gln, glu, asn and arg, and are all capable of downregulating Nrt2;1At expression through effects on transcription or mRNA stability.
Growth of Arabidopsis
Arabidopsis thaliana (ecotype Columbia) seedlings were grown hydroponically under sterile conditions as described in an earlier paper ( Touraine & Glass 1997), with N supplied as 1 m m ammonium citrate or 1 m m KNO3. Light was provided from fluorescent tubes (150 (E m–2 sec–1) on a 16-h light and 8-h dark photoperiod.
Isolation of nucleic acids
DNA was isolated from leaves of A. thaliana using the CTAB method ( Ausubel et al. 1994 ). RNA was isolated using TRIzol Reagent (Life Technologies) according to the manufacturer’s method. The mRNA were isolated by use of oligotex (Qiagen) according to suggested protocols. Southern blot transfer was carried out according to Sambrook et al. (1989 ), using 10 μg of DNA for each lane. Total RNA, 10–15 μg, were used for Northern blot analysis and separated on 1.6% formaldehyde agarose gels. The DNA and RNA were transferred onto a Hybond+ nylon membrane (Amersham) and fixed by UV exposure. The Northern blots were probed with Arabidopsis Nrt2;1At, and Nrt2;2At genes. Hybridizations were carried out as previously described ( Sambrook et al. 1989 ).
Most of the PCR amplifications were carried out using Expand High Fidelity Tag DNA polymerase (Boehringer, Mannheim) under the following conditions: 50 m m KCl, 1.5 m m MgCl2 and 200 μm dNTP. Inversion PCR amplifications were carried using the Expand Long Template PCR System (Boehringer, Mannheim).
Identification and isolation of Arabidopsis Nrt2;1At and Nrt2;2At genes
Arabidopsis cDNA libraries were constructed using the Marathon cDNA Amplification Kit from 1 μg of pooled mRNA, isolated from roots exposed to 1 m m KNO3 for 5, 10 and 24 h. Primers, based on the highly conserved regions of predicted protein sequences of the crnA gene family ( Trueman et al. 1996 ), yielded a 0.7-kb PCR product. A second primer set was used to amplify the cDNA library and generated a 1.5-kb fragment. Primers based on these cDNA sequences were used to amplify total genomic DNA, generating only one Nrt2;1At genomic copy. The flanking regions of the Nrt2;1At gene were digested with four different enzymes and ligated for inverse PCR amplification. Nrt2;2At was identified adjacent to the Nrt2;1At gene.
Isolation of plasmid DNA and DNA sequencing
PCR products were cloned and sequenced by standard methods ( Sambrook et al. 1989 ). Plasmid DNA and PCR products were sequenced by AmpliTag DNA polymerase using an automated 373 DNA Sequencer. DNA sequence analyses were carried out by use of the McVector 4.1 program and BCM Search Launcher ( Smith et al. 1996 ).
13NO3– influx measurements
The radiotracer 13NO3– was produced by the cyclotron facility at the University of British Columbia. IHATS activity of roots of 3-week-old plants was measured using 100 μm K13NO3 in the standard nutrient solution employed for growth, as described previously ( Touraine & Glass 1997).
The authors gratefully acknowledge financial support provided to A.D.M.G. in the form of NSERC Strategic and ongoing Research grants.