Journal of Experimental Botany, Vol. 54, No. 389, pp. 1851-1864,
August 1, 2003
© 2003 Oxford University Press
Induction of nitrate uptake in maize roots: expression of a putative high-affinity nitrate transporter and plasma membrane H+-ATPase isoforms
Received 18 February 2003; Accepted 24 April 2003
Dipartimento di Produzione Vegetale e Tecnologie Agrarie, University of Udine, Via delle Scienze 208, I-33100 Udine, Italy
* To whom correspondence should be sent. Fax: +39 0432 55 86 03. E-mail: zeno.varanini{at}dpvta.uniud.it
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
An investigation was carried out to assess the effect of nitrate supply on the root plasma membrane (PM) H+-ATPase of etiolated maize (Zea mays L.) seedlings grown in hydroponics. The treatment induced higher uptake rates of the anion and the expression of a putative high-affinity nitrate transporter gene (ZmNRT2.1), the first to be identified in maize. Root PM H+-ATPase activity displayed a similar time-course pattern as that of net nitrate uptake and investigations were carried out to determine which of the two isoforms reported to date in maize, MHA1 and 2, responded to the treatment. MHA1 was not expressed under the conditions analysed. Genome analysis revealed that MHA2, described as the most abundant form in all maize tissues, was not present in the maize hybrid investigated, but a similar form was found instead and named MHA3. A second gene (named MHA4) was also identified and partially sequenced. Both genes, classified as members of the PM H+-ATPase subfamily II, responded to nitrate supply, although to different degrees: MHA4, in particular, proved more sensitive than MHA3, with a greater up- and down-regulation in response to the treatment. Increased expression of subfamily II genes resulted in higher steady-state levels of the enzyme in the root tissues and enhanced ATP-hydrolysing activity. The results support the idea that greater proton-pumping activity is required when nitrate inflow increases and suggest that nitrate may be the signal triggering the expression of the two members of PM H+-ATPase subfamily II.
Key words: Gene expression, H+-ATPase, maize roots, nitrate transporter, nitrate uptake, plasma membrane.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Nitrate is the main source of nitrogen for most plant species growing in aerobic soils (Reisenauer, 1966). Its fundamental role as a nutrient has been known for many decades, but only in recent years has its importance as a signal been fully acknowledged. Nitrate applications determine numerous changes in plant growth, metabolism, allocation and phenology that are brought about by the induction and/or repression of genes encoding for nitrate transporters (reviewed by Forde, 2000), enzymes involved in nitrate and carbon metabolism (Stitt, 1999; Scheible et al., 1997a), the determination of shoot/root ratio (Scheible et al., 1997b) and even lateral root elongation (Zhang and Forde, 1998, 2000).
Among the various processes regulated by nitrate, its uptake from the external solution has been the object of extensive investigations over the last few years (reviewed by Forde and Clarkson, 1999).
Nitrate transporters have been isolated and characterized in various plant species (reviewed in Forde, 2000), including Arabidopsis (Tsay et al., 1993; Huang et al., 1999), barley (Vidmar et al., 2000a, b), soybean (Yokoyama et al., 2001), rice (Lin et al., 2000), and Nicotiana plumbaginifolia (Fraisier et al., 2000, 2001).
A number of these transporters are constitutively expressed, whereas others are induced in the presence of external nitrate and display a higher affinity for the substrate. Nitrate application, therefore, results in higher influx rates, which of necessity require additional energization, since nitrate transport into the cell is an active process, even at the highest nitrate concentrations likely to be found in the soil solution (Zhen et al., 1991). The transport process into the cell has also been observed to occur via a 2 H+:1 NO3– symport (McClure et al., 1990a, b; Glass et al., 1992).
Proton-coupled transport and energy requirement suggest that the plasma membrane (PM) H+-ATPase may be actively involved in nitrate uptake and possibly up- and down-regulated by the same signals regulating the inflow of the anion. Previous investigations support this hypothesis: Santi et al. (1995) had in fact observed higher steady-state levels of this enzyme in the plasma membrane of root cells isolated from maize seedlings treated for 24 h with nitrate.
PM H+-ATPase plays a key role in many physiological processes. Its proton pumping activity is believed to activate secondary solute transport across the root surface, apoplastic phloem loading and, according to the acid growth theory, wall loosening and subsequent cell expansion (reviewed by Michelet and Boutry, 1995; Palmgren, 1998).
Given the variety of physiological roles, it can be questioned whether different isoforms are involved in the various processes. Molecular studies have in fact shown that plant PM H+-ATPase is encoded by a multigene family, and the more than thirty cDNA clones obtained to date can mainly be classified into two different gene subfamilies (I and II) (Moriau et al., 1999). Since different isoforms may be simultaneously expressed in the same organ (Moriau et al., 1999), it is difficult to determine the enzymatic and regulatory properties of the various PM H+-ATPases: the isoforms may in fact function in different cells and tissues or at different metabolic states and under different environmental conditions. Up to now, evidence has been found for the regulation of specific PM H+-ATPase genes by such factors as auxin (Frías et al., 1996), salt stress (Niu et al., 1993a, b; Kalampanayil and Wimmers, 2001), mechanical stress (Oufattole et al., 2000), and immersion (Michelet et al., 1994).
To date, only two PM H+-ATPases genes have been reported in maize: MHA1 (Jin and Bennetzen, 1994) and MHA2 (Frías et al., 1996). According to their nucleotide sequences, MHA1 and MHA2 belong, respectively, to subfamily I and subfamily II (Morsomme and Boutry, 2000). While MHA1 was observed to be scarcely expressed in roots and other tissues, MHA2 has been described as the most abundant isoform present in all the tissues examined and has been detected in phloem cells, stomatal guard cells and in the root epidermis (Frías et al., 1996).
In the present research, an attempt was made to investigate whether a specific isoform of PM H+-ATPase in the root tissues of maize might respond to nitrate supply and, at the same time, study the expression of a high-affinity nitrate transporter.
A partial cDNA for a putative IHATS transporter and three others encoding for MHA isoforms were thus isolated from maize roots.
Maize seedlings were grown in the presence of 1.5 mM NO3– and the activity, quantity, and expression of root PM H+-ATPases were measured during the first 24 h of contact with the anion. The treatment induced the expression of the high-affinity nitrate transporter, the first to be identified in maize, and also proved effective in increasing PM H+-ATPase activity with a similar trend to that observed for net nitrate uptake. Gene-specific probes revealed that MHA1 remained substantially insensitive to nitrate, which instead stimulated the expression of one or more members of PM H+-ATPase subfamily II. Gene analysis revealed that MHA2 was not present in the genome of the hybrid under investigation, but similar forms were identified, which responded to nitrate supply by increasing their expression.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Growth conditions
Maize seeds (Zea mays L. cv. Cecilia, Pioneer Hibred Italia SpA), previously steeped in running tap water for 24 h, were germinated on a net suspended over an aerated 5 mM CaSO4 solution and placed in a dark growth chamber at 27 °C. After 72 h, the seedlings were transferred to an aerated solution containing either 0.75 mM Ca(NO3)2 or 1.5 mM CaSO4 (controls), and kept for up to 24 h in the dark at 27 °C.
Cloning of MHA and NRT2.1 cDNAs
Partial cDNAs for maize root PM H+-ATPases were obtained by 3'-RACE (Rapid Amplification of cDNA Ends), using the 3' RACE System Kit manufactured by Invitrogen (Invitrogen Co, Carlsbad, CA). Total RNA was isolated from maize roots using a guanidinium thiocyanate-phenol-chloroform method modified from Chomczynski and Sacchi (1987), and 2 µg of it were primed with the modified oligo-d(T): 5'-GGCCACGCGTCGACTAGTACTTT TTTTTTTTTTTTTT-3', following the manufacturer’s instructions. A specific probe for the MHA1 gene (Jin and Bennetzen, 1994; accession number U09989
[GenBank]
) was obtained by amplifying the cDNA with the forward primer: 5'-GAGGAACGAGAACGCAGATG-3', and the reverse primer (termed AUAP): 5'-GGCCACGCGTC GACTAGTAC-3'. A second nested amplification was carried out using the forward primer: 5'-GCATGCTCAGAGGTCTCTCC-3' and the above AUAP. The primers were designed on exon number 20 of the MHA1 gene sequence. The PCR product thus obtained had a 330 bp size (not considering the 20 bp AUAP) and included both the 3' untranslated region (UTR) and part of the coding sequence (199 bp) of MHA1.
In order to amplify a fragment of the MHA2 cDNA, a forward primer: 5'-CAGAGGATGAAGAACTACAC-3' was designed on this gene (Frías et al., 1996; accession number X85805 [GenBank] ) and used in combination with the AUAP primer. Two different partial cDNAs were obtained: one fragment, 1259 bp in size, corresponded to the MHA2 gene, although the identity was not total, while the second, 1359 bp in size, identified a novel MHA gene. Since the first fragment revealed numerous mismatches in a limited portion of the coding region, it was termed MHA3; while the latter was named MHA4. In order specifically to detect MHA2 and MHA3 transcripts in maize roots by PCR, two forward primers were designed on MHA2 and MHA3 sequences, respectively, HA2F: 5'-aagacctt cggaaaggagaga-3' and HA3f: 5'-GAGAACAAGACCGCC TTCAC-3', and used with a common reverse primer HA44R: 5'-AAGACGGGTACCCAACCATA-3'.
A partial cDNA encoding for a putative inducible, high-affinity, nitrate transporter (IHATS) was isolated by PCR-amplification of the cDNAs with the AUAP reverse primer and two forward degenerate primers: TIF1:[5'-GCTT(TC)CTCAT(TC)GGCTTCT C(GC)-3'] and TIF2:[5'-(TC)AGCAAGATCATCGGCAC(CA)G-3'], used in the first and in the nested PCR, respectively. The primers were devised from common, conserved amino acid domains of the HvNRT2.1 and HvNRT2.2 genes (Trueman et al., 1996; accession numbers U34198 [GenBank] and U34290 [GenBank] ).
All the PCR products, purified from a 1.2% (w/v) agarose gel, were cloned into pCR4-TOPO plasmids (TOPO TA Cloning Kit, Invitrogen CO, Carlsbad, CA) and sequenced.
Homology searches were carried out using the BLASTN and BLASTP algorithm (Altschul et al., 1997), identity percentages were calculated using GAP (Wisconsin GCG package, Accelrys; Devereux, 1991), and sequence alignment was performed using the ClustalX (1.5b) program (Thompson et al., 1997).
Southern blot analysis
Genomic DNA was purified from maize leaves using Plant DNAzol® Reagent (Invitrogen Co, Carlsbad, CA). Approximately 8 µg of total DNA were digested with the restriction enzyme indicated in the figure legend below in Results, ‘Southern blot analysis’, electrophoretically separated in a 0.8% agarose gel, and transferred by capillarity onto a nylon filter (Hybond-N, Amersham Biosciences Europe GmbH, Freiburg, Germany). The 3'-UTR of MHA3 was used as probe to detect MHA3 and gene homologues. The probe (347 bp) was obtained by PCR using the 1259 bp-long MHA3 cDNA clone as the template and 5'-ACCATCCAGCAGAACTACAC-3' and AUAP as the forward and reverse primers, respectively. The same primers were used to obtain the MHA4 probe (447 bp) from the 1359 bp cDNA clone. MHA1 and related genes were similarly detected using the 330 bp-long fragment of the MHA1 cDNA. The probes were labelled by including digoxigen-11-dUTP (Roche, Basel, Switzerland) in the PCR. Hybridization and washes were performed as described by Sambrook et al. (1989). The last washing step was carried out at 55 °C with 0.2x SSC (1x SSC=150 mM NaCl and 15 mM sodium citrate) containing 0.5% (w/v) SDS.
The digoxigenin-labelled probe was detected using CDP-Star® as the substrate (Roche, Basel, Switzerland) following the manufacturer’s instructions.
RNA analysis
Roots were harvested from maize seedlings after 0, 3 and 8 h of contact with nitrate.
For northern blot analyses, total RNA was isolated with TRIzol® Reagent (Invitrogen Co, Carlsbad, CA), separated (10–12 µg) in a 1.2% agarose denaturating gel and transferred onto a nylon filter (Hybond-N, Amersham Biosciences Europe GmbH, Freiburg, Germany) by capillarity (Sambrook et al., 1989).
Hybridization was carried out overnight at 42 °C in a SDS-buffer (50 mM sodium phosphate, pH 7.0, 50% (v/v) formamide, 7% (w/v) SDS, 2% (w/v) blocking reagent (Roche, Basel, Switzerland), 0.1% (w/v) sodium lauroylsarcosinate and 5x SSC [1x SSC=150 mM NaCl and 15 mM sodium citrate]). Filters were washed twice for 15 min at room temperature with 2x SSC containing 0.5% (w/v) SDS, then for further 60 min with SSC containing 0.5% (w/v) SDS. Temperature and SSC concentration varied according to stringency, and are indicated in the figure legend below in Results, ‘Analysis of gene expression’. When more hybridizations were performed on the same blot, the latter was stripped with four 5 min washes with 0.1% SDS at 90 °C.
The digoxigenin-labelled probe was detected using CDP-Star® as substrate (Roche, Basel, Switzerland) and following the manufacturer’s protocol. Hybridization signals on autoradiography films (Kodak X-Omat AR, Eastman Kodak Co, Rochester, NY) were quantified using Adobe Photoshop 5.0 (Adobe Systems, Mountain View, CA). A partial cDNA probe for maize 
-tubulin 5 (TUA5 gene, accession number X63177
[GenBank]
) was used as loading control.
For RT-PCR analyses, 2 µg of total DNase-treated RNA was reverse-transcribed using an oligo-dT primer and the SuperscriptTM II Reverse Transcriptase (Invitrogen Co, Carlsbad, CA) in a 20 µl total volume. 1 µl of the reaction mixture was then PCR-amplified with AccuprimeTM Taq DNA Polymerase (Invitrogen Co, Carlsbad, CA) in a 25 µl volume, using MHA3- and MHA4-specific primers, respectively, and specific primers for the maize -tubulin (TUA5 gene). The primers used to amplify MHA3 were HA3F and HA44R (described above); the product was 436 bp in length. The gene-specific primers for MHA4 were HA3F and HA42R (5'-CTTGTTGTTCTTGCGACGAC-3'), and amplified a 380 bp cDNA region (see Results). Reactions were run in an Eppendorf Mastercycler gradient (Eppendorf AG, Hamburg, Germany) for 23 cycles at an annealing temperature of 58 °C. Under these conditions, PCR amplification occurred in a linear range. The positive control templates were the clones recombining the cDNA of MHA3 and MHA4, respectively. The specificity of the MHA3 primers was checked on the MHA4 cDNA clone, and vice versa. PCR products were separated on a 1.5% agarose gel. Images were taken with a Kodak EDAS290 system (Eastman Kodak Co, Rochester, NY) and signals were quantified as described previously.
Measurement of net NO3– uptake
The roots of intact seedlings were immersed in 40 ml aerated solution containing 0.2 mM KNO3. Samples (0.2 ml) for NO3– determination were removed every 1.5 min, for 9 min, the period during which uptake was observed to have a linear trend, and the net uptake was measured as NO3– depletion from the solution per unit of time (Cataldo et al., 1975).
Isolation of the plasma membrane vesicles
Plasma membrane vesicles were isolated using a small scale procedure modified from Giannini et al. (1988). Briefly, the roots of NO3–-induced and control maize seedlings were cut separately and ground with a pestle in an ice-cold homogenization medium containing 250 mM sucrose, 10% (v/v) glycerol, 10 mM glycerol-1-phosphate, 2 mM MgSO4, 2 mM EDTA, 2 mM EGTA, 2 mM ATP, 2 mM DTT (dithiothreitol), 5.7% (w/v) choline-iodide, 1 mM PMSF, 20 µg ml–1 chymostatin, and 25 mM BTP (1,3-bis[TRIS (hydroxymethyl)methylamino]propane) buffered to pH 7.6 with MES. Approximately 2.5 ml g–1 fresh weight of root tissues were used.
The homogenates were filtered through four layers of cheese-cloth and subjected to 3 min of centrifugation at 13 000 g in an Eppendorf 5402 microcentrifuge (1.5 ml Eppendorf tubes) at 4 °C; the pellets were discarded and the suspension was centrifuged for a further 25 min under the same conditions. The pellets were then recovered, gently resuspended in 400 µl homogenization medium, and loaded onto discontinuous density gradients made by layering 700 µl of 25% (w/w) sucrose over a 300 µl 38% (w/w) sucrose cushion in 1.5 ml tubes. Both sucrose solutions were prepared in 5 mM BTP-MES, pH 7.4, and contained all the protectants present in the homogenization medium. The gradients were centrifuged for 1 h at 13 000 g, and the vesicles banding at the 25/38% interface were collected, diluted and centrifuged for a further 30 min at 316 000 g in a Centrikon T-1160 ultracentrifuge. The pellets, resuspended in a medium containing 20% glycerol, 5 mM DTT, 0.5 mM ATP, 50 µg ml–1 chymostatin, 2 mM EDTA, 2 mM EGTA, and 2 mM BTP titrated to pH 7.0 with MES, were immediately frozen in liquid N2 and stored at –80 °C until use.
Protein assay
Membranes were solubilized with the addition of NaOH (final concentration 0.5 M) to the suspension (Gogstad and Krutnes, 1982), and the protein content was determined as described by Bradford (1976), using BSA as standard.
PM H+-ATPase activity
ATP-hydrolysing activity was measured by determining the release of inorganic phosphate, as described by Forbusch (1983). Assays were performed at 38 °C in a 0.6 ml reaction vol. containing 50 mM MES-BTP, pH 6.5, 5 mM MgSO4, 100 mM KNO3, 600 µM Na2MoO4, 1.5 mM NaN3, 5 mM ATP-BTP, pH 6.5, 0.01% (w/v) Brij 58 (polyoxyethylene 20 cetyl ether), plus or minus 100 µM V2O5. The reaction was started with the addition of membrane vesicles (0.5 µg of total protein); after 30 min, the reaction was blocked and colour developed as previously described (Santi et al., 1995). Inorganic phosphate content was determined spectrophotometrically at 705 nm, and PM H+-ATPase activity was expressed as that inhibited by 100 µM vanadate.
Western analysis
Polyclonal antibodies were raised against synthetic peptides designed on specific regions of the C-terminal amino acid sequences of the maize PM H+-ATPase subfamilies. One 15-mer was designed on the C-terminal domain of the MHA1 sequence (Jin and Bennetzen, 1994): 901-CMFENKTSFSEVNQL-915 (Cys residue was added at the N terminus for coupling). The peptide was synthesized and conjugated to keyhole limpet haemocyanine by Sigma-Genosys (Sigma-Genosys Ltd, Cambridge UK). The same manufacturer produced the antisera. An antiserum obtained against the synthetic 18 aa peptide 273-AKRRAEIARLRELHTLKG-290 was used to detect MHA3 and MHA4.
Membrane vesicle proteins were separated by denaturing 8% SDS-PAGE as previously described (Santi et al., 1995), then stained with Coomassie Brilliant Blue or electro-transferred to PVDF membrane filters (Immobilon, Millipore Co, Bedford, MA) overnight at 4 °C for western analysis. Filters were blocked for 1 h in TBS-buffer (20 mM TRIS-HCl, pH 7.5, 150 mM NaCl) containing 1% (w/v) skimmed milk powder, then incubated for 3 h with the antiserum. After a 1 h incubation with the secondary antibody (Horseradish Peroxydase-conjugate antirabbit IgG, Sigma-Aldrich Co, St Louis, MO), the band corresponding to PM H+-ATPase was detected by chemiluminescence (ECL Detection Reagents, Amersham Biosciences Europe GmbH, Freiburg, Germany) on autoradiography film (Kodak X-Omat AR, Eastman Kodak Co, Rochester, NY). The film was scanned and the signals quantified as for northern analysis.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Isolation of NRT2.1 and MHA partial cDNAs
In order to investigate the expression of maize IHATS members and PM H+-ATPase isoforms in response to nitrate supply, a partial cDNA for a putative inducible-high-affinity nitrate transporter and three partial cDNAs corresponding to three different MHA genes were isolated from maize roots.
A 1200 bp partial cDNA was isolated by 3' RACE using degenerate oligonucleotides designed on the sequences of barley inducible-high-affinity transporters (IHATS) HvNRT2.1 and 2 (Trueman et al., 1996; accession numbers U34198 [GenBank] and U34290 [GenBank] ). The alignment of the deduced amino acidic sequence with members of the IHATS family of transporters (Fig. 1) revealed an 89%, 81%, 82%, and 84% identity (92%, 85%, 86%, and 88% similarity) with the corresponding amino acidic regions of OsNRT2, HvNRT2.2, HvNRT2.4, and HvNRT2.1, respectively, thus suggesting the isolation of a putative inducible-high-affinity nitrate transporter, that was named ZmNRT2.1 and deposited in the EMBL database (accession number AJ344451 [GenBank] ).
|
As regards PM H+-ATPase, partial cDNAs were obtained through sequence information relative to the isoforms identified to date in maize, namely MHA1 (Jin and Bennetzen, 1994; accession number U09989 [GenBank] ) and MHA2 (Frías et al., 1996; accession number X85805 [GenBank] ). In particular, a partial cDNA corresponding to MHA1 was obtained by 3' RACE using specific oligonucleotides designed on the 3' region of the sequence. The fragment was 330 bp in size, and corresponded to part (199 bp) of exon No. 20 and the 3' untranslated region (UTR) sequence of the MHA1 gene.
In a similar manner, a forward oligonucleotide designed on the MHA2 sequence was used in 3' RACE experiments to amplify a partial cDNA of this gene. Two cDNAs were thus obtained: one 1259 bp-long product that, although similar to MHA2, did not fully coincide with it and a second cDNA (1359 bp in size) that identified a novel MHA gene.
Sequence alignment of the 1259 bp-long cDNA (indicated as MHA3 in Fig. 2) with MHA2 revealed the presence of numerous mismatches, mostly clustered in a limited coding region of approximately 100 bp in length (between nucleotide numbers 645 and 758 of MHA3). The identity shared by MHA3 and the corresponding sequence of MHA2 was 95%. A homology search using NCBI’s (National Center for Biotechnology Information) BLASTN revealed the highest score alignment for MHA3 with other maize sequences and, in particular, it shared a 100% identity with the corresponding coding regions of the gene sequence AF480431 [GenBank] (GenBank accession number). The MHA3 sequence was confirmed by a 100% identity with several EST clones found in the EST divisions of the NCBI databases, among which EST BQ538480 [GenBank] (GenBank accession number), a sequence identified in a B73 maize seedling library, which corresponds to the 3'-UTR and the last 312 coding nucleotides (where most differences with MHA2 were observed) of MHA3. Moreover, a 100% identity was calculated between MHA3 and the TIGR Maize Gene Index (ZmGI) sequence TC140186 (TIGR database, www.tigr.org).
|
In order to verify whether MHA2 transcripts were present in the maize roots, two specific forward primers (HA2F and HA3F) and a common reverse primer (HA44R) were designed on the regions of MHA2 and MHA3 sequences where the mutations were clustered (Fig. 2). While MHA3 PCR amplification resulted in a clear band on the gel, no fragment was obtained for MHA2, either from root cDNA or from genomic DNA (data not shown). It is therefore possible that MHA3 corresponds to the major root isoform MHA2, previously characterized by Frías et al. (1996). The EMBL accession number for the MHA3 partial cDNA is AJ441084 [GenBank] .
As regards the 1359 bp partial cDNA (termed MHA4 in Fig. 2), this sequence was also confirmed by alignment with several EST clones in NCBI EST divisions, including sequences from a maize tassel primordium library (GenBank accession number BE345084 [GenBank] ) and germinating embryos (AW261317 [GenBank] ), that shared a 100% identity with its 3'-UTR. Alignment of MHA4 partial cDNA at nucleotide level (Fig. 2) revealed an 82% identity with MHA3. This cDNA was attributed to a novel PM H+-ATPase maize gene and submitted to EMBL database under accession number AJ539534 [GenBank] .
The alignment between the deduced amino acid sequences of MHA1, MHA3 and MHA4 is shown in Fig. 3: MHA3 shares 98% identity (100% similarity) with MHA4, and the two isoforms are 85% and 84% identical, respectively, with the corresponding region of tobacco PMA4 (Moriau et al., 1999), which, together with Arabidopsis AHA1–3 (Harper et al., 1989) and other genes, defines a specific PM H+-ATPase subfamily (Morsomme and Boutry, 2000). MHA3 and MHA4 were thus grouped in the same subfamily (termed subfamily II). The latter does not include MHA1, which belongs to the PM H+-ATPase gene subfamily I (Jin and Bennetzen, 1994), and only shares a 65% and 66% identity (84% similarity) with MHA3 and MHA4, respectively.
|
Southern blot analysis
Southern blot analysis was performed on total genomic DNA, using probes obtained from the 3' regions of the three different MHA cDNAs identified in the plant material (MHA1, MHA3 and MHA4).
The restriction enzymes used generated a strong signal and three or four weaker ones, as detected by the 330 bp MHA1 probe (Fig. 4A). On the other hand, the 347 bp-long 3'-UTR of MHA3 clearly detected at least two different genome fragments in all the lanes (Fig. 4B). Under the same conditions, the 3'-UTR (447 bp) of MHA4 recognized at least three genomic fragments in all the lanes and produced a pattern almost completely overlapping that obtained with the MHA3 probe (Fig. 4C). The same result was obtained even when washes were more stringent (not shown). Clearly, the 3'-UTR of MHA3 and MHA4 cDNA were not able to detect the corresponding genes specifically. It is, however, noteworthy that none of the bands detected by the MHA1 probe were recognized by the MHA3 and MHA4 probes, a result that confirmed the specificity of each probe at a subfamily level.
|
Since none of the restriction enzymes used cut any of the cDNA probes, Southern blot experiments confirmed the presence of MHA1, MHA3 and MHA4, and suggest that there may be more than three MHA genes in the maize genome.
Analysis of gene expression
The temporal expression pattern of PM H+-ATPase and IHATS genes in response to nitrate supply was investigated by northern blotting. The analysis was performed on total RNA purified from the roots of maize seedlings after 0 h, 3 h and 8 h of contact with 1.5 mM NO3–, using the cDNA probes for the putative high-affinity nitrate transporter ZmNRT2.1, and those for the PM H+-ATPase genes (Fig. 5). In the latter case, the MHA1 and MHA4 3'-UTR probes were used under the same stringency conditions as the Southern blot analyses, so as to cross-hybridize with all the related genes within the same subfamily. The MHA4 3'-UTR probe was chosen to investigate the expression of the MHA2-like subfamily as it could detect more elements than the MHA3 3'-UTR.
|
The expression of the ZmNRT2.1 gene was only induced in the presence of the anion: transcripts were barely detectable at the beginning of the experiment (less than 2% with respect to 3 h), but accumulated greatly after 3 h of treatment and remained high, even after 8 h, with only a 20% decrease as compared to the reference signal at 3 h. These results are consistent with the hypothesis of a high-affinity nitrate carrier belonging to the IHATS family, the expression of which would lead to higher inflow rates of the anion into the root cells.
Northern blot experiments performed using the MHA1 probe failed to reveal any signal at all time intervals (not shown). On the other hand, when investigating the expression of MHA3, MHA4 and related genes, a marked up-regulation of the sub-family was observed following root contact with nitrate. After 3 h, the signal showed an over three-fold increase with respect to that observed at 0 h, and remained high even after 8 h of treatment (+120% as compared to 0 h). No significant changes were observed in control roots during the treatment (not shown).
In order to analyse the expression of the identified members of PM H+-ATPase subfamily II specifically, RT-PCR was performed using specific primers for MHA3, MHA4 (reference in Fig. 2 caption) and the constitutively expressed maize -tubulin (TUA5 gene).
The expression of both forms was enhanced by nitrate supply (Fig. 6), although the response was different in the two cases. Quantitation of MHA3 transcript abundance relative to maize -tubulin transcripts revealed a 50–70% increase in the transcript level after 3 h of exposure to nitrate and only a slight down-regulation was observed after 8 h of treatment. MHA4, on the other hand, showed an over 120% increase after 3 h, and a marked decrease after 8 h (+50% with respect to 0 h).
|
Net nitrate uptake
Net nitrate uptake measurements were made after 0, 2, 4, 6, 8, 12, and 24 h of contact with the growth solutions (Fig. 7A). The results show that the uptake rates in seedlings grown in the presence of nitrate were higher than those in control plants, with maximum differences after 4–6 h of contact with the anion (+60–70%). Net uptake rates remained significantly higher in induced roots throughout the entire trial period, even as values declined, presumably due to increased nitrate efflux from the root cells and/or processes of feedback regulation. Net uptake displayed a typical time-course response, thus confirming earlier reports on nitrate influx in other plant species (Siddiqi et al., 1990; Zhuo et al., 1999).
|
H+-ATPase activity
Preliminary experiments aimed at determining the composition of membrane vesicles revealed that the procedure described in Materials and methods was particularly efficient in isolating plasma membrane-enriched vesicles. The ATP-hydrolysing activity measured at pH 6.5 was, in fact, inhibited to a great extent (>90%) by vanadate and was almost insensitive to NO3– and NaN3 (a 15% and 7% inhibition, respectively). No significant differences were observed in the composition of vesicles isolated from nitrate-treated and control roots (not shown).
Figure 7B depicts the phospho-hydrolysing activity of vanadate-sensitive H+-ATPase in membrane vesicles isolated from the control and nitrate-exposed maize roots after different lengths of time. Significant differences were detected after 6 h of treatment and remained so throughout the entire period of observation. The peak in enzyme activity was observed after 8 h of nitrate supply, with a 160% increase in respect to the initial values.
Western analysis
Immunoblotting experiments were performed with polyclonal antibodies directed against amino acid stretches (Fig. 3) in the C-terminal domains of the two maize PM H+-ATPase subfamilies. No signal was detected in the membrane proteins isolated from the nitrate-exposed and control roots when Western blot experiments were performed with the antibodies raised against the MHA1 form (not shown).
On the other hand, a clear signal was evident in both membrane preparations when the antibody against the identified members of subfamily II was used (Fig. 8A). While remaining almost constant in the controls, PM H+-ATPase levels were enhanced instead by contact with nitrate, and signal quantitation (Fig. 8B) revealed significant differences after 6 h of treatment. Peak levels were observed after 8 h of contact with nitrate, although proteins remained significantly more abundant than in the controls throughout the remaining period of investigation.
|
The similarity between enzyme activity and signal quantitation of MHA3 and MHA4 (Figs 7B, 8B) suggests that the enhancement of root PM H+-ATPase activity following exposure to nitrate can be mostly ascribed to accumulation of these isoforms at the plasma membrane.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The application of nitrate is known to induce the expression of transport systems mediating the inflow of higher rates of the anion across the plasma membrane of root cells. Since nitrate uptake requires energy (Clarkson, 1986) and electrophysiological studies (McClure et al., 1990a; Meharg and Blatt, 1995) have shown that its transport into the cell occurs in symport with protons, it seems reasonable to suppose an involvement of PM H+-ATPase in this process. Supporting evidence had already been supplied by Santi et al. (1995), who observed increased activity and steady-state levels of the enzyme in plasma membrane vesicles isolated from maize roots exposed for 24 h to this anion. The aim of this research was to clarify the relationship between the plasma membrane proton pump and nitrate transport, paying particular attention to the possible involvement of specific isoforms of maize PM H+-ATPase in response to nitrate supply.
So far only two PM H+-ATPase genes have been isolated and characterized in maize, namely MHA1 (Jin and Bennetzen, 1994) and MHA2 (Frías et al., 1996). MHA1, a gene partially captured by the maize retroelement Bs1, was reported to have very low expression levels in all maize tissues (Jin and Bennetzen, 1994). This result was confirmed by the present research: although this gene was present in the genome, transcrips were undetected by northern blot analysis, even when the roots were treated with nitrate. Similarly, no signal was detected in immunological experiments carried out using specific MHA1 antibodies.
MHA2, on the other hand, has been described as a major isoform of PM H+-ATPase, with the highest expression levels in the roots (Frías et al., 1996). 3'-RACE experiments and PCR amplifications with gene-specific primers, however, failed to detect this gene in the hybrid investigated, but always revealed the presence of a partial sequence 95% identical to the corresponding region of that characterized by Frías et al. (1996). This sequence, which was termed MHA3, shares an almost identical 3'-UTR with MHA2, but presents several mismatches mainly clustered in a limited coding region. The differences are confirmed by BLASTN alignment with several sequences found in various maize cultivars and tissues. The great similarity between MHA2 and MHA3, and the existence of several MHA3 EST sequences seem to imply that the two forms may actually be the same, and the differences in the sequence of MHA2 may be due to cDNA transcription errors. Experiments are under way to verify this hypothesis, and preliminary investigations on the maize inbred line characterized by Frías et al. (1996) support the suggestion (data not shown).
The plasma membrane H+-ATPase gene family in maize appears to be as numerous as that observed in other plant species: the probes used in Southern experiments clearly detected more than three MHA genes in the maize genome. The subfamily II of PM H+-ATPases, in particular, includes at least two genes, MHA3 and MHA4. The latter is a novel gene that, although similar to MHA3, differs in particular from the former for its 3'-UTR. It is worth mentioning that although Frías et al. suggested that the major maize PM H+-ATPase, MHA2 (presumed now to be MHA3), was a single-copy gene, they also observed that less specific probes recognized a related gene in the maize genome, especially under conditions of reduced stringency.
In order to investigate the relationship between nitrate uptake and members of the two PM H+-ATPase subfamilies, experiments were carried out over the first 24 h of maize seedling exposure to the nutrient.
Root contact with nitrate induced the expression of a putative high-affinity nitrate transporter with a time-course similar to that observed for barley (Vidmar et al., 2000a), tomato (Wang et al., 2001), Arabidopsis (Zhuo et al., 1999), and tobacco (Quesada et al., 1997) genes. The partial cDNA was termed ZmNRT2.1, as it appears to belong to the NRT2 family of high-affinity transporters, the expression of which is induced by nitrate. ZmNRT2.1, which is the first nitrate transporter found to date in maize, reveals an 89% homology with the corresponding amino acid region of the rice nitrate carrier OsNRT2, and an over 81% homology with barley HvNRT2 members. According to Cerezo et al. (2001), the NRT2 family plays an important role in the induction of nitrate uptake: Arabidopsis mutants lacking these genes were incapable of responding to nitrate supply by increasing its uptake.
These data confirmed that net nitrate uptake was indeed enhanced by exposure to the nutrient and, likewise, also root PM H+-ATPase activity.
Northern blot analyses also revealed that the expression of members of the PM H+-ATPase subfamily II was up-regulated in response to the treatment. The similarity in the temporal expression pattern of ZmNRT2.1 and the subfamily II genes suggests that the latter may also be induced by nitrate supply. The rapid increase in transcript levels and subsequent decline are in fact similar to that of other nitrate uptake and assimilation-related genes (e.g. NRT2, NIA and NII) (Forde, 2000, and references therein) which are expressed after exposure to the anion.
On the other hand, microarray experiments aimed at screening nitrate-induced genes in Arabidopsis (Wang et al., 2000) and tomato (Wang et al., 2001) roots failed to reveal any significant increase in PM H+-ATPase mRNA levels after contact with the nutrient. These seemingly contrasting results can be explained, however, by the growth conditions adopted: in both cases, the plants were subjected to prolonged exposure to NH4+, a condition that appears to stimulate higher steady-state levels of the enzyme in the roots (Sasakawa, 1995; Yamashita et al., 1995). Moreover, various authors (Siddiqi et al., 1990; Aslam et al., 1992, 1997) have observed great differences in the magnitude of the response to nitrate supply even within the same plant species. Investigations on Arabidopsis seedlings (Crawford and Glass, 1998) revealed that the levels of nitrate uptake were high even in the uninduced plants and that the up-regulation of influx rates, determined by exposure to the nutrient, was relatively small. The need to increase the electrochemical proton gradient across the plasma membrane would, therefore, be different and depend greatly on the plant species and its metabolism.
What remains undisputed is that changes in nitrate uptake rates also determine similar variations in the activity and steady-state levels of the PM H+-ATPase. In particular, it is the subfamily II that is involved in the response, whereas subfamily I appears to play a negligible role. RT-PCR experiments aimed at assessing the contribution of the two identified genes of the subfamily II in maize, MHA3 and MHA4, showed that both responded to the treatment, although to different degrees. MHA4 in particular appears to be more sensitive to nitrate, with a more marked up- and down-regulation than MHA3. It would be interesting to investigate whether its expression is tissue-specific and to identify other members of the subfamily that may also respond to nitrate supply.
The modulation of the enzyme forms appears to occur mostly at the translational level, although a post-translational regulation cannot be completely ruled out. Interestingly, both MHA3 and MHA4 possess in their C-terminal domain the Tyr-Thr-Val motif needed for the binding of 14-3-3 regulatory proteins (Fuglsang et al., 1999; Borch et al., 2002), the expression of which also appears to be up-regulated by nitrate (Wang et al., 2001).
The fundamental role of PM H+-ATPase in plant nutrition has been acknowledged for many years and, indeed, the question of improving crop-nutrient uptake by genetic manipulation of this enzyme has already been raised (Palmgren, 2001). However, before now, no investigation had been made on such an important issue as the relationship between nitrate uptake and PM H+-ATPase activity. This contribution to the subject sheds some light on the close link between the two processes and underlines the plant’s need to modulate root cell energization readily in response to the rapid changes in nitrate availability that occur at the rhizosphere.
|
Acknowledgements |
|---|
The research was supported by an Italian MIUR-PRIN 2002 grant to Zeno Varanini in the frame of the project ‘Acquisition of oxoanionic nutrients by crop plants: physiological and molecular analysis of the effect of modulators and interfering substances present at the rhizosphere’ and by CNR (NITCAR and Agenzia 2000 projects).
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25, 3389–3402.
Aslam M, Kielson K, Travis RL, Rains DW. 1997. Nitrate uptake, efflux, and in vivo reduction by Pima and Acala cotton cultivars. Crop Science 37, 1795–1801.
Aslam M, Travis RL, Huffaker RC. 1992. Comparative kinetics and reciprocal inhibition of nitrate and nitrite uptake in roots of uninduced and induced barley seedlings. Plant Physiology 109, 319–326.
Borch J, Bych K, Roepstorff P, Palmgren MG, Fuglsang AT. 2002. Phosphorylation-independent interaction between 14-3-3 protein and the plant plasma membrane H+-ATPase. Biochemical Society Transactions 30, 411–415.[CrossRef][ISI][Medline]
Bradford MM. 1976. A rapid and sensitive method of quantitation of microgram quantities of protein utilizing the principle of protein binding. Analytical Biochemistry 72, 248–254.[CrossRef][ISI][Medline]
Cataldo DA, Haaron M, Schrader LF, Youngs VL. 1975. Rapid colorimetric determination of nitrate in plant tissue by nitration of salicylic acid. Communications in Soil Science and Plant Analysis 6, 71–80.
Cerezo M, Tillard P, Filleur S, Munos S, Daniel-Vedèle F, Gojon A. 2001. Major alterations of the regulation of root NO3-uptake are associated with the mutation of Nrt2.1 and Nrt2.2 genes in Arabidopsis. Plant Physiology 127, 262–271.
Chomczynski P, Sacchi N. 1987. Acid-Guanidinium Thiocianate-Phenol-Chloroform extraction method. Analytical Biochemistry 162, 156–159.[ISI][Medline]
Clarkson DT. 1986. Regulation of the absorption and release of nitrate by plant cells: a review of current ideas and methodology. In: Lambers H, Neeteson JJ, Stulen I, eds. Fundamental, ecological and agricultural aspects of nitrogen metabolism in higher plants. Dordrecht, NL: Martinus Nijhoff International, 3–27.
Crawford NM, Glass ADM. 1998. Molecular and physiological aspects of nitrate uptake in plants. Trends in Plant Science 3, 389–395.[CrossRef][ISI]
Devereux J. 1991. The GCG sequence analysis software package, version 7.0. Madison, WI: Genetic Computer Group, University Research Park.
Forbusch B. 1983. Assay of the Na+-, K+-ATPase in plasma membrane preparations: increasing the permeability of membrane vesicles using sodium dodecylsulfate buffered with bovine serum albumine. Analytical Biochemistry 128, 159–163.[CrossRef][ISI][Medline]
Forde BG. 2000. Nitrate transporters in plants: structure, function and regulation. Biochimica et Biophysica Acta 1465, 219–235.[Medline]
Forde BG, Clarkson DT. 1999. Nitrate and ammonium nutrition of plants: physiological and molecular perspectives. Advances in Botanical Research 30, 1–90.
Fraisier V, Dorbe MF, Daniel-Vedèle F. 2001. Identification and expression analyses of two genes encoding putative low-affinity nitrate transporters from Nicotiana plumbaginifolia. Plant Molecular Biology 45, 181–190.[CrossRef][ISI][Medline]
Fraisier V, Gojon A, Tillard P, Daniel-Vedèle F. 2000. Constitutive expression of a putative high-affinity nitrate transporter in Nicotiana plumbaginifolia: evidence for post-transcriptional regulation by a reduced nitrogen source. The Plant Journal 23, 489–496.[CrossRef][ISI][Medline]
Frías I, Caldeira MT, Pérez-Castiñeira JR, Navarro-Aviñó JP, Culiañez-Maciá FA, Kuppinger O, Stransky H, Pagés M, Hager A, Serrano R. 1996. A major form of the maize plasma membrane H+-ATPase: characterization and induction by auxin in coleoptiles. The Plant Cell 8, 1533–1544.[Abstract]
Fuglsang AT, Visconti S, Drumm K, Jahn T, Stensballe A, Mattei B, Jensen ON, Aducci P, Palmgren MG. 1999. Binding of 14-3-3 protein to the plasma membrane H+-ATPase AHA2 involves the three C-terminal residues Tyr(946)-Thr-Val and requires phosphorylation of Thr(947). Journal of Biological Chemistry 274, 36774–36780.
Giannini JL, Ruiz-Cristin J, Briskin DP. 1988. A small scale procedure for the isolation of transport competent vesicles from plant tissues. Analytical Biochemistry 174, 561–567.[CrossRef][ISI][Medline]
Glass ADM, Shaff JE, Kochian LV. 1992. Studies on the uptake of nitrate in barley. IV. Electrophysiology. Plant Physiology 99, 456–463.
Gogstad GO, Krutnes MB. 1982. Measurement of protein in cell suspensions using the Coomassie brilliant blue die-binding assay. Analytical Biochemistry 126, 355–359.[CrossRef][ISI][Medline]
Harper JF, Surowy TK, Sussmann MR. 1989. Molecolar cloning and sequence of a cDNA encoding the plasma membrane proton pump (H+-ATPase) of Arabidopsis thaliana. Proceedings of the National Academy of Sciences, USA 86, 1234–1238.
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-affinity uptake. The Plant Cell 11, 1381–1392.
Jin YK, Bennetzen JL. 1994. Integration and non random mutation of a plasma membrane proton ATPase gene fragment within the BS1 retroelement of maize. The Plant Cell 6, 1177–1186.[Abstract]
Kalampanayil BD, Wimmers LE. 2001. Identification and characterization of a salt-stress-indiced plasma membrane H+-ATPase in tomato. Plant, Cell and Environment 24, 999–1005.[CrossRef]
Lin CM, Koh S, Stacey G, Yu SM, Lin TY, Tsay YF. 2000. Cloning and functional characterization of a constitutively expressed nitrate transporter gene, OsNRT1, from rice. Plant Physiology 122, 379–388.
McClure PR, Kochian LV, Spanswick RM, Shaff JE. 1990a. Evidence for cotransport of nitrate and protons in maize roots. I. Effect of nitrate on the membrane potential. Plant Physiology 93, 281–289.
McClure PR, Kochian LV, Spanswick RM, Shaff JE. 1990b. Evidence for cotransport of nitrate and protons in maize roots. II. Measurements of NO3– and H+ fluxes with ion-selective microelectrodes. Plant Physiology 93, 290–294.
Meharg AA, Blatt MR. 1995. NO3– transport across the plasma membrane of Arabidopsis thaliana root hairs: kinetic control by pH and membrane voltage. Journal of Membrane Biology 145, 49–66.[ISI][Medline]
Michelet B, Boutry M. 1995. The plasma membrane H+-ATPase. A highly regulated enzyme with multiple physiological functions. Plant Physiology 108, 1–6.[ISI][Medline]
Michelet B, Lukaszewicz M, Dupriez V, Boutry M. 1994. A plant plasma membrane proton-ATPase gene is regulated by development and environment and shows signs of a translational regulation. The Plant Cell 6, 1375–1389.[Abstract]
Moriau L, Michelet B, Bogaerts P, Lambert L, Michel A, Oufattole M, Boutry M. 1999. Expression analysis of two gene subfamilies encoding the plasma membrane H+-ATPase in Nicotiana plumbaginifolia reveals the major transport functions of this enzyme. The Plant Journal 19, 31–41.[CrossRef][ISI][Medline]
Morsomme P, Boutry M. 2000. The plant plasma membrane H+-ATPase: structure, function and regulation. Biochimica et Biophysica Acta 1465, 1–16.[Medline]
Niu X, Narasimhan ML, Salzman RA, Bressan RA, Hasegawa PM. 1993a. NaCl regulation of plasma membrane H+-ATPase gene expression in a glycophyte and a halophyte. Plant Physiology 103, 713–718.[Abstract]
Niu X, Zhu JK, Narasimhan ML, Bressan RA, Hasegawa PM. 1993b. Plasma membrane H+-ATPase gene expression is regulated by NaCl in cells of the halophyte Atriplex nummularia L. Planta 190, 433–438.[ISI][Medline]
Oufattole M, Arango M, Boutry M. 2000. Identification and expression of three new Nicotiana plumbaginifolia genes which encode isoforms of a plasma membrane H+-ATPase, and one of which is induced by mechanical stress. Planta 210, 715–722.[CrossRef][ISI][Medline]
Palmgren MG. 1998. Proton gradients and plant growth: role of the plasma membrane H+-ATPase. Advances in Botanical Research 28, 1–69.
Palmgren MG. 2001. Plant plasma membrane H+-ATPases: powerhouses for nutrient uptake. Annual Review of Plant Physiology and Plant Molecular Biology 52, 817–845.[CrossRef][ISI][Medline]
Quesada A, Krapp A, Trueman LJ, Daniel-Vedele F, Fernandez E, Forde B, Caboche M. 1997. PCR-identification of a Nicotiana plumbaginifolia cDNA homologous to the high-affinity nitrate transporters of the crnA family. Plant Molecular Biology 34, 265–274.[CrossRef][ISI][Medline]
Reisenauer HM. 1966. Mineral nutrients in soil solution. In: Altman PL, Dittmer DS, eds. Environmental biology. Bethesda, MD: Federation of American Societies for Experimental Biology, 507–508.
Sambrook J, Fritsch EF, Maniatis T. 1989. Transfer of denaturated RNA to nitrocellulose filters. In: Ford N, Nolan C, Ferguson M, eds. Molecular cloning. A laboratory manual, 2nd edn, Vol. 1. Cold Spring Harbor, NY: CSHL Press, 7.46–7.48.
Santi S, Locci G, Pinton R, Cesco S, Varanini Z. 1995. Plasma membrane H+-ATPase in maize roots induced for NO3– uptake. Plant Physiology 109, 1277–1283.[Abstract]
Sasakawa H. 1995. Stimulation of H+ extrusion and plasma membrane H+-ATPase activity of barley roots by ammonium treatment. Soil Science and Plant Nutrition 41, 133–140.
Scheible WR, Gonzales-Fontes A, Lauerer M, Muller-Rober B, Caboche M, Stitt M. 1997a. Nitrate acts as a signal to induce organic acid metabolism and repress starch metabolism in tobacco. The Plant Cell 9, 1–17.[CrossRef][ISI]
Scheible WR, Lauerer M, Schulze ED, Caboche M, Stitt M. 1997b. Accumulation of nitrate in the shoot acts as a signal to regulate shoot-root allocation in tobacco. The Plant Journal 11, 671–691.[CrossRef][ISI]
Siddiqi MY, Glass ADM, Ruth TJ, Rufty Jr TW. 1990. Studies of the uptake of nitrate in barley. I. Kinetics of 13NO3– influx. Plant Physiology 93, 1426–1432.
Stitt M. 1999. Nitrate regulation of metabolism and growth. Current Opinion in Plant Biology 2, 178–186.[CrossRef][ISI][Medline]
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. 1997. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 24, 4876–4882.
Trueman LJ, Richardson A, Forde BG. 1996. Molecolar cloning of higher plant homologues of the high-affinity nitrate transporters of Chlamydomonas reinhardtii and Aspergillus nidulans. Gene 175, 223–231.[CrossRef][ISI][Medline]
Tsay YF, Schroeder JI, Feldmann KA, Crawford NM. 1993. The herbicide sensitivity gene CHL1 of Arabidopsis encodes a nitrate-inducible nitrate transporter. Cell 72<