Putative High-affinity Nitrate Transporter Research Articles

Identification and molecular characterization of the Brachypodium distachyon NRT2 family, with a major role of BdNRT2.1.

The small monocot plant Brachypodium distachyon is rapidly emerging as a powerful model system to study questions unique to the monocot crops. An extensive BLAST search was carried to identify putative orthologues of the Arabidopsis NRT2 genes in the fully sequenced Brachypodium genome. Seven genes encoding putative high-affinity nitrate transporters (BdNRT2) were identified. Transcriptional analysis of individual BdNRT2 gene under various nitrogen sources and levels in the wild-type and a T-DNA mutant of BdNRT2.1 were performed. A transgenic approach was taken to complement the bdnrt2.1 mutant. BdNRT2.1 and BdNRT2.2 were strongly induced by nitrate resupply to nitrogen-starved plants and were classified as inducible genes. BdNRT2.5 was found to be repressed by nitrate resupply whereas other members were constitutively expressed in the root. Interestingly, higher ammonium concentrations also triggered similar gene expression regulation, suggesting BdNRT2 gene expression was also governed by internal nitrogen status, not just external nitrate concentrations. In bdnrt2.1 mutant, the high-affinity transporter system (HATS) was reduced by 30% and BdNRT2.2 and BdNRT2.6 were differentially regulated. This pioneering research demonstrates that genes in the BdNRT2 family have diverse roles, differing from the Arabidopsis AtNRT2 family, in response to various nitrogen conditions. BdNRT2.1 serves as a key member of the family.

Abscission of flowers and floral organs is closely associated with alkalization of the cytosol in abscission zone cells.

In vivo changes in the cytosolic pH of abscission zone (AZ) cells were visualized using confocal microscopic detection of the fluorescent pH-sensitive and intracellularly trapped dye, 2',7'-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF), driven by its acetoxymethyl ester. A specific and gradual increase in the cytosolic pH of AZ cells was observed during natural abscission of flower organs in Arabidopsis thaliana and wild rocket (Diplotaxis tenuifolia), and during flower pedicel abscission induced by flower removal in tomato (Solanum lycopersicum Mill). The alkalization pattern in the first two species paralleled the acceleration or inhibition of flower organ abscission induced by ethylene or its inhibitor 1-methylcyclopropene (1-MCP), respectively. Similarly, 1-MCP pre-treatment of tomato inflorescence explants abolished the pH increase in AZ cells and pedicel abscission induced by flower removal. Examination of the pH changes in the AZ cells of Arabidopsis mutants defective in both ethylene-induced (ctr1, ein2, eto4) and ethylene-independent (ida, nev7, dab5) abscission pathways confirmed these results. The data indicate that the pH changes in the AZ cells are part of both the ethylene-sensitive and -insensitive abscission pathways, and occur concomitantly with the execution of organ abscission. pH can affect enzymatic activities and/or act as a signal for gene expression. Changes in pH during abscission could occur via regulation of transporters in AZ cells, which might affect cytosolic pH. Indeed, four genes associated with pH regulation, vacuolar H(+)-ATPase, putative high-affinity nitrate transporter, and two GTP-binding proteins, were specifically up-regulated in tomato flower AZ following abscission induction, and 1-MCP reduced or abolished the increased expression.

The poplar NRT2 gene family of high affinity nitrate importers: Impact of nitrogen nutrition and ectomycorrhiza formation

As a result of natural or artificial disturbances, elevated nitrate concentrations are found in forest soils and could thus be important for tree nutrition. In an EST project of Populus tremula×tremuloides/Amanita muscaria ectomycorrhizas three putative high-affinity nitrate transporters of the NRT2 gene family were discovered of which PttNRT2.4A and PttNRT2.5B were expressed highest in fine roots at comparable levels.Ectomycorrhiza formation resulted in a nitrate independent enhanced PttNRT2.5B gene expression. However, as characterization of transport properties by heterologous expression failed, PttNRT2.5B function still remains unclear.With a KM value of about 26μM, as determined by heterologous expression in Xenopus laevis oocytes, PttNRT2.4A is a high affinity nitrate importer. In fine roots of nitrate depleted poplar plants PttNRT2.4A expression was very low. Transcript levels increased 75-fold when plants were exposed to low nitrate concentrations (10–100μM). This increase was lower at elevated nitrate concentrations or in the presence of ammonium. By generating P. tremula×tremuloides plants transformed with a PttNRT2.4A promoter GFP fusion construct, nitrate induced PttNRT2.4A expression was localized in root hairs but not in underlying cortex cells.In ectomycorrhizas of ammonium grown plants PttNRT2.4A expression decreased compared to non-mycorrhizal fine roots. However, nitrate induced PttNRT2.4A expression in ectomycorrhizas indicates nitrate permeability of the fungal mantle under certain conditions.

Comparative Transcriptomic Profiling of a Salt-Tolerant Wild Tomato Species and a Salt-Sensitive Tomato Cultivar

Wild halophytic tomato has long been considered as an ideal gene donor for improving salt tolerance in tomato cultivars. Extensive research has been focused on physiological and quantitative trait locus (QTL) characterization of wild tomato species in comparison with cultivated tomato. However, the global gene expression modification of wild tomato in response to salt stress is not well known. A wild tomato genotype, Solanum pimpinellifolium 'PI365967' is significantly more salt tolerant than the cultivar, Solanum lycopersicum 'Moneymaker', as evidenced by its higher survival rate and lower growth inhibition at the vegetative stage. The Affymetrix Tomato Genome Array containing 9,200 probe sets was used to compare the transcriptome of PI365967 and Moneymaker. After treatment with 200 mM NaCl for 5 h, PI365967 showed relatively fewer responsive genes compared with Moneymaker. The salt overly sensitive (SOS) pathway was found to be more active in PI365967 than in Moneymaker, coinciding with relatively less accumulation of Na(+) in shoots of PI365967. A gene encoding salicylic acid-binding protein 2 (SABP2) was induced by salinity only in PI365967, suggesting a possible role for salicylic acid signaling in the salt response of PI365967. The fact that two genes encoding lactoylglutathione lyase were salt inducible only in PI365967, together with much higher basal expression of several glutathione S-transferase genes, suggested a more effective detoxification system in PI365967. The specific down-regulation in PI365967 of a putative high-affinity nitrate transporter, known as a repressor of lateral root initiation, may explain the better root growth of this genotype during salt stress.

Expression of two maize putative nitrate transporters in response to nitrate and sugar availability

A full-length cDNA encoding a putative high-affinity nitrate transporter (ZmNrt2.2) from maize was isolated and characterised, together with another previously identified transporter (ZmNrt2.1), in terms of phylogenesis, protein structure prediction and regulation of transcript accumulation in response to nitrate and sugar availability. The expression of both genes was evaluated by quantitative and semi-quantitative RT-PCR in response to nitrate and sugar supply and the in planta localisation of mRNA was studied by in situ hybridisation. Data obtained suggested similar genetic evolution and identical transmembrane structure prediction between the two deduced proteins, and differences in both regulation of their expression and mRNA localisation in response to nitrate, leading us to hypothesise a principal role for ZmNRT2.1 in the influx activity and the major involvement of ZmNRT2.2 in the xylem loading process. Our data suggest opposing sugar regulation by ZmNrt2.1 and ZmNrt2.2 transcription in the presence or absence of nitrate and the existence of both hexokinase-dependent and hexokinase-independent transduction mechanisms for the regulation of ZmNrt2.1 and ZmNrt2.2 expression by sugars.

Molecular cloning and expression analysis of cDNAs encoding a putative Nrt2 nitrate transporter from peach

Two cDNA clones encoding a putative high-affinity nitrate transporter (Nrt2) were isolated from a cDNA library of peach (Prunus persica (L.) Batsch) roots. The nucleotide sequences of the clones, named PpNrt2.1 and PpNrt2.2, showed 99% identity in their coding regions, but their 3'-untranslated sequences showed only 40% identity. The deduced amino acid sequences of the clones showed 44-90% homology to NRT2 proteins from other plants. Both clones contained open reading frames encoding polypeptides of 530 amino acid residues, each with a predicted molecular mass of about 57 kDa. Both polypeptides had 12 putative transmembrane domains and a long hydrophilic C-terminal domain like other members of this protein family. Analyses of the genomic structures and of Southern blots suggested that PpNrt2.1 and PpNrt2.2 originated from different loci in the peach genome. In Northern blots, transcripts of PpNrt2.1 and PpNrt2.2 were detected in roots but not shoots of seedlings after nitrate application. They were not detected in leaves, stems, flowers, fruits or seeds of mature trees in the field, indicating root-specific expression of PpNrt2.1 and PpNrt2.2. Application of nitrate to roots of nitrogen-deprived seedlings strongly induced expression of PpNrt2.1 within 0.5 h, and gradually induced expression of PpNrt2.2. Application of ammonium to roots of nitrogen-deprived seedlings repressed expression of PpNrt2.1 and PpNrt2.2. The relationship of PpNrt2.1 and PpNrt2.2 to the inducible high-affinity transport system (iHATS) involved in the uptake of nitrate in peach roots is discussed.

Distinct Roles of Nitrate and Nitrite in Regulation of Expression of the Nitrate Transport Genes in the Moss Physcomitrella patens

Five NRT2 genes and three Nar2 genes, encoding putative high-affinity nitrate transporters, and the respective cDNAs were identified and characterized in Physcomitrella patens. The deduced moss NRT2 and NAR2 proteins were more similar to the corresponding proteins of higher plants than to those of the green alga Chlamydomonas reinhardtii. Expression of all the genes was inhibited by ammonium added to the medium. The regulation by ammonium was abolished by an inhibitor of glutamine synthetase, but the effect of this inhibitor was counteracted by an inhibitor of glutamate synthase. Negative correlation was observed between the glutamine content of protonemata and the transcript levels of PpNRT2 and PpNar2. These results indicated that glutamine is the signal for repression of the genes. All the genes except PpNRT2;5 showed transient expression stimulated by nitrate but not by nitrite, peaking at 2-4 h after the medium was deprived of ammonium. When the glutamine synthetase inhibitor was used to inhibit assimilation of the ammonium generated intracellularly from nitrate or nitrite, the second phase of activation of genes became manifest at approximately 8 h after the medium was deprived of ammonium. Surprisingly, both nitrate and nitrite stimulated gene expression at this stage. PpNRT2;5 was distinct from the other genes in that its expression is sharply induced by nitrite, is strictly dependent on nitrite or nitrate, and is much less susceptible to the feedback regulation, retaining a constant level in nitrate-containing medium. These results indicated that P. patens has multiple mechanisms for sensing nitrate and nitrite.

The putative high-affinity nitrate transporter NRT2.1 represses lateral root initiation in response to nutritional cues

Lateral root initiation is strongly repressed in Arabidopsis by the combination of high external sucrose and low external nitrate. A previously isolated mutant, lin1, can overcome this repression. Here, we show that lin1 carries a missense mutation in the NRT2.1 gene. Several allelic mutants, including one in which the NRT2.1 gene is completely deleted, show similar phenotypes to lin1 and fail to complement lin1. NRT2.1 encodes a putative high-affinity nitrate transporter that functions at low external nitrate concentrations. Direct measurement of nitrate uptake and nitrate content in the lin1 mutant seedlings established that both are indeed reduced. Because repression of lateral root initiation in WT plants can be relieved by increased concentrations of external nitrate, it is surprising to find that repression is also relieved by a defect in a component of the high-affinity nitrate uptake system. Furthermore, lateral root initiation is increased in lin1 relative to WT even when seedlings are grown on nitrate-free media, suggesting that the mutant phenotype is nitrate-independent. These results indicate that NRT2.1 is a repressor of lateral root initiation and that this role is independent of nitrate uptake. We propose that Arabidopsis NRT2.1 acts either as a nitrate sensor or signal transducer to coordinate the development of the root system with nutritional cues.

Cloning and expression study of a putative high-affinity nitrate transporter gene from Dunaliella salina

A nitrate transporter gene, named DsNRT2.1 (GeneBank accession number AY621079), from Dunaliella salina has been cloned by screening a cDNA library, which was constructed with mRNAs from D. salina after 60 min treatment with 5 mM nitrate, with a 342 bp NRT2 cDNA fragment from D. salina as a probe. DsNRT2.1 exhibits sequence similarity to those known nitrate transporters of the NRT2 family. A hydrophobicity blot indicated that DsNRT2.1 belongs to the major facilitator superfamily (MFS). Northern analysis showed that an mRNA species of 1.9 kb can be rapidly induced by NO−3, but not by NH+4. Northern analysis also showed that NaCl could significantly increase the expression of DsNRT2.1.

Induction of nitrate uptake in maize roots: expression of a putative high-affinity nitrate transporter and plasma membrane H+-ATPase isoforms

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.

Expression of a putative high-affinity NO3- transporter and of an H+-ATPase in relation to whole plant nitrate transport physiology in two maize genotypes differently responsive to low nitrogen availability.

Two maize genotypes differently responsive to nitrogen availability were characterized for their efficiency in nitrate accumulation via both the LATS (Low-Affinity Transport System) and HATS (High-Affinity Transport System) nitrate uptake systems. In addition, a full-length cDNA encoding a putative high-affinity nitrate transporter (ZmNrt2.1) was isolated and its expression evaluated in both the roots and leaves of the two maize genotypes, together with the expression of a maize H(+)-ATPase isoform (Mha1). The data showed the importance of the iHATS (Inducible High-Affinity System) system efficiency as a physiological marker of adaptation to low input and suggested that the transcript accumulation of ZmNrt2.1 might be a key step for the regulation of iHATS. However, ZmNrt2.1 transcription cannot account for the differences found between the two hybrids in terms of the activity of their respective iHATS and, as a consequence, of their adaptation to low input. Therefore, the involvement of some other transporter(s) or of some post-transcriptional/post-translational mechanism of regulation affecting the efficiency of iHATS may be hypothesized. In addition, the data suggest that the transcription of the Mha1 gene may also be involved in the global efficiency of the iHATS system.

Constitutive expression of a putative high-affinity nitrate transporter in Nicotiana plumbaginifolia: evidence for post-transcriptional regulation by a reduced nitrogen source.

The NpNRT2.1 gene encodes a putative inducible component of the high-affinity nitrate (NO3-) uptake system in Nicotiana plumbaginifolia. Here we report functional and physiological analyses of transgenic plants expressing the NpNRT2.1 coding sequence fused to the CaMV 35S or rolD promoters. Irrespective of the level of NO3- supplied, NO3- contents were found to be remarkably similar in wild-type and transgenic plants. Under specific conditions (growth on 10 mM NO3-), the steady-state NpNRT2. 1 mRNA level resulting from the deregulated transgene expression was accompanied by an increase in 15NO3- influx measured in the low concentration range. This demonstrates for the first time that the NRT2.1 sequence codes a limiting element of the inducible high-affinity transport system. Both 15NO3- influx and mRNA levels decreased in the wild type after exposure to ammonium, in agreement with previous results from many species. Surprisingly, however, influx was also markedly decreased in transgenic plants, despite stable levels of transgene expression in independent transformants after ammonium addition. We conclude that the conditions associated with the supply of a reduced nitrogen source such as ammonium, or with the generation of a further downstream metabolite, probably exert a repressive effect on NO3- influx at both transcriptional and post-transcriptional levels.

Regulation of a putative high-affinity nitrate transporter (Nrt2;1At) in roots of Arabidopsis thaliana.

Putative high-affinity nitrate (NO3-) transporter genes, designated Nrt2;1At and Nrt2;2At, were isolated from Arabidopsis thaliana by 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 high-affinity NO3- transporters. Both genes were induced by NO3-, but Nrt2;1At gene expression was not apparent in 2- and 5-day-old plants. By 10 days, and thereafter, Nrt2;1At gene expression in roots was substantially higher than for the Nrt2;2At gene. Root Nrt2;1At expression 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 of Nrt2;1At expression was mediated by NH4+, gln and other amino acids.

Expression analysis of a high-affinity nitrate transporter isolated from Arabidopsis thaliana by differential display.

We used the differential display technique on total RNAs from roots of Arabidopsis thaliana (L.) Heynh. plants which had or had not been induced for 2 h by nitrate. One isolated cDNA clone, designated Nrt2:1At, was found to code for a putative high-affinity nitrate transporter. Two genomic sequences homologous to Nrt2:1At were found to be localized on the same fragment of chromosome 1 in the Arabidopsis genome. Expression analyses of both low- and high-affinity nitrate transporter genes, respectively Nrt1:1At (previously named Chl1) and Nrt2:1At, were carried out on plants grown under different nitrogen regimes. In this paper, we show that both genes are induced by very low levels of nitrate (50 microM KNO3). However, stronger induction was observed with Nrt2:1At than with Nrt1:1At. Moreover, these two genes, although both over-expressed in a nitrate-reductase-deficient mutant, were differently regulated when N-sufficient wild-type or mutant plants were transferred to an N-free medium. Indeed, the steady-state amounts of Nrt1:1At mRNA declined whereas the amount of Nrt2:1At mRNA increased, probably reflecting the de-repression of the high-affinity transport system during N-starvation.

Regulation of GmNRT2 expression and nitrate transport activity in roots of soybean (Glycine max).

A full-length cDNA, GmNRT2, encoding a putative high-affinity nitrate transporter was isolated from a Glycine max (L.) root cDNA library and sequenced. The deduced GmNRT2 protein is 530 amino acids in length and contains 12 putative membrane-spanning domains and a long, hydrophilic C-terminal domain. GmNRT2 is related to high-affinity nitrate transporters in the eukaryotes Chlamydomonas reinhardtii and Aspergillus nidulans, and to putative high-affinity nitrate transporters in barley and tobacco. Southern blot analysis indicated that GmNRT2 is part of a small, multigene family in soybean. Expression of GmNRT2 in roots was regulated by the type of nitrogen source provided to plants: GmNRT2 mRNA levels were barely detectable in ammonium-grown plants, higher in nitrogen-deprived plants, and highest in nitrate-grown plants. Induction of GmNRT2 mRNA levels in roots occurred within 1 h after exposure of plants to nitrate. Nitrate induction of GmNRT2 mRNA levels was accompanied by a fourfold increase in net nitrate uptake by soybean roots at 100 microM external nitrate. The molecular and physiological evidence indicates that GmNRT2 is probably a high-affinity nitrate transporter involved in nitrate uptake by soybean roots.