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At least 19 recordsLinked to original sources

PubMed2004

[Impact of localized compaction and ridge fertilization on field nitrate transport and nitrate use efficiency].

The primary season of nitrate leaching in the Loess Plateau region is the monsoon, which is caused by heavy rainfall during the growth season of corn (Zea mays L.). Nitrate leaching to groundwater is an increasing concern in agriculture, and is one of the major nitrogen losing ways in dryland farming system. Localized compaction and ridge fertilization is a method for nitrogen fertilizer application, by which, less fertilizer leaching would occur. The NO3(-)-N transport in soil profile, corn yields and nitrogen use efficiency under localized compaction and ridge fertilization were investigated through two years field study. The factors that affect NO3(-)-N transport under localized compaction and ridge fertilization were studied, combined with simulated experiment. The results showed that NO3(-)-N was leached to below 90 cm in plat fertilization in the year of about 370 mm rainfall, a mean precipitation during the season, while the NO3(-)-N leakage of the fertilizer zone was reduced by localized compaction and ridge fertilization, as a result that the NO3(-)-N concentration below 60 cm was less than 10 mg.kg-1, and NO3(-)-N accumulated in 20-40 cm with a concentration 80-90 mg.kg-1. There was no significant difference in yield between application methods with 240.0 kg N.hm-2. However, the absorbed amount of nitrogen was improved significantly by localized compaction and ridge fertilization, and the nitrogen use efficiency was increased by 9%. The bulk density of the barriers had an evident effect on NO3(-)-N transport under localized compaction and ridge fertilization, but the effect of ridge slope was insignificant.

Biological Transport↗

PubMed2004

Transcript profiling in the chl1-5 mutant of Arabidopsis reveals a role of the nitrate transporter NRT1.1 in the regulation of another nitrate transporter, NRT2.1.

Arabidopsis thaliana mutants deficient for the NRT1.1 NO(3)(-) transporter display complex phenotypes, including lowered NO(3)(-) uptake, altered development of nascent organs, and reduced stomatal opening. To obtain further insight at the molecular level on the multiple physiological functions of NRT1.1, we performed large-scale transcript profiling by serial analysis of gene expression in the roots of the chl1-5 deletion mutant of NRT1.1 and of the Columbia wild type. Several hundred genes were differentially expressed between the two genotypes, when plants were grown on NH(4)NO(3) as N source. Among these genes, the N satiety-repressed NRT2.1 gene, encoding a major component of the root high-affinity NO(3)(-) transport system (HATS), was found to be strongly derepressed in the chl1-5 mutant (as well as in other NRT1.1 mutants). This was associated with a marked stimulation of the NO(3)(-) HATS activity in the mutant, suggesting adaptive response to a possible N limitation resulting from NRT1.1 mutation. However, derepression of NRT2.1 in NH(4)NO(3)-fed chl1-5 plants could not be attributed to lowered production of N metabolites. Rather, the results show that normal regulation of NRT2.1 expression is strongly altered in the chl1-5 mutant, where this gene is no more repressible by high N provision to the plant. This indicates that NRT1.1 plays an unexpected but important role in the regulation of both NRT2.1 expression and NO(3)(-) HATS activity. Overexpression of NRT2.1 was also found in wild-type plants supplied with 1 mM NH(4)(+) plus 0.1 mM NO(3)(-), a situation where NRT1.1 is likely to mediate very low NO(3)(-) transport. Thus, we suggest that it is the lack of NRT1.1 activity, rather than the absence of this transporter, that derepresses NRT2.1 expression in the presence of NH(4)(+). Two hypotheses are discussed to explain these results: (1) NRT2.1 is upregulated by a NO(3)(-) demand signaling, indirectly triggered by lack of NRT1.1-mediated uptake, which overrides feedback repression by N metabolites, and (2) NRT1.1 plays a more direct signaling role, and its transport activity generates an unknown signal required for NRT2.1 repression by N metabolites. Both mechanisms would warrant that either NRT1.1 or NRT2.1 ensure significant NO(3)(-) uptake in the presence of NH(4)(+) in the external medium, which is crucial to prevent the detrimental effects of pure NH(4)(+) nutrition.

Anion Transport Proteins↗

PubMed1989

Genetically engineered mutant of the cyanobacterium Synechococcus PCC 7942 defective in nitrate transport.

Nitrate-grown cells of Synechococcus PCC 7942 (Anacystis nidulans R2) contain a 45-kDa protein as a major protein in the cytoplasmic membrane but ammonium-grown cells lack it. A mutant (M45) was constructed by inactivating the gene encoding the 45-kDa protein. M45 did not grow under low concentrations of nitrate but high concentrations of nitrate could support its growth, with the optimal concentration being 40-70 mM. The growth rate of M45 was as high as that of the wild-type cells when ammonium was the nitrogen source. The 45-kDa protein was absent in M45 irrespective of the growth conditions. The activities of nitrate and nitrite reductases were higher in M45 than in wild type. The rate of nitrate-dependent O(2) evolution in wild type measured in the presence of L-methionine D,L-sulfoximine and D,L-glyceraldehyde showed saturation kinetics with respect to nitrate concentration in the external medium. The nitrate concentration required to produce half the maximal rate was 1 muM. In M45, the rate of nitrate-dependent O(2) evolution was nearly zero at nitrate concentrations <1 mM and was linearly increased as the concentration increased. The presumed absence of nitrate transport in M45 demonstrated by these results suggested that the 45-kDa protein is a nitrate transporter.

Journal Article↗

PubMed1974

Nitrate transport system in Neurospora crassa.

Nitrate uptake in Neurospora crassa has been investigated under various conditions of nitrogen nutrition by measuring the rate of disappearance of nitrate from the medium and by determining mycelial nitrate accumulation. The nitrate transport system is induced by either nitrate or nitrite, but is not present in mycelia grown on ammonia or Casamino Acids. The appearance of nitrate uptake activity is prevented by cycloheximide, puromycin, or 6-methyl purine. The induced nitrate transport system displays a K(m) for nitrate of 0.25 mM. Nitrate uptake is inhibited by metabolic poisons such as 2,4-dinitrophenol, cyanide, and antimycin A. Furthermore, mycelia can concentrate nitrate 50-fold. Ammonia and nitrite are non-competitive inhibitors with respect to nitrate, with K(i) values of 0.13 and 0.17 mM, respectively. Ammonia does not repress the formation of the nitrate transport system. In contrast, the nitrate uptake system is repressed by Casamino Acids. All amino acids individually prevent nitrate accumulation, with the exception of methionine, glutamine, and alanine. The influence of nitrate reduction and the nitrate reductase protein on nitrate transport was investigated in wild-type Neurospora lacking a functional nitrate reductase and in nitrate non-utilizing mutants, nit-1, nit-2, and nit-3. These mycelia contain an inducible nitrate transport system which displays the same characteristics as those found in the wild-type mycelia having the functional nitrate reductase. These findings suggest that nitrate transport is not dependent upon nitrate reduction and that these two processes are separate events in the assimilation of nitrate.

Amino Acids↗

PubMed2006

Functional properties and differential mode of regulation of the nitrate transporter from a plant symbiotic ascomycete.

Nitrogen assimilation by plant symbiotic fungi plays a central role in the mutualistic interaction established by these organisms, as well as in nitrogen flux in a variety of soils. In the present study, we report on the functional properties, structural organization and distinctive mode of regulation of TbNrt2 (Tuber borchii NRT2 family transporter), the nitrate transporter of the mycorrhizal ascomycete T. borchii. As revealed by experiments conducted in a nitrate-uptake-defective mutant of the yeast Hansenula polymorpha, TbNrt2 is a high-affinity transporter (K(m)=4.7 microM nitrate) that is bispecific for nitrate and nitrite. It is expressed in free-living mycelia and in mycorrhizae, where it preferentially accumulates in the plasma membrane of root-contacting hyphae. The TbNrt2 mRNA, which is transcribed from a single-copy gene clustered with the nitrate reductase gene in the T. borchii genome, was specifically up-regulated following transfer of mycelia to nitrate- (or nitrite)-containing medium. However, at variance with the strict nitrate-dependent induction commonly observed in other organisms, TbNrt2 was also up-regulated (at both the mRNA and the protein level) following transfer to a nitrogen-free medium. This unusual mode of regulation differs from that of the adjacent nitrate reductase gene, which was expressed at basal levels under nitrogen deprivation conditions and required nitrate for induction. The functional and expression properties, described in the present study, delineate TbNrt2 as a versatile transporter that may be especially suited to cope with the fluctuating (and often low) mineral nitrogen concentrations found in most natural, especially forest, soils.

Amino Acid Sequence↗

PubMed1989

The narK gene product participates in nitrate transport induced in Escherichia coli nitrate-respiring cells.

The nucleotide sequence of the Escherichia coli narK gene, which is located in the upstream region of the narCHJI operon, was determined. The narK gene encodes a very hydrophobic protein with 463 amino acid residues (Mr 49,693). A narK deletion mutant, under conditions for the induction of nitrate respiration, was unable to perform nitrate transport. Loss of transport activity was recovered by transforming the mutant with a narK+ plasmid. Thus, we conclude that the narK gene encodes a transmembrane protein participating in nitrate transport. In the narK promoter region, we defined a unique sequence that we designate as a 'nitrate box', functioning as a putative NarL-binding site, in addition to the consensus sequence of the 'anaero-box'.

Amino Acid Sequence↗

PubMed2004

Missense mutations that inactivate the Aspergillus nidulans nrtA gene encoding a high-affinity nitrate transporter.

The transport of nitrate into prokaryotic and eukaryotic cells, of considerable interest to agriculture, ecology, and human health, is carried out by members of a distinct cluster of proteins within the major facilitator superfamily. To obtain structure/function information on this important class of nitrate permeases, a collection of chemically induced mutations in the nrtA gene encoding a 12-transmembrane domain, high-affinity nitrate transporter from the eukaryote Aspergillus nidulans was isolated and characterized. This mutational analysis, coupled with protein alignments, demonstrates the utility of the approach to predicting peptide motifs and individual residues important for the movement of nitrate across the membrane. These include the highly conserved nitrate signature motif (residues 166-173) in Tm 5, the conserved charged residues Arg87 (Tm 2) and Arg368 (Tm 8), as well as the aromatic residue Phe47 (Tm 1), all within transmembrane helices. No mutations were observed in the large central loop (Lp 6/7) between Tm 6 and Tm 7. Finally, the study of a strain with a conversion of Trp481 (Tm 12) to a stop codon suggests that all 12 transmembrane domains and/or the C-terminal tail are required for membrane insertion and/or stability of NrtA.

Amino Acid Sequence↗

PubMed2002

The activity of the high-affinity nitrate transport system I (NRT2;1, NAR2) is responsible for the efficient signalling of nitrate assimilation genes in Chlamydomonas reinhardtii.

The expression of nitrite uptake activity and the induction of transcripts from several nitrate assimilation genes (Nii1, Nrt2;1, Nrt2;3, and Nar1) have been analysed in Chlamydomonas reinhardtii Dang. strains bearing several sets of genes encoding nitrate transport systems. Different nitrate concentrations resulted in a differential induction pattern of nitrite uptake activity depending on which particular nitrate transport system was present in the cells, and that was directly related to their relative efficiency for nitrate transport. The presence of the high-affinity nitrate transport system I (NRT2;1, NAR2) made cells able to sense the very low concentrations of nitrate present in culture medium with no added nitrate and to express optimally Nii1, Nrt2;1, Nrt2;3, and Nar1 genes involved in nitrate/nitrite assimilation. In addition, strains lacking nitrate reductase activity overexpressed these gene transcripts as a result of continuous signalling by nitrate, but only those bearing an active system I. This study supports the hypothesis that signalling of nitrate assimilation genes occurs intracellularly in a process dependent on the nitrate uptake activity. The bispecific nitrate/nitrite transport system I with the highest affinity for nitrate is the most efficient one for signalling the expression of nitrate/nitrite assimilation genes in C. reinhardtii.

Algal Proteins↗

PubMed1992

Nitrate transport in the cyanobacterium Anacystis nidulans R2. Kinetic and energetic aspects.

Nitrate transport has been studied in the cyanobacterium Anacystis nidulans R2 by monitoring intracellular nitrate accumulation in intact cells of the mutant strain FM6, which lacks nitrate reductase activity and is therefore unable to reduce the transported nitrate. Kinetic analysis of nitrate transport as a function of external nitrate concentration revealed apparent substrate inhibition, with a peak velocity at 20-25 microM-nitrate. A Ks (NO3-) of 1 microM was calculated. Nitrate transport exhibited a stringent requirement for Na+. Neither Li+ nor K+ could substitute for Na+. Monensin depressed nitrate transport in a concentration-dependent manner, inhibition being more than 60% at 2 microM, indicating that the Na(+)-dependence of active nitrate transport relies on the maintenance of a Na+ electrochemical gradient. The operation of an Na+/NO3- symport system is suggested. Nitrite behaved as an effective competitive inhibitor of nitrate transport, with a Ki (NO2-) of 3 microM. The time course of nitrite inhibition of nitrate transport was consistent with competitive inhibition by mixed alternative substrates. Nitrate and nitrite might be transported by the same carrier.

Biological Transport↗

PubMed2003

Regulation of the nitrate transporter gene AtNRT2.1 in Arabidopsis thaliana: responses to nitrate, amino acids and developmental stage.

The NR72.1 gene codes for a high-affinity nitrate transporter in Arabidopsis thaliana. To examine the regulation of NRT2.1 gene expression, we used a promoter-beta-glucuronidase (GUS) fusion and found that the NRT2.1 promoter directs expression to the epidermal, cortical and endodermal cell layers of mature root parts. The gene appeared to be expressed essentially in roots, but was also present in the leaf hydathodes. Investigation of NRT2.1 expression pattern during the plant developmental cycle showed that it increased rapidly during early vegetative growth, peaked prior to floral stem emergence, and decreased to very low levels in flowering and silique-bearing plants. Experiments with various nitrogen supply regimes demonstrated the induction of NRT2.1 expression by nitrate and repression by amino acids. Amino acid analysis showed that this repression was specifically related to increased internal glutamine, suggesting a role for this particular amino acid in nitrogen signalling responsible for nitrate uptake regulation. Taken together, our results support the hypothesis that the NRT2.1 gene codes for a major component of the inducible high-affinity transport system for nitrate, which is spatially and developmentally controlled at the transcriptional level. Surprisingly, NRT2.1 was not expressed in younger root parts, although a similar rate of nitrate influx was observed in both young and old root samples. This lack of correlation between nitrate influx and NRT2.1 expression suggests that another high-affinity nitrate transporter operates in root tips.

Amino Acids↗

PubMed2004

Mutation of a nitrate transporter, AtNRT1:4, results in a reduced petiole nitrate content and altered leaf development.

Unlike nitrate uptake of plant roots, less is known at the molecular level about how nitrate is distributed in various plant tissues. In the present study, characterization of the nitrate transporter, AtNRT1:4, revealed a special role of petiole in nitrate homeostasis. Electrophysiological studies using Xenopus oocytes showed that AtNRT1:4 was a low-affinity nitrate transporter. Whole-mount in situ hybridization and RT-PCR demonstrated that AtNRT1:4 was expressed in the leaf petiole. In the wild type, the leaf petiole had low nitrate reductase activity, but a high nitrate content, indicating that it is the storage site for nitrate, whereas, in the atnrt1:4 mutant, the petiole nitrate content was reduced to 50-64% of the wild-type level. Moreover, atnrt1:4 mutant leaves were wider than wild-type leaves. This study revealed a critical role of AtNRT1:4 in regulating leaf nitrate homeostasis, and the deficiency of AtNRT1:4 can alter leaf development.

Anion Transport Proteins↗

PubMed1989

Nitrate transport is independent of NADH and NAD(P)H nitrate reductases in barley seedlings.

Barley (Hordeum vulgare L.) has NADH-specific and NAD(P)H-bispecific nitrate reductase isozymes. Four isogenic lines with different nitrate reductase isozyme combinations were used to determine the role of NADH and NAD(P)H nitrate reductases on nitrate transport and assimilation in barley seedlings. Both nitrate reductase isozymes were induced by nitrate and were required for maximum nitrate assimilation in barley seedlings. Genotypes lacking the NADH isozyme (Az12) or the NAD(P)H isozyme (Az70) assimilated 65 or 85%, respectively, as much nitrate as the wild type. Nitrate assimilation by genotype (Az12;Az70) which is deficient in both nitrate reductases, was only 13% of the wild type indicating that the NADH and NAD(P)H nitrate reductase isozymes are responsible for most of the nitrate reduction in barley seedlings. For all genotypes, nitrate assimilation rates in the dark were about 55% of the rates in light. Hypotheses that nitrate reductase has direct or indirect roles in nitrate uptake were not supported by this study. Induction of nitrate transporters and the kinetics of net nitrate uptake were the same for all four genotypes indicating that neither nitrate reductase isozyme has a direct role in nitrate uptake in barley seedlings.

Biological Transport↗

PubMed1987

Molecular oxygen controls nitrate transport of Escherichia coli nitrate-respiring cells.

Escherichia coli cells grown anaerobically in the presence of nitrate reduce the nitrate as a terminal electron acceptor in place of molecular oxygen by an induced respiratory-type electron transferring system residing in the inner membrane structure. When oxygen is introduced to a suspension of nitrate-respiring cells, the oxygen is immediately reduced preferentially and the cellular uptake of nitrate ceases abruptly. In contrast, we found that the cells exhibited no oxygen control on uptake of chlorate, a competitive substrate analogue, indicating operation of an oxygen-sensitive transport system specific to nitrate. This was further evidenced by the fact that chlorate inhibition of reduction of nitrate was brought about only when the transport of both chlorate and nitrate was facilitated by the aid of carrier-type chlorate (or nitrate) ionophore. We demonstrated that oxygen inhibition on reduction of nitrate was abolished within the cells treated by octyl glucoside resulting in a removal of permeability barrier specific to nitrate. We conclude that the transient control by molecular oxygen is primarily due to the inhibition of nitrate transport into the cytoplasmic side. Since nitrate induces the nitrate-respiring system, the repression of the nitrate reductase operon by molecular oxygen is consistently interpreted on the basis of the "inducer exclusion mechanism."

Aerobiosis↗

PubMed1994

Identification of nitrate transporter genes in Chlamydomonas reinhardtii.

The Chlamydomonas reinhardtii nar-2, nar-3, and nar-4 genes, which are within a nitrate-regulated gene cluster containing the nitrate reductase structural gene nit-1, have been related to nitrate transport. Mutant strains defective in nitrate transport and having an active nitrate reductase have been genetically constructed. Their nitrate non-utilizing phenotype has been directly complemented by transformation using the pCO-5 plasmid which carries the nar-2, nar-3, and nar-4 clustered genes. Integration of pCO-5 DNA in the genome of nitrate transport mutants resulted in the expression of these nar transcripts and the recovery of a high affinity nitrate transport activity. Complementation of the nitrate non-utilizing phenotype of the constructed strains was also achieved by co-transformation with plasmids containing nar-2 and nar-3 genes or nar-2 and nar-4, but not with single plasmids containing each individual gene. In addition, DNA sequences of a practically complete cDNA of nar-3 and a partial one of nar-4 have been generated and the deduced amino acid sequences showed a very significant identity with that of the nitrate transporter gene (crnA) from Aspergillus nidulans. These data strongly support the hypothesis that the nitrate transport system in C. reinhardtii contains at least two protein components encoded by the nar-2 and nar-3 genes. The nar-4 gene would produce a protein with a high identity to that of nar-3.

Algal Proteins↗

PubMed2000

A high affinity nitrate transport system from Chlamydomonas requires two gene products.

A nitrate-regulated cluster of genes involved in nitrate transport and assimilation has been identified in Chlamydomonas reinhardtii. Mutant strains of the alga, which are defective in some aspect of transport and assimilation have been used to assign functions to these genes. This analysis has suggested that two gene products are necessary to obtain a functional high affinity nitrate system in Chlamydomonas [Quesada et al. (1994) Plant J. 5, 407-419]. In this paper we have tested this hypothesis by injecting Xenopus oocytes with mRNA prepared from these two cDNAs, Nrt2;1 and Nar2, and then assaying the oocytes for nitrate transport activity. Oocytes injected with single types of mRNA did not show any nitrate transport activity. Furthermore, Nar2 mRNA was toxic to oocytes, with nearly 60%, of the oocytes dead 3 days after the injection. However, when oocytes were injected with a mixture of two mRNAs prepared from Nrt2;1 and Nar2, a high affinity nitrate transport activity could be measured. However, the Km for nitrate of this transport system was 28 microM which is higher than the value of 1.6 microM which had been obtained by the analysis of mutant phenotypes. The pH-dependence of the nitrate-elicited currents was consistent with a proton-cotransport mechanism. These results prove that two gene products are required to produce a functional high affinity nitrate transport system and that this process does not involve transcriptional regulation.

Animals↗

PubMed1998

Independence of carbon and nitrogen control in the posttranslational regulation of nitrate transport in the cyanobacterium Synechococcus sp. strain PCC 7942.

Nitrate transport by Synechococcus sp. strain PCC 7942 cells was inhibited by ammonium and by inhibitors of CO2 fixation. Ammonium assimilation inhibitors, such as L-methionine D,L-sulfoximine, were known to prevent the negative effects of ammonium and of inhibitors of CO2 fixation on nitrate uptake, leading to propose that CO2 fixation was required to counteract the feed-back inhibition of nitrate assimilation. In NR-less mutants, L-methionine D,L-sulfoximine prevented the negative effects of ammonium on nitrate transport, but not always prevented those of inhibiting CO2 fixation. The carboxy-terminal domain of the NrtC subunit of the nitrate transporter has recently been identified as a regulatory domain involved in N-control. The mutant strain NC2, constructed by deleting the 3' portion of nrtC, showed high nitrate transport activity insensitive to ammonium but sensitive to inhibitors of CO2 fixation. These findings indicate that the C-control and the N-control of nitrate transport are independent at both the physiological and the molecular level.

Biological Transport↗

PubMed2002

Nitrate signalling on the nitrate reductase gene promoter depends directly on the activity of the nitrate transport systems in Chlamydomonas.

Nitrate signalling on the nitrate reductase (Nia1) gene promoter from Chlamydomonas reinhardtii has been studied by using a construct of the Nia1 promoter transcriptionally fused to the Chlamydomonas arylsulphatase gene as a reporter in strains bearing different sets of nitrate/nitrite transport genes. The high-affinity nitrate transport (HANT) system I is required for efficient signalling by nitrate, even at submicromolar concentrations of the anion. In addition, the autogenous regulation of nitrate reductase has been found to depend on the presence of system I. The low-affinity nitrate transport system III promoted signalling optimally on the promoter at millimolar nitrate concentrations. The HANT system IV, which is insensitive to ammonium and active at low CO2, allowed nitrate signalling at micromolar concentrations even in the presence of ammonium, suggesting that the balance of these two effectors controls Nia1 transcription. Our data indicate that nitrate signalling on the Nia1 gene promoter occurs intracellularly and depends on the activity of nitrate transporters.

Animals↗

PubMed2000

Cloning and functional characterization of a constitutively expressed nitrate transporter gene, OsNRT1, from rice.

Elucidating how rice (Oryza sativa) takes up nitrate at the molecular level could help improve the low recovery rate (<50%) of nitrogen fertilizer in rice paddies. As a first step toward that goal, we have cloned a nitrate transporter gene from rice called OsNRT1. OsNRT1 is a new member of a growing transporter family called PTR, which consists not only of nitrate transporters from higher plants that are homologs of the Arabidopsis CHL1 (AtNRT1) protein, but also peptide transporters from a wide variety of genera including animals, plants, fungi, and bacteria. However, despite the fact that OsNRT1 shares a higher degree of sequence identity with the two peptide transporters from plants (approximately 50%) than with the nitrate transporters (approximately 40%) of the PTR family, no peptide transport activity was observed when OsNRT1 was expressed in either Xenopus oocytes or yeast. Furthermore, contrasting the dual-affinity nitrate transport activity of CHL1, OsNRT1 displayed only low-affinity nitrate transport activity in Xenopus oocytes, with a K(m) value of approximately 9 mM. Northern-blot and in situ hybridization analysis indicated that OsNRT1 is constitutively expressed in the most external layer of the root, epidermis and root hair. These data strongly indicate that OsNRT1 encodes a constitutive component of a low-affinity nitrate uptake system for rice.

Amino Acid Sequence↗