High-affinity Nitrate Transporter Gene Research Articles

Positive feed-forward regulation of nitrate uptake by rice roots and its molecular mechanism

To investigate the positive feed-forward regulatory mechanism of nitrate uptake by rice, its responses to various light and carbohydrates were compared. In order to measure nitrate uptake in real time, the non-invasive method was used. The results showed that net nitrate uptake increased in the light and decreased in the dark, and finally reached a steady state after about 5 h. Based on it, carbohydrates effects could be investigated without considering light effects. After sucrose addition for 2 h, net nitrate uptake increased by about 80% without a lag, while glucose, fructose and raffinose had a slight effect with a lag and other sugars had no effect. It provided an evidence that sucrose was a positive feed-forward signal molecule of nitrate uptake by rice roots. To further analyze the effect of sucrose on the expression of high affinity nitrate transporter genes OsNRT2.1, OsNRT2.2, OsNRT2.3a and OsNRT2.3b, qRT-PCR was used to further verify after treated with 10 mM sucrose. The results revealed that these genes expression was immediately up-regulated, which indicated that these genes were post transcriptionally regulated. Further, 15N exchange dynamics analyzed N transport. It is benefit for increasing nitrate uptake by rice and improving its yield.

Dof1.7 and NIGT1 transcription factors mediate multilayered transcriptional regulation for different expression patterns of NITRATE TRANSPORTER2 genes under nitrogen deficiency stress.

Elucidating the mechanisms regulating nitrogen (N) deficiency responses in plants is of great agricultural importance. Previous studies revealed that decreased expression of NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR1 (NIGT1) transcriptional repressor genes upon N deficiency is involved in N deficiency-inducible gene expression in Arabidopsis thaliana. However, our knowledge of the mechanisms controlling N deficiency-induced changes in gene expression is still limited. Through the identification of Dof1.7 as a direct target of NIGT1 repressors and a novel N deficiency response-related transcriptional activator gene, we here show that NIGT1 and Dof1.7 transcription factors (TFs) differentially regulate N deficiency-inducible expression of three high-affinity nitrate transporter genes, NRT2.1, NRT2.4, and NRT2.5, which are responsible for most of the soil nitrate uptake activity of Arabidopsis plants under N-deficient conditions. Unlike NIGT1 repressors, which directly suppress NRT2.1, NRT2.4, and NRT2.5 under N-sufficient conditions, Dof1.7 directly activated only NRT2.5 but indirectly and moderately activated NRT2.1 and NRT2.4 under N-deficient conditions, probably by indirectly decreasing NIGT1 expression. Thus, Dof1.7 converted passive transcriptional activation into active and potent transcriptional activation, further differentially enhancing the expression of NRT2 genes. These findings clarify the mechanism underlying different expression patterns of NRT2 genes upon N deficiency, suggesting that time-dependent multilayered transcriptional regulation generates complicated expression patterns of N deficiency-inducible genes.

The Functional Diversity of the High-Affinity Nitrate Transporter Gene Family in Hexaploid Wheat: Insights from Distinct Expression Profiles.

High-affinity nitrate transporters (NRT) are key components for nitrogen (N) acquisition and distribution within plants. However, insights on these transporters in wheat are scarce. This study presents a comprehensive analysis of the NRT2 and NRT3 gene families, where the aim is to shed light on their functionality and to evaluate their responses to N availability. A total of 53 NRT2s and 11 NRT3s were identified in the bread wheat genome, and these were grouped into different clades and homoeologous subgroups. The transcriptional dynamics of the identified NRT2 and NRT3 genes, in response to N starvation and nitrate resupply, were examined by RT-qPCR in the roots and shoots of hydroponically grown wheat plants through a time course experiment. Additionally, the spatial expression patterns of these genes were explored within the plant. The NRT2s of clade 1, TaNRT2.1-2.6, showed a root-specific expression and significant upregulation in response to N starvation, thus emphasizing a role in N acquisition. However, most of the clade 2 NRT2s displayed reduced expression under N-starved conditions. Nitrate resupply after N starvation revealed rapid responsiveness in TaNRT2.1-2.6, while clade 2 genes exhibited gradual induction, primarily in the roots. TaNRT2.18 was highly expressed in above-ground tissues and exhibited distinct nitrate-related response patterns for roots and shoots. The TaNRT3 gene expression closely paralleled the profiles of TaNRT2.1-2.6 in response to nitrate induction. These findings enhance the understanding of NRT2 and NRT3 involvement in nitrogen uptake and utilization, and they could have practical implications for improving nitrogen use efficiency. The study also recommends a standardized nomenclature for wheat NRT2 genes, thereby addressing prior naming inconsistencies.

Physiological and Transcriptomic Analysis Provides Insights into Low Nitrogen Stress in Foxtail Millet (Setaria italica L.).

Foxtail millet (Setaria italica (L.) P. Beauv) is an important food and forage crop that is well adapted to nutrient-poor soils. However, our understanding of how different LN-tolerant foxtail millet varieties adapt to long-term low nitrogen (LN) stress at the physiological and molecular levels remains limited. In this study, two foxtail millet varieties with contrasting LN tolerance properties were investigated through analyses of physiological parameters and transcriptomics. The physiological results indicate that JG20 (high tolerance to LN) exhibited superior biomass accumulation both in its shoots and roots, and higher nitrogen content, soluble sugar concentration, soluble protein concentration, zeatin concentration in shoot, and lower soluble sugar and soluble protein concentration in its roots compared to JG22 (sensitive to LN) under LN, this indicated that the LN-tolerant foxtail millet variety can allocate more functional substance to its shoots to sustain aboveground growth and maintain high root activity by utilizing low soluble sugar and protein under LN conditions. In the transcriptomics analysis, JG20 exhibited a greater number of differentially expressed genes (DEGs) compared to JG22 in both its shoots and roots in response to LN stress. These LN-responsive genes were enriched in glycolysis metabolism, photosynthesis, hormone metabolism, and nitrogen metabolism. Furthermore, in the shoots, the glutamine synthetase gene SiGS5, chlorophyll apoprotein of photosystem II gene SiPsbQ, ATP synthase subunit gene Sib, zeatin synthesis genes SiAHP1, and aldose 1-epimerase gene SiAEP, and, in the roots, the high-affinity nitrate transporter genes SiNRT2.3, SiNRT2.4, glutamate synthase gene SiGOGAT2, fructose-bisphosphate aldolase gene SiFBA5, were important genes involved in the LN tolerance of the foxtail millet variety. Hence, our study implies that the identified genes and metabolic pathways contribute valuable insights into the mechanisms underlying LN tolerance in foxtail millet.

Integrated BSA-seq and RNA-seq analysis to identify candidate genes associated with nitrogen utilization efficiency (NUtE) in rapeseed (Brassica napus L.)

Rapeseed (Brassica napus L.) is one of the important oil crops, with a high demand for nitrogen (N). It is essential to explore the potential of rapeseed to improve nitrogen utilization efficiency (NUtE). Rapeseed is an allotetraploid crop with a relatively large and complex genome, and there are few studies on the mapping of genes related to NUtE regulation. In this study, we used the combination of bulk segregant analysis sequencing (BSA-Seq) and RNA sequencing (RNA-Seq) to analyze the N-efficient genotype ‘Zheyou 18’ and N-inefficient genotype ‘Sollux’, to identify the genetic regulatory mechanisms. Several candidate genes were screened, such as the high-affinity nitrate transporter gene NRT2.1 (BnaC08g43370D) and the abscisic acid (ABA) signal transduction-related genes (BnaC02g14540D, BnaA03g20760D, and BnaA05g01330D). BnaA05g01330D was annotated as ABA-INDUCIBLE bHLH-TYPE TRANSCRIPTION FACTOR (AIB/bHLH17), which was highly expressed in the root. The results showed that the primary root length of the ataib mutant was significantly longer than that of the wild type under low N conditions. Overexpression of BnaA5.AIB could reduce the NUtE under low N levels in Arabidopsis (Arabidopsis thaliana). Candidate genes identified in this study may be involved in the regulation of NUtE in rapeseed, and new functions of AIB in orchestrating N uptake and utilization have been revealed. It is indicated that BnaA5.AIB may be the key factor that links ABA to N signaling and a negative regulator of NUtE. It will provide a theoretical basis and application prospect for resource conservation, environmental protection, and sustainable agricultural development.

Linking glucose signaling to nitrogen utilization by the OsHXK7-ARE4 complex in rice

How reciprocal regulation of carbon and nitrogen metabolism works is a long-standing question. In plants, glucose and nitrate are proposed to act as signaling molecules, regulating carbon and nitrogen metabolism via largely unknown mechanisms. Here, we show that the MYB-related transcription factor ARE4 coordinates glucose signaling and nitrogen utilization in rice. ARE4 is retained in the cytosol in complexing with the glucose sensor OsHXK7. Upon sensing a glucose signal, ARE4 is released, is translocated into the nucleus, and activates the expression of a subset of high-affinity nitrate transporter genes, thereby boosting nitrate uptake and accumulation. This regulatory scheme displays a diurnal pattern in response to circadian changes of soluble sugars. The are4 mutations compromise in nitrate utilization and plant growth, whereas overexpression of ARE4 increases grain size. We propose that the OsHXK7-ARE4 complex links glucose to the transcriptional regulation of nitrogen utilization, thereby coordinating carbon and nitrogen metabolism.

Cloning and expression study of a high-affinity nitrate transporter gene from Zea mays L

ABSTRACT A nitrate transporter gene, named B46NRT2.1, from salt-tolerant Zea mays L. B46 has been cloned. B46NRT2.1 contained the same domain belonging to the major facilitator superfamily (PLN00028). The results of the phylogenetic tree indicated that B46NRT2.1 exhibits sequence similarity and the closest relationship with those known nitrate transporters of the NRT2 family. Through RT-qPCR, we found that the expression of B46NRT2.1 mainly happens in the root and leaf. Moreover, the treatment with NaCl, Na2CO3, and NaHCO3 could significantly increase the expression of B46NRT2.1. B46NRT2.1 was located in the plasma membrane. Through the study of yeast and plant salt response brought by B46NRT2.1 overexpression, we have preliminary knowledge that the expression of B46NRT2.1 makes yeast and plants respond to salt shock. There are 10 different kinds of cis-acting regulatory elements (CRES) in the promotor sequences of B46NRT2.1 gene using the PlantCARE web server to analyze. It mainly includes hormone response, abscisic acid, salicylic acid, gibberellin, methyl jasmonate, and auxin. The B46NRT2.1 gene’s co-expression network showed that it was co-expressed with a number of other genes in several biological pathways, including regulation of NO3 long-distance transit, modulation of nitrate sensing and metabolism, nitrate assimilation, and transduction of Jasmonic acid-independent wound signal. The results of this work should serve as a good scientific foundation for further research on the functions of the NRT2 gene family in plants (inbred line B46), and this research adds to our understanding of the molecular mechanisms under salt tolerance.

Growth stage and nitrate limiting response of NRT2 and NAR2 gene families of bread wheat, and complementation and retrieval of nitrate uptake of atnrt2.1 mutant by a wheat NRT2 gene

The Nitrate Transporter 2 (NRT2), together with accessory protein (NAR), form high-affinity nitrate transport systems (HATS) responsible for nitrate uptake and translocation under its limited availability in plants. In the present study, we aimed to identify members of these two families’ genes in the bread wheat genome on a genome-wide scale, the evolutionary relationship of these families to major cereals, and their expression dynamics. A total of 46 and 8 NRT2 and NAR2 family genes, respectively, have been identified, which showed tissue and growth-stage specific nitrate limiting responses in all major tissues with a tendency of homeolog expression bias in two wheat genotypes. The high-affinity transport capacity of four wheat genotypes using 15N influx at three major physiological stages also showed significant roles of HATS at tissue and growth stage levels. The complementation of atnrt2.1 mutant with one of the high-affinity nitrate transporter genes, i.e., TaNRT2.1-B6, caused recovery of about 1.5 times 15N influx in N + and N- conditions in Arabidopsis mutant than wild type. The present study contributes to the genomic and functional understanding of NRT2 and NAR2 genes of bread wheat, providing a platform for further learning the underlying mechanism of nitrate uptake in bread wheat.

Comparative genomic and transcriptomic analyses uncover the molecular basis of high nitrogen-use efficiency in the wheat cultivar Kenong 9204

Studying the regulatory mechanisms that drive nitrogen-use efficiency (NUE) in crops is important for sustainable agriculture and environmental protection. In this study, we generated a high-quality genome assembly for the high-NUE wheat cultivar Kenong 9204 and systematically analyzed genes related to nitrogen uptake and metabolism. By comparative analyses, we found that the high-affinity nitrate transporter gene family had expanded in Triticeae. Further studies showed that subsequent functional differentiation endowed the expanded family members with saline inducibility, providing a genetic basis for improving the adaptability of wheat to nitrogen deficiency in various habitats. To explore the genetic and molecular mechanisms of high NUE, we compared genomic and transcriptomic data from the high-NUE cultivar Kenong 9204 (KN9204) and the low-NUE cultivar Jing 411 and quantified their nitrogen accumulation under high- and low-nitrogen conditions. Compared with Jing 411, KN9204 absorbed significantly more nitrogen at the reproductive stage after shooting and accumulated it in the shoots and seeds. Transcriptome data analysis revealed that nitrogen deficiency clearly suppressed the expression of genes related to cell division in the young spike of Jing 411, whereas this suppression of gene expression was much lower in KN9204. In addition, KN9204 maintained relatively high expression of NPF genes for a longer time than Jing 411 during seed maturity. Physiological and transcriptome data revealed that KN9204 was more tolerant of nitrogen deficiency than Jing 411, especially at the reproductive stage. The high NUE of KN9204 is an integrated effect controlled at different levels. Taken together, our data provide new insights into the molecular mechanisms of NUE and important gene resources for improving wheat cultivars with a higher NUE trait.

Identification of a novel D3 allele playing a role in nitrogen utilization

Tillering is an important agricultural trait in rice and can affect grain yield. Therefore, screening and identification of mutants in tiller-associated traits are crucial for understanding the development of rice tillers. In this study, we identified a new allelic mutant of d3 with phenotypic characteristics influenced by exogenous nitrogen. The j30 mutant harbors a G-to-A base substitution at position 1900 of the coding sequence for LOC_Os06g06050, resulting in an amino acid conversion from Asp to Asn, being a new allelic mutant of d3. During the whole growth period, j30 displayed multiple characteristic variations, including dwarfism, thinner stems, narrower leaves, more tillers, etc. Compared with wild-type (WT) counterparts, the leaf blades and culms of j30 consisted of fewer vascular bundles with shorter distances between them. Quantitative polymerase chain reaction (qPCR) analysis revealed that, in the mutant, N-fertilizer could extremely upregulate expression of the N-pathway-related glutamine synthetase gene GS1;3 and reduce the transcription of high-affinity nitrate transporter genes (OsNRT2.2 and OsNRT2.3a were downregulated before N treatment) to sparse expression. The catalase-encoding gene CATA alone displayed a lower transcriptional level in j30 when treated with N-fertilizer, while CATB and CATC showed higher expression and CATA no difference compared with before N-fertilizer treatment.

Bacillus subtilis strain L1 promotes nitrate reductase activity in Arabidopsis and elicits enhanced growth performance in Arabidopsis, lettuce, and wheat.

Plant growth promoting rhizobacteria (PGPR) are a group of bacteria that promote plants growth in the rhizosphere. PGPRs are involved in various mechanisms that reinforce plant development. In this study, we screened for PGPRs that were effective in early growth of Arabidopsis thaliana when added to the media and one Bacillus subtilis strain L1 (Bs L1) was selected for further study. When Bs L1 was placed near the roots, seedlings showed notably stronger growth than that in the control, particularly in biomass and root hair. Quantitative reverse transcription polymerase chain reaction analysis revealed a high level of expression of the high affinity nitrate transporter gene, NRT2.1 in A. thaliana treated with Bs L1. After considering how Bs L1 could promote plant growth, we focused on nitrate, which is essential to plant growth. The nitrate content was lower in A. thaliana treated with Bs L1. However, examination of the activity of nitrate reductase revealed higher activity in plants treated with PGPR than in the control. Bs L1 had pronounced effects in representative crops (wheat and lettuce). These results suggest that Bs L1 promotes the assimilation and use of nitrate and plant growth.

Transcriptome and physiological analysis of a lutein-producing alga Desmodesmus sp. reveals the molecular mechanisms for high lutein productivity

A green alga Desmodesmus sp. JSC3 is a lutein producer that appears high in lutein production in batch culture under 200μmol·m−2·s−1 condition using 1g∙L−1 sodium nitrate as nitrogen source. Because lutein content increased as growth advanced with the maximum (5.18mg∙g−1) on day 6, comparative transcriptome and physiological analyses on day 1, 6, and 8 were thus employed to uncover the molecular mechanisms for lutein production in this lutein producer. The gene set enrichment analysis using the False Discovery Rate (FDR) test showed that chlorophyll synthesis and photosynthesis were significantly changed when lutein accumulated. The up-regulation of the carotenogenesis genes explains the preference of this alga in lutein accumulation by enhancing lycopene synthesis and promoting metabolic flux towards synthesis of α-carotene and then hydroxylation of the β- and ε-ring of α-carotene, in the late exponential growth period (4–6days). Chlorophyll content and the expression of the genes encoding the enzymes in tetrapyrrole biosynthesis also increased during 0–6days, although chlorophyll a/chlorophyll b ratio decreased on day 6 due to light attenuation caused by high cell density. Photosynthetic activity and the expression of the genes encoding light-harvesting complex (LHC) proteins increased during 0–4days but decreased after that due to nitrogen deficiency (up-regulation of high-affinity nitrate transporter gene and low medium nitrate concentration). It suggests that LHC synthesis and photosynthesis are sensitive to nitrogen deficiency occurring on day 6 while pigment synthesis remains active. Because the expression of the genes encoding LHC-like proteins (early light-inducible proteins) that bind lutein and chlorophyll increased on day 6, LHC-like proteins could be temporarily stores of accumulating pigments. In conclusion, coordinate up-regulation of LHC-like proteins with pigment synthesis for the acclimation to decreasing light availability is believed as the molecular mechanism for high lutein production in Desmodesmus sp. JSC3.

The calcium sensor CBL7 modulates plant responses to low nitrate in Arabidopsis

Calcium (Ca2+) serves as a critical messenger in a number of adaptation and developmental processes. In plants, CBL family represents a unique group of calcium sensors that decodes calcium signals. Several CBL members have been functionally characterized in the model plant Arabidopsis thaliana, but the role of CBL7 remains unknown. Here, we report that CBL7 is involved in the regulation of low-nitrate response in Arabidopsis. Expression of CBL7 was predominant in the root of young seedlings and substantially induced by nitrate starvation. Cbl7 mutant was more inhibited in root growth upon nitrate starvation compared to the wild-type. Interestingly, the growth arrest of cbl7 under low-nitrate conditions relied on acidic pH. Further analyses revealed that expression of two high-affinity nitrate transporter genes, NRT2.4 and NRT2.5, was down-regulated in cbl7 under nitrogen-starvation condition. Accordingly, the cbl7 mutant plants retained lower nitrate content than wild-type plants under low-nitrate condition. Taken together, our results uncover a novel role of CBL7 in the response to nitrate deficiency in Arabidopsis.

Phytosiderophores revisited: 2′-deoxymugineic acid-mediated iron uptake triggers nitrogen assimilation in rice (Oryza sativa L.) seedlings

Poaceae plants release phytosiderophores into the rhizosphere in order to chelate iron (Fe), which often exists in insoluble forms especially under high pH conditions. The impact of phytosiderophore treatment at the physiological and molecular levels in vivo remains largely elusive, although the biosynthesis of phytosiderophores and the transport of phytosiderophore-metal complexes have been well studied. We recently showed that the application of 30 μM of the chemically synthesized phytosiderophore 2′-deoxymugineic acid (DMA) was sufficient for apparent full recovery of otherwise considerably reduced growth of hydroponic rice seedlings at high pH. Moreover, unexpected induction of high-affinity nitrate transporter gene expression as well as nitrate reductase activity indicates that the nitrate response is linked to Fe homeostasis. These data shed light on the biological relevance of DMA not simply as a Fe chelator, but also as a trigger that promotes plant growth by reinforcing nitrate assimilation.

Cloning and molecular characterization of high-affinity nitrate transporter gene BjNRT2.1 from Indian mustard

Inducible high-affinity nitrate transporter gene (BjNRT2.1) was isolated and structurally characterized from Indian mustard (Brassica juncea L.) seedlings grown in 10 µM nitrate solution. By performing 5′RACE-PCR, the full length cDNA of 1593 bp was obtained. Alignment and comparative analyses with other NRT2 proteins showed that BjNRT2.1 encodes a member of HATS family. Highly conserved motifs between AtNRT2.1 and BjNRT2.1 suggested that it is an orthologue of AtNRT2.1. Hydropathy plot of BjNRT2.1 predicted 12 membrane spanning α-helices with both C- and N-terminal inside. Reliability of this model was supported by the presence of major facilitator superfamily and nitrate-nitrite porter signatures associated with NRT2 transporter and conserved arginine residues. Induction of BjNRT2.1 transcripts at 10 µM nitrate concentration only in roots confirmed that it is root-specific and a member of inducible HATS gene family.

AtTGA4, a bZIP transcription factor, confers drought resistance by enhancing nitrate transport and assimilation in Arabidopsis thaliana

To cope with environmental stress caused by global climate change and excessive nitrogen application, it is important to improve water and nitrogen use efficiencies in crop plants. It has been reported that higher nitrogen uptake could alleviate the damaging impact of drought stress. However, there is scant evidence to explain how nitrogen uptake affects drought resistance. In this study we observed that bZIP transcription factor AtTGA4 (TGACG motif-binding factor 4) was induced by both drought and low nitrogen stresses, and that overexpression of AtTGA4 simultaneously improved drought resistance and reduced nitrogen starvation in Arabidopsis. Following drought stress there were higher nitrogen and proline contents in transgenic AtTGA4 plants than in wild type controls, and activity of the key enzyme nitrite reductase (NIR) involved in nitrate assimilation processes was also higher. Expressions of the high-affinity nitrate transporter genes NRT2.1 and NRT2.2 and nitrate reductase genes NIA1 and NIA2 in transgenic plants were all higher than in wild type indicating that higher levels of nitrate transport and assimilation activity contributed to enhanced drought resistance of AtTGA4 transgenic plants. Thus genetic transformation with AtTGA4 may provide a new approach to simultaneously improve crop tolerance to drought and low nitrogen stresses.

Root aeration improves growth and nitrogen accumulation in rice seedlings under low nitrogen.

In wetland soils, changes in oxygen (O2) level in the rhizosphere are believed to influence the behaviour of nutrients and their usage by plants. However, the effect of aeration on nitrogen (N) acquisition under different N supply conditions remains largely unknown. In this study, the rice cultivars Yangdao 6 (YD6, with higher root aerenchyma abundance) and Nongken 57 (NK57, with lower root aerenchyma abundance) were used to evaluate the effects of aeration on rice growth and N accumulation. Our results showed that the number of adventitious roots and the root surface area increased significantly, and ethylene production and aerenchyma formation decreased in both cultivars after external aeration (EA). Five N treatments, including no N (-N), 0.125 mM NH4NO3 (LN), 1.25 mM Ca(NO3)2 (NO3-N), 1.25 mM (NH4)2SO4 (NH4-N) and 1.25 mM NH4NO3 (N/N), were applied to YD6 and NK57 for 2 days under internal aeration or EA conditions. External aeration increased the root biomass in both cultivars and the shoot biomass in NK57 by 18-50 %. The total N concentrations in roots of YD6 grown under -N and LN and of NK57 grown under NO3-N were increased by EA. Expression of OsPAD4, one of four putative genes regulating aerenchyma formation, showed a similar pattern alongside changes in the ethylene level in the EA-treated rice irrespective of the N treatments. Furthermore, expression of the high-affinity nitrate transporter gene OsNRT2.1 was increased by EA under -N, LN and NO3-N conditions. Our data provide evidence of an interaction between O2 and the supply of N in ethylene production, aerenchyma formation and N nutrition through modification of the expression of OsPAD4 and OsNRT2.1.

Influences of external nutrient conditions on the transcript levels of a nitrate transporter gene in Skeletonema costatum

To verify the feasibility of high-affinity nitrate transporter gene (Nrt2) as an indicator of nitrogen status, changes in the transcript levels of transcripts associated with phosphate starvation and different nitrate concentrations were studied using real-time quantitative reverse-transcription PCR (QRT-PCR) technology in batch cultures of Skeletonema costatum. The results show that compared with P-replete condition, P starvation could reduce the Nrt2 transcript levels apparently. Nrt2 transcript levels had a significant negative linear correlation with nitrate concentrations below 40 mu mol/L. The results of 48 h short-term incubation experiment under different nitrate concentrations confirmed this correlation, and the following regression equation is built: y = -3.305x + 98.95, R-2 = 0.988, where x represents nitrate concentrations (<40 mu mol/L) and y represents the Nrt2 transcript levels.

Production and characterization of transgenic rice plants carrying a high-affinity nitrate transporter gene (OsNRT2.1)

Enhancing nitrogen utilization is important in maintaining crop yield under low nitrogen conditions. Therefore, rice plants (Oryza sativa L.) were transformed with pMLH7133-RNRT2, which bears OsNRT2.1, a major gene involved in nitrate uptake under low nitrate concentrations. Two transgenic plants harboring an OsNRT2.1 transgene were identified and used for further study. In transgenic plants, OsNRT2.1 was expressed constitutively in roots, while in wild-type plants, the transcript was detected 30 min after starting to supply nitrate. No co-suppression between transgene and host gene was observed, as was observed on introduction of a nitrate reductase gene from the same species. One of the transformants grew faster than the wild-type cultivars when KNO3 or NH4NO3 was supplied as a nitrogen source. However, no increase in nitrate uptake by young plants was observed. These results suggest that constitutive expression of OsNRT2.1 might enhance vegetative growth and that introduction of genes involved in nitrate uptake from the same species may be useful for genetic improvement of plant growth under low nitrogen conditions. Further studies are necessary to understand the molecular and physiological bases of expression of NRT2.1 in relation to plant growth.

Nitrite transport is mediated by the nitrite-specific high-affinity NitA transporter and by nitrate transporters NrtA, NrtB in Aspergillus nidulans

Disruption of the Aspergillus nidulans high-affinity nitrate transporter genes ( nrtA and nrtB) prevents growth on nitrate but not nitrite. We identified a distinct nitrite transporter ( K m = 4.2 ± 1 μM, V max = 168 ± 21 nmol mg −1 DW −1 h −1), designated NitA. Disruption of nrtA, nrtB and nitA blocked growth on nitrite, despite low rates of nitrite depletion we ascribe to passive nitrous acid permeation. Growth of the single mutant nitA16 on nitrite was wild-type, suggesting that NrtA and/or NrtB transports nitrite as well as nitrate. Indeed, NrtA and NrtB transport nitrite at higher rates than NitA; K m and V max values were 16 ± 4 μM and 808 ± 67 nmol mg −1 DW −1 h −1 (NrtA) and 11 ± 1 μM and 979 ± 17 nmol mg −1 DW −1 h −1 (NrtB). We suggest that NrtA is a nitrate/nitrite transporter, NrtB absorbs nitrite in preference to nitrate and NitA is exclusively a nitrite transporter.