Characterization of a two-component high-affinity nitrate uptake system in Arabidopsis. Physiology and protein-protein interaction

The identification of a family of NAR2-type genes in higher plants showed that there was a homolog in Arabidopsis (Arabidopsis thaliana), AtNAR2.1. These genes encode part of a two-component nitrate high-affinity transport system (HATS). As the Arabidopsis NRT2 gene family of nitrate transporters has been characterized, we tested the idea that AtNAR2.1 and AtNRT2.1 are partners in a two-component HATS. Results using the yeast split-ubiquitin system and Xenopus oocyte expression showed that the two proteins interacted to give a functional HATS. The growth and nitrogen (N) physiology of two Arabidopsis gene knockout mutants, atnrt2.1-1 and atnar2.1-1, one for each partner protein, were compared. Both types of plants had lost HATS activity at 0.2 mm nitrate, but the effect was more severe in atnar2.1-1 plants. The relationship between plant N status and nitrate transporter expression revealed a pattern that was characteristic of N deficiency that was again stronger in atnar2.1-1. Plants resulting from a cross between both mutants (atnrt2.1-1 x atnar2.1-1) showed a phenotype like that of the atnar2.1-1 mutant when grown in 0.5 mm nitrate. Lateral root assays also revealed growth differences between the two mutants, confirming that atnar2.1-1 had a stronger phenotype. To show that the impaired HATS did not result from the decreased expression of AtNRT2.1, we tested if constitutive root expression of a tobacco (Nicotiana plumbaginifolia) gene, NpNRT2.1, previously been shown to complement atnrt2.1-1, can restore HATS to the atnar2.1-1 mutant. These plants did not recover wild-type nitrate HATS. Taken together, these results show that AtNAR2.1 is essential for HATS of nitrate in Arabidopsis.     

Multiple roles of nitrate transport accessory protein NAR2 in plants

Two component high affinity nitrate transport system, NAR2/NRT2, has been defined in several plant species. In Arabidopsis, AtNAR2.1 has a role in the targeting of AtNRT2.1 to the plasma membrane. The gene knock out mutant atnar2.1 lacks inducible high-affinity transport system (IHATS) activity, it also shows the same inhibition of lateral root (LR) initiation on the newly developed primary roots as the atnrt2.1 mutant in response to low nitrate supply. In rice, OsNAR2.1 interacts with OsNRT2.1, OsNRT2.2 and OsNRT2.3a to provide nitrate uptake over high and low concentration ranges. In rice roots OsNAR2.1 and its partner NRT2s show some expression differences in both tissue specificity and abundance. It can be predicted that NAR2 plays multiple roles in addition to being an IHATS component in plants.Key words: NAR2, NRT2, nitrate transporter, root     

Characterization of an intact two‐component high‐affinity nitrate transporter from Arabidopsis roots

AtNRT2.1, a polypeptide of the Arabidopsis thaliana two‐component inducible high‐affinity nitrate transport system (IHATS), is located within the plasma membrane. The monomeric form of AtNRT2.1 has been reported to be the most abundant form, and was suggested to be the form that is active in nitrate transport. Here we have used immunological and transient protoplast expression methods to demonstrate that an intact two‐component complex of AtNRT2.1 and AtNAR2.1 (AtNRT3.1) is localized in the plasma membrane. A. thaliana mutants lacking AtNAR2.1 have virtually no IHATS capacity and exhibit extremely poor growth on low nitrate as the sole source of nitrogen. Near‐normal growth and nitrate transport in the mutant were restored by transformation with myc‐tagged AtNAR2.1 cDNA. Membrane fractions from roots of the restored myc‐tagged line were solubilized in 1.5% dodecyl‐β‐maltoside and partially purified in the first dimension by blue native gel electrophoresis. Using anti‐NRT2.1 antibodies, an oligomeric polypeptide (approximate molecular mass 150 kDa) was identified, but monomeric AtNRT2.1 was absent. This oligomer was also observed in the wild‐type, and was resolved, using SDS–PAGE for the second dimension, into two polypeptides with molecular masses of approximately 48 and 26 kDa, corresponding to AtNRT2.1 and myc‐tagged AtNAR2.1, respectively. This result, together with the finding that the oligomer is absent from NRT2.1 or NAR2.1 mutants, suggests that this complex, rather than monomeric AtNRT2.1, is the form that is active in IHATS nitrate transport. The molecular mass of the intact oligomer suggests that the functional unit for high‐affinity nitrate influx may be a tetramer consisting of two subunits each of AtNRT2.1 and AtNAR2.1.     

A central role for the nitrate transporter NRT2.1 in the integrated morphological and physiological responses of the root system to nitrogen limitation in Arabidopsis

Up-regulation of the high-affinity transport system (HATS) for NO(3)(-) and stimulation of lateral root (LR) growth are two important adaptive responses of the root system to nitrogen limitation. Up-regulation of the NO(3)(-) HATS by nitrogen starvation is suppressed in the atnrt2.1-1 mutant of Arabidopsis (Arabidopsis thaliana), deleted for both NRT2.1 and NRT2.2 nitrate transporter genes. We then used this mutant to determine whether lack of HATS stimulation affected the response of the root system architecture (RSA) to low NO(3)(-) availability. In Wassilewskija (Ws) wild-type plants, transfer from high to low NO(3)(-) medium resulted in contrasting responses of RSA, depending on the level of nitrogen limitation. Moderate nitrogen limitation (transfer from 10 mm to 1 or 0.5 mm NO(3)(-)) mostly led to an increase in the number of visible laterals, while severe nitrogen stress (transfer from 10 mm to 0.1 or 0.05 mm NO(3)(-)) promoted mean LR length. The RSA response of the atnrt2.1-1 mutant to low NO(3)(-) was markedly different. After transfer from 10 to 0.5 mm NO(3)(-), the stimulated appearance of LRs was abolished in atnrt2.1-1 plants, whereas the increase in mean LR length was much more pronounced than in Ws. These modifications of RSA mimicked those of Ws plants subjected to severe nitrogen stress and could be fully explained by the lowered NO(3)(-) uptake measured in the mutant. This suggests that the uptake rate of NO(3)(-), rather than its external concentration, is the key factor triggering the observed changes in RSA. However, the mutation of NRT2.1 was also found to inhibit initiation of LR primordia in plants subjected to nitrogen limitation independently of the rate of NO(3)(-) uptake by the whole root system and even of the presence of added NO(3)(-) in the external medium. This indicates a direct stimulatory role for NRT2.1 in this particular step of LR development. Thus, it is concluded that NRT2.1 has a key dual function in coordinating root development with external NO(3)(-) availability, both indirectly through its role as a major NO(3)(-) uptake system that determines the nitrogen uptake-dependent RSA responses, and directly through a specific action on LR initiation under nitrogen-limited conditions.     

Nitrate transport capacity of the Arabidopsis thaliana NRT2 family members and their interactions with AtNAR2.1

? Interactions between the Arabidopsis NitRate Transporter (AtNRT2.1) and Nitrate Assimilation Related protein (AtNAR2.1, also known as AtNRT3.1) have been well documented, and confirmed by the demonstration that AtNRT2.1 and AtNAR2.1 form a 150-kDa plasma membrane complex, thought to constitute the high-affinity nitrate transporter of Arabidopsis thaliana roots. Here, we have investigated interactions between the remaining AtNRT2 family members (AtNRT2.2 to AtNRT2.7) and AtNAR2.1, and their capacity for nitrate transport. ? Three different systems were used to examine possible interactions with AtNAR2.1: membrane yeast split-ubiquitin, bimolecular fluorescence complementation in A. thaliana protoplasts and nitrate uptake in Xenopus oocytes. ? All NRT2s, except for AtNRT2.7, restored growth and β-galactosidase activity in the yeast split-ubiquitin system, and split-YFP fluorescence in A. thaliana protoplasts only when co-expressed with AtNAR2.1. Thus, except for AtNRT2.7, all other NRT2 transporters interact strongly with AtNAR2.1. ? Co-injection into Xenopus oocytes of cRNA of all NRT2 genes together with cRNA of AtNAR2.1 resulted in statistically significant increases of uptake over and above that resulting from single cRNA injections.     

High-affinity nitrate transport in roots of Arabidopsis depends on expression of the NAR2-like gene AtNRT3.1

The NAR2 protein of Chlamydomonas reinhardtii has no known transport activity yet it is required for high-affinity nitrate uptake. Arabidopsis (Arabidopsis thaliana) possesses two genes, AtNRT3.1 and AtNRT3.2, that are similar to the C. reinhardtii NAR2 gene. AtNRT3.1 accounts for greater than 99% of NRT3 mRNA and is induced 6-fold by nitrate. AtNRT3.2 was expressed constitutively at a very low level and did not compensate for the loss of AtNRT3.1 in two Atnrt3.1 mutants. Nitrate uptake by roots and nitrate induction of gene expression were analyzed in two T-DNA mutants, Atnrt3.1-1 and Atnrt3.1-2, disrupted in the AtNRT3.1 promoter and coding regions, respectively, in 5-week-old plants. Nitrate induction of the nitrate transporter genes AtNRT1.1 and AtNRT2.1 was reduced in Atnrt3.1 mutant plants, and this reduced expression was correlated with reduced nitrate concentrations in the tissues. Constitutive high-affinity influx was reduced by 34% and 89%, respectively, in Atnrt3.1-1 and Atnrt3.1-2 mutant plants, while high-affinity nitrate-inducible influx was reduced by 92% and 96%, respectively, following induction with 1 mm KNO(3) after 7 d of nitrogen deprivation. By contrast, low-affinity influx appeared to be unaffected. Thus, the constitutive high-affinity influx and nitrate-inducible high-affinity influx (but not the low-affinity influx) of higher plant roots require a functional AtNRT3 (NAR2) gene.     

Reciprocal regulation between AtNRT2.1 and AtAMT1.1 expression and the kinetics of NH(4)(+) and NO(3)(-) influxes

Our results show that AtNRT2.1 expression has a positive effect on the NH₄⁺ ion influx, mediated by the HATS, as also occurs with AtAMT1.1 expression on the NO₃⁻ ion influx. AtNRT2.1 expression plays a key role in the regulation of AtAMT1.1 expression and in the NH₄⁺ ion influx, differentiating the nitrogen source, and particularly, the lack of it. Nitrogen starvation produces a compensatory effect by AtAMT1.1 when there is an absence of the AtNRT2.1 gene. Our results also show that, in the atnrt2 mutant lacking both AtNRT2.1 and AtNRT2.2, gene functions present different kinetic parameters on the NH₄⁺ ion influx mediated by the HATS, according to the source and availability of nitrogen. Finally, the absence of AMT1.1 also produces changes in the kinetic parameters of the NO₃⁻ influx, showing different V_max values depending on the source of nitrogen available.     

Regulation of the High-Affinity Nitrate Transport System in Wheat Roots by Exogenous Abscisic Acid and Glutamine

Nitrate is a major nitrogen （N） source for most crops. Nitrate uptake by root cells is a key step of nitrogen metabolism and has been widely studied at the physiological and molecular levels. Understanding how nitrate uptake is regulated will help us engineer crops with improved nitrate uptake efficiency. The present study investigated the regulation of the high-affinity nitrate transport system （HATS） by exogenous abscisic acid （ABA） and glutamine （Gin） in wheat （Triticum aestivum L.） roots. Wheat seedlings grown in nutrient solution containing 2 mmol/L nitrate as the only nitrogen source for 2weeks were deprived of N for 4d and were then transferred to nutrient solution containing 50 μmol/L ABA, and 1 mmol/L Gin in the presence or absence of 2 mmol/L nitrate for 0, 0.5, 1, 2, 4, and 8 h. Treated wheat plants were then divided into two groups. One group of plants was used to investigate the mRNA levels of the HATS components NRT2 and NAR2 genes in roots through semi-quantitative RT-PCR approach, and the other set of plants were used to measure high-affinity nitrate influx rates in a nutrient solution containing 0.2 mmol/L ^15N-labeled nitrate. The results showed that exogenous ABA induced the expression of the TaNRT2.1, TaNRT2.2, TaNRT2.3, TaNAR2.1, and TaNAR2.2 genes in roots when nitrate was not present in the nutrient solution, but did not further enhance the induction of these genes by nitrate. Glutamine, which has been shown to inhibit the expression of NRT2 genes when nitrate is present in the growth media, did not inhibit this induction. When Gin was supplied to a nitrate-free nutrient solution, the expression of these five genes in roots was induced. These results imply that the inhibition by Gin of NRT2 expression occurs only when nitrate is present in the growth media. Although exogenous ABA and Gin induced HATS genes in the roots of wheat, they did not induce nitrate influx.     

Nitrate transporters and peptide transporters

In higher plants, two types of nitrate transporters, NRT1 and NRT2, have been identified. In Arabidopsis, there are 53 NRT1 genes and 7 NRT2 genes. NRT2 are high-affinity nitrate transporters, while most members of the NRT1 family are low-affinity nitrate transporters. The exception is CHL1 (AtNRT1.1), which is a dual-affinity nitrate transporter, its mode of action being switched by phosphorylation and dephosphorylation of threonine 101. Two of the NRT1 genes, CHL1 and AtNRT1.2, and two of the NRT2 genes, AtNRT2.1 and AtNRT2.2, are known to be involved in nitrate uptake. In addition, AtNRT1.4 is required for petiole nitrate storage. On the other hand, some members of the NRT1 family are dipeptide transporters, called PTRs, which transport a broad spectrum of di/tripeptides. In barley, HvPTR1, expressed in the plasma membrane of scutellar epithelial cells, is involved in mobilizing peptides, produced by hydrolysis of endosperm storage protein, to the developing embryo. In higher plants, there is another family of peptide transporters, called oligopeptide transporters (OPTs), which transport tetra/pentapeptides. In addition, some OPTs transport GSH, GSSH, GSH conjugates, phytochelatins, and metals.     

A 150 kDa plasma membrane complex of AtNRT2.5 and AtNAR2.1 is the major contributor to constitutive high‐affinity nitrate influx in Arabidopsis thaliana

In plants that have been deprived of nitrate for a significant length of time, a constitutive high‐affinity nitrate transport system (cHATS) is responsible for initial nitrate uptake. This absorbed nitrate leads to the induction of the major nitrate transporters and enzymes involved in nitrate assimilation. By use of ¹³NO₃^– influx measurements and Blue Native polyacrylamide gel electrophoresis we examined the role of AtNRT2.5 in cHATS in wild type (WT) and various T‐DNA mutants of Arabidopsis thaliana. We demonstrate that AtNRT2.5 is predominantly expressed in roots of nitrate‐deprived WT plants as a 150 kDa molecular complex with AtNAR2.1. This complex represents the major contributor to cHATS influx, which is reduced by 63% compared with WT in roots of Atnrt2.5 mutants. The remaining cHATS nitrate influx in these mutants is due to a residual contribution by the inducible high‐affinity transporter encoded by AtNRT2.1/AtNAR2.1. Estimates of the kinetic properties of the NRT2.5 transporter reveal that its low K_m for nitrate makes this transporter ideally suited to detect and respond to trace quantities of nitrate in the root environment.     

Regulation of the high-affinity NO3- uptake system by NRT1.1-mediated NO3- demand signaling in Arabidopsis

The NRT2.1 gene of Arabidopsis thaliana encodes a major component of the root high-affinity NO(3)(-) transport system (HATS) that plays a crucial role in NO(3)(-) uptake by the plant. Although NRT2.1 was known to be induced by NO(3)(-) and feedback repressed by reduced nitrogen (N) metabolites, NRT2.1 is surprisingly up-regulated when NO(3)(-) concentration decreases to a low level (<0.5 mm) in media containing a high concentration of NH(4)(+) or Gln (>or=1 mm). The NRT3.1 gene, encoding another key component of the HATS, displays the same response pattern. This revealed that both NRT2.1 and NRT3.1 are coordinately down-regulated by high external NO(3)(-) availability through a mechanism independent from that involving N metabolites. We show here that repression of both genes by high NO(3)(-) is specifically mediated by the NRT1.1 NO(3)(-) transporter. This mechanism warrants that either NRT1.1 or NRT2.1 is active in taking up NO(3)(-) in the presence of a reduced N source. Under low NO(3)(-)/high NH(4)(+) provision, NRT1.1-mediated repression of NRT2.1/NRT3.1 is relieved, which allows reactivation of the HATS. Analysis of atnrt2.1 mutants showed that this constitutes a crucial adaptive response against NH(4)(+) toxicity because NO(3)(-) taken up by the HATS in this situation prevents the detrimental effects of pure NH(4)(+) nutrition. It is thus hypothesized that NRT1.1-mediated regulation of NRT2.1/NRT3.1 is a mechanism aiming to satisfy a specific NO(3)(-) demand of the plant in relation to the various specific roles that NO(3)(-) plays, in addition to being a N source. A new model is proposed for regulation of the HATS, involving both feedback repression by N metabolites and NRT1.1-mediated repression by high NO(3)(-).     

Regulation of root nitrate uptake at the NRT2.1 protein level in Arabidopsis thaliana

In Arabidopsis the NRT2.1 gene encodes a main component of the root high-affinity nitrate uptake system (HATS). Its regulation has been thoroughly studied showing a strong correlation between NRT2.1 expression and HATS activity. Despite its central role in plant nutrition, nothing is known concerning localization and regulation of NRT2.1 at the protein level. By combining a green fluorescent protein fusion strategy and an immunological approach, we show that NRT2.1 is mainly localized in the plasma membrane of root cortical and epidermal cells, and that several forms of the protein seems to co-exist in cell membranes (the monomer and at least one higher molecular weight complex). The monomer is the most abundant form of NRT2.1, and seems to be the one involved in NO(3)(-) transport. It strictly requires the NAR2.1 protein to be expressed and addressed at the plasma membrane. No rapid changes in NRT2.1 abundance were observed in response to light, sucrose, or nitrogen treatments that strongly affect both NRT2.1 mRNA level and HATS activity. This suggests the occurrence of post-translational regulatory mechanisms. One such mechanism could correspond to the cleavage of NRT2.1 C terminus, which results in the presence of both intact and truncated proteins in the plasma membrane.     

Nitrate transporter NPF7.3/NRT1.5 plays an essential role in regulating phosphate deficiency responses in Arabidopsis

AtNPF7.3/AtNRT1.5, which is a nitrate transporter that drives root-to-shoot transport of NO₃^?, is also involved in modulating the response to K⁺ deprivation in Arabidopsis by affecting root development and K⁺ transport. However, whether NPF7.3/NRT1.5 functions in regulating plant responses to deficiencies of other nutrients remains unknown. In this study, we found that the expression of AtNPF7.3/AtNRT1.5 was predominant in the roots and was substantially induced by phosphate (Pi) starvation. The atnrt1.5 mutants displayed conspicuously longer primary roots along with a significantly reduced lateral root density under Pi-deficient conditions than did the wild-type plants, and these morphological differences in the roots were eliminated to a certain extent by the ethylene synthesis antagonist Co²⁺. Further analyses revealed that the expression of important Pi starvation-induced genes, which are directly involved in Pi transport, mobilization and distribution, were significantly higher in the atnrt1.5 mutants than that in the wild-type plants under Pi-starvation conditions; therefore, the atnrt1.5 mutants retained higher tissue Pi concentrations. Taken together, our results suggest that NPF7.3/NRT1.5 is an important component in the regulation of phosphate deficiency responses in Arabidopsis.     

Gene structure and expression of the high-affinity nitrate transport system in rice roots

Rice has a preference for uptake of ammonium over nitrate and can use ammonium-N efficiently. Consequently, transporters mediating ammonium uptake have been extensively studied, but nitrate transporters have been largely ignored. Recently,some reports have shown that rice also has high capacity to acquire nitrate from growth medium, so understanding the nitrate transport system in rice roots is very important for improving N use efficiency in rice. The present study identified four putative NRT2 and two putative NAR2 genes that encode components of the high-affinity nitrate transport system (HATS) in the rice (Oryza sativa L. subsp, japonica cv. Nipponbare) genome. OsNRT2.1 and OsNRT2.2 share an identical coding region sequence, and their deduced proteins are closely related to those from monocotyledonous plants. The two NAR2 proteins are closely related to those from mono-cotyledonous plants as well. However, OsNRT2.3 and OsNRT2.4 are more closely related to Arabidopsis NRT2 proteins. Relative quantitative reverse tranecdption-polymerase chain reaction analysis showed that all of the six genes were rapidly upregulated and then downregulated in the roots of N-starved rice plants after they were re-supplied with 0.2 mM nitrate, but the response to nitrate differed among gene members.The results from phylogenetic tree, gene structure and expression analysis implied the divergent roles for the individual members of the rice NRT2 and NAR2 families. High-affinity nitrate influx rates associated with nitrate induction in rice roots were investigated and were found to be regulated by external pH. Compared with the nitrate influx rates at pH 6.5, alkaline pH (pH 8.0) inhibited nitrate Influx, and acidic pH (pH 5.0) enhanced the nitrate influx In I h nitrate induced roots, but did not significantly affect that in 4 to 8 h nitrate induced roots.     

An arabidopsis T-DNA mutant affected in Nrt2 genes is impaired in nitrate uptake

Expression analyses of Nrt2 plant genes have shown a strict correlation with root nitrate influx mediated by the high-affinity transport system (HATS). The precise assignment of NRT2 protein function has not yet been possible due to the absence of heterologous expression studies as well as loss of function mutants in higher plants. Using a reverse genetic approach, we isolated an Arabidopsis thaliana knock-out mutant where the T-DNA insertion led to the complete deletion of the AtNrt2.1 gene together with the deletion of the 3' region of the AtNrt2.2 gene. This mutant is impaired in the HATS, without being modified in the low-affinity system. Moreover, the de-regulated expression of a Nicotiana plumbaginifolia Nrt2 gene restored the mutant nitrate influx to that of the wild-type. These results demonstrate that plant NRT2 proteins do have a role in HATS.     

Disruption of the nitrate transporter genes<Emphasis Type="Italic"> AtNRT2.1</Emphasis> and<Emphasis Type="Italic"> AtNRT2.2</Emphasis> restricts growth at low external nitrate concentration

The high-affinity transport systems in Arabidopsis thaliana (L.) Heynh. involve potentially seven genes. Among these, the AtNRT2.1 and/or AtNRT2.2 genes have been shown to play a major role in the inducible component of this transport system. The physiological impact of a disruption of AtNRT2.1 and AtNRT2.2 on plant growth and N-metabolism was investigated. The reduced nitrate uptake in the mutant under a limiting N-regime was found to correlate with a significant difference in shoot/root ratio between wild type and mutant and a drastically reduced nitrate level in the shoot of the mutant. Carbohydrate analyses of plants under a low nitrate supply revealed a slight increase in glucose and fructose in the mutant shoots as well as an increase in sucrose and starch contents in mutant shoots. Interestingly, the AtNRT2.4 and AtNRT2.5 genes were over-expressed in the mutant growing in reduced N-conditions, without any compensation by root nitrate influx. These results are discussed in the context of the putative role of the different NRT2 genes.Abbreviations DW Dry weight - FW Fresh weight - HATS High-affinity transport system - LATS Low-affinity transport system - NRT Nitrate transporter - WT Wild type