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.     

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)(-).     

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.     

Major alterations of the regulation of root NO(3)(-) uptake are associated with the mutation of Nrt2.1 and Nrt2.2 genes in Arabidopsis

The role of AtNrt2.1 and AtNrt2.2 genes, encoding putative NO(3)(-) transporters in Arabidopsis, in the regulation of high-affinity NO(3)(-) uptake has been investigated in the atnrt2 mutant, where these two genes are deleted. Our initial analysis of the atnrt2 mutant (S. Filleur, M.F. Dorbe, M. Cerezo, M. Orsel, F. Granier, A. Gojon, F. Daniel-Vedele [2001] FEBS Lett 489: 220-224) demonstrated that root NO(3)(-) uptake is affected in this mutant due to the alteration of the high-affinity transport system (HATS), but not of the low-affinity transport system. In the present work, we show that the residual HATS activity in atnrt2 plants is not inducible by NO(3)(-), indicating that the mutant is more specifically impaired in the inducible component of the HATS. Thus, high-affinity NO(3)(-) uptake in this genotype is likely to be due to the constitutive HATS. Root (15)NO(3)(-) influx in the atnrt2 mutant is no more derepressed by nitrogen starvation or decrease in the external NO(3)(-) availability. Moreover, the mutant also lacks the usual compensatory up-regulation of NO(3)(-) uptake in NO(3)(-)-fed roots, in response to nitrogen deprivation of another portion of the root system. Finally, exogenous supply of NH(4)(+) in the nutrient solution fails to inhibit (15)NO(3)(-) influx in the mutant, whereas it strongly decreases that in the wild type. This is not explained by a reduced activity of NH(4)(+) uptake systems in the mutant. These results collectively indicate that AtNrt2.1 and/or AtNrt2.2 genes play a key role in the regulation of the high-affinity NO(3)(-) uptake, and in the adaptative responses of the plant to both spatial and temporal changes in nitrogen availability in the environment.     

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.     

Dichotomy in the NRT gene families of dicots and grass species

A large proportion of the nitrate (NO(3)(-)) acquired by plants from soil is actively transported via members of the NRT families of NO(3)(-) transporters. In Arabidopsis, the NRT1 family has eight functionally characterised members and predominantly comprises low-affinity transporters; the NRT2 family contains seven members which appear to be high-affinity transporters; and there are two NRT3 (NAR2) family members which are known to participate in high-affinity transport. A modified reciprocal best hit (RBH) approach was used to identify putative orthologues of the Arabidopsis NRT genes in the four fully sequenced grass genomes (maize, rice, sorghum, Brachypodium). We also included the poplar genome in our analysis to establish whether differences between Arabidopsis and the grasses may be generally applicable to monocots and dicots. Our analysis reveals fundamental differences between Arabidopsis and the grass species in the gene number and family structure of all three families of NRT transporters. All grass species possessed additional NRT1.1 orthologues and appear to lack NRT1.6/NRT1.7 orthologues. There is significant separation in the NRT2 phylogenetic tree between NRT2 genes from dicots and grass species. This indicates that determination of function of NRT2 genes in grass species will not be possible in cereals based simply on sequence homology to functionally characterised Arabidopsis NRT2 genes and that proper functional analysis will be required. Arabidopsis has a unique NRT3.2 gene which may be a fusion of the NRT3.1 and NRT3.2 genes present in all other species examined here. This work provides a framework for future analysis of NO(3)(-) transporters and NO(3)(-) transport in grass crop species.     

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     

Nitrate transport and signalling

Physiological measurements of nitrate (NO(3)(-)) uptake by roots have defined two systems of high and low affinity uptake. In Arabidopsis, genes encoding both of these two uptake systems have been identified. Most is known about the high affinity transport system (HATS) and its regulation and yet measurements of soil NO(3)(-) show that it is more often available in the low affinity range above 1 mM concentration. Several different regulatory mechanisms have been identified for AtNRT2.1, one of the membrane transporters encoding HATS; these include feedback regulation of expression, a second component protein requirement for membrane targeting and phosphorylation, possibly leading to degradation of the protein. These various changes in the protein may be important for a second function in sensing NO(3)(-) availability at the surface of the root. Another transporter protein, AtNRT1.1 also has a role in NO(3)(-) sensing that, like AtNRT2.1, is independent of their transport function. From the range of concentrations present in the soil it is proposed that the NO(3)(-)-inducible part of HATS functions chiefly as a sensor for root NO(3)(-) availability. Two other key NO(3)(-) transport steps for efficient nitrogen use by crops, efflux across membranes and vacuolar storage and remobilization, are discussed. Genes encoding vacuolar transporters have been isolated and these are important for manipulating storage pools in crops, but the efflux system is yet to be identified. Consideration is given to how well our molecular and physiological knowledge can be integrated as well to some key questions and opportunities for the future.     

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.     

Nitrate Signaling and the Two Component High Affinity Uptake System in Arabidopsis

Nitrate transporters are important for nitrogen acquisition by plants and in algae some require two gene products, NRT2 and NAR2, for function. The NRT2 family was already described and the recent identification of a family of the NAR2-type genes in higher plants showed that there was a homologue in Arabidopsis, AtNAR2.1. Using heterologous expression in yeast and oocytes we showed that the two Arabidopsis AtNRT2.1 and AtNAR2.1 proteins interacted to give a functional high affinity nitrate transport system (HATS). The gene knock out mutant atnar2.1-1 is deficient specifically for HATS activity and the resulting growth phenotype on low nitrate concentration is more severe than for the atnrt2.1-1 knock out mutant. Physiological characterisation of the plant N status and gene expression revealed a pattern that was characteristic of severe nitrogen deficiency. Consistent with the down regulation of AtNRT2.1 expression, the atnar2.1-1 plants also displayed the same phenotype as atnrt2.1 mutants in lateral root (LR) response to low nitrate supply. Using atnar2.1-1 plants constitutively expressing the NpNRT2.1 gene, we now show a specific role for AtNAR2.1 in LR response to low nitrate supply. AtNAR2.1 is also involved in the repression of LR initiation in response to high ratios of sucrose to nitrogen in the medium. Therefore the two component system itself is likely to be involved in the signaling pathway integrating nutritional cues for LR architecture regulation. Using a green fluorescent protein-NRT2.1 protein fusion we show the essential role of AtNAR2.1 for the presence of AtNRT2.1 to the plasma membrane.Key Words: high affinity nitrate transport, nitrate transporter, nitrate signalling, root growth     

Rice OsNAR2.1 interacts with OsNRT2.1, OsNRT2.2 and OsNRT2.3a nitrate transporters to provide uptake over high and low concentration ranges

Plants take up both nitrate and ammonium as main nitrogen (N) sources. Although ammonium is the predominant form in anaerobic-flooded paddy soil, it has been proposed that rice and other wetland plants may take up significant amounts of nitrate formed by nitrification of ammonium in the rhizosphere. A two-component system for nitrate transport including NRT2s with a partner protein (NAR2 or NRT3.1) has been identified in Arabidopsis. We report the physiological function of another member of the NAR2 family, OsNAR2.1 in rice (Oryza sativa, ssp. Japonica, cv. Nipponbare). OsNAR2.1 was mainly expressed in roots and induced by nitrate and suppressed by ammonium and some amino acids. Knockdown of OsNAR2.1 by RNA interference synchronously suppressed expression of OsNRT2.1, OsNRT2.2 and OsNRT2.3a in the osnar2.1mutants. Both high- and low-affinity nitrate transports were greatly impaired by OsNAR2.1 knockdown. Yeast two hybridization showed that OsNAR2.1 not only interacted with OsNRT2.1/OsNRT2.2, but also with OsNRT2.3a. Taken together, the data demonstrate that OsNAR2.1 plays a key role in enabling the plant to cope with variable nitrate supply.     

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.