Mutation of the Arabidopsis NRT1.5 nitrate transporter causes defective root-to-shoot nitrate transport

Little is known about the molecular and regulatory mechanisms of long-distance nitrate transport in higher plants. NRT1.5 is one of the 53 Arabidopsis thaliana nitrate transporter NRT1 (Peptide Transporter PTR) genes, of which two members, NRT1.1 (CHL1 for Chlorate resistant 1) and NRT1.2, have been shown to be involved in nitrate uptake. Functional analysis of cRNA-injected Xenopus laevis oocytes showed that NRT1.5 is a low-affinity, pH-dependent bidirectional nitrate transporter. Subcellular localization in plant protoplasts and in planta promoter-β-glucuronidase analysis, as well as in situ hybridization, showed that NRT1.5 is located in the plasma membrane and is expressed in root pericycle cells close to the xylem. Knockdown or knockout mutations of NRT1.5 reduced the amount of nitrate transported from the root to the shoot, suggesting that NRT1.5 participates in root xylem loading of nitrate. However, root-to-shoot nitrate transport was not completely eliminated in the NRT1.5 knockout mutant, and reduction of NRT1.5 in the nrt1.1 background did not affect root-to-shoot nitrate transport. These data suggest that, in addition to that involving NRT1.5, another mechanism is responsible for xylem loading of nitrate. Further analyses of the nrt1.5 mutants revealed a regulatory loop between nitrate and potassium at the xylem transport step.     

The Arabidopsis Nitrate Transporter NRT1.8 Functions in Nitrate Removal from the Xylem Sap and Mediates Cadmium Tolerance

Long-distance transport of nitrate requires xylem loading and unloading, a successive process that determines nitrate distribution and subsequent assimilation efficiency. Here, we report the functional characterization of NRT1.8, a member of the nitrate transporter (NRT1) family in Arabidopsis thaliana. NRT1.8 is upregulated by nitrate. Histochemical analysis using promoter-β-glucuronidase fusions, as well as in situ hybridization, showed that NRT1.8 is expressed predominantly in xylem parenchyma cells within the vasculature. Transient expression of the NRT1.8:enhanced green fluorescent protein fusion in onion epidermal cells and Arabidopsis protoplasts indicated that NRT1.8 is plasma membrane localized. Electrophysiological and nitrate uptake analyses using Xenopus laevis oocytes showed that NRT1.8 mediates low-affinity nitrate uptake. Functional disruption of NRT1.8 significantly increased the nitrate concentration in xylem sap. These data together suggest that NRT1.8 functions to remove nitrate from xylem vessels. Interestingly, NRT1.8 was the only nitrate assimilatory pathway gene that was strongly upregulated by cadmium (Cd²⁺) stress in roots, and the nrt1.8-1 mutant showed a nitrate-dependent Cd²⁺-sensitive phenotype. Further analyses showed that Cd²⁺ stress increases the proportion of nitrate allocated to wild-type roots compared with the nrt1.8-1 mutant. These data suggest that NRT1.8-regulated nitrate distribution plays an important role in Cd²⁺ tolerance.     

Arabidopsis nitrate transporter NRT1.9 is important in phloem nitrate transport

This study of the Arabidopsis thaliana nitrate transporter NRT1.9 reveals an important function for a NRT1 family member in phloem nitrate transport. Functional analysis in Xenopus laevis oocytes showed that NRT1.9 is a low-affinity nitrate transporter. Green fluorescent protein and β-glucuronidase reporter analyses indicated that NRT1.9 is a plasma membrane transporter expressed in the companion cells of root phloem. In nrt1.9 mutants, nitrate content in root phloem exudates was decreased, and downward nitrate transport was reduced, suggesting that NRT1.9 may facilitate loading of nitrate into the root phloem and enhance downward nitrate transport in roots. Under high nitrate conditions, the nrt1.9 mutant showed enhanced root-to-shoot nitrate transport and plant growth. We conclude that phloem nitrate transport is facilitated by expression of NRT1.9 in root companion cells. In addition, enhanced root-to-shoot xylem transport of nitrate in nrt1.9 mutants points to a negative correlation between xylem and phloem nitrate transport.     

Nitrate-Dependent Control of Shoot K Homeostasis by the Nitrate Transporter1/Peptide Transporter Family Member NPF7.3/NRT1.5 and the Stelar K+ Outward Rectifier SKOR in Arabidopsis

Root-to-shoot translocation and shoot homeostasis of potassium (K) determine nutrient balance, growth, and stress tolerance of vascular plants. To maintain the cation-anion balance, xylem loading of K⁺ in the roots relies on the concomitant loading of counteranions, like nitrate (NO₃⁻). However, the coregulation of these loading steps is unclear. Here, we show that the bidirectional, low-affinity Nitrate Transporter1 (NRT1)/Peptide Transporter (PTR) family member NPF7.3/NRT1.5 is important for the NO₃⁻-dependent K⁺ translocation in Arabidopsis (Arabidopsis thaliana). Lack of NPF7.3/NRT1.5 resulted in K deficiency in shoots under low NO₃⁻ nutrition, whereas the root elemental composition was unchanged. Gene expression data corroborated K deficiency in the nrt1.5-5 shoot, whereas the root responded with a differential expression of genes involved in cation-anion balance. A grafting experiment confirmed that the presence of NPF7.3/NRT1.5 in the root is a prerequisite for proper root-to-shoot translocation of K⁺ under low NO₃⁻ supply. Because the depolarization-activated Stelar K⁺ Outward Rectifier (SKOR) has previously been described as a major contributor for root-to-shoot translocation of K⁺ in Arabidopsis, we addressed the hypothesis that NPF7.3/NRT1.5-mediated NO₃⁻ translocation might affect xylem loading and root-to-shoot K⁺ translocation through SKOR. Indeed, growth of nrt1.5-5 and skor-2 single and double mutants under different K/NO₃⁻ regimes revealed that both proteins contribute to K⁺ translocation from root to shoot. SKOR activity dominates under high NO₃⁻ and low K⁺ supply, whereas NPF7.3/NRT1.5 is required under low NO₃⁻ availability. This study unravels nutritional conditions as a critical factor for the joint activity of SKOR and NPF7.3/NRT1.5 for shoot K homeostasis.The macronutrient potassium (K) is essential for plant growth and development because of its crucial roles in various cellular processes (i.e. regulation of enzyme activities), stabilization of protein synthesis, and neutralization of negative charges. In addition, it is a major component of the cation-anion balance and osmoregulation in plants, thereby influencing cellular turgor, xylem and phloem transport, pH homeostasis, and the setting of membrane potentials (Maathuis, 2009; Marschner, 2012; Sharma et al., 2013). K⁺ uptake and distribution in Arabidopsis (Arabidopsis thaliana) are accomplished by a total of 71 membrane proteins that have been assigned to five gene families: the Shaker and Tandem-Pore K⁺ channels (now also including the inward-rectifier K-like (K_ir-like) channels), the K⁺ uptake permeases (KUP/HAK/KT), the K⁺ transporter (HKT) family, and the cation proton antiporters (CPA; Gierth and Mäser, 2007; Gomez-Porras et al., 2012; Sharma et al., 2013).Root xylem loading is a key step for the delivery of nutrients to the shoot (Poirier et al., 1991; Engels and Marschner, 1992a; Gaymard et al., 1998; Takano et al., 2002; Park et al., 2008). Root-to-shoot translocation of K⁺ is mediated by the voltage-dependent Shaker family K⁺ channel Stelar K⁺ Outward Rectifier (SKOR). The gene is primarily expressed in pericycle and root xylem parenchyma cells, and it is down-regulated upon K shortage and in response to treatments with the phytohormones abscisic acid, cytokinin, and auxin. Such gene expression changes are thought to control K⁺ secretion into the xylem sap and K⁺ reallocation through the phloem to adjust root K⁺ transport activity to K⁺ availability and shoot demand (Pilot et al., 2003). SKOR is activated upon membrane depolarization, and it is in a closed state when the driving force for K⁺ is inwardly directed. It elicits outward K⁺ currents, facilitating the release of the cation from the cells into the xylem. The voltage dependency of the channel is modulated by the external K⁺ concentration to minimize the risk of an undesired K⁺ influx under high K⁺ availability (Johansson et al., 2006). Root-to-shoot K⁺ transfer was strongly reduced in the knockout mutant skor-1, resulting in a decreased shoot K content, whereas the root K content remained unaffected (Gaymard et al., 1998).Root xylem loading is subject to the maintenance of a cation-anion balance, and nitrate (NO₃⁻) is the quantitatively most important anion counterbalancing xylem loading of K⁺ (Engels and Marschner, 1993). Members of the Nitrate Transporter1 (NRT1)/Peptide Transporter (PTR) transporter family (NPF) play a prominent role in NO₃⁻ uptake and allocation in Arabidopsis (summarized in Krouk et al., 2010; Wang et al., 2012; and Léran et al., 2014). Two of them have recently been reported to control xylem NO₃⁻ loading and unloading. The low-affinity, pH-dependent bidirectional NO₃⁻ transporter NPF7.3/NRT1.5 (subsequently termed NRT1.5) mediates NO₃⁻ efflux from pericycle cells to the xylem vessels, whereas the low-affinity influx protein NPF7.2/NRT1.8 removes NO₃⁻ from the xylem sap and transfers it into xylem parenchyma cells (Lin et al., 2008; Li et al., 2010; Chen et al., 2012). Accordingly, the expression of both genes is oppositely regulated under various stress conditions (Li et al., 2010). In nrt1.5 mutants, NRT1.8 expression is increased, which is thought to enhance NO₃⁻ reallocation to the root (Chen et al., 2012).The NRT1.5 gene is mainly expressed in root pericycle cells close to the xylem, and the protein localizes to the plasma membrane. In nrt1.5 mutants, less NO₃⁻ is transported from the root to the shoot, and the NO₃⁻ concentration in the xylem sap is reduced. However, root-to-shoot NO₃⁻ transport is not completely abolished in these mutants, indicating the existence of additional xylem-loading activities for NO₃⁻ (Lin et al., 2008; Wang et al., 2012). The recent observation that NPF6.3/NRT1.1/CHL1 and NPF6.2/NRT1.4 are also capable of mediating bidirectional NO₃⁻ transport in Xenopus laevis oocytes might indicate that more NPF family members are contributing to xylem loading with NO₃⁻ (Léran et al., 2013).Electrophysiological studies with NRT1.5-expressing X. laevis oocytes revealed that NO₃⁻ excited an inward current at pH 5.5, which would be expected for a proton-coupled nitrate transporter with a proton to nitrate ratio larger than one (Lin et al., 2008). The inward currents elicited by exposure to nitrate were pH dependent, and Lin et al. (2008) observed that NRT1.5 can also facilitate nitrate efflux when the oocytes were incubated at pH 7.4. Lin et al. (2008) concluded that NRT1.5 can transport nitrate in both directions, presumably through a proton-coupled mechanism. Interestingly, a K⁺ gradient was not sufficient to drive NRT1.5-mediated NO₃⁻ export. However, the determination of root and shoot cation concentrations in the nrt1.5-1 mutant revealed that the amount of K⁺ translocated to the shoot was reduced when NO₃⁻ but not NH₄⁺ was supplied as the N source. Therefore, Lin et al. (2008) suggested a regulatory loop between NO₃⁻ and K⁺ at the xylem loading step.A close relationship between these two nutrients concerning uptake, translocation, recycling, and reduction (of NO₃⁻) has been described in physiological studies since the 1960s (e.g. Ben Zioni et al., 1971; Blevins et al., 1978; Barneix and Breteler, 1985), but only recently, common components in the NO₃⁻ and K⁺ uptake pathways were identified and led to the first ideas of how such a cross talk might be coordinated on the molecular level. The uptake activity of the K⁺ channel AKT1 as well as the affinity of the NO₃⁻ transporter NPF6.3/NRT1.1/CHL1 are both modulated by the activity of CALCINEURIN B-LIKE PROTEIN-INTERACTING PROTEIN KINASE23 (CIPK23), which itself is regulated by CALCINEURIN B-LIKE PROTEIN9 (CBL9) under both deficiencies (Xu et al., 2006; Ho et al., 2009). Yet, the details of this interaction in root K⁺ uptake, the (regulation of) xylem loading with K⁺ and NO₃⁻, and the involvement of SKOR and NRT1.5 in this process are unknown.In this study, we approached this problem by investigating the molecular and physiological responses of Arabidopsis wild-type (Columbia-0 [Col-0]), nrt1.5, and skor transfer DNA (T-DNA) insertion lines to varying NO₃⁻ and K⁺ regimes. The nrt1.5 mutant developed an early senescence phenotype under low NO₃⁻ nutrition, which could be attributed to a reduced K⁺ translocation to the shoot. The assessment of nrt1.5 and skor single- and double-knockout lines disclosed an interplay of the two proteins in the NO₃⁻-dependent control of shoot K homeostasis. The presented data indicate that SKOR mediates K⁺ root-to-shoot translocation under high NO₃⁻ and low K⁺ availability, whereas NRT1.5 is important for K⁺ translocation under low NO₃⁻ availability, irrespective of the K⁺ supply.     

Characterization of the Arabidopsis Nitrate Transporter NRT1.6 Reveals a Role of Nitrate in Early Embryo Development

This study of the Arabidopsis thaliana nitrate transporter NRT1.6 indicated that nitrate is important for early embryo development. Functional analysis of cDNA-injected Xenopus laevis oocytes showed that NRT1.6 is a low-affinity nitrate transporter and does not transport dipeptides. RT-PCR, in situ hybridization, and β-glucuronidase reporter gene analysis showed that expression of NRT1.6 is only detectable in reproductive tissue (the vascular tissue of the silique and funiculus) and that expression increases immediately after pollination, suggesting that NRT1.6 is involved in delivering nitrate from maternal tissue to the developing embryo. In nrt1.6 mutants, the amount of nitrate accumulated in mature seeds was reduced and the seed abortion rate increased. In the mutants, abnormalities (i.e., excessive cell division and loss of turgidity), were found mainly in the suspensor cells at the one- or two-cell stages of embryo development. The phenotype of the nrt1.6 mutants revealed a novel role of nitrate in early embryo development. Interestingly, the seed abortion rate of the mutant was reduced when grown under N-deficient conditions, suggesting that nitrate requirements in early embryo development can be modulated in response to external nitrogen changes.     

Arabidopsis NRT1.5 Is Another Essential Component in the Regulation of Nitrate Reallocation and Stress Tolerance

Nitrate reallocation to plant roots occurs frequently under adverse conditions and was recently characterized to be actively regulated by Nitrate Transporter1.8 (NRT1.8) in Arabidopsis (Arabidopsis thaliana) and implicated as a common response to stresses. However, the underlying mechanisms remain largely to be determined. In this study, characterization of NRT1.5, a xylem nitrate-loading transporter, showed that the mRNA level of NRT1.5 is down-regulated by salt, drought, and cadmium treatments. Functional disruption of NRT1.5 enhanced tolerance to salt, drought, and cadmium stresses. Further analyses showed that nitrate, as well as Na(+) and Cd(2+) levels, were significantly increased in nrt1.5 roots. Important genes including Na(+)/H(+) exchanger1, Salt overly sensitive1, Pyrroline-5-carboxylate synthase1, Responsive to desiccation29A, Phytochelatin synthase1, and NRT1.8 in stress response pathways are steadily up-regulated in nrt1.5 mutant plants. Interestingly, altered accumulation of metabolites, including proline and malondialdehyde, was also observed in nrt1.5 plants. These data suggest that NRT1.5 is involved in nitrate allocation to roots and the consequent tolerance to several stresses, in a mechanism probably shared with NRT1.8.     

The Arabidopsis Nitrate Transporter NRT1.7, Expressed in Phloem,Is Responsible for Source-to-Sink Remobilization of Nitrate

Several quantitative trait locus analyses have suggested that grain yield and nitrogen use efficiency are well correlated with nitrate storage capacity and efficient remobilization. This study of the Arabidopsis thaliana nitrate transporter NRT1.7 provides new insights into nitrate remobilization. Immunoblots, quantitative RT-PCR, β-glucuronidase reporter analysis, and immunolocalization indicated that NRT1.7 is expressed in the phloem of the leaf minor vein and that its expression levels increase coincidentally with the source strength of the leaf. In nrt1.7 mutants, more nitrate was present in the older leaves, less ¹⁵NO₃⁻ spotted on old leaves was remobilized into N-demanding tissues, and less nitrate was detected in the phloem exudates of old leaves. These data indicate that NRT1.7 is responsible for phloem loading of nitrate in the source leaf to allow nitrate transport out of older leaves and into younger leaves. Interestingly, nrt1.7 mutants showed growth retardation when external nitrogen was depleted. We conclude that (1) nitrate itself, in addition to organic forms of nitrogen, is remobilized, (2) nitrate remobilization is important to sustain vigorous growth during nitrogen deficiency, and (3) source-to-sink remobilization of nitrate is mediated by phloem.     

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.     

Nitrate transceptor(s) in plants

The availability of mineral nutrients in the soil dramatically fluctuates in both time and space. In order to optimize their nutrition, plants need efficient sensing systems that rapidly signal the local external concentrations of the individual nutrients. Until recently, the most upstream actors of the nutrient signalling pathways, i.e. the sensors/receptors that perceive the extracellular nutrients, were unknown. In Arabidopsis, increasing evidence suggests that, for nitrate, the main nitrogen source for most plant species, a major sensor is the NRT1.1 nitrate transporter, also contributing to nitrate uptake by the roots. Membrane proteins that fulfil a dual nutrient transport/signalling function have been described in yeast and animals, and are called 'transceptors'. This review aims to illustrate the nutrient transceptor concept in plants by presenting the current evidence indicating that NRT1.1 is a representative of this class of protein. The various facets, as well as the mechanisms of nitrate sensing by NRT1.1 are considered, and the possible occurrence of other nitrate transceptors is discussed.     

Nitrate does not compete with abscisic acid as a substrate of AtNPF4.6/NRT1.2/AIT1 in Arabidopsis

We identified a member of the Arabidopsis NRT1/PTR FAMILY (NPF), AtNPF4.6, as an abscisic acid (ABA) transporter, AIT1. AtNPF4.6 was originally characterized as a low-affinity nitrate transporter NRT1.2. We hypothesized that the competition between nitrate and ABA as substrates for AtNPF4.6 might be involved in the interactions between nitrate and ABA signaling. However, the ABA transport activity of AtNPF4.6 was not inhibited by an excess amount of nitrate. In addition, the npf4.6 mutant was less sensitive to ABA than the wild type during germination irrespective of nitrate concentrations in the media. Furthermore, nitrate promoted germination of both wild type and npf4.6 in the presence of ABA. These results do not support the idea of a physiological linkage between nitrate and ABA signals through AtNPF4.6.     

A deletion in the nitrate high affinity transporter NRT2.1 alters metabolomic and transcriptomic responses to Pseudomonas syringae

A deletion in the high affinity nitrate trasporter NRT2.1 in Arabidopsis results in a reduced susceptibility to Pseudomonas syringae by two different mechanisms, the SA priming and an interference in the effector triggered susceptibility. In the present research we further characterized the metabolic and genetic profiles of the mutant nrt2 in the interaction with P. syringae. Despite the priming found in the SA-dependent pathway, the metabolic changes in nrt2 compared with wild-type plants are more remarkable prior infection. This is associated mainly to a pre-existing over representation of signals attributed to aromatic amino acids and phenylpropanoids in the nrt2. Genomic analysis confirms the implication of aromatic aminoacids and phenylpropanoids, but additionally, suggests a new role in ribosomal proteins as the major changes observed in nrt2 upon infection by the bacterium.     

Characterization of the Nrt2.6 Gene in Arabidopsis thaliana: A Link with Plant Response to Biotic and Abiotic Stress

The high affinity nitrate transport system in Arabidopsis thaliana involves one gene and potentially seven genes from the NRT1 and NRT2 family, respectively. Among them, NRT2.1, NRT2.2, NRT2.4 and NRT2.7 proteins have been shown to transport nitrate and are localized on the plasmalemma or the tonoplast membranes. NRT2.1, NRT2.2 and NRT2.4 play a role in nitrate uptake from soil solution by root cells while NRT2.7 is responsible for nitrate loading in the seed vacuole. We have undertaken the functional characterization of a third member of the family, the NRT2.6 gene. NRT2.6 was weakly expressed in most plant organs and its expression was higher in vegetative organs than in reproductive organs. Contrary to other NRT2 members, NRT2.6 expression was not induced by limiting but rather by high nitrogen levels, and no nitrate-related phenotype was found in the nrt2.6-1 mutant. Consistently, the over-expression of the gene failed to complement the nitrate uptake defect of an nrt2.1-nrt2.2 double mutant. The NRT2.6 expression is induced after inoculation of Arabidopsis thaliana by the phytopathogenic bacterium Erwinia amylovora. Interestingly, plants with a decreased NRT2.6 expression showed a lower tolerance to pathogen attack. A correlation was found between NRT2.6 expression and ROS species accumulation in response to infection by E. amylovora and treatment with the redox-active herbicide methyl viologen, suggesting a probable link between NRT2.6 activity and the production of ROS in response to biotic and abiotic stress.     

Genomic survey of NPF and NRT2 transporter gene families in five inbred maize lines and their responses to pathogens infection

Besides manipulating nitrate uptake and allocation, nitrate transporters (NRTs) are also known to play crucial roles in pathogen defense and stress response. By blasting with the model NRT genes of poplar and Arabidopsis, a total of 408 gene members were identified from 5 maize inbred lines in which the number of NRTs ranged from 72 to 88. Phylogenetic analysis showed that the NRT genes of maize were classified into NRT1/PTR (NPF), NRT2 and NRT3 subfamilies, respectively. Marked divergence of the duplication patterns of NRT genes were identified, which may be a new basis for classification and identification of maize varieties. In terms of biotic stress, NRT2.5A showed an enhanced expression during the pathogen infection of Colletotrichum graminicola, while NRT1c4C was down-regulated, suggesting that maize NRT transporters may have both positive and negative roles in the disease resistance response. This work will promote the further studies of NRT gene families in maize, as well as be beneficial for further understanding of their potential roles in plant-pathogen interactions.     

The Arabidopsis nitrate transporter NRT2.4 plays a double role in roots and shoots of nitrogen-starved plants

Plants have evolved a variety of mechanisms to adapt to N starvation. NITRATE TRANSPORTER2.4 (NRT2.4) is one of seven NRT2 family genes in Arabidopsis thaliana, and NRT2.4 expression is induced under N starvation. Green fluorescent protein and β-glucuronidase reporter analyses revealed that NRT2.4 is a plasma membrane transporter expressed in the epidermis of lateral roots and in or close to the shoot phloem. The spatiotemporal expression pattern of NRT2.4 in roots is complementary with that of the major high-affinity nitrate transporter NTR2.1. Functional analysis in Xenopus laevis oocytes and in planta showed that NRT2.4 is a nitrate transporter functioning in the high-affinity range. In N-starved nrt2.4 mutants, nitrate uptake under low external supply and nitrate content in shoot phloem exudates was decreased. In the absence of NRT2.1 and NRT2.2, loss of function of NRT2.4 (triple mutants) has an impact on biomass production under low nitrate supply. Together, our results demonstrate that NRT2.4 is a nitrate transporter that has a role in both roots and shoots under N starvation.