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.     

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.     

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 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.     

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     

The K+ and NO3− Interaction Mediated by NITRATE TRANSPORTER1.1 Ensures Better Plant Growth under K+-Limiting Conditions

K⁺ and NO₃⁻ are the major forms of potassium and nitrogen that are absorbed by the roots of most terrestrial plants. In this study, we observed that a close relationship between NO₃⁻ and K⁺ in Arabidopsis (Arabidopsis thaliana) is mediated by NITRATE TRANSPORTER1.1 (NRT1.1). The nrt1.1 knockout mutants showed disturbed K⁺ uptake and root-to-shoot allocation, and were characterized by growth arrest under K⁺-limiting conditions. The K⁺ uptake and root-to-shoot allocation of these mutants were partially recovered by expressing NRT1.1 in the root epidermis-cortex and central vasculature using SULFATE TRANSPORTER1;2 and PHOSPHATE1 promoters, respectively. Two-way analysis of variance based on the K⁺ contents in nrt1.1-1/K⁺ transporter1, nrt1.1-1/high-affinity K⁺ transporter5-3, nrt1.1-1/K⁺ uptake permease7, and nrt1.1-1/stelar K⁺ outward rectifier-2 double mutants and the corresponding single mutants and wild-type plants revealed physiological interactions between NRT1.1 and K⁺ channels/transporters located in the root epidermis–cortex and central vasculature. Further study revealed that these K⁺ uptake-related interactions are dependent on an H⁺-consuming mechanism associated with the H⁺/NO₃⁻ symport mediated by NRT1.1. Collectively, these data indicate that patterns of NRT1.1 expression in the root epidermis–cortex and central vasculature are coordinated with K⁺ channels/transporters to improve K⁺ uptake and root-to-shoot allocation, respectively, which in turn ensures better growth under K⁺-limiting conditions.

Potassium (K) is an essential element for plant growth and development and contributes to determining the yield and quality of crops in agriculture production (Wang and Wu, 2013). However, the concentrations of soluble K⁺ in most soils are relatively low, which often limits plant growth (Maathuis, 2009). Although crop production can be increased by applying large amounts of potassic fertilizers to agricultural fields, only approximately one-half of the applied fertilizers is available to plants; the remainder accumulates as residues in soils, consequently leading to environmental contamination (Meena et al., 2016). Therefore, there is a pressing need to gain a more complete understanding of the molecular mechanisms underlying K⁺ transport and regulation in order to enhance the K⁺ utilization efficiency of plants. Accordingly, in the past few decades, researchers have focused on identifying K⁺ channels and transporters in plants, as well as the mechanisms underlying their regulation.In Arabidopsis (Arabidopsis thaliana), 71 K⁺ channels and transporters have been identified and categorized into three channel (Shaker, Tandem-Pore K⁺, and K_ir-like) and three transporter (K⁺ uptake permeases [KT/HAK/KUP], High-affinity K⁺ transporters [HKT], and cation/proton antiporter [CPA]) families (Wang and Wu, 2010). Among these, the shaker inward K⁺ channel K⁺ TRANSPORTER1 (AKT1) and the KT/HAK/KUP K⁺ transporter HIGH-AFFINITY K⁺ TRANSPORTER5 (HAK5) have been characterized as the two major components that contribute to K⁺ uptake in roots, although they have been found to operate at different K⁺ levels (Pyo et al., 2010; Wang and Wu, 2013). AKT1 functions in plant K⁺ uptake over a wide range of K⁺ concentrations, whereas HAK5 shows high-affinity K⁺ transport activity (Gierth et al., 2005). Following its uptake into root epidermal cells, K⁺ is distributed to different plant organs or tissues. The Arabidopsis shaker-like outward-rectifying K⁺ channel STELAR K⁺ OUTWARD RECTIFIER (SKOR), the expression of which was first identified in stelar tissues, has been shown to facilitate K⁺ secretion into xylem sap, which is a critical step in long-distance K⁺ transport from roots to shoots (Gaymard et al., 1998). Recently, K⁺ UPTAKE PERMEASE7 (KUP7), a member of the KT/HAK/KUP family, was functionally characterized as a K⁺ transporter participating in both root K⁺ uptake and root-to-shoot K⁺ allocation, particularly under K⁺-limiting conditions (Han et al., 2016). However, the uptake affinity for K⁺ has been found to be considerably lower in KUP7 than in HAK5 (Wang and Wu, 2017).In addition to the aforementioned K⁺ channels and transporters, other mineral elements, including Na⁺, Ca²⁺, and N, are known to have pronounced effects on K⁺ nutrition in plants. Given that N is the nutrient that is required in the greatest quantity by most plants and is the most widely used fertilizer nutrient in crop production, the relationships between N and K have long been investigated (Fageria and Baligar, 2005; Wang and Wu, 2013; Meng et al., 2016; Shin, 2017). Since the 1960s, physiological studies have revealed a close relationship between NO₃⁻ and K⁺ with regard to uptake and translocation (Zioni et al., 1971; Blevins et al., 1978; Barneix and Breteler, 1985; Drechsler et al., 2015). However, the coordination between these two nutrients in plant transport pathways remains to be extensively studied at the molecular level. We hypothesized that transporters involved in the transference of NO₃⁻ across cell membranes may play a role in controlling K⁺ nutrition in plants. Recently, NITRATE TRANSPORTER1.5 (NRT1.5), a member of the nitrate transporter1/peptide transporter family (NPF), initially identified as a pH-dependent bidirectional NO₃⁻ transporter (Lin et al., 2008), was shown to be involved in the control of K⁺ allocation in plants (Drechsler et al., 2015; Li et al., 2017; Du et al., 2019). Nevertheless, it was subsequently established that this function was merely associated with its role as a proton-coupled H⁺/K⁺ antiporter for K⁺ loading into the xylem (Li et al., 2017; Du et al., 2019), which is not associated with the transport of NO₃⁻. In this study, we showed that the loss of another nitrate transporter1 member, NRT1.1/NPF6.3, in nrt1.1 mutants led to the development of a more pronounced K⁺-deficiency phenotype under conditions of low-K⁺ stress. Further physiological and genetic evidence revealed that both the uptake and root-to-shoot allocation of K⁺ in plants require NRT1.1. However, NRT1.1 acts as a coordinator rather than a K⁺ channel/transporter in K⁺ uptake and root-to-shoot allocation, which could depend on its NO₃⁻-related transport activity. Our findings highlight the significance of nutrients and nutrient interactions in ensuring plant growth, and indicate that the modification of NRT1.1 homolog activity in crops using biological engineering techniques might be a promising approach that could simultaneously contribute to enhancing the utilization efficiencies of K and N fertilizers in agricultural production.     

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.     

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.     

Potassium and Phosphate Uptake in Corn Roots: Further Evidence for an Electrogenic H/K Exchanger and an OH/Pi Antiporter

Evidence is presented that K⁺ uptake in corn root segments is coupled to an electrogenic H⁺/K⁺ -exchanging plasmalemma ATPase while phosphate uptake is coupled to an OH⁻/Pi antiporter. The plasmalemma ATPase inhibitor, diethylstilbestrol, or the stimulator, fusicoccin, altered K⁺ uptake directly and phosphate uptake indirectly. On the other hand, mersalyl, an OH⁻/Pi antiporter inhibitor, inhibited phosphate uptake instantly but only slightly affected K⁺ uptake. Collapse of the proton gradient across the membrane by (p-trifluoromethoxy) carbonyl cyanide phenylhydrazone resulted in immediate inhibition of K⁺ uptake but only later inhibited phosphate uptake. Changing the pH of the absorption solution had opposite effects on K⁺ and phosphate uptake. In addition, a 4-hour washing of corn root tissue induced a 5-fold increase in the rate of K⁺ uptake with little or no lag, but only a 2- to 3-fold increase in phosphate uptake with a 30- to 45-minute lag. Collectively these differences strongly support the coupling of an electrogenic H⁺/K⁺ -exchanging ATPase to an OH⁻/Pi antiporter in corn root tissue.     

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.     

Uptake and translocation of phosphate by pho2 mutant and wild-type seedlings of Arabidopsis thaliana

The pho2 mutant of Arabidopsis thaliana (L.) Heynh. accumulates excessive Pi (inorganic phosphate) concentrations in shoots compared to wild-type plants (E. Delhaize and P. Randall, 1995, Plant Physiol. 107: 207–213). In this study, a series of experiments was conducted to compare the uptake and translocation of Pi by pho2 with that of wild-type plants. The pho2 mutants had about a twofold greater Pi uptake rate than wild-type plants under P-sufficient conditions and a greater proportion of the Pi taken up accumulated in shoots of pho2. When shoots were removed, the uptake rate by roots was found to be similar for both genotypes, suggesting that the greater Pi uptake by the intact pho2 mutant is due to a greater shoot sink for Pi. Although pho2 mutants could recycle ³²Pi from shoots to roots through phloem the proportion of ³²Pi translocated to roots was less than half of that found in wild-type plants. When transferred from P-sufficient to P-deficient solutions, Pi concentrations in pho2 roots had a similar depletion rate to wild-type roots despite pho2 shoots having a fourfold greater Pi concentration than wild-type shoots throughout the experiment. We suggest that the pho2 phenotype could result from a partial defect in Pi transport in the phloem between shoots and roots or from an inability of shoot cells to regulate internal Pi concentrations. Received: 20 August 1997 / Accepted: 4 October 1997     

Potential transceptor AtNRT1.13 modulates shoot architecture and flowering time in a nitrate-dependent manner

Compared with root development regulated by external nutrients, less is known about how internal nutrients are monitored to control plasticity of shoot development. In this study, we characterize an Arabidopsis thaliana transceptor, NRT1.13 (NPF4.4), of the NRT1/PTR/NPF family. Different from most NRT1 transporters, NRT1.13 does not have the conserved proline residue between transmembrane domains 10 and 11; an essential residue for nitrate transport activity in CHL1/NRT1.1/NPF6.3. As expected, when expressed in oocytes, NRT1.13 showed no nitrate transport activity. However, when Ser 487 at the corresponding position was converted back to proline, NRT1.13 S487P regained nitrate uptake activity, suggesting that wild-type NRT1.13 cannot transport nitrate but can bind it. Subcellular localization and β-glucuronidase reporter analyses indicated that NRT1.13 is a plasma membrane protein expressed at the parenchyma cells next to xylem in the petioles and the stem nodes. When plants were grown with a normal concentration of nitrate, nrt1.13 showed no severe growth phenotype. However, when grown under low-nitrate conditions, nrt1.13 showed delayed flowering, increased node number, retarded branch outgrowth, and reduced lateral nitrate allocation to nodes. Our results suggest that NRT1.13 is required for low-nitrate acclimation and that internal nitrate is monitored near the xylem by NRT1.13 to regulate shoot architecture and flowering time.

Nitrate transporter/transceptor NRT1.13 monitors xylem 12 nitrate level to regulate shoot architecture and flowering time.     

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.     

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.     

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.     

盐胁迫对二穗短柄草幼苗生长及不同器官中盐离子稳态的影响

采用4种浓度的NaCl溶液(50、100、150、200 mmol/L)处理二穗短柄草和拟南芥(对照)幼苗,测定不同浓度盐胁迫下2种植物幼苗的生长指标和离子分布,以探讨二穗短柄草在盐胁迫下主要阳离子平衡机制.结果表明:(1)盐胁迫显著抑制二穗短柄草根叶的生物量积累.(2)根冠比数据显示,在盐胁迫条件下二穗短柄草能够更好地维系地下部分的生物量积累.(3)在4种浓度盐胁迫下,二穗短柄草叶中Na+含量低于根系,而且K+、Cl-含量和K+/Na+比值始终高于根系,说明在二穗短柄草中Na+从地下到地上的转运受到抑制,但对Cl-的转运缺乏有效的调控.(4)回归分析发现,盐胁迫下二穗短柄草和拟南芥根部Na+与K+含量变化呈正相关关系,而在叶部则不相关,说明二穗短柄草和拟南芥Na+与K+在根部具有相同的离子通道,而在叶部却具有各自独立的转运途径.     

AtPTR1 and AtPTR5 transport dipeptides in planta

Transporters for di- and tripeptides belong to the large and poorly characterized PTR/NRT1 (peptide transporter/nitrate transporter 1) family. A new member of this gene family, AtPTR5, was isolated from Arabidopsis (Arabidopsis thaliana). Expression of AtPTR5 was analyzed and compared with tissue specificity of the closely related AtPTR1 to discern their roles in planta. Both transporters facilitate transport of dipeptides with high affinity and are localized at the plasma membrane. Mutants, double mutants, and overexpressing lines were exposed to several dipeptides, including toxic peptides, to analyze how the modified transporter expression affects pollen germination, growth of pollen tubes, root, and shoot. Analysis of atptr5 mutants and AtPTR5-overexpressing lines showed that AtPTR5 facilitates peptide transport into germinating pollen and possibly into maturating pollen, ovules, and seeds. In contrast, AtPTR1 plays a role in uptake of peptides by roots indicated by reduced nitrogen (N) levels and reduced growth of atptr1 mutants on medium with dipeptides as the sole N source. Furthermore, overexpression of AtPTR5 resulted in enhanced shoot growth and increased N content. The function in peptide uptake was further confirmed with toxic peptides, which inhibited growth. The results show that closely related members of the PTR/NRT1 family have different functions in planta. This study also provides evidence that the use of organic N is not restricted to amino acids, but that dipeptides should be considered as a N source and transport form in plants.     

Resveratrol is an inhibitor of sodium-dependent inorganic phosphate transport in triple-negative MDA-MB-231 breast cancer cells

Metastasis is a major cause of death in patients with breast cancer. A growing body of evidence has demonstrated the antitumour effects of resveratrol, a non-flavonoid polyphenol. Resveratrol inhibits metastatic processes, such as the migration and invasion of cancer cells. In several cancer types, the importance of inorganic phosphate (Pi) for tumor progression has been demonstrated. The metastatic process in breast cancer is associated with Na⁺-dependent Pi transporters. In this study, we demonstrate, for the first time, that resveratrol inhibits the Na⁺-dependent Pi transporter. Results from kinetic analysis shows that resveratrol inhibits Na⁺-dependent Pi transport non-competitively. Resveratrol also inhibits adhesion/migration in MDA-MB-231 cells, likely related to inhibition of the Na⁺-dependent Pi transporter.     

Corn Root Protoplasts: ISOLATION AND GENERAL CHARACTERIZATION OF ION TRANSPORT

A method was developed for the large scale and rapid isolation of intact viable corn root protoplasts. Pure and metabolically active protoplasts were collected using a flotation technique. Vital staining tests, light and electron microscopy, and measurements of basic metabolic processes indicated that the isolated protoplasts were metabolically active, and that the plasmalemma and other organelles were well preserved. The isolated protoplasts performed normal, active ion transport functions. Time course of K⁺ and inorganic phosphate (H₂PO₄⁻) influx and the effects of external pH, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, fusicoccin, and diethylstilbestrol on K⁺ and inorganic phosphate influx and net H⁺ efflux in isolated protoplasts correlated well with data obtained on root segments. Data presented indicated that isolated protoplasts from roots can be used to gain additional insights into the mechanism of ion transport in plant cells.