Research ArticlePlant biology

Nitrate sensing and uptake in Arabidopsis are enhanced by ABI2, a phosphatase inactivated by the stress hormone abscisic acid

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Science Signaling  05 May 2015:
Vol. 8, Issue 375, pp. ra43
DOI: 10.1126/scisignal.aaa4829

Phosphatase promotes nitrate uptake

Plants require nitrogen, either from the soil mostly in the form of nitrate (NO3) or from a symbiotic relationship with nitrogen-fixing bacteria or fungi. Léran et al. identified ABI2, a phosphatase that is inhibited by the stress hormone abscisic acid (ABA), as a key positive regulator of the nitrate transporter NPF6.3. ABI2 dephosphorylated components of a calcium-sensing and kinase complex that phosphorylated and inhibited NPF6.3-dependent NO3 uptake, sensing, and signaling in roots. Because when dephosphorylated by ABI2 a related calcium-sensing kinase complex also stimulates K+ influx, the identification of this ABA-regulated phosphatase suggests a mechanism for integrating regulation of ionic balance and energy-consuming nitrate reduction during periods of plant stress.

Abstract

Living organisms sense and respond to changes in nutrient availability to cope with diverse environmental conditions. Nitrate (NO3) is the main source of nitrogen for plants and is a major component in fertilizer. Unraveling the molecular basis of nitrate sensing and regulation of nitrate uptake should enable the development of strategies to increase the efficiency of nitrogen use and maximize nitrate uptake by plants, which would aid in reducing nitrate pollution. NPF6.3 (also known as NRT1.1), which functions as a nitrate sensor and transporter; the kinase CIPK23; and the calcium sensor CBL9 form a complex that is crucial for nitrate sensing in Arabidopsis thaliana. We identified two additional components that regulate nitrate transport, sensing, and signaling: the calcium sensor CBL1 and protein phosphatase 2C family member ABI2, which is inhibited by the stress-response hormone abscisic acid. Bimolecular fluorescence complementation assays and in vitro kinase assays revealed that ABI2 interacted with and dephosphorylated CIPK23 and CBL1. Coexpression studies in Xenopus oocytes and analysis of plants deficient in ABI2 indicated that ABI2 enhanced NPF6.3-dependent nitrate transport, nitrate sensing, and nitrate signaling. These findings suggest that ABI2 may functionally link stress-regulated control of growth and nitrate uptake and utilization, which are energy-expensive processes.

INTRODUCTION

Nitrogen is a component of most biological macromolecules, including DNA, RNA, and proteins. Consequently, transmembrane transport of nitrogen forms is a key step for the survival of living organisms. The peptide transporter (PTR) family is present in prokaryotic and eukaryotic organisms (1) and is involved in transport of nitrogen-containing molecules. In animals, the SLC15 family (2) of di- or tripeptide transporters (3) provides the major routes for absorption of dietary nitrogen (1). In bacteria, PTRs are also involved in uptake of dipeptides (3). In plants, PTRs are represented by the large and diverse NPF family, members of which transport various substrates, such as nitrate, nitrite, dipeptides, amino acids, auxin, abscisic acid (ABA), gibberellin, glucosinolates, or jasmonoyl-isoleucine (47).

Most plants use nitrate (NO3) as their major nitrogen source. The NO3 uptake capacity of a plant is determined by three interdependent factors that are sensitive to NO3 availability: (i) the functional properties of the transporters in the roots that contribute to the acquisition of NO3 from the external medium, (ii) the density of functional transporters at the plasma membrane of root cells, and (iii) the surface and architecture of the root system [reviewed in (811)]. The identification of factors that regulate the NO3-sensing systems is important for both fundamental and applied science, because these factors control the capacity of plants to use the available NO3, a process known as the “nitrate use efficiency.”

The protein known as NPF6.3, NRT1.1, or CHL1 (hereafter referred to as NPF6.3) is involved in NO3 transport and sensing in Arabidopsis thaliana (5, 1218). NPF6.3 triggers specific plant signaling pathways in response to NO3 availability. Analysis of three different npf6.3 knockout (KO) lines (12, 19, 20) revealed that NO3 stimulates NPF6.3-dependent changes in gene expression. Moreover, NO3 uptake in roots depends on NPF6.3 function (12, 21, 22), and NPF6.3 plays a key role in the NO3-dependent regulation of root development (13, 14, 16). The phosphorylation state of Thr101 may determine whether NPF6.3 functions as a high- or low-affinity NO3 transporter (15, 2226).

NPF6.3 is a component of a macromolecular complex that also contains a protein kinase, CIPK23, and a calcium sensor, CBL9 (15). CBL9 interacts with and activates CIPK23 that, in turn, phosphorylates and thereby inhibits NO3 transport activity of NPF6.3 in high external NO3 concentration and reduces the NO3-dependent induction of NRT2.1, which encodes a NO3 transporter (15). CIPK23 belongs to a family of protein kinases [calcineurin B–like (CBL)–interacting protein kinase (CIPK)] encompassing 26 members in Arabidopsis (27). Each CIPK specifically interacts with one or several of the 10 CBL calcium sensors to specifically decode Ca2+ signals (28). Specific roles for some CIPK-CBL pairs have been elucidated, and several targets have been identified (2932).

The NO3 transport activity of NPF6.3 can be assessed by expression in Xenopus oocytes (33). In this system and in the presence of high external NO3 concentration (10 mM), coexpression of CBL9 and CIPK23 inhibits NPF6.3 activity (15). Interaction studies using yeast two-hybrid and bimolecular fluorescence complementation (BiFC) experiments indicated that CIPK23 also interacts with CBL1 (15). In the Xenopus expression system, CIPK23 coexpressed with either CBL1 or CBL9 activates the potassium channel AKT1 (30). Therefore, we used NPF6.3 expressed in Xenopus oocytes to identify additional regulators of this NO3 sensor and transporter. We identified the protein phosphatase ABI2 [ABA-insensitive 2] as a component of the regulatory network involved in NPF6.3-dependent NO3 transport, and we verified the relevance of this interaction in NO3 responses in Arabidopsis.

RESULTS

NPF6.3 regulation by CIPK23 and CBL1

Because CIPK23 can interact with either CBL9 or CBL1 to regulate other channels (15), we monitored NO3 transport activity as the accumulation of 15N-labeled NO3 in oocytes that coexpressed either CBL9, CIPK23, and NPF6.3 or CBL1, CIPK23, and NPF6.3. These studies confirmed the inhibitory effect of CBL9 and CIPK23 on NPF6.3-dependent NO3 accumulation (15) and identified CBL1 as additional calcium sensor that, in combination with CIPK23, reduced NPF6.3-dependent NO3 accumulation in oocytes (Fig. 1).

Fig. 1 Inhibition of NPF6.3 nitrate transport activity by coexpression with CIPK23 and CBL1 or CIPK23 and CBL9 in Xenopus oocytes.

Nitrate uptake in oocytes injected with complementary RNAs (cRNAs) expressing the indicated proteins is presented as the percent of 15N accumulation relative to NPF6.3-expressing oocytes (n = 8 to 10). Values are means ± SE. ***P < 0.001, two-sided t test after comparison with NPF6.3-expressing oocytes.

Identification of a PP2C that regulates NPF6.3-mediated nitrate uptake

To identify protein phosphatases that counteracted the inhibitory effect of CBL1-CIPK23 on NPF6.3-dependent NO3 transport activity, we coexpressed phosphatases of the clade-A protein phosphatase 2C (PP2C) family (34), ABI1, ABI2, HAB1, or PP2CA with NPF6.3, CBL1, and CIPK23 in oocytes and monitored NO3 accumulation. Expression of ABI1 or ABI2 counteracted the inhibitory effect of CBL1 and CIPK23 on NPF6.3-mediated NO3 accumulation, whereas expression of HAB1 or PP2CA had no effect (Fig. 2A). Furthermore, NO3 accumulation in oocytes expressing NPF6.3 or ABI2 and NPF6.3 was similar, suggesting that ABI2 influenced the activity of the CBL1-CIPK23 complex in the oocyte system (Fig. 2A).

Fig. 2 Involvement of ABI2 in nitrate transport.

(A) Nitrate uptake in oocytes injected with cRNAs expressing the indicated proteins is presented in percent of 15N accumulation in NPF6.3-expressing oocytes (n = 8 to 10). Values are means ± SE. Bars with different letters are significantly different (P < 0.05, Duncan’s multiple range tests). (B) Nitrate influx in root of the indicated Arabidopsis genotypes is presented in μmol of 15N per gram of root dry weight per hour (n = 25 to 30 from four independent experiments). Values are means ± SE. ***P < 0.001, two-sided t test after comparison with wild-type (Col) plants.

To evaluate the role of these phosphatases in planta, we quantified NO3 influx into roots of wild type A. thaliana lacking various PP2C family members or lacking NPF6.3: wild type (Col-0), npf6.3 KO (chl1-5), abi1 KO (abi1-2), abi2 KO (abi2-2), hab1 KO (hab1-1), and pp2ca KO (pp2ca-1). The chl1-5 roots exhibited impairment in NO3 influx (Fig. 2B) as expected (12, 21, 22). Roots of the abi2-2 plants exhibited a similarly reduced NO3 influx, whereas the roots of the other phosphatase KO mutants displayed influx like that of wild-type plant roots (Fig. 2B).

Role of ABI2 in nitrate sensing and signaling

The Xenopus oocyte coexpression study and the phenotype of the abi2 KO roots supported a role for ABI2 in the regulation of NPF6.3-dependent NO3 uptake. NPF6.3 also mediates the induction of NRT2.1 expression in response to NO3 (an example of nitrate signaling) and enables differential lateral root development in the presence of low or high concentrations of NO3 (an example of nitrate sensing) (16).To test a role for ABI2 on the regulation of NO3 signaling and sensing (11, 12, 14, 16), we analyzed these two NPF6.3-dependent responses in plants of various genotypes.

For analysis of NRT2.1 expression, we grew plants in the absence of NO3 (with NH4-succinate as nitrogen source) in hydroponic conditions for 15 days before supplying 1 mM KNO3 (or KCl as control) for 30 min and then quantified NRT2.1 mRNA by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Wild-type plants, abi1-2, and hab1-1 exhibited similar significant increases in NRT2.1 mRNA in response to the addition of NO3 (Fig. 3A). In agreement with previous reports (35), loss of NPF6.3 function in chl1-5 or chl1-12 reduced induction of NRT2.1 expression upon addition of NO3. NO3-stimulated induction of NRT2.1 expression was abolished in abi2-2 (Fig. 3A), supporting the notion that ABI2 function is required for proper NO3 signaling.

Fig. 3 Involvement of ABI2 in nitrate sensing.

(A) Relative expression of NRT2.1 in Arabidopsis roots for indicated genotypes. Plants were grown for14 days in Phytatray. Thirty minutes after KCl (dark gray bars) or KNO3 (gray bars) treatment, roots were collected and RNA was extracted. qRT-PCR was performed with NRT2.1 primers and with CLATHRIN and ACTIN to normalize gene expression. Values are means of three independent experiments ± SE. Bars with different letters are significantly different (P < 0.05, Duncan’s multiple range tests). (B and C) Role of ABI2 in nitrate-dependent lateral root growth. (B) Vertical agar plate split-root experiments with two plants per petri dish. Plants were grown in split-root system containing 10 mM nitrate (HN) in the center and 0.05 mM nitrate (LN) in the border sides. (C) Ratio of total root length between HN and LN for different genotypes. The ratio between total length of second-order lateral root in HN and LN was measured 7 days after transferring plants in this split-root system (n = 25 to 30). Values are means ± SE. ***P < 0.001, two-sided t test after comparison with wild-type (Col) plants.

We analyzed the role of ABI2 in regulating root development of Arabidopsis seedlings with the split-root system. In this assay, the root system is divided in two (Fig. 3B): one side grows in low NO3 (LN; 0.05 mM) and the other side in high NO3 (HN; 10 mM). We analyzed the length of second-order lateral roots and calculated the ratio of lateral root length between the HN and LN sides. For wild-type plants and hab1-1 plants, the total lateral root length was higher in the HN side resulting in a ratio >1 (Fig. 3, B and C), indicating preferential lateral root growth in the HN side. Similar to the reported phenotype of chl1-5 mutants (16), abi2-2 plants also exhibited similar lateral root growth on both sides resulting in a ratio close to 1, indicating a defect in NO3 sensing and confirming the involvement of ABI2 in NO3 perception.

Dephosphorylation of CBL1 and CIPK23 by ABI2

The effects of ABI2 on NO3 transport in both Xenopus oocytes and planta established a functional link between NPF6.3, CIPK23, CBL1, and ABI2. NPF6.3 directly interacts with CIPK23 (15), and CIPK23 interacts with CBL1 and CBL9 (30). We studied the interaction between CBL1, CIPK23, and ABI2 or PP2CA with BiFC (Fig. 4A). Whereas CIPK23-YN [CIPK23 fused to the N terminus of yellow fluorescent protein (YFP)] produced a complementation signal with both ABI2 and PP2CA fused to the C terminus of YFP, the location of the interaction was different. ABI2-YC and CIPK23-YN interacted in a pattern that partially overlapped with a marker of the plasma membrane, consistent with an association with proteins at the plasma membrane, and PP2CA-YC interacted with CIPK23-YN in the nucleus. No complementation signal was detectable with PP2CA-YN and CBL1-YC or NPF6.3-YC, indicating that these two proteins do not interact with PP2CA. ABI2-YN produced a complementation signal with CBL1-YC and NPF6.3-YC that colocalized with the plasma membrane marker.

Fig. 4 Physical and functional interaction between ABI2, CIPK23, and CBL1.

(A) BiFC protein-protein interaction analysis of Nicotiana benthamiana epidermal cells transiently expressing the indicated plasmids and analyzed by confocal microscopy. Positive interactions are represented by green; PM-OFP coexpression (red) marks the plasma membrane. Scale bars, 100 μm. (B) In vitro phosphorylation assays. Lane 1: CIPK23 + CBL1 for 60 min; lane 2: CIPK23 + CBL1 + K252a (kinase inhibitor) for 60 min; lane 3: CIPK23 + CBL1 + ABI2 for 60 min; lane 4: CIPK23 + CBL1 for 60 min, stop kinase reaction by K252a, then further incubation for 30 min; lane 5: CIPK23 + CBL1 for 60 min, stop kinase reaction by K252a, then + ABI2 for 30 min. The STREPII-ABI2 migrated closely with STREPII-CIPK23, and they appeared as overlapped bands on the SDS–polyacrylamide gel electrophoresis (SDS-PAGE). Data are representative of three experiments.

Efficient activation of the Ca2+ sensor/kinase complexes (CBL/CIPK) toward their target proteins often requires CIPK autophosphorylation and CIPK-dependent phosphorylation of the Ca2+-sensor moiety in the associated CBL (36). We therefore investigated the phosphorylation status of CIPK23 and CBL1 in the presence and absence of ABI2 (Fig. 4B). We observed CIPK23 autophosphorylation and CIPK23-dependent phosphorylation of CBL1 (Fig. 4B, lane 1). The amount of phosphorylated CIPK23 and CBL1 was substantially reduced when the kinase reactions also contained a kinase inhibitor (K252a) or ABI2 (Fig. 4B, lanes 2 and 3, respectively). Because the bands for ABI2 and CIPK23 overlap, in the presence of ABI2 (lane 3), we cannot exclude phosphorylation of ABI2 by CIPK23, but, if so, this would mean that CIPK23 phosphorylation was even lower. Adding a kinase inhibitor after phosphorylation assay had no effect (Fig. 4B, lane 4), whereas adding ABI2 after this reaction decreased phosphorylation of CIPK23 and CBL1 (Fig. 4B, lane 5). Here, we could exclude ABI2 phosphorylation because of the presence of the kinase inhibitor. Thus, these experiments revealed that ABI2 effectively dephosphorylated CBL1 and CIPK23. These data suggested that the effect of ABI2 on NO3 transport results, at least in part, from inactivation of CBL1-CIPK23 complexes by dephosphorylation of both the Ca2+ sensor and the kinase.

DISCUSSION

Plants have NO3-sensing systems, enabling them to perceive changes in external (soil) NO3 concentration and to translate this information into specific biological responses. These sensing mechanisms are specific to NO3 and not the products that incorporate nitrogen because NO3 sensing and responsiveness occurs in nitrate reductase–null mutants that cannot assimilate NO3 (9). When an increase in NO3 is detected, the plants increase the expression of genes involved in NO3 uptake and assimilation and enhance root growth, resulting in an increase of nutrient uptake capacity in the NO3-rich area (37). Here, we provide evidence that the protein phosphatase PP2C family member ABI2 is a component of the macromolecular NO3-sensing complex that governs this important physiological process.

The protein kinase CIPK23 is a key regulator of NPF6.3-dependent NO3 sensing, signaling, and uptake. Upon interaction with and activation by CBL9, CIPK23 phosphorylates NPF6.3 and decreases its uptake capacity at high external NO3 concentration (15). Because CIPK23 also interacts with CBL1 and their encoding genes have an overlapping expression pattern (38), we tested the functional effect of coexpression of CBL1 with CIPK23 and NPF6.3 in Xenopus oocytes. Similar to the reported regulation of the plant potassium channel AKT1 (30) and the plant anion channels SLAC1 and SLAH3 (39), we found that CBL1 is as efficient as CBL9 in enabling CIPK23-mediated inhibition of the NO3 uptake activity of NPF6.3. Whereas the CBL1-CIPK23 and CBL9-CIPK23 complexes activate the K+ channel AKT1 and the anion channels SLAC1 and SLAH3, both of these Ca2+ sensor/kinase complexes inhibited the NO3 uptake activity of NPF6.3.

Because of the importance of phosphorylation in the function of CBL-CIPK complexes, we focused on identifying protein phosphatase(s) that regulated NO3 transport and determined that the clade-A PP2C family members ABI1 and ABI2 counteracted the inhibition of NPF6.3 NO3 uptake activity by CBL1-CIPK23 in the Xenopus oocyte expression system. However, in plants, only loss of ABI2 function reduced root NO3 uptake; loss of ABI1 function did not result in a discernable phenotype. The lack of an effect on NPF6.3-dependent NO3 responses is consistent with evidence that ABI1 is not expressed in the same cells or tissues as CBL1, CIPK23, or NPF6.3 (eFP Browser, http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi). In the absence of ABI2 protein in the abi2-2 mutant, the CBL1-CIPK23 complex inhibited NPF6.3 activity, leading to a similar reduction in NO3 uptake in the phosphatase mutant abi2-2 and in the NO3 transporter mutant chl1-5.

NPF6.3 is essential to repress lateral root growth in areas poor in NO3 and to stimulate growth in areas rich in NO3 (13, 16), thereby optimizing NO3 acquisition by the plant. Indeed, just as chl1-5 plants did not properly sense external NO3 and, therefore, developed lateral roots to a similar extent in both NO3-rich and NO3-poor media, abi2-2 mutant plants displayed the same phenotype, which supports the conclusion that ABI2 is also involved in NO3 sensing. Using the ABA-insensitive mutant abi2-1, a role of ABI2 in nitrate- and ABA-dependent lateral root development has been hypothesized (40, 41).

Our expression analyses showed the importance of ABI2, but not other PP2C family members, in NO3 signaling. Although NO3 induced NRT2.1 expression in wild-type plants, NO3 did not induce expression in chl1-5 plants and the same phenotype was observed in abi2-2 mutant plants, but not in the PP2C mutant plants abi1-2 or hab1-1.

Specific molecular interactions with clade-A PP2C members appear to be a general feature of CIPKs. During the regulation of K+ uptake, the positive effect of CBL1-CIPK23 on AKT1 activity is counteracted by the phosphatases HAI2 (also known as AIP1) (31) and HAI3 (42), both of which can interact with CIPK23. ABI2 interacts with CIPK8, CIPK14, CIPK24 (also known as SOS2), and CIPK26; ABI1 with CIPK20 and CIPK26; and AtPP2CA with CIPK1 and CIPK6 (31, 4244). Here, we observed that ABI2 interacts with CBL1 and CIPK23 and dephosphorylates CBL1 and CIPK23. Therefore, we propose a model in which the activity of the phosphatase ABI2 prevents full phosphorylation of both the Ca2+-sensor subunit CBL1 and the kinase CIPK23 subunit of CBL1-CIPK23 complexes, thereby suppressing activity of the kinase complex toward its substrate NPF6.3, enabling increased activity of the transporter.

Upon exposure to abiotic stresses, like drought and salt stress, plants produce the hormone ABA that binds to receptors of the RCAR/PYL/PYR family, which then interacts with and inhibits the activity of clade-A PP2C phosphatases, including ABI2 (45). Consequently, our results suggest a mechanism plant stress signaling and nutrient uptake and consequently energy expenditure may be coordinated. Upon stress exposure, ABA accumulation would inactivate ABI2 by RCAR/PYL/PYR interaction and thereby enhance phosphorylation of NPF6.3 by CBL1-CIPK23, resulting in reduced NO3 uptake, and also enhance phosphorylation of AKT1 by CBL1-CIPK23 to stimulate K+ uptake. Thus, ABA accumulation during, for example, drought and osmotic stress would inactivate ABI2, thereby enhancing the uptake of K+ ions that function as cellular osmolytes while also reducing the uptake of NO3 to shut down energy-consuming processes, like NO3 reduction. This coordinated response would enable the plant to allocate resources required to respond and adapt to the stress.

MATERIALS AND METHODS

Expression in Xenopus oocytes

For expression in Xenopus oocytes, we used the vector pGEMKN, which is a derivate of the previously described pGEMHE (46). The coding sequence of the analyzed phosphatases was amplified by PCR and subsequently inserted into pGEMKN (Table 1). Construction of pGEMKN clones containing CBL1, CBL9, or CIPK23 coding sequences was described previously (32, 47). Oocytes were obtained and injected as previously described (14). Control oocytes were noninjected. Briefly, cDNAs [NPF6.3 (At1g12110); CIPK23 (At1g30270); CBL1 (At4g17615); CBL9 (At5g47100); ABI1 (At4g26080); ABI2 (At5g57050); HAB1 (At1g72770); and PP2CA (At3g11410)] were transcribed in vitro using mMessage mMachine T7 Ultra Kit (Life Technologies) following the manufacturer’s instructions, the concentration was adjusted to 500 ng/μl, and 10 nl was injected in oocytes. After 3 days, oocytes were incubated for 2 hours in 2 ml of ND96 medium (pH 6.5) containing 10 mM 15N-nitrate (atom % 15N abundance, 99.9%). Oocytes were then washed five times in 15 ml of ND96 medium at 4°C. Batches of two oocytes were then analyzed for total nitrogen content and atom % 15N abundance by continuous-flow mass spectrometry, using a Euro-EA EuroVector elemental analyzer coupled with an IsoPrime mass spectrometer (GV Instruments).

Table 1 List of oligonucleotides used in this work.

Indicated are the names, 5′-3′ sequences, and information about the purpose of use. Restriction sites are indicated in capital, italic letters, and start and stop codons are underlined.

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Plant material

The A. thaliana accession used is Columbia (Col-0). The npf6.3 mutants are chl1-5 (33) and chl1-12. The hab1-1, abi1-2, pp2ca-1, and abi2-2 mutants have been described previously (4850).

Root nitrate influx

Root 15NO3 influx was assayed as described previously (12). Briefly, the plants were grown for 8 days on vertical agar plates containing nutrient solution and 1 mM NH4NO3. Plants were transferred to 0.1 mM CaSO4 solution in petri dishes for 1 min, then to nutrient solution (pH 5.7) containing 30 mM 15NO3 (99% atom excess 15N) for 5 min, and finally washed in 0.1 mM CaSO4 for 1 min. Roots were then separated from shoots, and the organs were dried at 70°C for at least 48 hours. After determination of their dry weight, the samples were analyzed for total nitrogen and atom % 15N using a Euro-EA EuroVector elemental analyzer coupled with an IsoPrime mass spectrometer (GV Instruments).

BiFC experiments

BiFC experiments were performed in transiently transformed N. benthamiana plants. All constructs used for BiFC protein-protein interaction studies were cloned into the pGPTVII backbone described previously (51). Details for the construction of CBL1-SPYCE(M) and SPYNE(R)-CIPK23 were discussed previously (38, 52). The plasma membrane marker PM-OFP consists of the first 12 amino acids of CBL1 fused N-terminal to an OFP as described before (52). It has been confirmed that this first 12 amino acids do not interact with CIPKs and thereby will not influence any analysis (52, 53). The transformation of the 4- to 5-week-old plants was conducted as described previously (54). Plants were exposed to continuous light for 3 days subsequent to the Agrobacterium tumefaciens (strain GV3101/pMP90)–mediated transformation. Confocal microscopy of abaxial leaf epidermal cells was performed with a Leica TCS SP5 Confocal Laser Scanning Microscope equipped with a HCX APO L 63×/0.90 W U-V-I objective according to previously described procedures (52, 53).

In vitro phosphorylation

Constructs used for protein synthesis were cloned in either pIVEX 1.3 WG vector (5 PRIME; CIPK23 and CBL1) or a modified pET24b vector (Novagene; ABI2) as described in (36). For all constructs, the coding sequence of the respective genes was PCR-amplified and inserted in the vectors by ligation after enzymatic restriction (Table 1). All constructs were subsequently checked by sequencing to ensure correctness. Protein synthesis and purification and in vitro phosphorylation assays were performed according to (36). Briefly, proteins were either produced in Wheat Germ–based Cell-Free Protein Synthesis Kits (RTS 500 Wheat Germ CECF Kit; 5 PRIME used for STREPII-CIPK23 and CBL1-STREPII) following the manufacturer’s instructions or isolated from pET24b-STREPII-ABI2 transformed BL21-CodonPlus(DE3)-RIL E. coli (Stratagene) as described in (36). For affinity purification, samples were mixed with Strep-Tactin MacroPrep (IBA) and incubated for 60 min at 4°C. After washing, STREPII-tagged proteins were eluted by gravity flow in elution buffer [100 mM tris (pH 8.0), 150 mM NaCl, and 2.5 mM desthiobiotin], collected, and quantified. Purified proteins were incubated for 60 min at 30°C in 24 μl reaction that contained 66.7 mM tris (pH 8.0), 100 mM NaCl, 5 mM MnSO4, 0.5 mM CaCl2, 2 mM dithiothreitol, 10 μM adenosine triphosphate (ATP), and 4 μCi of [γ-32P] ATP (3000 Ci/mmol). Reactions were stopped by adding 20 mM EDTA and afterward subjected for SDS-PAGE. SDS gels were fixed by Coomassie staining, and radioactively labeled proteins were visualized by autoradiography.

Split-root experiments

Split-root experiments were performed as described by Mounier et al. (16). Basal medium contains 0.5 mM CaSO4, 0.5 mM MgCl2, 1 mM KH2PO4, 2.5 mM MES (Sigma) (pH 5.7), 50 μM NaFeEDTA, 50 μM H3BO3, 12 μM MnCl2, 1 μM CuCl2, 1 μM ZnCl2, and 0.03 μM NH4MoO4. This basal medium was supplemented with KNO3 as a sole nitrogen source at the concentrations indicated for each individual experiment.

Relative gene expression

Roots from 14-day-old plants grown on hydroponic system (Phytatray) were treated with 1 mM KCl (as control) or 1 mM KNO3 and collected after 30 min of treatment, as described by Krouk et al. (55). Gene expression was determined by qRT-PCR (LightCycler 480; Roche Diagnostics) using gene-specific primers [NRT2.1: forward, 5′-aacaagggctaacgtggatg-3′; reverse, 5′-ctgcttctcctgctcattcc-3′; ACT2/8: forward, 5′-ggtaacattgtgctcagrggtgg-3′; reverse, 5′-aacgaccttaatcttcatgctgc-3′; CLATHRIN: forward, 5′-agcatacactgcgtgcaaag-3′; reverse, 5′-tcgcctgtcacatatctc-3′] and LightCycler FastStart DNA Master SYBR Green (Roche Diagnostics). Expression of NRT2.1 was normalized to ACT2/8 and CLATHRIN.

REFERENCES AND NOTES

Funding: This work was supported by the Institut National de la Recherche Agronomique (CJS PhD Fellowship to S.L. and Projet Département BAP, BAP2013-33-NITSE to B.L.), Agence Nationale de la Recherche (ANR-11-JSV6-002-01-NUTSE to B.L.), Agropolis Fondation (RHIZOPOLIS grant 07024 to A.G.), and the Région Languedoc-Roussillon (Chercheur d’Avenir to B.L.) and by grants from the Deutsche Forschungsgemeinschaft (FOR964 and SFB629 to J.K.). K.H. was supported by a fellowship of the Humboldt Foundation. Author contributions: S.L., J.K., and B.L. designed the research. S.L. and C.C.-F. performed uptake experiments. K.H.E. and J.N.O. performed BiFC analysis in N. benthamiana. S.L. and M.P. performed transcript and root development analysis. P.T. performed the 15N analysis. K.H. performed in vitro kinase and phosphatase assays. S.L., K.H.E., M.P., C.C.-F., J.N.O., A.G., J.K., and B.L. analyzed the data. S.L., K.H.E., J.K., and B.L. wrote the paper. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All materials are available upon requests.
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