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The Plant Cell, Vol. 28: 485–504, February 2016, www.plantcell.org ã 2016 American Society of Plant Biologists. All rights reserved. The Arabidopsis NRG2 Protein Mediates Nitrate Signaling and Interacts with and Regulates Key Nitrate Regulators OPEN Na Xu,a,1,2 Rongchen Wang,b,c,1 Lufei Zhao,a Chengfei Zhang,a Zehui Li,a Zhao Lei,a Fei Liu,a Peizhu Guan,c Zhaohui Chu,d Nigel M. Crawford,c and Yong Wanga,3 a State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Tai’an, Shandong 271018, China Key Laboratory of Crop Genetic Improvement, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China c Section of Cell and Developmental Biology, Division of Biological Sciences, University of California at San Diego, La Jolla, California 92093-0116 d State Key Laboratory of Crop Biology, College of Agronomic Sciences, Shandong Agricultural University, Tai’an, Shandong 271018, China b National ORCID IDs: 0000-0003-0896-4296 (N.X.); 0000-0002-6378-7504 (R.W.); 0000-0001-8320-7872 (Z.C.); 0000-0003-0219-4403 (Y.W.) We show that NITRATE REGULATORY GENE2 (NRG2), which we identified using forward genetics, mediates nitrate signaling in Arabidopsis thaliana. A mutation in NRG2 disrupted the induction of nitrate-responsive genes after nitrate treatment by an ammonium-independent mechanism. The nitrate content in roots was lower in the mutants than in the wild type, which may have resulted from reduced expression of NRT1.1 (also called NPF6.3, encoding a nitrate transporter/receptor) and upregulation of NRT1.8 (also called NPF7.2, encoding a xylem nitrate transporter). Genetic and molecular data suggest that NRG2 functions upstream of NRT1.1 in nitrate signaling. Furthermore, NRG2 directly interacts with the nitrate regulator NLP7 in the nucleus, but nuclear retention of NLP7 in response to nitrate is not dependent on NRG2. Transcriptomic analysis revealed that genes involved in four nitrogen-related clusters including nitrate transport and response to nitrate were differentially expressed in the nrg2 mutants. A nitrogen compound transport cluster containing some members of the NRT/ PTR family was regulated by both NRG2 and NRT1.1, while no nitrogen-related clusters showed regulation by both NRG2 and NLP7. Thus, NRG2 plays a key role in nitrate regulation in part through modulating NRT1.1 expression and may function with NLP7 via their physical interaction. INTRODUCTION Nitrogen is an important macronutrient required by plants for normal growth and development. Most plants grown under aerobic conditions absorb nitrogen mainly in the form of nitrate. Nitrate serves not only as a nutrient, but also as an important signaling molecule. Transcriptome analyses have revealed that the expression of more than 1000 genes is altered within 3 h of nitrate treatment. Among these genes, those involved in nitrate transport and assimilation, such as several members of the NITRATE TRANSPORT (NRT) gene families and the genes for nitrate and nitrite reductase (NIA and NiR, respectively), are quickly induced (Bi et al., 2007; Wang et al., 2007). In addition, some genes required for controlling carbon metabolism and for providing chemical energy used in reduction and assimilation are induced as well (Price et al., 2004; Scheible et al., 2004; Wang et al., 2004, 2007; Fritz et al., 2006; Gutiérrez et al., 2007). Nitrate signaling also 1 These authors contributed equally to this work. address: School of Biological Science, Jining Medical University, Rizhao, Shandong 276826, China. 3 Address correspondence to [email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Yong Wang (wangyong@ sdau.edu.cn). OPEN Articles can be viewed online without a subscription. www.plantcell.org/cgi/doi/10.1105/tpc.15.00567 2 Current influences root growth, development and architecture, seed dormancy, and leaf expansion (Walch-Liu et al., 2000, 2006; Forde, 2002; Alboresi et al., 2005; Bi et al., 2007; Forde and Walch-Liu, 2009). However, our understanding of the regulatory mechanisms and genes involved in nitrate signaling in plants is incomplete. In the last few years, several nitrate regulatory genes functioning in the primary nitrate response have been characterized. One key regulator is NRT1.1 (also called NPF6.3 and CHL1), which functions not only as a dual-affinity nitrate transporter, but also as a nitrate sensor (Tsay et al., 1993; Wang et al., 1998, 2009; Liu et al., 1999; Alboresi et al., 2005; Remans et al., 2006; Walch-Liu and Forde, 2008; Ho et al., 2009; Léran et al., 2014; Muños et al., 2004). Recent crystal structure studies on NRT1.1 provide further insights into its transport mechanisms (Parker and Newstead, 2014; Sun et al., 2014; Tsay, 2014); however, little is known about how the NRT1.1 gene itself is regulated. Other nitrate regulators include two members of the CBL-interacting protein kinase family, CIPK8 and CIPK23, which are themselves regulated by NRT1.1 and are involved in the primary nitrate response with CIPK8 acting as a positive regulator and CIPK23 as a negative regulator (Ho et al., 2009; Hu et al., 2009; Krouk et al., 2010a). In addition, CIPK23 can interact with and phosphorylate NRT1.1 at amino acid Thr-101 to maintain the high-affinity response under low-nitrate conditions (Ho et al., 2009). So far, several transcription factors (ANR1, LBD37/38/39, NLP6, NLP7, SPL9, TGA1, TGA4, and NAC4) have 486 The Plant Cell been identified to be nitrate regulatory genes (Zhang and Forde, 1998; Remans et al., 2006; Castaings et al., 2009; Rubin et al., 2009; Wang et al., 2009; Krouk et al., 2010b; Vidal et al., 2010, 2013; Gan et al., 2012; Konishi and Yanagisawa, 2013; Marchive et al., 2013; Alvarez et al., 2014). The Arabidopsis thaliana MADS box transcription factor ANR1 was the first to be characterized to regulate lateral root growth in response to nitrate treatment (Zhang and Forde, 1998; Gan et al., 2005, 2012). Reverse genetics has revealed that three members of LATERAL ORGAN BOUNDARY DOMAIN (LBD) transcription factor family LBD37/38/39 are negative regulators for nitrateresponsive genes and the mutants show a constitutive nitrogen starvation response (Rubin et al., 2009). Arabidopsis NINLIKE PROTEIN7 (NLP7) has been found to function as a master regulator in the early nitrate response. Disruption of NLP7 results in a nitrogen-starved phenotype and impaired nitrate signaling in the mutants (Castaings et al., 2009). The nuclear retention of NLP7 is regulated by nitrate (Marchive et al., 2013). All nine NLPs can bind the nitrate-responsive cis-element NRE and activate NRE-dependent and nitrate-responsive gene expression (Konishi and Yanagisawa, 2013). A suppression study of NLP6 demonstrated that this gene plays an important role in nitrate signaling and other NLP members are also speculated to have similar function (Konishi and Yanagisawa, 2013). Using systems biology, SPL9, TGA1, TGA4, AFB3, and NAC4 have been identified as nitrate regulators involved in early nitrate response signaling (Krouk et al., 2010b; Vidal et al., 2010, 2013; Alvarez et al., 2014). A forward genetic screen for nitrate regulatory mutants was developed by transforming a nitrate-responsive promoter (NRP) and YFP marker into wild-type plants (Wang et al., 2009, 2010). The transgenic plants harboring this NRP-YFP construct show strong YFP fluorescence in the presence of nitrate. Two sets of mutants that showed low YFP fluorescence in the presence of nitrate were isolated and mapped to NRT1.1 and NLP7, respectively (Wang et al., 2009). Thus, this NRP-based mutant screen system can be used to screen for nitrate regulatory mutants, providing an effective forward genetic approach for discovering new genes involved in nitrate signaling. In this study, we performed a forward genetic screen using the NRP-YFP plants and isolated a mutant, Mut75. The mutation was mapped to the gene At3g60320, designated as NITRATE REGULATORY GENE2 (NRG2; as NRG1 has been used for NRT1.1; Wang et al., 2009), and further characterization showed that it plays a key role in nitrate signaling. Genetic and molecular analyses revealed that NRG2 modulates the expression of NRT1.1 and functions upstream of NRT1.1. Moreover, biochemical and in planta experiments showed that NRG2 can directly interact with NLP7. Our findings support a model in which NRG2 regulates the expression of NRT1.1 and directly interacts with NLP7 in nitrate signaling transduction. These results not only establish the key role of NRG2 in transcriptional control, but also demonstrate a direct involvement of NRG2 in central nitrate signaling and offer insights into the mechanism of nitrate regulation in plants. In addition, our findings provide the first insights into the functions of an uncharacterized, 15-member gene family in Arabidopsis, to which NRG2 belongs. RESULTS Defects in Nitrate Signaling in Mut75 Are Caused by a Mutation in At3g60320 To identify regulators in nitrate signaling, we performed a forward genetic screen. The seeds from homozygous transgenic plants containing the nitrate-responsive promoter NRP fused to a YFP marker (Wang et al., 2009) were treated with ethyl methanesulfonate, and M2 population seedlings grown on nitrate medium were checked for YFP fluorescence in roots under a fluorescence microscope. The transgenic wild-type seedling roots showed strong YFP signal in the presence of nitrate, as they are responsive to nitrate (Figure 1Aa). One mutant, Mut75, exhibiting much lower YFP fluorescence than the wild type in the presence of nitrate, was isolated (Figures 1Ab and 1B). The location of the mutation in Mut75 was narrowed down to the end of chromosome 3 in a 110-kb region (Figure 1C) by a map-based cloning strategy. Unexpectedly, the sequencing results showed two point mutations in this region with one (G to A) in At3g60320 that converted Trp at position 638 to a stop codon (Figure 1C) and another one (C to T) in At3g60240 that changed Gln at position 332 to a stop codon (Supplemental Figure 1A). At3g60320 is an uncharacterized gene, while At3g60240 encodes PROTEIN SYNTHESIS INITIATION FACTOR 4G (EIF4G), which is involved in virus resistance (Yoshii et al., 2004; Nicaise et al., 2007). To determine which mutation results in the weak YFP fluorescence phenotype of the mutant, several genetic tests were performed. For the gene At3g60320, transforming wild-type cDNA for this gene driven by a 35S promoter into the Mut75 mutant restored strong YFP fluorescence in the roots on nitrate medium (Figure 2A), indicating that the gene At3g60320 can rescue the YFP phenotype of Mut75. In addition, two homozygous T-DNA insertion mutant lines for this gene were isolated from the ABRC T-DNA population (Alonso et al., 2003). The transcript levels of At3g60320 were very low in SALK_014743, which has a T-DNA insertion in the promoter region, and undetectable in SALK_079096, which contains a T-DNA insertion in the second exon, when tested by RT-PCR (Figures 2B and 2C). Mut75 was crossed with these two lines. Both F1 plants exhibited lower YFP fluorescence in roots when grown on nitrate medium (Figure 2D), confirming that the weak YFP phenotype of Mut75 is caused by the mutation in At3g60320. Therefore, we designated At3g60320 as NRG2, its T-DNA mutants SALK_014743 as nrg2-1 and SALK_079096 as nrg2-2, and Mut75 as nrg2-3. To test if the phenotype of nrg2-3 could have resulted from the disruption of the gene EIF4G, a knockout T-DNA insertion mutant SALK_002002 with the T-DNA inserted in the seventh exon of EIF4G was identified (Supplemental Figures 1A and 1B) and then crossed with nrg2-3. The F1 plants grown on nitrate medium showed strong YFP fluorescence in the roots (Supplemental Figure 1C), suggesting that the weak YFP phenotype of nrg2-3 is not caused by the mutation in EIF4G. qPCR results showed that the expression of several nitrate-responsive genes (NIA1, NiR, and NRT2.1) was induced by nitrate in the mutant to a similar level as in the wild type (Supplemental Figure 1D). These data imply that EIF4G is not involved in nitrate regulation. NRG2 Plays an Essential Role in Nitrate Signaling 487 Figure 1. Identification and Mapping of Mut75. (A) Nitrate induction of NRP-YFP in wild type (WT) and Mut75 roots. Fluorescence and light images of 4-d-old seedlings grown on KCl/KNO3 media (a) and on KNO3 (b) were captured with a fluorescence microscope. (B) Quantification of root fluorescence of wild-type and Mut75 seedlings grown on the same conditions as (A). Error bars represent SD (n = 60). Asterisks indicate significant differences (P < 0.05) compared with the wild type (t test). (C) Mapping of NRG2 (Mut75). The schematic map shows that the mutation in Mut75 was located in the gene NRG2 on chromosome 3. Amino acid and nucleotide changes found in Mut75 are also shown. 488 The Plant Cell Figure 2. The Mut75 Phenotype Is Caused by the Mutation in NRG2. (A) Complementation test of NRG2 in Mut75. Fluorescence and light images of 4-d-old seedlings grown on nitrate media were captured with a fluorescence microscope. (B) Schematic map of the T-DNA insertion sites in nrg2-1 and nrg2-2 mutants. Exons, introns, and untranslated regions are represented by black boxes, lines, and white boxes, respectively. The locations of the T-DNA insertion in the two nrg2 alleles from SALK are indicated with triangles. Black arrow indicates the mutation site in Mut75. (C) RT-PCR analysis of NRG2 mRNA levels in the wild type and the nrg2 mutants. Total RNA isolated from 7-d-old seedlings grown on ammonium nitrate was analyzed by RT-PCR and a program based on 25 cycles of PCR amplifications was performed to test the expression of NRG2. TUB2 serves as a control to show the equal amount of cDNA in each reaction. (D) Root fluorescence of F1 plants from nrg2-3 crossed with nrg2-1 and nrg2-2, respectively. Fluorescence and light images of 4-d-old seedlings grown on nitrate media were captured with a fluorescence microscope. Taking together, these results indicate that the weak YFP phenotype of Mut75 is caused by the mutation in NRG2, but not by the mutation in EIF4G. Therefore, we focused on the functional characterization of NRG2 in the following analyses. NRG2 Is Required for Nitrate-Regulated Gene Expression The defect in responding to nitrate with NRP-YFP expression in nrg2-3 suggests that NRG2 plays an important role in nitrate signaling. To test if NRG2 also regulates endogenous genes, the expression of the nitrate-responsive genes NIA1, NiR, and NRT2.1 was investigated. qPCR results showed that the nitrate induction of these genes in the roots of both nrg2-1 and nrg2-2 mutants was significantly inhibited (Figure 3A; Supplemental Figures 2A and 2B). No difference was found for the expression of these genes among wild type and the mutants when grown on ammonium succinate without any nitrate treatment (Supplemental Figure 2C) or after KCl treatment (Supplemental Figure 2D), while a significant decrease in the mutants was seen after KNO3 treatment (Supplemental Figure 2E). The inhibited nitrate induction in the mutants was also observed when the seedlings were treated with a low concentration of nitrate (Supplemental Figure 2F). The above results demonstrate that NRG2 functions in nitrate signaling in plants. NRG2 Plays an Essential Role in Nitrate Signaling 489 Figure 3. The mutants of NRG2 Are Defective in Response to Nitrate. (A) Nitrate induction of endogenous genes without nitrogen starvation. Wild-type and nrg2 plants were grown on medium with 2.5 mM ammonium succinate as the sole nitrogen source for 7 d and then treated with 10 mM KNO3 or KCl as a control for 2 h. Roots were collected for RNA extraction. The transcripts of nitrate-responsive genes were determined by qPCR. Error bars represent SD of biological replicates (n = 4). Asterisks indicate significant differences (P < 0.05) compared with the wild type (t test). (B) Nitrate induction of endogenous genes after nitrogen starvation. Plants were grown, treated, and analyzed as described in (A), except plants at day 6 were transferred to nitrogen-free medium for 24 h then treated with 10 mM KNO3 or KCl for 2 h. The transcripts of nitrate-responsive genes in roots were determined by qPCR. Previous studies have shown that NRT1.1 acts as a nitrate sensor and mediates nitrate responses as evidenced by the fact that nrt1.1 mutants (chl1-5 and chl1-13) exhibited decreased nitrate induction of the nitrate-responsive genes (NIA1, NiR, and NRT2.1) (Ho et al., 2009; Wang et al., 2009). However, this phenotype is dependent on nitrogen pretreatment, as nitrogen deprivation restores the wild-type phenotype in nrt1.1 mutants (Wang et al., 2009; Krouk et al., 2010a). We tested nrg2 mutants under both nitrate-replete and nitrogen-deprived conditions and found that the induction levels for these nitrate-responsive genes were reduced under both conditions compared with the wild type (Figures 3A and 3B), indicating that NRG2 functions in nitrate signaling regardless of nitrogen starvation. This finding contrasts with that of NRT1.1, whose nitrate regulatory function is lost after nitrogen starvation and shows that NRG2 functions in both types of nitrogen conditions (nitrogen replete and nitrogen deprived). To test if NRG2 is regulated by different nitrogen conditions, we investigated its expression levels after nitrate, ammonium, and nitrogen starvation treatments. The results did not show significant changes in the expression of this gene after these treatments (Supplemental Figure 2G), indicating that the expression of NRG2 is not modulated by nitrate, ammonium, and nitrogen starvation. NRG2 Is Predominantly Expressed in the Vascular Tissue of Leaves and Roots, and NRG2 Protein Is Localized in the Nucleus The expression profile of NRG2 in wild-type plants was examined using qPCR. Tissues were harvested either from plants grown in soil (leaves, stems, flowers, and siliques) or from plants grown on ammonium nitrate medium (seedlings and roots). NRG2 was expressed in all tested tissues, with highest levels in leaves and roots and lowest levels in flowers and siliques (Figure 4A). The expression profile of NRG2 was further analyzed by the promoterGUS approach. The GUS staining profile is largely consistent with the results obtained from qPCR, confirming the expression pattern of NRG2 in the tissues tested. Moreover, GUS staining revealed that NRG2 is predominantly expressed in the vascular bundles of leaves and roots (Figures 4Ba to 4Bh). In addition, expression of NRG2 was also found in stomata (Figure 4Bi), flowers (Figures 4Bj to 4Bm), and young siliques (Figure 4Bn). In flowers, GUS expression was observed in the pistil (Figure 4Bk), junction of filament and anther (Figure 4Bl), and vascular tissue of sepals and petals (Figure 4Bm). To more precisely determine the cells that express NRG2, the GUS staining in vascular bundles was further analyzed. Cross sections of the roots showed GUS expression in the stelar cells, including the pericycle, phloem, and parenchyma cells (Figure 4 Ca). Longitudinal and transversal sections of the leaves revealed GUS expression in the bundle sheath, phloem, and parenchyma cells of the vascular tissues (Figures 4Cb and 4Cc). This expression profile suggests that NRG2 may function in regulating nitrate transport in the vasculature. NRG2 contains two uncharacterized functional domains: DUF630 and DUF632, which are shared by all 15 members in this family of unknown proteins (Supplemental Figure 3). To explore the subcellular localization of the NRG2, several subcellular localization prediction tools were used to analyze its protein sequence. NRG2 was predicted to be localized in the nucleus by SubLoc (Chen et al., 2006) and WoLFPSORT (Horton et al., 2007) tools, but in mitochondria by MitoPred (Guda et al., 2004). To determine the bona fide localization of the protein, we cloned the NRG2 cDNA and ligated the fragment in frame to be expressed with the GFP reporter at the N-terminal position (Pro35S:GFPNRG2). The construct was transformed into Arabidopsis wild-type plants and the protein was observed in the nucleus in stable transgenic lines (Figure 4D). Thus, we conclude that NRG2 protein is targeted to the nucleus. Nitrate Content in nrg2 Mutants Is Lower Than the Wild Type in Roots, but Not in Leaves We demonstrated that the induction of the nitrate-responsive genes is inhibited in the nrg2 mutants. We then tested if this 490 The Plant Cell Figure 4. NRG2 Is Predominantly Expressed in Vascular Tissues and the NRG2 Protein Is Localized in the Nucleus. (A) Analysis of the relative expression level of NRG2 in different organs of Arabidopsis by qPCR. Tissues were harvested either from 45-d-old plants grown in soil (leaves, stems, flowers, and siliques) or from 7-d-old plants grown in NH4NO3 liquid medium (seedlings and roots). Error bars represent SD of biological replicates (n = 4). (B) Histochemical staining of GUS activity in transgenic plants expressing ProNRG2:GUS. GUS activity was detectable in root (a), root tip (d), root vascular system ([c] to [e]), vascular system of cotyledon (f) and cauline leaves ([b], [g], and [h]), stomata (i), flower (j), pistil (k), junction of filament and anther (l), vascular tissue of sepals and petals (m), and young silique (n). (C) GUS staining for NRG2 promoter-driven activity in vascular bundles. Cross section of the roots (a) revealed GUS expression in the stelar cells, including pericycle, phloem, and parenchyma cells. Longitudinal section (b) and cross section of the leaves (c) revealed that the NRG2 promoter drives expression mainly in vascular bundles including bundle sheath, phloem, and parenchyma cells. XV, xylem vessels; PL, phloem; PR, parenchyma; BS, bundle sheath. Bars = 50 mm. (D) Subcellular localization of NRG2 protein. (a) Confocal laser scanning microscopy and corresponding bright-field images of Arabidopsis roots. (b) Higher magnifications of the red squares in (a); red arrows indicate the nucleus. Bars = 50 mm. NRG2 Plays an Essential Role in Nitrate Signaling molecular defect results in any phenotype at the morphological and physiological levels in the mutants. Under a high nitrate concentration, the mutant seedlings were slightly smaller and displayed later flowering compared with the wild type (Supplemental Figures 4A and 4B). Under low nitrate condition, no obvious phenotype was observed (Supplemental Figure 4C). Previous studies on several known nitrate regulatory genes (NRT1.1, NLP7, and LBD37/38/39) have shown that the nitrate levels in their respective mutant plants are altered (Castaings et al., 2009; Rubin et al., 2009; Wang et al., 2009). Here, we found that the nitrate accumulation in nrg2 mutant seedlings was significantly lower than in the wild type (Figure 5A). Further investigation 491 revealed that the nitrate accumulation in roots was significantly lower with each mutant allele than in the wild type (Figure 5B). However, no difference between the mutants and the wild type was found in leaves (Figure 5C). These data indicate that the nitrate accumulation in roots is defective, while the accumulation of nitrate in leaves is normal in nrg2 mutants. We further assayed the nitrate content in whole seedlings treated with various concentrations of nitrate (0.25 to 20 mM) for 2 h in the presence of ammonium and found that the nitrate accumulation in the mutants (including the chl1-13 mutant as a control) was significant lower in all concentrations tested (Figure 5D). We also tested the time course of nitrate accumulation in whole seedlings treated with Figure 5. NRG2 Affects Nitrate Accumulation and Uptake. (A) to (C) Nitrate content in seedlings (A), roots (B), and leaves (C). Wild-type and nrg2 mutant plants were grown on ammonium nitrate medium for 7 d and collected for nitrate concentration test. Error bars represent SD of biological replicates (n = 4). Asterisks indicate significant differences (P < 0.05) compared with the wild type (t test). FW, fresh weight. (D) and (E) Nitrate accumulation in wild type, nrg2, and chl1 mutants. Seedlings grown with 2.5 mM ammonium succinate for 7 d were treated with various concentrations of KNO3 for 2 h (D) or treated with 5 mM KNO3 for different times in the presence of ammonium succinate (E) and then collected for nitrate concentration test. The chl1-13 mutant was used as a control. Asterisks indicate significant differences (P < 0.05) between the wild type and two mutants. (F) and (G) Relative expression of NRT1.1 (F) and NRT1.8 (G). Wild-type and nrg2 mutant plants were grown on ammonium nitrate medium for 7 d, and then roots and shoots were collected separately for RNA extraction. The transcription levels of NRT1.1 and NRT1.8 were determined by qPCR. Error bars represent SD of biological replicates (n = 4). Asterisks indicate significant differences (P < 0.05) compared with the wild type (t test). 492 The Plant Cell 5 mM KNO3, and the results showed that the nitrate uptake in the mutants was significantly lower than in the wild type at all time points tested (Figure 5E). These findings suggest that nitrate uptake is affected in nrg2 mutants. To investigate the mechanisms for the lower nitrate accumulation in mutant roots, the expression of several known nitrate transport genes was studied. Among the 13 transport genes tested, only the expression of NRT1.1 was significantly decreased under ammonium nitrate conditions (Figure 5F), while no change was found for the other 12 tested genes (Supplemental Figure 5) in the mutant roots. In leaves, only the transcript levels of NRT1.8 were significantly increased (Figure 5G), and there was no significant change in the expression of the other genes (Supplemental Figure 6) in the mutants. Previous studies have shown that NRT1.1 functions as a dual-affinity nitrate transporter involved in transporting nitrate from the environment into roots (Tsay et al., 1993; Wang et al., 1998; Liu and Tsay, 2003). Thus, the decreased nitrate content in mutant seedlings may be caused by the decreased expression of NRT1.1. NRT1.8 functions in removing nitrate from xylem vessels, as the functional disruption of NRT1.8 increased nitrate concentration in xylem sap (Li et al., 2010). Thus, it is possible that the increased expression of NRT1.8 may direct more nitrate to be unloaded from xylem vessels resulting in similar nitrate levels in the mutant leaves to those in wild-type leaves. The expression of several key nitrate assimilatory genes (NIA1, NIA2, NiR, GLN1.1, and GLN1.3) was also detected by qPCR, and no significant difference was found between the wild type and nrg2 mutants (Supplemental Figure 7). Therefore, these results imply that the lower nitrate content in mutant roots may be correlated with the reduced expression of NRT1.1 in roots and the increased transcripts of NRT1.8 in leaves. NRG2 Regulates the Expression and Works Upstream of NRT1.1 To understand the relationship of NRG2 and the characterized nitrate regulators, we first investigated the expression levels of several known nitrate regulators in the nrg2 mutants under different nitrogen conditions. The results showed that, among the investigated known regulatory genes, the expression of NRT1.1 in the nrg2 mutants was significantly decreased, to <40% of the expression in wild-type plants under potassium nitrate or Figure 6. The Expression of NRT1.1 in the nrg2 Mutants Was Reduced. (A) Relative expression of NRT1.1 in nrg2 mutants. Wild-type and nrg2 mutant plants were grown on media with KNO3 or NH4NO3 for 7 d, and whole seedlings were collected for gene expression detection. Error bars represent SD of biological replicates (n = 4). Asterisks indicate significant differences (P < 0.05) compared with the wild type (t test). (B) Relative expression of NRG2 in nrt1.1 mutants. Wild-type, chl1-5, and chl-13 plants were grown on media with KNO3 or NH4NO3 for 7 d, and whole seedlings were collected for gene expression detection. Error bars represent SD of biological replicates (n = 4). (C) Nitrate induction of NRT1.1 in the wild type and the nrg2 mutants. Plants were grown on medium with 2.5 mM ammonium succinate as the sole nitrogen source for 7 d and then treated with 10 mM KNO3 or KCl as a control for 2 h. Roots were collected for RNA extraction. The relative expression of NRT1.1 was determined by qPCR. Error bars represent SD of biological replicates (n = 4). Asterisks indicate significant differences (P < 0.05) compared with the wild type (t test). NRG2 Plays an Essential Role in Nitrate Signaling ammonium nitrate conditions (Figure 6A). The expression of other known nitrate regulatory genes tested was not changed (Supplemental Figure 8). This indicates that the expression of NRT1.1 is regulated by NRG2. To test if NRG2 is regulated by known nitrate regulators, we measured NRG2 transcript levels in the mutants of several 493 identified regulatory genes (NRT1.1, NLP7, CIPK8, and CIPK23) in nitrate or ammonium nitrate media. No change was found for the expression of NRG2 in these mutants (Supplemental Figure 9), including in nrt1.1 mutants (Figure 6B). These results imply that NRG2 may not be regulated by these four genes. Figure 7. NRG2 and NRT1.1 Work in the Same Nitrate Signaling Pathway. (A) Root fluorescence phenotypes of wild-type, nrg2-3, chl1-13, and chl1-13 nrg2-3 plants on KNO3 medium. The plants were grown on KNO3 medium for 4 d. Fluorescence and light images were captured with a fluorescence microscope to visualize YFP expression. (B) Quantification of root fluorescence of wild-type, nrg2-3, chl1-13, and chl1-13 nrg2-3 plants. The plants were grown under the same conditions as in (A). Error bars represent SD (n = 60). Different letters indicate statistically significant difference (P < 0.05, t test). (C) Root fluorescence phenotypes of wild-type, nrg2-3, chl1-13, and chl1-13 nrg2-3 plants on NH4NO3 medium. The plants were grown on NH4NO3 medium for 4 d. Fluorescence and light images were captured with a fluorescence microscope to visualize YFP expression. (D) Quantification of root fluorescence of wild-type, nrg2-3, chl1-13, and chl1-13 nrg2-3 plants on NH4NO3 medium. The seedlings were grown on the same NH4NO3 medium as (C). Error bars represent SD (n = 60). Different letters indicate statistically significant difference (P < 0.05, t test). (E) Nitrate induction of gene expression in wild-type, nrg2-3, chl1-13, and chl1-13 nrg2-3 plants. The seedlings were grown and treated as described in Figure 3A. The transcripts of nitrate-responsive genes in roots were measured by qPCR. Error bars represent SD of biological replicates (n = 4). Different letters indicate statistically different means (P < 0.05, t test). 494 The Plant Cell To study further the effects of NRG2 on NRT1.1, we examined the nitrate induction of NRT1.1 in the wild type and the nrg2 mutants. The results showed a significant decrease in the nitrate induction levels in the mutants (Figure 6C), indicating that NRG2 affects the nitrate induction of NRT1.1. We also tested the expression of NRT1.1 in the absence of nitrate and found that the expression of NRT1.1 was significantly lower in the mutants compared with that in the wild type (Supplemental Figure 10A). When seedlings grown on NH4NO3 medium were subjected to nitrogen starvation, the expression of NRT1.1 increased during the first 24 h in both the wild type and the mutants and no significant differences in expression levels were found for the time points tested between wild type and the nrg2 mutants (Supplemental Figure 10B). To understand better the relationship of NRG2 and NRT1.1, a double mutant of the two genes was obtained by crossing the single mutants of each gene: nrg2-3 and chl1-13 isolated by our mutant screens. The YFP signal from the NRP-YFP transgene in roots of the double mutant seedlings grown on nitrate medium (with no ammonium) was detected and found to be much weaker than in the wild type and similar to nrg2-3 while weaker than chl1-13 (Figure 7A). Quantifying the root fluorescence signal confirmed the weaker YFP signal in double mutant than in the wild type and chl1-13 and was similar to nrg2-3 (Figure 7B). Notably, the signal in chl1-13 was much higher than in nrg2-3 and double mutant and mildly lower than in the wild type. We also tested the YFP levels in roots of the single and double mutants grown on ammonium nitrate medium to investigate the function of the genes in the presence of ammonium. The observation and fluorescence quantification data showed that the YFP levels in the double mutant were similar to those of chl1-13 while lower than of nrg2-3 (Figures 7C and 7D). Interestingly, the chl1-13 exhibited much lower signal than the wild type, confirming that NRT1.1 function in the nitrate signaling pathway is ammonium dependent. To provide further molecular evidence, we inspected the expression of nitrate-responsive genes in the wild type and these single and double mutants. The qPCR results showed that the expression levels of the nitrate-responsive genes in the double mutant chl1-13 nrg2-3 were similar to those in single mutant chl1-13 and much lower than in the wild type (Figure 7E). Additionally, we transformed the cDNA of NRT1.1 into the nrg2-2 mutant to investigate further the relationship between NRT1.1 and NRG2. The nitrate content in roots of nrg2-2 was recovered to the wild-type level when NRT1.1 was overexpressed in the mutant (Figure 8A; Supplemental Figure 10C). We also detected the expression levels of the nitrate-responsive genes NIA1, NiR, and NRT2.1 and found that nitrate induction of these genes was recovered to the wild-type phenotype in NRT1.1/nrg2-2 (Figure 8B). Taken together, these results suggest that NRG2 and NRT1.1 work in the same nitrate signaling pathway and that NRG2 functions upstream of NRT1.1. Genetic and Molecular Analysis Reveals that NRG2 and NLP7 Have Nonoverlapping Functions in Nitrate Regulation NLP7 is an important nitrate regulator in nitrate signaling (Castaings et al., 2009; Konishi and Yanagisawa, 2013; Marchive et al., 2013). To investigate the relationship between NRG2 and NLP7 in the process of regulating nitrate response, a double mutant of the two genes was generated by crossing the respective single mutants nrg2-3 and nlp7-4 isolated by our mutant screen system. The mutant nlp7-4 harbors a mutation (C to T) in NLP7 that converts Gln at the position 62 to a stop codon, resulting in lower YFP fluorescence in roots when grown on nitrate-containing media (Figures 9A to 9D). In the presence of nitrate without ammonium, both single mutants showed much lower YFP fluorescence in roots than the wild type, with 23% of the wild type for nlp7-4 and 35% of the wild type for nrg2-3 in terms of fluorescence intensity, respectively. Interestingly, the double mutant plants exhibited even lower YFP signal than the individual single mutants, with only 13% of the wild type (Figures 9A and 9B). Under ammonium nitrate conditions, similar results were obtained as under Figure 8. NRG2 Functions Upstream of NRT1.1. (A) Nitrate content in roots. Wild-type, nrg2-2, and NRT1.1/nrg2-2 plants were grown on ammonium nitrate medium for 7 d, and the roots were collected for nitrate concentration analysis. Error bars represent SD of biological replicates (n = 4). Asterisks indicate significant differences (P < 0.05) compared with the wild type (t test). (B) Nitrate induction of the endogenous genes. Wild-type, nrg2-2, and NRT1.1/nrg2-2 plants were grown on medium with 2.5 mM ammonium succinate as the sole nitrogen source for 7 d and then treated with 10 mM KNO3 or KCl as a control for 2 h. The roots were collected for RNA extraction. The transcript levels of nitrate-responsive genes were determined by qPCR. Error bars represent SD of biological replicates (n = 4). Asterisks indicate significant differences (P < 0.05) compared with the wild type (t test). NRG2 Plays an Essential Role in Nitrate Signaling nitrate conditions, with 20, 36, and 15% of the wild type for nlp7-4, nrg2-3, and nlp7-4 nrg2-3 mutants, respectively (Figures 9C and 9D). These results suggest that NRG2 and NLP7 play important roles in nitrate regulation in nonoverlapping ways. Our qPCR results showed that the expression of NLP7 was not altered in nrg2 mutants (Supplemental Figure 8) and the expression of NRG2 was not changed in nlp7 mutant either (Supplemental Figure 9), so that there was no evidence for transcriptional regulation of these genes by each other. We also detected the expression of the nitrate-responsive genes in the wild type and in single and double mutants. The qPCR 495 results revealed that the transcripts of the three tested genes in double mutant nlp7-4 nrg2-2 were significantly lower than in both single mutants (Figure 9E), further showing that NRG2 and NLP7 function in nonoverlapping ways to regulate nitrate responses. NRG2 Interacts with NLP7 but Does Not Affect the Nuclear Retention of NLP7 in Response to Nitrate Previous studies have shown that NLP7 is mainly expressed in the vascular tissue and the protein is targeted to the nucleus after nitrate treatment (Castaings et al., 2009; Marchive et al., 2013), Figure 9. Analysis of Nitrate Regulation in nrg2 nlp7 Double Mutants. (A) Root fluorescence phenotypes of the wild type, nrg2-3 and nlp7-4 single mutants, and nlp7-4 nrg2-3 plants on KNO3 medium. The plants were grown on KNO3 medium for 4 d. Fluorescence and light images were captured with a fluorescence microscope to visualize YFP expression. (B) Quantification of root fluorescence of wild-type, nrg2-3, nlp7-4, and nlp7-4 nrg2-3 plants. The plants were grown on the same condition as (A). Error bars represent SD (n = 60). Different letters indicate statistically significant difference (P < 0.05, t test). (C) Root fluorescence phenotypes of wild-type, nrg2-3, nlp7-4, and nlp7-4 nrg2-3 plants on NH4NO3 medium. The plants were grown on NH4NO3 medium for 4 d. Fluorescence and light images were captured with a fluorescent microscope to visualize YFP expression. (D) Quantification of root fluorescence of wild-type, nrg2-3, nlp7-4, and nlp7-4 nrg2-3 plants on NH4NO3 medium. The seedlings were grown on the same NH4NO3 medium as (C). Error bars represent SD (n = 60). Different letters indicate statistically significant difference (P < 0.05, t test). (E) Nitrate induction of gene expression in wild-type, nrg2-2, nlp7-4, and nlp7-4 nrg2-2 plants. The seedlings were grown and treated as described in Figure 3A. The transcripts of nitrate-responsive genes in roots were determined by qPCR. Error bars represent SD of biological replicates (n = 5). Different letters indicate statistically different means (P < 0.05, t test). 496 The Plant Cell which is similar to the expression pattern of NRG2. Even though these two proteins show no evidence of genetic or transcriptional interaction, it is possible that the two proteins can interact at the protein level. To test this idea, yeast two-hybrid assays were performed. Indeed, NRG2 and NLP7 cotransformed yeast (Saccharomyces cerevisiae) cells grew well, while each gene and empty vector cotransformed yeast cells used as negative controls could not grow on the selective media (Figure 10A), indicating that NRG2 protein can directly interact with NLP7 in vitro. To confirm the interaction between NRG2 and NLP7, in vivo tests with bimolecular fluorescence complementation (BiFC) assays on Nicotiana benthamiana leaves was performed. A direct Figure 10. NLP7 Interacts with NRG2. (A) Yeast two-hybrid assay of the NRG2 and NLP7 interaction. Serial dilution of yeast cells containing the indicated constructs was spotted on the indicated medium for lacZ and His reporter assays (four independent experiments). pGADT7, empty prey vector; pGBKT7, empty bait vector; NRG2, a bait vector containing cDNA of NRG2; NLP7, a prey vector containing cDNA of NLP7. SD/TW-, SD medium lacking tryptophan and leucine; SD/LWHA-, SD medium lacking tryptophan, leucine, histidine, and adenine with X-a-Gal. (B) BiFC analysis for interaction between NRG2 and NLP7. N- and C-terminal fragments of YFP were fused to NRG2 and NLP7, respectively. Different combinations of expression vectors encoding NRG2-YFPN and NLP7-YFPC and controls (indicated on the left of the panel) were transformed into leaves of N. benthamiana grown on NH4NO3 medium. Presence of YFP signal indicates reconstitution of YFP through protein interaction of the tested pairs. N. benthamiana cells showing YFP fluorescence in the nucleus were observed and marked by red arrows. Bar = 5 mm. NRG2 Plays an Essential Role in Nitrate Signaling interaction was observed between NRG2 and NLP7 in the nucleus of plant cells, where coexpression of NRG2-YFPN and NLP7-YFPC reconstituted a functional YFP, whereas no significant signals were found in controls lacking NRG2 or NLP7 (Figure 10B). The direct interaction between NRG2 and NLP7 proteins in the nucleus was also observed on infiltrated leaves from plants with starvation pretreatment, while no significant signals were seen in controls lacking NRG2 or NLP7 (Supplemental Figure 11). These in vitro and in vivo results demonstrate the direct interaction of NRG2 and NLP7 in the nucleus. As the nuclear retention of NLP7 is regulated by nitrate (Marchive et al., 2013), we wanted to determine if NRG2 is involved in the nitrate-induced nuclear retention of NLP7. Therefore, we checked the NLP7 subcellular localization in nrg2-2 mutant lines transformed with the NLP7-YFP construct and grown in the presence or absence of nitrate. The confocal images showed that localization of NLP7 protein was indistinguishable between wild-type and nrg2 mutant plants (Figure 11). This finding indicates that the nuclear retention of NLP7 is not dependent on NRG2. Transcriptomic Analysis of Nitrate Response in nrg2, chl1, and nlp7 Mutants To investigate systematically the molecular mechanism by which NRG2 mediates plant responses to nitrate, and to probe the relationships among NRG2, NRT1.1, and NLP7, we performed 497 a comparative RNA-seq analysis. The seedlings of the wild type and nrg2-2, chl1-13, and nlp7-4 mutants were grown on medium with 2.5 mM ammonium succinate for 7 d and then treated with either 10 mM KNO3 or KCl for 2 h. The total root RNA analyzed using an Illumina HiSeq 2500. For each genotype and NO32 treatment, three biological replicates were tested. After filtering low-quality reads and removing reads that aligned to rRNA or tRNA, we selected 435,055,962 reads for analysis (Supplemental Data Set 1). Twofold change in gene expression levels and adjusted P value < 0.05 were used as a cutoff value to select differentially expressed transcripts. We first compared the gene expression in the roots of wild-type and nrg2 mutant plants in response to nitrate treatments. The results (Figure 12A; Supplemental Data Set 2) showed that the transcripts of 276 genes (including 117 upregulated and 159 downregulated) were altered in the wild type after nitrate treatment, but not in nrg2 mutant. In other words, the expression of these nitrate-responsive genes in the wild type was suppressed in nrg2 mutant. The transcripts of 131 genes (88 induced and 43 suppressed) were changed in the nrg2 mutant, but not in the wild type. In addition, the expression of 314 genes were regulated by nitrate in both the wild type and nrg2 mutant, among which 148 genes (107 suppressed and 41 induced) were differentially expressed by more than 25% in the wild type and the mutant (Supplemental Data Set 3). Therefore, the mutation in NRG2 results in a total of 555 genes with altered expression after nitrate Figure 11. The Nuclear Retention of NLP7-YFP Is Not Regulated by NRG2. Subcellular localization of NLP7-YFP in the wild type ([A] and [C]) and nrg2-2 ([B] and [D]) mutants. Seedlings in (A) and (B) were grown on nitrate medium, and seedlings in (C) and (D) were treated with nitrogen deprivation. Fluorescence and corresponding bright-field pictures were captured by confocal laser scanning microscopy. Bar = 50 mm. 498 The Plant Cell To clarify the relationship among NRG2, NRT1.1, and NLP7 genes, transcriptomic analysis was performed using roots treated with nitrate. The genes with differentially induced expression in nrg2, nrt1.1, and nlp7 mutants compared with the wild type were analyzed and are shown in the Venn diagram in Figure 12B. The expression of 235 genes was found to be changed in all three mutants with 57.7, 48.5, and 44.5% of the total differentially expressed genes in nrg2, nrt1.1 (chl1), and nlp7 mutants, respectively (Supplemental Data Set 5), indicating that these three genes are closely involved in nitrate regulation in plants. In the nrt1.1 mutant, the transcripts of 485 genes were altered compared with those in the wild type, among which 277 genes (57.1% of 485) were also regulated by NRG2 (Figure 12B; Supplemental Data Set 6). Those genes that were regulated by both NRT1.1 and NRG2 were further investigated by GO analysis, and the results showed that a nitrogen compound transport cluster was involved, including some members of NRT/PTR family (Table 3; Supplemental Data Set 6). These data support the conclusion that NRG2 works in the same nitrate signaling pathway as NRT1.1. For the mutant nlp7, 276 genes were found to be regulated by both NRG2 and NLP7 (Figure 12B; Supplemental Data Set 7); however, 252 NLP7-regulated genes and 131 NRG2-regulated genes were not (i.e., were regulated by only NLP7 or NRG2 and not the other; Figure 12B). In addition, no nitrate-related cluster in these genes was found by GO analysis (Supplemental Data Set 7). This result provides further evidence that NRG2 and NLP7 have some independent functions in nitrate regulation. DISCUSSION Figure 12. RNA-Seq Analyses of Differentially Expressed Transcripts in the Roots of Wild-Type, nrg2-2, chl1-13, and nlp7-4 Seedlings Grown on Ammonium Succinate Followed by Nitrate Treatment. (A) Venn diagram showing the number of the genes up- or downregulated by nitrate treatment in the wild type and nrg2-2 mutant. (B) Diagram showing the number of the genes differentially expressed in nrg2-2, chl1-13, and nlp7-4 mutants compared with the wild type. treatment. Many known nitrate-inducible and regulatory genes, including NiR, NRT2.1, HHO1, UPM1, LBD37, LBD38, NRT1.1, TGA1, and TGA4, showed reduced nitrate induction in the mutant (Table 1). To explore the data further, we performed Gene Ontology (GO) analysis using Panther (http://www.pantherdb.org/ pathway) for these differentially expressed 555 genes. Major GO clusters for all analyzed genes are listed in Table 2, and four clusters were found to be related to nitrogen, including response to nitrogen compound, nitrogen compound transport, response to nitrate, and nitrate transport (Supplemental Data Set 4). These data strongly support our conclusion that NRG2 functions in the nitrate signaling. In addition, GO analysis revealed that genes most affected by the mutation in NRG2 were overrepresented in 20 clusters (P value < 0.001), including response to stimulus, response to chemical, ion transport, organic substance, oxygen-containing compound, stress, and hormone (Table 2). To adapt to the changing nitrate conditions in the environment, plants have evolved diverse mechanisms to maintain normal growth and development. A sophisticated gene network is thought to regulate the responses to nitrate in plants. However, only the several nitrate regulatory genes mentioned in the Introduction have been characterized thus far using systems biology and reverse genetics approaches. In this article, we performed a forward genetic screen and isolated the mutant Mut75 defective in nitrate signaling. Mapping revealed that the mutation in the gene At3g60320 (designated NRG2) resulted in the Table 1. Known Nitrate-Inducible and Regulatory Genes with Different Levels of Nitrate-Responsive Expression in the Wild Type and nrg2-2 Mutant Gene Fold Change in the Wild Type P Value Fold Change in Mutant P Value NiR NRT2.1 HHO1 UPM1 LBD37 LBD38 NRT1.1 TGA1 TGA4 26.35 11.25 400.49 14.19 11.01 3.28 3.63 4.17 3.55 3.03E-30 5.10 E-4 3.12 E-14 2.48E-55 6.19E-35 1.51 E-11 4.99 E-15 9.70 E-17 6.11 E-18 10.91 3.38 6.18 7.25 6.92 2.23 2.69 3.20 2.65 9.69 2.52 4.66 6.08 5.02 1.63 7.11 6.40 8.65 E-17 E-3 E-11 E-18 E-24 E-5 E-12 E-25 E-10 NRG2 Plays an Essential Role in Nitrate Signaling Table 2. GO Cluster Analysis for Genes Differentially Expressed in the Wild Type and the nrg2 Mutant after Nitrate Treatment GO Term P Value Response to nitrogen compound Response to stimulus Response to chemical Anion transport Response to endogenous stimulus Response to organic substance Inorganic anion transport Response to oxygen-containing compound Ion transport Nitrogen compound transport Response to nitrate Nitrate transport Response to stress Response to acid chemical Response to hormone Cellular hormone metabolic process Response to external stimulus Response to other organism Response to external biotic stimulus 5.61E-09 1.19E-07 2.18E-07 1.3 E-06 2.32E-06 3.98E-06 4.43E-06 4.47E-06 7.53E-06 8.29E-06 1.19E-05 1.57E-05 9.35E-05 9.72E-05 2.24E-04 4.29E-04 6.13E-04 7.34E-04 7.34E-04 phenotype. This gene NRG2 belongs to a gene family with 15 members in Arabidopsis (Supplemental Figure 3). Each member contains DUF632 and DUF630 domains whose functions are still unknown, as none of the proteins in this family have been characterized thus far. Our results show that the induction of nitrate-responsive genes in nrg2 mutants is inhibited when plants are treated with nitrate in the presence of ammonium (Figure 3A), indicating that NRG2 is a nitrate-regulatory gene. Remarkably, this phenotype was not restored after nitrogen starvation, which is different from nrt1.1 mutants (Wang et al., 2009). Although nrt1.1 mutants have been studied for more than 10 years, the inhibition of nitrate induction in the mutants had not been found until it was tested in the presence of ammonium (Tsay et al., 1993; Ho et al., 2009; Wang et al., 2009). It has also been reported that the expression of nitrate-induced genes in the presence of ammonium was inhibited in cipk8 mutants but enhanced in cipk23 mutants compared with the wild type after nitrate treatment (Ho et al., 2009; Hu et al., 2009). Overexpression lines of SPL9 have been monitored as well in the presence of ammonium, and the induction of the nitrateresponsive genes was increased after nitrate treatment (Krouk et al., 2010b). Nevertheless, it has not been tested if this phenotype can be recovered after nitrogen depletion for these mutants. On the contrary, nlp7, tga1/tga4, and lbd37/38/39 mutants have been analyzed after nitrogen starvation and the results showed inhibited induction of nitrate-responsive genes in nlp7 and tga1/tga4 mutants but higher induction in lbd37/38/39 mutants after nitrate treatments (Castaings et al., 2009; Rubin et al., 2009; Alvarez et al., 2014). However, the expression of nitrate-induced genes in these mutants has not been investigated without nitrogen starvation to date. The mutant nlp7-4 showed weaker YFP fluorescence in roots when grown on ammonium nitrate medium and reduced induction of the nitrate-responsive genes after nitrate treatments than in the wild type (Figures 9A to 499 9E), suggesting that NLP7 modulates the nitrate signaling in the presence of ammonium. Our results, combined with previous studies, reveal that some genes function as nitrate-regulatory players in an ammonium-dependent manner, while some play important roles in nitrate signaling regardless of ammonium. Therefore, we propose that nitrate regulators may work in at least two different ways: (1) regulating nitrate responses in the presence of ammonium, such as NRT1.1, and (2) functioning as nitrate regulators regardless of ammonium, as represented by NRG2 and NLP7. A third way may exist that modulates nitrate response only in the absence of ammonium. The different signaling mechanisms under conditions with and without ammonium reflect the complexity with which plants adapt to the changing environments. To understand the physiological effects caused by the mutation in NRG2, the nitrate accumulation in nrg2 mutants was tested. Our results showed that the nitrate content in seedlings was significantly lower than that in the wild type. This defect may result from reduced uptake and/or increased reduction and assimilation. Further analysis by determining nitrate content in both leaves and roots revealed lower nitrate levels in roots, indicating that NRG2 is involved in regulating nitrate accumulation in roots. Previous studies have shown that several characterized nitrate regulatory genes play important roles in plant nitrate homeostasis. In nrt1.1 mutant seedlings, the nitrate concentration is lower than in wild-type plants (Wang et al., 2009). On the contrary, the nitrate content in nlp7 mutants was found to be higher than in the wild type, which might result from the decreased nitrate reduction and assimilation (Castaings et al., 2009). In addition, LBD37, 38, and 39 overexpression lines displayed lower nitrate content and Table 3. Genes Involved in a Nitrogen-Related Cluster Regulated by Both NRG2 and NRT1.1 AGI Description AT5G47330 AT5G46050 AT5G11570 AT2G02990 AT1G33440 AT1G30840 AT4G19680 AT5G41800 AT1G14780 At5g47330; AT5G47330; ortholog Protein NRT1/PTR FAMILY 5.2; NPF5.2; ortholog Protein NRT1/PTR FAMILY 1.3; NPF1.3; ortholog Ribonuclease 1; RNS1; ortholog Protein NRT1/PTR FAMILY 4.4; NPF4.4; ortholog Probable purine permease 4; PUP4; ortholog Fe2+ transport protein 2; IRT2; ortholog Probable GABA transporter 2; At5g41800; ortholog MACPF domain-containing protein At1g14780; At1g14780; ortholog Alpha-dioxygenase 1; DOX1; ortholog UDP-glycosyltransferase 71C1; UGT71C1; ortholog Monothiol glutaredoxin-S13; GRXS13; ortholog Protein NRT1/PTR FAMILY 8.1; NPF8.1; ortholog Peroxidase 69; PER69; ortholog Protein NRT1/PTR FAMILY 5.12; NPF5.12; ortholog Protein GLUTAMINE DUMPER 3; GDU3; ortholog Probable purine permease 18; PUP18; ortholog Berberine bridge enzyme-like protein; AT5G44390; ortholog 1-Aminocyclopropane-1-carboxylate oxidase homolog 12; At5g59540; ortholog Organic cation/carnitine transporter 3; OCT3; ortholog Cytokinin riboside 59-monophosphate phosphoribohydrolase LOG7; LOG7; ortholog AT3G01420 AT2G29750 AT1G03850 AT3G54140 AT5G64100 AT1G72140 AT5G57685 AT1G57990 AT5G44390 AT5G59540 AT1G16390 AT5G06300 500 The Plant Cell Figure 13. NRG2 Plays a Key Role in Nitrate Regulation. (A) NRG2 regulates nitrate response in the presence of ammonium. Under the conditions with NH4+, NRG2 regulates the expression of NRT1.1 and NRT1.1 modulates the expression of CIPK8 and CIPK23. CIPK23 negatively affects the expression of nitrate-responsive genes. The proteins NRT1.1 and CIPK23 interact with each other, and NRT1.1 is phosphorylated by CIPK23 under low-nitrate conditions to maintain its high affinity for nitrate. NLP7 is a positive regulator involved in the nitrate signaling. NRG2 and NLP7 work in different pathways in nitrate signaling whereas both proteins can interact with each other. (B) NRG2 regulates nitrate signaling after nitrogen starvation (no ammonium). After experiencing nitrogen starvation, NRG2 can still interact NLP7 and both proteins play key roles in the primary nitrate response. decreased maximal nitrate reductase activity compared with wild-type plants. The defects in nitrate content may be caused by the reduced nitrate transport activity as the expression of several high-affinity nitrate transport genes was strongly decreased (Rubin et al., 2009). Among these characterized nitrate transport and assimilation genes tested, only NRT1.1 exhibited decreased expression in nrg2 mutant roots, and NRT1.8 displayed increased expression in mutant leaves. NRT1.1 has been characterized as a dual affinity nitrate transporter involved in absorbing nitrate from the environment (Wang et al., 1998; Liu et al., 1999; Liu and Tsay, 2003). Thus, the lower nitrate content phenotype in nrg2 mutants may be caused, at least partially, by the reduced expression of NRT1.1. NRT1.8 has been identified to be a low-affinity nitrate transporter with a function in unloading nitrate from xylem. The higher expression of NRT1.8 in mutant leaves may lead to relatively more nitrate transport into leaves despite the relatively lower nitrate absorption from the medium, resulting in decreased nitrate levels in roots but similar levels in leaves compared with the wild type. Taken together, these data suggest that NRG2 is involved in nitrate accumulation in plants and the altered nitrate accumulation in the mutants may result from modulated expression of NRT1.1 and NRT1.8. It is also possible that some other uncharacterized nitrate transporters contribute to the modified nitrate concentration in the mutants. NRT1.1 plays an essential role in nitrate regulation through its functions in dual-affinity nitrate transport, nitrate sensing, and auxin transport. Nevertheless, how it is regulated, i.e., what genes can modulate the expression of NRT1.1, remains to be characterized. Our molecular and genetic data demonstrated that NRG2 can regulate the expression of NRT1.1 and both genes may work in the same pathway of nitrate signaling. This finding is of great importance for further understanding of the regulation of NRT1.1 and adds a key component into the nitrate signaling network. NLP7 acts as a master regulator in response to nitrate (Castaings et al., 2009; Konishi and Yanagisawa, 2013; Marchive et al., 2013). As a transcription factor, NLP7 can bind the promoter of many genes involved in nitrate signaling and assimilation, modulate the expression of nitrate responsive genes, and regulate nitrogen assimilation genes (Konishi and Yanagisawa, 2013). Whether NLP7 functions with other proteins by interacting or acts solely in the nitrate regulation is still unclear. Our molecular and genetic analysis showed that NRG2 and NLP7 have some nonoverlapping functions as the phenotype of the double mutant is more severe than that of either single mutant. However, both proteins can physically interact in vitro and in vivo as revealed by yeast two-hybrid and BiFC assays, indicating that these proteins likely converge on part of the nitrate signaling pathways as well as functioning independently. In addition, the nuclear retention of NLP7 in response to nitrate is not affected by the mutation in NRG2. These results further strengthen our understanding of nitrate signaling mechanisms. Our comparative RNA-seq analysis of the roots in response to nitrate showed that many genes involved in nitrogen-related clusters, including nitrate transport and response to nitrate, were differentially expressed in the nrg2 mutant, providing further evidence that NRG2 plays an important role in nitrate signaling. Molecular and genetic evidence indicates that NRG2 and NRT1.1 works in the same pathway in nitrate regulation. This would lead us to predict that both genes may regulate some common nitraterelated genes. Indeed, the transcriptomic analysis revealed that a group of genes involved in a nitrogen compound transport cluster were modulated by NRG2 and NRT1.1 coordinately. No common group of genes involved in nitrogen-related clusters was found to be regulated by NRG2 and NLP7, in accordance with the conclusion that both genes function independently in nitrate signaling. NRG2 Plays an Essential Role in Nitrate Signaling Taken together, the regulation of NRT1.1 by NRG2 and the physical interaction of NRG2 and NLP7 highlight the importance of NRG2 as a key player in the nitrate regulatory network. Thus, we propose the working model shown in Figure 13. In the presence of ammonium, NRG2 regulates the expression of NRT1.1, while NRT1.1 modulates the expression of other downstream genes including CIPK8 and CIPK23. NRG2 and NLP7 both act as positive regulators of nitrate assimilatory genes with some independent functions, and they physically interact, suggesting they converge in part of the nitrate signaling pathway. After nitrogen starvation (no ammonium), NRG2 and NLP7 appear to function in a similar manner, acting as positive regulators with some independent functions while physically interacting. The relationship between NRG2 and other known regulatory players remains to be investigated. In the meantime, NRG2 is the first member of a 15member Arabidopsis gene family (Supplemental Figure 3) to be characterized. What roles other members may play in nitrate signaling and what functions the two DUF domains shared by each member carry out are interesting questions for future work. Using the amino acid sequence of NRG2 as a query to search different species revealed that this family exists broadly in plants from moss to crops (rice, maize, soybean, etc.) and trees (apple, peach, poplar, etc.) (www.greenphyl.org/cgi-bin/blast.cgi), but no homologs were found in microbes or animals (http://blast.ncbi. nlm.nih.gov/Blast.cgi), indicating that this family exists specifically in plants. The characterization of the NRG2 opens a door to reveal the roles of these family members. 501 microscope (Nikon Eclipse Ti-S). The fluorescence of roots was quantified using ImageJ. qPCR Analysis RNA samples were prepared using a total RNA miniprep kit (CWBIO). Real-time PCR was performed using the reagent kit ABI7500 Fast (Applied Biosystems). Template cDNA samples were prepared using the RevertAid first-strand synthesis system kit (Thermo Scientific) with 1 mg of total RNA in a reaction volume of 20 mL. The cDNA synthesis reaction mixture was diluted 20-fold before being used for qPCR. The FastStart Universal SYBR Green Master Q-PCR kit (Roche Diagnostics) was used in the qPCR reaction following the instructions provided by the manufacturer. TUB2 (At5g62690) was used as the internal reference gene. Expression Analysis by Promoter-GUS Assay The 2951-bp promoter fragment located immediately upstream of the NRG2 start codon was cloned in front of the GUS gene in the binary vector pMDC162 (Invitrogen). Transgenic Arabidopsis (Col-0) plants expressing the GUS gene were obtained and GUS activity in different organs was detected as described (Dai et al., 2014). For section observation, roots and leaves of the transgenic plants were fixed and embedded in paraffin (Sigma-Aldrich). Sections were cut at 8 mm using a microtome (Leica RM2235) and mounted on glass slides. Ruthenium red (100 mg/L) solution was added onto the sectioned samples on slides for 1 min and then the slides were observed and photographed with a microscope (Nikon Eclipse Ni) equipped with a camera (Nikon Digital Sight DS-Qi1Mc). Subcellular Localization Test METHODS Plant Materials The wild-type Arabidopsis thaliana ecotype used in this study is Col-0. The mutant lines chl1-13 (original name Mut21) (Wang et al., 2009), cipk8-1 (Hu et al., 2009), and cipk23-3 (Ho et al., 2009) were described previously. The full-length cDNA of NRG2 was introduced in frame with the GFP reporter gene in the binary vector pMDC43 (Invitrogen) to generate a fusion protein with GFP at the N-terminal position. The construct was transformed into Arabidopsis (Col-0) plants as described previously (Feng et al., 2008). The images were captured using confocal microscope (Leica TCS SP5II). Nitrate Assay Mutagenesis and Mutant Screen Homologous backcrossed transgenic seeds containing the NRP-YFP construct were treated with ethyl methanesulfonate (Wang et al., 2009), and M2 seedlings were screened on nitrate medium (initial medium with 10 mM KNO3) based on the previous report (Wang et al., 2009). Mutants were selfed and retested. Confirmed mutants were backcrossed to the transgenic wild type twice and homozygous lines were identified and analyzed. Growth and Treatment Conditions Plants used for qPCR analysis of the gene expression induced by nitrate treatment were grown in aseptic hydroponics (initial medium with 2.5 mM ammonium succinate) as described (Wang et al., 2007) for 7 d and then treated with 10 mM KNO3 or KCl as a control for 2 h followed by the roots being collected. For nitrate treatment on plants after nitrate starvation, seedlings were grown in aseptic hydroponics for 6 d and then transferred to the same fresh medium except without ammonium succinate to grow for 24 h. The roots of the seedlings treated with 10 mM KNO3 or KCl for 2 h were harvested separately for RNA extraction. For testing the YFP fluorescence of transgenic plants harboring NRP-YFP construct in response to nitrate, seedlings were grown on plates with either nitrate medium or ammonium nitrate medium (initial medium with 10 mM NH4NO3) for 4 d followed by observation under a fluorescence Nitrate was measured using the salicylic acid method (Cataldo et al., 1975; Vendrell and Zupancic, 1990). Briefly, weighed samples (;0.1 g) in a 1.5-mL tube were frozen by liquid nitrogen and milled into powder using a RETCH MM400.Then,1 mL of deionized water was added into the tube followed by boiling at 100°C for 20 min. The samples were centrifuged at 15,871g for 10 min, and 0.1 mL supernatant was transferred into a 12-mL tube. Next, 0.4 mL of salicylic acid-sulfate acid (5 g salicylic acid in 100 mL sulfate acid) was added and the sample was mixed well. The reactions were incubated at room temperature for 20 min, and 9.5 mL of 8% NaOH solution was added. After cooling the tube to room temperature, the OD410 value was determined. For the control, 0.1 mL of deionized water was used instead of 0.1 mL supernatant. The nitrate content was calculated using the following equation: Y = C$V/W (Y, nitrate content; C, nitrate concentration calculated with OD410 into regression equation; V, total volume of extracted sample; W, weight of sample). Standard curve was made with KNO3 at concentrations between 10 to 120 mg/L and regression equation was obtained based on standard curve. Yeast Two-Hybrid Assays A full-length fragment of cDNA for NRG2 was ligated into the pGBKT7 vector (Clontech), and full-length cDNA fragments of tested genes were introduced into pGADT7 AD vector (Clontech). The two-hybrid interaction 502 The Plant Cell assays were performed according to the instruction provided by the manufacturer (Clontech). Supplemental Figure 3. Sequence alignment of 15-member Arabidopsis gene family containing NRG2. BiFC Analysis Supplemental Figure 4. Morphological phenotype of the nrg2 mutant under different concentrations of nitrate. Transient BiFC assays in Nicotiana benthamiana were performed on the leaves as described (Walter et al., 2004). Briefly, full-length cDNAs of NRG2 and NLP7 were cloned into the binary vectors pSPYNE-35S and pSPYCE35S containing the N- and C-terminal fragments of YFP (YFPN and YFPC), respectively. N. benthamiana plants were grown on perlite watered with ammonium nitrate solution for 5 weeks. For nitrogen starvation treatment, plants were grown on perlite watered with ammonium nitrate solution for 3 weeks and then watered with the initial medium without nitrogen for another 2 weeks. The two constructs NRG2-YFPN and NLP7-YFPC were cotransfected into the fourth to fifth leaves and the empty vectors YFPC and YFPN in combination with NRG2-YFPN and NLP7-YFPC, respectively, were used as negative controls. Transfected plants were watered with ammonium nitrate solution for 3 to 4 d followed by harvesting the infiltrated leaves for observation using confocal microscope (Leica TCS SP5II). RNA-Seq Data Analysis The seeds of the wild type, nrg2-2, chl1-13, and nlp7-4 were grown on ammonium succinate for 7 d and then treated with either 10 mM KNO3 or KCl (as a control) for 2 h. Total RNA of the roots was prepared using a RNA miniprep kit, and the concentrations were measured using a NanoDrop 2000 spectrophotometer (Thermo). The libraries were constructed and then sequenced using a HiSeq 2500 (Illumina), which generated ;21 million read pairs per sample (Annoroad). Raw reads containing adapter, poly-N, and low-quality reads were filtered and the effective data were mapped with the Arabidopsis TAIR 10.2 reference genome using TopHat (version 2.0.12). After excluding the rRNA or tRNA, we estimated the abundance of the transcripts using RPKM (reads per kilobases per million reads) (Wagner et al., 2012). The P values were adjusted using the Benjamini and Hochberg method (Benjamini and Hochberg, 1995). Corrected P value < 0.05 and fold change more than 2 were set as the threshold for significant difference in expression. GO annotations of the data provided by our RNA-seq analysis were performed using Panther (www.pantherdb.org/pathway/; Mi et al., 2013). Accession Numbers Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: NiR (AT2G15620), NIA1 (AT1G77760), NRT2.1 (AT1G08090), CIPK8 (AT4G24400), CIPK23 (AT1G30270), LBD37 (AT5G67420), LBD38 (AT3G49940), LBD39 (AT4G37540), NRT1.1 (AT1G12110), NLP7 (AT4G24020), NRT1.2 (AT1G69850), NRT1.4 (AT2G26690), NRT1.5 (AT1G32450), NRT1.6 (AT1G27080), NRT1.7 (AT1G69870), NRT1.8 (AT4G21680), NRT1.9 (AT1G18880), NRT1.11 (AT1G52190), NRT1.12 (AT3G16180), NRT2.6 (AT3G45060), NRT2.7 (AT5G14570), GLN1.1 (AT5G37600), and GLN1.3 (AT3G17820). The RNA-seq data discussed in this article have been deposited in the National Center for Biotechnology Information database (www.ncbi.nlm.nih.gov/sra; accession number SRP067979). Supplemental Figure 5. The expression of additional nitrate transport genes in roots is not affected in nrg2 mutants. Supplemental Figure 6. The expression of additional nitrate transport genes in leaves is not affected by disruption of NRG2. Supplemental Figure 7. The expression of nitrate reduction genes in nrg2 mutants is not altered compared with that in the wild type. Supplemental Figure 8. The expression of some characterized nitrate regulatory genes tested is not altered in nrg2 mutants compared with that in the wild type. Supplemental Figure 9. The expression of NRG2 is not changed in characterized nitrate regulatory gene mutants. Supplemental Figure 10. The expression of NRT1.1 in wild-type, nrg2 mutant, and NRT1.1/nrg2-2 lines. Supplemental Figure 11. BiFC assays revealed direct interaction between NRG2 and NLP7 when plants were treated with nitrate after nitrogen starvation. Supplemental Data Set 1. Read numbers of the 24 samples. Supplemental Data Set 2. Genes whose expression changed more than 2-fold in the wild type and nrg2 mutant after nitrate treatment. Supplemental Data Set 3. Genes with differentially induced expression levels in the mutant compared with the wild type after nitrate treatment. Supplemental Data Set 4. Four nitrogen-related clusters for genes differentially expressed in the wild type and nrg2 mutant after nitrate induction. Supplemental Data Set 5. Genes that are differentially expressed in the mutants compared with the wild type and commonly regulated by NRG2, NRT1.1, and NLP7. Supplemental Data Set 6. Genes regulated by both NRG2 and NRT1.1. Supplemental Data Set 7. Genes regulated by both NRG2 and NLP7. Supplemental Data Set 8. Primers used in this article. ACKNOWLEDGMENTS We thank Yi-Fang Tsay for the cipk8-1 and cipk23-3 seeds, Lei Ge and Gang Li for discussion of unpublished data, and Xiansheng Zhang, Chengchao Zheng, and Daolin Fu for comments on the manuscript. This research was supported by an NSFC grant (31170230) and the Taishan Scholar Foundation to Y.W. AUTHOR CONTRIBUTIONS Supplemental Data Supplemental Figure 1. 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The Arabidopsis NRG2 Protein Mediates Nitrate Signaling and Interacts with and Regulates Key Nitrate Regulators Na Xu, Rongchen Wang, Lufei Zhao, Chengfei Zhang, Zehui Li, Zhao Lei, Fei Liu, Peizhu Guan, Zhaohui Chu, Nigel M. Crawford and Yong Wang Plant Cell 2016;28;485-504; originally published online January 7, 2016; DOI 10.1105/tpc.15.00567 This information is current as of August 3, 2017 Supplemental Data /content/suppl/2016/01/07/tpc.15.00567.DC1.html References This article cites 54 articles, 19 of which can be accessed free at: /content/28/2/485.full.html#ref-list-1 Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X eTOCs Sign up for eTOCs at: http://www.plantcell.org/cgi/alerts/ctmain CiteTrack Alerts Sign up for CiteTrack Alerts at: http://www.plantcell.org/cgi/alerts/ctmain Subscription Information Subscription Information for The Plant Cell and Plant Physiology is available at: http://www.aspb.org/publications/subscriptions.cfm © American Society of Plant Biologists ADVANCING THE SCIENCE OF PLANT BIOLOGY