The Arabidopsis NRG2 Protein Mediates Nitrate Signaling andInteracts with and Regulates Key Nitrate RegulatorsOPEN

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, ChinabNational Key Laboratory of Crop Genetic Improvement, College of Life Science and Technology, Huazhong Agricultural University,Wuhan, Hubei 430070, Chinac Section of Cell and Developmental Biology, Division of Biological Sciences, University of California at San Diego, La Jolla, California92093-0116d State Key Laboratory of Crop Biology, College of Agronomic Sciences, Shandong Agricultural University, Tai’an, Shandong 271018,China

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 signalingin Arabidopsis thaliana. A mutation in NRG2 disrupted the induction of nitrate-responsive genes after nitrate treatment by anammonium-independent mechanism. The nitrate content in roots was lower in the mutants than in the wild type, which mayhave resulted from reduced expression of NRT1.1 (also called NPF6.3, encoding a nitrate transporter/receptor) andupregulation of NRT1.8 (also called NPF7.2, encoding a xylem nitrate transporter). Genetic and molecular data suggest thatNRG2 functions upstream of NRT1.1 in nitrate signaling. Furthermore, NRG2 directly interacts with the nitrate regulator NLP7in the nucleus, but nuclear retention of NLP7 in response to nitrate is not dependent on NRG2. Transcriptomic analysisrevealed that genes involved in four nitrogen-related clusters including nitrate transport and response to nitrate weredifferentially 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 andNLP7. Thus, NRG2 plays a key role in nitrate regulation in part through modulating NRT1.1 expression and may function withNLP7 via their physical interaction.

INTRODUCTION

Nitrogen is an important macronutrient required by plants fornormal growth and development. Most plants grown under aer-obic conditions absorb nitrogen mainly in the form of nitrate.Nitrate serves not only as a nutrient, but also as an importantsignaling molecule. Transcriptome analyses have revealed thatthe expression of more than 1000 genes is altered within 3 h ofnitrate treatment. Among these genes, those involved in nitratetransport and assimilation, such as several members of theNITRATE TRANSPORT (NRT) gene families and the genes fornitrateandnitrite reductase (NIAandNiR, respectively), arequicklyinduced (Bi et al., 2007;Wangetal., 2007). Inaddition, somegenesrequired for controlling carbon metabolism and for providingchemical energyused in reductionandassimilationare inducedaswell (Price et al., 2004; Scheible et al., 2004; Wang et al., 2004,2007; Fritz et al., 2006; Gutiérrez et al., 2007). Nitrate signaling also

influences root growth, development and architecture, seeddormancy, and leaf expansion (Walch-Liu et al., 2000, 2006;Forde, 2002; Alboresi et al., 2005; Bi et al., 2007; Forde andWalch-Liu, 2009).However, our understanding of the regulatorymechanisms and

genes involved in nitrate signaling in plants is incomplete. In thelast few years, several nitrate regulatory genes functioning in theprimary nitrate response have been characterized. One key reg-ulator is NRT1.1 (also called NPF6.3 and CHL1), which functionsnot only as a dual-affinity nitrate transporter, but also as a nitratesensor (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;Hoetal., 2009;Léranetal., 2014;Muñosetal., 2004).Recentcrystal structure studiesonNRT1.1provide further insights into itstransport mechanisms (Parker and Newstead, 2014; Sun et al.,2014; Tsay, 2014); however, little is known about how theNRT1.1gene itself is regulated. Other nitrate regulators include twomembers of the CBL-interacting protein kinase family, CIPK8 andCIPK23, which are themselves regulated by NRT1.1 and are in-volved in the primary nitrate response with CIPK8 acting asa positive regulator and CIPK23 as a negative regulator (Ho et al.,2009; Hu et al., 2009; Krouk et al., 2010a). In addition, CIPK23 caninteract with and phosphorylate NRT1.1 at amino acid Thr-101 tomaintain 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, andNAC4) have

1 These authors contributed equally to this work.2 Current address: School of Biological Science, Jining Medical Univer-sity, Rizhao, Shandong 276826, China.3 Address correspondence to wangyong@sdau.edu.cn.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Yong Wang (wangyong@sdau.edu.cn).OPENArticles can be viewed online without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.15.00567

The Plant Cell, Vol. 28: 485–504, February 2016, www.plantcell.org ã 2016 American Society of Plant Biologists. All rights reserved.

been identified to benitrate regulatory genes (ZhangandForde,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 ArabidopsisthalianaMADSbox transcription factor ANR1was the first to becharacterized to regulate lateral root growth in response tonitrate treatment (Zhang and Forde, 1998; Gan et al., 2005,2012). Reverse genetics has revealed that three members ofLATERAL ORGAN BOUNDARY DOMAIN (LBD) transcriptionfactor family LBD37/38/39 are negative regulators for nitrate-responsive genes and themutants showa constitutive nitrogenstarvation response (Rubin et al., 2009). Arabidopsis NIN-LIKE PROTEIN7 (NLP7) has been found to function as a masterregulator in the early nitrate response. Disruption of NLP7results in a nitrogen-starved phenotype and impaired nitratesignaling in the mutants (Castaings et al., 2009). The nuclearretention of NLP7 is regulated by nitrate (Marchive et al., 2013).All nine NLPs can bind the nitrate-responsive cis-element NREand activate NRE-dependent and nitrate-responsive geneexpression (Konishi and Yanagisawa, 2013). A suppressionstudy of NLP6 demonstrated that this gene plays an importantrole in nitrate signaling and other NLP members are alsospeculated to have similar function (Konishi and Yanagisawa,2013). Using systems biology, SPL9, TGA1, TGA4, AFB3, andNAC4 have been identified as nitrate regulators involved inearly nitrate response signaling (Krouk et al., 2010b; Vidal et al.,2010, 2013; Alvarez et al., 2014).

A forward genetic screen for nitrate regulatory mutants wasdeveloped 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 showstrong YFP fluorescence in the presence of nitrate. Two sets ofmutants that showed low YFP fluorescence in the presence ofnitrate were isolated and mapped to NRT1.1 and NLP7, re-spectively (Wang et al., 2009). Thus, this NRP-based mutantscreen system can be used to screen for nitrate regulatorymutants, providing an effective forward genetic approach fordiscovering new genes involved in nitrate signaling.

In this study, we performed a forward genetic screen usingthe NRP-YFP plants and isolated a mutant, Mut75. The mu-tation was mapped to the gene At3g60320, designated asNITRATE REGULATORY GENE2 (NRG2; as NRG1 has beenused for NRT1.1; Wang et al., 2009), and further character-ization showed that it plays a key role in nitrate signaling.Genetic andmolecular analyses revealed thatNRG2modulatesthe expression of NRT1.1 and functions upstream of NRT1.1.Moreover, biochemical and in planta experiments showed thatNRG2 can directly interact with NLP7. Our findings supportamodel in whichNRG2 regulates the expression ofNRT1.1 anddirectly interacts with NLP7 in nitrate signaling transduction.These results not only establish the key role of NRG2 in tran-scriptional control, but also demonstrate a direct involvementof NRG2 in central nitrate signaling and offer insights into themechanism of nitrate regulation in plants. In addition, ourfindings provide the first insights into the functions of anuncharacterized, 15-member gene family in Arabidopsis, towhich NRG2 belongs.

RESULTS

Defects in Nitrate Signaling in Mut75 Are Caused bya Mutation in At3g60320

To identify regulators in nitrate signaling, we performed a forwardgenetic screen. The seeds from homozygous transgenic plantscontaining the nitrate-responsive promoter NRP fused to a YFPmarker (Wang et al., 2009)were treatedwith ethylmethanesulfonate,and M2 population seedlings grown on nitrate medium werechecked for YFP fluorescence in roots under a fluorescencemicroscope. The transgenic wild-type seedling roots showedstrongYFPsignal in thepresenceof nitrate, as theyare responsiveto nitrate (Figure 1Aa). One mutant, Mut75, exhibiting much lowerYFP fluorescence than thewild type in thepresenceof nitrate,wasisolated (Figures 1Ab and 1B). The location of the mutation inMut75 was narrowed down to the end of chromosome 3 ina 110-kb region (Figure 1C) by a map-based cloning strategy.Unexpectedly, the sequencing results showed two pointmutations in this region with one (G to A) in At3g60320 thatconverted Trp at position 638 to a stop codon (Figure 1C) andanother one (C toT) inAt3g60240 that changedGlnatposition332to a stop codon (Supplemental Figure 1A). At3g60320 is anuncharacterized gene, while At3g60240 encodes PROTEINSYNTHESIS INITIATIONFACTOR4G (EIF4G),which is involved invirus resistance (Yoshii et al., 2004; Nicaise et al., 2007).To determine which mutation results in the weak YFP fluo-

rescence phenotype of the mutant, several genetic tests wereperformed. For the gene At3g60320, transforming wild-typecDNA for this gene driven by a 35S promoter into the Mut75mutant restored strong YFP fluorescence in the roots on nitratemedium (Figure 2A), indicating that the gene At3g60320 canrescue the YFP phenotype of Mut75. In addition, two homozy-gous T-DNA insertion mutant lines for this gene were isolatedfrom the ABRC T-DNA population (Alonso et al., 2003). Thetranscript levels of At3g60320 were very low in SALK_014743,which has a T-DNA insertion in the promoter region, and un-detectable inSALK_079096,whichcontainsaT-DNA insertion inthe second exon, when tested by RT-PCR (Figures 2B and 2C).Mut75 was crossed with these two lines. Both F1 plantsexhibited lower YFP fluorescence in roots when grown on nitratemedium (Figure 2D), confirming that the weak YFP phenotypeof Mut75 is caused by the mutation in At3g60320. Therefore,we designated At3g60320 as NRG2, its T-DNA mutantsSALK_014743 as nrg2-1 and SALK_079096 as nrg2-2, andMut75 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 mutantSALK_002002 with the T-DNA inserted in the seventh exon ofEIF4G was identified (Supplemental Figures 1A and 1B) and thencrossed with nrg2-3. The F1 plants grown on nitrate mediumshowed strong YFP fluorescence in the roots (SupplementalFigure 1C), suggesting that the weak YFP phenotype of nrg2-3 isnot caused by the mutation in EIF4G. qPCR results showed thattheexpressionofseveral nitrate-responsivegenes (NIA1,NiR, andNRT2.1) was inducedbynitrate in themutant to a similar level as inthe wild type (Supplemental Figure 1D). These data imply thatEIF4G is not involved in nitrate regulation.

486 The Plant Cell

Figure 1. Identification and Mapping of Mut75.

(A)Nitrate inductionofNRP-YFP inwild type (WT)andMut75 roots.Fluorescenceand light imagesof4-d-oldseedlingsgrownonKCl/KNO3media (a)andonKNO3 (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). Asterisksindicate 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 andnucleotide changes found in Mut75 are also shown.

NRG2 Plays an Essential Role in Nitrate Signaling 487

Taking together, these results indicate that the weak YFPphenotype of Mut75 is caused by the mutation in NRG2, butnot by the mutation in EIF4G. Therefore, we focused on thefunctional 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 innrg2-3 suggests that NRG2 plays an important role in nitratesignaling. To test if NRG2 also regulates endogenous genes, theexpression of the nitrate-responsive genes NIA1, NiR, and NRT2.1was investigated. qPCR results showed that the nitrate induction

of these genes in the roots of bothnrg2-1 and nrg2-2mutantswassignificantly inhibited (Figure 3A; Supplemental Figures 2A and2B). No difference was found for the expression of these genesamong wild type and the mutants when grown on ammoniumsuccinate without any nitrate treatment (Supplemental Figure 2C)or after KCl treatment (Supplemental Figure 2D), while a signifi-cant decrease in the mutants was seen after KNO3 treatment(Supplemental Figure 2E). The inhibited nitrate induction in themutants was also observed when the seedlings were treated witha low concentration of nitrate (Supplemental Figure 2F). Theabove results demonstrate that NRG2 functions in nitratesignaling in plants.

Figure 2. The Mut75 Phenotype Is Caused by the Mutation in NRG2.

(A)Complementation test ofNRG2 inMut75. Fluorescence and light images of 4-d-old seedlings grownonnitratemediawere capturedwith a fluorescencemicroscope.(B) Schematic map of the T-DNA insertion sites in nrg2-1 and nrg2-2mutants. Exons, introns, and untranslated regions are represented by black boxes,lines, andwhiteboxes, respectively. The locationsof theT-DNA insertion in the twonrg2alleles fromSALKare indicatedwith triangles.Blackarrow indicatesthe mutation site in Mut75.(C)RT-PCRanalysisofNRG2mRNA levels in thewild typeand thenrg2mutants. TotalRNA isolated from7-d-oldseedlingsgrownonammoniumnitratewasanalyzed by RT-PCR and a program based on 25 cycles of PCR amplifications was performed to test the expression ofNRG2. TUB2 serves as a control toshow the equal amount of cDNA in each reaction.(D)Root fluorescence of F1 plants from nrg2-3 crossedwith nrg2-1 and nrg2-2, respectively. Fluorescence and light images of 4-d-old seedlings grown onnitrate media were captured with a fluorescence microscope.

488 The Plant Cell

Previous studies have shown that NRT1.1 acts as a nitratesensor and mediates nitrate responses as evidenced by the factthat nrt1.1 mutants (chl1-5 and chl1-13) exhibited decreasednitrate induction of the nitrate-responsive genes (NIA1, NiR, andNRT2.1) (Ho et al., 2009; Wang et al., 2009). However, this phe-notype is dependent on nitrogen pretreatment, as nitrogendeprivation restores the wild-type phenotype in nrt1.1 mutants(Wang et al., 2009; Krouk et al., 2010a). We tested nrg2 mutantsunder both nitrate-replete and nitrogen-deprived conditions andfound that the induction levels for these nitrate-responsive geneswere reduced under both conditions compared with the wild type(Figures 3A and 3B), indicating that NRG2 functions in nitratesignaling regardless of nitrogen starvation. This finding contrastswith that of NRT1.1, whose nitrate regulatory function is lost afternitrogen starvation and shows that NRG2 functions in both typesof nitrogen conditions (nitrogen replete and nitrogen deprived).

To test ifNRG2 is regulated by different nitrogen conditions, weinvestigated its expression levels after nitrate, ammonium, andnitrogen starvation treatments. The results did not show signifi-cant changes in the expression of this gene after these treatments(Supplemental Figure 2G), indicating that the expression ofNRG2is not modulated by nitrate, ammonium, and nitrogen starvation.

NRG2 Is Predominantly Expressed in the Vascular Tissue ofLeaves and Roots, and NRG2 Protein Is Localized inthe Nucleus

The expression profile ofNRG2 in wild-type plants was examinedusing qPCR. Tissues were harvested either from plants grown insoil (leaves, stems, flowers, and siliques) or from plants grown onammonium nitrate medium (seedlings and roots). NRG2 wasexpressed in all tested tissues, with highest levels in leaves androots and lowest levels in flowers and siliques (Figure 4A). Theexpression profile ofNRG2was further analyzed by the promoter-GUS approach. TheGUS staining profile is largely consistent withthe results obtained from qPCR, confirming the expression patternof NRG2 in the tissues tested. Moreover, GUS staining revealed

that NRG2 is predominantly expressed in the vascular bundles ofleaves and roots (Figures 4Ba to 4Bh). In addition, expression ofNRG2was also found in stomata (Figure 4Bi), flowers (Figures 4Bjto 4Bm), and young siliques (Figure 4Bn). In flowers, GUS ex-pression was observed in the pistil (Figure 4Bk), junction of fila-ment and anther (Figure 4Bl), and vascular tissue of sepals andpetals (Figure 4Bm).To more precisely determine the cells that express NRG2, the

GUS staining in vascular bundles was further analyzed. Crosssections of the roots showed GUS expression in the stelar cells,including the pericycle, phloem, and parenchyma cells (Figure 4Ca). Longitudinal and transversal sections of the leaves revealedGUS expression in the bundle sheath, phloem, and parenchymacells of the vascular tissues (Figures 4Cb and 4Cc). This ex-pression profile suggests that NRG2 may function in regulatingnitrate transport in the vasculature.NRG2 contains two uncharacterized functional domains:

DUF630 and DUF632, which are shared by all 15 members in thisfamily of unknown proteins (Supplemental Figure 3). To explorethe subcellular localization of the NRG2, several subcellular lo-calization prediction tools were used to analyze its protein se-quence. NRG2 was predicted to be localized in the nucleus bySubLoc (Chen et al., 2006) andWoLFPSORT (Horton et al., 2007)tools, but in mitochondria by MitoPred (Guda et al., 2004). Todetermine the bona fide localization of the protein, we cloned theNRG2 cDNA and ligated the fragment in frame to be expressedwith the GFP reporter at the N-terminal position (Pro35S:GFP-NRG2). Theconstructwas transformed intoArabidopsiswild-typeplants and the protein was observed in the nucleus in stabletransgenic lines (Figure 4D). Thus,we conclude thatNRG2proteinis targeted to the nucleus.

Nitrate Content in nrg2Mutants Is Lower Than the Wild Typein Roots, but Not in Leaves

We demonstrated that the induction of the nitrate-responsivegenes is inhibited in the nrg2 mutants. We then tested if this

Figure 3. The mutants of NRG2 Are Defective in Response to Nitrate.

(A)Nitrate inductionof endogenousgeneswithoutnitrogenstarvation.Wild-typeandnrg2plantsweregrownonmediumwith2.5mMammoniumsuccinateas the sole nitrogen source for 7 d and then treatedwith 10mMKNO3 or KCl as a control for 2 h. Roots were collected for RNA extraction. The transcripts ofnitrate-responsivegenesweredeterminedbyqPCR.Errorbars represent SDofbiological replicates (n=4).Asterisks indicatesignificantdifferences (P<0.05)compared with the wild type (t test).(B)Nitrate inductionofendogenousgenesafternitrogenstarvation.Plantsweregrown, treated,andanalyzedasdescribed in (A), exceptplantsatday6weretransferred 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 weredetermined by qPCR.

NRG2 Plays an Essential Role in Nitrate Signaling 489

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 ofNRG2 in different organsofArabidopsis byqPCR. Tissueswereharvested either from45-d-oldplants grown insoil (leaves, stems, flowers, and siliques) or from 7-d-old plants grown in NH4NO3 liquid medium (seedlings and roots). Error bars represent SD of biologicalreplicates (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 vascularsystem ([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)GUSstaining forNRG2promoter-driven activity in vascular bundles. Cross section of the roots (a) revealedGUSexpression in the stelar cells, includingpericycle, phloem, and parenchyma cells. Longitudinal section (b) and cross section of the leaves (c) revealed that the NRG2 promoter drives expressionmainly 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 ofNRG2protein. (a)Confocal laser scanningmicroscopy andcorrespondingbright-field imagesof Arabidopsis roots. (b)Highermagnifications of the red squares in (a); red arrows indicate the nucleus. Bars = 50 mm.

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molecular defect results in any phenotype at the morphologicaland physiological levels in the mutants. Under a high nitrateconcentration, the mutant seedlings were slightly smaller anddisplayed later flowering compared with the wild type (SupplementalFigures 4A and 4B). Under low nitrate condition, no obviousphenotype was observed (Supplemental Figure 4C).

Previous studies on several known nitrate regulatory genes(NRT1.1, NLP7, and LBD37/38/39) have shown that the nitratelevels in their respective mutant plants are altered (Castaings et al.,2009;Rubin et al., 2009;Wanget al., 2009).Here,we found that thenitrate accumulation in nrg2 mutant seedlings was significantlylower than in the wild type (Figure 5A). Further investigation

revealed that the nitrate accumulation in roots was significantlylower with each mutant allele than in the wild type (Figure 5B).However, no difference between the mutants and the wild typewas found in leaves (Figure5C).Thesedata indicate that thenitrateaccumulation in roots is defective, while the accumulation ofnitrate in leaves is normal in nrg2 mutants. We further assayedthe nitrate content in whole seedlings treated with variousconcentrations of nitrate (0.25 to 20mM) for 2 h in the presence ofammonium and found that the nitrate accumulation in the mutants(including the chl1-13mutant as a control) was significant lower inall concentrations tested (Figure 5D). We also tested the timecourse 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 nrg2mutant plants were grown on ammonium nitrate medium for 7 d andcollected for nitrate concentration test. Error bars represent SD of biological replicates (n = 4). Asterisks indicate significant differences (P < 0.05) comparedwith the wild type (t test). FW, fresh weight.(D) and (E)Nitrate accumulation in wild type, nrg2, and chl1mutants. Seedlings grown with 2.5mM ammonium succinate for 7 d were treated with variousconcentrations of KNO3 for 2 h (D) or treated with 5 mMKNO3 for different times in the presence of ammonium succinate (E) and then collected for nitrateconcentration test. The chl1-13mutant was used as a control. Asterisks indicate significant differences (P < 0.05) between the wild type and twomutants.(F) and (G)Relative expression ofNRT1.1 (F) andNRT1.8 (G).Wild-type and nrg2mutant plantswere grown on ammonium nitratemedium for 7 d, and thenroots and shoots were collected separately for RNA extraction. The transcription levels of NRT1.1 and NRT1.8 were determined by qPCR. Error barsrepresent 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 491

5 mM KNO3, and the results showed that the nitrate uptake in themutants was significantly lower than in the wild type at all timepoints tested (Figure 5E). These findings suggest that nitrateuptake is affected in nrg2 mutants.

To investigate the mechanisms for the lower nitrate accumu-lation in mutant roots, the expression of several known nitratetransport genes was studied. Among the 13 transport genestested, only the expressionofNRT1.1wassignificantly decreasedunder ammonium nitrate conditions (Figure 5F), while no changewas found for the other 12 tested genes (Supplemental Figure 5)in the mutant roots. In leaves, only the transcript levels ofNRT1.8 were significantly increased (Figure 5G), and there wasno significant change in the expression of the other genes(Supplemental Figure 6) in the mutants. Previous studies haveshown that NRT1.1 functions as a dual-affinity nitrate transporterinvolved in transporting nitrate from the environment into roots(Tsay et al., 1993;Wang et al., 1998; Liu andTsay, 2003). Thus, thedecreased nitrate content in mutant seedlings may be caused bythe decreased expression of NRT1.1. NRT1.8 functions in re-moving nitrate from xylem vessels, as the functional disruption ofNRT1.8 increasednitrateconcentration inxylemsap (Li et al., 2010).

Thus, it is possible that the increased expression of NRT1.8 maydirect more nitrate to be unloaded from xylem vessels resulting insimilar nitrate levels in the mutant leaves to those in wild-typeleaves. The expression of several key nitrate assimilatory genes(NIA1, NIA2, NiR, GLN1.1, and GLN1.3) was also detected byqPCR, and no significant difference was found between the wildtype and nrg2mutants (Supplemental Figure 7). Therefore, theseresults imply that the lower nitrate content in mutant roots may becorrelatedwith the reduced expression ofNRT1.1 in roots and theincreased transcripts of NRT1.8 in leaves.

NRG2 Regulates the Expression and Works Upstreamof NRT1.1

To understand the relationship of NRG2 and the characterizednitrate regulators, we first investigated the expression levels ofseveral known nitrate regulators in the nrg2 mutants under dif-ferent nitrogen conditions. The results showed that, among theinvestigated known regulatory genes, the expression of NRT1.1in the nrg2 mutants was significantly decreased, to <40% ofthe expression in wild-type plants under potassium nitrate or

Figure 6. The Expression of NRT1.1 in the nrg2 Mutants Was Reduced.

(A)RelativeexpressionofNRT1.1 innrg2mutants.Wild-typeandnrg2mutantplantsweregrownonmediawithKNO3orNH4NO3 for7d, andwholeseedlingswere 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 wholeseedlings were collected for gene expression detection. Error bars represent SD of biological replicates (n = 4).(C)Nitrate induction ofNRT1.1 in thewild type and the nrg2mutants. Plantswere grown onmediumwith 2.5mMammonium succinate as the sole nitrogensource for 7 d and then treatedwith 10mMKNO3 or KCl as a control for 2 h. Rootswere collected for RNA extraction. The relative expression ofNRT1.1wasdetermined by qPCR. Error bars represent SD of biological replicates (n= 4). Asterisks indicate significant differences (P < 0.05) comparedwith thewild type(t test).

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ammonium nitrate conditions (Figure 6A). The expression ofother known nitrate regulatory genes tested was not changed(Supplemental Figure 8). This indicates that the expression ofNRT1.1 is regulated by NRG2.

To test if NRG2 is regulated by known nitrate regulators, wemeasured NRG2 transcript levels in the mutants of several

identified regulatory genes (NRT1.1, NLP7, CIPK8, andCIPK23) in nitrate or ammonium nitrate media. No changewas found for the expression of NRG2 in these mutants(Supplemental Figure 9), including innrt1.1mutants (Figure 6B).These results imply that NRG2 may not be regulated by thesefour genes.

Figure 7. NRG2 and NRT1.1 Work in the Same Nitrate Signaling Pathway.

(A)Root fluorescencephenotypesofwild-type,nrg2-3, chl1-13, and chl1-13 nrg2-3plants onKNO3medium. Theplantswere grownonKNO3medium for 4d. 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)Rootfluorescencephenotypesofwild-type,nrg2-3,chl1-13, andchl1-13nrg2-3plants onNH4NO3medium.TheplantsweregrownonNH4NO3mediumfor 4 d. Fluorescence and light images were captured with a fluorescence microscope to visualize YFP expression.(D)Quantification of root fluorescenceofwild-type,nrg2-3, chl1-13, and chl1-13 nrg2-3plants onNH4NO3medium. The seedlingsweregrownon the sameNH4NO3 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 inFigure 3A. The transcripts of nitrate-responsive genes in roots were measured by qPCR. Error bars represent SD of biological replicates (n = 4). Differentletters indicate statistically different means (P < 0.05, t test).

NRG2 Plays an Essential Role in Nitrate Signaling 493

To study further the effects of NRG2 on NRT1.1, we examinedthe nitrate induction of NRT1.1 in the wild type and the nrg2mutants. The results showed a significant decrease in the nitrateinduction levels in the mutants (Figure 6C), indicating that NRG2affects the nitrate induction of NRT1.1. We also tested the ex-pression of NRT1.1 in the absence of nitrate and found that theexpression of NRT1.1 was significantly lower in the mutantscompared with that in the wild type (Supplemental Figure 10A).When seedlings grown on NH4NO3 medium were subjected tonitrogenstarvation, theexpressionofNRT1.1 increasedduring thefirst 24 h in both the wild type and the mutants and no significantdifferences in expression levels were found for the time pointstested between wild type and the nrg2 mutants (SupplementalFigure 10B).

To understand better the relationship of NRG2 and NRT1.1,a double mutant of the two genes was obtained by crossing thesingle mutants of each gene: nrg2-3 and chl1-13 isolated by ourmutant screens. The YFP signal from the NRP-YFP transgene inroots of the double mutant seedlings grown on nitrate medium(with no ammonium) was detected and found to be much weakerthan in the wild type and similar to nrg2-3 while weaker thanchl1-13 (Figure 7A). Quantifying the root fluorescence signalconfirmed theweaker YFP signal in doublemutant than in thewildtype 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 doublemutant and mildly lower than in the wild type. We also tested theYFP levels in roots of the single and double mutants grown onammoniumnitratemediumto investigate the functionof thegenesin the presence of ammonium. The observation and fluorescencequantification data showed that the YFP levels in the doublemutant were similar to those of chl1-13while lower than of nrg2-3(Figures 7C and 7D). Interestingly, the chl1-13 exhibited muchlower signal than the wild type, confirming thatNRT1.1 function inthe nitrate signaling pathway is ammonium dependent.

To provide further molecular evidence, we inspected the ex-pression of nitrate-responsive genes in the wild type and these

single and double mutants. The qPCR results showed that theexpression levels of the nitrate-responsive genes in the doublemutant chl1-13 nrg2-3 were similar to those in single mutantchl1-13 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 betweenNRT1.1 and NRG2. The nitrate content in roots of nrg2-2 was re-covered to the wild-type level when NRT1.1was overexpressed inthemutant (Figure8A;Supplemental Figure 10C).Wealsodetectedtheexpression levelsof thenitrate-responsivegenesNIA1,NiR, andNRT2.1 and found that nitrate induction of these genes was re-covered 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 NRG2functions upstream of NRT1.1.

Genetic and Molecular Analysis Reveals that NRG2 andNLP7 Have Nonoverlapping Functions in Nitrate Regulation

NLP7 is an important nitrate regulator in nitrate signaling(Castaings et al., 2009; Konishi and Yanagisawa, 2013; Marchiveet al., 2013). To investigate the relationship between NRG2 andNLP7 in the process of regulating nitrate response, a doublemutant of the twogeneswasgeneratedbycrossing the respectivesingle mutants nrg2-3 and nlp7-4 isolated by our mutant screensystem.Themutantnlp7-4harborsamutation (C toT) inNLP7 thatconverts Gln at the position 62 to a stop codon, resulting in lowerYFP fluorescence in roots when grown on nitrate-containingmedia (Figures 9A to 9D). In the presence of nitrate without am-monium, both single mutants showed much lower YFP fluores-cence in roots than the wild type, with 23% of the wild type fornlp7-4and35%of thewild type fornrg2-3 in termsoffluorescenceintensity, respectively. Interestingly, the double mutant plantsexhibitedeven lowerYFPsignal than the individual singlemutants,with only 13% of the wild type (Figures 9A and 9B). Under am-monium 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, andNRT1.1/nrg2-2 plantswere grown on ammoniumnitratemedium for 7 d, and the rootswere collected fornitrate concentration analysis. Error bars represent SD of biological replicates (n = 4). Asterisks indicate significant differences (P < 0.05) compared with thewild type (t test).(B)Nitrate induction of the endogenous genes.Wild-type, nrg2-2, andNRT1.1/nrg2-2 plants were grown onmediumwith 2.5mMammonium succinate asthesolenitrogensource for7dand then treatedwith10mMKNO3orKClasacontrol for2h.The rootswerecollected forRNAextraction.The transcript levelsof nitrate-responsive geneswere 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).

494 The Plant Cell

nitrate conditions,with 20, 36, and15%of thewild type fornlp7-4,nrg2-3, and nlp7-4 nrg2-3 mutants, respectively (Figures 9C and9D). These results suggest that NRG2 and NLP7 play importantroles in nitrate regulation in nonoverlapping ways. Our qPCRresults showed that theexpressionofNLP7wasnotaltered innrg2mutants (Supplemental Figure 8) and the expression ofNRG2wasnot changed in nlp7mutant either (Supplemental Figure 9), so thattherewasnoevidence for transcriptional regulationof thesegenesby each other.

We also detected the expression of the nitrate-responsivegenes in the wild type and in single and double mutants. The qPCR

results revealed that the transcripts of the three tested genes indouble mutant nlp7-4 nrg2-2were significantly lower than in bothsingle mutants (Figure 9E), further showing that NRG2 and NLP7function in nonoverlapping ways to regulate nitrate responses.

NRG2 Interacts with NLP7 but Does Not Affect the NuclearRetention of NLP7 in Response to Nitrate

Previous studies have shown thatNLP7 ismainly expressed in thevascular tissue and the protein is targeted to the nucleus afternitrate treatment (Castaings et al., 2009; Marchive et al., 2013),

Figure 9. Analysis of Nitrate Regulation in nrg2 nlp7 Double Mutants.

(A)Root fluorescencephenotypesof thewild type, nrg2-3 andnlp7-4 singlemutants, andnlp7-4 nrg2-3plants onKNO3medium. Theplantswere grownonKNO3 medium for 4 d. Fluorescence and light images were captured with a fluorescence microscope to visualize YFP expression.(B)Quantificationof root fluorescenceofwild-type,nrg2-3,nlp7-4, andnlp7-4 nrg2-3plants. Theplantsweregrownon the samecondition as (A). Error barsrepresent SD (n = 60). Different letters indicate statistically significant difference (P < 0.05, t test).(C)Rootfluorescencephenotypesofwild-type,nrg2-3,nlp7-4, andnlp7-4nrg2-3plantsonNH4NO3medium.TheplantsweregrownonNH4NO3mediumfor4 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 sameNH4NO3 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 inwild-type, nrg2-2,nlp7-4, and nlp7-4 nrg2-2plants. The seedlingswere grown and treated as described in Figure3A. The transcripts of nitrate-responsive genes in roots were determined by qPCR. Error bars represent SD of biological replicates (n = 5). Different lettersindicate statistically different means (P < 0.05, t test).

NRG2 Plays an Essential Role in Nitrate Signaling 495

which is similar to the expression pattern of NRG2. Even thoughthese two proteins show no evidence of genetic or transcrip-tional interaction, it is possible that the two proteins can interactat the protein level. To test this idea, yeast two-hybrid assayswere performed. Indeed, NRG2 and NLP7 cotransformed yeast(Saccharomyces cerevisiae) cells grew well, while each gene and

empty vector cotransformed yeast cells used as negative controlscould not growon the selectivemedia (Figure 10A), indicating thatNRG2 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 onNicotiana benthamiana leaves was performed. A direct

Figure 10. NLP7 Interacts with NRG2.

(A)Yeast two-hybrid assay of theNRG2andNLP7 interaction. Serial dilution of yeast cells containing the indicated constructswas spotted on the indicatedmedium for lacZ and His reporter assays (four independent experiments). pGADT7, empty prey vector; pGBKT7, empty bait vector; NRG2, a bait vectorcontaining cDNA of NRG2; NLP7, a prey vector containing cDNA of NLP7. SD/TW-, SD medium lacking tryptophan and leucine; SD/LWHA-, SD mediumlacking 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. Differentcombinationsof expression vectors encodingNRG2-YFPNandNLP7-YFPCandcontrols (indicated on the left of thepanel) were transformed into leaves ofN. 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.

496 The Plant Cell

interactionwasobservedbetweenNRG2andNLP7 in thenucleusof plant cells, where coexpression of NRG2-YFPN andNLP7-YFPC reconstituted a functional YFP, whereas no signifi-cant signals were found in controls lacking NRG2 or NLP7 (Figure10B). The direct interaction between NRG2 and NLP7 proteins inthe nucleus was also observed on infiltrated leaves from plantswith starvation pretreatment, while no significant signals wereseen in controls lacking NRG2 or NLP7 (Supplemental Figure 11).These in vitro and in vivo results demonstrate thedirect interactionof NRG2 and NLP7 in the nucleus.

As the nuclear retention of NLP7 is regulated by nitrate(Marchive et al., 2013),wewanted todetermine ifNRG2 is involvedin the nitrate-induced nuclear retention of NLP7. Therefore, wechecked the NLP7 subcellular localization in nrg2-2 mutant linestransformed with the NLP7-YFP construct and grown in thepresence or absence of nitrate. The confocal images showed thatlocalization of NLP7 protein was indistinguishable betweenwild-typeandnrg2mutantplants (Figure11). Thisfinding indicatesthat 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 whichNRG2 mediates plant responses to nitrate, and to probe the re-lationships among NRG2, NRT1.1, and NLP7, we performed

a comparative RNA-seq analysis. The seedlings of the wild typeand nrg2-2, chl1-13, and nlp7-4mutants were grown on mediumwith 2.5 mM ammonium succinate for 7 d and then treated witheither 10 mM KNO3 or KCl for 2 h. The total root RNA analyzedusing an Illumina HiSeq 2500. For each genotype and NO3

2

treatment, three biological replicates were tested. After filteringlow-quality reads and removing reads that aligned to rRNA ortRNA, we selected 435,055,962 reads for analysis (SupplementalData Set 1). Twofold change in gene expression levels and ad-justed P value < 0.05 were used as a cutoff value to select dif-ferentially expressed transcripts.We first compared the gene expression in the roots of wild-type

and nrg2 mutant plants in response to nitrate treatments. Theresults (Figure 12A; Supplemental Data Set 2) showed that thetranscripts of 276 genes (including 117 upregulated and 159downregulated) were altered in the wild type after nitrate treat-ment, but not in nrg2 mutant. In other words, the expression ofthese nitrate-responsive genes in thewild typewas suppressed innrg2 mutant. The transcripts of 131 genes (88 induced and 43suppressed) were changed in the nrg2mutant, but not in the wildtype. In addition, the expression of 314 genes were regulated bynitrate in both the wild type and nrg2 mutant, among which 148genes (107 suppressed and 41 induced) were differentiallyexpressed by more than 25% in the wild type and the mutant(Supplemental Data Set 3). Therefore, the mutation in NRG2results 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 laserscanning microscopy. Bar = 50 mm.

NRG2 Plays an Essential Role in Nitrate Signaling 497

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 themutant (Table 1). Toexplore thedata further,weperformedGeneOntology (GO) analysis using Panther (http://www.pantherdb.org/pathway) for these differentially expressed 555 genes.Major GOclusters for all analyzed genes are listed in Table 2, and fourclusters were found to be related to nitrogen, including re-sponse to nitrogen compound, nitrogen compound transport,response to nitrate, and nitrate transport (Supplemental DataSet 4). These data strongly support our conclusion that NRG2functions in the nitrate signaling. In addition, GO analysisrevealed that genesmost affectedby themutation inNRG2wereoverrepresented in 20 clusters (P value < 0.001), including re-sponse to stimulus, response to chemical, ion transport, or-ganic substance, oxygen-containing compound, stress, andhormone (Table 2).

To clarify the relationship among NRG2, NRT1.1, and NLP7genes, transcriptomic analysiswas performed using roots treatedwith nitrate. The genes with differentially induced expression innrg2, nrt1.1, and nlp7mutants compared with the wild type wereanalyzed and are shown in the Venn diagram in Figure 12B. Theexpression of 235 genes was found to be changed in all threemutants with 57.7, 48.5, and 44.5% of the total differentiallyexpressed genes in nrg2, nrt1.1 (chl1), and nlp7 mutants, re-spectively (Supplemental Data Set 5), indicating that these threegenes 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 bybothNRT1.1 andNRG2were further investigated byGO analysis,and the results showed that a nitrogen compound transportclusterwas involved, including somemembers ofNRT/PTR family(Table 3; Supplemental Data Set 6). These data support theconclusion thatNRG2works in thesamenitrate signalingpathwayas 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-regulatedgeneswerenot (i.e.,were regulatedbyonlyNLP7orNRG2andnotthe other; Figure 12B). In addition, no nitrate-related cluster inthese geneswas foundbyGOanalysis (Supplemental DataSet 7).This result provides further evidence that NRG2 and NLP7 havesome independent functions in nitrate regulation.

DISCUSSION

To adapt to the changing nitrate conditions in the environment,plants have evolved diverse mechanisms to maintain normalgrowth and development. A sophisticated gene network isthought to regulate the responses to nitrate in plants. However,only the several nitrate regulatory genes mentioned in the In-troduction have been characterized thus far using systems bi-ology and reverse genetics approaches. In this article, weperformed a forward genetic screen and isolated the mutantMut75 defective in nitrate signaling. Mapping revealed that themutation in thegeneAt3g60320 (designatedNRG2) resulted in the

Figure 12. RNA-Seq Analyses of Differentially Expressed Transcripts inthe Roots of Wild-Type, nrg2-2, chl1-13, and nlp7-4 Seedlings Grown onAmmonium Succinate Followed by Nitrate Treatment.

(A) Venn diagram showing the number of the genes up- or downregulatedby nitrate treatment in the wild type and nrg2-2 mutant.(B) Diagram showing the number of the genes differentially expressed innrg2-2, chl1-13, and nlp7-4 mutants compared with the wild type.

Table 1. Known Nitrate-Inducible and Regulatory Genes with DifferentLevels of Nitrate-Responsive Expression in the Wild Type and nrg2-2Mutant

GeneFold Change inthe Wild Type P Value

Fold Changein Mutant P Value

NiR 26.35 3.03E-30 10.91 9.69 E-17NRT2.1 11.25 5.10 E-4 3.38 2.52 E-3HHO1 400.49 3.12 E-14 6.18 4.66 E-11UPM1 14.19 2.48E-55 7.25 6.08 E-18LBD37 11.01 6.19E-35 6.92 5.02 E-24LBD38 3.28 1.51 E-11 2.23 1.63 E-5NRT1.1 3.63 4.99 E-15 2.69 7.11 E-12TGA1 4.17 9.70 E-17 3.20 6.40 E-25TGA4 3.55 6.11 E-18 2.65 8.65 E-10

498 The Plant Cell

phenotype. This gene NRG2 belongs to a gene family with 15members in Arabidopsis (Supplemental Figure 3). Each membercontains DUF632 and DUF630 domains whose functions arestill unknown, as none of the proteins in this family have beencharacterized thus far.

Our results show that the induction of nitrate-responsive genesin nrg2mutants is inhibited when plants are treated with nitrate inthe presence of ammonium (Figure 3A), indicating that NRG2 isa nitrate-regulatory gene. Remarkably, this phenotype was notrestored after nitrogen starvation, which is different from nrt1.1mutants (Wang et al., 2009). Although nrt1.1 mutants have beenstudied formore than 10 years, the inhibition of nitrate induction inthemutants had not been found until it was tested in the presenceof ammonium (Tsay et al., 1993;Hoet al., 2009;Wang et al., 2009).It has also been reported that the expression of nitrate-inducedgenes in the presence of ammonium was inhibited in cipk8mutants but enhanced in cipk23mutants compared with the wildtype after nitrate treatment (Ho et al., 2009; Hu et al., 2009).Overexpression lines of SPL9 have been monitored as well in thepresence of ammonium, and the induction of the nitrate-responsive genes was increased after nitrate treatment (Krouket al., 2010b). Nevertheless, it has not been tested if this phe-notype can be recovered after nitrogen depletion for thesemutants. On the contrary, nlp7, tga1/tga4, and lbd37/38/39mutants have been analyzed after nitrogen starvation and theresults showed inhibited induction of nitrate-responsive genes innlp7 and tga1/tga4 mutants but higher induction in lbd37/38/39mutants after nitrate treatments (Castaings et al., 2009; Rubinet al., 2009; Alvarez et al., 2014). However, the expression ofnitrate-induced genes in thesemutants hasnot been investigatedwithout nitrogen starvation to date. The mutant nlp7-4 showedweaker YFP fluorescence in roots when grown on ammoniumnitrate medium and reduced induction of the nitrate-responsivegenes after nitrate treatments than in the wild type (Figures 9A to

9E), suggesting that NLP7 modulates the nitrate signaling in thepresence of ammonium. Our results, combined with previousstudies, reveal that some genes function as nitrate-regulatoryplayers in an ammonium-dependent manner, while some playimportant roles in nitrate signaling regardless of ammonium.Therefore, we propose that nitrate regulators may work in at leasttwodifferentways: (1) regulating nitrate responses in thepresenceof ammonium, such as NRT1.1, and (2) functioning as nitrateregulators regardless of ammonium, as representedbyNRG2andNLP7. A third way may exist that modulates nitrate response onlyin the absence of ammonium. The different signalingmechanismsunder conditions with and without ammonium reflect the com-plexity with which plants adapt to the changing environments.To understand thephysiological effects causedby themutation

inNRG2, thenitrate accumulation innrg2mutantswas tested.Ourresults showed that the nitrate content in seedlings was signifi-cantly lower than that in the wild type. This defect may result fromreduced uptake and/or increased reduction and assimilation.Further analysis by determining nitrate content in both leaves androots revealed lower nitrate levels in roots, indicating thatNRG2 isinvolved in regulating nitrate accumulation in roots. Previousstudies have shown that several characterized nitrate regulatorygenes play important roles in plant nitrate homeostasis. In nrt1.1mutant seedlings, the nitrate concentration is lower than inwild-type plants (Wang et al., 2009). On the contrary, the nitratecontent in nlp7 mutants was found to be higher than in the wildtype, whichmight result from the decreased nitrate reduction andassimilation (Castaings et al., 2009). In addition, LBD37, 38,and 39 overexpression lines displayed lower nitrate content and

Table 2. GO Cluster Analysis for Genes Differentially Expressed in theWild Type and the nrg2 Mutant after Nitrate Treatment

GO Term P Value

Response to nitrogen compound 5.61E-09Response to stimulus 1.19E-07Response to chemical 2.18E-07Anion transport 1.3 E-06Response to endogenous stimulus 2.32E-06Response to organic substance 3.98E-06Inorganic anion transport 4.43E-06Response to oxygen-containing compound 4.47E-06Ion transport 7.53E-06Nitrogen compound transport 8.29E-06Response to nitrate 1.19E-05Nitrate transport 1.57E-05Response to stress 9.35E-05Response to acid chemical 9.72E-05Response to hormone 2.24E-04Cellular hormone metabolic process 4.29E-04Response to external stimulus 6.13E-04Response to other organism 7.34E-04Response to external biotic stimulus 7.34E-04

Table 3. Genes Involved in a Nitrogen-Related Cluster Regulated byBoth NRG2 and NRT1.1

AGI Description

AT5G47330 At5g47330; AT5G47330; orthologAT5G46050 Protein NRT1/PTR FAMILY 5.2; NPF5.2; orthologAT5G11570 Protein NRT1/PTR FAMILY 1.3; NPF1.3; orthologAT2G02990 Ribonuclease 1; RNS1; orthologAT1G33440 Protein NRT1/PTR FAMILY 4.4; NPF4.4; orthologAT1G30840 Probable purine permease 4; PUP4; orthologAT4G19680 Fe2+ transport protein 2; IRT2; orthologAT5G41800 Probable GABA transporter 2; At5g41800; orthologAT1G14780 MACPF domain-containing protein At1g14780;

At1g14780; orthologAT3G01420 Alpha-dioxygenase 1; DOX1; orthologAT2G29750 UDP-glycosyltransferase 71C1; UGT71C1; orthologAT1G03850 Monothiol glutaredoxin-S13; GRXS13; orthologAT3G54140 Protein NRT1/PTR FAMILY 8.1; NPF8.1; orthologAT5G64100 Peroxidase 69; PER69; orthologAT1G72140 Protein NRT1/PTR FAMILY 5.12; NPF5.12; orthologAT5G57685 Protein GLUTAMINE DUMPER 3; GDU3; orthologAT1G57990 Probable purine permease 18; PUP18; orthologAT5G44390 Berberine bridge enzyme-like protein; AT5G44390;

orthologAT5G59540 1-Aminocyclopropane-1-carboxylate oxidase

homolog 12; At5g59540; orthologAT1G16390 Organic cation/carnitine transporter 3; OCT3; orthologAT5G06300 Cytokinin riboside 59-monophosphate

phosphoribohydrolase LOG7; LOG7; ortholog

NRG2 Plays an Essential Role in Nitrate Signaling 499

decreased maximal nitrate reductase activity compared withwild-type plants. The defects in nitrate content may be caused bythe reduced nitrate transport activity as the expression of severalhigh-affinity nitrate transport genes was strongly decreased(Rubin et al., 2009). Among these characterized nitrate transportand assimilation genes tested, only NRT1.1 exhibited decreasedexpression in nrg2mutant roots, andNRT1.8 displayed increasedexpression in mutant leaves. NRT1.1 has been characterized asa dual affinity nitrate transporter involved in absorbing nitrate fromthe environment (Wang et al., 1998; Liu et al., 1999; Liu and Tsay,2003). Thus, the lower nitrate content phenotype in nrg2mutantsmay be caused, at least partially, by the reduced expression ofNRT1.1. NRT1.8 has been identified to be a low-affinity nitratetransporter with a function in unloading nitrate from xylem. Thehigher expression of NRT1.8 in mutant leaves may lead to rela-tivelymore nitrate transport into leaves despite the relatively lowernitrate absorption from themedium, resulting in decreased nitratelevels in roots but similar levels in leaves compared with the wildtype. Taken together, these data suggest thatNRG2 is involved innitrate accumulation inplants and thealterednitrate accumulationin the mutants may result from modulated expression of NRT1.1and NRT1.8. It is also possible that some other uncharacterizednitrate transporters contribute to the modified nitrate concen-tration in the mutants.

NRT1.1 plays an essential role in nitrate regulation through itsfunctions in dual-affinity nitrate transport, nitrate sensing, andauxin transport. Nevertheless, how it is regulated, i.e., whatgenes can modulate the expression of NRT1.1, remains to becharacterized. Our molecular and genetic data demonstratedthat NRG2 can regulate the expression of NRT1.1 and bothgenes may work in the same pathway of nitrate signaling. Thisfinding is of great importance for further understanding of theregulation of NRT1.1 and adds a key component into the nitratesignaling 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 bindthe promoter of many genes involved in nitrate signaling andassimilation, modulate the expression of nitrate responsivegenes, and regulate nitrogen assimilation genes (Konishi andYanagisawa, 2013).WhetherNLP7 functionswith other proteinsby interacting or acts solely in the nitrate regulation is still un-clear. Our molecular and genetic analysis showed that NRG2and NLP7 have some nonoverlapping functions as the phe-notype of the double mutant is more severe than that of eithersingle mutant. However, both proteins can physically interact invitro and in vivo as revealed by yeast two-hybrid and BiFCassays, indicating that these proteins likely converge on part ofthe nitrate signaling pathways as well as functioning inde-pendently. In addition, the nuclear retentionofNLP7 in responseto nitrate is not affected by the mutation in NRG2. Theseresults further strengthen our understanding of nitrate signalingmechanisms.Our comparative RNA-seq analysis of the roots in response to

nitrate showed that many genes involved in nitrogen-relatedclusters, including nitrate transport and response to nitrate, weredifferentially expressed in the nrg2 mutant, providing further ev-idence that NRG2 plays an important role in nitrate signaling.Molecular and genetic evidence indicates thatNRG2 andNRT1.1works in the samepathway innitrate regulation. Thiswould leadusto predict that both genes may regulate some common nitrate-related genes. Indeed, the transcriptomic analysis revealed thata group of genes involved in a nitrogen compound transportcluster were modulated by NRG2 and NRT1.1 coordinately. Nocommon group of genes involved in nitrogen-related clusterswas found to be regulated by NRG2 and NLP7, in accordancewith the conclusion that both genes function independently innitrate signaling.

Figure 13. NRG2 Plays a Key Role in Nitrate Regulation.

(A)NRG2 regulates nitrate response in the presence of ammonium.Under the conditionswithNH4+,NRG2 regulates the expression ofNRT1.1 andNRT1.1

modulates the expression ofCIPK8 andCIPK23.CIPK23 negatively affects the expression of nitrate-responsive genes. The proteins NRT1.1 and CIPK23interact with each other, and NRT1.1 is phosphorylated by CIPK23 under low-nitrate conditions to maintain its high affinity for nitrate. NLP7 is a positiveregulator involved in the nitrate signaling.NRG2andNLP7work in different pathways in nitrate signalingwhereasboth proteins can interactwith eachother.(B)NRG2 regulates nitrate signaling after nitrogen starvation (no ammonium). After experiencing nitrogen starvation, NRG2 can still interact NLP7 and bothproteins play key roles in the primary nitrate response.

500 The Plant Cell

Taken together, the regulation of NRT1.1 by NRG2 and thephysical interaction of NRG2 and NLP7 highlight the importanceofNRG2as a keyplayer in the nitrate regulatory network. Thus,wepropose theworkingmodel shown in Figure 13. In the presence ofammonium, NRG2 regulates the expression of NRT1.1, whileNRT1.1 modulates the expression of other downstream genesincludingCIPK8andCIPK23.NRG2andNLP7bothactaspositiveregulators of nitrate assimilatory genes with some independentfunctions, and they physically interact, suggesting they convergein part of the nitrate signaling pathway. After nitrogen starvation(no ammonium), NRG2 and NLP7 appear to function in a similarmanner, acting as positive regulators with some independentfunctions while physically interacting. The relationship betweenNRG2 and other known regulatory players remains to be in-vestigated. In the meantime, NRG2 is the first member of a 15-member Arabidopsis gene family (Supplemental Figure 3) to becharacterized. What roles other members may play in nitratesignalingandwhat functions the twoDUFdomainssharedbyeachmember carry out are interesting questions for future work. Usingthe amino acid sequence of NRG2 as a query to search differentspecies revealed that this family exists broadly in plants frommoss to crops (rice, maize, soybean, etc.) and trees (apple,peach, poplar, etc.) (www.greenphyl.org/cgi-bin/blast.cgi), but nohomologs were found in microbes or animals (http://blast.ncbi.nlm.nih.gov/Blast.cgi), indicating that this familyexists specificallyin plants. The characterization of theNRG2 opens a door to revealthe roles of these family members.

METHODS

Plant Materials

The wild-type Arabidopsis thaliana ecotype used in this study is Col-0. Themutant lines chl1-13 (original nameMut21) (Wang et al., 2009), cipk8-1 (Huet al., 2009), and cipk23-3 (Ho et al., 2009) were described previously.

Mutagenesis and Mutant Screen

Homologous backcrossed transgenic seeds containing the NRP-YFPconstructwere treatedwithethylmethanesulfonate (Wangetal., 2009), andM2 seedlingswere screened on nitratemedium (initial mediumwith 10mMKNO3) based on the previous report (Wang et al., 2009). Mutants wereselfed and retested. Confirmed mutants were backcrossed to the trans-genic 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 nitratetreatment were grown in aseptic hydroponics (initial medium with 2.5 mMammonium succinate) as described (Wang et al., 2007) for 7 d and thentreated with 10 mM KNO3 or KCl as a control for 2 h followed by the rootsbeing collected. For nitrate treatment on plants after nitrate starvation,seedlingsweregrown inaseptic hydroponics for 6dand then transferred tothe same fresh medium except without ammonium succinate to grow for24h.The rootsof theseedlings treatedwith10mMKNO3orKCl for 2hwereharvested separately for RNA extraction.

For testing the YFP fluorescence of transgenic plants harboringNRP-YFP construct in response to nitrate, seedlingswere grown on plateswith either nitrate medium or ammonium nitrate medium (initial mediumwith 10mMNH4NO3) for 4 d followed by observation under a fluorescence

microscope (Nikon Eclipse Ti-S). The fluorescence of roots was quantifiedusing ImageJ.

qPCR Analysis

RNA samples were prepared using a total RNA miniprep kit (CWBIO).Real-time PCRwas performed using the reagent kit ABI7500 Fast (AppliedBiosystems). Template cDNA samples were prepared using the RevertAidfirst-strand synthesis system kit (Thermo Scientific) with 1 mg of total RNAin a reaction volume of 20 mL. The cDNA synthesis reaction mixture wasdiluted 20-fold before being used for qPCR. The FastStart Universal SYBRGreen Master Q-PCR kit (Roche Diagnostics) was used in the qPCR re-action 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 theNRG2 start codonwas cloned in front of theGUS gene in the binary vectorpMDC162 (Invitrogen). Transgenic Arabidopsis (Col-0) plants expressingthe GUS gene were obtained and GUS activity in different organs wasdetected as described (Dai et al., 2014). For section observation, roots andleaves of the transgenic plants were fixed and embedded in paraffin(Sigma-Aldrich). Sections were cut at 8 mm using a microtome (LeicaRM2235) andmounted on glass slides. Ruthenium red (100mg/L) solutionwas added onto the sectioned samples on slides for 1 min and then theslides were observed and photographedwith amicroscope (Nikon EclipseNi) equipped with a camera (Nikon Digital Sight DS-Qi1Mc).

Subcellular Localization Test

The full-length cDNA of NRG2 was introduced in frame with the GFPreporter gene in the binary vector pMDC43 (Invitrogen) to generatea fusion protein with GFP at the N-terminal position. The construct wastransformed into Arabidopsis (Col-0) plants as described previously(Feng et al., 2008). The imageswere capturedusing confocalmicroscope(Leica TCS SP5II).

Nitrate Assay

Nitrate wasmeasured using the salicylic acidmethod (Cataldo et al., 1975;Vendrell and Zupancic, 1990). Briefly, weighed samples (;0.1 g) ina 1.5-mL tube were frozen by liquid nitrogen and milled into powder usinga RETCH MM400.Then,1 mL of deionized water was added into the tubefollowed by boiling at 100°C for 20 min. The samples were centrifuged at15,871g for 10 min, and 0.1 mL supernatant was transferred into a 12-mLtube. Next, 0.4 mL of salicylic acid-sulfate acid (5 g salicylic acid in 100mLsulfate acid)wasaddedand thesamplewasmixedwell. The reactionswereincubatedat roomtemperature for 20min, and9.5mLof8%NaOHsolutionwas added. After cooling the tube to room temperature, the OD410 valuewas determined. For the control, 0.1 mL of deionized water was usedinsteadof0.1mLsupernatant. Thenitrate contentwascalculatedusing thefollowing equation: Y = C$V/W (Y, nitrate content; C, nitrate concentrationcalculatedwithOD410 into regression equation; V, total volumeof extractedsample; W, weight of sample). Standard curve was made with KNO3 atconcentrations between 10 to 120 mg/L and regression equation wasobtained based on standard curve.

Yeast Two-Hybrid Assays

A full-length fragment of cDNA for NRG2 was ligated into the pGBKT7vector (Clontech), and full-length cDNA fragments of tested genes wereintroduced into pGADT7 AD vector (Clontech). The two-hybrid interaction

NRG2 Plays an Essential Role in Nitrate Signaling 501

assays were performed according to the instruction provided by themanufacturer (Clontech).

BiFC Analysis

Transient BiFC assays in Nicotiana benthamiana were performed on theleaves asdescribed (Walter et al., 2004). Briefly, full-length cDNAsofNRG2andNLP7were cloned into the binary vectors pSPYNE-35S and pSPYCE-35S containing the N- and C-terminal fragments of YFP (YFPN and YFPC),respectively. N. benthamiana plants were grown on perlite watered withammonium nitrate solution for 5 weeks. For nitrogen starvation treatment,plants were grown on perlite watered with ammonium nitrate solution for3 weeks and then watered with the initial medium without nitrogen for an-other 2 weeks. The two constructs NRG2-YFPN and NLP7-YFPC werecotransfected into the fourth tofifth leavesand theemptyvectorsYFPCandYFPN incombinationwithNRG2-YFPNandNLP7-YFPC, respectively,wereused as negative controls. Transfected plants were watered with am-monium nitrate solution for 3 to 4 d followed by harvesting the infiltratedleaves 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 onammoniumsuccinate for 7dand then treatedwitheither 10mMKNO3orKCl(as a control) for 2 h. Total RNA of the roots was prepared using a RNAminiprepkit, and the concentrationsweremeasuredusing aNanoDrop2000spectrophotometer (Thermo). The libraries were constructed and then se-quenced using a HiSeq 2500 (Illumina), which generated ;21 million readpairs per sample (Annoroad). Raw reads containing adapter, poly-N, andlow-quality reads were filtered and the effective data were mapped with theArabidopsisTAIR10.2referencegenomeusingTopHat (version2.0.12).Afterexcluding the rRNA or tRNA, we estimated the abundance of the transcriptsusingRPKM(readsper kilobasespermillion reads) (Wagner et al., 2012). TheP values were adjusted using the Benjamini and Hochberg method(Benjamini and Hochberg, 1995). Corrected P value < 0.05 and fold changemore than2wereset as the threshold for significant difference inexpression.GO annotations of the data provided by our RNA-seq analysis wereperformed using Panther (www.pantherdb.org/pathway/; Mi et al., 2013).

Accession Numbers

Sequence data from this article can be found in the Arabidopsis GenomeInitiative or GenBank/EMBL databases under the following accessionnumbers: 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 inthis article have been deposited in the National Center for BiotechnologyInformation database (www.ncbi.nlm.nih.gov/sra; accession numberSRP067979).

Supplemental Data

Supplemental Figure 1. The weak fluorescence phenotype of Mut75is not caused by the mutation in At3g60240.

Supplemental Figure 2. Nitrate induction of the endogenous genestested is inhibited in the nrg2 mutants, and the expression of NRG2is not regulated by nitrate, ammonium, and nitrogen starvationtreatments.

Supplemental Figure 3. Sequence alignment of 15-memberArabidopsis gene family containing NRG2.

Supplemental Figure 4. Morphological phenotype of the nrg2 mutantunder different concentrations of nitrate.

Supplemental Figure 5. The expression of additional nitrate transportgenes in roots is not affected in nrg2 mutants.

Supplemental Figure 6. The expression of additional nitrate transportgenes in leaves is not affected by disruption of NRG2.

Supplemental Figure 7. The expression of nitrate reduction genes innrg2 mutants is not altered compared with that in the wild type.

Supplemental Figure 8. The expression of some characterized nitrateregulatory genes tested is not altered in nrg2 mutants compared withthat in the wild type.

Supplemental Figure 9. The expression of NRG2 is not changed incharacterized nitrate regulatory gene mutants.

Supplemental Figure 10. The expression of NRT1.1 in wild-type, nrg2mutant, and NRT1.1/nrg2-2 lines.

Supplemental Figure 11. BiFC assays revealed direct interactionbetween NRG2 and NLP7 when plants were treated with nitrate afternitrogen starvation.

Supplemental Data Set 1. Read numbers of the 24 samples.

Supplemental Data Set 2. Genes whose expression changed morethan 2-fold in the wild type and nrg2 mutant after nitrate treatment.

Supplemental Data Set 3. Genes with differentially induced expres-sion levels in the mutant compared with the wild type after nitratetreatment.

Supplemental Data Set 4. Four nitrogen-related clusters for genesdifferentially expressed in the wild type and nrg2 mutant after nitrateinduction.

Supplemental Data Set 5. Genes that are differentially expressed inthe mutants compared with the wild type and commonly regulated byNRG2, NRT1.1, and NLP7.

Supplemental Data Set 6. Genes regulated by both NRG2 andNRT1.1.

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 andGang 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 theTaishan Scholar Foundation to Y.W.

AUTHOR CONTRIBUTIONS

Y.W., N.M.C., N.X., R.W., and Z.C. designed the research. N.X., L.Z.,C.Z., Z. Li, Z. Lei, F.L., and P.G. performed research. N.X., Y.W., andR.W. analyzed data. Y.W., N.M.C., and N.X. wrote the article.

Received June 30, 2015; revisedDecember 11, 2015; accepted January 3,2016; published January 7, 2016.

502 The Plant Cell

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DOI 10.1105/tpc.15.00567; originally published online January 7, 2016; 2016;28;485-504Plant Cell

Zhaohui Chu, Nigel M. Crawford and Yong WangNa Xu, Rongchen Wang, Lufei Zhao, Chengfei Zhang, Zehui Li, Zhao Lei, Fei Liu, Peizhu Guan,

Nitrate RegulatorsThe Arabidopsis NRG2 Protein Mediates Nitrate Signaling and Interacts with and Regulates Key

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