The Arabidopsis NRG2 protein mediates nitrate

Plant Cell Advance Publication. Published on January 7, 2016, doi:10.1105/tpc.15.00567

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The Arabidopsis NRG2 protein mediates nitrate signaling and interacts

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with and regulates key nitrate regulators

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Na Xua,1,2, Rongchen Wangb,c,1, Lufei Zhaoa, Chengfei Zhanga, Zehui Lia, Zhao Leia, Fei Liua,

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Peizhu Guanc, Zhaohui Chud, Nigel M. Crawfordc, Yong Wanga,3

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a

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Tai’an, Shandong 271018, China

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b

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Huazhong Agricultural University, Wuhan, Hubei 430070, China

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c

State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University,

National Key Laboratory of Crop Genetic Improvement, College of Life Science and Technology,

Section of Cell and Developmental Biology, Division of Biological Sciences, University of

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California at San Diego, La Jolla, California 92093-0116, USA

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d

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University, Tai’an, Shandong 271018, China

State Key Laboratory of Crop Biology, College of Agronomic Sciences, Shandong Agricultural

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1 These authors contributed equally to this work.

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2 Current address: School of Biological Science, Jining Medical University, Rizhao, Shandong 276826, China

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3 Address correspondence to [email protected]

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Synopsis Arabidopsis NRG2, which regulates NRT1.1 and interacts with NLP7, mediates nitrate signaling.

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Abstract

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We show that NITRATE REGULATORY GENE 2 (NRG2), which we identified using

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forward genetics, mediates nitrate signaling in Arabidopsis thaliana. A mutation in NRG2

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disrupted the induction of nitrate-responsive genes after nitrate treatment by an

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ammonium-independent mechanism. The nitrate content in roots was lower in the

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mutants than in WT, which may have resulted from reduced expression of NRT1.1 (also

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called NPF6.3, encoding a nitrate transporter/receptor) and up-regulation of NRT1.8 (also 1 / 48

©2016 American Society of Plant Biologists. All Rights Reserved.

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called NPF7.2, encoding a xylem nitrate transporter). Genetic and molecular data suggest

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that NRG2 functions upstream of NRT1.1 in nitrate signaling. Furthermore, NRG2

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directly interacts with the nitrate regulator NLP7 in the nucleus, but nuclear retention of

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NLP7 in response to nitrate is not dependent on NRG2. Transcriptomic analysis revealed

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that genes involved in four nitrogen-related clusters including nitrate transport and

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response to nitrate were differentially expressed in the nrg2 mutants. A nitrogen

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-compound-transport cluster containing some members of the NRT/PTR family was

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regulated by both NRG2 and NRT1.1, while no nitrogen-related clusters showed

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regulation by both NRG2 and NLP7. Thus, NRG2 plays a key role in nitrate regulation in

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part through modulating NRT1.1 expression and may function with NLP7 via their

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physical interaction.

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INTRODUCTION

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Nitrogen is an important macronutrient required by plants for normal growth and

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development. Most plants grown under aerobic conditions absorb nitrogen mainly in the

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form of nitrate. Nitrate serves not only as a nutrient, but also as an important signaling

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molecule. Transcriptome analyses have revealed that the expression of more than 1,000

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genes is altered within 3 hours of nitrate treatment. Among these genes, those involved in

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nitrate transport and assimilation such as several members of the NRT (NITRATE

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TRANSPORT) gene families and the genes for nitrate and nitrite reductase (NIA and NiR,

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respectively) are quickly induced (Bi et al., 2007; Wang et al., 2007). In addition, some

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genes required for controlling carbon metabolism and for providing chemical energy used

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in reduction and assimilation are induced as well (Fritz et al., 2006; Gutierrez et al., 2007;

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Price et al., 2004; Scheible et al., 2004; Wang et al., 2004; Wang et al., 2007). Nitrate

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signaling also influences root growth, development and architecture, seed dormancy, and

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leaf expansion (Alboresi et al., 2005; Bi et al., 2007; Forde, 2002; Forde and Walch-Liu,

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2009; Walch-Liu et al., 2006; Walch-Liu et al., 2000). 2 / 48

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However, our understanding of the regulatory mechanisms and genes involved in nitrate

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signaling in plants is incomplete. In the last few years, several nitrate regulatory genes

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functioning in the primary nitrate response have been characterized. One key regulator is

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NRT1.1 (also called NPF6.3 and CHL1), which functions not only as a dual-affinity

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nitrate transporter, but also as a nitrate sensor (Alboresi et al., 2005; Ho et al., 2009;

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Leran et al., 2014; Liu et al., 1999; Munos et al., 2004; Remans et al., 2006; Tsay et al.,

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1993; Walch-Liu and Forde, 2008; Wang et al., 1998; Wang et al., 2009). Recent crystal

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structure studies on NRT1.1 provide further insights into its transport mechanisms

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(Parker and Newstead, 2014; Sun et al., 2014; Tsay, 2014); however, little is known about

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how the NRT1.1 gene itself is regulated. Other nitrate regulators include two members of

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the CBL-interacting protein kinase family, CIPK8 and CIPK23, which are themselves

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regulated by NRT1.1 and are involved in the primary nitrate response with CIPK8 acting

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as a positive regulator and CIPK23 as a negative regulator (Ho et al., 2009; Hu et al.,

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2009; Krouk et al., 2010a). In addition, CIPK23 can interact with and phosphorylate

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NRT1.1 at amino acid T101 to maintain the high-affinity response under low-nitrate

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conditions (Ho et al., 2009). So far, several transcription factors (ANR1, LBD37/38/39,

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NLP6, NLP7, SPL9, TGA1, TGA4, and NAC4) have been identified to be nitrate

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regulatory genes (Alvarez et al., 2014a; Castaings et al., 2009; Gan et al., 2012; Konishi

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and Yanagisawa, 2013; Krouk et al., 2010b; Marchive et al., 2013; Remans et al., 2006;

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Rubin et al., 2009; Vidal et al., 2010; Vidal et al., 2013; Wang et al., 2009; Zhang and

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Forde, 1998). The Arabidopsis MADS-box transcription factor ANR1 was the first to be

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characterized to regulate lateral root growth in response to nitrate treatment (Gan et al.,

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2012; Gan et al., 2005; Zhang and Forde, 1998). Reverse genetics has revealed that three

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members of LATERAL ORGAN BOUNDARY DOMAIN (LBD) transcription factor

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family LBD37/38/39 are negative regulators for nitrate-responsive genes and the mutants

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show a constitutive nitrogen-starvation response (Rubin et al., 2009). Arabidopsis

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NIN-LIKE PROTEIN 7 (NLP7) has been found to function as a master regulator in the 3 / 48

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early nitrate response. Disruption of NLP7 results in a nitrogen-starved phenotype and

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impaired nitrate signaling in the mutants (Castaings et al., 2009). The nuclear retention of

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NLP7 is regulated by nitrate (Marchive et al., 2013). All nine NLPs can bind the

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nitrate-responsive cis-element NRE and activate NRE-dependent and nitrate-responsive

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gene expression (Konishi and Yanagisawa, 2013). A suppression study of NLP6

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demonstrated that this gene plays an important role in nitrate signaling and other NLP

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members are also speculated to have similar function (Konishi and Yanagisawa, 2013).

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Using systems biology, SPL9, TGA1, TGA4, AFB3, and NAC4 have been identified as

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nitrate regulators involved in early nitrate response signaling (Alvarez et al., 2014b;

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Krouk et al., 2010b; Vidal et al., 2010; Vidal et al., 2013).

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A forward genetic screen for nitrate regulatory mutants was developed by transforming a

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nitrate-responsive promoter (NRP) and YFP marker into WT plants (Wang et al., 2010;

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Wang et al., 2009). The transgenic plants harboring this NRP-YFP construct show strong

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YFP fluorescence in the presence of nitrate. Two sets of mutants that showed low YFP

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fluorescence in the presence of nitrate were isolated and mapped to NRT1.1 and NLP7,

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respectively (Wang et al., 2009). Thus, this NRP-based mutant screen system can be used

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to screen for nitrate regulatory mutants, providing an effective forward genetic approach

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for discovering new genes involved in nitrate signaling.

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In this study, we performed a forward genetic screen using the NRP-YFP plants and

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isolated a mutant, Mut75. The mutation was mapped to the gene At3g60320, designated

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as NITRATE REGULATORY GENE 2 (NRG2, as NRG1 has been used for NRT1.1 (Wang

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et al., 2009)), and further characterization showed that it plays a key role in nitrate

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signaling. Genetic and molecular analyses revealed that NRG2 modulates the expression

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of NRT1.1 and functions upstream of NRT1.1. Moreover, biochemical and in planta

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experiments showed that NRG2 can directly interact with NLP7. Our findings support a

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model in which NRG2 regulates the expression of NRT1.1 and directly interacts with

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NLP7 in nitrate signaling transduction. These results not only establish the key role of 4 / 48

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NRG2 in transcriptional control, but also demonstrate a direct involvement of NRG2 in

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central nitrate signaling and offer insights into the mechanism of nitrate regulation in

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plants. In addition, our findings provide the first insights into the functions of an

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uncharacterized, 15-member gene family in Arabidopsis, to which NRG2 belongs.

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RESULTS

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Defects in Nitrate Signaling in Mut75 Are Caused by a Mutation in At3g60320

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To identify regulators in nitrate signaling, we performed a forward genetic screen. The

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seeds from homozygous transgenic plants containing the nitrate-responsive promoter

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NRP fused to a YFP marker (Wang et al., 2009) were treated with ethyl methanesulfonate

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(EMS), and M2 population seedlings grown on nitrate medium were checked for YFP

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fluorescence in roots under a fluorescence microscope. The transgenic WT seedling roots

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showed strong YFP signal in the presence of nitrate, as they are responsive to nitrate

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(Figure 1 Aa). One mutant, Mut75, exhibiting much lower YFP fluorescence than WT in

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the presence of nitrate, was isolated (Figure 1 Ab, and 1 B). The location of the mutation

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in Mut75 was narrowed down to the end of chromosome 3 in a 110-kb region (Figure 1 C)

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by a map-based cloning strategy. Unexpectedly, the sequencing results showed two point

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mutations in this region with one (G to A) in At3g60320 that converted Trp at position

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638 to a stop codon (Figure 1 C) and another one (C to T) in At3g60240 that changed Gln

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at position 332 to a stop codon (Supplemental Figure 1 A). At3g60320 is an

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uncharacterized gene, while At3g60240 encodes PROTEIN SYNTHESIS INITIATION

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FACTOR 4G (EIF4G), which is involved in virus resistance (Nicaise et al., 2007; Yoshii

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et al., 2004).

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To determine which mutation results in the weak YFP fluorescence phenotype of the

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mutant, several genetic tests were performed. For the gene At3g60320, transforming WT

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cDNA for this gene driven by a 35S promoter into the Mut75 mutant restored strong YFP

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fluorescence in the roots on nitrate medium (Figure 2 A), indicating that the gene

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At3g60320 can rescue the YFP phenotype of Mut75. In addition, two homozygous

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T-DNA insertion mutant lines for this gene were isolated from the ABRC T-DNA

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population (Alonso et al., 2003). The transcript levels of At3g60320 were very low in

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SALK_014743, which has a T-DNA insertion in the promoter region, and undetectable in 6 / 48

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SALK_079096, which contains a T-DNA insertion in the second exon, when tested by

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RT-PCR (Figure 2 B and 2 C). Mut75 was crossed with these two lines. Both F1 plants

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exhibited lower YFP fluorescence in roots when grown on nitrate medium (Figure 2 D),

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confirming that the weak YFP phenotype of Mut75 is caused by the mutation in 7 / 48

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At3g60320. Therefore, we designated At3g60320 as NITRATE REGULATORY GENE 2

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(NRG2), its T-DNA mutants SALK_014743 as nrg2-1 and SALK_079096 as nrg2-2, and

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Mut75 as nrg2-3.

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To test if the phenotype of nrg2-3 could have resulted from the disruption of the gene

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EIF4G, a knock-out T-DNA insertion mutant SALK_002002 with the T-DNA inserted in

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the seventh exon of EIF4G was identified (Supplemental Figure 1 A and 1 B) and then 8 / 48

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crossed with nrg2-3. The F1 plants grown on nitrate medium showed strong YFP

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fluorescence in the roots (Supplemental Figure 1 C), suggesting that the weak YFP

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phenotype of nrg2-3 is not caused by the mutation in EIF4G. Quantitative PCR (qPCR)

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results showed that the expression of several nitrate-responsive genes (NIA1, NiR, and

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NRT2.1) was induced by nitrate in the mutant to a similar level as in WT (Supplemental

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Figure 1 D). These data imply that EIF4G is not involved in nitrate regulation.

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Taking together, these results indicate that the weak YFP phenotype of Mut75 is caused

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by the mutation in NRG2, but not by the mutation in EIF4G. Therefore, we focused on

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the functional characterization of NRG2 in the following analyses.

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NRG2 Is Required for Nitrate-regulated Gene Expression

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The defect in responding to nitrate with NRP-YFP expression in nrg2-3 suggests that

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NRG2 plays an important role in nitrate signaling. To test if NRG2 also regulates

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endogenous genes, the expression of the nitrate-responsive genes NIA1, NiR, and NRT2.1

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was investigated. qPCR results showed that the nitrate induction of these genes in the

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roots of both nrg2-1 and nrg2-2 mutants was significantly inhibited (Figure 3 A,

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Supplemental Figure 2 A and 2 B). No difference was found for the expression of these

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genes among WT and the mutants when grown on ammonium succinate without any

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nitrate treatment (Supplemental Figure 2 C) or after KCl treatment (Supplemental Figure

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2 D), while a significant decrease in the mutants was seen after KNO3 treatment

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(Supplemental Figure 2 E). The inhibited nitrate induction in the mutants was also

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observed when the seedlings were treated with a low concentration of nitrate

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(Supplemental Figure 2 F).The above results demonstrate that NRG2 functions in nitrate

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signaling in plants.

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Previous studies have shown that NRT1.1 acts as a nitrate sensor and mediates nitrate

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responses as evidenced by the fact that nrt1.1 mutants (chl1-5 and chl1-13) exhibited

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decreased nitrate induction of the nitrate-responsive genes (NIA1, NiR, and NRT2.1) (Ho 9 / 48

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et al., 2009; Wang et al., 2009). However, this phenotype is dependent on nitrogen

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pre-treatment, as nitrogen deprivation restores the WT phenotype in nrt1.1 mutants

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(Krouk et al., 2010a; Wang et al., 2009). We tested nrg2 mutants under both

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nitrate-replete and nitrogen-deprived conditions and found that the induction levels for

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these nitrate-responsive genes were reduced under both conditions compared to WT

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(Figure 3 A and 3 B), indicating that NRG2 functions in nitrate signaling regardless of

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nitrogen starvation. This finding contrasts with that of NRT1.1, whose nitrate regulatory

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function is lost after nitrogen starvation and shows that NRG2 functions in both types of

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nitrogen conditions (nitrogen replete and nitrogen deprived).

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To test if NRG2 is regulated by different nitrogen conditions, we investigated its

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expression levels after nitrate, ammonium, and nitrogen starvation treatments. The results

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did not show significant changes in the expression of this gene after these treatments

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(Supplemental Figure 2 G), indicating that the expression of NRG2 is not modulated by

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nitrate, ammonium, and nitrogen starvation.

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NRG2 Is Predominantly Expressed in the Vascular Tissue of Leaves and Roots, and 10 / 48

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NRG2 Protein Is Localized in the Nucleus

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The expression profile of NRG2 in WT plants was examined using qPCR. Tissues were

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harvested either from plants grown in soil (leaves, stems, flowers, and siliques) or from

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plants grown on ammonium nitrate medium (seedlings and roots). NRG2 was expressed

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in all tested tissues, with highest levels in leaves and roots and lowest levels in flowers

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and siliques (Figure 4 A). The expression profile of NRG2 was further analyzed by the

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promoter-GUS approach. The GUS staining profile is largely consistent with the results

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obtained from qPCR, confirming the expression pattern of NRG2 in the tissues tested.

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Moreover, GUS staining revealed that NRG2 is predominantly expressed in the vascular

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bundles of leaves and roots (Figures 4 Ba-h). In addition, expression of NRG2 was also

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found in stomata (Figure 4 Bi), flowers (Figures 4 Bj-m), and young siliques (Figure 4

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Bn). In flowers, GUS expression was observed in the pistil (Figure 4 Bk), junction of

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filament and anther (Figure 4 Bl), and vascular tissue of sepals and petals (Figure 4 Bm).

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To more precisely determine the cells that express NRG2, the GUS staining in vascular

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bundles was further analyzed. Cross-sections of the roots showed GUS expression in the

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stelar cells, including the pericycle, phloem and parenchyma cells (Figure 4 Ca).

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Longitudinal and transversal sections of the leaves revealed GUS expression in the

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bundle sheath, phloem and parenchyma cells of the vascular tissues (Figure 4 Cb and 4

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Cc). This expression profile suggests that NRG2 may function in regulating nitrate

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transport in the vasculature.

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NRG2 contains two uncharacterized functional domains: DUF630 and DUF632, which

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are shared by all 15 members in this family of unknown proteins (Supplemental Figure 3).

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To explore the subcellular localization of the NRG2, several subcellular localization

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prediction tools were used to analyze its protein sequence. NRG2 was predicted to be

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localized in the nucleus by SubLoc (Chen et al., 2006) and WoLFPSORT (Horton et al.,

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2007) tools, but in mitochondria by MitoPred (Guda et al., 2004). To determine the bona 11 / 48

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fide localization of the protein, we cloned the NRG2 cDNA and ligated the fragment

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in-frame to be expressed with the GFP reporter at the N-terminal position

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(Pro35S:GFP-NRG2). The construct was transformed into Arabidopsis WT plants and the

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protein was observed in the nucleus in stable transgenic lines (Figure 4 D). Thus, we 12 / 48

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conclude that NRG2 protein is targeted to the nucleus.

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Nitrate Content in nrg2 Mutants Is Lower than WT in Roots, but Not in Leaves

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We have demonstrated that the induction of the nitrate-responsive genes is inhibited in

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the nrg2 mutants. We then tested if this molecular defect results in any phenotype at the

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morphological and physiological levels in the mutants. Under a high nitrate concentration,

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the mutant seedlings were slightly smaller and displayed later flowering compared with

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WT (Supplemental Figure 4 A and 4 B). Under low nitrate condition, no obvious

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phenotype was observed (Supplemental Figure 4 C).

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Previous studies on several known nitrate-regulatory genes (NRT1.1, NLP7, and

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LBD37/38/39) have shown that the nitrate levels in their respective mutant plants are

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altered (Castaings et al., 2009; Rubin et al., 2009; Wang et al., 2009). Here, we found that

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the nitrate accumulation in nrg2 mutant seedlings was significantly lower than in WT

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(Figure 5 A). Further investigation revealed that the nitrate accumulation in roots was

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significantly lower with each mutant allele than in WT (Figure 5 B). However, no

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difference between the mutants and WT was found in leaves (Figure 5 C). These data

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indicate that the nitrate accumulation in roots is defective while the accumulation of

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nitrate in leaves is normal in nrg2 mutants. We further assayed the nitrate content in

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whole seedlings treated with various concentrations of nitrate (0.25 – 20 mM) for 2 hours

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in the presence of ammonium and found that the nitrate accumulation in the mutants

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(including the chl1-13 mutant as a control) was significant lower in all concentrations

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tested (Figure 5 D).We also tested the time-course of nitrate accumulation in whole

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seedlings treated with 5 mM KNO3 and the results showed that the nitrate uptake in the

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mutants was significantly lower than in WT at all time points tested (Figure 5 E). These

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findings suggest that nitrate uptake is affected in nrg2 mutants.

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To investigate the mechanisms for the lower nitrate accumulation in mutant roots, the

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expression of several known nitrate transport genes was studied. Among the 13 transport 13 / 48

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genes tested, only the expression of NRT1.1 was significantly decreased under

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ammonium nitrate conditions (Figure 5 F), while no change was found for the other

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twelve tested genes (Supplemental Figure 5) in the mutant roots. In leaves, only the

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transcript levels of NRT1.8 were significantly increased (Figure 5 G), and there was no

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significant change in the expression of the other genes (Supplemental Figure 6) in the

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mutants. Previous studies have shown that NRT1.1 functions as a dual-affinity nitrate 14 / 48

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transporter involved in transporting nitrate from the environment into roots (Liu and Tsay,

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2003; Tsay et al., 1993; Wang et al., 1998). Thus, the decreased nitrate content in mutant

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seedlings may be caused by the decreased expression of NRT1.1. NRT1.8 functions in

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removing nitrate from xylem vessels, as the functional disruption of NRT1.8 increased

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nitrate concentration in xylem sap (Li et al., 2010). Thus, it is possible that the increased

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expression of NRT1.8 may direct more nitrate to be unloaded from xylem vessels

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resulting in similar nitrate levels in the mutant leaves to those in WT leaves. The

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expression of several key nitrate assimilatory genes (NIA1, NIA2, NiR, GLN1.1, and

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GLN1.3) was also detected by qPCR and no significant difference was found between

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WT and nrg2 mutants (Supplemental Figure 7). Therefore, these results imply that the

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lower nitrate content in mutant roots may be correlated with the reduced expression of

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NRT1.1 in roots and the increased transcripts of NRT1.8 in leaves.

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NRG2 Regulates the Expression and Works upstream of NRT1.1

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To understand the relationship of NRG2 and the characterized nitrate regulators, we first

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investigated the expression levels of several known nitrate regulators in the nrg2 mutants

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under different nitrogen conditions. The results showed that, among the investigated

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known regulatory genes, the expression of NRT1.1 in the nrg2 mutants was significantly

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decreased, to less than 40% of the expression in WT plants under potassium nitrate or

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ammonium nitrate conditions (Figure 6 A). The expression of other known nitrate

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regulatory genes tested was not changed (Supplemental Figure 8). This indicates that the

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expression of NRT1.1 is regulated by NRG2.

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To test if NRG2 is regulated by known nitrate regulators, we measured NRG2 transcript

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levels in the mutants of several identified regulatory genes (NRT1.1, NLP7, CIPK8, and

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CIPK23) in nitrate or ammonium nitrate media. No change was found for the expression

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of NRG2 in these mutants (Supplemental Figure 9), including in nrt1.1 mutants (Figure 6

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B). These results imply that NRG2 may not be regulated by these four genes.

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To study further the effects of NRG2 on NRT1.1, we examined the nitrate induction of 15 / 48

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NRT1.1 in WT and the nrg2 mutants. The results showed a significant decrease in the

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nitrate induction levels in the mutants (Figure 6 C), indicating that NRG2 affects the

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nitrate induction of NRT1.1. We also tested the expression of NRT1.1 in the absence of

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nitrate and found that the expression of NRT1.1 was significantly lower in the mutants

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compared with that in WT (Supplemental Figure 10 A). When seedlings grown on

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NH4NO3 medium were subjected to nitrogen starvation, the expression of NRT1.1

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increased during the first 24 h in both WT and the mutants and no significant differences

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in expression levels were found for the time points tested between WT and the nrg2

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mutants (Supplemental Figure 10 B). 16 / 48

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To understand better the relationship of NRG2 and NRT1.1, a double mutant of the two

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genes was obtained by crossing the single mutants of each gene: nrg2-3 and chl1-13

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isolated by our mutant screens. The YFP signal from the NRP-YFP transgene in roots of

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the double mutant seedlings grown on nitrate medium (with no ammonium) was detected

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and found to be much weaker than in WT and similar to nrg2-3 while weaker than

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chl1-13 (Figure 7 A). Quantifying the root fluorescence signal confirmed the weaker YFP

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signal in double mutant than in WT and chl1-13 and was similar to nrg2-3 (Figure 7 B).

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Notably, the signal in chl1-13 was much higher than in nrg2-3 and double mutant, and

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mildly lower than in WT. We also tested the YFP levels in roots of the single and double

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mutants grown on ammonium nitrate medium to investigate the function of the genes in

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the presence of ammonium. The observation and fluorescence quantification data showed

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that the YFP levels in the double mutant were similar to those of chl1-13 while lower

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than of nrg2-3 (Figures 7 C and 7 D). Interestingly, the chl1-13 exhibited much lower

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signal than WT, confirming that NRT1.1 function in the nitrate signaling pathway is

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ammonium-dependent.

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To provide further molecular evidence, we inspected the expression of nitrate-responsive

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genes in WT and these single and double mutants. The qPCR results showed that the

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expression levels of the nitrate-responsive genes in the double mutant chl1-13 nrg2-3

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were similar to those in single mutant chl1-13 and much lower than in WT (Figure 7 E).

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Additionally, we transformed the cDNA of NRT1.1 into the nrg2-2 mutant to investigate

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further the relationship between NRT1.1 and NRG2. The nitrate content in roots of nrg2-2

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was recovered to the WT level when NRT1.1 was overexpressed in the mutant (Figure 8A,

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Supplemental Figure 10 C). We also detected the expression levels of the

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nitrate-responsive genes NIA1, NiR, and NRT2.1 and found that nitrate induction of these

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genes was recovered to the WT phenotype in NRT1.1/nrg2-2 (Figure 8 B).

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Taken together, these results suggest that NRG2 and NRT1.1 work in the same nitrate

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signaling pathway and that NRG2 functions upstream of NRT1.1. 17 / 48

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Genetic

and

Molecular

Analysis

Reveals

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Non-overlapping Functions in Nitrate Regulation

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NLP7 is an important nitrate regulator in nitrate signaling (Castaings et al., 2009; Konishi 18 / 48

that

NRG2

and

NLP7

Have

321

and Yanagisawa, 2013; Marchive et al., 2013). To investigate the relationship between

322

NRG2 and NLP7 in the process of regulating nitrate response, a double mutant of the two

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genes was generated by crossing the respective single mutants nrg2-3 and nlp7-4 isolated

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by our mutant screen system. The mutant nlp7-4 harbors a mutation (C to T) in NLP7 that

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converts Gln at the position 62 to a stop codon, resulting in lower YFP fluorescence in

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roots when grown on nitrate-containing media (Figure 9 A-9 D). In the presence of nitrate

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without ammonium, both single mutants showed much lower YFP fluorescence in roots

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than WT, with 23% of WT for nlp7-4 and 35% of WT for nrg2-3 in terms of fluorescence

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intensity, respectively. Interestingly, the double mutant plants exhibited even lower YFP

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signal than the individual single mutants, with only 13% of WT (Figure 9 A and 9 B).

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Under ammonium nitrate conditions, similar results were obtained as under nitrate

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conditions, with 20%, 36%, and 15% of WT for nlp7-4, nrg2-3, and nlp7-4 nrg2-3

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mutants, respectively (Figure 9 C and 9 D). These results suggest that NRG2 and NLP7

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play important roles in nitrate regulation in non-overlapping ways. Our qPCR results

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showed that the expression of NLP7 was not altered in nrg2 mutants (Supplemental

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Figure 8) and the expression of NRG2 was not changed in nlp7 mutant either

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(Supplemental Figure 9), so that there was no evidence for transcriptional regulation of

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these genes by each other.

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We also detected the expression of the nitrate-responsive genes in WT and in single and

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double mutants. The qPCR results revealed that the transcripts of the three tested genes in 23 / 48

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double mutant nlp7-4 nrg2-2 were significantly lower than in both single mutants (Figure

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9 E), further showing that NRG2 and NLP7 function in non-overlapping ways to regulate

343

nitrate responses.

344

NRG2 Interacts with NLP7 but Does Not Affect the Nuclear Retention of NLP7 in

345

Response to Nitrate

346

Previous studies have shown that NLP7 is mainly expressed in the vascular tissue and the

347

protein is targeted to the nucleus after nitrate treatment (Castaings et al., 2009; Marchive

348

et al., 2013), which is similar to the expression pattern of NRG2. Even though these two

349

proteins show no evidence of genetic or transcriptional interaction, it is possible that the

350

two proteins can interact at the protein level. To test this idea, yeast two-hybrid assays

351

were performed. Indeed, NRG2 and NLP7-cotransformed yeast (Saccharomyces

352

cerevisiae) cells grew well while each gene and empty vector-cotransformed yeast cells

353

used as negative controls could not grow on the selective media (Figure 10 A), indicating

354

that NRG2 protein can directly interact with NLP7 in vitro.

355

To confirm the interaction between NRG2 and NLP7, in vivo tests with bimolecular

356

fluorescence complementation (BiFC) assays on N. benthamiana leaves was carried out.

357

A direct interaction was observed between NRG2 and NLP7 in the nucleus of plant cells,

358

where coexpression of NRG2-YFPN and NLP7-YFPC reconstituted a functional YFP,

359

whereas no significant signals were found in controls lacking NRG2 or NLP7 (Figure 10

360

B). The direct interaction between NRG2 and NLP7 proteins in the nucleus was also

361

observed on infiltrated leaves from plants with starvation pre-treatment, while no

362

significant signals were seen in controls lacking NRG2 or NLP7 (Supplemental Figure

363

11). These in vitro and in vivo results demonstrate the direct interaction of NRG2 and

364

NLP7 in the nucleus.

365

As the nuclear retention of NLP7 is regulated by nitrate (Marchive et al., 2013), we

366

wished to determine if NRG2 is involved in the nitrate-induced nuclear retention of 24 / 48

367

NLP7. Therefore, we checked the NLP7 subcellular localization in nrg2-2 mutant lines

368

transformed with the NLP7-YFP construct and grown in the presence or absence of

369

nitrate. The confocal images showed that localization of NLP7 protein was

370

indistinguishable between WT and nrg2 mutant plants (Figure 11). This finding indicates 25 / 48

371

that the nuclear retention of NLP7 is not dependent on NRG2.

372

Transcriptomic Analysis of Nitrate Response in nrg2, chl1, and nlp7 Mutants

373

To investigate systematically the molecular mechanism by which NRG2 mediates plant

374

responses to nitrate, and to probe the relationships among NRG2, NRT1.1, and NLP7, we

375

performed a comparative RNA sequencing (RNA-seq) analysis. The seedlings of WT and

376

nrg2-2, chl1-13, and nlp7-4 mutants were grown on medium with 2.5 mM ammonium

377

succinate for 7 days and then treated with either 10 mM KNO3 or KCl for 2 h. The total

378

root RNA analyzed using an Illumina HiSeq 2500. For each genotype and NO3- treatment,

379

three biological replicates were tested. After filtering low-quality reads and removing

380

reads that aligned to ribosomal RNA or transfer RNA, we selected 435,055,962 reads for

381

analysis (Supplemental Data set 1). Two-fold change in gene expression levels and

382

adjusted p value ＜0.05 were used as a cutoff value to select differentially expressed

383

transcripts. 26 / 48

384

We first compared the gene expression in the roots of WT and nrg2 mutant plants in

385

response to nitrate treatments. The results (Figure 12 A, Supplemental Data set 2) showed

386

that the transcripts of 276 genes (including 117 up-regulated and 159 down-regulated)

387

were altered in WT after nitrate treatment, but not in nrg2 mutant. In other words, the

388

expression of these nitrate-responsive genes in WT was suppressed in nrg2 mutant. The

389

transcripts of 131 genes (88 induced and 43 suppressed) were changed in the nrg2 mutant,

390

but not in WT. In addition, the expression of 314 genes were regulated by nitrate in both

391

WT and nrg2 mutant, among which 148 genes (107 suppressed and 41 induced) were

392

differentially expressed by more than 25% in WT and the mutant (Supplemental Data set

393

3). Therefore, the mutation in NRG2 results in a total of 555 genes with altered

394

expression after nitrate treatment. Many known nitrate-inducible and -regulatory genes

395

including NiR, NRT2.1, HHO1, UPM1, LBD37, LBD38, NRT1.1, TGA1, and TGA4

396

showed reduced nitrate induction in the mutant (Table 1). To explore the data further, we

397

performed GO analysis using PANTHER (http://www.pantherdb.org/pathway) for these

398

differentially expressed 555 genes. Major GO clusters for all analyzed genes are listed in

399

Table 2, and 4 clusters were found to be related to nitrogen including response to nitrogen

400

compound, nitrogen compound transport, response to nitrate, and nitrate transport

401

(Supplemental Data set 4). These data strongly support our conclusion that NRG2

402

functions in the nitrate signaling. In addition, GO analysis revealed that genes most

403

affected by the mutation in NRG2 were overrepresented in 20 clusters (p value﹤0.001)

404

including response to stimulus, response to chemical, ion transport, organic substance,

405

oxygen-containing compound, stress, and hormone (Table 2).

406

To clarify the relationship among NRG2, NRT1.1, and NLP7 genes, transcriptomic

407

analysis was performed using roots treated with nitrate. The genes with differentially

408

induced expression in nrg2, nrt1.1, and nlp7 mutants compared with WT were analyzed

409

and are shown in the Venn diagram in Figure 12 B. The expression of 235 genes was

410

found to be changed in all three mutants with 57.7%, 48.5%, and 44.5% of the total 27 / 48

411

differentially expressed genes in nrg2, nrt1.1 (chl1), and nlp7 mutants, respectively

412

(Supplemental Data set 5), indicating that these three genes are closely involved in nitrate

413

regulation in plants.

414

In the nrt1.1 mutant, the transcripts of 485 genes were altered compared with those in WT,

415

among which 277 genes (57.1% of 485) were also regulated by NRG2 (Figure 12 B,

416

Supplemental Data set 6). Those genes that were regulated by both NRT1.1 and NRG2

417

were further investigated by GO analysis, and the results showed that a nitrogen

418

compound transport cluster was involved, including some members of NRT/PTR family

419

(Table 3, Supplemental Data set 6). These data support the conclusion that NRG2 works

420

in the same nitrate signaling pathway as NRT1.1.

421

For the mutant nlp7, 276 genes were found to be regulated by both NRG2 and NLP7

422

(Figure 12 B, Supplemental Data set 7); however, 252 NLP7-regulated genes and 131

423

NRG2-regulated genes were not (i.e. were regulated by only NLP7 or NRG2 and not the

424

other, Figure 12 B). In addition, no nitrate-related cluster in these genes was found by GO 28 / 48

425

analysis (Supplemental Data set 7). This result provides further evidence that NRG2 and

426

NLP7 have some independent functions in nitrate regulation.

427 428

29 / 48

429

DISCUSSION

430

To adapt to the changing nitrate conditions in the environment, plants have evolved

431

diverse mechanisms to maintain normal growth and development. A sophisticated gene

432

network is thought to regulate the responses to nitrate in plants. However, only the several

433

nitrate regulatory genes mentioned in the Introduction have been characterized thus far

434

using systems biology and reverse genetics approaches. In this paper, we performed a

435

forward genetic screen and isolated the mutant Mut75 defective in nitrate signaling.

436

Mapping revealed that the mutation in the gene At3g60320 (designated NRG2) resulted in

437

the phenotype. This gene NRG2 belongs to a gene family with 15 members in Arabidopsis

438

(Supplemental Figure 3). Each member contains DUF632 and DUF630 domains whose

439

functions are still unknown, as none of the proteins in this family have been characterized

440

thus far.

441

Our results show that the induction of nitrate-responsive genes in nrg2 mutants is

442

inhibited when plants are treated with nitrate in the presence of ammonium (Figure 3 A),

443

indicating that NRG2 is a nitrate-regulatory gene. Remarkably, this phenotype was not

444

restored after nitrogen starvation, which is different from nrt1.1 mutants (Wang et al.,

445

2009). Although nrt1.1 mutants have been studied for more than ten years, the inhibition

446

of nitrate induction in the mutants had not been found until it was tested in the presence

447

of ammonium (Ho et al., 2009; Tsay et al., 1993; Wang et al., 2009). It has also been

448

reported that the expression of nitrate-induced genes in the presence of ammonium was

449

inhibited in cipk8 mutants but enhanced in cipk23 mutants compared to WT after nitrate

450

treatment (Ho et al., 2009; Hu et al., 2009). Over-expression lines of SPL9 have been

451

monitored as well in the presence of ammonium, and the induction of the

452

nitrate-responsive genes was increased after nitrate treatment (Krouk et al., 2010b).

453

Nevertheless, it has not been tested if this phenotype can be recovered after nitrogen

454

depletion for these mutants. On the contrary, nlp7, tga1/tga4, and lbd37/38/39 mutants

455

have been analyzed after nitrogen starvation and the results showed inhibited induction of 30 / 48

456

nitrate-responsive genes in nlp7 and tga1/tga4 mutants but higher induction in

457

lbd37/38/39 mutants after nitrate treatments (Alvarez et al., 2014b; Castaings et al., 2009;

458

Rubin et al., 2009). However, the expression of nitrate-induced genes in these mutants

459

has not been investigated without nitrogen starvation to date. The mutant nlp7-4 showed

460

weaker YFP fluorescence in roots when grown on ammonium nitrate medium and

461

reduced induction of the nitrate-responsive genes after nitrate treatments than in WT

462

(Figure 9 A-9 E), suggesting that NLP7 modulates the nitrate signaling in the presence of

463

ammonium. Our results, combined with previous studies, reveal that some genes function

464

as nitrate-regulatory players in an ammonium-dependent manner, while some play

465

important roles in nitrate signaling regardless of ammonium. Therefore, we propose that

466

nitrate regulators may work in at least two different ways: 1) regulating nitrate responses

467

in the presence of ammonium, such as NRT1.1; 2) functioning as nitrate regulators

468

regardless of ammonium, as represented by NRG2 and NLP7. A third way may exist that

469

modulates nitrate response only in the absence of ammonium. The different signaling

470

mechanisms under conditions with and without ammonium reflect the complexity with

471

which plants adapt to the changing environments.

472

To understand the physiological effects caused by the mutation in NRG2, the nitrate

473

accumulation in nrg2 mutants was tested. Our results showed that the nitrate content in

474

seedlings was significantly lower than that in WT. This defect may result from reduced

475

uptake and/or increased reduction and assimilation. Further analysis by determining

476

nitrate content in both leaves and roots revealed lower nitrate levels in roots, indicating

477

that NRG2 is involved in regulating nitrate accumulation in roots. Previous studies have

478

shown that several characterized nitrate regulatory genes play important roles in plant

479

nitrate homeostasis. In nrt1.1 mutant seedlings, the nitrate concentration is lower than in

480

WT plants (Wang et al., 2009). On the contrary, the nitrate content in nlp7 mutants was

481

found to be higher than in WT, which might result from the decreased nitrate reduction

482

and assimilation (Castaings et al., 2009). In addition, LBD37, 38, and 39 over-expression 31 / 48

483

lines displayed lower nitrate content and decreased maximal nitrate reductase activity

484

compared to WT plants. The defects in nitrate content may be caused by the reduced

485

nitrate transport activity as the expression of several high-affinity nitrate transport genes

486

was strongly decreased (Rubin et al., 2009). Among these characterized nitrate transport

487

and assimilation genes tested, only NRT1.1 exhibited decreased expression in nrg2

488

mutant roots, and NRT1.8 displayed increased expression in mutant leaves. NRT1.1 has

489

been characterized as a dual affinity nitrate transporter involved in absorbing nitrate from

490

the environment (Liu et al., 1999; Liu and Tsay, 2003; Wang et al., 1998). Thus, the lower

491

nitrate content phenotype in nrg2 mutants may be caused, at least partially, by the

492

reduced expression of NRT1.1. NRT1.8 has been identified to be a low-affinity nitrate

493

transporter with a function in unloading nitrate from xylem. The higher expression of

494

NRT1.8 in mutant leaves may lead to relatively more nitrate transport into leaves despite

495

the relatively lower nitrate absorption from the medium, resulting in decreased nitrate

496

levels in roots but similar levels in leaves compared with WT. Taken together, these data

497

suggest that NRG2 is involved in nitrate accumulation in plants and the altered nitrate

498

accumulation in the mutants may result from modulated expression of NRT1.1 and

499

NRT1.8. It is also possible that some other uncharacterized nitrate transporters contribute

500

to the modified nitrate concentration in the mutants.

501

NRT1.1 plays an essential role in nitrate regulation through its functions in dual-affinity

502

nitrate transport, nitrate sensing, and auxin transport. Nevertheless, how it is regulated,

503

i.e., what genes can modulate the expression of NRT1.1, remains to be characterized. Our

504

molecular and genetic data demonstrated that NRG2 can regulate the expression of

505

NRT1.1 and both genes may work in the same pathway of nitrate signaling. This finding

506

is of great importance for further understanding of the regulation of NRT1.1 and adds a

507

key component into the nitrate signaling network.

508

NLP7 acts as a master regulator in response to nitrate (Castaings et al., 2009; Konishi and

509

Yanagisawa, 2013; Marchive et al., 2013). As a transcription factor, NLP7 can bind the 32 / 48

510

promoter of many genes involved in nitrate signaling and assimilation, modulate the

511

expression of nitrate responsive genes, and regulate nitrogen assimilation genes (Konishi

512

and Yanagisawa, 2013). Whether NLP7 functions with other protein(s) by interacting or

513

acts solely in the nitrate regulation is still unclear. Our molecular and genetic analysis

514

showed that NRG2 and NLP7 have some non-overlapping functions as the phenotype of

515

the double mutant is more severe than that of either single mutant. However, both

516

proteins can physically interact in vitro and in vivo as revealed by yeast-two hybrid and

517

BiFC assays, indicating that these proteins likely converge on part of the nitrate signaling

518

pathways as well as functioning independently. In addition, the nuclear retention of NLP7

519

in response to nitrate is not affected by the mutation in NRG2. These results further

520

strengthen our understanding of nitrate signaling mechanisms.

521

Our comparative RNA-seq analysis of the roots in response to nitrate showed that many

522

genes involved in nitrogen–related clusters, including nitrate transport and response to

523

nitrate, were differentially expressed in thenrg2 mutant, providing further evidence that

524

NRG2 plays an important role in nitrate signaling. Molecular and genetic evidence

525

indicates that NRG2 and NRT1.1 works in the same pathway in nitrate regulation. This

526

would lead us predict that both genes may regulate some common nitrate-related genes.

527

Indeed, the transcriptomic analysis revealed that a group of genes involved in a nitrogen

528

compound transport cluster were modulated by NRG2 and NRT1.1 coordinately. No

529

common group of genes involved in nitrogen-related clusters were found to be regulated

530

by NRG2 and NLP7, in accord with the conclusion that both genes function

531

independently in nitrate signaling.

532

Taken together, the regulation of NRT1.1 by NRG2 and the physical interaction of NRG2

533

and NLP7 highlight the importance of NRG2 as a key player in the nitrate regulatory

534

network. Thus, we propose the working model shown in Figure 13. In the presence of

535

ammonium, NRG2 regulates the expression of NRT1.1 while NRT1.1 modulates the

536

expression of other downstream genes including CIPK8 and CIPK23. NRG2 and NLP7 33 / 48

537

both act as positive regulators of nitrate assimilatory genes with some independent

538

functions, and they physically interact, suggesting they converge in part of the nitrate

539

signaling pathway. After nitrogen starvation (no ammonium), NRG2 and NLP7 appear to

540

function in a similar manner, acting as positive regulators with some independent

541

functions while physically interacting. The relationship between NRG2 and other known

542

regulatory players remains to be investigated. In the meantime, NRG2 is the first member

543

of a 15-member, Arabidopsis gene family (Supplemental Figure 3) to be characterized.

544

What roles other members may play in nitrate signaling and what functions the two DUF

545

domains shared by each member carry out are interesting questions for future work.

546

Using the amino acid sequence of NRG2 as a query to search different species revealed

547

that this family exists broadly in plants from moss to crops (rice, maize, soybean, etc.)

548

and trees (apple, peach, poplar, etc.) (www.greenphyl.org/cgi-bin/blast.cgi), but no

549

homologues were found in microbes or animals (http://blast.ncbi.nlm.nih.gov/Blast.cgi),

550

indicating that this family exists specifically in plants. The characterization of the NRG2

551

opens a door to reveal the roles of these family members. 34 / 48

552

35 / 48

553

METHODS

554

Plant Materials

555

The WT Arabidopsis ecotype used in this study is Columbia-0 (Col-0). The mutant lines

556

chl1-13 (original name Mut21) (Wang et al., 2009), cipk8-1 (Hu et al., 2009), and

557

cipk23-3 (Ho et al., 2009) were described previously.

558

Mutagenesis and Mutant Screen

559

Homologous backcrossed transgenic seeds containing the NRP-YFP construct were

560

treated with EMS (Wang et al., 2009) and M2 seedlings were screened on nitrate medium

561

(initial medium with 10 mM KNO3) based on the previous report (Wang et al., 2009).

562

Mutants were selfed and retested. Confirmed mutants were backcrossed to the transgenic

563

WT twice and homozygous lines were identified and analyzed.

564

Growth and Treatment Conditions

565

Plants used for qPCR analysis of the gene expression induced by nitrate treatment were

566

grown in aseptic hydroponics (initial medium with 2.5 mM ammonium succinate) as

567

described (Wang et al., 2007) for 7 d and then treated with 10 mM KNO3 or KCl as a

568

control for 2 h followed by the roots being collected. For nitrate treatment on plants after

569

nitrate starvation, seedlings were grown in aseptic hydroponics for 6 d then transferred to

570

the same fresh medium except without ammonium succinate to grow for 24 h. The roots

571

of the seedlings treated with 10 mM KNO3 or KCl for 2 h were harvested separately for

572

RNA extraction.

573

For testing the YFP fluorescence of transgenic plants harboring NRP-YFP construct in

574

response to nitrate, seedlings were grown on plates with either nitrate medium or

575

ammonium nitrate medium (initial medium with 10 mM NH4NO3) for 4 d followed by

576

observation under a fluorescence microscope (Nikon Eclipse Ti-S). The fluorescence of

577

roots was quantified using ImageJ. 36 / 48

578

qPCR Analysis

579

RNA samples were prepared using a total RNA miniprep kit (CWBIO). Real-time PCR

580

was performed using the reagent kit ABI7500 Fast (Applied Biosystems). Template

581

cDNA samples were prepared using the RevertAid first-strand synthesis system kit

582

(Thermo Scientific) with 1μg of total RNA in a reaction volume of 20 μl. The cDNA

583

synthesis reaction mixture was diluted 20 fold before being used for qPCR. The FastStart

584

Universal SYBR Green Master Q-PCR kit (Roche Diagnostics) was used in the qPCR

585

reaction following the instructions provided by the manufacturer. TUB2 (At5g62690) was

586

used as the internal reference gene.

587

Expression Analysis by Promoter-GUS Assay

588

The 2951 bp promoter fragment located immediately upstream of the NRG2 start codon

589

was cloned in front of the GUS gene in the binary vector pMDC162 (Invitrogen).

590

Transgenic Arabidopsis (Col-0) plants expressing the GUS gene were obtained and GUS

591

activity in different organs was detected as described (Dai et al., 2014). For section

592

observation, roots and leaves of the transgenic plants were fixed and embedded in

593

paraffin (Sigma-Aldrich). Sections were cut at 8 μm using microtome (Leica RM2235)

594

and mounted on glass slides. Ruthenium red (100 mg/L) solution was added onto the

595

sectioned samples on slides for 1 min and then the slides were observed and

596

photographed with a microscope (Nikon Eclipse Ni) equipped with a camera (Nikon

597

Digital Sight DS-Qi1Mc).

598

Subcellular Localization Test

599

The full-length cDNA of NRG2 was introduced in frame with the GFP reporter gene in

600

the binary vector pMDC43 (Invitrogen) to generate a fusion protein with GFP at the

601

N-terminal position. The construct was transformed into Arabidopsis (Col-0) plants as

602

described previously (Feng et al., 2008). The images were captured using confocal 37 / 48

603

microscope (Leica TCS SP5II).

604

Nitrate Assay

605

Nitrate was measured using the salicylic acid method (Cataldo et al., 1975; Vendrell and

606

Zupancic, 1990). Briefly, weighed samples (about 0.1 g) in a 1.5-ml tube were frozen by

607

liquid nitrogen and milled into powder using a RETCH MM400.Then,1 ml deionized

608

water was added into the tube followed by boiling at 100℃ for 20 min. The samples

609

were centrifuged at 15871 g for 10 min, and 0.1 ml supernatant was transferred into a

610

12-ml tube. Next, 0.4 ml salicylic acid-sulphate acid (5 g salicylic acid in 100 ml sulphate

611

acid) was added and the sample was mixed well. The reactions were incubated at room

612

temperature for 20 min, and 9.5 ml of 8% NaOH solution was added. After cooling the

613

tube to room temperature, the OD410 value was determined. For the control, 0.1 ml

614

deionized water was used instead of 0.1 ml supernatant. The nitrate content was

615

calculated using the following equation: Y=C·V/W (Y: nitrate content, C: nitrate

616

concentration calculated with OD410 into regression equation, V: total volume of

617

extracted sample, W: weight of sample). Standard curve was made with KNO3 at

618

concentrations between 10 to 120 mg/L and regression equation was obtained based on

619

standard curve.

620

Yeast Two-Hybrid Assays

621

A full-length fragment of cDNA for NRG2 was ligated into the pGBKT7 vector

622

(Clontech) and full-length cDNA fragments of tested genes were introduced into

623

pGADT7 AD vector (Clontech). The two-hybrid interaction assays were performed

624

according to the instruction provided by the manufacturer (Clontech).

625

Bimolecular Fluorescence Complementation (BiFC) Analysis

626

Transient BiFC assays in N. benthamiana were performed on the leaves as described 38 / 48

627

(Walter et al., 2004). Briefly, full-length cDNAs of NRG2 and NLP7 were cloned into the

628

binary vectors pSPYNE-35S and pSPYCE-35S containing the N- and C-terminal

629

fragments of YFP (YFPN and YFPC), respectively. N. benthamiana plants were grown on

630

perlite watered with ammonium nitrate solution for 5 weeks. For nitrogen starvation

631

treatment, plants were grown on perlite watered with ammonium nitrate solution for 3

632

weeks and then watered with the initial medium without nitrogen for another 2 weeks.

633

The two constructs NRG2-YFPN and NLP7-YFPC were co-transfected into the 4-5th

634

leaves and the empty vectors YFPC and YFPN in combination with NRG2-YFPN and

635

NLP7-YFPC respectively were used as negative controls. Transfected plants were watered

636

with ammonium nitrate solution for 3-4 days followed by harvesting the infiltrated leaves

637

for observation using confocal microscope (Leica TCS SP5II).

638

RNA-Seq data analysis

639

The seeds of WT, nrg2-2, chl1-13, and nlp7-4 were grown on ammonium succinate for 7

640

days and then treated with either 10 mM KNO3 or KCl (as a control) for 2 h. Total RNA

641

of the roots was prepared using a RNA miniprep kit (CWBIO) and the concentrations

642

were measured using NanoDrop 2000 spectrophotometer (Thermo). The libraries were

643

constructed and then sequenced using HiSeq 2500 (Illumina), which generated about 21

644

million read pairs per sample (ANNOROAD). Raw reads containing adapter, poly-N, and

645

low quality reads were filtered and the effective data were mapped with the Arabidopsis

646

(Arabidopsis thaliana) TAIR 10.2 reference genome using TopHat (version 2.0.12). After

647

excluding the ribosomal RNA or transfer RNA, we estimated the abundance of the

648

transcripts using RPKM (Reads per Kilo bases per Million reads) (Wagner et al., 2012).

649

The p values were adjusted using the Benjamini and Hochberg method (Benjamini and

650

Hochberg, 1995). Corrected p value﹤0.05 and fold change more than 2 were set as the

651

threshold for significant difference in expression. GO annotations of the data provided by

652

our RNA-Seq analysis were performed using PANTHER (www.pantherdb.org/pathway/)

653

(Mi et al., 2013). 39 / 48

654 655

Accession Numbers

656

Sequence data from this article can be found in the Arabidopsis Genome Initiative or

657

GenBank/EMBL databases under the following accession numbers: NiR (AT2G15620),

658

NIA1

659

(AT1G30270), LBD37 (AT5G67420), LBD38 (AT3G49940), LBD39 (AT4G37540),

660

NRT1.1

661

(AT2G26690), NRT1.5 (AT1G32450), NRT1.6 (AT1G27080), NRT1.7 (AT1G69870),

662

NRT1.8 (AT4G21680), NRT1.9 (AT1G18880), NRT1.11 (AT1G52190), NRT1.12

663

(AT3G16180), NRT2.6 (AT3G45060), NRT2.7 (AT5G14570), GLN1.1 (AT5G37600), and

664

GLN1.3 (AT3G17820). The RNA-seq data discussed in this paper have been deposited in

665

National Center for Biotechnology Information database (www.ncbi.nlm.nih.gov/sra;

666

accession number: XXXX).

667

Supplemental Data

668

Supplemental Figure 1. The weak fluorescence phenotype of Mut75 is not caused by the

669

mutation in At3g60240.

670

Supplemental Figure 2. Nitrate induction of the endogenous genes tested is inhibited in

671

the nrg2 mutants, and the expression of NRG2 is not regulated by nitrate, ammonium,

672

and nitrogen-starvation treatments.

673

Supplemental Figure 3. Sequence alignment of 15-member, Arabidopsis gene family

674

containing NRG2.

675

Supplemental Figure 4. Morphological phenotype of the nrg2 mutant under different

676

concentrations of nitrate.

677

Supplemental Figure 5. The expression of additional nitrate transport genes in roots is

(AT1G77760),

NRT2.1

(AT1G12110),

NLP7

(AT1G08090),

(AT4G24020),

40 / 48

CIPK8

NRT1.2

(AT4G24400),

(AT1G69850),

CIPK23

NRT1.4

678

not affected in nrg2 mutants.

679

Supplemental Figure 6. The expression of additional nitrate transport genes in leaves is

680

not affected by disruption of NRG2.

681

Supplemental Figure 7. The expression of nitrate reduction genes in nrg2 mutants is not

682

altered compared with that in WT.

683

Supplemental Figure 8. The expression of some characterized nitrate regulatory genes

684

tested is not altered in nrg2 mutants compared with that in WT.

685

Supplemental Figure 9. The expression of NRG2 is not changed in characterized nitrate

686

regulatory gene mutants.

687

Supplemental Figure 10. The expression of NRT1.1 in WT, nrg2 mutants and

688

NRT1.1/nrg2-2 lines.

689

Supplemental Figure 11. BiFC assays revealed direct interaction between NRG2 and

690

NLP7 when plants were treated with nitrate after nitrogen starvation.

691

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

692

Supplemental Data set 2. Genes whose expression changed more than 2-fold in WT and

693

nrg2 mutant after nitrate treatment.

694

Supplemental Data set 3. Genes with differentially induced expression levels in the

695

mutant compared with WT after nitrate treatment.

696

Supplemental Data set 4. Four nitrogen-related clusters for genes differentially

697

expressed in WT and nrg2 mutant after nitrate induction.

698

Supplemental Data set 5. Genes that are differentially expressed in the mutants

699

compared with WT and commonly regulated by NRG2, NRT1.1, and NLP7. 41 / 48

700

Supplemental Data set 6. Genes regulated by both NRG2 and NRT1.1.

701

Supplemental Data set 7. Genes regulated by both NRG2 and NLP7.

702

Supplemental Data set 8. Primers used in this paper.

703

ACKNOWLEDGMENTS

704

We thank Dr. Yi-Fang. Tsay for offering the cipk8-1, cipk23-3 seeds; Dr. Lei Ge and Dr.

705

Gang Li for discussion of unpublished data; Dr. Xiansheng Zhang, Dr. Chengchao Zheng,

706

and Dr. Daolin Fu for comments on the manuscript. This research was supported by

707

NSFC grant (31170230) and Taishan Scholar Foundation to Y.W.

708 709

AUTHOR CONTRIBUTIONS

710

Y.W., N.M.C., N.X., R.W., and Z.C. designed the research; N.X., L.Z.,C.Z., Z.L., Z.L,F.L,

711

and P.G. performed research; N.X., Y.W., and R.W. analyzed data; Y.W., N.M.C., and

712

N.X. wrote the paper.

713

Figure legends

714 715 716 717 718 719

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-day-old seedlings grown on KCl/KNO3 media (a) and on KNO3 (b) were captured with a fluorescence microscope.(B) Quantification of root fluorescence of WT and Mut75 seedlings grown on the same conditions as (A). Error bars represent SD (n = 60).Asterisks indicate significant

720

differences (P＜0.05) compared with the WT (t test). (C) Mapping of NRG2 (Mut75). The schematic

721 722 723 724 725 726 727 728 729

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. 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-day-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 42 / 48

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mutation site in Mut75.(C) RT-PCR analysis of NRG2 mRNA levels in WT and the nrg2 mutants. Total RNA isolated from 7-day-old seedlings grown on ammonium nitrate was analyzed by RT-PCR and a program based on 25 cycles of PCR amplifications was carried out 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-day-old seedlings grown on nitrate media were captured with a fluorescence microscope. Figure 3. The mutants of NRG2 are defective in response to nitrate. (A) Nitrate induction of endogenous genes without nitrogen starvation. WT and nrg2 plants were grown on medium with 2.5 mM ammonium succinate as the sole nitrogen source for 7 days 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 WT (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. 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-day-old plants grown in soil (leaves, stems, flowers, and siliques) or from 7-day-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, d, e,), vascular system of cotyledon (f) and cauline leaves (b, g, 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. Bar =50 μm. XV, xylem vessels; PL, phloem; PR, parenchyma; BS, bundle sheath.(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 square areas in (a), respectively, red arrows indicate the nucleus. Bar =50 μm. Figure 5. NRG2 affects nitrate accumulation and uptake. (A)-(C) Nitrate content in seedlings (A), roots (B) and leaves (C). WT and nrg2 mutant plants were grown on ammonium nitrate medium for 7 days and collected for nitrate concentration test. Error bars represent SD of biological replicates (n=4). Asterisks indicate significant differences (P＜0.05) 43 / 48

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compared with the WT (t test). (D) and (E) Nitrate accumulation in WT, nrg2, and chl1 mutants. Seedlings grown with 2.5 mM ammonium succinate for 7 days 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 WT and two mutants.(F) and (G) Relative expression of NRT1.1 (F) and NRT1.8 (G). WT and nrg2 mutant plants were grown on ammonium nitrate medium for 7 days, and then roots and shoots were collected separately for RNA extraction. The transcription levels of NRT1.1 and NRT1.8were determined by qPCR. Error bars represent SD of biological replicates (n=4). Asterisks indicate significant differences (P＜0.05) compared with the WT (t test). Figure 6. The expression of NRT1.1 in the nrg2 mutants was reduced. (A) Relative expression of NRT1.1 in nrg2 mutants. WT and nrg2 mutant plants were grown on media with KNO3 or NH4NO3 for 7 days 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 WT (t test). (B) Relative expression of NRG2 in nrt1.1 mutants. WT, chl1-5, and chl-13 plants were grown on media with KNO3 or NH4NO3 for 7 days 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 WT and the nrg2 mutants. Plants were grown on medium with 2.5 mM ammonium succinate as the sole nitrogen source for 7 days, 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 WT (t test). Figure 7. NRG2 and NRT1.1 work in the same nitrate signaling pathway. (A) Root fluorescence phenotypes of WT, nrg2-3, chl1-13 and chl1-13 nrg2-3 plants on KNO3 medium. The plants were grown on KNO3 medium for 4 days. Fluorescence and light images were captured with a fluorescence microscope to visualize YFP expression. (B) Quantification of root fluorescence of WT, 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 WT, 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 WT, 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 WT, nrg2-3, chl1-13, and chl1-13 nrg2-3 plants. The seedlings were grown and treated as described in Figure 3 A. 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).

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Figure 8. NRG2 functions upstream of NRT1.1. (A) Nitrate content in roots. WT, nrg2-2, and NRT1.1/nrg2-2 plants were grown on ammonium nitrate medium for 7 days 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 WT (t test). (B) Nitrate induction of the endogenous genes. WT, 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 days 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 WT (t test). Figure 9. Analysis of nitrate regulation in nrg2nlp7 double mutants. (A) Root fluorescence phenotypes of WT, 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 days. Fluorescence and light images were captured with a fluorescence microscope to visualize YFP expression. (B) Quantification of root fluorescence of WT, 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 WT, nrg2-3, nlp7-4, and nlp7-4 nrg2-3 plants on NH4NO3 medium. The plants were grown on NH4NO3 medium for 4 days. Fluorescence and light images were captured with a fluorescent microscope to visualize YFP expression. (D) Quantification of root fluorescence of WT, 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 WT, nrg2-2, nlp7-4, and nlp7-4 nrg2-2 plants. The seedlings were grown and treated as described in Figure 3 A. 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). Figure 10. NLP7 interacts with NRG2. (A) Yeast two-hybrid assay of the NRG2 and NLP7 interaction. Serial dilution of yeastcells containing the indicated constructs were 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-α-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 =5μm. 45 / 48

853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878

Figure 11. The nuclear retention of NLP7-YFP is not regulated by NRG2. Subcellular localization of NLP7-YFP in WT (A and C) and nrg2-2 (B and D) mutants. (A) and (B) seedlings were grown on nitrate medium, (C) and (D) seedlings were treated with nitrogen deprivation. Fluorescence and corresponding bright-field pictures were captured by confocal laser scanning microscopy. Bar =50 μm. Figure 12. RNA-seq analyses of differentially expressed transcripts in the roots of WT, 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 down-regulated by nitrate treatment in WT 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 WT. 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. Table 1. Known nitrate-inducible and regulatory genes with different levels of nitrate-responsive expression in WT and nrg2-2 mutant. Gene

Fold change in WT

p value

Fold change in mutant

p value

NiR

26.35

3.03E-30

10.91

9.69 E-17

NRT2.1

11.25

5.10 E-4

3.38

2.52 E-3

HHO1

400.49

3.12 E-14

6.18

4.66 E-11

UPM1

14.19

2.48E-55

7.25

6.08 E-18

LBD37

11.01

6.19E-35

6.92

5.02 E-24

LBD38

3.28

1.51 E-11

2.23

1.63 E-5

NRT1.1

3.63

4.99 E-15

2.69

7.11 E-12

TGA1

4.17

9.70 E-17

3.20

6.40 E-25

TGA4

3.55

6.11 E-18

2.65

8.65 E-10

879 .

880 881 882 46 / 48

883 884

Table 2. GO cluster analysis for genes differentially expressed in WT and the nrg2 mutant after nitrate treatment.

885 GO Term

p value

response to nitrogen compound

5.61E-09

response to stimulus

1.19E-07

response to chemical

2.18E-07

anion transport

1.3 E-06

response to endogenous stimulus

2.32E-06

response to organic substance

3.98E-06

inorganic anion transport

4.43E-06

response to oxygen-containing compound

4.47E-06

ion transport

7.53E-06

nitrogen compound transport

8.29E-06

response to nitrate

1.19E-05

nitrate transport

1.57E-05

response to stress

9.35E-05

response to acid chemical

9.72E-05

response to hormone

2.24E-04

cellular hormone metabolic process

4.29E-04

response to external stimulus

6.13E-04

response to other organism

7.34E-04

response to external biotic stimulus

7.34E-04

886 .

887 888 889 890 891

47 / 48

892

Table 3. Genes involved in a nitrogen-related cluster regulated by both NRG2 and NRT1.1.

893 AGI

DESCRIPTION

AT5G47330

At5g47330;AT5G47330;ortholog

AT5G46050

Protein NRT1/ PTR FAMILY 5.2;NPF5.2;ortholog

AT5G11570

Protein NRT1/ PTR FAMILY 1.3;NPF1.3;ortholog

AT2G02990

Ribonuclease 1;RNS1;ortholog

AT1G33440

Protein NRT1/ PTR FAMILY 4.4;NPF4.4;ortholog

AT1G30840

Probable purine permease 4;PUP4;ortholog

AT4G19680

Fe(2+) transport protein 2;IRT2;ortholog

AT5G41800

Probable GABA transporter 2;At5g41800;ortholog

AT1G14780

MACPF domain-containing protein At1g14780;At1g14780;ortholog

AT3G01420

Alpha-dioxygenase 1;DOX1;ortholog

AT2G29750 AT1G03850

UDP-glycosyltransferase Monothiol

71C1;UGT71C1;ortholog

glutaredoxin-S13;GRXS13;ortholog

AT3G54140

Protein NRT1/ PTR FAMILY 8.1;NPF8.1;ortholog

AT5G64100

Peroxidase 69;PER69;ortholog

AT1G72140

Protein NRT1/ PTR FAMILY 5.12;NPF5.12;ortholog

AT5G57685

Protein GLUTAMINE DUMPER 3;GDU3;ortholog

AT1G57990

Probable purine permease 18;PUP18;ortholog

AT5G44390

Berberine bridge enzyme-like protein;AT5G44390;ortholog

AT5G59540

1-aminocyclopropane-1-carboxylate oxidase homolog 12;At5g59540;ortholog

AT1G16390

Organic cation/carnitine transporter 3;OCT3;ortholog

AT5G06300

Cytokinin riboside 5'-monophosphate phosphoribohydrolase LOG7;LOG7;ortholog

894 895 .

896 897

48 / 48

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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 Crawford and Yong Wang Plant Cell; originally published online January 7, 2016; DOI 10.1105/tpc.15.00567 This information is current as of January 12, 2016 Supplemental Data

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