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
327
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
20 / 48
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intensity, respectively. Interestingly, the double mutant plants exhibited even lower YFP
330
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
335
showed that the expression of NLP7 was not altered in nrg2 mutants (Supplemental
336
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
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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
http://www.plantcell.org/content/suppl/2016/01/07/tpc.15.00567.DC1.html
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