Download The Arabidopsis NRG2 Protein Mediates Nitrate

The Plant Cell, Vol. 28: 485–504, February 2016, www.plantcell.org ã 2016 American Society of Plant Biologists. All rights reserved.
The Arabidopsis NRG2 Protein Mediates Nitrate Signaling and
Interacts with and Regulates Key Nitrate Regulators
OPEN
Na Xu,a,1,2 Rongchen Wang,b,c,1 Lufei Zhao,a Chengfei Zhang,a Zehui Li,a Zhao Lei,a Fei Liu,a Peizhu Guan,c
Zhaohui Chu,d Nigel M. Crawford,c and Yong Wanga,3
a State
Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Tai’an, Shandong 271018, China
Key Laboratory of Crop Genetic Improvement, College of Life Science and Technology, Huazhong Agricultural University,
Wuhan, Hubei 430070, China
c Section of Cell and Developmental Biology, Division of Biological Sciences, University of California at San Diego, La Jolla, California
92093-0116
d State Key Laboratory of Crop Biology, College of Agronomic Sciences, Shandong Agricultural University, Tai’an, Shandong 271018,
China
b National
ORCID IDs: 0000-0003-0896-4296 (N.X.); 0000-0002-6378-7504 (R.W.); 0000-0001-8320-7872 (Z.C.); 0000-0003-0219-4403 (Y.W.)
We show that NITRATE REGULATORY GENE2 (NRG2), which we identified using forward genetics, mediates nitrate signaling
in Arabidopsis thaliana. A mutation in NRG2 disrupted the induction of nitrate-responsive genes after nitrate treatment by an
ammonium-independent mechanism. The nitrate content in roots was lower in the mutants than in the wild type, which may
have resulted from reduced expression of NRT1.1 (also called NPF6.3, encoding a nitrate transporter/receptor) and
upregulation of NRT1.8 (also called NPF7.2, encoding a xylem nitrate transporter). Genetic and molecular data suggest that
NRG2 functions upstream of NRT1.1 in nitrate signaling. Furthermore, NRG2 directly interacts with the nitrate regulator NLP7
in the nucleus, but nuclear retention of NLP7 in response to nitrate is not dependent on NRG2. Transcriptomic analysis
revealed that genes involved in four nitrogen-related clusters including nitrate transport and response to nitrate were
differentially expressed in the nrg2 mutants. A nitrogen compound transport cluster containing some members of the NRT/
PTR family was regulated by both NRG2 and NRT1.1, while no nitrogen-related clusters showed regulation by both NRG2 and
NLP7. Thus, NRG2 plays a key role in nitrate regulation in part through modulating NRT1.1 expression and may function with
NLP7 via their physical interaction.
INTRODUCTION
Nitrogen is an important macronutrient required by plants for
normal growth and development. Most plants grown under aerobic conditions absorb nitrogen mainly in the form of nitrate.
Nitrate serves not only as a nutrient, but also as an important
signaling molecule. Transcriptome analyses have revealed that
the expression of more than 1000 genes is altered within 3 h of
nitrate treatment. Among these genes, those involved in nitrate
transport and assimilation, such as several members of the
NITRATE TRANSPORT (NRT) gene families and the genes for
nitrate and nitrite reductase (NIA and NiR, respectively), are quickly
induced (Bi et al., 2007; Wang et al., 2007). In addition, some genes
required for controlling carbon metabolism and for providing
chemical energy used in reduction and assimilation are induced as
well (Price et al., 2004; Scheible et al., 2004; Wang et al., 2004,
2007; Fritz et al., 2006; Gutiérrez et al., 2007). Nitrate signaling also
1 These
authors contributed equally to this work.
address: School of Biological Science, Jining Medical University, Rizhao, Shandong 276826, China.
3 Address correspondence to [email protected].
The author responsible for distribution of materials integral to the findings
presented in this article in accordance with the policy described in the
Instructions for Authors (www.plantcell.org) is: Yong Wang (wangyong@
sdau.edu.cn).
OPEN
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www.plantcell.org/cgi/doi/10.1105/tpc.15.00567
2 Current
influences root growth, development and architecture, seed
dormancy, and leaf expansion (Walch-Liu et al., 2000, 2006;
Forde, 2002; Alboresi et al., 2005; Bi et al., 2007; Forde and
Walch-Liu, 2009).
However, our understanding of the regulatory mechanisms and
genes involved in nitrate signaling in plants is incomplete. In the
last few years, several nitrate regulatory genes functioning in the
primary nitrate response have been characterized. One key regulator is NRT1.1 (also called NPF6.3 and CHL1), which functions
not only as a dual-affinity nitrate transporter, but also as a nitrate
sensor (Tsay et al., 1993; Wang et al., 1998, 2009; Liu et al., 1999;
Alboresi et al., 2005; Remans et al., 2006; Walch-Liu and Forde,
2008; Ho et al., 2009; Léran et al., 2014; Muños et al., 2004). Recent
crystal structure studies on NRT1.1 provide further insights into its
transport mechanisms (Parker and Newstead, 2014; Sun et al.,
2014; Tsay, 2014); however, little is known about how the NRT1.1
gene itself is regulated. Other nitrate regulators include two
members of the CBL-interacting protein kinase family, CIPK8 and
CIPK23, which are themselves regulated by NRT1.1 and are involved in the primary nitrate response with CIPK8 acting as
a positive regulator and CIPK23 as a negative regulator (Ho et al.,
2009; Hu et al., 2009; Krouk et al., 2010a). In addition, CIPK23 can
interact with and phosphorylate NRT1.1 at amino acid Thr-101 to
maintain the high-affinity response under low-nitrate conditions
(Ho et al., 2009). So far, several transcription factors (ANR1,
LBD37/38/39, NLP6, NLP7, SPL9, TGA1, TGA4, and NAC4) have
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been identified to be nitrate regulatory genes (Zhang and Forde,
1998; Remans et al., 2006; Castaings et al., 2009; Rubin et al.,
2009; Wang et al., 2009; Krouk et al., 2010b; Vidal et al., 2010,
2013; Gan et al., 2012; Konishi and Yanagisawa, 2013;
Marchive et al., 2013; Alvarez et al., 2014). The Arabidopsis
thaliana MADS box transcription factor ANR1 was the first to be
characterized to regulate lateral root growth in response to
nitrate treatment (Zhang and Forde, 1998; Gan et al., 2005,
2012). Reverse genetics has revealed that three members of
LATERAL ORGAN BOUNDARY DOMAIN (LBD) transcription
factor family LBD37/38/39 are negative regulators for nitrateresponsive genes and the mutants show a constitutive nitrogen
starvation response (Rubin et al., 2009). Arabidopsis NINLIKE PROTEIN7 (NLP7) has been found to function as a master
regulator in the early nitrate response. Disruption of NLP7
results in a nitrogen-starved phenotype and impaired nitrate
signaling in the mutants (Castaings et al., 2009). The nuclear
retention of NLP7 is regulated by nitrate (Marchive et al., 2013).
All nine NLPs can bind the nitrate-responsive cis-element NRE
and activate NRE-dependent and nitrate-responsive gene
expression (Konishi and Yanagisawa, 2013). A suppression
study of NLP6 demonstrated that this gene plays an important
role in nitrate signaling and other NLP members are also
speculated to have similar function (Konishi and Yanagisawa,
2013). Using systems biology, SPL9, TGA1, TGA4, AFB3, and
NAC4 have been identified as nitrate regulators involved in
early nitrate response signaling (Krouk et al., 2010b; Vidal et al.,
2010, 2013; Alvarez et al., 2014).
A forward genetic screen for nitrate regulatory mutants was
developed by transforming a nitrate-responsive promoter (NRP)
and YFP marker into wild-type plants (Wang et al., 2009, 2010).
The transgenic plants harboring this NRP-YFP construct show
strong YFP fluorescence in the presence of nitrate. Two sets of
mutants that showed low YFP fluorescence in the presence of
nitrate were isolated and mapped to NRT1.1 and NLP7, respectively (Wang et al., 2009). Thus, this NRP-based mutant
screen system can be used to screen for nitrate regulatory
mutants, providing an effective forward genetic approach for
discovering new genes involved in nitrate signaling.
In this study, we performed a forward genetic screen using
the NRP-YFP plants and isolated a mutant, Mut75. The mutation was mapped to the gene At3g60320, designated as
NITRATE REGULATORY GENE2 (NRG2; as NRG1 has been
used for NRT1.1; Wang et al., 2009), and further characterization showed that it plays a key role in nitrate signaling.
Genetic and molecular analyses revealed that NRG2 modulates
the expression of NRT1.1 and functions upstream of NRT1.1.
Moreover, biochemical and in planta experiments showed that
NRG2 can directly interact with NLP7. Our findings support
a model in which NRG2 regulates the expression of NRT1.1 and
directly interacts with NLP7 in nitrate signaling transduction.
These results not only establish the key role of NRG2 in transcriptional control, but also demonstrate a direct involvement
of NRG2 in central nitrate signaling and offer insights into the
mechanism of nitrate regulation in plants. In addition, our
findings provide the first insights into the functions of an
uncharacterized, 15-member gene family in Arabidopsis, to
which NRG2 belongs.
RESULTS
Defects in Nitrate Signaling in Mut75 Are Caused by
a Mutation in At3g60320
To identify regulators in nitrate signaling, we performed a forward
genetic screen. The seeds from homozygous transgenic plants
containing the nitrate-responsive promoter NRP fused to a YFP
marker (Wang et al., 2009) were treated with ethyl methanesulfonate,
and M2 population seedlings grown on nitrate medium were
checked for YFP fluorescence in roots under a fluorescence
microscope. The transgenic wild-type seedling roots showed
strong YFP signal in the presence of nitrate, as they are responsive
to nitrate (Figure 1Aa). One mutant, Mut75, exhibiting much lower
YFP fluorescence than the wild type in the presence of nitrate, was
isolated (Figures 1Ab and 1B). The location of the mutation in
Mut75 was narrowed down to the end of chromosome 3 in
a 110-kb region (Figure 1C) by a map-based cloning strategy.
Unexpectedly, the sequencing results showed two point
mutations in this region with one (G to A) in At3g60320 that
converted Trp at position 638 to a stop codon (Figure 1C) and
another one (C to T) in At3g60240 that changed Gln at position 332
to a stop codon (Supplemental Figure 1A). At3g60320 is an
uncharacterized gene, while At3g60240 encodes PROTEIN
SYNTHESIS INITIATION FACTOR 4G (EIF4G), which is involved in
virus resistance (Yoshii et al., 2004; Nicaise et al., 2007).
To determine which mutation results in the weak YFP fluorescence phenotype of the mutant, several genetic tests were
performed. For the gene At3g60320, transforming wild-type
cDNA for this gene driven by a 35S promoter into the Mut75
mutant restored strong YFP fluorescence in the roots on nitrate
medium (Figure 2A), indicating that the gene At3g60320 can
rescue the YFP phenotype of Mut75. In addition, two homozygous T-DNA insertion mutant lines for this gene were isolated
from the ABRC T-DNA population (Alonso et al., 2003). The
transcript levels of At3g60320 were very low in SALK_014743,
which has a T-DNA insertion in the promoter region, and undetectable in SALK_079096, which contains a T-DNA insertion in
the second exon, when tested by RT-PCR (Figures 2B and 2C).
Mut75 was crossed with these two lines. Both F1 plants
exhibited lower YFP fluorescence in roots when grown on nitrate
medium (Figure 2D), confirming that the weak YFP phenotype
of Mut75 is caused by the mutation in At3g60320. Therefore,
we designated At3g60320 as NRG2, its T-DNA mutants
SALK_014743 as nrg2-1 and SALK_079096 as nrg2-2, and
Mut75 as nrg2-3.
To test if the phenotype of nrg2-3 could have resulted from the
disruption of the gene EIF4G, a knockout T-DNA insertion mutant
SALK_002002 with the T-DNA inserted in the seventh exon of
EIF4G was identified (Supplemental Figures 1A and 1B) and then
crossed with nrg2-3. The F1 plants grown on nitrate medium
showed strong YFP fluorescence in the roots (Supplemental
Figure 1C), suggesting that the weak YFP phenotype of nrg2-3 is
not caused by the mutation in EIF4G. qPCR results showed that
the expression of several nitrate-responsive genes (NIA1, NiR, and
NRT2.1) was induced by nitrate in the mutant to a similar level as in
the wild type (Supplemental Figure 1D). These data imply that
EIF4G is not involved in nitrate regulation.
NRG2 Plays an Essential Role in Nitrate Signaling
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Figure 1. Identification and Mapping of Mut75.
(A) Nitrate induction of NRP-YFP in wild type (WT) and Mut75 roots. Fluorescence and light images of 4-d-old seedlings grown on KCl/KNO3 media (a) and on
KNO3 (b) were captured with a fluorescence microscope.
(B) Quantification of root fluorescence of wild-type and Mut75 seedlings grown on the same conditions as (A). Error bars represent SD (n = 60). Asterisks
indicate significant differences (P < 0.05) compared with the wild type (t test).
(C) Mapping of NRG2 (Mut75). The schematic map shows that the mutation in Mut75 was located in the gene NRG2 on chromosome 3. Amino acid and
nucleotide changes found in Mut75 are also shown.
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Figure 2. The Mut75 Phenotype Is Caused by the Mutation in NRG2.
(A) Complementation test of NRG2 in Mut75. Fluorescence and light images of 4-d-old seedlings grown on nitrate media were captured with a fluorescence
microscope.
(B) Schematic map of the T-DNA insertion sites in nrg2-1 and nrg2-2 mutants. Exons, introns, and untranslated regions are represented by black boxes,
lines, and white boxes, respectively. The locations of the T-DNA insertion in the two nrg2 alleles from SALK are indicated with triangles. Black arrow indicates
the mutation site in Mut75.
(C) RT-PCR analysis of NRG2 mRNA levels in the wild type and the nrg2 mutants. Total RNA isolated from 7-d-old seedlings grown on ammonium nitrate was
analyzed by RT-PCR and a program based on 25 cycles of PCR amplifications was performed to test the expression of NRG2. TUB2 serves as a control to
show the equal amount of cDNA in each reaction.
(D) Root fluorescence of F1 plants from nrg2-3 crossed with nrg2-1 and nrg2-2, respectively. Fluorescence and light images of 4-d-old seedlings grown on
nitrate media were captured with a fluorescence microscope.
Taking together, these results indicate that the weak YFP
phenotype of Mut75 is caused by the mutation in NRG2, but
not by the mutation in EIF4G. Therefore, we focused on the
functional characterization of NRG2 in the following analyses.
NRG2 Is Required for Nitrate-Regulated Gene Expression
The defect in responding to nitrate with NRP-YFP expression in
nrg2-3 suggests that NRG2 plays an important role in nitrate
signaling. To test if NRG2 also regulates endogenous genes, the
expression of the nitrate-responsive genes NIA1, NiR, and NRT2.1
was investigated. qPCR results showed that the nitrate induction
of these genes in the roots of both nrg2-1 and nrg2-2 mutants was
significantly inhibited (Figure 3A; Supplemental Figures 2A and
2B). No difference was found for the expression of these genes
among wild type and the mutants when grown on ammonium
succinate without any nitrate treatment (Supplemental Figure 2C)
or after KCl treatment (Supplemental Figure 2D), while a significant decrease in the mutants was seen after KNO3 treatment
(Supplemental Figure 2E). The inhibited nitrate induction in the
mutants was also observed when the seedlings were treated with
a low concentration of nitrate (Supplemental Figure 2F). The
above results demonstrate that NRG2 functions in nitrate
signaling in plants.
NRG2 Plays an Essential Role in Nitrate Signaling
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Figure 3. The mutants of NRG2 Are Defective in Response to Nitrate.
(A) Nitrate induction of endogenous genes without nitrogen starvation. Wild-type and nrg2 plants were grown on medium with 2.5 mM ammonium succinate
as the sole nitrogen source for 7 d and then treated with 10 mM KNO3 or KCl as a control for 2 h. Roots were collected for RNA extraction. The transcripts of
nitrate-responsive genes were determined by qPCR. Error bars represent SD of biological replicates (n = 4). Asterisks indicate significant differences (P < 0.05)
compared with the wild type (t test).
(B) Nitrate induction of endogenous genes after nitrogen starvation. Plants were grown, treated, and analyzed as described in (A), except plants at day 6 were
transferred to nitrogen-free medium for 24 h then treated with 10 mM KNO3 or KCl for 2 h. The transcripts of nitrate-responsive genes in roots were
determined by qPCR.
Previous studies have shown that NRT1.1 acts as a nitrate
sensor and mediates nitrate responses as evidenced by the fact
that nrt1.1 mutants (chl1-5 and chl1-13) exhibited decreased
nitrate induction of the nitrate-responsive genes (NIA1, NiR, and
NRT2.1) (Ho et al., 2009; Wang et al., 2009). However, this phenotype is dependent on nitrogen pretreatment, as nitrogen
deprivation restores the wild-type phenotype in nrt1.1 mutants
(Wang et al., 2009; Krouk et al., 2010a). We tested nrg2 mutants
under both nitrate-replete and nitrogen-deprived conditions and
found that the induction levels for these nitrate-responsive genes
were reduced under both conditions compared with the wild type
(Figures 3A and 3B), indicating that NRG2 functions in nitrate
signaling regardless of nitrogen starvation. This finding contrasts
with that of NRT1.1, whose nitrate regulatory function is lost after
nitrogen starvation and shows that NRG2 functions in both types
of nitrogen conditions (nitrogen replete and nitrogen deprived).
To test if NRG2 is regulated by different nitrogen conditions, we
investigated its expression levels after nitrate, ammonium, and
nitrogen starvation treatments. The results did not show significant changes in the expression of this gene after these treatments
(Supplemental Figure 2G), indicating that the expression of NRG2
is not modulated by nitrate, ammonium, and nitrogen starvation.
NRG2 Is Predominantly Expressed in the Vascular Tissue of
Leaves and Roots, and NRG2 Protein Is Localized in
the Nucleus
The expression profile of NRG2 in wild-type plants was examined
using qPCR. Tissues were harvested either from plants grown in
soil (leaves, stems, flowers, and siliques) or from plants grown on
ammonium nitrate medium (seedlings and roots). NRG2 was
expressed in all tested tissues, with highest levels in leaves and
roots and lowest levels in flowers and siliques (Figure 4A). The
expression profile of NRG2 was further analyzed by the promoterGUS approach. The GUS staining profile is largely consistent with
the results obtained from qPCR, confirming the expression pattern
of NRG2 in the tissues tested. Moreover, GUS staining revealed
that NRG2 is predominantly expressed in the vascular bundles of
leaves and roots (Figures 4Ba to 4Bh). In addition, expression of
NRG2 was also found in stomata (Figure 4Bi), flowers (Figures 4Bj
to 4Bm), and young siliques (Figure 4Bn). In flowers, GUS expression was observed in the pistil (Figure 4Bk), junction of filament and anther (Figure 4Bl), and vascular tissue of sepals and
petals (Figure 4Bm).
To more precisely determine the cells that express NRG2, the
GUS staining in vascular bundles was further analyzed. Cross
sections of the roots showed GUS expression in the stelar cells,
including the pericycle, phloem, and parenchyma cells (Figure 4
Ca). Longitudinal and transversal sections of the leaves revealed
GUS expression in the bundle sheath, phloem, and parenchyma
cells of the vascular tissues (Figures 4Cb and 4Cc). This expression profile suggests that NRG2 may function in regulating
nitrate transport in the vasculature.
NRG2 contains two uncharacterized functional domains:
DUF630 and DUF632, which are shared by all 15 members in this
family of unknown proteins (Supplemental Figure 3). To explore
the subcellular localization of the NRG2, several subcellular localization prediction tools were used to analyze its protein sequence. NRG2 was predicted to be localized in the nucleus by
SubLoc (Chen et al., 2006) and WoLFPSORT (Horton et al., 2007)
tools, but in mitochondria by MitoPred (Guda et al., 2004). To
determine the bona fide localization of the protein, we cloned the
NRG2 cDNA and ligated the fragment in frame to be expressed
with the GFP reporter at the N-terminal position (Pro35S:GFPNRG2). The construct was transformed into Arabidopsis wild-type
plants and the protein was observed in the nucleus in stable
transgenic lines (Figure 4D). Thus, we conclude that NRG2 protein
is targeted to the nucleus.
Nitrate Content in nrg2 Mutants Is Lower Than the Wild Type
in Roots, but Not in Leaves
We demonstrated that the induction of the nitrate-responsive
genes is inhibited in the nrg2 mutants. We then tested if this
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Figure 4. NRG2 Is Predominantly Expressed in Vascular Tissues and the NRG2 Protein Is Localized in the Nucleus.
(A) Analysis of the relative expression level of NRG2 in different organs of Arabidopsis by qPCR. Tissues were harvested either from 45-d-old plants grown in
soil (leaves, stems, flowers, and siliques) or from 7-d-old plants grown in NH4NO3 liquid medium (seedlings and roots). Error bars represent SD of biological
replicates (n = 4).
(B) Histochemical staining of GUS activity in transgenic plants expressing ProNRG2:GUS. GUS activity was detectable in root (a), root tip (d), root vascular
system ([c] to [e]), vascular system of cotyledon (f) and cauline leaves ([b], [g], and [h]), stomata (i), flower (j), pistil (k), junction of filament and anther (l),
vascular tissue of sepals and petals (m), and young silique (n).
(C) GUS staining for NRG2 promoter-driven activity in vascular bundles. Cross section of the roots (a) revealed GUS expression in the stelar cells, including
pericycle, phloem, and parenchyma cells. Longitudinal section (b) and cross section of the leaves (c) revealed that the NRG2 promoter drives expression
mainly in vascular bundles including bundle sheath, phloem, and parenchyma cells. XV, xylem vessels; PL, phloem; PR, parenchyma; BS, bundle sheath.
Bars = 50 mm.
(D) Subcellular localization of NRG2 protein. (a) Confocal laser scanning microscopy and corresponding bright-field images of Arabidopsis roots. (b) Higher
magnifications of the red squares in (a); red arrows indicate the nucleus. Bars = 50 mm.
NRG2 Plays an Essential Role in Nitrate Signaling
molecular defect results in any phenotype at the morphological
and physiological levels in the mutants. Under a high nitrate
concentration, the mutant seedlings were slightly smaller and
displayed later flowering compared with the wild type (Supplemental
Figures 4A and 4B). Under low nitrate condition, no obvious
phenotype was observed (Supplemental Figure 4C).
Previous studies on several known nitrate regulatory genes
(NRT1.1, NLP7, and LBD37/38/39) have shown that the nitrate
levels in their respective mutant plants are altered (Castaings et al.,
2009; Rubin et al., 2009; Wang et al., 2009). Here, we found that the
nitrate accumulation in nrg2 mutant seedlings was significantly
lower than in the wild type (Figure 5A). Further investigation
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revealed that the nitrate accumulation in roots was significantly
lower with each mutant allele than in the wild type (Figure 5B).
However, no difference between the mutants and the wild type
was found in leaves (Figure 5C). These data indicate that the nitrate
accumulation in roots is defective, while the accumulation of
nitrate in leaves is normal in nrg2 mutants. We further assayed
the nitrate content in whole seedlings treated with various
concentrations of nitrate (0.25 to 20 mM) for 2 h in the presence of
ammonium and found that the nitrate accumulation in the mutants
(including the chl1-13 mutant as a control) was significant lower in
all concentrations tested (Figure 5D). We also tested the time
course of nitrate accumulation in whole seedlings treated with
Figure 5. NRG2 Affects Nitrate Accumulation and Uptake.
(A) to (C) Nitrate content in seedlings (A), roots (B), and leaves (C). Wild-type and nrg2 mutant plants were grown on ammonium nitrate medium for 7 d and
collected for nitrate concentration test. Error bars represent SD of biological replicates (n = 4). Asterisks indicate significant differences (P < 0.05) compared
with the wild type (t test). FW, fresh weight.
(D) and (E) Nitrate accumulation in wild type, nrg2, and chl1 mutants. Seedlings grown with 2.5 mM ammonium succinate for 7 d were treated with various
concentrations of KNO3 for 2 h (D) or treated with 5 mM KNO3 for different times in the presence of ammonium succinate (E) and then collected for nitrate
concentration test. The chl1-13 mutant was used as a control. Asterisks indicate significant differences (P < 0.05) between the wild type and two mutants.
(F) and (G) Relative expression of NRT1.1 (F) and NRT1.8 (G). Wild-type and nrg2 mutant plants were grown on ammonium nitrate medium for 7 d, and then
roots and shoots were collected separately for RNA extraction. The transcription levels of NRT1.1 and NRT1.8 were determined by qPCR. Error bars
represent SD of biological replicates (n = 4). Asterisks indicate significant differences (P < 0.05) compared with the wild type (t test).
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5 mM KNO3, and the results showed that the nitrate uptake in the
mutants was significantly lower than in the wild type at all time
points tested (Figure 5E). These findings suggest that nitrate
uptake is affected in nrg2 mutants.
To investigate the mechanisms for the lower nitrate accumulation in mutant roots, the expression of several known nitrate
transport genes was studied. Among the 13 transport genes
tested, only the expression of NRT1.1 was significantly decreased
under ammonium nitrate conditions (Figure 5F), while no change
was found for the other 12 tested genes (Supplemental Figure 5)
in the mutant roots. In leaves, only the transcript levels of
NRT1.8 were significantly increased (Figure 5G), and there was
no significant change in the expression of the other genes
(Supplemental Figure 6) in the mutants. Previous studies have
shown that NRT1.1 functions as a dual-affinity nitrate transporter
involved in transporting nitrate from the environment into roots
(Tsay et al., 1993; Wang et al., 1998; Liu and Tsay, 2003). Thus, the
decreased nitrate content in mutant seedlings may be caused by
the decreased expression of NRT1.1. NRT1.8 functions in removing nitrate from xylem vessels, as the functional disruption of
NRT1.8 increased nitrate concentration in xylem sap (Li et al., 2010).
Thus, it is possible that the increased expression of NRT1.8 may
direct more nitrate to be unloaded from xylem vessels resulting in
similar nitrate levels in the mutant leaves to those in wild-type
leaves. The expression of several key nitrate assimilatory genes
(NIA1, NIA2, NiR, GLN1.1, and GLN1.3) was also detected by
qPCR, and no significant difference was found between the wild
type and nrg2 mutants (Supplemental Figure 7). Therefore, these
results imply that the lower nitrate content in mutant roots may be
correlated with the reduced expression of NRT1.1 in roots and the
increased transcripts of NRT1.8 in leaves.
NRG2 Regulates the Expression and Works Upstream
of NRT1.1
To understand the relationship of NRG2 and the characterized
nitrate regulators, we first investigated the expression levels of
several known nitrate regulators in the nrg2 mutants under different nitrogen conditions. The results showed that, among the
investigated known regulatory genes, the expression of NRT1.1
in the nrg2 mutants was significantly decreased, to <40% of
the expression in wild-type plants under potassium nitrate or
Figure 6. The Expression of NRT1.1 in the nrg2 Mutants Was Reduced.
(A) Relative expression of NRT1.1 in nrg2 mutants. Wild-type and nrg2 mutant plants were grown on media with KNO3 or NH4NO3 for 7 d, and whole seedlings
were collected for gene expression detection. Error bars represent SD of biological replicates (n = 4). Asterisks indicate significant differences (P < 0.05)
compared with the wild type (t test).
(B) Relative expression of NRG2 in nrt1.1 mutants. Wild-type, chl1-5, and chl-13 plants were grown on media with KNO3 or NH4NO3 for 7 d, and whole
seedlings were collected for gene expression detection. Error bars represent SD of biological replicates (n = 4).
(C) Nitrate induction of NRT1.1 in the wild type and the nrg2 mutants. Plants were grown on medium with 2.5 mM ammonium succinate as the sole nitrogen
source for 7 d and then treated with 10 mM KNO3 or KCl as a control for 2 h. Roots were collected for RNA extraction. The relative expression of NRT1.1 was
determined by qPCR. Error bars represent SD of biological replicates (n = 4). Asterisks indicate significant differences (P < 0.05) compared with the wild type
(t test).
NRG2 Plays an Essential Role in Nitrate Signaling
ammonium nitrate conditions (Figure 6A). The expression of
other known nitrate regulatory genes tested was not changed
(Supplemental Figure 8). This indicates that the expression of
NRT1.1 is regulated by NRG2.
To test if NRG2 is regulated by known nitrate regulators, we
measured NRG2 transcript levels in the mutants of several
493
identified regulatory genes (NRT1.1, NLP7, CIPK8, and
CIPK23) in nitrate or ammonium nitrate media. No change
was found for the expression of NRG2 in these mutants
(Supplemental Figure 9), including in nrt1.1 mutants (Figure 6B).
These results imply that NRG2 may not be regulated by these
four genes.
Figure 7. NRG2 and NRT1.1 Work in the Same Nitrate Signaling Pathway.
(A) Root fluorescence phenotypes of wild-type, nrg2-3, chl1-13, and chl1-13 nrg2-3 plants on KNO3 medium. The plants were grown on KNO3 medium for 4
d. Fluorescence and light images were captured with a fluorescence microscope to visualize YFP expression.
(B) Quantification of root fluorescence of wild-type, nrg2-3, chl1-13, and chl1-13 nrg2-3 plants. The plants were grown under the same conditions as in (A).
Error bars represent SD (n = 60). Different letters indicate statistically significant difference (P < 0.05, t test).
(C) Root fluorescence phenotypes of wild-type, nrg2-3, chl1-13, and chl1-13 nrg2-3 plants on NH4NO3 medium. The plants were grown on NH4NO3 medium
for 4 d. Fluorescence and light images were captured with a fluorescence microscope to visualize YFP expression.
(D) Quantification of root fluorescence of wild-type, nrg2-3, chl1-13, and chl1-13 nrg2-3 plants on NH4NO3 medium. The seedlings were grown on the same
NH4NO3 medium as (C). Error bars represent SD (n = 60). Different letters indicate statistically significant difference (P < 0.05, t test).
(E) Nitrate induction of gene expression in wild-type, nrg2-3, chl1-13, and chl1-13 nrg2-3 plants. The seedlings were grown and treated as described in
Figure 3A. The transcripts of nitrate-responsive genes in roots were measured by qPCR. Error bars represent SD of biological replicates (n = 4). Different
letters indicate statistically different means (P < 0.05, t test).
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The Plant Cell
To study further the effects of NRG2 on NRT1.1, we examined
the nitrate induction of NRT1.1 in the wild type and the nrg2
mutants. The results showed a significant decrease in the nitrate
induction levels in the mutants (Figure 6C), indicating that NRG2
affects the nitrate induction of NRT1.1. We also tested the expression of NRT1.1 in the absence of nitrate and found that the
expression of NRT1.1 was significantly lower in the mutants
compared with that in the wild type (Supplemental Figure 10A).
When seedlings grown on NH4NO3 medium were subjected to
nitrogen starvation, the expression of NRT1.1 increased during the
first 24 h in both the wild type and the mutants and no significant
differences in expression levels were found for the time points
tested between wild type and the nrg2 mutants (Supplemental
Figure 10B).
To understand better the relationship of NRG2 and NRT1.1,
a double mutant of the two genes was obtained by crossing the
single mutants of each gene: nrg2-3 and chl1-13 isolated by our
mutant screens. The YFP signal from the NRP-YFP transgene in
roots of the double mutant seedlings grown on nitrate medium
(with no ammonium) was detected and found to be much weaker
than in the wild type and similar to nrg2-3 while weaker than
chl1-13 (Figure 7A). Quantifying the root fluorescence signal
confirmed the weaker YFP signal in double mutant than in the wild
type and chl1-13 and was similar to nrg2-3 (Figure 7B). Notably,
the signal in chl1-13 was much higher than in nrg2-3 and double
mutant and mildly lower than in the wild type. We also tested the
YFP levels in roots of the single and double mutants grown on
ammonium nitrate medium to investigate the function of the genes
in the presence of ammonium. The observation and fluorescence
quantification data showed that the YFP levels in the double
mutant were similar to those of chl1-13 while lower than of nrg2-3
(Figures 7C and 7D). Interestingly, the chl1-13 exhibited much
lower signal than the wild type, confirming that NRT1.1 function in
the nitrate signaling pathway is ammonium dependent.
To provide further molecular evidence, we inspected the expression of nitrate-responsive genes in the wild type and these
single and double mutants. The qPCR results showed that the
expression levels of the nitrate-responsive genes in the double
mutant chl1-13 nrg2-3 were similar to those in single mutant
chl1-13 and much lower than in the wild type (Figure 7E).
Additionally, we transformed the cDNA of NRT1.1 into the
nrg2-2 mutant to investigate further the relationship between
NRT1.1 and NRG2. The nitrate content in roots of nrg2-2 was recovered to the wild-type level when NRT1.1 was overexpressed in
the mutant (Figure 8A; Supplemental Figure 10C). We also detected
the expression levels of the nitrate-responsive genes NIA1, NiR, and
NRT2.1 and found that nitrate induction of these genes was recovered to the wild-type phenotype in NRT1.1/nrg2-2 (Figure 8B).
Taken together, these results suggest that NRG2 and NRT1.1
work in the same nitrate signaling pathway and that NRG2
functions upstream of NRT1.1.
Genetic and Molecular Analysis Reveals that NRG2 and
NLP7 Have Nonoverlapping Functions in Nitrate Regulation
NLP7 is an important nitrate regulator in nitrate signaling
(Castaings et al., 2009; Konishi and Yanagisawa, 2013; Marchive
et al., 2013). To investigate the relationship between NRG2 and
NLP7 in the process of regulating nitrate response, a double
mutant of the two genes was generated by crossing the respective
single mutants nrg2-3 and nlp7-4 isolated by our mutant screen
system. The mutant nlp7-4 harbors a mutation (C to T) in NLP7 that
converts Gln at the position 62 to a stop codon, resulting in lower
YFP fluorescence in roots when grown on nitrate-containing
media (Figures 9A to 9D). In the presence of nitrate without ammonium, both single mutants showed much lower YFP fluorescence in roots than the wild type, with 23% of the wild type for
nlp7-4 and 35% of the wild type for nrg2-3 in terms of fluorescence
intensity, respectively. Interestingly, the double mutant plants
exhibited even lower YFP signal than the individual single mutants,
with only 13% of the wild type (Figures 9A and 9B). Under ammonium nitrate conditions, similar results were obtained as under
Figure 8. NRG2 Functions Upstream of NRT1.1.
(A) Nitrate content in roots. Wild-type, nrg2-2, and NRT1.1/nrg2-2 plants were grown on ammonium nitrate medium for 7 d, and the roots were collected for
nitrate concentration analysis. Error bars represent SD of biological replicates (n = 4). Asterisks indicate significant differences (P < 0.05) compared with the
wild type (t test).
(B) Nitrate induction of the endogenous genes. Wild-type, nrg2-2, and NRT1.1/nrg2-2 plants were grown on medium with 2.5 mM ammonium succinate as
the sole nitrogen source for 7 d and then treated with 10 mM KNO3 or KCl as a control for 2 h. The roots were collected for RNA extraction. The transcript levels
of nitrate-responsive genes were determined by qPCR. Error bars represent SD of biological replicates (n = 4). Asterisks indicate significant differences (P <
0.05) compared with the wild type (t test).
NRG2 Plays an Essential Role in Nitrate Signaling
nitrate conditions, with 20, 36, and 15% of the wild type for nlp7-4,
nrg2-3, and nlp7-4 nrg2-3 mutants, respectively (Figures 9C and
9D). These results suggest that NRG2 and NLP7 play important
roles in nitrate regulation in nonoverlapping ways. Our qPCR
results showed that the expression of NLP7 was not altered in nrg2
mutants (Supplemental Figure 8) and the expression of NRG2 was
not changed in nlp7 mutant either (Supplemental Figure 9), so that
there was no evidence for transcriptional regulation of these genes
by each other.
We also detected the expression of the nitrate-responsive
genes in the wild type and in single and double mutants. The qPCR
495
results revealed that the transcripts of the three tested genes in
double mutant nlp7-4 nrg2-2 were significantly lower than in both
single mutants (Figure 9E), further showing that NRG2 and NLP7
function in nonoverlapping ways to regulate nitrate responses.
NRG2 Interacts with NLP7 but Does Not Affect the Nuclear
Retention of NLP7 in Response to Nitrate
Previous studies have shown that NLP7 is mainly expressed in the
vascular tissue and the protein is targeted to the nucleus after
nitrate treatment (Castaings et al., 2009; Marchive et al., 2013),
Figure 9. Analysis of Nitrate Regulation in nrg2 nlp7 Double Mutants.
(A) Root fluorescence phenotypes of the wild type, nrg2-3 and nlp7-4 single mutants, and nlp7-4 nrg2-3 plants on KNO3 medium. The plants were grown on
KNO3 medium for 4 d. Fluorescence and light images were captured with a fluorescence microscope to visualize YFP expression.
(B) Quantification of root fluorescence of wild-type, nrg2-3, nlp7-4, and nlp7-4 nrg2-3 plants. The plants were grown on the same condition as (A). Error bars
represent SD (n = 60). Different letters indicate statistically significant difference (P < 0.05, t test).
(C) Root fluorescence phenotypes of wild-type, nrg2-3, nlp7-4, and nlp7-4 nrg2-3 plants on NH4NO3 medium. The plants were grown on NH4NO3 medium for
4 d. Fluorescence and light images were captured with a fluorescent microscope to visualize YFP expression.
(D) Quantification of root fluorescence of wild-type, nrg2-3, nlp7-4, and nlp7-4 nrg2-3 plants on NH4NO3 medium. The seedlings were grown on the same
NH4NO3 medium as (C). Error bars represent SD (n = 60). Different letters indicate statistically significant difference (P < 0.05, t test).
(E) Nitrate induction of gene expression in wild-type, nrg2-2, nlp7-4, and nlp7-4 nrg2-2 plants. The seedlings were grown and treated as described in Figure
3A. The transcripts of nitrate-responsive genes in roots were determined by qPCR. Error bars represent SD of biological replicates (n = 5). Different letters
indicate statistically different means (P < 0.05, t test).
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The Plant Cell
which is similar to the expression pattern of NRG2. Even though
these two proteins show no evidence of genetic or transcriptional interaction, it is possible that the two proteins can interact
at the protein level. To test this idea, yeast two-hybrid assays
were performed. Indeed, NRG2 and NLP7 cotransformed yeast
(Saccharomyces cerevisiae) cells grew well, while each gene and
empty vector cotransformed yeast cells used as negative controls
could not grow on the selective media (Figure 10A), indicating that
NRG2 protein can directly interact with NLP7 in vitro.
To confirm the interaction between NRG2 and NLP7, in vivo
tests with bimolecular fluorescence complementation (BiFC)
assays on Nicotiana benthamiana leaves was performed. A direct
Figure 10. NLP7 Interacts with NRG2.
(A) Yeast two-hybrid assay of the NRG2 and NLP7 interaction. Serial dilution of yeast cells containing the indicated constructs was spotted on the indicated
medium for lacZ and His reporter assays (four independent experiments). pGADT7, empty prey vector; pGBKT7, empty bait vector; NRG2, a bait vector
containing cDNA of NRG2; NLP7, a prey vector containing cDNA of NLP7. SD/TW-, SD medium lacking tryptophan and leucine; SD/LWHA-, SD medium
lacking tryptophan, leucine, histidine, and adenine with X-a-Gal.
(B) BiFC analysis for interaction between NRG2 and NLP7. N- and C-terminal fragments of YFP were fused to NRG2 and NLP7, respectively. Different
combinations of expression vectors encoding NRG2-YFPN and NLP7-YFPC and controls (indicated on the left of the panel) were transformed into leaves of
N. benthamiana grown on NH4NO3 medium. Presence of YFP signal indicates reconstitution of YFP through protein interaction of the tested pairs.
N. benthamiana cells showing YFP fluorescence in the nucleus were observed and marked by red arrows. Bar = 5 mm.
NRG2 Plays an Essential Role in Nitrate Signaling
interaction was observed between NRG2 and NLP7 in the nucleus
of plant cells, where coexpression of NRG2-YFPN and
NLP7-YFPC reconstituted a functional YFP, whereas no significant signals were found in controls lacking NRG2 or NLP7 (Figure
10B). The direct interaction between NRG2 and NLP7 proteins in
the nucleus was also observed on infiltrated leaves from plants
with starvation pretreatment, while no significant signals were
seen in controls lacking NRG2 or NLP7 (Supplemental Figure 11).
These in vitro and in vivo results demonstrate the direct interaction
of NRG2 and NLP7 in the nucleus.
As the nuclear retention of NLP7 is regulated by nitrate
(Marchive et al., 2013), we wanted to determine if NRG2 is involved
in the nitrate-induced nuclear retention of NLP7. Therefore, we
checked the NLP7 subcellular localization in nrg2-2 mutant lines
transformed with the NLP7-YFP construct and grown in the
presence or absence of nitrate. The confocal images showed that
localization of NLP7 protein was indistinguishable between
wild-type and nrg2 mutant plants (Figure 11). This finding indicates
that the nuclear retention of NLP7 is not dependent on NRG2.
Transcriptomic Analysis of Nitrate Response in nrg2, chl1,
and nlp7 Mutants
To investigate systematically the molecular mechanism by which
NRG2 mediates plant responses to nitrate, and to probe the relationships among NRG2, NRT1.1, and NLP7, we performed
497
a comparative RNA-seq analysis. The seedlings of the wild type
and nrg2-2, chl1-13, and nlp7-4 mutants were grown on medium
with 2.5 mM ammonium succinate for 7 d and then treated with
either 10 mM KNO3 or KCl for 2 h. The total root RNA analyzed
using an Illumina HiSeq 2500. For each genotype and NO32
treatment, three biological replicates were tested. After filtering
low-quality reads and removing reads that aligned to rRNA or
tRNA, we selected 435,055,962 reads for analysis (Supplemental
Data Set 1). Twofold change in gene expression levels and adjusted P value < 0.05 were used as a cutoff value to select differentially expressed transcripts.
We first compared the gene expression in the roots of wild-type
and nrg2 mutant plants in response to nitrate treatments. The
results (Figure 12A; Supplemental Data Set 2) showed that the
transcripts of 276 genes (including 117 upregulated and 159
downregulated) were altered in the wild type after nitrate treatment, but not in nrg2 mutant. In other words, the expression of
these nitrate-responsive genes in the wild type was suppressed in
nrg2 mutant. The transcripts of 131 genes (88 induced and 43
suppressed) were changed in the nrg2 mutant, but not in the wild
type. In addition, the expression of 314 genes were regulated by
nitrate in both the wild type and nrg2 mutant, among which 148
genes (107 suppressed and 41 induced) were differentially
expressed by more than 25% in the wild type and the mutant
(Supplemental Data Set 3). Therefore, the mutation in NRG2
results in a total of 555 genes with altered expression after nitrate
Figure 11. The Nuclear Retention of NLP7-YFP Is Not Regulated by NRG2.
Subcellular localization of NLP7-YFP in the wild type ([A] and [C]) and nrg2-2 ([B] and [D]) mutants. Seedlings in (A) and (B) were grown on nitrate medium,
and seedlings in (C) and (D) were treated with nitrogen deprivation. Fluorescence and corresponding bright-field pictures were captured by confocal laser
scanning microscopy. Bar = 50 mm.
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The Plant Cell
To clarify the relationship among NRG2, NRT1.1, and NLP7
genes, transcriptomic analysis was performed using roots treated
with nitrate. The genes with differentially induced expression in
nrg2, nrt1.1, and nlp7 mutants compared with the wild type were
analyzed and are shown in the Venn diagram in Figure 12B. The
expression of 235 genes was found to be changed in all three
mutants with 57.7, 48.5, and 44.5% of the total differentially
expressed genes in nrg2, nrt1.1 (chl1), and nlp7 mutants, respectively (Supplemental Data Set 5), indicating that these three
genes are closely involved in nitrate regulation in plants.
In the nrt1.1 mutant, the transcripts of 485 genes were altered
compared with those in the wild type, among which 277 genes
(57.1% of 485) were also regulated by NRG2 (Figure 12B;
Supplemental Data Set 6). Those genes that were regulated by
both NRT1.1 and NRG2 were further investigated by GO analysis,
and the results showed that a nitrogen compound transport
cluster was involved, including some members of NRT/PTR family
(Table 3; Supplemental Data Set 6). These data support the
conclusion that NRG2 works in the same nitrate signaling pathway
as NRT1.1.
For the mutant nlp7, 276 genes were found to be regulated by
both NRG2 and NLP7 (Figure 12B; Supplemental Data Set 7);
however, 252 NLP7-regulated genes and 131 NRG2-regulated
genes were not (i.e., were regulated by only NLP7 or NRG2 and not
the other; Figure 12B). In addition, no nitrate-related cluster in
these genes was found by GO analysis (Supplemental Data Set 7).
This result provides further evidence that NRG2 and NLP7 have
some independent functions in nitrate regulation.
DISCUSSION
Figure 12. RNA-Seq Analyses of Differentially Expressed Transcripts in
the Roots of Wild-Type, nrg2-2, chl1-13, and nlp7-4 Seedlings Grown on
Ammonium Succinate Followed by Nitrate Treatment.
(A) Venn diagram showing the number of the genes up- or downregulated
by nitrate treatment in the wild type and nrg2-2 mutant.
(B) Diagram showing the number of the genes differentially expressed in
nrg2-2, chl1-13, and nlp7-4 mutants compared with the wild type.
treatment. Many known nitrate-inducible and regulatory genes,
including NiR, NRT2.1, HHO1, UPM1, LBD37, LBD38, NRT1.1,
TGA1, and TGA4, showed reduced nitrate induction in the
mutant (Table 1). To explore the data further, we performed Gene
Ontology (GO) analysis using Panther (http://www.pantherdb.org/
pathway) for these differentially expressed 555 genes. Major GO
clusters for all analyzed genes are listed in Table 2, and four
clusters were found to be related to nitrogen, including response to nitrogen compound, nitrogen compound transport,
response to nitrate, and nitrate transport (Supplemental Data
Set 4). These data strongly support our conclusion that NRG2
functions in the nitrate signaling. In addition, GO analysis
revealed that genes most affected by the mutation in NRG2 were
overrepresented in 20 clusters (P value < 0.001), including response to stimulus, response to chemical, ion transport, organic substance, oxygen-containing compound, stress, and
hormone (Table 2).
To adapt to the changing nitrate conditions in the environment,
plants have evolved diverse mechanisms to maintain normal
growth and development. A sophisticated gene network is
thought to regulate the responses to nitrate in plants. However,
only the several nitrate regulatory genes mentioned in the Introduction have been characterized thus far using systems biology and reverse genetics approaches. In this article, we
performed a forward genetic screen and isolated the mutant
Mut75 defective in nitrate signaling. Mapping revealed that the
mutation in the gene At3g60320 (designated NRG2) resulted in the
Table 1. Known Nitrate-Inducible and Regulatory Genes with Different
Levels of Nitrate-Responsive Expression in the Wild Type and nrg2-2
Mutant
Gene
Fold Change in
the Wild Type
P Value
Fold Change
in Mutant
P Value
NiR
NRT2.1
HHO1
UPM1
LBD37
LBD38
NRT1.1
TGA1
TGA4
26.35
11.25
400.49
14.19
11.01
3.28
3.63
4.17
3.55
3.03E-30
5.10 E-4
3.12 E-14
2.48E-55
6.19E-35
1.51 E-11
4.99 E-15
9.70 E-17
6.11 E-18
10.91
3.38
6.18
7.25
6.92
2.23
2.69
3.20
2.65
9.69
2.52
4.66
6.08
5.02
1.63
7.11
6.40
8.65
E-17
E-3
E-11
E-18
E-24
E-5
E-12
E-25
E-10
NRG2 Plays an Essential Role in Nitrate Signaling
Table 2. GO Cluster Analysis for Genes Differentially Expressed in the
Wild Type and the nrg2 Mutant after Nitrate Treatment
GO Term
P Value
Response to nitrogen compound
Response to stimulus
Response to chemical
Anion transport
Response to endogenous stimulus
Response to organic substance
Inorganic anion transport
Response to oxygen-containing compound
Ion transport
Nitrogen compound transport
Response to nitrate
Nitrate transport
Response to stress
Response to acid chemical
Response to hormone
Cellular hormone metabolic process
Response to external stimulus
Response to other organism
Response to external biotic stimulus
5.61E-09
1.19E-07
2.18E-07
1.3 E-06
2.32E-06
3.98E-06
4.43E-06
4.47E-06
7.53E-06
8.29E-06
1.19E-05
1.57E-05
9.35E-05
9.72E-05
2.24E-04
4.29E-04
6.13E-04
7.34E-04
7.34E-04
phenotype. This gene NRG2 belongs to a gene family with 15
members in Arabidopsis (Supplemental Figure 3). Each member
contains DUF632 and DUF630 domains whose functions are
still unknown, as none of the proteins in this family have been
characterized thus far.
Our results show that the induction of nitrate-responsive genes
in nrg2 mutants is inhibited when plants are treated with nitrate in
the presence of ammonium (Figure 3A), indicating that NRG2 is
a nitrate-regulatory gene. Remarkably, this phenotype was not
restored after nitrogen starvation, which is different from nrt1.1
mutants (Wang et al., 2009). Although nrt1.1 mutants have been
studied for more than 10 years, the inhibition of nitrate induction in
the mutants had not been found until it was tested in the presence
of ammonium (Tsay et al., 1993; Ho et al., 2009; Wang et al., 2009).
It has also been reported that the expression of nitrate-induced
genes in the presence of ammonium was inhibited in cipk8
mutants but enhanced in cipk23 mutants compared with the wild
type after nitrate treatment (Ho et al., 2009; Hu et al., 2009).
Overexpression lines of SPL9 have been monitored as well in the
presence of ammonium, and the induction of the nitrateresponsive genes was increased after nitrate treatment (Krouk
et al., 2010b). Nevertheless, it has not been tested if this phenotype can be recovered after nitrogen depletion for these
mutants. On the contrary, nlp7, tga1/tga4, and lbd37/38/39
mutants have been analyzed after nitrogen starvation and the
results showed inhibited induction of nitrate-responsive genes in
nlp7 and tga1/tga4 mutants but higher induction in lbd37/38/39
mutants after nitrate treatments (Castaings et al., 2009; Rubin
et al., 2009; Alvarez et al., 2014). However, the expression of
nitrate-induced genes in these mutants has not been investigated
without nitrogen starvation to date. The mutant nlp7-4 showed
weaker YFP fluorescence in roots when grown on ammonium
nitrate medium and reduced induction of the nitrate-responsive
genes after nitrate treatments than in the wild type (Figures 9A to
499
9E), suggesting that NLP7 modulates the nitrate signaling in the
presence of ammonium. Our results, combined with previous
studies, reveal that some genes function as nitrate-regulatory
players in an ammonium-dependent manner, while some play
important roles in nitrate signaling regardless of ammonium.
Therefore, we propose that nitrate regulators may work in at least
two different ways: (1) regulating nitrate responses in the presence
of ammonium, such as NRT1.1, and (2) functioning as nitrate
regulators regardless of ammonium, as represented by NRG2 and
NLP7. A third way may exist that modulates nitrate response only
in the absence of ammonium. The different signaling mechanisms
under conditions with and without ammonium reflect the complexity with which plants adapt to the changing environments.
To understand the physiological effects caused by the mutation
in NRG2, the nitrate accumulation in nrg2 mutants was tested. Our
results showed that the nitrate content in seedlings was significantly lower than that in the wild type. This defect may result from
reduced uptake and/or increased reduction and assimilation.
Further analysis by determining nitrate content in both leaves and
roots revealed lower nitrate levels in roots, indicating that NRG2 is
involved in regulating nitrate accumulation in roots. Previous
studies have shown that several characterized nitrate regulatory
genes play important roles in plant nitrate homeostasis. In nrt1.1
mutant seedlings, the nitrate concentration is lower than in
wild-type plants (Wang et al., 2009). On the contrary, the nitrate
content in nlp7 mutants was found to be higher than in the wild
type, which might result from the decreased nitrate reduction and
assimilation (Castaings et al., 2009). In addition, LBD37, 38,
and 39 overexpression lines displayed lower nitrate content and
Table 3. Genes Involved in a Nitrogen-Related Cluster Regulated by
Both NRG2 and NRT1.1
AGI
Description
AT5G47330
AT5G46050
AT5G11570
AT2G02990
AT1G33440
AT1G30840
AT4G19680
AT5G41800
AT1G14780
At5g47330; AT5G47330; ortholog
Protein NRT1/PTR FAMILY 5.2; NPF5.2; ortholog
Protein NRT1/PTR FAMILY 1.3; NPF1.3; ortholog
Ribonuclease 1; RNS1; ortholog
Protein NRT1/PTR FAMILY 4.4; NPF4.4; ortholog
Probable purine permease 4; PUP4; ortholog
Fe2+ transport protein 2; IRT2; ortholog
Probable GABA transporter 2; At5g41800; ortholog
MACPF domain-containing protein At1g14780;
At1g14780; ortholog
Alpha-dioxygenase 1; DOX1; ortholog
UDP-glycosyltransferase 71C1; UGT71C1; ortholog
Monothiol glutaredoxin-S13; GRXS13; ortholog
Protein NRT1/PTR FAMILY 8.1; NPF8.1; ortholog
Peroxidase 69; PER69; ortholog
Protein NRT1/PTR FAMILY 5.12; NPF5.12; ortholog
Protein GLUTAMINE DUMPER 3; GDU3; ortholog
Probable purine permease 18; PUP18; ortholog
Berberine bridge enzyme-like protein; AT5G44390;
ortholog
1-Aminocyclopropane-1-carboxylate oxidase
homolog 12; At5g59540; ortholog
Organic cation/carnitine transporter 3; OCT3; ortholog
Cytokinin riboside 59-monophosphate
phosphoribohydrolase LOG7; LOG7; ortholog
AT3G01420
AT2G29750
AT1G03850
AT3G54140
AT5G64100
AT1G72140
AT5G57685
AT1G57990
AT5G44390
AT5G59540
AT1G16390
AT5G06300
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The Plant Cell
Figure 13. NRG2 Plays a Key Role in Nitrate Regulation.
(A) NRG2 regulates nitrate response in the presence of ammonium. Under the conditions with NH4+, NRG2 regulates the expression of NRT1.1 and NRT1.1
modulates the expression of CIPK8 and CIPK23. CIPK23 negatively affects the expression of nitrate-responsive genes. The proteins NRT1.1 and CIPK23
interact with each other, and NRT1.1 is phosphorylated by CIPK23 under low-nitrate conditions to maintain its high affinity for nitrate. NLP7 is a positive
regulator involved in the nitrate signaling. NRG2 and NLP7 work in different pathways in nitrate signaling whereas both proteins can interact with each other.
(B) NRG2 regulates nitrate signaling after nitrogen starvation (no ammonium). After experiencing nitrogen starvation, NRG2 can still interact NLP7 and both
proteins play key roles in the primary nitrate response.
decreased maximal nitrate reductase activity compared with
wild-type plants. The defects in nitrate content may be caused by
the reduced nitrate transport activity as the expression of several
high-affinity nitrate transport genes was strongly decreased
(Rubin et al., 2009). Among these characterized nitrate transport
and assimilation genes tested, only NRT1.1 exhibited decreased
expression in nrg2 mutant roots, and NRT1.8 displayed increased
expression in mutant leaves. NRT1.1 has been characterized as
a dual affinity nitrate transporter involved in absorbing nitrate from
the environment (Wang et al., 1998; Liu et al., 1999; Liu and Tsay,
2003). Thus, the lower nitrate content phenotype in nrg2 mutants
may be caused, at least partially, by the reduced expression of
NRT1.1. NRT1.8 has been identified to be a low-affinity nitrate
transporter with a function in unloading nitrate from xylem. The
higher expression of NRT1.8 in mutant leaves may lead to relatively more nitrate transport into leaves despite the relatively lower
nitrate absorption from the medium, resulting in decreased nitrate
levels in roots but similar levels in leaves compared with the wild
type. Taken together, these data suggest that NRG2 is involved in
nitrate accumulation in plants and the altered nitrate accumulation
in the mutants may result from modulated expression of NRT1.1
and NRT1.8. It is also possible that some other uncharacterized
nitrate transporters contribute to the modified nitrate concentration in the mutants.
NRT1.1 plays an essential role in nitrate regulation through its
functions in dual-affinity nitrate transport, nitrate sensing, and
auxin transport. Nevertheless, how it is regulated, i.e., what
genes can modulate the expression of NRT1.1, remains to be
characterized. Our molecular and genetic data demonstrated
that NRG2 can regulate the expression of NRT1.1 and both
genes may work in the same pathway of nitrate signaling. This
finding is of great importance for further understanding of the
regulation of NRT1.1 and adds a key component into the nitrate
signaling network.
NLP7 acts as a master regulator in response to nitrate
(Castaings et al., 2009; Konishi and Yanagisawa, 2013;
Marchive et al., 2013). As a transcription factor, NLP7 can bind
the promoter of many genes involved in nitrate signaling and
assimilation, modulate the expression of nitrate responsive
genes, and regulate nitrogen assimilation genes (Konishi and
Yanagisawa, 2013). Whether NLP7 functions with other proteins
by interacting or acts solely in the nitrate regulation is still unclear. Our molecular and genetic analysis showed that NRG2
and NLP7 have some nonoverlapping functions as the phenotype of the double mutant is more severe than that of either
single mutant. However, both proteins can physically interact in
vitro and in vivo as revealed by yeast two-hybrid and BiFC
assays, indicating that these proteins likely converge on part of
the nitrate signaling pathways as well as functioning independently. In addition, the nuclear retention of NLP7 in response
to nitrate is not affected by the mutation in NRG2. These
results further strengthen our understanding of nitrate signaling
mechanisms.
Our comparative RNA-seq analysis of the roots in response to
nitrate showed that many genes involved in nitrogen-related
clusters, including nitrate transport and response to nitrate, were
differentially expressed in the nrg2 mutant, providing further evidence that NRG2 plays an important role in nitrate signaling.
Molecular and genetic evidence indicates that NRG2 and NRT1.1
works in the same pathway in nitrate regulation. This would lead us
to predict that both genes may regulate some common nitraterelated genes. Indeed, the transcriptomic analysis revealed that
a group of genes involved in a nitrogen compound transport
cluster were modulated by NRG2 and NRT1.1 coordinately. No
common group of genes involved in nitrogen-related clusters
was found to be regulated by NRG2 and NLP7, in accordance
with the conclusion that both genes function independently in
nitrate signaling.
NRG2 Plays an Essential Role in Nitrate Signaling
Taken together, the regulation of NRT1.1 by NRG2 and the
physical interaction of NRG2 and NLP7 highlight the importance
of NRG2 as a key player in the nitrate regulatory network. Thus, we
propose the working model shown in Figure 13. In the presence of
ammonium, NRG2 regulates the expression of NRT1.1, while
NRT1.1 modulates the expression of other downstream genes
including CIPK8 and CIPK23. NRG2 and NLP7 both act as positive
regulators of nitrate assimilatory genes with some independent
functions, and they physically interact, suggesting they converge
in part of the nitrate signaling pathway. After nitrogen starvation
(no ammonium), NRG2 and NLP7 appear to function in a similar
manner, acting as positive regulators with some independent
functions while physically interacting. The relationship between
NRG2 and other known regulatory players remains to be investigated. In the meantime, NRG2 is the first member of a 15member Arabidopsis gene family (Supplemental Figure 3) to be
characterized. What roles other members may play in nitrate
signaling and what functions the two DUF domains shared by each
member carry out are interesting questions for future work. Using
the amino acid sequence of NRG2 as a query to search different
species revealed that this family exists broadly in plants from
moss to crops (rice, maize, soybean, etc.) and trees (apple,
peach, poplar, etc.) (www.greenphyl.org/cgi-bin/blast.cgi), but no
homologs were found in microbes or animals (http://blast.ncbi.
nlm.nih.gov/Blast.cgi), indicating that this family exists specifically
in plants. The characterization of the NRG2 opens a door to reveal
the roles of these family members.
501
microscope (Nikon Eclipse Ti-S). The fluorescence of roots was quantified
using ImageJ.
qPCR Analysis
RNA samples were prepared using a total RNA miniprep kit (CWBIO).
Real-time PCR was performed using the reagent kit ABI7500 Fast (Applied
Biosystems). Template cDNA samples were prepared using the RevertAid
first-strand synthesis system kit (Thermo Scientific) with 1 mg of total RNA
in a reaction volume of 20 mL. The cDNA synthesis reaction mixture was
diluted 20-fold before being used for qPCR. The FastStart Universal SYBR
Green Master Q-PCR kit (Roche Diagnostics) was used in the qPCR reaction following the instructions provided by the manufacturer. TUB2
(At5g62690) was used as the internal reference gene.
Expression Analysis by Promoter-GUS Assay
The 2951-bp promoter fragment located immediately upstream of the
NRG2 start codon was cloned in front of the GUS gene in the binary vector
pMDC162 (Invitrogen). Transgenic Arabidopsis (Col-0) plants expressing
the GUS gene were obtained and GUS activity in different organs was
detected as described (Dai et al., 2014). For section observation, roots and
leaves of the transgenic plants were fixed and embedded in paraffin
(Sigma-Aldrich). Sections were cut at 8 mm using a microtome (Leica
RM2235) and mounted on glass slides. Ruthenium red (100 mg/L) solution
was added onto the sectioned samples on slides for 1 min and then the
slides were observed and photographed with a microscope (Nikon Eclipse
Ni) equipped with a camera (Nikon Digital Sight DS-Qi1Mc).
Subcellular Localization Test
METHODS
Plant Materials
The wild-type Arabidopsis thaliana ecotype used in this study is Col-0. The
mutant lines chl1-13 (original name Mut21) (Wang et al., 2009), cipk8-1 (Hu
et al., 2009), and cipk23-3 (Ho et al., 2009) were described previously.
The full-length cDNA of NRG2 was introduced in frame with the GFP
reporter gene in the binary vector pMDC43 (Invitrogen) to generate
a fusion protein with GFP at the N-terminal position. The construct was
transformed into Arabidopsis (Col-0) plants as described previously
(Feng et al., 2008). The images were captured using confocal microscope
(Leica TCS SP5II).
Nitrate Assay
Mutagenesis and Mutant Screen
Homologous backcrossed transgenic seeds containing the NRP-YFP
construct were treated with ethyl methanesulfonate (Wang et al., 2009), and
M2 seedlings were screened on nitrate medium (initial medium with 10 mM
KNO3) based on the previous report (Wang et al., 2009). Mutants were
selfed and retested. Confirmed mutants were backcrossed to the transgenic wild type twice and homozygous lines were identified and analyzed.
Growth and Treatment Conditions
Plants used for qPCR analysis of the gene expression induced by nitrate
treatment were grown in aseptic hydroponics (initial medium with 2.5 mM
ammonium succinate) as described (Wang et al., 2007) for 7 d and then
treated with 10 mM KNO3 or KCl as a control for 2 h followed by the roots
being collected. For nitrate treatment on plants after nitrate starvation,
seedlings were grown in aseptic hydroponics for 6 d and then transferred to
the same fresh medium except without ammonium succinate to grow for
24 h. The roots of the seedlings treated with 10 mM KNO3 or KCl for 2 h were
harvested separately for RNA extraction.
For testing the YFP fluorescence of transgenic plants harboring
NRP-YFP construct in response to nitrate, seedlings were grown on plates
with either nitrate medium or ammonium nitrate medium (initial medium
with 10 mM NH4NO3) for 4 d followed by observation under a fluorescence
Nitrate was measured using the salicylic acid method (Cataldo et al., 1975;
Vendrell and Zupancic, 1990). Briefly, weighed samples (;0.1 g) in
a 1.5-mL tube were frozen by liquid nitrogen and milled into powder using
a RETCH MM400.Then,1 mL of deionized water was added into the tube
followed by boiling at 100°C for 20 min. The samples were centrifuged at
15,871g for 10 min, and 0.1 mL supernatant was transferred into a 12-mL
tube. Next, 0.4 mL of salicylic acid-sulfate acid (5 g salicylic acid in 100 mL
sulfate acid) was added and the sample was mixed well. The reactions were
incubated at room temperature for 20 min, and 9.5 mL of 8% NaOH solution
was added. After cooling the tube to room temperature, the OD410 value
was determined. For the control, 0.1 mL of deionized water was used
instead of 0.1 mL supernatant. The nitrate content was calculated using the
following equation: Y = C$V/W (Y, nitrate content; C, nitrate concentration
calculated with OD410 into regression equation; V, total volume of extracted
sample; W, weight of sample). Standard curve was made with KNO3 at
concentrations between 10 to 120 mg/L and regression equation was
obtained based on standard curve.
Yeast Two-Hybrid Assays
A full-length fragment of cDNA for NRG2 was ligated into the pGBKT7
vector (Clontech), and full-length cDNA fragments of tested genes were
introduced into pGADT7 AD vector (Clontech). The two-hybrid interaction
502
The Plant Cell
assays were performed according to the instruction provided by the
manufacturer (Clontech).
Supplemental Figure 3. Sequence alignment of 15-member
Arabidopsis gene family containing NRG2.
BiFC Analysis
Supplemental Figure 4. Morphological phenotype of the nrg2 mutant
under different concentrations of nitrate.
Transient BiFC assays in Nicotiana benthamiana were performed on the
leaves as described (Walter et al., 2004). Briefly, full-length cDNAs of NRG2
and NLP7 were cloned into the binary vectors pSPYNE-35S and pSPYCE35S containing the N- and C-terminal fragments of YFP (YFPN and YFPC),
respectively. N. benthamiana plants were grown on perlite watered with
ammonium nitrate solution for 5 weeks. For nitrogen starvation treatment,
plants were grown on perlite watered with ammonium nitrate solution for
3 weeks and then watered with the initial medium without nitrogen for another 2 weeks. The two constructs NRG2-YFPN and NLP7-YFPC were
cotransfected into the fourth to fifth leaves and the empty vectors YFPC and
YFPN in combination with NRG2-YFPN and NLP7-YFPC, respectively, were
used as negative controls. Transfected plants were watered with ammonium nitrate solution for 3 to 4 d followed by harvesting the infiltrated
leaves for observation using confocal microscope (Leica TCS SP5II).
RNA-Seq Data Analysis
The seeds of the wild type, nrg2-2, chl1-13, and nlp7-4 were grown on
ammonium succinate for 7 d and then treated with either 10 mM KNO3 or KCl
(as a control) for 2 h. Total RNA of the roots was prepared using a RNA
miniprep kit, and the concentrations were measured using a NanoDrop 2000
spectrophotometer (Thermo). The libraries were constructed and then sequenced using a HiSeq 2500 (Illumina), which generated ;21 million read
pairs per sample (Annoroad). Raw reads containing adapter, poly-N, and
low-quality reads were filtered and the effective data were mapped with the
Arabidopsis TAIR 10.2 reference genome using TopHat (version 2.0.12). After
excluding the rRNA or tRNA, we estimated the abundance of the transcripts
using RPKM (reads per kilobases per million reads) (Wagner et al., 2012). The
P values were adjusted using the Benjamini and Hochberg method
(Benjamini and Hochberg, 1995). Corrected P value < 0.05 and fold change
more than 2 were set as the threshold for significant difference in expression.
GO annotations of the data provided by our RNA-seq analysis were
performed using Panther (www.pantherdb.org/pathway/; Mi et al., 2013).
Accession Numbers
Sequence data from this article can be found in the Arabidopsis Genome
Initiative or GenBank/EMBL databases under the following accession
numbers: NiR (AT2G15620), NIA1 (AT1G77760), NRT2.1 (AT1G08090),
CIPK8 (AT4G24400), CIPK23 (AT1G30270), LBD37 (AT5G67420), LBD38
(AT3G49940), LBD39 (AT4G37540), NRT1.1 (AT1G12110), NLP7
(AT4G24020), NRT1.2 (AT1G69850), NRT1.4 (AT2G26690), NRT1.5
(AT1G32450), NRT1.6 (AT1G27080), NRT1.7 (AT1G69870), NRT1.8
(AT4G21680), NRT1.9 (AT1G18880), NRT1.11 (AT1G52190), NRT1.12
(AT3G16180), NRT2.6 (AT3G45060), NRT2.7 (AT5G14570), GLN1.1
(AT5G37600), and GLN1.3 (AT3G17820). The RNA-seq data discussed in
this article have been deposited in the National Center for Biotechnology
Information database (www.ncbi.nlm.nih.gov/sra; accession number
SRP067979).
Supplemental Figure 5. The expression of additional nitrate transport
genes in roots is not affected in nrg2 mutants.
Supplemental Figure 6. The expression of additional nitrate transport
genes in leaves is not affected by disruption of NRG2.
Supplemental Figure 7. The expression of nitrate reduction genes in
nrg2 mutants is not altered compared with that in the wild type.
Supplemental Figure 8. The expression of some characterized nitrate
regulatory genes tested is not altered in nrg2 mutants compared with
that in the wild type.
Supplemental Figure 9. The expression of NRG2 is not changed in
characterized nitrate regulatory gene mutants.
Supplemental Figure 10. The expression of NRT1.1 in wild-type, nrg2
mutant, and NRT1.1/nrg2-2 lines.
Supplemental Figure 11. BiFC assays revealed direct interaction
between NRG2 and NLP7 when plants were treated with nitrate after
nitrogen starvation.
Supplemental Data Set 1. Read numbers of the 24 samples.
Supplemental Data Set 2. Genes whose expression changed more
than 2-fold in the wild type and nrg2 mutant after nitrate treatment.
Supplemental Data Set 3. Genes with differentially induced expression levels in the mutant compared with the wild type after nitrate
treatment.
Supplemental Data Set 4. Four nitrogen-related clusters for genes
differentially expressed in the wild type and nrg2 mutant after nitrate
induction.
Supplemental Data Set 5. Genes that are differentially expressed in
the mutants compared with the wild type and commonly regulated by
NRG2, NRT1.1, and NLP7.
Supplemental Data Set 6. Genes regulated by both NRG2 and
NRT1.1.
Supplemental Data Set 7. Genes regulated by both NRG2 and NLP7.
Supplemental Data Set 8. Primers used in this article.
ACKNOWLEDGMENTS
We thank Yi-Fang Tsay for the cipk8-1 and cipk23-3 seeds, Lei Ge and
Gang Li for discussion of unpublished data, and Xiansheng Zhang,
Chengchao Zheng, and Daolin Fu for comments on the manuscript.
This research was supported by an NSFC grant (31170230) and the
Taishan Scholar Foundation to Y.W.
AUTHOR CONTRIBUTIONS
Supplemental Data
Supplemental Figure 1. The weak fluorescence phenotype of Mut75
is not caused by the mutation in At3g60240.
Supplemental Figure 2. Nitrate induction of the endogenous genes
tested is inhibited in the nrg2 mutants, and the expression of NRG2
is not regulated by nitrate, ammonium, and nitrogen starvation
treatments.
Y.W., N.M.C., N.X., R.W., and Z.C. designed the research. N.X., L.Z.,
C.Z., Z. Li, Z. Lei, F.L., and P.G. performed research. N.X., Y.W., and
R.W. analyzed data. Y.W., N.M.C., and N.X. wrote the article.
Received June 30, 2015; revised December 11, 2015; accepted January 3,
2016; published January 7, 2016.
NRG2 Plays an Essential Role in Nitrate Signaling
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The Arabidopsis NRG2 Protein Mediates Nitrate Signaling and Interacts with and Regulates Key
Nitrate Regulators
Na Xu, Rongchen Wang, Lufei Zhao, Chengfei Zhang, Zehui Li, Zhao Lei, Fei Liu, Peizhu Guan,
Zhaohui Chu, Nigel M. Crawford and Yong Wang
Plant Cell 2016;28;485-504; originally published online January 7, 2016;
DOI 10.1105/tpc.15.00567
This information is current as of August 3, 2017
Supplemental Data
/content/suppl/2016/01/07/tpc.15.00567.DC1.html
References
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