Advance Publication by J-STAGE
Plant 34,
Biotechnology
Plant Biotechnology
1–8 (2017)
http://www.jstage.jst.go.jp
DOI: 10.5511/plantbiotechnology.17.0727a
Short Communication
The chimeric repressor for the GATA4 transcription factor
improves tolerance to nitrogen deficiency in Arabidopsis
Ji Min Shin 1, KwiMi Chung 2, Shingo Sakamoto 2, Soichi Kojima 3,
Chuan-Ming Yeh 1, Miho Ikeda 1, Nobutaka Mitsuda 1,2, Masaru Ohme-Takagi 1,2*
1
Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan; 2 Bioproduction Institute,
Institute Advanced Industrial Science and Technology (AIST), Tsukuba 305-8562, Japan; 3 Graduate School of Agricultural
Science, Tohoku University, Sendai 980-0845, Japan
* E-mail: mtakagi@mail.saitama-u.ac.jp Tel: +81-48-858-3101
Received July 2, 2017; accepted July 27, 2017 (Edited by T. Mizoguchi)
Abstract Nitrogen limits crop yield, but application of nitrogen fertilizer can cause environmental problems and much
fertilizer is lost without being absorbed by plants. Increasing nitrogen use efficiency in plants may help overcome these
problems and is, therefore, an important and active subject of agricultural research. Here, we report that the expression
of the chimeric repressor for the GATA4 transcription factor (35S:GATA4-SRDX) improved tolerance to nitrogen
deficiency in Arabidopsis thaliana. 35S:GATA4-SRDX seedlings were significantly larger than wild type under nitrogensufficient and -deficient conditions (10 and 0.5 mM NH4NO3, respectively). 35S:GATA4-SRDX plants exhibited shorter
primary roots, fewer lateral roots, and higher root hair density compared with wild type. The expression levels of NITRATE
TRANSPORTER 2.1, ASPARAGINE SYNTHETASE and NITRATE REDUCTASE 1 were significantly higher in roots of
35S:GATA4-SRDX plants than in wild type under nitrogen-sufficient conditions. Under nitrogen-deficient conditions, the
expression of genes for cytosolic glutamine synthetases was upregulated in shoots of 35S:GATA4-SRDX plants compared
with wild type. This upregulation of nitrogen transporter and nitrogen assimilation-related genes might confer tolerance to
nitrogen deficiency in 35S:GATA4-SRDX plants.
Key words:
Arabidopsis, chimeric repressor, nitrogen use efficiency, transcription factor, tolerance.
Nitrogen is an essential macronutrient for plant growth
and development, and a major factor limiting agricultural
production. Plants absorb and utilize ammonium (NH+4 )
and nitrate (NO−3 ) from soil (Crawford and Forde 2002;
Kronzucker et al. 1997; Marschner 1995). Nitrogen levels
affect crop productivity by modulating the expression
of genes that affect leaf development, root architecture,
senescence, flowering, and metabolite biosynthesis (Diaz
et al. 2008; Rubin et al. 2009; Stitt et al. 2002; Walch-Liu
et al. 2000; Wang et al. 2004; Zhang and Forde 1998).
Adding nitrogen fertilizer can boost crop yields, but
such inputs are costly and plants fail to use 50–70% of
nitrogen provided as fertilizer. Nitrogen loss can cause
soil and water pollution, and may contribute to global
warming. Therefore, increasing nitrogen use efficiency
(NUE) remains a crucial issue for agriculture and plant
nutrient research.
Ongoing work has identified factors that regulate
nitrogen uptake, translocation, and assimilation
(Masclaux-Daubresse et al. 2010), including enzymes
such as NITRATE TRANSPORTERs (NRTs),
AMMONIUM TRANSPORTERs (ATMs), GLUTAMINE
SYNTHETASEs (GLNs) and ASPARAGINE
SYNTHETASEs (ASNs) (Masclaux-Daubresse et al. 2010;
Vidal and Gutiérrez 2008). In addition to these enzymes,
the transcription factors that regulate the expression of
nitrogen-responsive genes have been analyzed. NODULE
INCEPTION (NIN) functions as a key regulator of the
symbiotic nitrogen fixation pathway in legumes such as
Lotus japonicus, and NIN-Like proteins were identified
as transcription factors that interact with cis-elements
conserved in promoters of nitrate-responsive genes in
Arabidopsis thaliana (Konishi and Yanagisawa 2013).
Several studies have manipulated transcription factor
expression in attempts to enhance NUE. For example,
in Arabidopsis, ectopic expression of Dof1, which
regulates the expression of genes related to organic
acid metabolism, resulted in increased growth under
low-nitrogen conditions through the accumulation
of amino acids (Kurai et al. 2011; Yanagisawa et al.
2004). In this report, we attempted to identify novel
transcription factors involved in tolerance to nitrogen
deficiency by screening Arabidopsis lines that express
chimeric repressors (CRES-T) for transcription factors.
Many plant transcription factors are structurally and
functionally redundant and a single-gene knock-out
This article can be found at http://www.jspcmb.jp/
Published online September 16, 2017
Copyright © 2017 The Japanese Society for Plant Cell and Molecular Biology
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GATA4 regulates responses to nitrogen deficiency
Figure 1. Phenotype at 14 days after sowing (DAS) of 35S:GATA4-SRDX and wild type seedlings. (A) Comparison of shoot fresh weight (FW)
between wild type (WT) and 35S:GATA4-SRDX plants under nitrogen-sufficient (10 mM NH4NO3) and nitrogen-deficient (0.5 mM NH4NO3). Open
and closed bars indicate wild type and 35S:GATA4-SRDX plants, respectively. An Arabic numeral with ‘#’ symbol under each closed bar represents an
independent line of 35S:GATA4-SRDX plants. Values are means±SD (n=16–23). Double asterisks indicate significant differences at p<0.01 in t-test
when compared to wild type grown in each nitrogen condition. (B) Photos of 14 DAS wild type and independent line #30 of 35S:GATA4-SRDX plants
grown under nitrogen-deficient conditions. Scale bar; 2 mm.
often fails to exhibit an informative phenotype. However,
in the CRES-T gene silencing system, fusion to the SRDX
repression domain (SUPERMAN Repression Domain
X) converts a transcription factor to a strong repressor
that dominantly represses the target genes, producing
phenotypes similar to loss-of-function of its redundant
transcription factor genes (Hiratsu et al. 2003).
To screen our set of CRES-T lines for Arabidopsis
transcription factors, we used Murashige-Skoog solid
medium containing 0.5 mM NH 4NO 3 and 10 mM
NH 4NO 3 as deficient and sufficient conditions,
respectively. We screened for CRES-T lines that exhibit
different sizes from wild type under nitrogen-deficient
conditions. We identified a CRES-T line that produced
bigger seedlings under nitrogen-deficient conditions. The
fresh weight of 14-day-old seedlings of the CRES-T line
was significantly higher than that of wild type under both
nitrogen-sufficient and -deficient conditions (Figure 1).
Genome PCR analysis revealed the transcription factor
of the chimeric repressor to be GAT A4 (AT3G60530;
35S:GATA4-SRDX). Therefore, we considered that
35S:GATA4-SRDX plants are tolerant to nitrogen
Copyright © 2017 The Japanese Society for Plant Cell and Molecular Biology
Figure 2. GATA4 expression in response to different nitrogen
conditions. Relative expression of GATA4 in shoots (A) and roots (B)
of wild type at 13 DAS grown under different nitrogen conditions
analyzed by qRT-PCR, and all expression levels were normalized to that
of PP2AA3 (At1g3320), reference gene. Values are means±SD of four
biological replicates. Double asterisks indicate significant difference at
p< 0.01 in t-test when compared to plants grown in nitrogen-sufficient
conditions (10 mM NH4NO3).
J. M. Shin et al.
Figure 4. Nitrogen content in shoots and roots of wild type and
35S:GATA4-SRDX plants. Plants were grown under different nitrogen
conditions for 15 DAS. Values are means±SD of four independent
biological replicates. Single and double asterisks indicate significant
difference at p<0.05 and 0.01, respectively, in t-test when compared to
wild type grown in each nitrogen condition.
Figure 3. Transient effector–reporter analysis of GATA4
transcriptional activity. (A) Schematic representation of the constructs
used in transient assay in Arabidopsis leaf protoplasts. The reporter
construct contains firefly luciferase (LUC) driven by the promoter
containing 5×Gal4 DNA-binding sequence (GAL4BS) and a TATA
sequence. Each effector construct contains a Gal4 DNA-binding
domain (GALDB) and TMV Omega leader sequence (Ω) driven
by the CaMV 35 S promoter (p35S). The effector construct with the
SRDX-fused Gal4 DNA-binding domain (GALDB-SRDX) was used
as a positive control for repression. (B) Relative LUC reporter activity
when each effector was co-transfected into leaf protoplasts. Values
are means±SD of six technical replicates. Double asterisks indicate
significant difference at p< 0.001 in Dunnett’s test when compared with
negative control (GAL4DB).
deficiency.
GAT A4 is a transcription factor that belongs to the
GAT A family and possesses a C2C2 zinc finger DNA
binding domain that binds to the (A/T)GAT A(A/G)
motif (Orkin 1992). The Arabidopsis genome has 29
genes encoding GAT A transcription factors (Manfield et
al. 2006; Riechmann et al. 2000). Arabidopsis GATA4 is
expressed in all tissues including root, stem, leaf, flower,
and silique at the reproductive stage and especially
upregulated under darkness (Manfield et al. 2006).
We analyzed whether nitrogen deficiency affects the
expression level of GATA4 by qRT-PCR and found that
the expression of GATA4 was moderately suppressed
in roots under nitrogen-deficient conditions, but we
observed no reduction in shoots (Figure 2).
To analyze the molecular activity of GAT A4, we
performed transient expression assays using a luciferase
reporter gene (LUC) fused to a promoter containing the
Gal4 DNA binding site (GAL4:LUC) and an effector in
which the coding region of GATA4 was fused to the Gal4
DNA-binding domain (GAL4DB) driven by the CaMV
35S promoter (35S:GAL4BD-GATA4) in protoplasts
isolated from leaf epidermal cells of Arabidopsis. The
transient assay showed that the 35S:GAL4DB-GAT A4
effector significantly suppressed GAL4:LUC activity,
compared with the control 35S:GAL4DB effector (Figure
3), suggesting that GAT A4 appears to function as a
repressor.
The nitrogen content of 35S:GATA4-SRDX shoots
(about 8.5%) was significantly higher than that of wild
type (about 7.9%) under nitrogen-sufficient conditions
(Figure 4). By contrast, under nitrogen-deficient
conditions, the nitrogen content of 35S:GATA4-SRDX
shoots (about 2.0%) was lower than that of wild type
(about 2.4%, Figure 4). These results suggest that NUE is
enhanced in 35S:GATA4-SRDX plants to maintain larger
shoot biomass, even under low nitrogen.
To examine the mechanisms by which 35S:GATA4SRDX plants tolerate nitrogen deficiency, we analyzed
the expression of genes related to nitrogen transport
and assimilation. AMT1.1, which codes for a high-
Copyright © 2017 The Japanese Society for Plant Cell and Molecular Biology
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GATA4 regulates responses to nitrogen deficiency
Figure 5. Expression of nitrogen transporter genes. Gene expression in roots (A) and shoots (B) of 13 DAS plants grown under different nitrogen
conditions, measured by qRT-PCR, and all expression levels were normalized to that of PP2AA3 (At1g3320), reference gene. Values are means±SD
of four independent biological replicates. Single and double asterisks indicate significant differences at p< 0.05 and 0.01, respectively, in t-test when
compared to wild type grown in each nitrogen condition.
affinity ammonium transporter, is de-repressed in roots
under nitrogen-deficient conditions, but upregulated in
shoots under nitrogen-sufficient conditions (Engineer
and Kranz 2007; Gazzarrini et al. 1999). Nitrate
induces NRT1.1, which codes for a dual-affinity nitrate
transporter, and NRT2.1, which codes for a high-affinity
transporter, but they usually display opposite expression
patterns under nitrogen-deficient conditions. In roots
under nitrogen deficiency, NRT1.1 is down-regulated
and NRT2.1 is dramatically induced (Forde 2000; Liu
et al. 1999; Orsel et al. 2002; Wang et al. 1998). Our
qRT-PCR analyses showed that NRT1.1 and NRT2.1
were highly expressed in roots of 35S:GATA4-SRDX
plants compared to wild type under nitrogen-sufficient
conditions (Figure 5A). NRT1.1 expression was higher in
roots of 35S:GATA4-SRDX plants than in wild type, even
though its expression was suppressed in wild-type and
35S:GATA4-SRDX roots by nitrogen deficiency (Figure
5A). NRT2.1 expression was highly increased by nitrogen
deficiency in roots of wild-type and 35S:GATA4-SRDX
Copyright © 2017 The Japanese Society for Plant Cell and Molecular Biology
plants, but did not show a significant difference between
wild type and 35S:GTATA4-SRDX (Figure 5A). We also
found that the expression of AMT1.1 was upregulated in
shoots of 35S:GATA4-SRDX plants compared to wild type
even under nitrogen-deficient conditions, and NRT1.1
was induced higher in shoots of 35S:GATA4-SRDX plants
than in wild type (Figure 5B).
Glutamine synthetases assimilate ammonium to
glutamine and re-assimilate ammonia released by
photorespiration or protein modification during
senescence (Fuentes et al. 2001; Krapp 2015; Xu et al.
2012). The expression of genes encoding the cytosolic
glutamine synthetases GLN1;1, GLN1;2, GLN1;3,
and GLN1;4, was induced to higher levels in shoots of
35S:GATA4-SRDX plants than in wild type in response
to nitrogen deficiency (Figure 6A). In addition, the
absorbed nitrate from soil to plant roots is reduced to
nitrite by nitrate reductase, and NIA1 is one of isoforms
of nitrate reductases in Arabidopsis (Wilkinson and
Crawford 1993). The expression of NIA1 was higher in
J. M. Shin et al.
Figure 6. Expression of nitrogen assimilation-related genes. Gene expression in shoots (A) and roots (B) of 13 DAS plants grown under different
nitrogen conditions, measured by qRT-PCR, and all expression levels were normalized to that of PP2AA3 (At1g3320), reference gene. (A, B)
Relative expression of GLN1;1, GLN1;2, GLN1;3, and GLN1;4. (C, D) Relative expression of NIA1 (C) and ASN1 (D) in shoots and roots. Values are
means±SD of four independent biological replicates. Single and double asterisks indicate significant differences at p< 0.05 and 0.01, respectively, in
t-test when compared to wild type grown in each nitrogen condition.
roots of 35S:GATA4-SRDX plants than in wild type when
the plants were grown in nitrogen-sufficient conditions
(Figure 6C). This expression pattern is similar to the
upregulation of NRT1.1 and NRT2.1, which are induced
by nitrate in roots of 35S:GATA4-SRDX plants grown in
nitrogen-sufficient conditions (Figure 5B). ASN1, which
encodes asparagine synthetase, plays an important role
in nitrogen assimilation and translocation together with
glutamine synthetase (Carvalho et al. 2003; Good et al.
2004; Miflin and Lea 1976), was highly upregulated in
35S:GATA4-SRDX roots, compared with wild type under
nitrogen-sufficient conditions, but the two genotypes
showed similar expression levels under nitrogendeficient conditions (Figure 6D). These results indicate
that the tolerance of 35S:GATA4-SRDX plants to nitrogen
deficiency may be due to differential upregulation
of genes related to nitrogen transport and nitrogen
assimilation in response to nitrogen status.
Our data base analyses showed that the AMT1.1,
NRT1.1, GLN1;1-4, NIA and ASN1 genes have putative
GAT A binding sites in their 5’ upstream regions.
However, it is unlikely that those genes are the direct
targets of GAT A4, because they were upregulated in
35S:GATA4-SRDX plants. GAT A4-SRDX might repress
the expression of negative regulator(s) that suppress the
expression of several nitrogen metabolism-related genes.
Root architecture can be modified based on nitrogen
availability. Under mild nitrogen deficiency, the growth
Copyright © 2017 The Japanese Society for Plant Cell and Molecular Biology
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GATA4 regulates responses to nitrogen deficiency
Figure 7. Root architecture of wild type and 35S:GATA4-SRDX plants under different nitrogen conditions. (A) Photos of 15 DAS 35S:GATA4-SRDX
plants and wild type grown vertically under nitrogen-sufficient (left; 10 mM NH4NO3) and nitrogen-deficient (right; 0.2 mM NH4NO3) conditions.
Scale bar; 1 cm. (B) Root hair phenotype of 35S:GATA4-SRDX plants and wild type grown vertically under nitrogen-sufficient (top; 10 mM NH4NO3)
and nitrogen-deficient (bottom; 0.5 mM NH4NO3) conditions, at 11 DAS. (C) Primary root length of 15 DAS seedlings of 35S:GATA4-SRDX plants
and wild type. (D) Number of lateral root initiations. (E) Lateral root density (number of lateral roots divided by primary root length). Values are
means±SD (n=35–50). Total replicates of 35S:GATA4-SRDX plants contain four independent lines. Double asterisks indicate significant difference at
p< 0.01 in t-test when compared to wild type grown in each nitrogen condition.
of lateral roots is promoted to expand the root surface
area for nitrogen uptake, but under severe nitrogendeficient conditions, primary and lateral root growth
are suppressed (Giehl and von Wirén 2014; Gruber et
al. 2013). 35S:GATA4-SRDX plants had shorter primary
Copyright © 2017 The Japanese Society for Plant Cell and Molecular Biology
roots, fewer and shorter lateral roots, and more root
hairs compared to wild type (Figure 7A, B). The growth
of lateral and primary roots of 35S:GATA4-SRDX plants
was suppressed compared with wild type in nitrogensufficient and deficient conditions (Figure 7A, C–E). In
J. M. Shin et al.
this experiment, we used more severe nitrogen deficient
condition (0.2 mM instead of 0.5 mM), because such
severe nitrogen deficiency clearly promoted the effect on
root development and architecture of 35S:GATA4-SRDX
plants.
These results suggest that the GAT A4 transcription
factor is involved in the regulation of root development
and GAT A4-SRDX altered root architectures that affect
nitrogen availability for plants. Increased root hair
density may extend root surface area, thus increasing
absorption of nutrients and water from soil compared to
lateral roots (Gilroy and Jones 2000; Marschner 1995).
As we demonstrated above, the expression of ASN1 was
highly increased in roots of 35S:GATA4-SRDX plants
(Figure 6B). Considering that ASN1 is expressed in
root hairs and in the elongation and maturation zones
of the root (Brady et al. 2007; Schultz et al. 2017), the
upregulation of ASN1 in the roots of 35S:GATA4-SRDX
plants might be related to the root phenotype. Further
experiments will be required to elucidate the molecular
mechanisms responsible for relationship between
nitrogen assimilation and root development including
the roles of GAT A4 in root development and in
nitrogen metabolism regardless of the alteration of root
architecture.
Acknowledgements
We thank Ms. Yoshimi Sugimoto and Ms. Miyoko Yamada for their
technical assistance.
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