Repression of Nitrogen Starvation Responses by Members of the Arabidopsis GARP-Type Transcription Factor NIGT1/HRS1 Subfamily

Takatoshi Kiba; Jun Inaba; Toru Kudo; Nanae Ueda; Mineko Konishi; Nobutaka Mitsuda; Yuko Takiguchi; Youichi Kondou; Takeshi Yoshizumi; Masaru Ohme-Takagi; Minami Matsui; Kentaro Yano; Shuichi Yanagisawa; Hitoshi Sakakibara

doi:10.1105/tpc.17.00810

. 2018 Apr 5;30(4):925–945. doi: 10.1105/tpc.17.00810

Repression of Nitrogen Starvation Responses by Members of the Arabidopsis GARP-Type Transcription Factor NIGT1/HRS1 Subfamily^{^[OPEN]}

Takatoshi Kiba ^a,¹, Jun Inaba ^a, Toru Kudo ^b, Nanae Ueda ^a, Mineko Konishi ^c, Nobutaka Mitsuda ^d, Yuko Takiguchi ^d, Youichi Kondou ^e, Takeshi Yoshizumi ^a, Masaru Ohme-Takagi ^d,^f, Minami Matsui ^a, Kentaro Yano ^b, Shuichi Yanagisawa ^c, Hitoshi Sakakibara ^a,^g

PMCID: PMC5969275 PMID: 29622567

NIGT1/HRS1s are GARP-type transcription factors that repress nitrogen starvation responses in order to optimize nitrogen acquisition and utilization under fluctuating nitrogen availability and demand.

Abstract

Nitrogen (N) is often a limiting nutrient whose availability determines plant growth and productivity. Because its availability is often low and/or not uniform over time and space in nature, plants respond to variations in N availability by altering uptake and recycling mechanisms, but the molecular mechanisms underlying how these responses are regulated are poorly understood. Here, we show that a group of GARP G2-like transcription factors, Arabidopsis thaliana NITRATE-INDUCIBLE, GARP-TYPE TRANSCRIPTIONAL REPRESSOR1/HYPERSENSITIVE TO LOW Pi-ELICITED PRIMARY ROOT SHORTENING1 proteins (NIGT1/HRS1s), are factors that bind to the promoter of the N starvation marker NRT2.4 and repress an array of N starvation-responsive genes under conditions of high N availability. Transient assays and expression analysis demonstrated that NIGT1/HRS1s are transcriptional repressors whose expression is regulated by N availability. We identified target genes of the NIGT1/HRS1s by genome-wide transcriptome analyses and found that they are significantly enriched in N starvation response-related genes, including N acquisition, recycling, remobilization, and signaling genes. Loss of NIGT1/HRS1s resulted in deregulation of N acquisition and accumulation. We propose that NIGT1/HRS1s are major regulators of N starvation responses that play an important role in optimizing N acquisition and utilization under fluctuating N conditions.

INTRODUCTION

Nitrogen (N) is a key nutrient that is often the limiting factor in plant growth and productivity. Under natural conditions, plants often face N-poor environments (Jackson and Caldwell, 1993; Miller and Cramer, 2004), and they have developed a series of N starvation responses to efficiently acquire and utilize available N. They do this by sensing and signaling both external and internal N status (Good et al., 2004; Hermans et al., 2006; Nacry et al., 2013).

N starvation activates a series of metabolic changes for N recycling and remobilization that catabolizes proteins and nucleic acids through vacuolar proteolysis, autophagy, and purine ring catabolism (Müntz, 2007; Werner and Witte, 2011; Avila-Ospina et al., 2014). The ammonium that is released is reincorporated into amino acids by mechanisms proposed to involve cytosolic Gln synthetase (GLN1), glutamate dehydrogenase (GDH), and asparagine synthetase (ASN) and allows the ammonia to be reused and/or remobilized for de novo synthesis of nitrogenous biomolecules (Masclaux-Daubresse et al., 2008, 2010; Gaufichon et al., 2016).

Under low-N conditions, plants modulate root architecture and uptake activity to improve N acquisition in response to local and systemic N signals (Forde, 2014; Krapp et al., 2014; Kiba and Krapp, 2016). Several factors involved in the regulation of root morphology in response to N starvation have been described, including CLAVATA3/ESR-RELATED peptides, CALCINEURIN B-LIKE PROTEIN7 (CBL7), and TRYPTOPHAN AMINOTRANSFERASE RELATED2 (Araya et al., 2014; Ma et al., 2014, 2015). Uptake activity is enhanced in response to N starvation mostly through high-affinity uptake systems (Crawford and Forde, 2002; Liu et al., 2003; Yuan et al., 2007a). High-affinity uptake of urea and ammonium is mediated by DEGRADATION OF UREA3 (DUR3) and AMT/MEP/Rh (AMT) superfamily members, respectively (Liu et al., 2003; Kojima et al., 2007; Ludewig et al., 2007). Two families of nitrate transporters, NITRATE TRANSPORTER1/NRT1 PTR FAMILY (NRT1/NPF) and NITRATE TRANSPORTER2 (NRT2), have been implicated in high-affinity nitrate uptake (Nacry et al., 2013; Krapp et al., 2014). NRT1.1/NPF6.3/CHL1 (NRT1.1/NPF6.3) plays a role in high-affinity nitrate uptake as a dual-affinity transporter (Liu et al., 1999; Cerezo et al., 2001; Ho et al., 2009). NRT2 family members, in most cases, act together with NITRATE ASSIMILATION RELATED PROTEIN/NITRATE TRANSPORTER3 (NAR2/NRT3.1) as high-affinity nitrate transporters (Liu et al., 1999; Cerezo et al., 2001; Filleur et al., 2001; Kotur et al., 2012). NRT2.1, NRT2.2, NRT2.4, and NRT2.5 are expressed in the roots of N-starved Arabidopsis thaliana, and they account for ∼95% of high-affinity nitrate influx activity under N starvation conditions (Kiba et al., 2012; Lezhneva et al., 2014; Kiba and Krapp, 2016).

graphic file with name TPC_TPC201700810R2_fx1.jpg

Many of the adaptive responses to N starvation are regulated at the level of gene expression, and some molecular components involved in that regulation have been described. Arabidopsis miR169 inhibits some aspects of the N starvation response, including NRT2.1 expression and N content (Zhao et al., 2011). CBL7 plays a positive role in the regulation of NRT2.4 and NRT2.5, root architecture, and root nitrate content (Ma et al., 2015). Although members of the NIN-LIKE PROTEIN (NLP) family are master regulators of the primary nitrate response in Arabidopsis (Konishi and Yanagisawa, 2013; Marchive et al., 2013; Liu et al., 2017), they have been implicated in the regulation of some elements of N starvation-inducible gene expression (Castaings et al., 2009). Rubin et al. (2009) reported that LATERAL ORGAN BOUNDARY DOMAIN family transcription factors (LBD37-39) are involved in the repression of a number of N starvation-inducible genes, as well as anthocyanin accumulation. Despite these advances, the molecular mechanisms underlying transcriptional regulation of N starvation responses remain largely unknown.

Using yeast one-hybrid (Y1H) screening, we identified transcriptional regulators of Arabidopsis NRT2.4, a N starvation marker gene (Kiba et al., 2012). These regulators are named NIGT1/HRS1s because they are highly homologous to a rice (Oryza sativa) protein known as NITRATE-INDUCIBLE, GARP-TYPE TRANSCRIPTIONAL REPRESSOR1 (NIGT1) (Sawaki et al., 2013) and a member of them has already been named HYPERSENSITIVE TO LOW Pi-ELICITED PRIMARY ROOT SHORTENING1 (HRS1) (Liu et al., 2009). They belong to a small clade in the GARP-type transcription factor family, which are transcription factors widely distributed in eukaryotic organisms (Safi et al., 2017) and are expressed when N availability is high. They appear to act as direct repressors of NRT2.4 and possibly as direct repressors of an array of N starvation-regulated genes in order to repress N starvation responses under high N availability conditions. Our results also suggest that they indirectly sensitize phosphate (Pi) starvation responses such as activation of Pi starvation-induced gene expression (Puga et al., 2017) by directly repressing the repressors of the Pi starvation response. Together, these results define Arabidopsis NIGT1/HRS1s as important regulators of N starvation responses, with activity as balancers of N and Pi starvation responses.

RESULTS

Identification of Arabidopsis NIGT1/HRS1/HHO Family Members as NRT2.4 Promoter-Interacting Proteins

The full-length NRT2.4 promoter, extending 1886 bp upstream of the inferred initiation codon (Pro1886), is activated under N starvation and reports N availability (Kiba et al., 2012). To identify the regulatory factors of this promoter, we first defined the minimal NRT2.4 promoter region required for the N availability response by fusing a 5′-deletion series of the NRT2.4 promoter with sequences encoding GFP (Figure 1A). Stable Arabidopsis transformants harboring the Pro1886:GFP (P1886G), P639G, and P389G constructs had high levels of GFP fluorescence and transcript levels under N starvation conditions, whereas P254G did not respond to N availability (Figures 1A to 1C). This indicates that P389 includes the minimal sequence required for N availability-responsive expression.

The P389 fragment was used as bait in a Y1H screening experiment to identify factors that regulate this promoter (Figure 1D). The bait-reporter yeast strain, which harbors a construct consisting of three tandem copies of P389 fused to the aureobasidin A-resistance gene, was transformed with a prey library composed of 1498 Arabidopsis transcription factor cDNAs (Mitsuda et al., 2010). We sequenced prey plasmids from the 27 aureobasidin A-resistant colonies and found that 21 of them were GARP (GOLDEN2, ARR-B, Psr1) GOLDEN2-like (GARP-G2) family members AT1G25550, AT1G68670, and AT3G25790 (Figure 1D; Supplemental Table 1). Among 40 GARP-G2 family transcription factors in Arabidopsis, these three proteins fall into a subclade consisting of four members within a larger clade consisting of seven members (Supplemental Figure 1). Since the subclade members are highly homologous to a rice protein known as NITRATE-INDUCIBLE, GARP-TYPE TRANSCRIPTIONAL REPRESSOR1 (NIGT1) (Sawaki et al., 2013), we designate these proteins collectively as Arabidopsis NIGT1/HYPERSENSITIVE TO LOW Pi-ELICITED PRIMARY ROOT SHORTENING1 (NIGT1) subfamily members and individually as NIGT1.1/HRS1 HOMOLOG3 (NIGT1.1), NIGT1.2, NIGT1.3, and NIGT1.4. The other members of the larger clade have already been named HRS1 HOMOLOG4 (HHO4) to HHO6 (Liu et al., 2009; Medici et al., 2015). Thus, the larger clade with seven members was designated the NIGT1/HRS1/HHO family.

To further test the interactions between NIGT1s and the NRT2.4 promoter, we conducted particle bombardment-mediated transient assays in Arabidopsis seedlings (Figure 1E). Bombardment with an effector plasmid expressing each of the NIGT1s fused with two stringent transcriptional activation domains VP16 (VP) increased firefly luciferase (FLUC) activity under control of the NRT2.4 P389 promoter. HHO4-VP, HHO5-VP, or HHO6-VP fusion proteins also activated the promoter, but neither GFP nor GARP-G2 family members in other clades (e.g., PHR1 and AT3G10760) did. This indicates that, although there are differences in levels of activation, each of the NIGT1/HRS1/HHO family members interacts with the NRT2.4 promoter. In this study, we focused on the characterization of four highly homologous members NIGT1.1 through 1.4.

Arabidopsis NIGT1 Subfamily Members Are Transcriptional Repressors That Bind to the NRT2.4 Promoter

To determine the molecular function of the NIGT1s, we confirmed that these proteins are localized in the nuclei of transgenic plants expressing NIGT1.1-GFP or NIGT1.2-GFP fusions under the control of the CaMV 35S promoter (Figure 2) (Medici et al., 2015; Nagarajan et al., 2016). The localization pattern was not altered by N availability (Figure 2A).

Figure 2. — Arabidopsis NIGT1 Subfamily Members Are Nuclear-Localized Transcriptional Repressors and Interact with the *NRT2.4* Promoter in a Sequence-Specific Manner.

**(A)** Localization of GFP in roots of 5-d-old transgenic seedlings expressing NIGT1.1-GFP (NIGT1.1-GFP) or NIGT1.2-GFP (NIGT1.2-GFP) under the control of the CaMV 35S promoter grown on 0.5× MS agar plates with (+N) or without a nitrogen source (−N). BF/GFP represents merged images of bright field and GFP. Bar = 25 μm.

**(B)** The effect of NIGT1 subfamily members on *GAL4* promoter activity in transient assays. *GAL4* promoter activity from *ProGAL4:FLUC* was measured when cobombarded with the GAL4 DNA binding domain (GDB) fused to GFP or NIGT1.1-1.4. Promoter activities are given as FLUC/RLUC activity ratios. A vector containing *RLUC* driven by the CaMV 35S promoter (*ProCAMV35S:RLUC*) was used as an internal control for normalization of bombardment efficiency.

**(C)** to (G) Interactions between NIGT1 subfamily members and mutated *NRT2.4* promoters in transient assays.

**(C)** Schematic of the *NRT2.4* P389 promoter and mutated promoters (B1^m, B2^m, and B1^mB2^m).

**(D)** to **(G)** Transient assays were conducted using constructs harboring the promoters illustrated in **(C)** fused with *FLUC* or *FLUC* only (Empty) as reporters. As effectors, constructs expressing NIGT1.1 **(D)**, NIGT1.2 **(E)**, NIGT1.3 **(F)**, and NIGT1.4 **(G)** fused with two VP16 domains (VP) were used. *ProCAMV35S:RLUC* was used as an internal control for normalization of bombardment efficiency. The promoter activities are shown as FLUC/RLUC activity ratios. B1^m and B2^m represent mutated *NRT2.4* promoters at the B1 and B2 motifs, respectively. Different lowercase letters indicate statistically significant differences as indicated by Tukey’s HSD test (P < 0.05). Error bars represent sd of four (**[B]**, **[D]**, and **[E]**) and three (**[F]** and **[G]**) independent pools of more than eight seedlings. Each experiment was conducted twice with similar results.

To test whether NIGT1s have transcriptional activator or repressor activity, transient assays were conducted using a reporter plasmid harboring a GAL4 promoter:FLUC (ProGAL4:FLUC) fusion and an effector plasmid expressing NIGT1 fused with a GAL4 DNA binding domain at its N terminus (GAL4DB-NIGT1.1 through GAL4DB-NIGT1.4). Expression of GAL4DB-NIGT1s reduced GAL4 promoter activity compared with that of control (GAL4DB-GFP), indicating that NIGT1s act as transcriptional repressors (Figure 2B).

In vitro DNA binding site selection experiments and electrophoretic mobility shift assays had previously identified rice NIGT1 binding sequences as GAATATTC and GAATC (Sawaki et al., 2013). Recently, DNA affinity purification sequencing (DAP-seq) indicated that the binding motifs of Arabidopsis NIGT1.1, NIGT1.2, and NIGT1.4 are identical to those of rice NIGT1 (O’Malley et al., 2016). There is one GAATATTC (B1) and three GAATC (B2) motifs within the Arabidopsis NRT2.4 P389 promoter (Supplemental Figure 2). To determine whether the NIGT1 proteins bind to P389 at these motifs, we tested the interactions between mutant promoters and NIGT1s in transient assays in Arabidopsis seedlings (Figures 2C to 2G). Reporter plasmids harboring mutant NRT2.4 promoters that were mutated (as shown in Figure 2C) in either the B1 motif (B1^m), the B2 motifs (B2^m), or in both the B1 and B2 motifs (B1^mB2^m) were fused with FLUC, and effector plasmids expressing NIGT1.1-VP to NIGT1.4-VP were used to assay activity. Although mutations in either the B1 or B2 motifs resulted in significantly decreased FLUC expression, the B1^mB2^m double mutation resulted in complete loss of interaction (Figures 2D to 2G), indicating that full NIGT1 binding within the NRT2.4 promoter requires both motifs (NIGT1 binding motifs).

To confirm the interactions in vivo, transgenic plants constitutively overexpressing NIGT1.1-GFP (NIGT1.1ox), NIGT1.2-GFP (NIGT1.2ox) fusion genes, or GFP (GFPox) under control of the CaMV 35S promoter were used in chromatin immunoprecipitation (ChIP)-quantitative reverse transcription-PCR (qPCR) assays with an anti-GFP antibody. We analyzed two regions of the NRT2.4 promoter having multiple NIGT1 binding motifs (#2 and #3) and three regions without the motif (#1, #4, and #5) (Figure 3). In addition, promoter regions of previously reported qPCR reference genes At2g28390 (#6) and At4g34270 (#7) (Dekkers et al., 2012) were chosen as controls (Figure 3A). Regions #2 and #3 were enriched in NIGT1.1ox and NIGT1.2ox compared with GFPox, but no significant enrichment was found on any other regions tested (Figure 3B). When transgenic lines expressing NIGT1.3-FLAG (eNIGT1.3ox), NIGT1.4-FLAG (eNIGT1.4ox), or GFP-FLAG (eGFPox) fusion genes under control of the β-estradiol-inducible promoter were analyzed using ChIP-qPCR and an anti-FLAG antibody, we found enrichment of the same regions (#2 and #3) (Figure 3B). These results show that all NIGT1 subfamily members associate with NRT2.4 promoter in vivo.

Figure 3. — Arabidopsis NIGT1 Subfamily Members Are Associated with the *NRT2.4* Promoter.

NIGT1 interaction with the *NRT2.4* promoter was analyzed using ChIP assays.

**(A)** Diagrams of *NRT2.4* and control (*At2g28390* and *At4g34270*) loci and the locations of B1 (GAATATTC, red line) and B2 (GAATC, blue lines) motifs. Black bars and numbers 1 to 7 indicate DNA regions used in the ChIP assay.

**(B)** ChIP-qPCR assays were performed using 10-d-old transgenic seedlings. Left graph: Transgenic lines expressing *GFP* (GFPox), *NIGT1.1-GFP* (NIGT1.1ox), or *NIGT1.2-GFP* (NIGT1.2ox) fusion genes under control of the CaMV 35S promoter were used and immunoprecipitated with an anti-GFP antibody. Right graph: Transgenic lines expressing *GFP-FLAG* (eGFPox), *NIGT1.3-FLAG* (eNIGT1.3ox), or *NIGT1.4-FLAG* (eNIGT1.4ox) fusion genes under control of the β-estradiol-inducible promoter treated with 10 μM β-estradiol for 24 h were used and immunoprecipitated with an anti-FLAG antibody. The percentages of the DNA coimmunoprecipitated with antibody relative to input DNA are indicated. Numbers on the x axis indicate DNA regions shown in **(A)**. Error bars represent sd (n = 3 independent pools of more than 10 whole plants). Asterisks indicate statistically significant differences (*P < 0.01, Student’s t test). Each experiment was conducted twice with similar results.

Arabidopsis NIGT1 Subfamily Members Repress NRT2.4 Expression in Vivo

To investigate the biological significance of interactions between the NRT2.4 promoter and NIGT1 proteins, β-estradiol (Est)-inducible overexpression lines were generated (eNIGT1.1ox to eNIGT1.4ox), and expression was measured with quantitative real-time RT-PCR (qRT-PCR) after Est treatment (Figure 4). Each of the Est-inducible lines overexpresses the corresponding NIGT1s in response to exogenous Est treatment. Compared with the vector control line eVC, inducible overexpression of the NIGT1s reduced N starvation-inducible NRT2.4 expression (Figure 4A). A similar reduction in NRT2.4 expression was observed in constitutive overexpressors NIGT1.1ox and NIGT1.2ox (Supplemental Figure 3A). To examine whether the reduction occurs at the level of transcription, we introduced the P1886G reporter into eNIGT1.2ox by crossing (P1886G x eNIGT1.2ox) and observed that GFP fluorescence was completely absent from roots after Est treatment (Figure 4B). qRT-PCR assays confirmed that GFP transcription is reduced by NIGT1.2 overexpression in a manner similar to that of endogenous NRT2.4 (Supplemental Figure 3B). These results suggest that NIGT1s repress NRT2.4 promoter expression by directly binding to it in vivo.

Figure 4. — Expression of Arabidopsis *NRT2.4* Is Downregulated in Overexpressing Lines of *NIGT1.1-1.4* and Upregulated in *nigt1.1 nigt1.2 nigt1.3 nigt1.4* Quadruple Mutants.

**(A)** Expression of *NRT2.4* in roots of transgenic lines expressing *NIGT1.1* (eNIGT1.1ox-4), *NIGT1.2* (eNIGT1.2ox-4), *NIGT1.3* (eNIGT1.3ox-3), or *NIGT1.4* (eNIGT1.4ox-4) under control of the Est-inducible promoter. eVC-3 is an empty vector control. Seven-day-old 0.5× MS-grown seedlings were transferred and incubated on MGRL plates without an exogenous N source but containing 1 μM Est for 3 d. Error bars represent sd (n = 3 independent pools of more than 10 roots).

**(B)** *NRT2.4* promoter activity in transgenic lines harboring the 1886 bp *NRT2.4* promoter:GFP construct (P1886G) and eNIGT1.2ox-4 or eVC-3. Eight-day-old 0.5× MS-grown seedlings were transferred to and incubated on MGRL plates without an exogenous nitrogen source but containing 1 μM Est for 3 d. GFP fluorescence (green) was merged with chlorophyll autofluorescence (red) and representative pictures are presented. qRT-PCR analysis of *GFP* expression is presented in Supplemental Figure 3B. Bar = 1 cm.

**(C)** and **(D)** Expression levels of *NRT2.4* in *nigt1* multiple mutants.

**(C)** *NRT2.4* expression in whole seedlings of 10-d-old 0.5× MS-grown wild-type (Col-0), *nigt1* single, double, triple, and quadruple (*nigtQ*) mutants. Double and triple mutants are combinations of *nigt1.1-1*, *nigt1.2-1*, *nigt1.3-1*, and *nigt1.4-1*, except for *nigt124*. The *nigt124* represents *nigt1.1-1 nigt1.2-2 nigt1.4-1.* The *nigtQ-1*, *nigtQ-2*, *nigtQ-3*, and *nigtQ-4* represent *nigt1.1-1 nigt1.2-1 nigt1.3-2 nigt1.4-1*, *nigt1.1-1 nigt1.2-1 nigt1.3-1 nigt1.4-1*, *nigt1.1-1 nigt1.2-2 nigt1.3-2 nigt1.4-1*, and *nigt1.1-1 nigt1.2-2 nigt1.3-1 nigt1.4-1*, respectively.

**(D)** *NRT2.4* expression in the root of Col-0, *nigtQ-1*, and *nigtQ-4* seedlings incubated under 0.5× MS (high N availability) or N starvation conditions. Seven-day-old 0.5× MS-grown seedlings were transferred to and incubated on 0.5× MS or MGRL plates without nitrogen (N starvation) for 3 d before harvest. Error bars represent sd (n = 4 independent pools of indicated tissue from more than ten plants). Asterisks indicate statistically significant differences (*P < 0.01 and **P < 0.001, Student’s t test). Expression levels were analyzed by qRT-PCR and normalized using *At4g34270* as an internal control. Each experiment was conducted twice with similar results.

We isolated homozygous T-DNA insertion mutants in order to examine the effect of the loss of NIGT1 function on NRT2.4 expression (Supplemental Figure 4A). We tested to see if the homologous T-DNA insertions resulted in null mutants by RT-PCR. No full-length transcripts of corresponding NIGT1s were amplified by RT-PCR with saturating cycles in any single mutant (Supplemental Figure 4B), indicating that the T-DNA insertions resulted in null alleles. A series of double, triple, and quadruple mutants were produced by crossing the T-DNA mutants, and these mutants were analyzed using NRT2.4 expression assays (Figures 4C and 4D). There were no significant differences in NRT2.4 expression relative to wild-type levels in any of the single mutants, double mutants nigt1.1 nigt1.2, or nigt1.3 nigt1.4, or in any combination of triple mutants grown in half-strength Murashige and Skoog plates (0.5× MS containing ∼19.7 mM nitrate and 10.3 mM ammonium). However, expression was significantly upregulated (or derepressed) in all quadruple mutant (nigtQ) combinations (Figure 4C) unless plants were under N starvation conditions (Figure 4D).

We have confirmed that introduction of a genomic fragment containing either NIGT1.1 or NIGT1.2 complements the NRT2.4 expression phenotype in nigtQ (Supplemental Figure 5A). Collectively, these results indicate that NIGT1 proteins act redundantly to directly repress NRT2.4 expression under high N availability.

Expression of Arabidopsis NIGT1 Subfamily Genes Is Regulated by Nitrogen and Nitrate Availability

Because deregulation of NRT2.4 in nigtQ is N dependent (Figure 4D), we grew wild-type seedlings under different N conditions. Seedlings were incubated on agar plates containing 5 mM ammonium nitrate (NH₄NO₃) or without a nitrogen source for 3 d, and expression levels were assayed in shoots and roots by qRT-PCR (Figure 5). NIGT1.1 and NIGT1.2 were expressed in shoots and roots, and NIGT1.3 and NIGT1.4 were detected only in roots of NH₄NO₃-grown seedlings (Figure 5A). Under N starvation conditions, transcript levels of all NIGT1s except NIGT1.3 were significantly downregulated compared with NH₄NO₃-grown seedlings, and we observed no alteration of expression patterns in shoots or roots (Figure 5A). We then tested the effect of other N sources on NIGT1 expression in seedlings incubated with nitrate, ammonium, ammonium nitrate, Gln, urea, or without any added nitrogen (Figure 5B). Each of the nitrogen sources resulted in higher transcription levels of NIGT1.1 and NIGT1.2 than under N starvation conditions (Figure 5B), indicating that NIGT1.1 and NIGT1.2 expression is positively regulated by N availability. The expression levels of NIGT1.4 were higher when incubated with nitrate and ammonium nitrate but were lower with ammonium, Gln, and urea (reduced N metabolites) than under N starvation conditions. Compared with ammonium nitrate, nitrate induced the expression of NIGT1.3, whereas reduced N metabolites repressed it (Figure 5B). This observation agrees with a previous report indicating that NIGT1.3 and NIGT1.4 are nitrate-inducible (Medici et al., 2015). Given that the expression levels of NIGT1.3 and NIGT1.4 under N starvation conditions were higher than when grown with reduced N metabolites, it is likely that the expression of NIGT1.3 and NIGT1.4 is regulated by the balance between nitrate induction, repression, and derepression in response to reduced N metabolite availability, as has been reported for many nitrate-inducible genes, including NRT2.1 (Gansel et al., 2001; Nazoa et al., 2003; Widiez et al., 2011; Camañes et al., 2012).

Figure 5. — Expression Patterns of Arabidopsis *NIGT1* Subfamily Members.

**(A)** Expression of *NIGT1* subfamily members (*NIGT1*s) and *NRT2.4* in shoots (upper panel) and roots (lower panel). Seven-day-old 0.5× MS-grown wild-type (Col-0) seedlings were transferred to MGRL plates with either 5 mM NH₄NO₃ (NH₄NO₃) or no nitrogen (N starvation) and incubated for 3 d. Genes that are significantly downregulated under N starvation conditions compared with added NH₄NO₃ are marked with asterisks (P < 0.01, Student’s t test). U.Q., under the quantification detection limit. Error bars represent sd (n = 6 independent pools of indicated tissue from more than 10 plants).

**(B)** Expression of *NIGT1s* in whole seedlings in response to different nitrogen species. Seven-day-old 0.5× MS-grown Col-0 seedlings were transferred to MGRL plates containing 3 mM potassium nitrate (NO), 3 mM ammonium chloride (NH), 1.5 mM ammonium nitrate (NHNO), 1.5 mM glutamine (GLN), 1.5 mM urea (UREA), or no nitrogen (−N) and incubated for 3 d. Error bars represent sd (n = 4 independent pools of more than 10 whole plants). Different lowercase letters indicate statistically significant differences (P < 0.05, Tukey’s HSD test).

**(C)** and **(D)** Time-course analysis of expression of *NIGT1*s in roots after changes in nitrogen availability.

**(C)** Expression levels of *NIGT1*s and *NRT2.4* in roots during N starvation. Seven-day-old 0.5× MS-grown Col-0 seedlings were transferred to MGRL plates containing 5 mM NH₄NO₃ (NH₄NO₃) or no nitrogen (N starvation) and harvested at the indicated times.

**(D)** Expression levels of *NIGT1*s and *NRT2.4* in roots in response to nitrogen supplement. Ten-day-old Col-0 seedlings N starved for 3 d were transferred to NH₄NO₃ plates or exposed to N starvation conditions and harvested for the indicated times. Error bars represent sd (n = 3 independent pools of more than 10 roots). Expression levels were analyzed by qRT-PCR and normalized using *At4g34270* as an internal control. Each experiment was conducted twice with similar results.

In order to compare the patterns of NIGT1s and NRT2.4 expression in detail, we followed time-course expression patterns of them during N deprivation in roots. Seedlings grown in 0.5× MS were transferred to N-free media and expression was measured at daily intervals over the course of 5 d. Transcript levels of NIGT1.1, NIGT1.2, and NIGT1.4 gradually decreased after transfer, but NRT2.4 steadily increased (Figure 5C). Although there was no significant decrease in NIGT1.3 transcript levels when measured at daily intervals (Figure 5C), there was a transient decrease when measured at shorter intervals (Supplemental Figure 6A). This is consistent with a previous report that NIGT1.3 is an early nitrogen deprivation-downregulated gene (Menz et al., 2016).

We also analyzed time-course expression patterns in roots after N resupply. N-starved seedlings were transferred to agar plates containing NH₄NO₃, and expression was measured for 3 d. NIGT1.1, NIGT1.2, and NIGT1.4 expression increased rapidly after NH₄NO₃ resupply, whereas that of NRT2.4 dropped immediately (Figure 5D). Although no significant change in NIGT1.3 transcript levels after NH₄NO₃ resupplementation was observed under our time-course conditions (Figure 5D), NIGT1.3 expression increased in response to nitrate resupplementation as reported previously (Supplemental Figure 6B; Konishi and Yanagisawa, 2013; Marchive et al., 2013; Medici et al., 2015). Together, these results show that expression patterns of NIGT1s are positively correlated with N and/or nitrate availability and are inversely correlated with NRT2.4 expression under these experimental conditions.

We further analyzed the spatial expression patterns of NIGT1s in transgenic seedlings harboring a fusion between the NIGT1 promoter and the GUS reporter gene (Figure 6). GUS staining patterns in organs in response to N availability were consistent with our qRT-PCR results (compare Figures 5A and 6). In seedlings grown under high N availability, promoter activity of NIGT1s was observed not only in root epidermal cells, where NRT2.4 is expressed (Kiba et al., 2012), but also in many other cells and tissues. NIGT1.1 and NIGT1.2 expression was detected in most tissues, with hypocotyl and root cap being the exceptions. NIGT1.3 and NIGT1.4 appeared to be expressed in most cell types in the older part of the root, but not in the root tip region (Figure 6).

Identification of NIGT1 Target Genes

NIGT1s are expressed much more broadly than NRT2.4 (Figure 6), suggesting that there are other genes directly repressed by NIGT1s than NRT2.4. To identify NIGT1 direct target genes, we conducted combinatorial microarray analyses to allow a genome-wide search for genes bound and repressed by NIGT1s. We generated transgenic lines that overexpress NIGT1.1-VP or NIGT1.2-VP in an Est-inducible manner in the wild-type Col-0 background (pER-NIGT1.1-VP or pER-NIGT1.2-VP). These lines show upregulation of NRT2.4 with Est treatment under high N availability conditions (Supplemental Figure 7), suggesting that other NIGT1-bound genes would also be induced in these lines. Using the pER-NIGT1.2-VP and NIGT1.2ox transgenic plants, two sets of microarray data were produced. The first data set was obtained from pER-NIGT1.2-VP seedlings incubated with or without 10 μM Est for 24 h under high N availability. These seedlings had significantly upregulated probes in response to Est treatment and were recovered as NIGT1-bound genes (Welch’s t test, P < 0.05; Benjamini-Hochberg procedure, false discovery rate [FDR] < 0.1; fold change [FC] > 0). NIGT1.2ox and wild-type seedlings were incubated under N starvation conditions for 3 d. These seedlings had significantly downregulated probes in NIGT1.2ox and were recovered as NIGT1-repressed genes (P < 0.05, FDR < 0.1, FC < 0). Shoots and roots were analyzed separately. We expect that the genes delineated by combining these data are enriched in NIGT1 direct targets (Figure 7).

Figure 7. — Identification of Target Genes of NIGT1 Subfamily Members.

**(A)** and **(B)** Venn diagram of NIGT1.2ox downregulated and pER-NIGT1.2-VP upregulated probes in roots **(A)** and shoots **(B)**. To obtain NIGT1.2ox downregulated probes, 10-d-old NIGT1.2ox (overexpressing *NIGT1.2* under control of the CaMV 35S promoter) and wild-type seedlings incubated under N starvation conditions for 3 d were compared. Significantly downregulated probes in NIGT1.2ox were recovered (Welch’s t test, P < 0.05; Benjamini-Hochberg procedure, FDR < 0.1; FC < 0). To obtain pER-NIGT1.2-VP upregulated probes, 9-d-old pER-NIGT1.2-VP seedlings incubated with or without 10 μM Est for 24 h under high N availability were compared, and significantly upregulated probes by Est treatment were recovered (P < 0.05, FDR < 0.1, FC > 0).

**(C)** Representative significantly enriched gene ontology terms common in roots and shoots. Numbers represent ranked P values by Fisher’s exact test. See Supplemental Data Sets 7 and 8 for a complete list.

**(D)** Classification of NIGT1 direct target genes by their response to N starvation. Microarray data obtained from 10-d-old wild-type seedlings incubated in MGRL plates with 5 mM NH₄NO₃ or without a nitrogen source for 3 d were used. Genes were classified into upregulated (P < 0.05, FDR < 0.1, FC > 0), downregulated (P < 0.05, FDR < 0.1, FC < 0), and not changed (P ≥ 0.05, FDR ≥ 0.1).

In roots, 2390 probes were identified as upregulated in pER-NIGT1.2-VP with Est treatment and 1353 were downregulated in NIGT1.2ox. A comparison between these two probe lists resulted in 419 putative direct targets in roots (Figure 7A; Supplemental Data Sets 1 to 3). We identified 2807 probes that were upregulated in Est-treated pER-NIGT1.2-VP shoots and 2172 that were downregulated in NIGT1.2ox shoots, yielding 310 putative direct targets (Figure 7B; Supplemental Data Sets 4 to 6). To validate this search procedure, seven genes (GLN1;4, NRT2.5, GDH3, DUR3, AMT1;1, NLP3, and SPX1) were arbitrarily selected, and their expression was analyzed in NIGT1.1ox, NIGT1.2ox, and nigtQ plants (Figure 8). All of these genes were repressed in NIGT1 overexpression lines under N starvation conditions but were derepressed in nigtQ under high N availability (Figures 8A and 8B). Introduction of a genomic fragment containing either NIGT1.1 or NIGT1.2 into nigtQ complemented the derepression of these genes (Supplemental Figure 5B). Furthermore, ChIP-qPCR experiments confirmed the association of NIGT1 with the NRT2.5, GLN1;4, and GDH3 promoters at the NIGT1 binding motifs (Figures 8C and 8D). Using different methods, Safi et al. (2018) recently reported that NRT2.4, NRT2.5, and GDH3 are directly repressed by NIGT1s. These results strongly support the validity of our lists as enriched for NIGT1 direct targets.

Figure 8. — Validation of NIGT1 Target Genes.

**(A)** Expression of arbitrarily selected genes from the NIGT1 direct target list (*GLN1;4*, *NRT2.5*, *DUR3*, *AMT1;1*, *NLP3*, and *SPX1*) in transgenic plants constitutively expressing *NIGT1.1-GFP* (NIGT1.1ox) or *NIGT1.2-GFP* (NIGT1.2ox) fusion genes, or *GFP* (GFPox) under control of the CaMV 35S promoter under N starvation conditions. Seven-day-old 0.5× MS-grown seedlings were transferred to and incubated on MGRL plates without a nitrogen source for 3 d. Error bars represent sd (n = 3 independent pools of more than 10 whole plants).

**(B)** Expression levels of *GLN1;4*, *NRT2.5*, *DUR3*, *AMT1;1*, *NLP3*, and *SPX1* in wild type (Col-0) and *nigt1.1 nigt1.2 nigt1.3 nigt1.4* quadruple mutants (*nigtQ-1* and *nigtQ-4*) under high N availability. Ten-day-old 0.5× MS-grown seedlings were analyzed. Error bars represent sd (n = 5 independent pools of more than 10 whole plants).

**(C)** and **(D)** NIGT1s interactions with *NRT2.5*, *GLN1;4*, and *GDH3* promoters in ChIP assays.

**(C)** Diagrams of the *NRT2.5*, *GLN1;4*, and *GDH3* loci, the locations of B1 (GAATATTC, red lines) and B2 (GAATC, blue lines) motifs, and the locations of amplicons used in the ChIP assays.

**(D)** ChIP-qPCR assays were performed using 10-d-old transgenic seedlings. Left side: Transgenic lines expressing *GFP* (GFPox), *NIGT1.1-GFP* (NIGT1.1ox), or *NIGT1.2-GFP* (NIGT1.2ox) fusion genes under control of the CaMV 35S promoter were used and immunoprecipitated with anti-GFP antibody. Right side: Transgenic lines expressing *GFP-FLAG* (eGFPox), *NIGT1.3-FLAG* (eNIGT1.3ox), or *NIGT1.4-FLAG* (eNIGT1.3ox) fusion genes under control of the β-estradiol-inducible promoter treated with 10 μM β-estradiol for 24 h were used and immunoprecipitated with an anti-FLAG antibody. The percentages of the DNA coimmunoprecipitated with antibody relative to input DNA are indicated. Numbers on the x axis indicate DNA regions shown in **(C)**. Error bars represent sd (n = 3 independent pools of more than 10 whole plants). Asterisks indicate statistically significant differences (*P < 0.05 and **P < 0.01, Student’s t test). Each experiment was conducted twice with similar results.

Gene Ontology (GO) term enrichment analysis showed that nutrient-related GO terms were significantly overrepresented both in shoots and roots (Figure 7C; Supplemental Data Sets 7 and 8). Frequent ontology terms include “starvation,” “nutrient levels,” “homeostasis,” “transport,” “phosphate,” and “nitrogen.” When these direct targets were tested for N starvation responsiveness using microarray data obtained from wild-type seedlings incubated under N starvation or high N availability conditions, 53% of them were N starvation upregulated genes in roots and 66% in shoots (Figure 7D; Supplemental Data Sets 3 and 6). This significant enrichment in N starvation-inducible genes both in roots (P = 7.3 × 10⁻⁸¹, Fisher’s exact test) and shoots (P = 8.1 × 10⁻⁹⁴) suggests that NIGT1 proteins act as repressors of N starvation responses (Supplemental Figure 8A). N starvation-inducible targets included N uptake-related genes, N recycling- and remobilization-related genes, and N signaling-related genes, such as high-affinity nitrate transport-related genes (NRT2.4, NRT2.5, and NAR2.1/NRT3.1), an ammonium transporter (AMT1;1), amino acid metabolism genes (GDH3, GLN1;1, GLN1;4, ASN3, GLU2, and ASPGB1), purine ring catabolism-associated genes (ATALN, UPS1, UPS3, AAH1, and AAH2), vacuolar proteolysis-related genes (SASP and SCPL29), autophagy-related genes (ATG8F), and N-related transcription factor genes (NLP3, NLP4, NLP5, and NLP8) (Okamoto et al., 2003; Carter et al., 2004; Loqué et al., 2006; Rose et al., 2006; Masclaux-Daubresse et al., 2010; Werner and Witte, 2011; Lezhneva et al., 2014; Yan et al., 2016).

We also found N starvation downregulated genes, or N-inducible genes in the lists. They include well-known nitrate-inducible genes such as NRT1.1/NPF6.3, LBD37, and CIPK23 (Ho et al., 2009; Rubin et al., 2009), as well as important genes in the regulation of Pi-starvation responses such as class I SYG1/Pho81/XPR1 domain protein (SPX) genes, SPX1, SPX2, and SPX4, and PHOSPHATE2 (PHO2) (Liu et al., 2012; Huang et al., 2013; Puga et al., 2014) (Supplemental Data Sets 3 and 6). These results suggest that NIGT1 proteins have regulatory functions other than repressing N starvation-inducible genes.

Furthermore, all NIGT1s except for the overexpressed NIGT1.2 were found in the direct target list (Supplemental Data Sets 3 and 6). Overexpression of NIGT1.1 or NIGT1.2 represses endogenous NIGT1s (Figure 9A). ChIP-qPCR experiments confirmed NIGT1 association with all NIGT1 promoters around the NIGT1 binding motifs (Figures 9B and 9C), indicating that an auto-negative feedback-type regulation system, as has been reported for NIGT1 in rice (Sawaki et al., 2013), is also active in Arabidopsis.

Modulation of NIGT1 Activity Results in Altered N Uptake and N Content

Because nigtQ mutants were indistinguishable from the wild type in our high N availability/N-starved conditions (Supplemental Figure 9), we measured N-related physiological parameters in both NIGT1 overexpressors and nigtQ mutants (Figure 10; Supplemental Figure 10). NIGT1 overexpressors were analyzed under N starvation conditions, when most NIGT1 expression is downregulated in wild-type seedlings. The nigtQ mutant line was compared with the wild type under high N availability, when most NIGT1 expression is high in wild-type seedlings (Figure 5). N content was lower in NIGT1.1ox and NIGT1.2ox plants than in the GFPox line (Figure 10A). On the other hand, nigtQ had increased N content in shoots and roots compared with the wild type (Figure 10B). NIGT1.1ox and NIGT1.2ox contained less nitrate (Figure 10C). Although the nigtQ line had more nitrate than control seedlings (Figure 10D), there was no significant difference in soluble protein, chlorophyll, or total amino acid contents between nigtQ lines and wild-type seedlings (Supplemental Figures 10A to 10C). Because levels of glutamine and asparagine were higher but histidine, proline, and valine levels were reduced, the total amino acid levels were not significantly altered in the mutant (Supplemental Figures 10C and 10D).

Because many genes involved in high-affinity nitrate uptake were on the root tissue direct target list (Supplemental Data Set 3), high-affinity nitrate influx was measured (Figures 10E and 10F). In N-starved NIGT1.1ox and NIGT1.2ox plants, influx was reduced in the high-affinity range at 0.02 and 0.2 mM (Figure 10E). In contrast, nigtQ plants grown under high N availability showed increased high-affinity influx at 0.2 mM, but no significant difference in low-affinity influx was detected (Figure 10F). Not surprisingly, alterations in high-affinity influx were positively correlated with the expression levels of high-affinity transport-related genes, NRT2.4, NRT2.5, NAR2.1/NRT3.1, and NRT1.1/NPF6.3 (Figures 8A, 8B, 10G, and 10H). Expression was repressed in NIGT1 overexpressors under N starvation conditions and derepressed in nigtQ under high N availability. We also tested the expression of the major high-affinity nitrate transporter gene NRT2.1, though it was not in our direct target list. NRT2.1 expression was positively correlated with high-affinity nitrate influx in a way similar to other direct target genes (Figures 10G and 10H). Nitrogen content, nitrate uptake, nitrate accumulation, and high-affinity nitrate transport-related gene expression phenotypes of nigtQ were complemented by introducing a genomic fragment containing NIGT1.2 (Supplemental Figures 5C to 5E).

Given that nigtQ plants had higher N uptake and N content (Figure 10) without any positive effect on growth under high N availability conditions (Supplemental Figure 9), it could be that the nigtQ line is impaired in its effective use of N under high N availability conditions. Collectively, these results suggest that NIGT1s suppress N starvation responses, especially responses that enhance N acquisition and N reserves, by directly repressing their target genes under high N availability (Figure 12A).

Figure 12. — A Model for NIGT1 Functions.

**(A)** A model is proposed for NIGT1 function in the regulation of N starvation responses. Under N starvation, low *NIGT1* expression allows N starvation-responsive genes such as genes involved in N uptake, recycling, remobilization, and signaling to be upregulated, leading to N starvation responses. Upon N supplementation, *NIGT1*s are rapidly induced and directly repress N starvation-responsive genes and attenuate N starvation responses.

**(B)** A model for the role of NIGT1s in balancing N starvation and Pi starvation responses (PSR). *NIGT1* expression is positively regulated by Pi deficiency only under high N availability. *NIGT1*s have a dual role both as a direct repressor of N starvation responses and as an indirect sensitizer of Pi starvation responses by directly repressing repressors of PSR (*SPX1*, *SPX2*, *SPX4*, and *PHO2*) to balance N and Pi starvation responses.

DISCUSSION

Direct Repression of Nitrogen Starvation-Inducible Genes by Arabidopsis NIGT1 Subfamily Members under High Nitrogen Availability

In vitro DNA binding site selection experiments and ChIP-based techniques (ChIP-chip and ChIP-seq) have been used to map in vivo genome-wide transcription factor binding sites (Johnson et al., 2007; Liu et al., 2011; Franco-Zorrilla et al., 2014; O’Malley et al., 2016). However, it is becoming evident that not all bound genes are functional targets of the transcription factor (Nakamichi et al., 2012; Marchive et al., 2013). In this study, NIGT1 direct target genes were screened by combining microarray data obtained from NIGT1.2ox and pER-NIGT1.2-VP (Figure 7) and validated by expression analysis in nigtQ and by ChIP assays for arbitrarily selected genes (Figures 8 and 9). Our direct targets were significantly enriched in N starvation-inducible genes (Figure 7D; Supplemental Figure 8A), and they significantly overlapped with the NIGT1s binding targets recently identified by DAP-seq (P = 4.4 × 10⁻⁵⁸ and P = 2.3 × 10⁻²⁴ for root and shoot targets, respectively, with Fisher’s exact test; Supplemental Figure 8B) (O’Malley et al., 2016). This indicates that our screening strategy effectively enriches for genes bound and repressed by NIGT1s, namely, “NIGT1 direct targets.”

Although Arabidopsis LBD37, LBD38, and LBD39 (LBDs) are involved in the repression of a large number of N starvation-inducible genes, it is not known whether these genes are directly regulated by LBDs (Rubin et al., 2009). LBDs and NIGT1s are both regulated in response to N or a combination of N and nitrate availability, and expression of these genes is high under high N availability and low under low N availability (Figure 5) (Rubin et al., 2009). They also share many repression targets, including NRT1.1/NPF6.3, NRT2.1, NRT2.5, NAR2.1/NRT3.1, GDH3, GLN1;4, NLP5, and NAC55 (Supplemental Data Sets 3 and 6) (Rubin et al., 2009). The emerging model is that NIGT1 proteins and LBDs appear to act in the same pathway. Given that NIGT1 proteins directly repress the target genes, it can be hypothesized that LBDs act upstream of NIGT1s. However, microarray data published by Rubin et al. (2009) show that the expression of NIGT1s is not altered in the overexpressors and null mutants of LBDs, except that NIGT1.4 expression was lower in the LBD37 overexpressor under N starvation conditions (Rubin et al., 2009). This indicates that LBDs and NIGT1s do not function in the same pathway. Because our data indicate that LBD37 is a direct target of NIGT1s in roots (Supplemental Data Set 3), NIGT1s and LBDs likely function in different pathways but influence each other through negative regulatory interactions to repress overlapping sets of N starvation-inducible genes. This might explain why loss of NIGT1 activity did not result in full derepression of NIGT1 direct targets under high N availability conditions (Figures 4D and 8B). However, further experiments, such as characterization of mutants that have lost both LBD and NIGT1 functions, would be required to test this hypothesis.

NIGT1s Are Repressors of Nitrogen Starvation Responses in Arabidopsis

By combinatorial microarray analyses, we identified 419 root and 310 shoot direct target genes of NIGT1s (Figure 7; Supplemental Data Sets 3 and 6). Although direct targets were enriched with N starvation-inducible genes involved in various pathways, and N uptake and N content were significantly increased in nigtQ plants (Figure 10), we could not detect any alterations in growth or development of nigtQ (Supplemental Figure 9). A possible explanation could be that N starvation-inducible genes are redundantly repressed by NIGT1s and other factors, including LBDs (Rubin et al., 2009). Other members of the NIGT1/HRS1/HHO family could serve as redundant factors in this regulatory scheme because they also interact with the NRT2.4 promoter (Figure 1E). Alternatively, N starvation responses could also be regulated at the posttranscriptional level. Consistent with this idea, AMT1;1 and NRT2.1 are subjected to posttranscriptional regulation, a process that is dependent on N availability (Yuan et al., 2007b; Laugier et al., 2012), and many enzymes in primary metabolism are known to be regulated at the posttranslational level in response to environmental factors (MacKintosh, 1998). These observations suggest that N starvation responses are a critical biological process that is tightly controlled by multiple overlapping layers of regulation. In any case, we propose that the transcriptional repression of N starvation-inducible genes by NIGT1s is an important component of the N starvation response regulatory system (Figure 12A).

The Role of NIGT1s in Optimizing Nitrogen Acquisition and Utilization under Fluctuating Nutritional Conditions

We showed that the expression levels of NIGT1s decrease upon transfer to N starvation conditions, followed by a rapid increase after N and/or nitrate supplementation (Figures 5C and 5D; Supplemental Figure 6). Given that the NIGT1s are early nitrate-inducible genes and are directly regulated by NLPs and NRT1.1/NPF6.3 (Konishi and Yanagisawa, 2013; Marchive et al., 2013; Medici et al., 2015), the role of NIGT1s would likely be to repress N starvation-inducible genes immediately upon sensing the availability of nitrate. Because NIGT1.1 and NIGT1.2 expression is also induced by reduced N sources (Figure 5B), NIGT1.1 and NIGT1.2 should play a similar role upon detection of a reduced N source. Furthermore, data presented here show that nitrate-inducible genes are also repressed by NIGT1s (Figure 7D; Supplemental Data Sets 3 and 6), meaning that NIGT1s also participate in the feedback repression of nitrate-inducible genes. This sort of activity by NIGT1s would enable plants to swiftly respond to fluctuating N availability and demand in order to fine-tune N responses so that N acquisition and utilization can be optimized.

Several studies have implicated NIGT1s in the regulation of Pi responses, though no molecular mechanism has been pinpointed (Liu et al., 2009; Medici et al., 2015; Nagarajan et al., 2016). Liu et al. (2009) reported that NIGT1.4 (HRS1) expression is induced by Pi deficiency and that an NIGT1.4 overexpressor line is susceptible to Pi deficiency-elicited primary root growth repression and root hair differentiation enhancement. An overexpressor line and mutants of NIGT1.2 (HHO2) exhibit alterations in Pi starvation responsive genes and Pi content (Nagarajan et al., 2016). In our study, we identified three class I SPX and PHO2 genes as NIGT1 direct targets (Supplemental Data Sets 3 and 6), supporting the notion that NIGT1s are involved in the regulation of Pi responses. Since class I SPXs and PHO2 are known to be major negative regulators of Pi starvation responses (Liu et al., 2012; Huang et al., 2013; Puga et al., 2014), it is possible that NIGT1s sensitize Pi starvation responses indirectly through repression of the negative regulators (Figure 12B). As expected, NIGT1 overexpressor lines had higher levels of Pi content and Pi transporter expression (PHT1;1 and PHT1;4) than the wild type and the nigtQ mutant had lower levels than the wild type (Figures 11A to 11E). We also found that expression of each of the NIGT1s is induced by Pi starvation, but only under high N availability (Figure 11F), indicating that NIGT1 action in the regulation of Pi starvation responses is N dependent, as described by Medici et al. (2015) for NIGT1.3 (HHO1) and NIGT1.4 (HRS1) in primary root growth repression. Although further study will be required to fully understand the mechanism of Pi starvation response regulation by NIGT1s, we propose that NIGT1s have a dual function as direct repressors of N starvation responses and as indirect sensitizers of Pi starvation responses to balance N and Pi starvation responses (Figure 12B). Such a mechanism would enable plants to divert energy required for N starvation responses to Pi starvation responses under high N availability.

Figure 11. — Phosphate Starvation-Related Phenotypes Are Altered in *NIGT1* Overexpressors and in the *nigt1.1 nigt1.2 nigt1.3 nigt1.4* Quadruple Mutant.

**(A)** and **(B)** Phosphate (P) content in shoots and roots of the *NIGT1* overexpressor **(A)** and the *nigt1.1 nigt1.2 nigt1.3 nigt1.4* quadruple (*nigtQ*) mutant **(B)**. Fourteen-day-old 0.5× MS-grown seedlings were used in the assay.

**(C)** P content in wild-type (Col-0), the *nigt1.1 nigt1.2 nigt1.3 nigt1.4* quadruple mutant, and a complemented line (Comp1-1.2). Fourteen-day-old 0.5× MS-grown seedlings were used in the assay.

**(D)** and **(E)** Expression levels of phosphate transporter genes involved in phosphate acquisition in roots of 10-d-old 0.5× MS agar plate-grown *NIGT1* overexpressor **(D)** and N-replete *nigt1.1 nigt1.2 nigt1.3 nigt1.4* quadruple mutant **(E)**.

**(F)** Expression levels of *NIGT1.1* through *1.4* in roots in response to changes in N and P availability. Seven-day-old 0.5× MS-grown wild-type seedlings were transferred to and incubated on MGRL plates with 5 mM NH₄NO₃ and 1.75 mM phosphate (+N+P), without a nitrogen source (-N+P), without phosphate (+N-P), or without nitrogen and phosphate (-N-P) for 5 d.

**(A)** and **(D)** Transgenic seedlings constitutively expressing *NIGT1.1-GFP* (NIGT1.1ox) or *NIGT1.2-GFP* (NIGT1.2ox) fusion genes or *GFP* (GFPox) under the control of the CaMV 35S promoter were analyzed. Expression levels were analyzed by qRT-PCR and normalized using *At4g34270* as an internal control.

Asterisks indicate statistically significant differences (P < 0.05; Student’s t test). Different lowercase letters indicate statistically significant differences as indicated by Tukey’s HSD test (P < 0.01). Error bars represent sd of five (**[A]**, **[B]**, **[D]**, and **[E]**) and four (**[C]** and **[F]**) independent pools of indicated tissues from more than 10 plants. The experiment was conducted twice with similar results.

In conclusion, we propose a mechanism that, after N supplementation and/or under high N availability conditions, accounts for the direct repression of N starvation responsive genes by NIGT1s. This optimizes N acquisition and utilization under fluctuating N availability and demand (Figure 12A). We also found that NIGT1s act to balance N and Pi starvation responses (Figure 12B). NIGT1 homologs are widely conserved among higher plants but can be phylogenetically divided into monocot and dicot types (Supplemental Figure 11). Although the role of rice NIGT1 in the regulation of N starvation responses has not been studied, rice NIGT1 shares many features with Arabidopsis NIGT1s, such as transcriptional repressor activity, binding motifs, auto-negative feedback regulation, and nitrate-inducible expression (Sawaki et al., 2013). This mechanism might be conserved among monocots and dicots and thus may be a key factor for studying N responses among all of the important crop plants.

METHODS

Plant Materials and Growth Conditions

Arabidopsis thaliana ecotype Columbia (Col-0) was used as the wild type. T-DNA insertion lines SALK_044835 (nigt1.2-1/hho2-1), SALK_070096 (nigt1.2-2/hho2-2), WISCDSLOXHS231_10C (nigt1.3-2), SAIL_28_D03 (nigt1.3-1/hho1-1), GK-267G03 (nigt1.1-1), and SALK_067074 (nigt1.4-1/hrs1-1) were obtained from the Arabidopsis Biological Resource Center (https://abrc.osu.edu/) or the Nottingham Arabidopsis Stock Centre (http://arabidopsis.info/) (Supplemental Figure 4). Multiple null mutants were generated by crossing these lines. The quadruple mutants nigtQ-1, nigtQ-2, nigtQ-3, and nigtQ-4 represent nigt1.1-1 nigt1.2-1 nigt1.3-2 nigt1.4-1, nigt1.1-1 nigt1.2-1 nigt1.3-1 nigt1.4-1, nigt1.1-1 nigt1.2-2 nigt1.3-2 nigt1.4-1, and nigt1.1-1 nigt1.2-2 nigt1.3-1 nigt1.4-1, respectively. Genotypes were determined by genomic PCR using the primers shown in Supplemental Table 2.

For studies on seedlings, plant lines were grown on MGRL-based (Kiba et al., 2012) or 0.5× MS vertical agar plates containing 1% sucrose and 2.3 mM MES buffer (pH 5.8) at 22°C under fluorescent light (100 µmol m⁻² s⁻¹, 16 h light/8 h dark). For N-starved conditions, ion equilibrium of the MGRL-based media was accomplished by replacing KNO₃ and CaNO₃ with KCl and CaCl₂. For Pi starvation conditions, ion equilibrium of the MGRL-based media was ensured by replacing NaPi with NaCl. Adult plants were grown on nutrient-rich soil (Supermix A; Sakata Seed) watered every other day with MGRL media containing 10 mM nitrate at 22°C under fluorescent light (100 µmol m⁻² s⁻¹, 16 h light/8 h dark).

Binary Plasmid Construction

To generate constructs for truncated NRT2.4 promoter:GFP reporter lines, the full-length NRT2.4 promoter (Pro1886; 1886 bp upstream of the inferred initiation codon) and truncated promoters (P639, P389, and P254; 639, 389, and 254 bp upstream of the inferred initiation codon) were amplified with PrimeSTAR GXL DNA polymerase (Takara) using the specific primer sets in Supplemental Table 2. Amplicons were cloned into pENTR/D-TOPO vector (Invitrogen), sequenced, and then integrated into the Gateway binary vector pBA002a-GFP, which is a derivative of pBA002a (Kiba et al., 2012), to generate promoter:GFP fusion constructs (P1886G, P639G, P389G, and P254G), using LR clonase (Invitrogen).

Constructs expressing NIGT1.1-GFP or NIGT1.2-GFP translational fusion proteins under the control of their native promoters were used to observe intercellular localization of NIGT1 (Figure 2A) and in complementation tests using the multiple mutants (Supplemental Figure 5). These constructs were generated by amplifying a genomic fragment encompassing the promoter and coding region (without a stop codon) of NIGT1.1 or NIGT1.2. These amplicons were cloned into pENTR/D-TOPO vector (Invitrogen), sequenced, and integrated into the Gateway binary vector pBA002a-GFP to generate pNIGT1.1: NIGT1.1-GFP and pNIGT1.2:NIGT1.2-GFP.

To generate β-estradiol-inducible NIGT1 overexpression lines, genomic fragments encompassing the coding regions of NIGT1.1, NIGT1.2, NIGT1.3, or NIGT1.4 (without a stop codon) were amplified and cloned into pENTR/D-TOPO vector (Invitrogen), sequenced, and then integrated into the Gateway binary vector pER8-derivative pER8-GW-FLAG (Zuo et al., 2000), to generate a construct that expresses NIGT1s under control of the β-estradiol-inducible promoter (eNIGT1.1ox, eNIGT1.2ox, eNIGT1.3ox, and eIGT1.4ox). To create β-estradiol-inducible NIGT1-VP overexpression lines, the full-length coding regions of NIGT1.1 or NIGT1.2 without stop codons were amplified from Arabidopsis cDNA and cloned into pENTR/D-TOPO vector (Invitrogen), sequenced, and integrated into pER8-VP, a pER8-derivative, to generate constructs that express NIGT1.1 or NIGT1.2 fused with two stringent transcriptional activation domains VP16 (VP) at their C termini under control of the β-estradiol-inducible promoter (pER-NIGT1.1-VP or pER-NIGT1.2-VP).

To make constitutive NIGT1 overexpression lines, a genomic fragment encompassing the coding region of NIGT1.1 or NIGT1.2 (without a stop codon) was amplified and cloned into pENTR/D-TOPO vector (Invitrogen), sequenced, and then integrated into the Gateway binary vector pBA002-GFP (a derivative of pBA002-HA; Kiba et al., 2012), to generate constructs that express the NIGT1.1-GFP or NIGT1.2-GFP fusion genes or GFP alone under regulation of the CaMV 35S promoter (NIGT1.1ox, NIGT1.2ox, or GFPox).

For promoter:GUS constructs, the NIGT1.1 promoter (ProNIGT1.1; 2076 bp upstream of the inferred initiation codon), the NIGT1.2 promoter (ProNIGT1.2; 2056 bp upstream), the NIGT1.3 promoter (ProNIGT1.3; 1996 bp upstream), the NIGT1.3 3′ untranslated region (3′NIGT1.3; 2014 bp downstream of the inferred stop codon), and the NIGT1.4 promoter (ProNIGT1.4; 2008 bp upstream) fragments were amplified by PCR and assembled in the Gateway binary vector pBA002a-GUS to generate ProNIGT1.1:GUS, ProNIGT1.2:GUS, ProNIGT1.3:GUS:3′NIGT1.3, and ProNIGT1.4:GUS.

These constructs were transferred into Agrobacterium tumefaciens strain EHA105, and wild-type or nigtQ mutant plants were transformed by the floral dip method as described elsewhere (Clough and Bent, 1998). Transformants were selected on MS plates with 1% sucrose, containing 10 μg L^–1 bialaphos sodium salt (for pBA002 and pBA002a), or 50 μg L^–1 hygromycin (pER8).

GFP Imaging

For whole-plant GFP imaging, chlorophyll autofluorescence and GFP fluorescence were visualized using a FluorImager 595 (Molecular Dynamics). Chlorophyll autofluorescence and the GFP signal were obtained at 488-nm excitation using a 610RG filter and at 488-nm excitation using a 530DF30 filter.

For analysis by confocal microscopy, pNIGT1.1:NIGT1.1-GFP and pNIGT1.2:NIGT1.2-GFP seedlings grown on 0.5× MS for 10 d were observed with a Zeiss LSM700 confocal imaging system.

qRT-PCR

Total RNA was extracted from root and shoot samples using an RNeasy Plant Mini kit (Qiagen) with the RNase-Free DNase set (Qiagen). First-strand cDNA was synthesized from total RNA with the ReverTra Ace qPCR RT Master Mix (Toyobo). qRT-PCR was performed on a StepOnePlus Real-Time PCR system (Applied Biosystems) with the KAPA SYBR Fast qPCR kit (KAPA Biosystems). At4g34270 was used as an internal control because this gene is one of the most stably expressed genes in Arabidopsis (Dekkers et al., 2012). Similar results were obtained using other internal control genes (ACT8). Primer sets are listed in Supplemental Table 2.

Y1H Screening

Y1H screening was performed using the Matchmaker Gold Yeast One-Hybrid System (Clontech) according to the manufacturer’s protocol. Three tandem copies of the P389 NRT2.4 promoter were cloned into pAbAi. The resulting plasmid was linearized with BbsI (New England Bio Labs) and transformed into the Y1H Gold strain to generate a bait-reporter yeast. This strain was then transformed with the pGAD424-based Arabidopsis transcription factor library, which contains 1498 Arabidopsis transcription factor cDNAs (Mitsuda et al., 2010). Transformants were spread on SD/-Leu medium with 100 ng/mL aureobasidin A and colonies were picked based on their growth. Plasmids isolated from each colony were retransformed to confirm positive interactions. Validated plasmids were then sequenced

Transient Assays in Arabidopsis Seedlings

Reporter, effector, and reference plasmids were codelivered to 10-d-old MS-grown Arabidopsis Col-0 seedlings by particle bombardment (-1000/He; Bio-Rad) as described previously (Nakamichi et al., 2010). Reporter and reference values were measured using the Dual-Luciferase Reporter Assay System (Promega) and Mithras LB940 (Berthold). Promoter activity was calculated as the firefly luciferase (FLUC)/Renilla luciferase (RLUC) ratio. The reporter plasmid contains FLUC downstream of the promoter of interest, and the reference plasmid harbors the ProCaMV35S:RLUC fusion gene (Fujimoto et al., 2000; Nakamichi et al., 2010). For analysis of the NIGT1-NRT2.4 promoter interaction (Figures 1E and 2D to 2G), an effector plasmid that expresses each NIGT1 family member fused with two stringent transcriptional activation domains VP16 (VP) at its C terminus under the CaMV 35S promoter was constructed as described previously (Fujimoto et al., 2000; Nakamichi et al., 2010). To construct reporter plasmids harboring a mutant NRT2.4 promoter that was mutated at the B1 motif (B1^m), the B2 motifs (B2^m), or in both the B1 and B2 motifs (B1^mB2^m), mutant promoters were synthesized (Eurofins Genomics) and cloned into the reporter plasmid. For transcriptional repressor activity assays (Figure 2B), a reporter plasmid harboring GAL4 promoter:FLUC and an effector plasmid that expresses each NIGT1 family member fused with GAL4 DNA binding domain at its N terminus under the CaMV 35S promoter were constructed as described previously (Fujimoto et al., 2000; Nakamichi et al., 2010).

ChIP Assays

Transgenic lines expressing GFP (GFPox), NIGT1.1-GFP (NIGT1.1ox), or NIGT1.2-GFP (NIGT1.2ox) fusion genes under control of the CaMV 35S promoter were used for immunoprecipitation with an anti-GFP antibody (Abcam, ab290, lot GR186433-3). Transgenic lines expressing GFP-FLAG (eGFPox), NIGT1.3-FLAG (eNIGT1.3ox), or NIGT1.4-FLAG (eNIGT1.4ox) fusion genes under control of the β-estradiol-inducible promoter treated with 10 μM β-estradiol for 24 h were used for immunoprecipitation with an anti-FLAG antibody (MBL Life Science, M185, lot 011).

Two grams of seedlings were harvested and cross-linked in 40 mL of 1% formaldehyde under vacuum for 20 min at room temperature. Cross-linking was stopped by adding glycine to a final concentration of 0.125 M and incubating for 5 min under vacuum. The cross-linked sample was frozen in liquid N and ground to a powder with a mortar and pestle in liquid N. The powder was added to 30 mL of ice-cold extraction buffer (0.4 M sucrose, 10 mM Tris-HCl, pH 8.0, 10 mM MgCl₂, 5 mM 2-mercaptoethanol, and complete protease inhibitor cocktail tablets [Roche]), and the mixture was homogenized with a Polytron (Kinematica) at 10,000 rpm for 2 min. After filtration through two layers of Miracloth (Millipore), the solution was centrifuged at 3000g at 4°C for 20 min. The pellet was suspended in 1 mL of resuspension buffer 1 (0.25 M sucrose, 10 mM Tris-HCl, pH 8.0, 10 mM MgCl₂, 5 mM 2-mercaptoethanol, 1% Triton X-100, and complete protease inhibitor cocktail tablets). The resuspension was centrifuged at 12,000g at 4°C for 10 min and the pellet was suspended in 300 μL resuspension buffer 2 (1.7 M sucrose, 10 mM Tris-HCl, pH 8.0, 2 mM MgCl₂, 5 mM 2-mercaptoethanol, 0.15% Triton X-100, and complete protease inhibitor cocktail tablets [Roche]). The resuspension was layered over 300 μL of resuspension buffer 2 in a 1.5 mL microcentrifuge tube and centrifuged at 16,000g at 4°C for 60 min. The pellet was resuspended in 300 μL of sonication buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 0.5% SDS, and complete protease inhibitor cocktail tablets), sonicated five times for 15 s at setting 3 (TAITEC VP30S), and then centrifuged at 12,000g at 4°C for 5 min. The supernatant was recovered as a chromatin complex solution, and 10 μL of the solution was stored as the input fraction. The chromatin complex solution was diluted 10-fold with ChIP dilution buffer (16.7 mM Tris-HCl, pH 8.0, 1.2 mM EDTA, 0.2% Triton X-100, and 167 mM NaCl). Forty microliters of Dynabeads protein G (Thermo Fisher Scientific) was added to preclear the solution during an incubation for 1 h at 4°C. Then, 5 μL of anti-GFP antibody or FLAG antibody was added to 1 mL of precleared solution (1/200 dilution) and incubated overnight at 4°C. This was followed by incubation with 40 μL of Dynabeads protein G (Thermo Fisher Scientific). The beads were washed three times with 1 mL of low-salt buffer (50 mM Tris-HCl, pH 8.0, 0.1% Triton X-100, and 167 mM NaCl) and then three times with high-salt buffer (50 mM Tris-HCl, pH 8.0, 0.1% Triton X-100, and 400 mM NaCl) three times each. The immunocomplex was eluted with 100 μL of elution buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, and 1% SDS) at 65°C for 15 min. Eluent and input fractions were incubated at 65°C overnight followed by digestion with 5 μL Proteinase K (Takara) for 1 h at 37°C to reverse cross-linking and to remove proteins. DNA was purified with a QIAquick PCR purification kit (Qiagen), and the amount precipitated was determined by qPCR using the primer sets listed in Supplemental Table 2. qPCR was performed on a StepOnePlus Real-Time PCR system (Applied Biosystems) with a KAPA SYBR Fast qPCR kit (KAPA Biosystems). The enrichment value (%input) of each fragment was calculated by normalizing the amount of fragment in the immunoprecipitate against the amount in the input.

GUS Staining

Histochemical GUS staining was performed as described elsewhere (Kiba et al., 2012). Seedlings were vacuum infiltrated for 5 min in staining buffer (50 mM potassium phosphate buffer, pH 7.0, 0.05% Triton X-100, 2 mM ferro/ferricyanide, and 2 mM X-glucuronide). Samples were incubated overnight in the dark at 37°C. Stained plants were cleared by incubation in a 70% to 100% ethanol series and observed by light microscopy (Axioplan 2; Zeiss).

Microarray Analysis

Total RNA was extracted using an RNeasy Plant Mini kit with the RNase-Free DNase kit (Qiagen) from shoots and roots of wild-type Col-0, NIGT1.2ox line 1, and pER-NIGT1.2-VP line 2 plants (n = 3 independent pools of indicated tissues from more than 10 plants). Col-0 seedlings grown in 0.5× MS agar plates for 7 d were transferred to and incubated in MGRL plates with 5 mM NH₄NO₃ (WT_Nsuf) or without nitrogen source (WT_Nstv) for 3 d. NIGT1.2ox line1 seedlings grown in 0.5× MS agar plates for 7 d were transferred to and incubated in MGRL plates without a nitrogen source (OX_Nstv) for 3 d. pER-NIGT1.2-VP line 2 seedlings grown in MGRL plates containing 5 mM NH₄NO₃ for 9 d were transferred to and incubated in MGRL plates containing 5 mM NH₄NO₃ (VP_Mock) or 5 mM NH₄NO₃ and 10 μM β-estradiol (VP_+Est) for 24 h. Three biological replicates were analyzed with the Affymetrix Arabidopsis ATH1 Genome Array. RNA quality was determined on a Bioanalyzer (Agilent), and RNA amplification, labeling, and hybridization were conducted with the 3′ IVT Express Kit (Affymetrix) according to the manufacturer’s instructions. Processing and analyses of microarray data were performed using R software (version 3.2.3 or later; https://www.r-project.org/). Gene expression levels, represented by signal intensities of corresponding probes, were obtained as logarithms to base 2 after normalization among the 15 microarray chips by the robust multiarray average method applying the “affy” library (Gautier et al., 2004). After normalization, probes annotated as “control sequence” were excluded from the following analyses. The 22,746 probes annotated as “exemplar sequence” were applied. Probes showing statistically different signal intensities between conditions or strains were determined with a two-tailed Welch’s t test (P < 0.05) (Welch, 1947) following a multiple testing correction by the Benjamini-Hochberg procedure (FDR < 0.1) (Benjamini and Hochberg, 1995). Overrepresented GO terms were determined by Fisher’s exact test (Fisher, 1922) followed by the Benjamini-Hochberg procedure (FDR < 0.05). For this analysis, probes were annotated with the GO terms by referring probe-AGI code correspondence provided by the manufacturer and the GO Slim classification downloaded from the TAIR database (https://www.arabidopsis.org) as of April 21, 2016.

Chlorophyll, Soluble Protein, and Total Amino Acid Measurements

Chlorophyll measurements were performed as described previously (Porra et al., 1989). Briefly, chlorophyll was extracted with N,N-dimethylformamide for 24 h at 4°C in the dark followed by measurement of absorbance at 663.8 nm (for chlorophyll a) and 646.8 nm (for chlorophyll b) in a spectrophotometer (UV-1650PC; Shimadzu).

Determination of soluble protein content was conducted as previously described (Makino et al., 1985). Shoot and root samples were homogenized and suspended in 50 mM Na-phosphate buffer (pH 7.5) containing 5 mM dithiothreitol and 12.5% (v/v) glycerol. The homogenate was centrifuged at 12,000g at 4°C for 10 min to clear cellular debris. Soluble protein in the supernatant was measured using Bio-Rad Bradford Protein Assay according to the manufacturer’s instructions.

For determination of free amino acids, root samples were powdered in liquid nitrogen, mixed with 10 volumes of 10 mM HCl containing 0.2 mM methionine sulfone as an internal control, and centrifuged at 18,000g at 4°C for 5 min, and the supernatant was filtered through an Ultrafree-MC filter (0.22 μm; Millipore). The filtrate was derivatized using a derivatization regent (a mixture of methanol:water:triethylamine:phenylisothiocyanate at the volume ratio of 7:1:1:1). Derivatized amino acid levels were determined using a high performance liquid chromatography system (Alliance 2695; Waters) linked to a photodiode array detector (2995; Waters) with Pico-Tag eluents (Waters) and a PicoTag reverse phase column (3.9 mm × 300 mm; Waters) according to the manufacturer’s instructions.

Nitrogen Content and Root ¹⁵N Influx

Influx of ¹⁵NO₃ was assayed as previously described (Lezhneva et al., 2014) with some modifications. Seedling roots were submerged in 0.1 mM CaSO₄ for 1 min and then transferred to complete nutrient solution containing ¹⁵NO₃ (atom% ¹⁵N: 99%) at the indicated concentrations for 5 to 15 min. Roots were transferred to 0.1 mM CaSO₄ for 1 min before harvest. Whole seedlings were dried completely at 80°C and analyzed using a FlashEA1112 elemental analyzer (Thermo) and DELTA plus Advantage Isotope-ratio mass spectrometer (Themo) coupled via a ConFlo III interface (Thermo). Influx of ¹⁵NO₃^- was calculated from the total N and ¹⁵N content in whole seedlings.

Nitrate and Phosphate Measurements

Whole seedlings, shoots, or roots were weighed and collected in 2.0 mL Eppendorf Safe-Lock tubes (Eppendorf). Samples were pulverized in liquid N, and 1 mL of milli-Q water was added to each tube to extract nitrate and free phosphate (Pi). After centrifugation, supernatant nitrate concentrations were quantified by a high performance liquid chromatography system (alliance 2695; Waters) linked to a photodiode array detector (2995; Waters) using an anion exchange column (Waters Spherisorb S5 SAX; 50 mM K₂HPO₄/KH₂PO₄, pH 3.7 at a rate of 1 mL/min) (Kiba et al., 2012) or by a spectrophotometric method as previously described (Miranda et al., 2001). Similar results were obtained by both methods. Phosphate levels were measured using the Malachite Green Phosphate Assay Kit (POMG-25H; BioAssay Systems).

Statistical Analysis

Data are given as means ± sd of one representative experiment. In order to examine whether values such as promoter activity, gene expression, and nitrate influx were significantly different between treatments or genotypes, Student’s t tests and Tukey’s honestly significant difference (HSD) tests were performed using KaleidaGraph version 4.1 software (Synergy Software).

Accession Numbers

Arabidopsis Genome Initiative locus identifiers for genes mentioned in this article are as follows: AT5G60770 (NRT2.4), AT1G25550 (NIGT1.1/HHO3), AT1G68670 (NIGT1.2/HHO2), AT3G25790 (NIGT1.3/HHO1), AT1G13300 (NIGT1.4/HRS1), AT2G03500 (HHO4/EFM), AT4G37180 (HHO5/UIF1), AT1G49560 (HHO6), AT4G28610 (PHR1), AT1G12940 (NRT2.5), AT5G16570 (GLN1;4), AT4G38340 (NLP3), AT3G03910 (GDH3), AT5G45380 (DUR3), AT4G13510 (AMT1;1), AT5G20150 (SPX1), AT5G50200 (NAR2.1/NRT3.1), AT1G08090 (NRT2.1), AT1G12110 (NRT1.1/NPF6.3), AT1G69850 (NRT1.2/NPF4.6/AIT1), AT5G43350 (PHT1;1), AT2G38940 (PHT1;4), and At1G49240 (ACT8). Rice Genome Annotation Project locus identifiers for rice NIGT1 is Os02g032560 (LOC_Os02g22020). The microarray data from this study can be found in Gene Expression Omnibus under accession number GSE100903. Germplasm used is as follows: SALK_044835 (nigt1.2-1/hho2-1), SALK_070096 (nigt1.2-2/hho2-2), WISCDSLOXHS231_10C (nigt1.3-2), SAIL_28_D03 (nigt1.3-1/hho1-1), GK-267G03 (nigt1.1-1), and SALK_067074 (nigt1.4-1/hrs1-1).

Supplemental Data

Supplemental Figure 1. Phylogenetic Tree of the Arabidopsis GARP GOLDEN2-like Family Plus Rice NIGT1.
Supplemental Figure 2. Nucleotide Sequence of the Minimal NRT2.4 Promoter (P389).
Supplemental Figure 3. Expression of NRT2.4 in NIGT1-Overexpressing Lines.
Supplemental Figure 4. T-DNA Insertion Mutants of Arabidopsis NIGT1.1, NIGT1.2, NIGT1.3, and NIGT1.4.
Supplemental Figure 5. Complementation of Arabidopsis nigt1 Quadruple Mutant Phenotypes by Genomic Fragments Containing Either NIGT1.1 or NIGT1.2.
Supplemental Figure 6. Time-Course Analysis of Expression of Arabidopsis NIGT1s after Changes in Nitrogen Availability.
Supplemental Figure 7. Upregulation of NRT2.4 by Chimeric Protein NIGT1-VP.
Supplemental Figure 8. Comparisons between Arabidopsis NIGT1 Direct Targets and Either the Nitrogen Starvation Upregulated Transcriptome or DAP-Seq.
Supplemental Figure 9. Representative Images of the Wild Type and the Arabidopsis nigt1.1 nigt1.2 nigt1.3 nigt1.4 Quadruple Mutant under Different Nitrogen Conditions.
Supplemental Figure 10. Phenotypes of NIGT1 Overexpressors and the nigt1.1 nigt1.2 nigt1.3 nigt1.4 Quadruple Mutant.
Supplemental Figure 11. Phylogenetic Tree of Plant Homologs of Arabidopsis NIGT1.
Supplemental Table 1. List of Genes Isolated in Yeast One-Hybrid Screening against the NRT2.4 Minimal Promoter.
Supplemental Table 2. List of Primers Used in This Study.
Supplemental Data Set 1. List of Genes Upregulated in Roots of pER-NIGT1.2-VP upon β-Estradiol Treatment for 24 h.
Supplemental Data Set 2. List of Genes Downregulated in Roots of NIGT1.2ox under Nitrogen Starvation Compared with the Wild Type.
Supplemental Data Set 3. List of NIGT1 Target Genes in the Root.
Supplemental Data Set 4. List of Genes Upregulated in Shoots of pER-NIGT1.2-VP upon β-Estradiol Treatment for 24 h.
Supplemental Data Set 5. List of Genes Downregulated in Shoots of NIGT1.2ox under Nitrogen Starvation Compared with the Wild Type.
Supplemental Data Set 6. List of NIGT1 Target Genes in the Shoot.
Supplemental Data Set 7. Gene Ontology Term Analysis of NIGT1 Target Genes (NIGT1 Direct Targets) in the Root.
Supplemental Data Set 8. Gene Ontology Term Analysis of NIGT1 Target Genes (NIGT1 Direct Targets) in the Shoot.
Supplemental File 1. The Alignment Used for the Phylogenetic Analysis Shown in Supplemental Figure 1.
Supplemental File 2. The Alignment Used for the Phylogenetic Analysis Shown in Supplemental Figure 11.

Acknowledgments

We thank the Arabidopsis Biological Resource Center and Nottingham Arabidopsis Stock Centre for supplying T-DNA insertion lines. This work was supported in part by the Japan Society for the Promotion of Science KAKENHI Grants 26712009 and 17K07705 to T. Kiba., 25252014 and 26221103 to S.Y., and 15H05616 to M.K., and by JST CREST Grant JPMJCR15O5 to S.Y.

AUTHOR CONTRIBUTIONS

T. Kiba., S.Y., and H.S. designed the research. T. Kiba, J.I., N.U., T. Kudo, M.K., N.M., Y.T., Y.K., T.Y., M.O.-T., M.M., K.Y., and S.Y. performed research. T. Kiba., J.I., and N.M. analyzed data. H.S and T. Kiba. wrote the article.

Footnotes

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PERMALINK

Repression of Nitrogen Starvation Responses by Members of the Arabidopsis GARP-Type Transcription Factor NIGT1/HRS1 Subfamily[OPEN]

Takatoshi Kiba

Jun Inaba

Toru Kudo

Nanae Ueda

Mineko Konishi

Nobutaka Mitsuda

Yuko Takiguchi

Youichi Kondou

Takeshi Yoshizumi

Masaru Ohme-Takagi

Minami Matsui

Kentaro Yano

Shuichi Yanagisawa

Hitoshi Sakakibara

Abstract

INTRODUCTION

RESULTS

Identification of Arabidopsis NIGT1/HRS1/HHO Family Members as NRT2.4 Promoter-Interacting Proteins

Figure 1.

Arabidopsis NIGT1 Subfamily Members Are Transcriptional Repressors That Bind to the NRT2.4 Promoter

Figure 2.

Figure 3.

Arabidopsis NIGT1 Subfamily Members Repress NRT2.4 Expression in Vivo

Figure 4.

Expression of Arabidopsis NIGT1 Subfamily Genes Is Regulated by Nitrogen and Nitrate Availability

Figure 5.

Figure 6.