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. 2018 Feb 23;30(3):638–651. doi: 10.1105/tpc.17.00809

Expression of the Nitrate Transporter Gene OsNRT1.1A/OsNPF6.3 Confers High Yield and Early Maturation in Rice[OPEN]

Wei Wang a,1, Bin Hu a,1, Dingyang Yuan b, Yongqiang Liu a,c, Ronghui Che a, Yingchun Hu d, Shujun Ou e, Yongxin Liu, Zhihua Zhang a,c, Hongru Wang a, Hua Li a,c, Zhimin Jiang a, Zhengli Zhang a, Xiaokai Gao f, Yahong Qiu a,c, Xiangbing Meng a, Yongxin Liu a, Yang Bai a, Yan Liang a,c, Yiqin Wang a, Lianhe Zhang f, Legong Li g, Sodmergen d, Haichun Jing h, Jiayang Li a, Chengcai Chu a,2
PMCID: PMC5894839  PMID: 29475937

OsNRT1.1A displays functional divergence with previously reported NRT1.1s in plants and holds great potential in promoting both high yield and early maturation in rice.

Abstract

Nitrogen (N) is a major driving force for crop yield improvement, but application of high levels of N delays flowering, prolonging maturation and thus increasing the risk of yield losses. Therefore, traits that enable utilization of high levels of N without delaying maturation will be highly desirable for crop breeding. Here, we show that OsNRT1.1A (OsNPF6.3), a member of the rice (Oryza sativa) nitrate transporter 1/peptide transporter family, is involved in regulating N utilization and flowering, providing a target to produce high yield and early maturation simultaneously. OsNRT.1A has functionally diverged from previously reported NRT1.1 genes in plants and functions in upregulating the expression of N utilization-related genes not only for nitrate but also for ammonium, as well as flowering-related genes. Relative to the wild type, osnrt1.1a mutants exhibited reduced N utilization and late flowering. By contrast, overexpression of OsNRT1.1A in rice greatly improved N utilization and grain yield, and maturation time was also significantly shortened. These effects were further confirmed in different rice backgrounds and also in Arabidopsis thaliana. Our study paves a path for the use of a single gene to dramatically increase yield and shorten maturation time for crops, outcomes that promise to substantially increase world food security.

INTRODUCTION

Nitrogen (N) application is one of the most effective means for crop yield improvement. However, N fertilizers can have deleterious effects on the environment, such as eutrophication of water bodies. N fertilization can also delay flowering and thus prolong the maturation times of crops, which significantly increases the risk of yield losses, especially in high-latitude regions where late-season low temperatures would severely restrict grain filling (Withrow, 1945; Scott et al., 1973; Davenport et al., 2001; Dielen et al., 2001; Castro Marín et al., 2011; Li et al., 2017). Early flowering, the major factor contributing to early maturation, is a prerequisite for the expansion of crop production to higher latitude regions with shorter growing seasons, and for double/triple cropping systems each year (Izawa, 2007; Li et al., 2009). Therefore, the ability to increase yields by improving N use efficiency (NUE) while simultaneously reducing maturation times is viewed as one of the most desirable goals in crop breeding (Li et al., 2017), although to date, breeders have had little success.

Functional and structural studies have demonstrated AtNRT1.1 (AtNPF6.3), a member of the nitrate transporter 1/peptide transporter family, is the central component in nitrate signaling in Arabidopsis thaliana, as in addition to mediating nitrate uptake and transport, AtNRT1.1 functions as a sensor to trigger the primary nitrate response (Ho et al., 2009). Monocots and eudicots differ in the number of their NRT1.1 genes, with grass species typically having three to four NRT1.1 members and most eudicot species having only one NRT1.1 gene (Plett et al., 2010). Recently, Wen et al. (2017) reported that two putative homologs of AtNRT1.1 (AtNPF6.3) in maize (Zea mays ssp mays), ZmNPF6.4 and ZmNPF6.6, display distinct substrates affinity for nitrate and chloride, indicating a possible functional divergence among different members of the NRT1.1 family in grasses. However, the biological functions and the possible divergence of the NRT1.1 genes in grass species remain largely unknown. Previously, we showed that OsNRT1.1B (OsNPF6.5) is involved in nitrate utilization, and a single polymorphism in this gene contributes to the long-noted divergence in NUE between indica and japonica subspecies of Asian cultivated rice (Oryza sativa) (Hu et al., 2015). Similar expression and subcellular localization patterns, as well as conserved functions in nitrate uptake, transport, and signaling, strongly suggest that OsNRT1.1B is the functional homolog of AtNRT1.1. In this study, we revealed that OsNRT1.1A (OsNPF6.3), another member of rice NRT1.1 family, displays different N-responsive expression and subcellular localization patterns with OsNRT1.1B, showing an example of functional divergence among NRT1.1 paralogs in grass species. More importantly, overexpression of OsNRT1.1A dramatically increases grain yields by improving NUE, and strikingly, also shortens maturation times, providing a promising target for crop improvement.

graphic file with name PC_TPC2017RA00809R1_fx1.jpg

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RESULTS

OsNRT1.1A Displays Ammonium-Inducible Expression and OsNRT1.1A Predominantly Localizes to the Tonoplast

Rice has three putative homologs of AtNRT1.1: OsNRT1.1A, OsNRT1.1B, and OsNRT1.1C (OsNPF6.4) (Plett et al., 2010). Among these, OsNRT1.1A clusters closest to and shares the highest protein sequence identity with AtNRT1.1 (Figure 1A; Supplemental Figure 1 and Supplemental File 1). However, in contrast to OsNRT1.1B, which is strongly induced by nitrate, OsNRT1.1A displays an ammonium-induced expression pattern (Figure 1B). The ammonium-induced expression of OsNRT1.1A suggested that it is probably involved in ammonium utilization, which is particular important for rice, as ammonium is not only the major N form in the paddy field but also the preferred N source for rice (Arth et al., 1998; Liu et al., 2015). OsNRT1.1A shows a very similar tissue expression pattern to OsNRT1.1B, as seen by OsNRT1.1Apromoter:GUS transgenic plants with preferential expression in the epidermis and in the vascular tissues of roots, except that OsNRT1.1A is also highly expressed in the parenchyma cells of both culms and leaf sheaths (Figure 2).

Figure 1.

Figure 1.

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OsNRT1.1A Displays Ammonium-Inducible Expression.

(A) Phylogenetic analysis of amino acid sequences of AtNRT1.1 and OsNRT1.1A/B/C using maximum likelihood method by Mega 6.0. Bootstrap = 1000.

(B) Expression analysis of OsNRT1.1A and OsNRT1.1B under different N sources (ammonium or nitrate) in roots of rice seedlings assessed by RNA-sequencing. RPKM, reads per kilobase per million mapped reads.

Figure 2.

Figure 2.

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GUS Staining of OsNRT1.1Apromoter:GUS Transgenic Plants.

Tissues used for GUS staining include roots ([A] to [C]), culms (D), leaf sheaths (E), and leaf blades (F), showing cross sections in (B) to (F). (B) and (C) indicate the regions of root tip and root hair, respectively. Bars = 1 mm in (A), 0.3 mm in (B) and (C), and 1 mm in (D) to (F).

OsNRT1.1A predominantly localizes to the tonoplast, as seen by colocalization assays with a tonoplast marker and immunogold analysis (Figures 3A and 3B). Furthermore, fluorescence observation in Nicotiana benthamiana leaves and vacuole release from rice protoplasts also consistently supported the tonoplast localization of OsNRT1.1A (Supplemental Figure 2), demonstrating a clear difference from plasma membrane-localized AtNRT1.1 and OsNRT1.1B (Ho et al., 2009; Hu et al., 2015). This indicated that the additional member of the NRT1.1 family in rice may have undergone functional divergence, although OsNRT1.1A also has nitrate transporter activity (Figure 4).

Figure 3.

Figure 3.

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OsNRT1.1A Predominantly Localizes to the Tonoplast.

(A) The OsNRT1.1A-eGFP signal colocalizes with the tonoplast marker α-TIP (fused with RFP) in rice protoplasts. Bars = 20 µm.

(B) Immunogold analysis of OsNRT1.1A-eGFP in 35S:OsNRT1.1A-eGFP transgenic rice and wild-type DJ (WT). Arrows indicate the immunodetection signal. PM, plasma membrane; V, vacuole. Bars = 100 nm.

Figure 4.

Figure 4.

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OsNRT1.1A Shows Nitrate Transport Activity in Vitro.

Nitrate uptake assay in Xenopus laevis oocytes injected with water, OsNRT1.1A, and AtNRT1.1 using 15N-nitrate. AtNRT1.1 was used as the positive control. Twenty replicates were performed for each sample. Asterisks indicate the significant differences between water (negative control) and OsNRT1.1A as evaluated by Student’s t tests: **P < 0.01.

Loss of Function of OsNRT1.1A Reduces the Utilization of Both Nitrate and Ammonium

To further investigate the function of OsNRT1.1A in rice, a homozygous loss-of-function mutant (japonica variety Dongjin background [DJ]) was characterized (Supplemental Figures 3A to 3C). The seedlings of the osnrt1.1a mutant exhibited significant growth retardation compared with the wild type when grown in hydroponic culture with sufficient N supply (Figure 5A). Notably, the growth retardation of the osnrt1.1a mutant was significant when N was supplied as ammonium or nitrate (Figure 5A). By contrast, the seedlings of the osnrt1.1b mutant did not display obvious growth retardation compared with the wild type under ammonium or nitrate conditions (Figure 5B). Moreover, 15N quantification following 15N-nitrate or 15N-ammonium feeding showed that acquisition of both nitrate and ammonium was repressed in the osnrt1.1a mutant (Supplemental Figure 3D).

Figure 5.

Figure 5.

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OsNRT1.1A Displays Functional Divergence with OsNRT1.1B.

(A) Growth of wild-type DJ (WT) and osnrt1.1a mutant seedlings under different N sources.

(B) Growth of wild-type ZH11 (WT) and osnrt1.1b mutant seedlings under different N sources. NN, no N; A, ammonium (2 mM); N, nitrate (2 mM); AN, ammonium and nitrate (1 mM for each). Bars = 8 cm.

(C) RT-qPCR-based expression analyses of genes involved in utilization of nitrate and ammonium in roots of wild-type DJ and osnrt1.1a mutant plants under normal hydroponic cultivation.

(D) RT-qPCR-based nitrate induction assay in roots of osnrt1.1a mutant and osnrt1.1b mutant. Values are the means ±sd (n = 3). Asterisks indicate significant differences between wild-type plants and according mutants as evaluated by Student’s t tests: *P < 0.05 and **P < 0.01.

Given that AtNRT1.1 and OsNRT1.1B act as transceptors triggering nitrate-responsive gene expression (Ho et al., 2009; Hu et al., 2015), we hypothesized that OsNRT1.1A may also function in regulating N utilization-related gene expression. Under normal hydroponic cultivation with nitrate and ammonium supply, the expression of genes for nitrate uptake and transport such as OsNRT1.1B, OsNRT2.1, and OsNRT2.3a, as well as genes for nitrate assimilation such as OsNIA1 and OsNIR1, was significantly downregulated in the osnrt1.1a mutant (Figure 5C). Interestingly, the expression of genes for ammonium uptake and assimilation, such as OsAMT1.1, OsGS1.2, and OsGOGAT1, was also greatly repressed in the osnrt1.1a mutant (Figure 5C). By contrast, the expression of these N utilization-related genes was not repressed in osnrt1.1b mutant (Supplemental Figure 3E). Under short-term nitrate treatment, the induction of typical nitrate-responsive genes such as OsNRT2.1, OsNIA1, and OsNIA2 was greatly repressed in osnrt1.1b mutant (Figure 5D), whereas this primary nitrate-stimulated response was not repressed in osnrt1.1a mutant (Figure 5D). These results revealed that OsNRT1.1A plays a fundamental role in maintaining N utilization at high rates not only for nitrate but also for ammonium. OsNRT1.1B, closely related to AtNRT1.1, is responsive for sensing the nitrate stimulus and triggering downstream nitrate-induced gene expression in the short term.

Together, the altered patterns of N-responsive expression and subcellular localization of OsNRT1.1A indicated that a functional divergence has occurred between OsNRT1.1A and OsNRT1.1B and that OsNRT1.1A confers a substantial improvement in N utilization, especially for ammonium. To test whether OsNRT1.1B could complement the growth retardation of the osnrt1.1a mutant, we introduced OsNRT1.1B into osnrt1.1a mutant (osnrt1.1a/OsNRT1.1B) (Supplemental Figure 4A). Compared with the wild type, the seedlings of osnrt1.1a/OsNRT1.1B transgenic plants showed severe growth retardation (Supplemental Figure 4B), further supporting the functional divergence between OsNRT1.1A and OsNRT1.1B.

The osnrt1.1a Mutant Displays Severe Yield Loss and Late Flowering

In the field, osnrt1.1a mutants displayed substantial decreases in plant height, panicle size, seed-setting rate, and 1000-grain weight (Figure 6A; Supplemental Figure 4C), and grain yield thereby decreased by ∼80% compared with the wild type (Figure 6B). The osnrt1.1b mutant also exhibited defects in these agronomic traits but much slighter than osnrt1.1a, with ∼20% loss of grain yield (Supplemental Figure 4D). Since osnrt1.1a (DJ background) and osnrt1.1b (ZH11 background) were derived from different cultivars, the phenotype of the mutants might differ in different backgrounds. To exclude this possibility, we knocked down the expression of OsNRT1.1A in the ZH11 background using RNA interference (Supplemental Figure 4E); this also led to much more severe growth defects and yield loss compared with the osnrt1.1b mutant (Supplemental Figures 4D to 4G).

Figure 6.

Figure 6.

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Loss of Function of OsNRT1.1A Results in Severe Grain Yield Loss and Late Flowering.

(A) Gross morphological phenotypes and panicle phenotypes of wild-type DJ (WT) and osnrt1.1a mutants grown in the field. Bars = 20 cm (gross morphological phenotypes) and 6 cm (panicle).

(B) Grain yield per plant of wild-type DJ and osnrt1.1a mutant.

(C) Days to flowering of wild-type DJ and osnrt1.1a mutant.

(D) RT-qPCR-based expression analysis of flowering-promoting genes in leaves of wild-type DJ and osnrt1.1a mutant. Values are the means ± sd (10 replicates for grain yield per plant, 10 replicates for days to flowering, and 3 replicates for RT-qPCR). Asterisks indicate significant differences between wild-type DJ and osnrt1.1a mutant as evaluated by Student’s t tests: **P < 0.01.

Notably, we also observed that osnrt1.1a mutant showed significant late-flowering and, thus, prolonged maturation times (Figures 6A and 6C; Supplemental Figure 4C), whereas the flowering time was not significantly altered in the osnrt1.1b mutant (Supplemental Figure 4D). All of the growth defects and the late-flowering phenotypes were completely rescued by the introduction of OsNRT1.1A into the osnrt1.1a mutant background (Supplemental Figures 5A and 5B). We also observed that the expression of Heading date 3a (Hd3a), Early heading date 1 (Ehd1), and RICE FLOWERING LOCUS T1 (RFT1), critical genes known to promote flowering in rice (Doi et al., 2004; Komiya et al., 2009; Itoh et al., 2010), were significantly downregulated in osnrt1.1a mutants (Figure 6D). Furthermore, the expression of both OsNRT1.1A and Hd3a in the wild type was dramatically repressed by high N treatment, which is in accordance with the late-flowering phenotype caused by high N application (Supplemental Figures 5C to 5E). These data indicated that OsNRT1.1A is also involved in N-regulated flowering. Flowering time is one of the most important agronomic traits for determining the adaption to local environments and expansion of cultivation area, and high N application usually delays flowering (Withrow, 1945; Castro Marín et al., 2011; Li et al., 2017). Recalling the important effect of OsNRT1.1A in improving N utilization, our results suggested that OsNRT1.1A might provide a solution for rice to simultaneously promote N utilization and early maturation.

Overexpression of OsNRT1.1A Significantly Improves N Utilization

We next investigated whether OsNRT1.1A overexpression can facilitate N utilization. To this end, we generated transgenic rice overexpressing OsNRT1.1A under the control of either its native promoter or the constitutively active rice ACTIN1 promoter. RT-qPCR analysis showed that the expression of OsNRT1.1A was greatly increased in the overexpression (OE) transgenic lines (OEnp-3 and OEnp-4, overexpression driven by the native promoter; OEa-6, overexpression driven by the ACTIN1 promoter) (Supplemental Figure 6A). Compared with wild-type plants, OsNRT1.1A-OE plants exhibited significantly improved growth under long-term hydroponic culture, as indicated by increased plant height, chlorophyll content, and biomass (Figure 7A; Supplemental Figure 6B). The acquisition of nitrate and ammonium increased, as demonstrated by 15N-nitrate and 15N-ammonium feeding experiments, and the expression of genes for the utilization of nitrate and ammonium was also significantly upregulated in OsNRT1.1A-OE plants (Figures 7B and 7C), indicating that manipulation of OsNRT1.1A has great potential for improving N utilization of rice.

Figure 7.

Figure 7.

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OsNRT1.1A Overexpression Activates N Utilization.

(A) Growth of wild-type DJ (WT) and OsNRT1.1A-OE plants in long-term hydroponic culture under LN (400 μM) or HN (2 mM) conditions. Bars = 20 cm.

(B) 15N accumulation assays in shoots of wild-type DJ and OsNRT1.1A-OE plants labeled with 15N-nitrate or 15N-ammonium. Values are the means ± sd (n = 4).

(C) RT-qPCR-based expression analysis of N utilization genes in roots of wild-type DJ and OsNRT1.1A-OE plants. Numbers in table indicate the relative expression level, and the darker color represents the higher increased folds of gene expression in OsNRT1.1A-OE plants. Values are the means ± sd (n = 3). Asterisks indicate significant differences between wild-type DJ and OsNRT1.1A-OE plants as evaluated by one-way ANOVA with Tukey’s test: *P < 0.05 and **P < 0.01.

OsNRT1.1A Promotes Nuclear Localization of NLPs

The results from osnrt1.1a mutant and OsNRT1.1A-OE plants consistently demonstrated that OsNRT1.1A is involved in upregulating the expression of a great number of critical N utilization genes. In Arabidopsis, AtNLP7 is viewed as the central transcription factor in nitrate signaling and is involved in regulating the expression of N utilization genes. AtNLP7 is regulated by nitrate-dependent nuclear localization to trigger target gene expression (Marchive et al., 2013). We found that the two closest homologs of AtNLP7 in rice (Chardin et al., 2014), OsNLP3 and OsNLP4, also exhibited nitrate-promoted nuclear localization (Supplemental Figure 7). Notably, OsNRT1.1A can greatly promote the nuclear localization of OsNLP3 and OsNLP4 even under nitrate-absent condition (Figure 8). Moreover, the nuclear localization of OsNLP3 and OsNLP4 was also repressed in osnrt1.1a mutants compared with that in the wild type (Supplemental Figure 8), collectively indicating that OsNRT1.1A upregulates N utilization genes via facilitating cytoplasmic-nuclear shuttling of NLPs.

Figure 8.

Figure 8.

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OsNRT1.1A Promotes Nuclear Localization of NLPs.

The effect of OsNRT1.1A on subcellular localization of OsNLP3/4-eGFP in rice protoplasts under nitrate-absent condition. OsNLP3/4-eGFP were cotransformed with 35S:RFP (red fluorescent protein) or 35S:OsNRT1.1A-RFP. Bars = 15 µm.

OsNRT1.1A Overexpression Promotes Both High Yield and Early Maturation

As expected, the OsNRT1.1A-OE plants displayed improved growth in field trials with significantly earlier flowering and larger panicle (Figures 9A and 9B; Supplemental Figures 9A to 9C). In accordance with the early-flowering phenotype, the expression of Hd3a, Ehd1, and RFT1 was also significantly upregulated in OsNRT1.1A-OE plants (Supplemental Figure 9D). The improved growth and early-flowering phenotypes and the upregulation of early-flowering genes in OsNRT1.1A-OE plants are consistent with the results from the osnrt1.1a mutant, which collectively demonstrated that OsNRT1.1A plays important roles in promoting both N utilization and flowering.

Figure 9.

Figure 9.

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OsNRT1.1A Overexpression Promotes Early Flowering and Grain Yield Improvement in Rice.

(A) Growth of wild-type DJ (WT) and OsNRT1.1A-OE plants (OEnp-4) in the field at flowering (left) and grain-filling (right) stage. Bars = 50 cm.

(B) The panicles of wild-type DJ and OsNRT1.1A-OE plants. Bar = 8 cm.

(C) Grain yield per plant, actual yield per plot, NUE, and days to flowering of wild-type and OsNRT1.1A-OE plants under LN conditions in a field trial in Beijing (2015).

(D) As in (C), for HN conditions in a field trial in Beijing (2015). Values in (C) and (D) are the means ± sd (18 replicates for grain yield per plant, 4 replicates for actual yield per plot and NUE, and 41 replicates for days to flowering). Asterisks indicate significant differences between wild-type and OsNRT1.1A-OE plants as evaluated by one-way ANOVA with Tukey’s test: **P < 0.01.

To further test the potential of OsNRT1.1A, large-scale field trials of OsNRT1.1A-OE plants were performed in different years at three different locations across China: Beijing (E116°, N40°), Changsha (E112°, N28°), and Sanya (E108°, N18°). In Beijing, the field trials were performed in two years (2015 and 2016) under both low (LN) and high N (HN) conditions. In 2015, under the LN condition, OsNRT1.1A-OE plants showed significant increases in seed number per panicle, 1000-grain weight, and slight increase in tiller number, resulting in ∼32 to 50% increases in grain yield per plant (Supplemental Table 1; Figure 9C). Under the HN conditions, OsNRT1.1A-OE plants displayed significant increases in the seed number per panicle and in the 1000-grain weight, resulting in ∼25 to 45% increases in grain yield per plant (Supplemental Table 1; Figure 9D). Compared with wild-type plants, the actual yield per plot and the NUE (defined as the grain yield per unit of available N in the soil) (Moll et al., 1982, 1987) increased by ∼38 to 54% and ∼33 to 45% in OsNRT1.1A-OE plants under the LN and HN conditions, respectively (Figures 6C and 6D). More excitingly, the flowering time of OsNRT1.1A-OE plants was ∼9 to 13 and ∼10 to 18 d earlier than wild-type plants under LN and HN conditions, respectively (Figures 9C and 9D), which significantly shortened the maturation times (Supplemental Table 1). The results of the 2015 and 2016 field tests in Beijing were very similar (Supplemental Figure 10). In Changsha (2016), OsNRT1.1A-OE plants displayed significant improvements in grain yield and NUE (∼27–37% in LN and ∼16% in HN for both traits), these plants also exhibited earlier flowering than the wild type under both of the N conditions (∼3–6 d in LN and ∼4–6 d in HN) (Supplemental Figure 11A). Field tests in Sanya (tropical region) were performed from December 2015 to April 2016, and OsNRT1.1A-OE plants had greatly increased grain yields and NUE (∼47–63% in LN and ∼11–32% in HN for both traits) (Supplemental Figure 11B). However, there was no significant difference in flowering time between OsNRT1.1A-OE and wild-type plants grown in Sanya (Supplemental Figure 11B), which may be attributable to the short-day conditions in Sanya, as short days strongly promote flowering in japonica rice (Brambilla and Fornara, 2013). We also overexpressed OsNRT1.1A in a different rice background, Hejiang 19 (HJ19), a japonica cultivar that developed for the northernmost rice-growing areas of China (E130°, N46°). When grown in Beijing (E116°, N40°) and Harbin (E127°, N48°), HJ19/OsNRT1.1A-OE transgenic plants consistently displayed markedly increased grain yields (Supplemental Figures 12A to 12C). These OE transgenic plants also exhibited an early-flowering phenotype, which is impressive as HJ19 already has a very short flowering time (Supplemental Figures 12A to 12C).

As eudicot species usually have one NRT1.1 gene, it is possible that introduction of OsNRT1.1A would give rise to stronger effects. To test this, we overexpressed OsNRT1.1A in Arabidopsis. This resulted in dramatically increased plant size and earlier flowering (∼6–15 d earlier than the wild type) (Figure 10A). Strikingly, the seed weight per plant and biomass increased up to 90% in the transgenic plants (Figure 10B). These results suggested that overexpression of OsNRT1.1A has enormous potential as a strategy to increase yields and shorten maturation times in both monocot and eudicot crops.

Figure 10.

Figure 10.

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OsNRT1.1A Overexpression in Arabidopsis Also Improves Seed Weight and Affects Flowering Time.

(A) Growth of wild-type Arabidopsis (WT) and transgenic Arabidopsis overexpressing OsNRT1.1A (AtOE-4/12) at different growth stages. Bars = 5 cm (left) and 12 cm (middle and right).

(B) RT-qPCR-based OsNRT1.1A expression, days to flowering, seed weight per plant, and biomass of wild-type Arabidopsis and overexpression plants. Values are the means ± sd (3 replicates for RT-qPCR analysis, 17 replicates for days to flowering, and 10 replicates for seed weight per plant and biomass). Asterisks indicate the significant differences between wild-type Arabidopsis and AtOE plants as evaluated by one way ANOVA with Tukey’s test: **P < 0.01.

DISCUSSION

As a plasma membrane-localized transporter, besides mediating nitrate uptake and transport, AtNRT1.1 also functions as a sensor to perceive external nitrate and trigger the expression of nitrate-responsive genes in Arabidopsis (Ho et al., 2009). Given the central role of AtNRT1.1 in nitrate utilization, it was very attractive to further explore the function and application of the NRT1.1 homologs in NUE improvement, especially in crops. However, the function of NRT1.1s is still largely unknown in crops, even though it has been noted that the major grain crops like rice, wheat (Triticum aestivum), and maize possess additional members of the NRT1.1 family (Plett et al., 2010). Our previous work demonstrated that OsNRT1.1B is the functional homolog of AtNRT1.1 in rice (Hu et al., 2015). In this study, we found that OsNTR1.1A, another rice NRT1.1, displays an unexpected expression pattern with strong induction by ammonium. Additionally, functional characterization of the loss-of-function mutant and overexpression transgenic plants demonstrated that OsNRT1.1A can promote the utilization of both ammonium and nitrate by upregulating the expression of N utilization-related genes. In comparison with AtNRT1.1 and OsNRT1.1B mediating the nitrate-stimulated primary response, OsNRT1.1A seems to play a more fundamental role in managing N utilization under a relative stable N supply with both ammonium and nitrate. Possibly, this functional divergence between OsNRT1.1A and OsNRT1.1B is associated with their subcellular localizations. The plasma membrane localization allows AtNRT1.1 and OsNRT11.B to sense the extracellular N status, whereas the tonoplast localization allows OsNRT1.1A to sense the intracellular N status. Rice is traditionally planted in waterlogged fields where ammonium is the major N source; however, up to 40% of total N taken up by rice is absorbed as nitrate because of nitrification in the rhizosphere (Arth et al., 1998; Li et al., 2008; Xu et al., 2012). Thereby, rice has evolved with the ability to utilize both ammonium and nitrate at high rates. Notably, the action of OsNRT1.1A seems to contribute to this specified N utilization manner of rice, suggesting a vital role of OsNRT1.1A in determination of rice NUE. The severe growth retardation and yield loss in osnrt1.1a mutants further supports this conclusion.

Many attempts have been made to use the overexpression of N transporter genes to improve NUE and grain yields (Fang et al., 2013; Chen et al., 2016; Fan et al., 2016). All such efforts have required consideration of the influence of N in delaying maturation times. Our results demonstrate that overexpression of OsNRT1.1A can confer improvements in NUE and grain yield while simultaneously shortening maturation times. Therefore, OsNRT1.1A possibly provides a solution to the conflict between N nutrition and maturation time. Impressively, the effect of OsNRT1.1A was not only verified in different rice backgrounds but also in Arabidopsis, indicating the great potential for the use of this gene in crop improvement programs for multiple crop species. Further transgenic tests in eudicot crops such as soybean (Glycine max) and tomato (Solanum lycopersicum) will be greatly helpful to explore the application value of OsNRT1.1A.

OsNRT1.1A functions in promoting both high yield and early maturation by upregulating the expression of genes involved in N utilization and flowering. However, the regulating mechanism between OsNRT1.1A and these genes is still far from fully understood. We provide preliminary evidence to unveil the regulatory function of OsNRT1.1A, showing that OsNRT1.1A can facilitate the nuclear localization of NLP transcription factors, which play a central role in activating the expression of N utilization-related genes. Recently, Liu et al. (2017) revealed that the phosphorylation of AtNLP7 by Ca2+-sensor protein kinases can promote its nuclear localization. Given the tonoplast localization, it is intriguing to speculate that OsNRT1.1A might serve as an intracellular scaffold to recruit NLPs and Ca2+-sensor protein kinases together, which can facilitate the phosphorylation of NLPs and consequently promote their nuclear localization. To date, the mechanism underlying N-regulated flowering remains largely elusive. Recently, Yuan et al. (2016) reported that CRYPTOCHROME1 (CRY1), a blue light receptor, is involved in N-regulated flowering in Arabidopsis. N supply can affect the nuclear CRY1 protein level, which modulates the amplitude of the circadian clock and in turn controls flowering. Given the direct involvement of OsNRT1.1A in N signaling and its function in promoting nuclear localization of NLPs, it is possible that OsNRT1.1A could also facilitate the nuclear localization of a CRY1 homolog in rice, thus mediating N-regulated flowering. Interestingly, OsNRT1.1A was also highly expressed in the developing panicles (Supplemental Figure 13), further suggesting OsNRT1.1A might be directly involved in regulating flowering or panicle development.

Alternative to direct effects on the clock, the function of OsNRT1.1A in regulating flowering could reflect its significant role in improving N utilization. In general, high N supply can greatly delay flowering, but it is not clear how high N application is responsible for delayed flowering. Possibly, high accumulation of the primary N source like nitrate and ammonium might convey the signal of a rich N status to plants, which favors the vegetative growth and delays flowering. As the assimilation of primary N sources, this repression of flowering will be released. Our results showed that OsNRT1.1A not only upregulates the expression of nitrate and ammonium transporters, but also activates the expression of a set of genes involved in nitrate and ammonium assimilation, including NIA, NIR, GS, and GOGAT, which greatly speeds up N assimilation and thereby eliminates high N accumulation, which is the cause of flowering repression. Despite such hypotheses, our understanding of the mechanism of OsNRT1.1A is far behind our recognition of its vital contribution in determining N utilization and agronomic performance in rice. Further efforts concentrating on identifying OsNRT1.1A-interacting proteins will be of particular importance to get new insight into its regulatory mechanism.

METHODS

Plant Materials and Growth Conditions

The rice (Oryza sativa) varieties Dongjin (DJ), Zhonghua 11 (ZH11), and Hejiang 19 (HJ19) were used in this study. The homozygous osnrt1.1a T-DNA insertion mutant (PFG_1E-00433.L, DJ background) was ordered from the Korea Rice Mutant Center (Pohang, Korea) (Jeong et al., 2002). The osnrt1.1b mutant was identified in a previous study (Hu et al., 2015). For short-term hydroponic culture, rice seedlings were grown in modified Kimura B solution in a growth chamber with a 12-h-light (30°C)/12-h-dark (28°C) photoperiod, ∼200 μmol m−2 s−2 photon density, and ∼70% humidity. The modified Kimura B solution contained the following macronutrients (mM): (NH4)2SO4 (0.5), MgSO4·7H2O (0.54), KNO3 (1), CaCl2 (0.36), K2SO4 (0.09), KH2PO4 (0.18), and Na2SiO3·9H2O (1.6); and micronutrients (μM): MnCl2·4H2O (9.14), H3BO3 (46.2), (NH4)6Mo7O24·4H2O (0.08), ZnSO4·7H2O (0.76), CuSO4·5H2O (0.32), and Fe(II)-EDTA (40), with the pH adjusted to 5.8. The nutrient solution for culture was renewed every day. For long-term hydroponic culture, rice seedlings were grown in an artificial climate chamber (with 1 kW high-intensity discharge lamps) with a 12-h-light (28°C)/12-h-dark (25°C) photoperiod, ∼300 μmol m−2 s−1 photon density, and ∼40% humidity. The modified Kimura B solution [200 μM KNO3 together with 100 μM (NH4)2SO4 for LN and 1 mM KNO3 together with 0.5 mM (NH4)2SO4 for HN] with different N concentrations were used for long-term hydroponic cultivation. For osnrt1.1a mutant phenotyping under different N sources, the N was supplied with ammonium [1 mM (NH4)2SO4], nitrate (2 mM KNO3), or ammonium and nitrate [0.5 mM (NH4)2SO4 and 1 mM KNO3], respectively.

The Arabidopsis thaliana ecotype Columbia (Col-0) was used in this study. Arabidopsis seeds were surface-sterilized with 2.5% NaClO, rinsed five times with sterile distilled water, and placed on half-strength Murashige and Skoog medium containing 1.0% (w/v) sucrose and 0.7% (w/v) agar. After vernalization at 4°C for 2 d, seeds were germinated and grown in growth chambers at 22°C and 70% relative humidity. For measurement of flowering time, 7-d-old seedlings were transferred to pots containing a 2:1 vermiculite:soil mixture under the condition of an 8-h-light /16-h-dark photoperiod and 120 μmol m−2 s−2 photon density.

RNA-Sequencing Analysis

About 100 rice (japonica cultivar ZH11) seedlings of each treatment were grown hydroponically in a growth chamber with the condition described above. For long-term ammonium and nitrate treatments, the seedlings were cultured in modified Kimura B solution after germination with different ammonium or nitrate concentrations (0.2/2/10 mM), and the total concentrations of potassium in all treatments were adjusted for consistency. Roots of 10-d-old seedlings were sampled for RNA-sequencing. For each treatment, 15 seedlings were collected as a sample, and three independent biological replicates were conducted. RNA library construction and sequence analysis were conducted as described previously (Li et al., 2016).

15N-Nitrate Uptake Assay in Xenopus laevis Oocytes

The coding region of OsNRT1.1A was amplified and cloned into the pCS2+ vector between the restriction sites BamHI and EcoRI, and then was linearized by ApaI (Rupp et al., 1994). Complementary RNA of OsNRT1.1A was synthesized in vitro using the mMESSAGE mMACHINE kit (Ambion; AM1340) according to the manufacturer’s protocol. X. laevis oocytes at stage V-VI were harvested and defolliculated in a Ca2+-free solution (82 mM NaCl, 20 mM MgCl2, 2 mM KCl, and 5 mM HEPES, pH 7.4) containing 1 mg/mL collagenase type II A (Sigma-Aldrich) for 30 min at room temperature (22°C). Oocytes were injected on the same day with 46 ng of OsNRT1.1A complementary RNA using a Nanoject II injector (Drummond Scientific). After injection, oocytes were cultured in ND-96 medium (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 5 mM HEPES, and 50 μg/mL gentamicin sulfate, pH 7.4) at 18°C for 24 h and ready for 15N-nitrate uptake assay. High- and low-affinity uptake assays in oocytes were performed using 200 μM and 10 mM 15N-KNO3, respectively, as described previously (Almagro et al., 2008). For each sample, one oocyte was analyzed as an independent sample, and 20 replicates were conducted. AtNRT1.1 was used as the positive control. Primers used are listed in Supplemental Table 2.

Labeling with 15N-Nitrate or 15N-Ammonium for Determination of 15N Accumulation

15N-accumulation assay after 15N-nitrate or 15N-ammonium labeling was performed with 15N-labeled KNO3 (98 atom % 15N; Sigma-Aldrich; No. 335134) or 15N-labeled NH4Cl (98 atom % 15N; Sigma-Aldrich; No. 299251), respectively. For 15N-nitrate accumulation assay, rice seedlings were cultured in the Kimura B solution for 10 d. Next, the seedlings were pretreated with the Kimura B solution for 2 h and then transferred to modified Kimura B solution containing 5 mM 15N-KNO3 for 3 h. At the end of labeling, the roots were washed for 1 min in 0.1 mM CaSO4 and separated from the shoots. Roots and shoots were collected and dried at 70°C. Finally, the samples were ground and the 15N content was evaluated by an isotope ratio mass spectrometer with an elemental analyzer (Thermo Finnigan Delta Plus XP; Flash EA 1112). For 15N-ammonium accumulation assays, the treatment was conducted as above except that 5 mM 15N-KNO3 was replaced with 1 mM 15N-NH4Cl and 1 mM KNO3. For each sample, 20 seedlings were collected as a sample, and four biological replicates were used.

Nitrate Induction Assay

Rice seedlings were first cultured in the modified Kimura B solution with 0.25 mM (NH4)2SO4 as the sole N source for 3 weeks and then were induced with 5 mM KNO3 for 0.5 h according to the method as previously described (Hu et al., 2015).

Subcellular Localization Assay

To investigate the subcellular localization of OsNRT1.1A, the 35S:OsNRT1.1A-eGFP (enhanced green fluorescent protein) fusion constructs were produced by inserting the full open reading frame of OsNRT1.1A into the pCAMBIA2300-35S:eGFP vector. The gene-specific primers used for PCR amplification are listed in Supplemental Table 2. α-TIP was used as a tonoplast marker (Hunter et al., 2007). Plasmids were extracted and purified using the Plasmid Midi Kit (Qiagen; No. 12143) following the manufacturer’s manual. The rice protoplasts were isolated and transformed according to the published methods described previously (Bart et al., 2006; Zhang et al., 2011). For vacuole releasing, intact rice protoplasts were treated by vacuole lysis buffer (10 mM EDTA, 10 mM EGTA, and 95 mM mannitol or sorbitol, pH 8.0 adjusted by 1 M Tris) for 3 to 5 min, and ready for microscopy observation. To confirm the subcellular localization, the construct was also transiently expressed in Nicotiana benthamiana leaves by Agrobacterium tumefaciens-mediated infiltration (strain GV3101) as described previously (An et al., 2017).

Moreover, for analysis the nuclear-cytoplasmic shuttling of OsNLP3/4 and the effect of OsNLP3/4 nuclear retention promoted by OsNRT1.1A, the full-length open reading frames of OsNLP3/4 and OsNRT1.1A were cloned into pCAMBIA2300-35S:eGFP and pSAT6-mRFP-N1 (CD3-1108) vector, respectively. To investigate the nuclear-cytoplasmic shuttling of OsNLP3/4, the transformed rice protoplasts were treated for 30 min with 10 mM KCl or KNO3, respectively. In addition, to test the OsNRT1.1A-mediated nuclear retention of OsNLP3/4, pCAMBIA2300-35S:OsNLP3/4-eGFP was cotransformed into rice protoplasts with pSAT6-OsNRT1.1A-mRFP or pSAT6-mRFP-N1, respectively. To further analyze the OsNLP3/4-eGFP subcellular localization patterns in the backgrounds of the wild type and osnrt1.1a mutant, rice seedlings were germinated and grown on the modified Kimura B solution with 0.25 mM (NH4)2SO4 and 0.5 mM KNO3 as the N sources for 10 d and used for rice protoplast isolation and transient expression analysis. The fluorescence images were captured via a confocal laser-scanning microscope (TCS SP5; Leica).

Electron Microscopy and Immunolabeling

Root tips of transgenic plants harboring 35S:OsNRT1.1A-eGFP were collected and fixed in 0.1 M phosphate buffer (pH 7.4), containing freshly prepared 4% (w/v) paraformaldehyde for 4 h at room temperature, and then overnight at 4°C. Root samples were dehydrated through a graded ethanol series and embedded in LR White resin (Sigma-Aldrich). Blocks were polymerized under UV light (360 nm) at −20°C for 24 h. Ultrathin sections (80 nm) were cut with a diamond knife using an ultramicrotome (UC7; Leica Microsystems) and mounted on nickel grids with a single slot.

For immunolabeling, nickel grids carrying ultrathin root tip sections were incubated with anti-GFP antibody at room temperature for 1 h and then 4°C overnight. Goat anti-mouse IgG conjugated with 10-nm colloidal gold particles (Sigma-Aldrich; diluted 1:50) were used as secondary antibody. Grids were stained with 2% uranyl acetate and examined with FEI Tecnai G2 20 transmission electron microscope at 120 kV (Saito et al., 2009; Li et al., 2011).

Generation of Transgene Constructs and Plant Transformation

The OsNRT1.1A full-length coding region was amplified and cloned into pCAMBIA2301-ACTIN1 to generate pCAMBIA2301-ACTIN1:OsNRT1.1A overexpression construct. A 2.0-kb promoter region and the full-length coding sequence of OsNRT1.1A were amplified and cloned into pCAMBIA2300 to generate pCAMBIA2300-OsNRT1.1Apromoter:OsNRT1.1A overexpression construct. To generate the OsNRT1.1A-RNAi vector, a specific sequence of the OsNRT1.1A coding region was amplified. The resulting PCR product was inserted into the pUCC-RNAi vector (Luo et al., 2006) in both sense and antisense orientation and then the fragment containing an artificial inverted-repeat sequence of OsNRT1.1A was transferred into pCAMBIA2301-ACTIN1 for OsNRT1.1A-RNAi construct. All the primers used to generate the overexpression constructs above are listed in Supplemental Table 2, and all of the constructs were confirmed by sequencing. pCAMBIA2300-NRT1.1BGenomic fragment was used as described previously (Hu et al., 2015). The constructs were introduced into Agrobacterium strain AGL1. The wild type (DJ or HJ) was used as the recipient for Agrobacterium-mediated transformation as described previously to generate the transgenic rice (Hiei et al., 1994). The T-DNA copy numbers of relative transgenic rice plants were confirmed by RT-qPCR described previously (Bubner and Baldwin, 2004; Yang et al., 2005), and the results are presented in the Supplemental Table 3. Homozygous T3 or T4 plants were taken for the following field test.

For Arabidopsis transformation, the resulting construct pCAMBIA2300-35S:OsNRT1.1A-eGFP was transformed into Agrobacterium strain GV3101 and introduced into wild-type plants Col-0 by the floral dip method (Bent, 2006). The harvested seeds were selected on half-strength Murashige and Skoog plates containing 1% sucrose and 50 μg/mL kanamycin to acquire independent T1 transgenic lines, and then the T2 seeds based on the 3:1 segregation of the selection marker were obtained. The homozygous T3 lines were used for further phenotypic analyses.

RNA Extraction, cDNA Preparation, and RT-qPCR

Total RNA was extracted using TRIzol reagent (Invitrogen) from the indicated tissues of rice plants. Two micrograms of total RNA was used to synthesize cDNA using ReverTra Ace qPCR RT Master Mix (Toyobo). RT-qPCR assay was performed with SYBR Green Real-Time PCR Master Mix reagent (Toyobo) on a Chromo4 real-time PCR detection system according to manufacturer’s instructions (Bio-Rad CFX96). Data were analyzed by Opticon monitor software (Bio-Rad). Three technical replicates from one of the three biological replicates were performed for each gene. Rice Ubiquitin1 was used as the internal reference. The primers used for RT-qPCR are listed in Supplemental Table 2.

Promoter-GUS Assay

A 2.0-kb promoter region of OsNRT1.1A was amplified from DJ and cloned into pCAMBIA2391Z to generate OsNRT1.1Apromoter:GUS, and the resulting vector was transformed into DJ. For GUS staining, tissues from the root, leaf sheath, leaf blade, and culm of OsNRT1.1Apromoter:GUS transgenic rice were sampled and immersed in X-gluc (5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid/cyclohexyl ammonium salt) staining solution containing 100 mM sodium phosphate, pH 7.0, 0.1% Triton X-100, 10 mM EDTA (pH 8.0), 2% DMSO, 0.1% X-gluc, 1 mM K3[Fe(CN)6], 1 mM K4[Fe(CN)6]·3H2O, and 5% methanol (Jefferson, 1989). After staining for 1 h at 37°C, the samples were dehydrated in an ethanol series (70, 85, 95, and 100%) to remove the chlorophyll. The stained tissues were observed under a stereomicroscope (Olympus SZX16) and photographed using a digital camera (Nikon D700).

Field Tests of Rice

To investigate the application potential of OsNRT1.1A, large-scale field tests for rice using T4 generation OsNRT1.1A-OE plants in the DJ background were performed in the paddy field under natural growth condition during 2015-2016 at three experimental stations: the Institute of Genetics and Developmental Biology (IGDB, Beijing), the China National Hybrid Rice Research and Development Center (Changsha, Hunan Province), and the South China Experimental Station of IGDB (Sanya, Hainan Province). All the nutritional composition of the soils before rice was transplanted in each paddy field are summarized in Supplemental Table 4. For the field test in Beijing in 2015 (May to October), nitrate mixed with ammonium (60% nitrate mixed with 40% ammonium) was used as the N source with 1 kg N/100 m2 for low N and 2 kg N/100 m2 for high N. KNO3 and (NH4)2SO4 were used as sources for nitrate and ammonium, respectively. The plants were transplanted in 10 rows × 15 plants for each plot, and four replicates were used for each N condition. For field test in Sanya (December 2015 to April 2016), nitrate mixed with ammonium was used as the N source, which is the same as that used in Beijing in 2015, and the planting density was 8 rows × 20 plants with five replicates under each N condition. For field tests in Changsha and Beijing in 2016 (May to October), urea was used as the N source with 1 kg N/100 m2 for low N and 2 kg N/100 m2 for high N, respectively. The rice plants with four or five replicates were transplanted with a density of 8 rows × 15 plants or 10 rows × 20 plants in Changsha or Beijing, respectively. P2O5 was used as phosphorus fertilizer (0.5 kg P/100 m2) in the paddy field before transferring, and the spacing between rice plants at all three locations was 20 cm. Plants were flooded throughout their growth period and grown in blocks with a completely random design for each plot. To reduce the variability in field test, the fertilizers are evenly applied to every plot for each N application level. For the final field test, the edge lines of each plot were removed to avoid margin effects.

For evaluating the grain yield per plant of OsNRT1.1A-OE in HJ19 background, field tests were conducted during 2016 and 2017 at two locations: the IGDB (Beijing) and Northeast Institute of Geography and Agroecology of Chinese Academy of Sciences (Harbin, Heilongjiang Province). The soil nutrient status in Harbin in 2017 is also presented in Supplemental Table 4. For field test in Beijing in 2016, T3 generation transgenic lines were transplanted as 8 rows × 15 plants along with wild-type plants. A field test was also conducted using T4 generation lines in Harbin in 2017, with a planting density of 10 rows × 20 plants. The spacing between rice plants at the two locations was 20 cm (row space) × 20 cm (plant space).

Analysis of Agronomic Traits

All the agronomic traits including plant height, seed number per panicle, seed-setting rate, tiller number per plant, and grain yield per plant were measured on a single-plant basis. Detailed methods for measurement of these agronomic traits were described previously (Hu et al., 2015).

Statistical Analysis

For the comparisons between two groups of data, the Student’s t test was used. For the data sets of more than two groups, mainly for the transgenic plants phenotyping and large field tests, one-way ANOVA with Tukey’s test was used. The results of statistical analysis with ANOVA are listed in Supplemental Data Set 1.

Accession Numbers

Sequence data from this article can be found in the Arabidopsis TAIR database (https://www.arabidopsis.org) or Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/) under the following accession numbers: AtNRT1.1, AT1G12110.1; OsNRT1.1A, LOC_Os08g05910; OsNRT1.1B, LOC_Os10g40600; OsNRT2.1, LOC_Os02g02170; OsNRT2.3a, LOC_Os01g50820; OsNIA1, LOC_Os08g36480; OsNIA2, LOC_Os08g36500; OsNIR1, LOC_Os01g25484; OsNIR2, LOC_Os02g52730; OsAMT1.1, LOC_Os04g43070; OsGOGAT1, LOC_Os01g48960; OsGOGAT2, LOC_Os05g48200; OsGS1.1, LOC_Os02g50240; OsGS1.2, LOC_Os03g12290; Ehd1, LOC_Os10g32600; Hd3a, LOC_Os06g06320; and RFT1, LOC_Os06g06300.

Supplemental Data

Acknowledgments

We thank Yi-Fang Tsay (Institute of Molecular Biology, Academia Sinica) and Wenfeng Qian (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) for their critical comments and suggestions on this manuscript. This work was supported by grants from the Ministry of Science and Technology of the People’s Republic of China (2016YFD0100700 and 2015CB755702), by the Ministry of Agriculture (2014ZX08001-004), and by the Chinese Academy of Sciences (XDA08010400).

AUTHOR CONTRIBUTIONS

W.W. and B.H. performed the experiments and analyzed the data. B.H., W.W., and C.C. designed the research. B.H., C.C., and W.W. wrote the manuscript. D.Y., Y.Q.L., R.C., Y.H., H.L., Z.H.Z., Z.J., Z.L.Z., S.O., H.W., X.G., Y.Q., X.M., Y.X.L., Y.W., L.Z., L.L., S., and H.J. conducted the experiments. B.H., W.W., Y.X.L., and Y.B. analyzed the data. C.C. supervised the project.

Footnotes

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References

  1. Almagro A., Lin S.H., Tsay Y.F. (2008). Characterization of the Arabidopsis nitrate transporter NRT1.6 reveals a role of nitrate in early embryo development. Plant Cell 20: 3289–3299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. An C., Li L., Zhai Q., You Y., Deng L., Wu F., Chen R., Jiang H., Wang H., Chen Q., Li C. (2017). Mediator subunit MED25 links the jasmonate receptor to transcriptionally active chromatin. Proc. Natl. Acad. Sci. USA 114: E8930–E8939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Arth I., Frenzel P., Conrad R. (1998). Denitrification coupled to nitrification in the rhizosphere of rice. Soil Biol. Biochem. 30: 509–515. [Google Scholar]
  4. Bart R., Chern M., Park C.-J., Bartley L., Ronald P.C. (2006). A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts. Plant Methods 2: 13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bent A. (2006). Arabidopsis thaliana floral dip transformation method. Methods Mol. Biol. 343: 87–103. [DOI] [PubMed] [Google Scholar]
  6. Brambilla V., Fornara F. (2013). Molecular control of flowering in response to day length in rice. J. Integr. Plant Biol. 55: 410–418. [DOI] [PubMed] [Google Scholar]
  7. Bubner B., Baldwin I.T. (2004). Use of real-time PCR for determining copy number and zygosity in transgenic plants. Plant Cell Rep. 23: 263–271. [DOI] [PubMed] [Google Scholar]
  8. Castro Marín I., Loef I., Bartetzko L., Searle I., Coupland G., Stitt M., Osuna D. (2011). Nitrate regulates floral induction in Arabidopsis, acting independently of light, gibberellin and autonomous pathways. Planta 233: 539–552. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chardin C., Girin T., Roudier F., Meyer C., Krapp A. (2014). The plant RWP-RK transcription factors: key regulators of nitrogen responses and of gametophyte development. J. Exp. Bot. 65: 5577–5587. [DOI] [PubMed] [Google Scholar]
  10. Chen J., Zhang Y., Tan Y., Zhang M., Zhu L., Xu G., Fan X. (2016). Agronomic nitrogen-use efficiency of rice can be increased by driving OsNRT2.1 expression with the OsNAR2.1 promoter. Plant Biotechnol. J. 14: 1705–1715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Davenport T., Rao R., Zheng Q., Li Y. (2001). Nitrogen, flowering and production of lychee in Florida. Acta Hortic. 558: 221–224. [Google Scholar]
  12. Dielen V., Lecouvet V., Dupont S., Kinet J.M. (2001). In vitro control of floral transition in tomato (Lycopersicon esculentum Mill.), the model for autonomously flowering plants, using the late flowering uniflora mutant. J. Exp. Bot. 52: 715–723. [DOI] [PubMed] [Google Scholar]
  13. Doi K., Izawa T., Fuse T., Yamanouchi U., Kubo T., Shimatani Z., Yano M., Yoshimura A. (2004). Ehd1, a B-type response regulator in rice, confers short-day promotion of flowering and controls FT-like gene expression independently of Hd1. Genes Dev. 18: 926–936. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fan X., Tang Z., Tan Y., Zhang Y., Luo B., Yang M., Lian X., Shen Q., Miller A.J., Xu G. (2016). Overexpression of a pH-sensitive nitrate transporter in rice increases crop yields. Proc. Natl. Acad. Sci. USA 113: 7118–7123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fang Z., Xia K., Yang X., Grotemeyer M.S., Meier S., Rentsch D., Xu X., Zhang M. (2013). Altered expression of the PTR/NRT1 homologue OsPTR9 affects nitrogen utilization efficiency, growth and grain yield in rice. Plant Biotechnol. J. 11: 446–458. [DOI] [PubMed] [Google Scholar]
  16. Hiei Y., Ohta S., Komari T., Kumashiro T. (1994). Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 6: 271–282. [DOI] [PubMed] [Google Scholar]
  17. Ho C.H., Lin S.H., Hu H.C., Tsay Y.F. (2009). CHL1 functions as a nitrate sensor in plants. Cell 138: 1184–1194. [DOI] [PubMed] [Google Scholar]
  18. Hu B., et al. (2015). Variation in NRT1.1B contributes to nitrate-use divergence between rice subspecies. Nat. Genet. 47: 834–838. [DOI] [PubMed] [Google Scholar]
  19. Hunter P.R., Craddock C.P., Di Benedetto S., Roberts L.M., Frigerio L. (2007). Fluorescent reporter proteins for the tonoplast and the vacuolar lumen identify a single vacuolar compartment in Arabidopsis cells. Plant Physiol. 145: 1371–1382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Itoh H., Nonoue Y., Yano M., Izawa T. (2010). A pair of floral regulators sets critical day length for Hd3a florigen expression in rice. Nat. Genet. 42: 635–638. [DOI] [PubMed] [Google Scholar]
  21. Izawa T. (2007). Adaptation of flowering-time by natural and artificial selection in Arabidopsis and rice. J. Exp. Bot. 58: 3091–3097. [DOI] [PubMed] [Google Scholar]
  22. Jefferson R.A. (1989). The GUS reporter gene system. Nature 342: 837–838. [DOI] [PubMed] [Google Scholar]
  23. Jeong D.H., An S., Kang H.G., Moon S., Han J.J., Park S., Lee H.S., An K., An G. (2002). T-DNA insertional mutagenesis for activation tagging in rice. Plant Physiol. 130: 1636–1644. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Komiya R., Yokoi S., Shimamoto K. (2009). A gene network for long-day flowering activates RFT1 encoding a mobile flowering signal in rice. Development 136: 3443–3450. [DOI] [PubMed] [Google Scholar]
  25. Li C., Wang Y., Liu L., Hu Y., Zhang F., Mergen S., Wang G., Schläppi M.R., Chu C. (2011). A rice plastidial nucleotide sugar epimerase is involved in galactolipid biosynthesis and improves photosynthetic efficiency. PLoS Genet. 7: e1002196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Li H., Hu B., Chu C. (2017). Nitrogen use efficiency in crops: lessons from Arabidopsis and rice. J. Exp. Bot. 68: 2477–2488. [DOI] [PubMed] [Google Scholar]
  27. Li H., Hu B., Wang W., Zhang Z., Liang Y., Gao X., Li P., Liu Y., Zhang L., Chu C. (2016). Identification of microRNAs in rice root in response to nitrate and ammonium. J. Genet. Genomics 43: 651–661. [DOI] [PubMed] [Google Scholar]
  28. Li J., Xin Y., Yuan L. (2009). Hybrid Rice Technology Development. Ensuring China’s Food Security. (Washington, DC: International Food Policy Research Institute; ). [Google Scholar]
  29. Li Y.L., Fan X.R., Shen Q.R. (2008). The relationship between rhizosphere nitrification and nitrogen-use efficiency in rice plants. Plant Cell Environ. 31: 73–85. [DOI] [PubMed] [Google Scholar]
  30. Liu K.H., et al. (2017). Discovery of nitrate-CPK-NLP signalling in central nutrient-growth networks. Nature 545: 311–316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Liu Q., Chen X., Wu K., Fu X. (2015). Nitrogen signaling and use efficiency in plants: what’s new? Curr. Opin. Plant Biol. 27: 192–198. [DOI] [PubMed] [Google Scholar]
  32. Luo A., et al. (2006). EUI1, encoding a putative cytochrome P450 monooxygenase, regulates internode elongation by modulating gibberellin responses in rice. Plant Cell Physiol. 47: 181–191. [DOI] [PubMed] [Google Scholar]
  33. Marchive C., Roudier F., Castaings L., Bréhaut V., Blondet E., Colot V., Meyer C., Krapp A. (2013). Nuclear retention of the transcription factor NLP7 orchestrates the early response to nitrate in plants. Nat. Commun. 4: 1713. [DOI] [PubMed] [Google Scholar]
  34. Moll R., Kamprath E., Jackson W. (1982). Analysis and interpretation of factors which contribute to efficiency of nitrogen utilization. Agron. J. 74: 562–564. [Google Scholar]
  35. Moll R., Kamprath E., Jackson W. (1987). Development of nitrogen-efficient prolific hybrids of maize. Crop Sci. 27: 181–186. [Google Scholar]
  36. Plett D., Toubia J., Garnett T., Tester M., Kaiser B.N., Baumann U. (2010). Dichotomy in the NRT gene families of dicots and grass species. PLoS One 5: e15289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rupp R.A., Snider L., Weintraub H. (1994). Xenopus embryos regulate the nuclear localization of XMyoD. Genes Dev. 8: 1311–1323. [DOI] [PubMed] [Google Scholar]
  38. Saito Y., Kishida K., Takata K., Takahashi H., Shimada T., Tanaka K., Morita S., Satoh S., Masumura T. (2009). A green fluorescent protein fused to rice prolamin forms protein body-like structures in transgenic rice. J. Exp. Bot. 60: 615–627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Scott R., Ogunremi E., Ivins J., Mendham N. (1973). The effect of fertilizers and harvest date on growth and yield of oilseed rape sown in autumn and spring. J. Agric. Sci. 81: 287–293. [Google Scholar]
  40. Wen Z., Tyerman S.D., Dechorgnat J., Ovchinnikova E., Dhugga K.S., Kaiser B.N. (2017). Maize NPF6 proteins are homologs of Arabidopsis CHL1 that are selective for both nitrate and chloride. Plant Cell 29: 2581–2596. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Withrow A.P. (1945). The interrelationship of nitrogen supply and photoperiod on the flowering, growth and stem anatomy of certain long and short day plants. Butler Univ. Bot. Stud. 7: 40–64. [Google Scholar]
  42. Xu G., Fan X., Miller A.J. (2012). Plant nitrogen assimilation and use efficiency. Annu. Rev. Plant Biol. 63: 153–182. [DOI] [PubMed] [Google Scholar]
  43. Yang L., Ding J., Zhang C., Jia J., Weng H., Liu W., Zhang D. (2005). Estimating the copy number of transgenes in transformed rice by real-time quantitative PCR. Plant Cell Rep. 23: 759–763. [DOI] [PubMed] [Google Scholar]
  44. Yuan S., et al. (2016). Arabidopsis cryptochrome 1 functions in nitrogen regulation of flowering. Proc. Natl. Acad. Sci. USA 113: 7661–7666. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Zhang Y., Su J., Duan S., Ao Y., Dai J., Liu J., Wang P., Li Y., Liu B., Feng D., Wang J., Wang H. (2011). A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes. Plant Methods 7: 30. [DOI] [PMC free article] [PubMed] [Google Scholar]

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