Molecular Regulatory Networks for Improving Nitrogen Use Efficiency in Rice

Nitrogen is an important factor limiting the growth and yield of rice. However, the excessive application of nitrogen will lead to water eutrophication and economic costs. To create rice varieties with high nitrogen use efficiency (NUE) has always been an arduous task in rice breeding. The processes for improving NUE include nitrogen uptake, nitrogen transport from root to shoot, nitrogen assimilation, and nitrogen redistribution, with each step being indispensable to the improvement of NUE. Here, we summarize the effects of absorption, transport, and metabolism of nitrate, ammonium, and amino acids on NUE, as well as the role of hormones in improving rice NUE. Our discussion provide insight for further research in the future.


Introduction
Rice is the staple food for more than half of the world's population. Nitrogen is the most abundant mineral element in plants and the main limiting factor in rice production. The application of nitrogen fertilizer has greatly increased global crop yield, accompanied by increasingly severe economic and environmental costs [1]. Therefore, it is urgent to understand the molecular regulation mechanism of nitrogen absorption and utilization in rice and formulate strategies to improve nitrogen use efficiency (NUE). NUE is the guarantee of high yield and quality of rice. With the rapid development of agriculture in China, the input of nitrogen fertilizer has reached significant scale, and some new varieties of rice have demonstrated a problematically low utilization efficiency of fertilizer [1,2]. Today, China is the largest fertilizer consumer in the world, with the production and application of nitrogen fertilizer accounting for more than one third of global total (IFA annual report, 2015). According to statistics, from 1980 to 2013, the net amount of agricultural nitrogen fertilizer application increased by nearly 1.6 times. However, the total grain yield only increased by 87%, the seasonal (traditional) NUE of main crops was only about 35%, and the nitrogen efficiency (including soil residual fertilizer nitrogen) was only about 48%, which was significantly lower than the international average [2,3]. Excessive application of nitrogen fertilizer results in energy waste, higher costs, soil acidification, water eutrophication and greenhouse gas emissions, which seriously restricted the sustainable development of agricultural production [1,4]. Besides, in the pursuit of high yield, how to fully explore the genetic potential of nitrogen absorption and utilization in rice, to reduce the demand

Nitrate Relative Genes Play an Important Role in Improving NUE in Rice
Plants mainly absorb nitrogen from soil in two inorganic forms, nitrate and ammonium. Given the anaerobicity of paddy soil, it is generally believed that ammonium is the main form of nitrogen in rice. However, rice has developed aerenchyma, which can transport oxygen from the ground to the roots and release it into the rhizosphere microenvironment, leaving the rhizosphere soil in a certain oxidation state, thus enhancing the nitrification of soil bacteria to convert ammonium into nitrate, and converting 15-40% of nitrogen into nitrate [16]. A large number of experiments at home and abroad have shown that, compared with single ammonium nitrogen supply, partially nitrate nitrogen can significantly promote the nitrogen absorption and utilization efficiency of rice [17][18][19][20]. Therefore, the uptake and assimilation of nitrate has become an important factor affecting NUE in rice.
In recent years, nitrate transporter genes as a famous family have been used in crop breeding to improve NUE [6,19,[21][22][23][24][25][26]. LeNRT2.3 mediates the low affinity nitrate transport system in tomato. Overexpression of LeNRT2.3 can increase the uptake of NO 3 − in tomato roots and the transport of NO 3 − from root to shoot, thus increasing the biomass and fruit weight [27]. Tanrt2.1 can increase the uptake of NO 3 − in wheat after flowering [28]. TaNAC2-5a is a key transcription factor controlling the expression of nitrate transporter. Overexpression of TaNAC2-5a can increase wheat growth, grain yield and nitrogen harvest index [21]. Overexpression of NAC2 gene in wheat can increase the expression of TaNRT2.1 and TaGS2 [21].
The absorption and utilization of NO 3 − in rice is a complex process, including absorption, translocation and assimilation. In order to adapt to the changing environment, plants have evolved two nitrate transport systems, low affinity transport systems (LATS) and high affinity transport systems (HATS), which are responsible for NRT1/PTR (NPF) family members and NRT2 family members respectively. The NRT2 family in rice includes OsNRT2.1, OsNRT2.2, OsNRT2.3a, OsNRT2.3b, and OsNRT2.4 [20,[23][24][25][29][30][31][32][33]. OsNRT2.1, OsNRT2.2, and OsNRT2.3a need to interact with OsNAR2.1 protein to have NO 3 − transport activity [29,33]. OsNRT2.3a was involved in the transport of NO 3 − from root to shoot [34]. A subsequent study found that OsNRT2.3b did not need the interaction of OsNAR2.1 and could transport NO 3 − alone [30]. Overexpression of OsNRT2.3b increased the buffering capacity of cell pH and significantly improved the grain yield and NUE of rice [19,26] ( Figure 1). Our previous results showed that the expression of pOsNAR2.1:OsNAR2.1 could increase the NO 3 − uptake rate at seedling stage, significantly promote rice growth, and improve grain yield and agronomic NUE [22]. The expression of pOsNAR2.1:OsNRT2.1 increased the expression ratio of OsNRT2.1 to OsNAR2.1 in stem, and increased grain yield and NUE in rice [25]. Co-overexpression of OsNAR2.1 and OsNRT2.3a can improve the biomass and nitrate transport efficiency, and ultimately improve the grain yield and NUE in rice [23]. A putative nitrate transporter gene OsNPF4.5 was induced by mycorrhizal, which is exclusively expressed in the cells containing arbuscules and displayed a low-affinity NO 3 − transport activity involved in symbiotic N uptake [35].
Chlorate is a kind of nitrate analogue, which was in the same pathway as NO 3 − absorption and assimilation in plants. Therefore, chlorate sensitivity simulated nitrate uptake or assimilation in rice. Hu et al. [6] used BC 2 F 5 lines derived from the cross between IR24 and Nipponbare were screened for chlorate sensitivity, and the chromosome segment substitution line (CSSL) with the highest chlorate sensitivity was used for fine mapping. The nitrate transporter gene OsNRT1.1b was cloned from Indica, and then OsNRT1.1b-Indica was introduced into Japonica rice varieties, which could significantly improve the yield and NUE of Japonica rice. OsNRT1.1b can also interact with phosphate signal repressor OsSPX4, which in turn activates phosphate and nitrate response genes to achieve coordinated utilization of nitrogen and phosphorus [36]. Gao et al. [8] used RIL populations of 9311 and Nipponbare to detect a QTL for potassium chlorate resistance, qCR2. After fine mapping and cloning, it was found that it encodes a NAD (P) H-dependent nitrate reductase gene OsNR2, and the difference between arginine (Indica) and tryptophan (Japonica) in the NAD (P) H binding domain is the determinant of the enzyme activity difference between the two OsNR2 genes. OsNR2 also interacts with OsNRT1.1b in a positive feedback manner. In Japonica, aggregating these two genes from Indica can obtain higher yield and NUE than a single gene [8] [40][41][42][43]. A NIN-like protein OsNLP4 was identified to function in NUE. Loss of OsNLP4 dramatically reduced yield and NUE compared with wild type under different N regimes. In contrast, the OsNLP4 overexpression lines increased yield and NUE under moderate N levels compared with wild type. Transcriptomic analyses revealed that OsNLP4 orchestrates the expression of a majority of known N uptake, assimilation, and signalling genes by directly binding to the nitrate-responsive cis-element in their promoters to regulate their expression. Moreover, overexpression of OsNLP4 can rescue the phenotype of atnlp7 mutant and enhance its biomass [44]. Interestingly, the same results were found through GWAS analysis. OsNLP4 transactivated OsNiR encoding nitrite reductase that was critical in nitrogen assimilation in rice. Using constructed quadrupling NREs (Nitrate-responsive cis-elements) in the promoter of OsNiR (p4xNRE:OsNiR) enhanced nitrogen assimilation significantly. OsNLP4-OsNiR increased tiller number and yield through enhancing nitrogen assimilation and NUE [45]. The nitrate related genes involved in NUE are summarized in Table 1.
lines increased yield and NUE under moderate N levels compared with wild typ scriptomic analyses revealed that OsNLP4 orchestrates the expression of a ma known N uptake, assimilation, and signalling genes by directly binding to the responsive cis-element in their promoters to regulate their expression. Moreover pression of OsNLP4 can rescue the phenotype of atnlp7 mutant and enhance its [44]. Interestingly, the same results were found through GWAS analysis. OsNLP activated OsNiR encoding nitrite reductase that was critical in nitrogen assimi rice. Using constructed quadrupling NREs (Nitrate-responsive cis-elements) in moter of OsNiR (p4xNRE:OsNiR) enhanced nitrogen assimilation significantly. O OsNiR increased tiller number and yield through enhancing nitrogen assimila NUE [45]. The nitrate related genes involved in NUE are summarized in Table 1. OsNRT2.3a cooperating with OsNAR2.1 is responsible for root-to-shoot NO3 − transpo OsNRT2.3b is also responsible for transporting NO3 − to the shoot and remobilizing N into grain. OsNPF2.2 and OsNPF2.4 are involved in NO3 − translocation. OsNPF4.5 is induced b corrhizal, involved in symbiotic N uptake. Blue line means NO3 − uptake, yellow line mean transport NO3 − from root to shoot, red line means NO3 − translocation, orange line means N distribution.

Ammonium, Amino Acid and Nitrogen Assimilation Relative Genes Apply to NUE
Rice grown in paddy fields is an ammonium (NH 4 + )-preferring crop. Ammonium is uptaken by ammonium transporters (AMTs) as well as aquaporins or cation channels [61][62][63][64][65][66]. Three members of the rice OsAMT1 gene family of ammonium transporters showed distinct expression patterns. OsAMT1;1 was constitutive and ammonium-induced expression in shoots and roots. OsAMT1;2 was specific expressed in root and ammoniuminducible. OsAMT1;3 was also root-specific expressed and repressed by nitrogen supply, and glutamine had the same effect on the transcriptional regulation of OsAMT1 genes as ammonium, indicating that glutamine rather than ammonium controls the expression of ammonium transporter genes in rice [62]. OsAMT1;1 mediates NH 4 + uptake at relatively lower affinity and mediates NH 4 + uptake in a proton independent manner [67]. OsAMT1;1 significantly contributes to the NH 4 + uptake under both low and high NH 4 + conditions and increased seed yield under suboptimal and optimal N conditions [68,69]. Concomitant activation of OsAMT1;2 and OsGOGAT1 genes led to increased tolerance to nitrogen limitation and better ammonium uptake and N remobilization at the whole plant level [70]. Overexpression of OsAMT1;3 regulates rice growth and carbon-nitrogen metabolic status [71]. Many studies on the genetic manipulation of ammonium uptake of rice in improving NUE are limited. A recent study found that some transcription factors can regulate the expression of ammonium transporters to affect NUE [72]. OsNLP1 protein was localized in the nucleus and its transcription level was rapidly induced by nitrogen starvation. Overexpression of OsNLP1 increased plant growth, yield and NUE under different nitrogen conditions, while knockout of OsNLP1 decreased yield and NUE under nitrogen limitation. A further study suggests that OsNLP1 regulates ammonium and nitrate utilization by coordinating multiple nitrogen uptake and assimilation genes. Chromatin immunoprecipitation (CHIP) and yeast-one hybrid experiments showed that OsNLP1 could directly bind to the promoters of these genes and activate their expression. Overall, enhancing the absorption and remobilization of ammonium will provide a promising strategy to improve rice NUE in the future [73]. It has been proven that plants can obtain organic nitrogen, especially amino acids, through root localized transporters [73][74][75]. When amino acid in soil is relatively high, such as in organic agricultural soil or other planting systems relying on manure or compost for nitrogen nutrition, this absorption system is particularly important [76][77][78]. Some people think that compared with mineral nitrogen fertilizer, the application of high proportion of organic fertilizer can improve the nitrogen absorption, yield, and NUE of crops, and reduce the emission of organic nitrogen to the environment [79]. In rice, the overexpression of OsAAP1 resulted in increased N uptake and redistribution, as well as significantly increased tiller number and final yield [80]. OsAAP3 and OsAAP5 are mainly expressed in the vascular system of rice, and may participate in the transport of amino acids between xylem and phloem [81,82]. RNAi lines of OsAAP3 and OsAAP5 also showed significant improvements in tiller number and yield under sufficient nitrogen conditions. Genetic association analyses with 68 representatives Japonica or Indica germplasms identified that OsLHT1 has a natural variation in aspartate absorption between Japonica and Jndica, and OsLHT1 functions in a broad spectrum of amino acids, and effectively transported aspartate, asparagine, and glutamate in yeast cells. OsLHT1 is responsible for both the root uptake and root to shoot allocation of a broad spectrum of amino acids in rice [83].
After being absorbed into the plant, the assimilation process is the same except that nitrate needs to be reduced to NH 4 + (NH3). Nitrate is reduced to nitrite in the cytoplasm and transported to the plastid for further reduction to ammonium [84][85][86][87]. The twostep reduction was achieved by nitrate reductase (NR) and nitrite reductase (NIR). NR is the main regulatory step in the process of nitrogen assimilation, and its activity is highly regulated by nitrate, light or water availability [88][89][90][91]. The overexpression of NR or NIR often leads to increased N uptake. Recent studies on rice have shown that the overexpression of Indica OsNR2 in Japonica can increase tiller number, grain yield, and NUE under high nitrogen supply [8]. This effect was better when Indica OsNR2 and OsNRT1.1b were concurrently expressed, indicating that OsNR2 could regulate OsNRT1.1b and thus nitrate uptake [8]. The overexpression of OsNADH-GOGAT in rice by its native promoter also resulted in increased grain weight at low N fertilization [92]. Interestingly, the combined overexpression of OsAMT1;2 and OsNADH-GOGAT1 in rice can increase NUE in rice under both sufficient and low nitrogen conditions [54]. The results suggest that concurrently increasing N uptake and N assimilation could improve NUE. Some autophagy-related gene could enhance N remobilization and yield and NUE. For example, the overexpression of OsATG8a in rice could increase shoot biomass, yield, and NUE when transgenic plants were exposed to high N conditions [93], while overexpression of OsATG8b led to increased biomass, yield, and NUE at moderate and low N [94]. Similarly, the constitutive expression of OsATG8b in rice led to more biomass and higher grain yield under sufficient N conditions [95], and the overexpression of OsATG8c led to improvements in shoot biomass, grain yield, and NUE under moderate and low N fertilization [96].
Too much uptake with a slower assimilation of ammonium may lead to ammonium toxicity and damage to plant growth. Glutamine synthetase is the key enzyme of ammonia assimilation in plants. Ammonium was the main nitrogen source in rice plants, which was assimilated into glutamine and glutamic acid by glutamine synthetase (GS). Glutamine and Glutamic Acid were the fundamental nitrogen sources of other amino acids biosynthesis [97][98][99]. GS has two isomers, cytoplasmic GS1 and plastidic GS2, which have different functions in nitrogen assimilation [100,101]. GS1 isoforms are mainly involved in nitrogen reassimilation and reuse. GS2 participates in primary nitrogen assimilation [102,103]. OsGS1;1 is involved in ammonium assimilation in rice shoot and root [104]. The overexpression of glutamine synthetase genes GS1 and GS2 in transgenic rice plants increases nitrogen-deficiency tolerance [105] and the simultaneous overexpression of OsGS1 and OsGS2 genes enhanced the tolerance to osmotic and salinity stress at the seedling stage. There are many examples of using GS gene to improve NUE in multiple species, including Tobacco [106][107][108][109], Arabidopsis [110], Sorghum [111], Maize [112], common wheat [113,114], Barley [115], and Rice [98,116,117]. However, not all GS genes can play a positive role after overexpression, and in some cases, they may have negative effects on plants [106,[116][117][118][119][120]. In general, it is possible to use nitrogen assimilation genes to improve the NUE of rice, but this requires the coordination of all aspects of rice development and nutrient condition.

Plant Hormones Act on NUE
Strigolactones (SLs) have been reported to function in the nutrient-dependent regulation of root and shoot architecture. The biosynthesis mutant dwarf 10 (d10) had a higher N concentration in older leaves but a lower N concentration in younger leaves, while the SL-signaling mutant dwarf 3 (d3) mutant showed a considerably lower N concentration, especially in its younger leaves under normal N levels. Both d3 and d10 mutants possess higher N in their leaves under N-deficient conditions. The analysis of uptake and distribution of 15 N showed that the significant difference of nitrogen concentration between d3, d10, and WT plants occurred only in leaves, not in roots. The SLS synthesized by exogenous GR24 changed the leaf N distribution of d10 mutant but did not change the leaf N distribution of d3 mutant and WT, indicating that the effects of SLS on rice growth and development may be related to the transport of nitrogen to different shoot tissues [121]. AUXIN is a looping star in plant development [122]. A rice nitrogen relative QTL named DULL NITROGEN RESPONSE1 (qDNR1) involved in auxin homeostasis, reflects the differences in nitrate (NO 3 − ) uptake, N assimilation, and yield enhancement between Indica and Japonica rice varieties. Rice plants carrying the DNR1 Indica allele exhibit reduced N-responsive transcription and protein abundance of DNR1 and promotes auxin biosynthesis, inducing AUXIN RESPONSE FACTOR-mediated activation of NO 3 − transporter and N-metabolism genes, resulting in improved NUE and grain yield. Lossof-function mutation at the DNR1 locus increased N uptake and assimilation, resulting in improved rice yield under moderate levels of N fertilizer input [123]. An auxin efflux carrier OsPIN9 was induced by ammonium, compared to single nitrate, and involved in ammonium induced rice tillering in rice. The overexpression of OsPIN9 increased yield under low nitrogen condition [124]. Therefore, appropriate modulation of auxin response is also a promising strategy for achieving NUE in rice. Gibberellin acid (GA) plays a crucial role in agricultural green revolution. The rice sd1 allele is shorter than normal plants given the reduction of gibberellin, and this increases accumulation of the rice DELLA protein SLR1 (SLENDER RICE1) [125][126][127]. The plants are shorter and therefore more resistant to lodging [128]. Transcription factor NGR5 (N-mediated tiller growth response 5) is the target of gibberellin receptor gibberellin insensitive dwarf 1 (GID1), promoting proteasome destruction. NGR5 promotes the N-dependent recruitment of polycomb inhibitor complex 2 through H3K27me3 modification to inhibit branching inhibitors. DELLA protein competitively inhibited GID1-NGR5 interaction, and explained the reason for the tillering increase of green revolution varieties. The increase of NGR5 activity resulted in the decoupling of tillering and nitrogen regulation, thus increasing rice yield at low nitrogen level. Therefore, optimal use of GA could improve the NUE and improve the sustainability of agriculture in the future [129]. Abscisic acid (ABA) is a dominant regulator in plants facing abiotic stress. Osmotic stress/ABA-activated protein kinase 2 (SAPK2) is a member of SnRK2s in rice. The ossapk2 mutants were shorter and produced fewer grains per panicle, smaller grains, and lower grain yield under drought stress. Moreover, SAPK2 consider-ably influences the nitrogen contents of rice grains and NO 3 − influx rate, while nitrate concentration analysis indicated that SAPK2 promotes nitrate uptake and assimilation by regulating nitrate-related transporters. These results suggest that ABA related gene could also enhance grain production by regulating NUE under drought stress. Jasmonates (JAs) represent a kind of lipid derived plant hormone, which plays an important role in plant development and defense response to insects. The exogenous addition of methyl jasmonate (MeJA) to rice seedlings significantly reduced root N uptake and 15 N transport from root to leaf, possibly due to the downregulation of glutamine synthetase and nitrite ferredoxin. MeJA treatment of shoot resulted in the reactivation of endogenous nitrogen from leaf to root. MeJA treatment of root also increased the accumulation of 14 N in roots, but did not affect the accumulation of 14 N in leaves. Subsequent analysis of proteomics and RT-qPCR showed that the up regulation of GDH2, a plastid disintegration dehydrogenase mediated by JA, contributed to the release of nitrogen in leaves and supported the production of defense proteins/compounds under nitrogen limitation [130]. JA signaling mediates largescale systemic changes in nitrogen uptake and distribution in rice. Cytokinins affect many aspects of plant growth and development. The overexpression of cytokinin-activation enzyme-like gene OsLOGL5 significantly reduced primary root growth, tiller number, and yield. On the contrary, a mutation in the C-terminal of OsLOGL5 led to normal plant morphology, but rice yield increased under the conditions of sufficient water, drought, normal nitrogen, and low nitrogen in multiple geographical locations. It was found that the C-terminal of OsLOGL5 protein played an important role in regulating rice yield and improving NUE under different abiotic stresses [131]. In summary, these results suggest that the reasonable use of hormones can also significantly improve the NUE of rice under various growth conditions.

Future Perspectives
Increasing crop NUE can be achieved by increasing crop yield under the same nitrogen supply or using less nitrogen input for sustainable productivity. A lot of work is now devoted to the identification of candidate genes in improving NUE and their regulatory networks. The basic mechanisms of local and systematic N sensing, uptake, transport, assimilation, and remobilization have been well studied in Arabidopsis, which should be pursued in rice [132]. With the development of bioinformatics, using a high-quality genome sequence, assembly, annotation, and the genome-wide association study (GWAS) of natural genetic variation will further accelerate the discovery of key genes and pathways related to NUE traits in rice. Recently, a new genomic analysis of 33 rice genetic diversity revealed multiple structural variations and gene copy number variations [133]. In order to make use of the variation and phenotype information, researchers should speed up the molecular breeding of these important traits.
Using gene editing by CRISPR-Cas9 technology to change the expression pattern of some genes is a choice for precision agriculture [134]. Nitrate transporters can be effectively used to improve crop yield and through genome editing. Changing the expression of some nitrate transporters or their regulators can improve the NUE in crops [6,19,[21][22][23][24][25]135,136]. However, it is not the best way to improve NUE by simply aggregating single positive alleles in rice. The overexpression of multiple genes at the same time can improve the nitrogen use efficiency of more rice. For example, compared with WT, the influx rate of 15 NO 3 − , agronomic nitrogen use efficiency, and nitrogen recovery efficiency of OsNRT2.1 or OsNAR2.1 overexpression lines increased by 10%, 20%, and 30%, respectively, while the co-overexpression of OsNRT2.1 and OsNAR2.1 lines increased by 40%, 50%, and 60%, respectively ( Figure 2). The elite haplotype OsNPF6.1 HapB improves NUE under low nitrogen conditions, which was activated by OsNAC42 [37]. Indica OsNR2 promotes NO 3 − uptake by feedback interaction OsNRT1.1B (8), which increased effective tiller number, grain yield, and NUE. Therefore, the co-expression of OsNAC42-OsNPF6.1 HapB cascade, Indica OsNR2-OsNRT1.1B could improve NUE in rice. In the future molecular breeding, there must be a balance between these genes and pathways, which necessitates better coordination. A potential direction is to find out all possible expression patterns of some useful genes during rice development under various conditions. Using genomic and phenotypic data to explore the best coordinated expression pattern of the regulatory network will deepen our understanding of the determinants for improving NUE. The optimized NUE should meet the best nitrogen demand of rice and be environmentally friendly.
NRT2.1 or OsNAR2.1 overexpression lines increased by 10%, 20%, and 30%, respectively, while the co-overexpression of OsNRT2.1 and OsNAR2.1 lines increased by 40%, 50%, and 60%, respectively ( Figure 2). The elite haplotype OsNPF6.1 HapB improves NUE under low nitrogen conditions, which was activated by OsNAC42 [37]. Indica OsNR2 promotes NO3 − uptake by feedback interaction OsNRT1.1B (8), which increased effective tiller number, grain yield, and NUE. Therefore, the co-expression of OsNAC42-OsNPF6.1 HapB cascade, Indica OsNR2-OsNRT1.1B could improve NUE in rice. In the future molecular breeding, there must be a balance between these genes and pathways, which necessitates better coordination. A potential direction is to find out all possible expression patterns of some useful genes during rice development under various conditions. Using genomic and phenotypic data to explore the best coordinated expression pattern of the regulatory network will deepen our understanding of the determinants for improving NUE. The optimized NUE should meet the best nitrogen demand of rice and be environmentally friendly.   Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: All data generated or analyzed during this study are included in this published article.

Conflicts of Interest:
The authors declare no conflict of interest.