{'fraction_non_alphanumeric': 0.04330668825255489, 'fraction_numerical': 0.037134473338055245, 'mean_word_length': 4.6757274119448695, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Nitrate signaling and use efficiency in crops

Nitrate (NO3–) is not only an essential nutrient but also an important signaling molecule for plant growth. Low nitrogen use efficiency (NUE) of crops is causing increasingly serious environmental and ecological problems. Understanding the molecular mechanisms of NO3– regulation in crops is crucial for NUE improvement in agriculture. During the last several years, significant progress has been made in understanding the regulation of NO3– signaling in crops, and some key NO3– signaling factors have been shown to play important roles in NO3– utilization. However, no detailed reviews have yet summarized these advances. Here, we focus mainly on recent advances in crop NO3– signaling, including short-term signaling, long-term signaling, and the impact of environmental factors. We also review the regulation of crop NUE by crucial genes involved in NO3– signaling. This review provides useful information for further research on NO3– signaling in crops and a theoretical basis for breeding new crop varieties with high NUE, which has great significance for sustainable agriculture.


INTRODUCTION
Nitrogen (N) is an essential nutrient for plant growth and has profound effects on crop productivity. During the last several decades, large amounts of chemical N fertilizer have been applied to fields to obtain high crop yields (Godfray et al., 2010;Liu et al., 2013). However, only 30-40% of the applied N can be absorbed by crops, and a large portion is lost to the environment, resulting in severe environmental pollution and ecological damage (Good et al., 2004;Coskun et al., 2017;. N use efficiency (NUE) is a complex agronomic trait that describes the efficiency of N uptake and utilization by crops. It mainly includes N uptake efficiency (NUpE) and N utilization (assimilation) efficiency (NUtE) (Raun and Johnson, 1999;Good et al., 2004;Xu et al., 2012). Improving crop NUE is the key to solving these problems. The processes of N uptake and utilization are finely controlled by complex and delicate regulatory networks. Therefore, it is important to elucidate the regulatory factors and underlying mechanisms in order to promote sustainable agriculture.
Nitrate (NO 3 -) is the predominant form of N absorbed by most crops such as maize (Zea mays L.), wheat (Triticum aestivum L.), and barley (Hordeum vulgare L.) (Crawford and Glass, 1998). Plants absorb NO 3 mainly through NO 3 transporters (NRT1 and NRT2 families), and it can then be translocated, stored, or assimilated into amino acids by NO 3 assimilation proteins (NR, NiR, GS, and GLN) (Vidal and Gutierrez, 2008;Girin et al., 2014). NO 3 also functions as an important signaling molecule to regulate plant growth and development. According to the time period when plants respond to NO 3 -, NO 3 signaling can be divided into short-term and long-term effects (Medici and Krouk, 2014;Zhao et al., 2018b). More and more regulators involved in short-and long-term NO 3 signaling have been identified, and some have been found to coordinately regulate multiple processes, including NO 3 transport and assimilation, root system architecture, and interactions of plant and environmental factors to affect yield and NUE (Alboresi et al., 2005;Wilkinson et al., 2007;Krouk et al., 2010;Zhao et al., 2018b). Therefore, key regulators involved in NO 3 signaling may play important roles in the NUE of crops.
Over the past decade, an increasing number of factors that function in NO 3 signaling have been identified, mainly in the model plant Arabidopsis, and several fine review papers have summarized these advances (Wang et al., 2012Mu and Luo, 2019). Great progress has also been made in the last few years in identifying important regulators of crop NO 3 signaling, although this work has not been reviewed to date. In addition, scientists have discovered some critical factors that contribute to crop NUE, and several excellent review papers have recently summarized these findings (Xu et al., 2012;Han et al., 2015;Wang et al., 2018bWang et al., , 2020bYu et al., 2022). However, no recent reviews have focused on the functions of NO 3 signaling regulators in the NUE of crops. Here, we summarize major advances in NO 3 signaling of crops from three aspects: short-term signaling, long-term signaling, and the impact of environmental factors. We also review the functions of NO 3 --signaling regulators in the NUE of crops. This review provides important information for regulation research on crop NO 3 signaling and a theoretical basis for improving the NUE of crops.

SHORT-TERM NITRATE SIGNALING
Short-term NO 3 signaling is defined as the primary nitrate response (PNR) (Medici and Krouk, 2014;Liu et al., 2019). The PNR is a series of early events that occur in plants after NO 3 provision and cause changes in the expression of thousands of genes related to NO 3 metabolism (Stitt, 1999;Scheible et al., 2004). A few key components of the regulation of NO 3 signaling in crops have been discovered in the last several years.

NRT1.1
In Arabidopsis, NRT1/PTR FAMILY 6.3/NITRATE TRANSPORTER 1.1 (AtNPF6.3/NRT1.1) has been reported to be the first NO 3 sensor, and it plays an essential role in both NO 3 signaling and metabolism (Tsay et al., 1993;Liu and Tsay, 2003;Ho et al., 2009;Wang et al., 2009 (Wang et al., 2018a). In the osnrt1.1b mutant, the induction of OsNIA1 and OsNIA2 after NO 3 treatment is greatly repressed, indicating that OsNRT1.1B functions in NO 3 signaling (Hu et al., 2015). OsNRT1.1A is located mainly on the vacuole membrane. Unlike OsNRT1.1B, OsNRT1.1A functions mainly in upregulating the expression of N utilization-related genes under NO 3 and ammonium conditions (Wang et al., 2018a). In maize, ZmNPF6.6 and ZmNPF6.4 have been found to transport NO 3 and chloride (Wen et al., 2017). However, whether both genes are involved in NO 3 signaling has not been reported. In wheat, 331 homologous NPF genes have been identified (Wang et al., 2020a). Again, which of these wheat homologs regulates NO 3 signaling remains unknown.

Transcription factor (TF) NLPs
Increasing evidence has shown that Arabidopsis NIN-LIKE PROTEIN 7 (NLP7) plays a central role in the regulation of NO 3 signaling, and there has been great research interest in its regulatory mechanisms (Guan et al., 2017;Liu et al., 2017;Mu and Luo, 2019;Zhao et al., 2018b). OsNLP3 is the closest rice homolog to AtNLP7, and the osnlp3 mutant shows reduced expression of OsNRT2.1, OsNIA1,and OsNiR1 after NO 3 treatment . Moreover, OsNLP3 can directly bind to NO 3 response element (NRE)-like cis-elements in the promoters of OsNIA1, OsNIA3,OsNRT1.1B,OsNRT2.4,and OsGRF4 and activate their expression .
OsNLP4 also binds to the promoter of OsNiR and activates its expression . These results indicate that OsNLP3/4 are involved in regulating NO 3 signaling. Furthermore, OsSPX4, a phosphate (Pi)-signaling repressor, can interact with OsNLP3 to inhibit its NO 3 --induced cytoplasmic-nuclear shuttling . In the presence of NO 3 -, OsNRT1.1B is able to promote the ubiquitination and degradation of OsSPX4 by recruiting the E3 ubiquitin ligase NBIP1 (NRT1.1B interacting protein 1). Degradation of OsSPX4 then releases OsNLP3 to shuttle from the cytoplasm to the nucleus, thereby activating the NO 3 response . Thus, the OsNRT1.1B-SPX4-NLP3 module plays important roles in regulating NO 3 signaling from the cell membrane to the nucleus. Interestingly, OsNRT1.1A can promote the localization of OsNLP3 and OsNLP4 in the nucleus in the absence of NO 3 -, implying that OsNRT1.1A may regulate NO 3 signaling by promoting cytoplasmic-nuclear shuttling of NLPs (Wang et al., 2018a) ( Figure 1). In maize, ZmNLP3.1/5/6/8 can bind to NRE elements in the promoters of NO 3 response-related genes to activate their expression, thereby regulating NO 3 signaling (Cao et al., 2017;Wang et al., 2018cWang et al., , 2020cZhao et al., 2018b) (Figure 2A). In Triticeae crops, only barley HvNLP2 has been found to function in the regulation of NO 3 signaling by binding to and activating the expression of HvNR1, HvNiR,and HvNRT2.1 (Gao et al., 2022). Functional characterization of other NLPs in NO 3 signaling in rice, maize, and barley, as well as NLPs in other crops, has not yet been reported.

Other factors
In addition to the NRT1.1s and NLPs described above, a few key factors have been identified as regulating the short-term NO 3response in crops. Calcium (Ca 2+ ) serves as an essential nutrient and a signaling molecule, and CALCINEURIN B-LIKE proteins (CBLs) are a unique group of Ca 2+ sensors that have been found to participate in various stress responses and plant nutrient acquisition (Tang et al., 2020). After NO 3 treatment in rice, the expression of OsNRT2.1, OsNRT2.2,and OsNAR2.1 is decreased in the oscbl1 mutant, suggesting that OsCBL1 is required for the regulation of NO 3 signaling (Hu et al., 2021) (Figure 1).
Chromatin-remodeling complexes (CRCs) play essential roles in plant growth and development (Sarnowska et al., 2016;Song et al., 2021). ZmCHB101 is the core subunit of the SWI/SNFtype ATP-dependent CRCs in maize (Meng et al., 2020). In the absence of NO 3 -, ZmCHB101 binds to the NREs of ZmNRT2.1 and ZmNRT2.2, antagonizing the binding of ZmNLP3.1 to the NREs. In the presence of NO 3 -, the binding affinity of ZmCHB101 for the NREs is decreased, resulting in increased binding of ZmNLP3.1 to the NREs (Wang et al., 2018c;Meng et al., 2020) (Figure 2A).

LONG-TERM NITRATE SIGNALING
Long-term NO 3 signaling refers to NO 3 regulation of plant growth and development over a longer period of time and involves processes such as root development, tillering, and flowering Liu et al., 2019;O'Brien et al., 2016). Following the identification of important players involved in long-term NO 3 signaling in Arabidopsis (Lin and Tsay, 2017;Wang et al., 2018b;Zhao et al., 2018b; Jia and von Wiré n, 2020), significant advances have been made in this area for crops during the last several years.

Root development
Root system architecture directly affects the absorption and utilization of nutrients as well as plant growth. Root growth and development are regulated by NO 3 availability. The MADS-box family TF AtANR1 was the first gene identified in NO 3 regulation of root growth in Arabidopsis, and overexpression of AtANR1 increases lateral root (LR) growth in the presence of NO 3 - (Zhang and Forde, 1998). In rice, OsMADS25/27/57 have also been found to regulate root growth in response to NO 3 - (Yu et al., 2015;Chen et al., 2018;Huang et al., 2019). Under different NO 3 conditions, OsMADS25 overexpression can promote primary root (PR) and LR growth (Yu et al., 2015). Overexpression of OsMADS27, however, inhibits PR elongation and increases LR number and length under 10 mM NO 3 conditions . The osmads57 mutant shows reduced total root length after treatment with low NO 3 due to shorter seminal and adventitious roots. Further investigation has revealed that mutation of OsMADS57 inhibits the expression of CYCLINB1;1 (OsCYCB1;1) and induces the polar transport of auxin toward and within the roots, thereby repressing meristematic cell proliferation (Huang et al., 2019). In addition, OsmiR444 has been reported to target OsMADS23, OsMADS27a, OsMADS27b,and OsMADS57.
Overexpression of OsmiR444a represses the expression of these OsMADS genes and root growth under NO 3 supply (Yan et al., 2014). In maize, local supply of NO 3 can induce the expression of ZmTMM1, a truncated MIKC-type MADS-box TF, to promote LR growth . Although TaANR1 has been identified in wheat, its function in NO 3 --regulated root development has not been reported (Lei et al., 2018). These results suggest that the functions of ANR1 homologs in regulating root growth are highly conserved, but the underlying mechanisms may be diverse in different species (Figures 1 and 2, Table 1). N deficiency promotes plant root development and thus increases N uptake from soil. Some Arabidopsis NRT2 genes are mainly expressed in the roots and play a central role in regulating LR development under conditions of NO 3 limitation (Remans et al., 2006). In rice, overexpression of OsNRT2.1 increases total root length, including that of seminal and adventitious roots, under 0.5 mM NO 3 conditions (Naz et al., 2019). The expression of auxin transporter genes OsPIN1a/b/c and OsPIN2 also increased significantly in OsNRT2.1overexpressing lines, implying that OsNRT2.1 may modulate NO 3 --dependent root elongation by regulating auxin transport in roots. In addition, knockdown of OsNAR2.1, a partner of OsNRT2.1, suppresses LR formation under low NO 3 conditions Song et al., 2020). Further research has found that OsNAR2.1 can directly interact with NITRILASE 1 and 2 (OsNIT1 and OsNIT2). In the osnar2.1 mutant, the expression of OsNIT1 and OsNIT2 is decreased under NO 3 supply, thereby repressing the expression of OsPIN1c and OsPIN1d to alter the distribution of auxin in roots. Thus, osnit1 and osnit2 as well as os-nar2.1 show reduced PR length and LR density in the presence   (Figure 2A and Table 1). In addition, the rice ubiquitin ligase OsEL5 is involved in the maintenance of root meristematic viability and LR formation by participating in cytokinin-mediated N effects (Koiwai et al., 2007;Mochizuki et al., 2014). Moreover, low N can enhance strigolactone biosynthesis and signaling, which in turn reduce OsPIN1b-mediated auxin polar transport from shoot to root and attenuate seminal root growth (Sun et al., 2014b(Sun et al., , 2018. In maize, the knockout mutant zmchb101 shows accelerated root growth and increased biomass under low NO 3 conditions, suggesting that ZmCHB101 may negatively regulate root development in response to NO 3 - (Meng et al., 2020) ( Figure 2A and Table 1). In wheat, RNAi lines of TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1/TRYPTOPHAN AMINOTRANSFERASE-RELATED (TaTAA1/TAR) show reduced LR growth under both low-and high-N conditions (Shao et al., 2017) ( Figure 2B and Table 1). Furthermore, overexpression of the wheat NUCLEAR FACTOR-Y TF TaNFYA-B1 upregulates expression of TaTAR2-A1, TaTAR2-B1, and TaTAR2-D1 in roots, thus increasing LR growth .

Tillering
Increased tillering can contribute to high crop yields, and NO 3 is one of the important factors that regulate crop tillering. In rice, the indica NADH/NADPH-DEPENDENT NITRATE REDUCTASE 2 (OsNR2) promotes NO 3 uptake via feed-forward interaction with OsNRT1.1B and increases effective tiller number in japonica rice. The japonica OsNR2 also functions in increasing tiller number but not to the extent seen in the indica OsNR2-overexpression lines .
NITROGEN-MEDIATED TILLER GROWTH RESPONSE 5 (OsNGR5) is a positive regulator of rice growth and development in response to N . It interacts with POLYCOMB REPRESSIVE COMPLEX 2 (PRC2) to regulate the expression of DWARF14 (D14) and SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 14 (OsSPL14) by mediating histone methylation (H3K27me3) modification levels, thereby regulating rice tillering and bud outgrowth in the presence of N. OsNGR5 is also a target of the gibberellin (GA) receptor GIBBERELLIN INSENSITIVE DWARF1 (GID1), and GA can promote degradation of OsNGR5 protein. Further research has revealed that the plant-growthinhibitory factor DELLA can interact with OsNGR5, competing with OsGID1 at the binding site to inhibit GA-mediated degradation of OsNGR5 and thus increase OsNGR5 stability . Therefore, the GA-GID1-NGR5 and DELLA-NGR5  molecular modules may synergistically improve the growth and maintain the semi-dwarfing and high-yield characteristics of rice ( Figure 1 and Table 1). which affects the transport of auxin and finally controls tiller growth . In addition, OsTCP19 can directly bind to and repress the activity of the tiller-promoting gene DWARF AND LOW-TILLERING (DLT) and negatively regulate tillering in the presence of N . Further investigation has revealed that a 29-bp insertion and/or deletion in the OsTCP19 promoter confers a differential transcriptional response and variation in tillering in response to N among rice varieties ( Figure 1 and Table 1). In wheat, overexpression lines of the NO 3 --INDUC-IBLE AND CEREAL-SPECIFIC NAM, ATAF, AND CUC (NAC) TF TaNAC2-5A show significantly higher tiller numbers under low-N conditions , suggesting that TaNAC2-5A is a key gene for NO 3 --regulated tillering.

Flowering time
Flowering time has a critical impact on crop yield. Recent studies have shown that NO 3 concentrations affect plant flowering time and that lower NO 3 can promote flowering (Lin and Tsay, 2017).
In rice, overexpression of OsNRT1.1A upregulates the expression of HEADING DATE 3A (Hd3a), EARLY HEADING DATE1 (Ehd1), and RICE FLOWERING LOCUS T 1 (RFT1), which are known to promote flowering, resulting in an early-flowering phenotype (Wang et al., 2018a). N-MEDIATED HEADING DATE-1 (Nhd1) can directly activate the expression of OsHd3a and negatively regulate the expression and activity of OsFd-GOGAT, thus balancing flowering time and photoperiod in rice   (Figure 1 and Table 1). Wheat TaVRN-A1 can interact with TaANR1, and mutations in both TaVRN-A1 and TaANR1 result in delayed heading after N fertilization (Lei et al., 2018) ( Figure 2B and Table 1). osnhd1 mutant and TaANR1-RNAi lines show delayed flowering time and a longer growth period (Lei et al., 2018;Zhang et al., 2021;Li et al., 2022). In general, early plant flowering is closely related to shortened growth period and reduced yield (Roux et al., 2006;Lu et al., 2014;Zhao et al., 2018a). However, OsNRT1.1A-overexpression lines exhibit both earlier flowering and high yields. Therefore, OsNRT1.1A has great potential for overcoming the antagonism between early flowering and high yield, enabling the breeding of new rice varieties in which yield and flowering time are well balanced.  (Li et al., 2014;Hu et al., 2019). In rice, OsNRT1.1B plays an important role in regulating NO 3 signaling (Wang et al., 2020b). It can also interact with OsSPX4 to promote ubiquitination and degradation of OsSPX4 by recruiting NBIP1. This then causes the Pi-signaling TF OsPHR2 to translocate into the nucleus and initiate the expression of Pi utilization genes, resulting in highly coordinated utilization of N and Pi . OsCBL1 is not only involved in NO 3 signaling but also forms a complex with OsCIPK23 to enhance OsAKT1-mediated K + uptake (Li et al., 2014). These findings may provide insights into synergistically improving the uptake efficiency of N, Pi, and K + in rice.

INFLUENCE OF ENVIRONMENTAL FACTORS ON NITRATE SIGNALING
The rhizobial and arbuscular mycorrhizal symbioses are very important for efficient plant uptake of nutrients from the environment . Rhizobia can convert atmospheric N into ammonium for absorption and utilization by legumes, and in turn, rhizobia can obtain nutrients and carbon sources from the plants to maintain growth (M€ uller, 2021). Advances in our understanding of symbiotic N fixation have been well summarized in several excellent review papers (Oldroyd et al., 2011;Udvardi and Poole, 2013;Ferguson et al., 2019;Yang et al., 2022). However, few genes involved in coordinately regulating NO 3 signaling and the rhizobial symbiosis have been identified to date. In the presence of NO 3 -, Medicago MtNLP1 can enter the nucleus to form a complex with the NODULE INCEPTION (NIN) TF protein, thereby inhibiting expression of two downstream genes, CRE1 and NF-YA1, which are essential for nodule development (Lin et al., 2018). This ultimately leads to inhibition of nodule formation and N fixation. In addition, low  -Radzman et al., 2016;Gautrat et al., 2020). Furthermore, MtNLP1 can bind to the half NRE (hNRE) in the MtCEP1 promoter to repress its expression, resulting in reduced nodule formation in the presence of NO 3 - (Luo et al., 2022). Interestingly, MtNLP1 can also bind to the NRE in the MtCLE35 promoter to activate its expression, and its product is then transported to the shoot to activate SUPER NUMERIC NODULES (SUNN) systemic pathways and further restrict nodule number under NO 3 conditions . All the results above suggest that there is NLP1-NO 3 --mediated inhibition of nodulation via systemic and local regulatory pathways in Medicago. The arbuscular mycorrhizal fungi (AMF) symbiosis is the result of the co-evolution of plants with mycorrhizal fungi in the long-term survival process, and it can increase plant uptake of soil nutrients, especially Pi (Montesinos-Navarro et al., 2012). Recent work has shown that AMFs can induce the expression of the NO 3 transport genes OsNPF2.2/PTR2, OsNPF1.3,OsNPF6.4,and OsNPF4.12 to improve N uptake in rice (Drechsler et al., 2018), but the mechanisms by which AMFs and NO 3 signaling affect one another remain elusive. Recently, OsNRT1.1B and OsCIPK2 have been found to modulate the root microbiome in rice to increase N utilization (Khan et al., 2019;Zhang et al., 2019). Clearly, rhizosphere microorganisms strongly influence NO 3 signaling and utilization, although the underlying mechanisms require further investigation.

NUE OF CROPS
NUE is a complicated agronomic trait that involves the multiple interconnected steps of NO 3 absorption, assimilation, translocation, and remobilization. Previous reviews on NUE have focused mainly on the key genes that function in these processes (Han et al., 2015;Wang et al., 2018b). However, no reviews have focused specifically on NUE in terms of NO 3 signaling regulators. More and more data indicate that many factors involved in NO 3 signaling play important roles in regulating crop NUE, and we have therefore summarized advances in this area.
In rice, several NO 3 transporter genes have been found to play important roles in the regulation of NUE in addition to modulating NO 3 signaling. Phylogenetic analysis shows that OsNRT1.1B diverged between indica and japonica subspecies (Hu et al., 2015). OsNRT1.1B-indica can improve tiller number, grain yield, and NUE, and plants overexpressing OsNRT1.1Bindica grow better and show a greater NUE than plants overexpressing OsNRT1.1B-japonica under both high-and low-N conditions. These results indicate that OsNRT1.1B-indica plays a more important role in NUE than OsNRT1.1B-japonica. Furthermore, japonica plants carrying OsNRT1.1B-indica exhibit significantly increased tiller number, grain yield, and NUE, suggesting that OsNRT1.1B-indica can improve the NUE of japonica rice (Hu et al., 2015;Fan et al., 2016a). OsNRT1.1B can also interact with indica OsNR2 to synergistically improve grain yield and NUE of japonica rice . OsNRT1.1A can dramatically increase grain yield and NUE by improving panicle size (Wang et al., 2018a), and transgenic lines overexpressing OsNRT2.1 or OsNAR2.1 show increased grain yield and NUE (Araus et al., 2016;Chen et al., 2016Chen et al., , 2017. Nevertheless, rice NUE was decreased in plants overexpressing OsBT2, a member of the BRIC-A-BRAC/TRAMTRACK/BROAD gene family. The bt1bt2 double mutant displays higher expression of OsNRT2.1 and OsNRT2.4 and 20% higher NUE under low-N conditions (Araus et al., 2016). OsNAR2.1 can also interact with OsNRT2.3a, and co-overexpression of OsNAR2.1 and OsNRT2.3a increased grain yield and NUE by 24.6% and 28.56%, respectively Yan et al., 2011;Liu et al., 2014;Chen et al., 2020). Functional analysis indicates that overexpression of OsNRT2.3b can increase N uptake and improve grain yield and NUE of rice (Fan et al., 2016b;. Besides the OsNRTs, several other rice NO 3 --signaling regulators have also been found to participate in affecting NUE. OsDEP1overexpressing plants show increased N uptake and assimilation, resulting in improved harvest index and grain yield (Huang et al., 2009;Sun et al., 2014a). OsSPL18 can bind to the promoter of OsDEP1 and increase its expression to regulate panicle architecture (Yuan et al., 2019). The osspl18 mutant exhibits reductions in grain width and thickness, panicle length, and grain number, resulting in decreased yield, suggesting that OsSPL18 positively regulates grain yield in rice (Xie et al., 2006;Yuan et al., 2019). By contrast, rice osspl14 mutants show increased tiller number and lodging resistance and enhanced grain yield, indicating that OSSPL14acts as a negative regulator of rice growth and yield (Jiao et al., 2010). Overexpression of OsNGR5 can repress the expression of OsSPL14 and D14 to enhance rice tiller bud outgrowth, thereby increasing tiller number, grain yield, and NUE . In addition, both OsNLP3 and OsNLP4 can bind to the promoters and activate the expression of downstream N assimilation-related genes to increase tiller number, yield, and NUE Yu et al., 2021;Zhang et al., 2022).
Recently, studies on NO 3 --signaling regulators involved in modulating NUE in barley, maize, and wheat have also been reported successively. Barley HvNLP2 and maize ZmNLP6/8 can increase the expression of NR-regulated genes to enhance NR activity and amino acid content, thereby increasing grain yield and NUE (Cao et al., 2017;Gao et al., 2022). zmnlp5 mutants accumulate less N in the ear leaves and seed kernels under N deficiency, implying that ZmNLP5 may take part in improving NUE of maize (Ge et al., 2020). In wheat, TaNAC2-5A has been found to directly bind to the promoters and activate the expression of TaNRT2.  transport and assimilation, resulting in increased grain yield and NUE .
Overexpression of TaNFYA-B1 also increases wheat grain yield and NUE by increasing spike number under low-N conditions . In addition, TaANR1-RNAi lines exhibit fewer fertile tillers and decreased grain yield, whereas TaVRN-A1-RNAi lines show increased plant biomass after N application (Lei et al., 2018). This result implies that TaVRN-A1 may function oppositely to TaANR1 in regulating wheat grain yield and NUE, even though both proteins can interact with each other. Moreover, TaHOX1 (a homeobox protein involved in wheat heading date) can also interact with TaVRN-A1, and plants overexpressing TaHOX1 show lower grain yield, implying that TaHOX1 has a repressive effect on yield (Lei et al., 2018).
All the NO 3 --signaling regulators that function in affecting NUE characterized to date in rice, wheat, and maize are shown in Table 2. They modulate important plant characteristics including growth, grain development, and tiller number to improve crop yield and NUE.

Perspectives
Improving crop NUE is the most effective way to reduce the application of N fertilizer and the resulting environmental pollution. However, only limited advances have been made in achieving high NUE in crops, as the molecular mechanisms and networks of NO 3 signaling are still poorly understood. Although some key NO 3 --signaling regulators have been successfully characterized as described above, these efforts are just the start of deciphering the sophisticated NO 3 regulation in crops. Many more novel genes and regulatory mechanisms remain to be identified.
AtNRT1.1 is the first and the only one NO 3 sensor and plays a powerful role in regulating NO 3 signaling and transport as well as in transporting auxin in Arabidopsis (Ho et al., 2009;Mounier et al., 2014;Wang et al., 2020b). In rice, OsNRT1.1B has also been found to play a central role in regulating NO 3 signaling and plant growth, yield, and NUE (Hu et al., 2015Fan et al., 2016a;Zhang et al., 2019 regulation of crop development, including germination, root growth, tiller number, flowering, senescence, and seed development, is still in its infancy or even absent. All of the above subjects require more efforts to identify key genes involved in regulating the crosstalk between NO 3 signaling and other factors in the environment and the plants themselves. Forward genetics techniques such as QTL and GWAS have been successfully used to identify novel genes involved in NO 3 signaling in rice (Huang et al., 2009;Hu et al., 2015;Gao et al., 2019;Wu et al., 2020) but not yet in other crops like wheat and maize. Recently, reverse genetics has also been gradually applied to identify crop NO 3 regulators such as ZmNLP3.1/6/ 8 and HvNLP2 (Cao et al., 2017;Wang et al., 2018c;Gao et al., 2022). Compared with forward genetics, reverse genetics has been proven to be more accurate, efficient, and time saving for characterizing the roles of homologous genes in regulating NO 3 signaling in crops. In addition, systems biology is a powerful approach and has been successfully used to identify key factors in NO 3 signaling in Arabidopsis, although it is not being used in crops. Therefore, according to the characteristics of different crops, comprehensive use of these techniques to identify more NO 3 regulatory genes in crops is very important for accelerating our understanding of NO 3 signaling regulation.
Compared with our understanding of NO 3 signaling in the model plant Arabidopsis, much less is known in crops. More endeavors are needed to identify and characterize novel genes involved in regulating NO 3 signaling as well as the crosstalk between NO 3 and other environmental factors. Following the application of advanced techniques, including multi-omics and gene editing, the NO 3 --regulatory mechanisms and networks in crops will be clearly elucidated. This will lay a solid foundation for evaluating the potential of these genes and taking full advantage of them in breeding new crop varieties with high NUE.

AUTHOR CONTRIBUTIONS
All work was performed in collaboration. Y.W. and Y.G. wrote the original draft and prepared the figure. Y.W., Y.G. and S.Q. designed and finalized the manuscript. The authors have read and agreed to the published version of the article.