Although NH₄ ⁺ has long been recognized as the primary form of N absorbed by rice, research on NH₄ ⁺ transporters have remained relatively limited. With the advancement of genomics, at least 12 potential NH₄ ⁺ transporters (AMTs) have been identified in the rice genome. These transporters are classified into five subfamilies: OsAMT1 (OsAMT1.1, OsAMT1.2, and OsAMT1.3), OsAMT2 (OsAMT2.1, OsAMT2.2, and OsAMT2.3), OsAMT3 (OsAMT3.1, OsAMT3.2, and OsAMT3.3), OsAMT4 (OsAMT4.1), and OsAMT5 (OsAMT5.1 and OsAMT5.2) (Suenaga et al., 2003; Li et al., 2009a). The OsAMT1 subfamily functions as a high-affinity NH₄ ⁺ transporter, operating under low NH₄ ⁺ concentrations and exhibiting saturation kinetics. In contrast, the OsAMT2, OsAMT3, and OsAMT4 families are classified as low-affinity transporters, predominantly active under high NH₄ ⁺ concentrations (1–40 mM), and do not display saturation kinetics (Gaur et al., 2012). Studies have demonstrated that OsAMT1.1, OsAMT1.2, OsAMT1.3, OsAMT2.1, and OsAMT5.1 all possess NH₄ ⁺ transport capacity (Bu et al., 2011) ( Figure 1 ).

The uptake of low concentrations of NH₄ ⁺ in rice roots requires the coordinated activity of three OsAMT1 members, which share high amino acid sequence homology. Among these, OsAMT1.1 makes the largest contribution to N accumulation (Sonoda et al., 2003a; Zhao et al., 2014). Spatial expression analyses revealed that NH₄ ⁺ exposure induces the upregulation of OsAMT1.1 and OsAMT1.2, and the downregulation of OsAMT1.3 (Konishi and Ma, 2021). OsAMT1.1, OsAMT3.2, and OsAMT3.3 are constitutively expressed in both roots and stems, while OsAMT1.1 expression is promoted by NH₄ ⁺. OsAMT1.2 is root-specific and induced by NH₄ ⁺, whereas OsAMT1.3 is root-specific but suppressed by N. OsAMT2.1, OsAMT2.2, OsAMT2.3, and OsAMT3.1 are mainly expressed in aerial tissues, with relatively higher expression in stems than other genes. The expression of OsAMT3 family genes is generally higher in shoots than in roots, suggesting that AMT3 members may participate in the translocation and distribution of NH₄ ⁺ within leaves (Suenaga et al., 2003; Gaur et al., 2012; Sonoda et al., 2003a; Li et al., 2012; Li and Shi, 2006). Additionally, the expression patterns of OsAMT1 genes show strong correlations with Gln levels in root tissues (OsAMT1.1 and OsAMT1.2 positively correlated, OsAMT1.3 negatively correlated), but not with NH₄ ⁺ content (Sonoda et al., 2003b). OsAMT5.1 is specifically expressed in leaves, with its expression enhanced by increasing NH₄ ⁺ concentrations (Deng et al., 2007).

At present, research on the regulation of AMT genes in rice has primarily focused on three OsAMT1 family genes. Among them, OsAMT1.1 plays a central role in N absorption and utilization in rice. It significantly promotes NH₄ ⁺ uptake under both low and high NH₄ ⁺ conditions, maintains N-potassium (K) homeostasis, and enhances NUE, plant growth, and grain yield under suboptimal to optimal N supply. Moreover, this gene underwent strong selection from wild rice to cultivated rice in response to soil conditions ( Figure 1 ; Table 1 ) (Lee et al., 2020; Ranathunge et al., 2014; Ding et al., 2011). Simultaneous activation of OsAMT1.2 and the glutamate synthase gene (OsGOGAT1) improve tolerance to N limitation and enhances NH₄ ⁺ uptake and N remobilization at the whole-plant level ( Figure 1 ; Table 1 ) (Lee et al., 2020). In contrast, overexpression of OsAMT1.3 causes imbalances in carbon (C)-N metabolism, leading to poor plant growth and reduced yield (Bao et al., 2015).

The AMT gene family plays a critical role in NH₄ ⁺ uptake in rice. To date, studies on the phylogeny, expression patterns, and functions of rice AMT genes have provided preliminary insights into their roles in N absorption. However, the regulation of AMT genes is not limited to N uptake but may also be involved in N assimilation, translocation, and other N-related metabolic processes. Therefore, future studies should expand our understanding of AMT gene regulation, environmental adaptability, and especially their integrative roles in N transport and metabolism. Such research will help uncover the potential applications of these genes in improving NUE and crop productivity in rice, ultimately offering new strategies for sustainable agricultural production and environmental protection.

The number of NO₃ ^- transporter 1/peptide transporter (NRT1/PTR, also known as NPF) family members in rice has been confirmed by several genomic analyses, with approximately 93 NPF genes identified. However, the functions of only a few NPF family members have been characterized to date (Léran et al., 2014). Different members of the NPF family perform distinct functions in rice. NRT1.1 in rice, an important NO₃ ^- transporter belonging to the NPF6 subfamily, mainly includes three homologs: OsNRT1.1A (OsNPF6.3), OsNRT1.1B (OsNPF6.5), and OsNRT1.1C (OsNPF6.4), which play critical roles in N uptake, transport, signaling, and NUE (Wang et al., 2020b).

OsNRT1.1A exhibits NH₄ ⁺-induced expression and can significantly upregulate the expression of various genes related to NO₃ ^- and NH₄ ⁺ utilization. Overexpression of OsNRT1.1A significantly improves NUE and grain yield and also shortens the rice maturity period, providing a feasible approach for breeding high-yield, early-maturing rice varieties (Wang et al., 2018c). Among the NRT1 family members in rice, only OsNRT1.1B possesses dual-affinity transport properties and functions across a wide range of NO₃ ^- concentrations. Under low N (LN) conditions, OsNRT1.1B enables plants to accumulate more N and promotes rice growth, whereas OsNRT1.1A lacks such functionality in rice (Fan et al., 2016a). The NO₃ ^- uptake activity of indica rice is higher than that of japonica, and genetic variation in OsNRT1.1B significantly influences differences in NUE between indica and japonica by regulating NO₃ ^- uptake and rhizosphere microbiota. Moreover, introducing the NRT1.1B^indica allele into japonica could potentially enhance the NUE of japonica rice (Hu et al., 2015; Zhang et al., 2019) ( Figure 1 ; Figure 2 ; Table 1 ). The functional differentiation between OsNRT1.1A and OsNRT1.1B helps rice coordinate internal and external N signals and improve its adaptability to complex N environments (Wang et al., 2020b). OsNRT1.3 promoter responds to drought stress, potentially participating in basic NO₃ ^- uptake and stress responses (Hu et al., 2006).

Within the rice NPF family, OsNPF2.2 can unload NO₃ ^- from the xylem, thereby affecting NO₃ ^- transport in the root-stem and plant development (Li et al., 2015). OsNPF2.4 (OsNRT1.6) is a pH-dependent low-affinity transporter functioning in NO₃ ^- uptake, long-distance transport, and redistribution, while its altered expression indirectly affects K reutilization in roots and stems (Xia et al., 2015). A coding region mutation in OsNPF3.1 affects NUE differences between wild and cultivated rice and can improve NUE and biomass yield (Yang et al., 2023). OsNPF4.1 (SP1) encodes a putative peptide transporter highly expressed in the phloem of young panicle branches, controlling panicle size (Li et al., 2009b). Mycorrhizal rice could receive more than 40% of its N via the mycorrhizal pathway, and the arbuscular mycorrhizal-specific NO₃ ^- transporter OsNPF4.5 accounted for approximately 45% of the mycorrhizal NO₃ ^- uptake. Enhanced expression of NPF4.5 can significantly improve NUE and promote rice growth (Wang et al., 2020a). The NO₃ ^- transporter OsNPF5.16 positively regulates rice tillering and yield by regulating cytokinin levels (Wang et al., 2022). OsNPF6.1 is NO₃ ^–inducible and has two haplotypes: OsNPF6.1^HapA and OsNPF6.1^HapB . OsNPF6.1^HapB enhances NO₃ ^- absorption and improves NUE. Furthermore, OsNPF6.1 enhances viral resistance by upregulating the expression of NO₃ ^- reductase (OsNR2) and subsequently promoting nitric oxide (NO) biosynthesis (Tang et al., 2019; Xu et al., 2025) ( Figure 1 ; Table 1 ).

Among the OsNPF7 and OsNPF8 subfamilies, OsNPF7.1 (OsPTR4) and OsNPF7.4 show opposite expression patterns in tiller buds under different N concentrations. Overexpression of either OsNPF7.1 or OsNPF7.4 promotes NO₃ ^- absorption, although biomass is reduced in OsNPF7.4-overexpressing plants (Huang et al., 2019b). OsNPF7.2 acts as a positive regulator of NO₃ ^- influx and concentration, with overexpression lines showing significant increases in tiller number and yield (Hu et al., 2016; Wang et al., 2018a). OsNPF7.3 (OsPTR6) mainly transports di- and tripeptides (e.g., Gly-His, Gly-His-Gly). While its overexpression promotes rice growth, its effect on NUE is limited (Fan et al., 2014); however, subsequent studies revealed that overexpression of both OsNPF7.3 and OsNPF7.7 increases rice tiller number, NUE, and yield (Fang et al., 2017; Huang et al., 2018). OsNPF8.1 (OsPTR7) mediates stress-induced organic N transport, contributing to balanced plant growth and enhanced tolerance to salt/drought stress and N deficiency (Qiu et al., 2023). OsNPF8.9 (OsNRT1) is the first low-affinity NO₃ ^- transporter identified in rice, functioning under high NO₃ ^- conditions (Lin et al., 2000). The di-/tripeptide and low-affinity NO₃ ^- transporter OsNPF8.20 (OsPTR9) enhances NH₄ ⁺ uptake, promotes lateral root formation, and increases grain yield when its expression is upregulated (Fang et al., 2013) ( Figure 1 ; Table 1 ).

The HATS play a crucial role in rice N uptake. To date, five NRT2 (OsNRT2.1, OsNRT2.2, OsNRT2.3a, OsNRT2.3b, OsNRT2.4) and two NAR2 (OsNAR2.1, OsNAR2.2) high-affinity NO₃ ^- transporters have been identified in rice (Araki and Hasegawa, 2006; Feng et al., 2011; Cai et al., 2008). The rice NRT2 and NAR2 family members exhibit distinct functions. Some NRT2 members require the partner protein NAR2 for NO₃ ^- transport within relatively low concentration ranges (Cai et al., 2008).

OsNRT2.3 generates two transcripts, OsNRT2.3a and OsNRT2.3b, through mRNA splicing, with 94.2% amino acid sequence identity and identical coding regions but different 5′ and 3′ untranslated regions. OsNRT2.3a is primarily expressed in roots and induced by NO₃ ^-, whereas OsNRT2.3b is mainly expressed in shoots. Further research revealed that under LN supply, OsNRT2.3a plays a key role in long-distance NO₃ ^- transport from roots to shoots, and OsMADS57 regulates NO₃ ^- transport through OsNRT2.3a (Huang et al., 2019a; Tang et al., 2012). Allelic variation in the 5’ untranslated region of OsNRT2.3 leads to elevated OsNRT2.3b protein levels under high-temperature stress, increasing yield (Zhang et al., 2022). Additionally, high OsNRT2.3b expression enhances pH buffering capacity and improves the uptake of N, iron, and phosphorus (Fan et al., 2016b; Feng et al., 2017).

OsNAR2.1 interacts with OsNRT2.1/2.2 and OsNRT2.3a to mediate NO₃ ^- uptake (Feng et al., 2011; Yan et al., 2011). OsNAR2.2, localized to the endoplasmic reticulum (ER), was recently shown to regulate NO₃ ^- transport from roots to stems and control spikelet number, yield, and NUE in rice (Hou et al., 2025). OsNRT2.1, OsNRT2.2, and OsNAR2.1 are promising candidate genes for breeding high NUE rice cultivars (Araki and Hasegawa, 2006). Enhancing OsNAR2.1 expression via its native promoter, or increasing OsNRT2.1 expression under the control of the OsNAR2.1 promoter, or co-overexpressing OsNAR2.1 and OsNRT2.3a can all improve NO₃ ^- uptake, yield, and NUE in rice ( Figure 1 ; Table 1 ) (Chen et al., 2016, Chen et al., 2017, Chen et al., 2020). OsNRT2.4, a dual-affinity NO₃ ^- transporter, participates in regulating NO₃ ^- uptake and allocation between roots and shoots and promotes plant growth and development under NO₃ ^- regulation (Wei et al., 2018).

In rice, NO₃ ^- acts not only as a nutrient but also as a signaling molecule. OsNRT1.1B has been confirmed to sense external NO₃ ^- signal (Hu et al., 2015). Additionally, studies have shown that the NO₃ ^- sensor OsNRT1.1B physically interacts with the phosphate signaling repressor OsSPX4; the presence of NO₃ ^- enhances this interaction and promotes the recruitment of NRT1.1B-Interacting Protein 1 (NBIP1, an E3 ubiquitin ligase), leading to the ubiquitination and degradation of OsSPX4. The core NO₃ ^- signaling transcription factor NLP3 is also regulated by SPX4. This OsNRT1.1B-OsSPX4-OsNLP3 regulatory module fills the gap between plasma membrane NO₃ ^- sensing and downstream NO₃ ^- responses in the nucleus ( Figure 2 ) (Wang et al., 2020b; Hu et al., 2019).

During crop domestication, many advantageous variant loci are retained by natural or artificial selection. Identifying these natural variant loci can provide theoretical support for crop genetic improvement (Hu et al., 2015; Li et al., 2018). In modern rice cultivars, NUE-related quantitative trait loci (QTL) or genes have been identified through map-based cloning methods. According to the varying N absorption capacity among different varieties, key genes controlling NUE, such as OsNRT1.1B, OsNR2, DNR1, and OsWRKY23, have been cloned (Hu et al., 2015; Li et al., 2018; Zhang et al., 2021b; Gao et al., 2019; Zhang et al., 2025b).

OsNRT1.1B and OsNR2 in indica have significant improvement in NUE and grain yield than those in japonica (Hu et al., 2015; Gao et al., 2019). Auxin Response Factor OsARFs mediate the promotion of N metabolism by auxin, DNR1 participates in the regulation of auxin homeostasis, and reflects differences in NO₃ ^- uptake, N assimilation, and yield enhancement between indica and japonica. The variation in the promoter of DNR1 in indica decreased expression levels and a higher auxin content, which triggers ARF-activated the transcription of NO₃ ^- uptake and assimilation-related genes, leading to improving the grain yield and NUE (Zhang et al., 2021b). RNR10 encodes an F-box protein that interacts with DNR1. RNR10 monoubiquitinates DNR1 and inhibits its degradation, thus antagonizing auxin accumulation, which results in reduced root responsivity to N and NO₃ ^- uptake (Huang et al., 2023). SOD5 directly binds to the DNR1 promoter, activates its expression, and further inhibits auxin accumulation. Notably, knockout of SOD5 significantly improves NUE and grain yield, especially under LN conditions (Zhang et al., 2025a). OsWRKY23 is a key regulator of the differences in NO₃ ^- absorption rate and NUE between indica and japonica rice. OsWRKY23^indica exhibits reduced transcriptional activation of DNR1, leading to higher auxin levels, improved NO₃ ^- absorption and assimilation, and ultimately enhanced NUE and yield (Zhang et al., 2025b).

Different varieties exhibit developmental differences due to their varying sensitivity to N supply, which can be observed in factors such as root length, biomass, and yield. Map-based cloning has been used to identify the genetic loci responsible for N modulation of plant growth, such as MYB61, through analysis of phenotypic values of these traits or ratios under different N levels supplied (Gao et al., 2020; Obara et al., 2010; Lian et al., 2005). The transcription factor MYB61 is regulated by GROWTH-REGULATING FACTOR4 (GRF4) and coordinates the production of cellulosic biomass and N utilization. The indica allele of MYB61 shows strong transcriptional activity, leading to improved NUE and higher grain yield under reduced N supply compared to the japonica allele (Gao et al., 2020) ( Figure 2 ; Table 1 ).

In recent years, genome-wide association study (GWAS) has also been used to locate some N-efficient genes. For example, using NUE-related agronomic traits, the GWAS identified the excellent variation of OsNPF6.1^HapB , which originated from the variation of wild rice. This excellent allele was transcriptionally activated by NAC42, and it enhances the ability of N absorption capacity and improves NUE under LN conditions (Tang et al., 2019). Through GWAS analysis of NUE-related traits (effective panicle number and yield per plant) in natural populations of rice, combined with transcript data under high N (HN) and LN conditions, OsNLP4 was identified as a transcription factor that regulates NUE. Simultaneously, OsNLP4 can promote the transcription of nitrite reductase gene OsNiR and N transport-related genes (NRTs and AMT1.1) (Yu et al., 2021; Wu et al., 2021), achieving coordinated regulation of N uptake and assimilation in rice. Accordingly, the localization of OsNLP3 and OsNLP4 in rice cells is affected by NO₃ ^- supply levels. With the application of NO₃ ^-, the localization of OsNLP3 and OsNLP4 in cells is shifted from cytoplasm to nucleus (Hu et al., 2019; Wu et al., 2021), indicating that OsNLP3 and OsNLP4 are central regulatory factors in the N signaling pathway of rice.

Using a multiparent advanced generation intercross (MAGIC) population, GWAS of NUE-related traits (tillering) under HN and LN conditions identified the OsSTP28 as a key regulator of N-responsive tillering and yield formation in rice (Zhang et al., 2024). A GWAS for N-responsive tillering in rice identified OsTCP19 as a regulatory factor; a 29-bp indel in the promoter of OsTCP19 represents a natural variation that determines tiller number under LN conditions in different rice varieties. OsTCP19 as a modulator of tillering response to N through its transcriptional response to N and its targeting to the tiller-promoting gene DWARF AND LOW-TILLERING (DLT), the OsLBD37/39-OsTCP19-DLT pathway is a key regulatory cascade governing N response and tillering in rice (Liu et al., 2021). Furthermore, the transcription factor OsGATA8 was identified as a critical regulator of N uptake and tiller formation in rice. OsGATA8 negatively regulates N absorption by repressing OsAMT3.2 transcription, while promoting tiller formation by inhibiting the transcription of the negative tillering regulator OsTCP19. The OsGATA8^H haplotype displays high NUE, with enhanced N uptake and a higher proportion of productive tillers (Wu et al., 2024) ( Figure 2 ; Table 1 ).

In recent years, several NUE-related genes have been cloned from mutant identification, such as ARE1, ARE4, OsELF3-1, and OsDOF18. ARE1 is a negative regulator of N assimilation, encoding a chloroplast-localized protein, and is transcriptionally inhibited by Ghd7. Loss-of-function mutations in ARE1 cause delayed senescence and grain yield increases, hence enhance NUE under LN conditions (Wang et al., 2018b). OsELF3–1 forms a ternary complex (OsEC) with OsELF4s and OsLUX, repressing the expression of Ghd7, which in turn directly inhibits ARE1 expression and promotes N absorption (Wang et al., 2021; Tsednee, 2024; Sun et al., 2024). ARE4, a MYB-related transcription factor, coordinates glucose signaling with NUE in rice. It is kept in the cytosol by interacting with the glucose sensor OsHXK7. Upon sensing a glucose signal, ARE4 is released, translocated into the nucleus, and activates the expression of a group of high-affinity NO₃ ^- transporter genes, resulting in increased NO₃ ^- uptake and accumulation (Ma et al., 2023). In the osdof18 mutant, the expression of OsAMT1.1, OsAMT1.3, OsAMT2.1, and OsAMT4.1 is reduced, indicating that these NH₄ ⁺ transporter genes function downstream of the transcription factor OsDOF18. The findings demonstrate that OsDOF18 mediates NH₄ ⁺ transport and N allocation, thereby influencing NUE (Wu et al., 2017) ( Figure 2 ; Table 1 ).

Excessive absorption of NH₄ ⁺ by plants leads to cellular acidification, while excessive NO₃ ^- uptake leads to cellular alkalization. Therefore, excessive uptake of a single N source affects the pH balance in plant cells, causing enzyme dysfunction and ultimately impacting crop growth and yield. In a recent study, overexpression of the N transport gene OsNRT2.3b helps counteract pH changes in rice plants, thus improving NUE and rice yield (Fan et al., 2016b). Plasma membrane (PM) H⁺-ATPase facilitates the transport of various nutrients, such as NO₃ ^-, phosphate (Pi), and K, and maintains cytosolic H⁺ homeostasis by pumping H⁺ outside the cells. In previous studies, overexpression of Oryza sativa PM H⁺-ATPase 1 (OSA1) in rice enhances NH₄ ⁺ uptake and assimilation, leading to increased grain yield and NUE (Zhang et al., 2021a). The assimilation of NH₄ ⁺ in root cells requires a C skeleton as the substrate for the synthesis of amino acids through the GS/GOGAT cycle. The assimilation of one molecule of NH₄ ⁺ generates two molecules of H⁺ in the cytoplasm. This inhibits the growth and development of plant roots and reduces NUE (Jia et al., 2020; Hachiya et al., 2021). A recent study identified a mutant that exhibited root hypersensitivity to NH₄ ⁺ due to a missense mutation in the gene encoding argininosuccinate lyase (ASL), which localizes to plastids and mitigates NH₄ ⁺-induced inhibition of root elongation by converting excess glutamine into arginine. Natural variations in ASL alleles between the japonica and indica subspecies of rice demonstrate ASL expression is positively correlated with NUE and yield (Xie et al., 2023). These results suggest that the H⁺ produced during the mitigation of NH₄ ⁺ assimilation can improve NUE in rice ( Figure 2 ; Table 1 ).

The transcription factor OsDREB1C is identified through RNA-seq as co-induced by light and LN supply, directly targets OsNR2, OsNRT2.4, and OsNRT1.1B, and simultaneously enhances the efficiency of photosynthesis and NUE, significantly improving rice yield (Wei et al., 2022). The bZIP transcription factor OsRF2b, identified through biochemical screening, interacts with OsbZIP61 to form heterodimers. This complex directly binds to the OsNRT1.1B promoter region and represses its expression, acting as a negative regulator of NUE and grain yield (Li et al., 2025). OsNR1.2 encodes an NADH-dependent NO₃ ^- reductase, essential for achieving high NUE in rice (Han et al., 2022). Furthermore, a batch of key genes involved in NUE has also been identified, such as rice OsBT1, OsBT2, and AAP genes (Araus et al., 2016; Lu et al., 2018; Pereira et al., 2022), crop yield and NUE can be improved by changing the expression levels of these genes ( Figure 2 ; Table 1 ).

Apart from using the methods mentioned above to identify N-efficient genes, analyzing the mechanism of low NUE in ‘Green Revolution’ varieties (GRVs) that limit efficient N use, and mining N-efficient genes from such varieties have also proven effective. The ‘Green Revolution’ gene sd1, which encodes the GA20 oxidase 2 (GA20ox2) enzyme, an important synthetic enzyme in the gibberellin (GA) synthesis pathway, is widely used in indica breeding. The mutated type of sd1 causes a decrease in endogenous GA activity and GA signal suppressor DELLAs protein (SLR1) accumulation in rice, which leads to a reduction in rice plant height (Ashikari et al., 2002).

A rice transcription factor GRF4 interacts with the transcriptional activator GIF1 to promote the expression of N transport and assimilation-related genes (such as OsAMT1.1, OsGS1.2, OsNRT1.1B) (Li et al., 2018). SLR1 competitively represses the GRF4-GIF1 interaction, inhibiting the formation of the GRF4-GIF1 protein complex, which leads to a reduction in NUE of rice. Introduction of the excellent allele gene GRF4^ngr2 into semi-dwarf and high-yielding rice varieties can achieve a coordinated increase in the yield and NUE of rice without changing their plant height (Li et al., 2018). The key repressor DWARF 53 (D53) of the SL signalling interacts with GRF4 and prevents GRF4 from binding to its target gene promoters, and negatively regulates NUE (Sun et al., 2023).

Subsequently, NGR5 is a new target of GA-GIBBERELLIN-INSENSITIVE DWARF1 (GID1)-mediated proteasomal destruction, and SLR1 competes with NGR5 for interaction with GID1, in the case of NGR5, with stabilized SLR1 of rice GRVs promoting stabilization of NGR5, thus explaining why GRVs exhibit increased tillering. N status affects chromatin function through modification of histones, a process in which the transcription factor NGR5 recruits polycomb repressive complex 2 (PRC2) to inhibit tiller genes, including OsD14 and OsSPL14, through repressive H3K27me3 modifications (Wu et al., 2020). Additionally, SLR1 competes with NGR5 for interaction with GID1. In the case of NGR5 with stabilized SLR1 of rice (GRVs), promoting the stabilization of NGR5 leads to increased tillering, explaining why GRVs exhibit enhanced tillering. Furthermore, pyramiding of sd1 elite NGR5 alleles can enhance NUE, leading to reduced N fertilizer usage and increased grain yield, without affecting the beneficial semi-dwarfism (Wu et al., 2020) ( Figure 2 ; Table 1 ). This suggests that manipulation of plant development and NUE co-modulation would drive modern breeding for sustainable food security.

Over the past two decades, one of the most significant advances in understanding N utilization regulation in rice has been the identification of NO₃ ^- and NH₄ ⁺ transporters, as well as transcription factors involved in NUE. In rice, OsNRT1, OsNRT1.1B, OsNRT1.6, OsNRT2.1, OsNRT2.2 and OsNRT2.4 are responsible for NO₃ ^- uptake. OsAMT1.1, OsAMT1.2, OsAMT1.3, OsAMT2.1, OsAMT2.3, OsAMT3.1 and OsAMT5.1 are principal for NH₄ ⁺ uptake. OsNRT1.1B, OsNRT1.6, OsNPF2.2, OsNRT2.3a and OsNAR2.2 are crucial for NO₃ ^- translocation to shoots. In addition, among the currently identified members of the rice NRT/NPF family, OsNRT1.1A, OsNRT1.1B, OsNRT2.1, OsNAR2.1, OsNRT2.3a, OsNRT2.3b, OsNPF3.1, OsNPF4.5, OsNPF6.1, OsNPF7.7, and OsNPF8.20 have all been shown to enhance NUE, whereas OsNPF7.3 decreased NUE at high NH₄ ⁺ supply. Compared to the substantial advances made in NO₃ ^- transporters, the progress achieved with NH₄ ⁺ transporter proteins in improving NUE has remained relatively limited. In the AMT family, only OsAMT1.1 and OsAMT1.2 have been identified as capable of improving NUE ( Figure 1 ; Table 1 ).

Significant genetic variation in NUE exists within rice germplasm resources, providing a valuable foundation for the precise breeding of cultivars with enhanced NUE. During rice domestication, natural variations in several key loci genes have been identified (including OsNRT1.1B, OsNR2, DNR1, OsWRKY23, and MYB61), playing important roles in regulating NUE and contributing to yield differences between indica and japonica cultivars. In the future, it remains essential to further dissect the candidate genes responsible for NUE variation among rice germplasm resources. GWAS has already cloned multiple key NUE-regulating genes, such as OsNPF6.1, OsNLP4, OsSTP28, OsTCP19, and OsGATA8, which enhance N uptake, assimilation, and tillering ability in rice, thereby improving both NUE and grain yield under varying N conditions. These findings have provided important genetic resources for the molecular breeding of rice cultivars with improved NUE. Moreover, maintaining cellular pH homeostasis is crucial for achieving high NUE in rice. Genes such as OsNRT2.3b, OSA1, and ASL play pivotal roles in regulating proton flux, nutrient transport, and N assimilation, ultimately enhancing NUE and grain yield under N stress conditions. Moreover, introducing advantageous alleles such as GRF4^ngr2 or NGR5 into the genetic background of the ‘Green Revolution’ sd1 can simultaneously enhance NUE and grain yield while maintaining a desirable dwarf plant architecture. The synergistic effect of sd1-GRF4-NGR5 enables the coordinated improvement of both NUE and yield, representing an ideal strategy for future molecular breeding ( Figure 2 ; Table 1 ).

Despite the successive identification and characterization of key genes involved in N uptake, transport, and utilization in rice, the effect of a single gene on improving N absorption or NUE remains limited. By adopting a multi−gene co−regulation strategy, rice NUE and yield can be further enhanced. Studies have shown that co−overexpression of genes for N transport, uptake, and assimilation increases both rice yield and NUE. For instance, the OsNPF8.9a×OsNR2, OsAMT1.2×OsGS1.2×OsAS1, and OsGS2×OsAS2×OsANT3 combinations respectively optimize NO₃ ^- uptake, NH₄ ⁺ conversion, and N recycling. Notably, combining OsAMT1.2, OsGS1.2, and OsAS1 overexpression represents a promising breeding strategy (Luo et al., 2023).

Looking forward, the success of such multi-gene strategies will benefit greatly from the integration of advanced biotechnological tools. CRISPR/Cas-based multiplex genome editing allows for precise and simultaneous modification of multiple target genes, while transgenic stacking enables coordinated expression of gene cassettes. When combined with high-throughput phenotyping and omics-assisted selection, these approaches provide a robust framework for the rational design of rice cultivars with enhanced NUE and yield potential. This systems-level breeding strategy offers an effective path toward reducing N fertilizer input while maintaining high productivity, contributing to more sustainable and environmentally friendly rice production.

In the context of global climate change and increasingly variable field conditions, the regulation of NUE in rice must be understood not only under optimal environments but also under abiotic stress conditions. Recent studies have demonstrated that N uptake and assimilation are not only genetically regulated but also highly responsive to environmental cues. Under abiotic stresses such as drought, salinity, and extreme temperatures, N transporter expression and NR activity or other enzymes involved in N metabolism are frequently suppressed, leading to reduced NUE (Henckel, 1964; Plaut, 1974; Guo et al., 2003; Robredo et al., 2011; Goel and Singh, 2015; Han et al., 2015; Meng et al., 2016).

However, the application of key genes has been shown to be significant potential for improving NUE under stress conditions. For instance, DST (Drought and Salt Tolerance)-OsNR1.2 regulatory module has been shown to be involved in the suppression of NO₃ ^- assimilation under drought tolerance. Given that DST negatively regulates stomatal closure while positively regulating N assimilation, it likely mediates a coupling between N metabolism and stomatal movement. This mechanism offers a promising target for developing drought-tolerant crops with improved NUE (Han et al., 2022). OsDREB1C, a member of the AP2/EREBP transcription factor family, was initially identified for its role in cold stress responses in rice, recent studies have demonstrated that overexpression of OsDREB1C shortens the growth duration, enhances NUE, and promotes more effective resource allocation, suggesting a potential regulatory link between stress response pathways and nutrient efficiency (Mao and Chen, 2012; Wei et al., 2022).

Moreover, agronomic practices such as irrigation regimes, fertilization strategies, and soil amendments can influence the expression and function of key N-related genes, thereby affecting NUE in crops (Shoji et al., 2001; Yang et al., 2015; Sajjad et al., 2024). Furthermore, in paddy fields, NO₃ ^- and NH₄ ⁺ availability fluctuates significantly in time and space, necessitating root responses to diverse and changing environmental cues. It has been found that NO₃ ^- supply enhances NH₄ ⁺ uptake in rice (Zhao et al., 2008). Therefore, a deeper understanding of the interaction between NO₃ ^- and NH₄ ⁺, and their roles in physiological and biochemical regulation of N uptake, is crucial for improving NUE. In summary, a systematic understanding of the dynamic interactions between genetic regulatory networks and management practices will provide a theoretical foundation and practical guidance for developing N management strategies that are both high-yielding and environmentally sustainable under variable environmental conditions.

In summary, we have outlined the genetic regulatory factors involved in the transport of NO₃ ^- and NH₄ ⁺, which contribute to efficient N absorption and translocation. We further discussed the key genes regulating NUE in rice, highlighting their potential to significantly improve both crop yield and NUE. Although substantial progress has been made in understanding the genetic architecture and molecular mechanisms underlying NUE in rice, there remain significant gaps in our knowledge of the complex genetic networks governing NUE regulation. This calls for an in-depth exploration of the genes and regulatory elements affecting NUE through advanced genomic technologies and bioinformatics tools. Future studies should focus on elucidating the functions of these genes, their interactions, and their responses to nitrogen availability under varying environmental conditions. Ultimately, by leveraging strategies such as multi-gene pyramiding, in-depth analysis of signaling regulatory networks, and the mining of elite genetic resources, it is expected that rice NUE can be further improved, facilitating the development of high-yield, environmentally sustainable rice varieties.