Nitrogen (N) is an essential element required for plant growth and overall yield; hence, the demand and use of N-based chemical fertilizers have consistently increased over the years. Approximately 60-70% of the applied N fertilizers are lost to the environment (Mohanty et al., 2020), causing severe environmental havoc such as pollution, global warming, biodiversity loss, and major plant physiological disorders. Since the increasing rate of N application is becoming increasingly alarming, minimizing fertilizer use while maintaining a high crop yield would be imperative. Thus, improving plants’ nitrogen use efficiency (NUE) is one of the inherent ways of overcoming these crises associated with crop production. Efficient N utilization is a critical factor in crop yield improvement, and research has shown that over 1.0 billion US dollars might be saved with a one percent NUE increment (Kant et al., 2011a).

Crop NUE is the measure of seed yield, grain, or fruit corresponding to a unit of soil N supplied, depending on the individual species of plant. NUE can also be expressed in terms of N uptake efficiency (NUpE), N transport efficiency (NTE), N remobilization efficiency (NRE), and N utilization (assimilation) efficiency (NUtE) (Bharati and Mandal, 2019), all of which are key determinant factors of NUE in plants. N is made available to plants in organic and inorganic forms; nitrate ( NO 3 - ) and ammonium. Due to the mobility nature of NO 3 - , it gets easily leached; thus, its availability to plants becomes limiting (Jin et al., 2015). NO 3 - functions as a signaling molecule, inducing the expression of NO₃ ^--related genes involved in its uptake, transport, assimilation, vegetative and reproductive development. Plants take up NO 3 - from the root, assimilate NO 3 - , and subsequently transport it to the shoot, where it can be remobilized to sink organs (Iqbal et al., 2020). NO 3 - transporters are the main drivers involved in the uptake of NO 3 - to the remobilization stage.

Indeed, several studies have discussed the relationship between NO 3 - uptake transport activities in plants while addressing the mechanisms involved in transport, sensing, and signaling processes (Fan et al., 2017; Zuluaga and Sonnante, 2019; Vidal et al., 2020). Therefore, optimizing the activities of NO 3 - transporters is a prerequisite for plants to utilize N supplies. Some studies have elucidated the functional roles of these NO 3 - transporters in plant NUE improvement. However, less is known about the influence of essential nutrients and environmental cues on the expression and activities of NO 3 - transporters. To better understand the extent to which these transporters can function in improving plant NUE, an illustration of their response to changes in plant environmental cues, including salinity, pathogenic and drought stress, and contamination from heavy metals, becomes expedient. Even if these conditions are being optimized, it is crucial to explore the possible aftermath effect of these NO 3 - transporters on the efficiency of other plant nutrient elements and related factors. These necessities ignite a few questions: 1) Does stress affect NO 3 - transporter activities directly or indirectly? and 2) Do the activities of these NO 3 - transporters exert a positive or negative effect on the uptake of other nutrients? To resolve these issues, this review critically summarized the roles of NO 3 - transporters in N uptake, transport, and distribution processes and their functions in crop productivity and NUE, especially when coexpressed with other transcription factors. This review focuses on the functional roles of these nitrate transporters in assisting plants in adverse environmental conditions. We also discussed the impact of these NO 3 - transporters on the uptake and utilization efficiency of other plant nutrients while describing possible strategic approaches to improving NUE in plants. The contribution of nitrate transporters in nitrate and auxin crosstalk for root growth and NUE is also reviewed. Understanding the specificity of all these factors is crucial for better N utilization efficiency of crops.

Most agricultural fields, especially, those used for commercial crop production, are NO 3 - deficient with significant spatiotemporal fluctuations, inhibiting N utilization (Kant, 2018). Plants have evolved two major NO 3 - uptake mechanisms to survive. The first is the low-affinity transport system (LATS), which facilitates nitrate uptake under high soil-N (millimolar concentration; > 0.5 mM), while the other is the high-affinity transport system (HATS), which drives nitrate under insufficient soil-N (micromolar range) (Léran et al., 2014; Iqbal et al., 2020; Raddatz et al., 2020). Four families of NO 3 - transporters have been widely known to participate in plant nitrate uptake and transport: nitrate transporter 1/or peptide transporter NPF (NRT1), nitrate transporter 2/nitrate-nitrite-porter NRT2/NNP, slow anion channel-associated homologs (SLAC/SLAH), and chloride channel (CLC) (Tsay et al., 1993; Bergsdorf et al., 2009; Maierhofer et al., 2014; Von Wittgenstein et al., 2014). Among them, NPF (NRT1) and NRT2 and homologs have been identified as the major channels actively involved in root nitrate uptake and long-distance transport between and within plant organs (Hsu and Tsay, 2013; Wang et al., 2021b). In this review, proteins or genes void of prefixes connote Arabidopsis plant species.

Phylogenetic studies revealed that the NPF family comprises 53 identified Arabidopsis genes, and over 130 genes exist in higher plants (Zhang et al., 2020). Generally, NPF transporter genes have low affinity for NO 3 - , except for Chlorate resistant 1/nitrate transporter 1 (CHL1 / NRT1.1), also called NPF6.3, a dual-affinity nitrate transporter that operates as both a low- and high-affinity transporter (Liu and Tsay, 2003). The regulatory mechanism involved in the dual-affinity system enables the rapid switch between these two affinity modes. Under a low external supply of NO 3 - , NPF6.3 (CHL1 / NRT1.1) functions as a high-affinity NO 3 - transporter and is phosphorylated, whereas it becomes dephosphorylated under a high NO 3 - supply to perform a low-affinity transporter role (Liu and Tsay, 2003; Noguero et al., 2018). Thus, the affinity of the NPF6.3 transporter for NO 3 - uptake depends on the phosphorylation state at the T101 residue, which is subject to the status of N in the medium NPF6.3 (CHL1/NRT1.1) is expressed in various plant tissues, including younger leaves, flower buds, and roots, where it participates in root NO 3 - uptake and translocation (Noguero et al., 2018). In addition to NPF6.3 (CHL1/NRT1.1), NPF4.6 (NRT1.2) and NPF2.7 (NAXT1) are the two putative NPF genes that coordinate NO 3 - influx and efflux in plant roots, respectively ( Figure 1 ). NPF4.6 (NRT1.2) is primarily expressed at the root tip where it takes up NO 3 - (Huang et al., 1999), whereas NPF2.7 (NAXT1), is expressed in the root zone but in the cortex, performs NO 3 - -efflux functions (Segonzac et al., 2007). A considerable amount of NRT1 family members have been identified in other crops, including wheat (Triticum aestivum) (Kumar et al., 2022), rice (Oryza sativa) (Yang et al., 2020), cucumber (Cucumis sativus) (Migocka et al., 2013), potato (Solanum tuberosum) (Zhang et al., 2021a), and apple (Malus × domestica Borkh.) (Wang et al., 2018b), with their unique expression at either the root or shoot of plants. The expression pattern of these transporters is a clear indication of their active involvement in uptake and long-distance NO 3 - transport.

Unlike the NRT1 family, NRT2 family members are high-affinity NO 3 - transporters (HATs). There are eight identified NRT2 family members, of which seven have been characterized (Von Wittgenstein et al., 2014). Four (NRT2.1, NRT2.2, NRT2.4, and NRT2.5) out of the seven characterized NRT2 transporters have been actively involved in the influx of NO 3 - into Arabidopsis root cells (O’Brien et al., 2016). Detailed functions of these transporters in uptake of NO 3 – are presented in ( Figure 1 ).

Nitrate transporters are the major channels mediating root-to-shoot NO 3 - transport. Transport is predominantly mediated by NRT1 and NRT2 transporters, such as NPF7.3 (NRT1.5), NPF7.2 (NRT1.8), NPF2.3, and NPF2.9 (NRT1.9). NPF7.3 (NRT1.5) is expressed in pericycle cells, where it facilitates xylem loading of NO 3 - ( Figure 1 ). Knockout nrt1.5 mutant plants had reduced amounts of NO 3 - translocated from the roots to the shoots. However, when NRT1.5 was reduced in nrt1.5, no NO 3 - translocation defect was observed, suggesting the existence of another mechanism facilitating nitrate xylem loading (Lin et al., 2008). The low-affinity nitrate transporters NRT1.8 and NRT1.9 perform similar roles of unloading NO 3 - from the xylem ( Figure 1 ), consequently reducing NO 3 - concentration within the xylem. Knockout mutants of such transporters (NRT1.8 and NRT 1.9) exhibited increased amounts of NO 3 - in the xylem and, by implication, accelerated root-shoot transport of nitrate (Li et al., 2010; Wang and Tsay, 2011). In addition, the uptake and transport function of the NRT1 and NRT2 homologs have also been revealed in rice (OsNRT1.1B and OsNRT2.3, respectively) (Tang et al., 2012; Hu et al., 2015; Fan et al., 2017), and tomato, LeNRT2.3 (Fu et al., 2015).

While NO 3 - is relocated to the shoot, a larger proportion of N is delivered to the sink organs (e.g., seeds, fruits, roots, and younger leaves), especially for the anabolic development of new tissues, prioritized by the growth stage or physiological condition of individual plants, a process called N remobilization (Snyder and Tegeder, 2021). NRT1.4, localized in the leaf petiole, regulates NO 3 - accumulation within the petiole while maintaining the homeostasis of available NO 3 - between the leaf lamina and petiole ( Figure 1 ). The nrt1.4 mutant had a low NO 3 - content in its petiole, a major NO 3 - storage organ, indicating the involvement of NRT1.4 in nitrate homeostasis and leaf development (Chiu et al., 2004). Another NO 3 - transporter, NRT1.7, predominantly expressed in the phloem of minor veins, enhances nitrate relocation from older to younger leaves ( Figure 1 ) (Fan et al., 2009). However, the extent of NO 3 - transfer and the proportion of NO 3 - remobilized to the sink organ remain unclear. NO 3 - storage in seeds is mediated by specific NO 3 - transporters that remobilize NO 3 - into embryos during seed formation. The expression of NRT1.6 within the host embryo and seed coat demonstrates a potential role of this transporter in mediating embryonic NO 3 - relocation at the reproductive phase of the parent plant ( Figure 1 ) (Almagro et al., 2008). Similar to NRT1.6, NPF5.5 also mediates NO 3 - transport into the embryo ( Figure 1 ) (Léran et al., 2015; Iqbal et al., 2020). NRT2.7, a high-affinity NO 3 - transporter in the tonoplast, plays specific NO 3 - storage roles in the seed vacuole (Chopin et al., 2007). In the tonoplast, CLCa and CLCb were observed to perform a similar localization pattern, where they also participate in NO 3 - storage (Von Der Fecht-Bartenbach et al., 2010). While NO 3 - accumulation in seed vacuoles has been well documented, relatively less is understood about the characterization of transporter genes involved in NO 3 - efflux out of the vacuole. An in-depth understanding of the specificity of these N transporters, from chronological studies, is the first step toward exploiting and optimizing NUE in plants.

For efficient NO 3 - assimilation, a larger proportion of NO 3 - assimilated after root uptake is diverted back to the cytosol, where it is converted to nitrite by nitrate reductase (NR). The nitrite obtained is relocated to plastids for subsequent reduction. At this stage, nitrite is converted to ammonium ( NH 4 + ) by the nitrite-reducing enzyme nitrite reductase (NiR) and then finally incorporated as an amino acid through the glutamine synthetase (GS) and glutamate synthase (GOGAT) cycle (Wilkinson and Crawford, 1993; Li et al., 2017a). Nitrogenous compounds incorporated via glutamine (free amino acid) and glutamate serve as a major checkpoint for regulating N utilization efficiency and are further enhanced by the synergetic expression of NR and NO 3 - transporters (Li et al., 2020; Snyder and Tegeder, 2021). However, a recent study opined an improved grain yield and NUE on concurrent coexpression of OsNRT1.1B and indica OsNR2, indicating the positive regulatory roles of OsNR2 and OsNRT1.1B in uptake of N in rice (Gao et al., 2019b).

The two functionally similar forms of GS, cytosolic GS1, and plastidic GS2, encoded by single or multiple gene families, have been reported to significantly influence N assimilation (Miflin and Habash, 2002). While cytosolic GS1 facilitates root N reassimilation and remobilization during protein turnover, GS2 isoforms primarily assimilate NH 4 + produced during chloroplast photorespiration (Ferreira et al., 2019). Although GS1 is responsible for NH 4 + reassimilation, some GS family members drive N assimilation when NO 3 - is abundant. A good example is GLN1;2 in Arabidopsis, which drives N assimilation when NO 3 - is abundant, compared to the gln1;2 mutant, which exhibits reduced GS activity, rosette biomass, and higher NH 4 + concentration under such conditions. Due to the principal roles of GS in N assimilation, specific focus has been directed toward overexpressing GS family members to improve N assimilation in different plant species, such as Triticum aestivum (Hu et al., 2018), and Oryza sativa (Bao et al., 2014).

Despite the fundamental roles of GS in improving NH 4 + assimilation, seed yield, and NUE (Hu et al., 2018; Gao et al., 2019a), attempts to improve NUE by overexpressing GS1 have yielded inconsistent results (Check Table 1 for details). For instance, TaGS2-2Ab-overexpressing lines in wheat had increased spike number, seed yield, and NUE under poor and rich N supply compared to their wild type, due to an increased root N uptake and remobilization capacity (Hu et al., 2018). Following a similar trend, overexpressing HvGS1-1 using its promoter confers improved grain yield and NUE on barley subjected to low and high N conditions (Gao et al., 2019a). In contrast, Bao et al. (2014) opined a drastic reduction in fresh and dry weight of OsGS1;1- and OsGS1;2-overexpressing lines in rice seedlings, with a further poor growth phenotype at the tillering and heading stages under limited and sufficient N conditions. The results suggest that the GS-overexpressing lines and plant biomass are negatively correlated. Further research is required to understand the underlying mechanisms of GS activity to improve NUE in plants.

Unlike GS, relatively few studies have addressed alterations in the expression of genes encoding NADH-dependent GOGAT (a key enzyme in N assimilation) and plastid-localized ferredoxin-dependent (Fd-GOGAT) (Good et al., 2004; Xu et al., 2012). The two kinds of GOGAT differ in their electron donor specificity. Fd-GOGAT is predominantly involved in the reassimilation of photorespiratory NH 4 + . In contrast, NADH-GOGAT participates in the assimilation of non-photorespiratory NH 4 + and the synthesis of glutamate needed for plant development (Lee et al., 2020). Many attempts have been devoted to studies on the fundamental roles of both NADH-GOGAT and Fd-GOGAT in the growth and seed development of Arabidopsis (Somerville and Ogren, 1980), Hordeum vulgare L. (Kendall et al., 1986), and Oryza sativa (Zeng et al., 2017). However, few research studies have altered the genetic expression of GOGAT to promote seed yield and NUE, while those that focused on NADH-GOGAT had rather limiting outcomes. For example, overexpression of ZmNADH - GOGAT in maize confers drastic reduction on shoot biomass with no considerable alterations in kernel yield when N is abundant (Cañas et al., 2020). Meanwhile, the overexpression lines of OsNADH - GOGAT resulted in an increase in rice grain weight under limited N (Yamaya et al., 2002). Interestingly, Lee et al. (2020) recently revealed that the synergetic expression of OsNADH - GOGAT1 and OsAMT1;2 confers an increase in NUE under both high and low N supply. While transgenic lines had improved seed protein levels without any yield alteration under N-sufficient conditions, seed quality and overall yield increased under N starvation. These observations imply that the combined expression of N-transporters and GOGAT improves N uptake, N assimilation, and NUE rather than the negative effect of the expression of AMT or GOGAT alone. Consequently, understanding the factors involved in the synergetic expression of NO 3 - transporters and GOGAT under rich and poor N conditions in plants is imperative to augment NUE.

In addition to its nutritional roles, NO 3 - functions as a major signaling element regulating several plant physiological processes, such as leaf expansion (Walch‐Liu et al., 2000), induction of root architectural changes (Walch‐Liu and Forde, 2008), regulation of root development, and regulation of floral induction (Marín et al., 2011). The first step in signaling is through external nitrate perception by the dual affinity NO 3 - transporter NPF6.3 (NRT1.1), induced immediately after NO 3 - treatment. NRT1.1 switches between two states of nitrate conditions (low and high NO 3 - conditions) (Wang et al., 1998; Bouguyon et al., 2015; Hu et al., 2015).

Nitrate transporters have been shown to play diverse NUE and yield improvement roles in plants (Check Table 4 for details). In Arabidopsis, NRT1.1 transgenic lines habouring Cauliflower Mosaic Virus (CaMV) 35S promoter were observed to increase the uptake of NO 3 - , however, this did not necessarily improve seed yield (Liu et al., 1999). In contrast, the expression of the NRT1.1 homolog OsNRT1.1B driven by the CaMV-35S promoter or its native promoter increased NUE and grain yield in rice. The key regulatory roles in NO 3 - nitrate signaling, absorption, and assimilation enable OsNRT1.1B to be a major contributor of rice NUE (Hu et al., 2015). Although, the crucial roles of OsNRT1.1B in NUE and yield improvement have been well studied, the underlying regulatory mechanism has not been elucidated. Similar to OsNRT1.1B, overexpression of the spliced form OsNRT1.1A also exhibits an approximately 50% grain yield and NUE increase, coupled with shortened maturation times (Wang et al., 2018c). The observations of this latter experiment could be successfully used to develop early maturing and high-yielding varieties in some other crops. The elevated expression of OsNPF8.20 (OsPTR9) leads to increased NH 4 + uptake, better root formation, and ultimately, an increased tiller and panicle number, indicating that OsNPF8.20 improves grain yield and NUE in rice breeding (Fang et al., 2013). Similarly, OsNPF7.20-overexpressing lines exhibited a drastic increase in rice tiller number, fresh weight, dry weight, and grain yield. In contrast, an opposite effect was conferred on the RNA interference (Ri) lines and osnpf7.2 mutant line under mixed nitrate supply (0.5-8 mM NO 3 - ) (Wang et al., 2018a). In their experiment on the modification of NO 3 - transporters in Arabidopsis and rice, Liu et al. (1999) and Hu et al. (2015) reported some discrepancies in the response of these plants to the modified transporters. This may be due to the tolerance and sensitivity of both crops to NH 4 + and NO 3 - . Arabiodopsis thrives under aerobic conditions where the NO 3 - transport system is well optimized, whereas rice thrives best in anaerobic environments where the NH 4 + transport system is optimized. Hence, manipulating NO 3 - and NH 4 + transporters for improved efficiency in Arabidopsis and rice, respectively, would generate little or no effect on their NUE. Several NO 3 - transporter genes in plants whose expression and subcellular localization pattern greatly determine the gene’s function are essential in genetic manipulations of plant traits. As such, deep insight into the function of a gene and the environment to which plants are better adapted can encourage precise manipulation of NUE in crops. The influence of nitrate transporters on crop yield was also reported in tomatoes, where overexpression of LeNRT2.3 improved NO 3 - uptake, root-to-shoot NO 3 - transport, plant biomass, and fruit weight (Fu et al., 2015).

The expression of several NRT2 transporters has also been found to influence yield and NUE under N-starved conditions. NRT2.2 was upregulated to improve N uptake, assimilation, and plant growth under low NO 3 - conditions (Li et al., 2007). Under the same NO 3 – stressed conditions, TaNRT2.5, highly expressed in wheat, increases NO 3 - uptake and root growth (Guo et al., 2014). Chen et al. (2016) conducted a study on transgenic rice and observed that OsNRT2.1, which has the OsNAR2.1 promoter (pOsNAR2.1: OsNRT2.1), was upregulated in the roots and culms. This upregulation significantly increases the overall yield, biomass, and NUE in transgenic lines harboring OsNAR2.1 (pOsNAR2.1: OsNRT2.1). However, the reverse (decrease in NUE) was obtained with the constitutive promoter of OsNRT2.1 (pUbi: OsNRT2.1). These variations could be accrued to alterations in the localization and abundance of OsNRT2.1 in the plant tissue (Chen et al., 2016). Further investigations regarding the importance of the NRT2 gene in NUE showed that two variants, OsNRT2.3a and OsNRT2.3b, were identified in rice. The elevated expression of OsNRT2.3b enhances intracellular pH balance under the synergetic supply of NH 4 + and NO 3 - , thereby increasing the uptake capacity of other nutrients (P, N, and Fe) and ultimately increasing grain yield and NUE by 40% (Fan et al., 2016b). This result demonstrates the importance of pH sensing by OsNRT2.3b in improving plant NUE and adaptation of rice to changes due to different NH 4 + - NO 3 - supplies. However, this N uptake and transport function observed in OsNRT2.3b was lost in OsNRT2.3a (Fan et al., 2016b; Chen et al., 2020a). OsNRT2.3a cannot independently improve crop yield and NUE due to its inability to increase the expression of OsNAR2.1 (Chen et al., 2020a). Thus, the coexpression of OsNRT2.3a with the OsNAR2.1 promoter becomes imperative to enhance rice N use. The literature reviewed thus far has demonstrated a need for most NRT family members to be coexpressed with specific promoters to effectively enhance plant growth, biomass, and NUE, especially in Arabidopsis and rice; however less in known in other crop species.

Numerous studies have investigated the crucial roles of NO 3 - transporters in mediating the uptake and long-distance transport of NO 3 - ; however, less is known towards understanding transport systems involved in NO 3 - reallocation under biotic and abiotic stresses. NO 3 - transporters play crucial roles in the plants’ response to adverse environmental conditions. Indeed, plants acclimatize better to environmental stress when less NO 3 - is allocated to the shoot. Thus, this section examines the contribution of NO 3 - transporters in assisting plants to strive in adverse environmental conditions.

The quantity of NO 3 - translocated from roots to shoots varies under diverse environmental conditions, as this could positively or negatively affect plant NUE. Hence, NO 3 - redistribution in plants is a prerequisite to improved plant growth under N shortages and adverse conditions (Fan et al., 2017). Stressed plants tend to uptake and transport less NO 3 - to the shoot while retaining more nitrate in its root than required ( Figure 2 ). Such NO 3 - allocation to the root as induced by environmental fluctuations (including biotic and abiotic stress) is referred to as “stress-initiated nitrate allocation to roots” (SINAR) (Zhang et al., 2018). Over two decades ago, Hernandez et al. (1997) investigated the inherent effects of cadmium (Cd²⁺) on NO 3 - uptake, and distribution in pea plants. They found that NO 3 - was increasingly retained at the plant root, and fewer NO 3 - were reallocated to the shoot of Cd-treated pea compared with the control, thereby disrupting the NUE of plants ( Figure 2 ). However, the study could not elucidate the mechanism underlying the fluctuation in the root-to-shoot transport of NO 3 - . Many years later, several research investigations have shown the active involvement of NO 3 - transporters in regulating Cd²⁺ uptake and other SINAR-related stress conditions (Lin et al., 2008; Zhang et al., 2014). Mao et al. (2014) reported NRT1.1 as a potential regulator of Cd²⁺ uptake in plants. They observed that plants exposed to Cd²⁺ stress exhibit repression of NRT1.1 and, as such, exert a negative influence on plant N nutrition ( Figure 2 ). Thus, the loss of NRT1.1 function reduced Cd²⁺ in the roots and shoots, improving plant biomass production under Cd²⁺ stress ( Figure 2 ). Although the disruption of NRT1.1 activity induced by Cd²⁺ stress negates NO 3 - uptake, it enhances plant tolerance to Cd²⁺ stress by reducing Cd²⁺ influx into the root. A recent study by Jian et al. (2019) opined that overexpression of NRG2 (which functions downstream of NRT1.1) in wild-type and nrt1.1 increased root NO 3 - over shoot nitrate, thus alleviating Cd²⁺ toxicity. These findings demonstrate the involvement of NRT1.1 in regulating cadmium uptake while coordinating nitrate allocation to the root. NRT1.1 also regulates Zn accumulation in Arabidopsis by improving NO 3 - uptake in the wild type through a NO 3 – dependent pathway under Zn stress ( Figure 2 ) (Pan et al., 2020).

In addition to NRT1.1, NRT1.5 and NRT1.8 regulate the acropetal reallocation of NO 3 - to shoots under cadmium and salinity stress (Fan et al., 2017a). Such stresses activate antagonistic expression of the two latter genes (NRT1.5 and NRT1.8), with reduced expression of NRT1.5/NPF7.3 (Chen et al., 2012) and increased expression of NRT1.8/NPF7.2 ( Figure 2 ) (Li et al., 2010). From the study conducted by Li et al. (2010), loss of NRT1.8 function displays greater sensitivity to Cd²⁺ stress than wild-type plants under high NO 3 - conditions. However, an opposite effect was observed, with nrt1.5 mutants having greater Cd²⁺ tolerance in relation to the control. The Cd²⁺ sensitivity observed with the ntr1.8 mutants could be due to Cd²⁺ translocation to its shoots, thus counteracting the plant adaptive strategy that supports Cd²⁺ accumulation in plant roots. The upregulation of NRT1.8 expression triggers nitrate removal from the xylem under Cd²⁺-stressed conditions. This result suggests a strong link between Cd²⁺ tolerance and NO 3 - allocation.

In addition to NRT1.5 and NRT1.8, NPF2.3 also contributes to the SINAR response under salt stress. Nitrate allocation to the shoot was drastically reduced under salt-stressed conditions due to the unaltered expression of NPF2.3 and partial expression of the NPF7.3 gene in the root stele. However, the loss of NPF2.3 function led to the reduced root-to-shoot allocation of NO 3 - ( Figure 2 ) (Taochy et al., 2015). These data demonstrate the quantitative and physiological contribution of the NO 3 - efflux transporter NPF2.3 to NO 3 - allocation to the shoot under salinity (Taochy et al., 2015; Chao et al., 2021). Alvarez-Aragon and Rodriguez-Navarro (2017) also found Na⁺ accumulation to be partially defective in the nrt1.1 mutant, demonstrating the partial contribution of NRT1.1 to NO 3 – dependent Na⁺ transport ( Figure 2 ). Plants expressing these NO 3 – related genes in response to heavy metal or salt stress exhibit enhanced NO 3 - uptake, plant growth, and tolerance to heavy metal- or salt-stressed environments.

Previous physiological research investigations have shown varying impacts of NO 3 - and NH 4 + availability on water uptake and transport in plants subjected to water stress (Guo et al., 2007). They found that the assimilation rate and stomatal conductance of NH 4 + -fed plants surpassed those of NO₃ ^--fed plants; thus, NH 4 + nutrition improves rice seedling tolerance to drought (Guo et al., 2007). Li et al. (2016a) revealed that the high-affinity NO₃- transporter NRT2.1 alters NO 3 - accumulation to regulate root hydraulic conductivity ( Figure 2 ). They found NRT2.1 to be a positive regulator of plasma membrane intrinsic protein PIPs. This latter study unraveled the link between NO 3 − use, water stress, and NRT2.1 expression, indicating the potential roles of NRT2.1 in drought tolerance (Li et al., 2016a). However, a more recent investigation has shown how the high-affinity NO 3 - transporter partner protein OsNAR2.1 positively regulates drought-related responses to stress and enhances drought tolerance in rice ( Figure 2 ) (Chen et al., 2019).

Ample agronomic evidence exists regarding the impact of excessive N fertilizer use on the incidence rate of plant diseases (Fagard et al., 2014; Fan et al., 2017). For example, excessive N fertilizer application triggers the severity of powdery mildew caused by a biotrophic pathogen that saps plant nutrients. Interestingly, a reduction in N fertilizer application has been found to reduce Arabidopsis tolerance to Erwinia amylovora. These findings indicate a complex relationship between N uptake, metabolism, and disease infection processes. Thus, it is evident that N status affects plant tolerance or susceptibility to diseases under specific environmental conditions (Fagard et al., 2014). Unfortunately, the molecular mechanism underlying the impact of NO 3 - transporters on fungal infection or pathogenic attack is not fully understood. To investigate the possible mechanisms involved in N uptake by the biotrophic pathogen, Pike et al. (2014) characterized the low-affinity transporter VvNPF3.2 (in grapevine) and cloned Arabidopsis ortholog NPF3.1. In this study, powdery mildew pathogen infection was shown to upregulate the expression of VvNPF3.2 and NPF3.1 in vascular tissues, major and minor veins of leaves. The loss of NRT2.1 and NRT2.2 under N-deficient conditions resulted in increased resistance to Pseudomonas syringae pv tomato DC3000 infection ( Figure 2 ) (Li et al., 2007; Camanes et al., 2012). Additionally, in the NRT2 family, the roles of two putative high-affinity NO 3 - transporters, NRT2.5 and NRT2.6, were investigated in response to rhizospheric bacterium STM196 using single and double Arabidopsis mutants (Kechid et al., 2013). The study revealed that mutations in NRT2.5 and NRT2.6 inhibited plant growth and abolished root system architecture in response to STM196. Hence, Arabidopsis leaves expressing NRT2.5 and NRT2.6 appear to play crucial roles in the plant response to STM196 in a NO 3 - uptake-independent manner ( Figure 2 ). The expression of both genes (NRT2.5 and NRT2.6) is also crucial for promoting plant growth mediated by STM196 (Kechid et al., 2013). Recently, T-DNA mutants of NRT2.5 showed stronger resistance to Pseudomonas syringae pv. tomato DC3000 inoculation compared to its wild-type counterpart, an indication of NRT2.5 role in plant biotic defense (Du Toit et al., 2020; Devanna et al., 2021). These research findings have demonstrated the functional roles of NO 3 − transporters in the plant response to biotic stress, while suggesting safe, innovative, and sustainable means of controlling crop pathogens.Mycorrhizal colonization of rice root also appears to promote the expression of a putative nitrate transporter, OsNPF4.5. This result improved growth and yield properties in host plant (Wang et al., 2020c). However, inactivation of OsNPF4.5 resulted in the reduction of arbuscule incidence, as well as a depletion in symbiotic nitrogen uptake in rice (Wang et al., 2020c).

Another member of the nitrate and peptide transporters family (NPF), OsNPF8.1 (OsPTR7), a putative peptide transporter in rice (localized in the cell plasma membrane), has been reported as permeable to methylated arsenic species, especially, dimethylarsenate (DMA). OsNPF8.1 is involved in long-distance transport of arsenic in rice (Tang et al., 2017). However, the peptide-mediated transport of arsenic species has been linked with imbalance nutrient (especially, phosphate) supply in plants (Finnegan and Chen, 2012). Consequently, it is imperative to investigate the activity of OsNPF8.1 on N uptake, as well as the collateral accumulation of DMA, its clinical significance and nutrient imbalance in economically significant crops.

Balanced nutrition is paramount to maintaining good human health, and this is achievable by eating a balanced diet. In plants, maintaining an appropriate nutrient balance is also required because excessive accumulation of a specific nutrient might affect the uptake of the other and vice versa (Aluko et al., 2021). This nutritional balance ultimately affects crop growth and plant nutrient use efficiency (Bouain et al., 2019). Such nutritional crosstalk coexists between phosphorus (P) and N, the most limiting nutrient element required for crop growth and development. Phosphorus starvation reduces nitrate uptake capacity in tobacco (Rufty et al., 1990), maize (De Magalhães et al., 1998), and barley (Lee, 1982). These phenomena demonstrate the mechanisms involved in optimizing nutrient uptake and utilization to maintain plant homeostatic balance. Molecular evidence indicates that nitrogen limitation adaptation (NLA) ubiquitin offsets NO 3 - deficiency induced by excessive P via degradation of PHT1, the phosphate transporter (Kant et al., 2011b). The phenotypic analysis illustrated the functional role of nitrate-inducible garp-type transcriptional repressor 1.2 (NIGT1.2) in integrating N and P signals. Under sufficient P supply, NIGT1.2 was not activated due to the coexpression of PHR1 and SPXs, which are P-sensor proteins and repressors of PHR1, respectively (Medici et al., 2015). However, PHR1 was detached from the inhibitors SPX1/2/3/4 to promote the expression of NIGT1 clade genes under P-starved conditions. Thus, nitrate uptake is suppressed due to P deficiency through the PHR1-NIGT1-NRT2.1 pathway (Maeda et al., 2018). With such development, N uptake regulation via the PHR1-NIGT1 path could be a good adaptative mechanism under P starvation (Maeda et al., 2018). Another recent study found that NIGT1.2 increased the expression of phosphate transporters (PHT1;1 and PHT1;4) but repressed the nitrate transporter NRT1.1, an indication that NIGT1.2 could maintain a balance between N and P to improve N uptake and utilization under (phosphorus) P starvation (Wang et al., 2020b).

The highly NO 3 – inducible NRT1.1-controlled GARP transcription factor, HRS1, and its closest homolog, HHO1, function downstream of NRT1.1, NLP6, and NLP7. However, HRS1 and HHO1 act as major primary root growth inhibitors only when the media is P-starved in the presence of NO 3 - , indicating extensive integration of the N and P signaling networks (Medici et al., 2015). Following the previous discussion on how HRS1 mediates N and P crosstalk, Medici et al. (2019) found that PSR marker gene responses depend on the N supplied. Indeed, transcript levels of PHO2 were coordinated by nitrate availability accumulated during both high and low supplies of nitrate. Notably, this nitrate-induced strategy of PSR regulation is conserved in plants. However, several PSR genes were not regulated by NO 3 - in a pho2 mutant, indicating that PHO2 incorporates nitrate signals into PSR (Medici et al., 2019). Upon P starvation, NRT1.1 is downregulated, while PHO2 functions to positively regulate NRT1.1. In rice, the genes induced by P starvation OsIPS1, OsSPX1, and the P transporter OsPT1 only respond to P starvation when nitrate is present (Medici et al., 2019). On the overall assessment, these findings elucidate the complexity of nitrate and phosphorus responses while emphasizing the principal roles of NRT1.1 in regulating the interaction.

Another macronutrient required for plant health is potassium (K⁺), as it strongly coordinates nitrate ( NO 3 - ). Previous reports indicated that NRT1.5 facilitates the long-distance transport of NO 3 - and K⁺ in a nitrate-dependent manner (Meng et al., 2016; Zheng et al., 2016). NRT1.5, expressed in the pericycle of root cells, participates in the xylem loading of nitrate. When there is a K deficit, NRT1.5 directly triggers the movement of K⁺ to the root xylem for root-to-shoot transport. This investigation demonstrates the crucial role of NRT1.5 in root-to-shoot K⁺ transport and its involvement in the synergetic regulation of NO 3 - /K⁺ distribution in plants (Li et al., 2017b). Another study reported that MYB59 activates the expression of NRT1.5 and binds directly to its promoter to ensure a controlled nutrient distribution from root to shoot. When plants become deficient in NO 3 - /K⁺, the expression of MYB59 and NRT1.5 is repressed to maintain a balanced NO 3 - /K⁺ distribution between the roots and shoots (Du et al., 2019).

Evidence has shown the impact of changes in N status on auxin distribution in plants (Hou et al., 2021). Compared with moderate N supply, limited NO 3 - supply engenders auxin deposition in the roots of Arabidopsis, wheat, soybean, maize, and rapeseed (Caba et al., 2000; Tian et al., 2008; Asim et al., 2020), indicating the importance of in situ auxin synthesis in the root (Yang et al., 2022). Thus, the in situ auxin synthesis and the shoot-to-root polar transport jointly contributes to auxin deposition in the root under N limitation (Yang et al., 2022). In contrast, a 30% reduction in root indole-3-acetic acid (the putative among natural auxins) content was observed when the amount of NO 3 - supplied to rice dropped from 2.5mM to 0.01mM (Sun et al., 2014b). Perhaps, the discrepancies in N induced auxin response stems from varying plant growth conditions and the species involved. Nevertheless, all these findings demonstrate the importance of nitrate and auxin crosstalk in root development, and the mechanism of such responses are triggered by the activities of NO 3 - transporters.

In addition to the NO 3 - transport and signaling function, NRT1.1, among other transporters, facilitates basipetal transport of auxin and negatively regulates auxin biosynthetic genes, TAR2 and LAX3, under NO 3 - deficiency (Maghiaoui et al., 2020). As a consequence, NRT1.1 removes auxin (required for lateral root growth) deposited at the lateral root primordia, inhibiting lateral root growth under such condition. All these inhibitory effects of NRT1.1, including root growth reduction and patchy auxins are alleviated in response to high NO 3 - supply (Maghiaoui et al., 2020). Thus, NRT1.1-mediated auxin transport was disrupted and its (NRT 1.1) expression repressed, to facilitate lateral root growth and auxin accumulation at the root tip under increasing NO 3 - supply (Remans et al., 2006). These findings indicated that NRT1.1 functions in reprogramming root system architecture in response to NO 3 - availability. However, the integrated function of this molecular circuit is yet unraveled.

Although, it is understood that external N status regulates auxin biosynthetic genes and signaling pathways. However, less is known about the identities of auxin-related genes that are N-responsive, and whether these genes reprogram plant N metabolism to improve crop NUE is yet unexplored. To this end, Zhang et al. (2021b) identified DULL NITROGEN RESPONSE1 (DNR1) as an intriguing QTL regulating auxin and N crosstalk for NUE improvement in rice. DNR1 mediates plant N metabolism by counteracting the auxin deposited in response to N availability. This process enhances auxin biosynthesis and induces AUXIN RESPONSE FACTOR, a major regulator of N-responsive genes to improve NUE and grain yield.

Out of the identified NO 3 - transporters, the functions of the NO 3 – transceptor’s (NRT1.1) in auxin regulation has been the most investigated. However, less is known about the versatile functions of other NO 3 – related proteins in regulating other plant developmental traits.

Genetic modification of crops has been a promising strategy for improving plant N use through diverse breeding techniques during the past few decades. Indeed, several NO 3 - transporter genes, their regulators, and other NO 3 - responsive genes regulating NUE have been well studied. However, mechanisms involved in this regulation, which specifically describes the strategies involved in NUE improvement, have been overlooked due to difficulties in identifying N-specific phenotypes. (Hu et al., 2015) revealed that genetic variation of the major quantitative trait locus (QTL) NRT1.1B (OsNPF6.8) promotes NUE divergence between Indica and Japonica rice subspecies. They found that NRT1.1B from indica improved the tiller number, NUE, and grain yield of Japonica rice. Several other QTL-based approaches have generated signaling proteins, transcriptional regulators, and components of hormonal pathways that regulate plant NUE. One of these is a QTL study that used positional cloning and genetic complementation to map out DEP1 (Dense and erect panicles 1), a heterotrimeric G protein that confers a significant yield increase (Sun et al., 2014a). Under moderate N fertilization, plants harboring the dominant allele DEP1-1 display N-insensitive vegetative growth, as well as improved N uptake and assimilation, thereby increasing yield (Sun et al., 2014a). This result implies that modulating the activity of DEP1 could provide a lasting strategy for grain yield increases in rice. Another QTL study showed that the accumulation of the growth inhibitor DELLA confers semi-dwarfism and reduces NUE in rice (Li et al., 2018). However, the NUE and grain yield of green revolution varieties are restored by tilting the GRF4–DELLA stability toward an increased abundance of GRF4. This study indicated that regulating physiological activities and plant growth induced by efficient N use could open up innovative breeding ideas for sustainable food security (Li et al., 2018). Although QTL analysis has also informed the recent NUE gene identification strategy in crop species such as maize (Zhang et al., 2019), the importance of QTL analysis is yet unknown in some other higher plants.

In addition to QTL analysis, other analytical studies involving genome-wide association studies (GWAS) could be used to identify an array of NUE candidate genes in Arabidopsis (Atwell et al., 2010), maize (Li et al., 2013), rice (Si et al., 2016), and other crop species (Korte and Farlow, 2013; Ogura and Busch, 2015). An elite haplotype of the nitrate transporter OsNPF6.1HapB was recently identified using GWAS (Tang et al., 2019). This allele improved nitrate uptake, NUE, and grain yield under N-deficient conditions. In the same study, the NUE-related transcription factor OsNAC4 was used to transactivate OsNPF6.1^HapB , thereby increasing plant NUE and grain yield. This result suggests that the NAC42-NPF6.1 signaling cascade is a promising strategy for improving NUE and rice yield (Tang et al., 2019).

To further identify the genes enhancing NUE, Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9 along with the Cas9 nuclease (CRISPR/CAS9) system was developed. CRISPR/CAS9 has been deployed to facilitate easy and robust technology to edit genes for improved plant N use. Multiple applications of CRISPR/CAS9 technology have been demonstrated in major crops, including sorghum, rice, and tomatoes (Ito et al., 2015; Ma et al., 2015). Notably, CRISPR/CAS9 mostly mutates negative growth regulators instead of overexpressing positive regulators, thereby providing prospects for crop breeding (Tiwari et al., 2020). A related strategy described one of the Bric-a-Brac/Tramtrack/Broad gene family members, BT2, that downregulates the NRT2.1 and NRT2.4 genes (Araus et al., 2016), thus reducing NO 3 - uptake and NUE under low NO 3 - conditions. When this BT2 gene was mutated in Arabidopsis, a 65% increment in nitrate uptake was observed, while mutation of OsBT2 yielded a 20% increase in NUE compared to wild-type under poor NO 3 - supply (Araus et al., 2016). To date, the functions and features of a significant number of negative regulators or inhibitors of nitrate transporters have yet to be functionally characterized in plants. Hence, it is plausible that gene editing or mutating their expression by CRISPR/Cas9 appears to be a promising strategy for achieving future breeding goals (Tiwari et al., 2020).

It is essential to note that incorporating transcriptomics, proteomics, and metabolomics, which characterize the expression profile, could facilitate the identification of agronomically induced genes or pathways. Moreover, computational and system biology could aid in identifying candidate genes during domestication.

Nitrate transporters have not only been shown to function in plant uptake and transport capacity; their vital roles and potential in improving plant N use have also guaranteed the possibility of meeting future global food demands. Indeed, improved NO 3 - uptake and utilization ( NO 3 - transport, remobilization, and assimilation) through transporter activity is a prerequisite to attaining increased NUE and overall plant growth. With the understanding that the activities of these NO 3 - transporters are enhanced when co-expressed with their specific promoters or Tfs, it becomes imperative to select and integrate NO₃ ^– specific promoters with their transporters for efficient plant N utilization. An excellent way to improve NO 3 - utilization could be to carefully select senescence-specific promoters (primarily expressed in source organs or leaves) to facilitate phloem-expressed nitrate transporters. Most research works have successfully established the impact of nitrate transporters on adverse environmental conditions (biotic and abiotic stress). They have also addressed their relationships with other plant nutrients only under controlled conditions; however, field-based studies affirming these functions are scarce.

Moreover, relatively few NO 3 - transporters performing complex interplay functions have been identified, while the established ones were found to play multiple physiological roles in environmental and nutritional stresses. The underlying mechanisms behind these multipurpose functions are unknown, and the extent to which these transporters can mitigate abiotic stress is unresolved. Thus, to understand and manipulate the functional roles of nitrate transporters in enhancing plant NUE under diverse conditions, future research should address some critical questions, including the following, but not limited to:

Developing profound resolutions to these questions will afford us a better understanding of how nitrate transporters could be maximized to enhance plant NUE under adverse environmental conditions. Knowledge of these factors will also help settle crises related to plant nutritional imbalance and cross-talk, thereby achieving plant breeding goals for quality and sustainable food production.

Conceptualization, OOA, QW, and HL; writing-original draft, OOA; review and editing; SK and OMA; visualization, OOA, GY, and CL; validation, SK, OMA, QW, and HL; supervision, QW and HL; funding acquisition, QW and HL. All authors contributed to the article and approved the submitted version.