The gaseous loss of NH₃ is known as volatilization. Volatilization is a complex process that is controlled by the physical, chemical, and biological properties of soil and the environment (Fan et al., 2011). Agriculture activities account for 50% of the total annual global NH₃ loss (32 Tg year⁻¹) to the atmosphere through volatilization (Liu et al., 2019a). Fertilizer and manure application and livestock activity are the primary sources of NH₃ emissions in agriculture. Chemical N fertilizer alone is responsible for 34% of the loss (He et al., 2014). In particular, urea-based fertilizers are more susceptible than other N fertilizers because of the temporary increase in soil pH through the consumption of H⁺ ions during hydrolysis (Eq. 13). There is an equilibrium between NH₄ and NH₃ in soil solution (Eq. 16). The pK_a for equilibrium in Eq. 16 is 9.3. Therefore, alkaline conditions favor greater proportions of NH₃ (Havlin et al., 2014). When soil pH exceeds 7.5, temperatures increase up to 45°C, sufficient air movement is present to remove NH₃ gas at the soil–atmosphere interface, and losses of N as NH₃ are maximized (Bock and Kissel, 1988; Havlin et al., 2014). Application to acidic soils raises a little risk of volatilization. Application to sandy soils with low native cation exchange capacity (CEC) raises the risk. The common management approaches to improve the NUE of NH₄/NH₃ fertilizers include incorporation into the soil through injection or tillage to protect NH₄/NH₃ through the association of NH₄ with clay colloid cation exchange sites. When animal wastes are used as nutrient sources for crops, volatilization has been markedly diminished by incorporation or pretreatment with acidifying agents (Marshall et al., 1998; Choi and Moore, 2008; Doydora et al., 2011). Splitting applications between pre-plant and one or more subsequent applications later in the growing season is also commonly recommended to reduce the time that NH₄/NH₃ fertilizers are exposed to environmental conditions that promote loss.

Urea hydrolysis (Eq. 13) may be considered the final step in the mineralization of organic N. The urease enzymes (urea amidohydrolases, EC 3.5.1.5) are produced by a large number of organisms filling a variety of ecological niches including plants, bacteria, algae, fungi, and invertebrates (Sigurdarson et al., 2018). In most soils, the enzyme is more than sufficiently present and free in solution to rapidly hydrolyze urea to NH₃ (Klose and Tabatabai, 1999). Therefore, management to avoid losses of NH₃ through volatilization following urea application has commonly involved the inhibition of ureases to prevent the reaction from occurring until the urea itself may be safely incorporated into the subsurface soil.

Conventional urease inhibitors include N-(n-butyl) thiophosphoric triamide (NBPT), perhaps the most widely employed, with demonstrated effectiveness in rice, cotton, wheat, maize, and pasture grasses (Zaman et al., 2009; Kawakami et al., 2012; Marshall et al, 1998; Martins et al., 2017; Wallace et al., 2020). Urease inhibition with NBPT and cyclohexylphopshoric triamide (CHPT) may also be effective in preventing N losses from manure sources (Svane et al., 2020). Plant-based materials such as those isolated from Canavalia ensiformis (jack bean), Eucalyptus camaldulensis (eucalyptus), allicin from Allium sativum (garlic), and certain Acacia spp. have been shown to inhibit ureases in soil (Mathialagan et al., 2017; Rana et al., 2021). This raises the possibility of the increased entrance of plant biotechnologies into this area. Finally, as with any N source, urea may also be split applied and/or subsurface applied to prevent exposure to environmental conditions that lead to losses.

Higher rates of animal manure or commercial N fertilizer application increase NO₃⁻ leaching as a result of increased available N concentration in soil solution. Nitrate N is highly susceptible to leaching due to the negative charge associated with NO₃⁻ which prevents its association with negatively charged soil colloids, whereas NH₄⁺ is electrostatically attracted to colloids and therefore protected from leaching (Lodhi et al., 2009). Therefore, rain and irrigation would take the NO₃⁻ out of the system. Nitrate leaching takes place mainly after the heavy rainy season and the period of slow crop growth. Pande et al. (1985) reported that the N leaching process accounted for 2%–60% of the applied N loss. It has been estimated that the irrigated wheat fields account for 5 to 12.5 kg N ha⁻¹ N leaching loss, where farmers have applied 250 kg N ha⁻¹ with two splits in northern Mexico (Riley et al., 2001). Clay soil typically has lower NO₃⁻ leaching than sandy soil due to limited hydraulic conductivity. In clay soils, NO₃⁻ measured in soil samples to 60 cm can be subtracted from maize N fertilizer recommendations due to the reduced leaching potential (Fromme et al., 2017).

Nitrification is a microbial process (Eq. 14, Table 1), in which the ammonium is converted into nitrate by the oxidation process (Ward et al., 2011). It is a two-stage process (Eqs. 8 and 9) and is mediated by autotrophic bacteria.

The first stage is initiated by the ammonia-oxidizing bacteria like Nitrosospira and Nitrosomonas, which perform the oxidation of NH₄⁺ to nitrite (NO₂⁻) by means of the membrane-bound ammonia monooxygenase enzyme associated with hydroxylamine oxygenase (Jiang et al, 2018). The second step involves the conversion of NO₂⁻ to NO₃⁻ mediated by Nitrobacter. The last stage is much faster and more effective than the first stage; hence, nitrite rarely accumulates in the soil (Linn and Doran, 1984). Nitrification takes place in an aerobic soil environment with optimal soil moisture (60% water-filled pore space) (Linn and Doran, 1984). However, it is a very slow process in anaerobic soil environments (rice ecosystem) (Linn and Doran, 1984). The process is also regulated by soil temperature, pH, NH₄⁺/NH₃ concentration, and microbial population (Sharma and Ahlert, 1977). Nitrate produced by this process can be leached, absorbed by plants, and immobilized by soil microorganisms.

Denitrification is also a microbe-mediated, though strictly anaerobic, process (Eq. 15, Table 1) wherein NO₂⁻ is reduced to N₂ gas using intermediate products such as nitrogen dioxide [NO₂, nitric oxide (NO), and nitrous oxide (N₂O) (Figure 1)]. The production of N₂O is a major concern because of its greenhouse gas (GHG) function, with approximately 300 times the GHG potential of CO₂. Soil N loss through denitrification as a percentage of applied N varies widely and is a function of soil water content, soluble carbon (C), the presence of NO₃⁻, temperature, and time. Global loss of N from denitrification is estimated to be 96 Tg year⁻¹ in 2000 and would probably increase to 142 Tg year⁻¹ by 2050 (Bouwman et al., 2013). The process is carried out by a group of facultative anaerobic bacteria and catalyzed by nitrate reductase and nitrite reductase enzymes (Garbeva et al., 2007; Ranatunga et al., 2018). Two different electron acceptors are used during the denitrification process in aerobic conditions; oxygen acts as an electron acceptor, while NO₃⁻ is used as an electron acceptor in anaerobic conditions (Bock et al., 1995). Chemo-denitrification is another process responsible for nitrous oxide emission, but the quantity is smaller than biological production (Kool et al., 2011). Likewise, the nitrification process also releases N₂O through the spontaneous oxidation of hydroxylamine, which is an intermediate in the nitrification process (Kool et al., 2011).

Conventional management for the prevention of denitrification losses has conventionally been through inhibition of nitrification in soil. Nitrification inhibition prevents the formation of NO₃⁻, the substrate for denitrification, from NH₄⁺ (Eq. 14). There are a number of chemistries known and used in agriculture for nitrification inhibition. These include nitrapyrin (and various other pyridines), thiourea, thiophosphoryl triamide (also a urease inhibitor), 3,4-dimethylpyrazole phosphate (DMPP), and dicyandiamide (DCD) (McGinn et al, 2016; Ruser and Schulz, 2015; Alonso-Ayuso et al., 2016). Each of these chemistries is known to increase the production and release of nitrous oxide (N₂O) from soils, a fact that should be considered in all efforts to increase NUE.

Research into biological nitrification inhibition (BNI) is advancing rapidly (Coskun et al., 2017). The current state of BNI research suggests that both plant-derived compounds (direct inhibition) and indirect mechanisms may be simultaneously responsible. For a thorough review of isolated plant exudates and metabolomics responsible for BNI, please see Nardi et al. (2020). Harnessing BNI for agricultural scale use will continue to be a fecund area of research in the near future for plant biotechnology and breeding disciplines.

Slope, rainfall intensity, soil type, and vegetation are key determinants of soil and nutrient loss and transport (Kang et al., 2001). Soil nearest to the surface often contains the greatest concentrations of N and organic matter which can be readily transported through runoff and erosion. It is possible that up to 70% of surface-applied N fertilizer may be lost to a runoff if rain occurs on the same day (Mandal et al., 2012).

Management of cropping systems to reduce such physical losses of N will improve NUE. Conventional approaches to minimizing erosion and runoff include reduced tillage or no-tillage, cover cropping, surface residue retention (conservation tillage), contour tillage, terracing, and grassed waterways (Boincean and Dent, 2019; Farzadfar et al., 2021; Young et al., 2021). While no-till and reduced till systems tend to protect or increase soil organic matter, which includes organic nitrogen, Canisares et al. (2021) reported that no-till increased mineralization rates without affecting the optimal corn fertilization response. In this case, yields were greater under no-till (~1,000 kg ha⁻¹), though the response to N fertilizer was unchanged. Depending on how it is defined (Eqs. 1-7), NUE may or may not have been improved in this case. However, efforts to control erosion and loss of N through reduced tillage should improve soil stocks of N through both conservation and enhanced mineralization and continue to be the best recommended practices.

When cover crops are included in cropping systems, there are multiple mechanisms that can lead to increased NUE. Reduction of erosion caused by overland flow is more effective when covers with finer roots such as cereal rye or oats are used as opposed to covers with thick roots such as mustards or radishes (De Baets et al., 2011). Leguminous cover crops fix N₂ gas from the atmosphere into plant-available NH₃ (Eq. 10) and incorporated into the plant biomass. Upon senescence of the cover crop, the biomass N may then be remineralized (Eq. 18). Any measure of NUE which simply considers the reduction of fertilizer requirement will naturally be improved by increasing soil stores in this way. Cereal covers have the potential to reduce leaching by scavenging N from soils into biomass and releasing to the following cash crop through mineralization as well. Ranells and Wagger (1997) reported that the legumes hairy vetch and crimson clover could release 132 and 60 kg N ha⁻¹, respectively, while the non-legume cereal rye released 24 kg ha⁻¹.

Ammonium fixation occurs with 2:1 type of clay minerals such as illite, vermiculite, and smectite because they have negative charges and have the ability to expand interlayer spacing when soil water enters the basal oxygen plane (Nieder et al., 2011). The NH₄⁺ ion is comparable to that of K⁺ with respect to ionic radii and low energy of hydration (Nieder et al., 2011). Therefore, the NH₄⁺ ion is fitted exactly in the ditrigonal holes, or interlayers, in the basal oxygen plane of 2:1 clay mineral when soil water is present (Kunze and Jeffries, 1953). The clay mineral interlayers collapse approximately 1 nm upon drying, and NH₄⁺ ions are then trapped between silicate sheets and largely removed from further exchange reactions (Juang et al., 2001).

Manure and residues are applied to the soil as a source of nutrients (Figure 2). The first step after applying organic matter to the soil is mineralization (Eq. 17, Table 1), which converts the unavailable nutrient form into the available form NH₄⁺ (Chen et al., 2014). The C:N ratio of organic matter influences the N mineralization process because microbial biomass production requires both N and C (Chen et al., 2014). The wider the C:N ratio (e.g., >30:1) could hinder the mineralization process due to insufficient N content, and this condition leads to the immobilization of N (Eq. 18) (Quemada and Cabrera, 1995; Yassen et al, 2010). Immobilization is a process by which applied N can be incorporated into microbial biomass to provide for protein synthesis and reproduction. When mineral N + mineralizable organic N are insufficiently present to meet these needs, immobilization will remove plant-available N from the system (Sakala et al., 2000; Bird et al., 2001). Immobilization is considered negligible when the C:N ratio is <20:1.

In addition, when the N concentration is insufficient at the early stage of residue decomposition, the N content of the microbe’s own tissues may be increased through the remineralization process (Zelenev et al., 2006). Remineralization is a natural process by which the microbes requiring N can meet by mineralization of dead microorganisms using the enzymolysis process. Shindo and Nishio (2005) reported that the remineralization rates of wheat straw were 0.71, 0.55, and 0.29 mg N kg⁻¹ day⁻¹ after 7, 28, and 54 days, respectively. The high rate of remineralization is usually happening due to high consumption and low assimilation of N by microbes (Braun et al., 2018).

The level of soil disturbance induced by different tillage practices affects soil N dynamics and plant N availability (Power and Peterson, 1998). For example, Francis and Knight (1993) reported that compared with conventional tillage systems, conservation tillage techniques reduced nitrogen availability. The absence of soil disturbance under the conservation tillage system can reduce the N mineralization rate, thereby decreasing the N availability to crops as well as the loss of N. In the conventional tillage system, however, increased oxidation of soil organic matter due to disturbance and exposure, as well as increased soil erosion, hastens the loss of soil organic matter (Schillinger et al., 1999). Soil organic matter loss caused by conventional tillage systems results in poor soil quality and low N availability. Therefore, the role of the tillage system will be vital for improving NUE.

The relationship between the conservation tillage system and NUE varies between studies, but overall NUE is often improved by the conservation tillage system (McConkey et al., 2002; Giacomini et al., 2010). Long-term conservation tillage systems (10–15 years) enhance the quantity of soil organic matter and increase the concentration of mineralizable organic nutrients at the soil surface layer (Sirivedhin and Gray, 2006), thereby improving the nutrient-supplying capacity of the soil (Van Den Bossche et al., 2009). As a result, conservation tillage systems that retain crop residues often result in higher crop yields and NUE compared with conventional tillage systems with a similar N application level (Stahl et al., 2019).

A long-term (10-year) study conducted in the southern United States of America showed that with the optimum application of N, cotton yields were higher in conservation tillage than in conventional tillage plots (Boquet et al., 2004). However, without N fertilizer application, the yields were lower in the conservation tillage system as a result of slow mineralization and immobilization of soil N (Boquet et al., 2004). For instance, in a study conducted in Kentucky, Phillips et al. (1980) found that fertilizer N applied on well-drained soil in a no-tillage system produced a greater (13.2 kg greater) corn yield per kilogram of applied N than under conventional tillage, but without N fertilizer, the corn yield was lower under a no-tillage system. On the contrary, crop residue retention, wetter soil surface, and anaerobic environments in no-till systems promote N immobilization, NH₄ volatilization, and denitrification, negatively affecting N availability and NUE. In a wheat–fallow cropping system under the conventional tillage system, the N uptake was greater than that of stubble mulch systems. This is probably due to increased N immobilization in the stubble mulch system (Rasmussen and Rohde, 1991). Therefore, changes in N management, rate of application, and type of N fertilizer can improve NUE under conservation tillage systems. Overall, the role of conservation tillage and NUE requires more research to find practical compatibility.

It has been demonstrated that NUE could be improved through management practices such as timing, rate, source, and placement of fertilizer application. These practices are considered fundamentals to N management and may be refined or supplemented by emerging and future technologies, but not replaced.

Nitrogen use efficiency is also dependent on the ability of the cropping system (Ortiz-Monasterio et al., 1997; Reddy, 2011). Crop diversification can improve soil structure, soil health, vertical nutrient stratification, and mycorrhizal fungal interactions, as well as offer diversity in crop residues. A potential cropping system could help improve N availability and plant uptake (Tisdall and Oades, 1979; Lehman et al., 2012). Cereal- and legume-based cropping is the best system for leaving more residual N accumulation (Lehman et al., 2012). In a study with fallow followed by rice and legume followed by rice systems in Japan, the fertilizer NUE was higher for the legume (broad bean) followed by rice with 40 kg N application compared with fallow followed by rice in a clay loam soil (Rahman et al., 2009). Similarly, in a 20-year study on clay loam soil in Ontario, Canada, Gaudin et al. (2015) reported an increase in maize fertilizer NUE when winter wheat is inserted into a maize–soybean (especially when wheat is under-seeded with red clover) cropping system. In another study conducted in China, Li et al, 2022 found a higher N uptake and N harvest index in faba bean when intercropped with wheat compared with sole faba bean. The benefits associated with crop rotation and intercropping are mainly due to the facilitation through interaction between legumes and cereals and shallow-rooted and deep-rooted crops. Therefore, the rotation of crops with different depths of roots can improve soil structure and stability (Obalum and Obi, 2010) and enhance resource use efficiency (Halli et al, 2019). Tap-rooted crops can more easily penetrate compacted soil layers than shallow or fibrous-rooted crops, which serve to enhance the water and N use efficiency of the overall system (Chen and Ray, 2010). In Denmark, van Oosterom et al, 2010 observed a maximum mineralized N (81 kg N ha⁻¹) within the rooting zone of pea–cabbage compared with the onion–cauliflower cropping sequence, where the mineralized N was only 52 kg ha⁻¹ within the root zone. The selection of varieties/crops with different root systems, varied capacity to fix atmospheric N, and higher biomass production is an effective strategy to enhance NUE that deserves future research attention.

Nitrogen use efficiency of plants depends on the rate of soil N used by roots and accumulation in different plant parts such as the stem, leaf, and harvestable portions. Therefore, NUE is influenced by the inclusion of cover crops in a cropping system. The inclusion of high biomass-producing crops such as cover crops and dual-use forage crops can enhance the overall NUE of any system (Reicosky and Forcella, 1998). Cover crops are the crops planted in the off-season when the land is otherwise left uncultivated. Leaving land fallow increases the likelihood of soil erosion and nutrient leaching. Cover crops can help to protect the soil from loss, keep living roots in the soil as much of the year as possible, and recycle nutrients.

Cover crops with low C:N ratio residues (legume) can hasten the mineralization of organic N which may be responsible for the high NUE of the main crops (Franzluebbers et al., 2014). However, the cover crops with high biomass and high C:N ratio residues can lead to the immobilization of N, decreasing NUE for the following cash crop. A simulation model study using NLEAP (N Leaching and Economic Analysis Package) predicted that the inclusion of winter cover crops increased the NUE of lettuce by 3.1 kg per 4.5 kg of available N (Delgado, 1998). The cover crops in this study included winter wheat and rye, which were modeled to recover and retain soil NO₃-N in tissue, preventing leaching loss and fertilizing the next crops. Similarly, planting cereal rye crops after no-till corn has reduced N leaching by 100% (McCracken, 1989). The CERES-N model modified by Quemada and Cabrera (1995) includes important considerations outside of simple C:N ratios to predict the mineralization or immobilization potential of cover crop residues. The model requires inputs for water-soluble carbohydrates, cellulose/hemicellulose, lignin, total C, total N, and C:N ratio.

Forage crops (often perennials) also contribute to the reduction in N loss and improved NUE. For example, a study from the USA reported that a perennial, such as alfalfa, reduced NO₃-N leaching by 10-fold over a corn–soybean rotation or continuous corn systems (Randall and David, 2001). Moreover, persistent roots of forage grasses are important to bind the soil particles together to develop a stable soil structure and potentially capture N from 1.5 m deep in the soil. Thus, surface available N can be utilized by subsequent crops to improve NUE.

Although there has been a consensus on the need to increase the NUE of crop plants through breeding, practically, no breeding program is primarily dedicated worldwide for this purpose, to the best of our knowledge. Theoretically, there may be different ways to improve NUE through breeding, such as overall consideration of grain yield or biomass growth under limited N conditions, selection and improvement of specific traits that contribute to high NUE, or introduction of the foreign gene. However, indirect selection for yield has been the common method for achieving higher NUE (Cormier et al., 2016).

NUE is considered a complex trait. Modifications in traits such as plant height, tiller number, dry weight of shoots and roots, grain yield, spikelet number, number of filled grains per panicle, 1,000-grain weight, and chloroplasts were reported to improve NUE (Lawlor, 2002; Zhao et al., 2011a; Hamaoka et al., 2013). Breeding targets for genetic improvement of the plant may be grouped into two major categories: first, improving N uptake efficiency by increasing uptake capacity (Le Gouis et al., 2000) and breeding for ideal root morphology (Liao et al., 2006) and, second, improving N utilization efficiency by modifying the leaf area index, specific leaf N, and biomass yield (Gastal and Lemaire, 2002) and by delaying the senescence (Foulkes et al., 2009).

Before initiating the new breeding efforts to create genetic variability for high NUE in modern crop cultivars, the rich genetic resources conserved in different gene/seed banks of the world should be explored for screening high NUE lines. There is proven genetic diversity for root N uptake in plants (Pereira et al, 2010; Le Gouis et al., 2000), and exploiting this property requires researchers to understand the underlying mechanism of higher root uptake.

Root morphology plays a critical role in modulating N uptake by plants (Garnett and Rebetzke, 2013). Plants with rapid root growth can minimize N losses that occur through various field processes (Gastal and Lemaire, 2002). Anbessa and Juskiw (2012) observed that barley plants with higher root dry weight and volume assessed at the five-leaf stage showed higher NUE than normal plants. Improvements in root traits such as length of root, root-length density, the radius of the root, root surface area, and number, length, and density of root hairs (Wang et al., 2006) are associated with greater N uptake in plants. Breeding efforts for enhancing root-related traits are essential for improving NUE. However, the limited scope of large-scale and high-throughput root phenotyping creates obstacles in breeding programs for selecting and screening specifically for such beneficial root architecture (Fiorani and Schurr, 2013).

The uptake of additional N must match with the metabolism of the plants to avoid systemic feedback control of metabolites representative of the whole-plant N status (Nacry et al., 2013). The uptake and utilization of N for the entire plant growth period can be separated into two phases: pre-anthesis and post-anthesis (Cormier et al., 2016). At the pre-anthesis stage, plants take up N, and the whole-plant system utilizes it upon receiving fractional interception of light at the start of the stem elongation phase. However, at post-anthesis, once grains appear, plants begin partitioning available N for higher grain yield, jeopardizing the simultaneous improvement in grain yield and protein content (Oury and Godin, 2007). Higher N utilization is possible under low N supply through an increased specific leaf N area (SLN), which is reported to be associated with the embryo size of the plant (López-Castañeda et al., 1996) and earlier canopy closure (Rebetzke and Richards, 1999). Physiological conditions wherein N is more efficiently utilized are associated with the abundance of prostrate leaves during vegetative growth and semi-erect to erect leaves during later vegetative and reproductive stages. This can be difficult for plant architecture to manipulate (Cormier et al., 2016). Normally, at the post-anthesis stage, the grains draw N from the stem and rachis in cereals and then from leaves if necessary. However, the stay-green plant types are prone to supply N to growing grains slowly and thus impact the balance in the N demand–supply framework (van Oosterom et al., 2010). Researchers are in consensus that physiologically important traits that directly or indirectly improve N utilization are taken into consideration in breeding programs, in addition to the common target traits. However, assessing those traits on the bulk scale is a question of technological advancement, resources available to the breeders, and practical limitations (Cormier et al., 2016).

It is of utmost importance to identify genes or QTLs that govern NUE to enable the breeding of crops with high NUE using approaches such as marker-assisted selection (MAS) and genomic selection. Nutrient use efficiency is a complex trait, and as a result, several research groups have undertaken efforts to map the genetic loci in correlation with specific traits (Balyan et al., 2016; Kumar et al., 2018b; Mălinaş et al., 2022). In rice, 20 single QTLs (S-QTLs) and 58 pairs of epistatic loci (E-QTLs) were identified for the grain N, straw N, shoot N, harvest index, grain yield, straw yield, and PE in low N and ordinary N conditions. Harvest index and grain yield were positively correlated with PE in both conditions (Cho et al., 2007). In another study carried out with rice, four QTL clusters harboring QTLs for both NDT and NUE traits were identified (Wei et al., 2012). In European winter wheat, a genome-wide association study using 214 varieties identified 333 genomic regions associated with 28 traits related to NUE (Cormier et al., 2014). For the second approach, specific NUE-associated genes should be identified. Some of the efforts successfully mapped genes and identified QTLs. In maize, a meta-analysis of published NUE QTLs revealed 37 “consensus” QTLs, of which 18 were detected under low N conditions. Comparing expressed sequence tags (ESTs) associated with low N stress response, N uptake and transport, and assimilation with the QTL map has resulted in identifying candidate NUE-associated genes. Among those genes, nine candidates introgressed into Ye478 have significantly altered grain yield/yield components (Liu et al., 2012). Five significant QTL clusters associated with large-rooted architecture and high N uptake efficiency (NupE) were identified in maize. The root system architecture (RSA), such as that found in maize, has an essential role in N acquisition. NupE had significant phenotypic correlations with RSA (Li et al., 2015). Three QTLs, NUE1a, NUE1b, and NUE2, were identified in maize for NUE (Mandolino et al., 2018). Under N starvation, the expression of TaNLP7 displayed enhanced expression in root and shoot tissues of the high NUE genotype (Kumar et al., 2018a). Forty-seven genes are known to involve N uptake, metabolism, and distribution in maize (Wani et al., 2021). In barley, 10 independent mapping studies were screened and a number of NUE-associated genes that control complex physiological traits were mapped (Han et al., 2016). Even though a large number of reports claim to be identifying QTLs for NUE, some of them are yet to be validated. Since NUE involves a myriad of factors, the traditional breeding strategy combined with MAS will be cumbersome. Therefore, exploiting genomic selection for improving NUE will speed up the development of superior genotypes by combining high-throughput phenotyping and genotyping (Han et al., 2016; Kumar et al., 2018c; Stahl et al., 2019). In wheat, four QTLs, viz., QNue.151-1D, QNue.151-4A, QNue.151-6A, and QNue.151-7D, were associated with NUE; one QTL, QNupe.151-4A, was associated with N uptake efficiency; and one QTL, QNute.151-4A, was associated with N utilization efficiency (Brasier et al., 2020). The details of the QTLs identified in the crop plants are given in Table 4.

The role of small RNAs in regulating the nutrition assimilation/starvation response is well documented in many crops (Balyan et al., 2016). A total of 126 long non-coding RNAs (lncRNAs) were altered during N starvation, and these RNAs regulate various protein-coding genes involved in diverse cellular functions (Chen et al., 2016). Forty-four miRNAs are differentially regulated under high and low N conditions (Li et al., 2016). Most of these targets were found to be the genes encoding for the transcription factors. The important miRNAs and transcription factors involved in the N starvation response in Arabidopsis are shown in Figure 4. In Arabidopsis and maize, the expression of miR167 was enhanced under N starvation conditions (Xu et al., 2011; Balyan et al., 2016). miR167 regulates the lateral root growth response to N starvation in Arabidopsis (Gifford et al., 2008). Conversely, downregulation of the transcription factors ARF10, ARF16, and ARF17 by N-responsive miR160 regulates the process of seed germination and development of the seedling after post-germination under N-deficient conditions (Liu et al., 2007; Hao et al., 2022). Downregulation of miR169 enhances the expression of the NFYA transcription factors; these genes regulate the function of the nitrate transporter genes, viz., AtNRT1.1 and AtNRT2.1 (Zhao et al., 2011b). These studies showed the involvement of small RNAs and their functional importance in inducing/repressing multiple genes in response to N assimilation/deprivation and regulation of root development in plants. In wheat, simple sequence repeat markers developed from miR171a effectively group the panel of wheat genotypes into N-efficient and non-efficient markers. These markers can be employed to characterize the wheat germplasm/breeding lines in crop breeding programs (Sagwal et al., 2022).

Plants have evolved mechanisms to alter the molecular machinery in response to N availability (Gaudinier et al., 2018). Yang et al. (2017) identified 1,158 and 492 genes that were differentially expressed in leaf sheaths and roots, respectively, after 12 h of N starvation in rice. Conversely, in Dunaliella salina, 3,127 were differentially expressed (2,380 genes were upregulated and 747 were downregulated) under N starvation (Lv et al., 2019). In maize, ZmGLK5, bZIP108, CLC-a, and miRNA399b genes play a significant role in regulating genes in response to N (Jiang et al., 2018). The NIGT1/HRS1s transcriptional repressors are essential in regulating N starvation response during high N availability (Kiba et al., 2018). The CLE peptides and the CBL7 and TAR2 proteins regulate root architecture in response to N starvation (Kiba et al., 2018); the DUR3 and AMT family proteins play an important role in the uptake of urea and ammonium, respectively, during N starvation (Krapp et al., 2014). The nitrate transporters, NRT1/NPF and NRT2, regulate nitrate uptake (Krapp et al., 2014).

The availability of high-throughput genomics tools and efficient transformation systems in model crops further eases the functional validation of NUE (Muthusamy et al., 2016; Lenka et al., 2019). Several attempts have been made to develop transgenics with high NUE. Overexpression of AtDof1, AtGS1, and AtGS2 enhances the N assimilation in transgenic tobacco lines grown under N-starved conditions compared with wild-type plants (Wang et al., 2013). Transgenic overexpression of OsDof25 modulates C and N metabolism in transgenic Arabidopsis lines during an increased supply of N (Santos et al., 2012). Plant species comprising the C₄ photosynthetic pathway have evolved highly efficient molecular mechanisms of carbon fixation. C₄ plants exhibit high radiational, N, and water use efficiencies compared with species with the C₃ photosynthetic mechanism (Ghannoum et al., 2011; Muthusamy et al., 2019). Engineering the genes involved in the C₄ photosynthetic pathway in C₃ plants remains an essential strategy for enhancing the NUE in C₃ crops (Lin et al., 2019; Muthusamy et al., 2019). Moreover, the availability of N regulates the ethylene and jasmonic acid hormone signaling, thereby regulating the plant response to pathogen infection (Vega et al., 2015; Farjad et al., 2018). miRNAs are known to play an important role in regulating the function of N-responsive genes during N-limiting conditions (Nguyen et al., 2015; Zuluaga et al., 2017). Thus, the identification of gene regulatory networks, including small RNAs involved in regulating the stress response, will further help to understand the development of stress-responsive crops with high NUE (Muthusamy et al., 2017; Zuluaga et al., 2017; Farjad et al., 2018). The details of the QTLs identified in the crop plants are given in Table 5.