Nitrogen use efficiency—a key to enhance crop productivity under a changing climate

1A rapidly growing global population places considerable pressure on agricultural lands to produce more food and energy per unit area. For sustainable production, agricultural practices must both intensify productivity and simultaneously protect the environment and human and animal health. Improving nitrogen use efficiency (NUE) is an element of this framework. Nitrogen (N) is a key constituent of all living cells and is essential for the growth and development of plants. Fertilizer N is the second largest requirement after water in crop production, and N is the most common yield-limiting nutrient deficiency. The ratio of N taken up versus the unit applied to a crop is referred to as NUE. The low N use of the crop indicates that uptake is inefficient or higher than the plant’s requirement. Cereal crops like rice, wheat, and maize require large amounts of N for healthy growth and higher yields. Hence, varieties with higher NUE should be a priority for breeders developing new varieties.

2The global estimates of N stored in soil are 65 Pg to 30 cm depth and 92–140 Pg to 100 cm depth. The largest portion of stores is in the form of organic N, which is not directly plant available. Chemical fertilizers and manures add 200 Tg of N each year. Biological N fixation provides an additional input of 258 Tg of N. Ammonium (NH4+) and nitrate (NO3−) are the two forms of plant-available N. Globally, only 50% of applied N is converted and the rest is wasted.

3Crop NUE is influenced by environmental factors, plants’ physiological activity, and their interactions. Biochemical transformations of N in soil are complex and are best considered as being in a state of continual flux (Table 1). The fluxes of biochemical transformation in the soil system are primarily responsible for constraints to NUE. However, physical losses of N from the plant or soil system also decrease NUE. The major forms of N loss are the volatilization of ammonia (NH3) gas, leaching of dissolved NO3−, and overland runoff of all soluble forms. Changes in temperature and precipitation patterns affect biological and enzyme activity rates, which are important for most transformations listed in Table 1.

4The simplest approach to quantifying NUE is to divide the crop yield (Y) by the nitrogen inputs (N) (Eq. 1).

5However, several authors have suggested that yield may be defined in several ways, including the mass of the harvested portion of the crop, total (aboveground) biomass of the crop, N content contained in the harvestable portion, and N content of the total biomass. proposed a number of general “groups” of approaches to calculate NUE that may be considered (Eqs. 2-7).

6Where Gf and Gu are the grain yields (kg) of the fertilized and unfertilized plots, respectively, and Na is the rate of N applied (kg).

7Where Yf and Yu are the total aboveground biomass (kg) of the crop in fertilized and unfertilized plots, respectively, and Nf and Nu are the N contents (kg) of the aboveground biomass in the fertilized and unfertilized plots, respectively.

8Where Gf and Gu are the grain yield in fertilized and unfertilized plots, respectively.

9All of the above equations rely on the assumption that varied nitrogen rate as fertilizer input is the independent variable. Naturally, as the mass of N inputs decreases, the calculated efficiency increases in equations using N rate or difference in N accumulation in the denominator. It would therefore be quite easy to interpret these as suggesting that the lowest rates of N fertilizer inputs result in the best NUE. This outcome ignores the importance of crop productivity.

10Berendse and Aerts (1987) proposed a “biologically meaningful” definition of NUE as the product of nitrogen productivity (An/Ln) and the mean residence time (1/Ln) of nitrogen in the plant (Eq. 7).

11This approach avoids the same pitfalls in Eqs. 1-6 but somehow fails to provide an interpretation of NUE necessary to evaluate the direct effects of climate change or advancements in crop management to adapt to climate change. It is indeed likely that future studies will not employ varied rates of N inputs to study NUE but will instead evaluate changes in other practices, varieties, genetic enhancements, and emerging biotechnologies. In this case, new approaches to the calculation of NUE will be needed. Preferably, these will also include mass balances of native soil plant-available N (PAN) and potential PAN in addition to fertilizer or manure inputs.

12When considering the pressures of climate change, increased atmospheric carbon dioxide (CO2) will impact the ultimate equilibrium states of many of these processes. Higher temperatures will reduce soil N inventories by 5%–10% due to increased mineralization. With the twin pressures of population expansion and climate change, management and breeding will need to focus on fundamental problems to make progress in NUE. Consider, for example, that leaf expansion and photosynthetic rates are affected by low N and that root traits are chiefly responsible for N uptake and NUE in maize. Inbred maize lines exhibiting higher NUE were those with larger root diameters. Root-ABA1, a major quantitative trait locus (QTL) for root development in maize, plays a vital role in NUE along with four other QTLs, viz., Qaer3.10, Qaer5.05–6, aer9.07–8, and Qaer10.04, responsible for aerenchyma cell development. In rice, the transcriptomic approach has helped to identify 62 candidate NUE genes. SHORT ROOT and SCARECROW are root-patterning genes responsible for root development and architecture. AUX1 and PIN proteins regulate the auxin movement and lead to lateral root development. NUE is a complex trait governed by the crop’s agronomic, physiological, environmental, and genetic traits. The integration of association mapping and genomics approach accompanied by the phenomic approach will be a major contributor to improve the NUE of global crops. Therefore, it is increasingly important to improve our understanding of factors affecting NUE and possible management measures for improving the NUE of crops.

13This review focuses on describing different forms of N loss in the environment, analyzing the factors influencing NUE, discussing the consequences of poor NUE, and suggesting possible management practices for enhancing the NUE in various crops. Overall, better agronomic management of crops, genetic resources, breeding programs, and biotechnological tools to improve NUE are presented as potential solutions to low NUE of crops.

14The negative effect of N loss on water, the environment, and human and animal health has been well reported. Soil N is transient and moves rapidly away from the point of application through various mechanisms. The processes responsible for N loss include volatilization, nitrification, denitrification, leaching, surface runoff, ammonium fixation, and immobilization. Overall, the amount of mineral N in the soil at any given time can be described by the following N balance equation (Eq. 19).

15where Np is the precipitation and dry deposition, Nb is the biological fixation (Eq. 10, Table 1), Nf is the fertilizer, Nu is the urine and dung return to the soil, Nm is the mineralization, Npl is the plant uptake (Eqs. 11 and 12, Table 1), Ng is the gaseous losses, Ni is the immobilization, Nl is the leaching loss, and Ne is the erosion and surface runoff.

16The 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. Agriculture activities account for 50% of the total annual global NH3 loss (32 Tg year−1) to the atmosphere through volatilization. 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. 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 pKa for equilibrium in Eq. 16 is 9.3. Therefore, alkaline conditions favor greater proportions of NH3. 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. 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. 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.

17Urea 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. In most soils, the enzyme is more than sufficiently present and free in solution to rapidly hydrolyze urea to NH3. 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.

18Conventional 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. Urease inhibition with NBPT and cyclohexylphopshoric triamide (CHPT) may also be effective in preventing N losses from manure sources. 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. 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.

19Higher 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. 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. 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. 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.

20Nitrification is a microbial process (Eq. 14, Table 1), in which the ammonium is converted into nitrate by the oxidation process. It is a two-stage process (Eqs. 8 and 9) and is mediated by autotrophic bacteria.

21The 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. 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. Nitrification takes place in an aerobic soil environment with optimal soil moisture (60% water-filled pore space). However, it is a very slow process in anaerobic soil environments (rice ecosystem). The process is also regulated by soil temperature, pH, NH4+/NH3 concentration, and microbial population. Nitrate produced by this process can be leached, absorbed by plants, and immobilized by soil microorganisms.

22Denitrification 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. The process is carried out by a group of facultative anaerobic bacteria and catalyzed by nitrate reductase and nitrite reductase enzymes. 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. Chemo-denitrification is another process responsible for nitrous oxide emission, but the quantity is smaller than biological production. Likewise, the nitrification process also releases N2O through the spontaneous oxidation of hydroxylamine, which is an intermediate in the nitrification process.

23Conventional 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). 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.

24Research into biological nitrification inhibition (BNI) is advancing rapidly. 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. 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.

25Slope, rainfall intensity, soil type, and vegetation are key determinants of soil and nutrient loss and transport. 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.

26Management 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. While no-till and reduced till systems tend to protect or increase soil organic matter, which includes organic nitrogen, 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.

27When 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. 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. 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.

28Ammonium 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. The NH4+ ion is comparable to that of K+ with respect to ionic radii and low energy of hydration. 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. 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.

29Manure 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+. The C:N ratio of organic matter influences the N mineralization process because microbial biomass production requires both N and C. 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). 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. Immobilization is considered negligible when the C:N ratio is <20:1.

30In 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. Remineralization is a natural process by which the microbes requiring N can meet by mineralization of dead microorganisms using the enzymolysis process. 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.

31The agronomic N use efficiency of crops is greatly influenced by crop characteristics, environmental variability, and management practices.

32Crops and crop varieties differ considerably in their ability to uptake N per unit of biomass production. The agronomic NUE of major crops is given in Table 2. Crops grown in well-irrigated conditions have a greater agronomic NUE than in unirrigated/rainfed conditions. A study conducted on various irrigation regimes on wheat in China concluded that the nitrogen partial factor productivity was higher for 40 mm per irrigation (41.57 to 43.69 kg grain per kg N applied) compared with 20 mm per irrigation (32.24 to 32.47 kg grain per kg N applied). High crop growth rate, yield, and N uptake in crops can be achieved by maintaining optimal soil moisture conditions. Annual crops have a higher agronomic NUE than perennial crops due to the higher N uptake efficiency and N concentration. However, yield-specific N efficiency was more for perennial crops than wheat. Compared with food crops, fodder crops have a higher agronomic NUE because of the higher biomass production per unit area and time.

33Important environmental factors that affect the agronomic NUE are photosynthetic active radiation (PAR), temperature, and rainfall. Environmental factors that affect the agronomic NUE of crops in decreasing order are temperature > rainfall > irradiance. The temperature requirement of crops may vary greatly (Table 3). For crops like rice and wheat, NUE increased significantly with increasing growing season temperature, but it decreased for corn, which may be due to the variation in plant N demand and uptake responses to temperature. reported that when the crop suffers because of lower than optimal temperature, an increase in seasonal air temperature suddenly increases crop growth and nitrogen demand, which could increase NUE. At low temperatures, the ability to absorb N by the roots is greatly reduced due to the high affinity of the temperature and nitrate influx systems in the roots. However, the increase in temperature may lead to a high loss of N, thus reducing the NUE. The N loss and crop N uptake are highly influenced by the intensity, duration, and frequency of rainfall in a crop season. The occurrence of rainfall within a day of N fertilizer application had a positive impact on the NUE. A strong correlation between the total rainfall and NUE was observed for the dryland summer sorghum in Australia. The highest NUE was reported for 125% simulated rainfall for wheat and corn in a silt loam soil of Kentucky, USA. Photosynthetic active radiation is a major driving force affecting crop growth and N uptake. However, it is only important for tropical and subtropical regions but not for temperate regions. Studies have observed that crop growth and nitrogen uptake vary significantly during the dry and wet seasons, mainly due to variations in PAR in the tropics.

34Globally, 50% of the nitrogen applied to crops is lost to the environment, resulting in resource wastage and increased GHG emissions. The 50-year data from 124 countries suggest that increased N fertilization involved low agronomical benefits and higher environmental risks. Different management practices have resulted in reduced NUE. Basically, the selection of crops or varieties with poor N uptake and assimilation followed by inefficient utilization through reduced N remobilization resulted in a lower N use efficiency. Furthermore, it is responsible for the loss of N from the soil and plant residue after harvesting the economic part. reported that extensive crop cultivation over grasslands exposes the protected and stored soil organic carbon pool. Thus, it increases nitrate leaching and NH3 or NO2 and N2O emission, leading to environmental pollution. In South America, Africa, and Asia, reduced NUE was reported in areas devoid of cropping systems with biological N fixation such as soybean, beans, and groundnut. Similarly, intensive cropping without integration of livestock systems also reduced the N use efficiency at the local and global levels. The promotion of synthetic N fertilizers rather than symbiotic N fixation resulted in poor N use efficiency. Likewise, uncontrolled flood irrigation resulted in NO3− leaching due to a negative charge and high solubility; furthermore, it creates anoxic conditions which lead to the development of denitrifying microorganisms.

35Environmental factors, mainly higher temperature and wind speed, increase the risk of NH3 volatilization. It was found that an increase in soil temperature due to climate change increases the nitrification rate resulting in N loss and poor NUE. Higher soil compactness and wet conditions promote the denitrification process, whereas no-till and coarse soils showed higher leaching or volatilization/loss of nitrogen. In coarse soils, NH4NO3 fertilizer is subject to severe leaching and denitrification losses.

36Globally, the majority of countries are facing a decreasing trend of NUE (from 68% to 47%) over a period of five to six decades (1960–1970). Greater NUE in the initial years was probably due to higher native soil fertility, less use of additional nutrients, and favorable soil conditions (physical, chemical, and biological) (Figure 3). During the last decade, intensive management practices, monoculture, and increased use of off-farm input resources have resulted in low NUE.

37Modern agriculture is entirely dependent on excessive N fertilizer application leading to ecosystem degradation and environmental pollution. According to estimates, 70% of applied nitrogen fertilizer is lost in the biosphere and affects the local and global atmospheric chemistry. Nitrate pollution of groundwater in particular has led to numerous socioeconomic and environmental issues. Nitrate contamination of drinking water is a major concern, particularly for children. Continued consumption of NO3-contaminated drinking water (recommended limit of 10 mg NO3-N L−1) results in methemoglobinemia in children and gastric cancer among adults. Moreover, NO3 or NH4+ contamination of water bodies promotes the growth of algae and other aquatic plants, which lowers the water’s oxygen level.

38The oxide forms of N are highly reactive and harmful to the environment in many ways. Excessive emissions of nitrous oxide and nitric oxide contribute to the formation of nitric acid, which is the key component of acid rain. It significantly affects soil microbial communities and damages infrastructure. Moreover, the atmospheric pollutant ozone is created when nitrous oxide combines with volatile organic pollutants. In this way, the loss of N leads to serious health and environmental problems. To avoid these consequences, the NUE of crops needs to be improved on a global basis.

39The level of soil disturbance induced by different tillage practices affects soil N dynamics and plant N availability. For example, 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. 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.

40The relationship between the conservation tillage system and NUE varies between studies, but overall NUE is often improved by the conservation tillage system. 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, thereby improving the nutrient-supplying capacity of the soil. 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.

41A 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. 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. For instance, in a study conducted in Kentucky, 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. 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.

42It 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.

43The chemical composition of N fertilizers influences the NUE of crops. Urea-based N sources can be lost through volatilization when hydrolyzed to ammonia (Eq. 16) and the effect is intensified when urea is surface applied. Slow-release N fertilizers have the potential to minimize N leaching and denitrification losses and to improve the synchronization of N release and uptake in accordance with crop demand. Similarly, coated N sources such as neem-coated urea, sulfur-coated urea, and slow-release synthetic urea-based fertilizers such as isobutylidene diurea (IBDU) and crotobylidene diurea (CDU) have also improved the NUE. Polymer-coated urea was also found to reduce N volatilization loss (23%–62%) and ammonia emissions (51.3%–91.3%) and improve NUE (3%–34%). The combined application of 150 kg N through urea + 2,000 kg manure and 90 kg N + 2,000 kg manure under normal and dry years, respectively, has recorded maximum grain yield and NUE by improving the nitrogen nutritive index and nitrogen productivity of wheat in dry land area. Furthermore, the combined application of N fertilizer (276 kg ha−1) with biochar (15 t ha−1) produced the maximum yield of maize and the NUE (46.3%). demonstrated that integrated application of 180 kg N ha−1 + 900 g Se ha−1 utilized the maximum resources and recorded maximum apparent recovery efficiency of N, agronomic N use efficiency, partial factor productivity, NUE, and grain yield of wheat.

44Before determining the amount of fertilizer to apply, consider the soil’s nutrient-supplying capacity through a regionally appropriate soil testing program. Excessive fertilizer application leads to losses from the system, environmental problems, and economic losses to farmers. On the other hand, an insufficient nutrient application can exhaust soil fertility and lead to nutrient mining (degradation) and poor long-term soil productivity. Optimizing the nutrients’ rate based on soil status and crop requirement is the right way to improve NUE.

45Typically, N fertilizers are applied either in single or two split applications. Split application of N at various crop stages is effective at increasing NUE. The application of 120 kg N ha−1 proved optimal to produce a higher grain yield and NUE in direct-seeded rice than 60 and 180 kg N ha−1. A higher fodder maize seed yield (3.80 t ha−1) and N utilization were recorded for 120 kg N ha−1 in a semiarid region. Application of 180 kg ha−1 has been recommended to achieve higher grain yield, NUE, and protein yield of buckwheat. Hu et al. (2023) reported that among the rates of N studied (0, 150, 200, 250, and 300 kg ha−1), fertilization at the rate of 250 kg N ha−1 recorded a maximum grain yield, maximum grain N accumulation, improved aboveground dry biomass and N metabolism enzymes, and increased NUE in corn.

46Site-specific N scheduling could be an alternative option to the blanket application of N. Results from a study conducted in 107 farmers’ fields indicated that the leaf color chart (LCC)-guided N management in hybrid rice had decreased N requirement by 25% without compromising the crop yield. Therefore, LCC can be further explored as a diagnostic tool to help farmers make appropriate decisions about N fertilizer applications throughout the crop cycle. However, the use of sensor-based N application techniques is still at a nascent stage in many parts of the world.

47Fertilizer placement nearer to the root zone of crop plants, as opposed to even distribution in the field, has the potential to minimize N losses. The incorporation of fertilizers in the soil (via tillage or injection) is recommended over broadcasting. The placement of N fertilizer under the seeds at the time of planting, band application, and fertilizer injection increased the NUE and reduced the NH3 volatilization compared with broadcasting in winter wheat.

48Deep placement of the USG fertilizer resulted in better N recovery efficiency (49%) compared with the broadcasting method (37%) in Australia. Granular ammonium nitrate fertilization at the depth of 20 cm below the soil surface recorded the highest NUE (134%), N recovery efficiency (18.1%), and grain yield (11%) in spring wheat and barley. noted that placement of controlled release urea at the depth of 16 cm achieved maximum grain yield, water productivity, partial factor productivity, and NUE in rainfed spring maize in Northern China.

49Fertigation, or co-application of N with irrigation, is a viable option for the improvement of NUE. N fertilization at 15 cm depth increased grain yield (13.9%–98.9%), NUE (7.1%–44.3%), and N absorption (6.5%–38.0%) in summer maize. This approach gives the farmer with the proper equipment the flexibility to engage in multiple applications of low rates to minimize exposure to losses and optimize the opportunity of the crop to take up the right amount at the right time.

50The timing of fertilizer application should coincide as close as possible with crop nutrient demand to avoid nutrient loss. For instance, in single applications, part of the applied nutrient is absorbed by plants, while a substantial portion is vulnerable to loss. Improved N partial factor productivity, agronomic N efficiency, N recovery efficiency, physiological efficiency, grain yield, and N uptake may be optimized when N is applied in four splits at the sowing, 6th leaf stage, 12th leaf stage, and silking stage in maize. However, commercial-scale agriculture will likely avoid multiple trips across the field and traffic when the crop canopy has closed to reduce fuel, compaction, and crop damage. Likewise, the application of N fertilizer in three splits has increased the wheat grain yield and N recovery use efficiency.

51indicated that more than 6 t ha−1 grain yields can be achieved in dry direct-seeded rice production systems when urea application is delayed by 10 days after sowing or split application compared with the blanket application. Although optimized in this way, the commercial-scale application on flooded rice will be impossible without aerial application. Split application of N at the time of sowing and later stages (V12, R1, and R2) increased the plant uptake, photosynthetic efficiency, and grain yield and improved NUE in summer maize. Late and split application of N during jointing, booting, anthesis, and grain filling stages through microsprinkler irrigation increased grain yield, protein concentration, and NUE of wheat by 5.8%, 8.6%, and 15.8%, respectively, as compared with the conventional method of fertilization and irrigation. N application with basal to top dressing ratio of 2:8 between the sowing and jointing stages recorded maximum dry matter yield, crude protein, N recovery, water, and N use efficiency of forage maize in a semiarid region of China. Hence, the split application of N would be superior to the blanket application, though the number and timing of these applications will be limited due to the practical considerations mentioned above.

52Nitrogen use efficiency is also dependent on the ability of the cropping system. 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. Cereal- and legume-based cropping is the best system for leaving more residual N accumulation. 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. Similarly, in a 20-year study on clay loam soil in Ontario, Canada, 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, 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 and enhance resource use efficiency. 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. In Denmark, 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.

53Nitrogen 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. 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.

54Cover 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. 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. 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%. The CERES-N model modified by 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.

55Forage 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. 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.

56During the green revolution and post-green revolution, high fertilizer-responsive cultivars have been favored owing to low N-fertilizer costs. Though there are contradictory reports that under low N, more N-responsive modern varieties still perform better than historical varieties, breeding efforts to develop high fertilizer-responsive cultivars under high fertilizer conditions have resulted in high-yielding cultivars with poor NUE. As a consequence, yielding increases are fast approaching a theoretical limit with given physiological and genetic potential of crop cultivars under high N availability. To narrow down the demand–supply gap of food amid decreasing farmland and depleting soils around the globe without further magnifying environmental impacts, breeding strategies to improve the NUE of crop cultivars are becoming the prime focus of agricultural researchers. Breeding for high input-responsive cultivars, occurring during the last five to six decades, is different from breeding for NUE. For NUE, the inherent capacity of the plant has to be improved and selected to facilitate efficient uptake and to use N and produce higher yield under moderate or marginal N availability. Therefore, breeding for high NUE is mainly aimed at realizing maximum benefit by reducing the N application rate while maintaining the high yield level.

57Although 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.

58NUE 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. Breeding targets for genetic improvement of the plant may be grouped into two major categories: first, improving N uptake efficiency by increasing uptake capacity and breeding for ideal root morphology and, second, improving N utilization efficiency by modifying the leaf area index, specific leaf N, and biomass yield and by delaying the senescence.

59Before 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, and exploiting this property requires researchers to understand the underlying mechanism of higher root uptake.

60Root morphology plays a critical role in modulating N uptake by plants. Plants with rapid root growth can minimize N losses that occur through various field processes. 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 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.

61The 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. The uptake and utilization of N for the entire plant growth period can be separated into two phases: pre-anthesis and post-anthesis. 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. 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 and earlier canopy closure. 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. 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. 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.

62The integration of molecular tools, such as genomics and marker-assisted breeding, into traditional breeding programs has revolutionized genetic enhancements for various intricate traits in crops. The incorporation of these tools has significantly increased the efficiency of the selection process, resulting in a reduction in the time and resources required to develop improved varieties or hybrids. Recent advances in genomics have further accelerated the generation of genomic resources for many crops, providing breeders with more data and insights into the genetic makeup of crops, ultimately leading to more effective breeding strategies. Ultimately, these resources can be exploited for identifying, characterizing, and developing molecular markers linked to N-responsive genes in crop plants. Two molecular approaches can be explored for improving NUE in crops; one is through a traditional breeding strategy combined with genomic selection, and the other is a transgenic approach, which would target specific NUE-associated genes for the genetic engineering of the plant.

63It 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. 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. In another study carried out with rice, four QTL clusters harboring QTLs for both NDT and NUE traits were identified. In European winter wheat, a genome-wide association study using 214 varieties identified 333 genomic regions associated with 28 traits related to NUE. 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. 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. Three QTLs, NUE1a, NUE1b, and NUE2, were identified in maize for NUE. Under N starvation, the expression of TaNLP7 displayed enhanced expression in root and shoot tissues of the high NUE genotype. Forty-seven genes are known to involve N uptake, metabolism, and distribution in maize. In barley, 10 independent mapping studies were screened and a number of NUE-associated genes that control complex physiological traits were mapped. 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. 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. The details of the QTLs identified in the crop plants are given in Table 4.

64The role of small RNAs in regulating the nutrition assimilation/starvation response is well documented in many crops. 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. Forty-four miRNAs are differentially regulated under high and low N conditions. 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. miR167 regulates the lateral root growth response to N starvation in Arabidopsis. 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. 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. 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.

65Plants have evolved mechanisms to alter the molecular machinery in response to N availability. 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. In maize, ZmGLK5, bZIP108, CLC-a, and miRNA399b genes play a significant role in regulating genes in response to N. The NIGT1/HRS1s transcriptional repressors are essential in regulating N starvation response during high N availability. The CLE peptides and the CBL7 and TAR2 proteins regulate root architecture in response to N starvation; the DUR3 and AMT family proteins play an important role in the uptake of urea and ammonium, respectively, during N starvation. The nitrate transporters, NRT1/NPF and NRT2, regulate nitrate uptake.

66The availability of high-throughput genomics tools and efficient transformation systems in model crops further eases the functional validation of NUE. 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. Transgenic overexpression of OsDof25 modulates C and N metabolism in transgenic Arabidopsis lines during an increased supply of N. 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. Engineering the genes involved in the C4 photosynthetic pathway in C3 plants remains an essential strategy for enhancing the NUE in C3 crops. Moreover, the availability of N regulates the ethylene and jasmonic acid hormone signaling, thereby regulating the plant response to pathogen infection. miRNAs are known to play an important role in regulating the function of N-responsive genes during N-limiting conditions. 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. The details of the QTLs identified in the crop plants are given in Table 5.

67In global agriculture, the low-efficiency uptake by crops of applied N fertilizer is a major concern because of its negative impact on production costs and the environment. To improve NUE in crops, modern agronomic, breeding, and biotechnological strategies should be incorporated to supplement fundamental nutrient management. Agronomic practices such as precise timing and placement of N fertilizer, site-specific nutrient management, conservation tillage, crop residue retention, and cultivation of high biomass crops can enhance NUE under various soil and climatic conditions. NUE is a multifaceted trait that involves physiological, biochemical, and molecular regulations. Therefore, the engineering of N-responsive genes through genome editing has great potential for improving NUE in crops. To breed superior genotypes with high NUE, the use of genomic selection combined with speed breeding techniques in breeding programs is expected to be a valuable approach in the future.

68PG, SM, MB, RV, PJ, AM, HH, RR, SB, VP, GT and ML: manuscript writing and editing. All authors contributed to the article and approved the submitted version.