Integrating Agronomic and Molecular Advancements to Enhance Nitrogen Use Efficiency (NUE) and Promote Sustainable Rice Production

Abstract

Rice is a major crop for half of the world’s population, and nitrogen (N) fertilizers play a crucial role in its production. However, imbalanced N fertilizer uses and traditional fertilization practices have led to low nitrogen use efficiency (NUE), increased N footprints, and reduced rice yields and farmers’ income. There are limited studies where the integration of both agronomic and molecular advancements to enhance NUE is discussed, particularly in developing countries. This review highlights novel agronomic and molecular strategies to enhance NUE, rice yields, and profitability, while minimizing environmental impact. The agronomic strategies include the 4R Nutrient Stewardship framework, enhanced efficiency nitrogen fertilizers (EENFs), nano-fertilizers, biochar-based fertilizers, biological N fixation, and sensor-based fertilizer management in major rice-growing countries. The molecular mechanisms focus on N uptake, assimilation, and utilization, highlighting the role of hormones, key genes, transcription factors (TFs), and regulatory pathways. Moreover, we examine promising rice genotypes and cultivars with improved NUE and grain yield. Additionally, this paper offers deep insights into recent advancements in molecular genetics, such as multi-omics approaches (transcriptomics, metabolomics, and metagenomics), the Genome-Wide Association Study (GWAS), Quantitative Traits Loci mapping (QTLs), Single Nucleotide Polymorphisms (SNPs) analysis, and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas9)-mediated genome editing, which serve as valuable tools for developing rice cultivars with enhanced NUE and grain yield.

1. Introduction

Rice serves as a staple food for half of the world’s population, with an annual production of milled rice of around 480 million metric tons. About 90% of global rice is produced and consumed in Asia, making its production in this region crucial for global food security. Notably, China and India together account for approximately 50% of rice production and consumption. Together with China and India, other Asian countries such as Bangladesh, Vietnam, Myanmar, Thailand, the Philippines, Japan, Pakistan, Cambodia, the Republic of Korea, Nepal, and Sri Lanka account for 90% of global rice production. Moreover, rice is a significantly source of energy for 50% of people in Asia living in poverty [1,2,3,4]. Its significance extends beyond mere nourishment, intertwining with human civilizations, cultures, and traditions. Nitrogen (N) is crucial for synthesizing nucleotides, amino acids, and chlorophyll [5], making it an essential fertilizer for enhancing crop production. Nitrogen (N) fertilizer is intensively used in modern agriculture to increase crop production and meet growing food demands driven by rapid population growth, often without considering NUE and the associated environmental costs. Prolonged application of fertilizers and organic manures differentially influence mineral N and organic N fractions and total N in soils reduces NUE, leading to significant losses through volatilization, leaching, and runoff, emitting greenhouse gases like N₂O, which contribute to global warming and threaten food security [6,7,8].

Global nitrogen fertilizer consumption exceeds 110 million tons annually, with China and India accounting for nearly half [9]. Rice alone consumes “30 to 50%” of total agricultural nitrogen [10]. With the cost of chemical fertilizers rising and likely to increase further in the future, it is essential to prioritize improving nutrient usage efficiency through the adoption of best management practices (BMPs), resulting in reducing fertilizer costs and contributing to significant yield gaps [11]. Additionally, climate change increases crop N concentrations while reducing internal nitrogen use efficiency (iNUE). A meta-analysis of single-season irrigated rice across different zone and climatic conditions in China (northern middle and lower river basin, southwestern) found N concentrations rose by 5–9.6%, while iNUE dropped by 9–14%. To meet food demands, and as the population is predicted to be 9.7 billion [12,13] by 2050, iNUE must increase by 7.8–13.6% [14]. Another meta-analysis (1961 to 2013) in China and India showed N use in cereal production has increased significantly, ranging from 10 to 84%, but NUE declined to 20–24% by 2013. NUE in major rice-growing countries remains below 40%, lagging 15–20% behind the global average [15,16]. Despite decades of research on improving NUE, progress remains limited, primarily due to blanket fertilization recommendations that overlook site-specific needs, limited soil testing, imbalanced fertilization practices, fertilizer subsidies, and low adoption of BMPs across farming communities. Additionally, the lack of widespread adoption of proven research technologies has led to significant spatial-temporal variations in NUE in many developing countries. Despite a slight global NUE increase from 33% in 1999 to 35% in 2015, over 60% of applied N is still lost through leaching and emissions via volatilization and denitrification, which are primarily attributed to excessive application of N fertilizer and increased mechanization [9]. To meet global nitrogen sustainability goals, NUE must rise to 70–80% through precision nutrient management (PNM) and BMPs, reducing N losses, cutting costs, and improving yields [17].

Some of the promising and scalable fertilizer management practices to improve NUE include 4R Nutrient Stewardship (right time, right place, right method, and right proportion), enhanced efficiency fertilizers (EENFs), nano-fertilizers, biochar-enriched N fertilizers, and sensor-based precision nutrient management. Moreover, conservation agriculture (CA) practices, such as intercropping, green manuring, cover crops, and crop rotation, enhance biological nitrogen fixation (BNF) and reduce N input, ensuring climate-resilient production systems. Additionally, rice root architecture and genetic variability in NUE, particularly between indica and japonica varieties, offer opportunities for crop breeding and genetic improvement [18,19]. A combined agronomic and genetic approach is essential for sustainable NUE enhancement. Similarly, multi-disciplinary collaboration among researchers, farmers, and stakeholders is key to advancing smart fertilizer management technologies. Integrating genomic tools like GWAS, SNPs, genome editing, and multi-omics approaches can enhance NUE by identifying key traits for resilient rice cultivars, contributing to SDGs 2 (zero hunger) and 13 (climate action) [20].

In Asian countries, over 90% of rice is produced by smallholder farmers [21]. Fertilizer use among these farmers is heavily skewed toward excessive N application, while the use of phosphorous (P), potassium, K, and other secondary nutrients and micronutrients remain insufficient. This imbalance results in low NUE and is largely driven by high subsidies for urea and limited adoption of BMPs. Inefficient N fertilization is responsible for nearly 50–66% of the gap between actual and potential rice yields [22]. Additionally, the intensive use of inputs such as fertilizer and irrigation during the green revolution era has degraded soil health and reduced fertilizing efficiency. To narrow this yield gap and meet the growing food demands of a rising population, there is an urgent need to promote and scale efficient fertilizer management strategies that improve NUE and are widely adopted by farmers. Integrated studies on agronomic and molecular approaches are essential for enhancing NUE and yet such studies remain limited in many developing countries. Through this review, we aim to provide a comprehensive understanding of NUE in rice by synthesizing advancements from both agronomic and molecular perspectives. By bridging the knowledge gap between these two domains, this paper offers valuable insights into emerging innovations and outlines future research directions to support sustainable rice production. The structure of this review includes (i) an overview of nitrogen use, its efficiency, and yield gaps in rice production, (ii) innovative and scalable agronomic strategies to enhance NUE, (iii) molecular and genetic mechanisms of N uptake, assimilation and utilization, and (iv) molecular advancement and genomic tools that contribute to the development of superior rice cultivars aimed at enhancing NUE, yield, and sustainability. This review will serve as a valuable resource for researchers, academics, and stakeholders in the public–private sectors by presenting practical approaches and research-based strategies to enhance NUE in rice production systems.

2. Concepts of NUE

NUE, the ratio of grain yield to nitrogen applied, is determined by N uptake efficiency (NUpE) and N utilization efficiency (NUtE), where NUpE is the total amount of N taken up by the plant relative to the N supplied, and NUtE is the ratio of total grain yield to the N in the plant. NUE is influenced by various factors, and different forms of NUE can be calculated using the following equation [23].

• Recovery efficiency of N (REN) = (UNN − UN0)/(FN − FN0), kg N uptake per kg N applied;
• Agronomic efficiency of N (AEN) = (GYN − GY0)/(FN − FN0), kg grain yield increase per kg N applied;
• Partial factor productivity of N (PFPN) = GYN/FN, kg grain yield per kg N applied;
• Physiological efficiency of applied N (PEN) = (GYN − GY0)/(UNN − UN0), kg grain yield increase per kg N uptake;
• Internal efficiency of N (IEN) = GYN/UNN, kg grain per kg N uptake;
• Utilization efficiency (UEN) = Physiological efficiency × Apparent N recovery, kg kg⁻¹, where GYN is the total grain yield when N is applied, FN is total N fertilizer applied, GY0 is total grain yield without N application, UNN is total N uptake with N treatment, and UN0 is total N uptake without nitrogen.

Generally, PEN ranges from 30 to 60 kg/kg, PFPN from 40 to 70 kg/kg, AEN from 10 to 30 kg/kg, and REN from 30 to 50% [24]. The recovery efficiency of applied nitrogen (REN) in rice varies from 35.4 to 55% (average 43.9%) in northern Thailand in acidic sandy soils under both irrigated and rainfed conditions [25] and 48% in Nepal in silty loam soils under rainfed conditions [23]. Moreover, meta-analysis 1333 field experiments conducted across varying soil types and biophysical factors in China revealed only 28.3% of REN in rice, which was primarily attributed to over-use of chemical fertilizer [26].

Blanket N applications, often surface-broadcasted, lead to low AEN and REN due to volatilization [27,28], especially when applied early during underdeveloped root stages [21]. NUE is influenced by various factors such as fertilizer type, rate, timing, rice variety, soil properties, crop management, and interaction with environmental factors.

The IRRI has developed strategies to improve NUE, including variety selection, biological N fixation (BNF), deep placement techniques, and chlorophyll meters for optimal N application [29]. Optical sensors, such as a chlorophyll meter like the Soil–Plant Analysis Development (SPAD) and crop reflectance sensors, enable optimized N application by allowing precise timing and dosages. For instance, the use of the SPAD meter in irrigated rice cultivation in India exhibited threshold values of 37 in south India and 37.5 in northwest India, indicating the plant nitrogen status and supporting precision nitrogen management strategies [30,31]. Moreover, in the context of BNF, free-living blue-green algae (BGA) and azolla are key sources for fixing atmospheric N and reducing the need for external chemical N fertilizer in rice fields, maintaining similar or even higher yields. Additionally, site-specific nutrient management (SSNM) remains crucial for increasing NUE, reducing N losses and enhancing productivity [32,33,34].

3. Efficient Fertilizer Management in Farmlands

In this section, some of the innovative, promising, and scalable agronomic N management strategies are highlighted (Figure 1) and discussed, along with their agronomic (NUE and yields) and economic benefits in Table 1.

3.1. The 4Rs Nutrient Stewardship Framework

Optimizing NUE and yields can be achieved by adopting the 4Rs technology: applying fertilizer at the right rate, source, place, and time, often termed as “fertilizer BMPs” [35,36,37]. This approach, as shown in Figure 1, integrates both organic and inorganic fertilizers, ensuring balanced fertilization based on soil, crops, and environmental factors.

Urea (46% N) is widely used in rice due to its high N content and affordability compared to ammonium sulfate (21% N). Multilocation and multiyear fertilizer trials with varying N rates, considering agro-ecological zones, soil types, indigenous nutrient supply, crop varieties, climate, and crop management, are crucial for identifying optimal N rates to achieve attainable yields and NUE [33,38]. Timely application also plays a key roles: Splitting a 100 kg N ha⁻¹ application into three doses increased total N uptake by 42%, AEN by 146%, and rice yield (variety: Chenab basmati-2016) by 44% compared to a single basal application over two years in medium alkaline sandy clay loam soil under tropical conditions at the University of Agriculture, Faisalabad, Pakistan (Table 1) [39]. Application methods impact efficiency, with deep placement reducing N losses by 50% compared to surface broadcasting [40]. Field trials and demonstrations conducted across nine districts in Nepal ranging from tropical to sub-tropical regions have shown higher NUE, crop yields, income, and the growing interest of farmers in the adoption of 4Rs [35,41,42]. However, despite farmers’ understanding from farmer schools and training that the 4Rs can offset production cost through increased yields, widespread adoption is constrained by recurrent fertilizer shortages, limited farmers’ knowledge, and inadequate access to fertilizer-distributing cooperatives [42].

Although the 4Rs have been practiced for 30 years, the emerging 4R Nutrient Stewardship (4RNS) framework integrates cropping systems, landscapes, and sustainability metrics. The 4RNS framework is the adaptive management of plant mineral nutrients through the refinement of interconnected strategies based on location, soil, climate, and crop species. As 4RNS is still in the developmental stage, further research is needed to refine best practices for long-term soil health, water quality, and economic viability, emphasizing collaborative and multidisciplinary efforts [36,43].

3.2. Enhanced Efficiency Nitrogen Fertilizers (EENFs)

Enhanced efficiency nitrogen fertilizers (EENFs) are a key component of 4R Nutrient Stewardship, helping reduce N losses and improve NUE [44,45]. These fertilizers include slow and controlled-release coated fertilizers, nitrification, urease inhibitors, and urea briquettes (UB) with deep placement (UDP), etc. Coated fertilizers like polymer-coated urea (PCU) and sulfur-coated urea (SCU) enable slow-release mechanisms, providing nutrients to plants throughout the crop cycle and minimizing losses from volatilization, denitrification, and leaching [46,47,48,49,50,51]. In addition, EENFs are economically viable due to the single-time application, which saves time and labor cost compared to multiple splits of conventional urea [51]. One-time applications of PCU reduced labor requirements by six workers per hectare compared to two splits of urea application in rice fields in Nepal [49].

PCU has been shown to increase rice yield by 1.0–1.3 t ha⁻¹ and agronomic NUE by 5.2–5.9 kg/kg compared to conventional urea. Additionally, it enhances root morphological traits [52]. Controlled-release urea led to enhanced NUE and grain yield in rice by increasing the activity of nitrogen metabolism enzymes such as Nitrate Reductase (NR), Glutamine Synthetase (GS), and Glutamate Synthase (GOGAT) by 50–200%, and upregulating key genes (OsNIA, OsGS1, OsGS2, OsGLN1.1, and OsGLN1.2) [53].

Urea briquette (UB) deep placement involves compressing prilled or granular urea into large briquettes (1–3 g) and placing them 7–10 cm below the soil surface [49]. This technique improves nitrogen retention, making it available during critical crop growth stages [23,54,55]. Studies show that UB can reduce N input by 20–35% while increasing crop yield by 25% and improving PFPN and AEN by 44% and 34%, respectively [23,56]. Research from Fujian Province, China, found that deep placement of slow-release fertilizers increased rice yield by 8.8–23.5% while cutting CH₄ emissions by 25.34% and N₂O emissions by 48.97%, as shown in Table 1 [57].

Controlled-release nitrogen fertilizers (CRNFs) offer both economic and environmental benefits. In southwest China, CRNFs improved environmental sustainability by 2.82–4.61%, NUE by up to 43.96%, and profitability by 5.21–11.44%. [58]. Similarly, in southeast China, slow-release fertilizer with deep placement increased NUE by 41.47%, economic benefits by 76.43%, net energy, and ecological benefits by 43.83%, while reducing cumulative CH₄ accumulation by 25.34% [59].

3.3. Nanotechnology

Nanotechnology is an innovative and emerging technology to achieve SDGs by enhancing NUE and promoting sustainable agricultural practices under the circumstances of climate change and resource depletion. Nano-fertilizers are the nutrient carriers produced by encapsulating plant nutrients within nano-materials that are less than 100 nm in size [60,61]. Nano-fertilizers are considered more eco-friendly than conventional fertilizers as they are foliar spayed, which can minimize N losses through leaching and emissions, thereby reducing environmental pollution and ensuring sustainable production systems [60,62]. Nano-fertilizers improve nutrient absorption through the plant cuticle and are characterized by their excellent solubility and reactivity, enabling precise nutrient delivery to plant cells and efficient nitrogen utilization [63]. Nano-fertilizers have higher surface area to volume ratio and surface functionalization, which can reduce nutrient losses and release N for prolonged periods compared to conventional urea [64,65]. While the use of nano-fertilizers has shown promising results, their impact on specific crops and diverse agroecological zones remains unexplored [66]. Therefore, future studies should focus on understanding the mechanistic effects of nano-urea on various crops, including rice, across diverse biophysical conditions such as soil types, temperature, rainfall, and topography.

One of the most significant developments is the nano-urea produced by the Indian Farmers Fertilizer Cooperative (IFFCO). Given its higher NUE (80–90%), IFFCO claimed a 500 mL nano-urea bottle equivalent to a 45 kg sack of conventional urea. As nano-urea is applied in smaller quantities, it reduces the need for large quantities of urea, minimizing nitrogen losses, and improving crop productivity [67]. Results suggest that nano-fertilizers enhanced tomato yield by 30% and lowered nitrogen use by 50% compared to conventional urea, demonstrating tangible benefits for crop production [68]. Applying 75% of conventional nitrogen along with nano-nitrogen in wheat enhances NUE and crop yield, achieving a benefit–cost ratio of 5.51 [69]. Similarly, co-application of urea (50% as basal) and nano-urea two sprays at tillering and panicle initiation stages in rice significantly increased leaf area index, panicle weight, grain yield and NUE compared to other fertilizer treatments [70]. Similarly, in another study, urea 75% N and two sprays of nano-urea N showed significantly higher crop yields, NUE and reduced GHGs over 100 N [65].

The underlying mechanism is that the nano-urea particles are absorbed through stomatal uptake or surface absorption and the nutrients are slowly released and metabolized within the plant cells to support growth and development. These results demonstrate that nano-urea can reduce nitrogen input by 25 to 50%, fostering more sustainable agricultural practices. In addition to enhancing NUE, nano-urea is an eco-friendly product that supports improved soil health, reduces N leaching and emissions, and contributes to better air and water quality, thereby aiding in the mitigation of global warming compared to conventional urea. However, small amounts of nano-particles may leach from plant surfaces into the soil, although this is significantly lower than the N inputs from direct soil application of conventional urea in soil. This reduced N input may influence soil biogeochemical processes, altering nutrient dynamics and microbial activity. It is also more affordable to farmers compared to conventional urea, requiring less time and labor for application, which can reduce overall production cost and increase farmers’ income [71,72].

Although nanotechnology offers some promising results, it lacks comprehensive documentation on its utilization, potential, and application in agriculture. Additionally, it requires more careful consideration for its safe handling to minimize potential human health and environmental risks [61].

3.4. Digital and Sensor-Based Technologies for Enhancing Fertilizer Use Efficiency

The integration of digital and sensor-based technologies is transforming agriculture by enabling precision nutrient management and real-time fertilizer application, optimizing nitrogen (N) use, reducing nutrient losses, input costs, and enhancing crop yields. Technologies such as real-time sensors, drones, satellite remote sensing, GIS-GPS, and variable rate technology (VRT) provide precise fertilizer application during critical growth stages, boosting NUE and yields [73]. Proximal sensing and ICTs, like NDVI and Green Seeker, detect nitrogen stress and optimize N application rates [23,73,74]. Use of Green Seeker reduced N input by 54% without compromising rice yields and increased AEN, PFPN, REN, and UEN [23].

Digital tools such as digital soil maps (DSM) and apps like Crop Doctor offer precise fertilizer recommendations, improving NUE, farm profitability, and reducing environmental impact [75]. Real-time data obtained from various digital and sensor-based approaches can be fitted into the modeling applications to determine site-specific fertilizer management. For instance, soil fertility information in DSM can be utilized in the QUEFTS model (Quantitative Evaluation of Fertility of Tropical Soils) to determine site-specific fertilizer recommendations [76]. Variable-rate fertilizer application (VRFA) using a DSM in Kharagpur, India, was helpful in identifying optimum rates required for attainable yields while minimizing input cost and pollution [77]. However, sensor-based fertilizer management faces several challenges, including difficulties in sensor deployment, inconsistencies in data quality, and requirements for sustainable reliable energy sources, all of which underscore the complexity of integrating such technologies into precision farming systems [78]. Therefore, scaling up these digital and sensor-based methods at the farm level requires collaboration, hands-on training, and demonstration regarding its implementation among farmers and stakeholders to transition to precision nitrogen management and enhance NUE [73].

3.5. Conjoint Application of Biochar and Nitrogenous Fertilizers

Biochar, a carbon-rich material produced through pyrolysis, is globally known for enhancing soil fertility and mitigating climate change [79]. The combined application of biochar and N fertilizers has emerged as an effective strategy to enhance NUE, reduce N losses, and increase crop yields [80,81,82,83]. Studies have shown that biochar positively influences rice yield, harvest index, and straw production, with AEN increasing by 140% and grain nutrient recovery (REN) rising by 191% over two years [82].

Biochar’s high porosity, large surface area, negative surface charges, and high cation exchange capacity (CEC) contribute to enhanced N retention, improved NUE, and reduced N₂O emissions. These effects are primarily due to biochar’s ability to promote electrostatic adsorption and retention of NH₄⁺, while also minimizing NO₃^- leaching by increasing soil water-holding capacity and decreasing infiltration rates [84]. Moreover, biochar applications can mitigate N losses by reducing NH₃ volatilization and N₂O emissions by suppressing the enzymatic activities of urease and nitrate/nitrite reductase, inhibiting the activity of denitrifying microbes and enhancing N immobilization by soil microorganisms through the supply of labile carbon and the creation of a favorable microhabitat. Applying rice straw biochar (5 to 10 t ha⁻¹) significantly improves soil N availability and nutrient levels, resulting in higher NUE and rice productivity [85]. Enriching biochar with organic and inorganic fertilizers increases its effectiveness [86,87,88,89], as seen in Nepalese soils where biochar enriched with cattle urine boosted NPK uptake and crop NUE [86]. The enhanced agronomic performance of nutrient-enriched biochar can be attributed to the organic coatings formed on biochar, which restrict water movement, enhance nutrient retention, and enable a slow release mechanism [90]. Additionally, biochar is economically viable, with a benefit–cost ratio of 1.47 in southern China, and it mitigates CO₂ emissions by 6.22 tons per ton applied [91]. Unlike other organic and mineral fertilizers, biochar is typically applied only once, which helps reduce variable costs. Its recalcitrant nature allows it to persist in the soil for hundreds to thousands of years, offering long-term agronomic and environmental benefits.

3.6. Biological N Fixation

Biological nitrogen fixation (BNF) is the process by which specific microorganisms convert atmospheric nitrogen (N₂) into ammonia (NH₃) or similar compounds, making nitrogen available for plants. BNF contributes 17% of total N inputs in soil ecosystems globally [92], and can reduce reliance on chemical N fertilizers [93]. Microorganisms like diazotrophic iron-reducing bacteria play a key role in BNF, particularly in paddy fields. Cyanobacteria species such as Anabaena variabilis and Nostoc muscorum enhance soil N levels and NUE in rice by reducing ammonia volatilization [94]. Combined use of BNF and 50% chemical fertilizers can increase rice yields by 18.62% and NUE by 43.83% [94].

BNF practices like green manuring, cover cropping, and intercropping help reduce the need for chemical fertilizers, improve soil health, and increase crop productivity. Leguminous plants such as Sesbania rostrata and Crotalaria juncea are used as green manure, releasing nitrogen into the soil for subsequent crops. These leguminous green plants decompose and release nutrients, especially nitrogen for subsequent crops [95]. Approximately 40–60% of total N contained in legumes is available to succeeding crops, mostly in organic forms. The release of available N for successive crops largely depends on the presence of effective soil microorganisms, biological activity, C:N ratio, soil type, moisture, and temperature [96]. Most green manure N is mineralized within 4 weeks of incorporation [97].Green manuring can reduce nitrogen fertilizer use by 20–40%, minimizing nitrogen losses and fertilizer costs. For example, green manure with 20% less N (80 N) increased rice yield and nutrient recovery compared to full nitrogen application [98].

Another promising BNF practice is the adoption of intercropping, where cereals (main crops) are primarily combined with legumes (component crops) in definite cropping geometry to improve soil fertility and yields. In Asian region, farmers predominantly cultivate single crops, particularly cereals, throughout the year, leading to limited nutrient recycling, substantial soil nutrient depletion, and adverse effects on soil fertility and crop productivity. Due to rising population pressures and decreasing arable land, crop intensification is crucial for addressing food security issues and achieving SDGs. Intercropping offers benefits such as improved soil fertility, reduced fertilizer inputs, enhanced NUE, and increased yields [99,100]. For instance, intercropping rice with Neptunia oleracea reduced N inputs by 22% and improved yields compared to sole-cropping with conventional N [101]. Legumes fix N and contribute to nutrient recycling, thus supporting sustainable agriculture and improved food security [102]. While BNF is an innovative and profitable approach for crop production, its successful implementation depends on the availability of skilled labor, appropriate technology, and essential resources. However, labor shortages and limited access to key agri-inputs such as seeds, fertilizers, biofertilizers like azolla, and BGA poses significant barriers to the adoption of green manuring and intercropping in many developing countries.

Table 1. Comparative analysis highlighting enhancements in NUE, grain yield, economic benefits, and N losses with the utilization of different technologies and major cereal crops across different countries.

S.N.	Technologies	Country	Crop	NUE Increment	Grain Yield Increment	Economic Benefits	Reduction in Emissions	References
1.	4Rs Technology (right timing- three spilt doses)	Pakistan	Rice	42 (N uptake), 146 (AFN),	44%	Reduction in cost of production (31–43%)		[39]
2.	4Rs Technology (right timing)	Ethiopia	Rice	31.4 (PFPN)	5.6%	Reduction in N fertilizer use by 31.4%		[40]
3.	4Rs	Nepal	Maize		35%	40%		[35]
4.	Polymer-coated urea	China	Rice	5.2 to 5.9 kg/kg	1.0 to 1.3 t ha−1			[52]
5.	Urea briquette (UB)	Nepal	Rice		25%			[23]
6.	Deep placement of urea super granules (USGs)	Bangladesh	Rice	NUE −13%, agronomic NUE- 20%, recovery N use efficiency −19%	5.22%			[103]
7.	Slow-release fertilizer	China	Rice	N recovery efficiency by 62.50–91.57% and 24.38–64.24% (two rice cultivars Yongxian15 and Yongyou1540, respectively)	6.30–11.64% and 6.23–13.11% (two rice cultivars Yongxian15 and Yongyou1540, respectively)		CH4 emissions by 25.34% and N20 emissions by 48.97%	[57]
8.	Controlled release urea + conventional urea	China	Rice	24.4%	25.3%			[104]
9.	Polymer-coated urea	China	Wheat	3–34%			Reduce N volatilization loss (23–62%), ammonia emissions (51.3–91.3%)	[105]
10.	Controlled release urea	China	Rice	0.65–43.96%	5.21–11.44%			[58]
11.	Controlled release urea with deep placement	China	Rice	41.47%		76.43%	CH4 accumulation by 25.34%	[59]
12.	Joint use of PCU and urea briquette	Nepal	Maize and rice	46%		23%		[49]
13.	Nano-urea	India	Maize, wheat	80–90%				[67]
14.	Application of nano-urea in flooding-irrigated conditions	Bangladesh	Rice	95.38%	4.91%			[106]
15.	Application of N 150 kg/ha along with nano-urea (4%) in wheat	India	Wheat		7.8%	Gross return of $2542 and cost–benefit of 2.01		[107]
16.	Co-joint application of biochar (8 t/ha)	India	Peas rabi season and maize kharif season			Benefit–cost ratio of 1.47	One ton of biochar mitigates 6.22 tonnes of CO2	[91]
17.	NFC combined with 50% chemical fertilizers	China	Rice	18.62%		43.83%	NH3 volatilization losses by 41.63–45.34%	[94]

Table 1. Comparative analysis highlighting enhancements in NUE, grain yield, economic benefits, and N losses with the utilization of different technologies and major cereal crops across different countries.

S.N.	Technologies	Country	Crop	NUE Increment	Grain Yield Increment	Economic Benefits	Reduction in Emissions	References
1.	4Rs Technology (right timing- three spilt doses)	Pakistan	Rice	42 (N uptake), 146 (AFN),	44%	Reduction in cost of production (31–43%)		[39]
2.	4Rs Technology (right timing)	Ethiopia	Rice	31.4 (PFPN)	5.6%	Reduction in N fertilizer use by 31.4%		[40]
3.	4Rs	Nepal	Maize		35%	40%		[35]
4.	Polymer-coated urea	China	Rice	5.2 to 5.9 kg/kg	1.0 to 1.3 t ha−1			[52]
5.	Urea briquette (UB)	Nepal	Rice		25%			[23]
6.	Deep placement of urea super granules (USGs)	Bangladesh	Rice	NUE −13%, agronomic NUE- 20%, recovery N use efficiency −19%	5.22%			[103]
7.	Slow-release fertilizer	China	Rice	N recovery efficiency by 62.50–91.57% and 24.38–64.24% (two rice cultivars Yongxian15 and Yongyou1540, respectively)	6.30–11.64% and 6.23–13.11% (two rice cultivars Yongxian15 and Yongyou1540, respectively)		CH4 emissions by 25.34% and N20 emissions by 48.97%	[57]
8.	Controlled release urea + conventional urea	China	Rice	24.4%	25.3%			[104]
9.	Polymer-coated urea	China	Wheat	3–34%			Reduce N volatilization loss (23–62%), ammonia emissions (51.3–91.3%)	[105]
10.	Controlled release urea	China	Rice	0.65–43.96%	5.21–11.44%			[58]
11.	Controlled release urea with deep placement	China	Rice	41.47%		76.43%	CH4 accumulation by 25.34%	[59]
12.	Joint use of PCU and urea briquette	Nepal	Maize and rice	46%		23%		[49]
13.	Nano-urea	India	Maize, wheat	80–90%				[67]
14.	Application of nano-urea in flooding-irrigated conditions	Bangladesh	Rice	95.38%	4.91%			[106]
15.	Application of N 150 kg/ha along with nano-urea (4%) in wheat	India	Wheat		7.8%	Gross return of $2542 and cost–benefit of 2.01		[107]
16.	Co-joint application of biochar (8 t/ha)	India	Peas rabi season and maize kharif season			Benefit–cost ratio of 1.47	One ton of biochar mitigates 6.22 tonnes of CO2	[91]
17.	NFC combined with 50% chemical fertilizers	China	Rice	18.62%		43.83%	NH3 volatilization losses by 41.63–45.34%	[94]

4. Molecular Mechanisms of Nitrogen Uptake and Assimilation

4.1. Root System Architecture (RSA) and Genes Associated with Improving RSA

Root functions influence NUE in plants to absorb nitrogen from soil, and the distribution and availability of nitrogen play a crucial role in shaping root system architecture (RSA) [108,109]. Rice fibrous root systems consist of seminal and adventitious roots. Primary roots extend deeper into the soil to acquire nitrogen, while lateral roots spread out to cover a larger surface area, encompassing a greater soil volume [110]. Moreover, rice root morphology includes the number of roots, weight, total length, surface area, root branches, root diameter, and angle [29]. These root morphological traits, as extended root length, greater volume, high density, and increased surface area, significantly increase nitrogen absorption [111]. Nitrogen absorption from the soil is actively facilitated by specialized transporter proteins responsible for nitrogen uptake. The inorganic nitrogen form, NO₃⁻ (nitrate), is primarily located in the rhizosphere, where its concentration is significantly higher than that of NH₄⁺ (ammonium) [112]. Nitrogen absorption efficiency is directly proportional to the size and shape of roots. It has been observed that nitrates, a common form of nitrogen in soil, are highly prone to leaching. So, improving the root system’s architecture, such as its depth and density, enhances the nitrogen absorption rate [113]. NUE is significantly affected by plant root’s ability to uptake and utilize nitrogen from external sources.

The availability and distribution of these sources alter crop RSA to optimize nitrogen utilization. RSA and specific gene expression possess a significant role in nitrogen uptake and grain yield in low nitrogen conditions. For example, overexpression of the OsAMT1;1 and OsAMT1;2 genes increased root length and surface area and encoded ammonium transporters enhancing nitrogen accumulation capability, leading to higher grain productivity in rice variety YD6 [114]. Research suggests that RNR10, F-box protein, is a crucial gene that controls nitrogen-responsive RSA in rice, and provides new tools to improve NUE by manipulating the RNR10-DNR1-auxin module [109]. Quantitative trait locus (QTL) qRDWN6XB provides new insights for a potential genetic resource for improving rice RSA through marker-assisted selection (MAS) under low nitrogen conditions, thereby enhancing NUE and grain yield [115]. The OsMADS57 gene elongates roots and modulates nitrate translocation from rice roots to shoots under nitrogen-deprived conditions. Results suggest that the over-expression of this gene increases nitrate translocation twofold, thereby enhancing NUE [116]. Upregulation of the OsNPF2.4 gene enhances root surface area, length, and volume, and carbon and nitrogen translocation, synergistically improving NUE and grain yield in the dep1 rice line [117]. Application of biochar in the seedling stage of rice showed up-regulation of N-metabolism genes such as OsAMT1.1, OsGS1.1, and OsGS2, enhancing root system and nitrate uptake by 33.60% [118]. The use of slow-release nitrogen fertilizer (sulfur-coated urea) in rice exhibited significant improvement in root development, leaf structure, NUE, and grain yield [119].

4.2. Hormonal Regulation for Enhancing NUE

Comprehensive transcriptomic analysis of the OsAAP1 gene suggests that OsAAP1 may have a crucial role in nitrogen absorption and metabolism through signaling pathways of auxin, cytokinin, and strigolactone, which are essential for regulating tillering in rice [120]. An expression of DNR1 protein in indica rice varieties has antagonistic effects on auxin regulation, NUE, and crop yield. For instance, lower DNR1 abundance enhances auxin synthesis, activating nitrate transporter and nitrogen metabolism-related genes through the auxin response factor (ARF) mechanisms, enhancing both NUE and grain yield [121]. Similarly, results suggest that auxin plays a crucial role in root growth, N uptake, and assimilation in maize. The application of naphthalene acetic acid (NAA, 0.1 mmol/L) exhibited improvements in plant photosynthetic rate and biomass by 58.6% and 91.7%, respectively. Moreover, the activities of key N assimilation enzymes NR, GS, and GOGAT were enhanced by 32.3%, 22.9%, and 16.2% [122]. Enhanced lateral root elongation through auxin signaling under lower N conditions in wheat increases NUE by boosting crop nitrate acquisition capacity [123].

Gibberellins (GAs) influence the expression of genes involved in nitrogen assimilation, such as NR and GS, which play vital roles in nitrogen metabolism and enhance NUE [124]; in addition, GAs have recently been reported to be involved in saline–alkaline stress management in rice through increased ammonium uptake [125]. GAs regulates the expression of ZmNRT2.1 and ZmNRT2.2 in maize, thereby promoting N uptake [126]. The expression of primary NPF (nitrate transporter1/peptide transporter family) genes, specifically NPF6.3 and NRT2.1, regulated by auxin, ethylene, and cytokinin play a role in influencing nitrogen uptake [126,127]. Cytokinin modifies root behavior in rice in response to high ammonium levels [128] as the overexpression of the cytokinin activation enzyme-like gene OsLOGL5 has a vital role in primary root growth, tiller number, yield, and NUE under various conditions, including irrigated conditions, water stress, nitrogen sufficiency, nitrogen deficiency, etc. [129,130]. Brassinosteroids (BRs) play a key role in plant growth, development, and responses to environmental stresses and nutrient utilization in maize. Research suggests that BRs regulate the physiological response, and nitrogen uptake in maize enhances the growth of primary roots and plant biomass and up-regulates the expression of nitrogen transporter genes [131,132]. A nitrate, GA, and ABA transporter gene OsNPF3.1 are reported to regulate NUE [133].

4.3. Mechanisms of Nitrogen Uptake and Assimilation Through Transporters

Nitrate (NO₃^-) transporters are the most important components for facilitating the uptake, transport, assimilation, and re-mobilization of nitrogen, resulting in enhanced NUE. These transporters belong to four major gene families: NRT1/NPF (Nitrate transporter 1 or peptide transporter), NRT2/NNP (Nitrate transporter 2/Nitrate–Nitrite porter), CLC (Chloride channel), and SLAC1/SLAH (slow anion channel-associated homologs) [134,135]. The physiology of NO₃⁻ uptake can be described by low- and high-affinity transport. Popularly known as the high-affinity transport system (HATS) and low-affinity transport system (LATS), both of these mechanisms have been observed in Arabidopsis (Arabidopsis thaliana). For instance, AtNRT2.1 is a HATS transporter that facilitates the uptake of nitrate from the soil, particularly when nitrate concentrations are low. On the other hand, the AtNRT1.1 transporter can operate both low and high nitrogen affinity depending upon the environmental conditions. Similarly, chlorate-resistant 1/nitrate transporter1 (CHL1/NRT1.1), NPF6.3, also operates as both a low- and high-affinity transporter [134,136,137]. The NRT2 family, in contrast to the NRT1 family, consists of high-affinity NO₃^- transporters. Among the eight members of the NRT2 family, seven have been characterized. NRT2.1, NRT2.2, NRT2.4, and NRT2.5 are actively involved in the influx of NO₃^- into Arabidopsis root cells [138,139]. Similarly, in the high-affinity nitrate transporter family, OsNRT2.3 (Table 2 generates two transcripts, OsNRT2.3A, and OsNRT2.3B, via alternative splicing primarily expressed in the plant’s phloem. OsNRT2.3A mediates the long-distance transportation of nitrate from the root to the stem and encodes a phloem protein located at amino acid position 516 [140,141]. Similarly, research elucidates that OsNRT1.1B, OsSPX4, and OsNLP3 genes have a significant role in regulating nitrate uptake from the soil, influencing rice productivity [142].

Ammonia (NH₄⁺) predominantly exists in flooded and acidic soil conditions [143]. High-affinity transporters mainly located in the root hairs and epidermis, known as ammonia-mediated transporters (AMT), regulate its transport [144]. The Arabidopsis genome contains six AMT, each coding for high-affinity NH₄⁺ transporters upregulated in low nitrogen conditions [145]. Among the six AMTs, AMT1;1, AMT1;2, and AMT1;3 play critical roles in the high-affinity NH₄⁺ absorption, showing that plants deploy diverse NH₄⁺ transporters to efficiently uptake NH₄⁺ under conditions of low nitrogen availability [144]. AtAMT1;1, AtAMT1;2, and AtAMT1;3 are predominantly found in roots, whereas AtAMT1;1 and AtAMT1;2 can also be expressed in shoots, with higher expression observed in leaves. The enhanced uptake of ammonia is co-related to the up-regulation of AtAMT1;1 and AtAMT1;3 under nitrogen deficiency [146,147]. OsAMT1;1 is expressed on both roots and shoots. The mutants of OsAMT1;1 showed a decrease in ammonium uptake as well as impeded growth of roots and shoots. OsAMT1;2 expressed exclusively on the roots and OsAMT1;3 on nitrogen deficiency roots. Moreover, the expression of OsAMT1 genes does not depend on endogenous ammonium levels but its expression is regulated by glutamine concentration, where glutamine synthase is a key enzyme converting inorganic ammonium into glutamine [147,148,149,150]. Moreover, Nin-like protein 4 (NLPs) and circadian clock genes have been reported in transcriptional regulation of the OsAMT1 gene, which is associated with N uptake under N-deficient conditions in rice [151].

4.4. Nitrogen Assimilation and Utilization Pathways

The first step in nitrate assimilation includes the activation of nitrate reductase (NR) and its involvement in the reduction and conversion of nitrate into biologically active forms like nucleic acids, amino acids, and other nitrogen-containing biomolecules within cytoplasm. Along with NR, a molybdenum cofactor (MoCo) also plays a crucial role in catalyzing nitrate reduction. The next step includes the conversion of nitrate into ammonia (NO₂⁻ to NH₄⁺) by nitrite reductase in the chloroplast [143,152,153,154].

The GS-GOGAT (Figure 2) cycles are major regulatory pathways that have a significant role in nitrogen assimilation and remobilization, with various metabolic processes leading to enhanced NUE and crop yield [155]. Ammonium can be absorbed by plant roots, formed through the conversion of nitrates, or released during the breakdown of amino acids or through photorespiration. The breakdown and conversion processes are facilitated by two major enzymes, GS and GOGAT, interactively known as the GS/GOGAT cycle. GS catalyzes the conversion of ammonia and glutamate to glutamine, GOGAT transfers the amide amino group from glutamine to 2-oxoglutarate, forming glutamate. Cytosolic glutamine synthetase (GS1) is crucial in assimilating inorganic nitrogen from the soil, such as nitrate or NH₄⁺. Furthermore, GS1 plays a role in N reassimilation from NH₄⁺ and enhanced expression during senescence. An integration of the chimeric alfalfa NADH-GOGAT gene into tobacco showed higher total carbon and nitrogen content in shoots and increased shoot dry weight at the flowering stage, reflecting a higher capacity to assimilate nitrogen [155,156,157].

NR assimilates nitrogen in plants via post-translational phosphorylation. NIA1 protein dephosphorylation on rice growth and metabolism under different nitrogen sources reveals that specific phosphorylation site mutations enhanced nitrogen assimilation and tolerance to ammonium nitrogen deficiency. When ammonium nitrate was used as the main nitrogen source, there was weak growth of NIA1 phosphorylation site-directed mutant lines by excessive nitrite accumulation. Conversely, in the case of potassium nitrate, the assimilation rates of nitrate, nitrite, and ammonium salt increased significantly in NIA1 phosphorylation site-directed mutant lines [158]. OsNLP4, one homologous gene of Arabidopsis NLP7, has a crucial role in nitrogen uptake, assimilation, signaling, and utilization in rice. Overexpression of OsNLP4 significantly increased NUE, crop yield, and plant biomass. Under a nitrate-dependent rice cultivation system, OsNLP4 can influence plant growth by affecting NR activity, particularly in OsNLP4 mutant lines controlling the expression patterns of some nitrate signaling genes. Moreover, OsNLP4 showed a shoot reduction of 30%, suggesting enhanced translocation of N from the roots, resulting in enhanced NUE (47%) and grain yield (30%) [159,160]. Partial nitrate nutrition (PNN) enhances NUE in rice by increasing nitric oxide (NO) production mediated by NR. This mechanism adapts rice to fluctuating nitrate supplies, improving nitrogen acquisition and utilization [161].

4.5. Key Genes and Transcription Factors (TFs) Associated with Enhanced NUE in Rice

Japonica and indica are two important cultivated rice subspecies. Japonica varieties are well-suited to high-altitude, temperate regions due to their tolerance to cold and fertilizers. In contrast, indica varieties thrive in subtropical and tropical climates characterized by high temperatures, humidity, and light intensity, but display lesser tolerance to cold and fertilizers. Both varieties exhibit notable genetic and environmental differences in nitrogen absorption and utilization. Compared to japonica rice varieties, indica have higher NUE. Research indicates that indica varieties demonstrate a 14.1% greater NUE than japonica varieties. Additionally, hybrid rice varieties exhibit enhanced NUE over traditional strains. Specifically, hybrid indica varieties show a 22.8% increase in NUE compared to conventional indica, and hybrid japonica varieties achieve a 16.4% improvement over their traditional counterparts [19]. Also, some research findings indicate that indica varieties exhibit 30–40% higher NUE than japonica varieties. The genes NRT1.1B, ARE1, and NR2 display functional differences between these rice varieties [162]. The NRT1.1B gene is crucial for the absorption and movement of nitrogen within plants. This gene variant, identified in indica rice and known as NRT1.1B-Indica, boosts nitrogen uptake and activates genes responsive to nitrate. When NRT1.1B-Indica (Table 2) is introduced into japonica rice varieties, it enhances the grain yield and NUE by 10% with normal nitrogen levels and 30% when the nitrogen supply is halved [163]. Decreasing the expression of OsCBL1 in rice leads to a higher expression of the high-affinity nitrate gene OsNRT2.2. This effect could stem from suppressing the OsCCA1 TF, which results in improved NUE under low nitrogen conditions [164].

The presence of DNR1 in indica varieties underscores the variance in nitrate absorption and assimilation compared to japonica types. The OsNPF3.1 gene, found in the aerial parts of rice, influences plant height, the timing of the heading, and grain weight. It also regulates NUE in rice. Variations in this gene contribute to the differences in NUE observed between indica and japonica rice varieties [162].

The MYB TFs OsMYB305 (shown in Table 2) enhances nitrogen uptake and assimilation, shoot dry weight, and tiller number significantly under low N conditions in rice by encoding a transcriptional activator and its expression [165]. Similarly, the indica allele of MYB61 augments biomass and grain yield under a limited nitrogen supply. This finding is significant because MYB61 is pivotal in governing cellulose synthesis and nitrogen utilization [10].

BT1 and BT2 (bric-a-brac/tramtrack/broad) are a prominent group of TFs that play a crucial role in forming a network for nitrate assimilation by acting on multiple genes. Functional analysis has revealed that these TFs are responsible for regulating NUE in Arabidopsis orthologs, particularly in rice and other cereal crops. The expression of the nitrate transporter genes NRT2.1 and NRT2.4 was higher in the bt1/bt2 double mutant compared to wild-type plants, accompanied by a 65% increase in nitrate uptake under low nitrate conditions. In rice, NUE increased by 20% compared to wild types in low nitrogen conditions [166]. OsDOF18 TFs play a crucial role in ammonium uptake in rice by regulating the expression of ammonium transporter genes and influencing NUE (Table 2) [167].

Table 2. Regulatory genes and transcription factors with their functions controlling NUE in plants.

Category	Gene	Descriptions	Function	Crops	References
Root System Architecture	OsAMT1;1	Ammonium transporter 1 member 1	Increase root length and surface area and encode ammonium transporters, enhancing nitrogen accumulation.	Rice	[114]
	OsAMT1;2	Ammonium transporter 1 member 2
	RNR10	Ribonucleoside-diphosphate reductase	Nitrogen-responsive RSA.		[109]
Nitrate (NO3−) Transporters	AtNRT1.1	Nitrate transporter 1.1	Uptake of nitrate NO3− in both low and high nitrogen availability.	Arabidopsis	[134,136,137]
	NPF6.3	Nitrate/chlorate transporter
	AtNRT2.1	High-affinity nitrate transporter 2.1	Uptake of nitrate (NO3−) when nitrate is low in soil.
	NRT2.4	High-affinity nitrate transporter 3.1	Influx of NO3− into root.		[138,139]
	NRT2.5	High-affinity nitrate transporter 2.5
	OsNRT2.3	High-affinity nitrate transporter 2.3	Transportation of nitrate from the root to the stem.	Rice	[140,141]
Ammonia (NH4+) Transporters	AtAMT1;1	Ammonium transporter 1 member 1	Decreased uptake of ammonia (NH4+), impedes root and shoot growth.	Arabidopsis	[144,147,148]
	AtAMT1;2	Ammonium transporter 1 member 2	Uptake of ammonia (NH4+), found in root and shoot (leaves).
	AtAMT1;3	Ammonium transporter 1 member 3	Uptake of ammonia (NH4+), found in root in low nitrogen availability.
Nitrogen Assimilation and Utilization	GLN1-1	Glutamine synthetase cytosolic isozyme 1-1	Catalyzes synthesis of glutamine from ammonium and glutamate to glutamine synthesis in roots. Ammonium assimilation.	Arabidopsis and Rice	[168,169]
	NADH-GOGAT 1	Glutamate synthase 1 [NADH], chloroplast	Glutamate biosynthesis and ammonium ions assimilation in roots. Reutilization of glutamine in developing organs. Development of tillers.	Rice	[170]
	GLU2	Ferredoxin-dependent glutamate synthase 2, chloroplast	Nitrogen assimilation in roots.	Arabidopsis	[171]
Transcription Factors	OsNLP4	Transcription factor NLP4	Nitrogen uptake, assimilation, signaling, and utilization in rice.	Rice	[159,160]
	OsMYB305	MYB transcription factor	Enhance nitrogen uptake and assimilation, shoot dry weight, and tiller number significantly under low N conditions in rice.	Rice	[165]
	MYB61	Transcription factor MYB61	Increase biomass and grain yield, under limited nitrogen.	Rice	[10]
	OsDOF18	Dof family transcription factor	Ammonium uptake by regulating the expression of ammonium transporter genes and influencing NUE.		[167]

Table 2. Regulatory genes and transcription factors with their functions controlling NUE in plants.

Category	Gene	Descriptions	Function	Crops	References
Root System Architecture	OsAMT1;1	Ammonium transporter 1 member 1	Increase root length and surface area and encode ammonium transporters, enhancing nitrogen accumulation.	Rice	[114]
	OsAMT1;2	Ammonium transporter 1 member 2
	RNR10	Ribonucleoside-diphosphate reductase	Nitrogen-responsive RSA.		[109]
Nitrate (NO3−) Transporters	AtNRT1.1	Nitrate transporter 1.1	Uptake of nitrate NO3− in both low and high nitrogen availability.	Arabidopsis	[134,136,137]
	NPF6.3	Nitrate/chlorate transporter
	AtNRT2.1	High-affinity nitrate transporter 2.1	Uptake of nitrate (NO3−) when nitrate is low in soil.
	NRT2.4	High-affinity nitrate transporter 3.1	Influx of NO3− into root.		[138,139]
	NRT2.5	High-affinity nitrate transporter 2.5
	OsNRT2.3	High-affinity nitrate transporter 2.3	Transportation of nitrate from the root to the stem.	Rice	[140,141]
Ammonia (NH4+) Transporters	AtAMT1;1	Ammonium transporter 1 member 1	Decreased uptake of ammonia (NH4+), impedes root and shoot growth.	Arabidopsis	[144,147,148]
	AtAMT1;2	Ammonium transporter 1 member 2	Uptake of ammonia (NH4+), found in root and shoot (leaves).
	AtAMT1;3	Ammonium transporter 1 member 3	Uptake of ammonia (NH4+), found in root in low nitrogen availability.
Nitrogen Assimilation and Utilization	GLN1-1	Glutamine synthetase cytosolic isozyme 1-1	Catalyzes synthesis of glutamine from ammonium and glutamate to glutamine synthesis in roots. Ammonium assimilation.	Arabidopsis and Rice	[168,169]
	NADH-GOGAT 1	Glutamate synthase 1 [NADH], chloroplast	Glutamate biosynthesis and ammonium ions assimilation in roots. Reutilization of glutamine in developing organs. Development of tillers.	Rice	[170]
	GLU2	Ferredoxin-dependent glutamate synthase 2, chloroplast	Nitrogen assimilation in roots.	Arabidopsis	[171]
Transcription Factors	OsNLP4	Transcription factor NLP4	Nitrogen uptake, assimilation, signaling, and utilization in rice.	Rice	[159,160]
	OsMYB305	MYB transcription factor	Enhance nitrogen uptake and assimilation, shoot dry weight, and tiller number significantly under low N conditions in rice.	Rice	[165]
	MYB61	Transcription factor MYB61	Increase biomass and grain yield, under limited nitrogen.	Rice	[10]
	OsDOF18	Dof family transcription factor	Ammonium uptake by regulating the expression of ammonium transporter genes and influencing NUE.		[167]

5. Genomics and Recent Innovations for Improving NUE in Rice

5.1. Multi-Omics Approaches for Improving NUE in Crops

Integrated transcriptomics and physiological analysis of NUE in rice under drought conditions showed that water scarcity led to a notable reduction in NUE. Similarly, expression of the GAPA and GAPB genes at the vegetative stage and GLO4, GLO5, and OsGDCH reproductive phase facilitates nitrogen utilization and enhancement, making them suitable genetic tools to develop specific crop traits under drought conditions. Moreover, glyoxylate dicarboxylate metabolism and carbon fixation in photosynthetic organisms are major regulatory pathways governing NUE in rice under water-scarce conditions [172]. Integrated multi-omics (Figure 3) in rice regarding low nitrogen (LN) tolerance in the flowering stage revealed three genes (LOC_Os02g40010, LOC_Os11g25260, and LOC_Os11g47300) involved in purine metabolism and genes (LOC_Os02g36880 and LOC_Os08g05830) and metabolites 3-phosphoglyceric acid and sedoheptulose 7-phosphate) associated with glycolysis and the pentose phosphate pathway. Moreover, results demonstrate that purine metabolism is the major pathway governing LN during flowering in rice and five candidate genes can be utilized for molecular breeding strategies for LN tolerance in rice during flowering [173]. Integrative transcriptomics and proteomics revealed that activation of nitrogen transporters (NRT2.1, NRT2.2, NRT2.3, NRT2.4, NAR2.1, AMT1.3, AMT1.2, and AMT3.3) and nitrogen assimilators (like NR2, GS1;1, GS1;2, GS1;3, NADH-GOGAT2, and AS2), along with key metabolic pathways such as phenylpropanoid biosynthesis and glutathione metabolism, optimize nitrogen use efficiency in rice under low nitrogen conditions [174]. Bulk segregant analysis sequencing (BSA-Seq) and RNA-Seq identified candidate genes (BnaC02g14540D, BnaA03g20760D, and BnaA05g01330D) related to NUE in rapeseed (Brassica napus L.) [175].

5.2. MAS and QTLs Mediated NUE Enhancements in Crops

Integrating traditional breeding methods with molecular genetics facilitates understanding crop inheritance patterns, leading to improvements in specific crop traits. Advances in rice breeding, such as MAS, QTLs (Figure 3), gene discovery, and CRISPR/Cas9 technologies, enhance NUE and contribute to sustainable crop production. For instance, 190 out of 293 QTLs (64.8%) were found for the nutrient uptake and utilization of N, P, and K in wheat [143,176,177]. A meta-analysis of 506 QTLs from 18 studies has identified 12 meta-QTLs associated with NUE and grain yield per plant under nitrogen-deficit conditions in rice. The mQTLs were found on four rice chromosomes: 1, 3, 4, and 8. These mQTLs are widely distributed throughout the rice germplasm, suggesting the potential to use them as MAS in developing genotypes that have more NUE. This could be an innovative breeding tool for screening and developing rice varieties requiring less nitrogen [178]. QTLs identified 13 loci on various chromosomes linked to root system architecture and nitrogen-deficiency tolerance in rice. Particularly, a locus on chromosome 1 showed a significant correlation with increased root depth and nitrogen-deficiency tolerance, directly impacting grain yield. These identified QTLs could serve as valuable assets in rice breeding programs aiming to enhance RSA traits, ultimately improving rice performance under nitrogen deficiency [179]. Similarly, advancements in genetic tools like QTLs, genes linked to nitrogen metabolism, proteomics, association mapping, microarrays, miRNA, genetic transformation, and transcriptomic approaches can improve crop NUE [180].

SNPs from indica and japonica rice varieties can be crucial for improving NUE in rice. Precisely replacing the japonica NRT-1B allele with the indica allele in a single generation using CRISPR/Cas9 technology illustrates that substituting any gene with superior alleles with desired traits can enhance specific characteristics in crops, such as NUE, leading to increased crop production and productivity [181]. Deploying genome-editing technology can develop cultivars with higher yields and improved nitrogen uptake and utilization efficiency. This technology can be precisely modified in the plant genome with specific genes related to NUE. Using tools like CRISPR-Cas9, researchers can edit genes that regulate nitrogen uptake, assimilation, and utilization in cereals. Moreover, the application of genome editing in cereals has the potential to address the challenges created by N pollution and environmental degradation due to the non-scientific use of N in crop production contributing to sustainable agriculture production [182]. Researchers have been working to identify germplasms that efficiently utilize nutrients, with a specific focus on NUE and crop productivity. High-throughput technologies are being utilized to discover potential germplasms. Exciting progress has been made in the field of plant breeding, through the use of innovative plant breeding techniques (NPBTs) and genome editing technologies, with consideration to be made for the biosafety concerns as well as social, ethical, and environmental issues [183].

5.3. Genome-Wide Association Study (GWAS) for Improving NUE

The GWAS (Figure 3) is a widely accepted and powerful genetic tool for understanding the relationship between phenotype and genotype, which facilitates the identification of potential alleles governing specific crop traits [184,185]. Genetic tools such as GWAS genomic selection (GS) are employed for enhancing NUE in cereal crops by improving traits associated with NUE and altering functional adaptive traits. Additionally, the development of phenotypic approaches coupled with the GWAS and GS showed various genes and agronomic traits and their adoptive mechanisms associated with NUE [186]. The GWAS performed on 419 rice varieties using 208,993 SNPs revealed the gene LOC_Os10g33210, which encodes a peptide transporter, and the gene LOC_Os05g51690, which encodes a CCT domain protein function for NUE in rice. These can serve as new genetic resources for breeding and developing rice cultivars with high NUE [187]. The GWAS conducted in 325 rice-variety natural populations using 111,205 SNPs showed that OsOAT and MOC2 genes play a critical role in N use. Similarly, the gene LOC_Os12g41090, which encodes calcium/calmodulin-dependent protein kinases, shows signals near OsCPK12. OsCPK12 is involved in the signal transduction pathway under low-nitrogen (LN) stress response and enhances NUE in rice [188]. The GWAS of agronomic traits associated with NUE in 244 wheat accessions showed four major genes: TraesCS3A01G101900, TraesCS3A01G102200, TraesCS3A01G104100, and TraesCS3A01G105400 expressed in low nitrogen-tolerant wheat cultivars governing N uptake and assimilation. These can be valuable candidate genes for breeding cultivars for low nitrogen conditions [189]. Similarly, the GWAS performed on 282 barley accessions identified the HvNRT2.7 gene, a high-affinity nitrate transporter linked with shoot and root dry weight, playing a key role in enhancing NUE [190].

5.4. Genetic Modifications and Genome Editing for Improving NUE in Crops

The genome editing technology has great potential to transform agriculture production sustainability through enhanced NUE. By utilizing genome editing technology, increased NUE in crops can serve as a means of resilient food production and ensure the food security of present and future generations. The key components of this technology include target gene selection and identification, diverse genome editing tools, delivery methodologies, and the assessment of crops aimed to improve NUE [191]. In recent years, RNA-guided genome editing technology (CRISPR-Cas9), as displayed in Figure 3 has significantly transformed plant genome editing. Engineering of CRISPR/dead (d) Cas9 (dCas9) cytosine, adenine base editing, and prime editing has had great success in achieving desired traits in crops. Moreover, the dCas9-SunTag system is being employed in plants for the activation of target genes and DNA methylation. This genetic improvement will improve crop nutrient stress tolerance and contribute to food sustainability [192]. Epigenetic gene regulation by non-coding RNA is used as a new genome editing approach for the identification and validation of genes governing NUE in crops. Additionally, haplotyping, allele mining, spatial and temporal expression analysis, gene network, and validation can be employed for the development of high-yielding rice varieties with enhanced NUE under optimum nitrogen [193]. Identification of the abnormal cytokinin response1 repressor 1 (HvARE1) gene in barley (Hordeum vulgare) through the GWAS and Cas9 showed that it is expressed predominantly in leaves and shoots with low expression in roots under low nitrogen conditions, demonstrating increases in plant height, tiller numbers, grain protein content, and grain yield [194]. Genetic modifications of crops that target diversified traits governing nitrogen uptake and utilization showed improved NUE. Meta-analysis data revealed that genetic modifications increase crop production by 16.7% on average, which can be justified by the increase in PFPN (9.47%) and NUpE (16.2%) [195]. Overexpression of the high-affinity NRT gene (NpNRT2.1) in Nicotiana plumbaginifolia notably enhanced nitrate uptake in transgenic plants [196].

5.5. Metagenomics and Recent Advancements for Enhancing NUE in Crops

Metagenomics has transformed the study of soil microbial populations by facilitating whole genome analysis without culturing and capturing a broader range of genetic information than traditional methods. It offers deep insights into associated microbiota, highlighting their importance in understanding plant–soil–microbe interactions, improving nutrient use efficiency and sustainable crop production [197]. Plant microbiomes are an important component of biological climate, determining the genetic makeup of microorganisms within soil and plant systems. They are also associated with different mechanisms in plants, such as plant resistance, nutrient uptake, recycling, mobilization, and crop production and productivity. Metagenomics is a modern and advanced tool that facilitates designing the right microbiome for soil and plant well-being and can be targeted to enhance specific crop traits [198]. Optimizing nitrogen (N) application rates and planting densities is crucial for improving rice yields and quality. An integrated metagenomics conducted to investigate the effects of different levels of N fertilizer and plant densities on soil microbial communities and nitrogen uptake revealed that out of high-nitrogen low-density (HNLD), medium-nitrogen medium-density (MNMD), and low-nitrogen high-density (LNHD), the HNLD treatment demonstrated the highest absorption and utilization efficiency. Key bacterial groups, including Actinobacteria, Acidobacteria, Chloroflexi, and Proteobacteria, predominantly play a vital role in nitrogen uptake and assimilation. Moreover, other factors influencing N uptake and efficiency include root nitrite reductase (NIR) activity, soil enzyme activities (sucrose and catalase), and specific bacterial communities [199]. A metagenomic study revealed that fulvic acid increases nitrogen fixation and retention in paddy soils through the combined microbial N and carbon cycle [200]. Adequate N supply reduced the abundance of pathogens in the phyllosphere, and fungi more vitally regulate phyllosphere microbial functions than bacteria [201]. An imported biogeochemical cycle, dissimilatory nitrate reduction to ammonium (DNRA), has recently been reported to have significant potential in improving nitrogen use efficiency, (NUE) while reducing nitrogen loss and N₂O emissions in paddy rice [202]. Cyanobacterial strains are involved in increased amounts of nitrate and ammonium in rice. In silico analysis discovered 12 gene loci and 15 OsAMT isoforms in the rice genome [203]. Metagenomic sequencing of rhizospheric soil in comparison to indica and japonica resulted in dominant bacteria at the phylum level as Actinobacteria, Proteobacteria, Chloroflexi, Acidobacteria, Gemmatimonadetes, and Nitrospirae. Indica rice’s rhizosphere was more enriched with microbial communities involved in nitrogen metabolism, contributing to enhanced NUE. Insights into the correlation between deficiency effects contribute to heightened N uptake and utilization in rice production [204]. The metagenomic study identified root oxidation activity (ROA) in bacterial communities related to N management in rice, possessing the potential to increase NUE and yield [205] (

Table 3. Rice cultivars and genotypes demonstrate enhanced NUE and grain yield under varied soil conditions in major rice-growing countries of Asia.

S.N.	Country	Cultivars and Genotypes with High NUE	Characteristics	References
1.	China	Huaidao 5 (HD-5) and Lianjing 7 (LJ-7)	Deeper roots, higher root oxidation, and enhanced photosynthetic NUE at low N conditions	[206]
2.	Philippines	IR54790-B-B-38, BG380-2, BG90-2 (medium duration), IR3932-182-2-3-3-2, IR54853-B-B-318, and IR29723-88-2-3-3 (long duration)		[207]
3.	China	Wuyunjing 23 (W23) and Zhendao 11 (Z11)	Cultivars W23 and Z11 have 21–27% less NH3 volatilization, 23–26% less N2O emission, 23–33% less N leaching, and 13–24% less N in runoff	[208]
4.	China	HY549 and YY4949	NUE for grain production (NUEg) 35.2 to 62.0 kg kg−1	[209]
5.	Nepal	Arize 6444	Use of Arize 6444 along with fertilization with deep-placed briquette urea increased grain yield by 21–23%	[23]
6.	India	Rice genotypes (IRGC 6386-1, WAS 20, IRGC 116967-1 WAS 207), Variety (IR64)	Higher yield and NUE under low N conditions	[210]
7.	India	Dhala Heera	Higher yield and NUE across soil and various N conditions	[211]
8.	India	Birupa	High NUE and crop yield under low nitrogen	[212]
9.	India	Nidhi and Daya	Higher grain yield, NUE, and grain yield efficiency index GYEI	[213]
10.	India	R-83929-B-B-291-3-1-1, Kalinga-1, APO, Pusa Basmati-1, and Nerica-L-44	Higher grain yield and NUE under N-deficit conditions	[214]
11.	China	Nconv and Nweek	Higher leaf area index, stem numbers, and N uptake	[215]
12.	Thailand	Dawk Pa–yawm	Showed higher NUE and nitrogen agronomic efficiency (NAE) at application of 90 kg N ha−1	[216]
13.	India	Apo and Nerica-L-42	Change in gene expression and amino acid sequence of NLP3, NLP4, and NLP5 make them potential resources for genome editing for enhancing NUE	[217]
14.	Philippines	CT6510-24-1-2, IR55423-01, IR72, and IR57514-PMI5-B-1-2	Higher NUE under limited N and water supply	[218]

).

6. Conclusions

Improving NUE is essential for achieving sustainable agriculture amidst an increasing population, food demands, environmental degradation, global warming, and climate change. This review offers deep insights into the benefits of advanced agronomic practices over conventional nitrogen-use approaches and molecular mechanisms of N uptake and assimilation, demonstrating recent molecular and genomic innovations to optimize nitrogen uptake, assimilation, and utilization. The adoption of fertilizer best management practices (BMPs) in farmers’ fields, the integration of efficient N management strategies such as the 4Rs Nutrient Stewardship, enhanced efficiency nitrogen fertilizers (EENFs), nanotechnology, biochar, conservation agriculture (intercropping and green manuring), and sensor-based technologies have all proven to optimize N inputs and increase NUE and crop yields. These approaches are key in reducing yield gaps while promoting sustainable agriculture practices, improving the livelihood of the farmers. The paradigm of molecular mechanisms, including root system architecture (RSA) modulation, nitrogen transporters, signal transduction, and regulatory pathways such as phenylpropanoid biosynthesis, glutathione metabolism, and GOGAT provides a genetic foundation of NUE in rice. Advances and innovations in genomic technologies, including genome editing and CRISPR-Cas9, marker-assisted selection (MAS), genome-wide association studies (GWAS), multi-omics, and metagenomics have revealed key genes and regulatory networks that can be valuable tools for developing nitrogen-efficient rice varieties. Integrating agronomic and genetic strategies creates a robust framework for enhancing NUE, minimizing fertilizer reliance, mitigating environmental pollution, and ensuring global food security.

7. Future Prospects

Future approaches to improve NUE should prioritize the integration of innovative and precise agricultural technologies, such as sensor-based fertilizer management and application, the use of digital soil maps, mobile applications, and real-time decision-making tools, to optimize nitrogen application and NUE. Scaling up the adoption of nano-fertilizers, including nano-urea, can transform nitrogen management by significantly lowering inputs and costs without sacrificing productivity. Insights into multi-omic technologies, such as transcriptomics, proteomics, and metagenomics, will provide a deeper foundation for the molecular and microbial interactions that regulate nitrogen cycling in soils and crops. Genomic approaches, particularly CRISPR-Cas9, can be further utilized to introduce superior alleles and create rice cultivars with enhanced NUE suitable to multiple agroecological zones and soil conditions.

Rice varieties with enhanced NUE lead to higher grain yield. Integrating agronomic and molecular approaches significantly enhance NUE in rice, reduces nutrient losses, and increases crop productivity, leading to sustainable rice production.