Introduction

The last four decades witnessed a double enhancement in cereal production driven mainly by the use of high-yielding varieties and fertilizers in agriculture (Zhu et al. 2022). Nitrogen (N) shares 78% of the atmospheric gaseous composition and crops are unable to use it in this molecular form (Anas et al. 2020). Although leguminous crops fix the gaseous N into a utilizable form such as ammonia (NH3) and nitrate (NO3−), this fixed N is insufficient to meet the crop production demand of larger acreages (Ladha et al. 2022). Moreover, N is deficient in almost all agricultural soils of the world, and about 111.59 million tonnes of nitrogenous fertilizers are applied in world agricultural land to meet the crop’s N requirement (FAO 2022). To tackle N paucity, the farmers generally solicit N fertilizers externally in their fields to maintain higher crop yields (Dimkpa et al. 2020). However, the excessive N application incurs higher environmental and farmer inputs costs, with sometimes human health implications (Ren et al. 2022). Moreover, crop plants absorb only 30% of applied nitrogenous fertilizer (Pahalvi et al. 2021), and unutilized N is lost to water bodies and the atmosphere (Rahman et al. 2021). Presently, elevated demand and high cost of nitrogenous fertilizer in agriculture call for a “second green revolution,” which could be accomplished by designing nitrogen-use efficient crops. These nitrogen-use efficient crops could efficiently uptake, translocate, and assimilate N and allow a lesser quantity of cascading reactive N to be released into the ecosystem (Liu et al. 2022b). Hence, this review has been formulated to focus on both present and forthcoming nitrogen use efficiency (NUE) for sustainable agriculture.

N is essential for plant growth and development (Liu et al. 2022a; Muhammad et al. 2022), and it is a constituent of essential biomolecules such as nucleic acid, enzymes, protein, ATP, chlorophyll, and alkaloids. Also, N improves root system architecture, resulting in better water and nutrient absorption from the soil. (Jia et al. 2022; Sinha et al. 2020a). In chronic N deficiency, older leaves undergo senescence and detach from plants (Zakari et al. 2020). Moreover, a deficiency of N leads to altered metabolism in the plant (Saloner and Bernstein 2021) and ultimately results in reduced yield and biomass (Gong et al. 2019; Meise et al. 2019).

The movement of N in the environment is governed by the biogeochemical cycle, and it involves the dynamic interaction of soil and plant systems. The interaction is regulated by various processes, including N fixation, nitrification, denitrification, and leaching. (Liu et al. 2022a). The flow of chemical N in the environment leads to environmental deterioration (Huang et al. 2019). The primary reason for N leaching in the ecosystem is the low NUE of crop plants (Darjee et al. 2023). The rise of chemical N in the soil causes groundwater pollution (Shukla and Saxena 2020. The enrichment of inland and marine water bodies with nutrients, especially N and phosphorus (P), in water bodies causes eutrophication, and a rise in greenhouse gases like N2O contributes to global warming (Lenhart et al. 2019).

Genetic Approaches for Enhancing NUE

Various approaches are being utilized to enhance NUE to counteract the effects of low NUE on crop plants, economic yield, and the environment. The most prominent and efficient available method among the contemporary approaches includes conventional breeding and genetic manipulation of the transcription factors, transporters, assimilatory, and remobilization genes for nitrogen metabolism in crop plants to enhance the NUE (Lee et al. 2021; Kasemsap and Bloom 2022; Yadesa 2022).

The NUE is an inherently complex trait involving gene interaction networks that regulate N uptake, assimilation, translocation, and storage (Fig. 1). The NUE is governed by complex genetic makeup that mediates the N uptake and utilization (Mahboob et al. 2023). Several candidate genes in crop plants have been identified for improving NUE (McAllister et al. 2012; Li et al. 2022b), and through genetic manipulation, these identified genes have been modulated in crop plants to enhance NUE and crop productivity (Tegeder and Masclaux-Daubresse 2018; Luo et al. 2023; Table 1). In this regard, to the present date, many plant N–metabolism-associated transporters and assimilation enzymes have been overexpressed in crop plants resulting in enhanced plant growth even under N insufficient conditions (Fiaz et al. 2021; Yadav et al. 2021; Lal et al. 2023). Furthermore, it is critical to figure out the other probable limiting processes in N metabolism while engineering the crops for enhanced NUE in plants rather than N uptake under high N input agriculture. Advancements in genome editing tools like CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9) technology could be utilized for enhancing NUE in plants (Tiwari et al. 2020).

Fig. 1
figure 1

Schematic pathway depicting nitrogen uptake, assimilation, and translocation

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Table 1 Genes involved in NUE in crop plants
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The key assimilatory genes include glutamine synthetase (GS), Glutamine oxoglutarate aminotransferase (GOGAT), Alanine aminotransferase (AlaAT), Glutamate dehydrogenase (GDH), Aspartate aminotransferase (AspAT), and Asparagine synthase (ASN) play a vital role in plant N metabolism (Lebedev et al. 2021; Singh et al. 2022; Fig. 1). Further, transcription factors (TFs) include OsMAD25, OsDOF18, OsMYB305, and N transporter like NRT1, NRT2, AMT, AAP, and STP are intensively characterized for improving NUE in crop plants.

Breeding Approaches for Enhancing NUE

Plant breeders aim to develop crop cultivars that can efficiently uptake N from the soil and its utilization inside the plant system. The NUE is the economic yield per unit N supplied to the plant. In contrast, physiological N use efficiency (PNUE) is the yield in relation to the amount of N accumulated within the plant as shown in Fig. 2 (Moll et al. 1982). PNUE is being applied in rice to assess NUE (Huang et al. 2022a, b). There is the existence of genetic variabilities in cereal crops for NUE traits as revealed by field studies (Pujarula et al. 2021). At the time of grain filling in plants, carbon (C) and N substrates are required, and N molecules get remobilized from shoots to the grain (Hajibarat and Saidi 2022). About 64% of remobilized N comes from the leaf blade and leaf sheath, while the stem contributes 36% of remobilized N from vegetative tissue to panicle at the time of grain filling (Mae and Ohira 1981). Significant variation exists among cultivars for N remobilization during the reproductive stage, and prevailing variation in rice germplasm could be employed for efficient N management (He et al. 2021). NUE is well correlated with the translocation of metabolites from leaves to panicles during grain development in rice (Melino et al. 2022).

Fig. 2
figure 2

Different processes involved in nitrogen uptake, utilization, and NUE

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During the Green revolution era (1960s), semi-dwarf wheat and rice varieties were bred to exploit the higher doses of nitrogenous fertilizers resulting in higher yield and productivity. However, in the last few decades, excessive use of nitrogenous fertilizer has witnessed yield stagnation along with environmental hazards, which brought much attention to the plant breeders for developing N-use efficient crop varieties (Liu et al. 2022b).

NUE is defined broadly as a dual function of N uptake efficiency (NUpE) and utilization efficiency (NUtE). The availability of adequate genetic variability for component traits is a prerequisite for response to genetic gain under selection. Classical plant breeding methods have been wonderfully performed in the last three decades by selecting the most desirable features in terms of yield improvement. However, very little attention has been paid to understanding the physiological and genetic basis, particularly in relation to NUE. Most of the plant breeding efforts have been made under idealized conditions to enhance productivity, and it is quite obvious that valuable allelic diversity might have been significantly reduced in available breeding materials for tolerance to limiting N conditions.

In the past, most peer-reviewed research papers for important crops suggested that contemporary cultivars have sufficient genetic variation for selecting and enhancing NUE component traits (Hawkesford 2017; Hawkesford and Griffiths 2019). Another source of genetic diversity can be found in the secondary or tertiary gene pool or wild relatives of almost all other major food crops, and NUE has not been thoroughly characterized in these potential wild cultivars (Hawkesford 2017). therefore, there is a greater scope for the identification and introgression of the NUE-related genes in elite cultivars through backcross breeding programs.

Grain yield and biomass are primarily used to select promising phenotypes under low N and optimal N conditions. Despite the availability of adequate variability for the component traits under low N, lower overlapping values in normalized conditions make the selection more attractive (Gallais et al. 2006). However, direct selection under low input/low N status yielded desired results, mainly in wheat and maize (Presterl et al. 2003; Laperche et al. 2006). Simultaneous selection of genotypes based on grain yield in both N stress and optimal conditions has resulted in the overall improvement in both conditions. Designing an efficient breeding program is based on the kind of genetic materials, genetic parameters of variability, and stability of parameters across the environment. The ability of the genotype to efficiently take up N is a key component trait for plant NUE. The key traits for NUE include N uptake at stress conditions, harvest index (HI), N harvest index (NHI), and biomass potential are important for a breeder while developing nitrogen-use efficient lines.

Genetic variability for root size, distribution, and proliferation has been reported in several crop plants; however, it also varies with soil types and nutrient status (Matsunami et al. 2009, 2010). Genetic diversity for root traits was observed while screening spring wheat genotypes under pot and hydroponics conditions and they have found NUPE largely responsible for contributing towards NUE mainly because of high root proliferation and biomass production (Ranjan et al. 2019). Sufficient variability for NUE in modern wheat cultivars was observed under low N conditions (Hawkesford 2017). However, very little variation for grain yield suggests incorporating a more diverse range of genotypes for creating a wider range of variability for yield coupled with NUE. Some previous research with N metabolism and signalling pathway-governed genes show adequate variability in crop species for breeding the NUE efficient cultivars. The most striking variability of NUE-related genes in rice is for a GOGAT enzyme. This gene was originally reported in japonica rice and transferred in indica background and resulting in enhancement of yield (Yamaya et al. 2002).

Since NUE is an integrative complex trait, decided by the number of key traits and other morpho-physio traits that are linked or function independently. The presence of significant genotypes by environment (G × E) interactions reduces the overall effectiveness of selection using grain yield under low N conditions. In addition, the polygenic nature, linkage, and non-additive gene action coupled with low heritability further complicate the selection criterion. Indirect selection using the component traits is more realistic because of high heritability and more stability with respect to genetic and external environmental factors. The selection-based index is the most attractive approach for complex traits like NUE; it overcomes the limitation of single trait selection and allows simultaneous improvement of component traits. The appropriate selection of selection index may vary depending upon the conditions and usefulness of the breeders while working on a particular problem. N efficient wheat lines identified based on utilizing four selection indices: tolerance index (TOL), stress susceptibility index (SSI), geometric mean productivity (GMP) and yield susceptibility index (YSI) (Tyagi et al. 2020). Presently, conventional breeding methods have been greatly improved by integrating the knowledge of genetics, physiology, and biochemistry.

Exploiting genetic variability through innovative molecular breeding technology is in great demand because of the easy dissection of complex traits and assisting in phenotyping in less time. Precise phenotyping of NUE traits is cumbersome and requires vigorous exercise to monitor the component's traits. Advancement in developing next-generation markers/sequencing-based markers and bioinformatics improves the chances of locating and mapping QTLs governing the NUE traits. While breeding nitrogen-use efficient cultivars, key agronomic traits such as grain protein content, grain yield, test weight, and other NUE traits such as NHI, N remobilization capacity, and N content in grain were used in the breeding program (Karunarathne et al. 2020).

Molecular linkage genetic maps and quantitative trait locus (QTL) mapping technologies are commonly used to estimate loci’s number and precise location governing genetic variation in various genetic materials (segregating or fixed populations). The selection of appropriate parents, evaluation of effective population sizes, multi-location testing, and the availability of high-density genetic maps are all required for successful QTL mapping programs for complex traits like NUE (Han et al. 2015). N deficiency traits (NDT) have also been targeted as an indirect selection criterion for selecting nitrogen-use efficient phenes in rice (Shen et al. 2021), and several QTLs for NDT and NUE traits in rice have been identified. (Ogawa et al. 2016; Shen et al. 2021). A few of a total of six QTL contributing towards NUE or low N-responsive traits in winter wheat showed the association with photoperiod and disease resistance and suggested for exploitation of potential marker-assisted selection (MAS) for breeding soft red winter wheat (Brasier et al. 2020).

Li et al. (2015) identified 184 and 147 QTLs for NUE and root system architecture (RSA) related traits and established a genetic relationship with RSA. They also explored the genomics segment for MAS in maize to improve complex traits like NUE and others. A total of 15 QTLs showing significant effects under low N conditions were identified in a barley population (Prisma × Apex) (Kindu et al. 2014). Besides that, several other QTLs have also been identified through genome-wide association studies (GWAS) for key indicator traits of NUE in barley (Pasam et al. 2012). Fifty-two putative QTLs were reported for different NUE traits in a double haploid (DH) potato population and field trial at several locations with different N-levels, specific QTLs showing significant QTL × E interaction (Getahun et al. 2020).

NUE and its component traits are mostly polygenic and background-specific; thus, the ultimate target would be to converge all the small effect loci in one genetic background to advance the realized genetic gain (R). Genomic selection is one of the most appropriate strategies to target all the small-effect minor QTLs through a predictive equation based on genome-estimated breeding value (EBV) and facilitate overall genetic improvement of complex traits like NUE. In future research, there is a need to holistically target the key information on the agronomical aspect of NUE, physiological NUE, and absorption NUE in respect of breeding varieties for low input or low N conditions.

Engineering Root System Architecture for NUE

The gene reported to control root growth and to give a particular response towards nutrients in soil has shown to be a promising target for engineering plants for better nutrient use efficiency. OsTOND1 belongs to QTL, which confers tolerance under N deficiency in rice. It codes for a thaumatin protein and its role in the induction of primary root elongation in low N soil. OsTOND1 has been found in only some indica rice varieties, which is absent in the japonica rice varieties studied so far. Furthermore, overexpression of OsTOND1 in rice confers tolerance to nitrogen deprivation, and overexpressed rice plants growing in nitrogen-deficient conditions have increased shoot N content, grain production, panicle number, dry weight, chlorophyll content, plant height, and root length (Zhang et al. 2015; Sujata et al. 2019).

A longer root system meant for the acquisition of nutrients from deeper soil layer, a characterized QTL, DEEPER ROOTING1 (DRO1) responsible for longer root in upland land rice, affects root system architecture in Arabidopsis and Prunus species and also enhances yield under drought in rice (Uga et al. 2011, 2013; Arai-Sanoh et al. 2014; Guseman et al. 2017). The rice cultivar with the DRO1 gene exhibiting enhanced N uptake and grain yield increased by 10% compared to the rice variety with a dormant DRO1 allele (Arai-Sanoh et al. 2014; Kitomi et al. 2020). Rice transcription factor OsMADS25 is involved in N signalling and it is predominantly expressed in the root during the growth period. OsMADS25 overexpression in rice results in better root system architecture and higher shoot mass (Yu et al. 2015; Nazish et al. 2021).

Mild N deficiency enhances lateral root (LR) branching (Forde 2014). A study in Arabidopsis reveals that N deficiency causes an increase in lateral roots (Ma et al. 2014). Moreover, expression of tryptophan aminotransferase related 2 (TATAR2) was enhanced under low nitrogen in wheat, and knockdown mutant of TATAR2.1 exhibits impaired lateral root growth and phenotype defects under limiting and optimum N supply in wheat (Shao et al. 2017). Also, TaTAR2.1-3A overexpression in wheat enhanced plant growth, grain yield, primary root length, lateral root branching, and N accumulation at different N conditions while reducing expression of TaTAR2.1 have opposite effects (Shao et al. 2017; Teng et al. 2022).

Genetic Manipulation of N Transporters

The N transporters play a crucial role in N plant metabolism. Moreover, N transporters are extensively studied in rice, and its genome encodes 94 nitrate transporter 1 (NRT1)/peptide transporter (PTR), five nitrate transporter 2 (NRT2), five chloride channel (CLC), and nine slow anion channel-associated homologs (SLAC/SLAH) gene (Lee 2021). Transport of NO3− inside crops is mainly regulated through NRT1 and NRT2 transporters (Aluko et al. 2023). However, utilizing existing natural variations and overexpressing NO3– transporters resulted in enhanced NUE and grain yield in rice (Hu et al. 2015; Fan et al. 2016b; Zhang et al. 2022). The OsNPF7.6 is a member of the nitrate transporter 1/peptide transporter family (NPF) expressed in all plant tissues. Moreover, overexpression of OsNPF7.6 enhances the rice yield and NUE (Zhang et al. 2022). Furthermore, OsNPF7.3 of the NO3– NPF is expressed in lateral roots and stem and localized in the vacuolar membrane. The elevated expression of OsNPF7.3 in rice results in enhanced grain yield, tiller number, and grain N content (Fang et al. 2017). The NH4+ induces the expression of OsNRT1.1A, which belongs to the NO3– transporter1 family. In rice, OsNRT1.1A overexpression results in up to 50% enhancement in NUE, grain yield, and early maturity by 18 days. On the contrary, the OsNRT1.1A mutant shows growth retardation, yield loss, late flowering, and reduced utilization of NH4+ and NO3– (Wang et al. 2018).

Studies revealed diversification in rice genotypes for uptake efficiency of NO3–; the indica subspecies have higher NO3– uptake efficiency than the japonica subspecies that is due to variation of the gene sequence of OsNRT1.1B (Hu et al. 2015). Moreover, OsNRT1.1b overexpression in rice accumulated higher N and improved plant growth under low and sufficient N supply (Fan et al. 2016a). Moreover, rice contains a high-affinity transporter, OsNRT2.3 consisting of OsNRT2.3a and OsNRT2.3b, splice forms that code for plasma membrane protein. OsNRT2.3a function in association with nitrate transport accessory protein, OsNAR2.1 (Yan et al. 2011; Tang et al. 2012). The concurrently expressing OsNAR2.1 and OsNRT2.3a in rice results in enhanced uptake and transport of NO3– and NH4+, and the grain yield and NUE were enhanced by 24.6% and 28.6% in co-overexpressed lines (Chen et al. 2020). Also, overexpression of OsNRT2.3b in barley results in up to 43% enhancement in NUE. The grain weight and yield of overexpressed lines are higher than control plants (Luo et al. 2020). Furthermore, overexpression of OsNRT2.3b in rice results in up to 40% higher NUE and grain yield (Fan et al. 2016b). The study reveals that NRT2 plays a key function in increasing yield and NUE under a different N supply. However, the gene should be expressed with tissue-specific promoters to get the desired result.

The OsPTR6 and OsPTR69 belong to the PTR/NRT1 family. The OsPTR6 overexpression in rice enhances the activity of glutamine synthetase (GS), NUE, and growth of the plant as compared to control plants supplied with NH4+ at the optimum amount (Fan et al. 2014), and overexpression of OsPTR9 in rice enhances NH4+ absorption, which drives LR branching and boosts grain production. On the contrary, OsPTR9 T-DNA mutant and OsPTR9 RNAi lines exhibit growth retardation (Fang et al. 2013).

Ammonium is transported inside plants through ammonium transporter (AMT). Rice (Oryza sativa) genome encodes four AMT family members: OsAMT1, OsAMT2, OsAMT3;1, OsAMT3, and OsAMT4. The OsAMT1 members belong to high-affinity transporters, while the other three family members are low-affinity transporters (Wu et al. 2017). Crop plants primarily absorb N in NH4+ and NO3– forms. In rice, overexpression of OsAMT1-1 boosts NH4+ uptake and its translocation inside the plant at an optimum level, but under a high amount of NH4+ supply, it impairs plant growth (Ranathunge et al. 2014; Li et al. 2016). Also, overexpressing OsAMT1.3 in rice enhances plant growth, and plants able to adapt at a low concentration of NH4+ (Bao et al. 2015; Ferreira et al. 2015). In rice, activation tagging mutants co-expressing OsAMT1;2 and OsGOGAT had better grain yield and NUE under deficient N as that of wild type and double mutants of OsAMT1;2 and OsGOGAT1 exhibit higher protein and N concentration as compared to wild type (Lee et al. 2020a). Also, ZmAMT1;1a overexpression in maize shows a 17% increase in NH4+ uptake under low NH4+ supplied to plants as that of control plants (Zhao et al. 2018).

N uptake in crop plants is regulated by transcription factors (TFs) and signalling molecules (Zhang et al. 2020; Zuluaga and Sonnante 2019). Among the TFS, DNA binding with one finger 18 (OsDOF18) rice mutant has impaired NH4+ assimilation and reduced expression of NH4+ transporter genes. Rice mutant plants grow well on supplying NH4+ as NO3– (Wu et al. 2017) and N-mediated heading date-1 (Nhd1) activate the transcription of OsAMT1;3 and OsNRT2.4 and knockdown of the Nhd1 inhibits the root growth in rice (Li et al. 2022a).

The OsMYB305 encodes for transcription activator in rice. Under low nitrogen, this gene shows higher expression in the root. The overexpression of OsMYB305 in rice exhibits enhancement in tiller number, shoot dry weight, and increased N accumulation in the plant grown under low N conditions. The expression of a high-affinity NO3– transporter and nitrite reductase gene upregulated in OsMYB305 overexpressed lines (Wang et al. 2020).

The NIN-like protein (NLP) is involved in nitrate signalling by regulating the expression of several N uptakes and assimilatory genes (Mu and Luo 2019). The OsNLP4 overexpression in rice results in an enhancement in yield by 30% and increased NUE by 47% as that of the control plant. The loss of function rice mutant generated through CRISPR/Cas9 shows a significant reduction in yield under low nitrogen (LN), normal nitrogen (NN), and high nitrogen (HN) as that of wild type (Wu et al. 2021). Also, overexpressing OsNLP1 in rice improved the biomass, grain yield, and NUE on supplying with variant N supply. The knocking out of OsNLP1 reduces the grain yield and NUE (Alfatih et al. 2020). Moreover, Overexpression of AtNLP7 upregulates the expression of N uptake and assimilatory genes and improves the NUE and yield in cotton (Jan et al. 2022).

Genetic Manipulation of Key Genes of C and N Metabolism

The well-known RuBisCO plays an important role in C fixation by incorporating CO2 into Ribulose 1,5-bisphosphate (RuBP) and converting it into 3-phosphoglycerate (3-PGA). At a low concentration of CO2, RuBP combines with O2 to produce phosphoglycolate and later releases ammonium ions during photorespiration, hence affecting carbon fixation (Stitt et al. 2010). In C3 plants, released ammonium ions are returned to amino acid pools through assimilatory enzymes (McAllister et al. 2012). At a higher level of CO2, plants exhibit enhanced assimilation of N and C (Zong and Shangguan 2016). Knocking down the RuBisCO expression in crop plants affects the C and N metabolism, reducing grain yield, biomass, amino acid pools, and plant metabolites (Maheshwari et al. 2021).

Furthermore, overexpression of RuBisCO in rice causes an increase in photosynthesis and NUE under sufficient N fertilization (Yoon et al. 2020). PEPC (phosphoenolpyruvate carboxylase) is another enzyme involved in photosynthesis and N storage in plants; this enzyme is involved in vital metabolic processes in bacteria, algae, and other lower species. The product of this enzyme is oxaloacetic acid (OAA), having a vital role in other metabolic processes like tricarboxylic acid cycle (TCA) cycle, amino acid biosynthesis, gluconeogenesis, urea cycle, glyoxylate cycle, fatty acid synthesis, etc. (Doubnerová and Ryšlavá 2011). PEPC plays a crucial role in N assimilation as validated by the knockdown of chloroplastic PEPC isoforms (Masumoto et al. 2010). In rice, PEPC overexpression enhances photosynthesis, biomass, and grain yield (Behera et al. 2023). Moreover, ZmPEPC overexpression in wheat results in upregulating TCA cycle genes that signify more C generation under low and high N conditions. The total amino acid concentration was enhanced by 48.18% in overexpressed lines as that of wild-type plants (Peng et al. 2018).

Plants mostly uptake inorganic N in the form of NO3– and NH4+ from the soil, while plants take up organic N in the form of amino acids through amino acid transporter (AAT). The transporters are classified into three major families: ATF (amino acid transporter family), APC (amino acid-polyamine-choline transporter family), and UMAMIT (usually multiple acids move in and out transporter family). Among all these families, most research has focused on AAP (amino acid permeases) because of their multiple physiological functions, AAP transporters belong to ATF (Yang et al. 2020). The AAP1 gene overexpression is extensively studied in legumes and VfAAP1, specifically overexpressed in the seed of Vicia narbonensis and Pisum sativum results in up to 15% increase in total N content and enhancement in seed size up to 30% and also, co-overexpression of AAP1 and sucrose transporter, SUT1 in pea results in enhanced grain number, protein and sugar content (Grant et al. 2021; Rolletschek et al. 2005). Sugar transporters are associated with phloem loading and unloading in plants, and these transporters are involved in carbon distribution inside plants (Wen et al. 2022). Sugar transporter proteins (STPs) play crucial roles in plant sugar transport. Overexpressing STP13 (hexose transporter) in Arabidopsis results in an enhancement in glucose uptake, internal glucose amount, and biomass when plants are grown in a limiting N environment (Schofield et al. 2009). These studies suggested a link exists in plant systems for coordinating C and N metabolism. Improvement in NUE requires both C substrate and N availability, the lack of C substrate can compromise the NUE, and N availability affects C fixation (Sandhu et al. 2021). The GS/GOGAT cycle is also critical for maintaining the C/N ratio. The keto group contains amino acids involved in C metabolism utilizing glutamate as signalling molecules (Hildebrandt et al. 2015). The tuning of the C/N ratio is crucial for plant growth and development. Moreover, the NUE is regulated by the photosynthesis assimilation rate, and under the limitation of C substrate, NUE gets reduced (Liu et al. 2022a).

The primary enzyme in sucrose production is sucrose phosphate synthase (SPS). The overexpression of SPS in alfalfa plants results in an enhancement in growth and nodule development, and overexpression of SPS in sugarcane enhances growth and increases the height, sucrose and biomass content (Kaur et al. 2019; Anur et al. 2020). N assimilatory gene, AlaAT converts pyruvate to alanine, and this conversion is activated during hypoxia; thus, AlaAT links Glycolysis and TCA Cycle (Rocha et al. 2010). Further, the genetic manipulation of HvAlaAT using root and stress inducible promoter enhances NUE and grain yield in rice, barley, wheat, oilseed rape (Brassica napus) and sugarcane under low N conditions (Tiong et al. 2021; Good et al. 2007; Shrawat et al. 2008; Snyman et al. 2015).

The Dof gene of maize has been utilized for improving N assimilation by regulating the TCA cycle. The overexpression of Dof1 in rice enhances the C flow, which results in improved plant growth under low nitrogen (LN) environments (Kurai et al. 2011). Also, overexpression TaDof1 in wheat shows enhanced expression of citrate synthase, phosphoenolpyruvate carboxylase, isocitrate dehydrogenase, pyruvate kinase, and overexpressed wheat transgenic lines show improved agronomical traits under limiting N supply as compared to wild type (Hasnain et al. 2020).

Amino Acid Metabolism Regulates NUE

Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of ammonia and Glutamate (Glu) to Glutamine (Gln), and GOGAT transfers the amide group of Gln to 2-oxoglutarate to form two molecules of Glu. Plant encodes two forms of GS, the cytosolic (GS1) and chloroplastic (GS2). In plants, GOGAT exists in two ferredoxin-GOGAT (Fd-GOGAT) and NADH-GOGAT that are expressed in chloroplast and cytoplasm, respectively. GS enzyme is the prime target of researchers for enhancing the NUE of crop plants (Hakvoort et al. 2017). GS modulated in plants through genetic engineering and tobacco plants overexpressing Arabidopsis GS1, GS2 grown under low nitrogen exhibits higher sugar, protein, and chlorophyll content (Wang et al. 2013). The cisgenic HvGS1;1 overexpressed line of barley exhibited enhanced grain yield and NUE compared to control plants at different N-supplied (Gao et al. 2019). Studies in durum wheat reveal GS2 alleles co-localized with grain protein content QTL, which makes the possibility of GS2 imparting a role in enhancing grain protein content (Gadaleta et al. 2011). Also, GS-overexpressed rice contains enhanced protein, amino acid, and total nitrogen content compared to wild type (Cai et al. 2009). Moreover, transgenic wheat expressing the plastidic GS2 isoform under low and high N regime conditions shows higher yield, NUE, and grain N concentration like wild type (Hu et al. 2018). Furthermore, Concurrent co-overexpression of OsGS1;1 and OsGS2 increased NUE, yield, and biomass in indica rice (Lal et al. 2023).

Plant roots exhibit plasticity rather than preference in the acquisition of NH4+ and NO3– (Chalk and Smith 2021). On supply of NH4+ to Arabidopsis, NADH-GOGAT shows higher expression and accumulation in the roots. T-DNA mutant line having non-functional NADH-GOGAT shows a reduction in biomass when the major N source is supplied as NH4+ (Konishi et al. 2014). In addition, glutamate dehydrogenase (GDH) is also a key enzyme in ammonia assimilation that converts 2-oxoglutarate to glutamate. The rice genome encodes four putative genes for Glutamate dehydrogenase (OsGDH1-4). There is differential regulation of OsGDH family members that takes place under P and N deprivation (Qiu et al. 2009). Overexpression of Novel TrGDH (Trichurus GDH) in rice results in improved plant growth and grain weight per plant (Du et al. 2019).

Aspartate aminotransferase (AAT; aspartate:2-oxoglutarate aminotransferase) catalyzes the reversible transamination reaction of glutamate and oxaloacetate to form aspartate and 2-oxoglutarate. AAT plays a key role in the C and N metabolism in plants. In rice, overexpression of aspartate aminotransferase (AAT) using CaMV 35S promoter enhanced AAT activity in the leaf and higher protein and seed amino acid content (Zhou et al. 2009). Another enzyme, asparagine synthetase (ASN) forms asparagine by condensing ammonia and aspartate or through the transamination of glutamine and aspartate. In Arabidopsis, ASN is encoded by three genes: ASN1, ASN2, and ASN3 (Gaufichon et al. 2016). In Arabidopsis, Asparagine synthetase 2 (ASN2) overexpression leads to amino acid accumulation under NH4+ as a sole N source, which depicts the role of ASN2 in NH4+ detoxification and N assimilation. Asparagine content was reduced in plants that under-express ASN2 (Igarashi et al. 2009). On overexpressing OsASN1 in rice enhanced the expressions of OsAMT1;1, OsAMT1;2, and OsAMT1;3. In overexpressed lines, Asparagine content in grain was enhanced to 3.4-fold, and N content in grain increased to 140% as that of wild type. (Lee et al. 2020a, b). Also, asparagine synthetase 4 (ZmTHP9) overexpressed maize lines exhibit enhanced asparagine content and seed protein content (Huang et al. 2022a, b). Furthermore, alanine:2-oxoglutarate aminotransferase (AlaAT) catalyzes the reversible transamination of glutamate and pyruvate to produce 2-oxoglutarate and alanine in the plant cytoplasm and mitochondria. Map-based cloning reveals floury endosperm12 (flo12) encodes alanine aminotransferase1 (OsAlaAT1) in rice. However, the starch synthesizing genes downregulated in flo12 mutants. Further, the amylose content was reduced, and protein content was enhanced in flo12 grain. This study depicts how OsAlaAT1 regulates the C and N metabolism (Zhong et al. 2019).

Future Prospects

For developing nitrogen use-efficient crops, the genes involved in N uptake, mobilization, and translocation need to be stacked and expressed inside the crop plants (Fig. 3). For enhancing NUE in crop plants, tissue-specific, inducible promoters could be utilized. Further, studies have revealed that enhancement in NUE is linked with C metabolism (Behera et al. 2023; Jin et al. 2022; Rosolem et al. 2022). Therefore, the C and N metabolism genes could be stacked together to get desired and consistent results.

Fig. 3
figure 3

Targeted approaches for improving NUE in crop plants

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The microbial communities are involved in biological N fixation and its uptake. For example, mycorrhiza mobilizes N and P and is also a reservoir of C (Scartazza et al. 2023). Additionally, the microbial genes involved in N fixation and uptake could be extensively screened, and desired NUE genes could be isolated from microbes. Furthermore, these genes can be transferred to crop plants for enhancing NUE. In addition to that, the mutant alleles imparting high NUE in crop plants could be made using CRISPR/Cas9 gene editing technology and mutant alleles imparting high NUE could be transferred in elite crop cultivars through conventional breeding. However, field trials of reported candidate genes associated with NUE need to be extensively conducted to reach at a final conclusion. Moreover, this study aids in understanding recent advancements and developments in improving crop NUE and yield. Therefore, the genetic approaches mentioned in this study can be adopted to obtain “tailored crop plants” having efficient NUE capabilities.

Concluding Remarks

In the present scenario of high N input agriculture, one of the major bottlenecks that dampen the farmer’s confidence and increase the agriculture–associated costs are the low NUE of crop plants (Govindasamy et al. 2023). Thus, in this scenario, one of the suitable solutions for maintaining crop productivity without increasing fertilizer usage is the development of crop plants with improved NUE via transgenic and gene editing technology (Karunarathne et al. 2022; Zhang et al. 2021; Sinha et al. 2020b; Fiaz et al. 2021). Moreover, intensive cultivation, adoption of high-yielding crop varieties, and hybrids in agriculture require enormous application of nitrogenous fertilizers in soils (Shukla et al. 2022). The N-efficient and responsive crop varieties need to be developed to reduce the nitrogen requirement. Developing such varieties is a bit challenging and complex task for the breeders. The identification and incorporation of potent genes, alleles, and QTLs for efficient nitrogen use efficiency into cultivated varieties through genetic engineering methods could be a promising alternative to minimize the use of fertilizers in agriculture (Karunarathne et al. 2022; Dong et al. 2022).

The cross-talk pathway related to nitrogen use efficiency needs to be modified to enhance NUE in crop plants. The carbon–nitrogen and nitrogen-phosphorus could be the prime targets for improving nitrogen use efficiency in crop plants. The most potent target for enhancing NUE could be the nitrogen transport pathways; the key transporters and activators of such pathways must be modified to channel nitrogen molecules into plant systems. Apart from this, the prime target could be the signalling pathway that directly/indirectly regulates nitrogen uptake, transport, assimilation, and translocation The geneticist and plant biotechnologist’s role is crucial for modifying the pathway in a specific and tailored way. The key genes involved in the pathway could be stacked to get a better phenotype for nitrogen use efficiency in crop plants. The developed designer N uses efficient crop plants, so-called super crops could provide a path and road map for an evergreen revolution.