Improving Nitrogen Acquisition and Utilization Through Root Architecture Remodelling: Insight from Legumes

In crop species, nutrient deficiency severely damages plant growth and developmental processes, leading to end-yield penalties. Root architecture remodelling is considered a key factor underpinning nutrient-poor soil environments. Adequate nitrogen (N) supply can play a significant role in sustaining crop productivity on nutrient-deficient soils. However, excessive application of nitrogenous fertilizer may pollute the soil and increase the production cost for the growers. To tackle this problem, crop breeders have made tremendous efforts to improve the N-use efficiency of agricultural crops. This article summarizes the recent progress in identifying QTLs/genes, regulatory pathways, and hormonal crosstalk involved in the growth and development of legumes roots system. Moreover, we have described the progress in microbe–root symbiosis via QTLs/genes regulations, which results in improved N acquisition. Understanding the molecular mechanisms that regulate the root architecture in response to N availability may help to strengthen the root system of legumes and promote environmental friendly and sustainable agriculture.


Introduction
Legumes play an essential role in contributing to food security and environmental maintenance (Graham et al. 2003). They are usually intercrop with cereals to improve land productivity through soil amelioration. In crop rotation, legumes as N 2 -fixing plant contribute to diversify the cropping system. Although legumes fix nitrogen; about 50% of soilnitrogen still needs to utilize efficiently by plants to improve and maintain crops yield at optimal levels. Nitrogen (N) is an essential mineral required in massive quantity by plants to produce sufficient energy for sustaining vegetative growth and achieving economic grain yield (Zörb et al. 2018;Wang et al. 2018).
The root is the primary organ modified in plants to acquire nutrients and water from the soil (Ramakrishna et al. 2019). The soil medium in which plant roots penetrate is highly heterogeneous in the distribution of minerals and water reservoirs. Plants have adopted various tactics to alter their root architecture in response to these heterogeneous distributions (Xu et al. 2022;Poitout et al. 2018). The root system architecture is affected by various abiotic and biotic Handling Editor: Francesca Resentini.
Muhammad Nadeem and Muhammad Yahya have contributed equally to this work.

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factors (Najeeb et al. 2015), including water starvation, N, potassium, and at phosphorus levels. Plants have evolved to survive under low N levels through long-term evolution, which might have involved the modifications in the root configuration, such as the primary root (PR) length, lateral root (LR) system, and root hairs structure (Rao et al. 2016;Qin et al. 2021).
Untangling the molecular mechanism, including various regulatory networks, proteins, and genes responding to low N stress, significantly improve the low N stress tolerance crop. A large number of genes are involved in the modification of root systems to enhance minerals acquisition in different plant species, such as maize (Li et al. 2011, arabidopsis (Yu et al. 2016), soybean (Kong et al. 2019), and rice Kitomi et al. 2020). Furthermore, regulatory proteins (Araya et al. 2014), transcription factors (Devaiah et al. 2007;Architecture et al. 2012;Miura et al. 2020), and miRNAs also contribute to regulatory networks associated with the root system to improve nutrient-use efficiency (Meng et al. 2010;Vidal et al. 2010). In addition, the identification of numerous root architecture QTLs for N starvation in different crops including bean/common bean (Cichy et al. 2009), rice (Shimizu et al. 2008;Li et al.2009), maize , and soybean  highlights the potential to improve nutrient use efficiency via marker-assisted breeding. This review provides a comprehensive overview of root architectural alterations under N-deficit conditions. It will help to modulate N deficiency molecular mechanisms for developing N-use efficient varieties (NUE) with superior root architecture.

Importance of Legumes in Conservation Agriculture
Grain and forage legumes are grown on approximately 180 million ha or 12% to 15% of the earth's farmland (Graham et al. 2003). Legumes account for 27% of the total global primary crop production, contributing 33% to the human needs for dietary proteins (Graham et al. 2003). Further, legumes cultivation improve soil fertility and organic matter contents and enhance soil water retention and nutrients circulation (Rasheed et al. 2010;Graham et al. 2003). Therefore, sustainable increase in legumes production is critical to feed the world's growing population. Likewise, by adopting sustainable agricultural production methods, we can minimize greenhouse gas emissions, food losses, and wastes, improve crop productivity and the global supply chain, and provide nutritional food to communities suffering from hunger and malnutrition. Therefore, introducing legumes in sustainable agriculture may offer a solid foundation for food safety, and security along with environmental quality (Vanlauwe et al. 2019).
The key advantages of legumes are; they fix atmospheric N, high quality of organic matter released into the soil, and improve soil C/N ratio (carbon-to-nitrogen ratio). Additionally, grain legumes have a robust and deep root system that encourages mycorrhizal mineral solubilization, recycling/ uptake, and water transport in deeper soil layers (Graham et al. 2003). Recently, some legumes such as faba bean, pea, chickpea, soybean, and lentil have widely been adopted in sustainable cropping systems and conservation agriculture in Turkey, Australia, Brazil, and North America (Stagnari et al. 2017).

Nitrogen (N) and Sustainable Crop Production
Sustainable agriculture refers to crop production which does not harm biodiversity, quality of crops, and the environment. Sustainable crop production relies on minimizing the pesticide usage through integrated pest management. Thus, it protects biodiversity, improves soil health, and ensures food quality and safety. N is an essential constituent of a plant cell as a mineral element and directly promotes protein formation in grain seeds (Worku et al. 2007;Perchlik and Tegeder 2020). It is also linked with crop yield and its accumulated amount in the plant is an essential factor for crop productivity (Khan et al. 2020). Considering this, NUE is a crucial parameter for crops yield, unfortunately, its usage efficiency is relatively low, e.g. accounts for 30% in China, 50-60% in developed countries, and approximately 59% globally (Williamson 2011). This significant loss has been causing a worse impact on our natural environment in the form of acid rain, soil acidification, polluting freshwater streams, and even air pollution, which ultimately raises human health issues (Bouwman et al. 2013;Udvardi et al. 2015).
Recently, various new high-yielded cultivars have been developed and they are primarily dependent on high input of nitrogenous fertilizers. These cultivars have been introduced into the cropping systems according to the preferences of breeders and farmers. However, increased application of nitrogenous fertilizers led to several problems, such as decreased N-use efficiency, high input cost, environmental pollution, and N fertilizer loss . Consequently, it is now a consensus that it is necessary to balance the benefit of N usage to improve yield and reduce its adverse impacts by reducing the N fertilizer input and soil pollution. Hence, developing cultivars with tolerance to low N stress or improved NUE are critical aims for future crop breeding . Achieving these goals will necessitate a comprehensive knowledge and understanding of N metabolism under N deficiency. Root-related traits have considerable influence on N capturing from the soil. However, there are still discrepancies over the impact of the 1 3 N deficit on root growth, length, and LR. Therefore, new cultivars with improved root architecture that can be grown under low N are prerequisites for sustainable agriculture (Xu et al. 2012).

Root Architecture Remodelling for Better N Acquisition
Plants alter their root system to extract N from the soil (Lay-Pruitt and Takahashi 2020). They can adjust to low N supply by directing root development towards N-rich patches and increasing the root absorptive surface area. Considering the low nitrate availability and high mobility within a given soil area, the definition of a more efficient root system may not be specific. It may differ and depends on soil type, plant species, and other environmental factors (Postma et al. 2014). Modelling techniques proposed that an effective capturing of nitrate results from a trade-off between the total volume of soil explored and the speed of N acquisition (Dunbabin et al. 2003).
A superior root architecture helps plants to explore soil and promote nutrients absorption. Therefore, the improvement of root architecture has widely been documented and considered an essential strategy to improve N uptake under N-deficient environments, which may accelerate the performance of roots in low N soils. N is an integral component of organic compounds in plants comprising protein, chlorophyll, and nucleic acid (Amtmann and Armengaud 2009). Thus, N deficiency is the main factor inhibiting plant growth and development, resulting in a significant drop in crops yield and productivity. Plants use different N forms in the rhizosphere, including nitrate, organic compounds (soluble N-containing), and ammonium (Tegeder and Rentsch 2010). In arabidopsis, for instance, the roots are adapted to different N supply levels and types, including stimulation of root branching, inhibition of LR initiation under a high C/N ratio, systemic/local regulation of LR growth, and inhibition of PR growth induced by exogenous L-glutamate (Zhang et al. 2007). In response to low N supply, nutrient acquisition is mainly enhanced through root growth and the deeper root system to absorb nitrate, which is one of the most movable nutrient ions in the soil (Ju et al. 2015;Rasmussen et al. 2015;Yu et al. 2015). Literature suggests that genotypes with deeper roots are more efficient in absorbing N from N-deficient soils (Saengwilai et al. 2014). Therefore, an ideotype of root architecture with strong LR, deeper roots, and robust nitrate response was proposed for effective N acquisition in intensive cropping systems (Zhang et al. 2007). A field trial-based study supported this by revealing that a broad and deep root system is a prerequisite for high N consumption in Zea mays (Mi et al. 2010). In legumes, modifications in root system architecture facilitate N and phosphorus acquisition and nodule formation (Egamberdieva et al. 2017). In a study, Egamberdieva et al. (2017) observed a significant positive relationship between the N content in plant tissue, the number of nodules, and the root system architecture of soybean.

Molecular Mechanisms of Root Architecture Remodelling in Response to N
Understanding the molecular mechanism of modifications in the root architecture of plants in response to specific nutrients would facilitate the genetic improvement of nutritional efficiency. In past years, many experiments have been designed to identify multiple QTLs/genes in plants related to changes in root architecture to cope with nutrient supply (Table 1).
Plants absorb N from the soil in two forms: organic (amino acids) or inorganic (ammonium or nitrate) (Hao et al. 2020). In ammonium foam, root growth is controlled by ammonium transporters (ATMs), they are crucial to optimize ammonium concentration in plants and regulate root responses to avoid ammonium toxicity (Hao et al. 2020). Legumes AMTs have functionally been characterized, and their transport properties are similar to those identified in arabidopsis (D'Apuzzo et al. 2004). In arabidopsis, two ATM families (ATM1 and ATM2) were identified (Yuan et al. 2007) and ATM2 family is involved in regulatory mechanisms. This is essential in recovering ammonium lost from nodule cells by efflux, whilst ATM1 regulates ammonium acquisition and transport. Two ATM2s and three ATM1s have been identified in Lotus japonicus (Salvemini et al. 2001). Under N-deficit conditions, LjAMT1;1 and LjAMT1;2 are up-regulated, LjAMT1;3 is down-regulated by higher ammonium concentrations and it regulates the root response to ammonium toxicity (Apuzzo et al. 2004;Rogato et al. 2010). LjAMT2;1 controls ammonium loss from cellular efflux in nodules, whereas LjATM2;2 is required for N transport and acquisition during AMF associations (Simon-rosin et al. 2003). Ammonium negatively influences the nodule formation by repressing the expression of NIN (NODULE INCEPTION), an essential gene for nodulation (Barbulova et al. 2007). When ammonium is the sole N source, primary and LR elongation suppression is a commonly observed indicator of ammonium toxicity (Araya et al. 2016).
On the other hand, nitrate is the primary N source and absorbs through roots by various nitrate transporters. Several aspects of nitrate-dependent root architecture regulation have been discussed and reported in recent reviews (Imin et al. 2013;Forde 2014;Giehl et al. 2014). The modulation of root architecture in an N-deficit environment depends on the environmental conditions (day length and light intensity) and the strength of N limitation. The root development is restricted under severe N stress, leading to a reduced number of LR and shorter PR, whereas LR and PR length increased under mild N stress (Araya et al. 2014;Gruber et al. 2020). In a past study, arabidopsis transcriptome data comprising about 100 root growth-related genes revealed that N stress triggers MDR4/PGP4 and WAK4 expression levels (Lally et al. 2001;Terasaka et al. 2005;Giehl et al. 2014). Both genes modulate PR growth and LR development and might be involved in response to mild N stress. Whilst, down-regulation of ACR4 and AXR5 reduced LR formation, it might depict that these two genes are constraints to LR formation under severe N stress ( Fig. 1B; Yang et al. 2004;Smet et al. 2008). Two families of transporters genes (NRT1 and NRT2) participate in nitrate transport in root and shoot cells (Criscuolo et al. 2012). Nitrate transporters with sensing functions are involved in signalling pathways. For instance, NRT1.1 acts as a transceptor of NO 3 have a dual-sensor/ transporter role in signalling pathways and could regulate LR formation under the low/absence of NO 3 -. While in N starvation, AtNRT2.1 acts as NO 3 signalling component or sensor inhibiting LR initiation. Although the definite molecular mechanism is yet uncertain, negative role of NRT2.1 and NRT1.1 in LR initiation and formation may represent a distinct systematic pathway under N starvation. Nitrate sensor/ transporter NRT1.1 is also involved in the auxin-mediated signalling pathway related to root structure changes. In contrast, nitrate sensor/transporter NRT2.1 has dual functions relevant to the coordination of nitrate availability in nitrate absorption and LR growth (Remans et al. 2006b). The NRT1/NRT2 families are also reported in the model legumes, including L. japonicus (Criscuolo et al. 2012). L. japonicus was found to have 92 NRT1 genes and four NRT2 genes. The expression pattern of three NRT2-type transporters in another model legume species, namely M. truncatula have also been reported (Pellizzaro et al. 2014). Some family members require a protein partner (NAR2 or NRT3) to function in legumes (Tong et al. 2005). Moreover, in soybean, overexpression of GmNRT1.2a and GmNRT1.2b in leaves induced by nitrate reported to increase the nodule numbers (Guo-ji et al. 2020).
Recent literature also suggests that beta-expansion protein gene plays an important role in regulating nodulation and root architecture. For example, in soybean overexpression of GmEXPB2 resulted in a longer root, larger root hair Improve lateral root growth (Imin et al. 2013) CDC16 Improve lateral root growth (Kuppusamy et al. 2009) NOOT/COCH Improve root growth and development (Couzigou et al. 2012) CRA2 Involve in root growth (Huault et al. 2014) 1 3 Fig. 1 Improved root architecture for better N acquisition. A Diagrammatic representation of modified root architecture for enhanced N uptake and symbiotic host-microbe interactions in response to N availability. B Signalling pathways control LR initiation, emer-gence, and elongation in response to low NO-3 or N-deficit response. Blunted lines and arrows represent interactions that are either negative or positive 1 3 area, and dense root hair and significantly improved the frequency of root hairs attachment of rhizobia ). The auxin biosynthesis TAR2 gene regulates low N-mediated reprogramming of root architecture in arabidopsis (Ma et al. 2014), supporting the vital role of auxin in regulating root architecture in response to N accessibility. Despite the evidence from the literature that several signalling pathways link soil N accessibility and plant N status to root structure ( Fig. 1B; Walch -liu et al. 2005;Zhang et al. 2007), only a few genes were observed to play a role in this process. For example, in arabidopsis, plasma membrane proton pump along with CLE peptide, AHA2 and CLV1 regulate the response of root growth to N availability (Araya et al. 2014). Besides these mechanisms, NRT1.1 also signals to activate gene expression of a MADS-box transcription factor ANR1 to promote LR elongation in response to local NO 3 supply (Remans et al. 2006a). The induction of NRT2.1 and potentially ANR1 gene expression occurs downstream of NO 3 and NRT1.1-induced Ca 2+ signalling, promoting the nuclear localization of the transcription factor NLP7 ( Fig. 1B; Krapp et al. 2014;Zhang et al. 2020). In Lotus japonicus, MAMI gene is evolutionarily relevant to GARP transcription factor aided in root development (Volpe et al. 2013). Large number of genes have been identified which are involved in regulating legume nodulation and root architecture remodelling. For example, LjHAR1 in Lotus japonicas and SIN1 in common bean play an essential role in root and nodule development (Battaglia et al. 2020). Furthermore, many genes, including LATD (Bright et al. 2005), SUNN (Schnabel et al. 2005), LATD/NIP (Harris and Dickstein 2010;Yendrek et al. 2010), NOOT/COCH (Couzigou et al. 2012), microRNAs (miRNA160/166) (Boualem et al. 2008;Bustos-sanmamed et al. 2012), and genes encoding cell cycle proteins (cell division cycle 16/CEP1/CRA2) were identified, which affect the growth and development of roots and nodules in Medicago truncatula (Kuppusamy et al. 2009;Huault et al. 2014). Imin et al. (2013)  In addition, microRNAs also have role in response to nutrient deficiency stress. In arabidopsis, two microRNAs, miR393, and miR167, were reported to regulate AFB3 (miR393) and ARF8 (miR167) adjust the changes in root architecture to cope with the changes in N supply (Vidal et al. 2010). In soybean, miR172 regulates root nodulation (Yan et al. 2013). Furthermore, an exogenous application of synthetic miPEP172c has been proven to stimulate actual miR172c expression, increases the number of formed nodules (Couzigou et al. 2016). Though role of miRNAs in the regulation of nodulation has been examined in previous studies; however, a link between nodulation and N status in legumes has yet to be defined. This is a novel area of research, and the detailed function of miRNAs in regulating root structure remains to be explored.

Improvement of Root Architecture Remodelling Associated with Symbiosis
Globally, more than 80% of plants including agricultural crops colonized through arbuscular mycorrhizal fungi (AMF) (Gianinazzi-Pearson et al. 1995). They expand the ability of roots to excavate necessary micro-and macronutrients from the soil and establish a symbiotic relationship that can significantly promote plant growth. In symbiosis, hyphae pass through the root's cortical cells to form well-differentiated arbuscules and exchanges minerals with the host root and AMF ( Fig. 1A; Parniske 2008). Another crucial step in the symbiotic relationship is to promote the acquisition of N and formation of legume nodules, where rhizobia fix N and provides plants with N-containing organic compounds. The first symbiosis event is just an interaction of chemical signals between bacteria and roots. Afterwards, bacteria attach to the roots, potentially cause hairs to curl, and thus produce colonies and form N-fixing nodules (Fig. 1A) (Oldroyd and Downie 2008). Studies show that AMF colonization of deep root soybean genotypes were more significant under low phosphorus, and the nodulation effect was better than that of shallow root genotypes under high phosphorus conditions (Wang et al. 2011). These results may suggest that the root architecture is linked in forming a symbiotic relationship between root rhizobia and AMF.
Under low nutrient supply, there are two types of root architecture modifications associated with rhizobia or AMF (Fig. 1A). In type I, AMF promotes root growth, improves density and length of LR, and increases the dry weight of roots (Berta et al. 1995;Yu et al. 2015). In type II, rootrhizobium symbiotic association, like in soybean infected by AMF, inhibits root growth with reduced total root length, surface area, and root volume (Wang et al. 2011). This growth inhibition may be due to the carbon cost of nodule formation, maintaining N 2 fixation and the effect of AMF on the plant-or fungal species-dependent root architecture remodelling.
Further, the effect of AMF on plants probably fluctuates by the changes in root architecture. For example, in rice, larger rather than thinner LR were mainly colonized through AMF (Gutjahr et al. 2009), and the mycorrhizal colonization rate of taproots were higher than fibrous roots . These findings indicate that the taproots may be more suitable for mycorrhizal colonization than the fibrous roots. Also, rhizobia-inoculated legumes could change their root architecture, producing smaller roots than un-inoculated plants . One possible explanation is that nodules compete with host roots for carbohydrates because nodules and N fixation consume more carbohydrates and nutrients (Aleman et al. 2009;Reich et al. 2013). Therefore, in the nodulation process, better root growth and sufficient supply of mineral nutrients may contribute to develop healthy nodules and, thus, improve final crops yield.

Hormonal Remodelling of Root Architecture in Legumes in Response to N
Plant endogenous hormones perform a decisive role in regulating root growth and LR formation ( Fig. 2; Vissenberg et al. 2020;Ubeda-Toma's et al. 2020). Auxin and cytokinin were found to crosstalk antagonistically or synergistically during plant growth and development. For instance, auxin and cytokinin have a positive role in root/stem growth (Schaller et al. 2015;Liu et al. 2017). Contrastingly, both hormones have a hostile function during LR development.
Auxin is an important hormone that plays a noteable role in nitrogen-mediated root growth and regulates developmental processes in plants. A high N level is assumed to decrease the local auxin accumulation, suggesting that auxin is a central element in the shoot-root signal of N availability (Okushima et al. 2011). Higher shoot N levels are believed to inhibit shoot-root auxin transport, resulting in lower LR numbers in arabidopsis (Reed et al. 1998;Forde 2002). However, studies on Medicago truncatula show that higher N levels in the shoot have improved the auxin shootroot transport . This response was further emphasized by the sunn-1 Medicago mutant, which has insensitive auxin shoot-to-root transport regardless of N concentration. In sunn-1, no correlation was observed between LR regulation and N-mediated auxin transport. However, SUNN-mediated auxin shoot-root movement only applies to nitrate-dependent LR remodelling but not in nodulation, indicating that auxin-mediated N regulation of nodule acts locally in the root . Auxin-mediated root growth also includes interaction with other hormones, such as ethylene.
Ethylene, a gaseous phytohormone, is a positive regulator of roots and engages in local nitrate-mediated root development. In many plant species, a higher nitrate level promotes root-ethylene progression (Caba et al. 1998;Tian et al. 2009). On the other hand, high ethylene limits LR development and nodule formation, whilst low ethylene level improves the LR growth and nodulation ( Fig. 2; Nukui et al. 2000;Oldroyd et al. 2001). Many rhizobia species limit localized ethylene by producing ethylene precursor or creating aminocyclopropane deaminase, which breaks down aminocyclopropane to promote nodulation response (Ma et al. 2002). Additionally, ethylene imparts positional control of nodulation because nodule formation is favoured by higher ethylene levels in the opposing phloem poles (Heidstra et al. 1997). The regulation of nodules and LR by ethylene, most likely to occur through the regulation of cell cycle pathways (Dan et al. 2003;Spadafora et al. 2012). Ethylene influences the cell cycle by interacting with cytokinin, which is also involved in nitrate-mediated root development (Spadafora et al. 2012). Cytokinin, another crucial plant hormone that regulates the cell cycle and mediates N status between roots and shoots via the phosphorelay pathway (Sakakibara et al. 2000). The cytokinin synthesis in N-starved roots is improved by nitrate supply, which goes into the shoots and signals the N status in roots. Cytokinin is moved back to roots to signalling the low N level in shoots as shoots N supply decrease (Sakakibara et al. 2000;Ruffel et al. 2011). Cytokinin acts directly on LR founder cells to inhibit LR initiation ( Fig. 2; Li et al. 2006;Laplaze et al. 2007). However, higher cytokinin levels stimulate LR elongation once LR differentiation occurs (Li et al. 2006). Elongation of LR is also triggered via higher nitrate; it could be fascinating to study further nitrate-cytokinin crosstalk during LR formation. In legumes, exogenous cytokinin application triggers several genes related to nodulation, as cytokinin acts as an upstream component of the nodule formation pathway (Fang and Hirsch 1998;Gonzalez-rizzo et al. 2006). According to studies, Medicago and Lotus cytokinin mutants had diminished nodule formation, whereas mutants with repaired function showed increased nodule formation (Gonzalez-Rizzo et al. 2006;Murray et al. 2007;Tirichine et al. 2007).

Approaches for Improvement of low N Tolerance
Recent advancements in plant biotechnology could provide a way to identify genomic regions possessing natural genetic variations (NGV) and transform these targeting alleles into new elite breeding lines to generate NUE lines. QTLs associated with root architecture systems (RSA) have also been identified in many major legume crops, including soybean, chickpea, lentil, and alfalfa (Abdel-Haleem et al. 2011). Some QTLs/genes could have lost during domestication processes, maybe due to linkage with negative loci or a pleiotropic effect on yield. It is therefore necessary to carry out a fine mapping that may investigate the precise cause of the adverse impact on the yield, caused by pleiotropy influence or linkage with any other adverse loci. For that, a sizeable segregant population can be used to break the tight linkage amongst them. Furthermore, functional characterization of these genes using NGV could be an appropriate technique to thoroughly understand the physiological and molecular pathways of stress adaption mechanisms to improve N-use efficiency in legumes.
Novel high-throughput genotyping and phenotyping approaches have the potential to monitor RSA and are indispensable for enhancing a better understanding of N-use efficiency. These field-based, cost-effective approaches have recently been utilized in some major field crops such as maize, wheat, and rice for phenotyping of RSA (Trachsel et al. 2011;York and Lynch 2015;Richard et al. 2015;Wissuwa et al. 2016) and also in some legume species, namely cowpea, common bean, and soybean (Manavalan et al. 2015;Prince et al. 2015). Furthermore, the efficiency and accuracy of RSA phenotyping can be accelerated by adopting highthroughput image analysis through digital imaging of root traits (DIRT) (Das et al. 2015).
In the past decade, legumes were considered amongst minor crops with limited genomic resources. However, as sequencing technology has improved, the scenario for legume crops has entirely transformed, and they now belong to a group of crops that are rich in genomic resources. This has resulted in a new initiative to enhance the world food production by breeding legumes, which is mainly based on genomic-assisted breeding (GAB) projects. Marker-assisted selection (MAS) and genomic-based selections are the most prevalent methods in legumes breeding (Pandey et al. 2016). The MAS technique can effectively improve traits controlled by only one or two genes. But, MAS is not considered suitable for complex traits as a genomic technique. In comparison, genomic-based selection has recently been applied to improve complex breeding traits due to its efficiency.
Breakthroughs in high-precision genotyping and phenotyping have allowed a more accurate selection of targeted genotypes and reduced the breeding cycle time by eliminating unnecessary phenotyping observations. Genomic selection technique integrates the minor and massive impact on genetic factors, and it could be advantageous for speeding effective selection gain. Additionally, cost-effective genotyping has created a diversified gene pool in legume crops, which is crucial for breeding new genetic traits (Jain et al. 2013). With emerging gene-editing tools like the CRISPR/ Cas9 system, plant scientists have a better chance of reaching their goals, especially for the specific traits they want to change (Jacobs et al. 2015;Sun et al. 2015). CRISPR/ Cas9-mediated genome editing has been reported in Lotus japonicus , Vigna unguiculata (Ji et al. 2019), Medicago truncatula (Meng et al. 2016), and Glycine max with some desirable results Sun et al. 2015). So, this new revolution in genome editing could modify RSA-related alleles to enhance their functionality in the required breeding environment. With these advanced genomic sequences, a massive database for various legume crops has been generated to identify critical regulatory genes, transcripts, and proteins, as well as gene families that contribute in stress tolerance. The availability of wider genetic diversity for major legume crops may smooth the way for digging out traits that can facilitate haplotypeassisted breeding. Therefore, incorporating broader variability of targeted traits from newly found genomic resources and technologies into advance breeding programmes would improve crop yield under nutrient-deficit conditions.

Advances in Breeding for low N Tolerance
Plants with a robust root system can extract nutrients from the soil efficiently and enable them to maintain normal growth in N-deficit conditions. For instance, root architecture of soybean is the key characteristic for sustaining yield in N-deficient environments. A better subsoil exploration owing to stronger and deeper roots has been proven to enhance the yield of chickpeas under stress conditions (Kashiwagi et al. 2015). Several root traits, including root diameter, length, root volume, and surface area were studied to determine the importance of root architecture in sustaining legume yield in a nutrient-limited environment (Fried et al. 2019). Root traits directly impact legume productivity under N-deficit conditions, suggesting that root architecture is a desired trait for further breeding initiatives. Although many recent studies have been conducted to identify QTLs/ genes and genetic diversity that influence root architecture, but fewer attempts to exploit these resources in breeding programmes. Efforts are currently being made to introduce the genomic region to various other elite legume lines (Pandey et al. 2016). Legumes are revealed to have abundant genetic resources for root architecture. Unfortunately, these genetic resources have not yet been exploited in crop stress breeding programs to develop better lines. Thus, genotypes with a better and robust root system should be exploited to develop breeding lines with improved root architecture under nutrient-poor environments.

Conclusion and Future Prospects
Extensive and rapid root growth is crucial for plant survival in unfavourable environments, allowing plants to access nutrients and water more efficiently. Furthermore, root growth angle, which is connected to the extraction of water and nutrients; is a fundamentally significant characteristic impacting the root system architecture of legumes. Therefore, a greater understanding of key genes associated with root structure and their control would allow researchers to use marker-assisted selection to develop new genotypes with robust root system. Significant progress has recently been made in understanding the genetic bases of the root system in legumes, primarily through the identification of QTLs.
Many genes that regulate root architecture have been identified, but the molecular mechanisms of root hair formation, LR development, and root elongation are still poorly understood. With the rapid advancements in molecular approaches, including transcriptomics, genomics, phenomics, and proteomics, researchers will be able to understand the underlying molecular mechanisms which regulate the roots system. A proposed approach to integrate the above-mentioned multidisciplinary strategies for enhancing legumes NUE is summarized in Fig. 3. Despite the fact that identifying the targeted QTLs with moderate effects is difficult, but cloning these genes using QTL analysis is an effective strategy to select candidate genes for future breeding programs. In addition, genome-wide association studies, Fig. 3 A proposed strategy for improving N acquisition and utilization by integrating multidisciplinary approaches often known as GWAS, have been proven a realistic and feasible way to find alleles of existing genotypes, which can be exploited in molecular breeding. Using these new strategies, numerous QTLs have been identified comprising candidate genes for root development. Unfortunately, GWAS and QTL mapping have limitations in cloning genes which regulate the root development. The root phenotypes grown in agar/gel or hydroponic conditions do not precisely reflect their growth when sown in the soil; this constraint makes it tougher to perform root phenotyping in natural soil environment. Exploring the symbiotic relationship between microorganisms (rhizobia and AMF) and host plants would be another impotent approach to speed up NUE. Roots are the primary site of rhizobial and AMF infection; therefore, the root system may play a vital role in colonization. Although the molecular mechanisms of the rhizobial and AMF symbiosis and signalling transduction have been identified, complex regulatory mechanisms and various other components are still unknown. To develop NUE cultivars, it is necessary to combine omics approaches with traditional breeding and effective agronomic strategies (Fig. 3). For a more precise assessment of root system, it is also necessary to investigate the root architecture in complex and natural soils using cutting-edge techniques, like magnetic resonance imaging and X-ray microcomputed tomography.
Author contributions MN and MY, conceived and designed the contents. MN, MY, JT, LS SK, AA, AS, AW, and NU wrote and revised the manuscript. All authors read and approved the final manuscript.
Funding Open Access funding enabled and organized by CAUL and its Member Institutions.

Conflict of interest
The authors declare no competing financial interest.
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