Crops produce a large proportion of food and nutrients for humans in modern society. During the past 50 years, the application of chemical fertilizers and the Green Revolution have substantially increased crop production, maintaining food security for the increasingly growing population (Godfray et al., 2010). However, a continuous increase in crop output cannot sustainably be achieved by excessive amounts of fertilizer because of the limited nutrient use efficiency (NUE) of crop plants. Importantly, a large amount of wasted resources and environmental pollution resulting from excessive fertilizer applications cannot be ignored (Springmann et al., 2018). For these reasons, crop cultivars that have relatively high NUE are environmentally friendly and helpful to conserve resources for sustainable agriculture. Roots, the major organs for plant anchorage, nutrient and water absorption, and interactions with symbiotic organisms, have attracted increased amounts of attention in recent studies given the discovery of their correlations with nutrient use and crop yield (Gewin, 2010). In general, manipulation of root architecture traits, including root number, root length, root surface area (RA) and root volume (RV), especially the spatial configuration of the root system in the soil, is an essential approach to increase crop yields, which may represent an important component of a new green revolution (Lynch, 2007; Meister et al., 2014). Unfortunately, root traits have been chronically overlooked in the traditional breeding due to their lack of aboveground visibility (Maqbool et al., 2022).

Bread wheat (Triticum aestivum L.) is one of the most important crop species worldwide, providing ~20% of calories for human needs (Appels et al., 2018). Efforts have been made to explore the determinants of root traits that contribute to increase yield potential in wheat. In addition to traditional labor-intensive and time-consuming methods for root trait analysis (Kücke et al., 1995), artificial medium systems, such as hydroponic culture, have been used in many studies and revealed a series of quantitative trait loci (QTLs) that drive genetic variation in root traits (An et al., 2006; Cao et al., 2014; Kabir et al., 2015; Maccaferri et al., 2016; Merchuk-Ovnat et al., 2017; Fan et al., 2018; Zheng et al., 2019). However, it remains largely unclear of the mechanisms of these loci control root traits due to the complexity of the wheat genome. To date, a few genes regulating root morphology have been analyzed through reverse genetics studies in wheat. For example, the expressions of TaRSL2 (Han et al., 2017a) and TaLBD16 (Wang et al., 2018a) were shown to be positively correlated with root hair length and number of lateral roots, respectively. Heterogenous expressions of TaMPK4 (Hao et al., 2015) and TaMOR (Li et al., 2016) demonstrated their positive effects on root development. Overexpression of the transcription factor (TF)-encoding genes TaNAC2-5A (He et al., 2015), TaNFYA-B1 (Qu et al., 2015), TaNAC69-1 (Chen et al., 2016) and TaWRKY51 (Hu et al., 2018) resulted in enhanced root growth, while TabZIP60 (Yang et al., 2019) had the opposite effects, implying a complex genetic basis of the formation of root traits. In addition, circular RNAs have been shown to participate in root length regulation (Xu et al., 2019).

At present, a holistic view of the root growth regulatory network and how it affects root traits and yield potential is strongly needed for molecular breeding. RNA sequencing (RNA-seq) makes it possible to explore the consistency between root morphology and its underlying mechanisms at the whole-plant level through DEG analysis. For example, the transcriptomic analysis revealed how the root growth of salt-tolerant bermudagrass was maintained under salinity stress; the salt tolerance was attributed to appropriate expression levels of genes involved in reactive oxygen species (ROS) homeostasis and cell wall loosening, which were regulated in part by a number of TFs linked to stress responses and growth regulation (Hu et al., 2015). DEGs detected in maize cultured under different nitrogen (N) levels highlighted the network of root growth in response to N deficiency, where the LBD TF-encoding gene participated in the balance between nutrient metabolism and the stress response (He et al., 2016). RNA-seq data suggested crosstalk occurs among the roots (in terms of growth promotion), gibberellin (GA) biosynthesis, auxin signaling, cell division and cell wall modification in uniconazole-treated soybean (Han et al., 2017b). These findings enriched our understanding of the molecular mechanisms behind the interplay of root traits and other agronomic characteristics, helping to generate strategies for crops with improved root systems, increased NUE and improved environmental adaptation.

Previous studies have shown that Triticum aestivum KN9204has a larger root system than does cultivar J411 (Wang et al., 2011). Using a RIL population derived from the cross of KN9204 and J411 (KJ-RIL), researchers identified QMrl-7B as a major stable QTL that controls maximum root length (MRL) (Fan et al., 2018), and its contribution to large root systems was dissected (Liu et al., 2021), prompting the question of how QMrl-7B regulates root architecture and N uptake. In the present study, transcriptomic analysis of the pairwise QMrl-7B NILs was performed to further analyze the function of QMrl-7B in root development. The results will help reveal the pathways controlling root development that are affected by QMrl-7B in wheat, providing insights into the genetic and physiological basis underlying desirable root traits for agriculture.

Improving plant roots is an effective way to increase NUE and minimize fertilizer application for sustainable agricultural development (Lynch, 2007). A number of genes/QTLs have been reported to be related to root growth and development; as such, these genes/QTLs have potential application in crop genetic improvement (Zhuang et al., 2021; Chen et al., 2022; Li et al., 2022). However, the genetic network explaining how these genes interact to influence root architecture still needs to be elucidated to assess the beneficial agronomic traits from an overall perspective and to guide the use of beneficial genes or pyramid of allelic variation in future breeding efforts. Compared with J411, KN9204 had a larger root system (Wang et al., 2011), with which the stable QTL QMrl-7B was associated (Fan et al., 2018). Further studies involving QMrl-7B NILs showed that, as compare to the NIL^J411s, the NIL^KN9204s had superior root traits, N-related traits and yield traits in field trials (Liu et al., 2021). The superiority of root traits of the NIL^KN9204 at the seedling stage was also consistently proven in the present study. When these hydroponically cultured pairwise NILs were used for transcriptomic analysis, it was found that the DEGs detected by RNA-seq encoded proteins of various groups, such as transporters, enzymes, TFs, and components involved in hormone signaling. The important DEGs that might be involved in root trait regulation are discussed here.

The genes with opposite expression patterns between the pairwise NILs under the NN and LN conditions were very likely to be involved in N-related root growth ( Tables 1 , S5 and S8 ). For example, the expression level of the class III peroxidase gene (TraesCS2B02G125500) was higher in the NIL^J411 under NN condition but was higher in the NIL^KN9204 under LN condition. Class III peroxidase uses H₂O₂ as a substrate to produce OH^- radicals that enhance cell wall plastic extensibility and elongation by nonenzymatic cleavage of cell wall polysaccharides (Cosio and Dunand, 2009). Since root development is directly related to cell wall loosening and elongation, the upregulated expression of this gene by LN in the NIL^KN9204 ( Table S14 ) was proposed to participate in LN-promoted root growth by affecting these processes. Another most enriched process of the DEGs between the pairwise NILs was phenylpropanoid biosynthesis that might affect root enlargement (Muro-Villanueva et al., 2019; Liu et al., 2020). Interestingly, the expression levels of a series of phenylalanine ammonia-lyase and cinnamoyl-CoA reductase genes increased in the NIL^J411 under both NN and LN conditions ( Tables S5 and S8 ), implying that the biosynthesis of cinnamate and lignin monomers increased, which cannot directly explain the reason for the smaller roots.

Nitrate is substantially absorbed and transported by high-affinity (NRT2s) and low-affinity (NPFs) nitrate transporters for assimilation and storage (Xu et al., 2012; Wang et al., 2018b). N stress tolerant sorghum genotypes exhibit higher expression levels of high-affinity NRTs than do the sensitive genotypes under N-limiting conditions (Gelli et al., 2014). We previously compared the expression profiles of 44 NRT2 and 217 NPF genes in the roots of KN9204 and J411 cultivated hydroponically with and without N supply and found that up to 79.2% and 43.7% of the root-expressed NRT2 and NPF genes, respectively, displayed differential expression between KN9204 and J411. Under NN conditions, more than two-thirds of the differentially expressed NRT2 and NPF genes exhibited higher expression in KN9204 than in J411, but the proportion of NRT2 and NPF genes with higher expression levels in KN9204 decreased under LN treatment, as the upregulated expression levels of most nitrate transporter genes, NRT2 family in particular, were higher in J411 than in KN9204 (Shi et al., 2022). Similarly, two high-affinity NRT genes (TraesCS6B02G044300 and TraesCS6D02G035900) exhibited lower expression levels in the NIL^J411 under NN condition, but higher expression levels in the NIL^J411 than in the NIL^KN9204 under LN condition ( Table 1 ). In addition, the expressions of four high-affinity NRT genes (TraesCS6A02G031000, TraesCS6A02G031100, TraesCS6B02G044100 and TraesCS6B02G044500) were induced specifically by LN in the NIL^J411 but not in the NIL^KN9204 ( Table 2 ). Our present study showed that LN led to altere expression of a number of genes encoding both low-affinity and high-affinity NRTs ( Table S4 ), in accordance with the viewpoint that different NRTs function at different N levels (Zhang and Forde, 1998).

Dual-affinity nitrate transporter 1 (NRT1) not only functions in N acquisition but also is a N O 3 − sensor; this action is independent of its uptake activity that controls root development (Ho et al., 2009). The cotransport of N O 3 − , peptides and hormones through the NRT1 family of transporters implies the occurrence of crosstalk between N O 3 − and other signaling pathways (Krouk et al., 2010; Tal et al., 2016). In our study, the expression levels of two and eleven putative NRT1 genes in the NIL^KN9204 were higher and lower than those in the NIL^J411 under both NN and LN conditions ( Table 3 ), respectively, indicating that the differences in root traits may result mainly from NRT1 functional differentiation. Additionally, the higher expression level of the CBL-interacting protein kinase 23 (CIPK23) gene (TraesCS2A02G251800) in the NIL^J411 compared to the NIL^KN9204 was also independent of N status ( Tables S5 and S8 ); this phenomenon may affect root formation together with NRT1 by phosphorylating T101 to regulate its primary N O 3 − response and high-affinity N O 3 − transport (Ho et al., 2009). Taken together, these results implied that a more sensitive response of roots to N acquisition occurred in the NIL^J411 compared with the NIL^KN9204, which cannot be fully explained.

Phytohormones, together with their receptors and signaling components, constitute the main intrinsic regulatory pathways of root traits. Ethylene, whose biosynthesis is regulated by several key enzymes, such as 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) and ACC oxidase (ACO), usually plays a negative regulatory role in root growth, inhibiting root enlargement and root meristem cell proliferation (Pattyn et al., 2020). In addition, ethylene regulates the expression of glutathione S-transferase (GST)-encoding genes. GST catalyses the conjugation of glutathione to various electrophilic compounds involved in the oxidative stress response, crosstalk with other hormones and drought-induced root growth (Dalal et al., 2018). Under both NN and LN conditions, significant increases in expression levels of genes encoding ACOs and GSTs were detected in the NIL^J411, with parallel increases in expression levels of genes encoding ethylene-responsive TFs ( Table 4 ), implying that enhanced ethylene signaling together with glutathione metabolism negatively regulated root growth in the NIL^J411. GA, whose catabolism is catalysed by GA2-oxidase, is considered a negative regulator of lateral root formation (Gou et al., 2010). However, overexpression of GA2-oxidase-encoding genes led to shortened primary root length, and mutations in these kinds of genes led to somewhat longer roots (Li et al., 2017), indicating a negative correlation between GA2-oxidase-encoding genes and root length. Compared to the NIL^KN9204, the NIL^J411 displayed upregulated expression levels of several GA2-oxidase-encoding genes under both NN and LN conditions ( Table 4 ); these upregulated levels may have inhibitory effects on root development. Brassinosteroids (BRs) regulate both root meristem size and cell expansion in controlling root growth. Deficiency or insensitivity of BRs leads to reduced root growth and lateral root formation (Wei and Li, 2016). The decreased expression of receptor-like protein kinase BRI1-like 3 genes in the NIL^J411 might impair BR signaling and inhibited root enlargement ( Table 4 ). Auxin facilitates lateral root initiation and development (Fukaki and Tasaka, 2009), while cytokinin antagonistically negatively regulates root growth (Moubayidin et al., 2009). In rice, crown root primordium initiation was promoted by auxin signaling, whereas cytokinin signaling inhibited it (Neogy et al., 2021). Members of the GH3 gene family respond to auxin and their encoding products use various amino acids as substrates to form indole-acetic acid (IAA)-amido conjugates for temporary storage and degradation (Staswick et al., 2005). Arabidopsis GH3.17 gene regulates environment-induced hypocotyl elongation (Zheng et al., 2016) and it is also responsible for root elongation, whereas gh3 mutants accumulate more free-IAA at the expense of any IAA-Glu and IAA-Asp auxin conjugates which causes defects in root development, demonstrating the critical roles of GH3 genes in auxin homeostasis and plant development (Guo et al., 2022). Cytokinin is activated by phosphoribohydrolase, which catalyses the hydrolysis of the bond between N ⁶ -substituted bases and ribose 5’-monophosphates in cytokinin precursors during its biosynthesis process (Kurakawa et al., 2007). Rice GH3-8 gene functions as a repressor of auxin-dependent developmental signaling, overexpression of GH3-8 suppressed auxin action, which resulted in abnormal morphology similar to that reported in auxin-deficient plants i.e. shorter roots and fewer adventitious roots (Ding et al., 2008). Significant increases in expression levels were observed for genes encoding GH3.8 and phosphoribohydrolase in the NIL^J411, accompanied by decreased expression levels of a gene encoding an auxin-responsive protein ( Table 4 ), implying the presence of weakened auxin signaling and accelerated cytokinin flux in the NIL^J411, which may be related to the differences in root traits of the pairwise NILs.

Several types of TFs play essential roles in root growth by regulating the expression of downstream genes that are usually closely associated with nutrient acquisition. Overexpression of TaNFYA-B1 was shown to increase both primary root length and total lateral root length in wheat (Qu et al., 2015). The expression levels of genes encoding NF-Y subunit C-3-like proteins (TraesCS1A02G354900, TraesCS1B02G366800 and TraesCS1D02G355600) were higher in the NIL^KN9204 than in the NIL^J411 under LN condition ( Table 5 ), indicating their potential contribution to LN-induced root enlargement. NAC TFs function in response to N O 3 − , depending on the NRT1.1 N O 3 − transport function (Vidal et al., 2014), to increase root biomass (He et al., 2015). Two genes encoding NAC TFs (TraesCS3A02G078400 and TraesCS3D02G078900) exhibited higher expression levels in the NIL^KN9204 than in the NIL^J411 under both NN and LN conditions ( Table 5 ), implying that they had similar effects on root growth. Ethylene responsive factors (ERFs) belong to the AP2/ERF (APETALA2/ERF) superfamily of transcription factors associated with various developmental processes and stress tolerance. It was reported that ERF transcription factor gene TaSRL1 resulted in short root length when ectopically expressed in rice (Zhuang et al., 2021). In the present study, expressions of ERF-like genes were significantly down-regulated in the NIL^KN9204 compared to the NIL^J411 under both NN and LN conditions, which may contribute to longer root length in NIL^KN9204.

In conclusion, transcriptomic analysis in the present study revealed the landscape of the DEGs between the QMrl-7B NILs under both NN and LN conditions, reflecting the physiological basis of the large roots of the superior parent KN9204. This study used pairwise NILs for analysis to dampen the effects from different genetic backgrounds; as such, the DEGs are more likely to be related to QMrl-7B, either directly or indirectly. These results revealed many pathways regulated by genes that might interact genetically with QMrl-7B, which may provide a foundation for the application of QMrl-7B in molecular breeding. However, since the materials used for RNA-seq were seedlings and not mature plants, much work is still needed to elucidate the overall genetic network that controls ideal root traits of wheat plants at different stages.

The data presented in the study are deposited in the NCBI repository, accession number PRJNA889686. The accession number PRJNA889686 (http://www.ncbi.nlm.nih.gov/bioproject/889686).

LS, JJL, LZ, NZ, WZ, DM, XR, JJ and JML conducted the hydroponic culture and field experiments. LS, JJL, LZ and AB analyzed the data. LS, JJL, YN and AB wrote the manuscript. RL and JML revised the manuscript. All authors contributed to the article and approved the submitted version.

This research was jointly supported by grants from the National Key Research and Development Program of China (2021YFF1000403), the National Natural Science Foundation of China (U22A6009), Hebei Natural Science Foundation (C2021205013) and China Agriculture Research System of MOF and MARA (CARS-03).

We are very grateful to Dr. Xigang Liu from Hebei Normal University, Shijiazhuang, China for his critical review of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.