Improving plant roots is an effective way to increase nutrient use efficiency and minimize fertilizer application for sustainable agricultural development. A number of genes/QTLs have been reported to be related to root growth and development; as such, these genes/QTLs have potential application for use in crop genetic improvement. However, the genetic network explaining how these genes interact to influence root architecture still needs to elucidated to assess the beneficial agronomic traits from an overall perspective and to guide the use of beneficial genes or pyramiding of allelic variation in future breeding efforts. Compared with J411, KN9204 has a larger root system, with which the stable QTL QMrl-7B is associated [15, 30]. Further studies involving QMrl-7B NILs showed that, as compare to the B-NILs, the A-NILs had superior root traits, N-related traits and yield traits in field trials (Liu et al., Frontiers Plant Sci, in revision). The superiority of root traits of the A-NIL 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 encode proteins of various groups, such as transporters, enzymes, TFs, and components involved in hormone signalling. 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 are 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 B-NIL under NN condition but was higher in the A-NIL 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 [31–33]. Because root development is directly related to cell wall loosening and elongation [34], the upregulated expression of this gene by LN in the A-NIL (Table S14) may participate in LN-promoted root growth by affecting these processes. Since the most enriched process of the DEGs between the pairwise NILs is phenylpropanoid biosynthesis, the relation of phenylpropanoid and root traits is worthy of attention. Phenylpropanoids are a source of many secondary metabolites, such as lignin, coumarin and flavonoids. Phenylalanine ammonia-lyase and cinnamoyl-CoA reductase catalyse the biosynthesis of cinnamate and lignin monomers, which are important metabolites in phenylpropanoid metabolism [35]. It was found that enhanced phenylpropanoid biosynthesis promotes root growth in rice [36], implying that there are positive effects of this metabolite on plant growth, including root enlargement [37]. Interestingly, the expression levels of a series of phenylalanine ammonia-lyase and cinnamoyl-CoA reductase genes increased in the B-NIL 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.
Low-affinity NO3− transporters and high-affinity NO3− transporters work under conditions of different N levels; the latter specifically transports NO3− and thus is more likely to reflect a response to N stress [38]. Dual-affinity nitrate transporter 1 (NRT1) not only functions in N acquisition but also is a NO3− sensor; this action is independent of its uptake activity that controls root development [39–41]. The cotransport of NO3−, peptides and hormones through the NRT1 family of transporters implies the occurrence of crosstalk between NO3− and other signalling pathways [42–44]. In addition, N stress tolerant genotypes exhibit higher expression levels of high-affinity NRTs than do the sensitive genotypes under N-limiting conditions [45]. Among these NRTs, NRT2.1 coordinates root development with external NO3− availability directly through stimulation of lateral root initiation under N-limiting conditions and indirectly through its NO3− transport function, which affects the N-dependent root system [40]. Our results showed that LN led to altered expression of a number of genes encoding both low-affinity and high-affinity NRTs, in accordance with the viewpoint that different NRTs function at different N levels (Table S4). Among these genes, the expression levels of 2 and 11 putative NRT1 genes in the A-NIL were higher and lower than those in the B-NIL under both NN and LN conditions (Table 2), indicating that the differences in root traits may originated mainly from NRT1 functional differentiation. Additionally, the higher expression level of the CBL-interacting protein kinase 23 (CIPK23) gene (TraesCS2A02G251800) in the B-NIL compared with the A-NIL 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 NO3− response and high-affinity NO3− transport [41]. Contrastingly, the expression of 4 high-affinity NRT genes (TraesCS6A02G031000, TraesCS6A02G031100, TraesCS6B02G044100 and TraesCS6B02G044500) was induced specifically by LN in the B-NIL but not in the A-NIL (Table 3). In addition, 2 high-affinity NRT genes (TraesCS6B02G044300 and TraesCS6D02G035900) exhibited higher expression levels in the B-NIL than in the A-NIL under LN condition, although under NN condition, their expression levels in the B-NIL were lower (Table 1). Taken together, these results implied that a more sensitive response of roots to N acquisition occurred in the B-NIL compared with the A-NIL, which cannot be fully explained.
Table 2
Differentially expressed NRT genes between the pairwise NILs
Gene_id | log2FC (NN_AA/NN_BB) | p-adjusted | log2FC (LN_AA/LN_BB) | p-adjusted | NR_description |
TraesCS2A02G309100 | -1.43 | 9.78E-12 | -1.24 | 2.60E-12 | protein NRT1/ PTR FAMILY 4.3-like |
TraesCS2A02G572200 | 1.13 | 0.04 | 1.20 | 0.003 | protein NRT1/ PTR FAMILY 5.10-like |
TraesCS5A02G485200 | -1.03 | 0.006 | -1.12 | 0.01 | protein NRT1/ PTR FAMILY 5.6-like |
TraesCS5D02G498700 | -2.07 | 4.20E-15 | -2.15 | 1.62E-11 | protein NRT1/ PTR FAMILY 5.6-like |
TraesCS6A02G267600 | -1.44 | 0.0001 | -1.92 | 0.002 | protein NRT1/ PTR FAMILY 8.3-like |
TraesCS6A02G267700 | -3.00 | 1.90E-24 | -1.56 | 6.45E-06 | protein NRT1/ PTR FAMILY 8.3-like |
TraesCS6A02G267800 | -3.23 | 3.26E-05 | -2.90 | 0.006 | protein NRT1/ PTR FAMILY 8.3-like |
TraesCS6A02G268500 | -1.09 | 7.42E-05 | -1.56 | 0.005 | protein NRT1/ PTR FAMILY 8.3-like |
TraesCS6B02G295000 | -2.41 | 1.52E-13 | -2.40 | 5.56E-08 | protein NRT1/ PTR FAMILY 8.3-like |
TraesCS6B02G295600 | -1.85 | 1.83E-12 | -1.78 | 0.005 | protein NRT1/ PTR FAMILY 8.3-like |
TraesCS6D02G247000 | -4.10 | 4.91E-25 | -2.03 | 0.0001 | protein NRT1/ PTR FAMILY 8.3-like |
TraesCS6D02G247100 | -1.45 | 9.32E-05 | -1.88 | 5.83E-05 | protein NRT1/ PTR FAMILY 8.3-like isoform X1 |
TraesCS7D02G378300 | 1.53 | 0.0004 | 1.04 | 0.008 | protein NRT1/ PTR FAMILY 8.3-like isoform X1 |
NN and LN indicate normal- and low-nitrogen conditions, respectively. AA and BB indicate the A-NIL and the B-NIL, respectively. The expression levels between the A-NIL and the B-NIL were compared under each condition, with the latter as a control. The expression ratios are presented as log2FC values. Genes with a log2FC ≤ -1 (p-adjusted < 0.05, coloured blue) represent the genes whose expression was downregulated in the A-NIL, and genes with log2FC ≥ 1 (p-adjusted < 0.05, coloured red) represent the genes whose expression was upregulated in the A-NIL. |
Table 3
NRT genes specifically expressed in the A-NIL or B-NIL in response to LN
Gene_id | log2FC (LN_AA/NN_AA) | p-adjusted | log2FC (LN_BB/NN_BB) | p-adjusted | NR_description |
TraesCS1A02G150200 | 1.03 | 0.004 | 0.14 | 0.84 | Peptide transporter PTR3-A |
TraesCS1A02G150400 | 1.18 | 2.29E-07 | 0.64 | 0.08 | protein NRT1/ PTR FAMILY 5.2-like |
TraesCS1B02G168100 | 1.60 | 1.11E-12 | 0.90 | 0.01 | protein NRT1/ PTR FAMILY 5.2-like |
TraesCS1B02G225000 | -1.39 | 2.12E-06 | -0.14 | 0.81 | protein NRT1/ PTR FAMILY 6.3-like |
TraesCS1B02G267900 | 1.29 | 0.01 | 1.15 | 0.18 | protein NRT1/ PTR FAMILY 3.1-like |
TraesCS1D02G201100 | 1.13 | 2.34E-11 | 0.81 | 0.0004 | uncharacterized protein LOC109744571 |
TraesCS1D02G214300 | -1.47 | 7.24E-08 | 0.20 | 0.72 | protein NRT1/ PTR FAMILY 6.3-like |
TraesCS2A02G007500 | 0.60 | 0.08 | 1.05 | 1.54E-09 | protein NRT1/ PTR FAMILY 8.5-like |
TraesCS2A02G074800 | -1.89 | 0.003 | -0.58 | 0.45 | high-affinity nitrate transporter 2.1-like |
TraesCS2A02G264500 | 0.99 | 8.86E-05 | 1.06 | 0.0006 | protein NRT1/ PTR FAMILY 4.5-like |
TraesCS2B02G277600 | 0.71 | 0.003 | 1.27 | 6.40E-09 | protein NRT1/ PTR FAMILY 4.5-like |
TraesCS2B02G626000 | 1.02 | 1.30E-05 | 0.43 | 0.19 | protein NRT1/ PTR FAMILY 5.10-like |
TraesCS2B02G626700 | 1.76 | 1.07E-08 | 0.97 | 0.01 | protein NRT1/ PTR FAMILY 5.10-like |
TraesCS2D02G413900 | 1.50 | 0.02 | 1.74 | 0.08 | protein NRT1/ PTR FAMILY 8.2-like |
TraesCS2D02G583500 | 0.99 | 0.01 | 1.30 | 0.0005 | protein NRT1/ PTR FAMILY 5.10-like |
TraesCS3A02G382100 | -2.55 | 6.06E-20 | -0.94 | 0.34 | protein NRT1/ PTR FAMILY 5.1-like isoform X1 |
TraesCS3A02G382200 | -0.12 | 0.90 | 1.57 | 0.003 | unnamed protein product |
TraesCS3B02G095900 | 0.20 | 0.62 | 1.12 | 0.02 | unnamed protein product |
TraesCS3B02G414900 | -3.35 | 5.34E-07 | 0.20 | 0.90 | unnamed protein product |
TraesCS3B02G415700 | 0.81 | 0.12 | 1.01 | 0.02 | predicted protein |
TraesCS3D02G056700 | 0.85 | 0.25 | 1.43 | 0.048 | protein NRT1/ PTR FAMILY 8.2-like isoform X1 |
TraesCS4A02G225400 | -1.07 | 6.53E-06 | -0.97 | 0.007 | protein NRT1/ PTR FAMILY 4.3-like |
TraesCS4A02G283900 | 1.20 | 6.27E-15 | 0.93 | 9.83E-06 | protein NRT1/ PTR FAMILY 2.11-like |
TraesCS4D02G026800 | 1.01 | 1.57E-08 | 0.85 | 1.76E-05 | protein NRT1/ PTR FAMILY 2.11-like |
TraesCS4D02G087900 | -1.39 | 2.77E-07 | -0.997 | 0.002 | protein NRT1/ PTR FAMILY 4.3-like |
TraesCS5B02G039100 | 3.41 | 0.02 | 2.02 | 0.22 | protein NRT1/ PTR FAMILY 2.11-like |
TraesCS5B02G152000 | 1.42 | 0.31 | 3.72 | 0.04 | putative nitrate excretion transporter 3 |
TraesCS5B02G245300 | 2.48 | 8.76E-05 | 1.89 | 0.09 | predicted protein |
TraesCS5B02G393100 | 1.46 | 0.39 | 4.38 | 0.03 | protein NRT1/ PTR FAMILY 4.5-like |
TraesCS5B02G414000 | 0.79 | 0.008 | 1.14 | 2.22E-05 | protein NRT1/ PTR FAMILY 6.4 isoform X2 |
TraesCS5B02G498700 | -0.82 | 0.02 | -1.09 | 0.01 | protein NRT1/ PTR FAMILY 5.6-like |
TraesCS5D02G419200 | 0.89 | 0.03 | 1.91 | 0.0007 | protein NRT1/ PTR FAMILY 6.4 isoform X1 |
TraesCS5D02G498700 | -1.03 | 0.009 | -0.95 | 0.0004 | protein NRT1/ PTR FAMILY 5.6-like |
TraesCS6A02G030700 | -2.93 | 7.26E-18 | -0.63 | 0.17 | high-affinity nitrate transporter 2.1-like |
TraesCS6A02G030800 | -2.84 | 1.38E-27 | -0.38 | 0.50 | high-affinity nitrate transporter 2.1-like |
TraesCS6A02G030900 | -2.75 | 1.36E-29 | -0.43 | 0.33 | High-affinity nitrate transporter 2.1 |
TraesCS6A02G031000 | 0.20 | 0.78 | 1.63 | 0.0002 | High-affinity nitrate transporter 2.1 |
TraesCS6A02G031100 | 0.01 | 0.98 | 1.45 | 3.00E-05 | high affinity nitrate transporter |
TraesCS6A02G033100 | -6.88 | 4.09E-06 | -2.71 | 1 | high-affinity nitrate transporter 2.1-like |
TraesCS6A02G033200 | -6.79 | 8.35E-06 | -3.36 | 1 | High affinity nitrate transporter 2.4 |
TraesCS6A02G267700 | 0.30 | 0.62 | -1.13 | 4.90E-05 | protein NRT1/ PTR FAMILY 8.3-like |
TraesCS6A02G369900 | 4.75 | 0.01 | 0.22 | 0.93 | protein NRT1/ PTR FAMILY 8.3-like isoform X1 |
TraesCS6B02G044100 | 0.06 | 0.90 | 1.51 | 4.09E-05 | high affinity nitrate transporter |
TraesCS6B02G044200 | -1.21 | 2.49E-05 | 0.42 | 0.39 | high-affinity nitrate transporter 2.1-like |
TraesCS6B02G044400 | -1.37 | 6.30E-07 | 0.93 | 0.01 | high-affinity nitrate transporter 2.1-like |
TraesCS6B02G044500 | 0.17 | 0.72 | 3.46 | 2.47E-36 | high affinity nitrate transporter |
TraesCS6B02G309200 | -1.09 | 0.10 | -2.04 | 0.008 | protein NRT1/ PTR FAMILY 7.3-like |
TraesCS6D02G035900 | -2.25 | 6.07E-16 | -0.22 | 0.69 | high-affinity nitrate transporter 2.1-like |
TraesCS6D02G037800 | -3.88 | 0.02 | -3.59 | 0.23 | high-affinity nitrate transporter 2.1-like |
TraesCS6D02G132100 | 1.45 | 0.008 | 1.18 | 0.09 | protein NRT1/ PTR FAMILY 8.3-like |
TraesCS7A02G121600 | 1.87 | 4.14E-10 | 0.60 | 0.24 | protein NRT1/ PTR FAMILY 2.3-like |
TraesCS7A02G301700 | -1.26 | 5.33E-12 | -0.96 | 3.48E-08 | protein NRT1/ PTR FAMILY 6.3-like |
TraesCS7B02G262200 | 0.99 | 2.59E-05 | 1.16 | 2.16E-08 | protein NRT1/ PTR FAMILY 4.6-like |
TraesCS7B02G283400 | 0.93 | 0.0001 | 1.31 | 0.0001 | protein NRT1/ PTR FAMILY 8.3-like |
TraesCS7D02G049300 | 0.71 | 0.16 | 1.80 | 0.004 | protein NRT1/ PTR FAMILY 2.3-like |
TraesCS7D02G357300 | 1.11 | 2.26E-06 | 0.83 | 0.0003 | protein NRT1/ PTR FAMILY 4.6-like |
NN and LN indicate normal- and low-nitrogen conditions, respectively. AA and BB indicate the A-NIL and the B-NIL, respectively. The expression levels between the A-NIL and the B-NIL were compared under each condition, with the latter as a control. The expression ratios are presented as log2FC values. Genes with a log2FC ≤ -1 (p-adjusted < 0.05, coloured blue) represent the genes whose expression was downregulated under LN conditions, and genes with log2FC ≥ 1 (p-adjusted < 0.05, coloured red) represent the genes whose expression was upregulated under LN conditions. |
Phytohormones, together with their receptors and signalling 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 [46–49]. 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 [50–51]. Under both NN and LN conditions, significant increases in expression levels of genes encoding ACOs and GSTs were detected in the B-NIL, with parallel increases in expression levels of genes encoding ethylene-responsive TFs (Table 4), implying that enhanced ethylene signalling together with glutathione metabolism negatively regulates root growth in the B-NIL. GA, whose catabolism is catalysed by GA2-oxidase, is considered a negative regulator of lateral root formation [52–53]. 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 [54–55], indicating a negative correlation between GA2-oxidase-encoding genes and root length. Compared to the A-NIL, the B-NIL 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 [56]. The decreased expression of receptor-like protein kinase BRI1-like 3 genes in the B-NIL may impair BR signalling and inhibit root enlargement (Table 4). Auxin facilitates lateral root initiation and development [57–58], while cytokinin antagonistically negatively regulates root growth [59]. 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 [60]. Cytokinin is activated by phosphoribohydrolase, which catalyses the hydrolysis of the bond between N6-substituted bases and ribose 5’-monophosphates in cytokinin precursors during its biosynthesis process [61–62] and is irreversibly degraded by cytokinin oxidase dehydrogenase [63]. Significant increases in expression levels were observed for genes encoding GH3.8 and phosphoribohydrolase in the B-NIL, accompanied by decreased expression levels of a gene encoding an auxin-responsive protein (Table 4), implying the presence of weakened auxin signalling and accelerated cytokinin flux in the B-NIL, which may be related to the differences in root traits.
Table 4
DEGs involved in phytohormone metabolism and signalling between the pairwise NILs
Gene_id | log2FC (NN_AA/NN_BB) | p-adjusted | log2FC (LN_AA/LN_BB) | p-adjusted | NR_description |
Ethylene | | | | | |
TraesCS1A02G370400 | -5.05 | 0.0004 | -2.53 | 5.63E-05 | ethylene-responsive transcription factor ERF109-like |
TraesCS1A02G370600 | -3.55 | 4.19E-17 | -1.44 | 8.02E-05 | AP2 domain containing protein |
TraesCS1A02G370700 | -5.28 | 1.08E-51 | -2.08 | 8.05E-11 | ethylene-responsive transcription factor ERF109-like |
TraesCS1B02G117400 | -1.32 | 5.13E-05 | -1.44 | 6.19E-09 | 1-aminocyclopropane-1-carboxylate oxidase-like |
TraesCS1B02G389700 | -3.15 | 3.17E-20 | -1.49 | 6.98E-06 | ethylene responsive transcription factor 6 |
TraesCS1B02G389800 | -3.64 | 1.61E-26 | -1.78 | 1.94E-07 | ethylene-responsive transcription factor ERF109-like |
TraesCS1D02G098000 | -1.80 | 2.67E-11 | -1.77 | 5.13E-10 | 1-aminocyclopropane-1-carboxylate oxidase-like |
TraesCS1D02G098100 | -1.10 | 8.10E-06 | -1.51 | 7.25E-09 | 1-aminocyclopropane-1-carboxylate oxidase-like |
TraesCS1D02G376500 | -4.11 | 8.13E-33 | -1.79 | 7.68E-08 | ethylene-responsive transcription factor ERF109-like |
TraesCS1D02G376600 | -3.26 | 2.22E-37 | -1.33 | 0.0001 | ethylene-responsive transcription factor ERF109-like |
TraesCS1D02G376700 | -3.34 | 1.30E-23 | -1.36 | 2.22E-05 | ethylene-responsive transcription factor ERF109-like |
TraesCS1D02G376800 | -4.79 | 1.53E-42 | -2.08 | 0.0005 | ethylene-responsive transcription factor ERF109-like |
TraesCS2D02G397100 | -5.47 | 0.0002 | -2.92 | 4.59E-09 | ethylene-responsive transcription factor ERF025-like |
TraesCS3B02G158800 | 1.56 | 0.037 | 1.89 | 0.003 | AP2 domain transcription factor TaDREB2 |
TraesCS3B02G412500 | -2.39 | 4.76E-05 | -1.17 | 0.046 | F-box domain containing protein, expressed |
TraesCS4A02G256500 | -4.94 | 5.60E-08 | -4.57 | 5.50E-19 | 1-aminocyclopropane-1-carboxylate synthase 1-like |
TraesCS4B02G058200 | -2.51 | 7.59E-09 | -3.30 | 5.16E-23 | 1-aminocyclopropane-1-carboxylate synthase 1-like |
TraesCS4D02G058200 | -2.67 | 1.16E-12 | -2.89 | 1.08E-23 | 1-aminocyclopropane-1-carboxylate synthase 1-like |
TraesCS4D02G096400 | -1.62 | 8.07E-10 | -1.25 | 2.91E-11 | lipase-like PAD4 |
TraesCS5B02G236900 | -2.68 | 6.45E-17 | -1.25 | 0.0009 | ethylene-responsive transcription factor ERF109-like |
TraesCS5D02G245300 | -2.37 | 2.30E-10 | -1.34 | 0.002 | ethylene-responsive transcription factor ERF109-like |
TraesCS6A02G235100 | 1.95 | 5.71E-07 | 1.08 | 0.004 | predicted protein |
TraesCS6A02G256600 | -3.44 | 9.28E-14 | -2.30 | 4.23E-12 | ethylene-responsive transcription factor ERF027-like |
TraesCS6A02G319500 | -3.88 | 2.62E-11 | -2.36 | 1.01E-09 | ethylene-responsive transcription factor ERF109-like |
TraesCS6A02G330500 | -1.83 | 5.11E-16 | -1.26 | 1.25E-06 | ethylene-responsive transcription factor ERF014-like |
TraesCS6B02G268700 | -3.01 | 3.89E-17 | -2.22 | 1.77E-19 | ethylene-responsive transcription factor ERF027-like |
TraesCS6B02G281000 | -1.76 | 4.23E-09 | -1.36 | 1.78E-07 | ethylene-responsive transcription factor 1-like |
TraesCS6B02G350100 | -4.04 | 9.00E-12 | -2.71 | 5.23E-10 | ethylene-responsive transcription factor ERF109-like |
TraesCS6B02G350400 | -3.41 | 7.85E-42 | -1.71 | 5.20E-07 | ethylene-responsive transcription factor ERF109-like |
TraesCS6B02G361400 | -2.21 | 9.84E-22 | -1.05 | 9.84E-08 | ethylene-responsive transcription factor ERF014-like |
TraesCS6D02G217800 | 1.72 | 1.14E-05 | 1.02 | 0.0004 | ethylene-responsive transcription factor ERF054-like |
TraesCS6D02G237800 | -3.23 | 2.79E-19 | -2.32 | 1.03E-21 | ethylene-responsive transcription factor ERF027-like |
TraesCS6D02G298700 | -3.68 | 1.54E-06 | -2.41 | 3.40E-09 | ethylene-responsive transcription factor ERF109-like |
TraesCS6D02G299000 | -4.6422 | 0.007 | -2.09 | 0.0022 | ethylene-responsive transcription factor ERF109-like |
TraesCS6D02G299100 | -3.71 | 2.77E-37 | -1.96 | 2.11E-06 | ethylene-responsive transcription factor ERF109-like |
TraesCS6D02G309600 | -1.66 | 1.08E-15 | -1.13 | 3.56E-05 | ethylene-responsive transcription factor ERF014-like |
glutathione S-transferase | | | | |
TraesCS1B02G194100 | -1.22 | 0.002 | -1.11 | 0.0006 | putative glutathione S-transferase GSTU6 |
TraesCS1B02G194500 | -1.36 | 0.0005 | -1.25 | 0.046 | probable glutathione S-transferase GSTU6 |
TraesCS2A02G218700 | -4.72 | 9.96E-19 | -2.78 | 5.22E-20 | probable glutathione S-transferase GSTU6 |
TraesCS2B02G244100 | -3.87 | 3.27E-19 | -2.39 | 6.06E-19 | probable glutathione S-transferase GSTU6 |
TraesCS2B02G322800 | -1.38 | 1.14E-06 | -1.19 | 9.82E-05 | glutathione S-transferase TCHQD |
TraesCS2D02G044100 | -3.99 | 0.03 | -2.58 | 0.002 | probable glutathione S-transferase GSTF1 |
TraesCS2D02G224700 | -1.83 | 0.001 | -1.72 | 5.55E-08 | probable glutathione S-transferase GSTU6 |
TraesCS2D02G304500 | -1.58 | 9.35E-06 | -1.45 | 8.40E-12 | glutathione S-transferase TCHQD |
TraesCS3A02G309100 | -1.92 | 5.18E-06 | -1.14 | 4.15E-05 | unnamed protein product |
TraesCS3D02G130700 | 1.39 | 0.006 | 1.16 | 0.0006 | probable glutathione S-transferase GSTU6 |
TraesCS3D02G486100 | -1.15 | 9.69E-05 | -1.07 | 3.18E-07 | glutathione transferase GST 23-like |
Gibberellin | | | | | |
TraesCS1A02G334400 | -1.81 | 2.73E-09 | -2.32 | 3.52E-16 | gibberellin 2-oxidase |
TraesCS2A02G189600 | -1.61 | 3.52E-11 | -1.29 | 5.11E-09 | chitin-inducible gibberellin-responsive protein 2-like |
TraesCS2B02G217500 | -1.59 | 8.03E-14 | -1.32 | 1.55E-09 | chitin-inducible gibberellin-responsive protein 2-like |
TraesCS2B02G239400 | -1.24 | 5.27E-08 | -1.03 | 3.57E-05 | chitin-inducible gibberellin-responsive protein 1 |
TraesCS2D02G198200 | -1.36 | 5.50E-11 | -1.46 | 1.23E-10 | chitin-inducible gibberellin-responsive protein 2-like |
TraesCS2D02G220200 | -1.33 | 2.10E-10 | -1.03 | 9.21E-07 | chitin-inducible gibberellin-responsive protein 1 |
TraesCS3A02G122600 | 1.25 | 0.04 | 1.67 | 3.54E-07 | gibberellin 3-oxidase 2 − 1 |
TraesCS3A02G294000 | -1.73 | 2.71E-09 | -1.45 | 9.93E-17 | gibberellin 2-oxidase |
TraesCS3B02G328700 | -1.86 | 1.62E-08 | -1.71 | 3.62E-07 | gibberellin 2-oxidase 3 isozyme B1 |
TraesCS6A02G221900 | -2.35 | 1.36E-13 | -2.00 | 1.46E-10 | gibberellin-2-oxidase-A9 |
TraesCS6D02G213100 | -2.84 | 2.58E-08 | -3.35 | 3.80E-17 | gibberellin 2-beta-dioxygenase 8-like |
Brassinosteroid | | | | | |
TraesCS2A02G142300 | 1.42 | 4.85E-05 | 1.077 | 7.91E-05 | probable cytochrome P450 313a4 |
TraesCS3B02G550900 | -2.31 | 0.004 | -1.70 | 0.0004 | protein BRASSINOSTEROID INSENSITIVE 1-like isoform X1 |
TraesCS5B02G174400 | 1.24 | 9.18E-07 | 1.43 | 1.95E-07 | receptor-like protein kinase BRI1-like 3 |
TraesCS5D02G181500 | 1.32 | 1.42E-07 | 1.56 | 4.46E-14 | receptor-like protein kinase BRI1-like 3 |
Auxin | | | | | |
TraesCS2A02G183900 | -1.73 | 1.42E-12 | -1.43 | 1.77E-13 | probable indole-3-acetic acid-amido synthetase GH3.8 |
TraesCS5A02G378300 | 1.42 | 0.0498 | 1.12 | 3.10E-05 | Auxin-responsive protein IAA30 |
TraesCS6B02G359400 | -1.38 | 0.001 | -1.07 | 0.005 | predicted protein, partial |
Cytokinin | | | | | |
TraesCS1A02G156100 | -3.19 | 2.83E-07 | -2.22 | 3.79E-14 | probable cytokinin riboside 5'-monophosphate phosphoribohydrolase LOGL10 isoform X1 |
TraesCS3D02G475800 | -2.54 | 4.52E-07 | -1.43 | 6.95E-05 | cytokinin dehydrogenase 4-like |
NN and LN indicate normal- and low-nitrogen conditions, respectively. AA and BB indicate the A-NIL and the B-NIL, respectively. The expression levels between the A-NIL and the B-NIL were compared under each condition, with the latter as a control. The expression ratios are presented as log2FC values. Genes with a log2FC ≤ -1 (p-adjusted < 0.05, coloured blue) represent the genes whose expression was downregulated in the A-NIL, and genes with log2FC ≥ 1 (p-adjusted < 0.05, coloured red) represent the genes whose expression was upregulated in the A-NIL. |
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. Conserved nuclear transcription factor Y (NF-Y) comprises 3 subunits, NF-YA, NF-YB and NF-YC, and regulates photosynthesis [64–65], nodule development [66], plant flowering [67–68], seed development [69] and tolerance to abiotic stresses [70–72] in eukaryotes. Overexpression of TaNFYA-B1 was shown to increase both primary root length and total lateral root length in wheat [22]. The expression levels of genes encoding NF-Y subunit C-3-like proteins (TraesCS1A02G354900, TraesCS1B02G366800 and TraesCS1D02G355600) were higher in the A-NIL than in the B-NIL under LN condition (Table 5), indicating their potential contribution to LN-induced root enlargement. Plant-specific NAM, ATAF and CUC (NAC) TFs are characterized by a highly conserved DNA-binding domain and variable C-terminal domains [73]. NAC TFs function not only in response to stresses [74] but also in response to NO3-, depending on the NRT1.1 NO3− transport function [75], and to increase root biomass [21]. Two genes encoding NAC TFs (TraesCS3A02G078400 and TraesCS3D02G078900) exhibited higher expression levels in the A-NIL than in the B-NIL under both NN and LN conditions (Table 5), implying that they have similar effects on root growth.
Table 5
Differentially expressed TF-encoding genes between the pairwise NILs
Gene_id | log2FC (NN_AA/NN_BB) | p-adjusted | log2FC (LN_AA/LN_BB) | p-adjusted | NR_description |
NTF | | | | | |
TraesCS1A02G354900 | 0.70 | 1 | 2.16 | 0.02 | nuclear transcription factor Y subunit C-3-like |
TraesCS1B02G366800 | 0.64 | 0.89 | 1.83 | 0.0007 | nuclear transcription factor Y subunit C-3-like |
TraesCS1D02G355600 | 0.45 | 0.95 | 2.60 | 0.003 | nuclear transcription factor Y subunit C-3-like |
NAC | | | | | |
TraesCS3A02G078400 | 1.48 | 0.004 | 1.37 | 3.04E-07 | NAC transcription factor |
TraesCS3A02G339600 | -3.07 | 3.15E-22 | -3.01 | 1.27E-27 | NAC transcription factor 4 |
TraesCS3D02G078900 | 1.79 | 6.26E-06 | 1.39 | 5.36E-05 | NAC transcription factor |
NN and LN indicate normal- and low-nitrogen conditions, respectively. AA and BB indicate the A-NIL and the B-NIL, respectively. The expression levels between the A-NIL and the B-NIL were compared under each condition, with the latter as a control. The expression ratios are presented as log2FC values. Genes with a log2FC ≤ -1 (p-adjusted < 0.05, coloured blue) represent the genes whose expression was downregulated in the A-NIL, and genes with log2FC ≥ 1 (p-adjusted < 0.05, coloured red) represent the genes whose expression was upregulated in the A-NIL. |
In conclusion, transcriptomic analysis in the present study revealed the landscape of the genes differentially expressed between the NILs with different QMrl-7B alleles 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 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 plants at different stages.