The focus of our study is to identify common genes that are differentially expressed between low-N tolerant and sensitive genotypes having different genetic backgrounds with differential response to N-stress. To select the genotypes with differential response to N, five sorghum genotypes (CK60, BTx623, San Chi San, China17 and KS78) and RILs from CK60 × San Chi San were evaluated under field conditions provided with full N (100 Kg ha-1 fertilizer) and N-stress (0 Kg ha-1). The phenotypes of five sorghum genotypes, five best and worst performing RILs tested under contrasting N-regimes showed that the mean values of plant height, biomass and grain yields were reduced from NN to LN field conditions (Table 1). Under controlled conditions, the average weights of roots and shoots of three week-old seedlings were also reduced from full N (100% Hoagland solution) to N-stress (Table 2). In maize, a 38% reduction in grain yield was observed from high-N to low-N conditions , which likely results from limitation of photosynthetic output caused by lower production of proteins like Ribisco . Under N-stress conditions, the lower root and shoot weights of three week old seedlings and lower biomass and grain yields of CK60, BTx623 and RILs 1-5 from field conditions, indicates their sensitivity to the limited N. San Chi San, China17 and RILs 6-10 grow taller and have higher biomass and grain yields in the field conditions and had higher root and shoot weights in the seedling stage, indicating their greater tolerance to the limited N. The RILs showed transgressive segregation and this suggested a polygenic inheritance of the traits. Maranville and Madhavan  showed that assimilation efficiency indices were significantly greater for the tolerant Chinese lines (San Chi San and China17) compared to sensitive US-lines (CK60 and BTx623) at both low and high N levels and the Chinese lines retained greater phosphoenolpyruvate carboxylase (PEPcase) activity under N-stress. This suggests that PEPcase and enzymes associated with PEP synthesis are perhaps responsible for maintaining relatively high photosynthesis under N-stress, and resulted in greater biomass accumulation of the tolerant genotypes .
Comparison of transcriptomes between sorghum genotypes
To identify common DEGs between genotypes having differential response to N-stress, RNA-seq was used to compare the transcriptomes of root tissues of genotypes grown under N-stress. From RNA-seq data, a total of 12 pair-wise comparisons were made by comparing three sensitive genotypes with each of the four tolerant genotypes to find common DEG transcripts across all genotypes. In order to differentiate non-specific DEG transcripts from those related to N-stress, the transcripts between four tolerant genotypes and three sensitive genotypes were inter-compared one by one. The transcripts that showed differential expression among tolerant (Additional file 3) and sensitive (Additional file 4) genotypes were discarded from the list of DEGs between 12 pair-wise comparisons. A total of 115 common DEG transcripts were observed between three sensitive and four tolerant genotypes, which could be related to N-stress (Additional file 7).
Expression analysis using qRT-PCR of selected genes confirmed their differential expression under low-N conditions (Additional file 5b). Furthermore, the differential expression of these genes was either absent, reduced or even reversed when plants were grown under full-N conditions (Additional file 5c). This is consistent with the suggestion that the selected genes are differentially expressed as a specific response to N-deficiency.
Differential expression of known nitrogen metabolism genes in sorghum genotypes
In general, N-starvation increases the expression of high-affinity transport systems for nitrate and ammonium . Here, N-stress increased the abundance of high affinity nitrate transporter gene transcripts (NRT2.5 or NRT2.7, NRT2.2, NRT2.3, and NRT2.6) in tolerant genotypes one to four-fold relative to sensitive genotypes (Table 4). Earlier reports showed that high affinity nitrate transporters were expressed in N-starved seedlings of Arabidopsis[38, 39]. In rice, the nitrate transporter (OsNRT2.2) in association with OsNAR2.1 transports nitrate in the high affinity concentration range in roots . The increased nitrate could promote the elongation of lateral roots . Conversely, the abundance of nitrate assimilatory gene transcripts, NR-1 and NiR, and ammonia assimilatory gene, GS-2 was higher in sensitive genotypes. GS-2 transcript increased in CK60 compared to China17, KS78 and the high-NUE bulk. However, San Chi San had higher levels of GS-2 transcript compared to BTx623 and low-NUE bulk, indicating a lack of functional redundancy in the expression of gene transcripts. The nitrate assimilation genes and GS-2 could be highly expressed to sustain the stress conditions. Overall, known nitrate transporter and assimilation genes showed very little change in expression between the tolerant and sensitive genotypes, indicating that the expression of basic N metabolism genes may be genotype independent. In the analysis of gene expression profile comparisons of rice using microarray, Lian et al  observed similar results; genes involved in N uptake and assimilation showed little response to N-stress.
Abundance of transcripts in sensitive genotypes under N-stress
DEG transcripts associated with secondary metabolism like flavonoids and anthocyanin biosynthesis, as well as those associated with abiotic stress responses, were abundant in sensitive genotypes (Table 5). Such expression changes may be involved in the plant’s tolerance to N-stress. The role flavonoids play in the sensitive genotypes under N-stress is not known. However, expression of flavonoid biosynthetic pathway genes was also reported in soybean  and Arabidopsis when genotypes grown under severe N-stress. In addition, the transcripts encoding Cytochrome P450s were abundant in sensitive genotypes (Table 5). Cytochrome P450s catalyze oxidation of a wide range of chemical reactions by activating dioxygen  and were reported to play an important role in biosynthesis of anthocyanin’s in response to stress . Similarly, four Cytochrome P450s were expressed higher in rice seedlings under N-stress .
A transcript encoding putative MYB transcription factor was abundant in sensitive genotypes (Table 5). It was reported that MYB genes contribute to the control of flavonoid biosynthesis in a wide range of plant species (maize, petunia) often in combination with other regulatory genes . A DEG transcript encoding choline monooxygenase gene, an iron sulphur enzyme involved in synthesis of glycine betaine in plants , was abundant in low-N sensitive genotypes CK60 and BTx623. It was reported that many species (maize, soybean, rice, and wheat) of transgenic plants with its over-expression had significantly increased glycine betaine content. Glycine betaine is a nitrogenous compound and acts as an osmoprotectant and its accumulation was associated with abiotic stress tolerance . In addition, transcript encoding Glutathione-S-transferase (GST) was also abundant in sensitive genotypes. GST catalyzes the glutathione-dependent detoxification reactions and the reduction of hydroperoxides. GSTs may act as binding proteins that sequestrate flavonoids in the vacuole for protection against environmental stresses . Therefore, induction of the flavonoid pathway may be a characteristic response of genotypes sensitive to N-stress.
Alteration in the lipid composition of plant cell membranes is one of the multiple defense strategies . Here, the transcripts encoding genes involved cell wall modification like peroxidases, peroxin-13, hydrolases like glycosyl hydrolase 17, were abundant in sensitive genotypes CK60 and BTx623. These proteins may be important for wall assembly, remodeling during growth, development and stress responses. Since nitrogen stress causes reduction in cell growth, it was not surprising to find abundance of a β-expansin gene transcript. Expansins play important roles in root growth and development under nutrient and abiotic stress conditions and are also involved in cell wall expansion [49, 50]. Therefore, the sensitive genotypes defend the stress and maintain the growth by altering the cell wall.
Phytohormones such as auxins and cytokinins were also reported to play important roles during the adaptation to limited N . The transcripts encoding auxin response factors (SAUR-like, ARF2) and auxin inducible proteins, 5NG4, were abundant in CK60 and BTx623 compared to tolerant genotypes (Table 5) under stress. Earlier reports showed that inhibition of auxin transport resulted in increased levels of MtN21-like-a/b and 5NG4 , led to localized increase in auxin concentration through a blockage of the PIN1 cycling , and resulted in reduced number of emerging lateral roots. The abundance of transcripts encoding auxin inducible proteins in sensitive genotypes could have resulted in their reduced root mass under N-stress (Table 2).
Kinases play important roles in the development of eukaryotic cells, such as cell cycle control and cell-type determination and differentiation . Kinases help the organism to cope with changing conditions and stresses in the environment. Because some of their targets are transcription factors, they also play a role in regulating transcription . In this study, DEG transcripts encoding five kinases were abundant in sensitive genotypes, which include cysteine-rich receptor like kinases (Table 5). Previous research indicated that receptor-like kinases play important roles in plant growth and development  and had differential expression in soybean genotypes grown under N-stress . Therefore, we hypothesize that these kinases might be important for adaptation to N-stress in sensitive genotypes of sorghum.
Abundance of transcripts in tolerant genotypes under N-stress
Under N-stress, plants tend to increase their N uptake ability by regulating physiological, biochemical activities and by changing root morphology including increased root length, root hair density and lateral root number . We found that tolerant genotypes adapt to N deficiency by producing higher root mass compared to sensitive genotypes (Table 2). Also, many gene transcripts involved in nitrate transport (Table 4) were present at higher levels in tolerant genotypes. It is proposed that N-metabolism related gene transcripts especially those encoding transporters, were increased in tolerant genotypes in order to uptake nitrate or amino acids from soil more efficiently and to produce more nitrogen containing metabolites required for their survival under N-stress.
The soil contains significant amounts of organic nitrogen derived from decomposition of organic matter by microorganisms, which is rich in amino acids. Plants have different capacities to take up these amino acids through putative amino acid transporters localized on the root epidermal cells . In this study, a DEG transcript encoding high affinity amino acid transporter, LYSINE HISTIDINE TRANSPORTER1 (LHT1), was massively expressed in San Chi San and China17 compared to sensitive genotypes (Table 6). It was reported that being expressed in the root, LHT1 is responsible for uptake of amino acids from soil into root tissue , and distributes from roots to shoots through xylem  for further metabolism especially under N-limited conditions. The amino acid uptake, and thus nitrogen use efficiency of the tolerant genotypes, could be higher with increased LHT1 expression under limited inorganic N supply.
To survive under N-stress, some genes involved in alleviating the detrimental effect of stress are abundantly expressed, which could facilitate tolerance to the stress. In this study, cell wall invertase-2 (CWINV2) transcript was massively increased in San Chi San and China17 (Table 6), indicating that sucrose degradation was increased in tolerant genotypes. A similar observation was made in the leaves of a water stress resistant cultivar of wheat . It is believed that the enhanced invertase expression in the roots of tolerant genotypes may contribute to the rapid cycling of sucrose, thus promoting carbon partitioning in favor of sucrose accumulation for counteracting the stress condition . In addition, the transcript of SEC14 cytosolic factor family protein was abundantly expressed in tolerant genotypes compared to CK60 and BTx623 (Table 6). It is also known as phosphatidylinositol/phosphatidylcholine transfer protein, and is located in the Golgi membrane. There, it acts as a signal precursor and activates stress responsive genes, phospholipids and galactolipids , which increase the membrane stability and provides stress tolerance . Gene transcripts responsible for numerous cellular activities, including protein biosynthesis, modification, and degradation enzymes were abundantly expressed in tolerant genotypes. Transcripts encoding ribosomal genes involved in protein biosynthesis, including structural constituent of ribosome L16p/L10 and translation elongation factors (EF1A) were also abundant in tolerant genotypes (Table 6).