Morphological and physiological changes of wheat under nitrogen deficiency
The morphological and physiological changes of wheat were shown in Fig. 1. The crop height of N0 was 0.75 times lower than that of N1; the leaf area per plant of N0 was 0.70 times lower than that of N1 (6.77 cm2); the specific leaf area of N0 and N1 had no significant difference; the net photosynthetic rate (Pn) of N0 was 0.47 times lower than that of N1; the shoot fresh wight of N0 was 0.61 times lower than that of N1.
The root length per plant of N0 was 1.61 times higher than that of N1; the root volume per plant of N0 was 0.61 times lower than that of N1; the root surface area per plant of N0 was 1.04 times higher than that of N1; the root fresh wight of N0 was 0.82 times lower than that of N1. The root shoot ratio of N0 was 1.36 times higher than that of N1.
Global analysis of RNA-seq data resulting from nitrogen deficiency
The number of genes expressed in different regions were calculated, and stacked histogram was drawn (Fig. 2a). There were 72487–78729 genes expressed in wheat shoots, and 17116–22418 genes had Fragments Per Kilobase of transcript per Million fragments mapped (FPKM) values greater than 1. There were 63273–64413 genes expressed in wheat roots, and 27785–29233 genes had FPKM values greater than 1.
Principal component analysis (PCA) was applied to explore the relationship between samples by locating the samples at different dimensions (Fig. 2b). The closer the clustering distance was, the more similar the samples were. The results of PCA analysis showed that PCA1 reflected the difference of root and shoot, accounting for 99.41% of the total variation; PCA2 reflected shoot transcription difference under N0 and N1, accounting for 0.21% of the total variation; PCA3 reflected root transcription difference under N0 and N1, accounting for 0.11% of the total variation.
The volcanogram (Fig. 3ab) and cluster map (Fig. 3 cd) of P value and log2FC were applied to screen the differentially expressed genes (DEGs) under nitrogen deficiency treatment (N0) as compared to control (N1). There were 3949 DEGs in shoot, 1535 of them were up-regulated, 2414 of them were down-regulated. There were 3911 DEGs were screened in roots, 1236 of them were up-regulated, 2675 were down-regulated (Fig. 3e). The venn map (Fig. 3f) revealed that 1535 DEGs were up-regulated and 2414 were down regulated in both shoot and root. There were 372 DEGs differentially expressed in roots and shoot.
Functional analysis of DEGs identified under nitrogen deficiency
The gene ontology classification (Fig. 4) enriched 1205 up-regulated genes and 1888 down-regulated genes in shoots, while 961 up-regulated genes and 1883 down-regulated genes in roots. The enriched genes were classified into 3 major classes and 64 sub-classes. Some genes belongs to the two or more classifications. The largest four classifications (more than 980 DEGs) were cellular process, metabolic process, binding, and catalytic activity (Table 1).
Table 1
The number of differentially expressed genes (DEGs) in the four pathways with the largest number of genes under nitrogen deficiency
|
GO_classify 1
|
GO_classify 2
|
DEGs-Up
|
DEGs-Down
|
Shoot
|
Biological process
|
Cellular process
|
385
|
947
|
Biological process
|
Metabolic process
|
498
|
1029
|
Molecular function
|
Binding
|
672
|
1031
|
Molecular function
|
Catalytic activity
|
560
|
854
|
Root
|
Biological process
|
Cellular process
|
312
|
669
|
Biological process
|
Metabolic process
|
390
|
891
|
Molecular function
|
Binding
|
527
|
893
|
Molecular function
|
Catalytic activity
|
429
|
936
|
Analysis of gene families associated with cellular process |
Expansin family members were mainly belonged to the cellular process in gene ontology (GO) classification. Under nitrogen deficiency, there were 3 DEGs (Fig. 5) of wheat Expansin family in shoot, including TreasCS2B02G411700 (up-regulated), TreasCS1A02G30020 (down-regulated) and TreasCS1B02G310300 (down-regulated); there were 6 down-regulated wheat Expansin family members (TreasCS6A02G307900 and so on) and 24 up-regulated wheat Expansin family members (TreasCS5B02G528400 and so on) in root. |
Analysis of gene families associated with metabolic process
Pet and Psb family members were important proteins in photosystem in wheat shoot, which mainly belonged to metabolic process in gene ontology (GO) classification. Under nitrogen deficiency (N0), there were 3 down-regulated DEGs (Fig. 6) of wheat Pet family (TreasCS7A02G325500 and so on); there were 8 down-regulated wheat Psb family members (TreasCS3D02G523300 and so on) and 1 up-regulated wheat Psb family members (TreasCS6B02G412100).
Nar and Nrt family members had functions related to nitrogen metabolism, and mainly belonged to metabolic process in gene ontology (GO) classification. Under nitrogen deficiency, there were 3 down-regulated DEGs (Fig. 7) of wheat Nar family (TreasCS6A02G326200, TreasCS6B02G356800, and TreasCS6D02G306000) in both shoot and root of wheat; but there were other 2 up-regulated wheat Nar family members (TreasCS6A02G210000 and TreasCS6D02G193100) in root; there were 9 up-regulated DEGs of wheat Nrt family in root (TreasCS6A02G031100).