Morphological and physiological changes in wheat grown under the nitrogen-deficient condition
The altered morphological and physiological states of wheat are depicted in Fig. 1. The height of the wheat plant in the N0 group was 0.75 times significantly lower than the wheat plants in the N1 group (Fig. 1a). The leaf area per plant of the wheat plants in the N0 group was 0.70 times significantly smaller than the wheat plants in the N1 group. However, no significant differences were observed in the specific leaf area of wheat plants in the N0 and N1 groups. The net photosynthetic rate (Pn) and fresh shoot weight of wheat plants in the N0 group were 0.47 and 0.61 times significantly lower, respectively, than the wheat plants in the N1 group (Fig. 1d). It showed that nitrogen deficiency led to reduced crop height, leaf area per plant, Pn, and fresh shoot weight in wheat plants.
The root length per plant of wheat plants in the N0 group was 1.61 times significantly more than wheat plants in the N1 group (Fig. 1b). Besides, the root volume per plant of wheat plants in the N0 group was 0.61 times lower than wheat plants in the N1 group. However, the root surface area per plant, fresh root weight, root shoot ratio of wheat plants in the N0 group was 1.04, 0.82, and 1.36 times higher, respectively, than wheat plants in the N1 group (Fig. 1c). It showed that nitrogen deficiency resulted in an increased root length, root surface area per plant, fresh root weight, root shoot ratio, and reduced root volume per plant.
Global analysis of RNA-seq data of wheat plants grown under the nitrogen-deficient condition
The number of genes expressed in different parts of N0 group wheat plants was calculated to construct the stacked histogram (Fig. S1a). A total of 72487-78729 genes were identified in the wheat shoot of the N0 group, out of which 17116-22418 genes had FPKM (Fragments Per Kilobase of transcript per Million fragments mapped) values > 1. Besides, 63273-64413 genes were identified in the wheat root of the N0 group, out of which 27785-29233 genes had FPKM values > 1.
Principal component analysis (PCA) was applied to explore the relationship between samples by locating the samples at different dimensions (Fig. S1b). Less clustering distance indicated more identical samples. PCA1 reflected the difference in root and shoot, accounting for 99.41% of the total variation. Besides, shoot and root transcription differences between the N0 and N1 groups of wheat plants were deduced by PCA2 and PCA3, which accounted for 0.21% and 0.11% of the total variation. PCA3 reflected the root transcription difference between the N0 and N1 groups of wheat plants, accounting for a total variation of 0.11%.
The volcanogram (Fig. 2a, b) and cluster map (Fig. 2c, d) of p-values and log2FC were applied to screen the differentially expressed genes (DEGs) in the N0 group of wheat plants as compared to the control (N1) wheat plants. We identified a total of 3949 DEGs in the shoots of wheat plants grown under NDC, out of which 1535 were up-regulated, and 2414 were down-regulated. Besides, we identified a total of 3911 DEGs in roots of wheat plants grown under NDC, 1236 of which were up-regulated, and 2675 were down-regulated (Fig. 2e). The Venn map (Fig. 2f) revealed that 1535 DEGs were up-regulated and 2414 were down-regulated in both shoot and root of wheat plants grown under NDC, and a total of 372 DEGs were identified in roots and shoot.
Functional analysis of DEGs identified in wheat grown under the nitrogen-deficient condition
1205 up-regulated genes and 1888 down-regulated genes in shoots, while 961 up-regulated genes and 1883 down-regulated genes identified in roots of wheat plants grown under NDC, were enriched in Gene Ontology (GO) analysis (Fig. S2). The enriched genes were classified into 3 major classes and 64 sub-classes, and some of these genes belonged to two or more categories. Cellular process, metabolic process, binding, and catalytic activity were the top enriched categories, which included more than 980 DEGs (Table 1).
We performed KEGG pathway enrichment analysis of (Fig. 3a, b) DEGs from shoots and roots of wheat plants grown under NDC, and the pathways that showed enrichment of the highest number of DEGs are discussed here. Root DEGs showed enrichment of the gene information processing-translation pathway (142 down-regulated genes), and metabolism-biosynthesis of other secondary metabolites pathway (54 up-regulated genes). Root DEG’s KEGG pathway analysis led to the enrichment of the metabolism-carbohydrate metabolism pathway (118 down-regulated genes) and the metabolism-biosynthesis of other secondary metabolites pathway (78 up-regulated genes). Shoot DEG’s KEGG pathway analysis showed enrichment of monobactam biosynthesis (Fig. 3c) and the nitrogen metabolism pathway (Fig. 3d).
Table 1. The number of differentially expressed genes (DEGs) in the four pathways with the largest number of genes under the nitrogen-deficient condition
|
GO category
|
Sub-category
|
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 primarily belong to the GO category-cellular process. 3 DEGs in shoot of wheat plant grown under NDC belonged to (Fig. 4) expansin family, including TreasCS2B02G411700 (up-regulated), TreasCS1A02G30020 (down-regulated) and TreasCS1B02G310300 (down-regulated). Also, 6 down-regulated genes (TreasCS6A02G307900 and so on) and 24 up-regulated genes (TreasCS5B02G528400 and so on) in roots of the wheat plant grown under NDC belonged to the expansin family.
Analysis of gene families associated with metabolic process
Pet and Psb family members serve as crucial photosystem members in the wheat shoot and belong to the GO category-metabolic process. In wheat plants grown under NDC, 3 down-regulated DEGs (Fig. 5) belonged to the Pet family (TreasCS7A02G325500 and so on), 8 down-regulated DEGs belonged to the Psb family (TreasCS3D02G523300 and so on), and 1 up-regulated DEG belonged to the Psb family (TreasCS6B02G412100).
Nar and Nrt family members are involved in nitrogen metabolism and belong to the GO category-metabolic process. In wheat plants grown under NDC, 3 down-regulated genes from root and shoot (Fig. 6) belonged to Nar family (TreasCS6A02G326200, TreasCS6B02G356800, and TreasCS6D02G306000), 2 up-regulated genes from root belonged to Nar family members (TreasCS6A02G210000 and TreasCS6D02G193100), and 9 up-regulated DEGs from root belonged to Nrt family (TreasCS6A02G031100).
Validation of transcriptomic data
As per the RT-qPCR based validation study, expression levels of 94 (46 shoot genes and 48 root genes) out of 100 candidate genes were in line with the FPKM values of transcriptomic data (Fig. 7a). It showed that around 94% of the transcriptomic data were reliable. The coefficients of X of regression lines were 0.93 and 1.05 for shoot and root, respectively, which indicated high accuracy of the transcriptomic data. The RT-qPCR data of 50 candidate genes from root and shoot are depicted in Fig. 7b, and Fig. 7c, respectively. The comparison between RT-qPCR and transcriptomic data of each gene can be queried using Table S1 and Table S2.