Genome-Wide Systematic Characterization of the NPF Family Genes and Their Transcriptional Responses to Multiple Nutrient Stresses in Allotetraploid Rapeseed

Abstract

NITRATE TRANSPORTER 1 (NRT1)/PEPTIDE TRANSPORTER (PTR) family (NPF) proteins can transport various substrates, and play crucial roles in governing plant nitrogen (N) uptake and distribution. However, little is known about the NPF genes in Brassica napus. Here, a comprehensive genome-wide systematic characterization of the NPF family led to the identification of 193 NPF genes in the whole genome of B. napus. The BnaNPF family exhibited high levels of genetic diversity among sub-families but this was conserved within each subfamily. Whole-genome duplication and segmental duplication played a major role in BnaNPF evolution. The expression analysis indicated that a broad range of expression patterns for individual gene occurred in response to multiple nutrient stresses, including N, phosphorus (P) and potassium (K) deficiencies, as well as ammonium toxicity. Furthermore, 10 core BnaNPF genes in response to N stress were identified. These genes contained 6-13 transmembrane domains, located in plasma membrane, that respond discrepantly to N deficiency in different tissues. Robust cis-regulatory elements were identified within the promoter regions of the core genes. Taken together, our results suggest that BnaNPFs are versatile transporters that might evolve new functions in B. napus. Our findings benefit future research on this gene family.

Keywords: Brassica napus; NPF gene family; expression profile; genome-wide; nutrient stress; subcellular localization; transcriptional regulation.

Figure 1

Phylogenetic tree of coding nucleotide sequences of the NITRATE TRANSPORTER 1 (NRT1)/PEPTIDE TRANSPORTER (PTR) family (NPF) in Brassica napus and Arabidopsis thaliana. The phylogenetic tree was constructed by Molecular Evolutionary Genetics Analysis (MEGA) 5.1 with neighbor-joining method and 1000 replicates. A total of 246 nucleotide sequences including 193 from B. napus (pink circular), and 53 from Arabidopsis (green circular) were involved in the analysis.

Figure 2

Molecular characterization of the NPF proteins in Brassica napus. (a) molecular weight (MW); (b) theoretical isoelectric point (PI); (c) grand average of hydropathy (GRAVY); (d) Ka/Ks values.

Figure 3

Schematic representations for the chromosomal distribution and interchromosomal relationships of rapeseed NPF genes. Gray lines in the background indicate all syntenic blocks in the Brassica napus genome, and the blue lines indicate syntenic NPF gene pairs. The chromosome number is indicated at the bottom of each chromosome. R, random.

Figure 4

Synteny analysis of the NPF genes in Brassica napus, B. rapa, B. oleracea and Arabidopsis thaliana chromosomes. Gray lines in the background indicate the collinear blocks within B. napus and other plant genomes, while the red lines highlight the syntenic NPF gene pairs. Genes located on B. napus A genome are syntenic with genes of B. rapa and A. thaliana, while genes located on B. napus C genome are syntenic with genes of B. oleracea and A. thaliana.

Figure 5

Expression profiles of the NPF family differentially expressed genes (DEGs) in the leaves and roots of Brassica napus under nitrogen (N), phosphorus (P), potassium (K) stress (a–c) and ammonium toxicity (d) environments. For nutrient stress treatments, 14-day-old seedlings were exposed to N, P and K for seven days. For ammonium toxicity assay, 14-day-old seedlings were treated with 6 mM NO₃⁻ (control) and NH₄⁺ only for nine days. The fully expanded leaves and roots were sampled separately for RNA-seq analysis. CK, normal nutrient supply. LN, N deficiency. LP, P deficiency. LK, K deficiency. NO₃⁻, NaNO₃. NH₄⁺, NH₄Cl. The color scale is shown in the middle. Heat maps of gene expression profiles were generated using TBtools after data normalization (Z-score).

Figure 6

Venn diagram showing the transcriptional responses of the BnaNPF genes in leaves (a) and roots (b) of Brassica napus under diverse nutrient supplies. Each color represents a different treatment and the number in brackets represent the differentially expressed genes between the control and treatments.

Figure 7

Coexpression networks of the BnaNPF family genes. Cycle nodes represent genes, and the size of the nodes represents the power of the interrelation among the nodes by degree value. The width of the lines between two nodes represents the strength of the interactions between two genes. The hub NPF genes located in the center of the network, while the 10 most coexpressed genes were displayed in network.

Figure 8

The expression profiles of the 10 core BnaNPF genes in different tissues under nitrogen (N) stress by quantitative Real-Time PCR (qRT-PCR). Seedlings of 14 days’ old were exposed to N-free nutrient solution for six days. The roots, hypocotyl, basal node, petioles, fully expanded leaves (expanded L) and new leaves (new L) were sampled separately for RNA extraction. CK, normal nutrient supply; LN, N stress (0 μM N) condition. The color scale was shown on the right side. The heat map was generated using TBtools after data normalization (Z-score). * and ** indicates significant difference at P < 0.05 and P < 0.01 by student’s t test, respectively.

Figure 9

Subcellular localization of BnaC08.NPF2;9a and BnaC06.NPF2;13a. 35S::BnaNPFs-GFP and 35S::AtNIP5;1-mCherry constructs were introduced into Arabidopsis protoplasts. The GFP and mChery fluorescence was observed with a confocal laser-scanning microscope. The images were taken in the dark and white field.

Figure 10

Identification of the cis-regulatory elements (CREs) in the 2.0-kb promoter region of the 10 core BnaNPF genes. (a) Over-presentation of the CREs in the promoters of the 10 core BnaNPF genes. The more the CREs, the bigger the typeface size. (b) Nitrogen responsive CREs (double sided wedge) and phytohormone responsive CREs (round-corner rectangle) were showed in the promoter regions of the 10 core BnaNPF genes. Different CREs are indicated with different colors.