Genome-Wide Identification and Analysis of Apple NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER Family (NPF) Genes Reveals MdNPF6.5 Confers High Capacity for Nitrogen Uptake under Low-Nitrogen Conditions

Abstract

The NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER family (NPF) proteins play important roles in moving substrates such as nitrate, peptides, amino acids, dicarboxylates, malate, glucosinolates, indole acetic acid (IAA), abscisic acid (ABA), and jasmonic acid. Although a unified nomenclature of NPF members in plants has been reported, this gene family has not been studied as thoroughly in apple (Malus × domestica Borkh.) as it has in other species. Our objective was to provide general information about apple MdNPFs and analyze the transcriptional responses of some members to different levels of nitrate supplies. We identified 73 of these genes from the apple genome and used phylogenetic analysis to organize them into eight major groups. These apple NPFs are structurally conserved, based on alignment of amino acid sequences and analyses of phylogenetics and conserved domains. Examination of their genomic structures indicated that these genes are highly conserved among other species. We monitored 14 cloned MdNPFs that showed varied expression patterns under different nitrate concentrations and in different tissues. Among them, NPF6.5 was significantly induced by both low and high levels of nitrate. When compared with the wild type, 35S:MdNPF6.5 transgenic apple calli were more tolerant to low-N stress, which demonstrated that this gene confers greater capacity for nitrogen uptake under those conditions. We also analyzed the expression patterns of those 73 genes in various tissues. Our findings benefit future research on this family of genes.

Keywords: NPF gene family; apple; expression analysis; genome-wide; nitrate concentration.

Figure 1

Phylogenetic tree and subfamily information for MdNPFs, AtNPFs, and OsNPFs. Neighbor-Joining method was used in tree construction with MEGA 5 software for 205 full-length amino acid sequences from apple, Arabidopsis, and rice. Eight subfamilies are indicated with Roman numerals. The numbers at nodes of the phylogenetic tree indicate the bootstrap values expressing branching probability per 1000 replicates; the bootstrap values of the confidence levels are shown as percentages.

Figure 2

Chromosome positions for MdNPF genes, marked with solid black lines. Scale on left is in Mb. Chromosome numbers are indicated on top of bar.

Figure 3

Sequence analysis of conserved domains from apple NPF proteins. X-axis, sequence of conserved motif; Y-axis, relative entropy that reflects rate of conservation for each amino acid.

Figure 4

Structure analysis of apple MdNPF family. Rectangle filled with blue, exon; solid black line, intron. Scale at bottom is in kb.

Figure 5

Exon-length distribution for NPF5.1, NPF6.3, NPF5.13, NPF5.14, and NPF8.1 in different plant species. Analysis was based on Boxplot depictions in SigmaPlot 12.0 program. Each box represents exon size range in which 50% of values for particular exon are grouped. Mean value is indicated by long red line.

Figure 6

Relative expression levels for 14 cloned apple NPFs under different nitrate concentrations, calculated by 2^−ΔΔCt method with respect to control samples (i.e., 6 mM NO₃⁻): (A) the relative expression levels for 14 cloned apple NPFs of roots under different nitrate concentrations; and (B) the relative expression levels for 14 cloned apple NPFs of leaves under different nitrate concentrations. Different letters on the bars within a group indicate significant differences (p < 0.05), based on Tukey’s multiple range tests.

Figure 6

Relative expression levels for 14 cloned apple NPFs under different nitrate concentrations, calculated by 2^−ΔΔCt method with respect to control samples (i.e., 6 mM NO₃⁻): (A) the relative expression levels for 14 cloned apple NPFs of roots under different nitrate concentrations; and (B) the relative expression levels for 14 cloned apple NPFs of leaves under different nitrate concentrations. Different letters on the bars within a group indicate significant differences (p < 0.05), based on Tukey’s multiple range tests.

Figure 7

Influence of overexpression by MdNPF6.5 on tolerance by apple calli to low-nitrogen supply. (A) Quantitative real time RT-PCR of samples from WT and MdNPF6.5-overexpressors. (B) Assay of low-nitrogen tolerance by WT and MdNPF6.5-overexpressors. Calli were transferred to MS medium or low-nitrogen medium, and photographed at 20 days after treatment began. (C) Comparison of fresh weights from WT and MdNPF6.5-overexpressors in response to low nitrogen. Values are means ± standard deviation. Different letters on the bars indicate significant differences (p < 0.05), based on Tukey’s multiple range tests.