Genome-wide identification, evolution, and expression analysis of the NPR1- like gene family in pears

Abstract

The NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1) plays a master regulatory role in the salicylic acid (SA) signal transduction pathway and plant systemic acquired resistance (SAR). Members of the NPR1-like gene family have been reported to the associated with biotic/abiotic stress in many plants, however the genome-wide characterization of NPR1-like genes has not been carried out in Chinese pear (Pyrus bretschneideri Reld). In this study, a systematic analysis was conducted on the characteristics of the NPR1-like genes in P. bretschneideri Reld at the whole-genome level. A total nine NPR1-like genes were detected which eight genes were located on six chromosomes and one gene was mapped to scaffold. Based on the phylogenetic analysis, the nine PbrNPR1-like proteins were divided into three clades (Clades I-III) had similar gene structure, domain and conserved motifs. We sorted the cis-acting elements into three clades, including plant growth and development, stress responses, and hormone responses in the promoter regions of PbrNPR1-like genes. The result of qPCR analysis showed that expression diversity of PbrNPR1-like genes in various tissues. All the genes were up-regulated after SA treatment in leaves except for Pbrgene8896. PbrNPR1-like genes showed circadian rhythm and significantly different expression levels after inoculation with Alternaria alternata. These findings provide a solid insight for understanding the functions and evolution of PbrNPR1-like genes in Chinese pear.

Keywords: Expression; NPR1; Pear; Phylogenetic; SAR; Salicylic acid.

Figure 1. Phylogenetic analysis of the NPR1-like proteins from P. bretschneideri Rehd and other species.

A phylogenetic tree of nine NPR1-like proteins from P. bretschneideri Rehd as well as other monocotyledons and dicotyledons plant species. The red star remark the NPR1-like protein in P. bretschneider Rehd. The blue circle remark the NPR1-like protein in A. thaliana. The tree was reconstructed in MEGA 6.0 software using the Neighbor-joining (NJ) tree. A thousand bootstrap replicates were performed to assess the tree reliability.

Figure 2. Gene structure and protein sequence comparison of PbrNPR1-like genes with AtNPR1-like sequences.

(A) Phylogenetic relationship of NPR1-like genes in A. thaliana and P. bretschneideri Reld. (B) Exons, introns, and UTRs are showed by yellow boxes, grey lines, and blue boxes, respectively. (C) Conserved domain of the BTB/POZ, Ank, and NPR1-like C-terminal region in A. thaliana and P. bretschneideri Rehd. (D) The motifs were identified by MEME database with the protein sequences. (E) A multiple alignment of amino acid sequences of P. bretschneideri Rehd NPR1-like proteins (PbrNPR1 to PbrNPR6) and A. thaliana NPR1-like proteins (AtNPR1 to AtNPR6). npr1-1, npr1-2, nim1-2, and nim1-4 in AtNPR1 mutation sites, and highly conserved cysteines residues (C82, C216, and C156 in AtNPR1) are indicated with black arrows. The conserved domains, BTB/POZ, ANK, and some important motifs, putative hinge region (LENRV), EAR-like repression motif (VDLNETP), NIMIN-binding region, and nuclear localization signal (NLS), are indicated with solid lines.

Figure 3. Statistics of cis-acting element numbers in NPR1-like gene family of pear. The different numbers and colors of the grid demonstrated the numbers of different class promoter elements in these genes.

The different numbers and colors of the grid demonstrated the numbers of different class promoter elements in these genes.

Figure 4. Expression profile of the PbrNPR1-like genes in 10 different tissues.

The expression patterns of eight PbrNPR1-like genes in flower, flower bud, young leaf, mature leaf, young stem and mature stem, young fruit, mature fruit, seed tissues were examined by qPCR assay. FR, flower; FB, flower bud; YL, young leaf; ML, mature leaf; YS, young stem; MS, mature stem; YF, young fruit; MF, mature fruit; SD, seed; BD, bud. The error bars show the standard deviations of the three independent biological replicates. The same letter shows no significantly difference at P < 0.05 by Duncan’s multiple range test.

Figure 5. Expression of PbrNPR1-like genes after SA treatment in Yali leaves.

The leaves were harvest at 0, 1, 3, 6, 12, 24, 48, 72 h after SA treatment. Data is means ± SD of n = 3 biological replicates. The same letter shows no significantly difference at P < 0.05 as determined by Duncan’s multiple range test.

Figure 6. Expression of PbrNPR1-like genes in Yali leaves after inoculation with A. alternata.

The leaves were harvest at 0, 6, 12, 24, 48, 72, 96, 120 h after A. alternata treatment. Different letters associated with each time point indicate statistically significant differences at the 5% level. The same letters indicate that the statistics did not differ significantly at P < 0.05 according to Duncan’s multiple range tests.

Figure 7. Expression profile of PbrNPR1-like genes in Yali leaves during a day.

The leaves were collected from eight different times of a day on 25 May 2019, 9:00 am, 12:00 am, 15:00 pm, 18:00 pm, 21:00 pm, 24:00, 3:00 am and 6:00 am. Different letters associated with each time point indicate statistically significant differences at the 5% level. The same the same letters indicate that the statistics did not differ significantly at P < 0.05 according to Duncan’s multiple range tests.