Genome-Wide Identification and Expression Profiling of the BZR Transcription Factor Gene Family in Nicotiana benthamiana

Abstract

Brassinazole-resistant (BZR) family genes encode plant-specific transcription factors (TFs), play essential roles in the regulation of plant growth and development, and have multiple stress-resistance functions. Nicotiana benthamiana is a model plant widely used in basic research. However, members of the BZR family in N. benthamiana have not been identified, and little is known about their function in abiotic stress. In this study, a total of 14 BZR members were identified in the N. benthamiana genome, which could be divided into four groups according to a phylogenetic tree. NbBZRs have similar exon-intron structures and conserved motifs, and may be regulated by cis-acting elements such as STRE, TCA, and ARE, etc. Organ-specific expression analysis showed that NbBZR members have different and diverse expression patterns in different tissues, and most of the members are expressed in roots, stems, and leaves. The analysis of the expression patterns in response to different abiotic stresses showed that all the tested NbBZR members showed a significant down-regulation after drought treatment. Many NbBZR genes also responded in various ways to cold, heat and salt stress treatments. The results imply that NbBZRs have multiple functions related to stress resistance.

Keywords: Brassinazole-resistant (BZR) transcription factor; Nicotiana benthamiana; abiotic stress; genome-wide expression analysis.

Figure 1

Phylogenetic tree of BZR transcription factors in N. benthamiana and A. thaliana. The phylogenetic tree was constructed using BZR amino acid sequences by the neighbor-joining method in MEGA X with 1000 bootstrap replicates. The phylogenetic tree was divided into four groups, which were shown in different colors, and identified by red Roman numerals.

Figure 2

Analysis of conserved motifs, gene structure and domains in the BZR genes of N. benthamiana. (A) Phylogenetic tree constructed using the NbBZR protein sequences. (B) Ten types of conserved motifs are predicted in the NbBZR protein sequences. The different motifs are shown in different color boxes. The sequence information for each motif is provided in Supplementary Table S2. (C) The gene structure of NbBZR members (untranslated regions, exons, and introns are shown as light green boxes, yellow boxes and horizontal lines, respectively. The red boxes represent the BES1_N domain).

Figure 3

Prediction of cis-acting elements in the NbBZR promoter regions. (A) Schematic representation of the numbers of four types of cis-acting elements predicted in the promoter region of each NbBZR member. (B,C) The type, quantity and position of environmental stress-related elements (B) and hormone-response elements (C) in the NbBZR promoter region.

Figure 4

Expression levels of NbBZRs in different tissues (A–K). The mean expression values were calculated from three independent biological replicates relative to that in young leaves. YL: young leaf; MF: mature leaf; ST: stem; RO: root; FL: flower. Red represents a high expression level and green represents a low expression level. The raw data of relative expression values and standard errors are provided in Supplementary Figure S1.

Figure 5

Expression analysis of NbBZRs after drought (0–14 days) treatment. Gene expression was normalized to control unstressed expression level, which was assigned a value of 1. Data represent the averages of three independent experiments ± SD. Standard errors are shown as bars above columns. Different letters indicate significant differences according to Tukey’s multiple comparisons test. Columns with different marked letters are significantly different (p < 0.05), and those with the same marked letter or share a letter are not significantly different.

Figure 6

Expression analysis of NbBZRs after cold treatment (0–48 h). Gene expression was normalized to control unstressed expression level, which was assigned a value of 1. Data represent averages of three independent experiments ± SD. Standard errors are shown as bars above columns. Different letters indicate significant differences according to Tukey’s multiple comparisons test. Columns with different marked letters are significantly different (p < 0.05), and those with the same marked letter or share a letter are not significantly different.

Figure 7

Expression analysis of NbBZRs after heat (0–5 days) stress. Gene expression was normalized to control unstressed expression level, which was assigned a value of 1. Data represent averages of three independent experiments ± SD. Standard errors are shown as bars above columns. Different letters indicate significant differences according to Tukey’s multiple comparisons test. Columns with different marked letters are significantly different (p < 0.05), and those with the same marked letter or share a letter are not significantly different.

Figure 8

Expression analysis of NbBZRs after salt (0–48 h) treatment. Gene expression was normalized to control the unstressed expression level, which was assigned a value of 1. Data represent averages of three independent experiments ± SD. Standard errors are shown as bars above columns. Different letters indicate significant differences according to Tukey’s multiple comparisons test. Columns with different marked letters are significantly different (p < 0.05), and those with the same marked letter or share a letter are not significantly different.