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. 2020 Oct 20:8:e10206.
doi: 10.7717/peerj.10206. eCollection 2020.

Structure and expression analysis of seven salt-related ERF genes of Populus

Juanjuan Huang #  1 , Shengji Wang #  1 , Xingdou Wang  1 , Yan Fan  1 , Youzhi Han  1
Affiliations

Structure and expression analysis of seven salt-related ERF genes of Populus

Juanjuan Huang et al. PeerJ. .

Abstract

Ethylene response factors (ERFs) are plant-specific transcription factors (TFs) that play important roles in plant growth and stress defense and have received a great amount of attention in recent years. In this study, seven ERF genes related to abiotic stress tolerance and response were identified in plants of the Populus genus. Systematic bioinformatics, including sequence phylogeny, genome organisation, gene structure, gene ontology (GO) annotation, etc. were detected. Expression-pattern of these seven ERF genes were analyzed using RT-qPCR and cross validated using RNA-Seq. Data from a phylogenetic tree and multiple alignment of protein sequences indicated that these seven ERF TFs belong to three subfamilies and contain AP2, YRG, and RAYD conserved domains, which may interact with downstream target genes to regulate the plant stress response. An analysis of the structure and promoter region of these seven ERF genes showed that they have multiple stress-related motifs and cis-elements, which may play roles in the plant stress-tolerance process through a transcriptional regulation mechanism; moreover, the cellular_component and molecular_function terms associated with these ERFs determined by GO annotation supported this hypothesis. In addition, the spatio-temporal expression pattern of these seven ERFs, as detected using RT-qPCR and RNA-seq, suggested that they play a critical role in mediating the salt response and tolerance in a dynamic and tissue-specific manner. The results of this study provide a solid basis to explore the functions of the stress-related ERF TFs in Populus abiotic stress tolerance and development process.

Keywords: Gene express; Salt stress; ERF; Populus; Transcription factor.

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Conflict of interest statement

The authors declare there are no competing interests.

Figures

Figure 1
Figure 1. Phylogenetic tree and gene structure analysis of seven ERF TFs.
(A) Phylogenetic tree of full-length amino acid sequences of seven PtERFs was constructed using MEGA 10.0.5 with the NJ method. (B) Gene structure and conserved motif were detected using the programme TBtools. The black lines indicate the introns. Conserved motifs were discovered using the MEME programme and were shown by colourful boxes. The sizes of conserved motifs, UTR, CDS and introns are estimated using the scale at the bottom. (C) Multiple alignments of the AP2 domain of ERFs were performed using the programme BioEdit 7.0.5.3.
Figure 2
Figure 2. Syntenic relationships of seven ERF genes among 19 Populus LGs are detected using the MCScanX programme.
The colinear blocks of Populus genome and ERFs are marked by grey and red connecting lines, respectively. The scales represent the distance of chromosomes. Bar and Heatmap graphs indicate the density of genes on each of LGs. The seven ERFs were mapped on 7 LGs. Whole-genome wide and tandemly duplicated genes are labelled in red and green, respectively.
Figure 3
Figure 3. GO annotation and enrichment of the seven ERF genes.
(A) GO annotation of seven ERF TFs for their involvement in cellular components and molecular functions. The abscissa represents the secondary classification term of GO, the left ordinate represents the percentage of the total number of genes included in the secondary classification, the right ordinate represents the number of mapped to the secondary classification, and the two colors represent the two major classifications. (B) GO enrichment of the seven ERF genes. The vertical axis represents the GO Term, while the horizontal axis represents the ratio of the Rich factor (the number of genes enriched in the GO Term to the Background number. The larger the Rich factor is, the greater the enrichment degree is). The size of the points represents the number of genes in the GO Term, and the color of the points corresponds to different FDR (Pvaule_corrected) ranges.
Figure 4
Figure 4. Spatio-temporal expression analysis of seven ERF genes under salt stress conditions based on RTq-PCR of Populus, respectively.
The time point hour 0 serves as a control. Data represents Mean ± SD. (A) PtERF001, (B) PtERF002, (C) PtERF003, (D) PtERF004, (E) PtERF005, (F) PtERF006, (G) PtERF007.
Figure 5
Figure 5. Tissue-specific expression analysis of seven ERF genes based on RT-qPCR of Populus.
Expression level of each genes in the root tissues under the normal (0 h) and salt stress (24 h) conditions was used as the control and normalized as 1.0, respectively. Black indicates treated with NaCl for 24 h and red denotes the control (normal condition). Data represents Mean ± SD. (A) PtERF001, (B) PtERF002, (C) PtERF003, (D) PtERF004, (E) PtERF005, (F) PtERF006, (G) PtERF007.
Figure 6
Figure 6. Expression analysis of seven ERFs under salt stress using RNA-Seq data.
The expression is measured as TPM (Transcripts Per Million reads). Data represents Mean ± SD. S indicates treated with NaCl for 24 hours and W denotes the control (normal condition). (A) PtERF001, (B) PtERF002, (C) PtERF003, (D) PtERF004, (E) PtERF005, (F) PtERF006, (G) PtERF007.
Figure 7
Figure 7. Heatmap clustering and correlations of the seven ERF genes based on RNA-Seq.
(A) Heatmap clustering of the seven ERF genes. Each column represents a sample, and each row represents a gene. The color in the figure represents the expression level of the gene in the sample [log10(TPM+1)]. Red represents the high expression level of the gene in the sample, while blue represents the low expression level. On the left are the tree diagram of gene clustering and the module diagram of sub-clustering, and on the right are the names of genes. The upper part is the tree diagram of sample clustering, and the lower part is the name of samples. The closer the branches of the two samples are, the closer the expression patterns of all genes in the two samples are, that is, the closer the variation trend of gene expression is. (B) Visualization of inter-gene expression correlations. Each node in the figure represents a gene, and the internode line represents the correlation of gene expression. The larger the node, the more the number of expression correlations between this gene and other genes.
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Grants and funding

This research was funded by the National Natural Science Foundation of China (31800564); the University Science and Technology Innovation Project of Shanxi Province (2019L0392); the Excellent Doctoral Program of Shanxi Province (SXYBKY201727); and the Funding for Doctoral Research of Shanxi Agricultural University (2017YJ21). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.