Chromosomal locations of <i>Populus Dof</i> genes.

<p>Forty-one <i>Dof</i> genes were mapped to 19 linkage groups (LG). A schematic view of chromosome reorganization caused by recent whole-genome duplication in <i>Populus</i> is shown. Segmental duplicated homologous blocks are indicated by the same color. The scale represents mega bases (Mb). The LG numbers are indicated above each bar.</p

Expression analysis of 31 selected <i>PtrDof</i> genes in leaves under osmotic stress using qRT-PCR.

<p>The relative mRNA abundance of 31 selected <i>PtrDo</i><b><i>f</i></b> genes was normalized with respect to the reference gene (<i>Actin1</i>). Error bars represent the standard deviations of three biological replicates. Asterisks indicate stress treatment groups that showed a significant difference in transcript abundance compared with the control group (* P < 0.05, ** P < 0.01).</p

Phylogenetic relationships, gene structure and motif compositions of <i>Populus Dof</i> genes.

<p>A. Multiple alignment of 41 full-length amino acid sequences of <i>Populus Dof</i> genes, executed by ClustalX 1.83. The phylogenetic tree was constructed using MEGA 5.0 and the neighbor-joining method. Support values from a bootstrap analysis with 1,000 replicates are specified at each node. The four major phylogenetic subgroups are marked with different colored backgrounds. B. Exon/intron structures. Exons and introns are represented by particular colored boxes and black lines, respectively. C. Schematic representation of the conserved motifs identified by MEME. Each colored box represents a motif and black lines represent non-conserved sequences.</p

Characterization of Dof Transcription Factors and Their Responses to Osmotic Stress in Poplar (<i>Populus trichocarpa</i>)

<div><p>The DNA-binding One Zinc Finger (<i>Dof</i>) genes are ubiquitous in many plant species and are especial transcription regulators that participate in plant growth, development and various procedures, including biotic and abiotic stress reactions. In this study, we identified 41 <i>PtrDof</i> members from <i>Populus trichocarpa</i> genomes and classified them into four groups. The conserved motifs and gene structures of some <i>PtrDof</i> genes belonging to the same subgroup were almost the same. The 41 <i>PtrDof</i> genes were dispersed on 18 of the 19 <i>Populus</i> chromosomes. Many key stress- or phytohormone-related <i>cis</i>-elements were discovered in the <i>PtrDof</i> gene promoter regions. Consequently, we undertook expression profiling of the <i>PtrDof</i> genes in leaves and roots in response to osmotic stress and abscisic acid. A total of seven genes (<i>PtrDof14</i>, <i>16</i>, <i>25</i>, <i>27</i>, <i>28</i>, <i>37</i> and <i>39</i>) in the <i>Populus Dof</i> gene family were consistently upregulated at point in all time in the leaves and roots under osmotic and abscisic acid (ABA) stress. We observed that 12 <i>PtrDof</i> genes could be targeted by 15 miRNAs. Moreover, we mapped the cleavage site in <i>PtrDof30</i> using the 5’RLM-RACE. The results showed that <i>PtrDofs</i> may have a role in resistance to abiotic stress in <i>Populus trichocarpa</i>.</p></div

Mapping of mRNA cleavage sites confirmed by 5′ RLM-RACE.

<p>Arrows indicate the 5′ ends of the mRNA fragments, as identified by cloned 5’ RLM-RACE products, with the frequency of clones shown.</p

Expression analysis of 14 selected <i>PtrDof</i> genes in roots under osmotic stress using qRT-PCR.

<p>The relative mRNA abundance of 33 selected <i>PtrDo</i><b><i>f</i></b> genes was normalized with respect to the reference gene (<i>Actin1</i>). These genes were upregulated in roots under drought stress according to the HeatMap. Error bars represent the standard deviations of three biological replicates. Asterisks indicate stress treatment groups that showed a significant difference in transcript abundance compared with the control group (* P < 0.05, ** P < 0.01).</p

Gene Ontology (GO) results for <i>Populus</i> Dof proteins.

<p>GO analysis of 41 <i>Dofs</i> sequences predicted for their involvement in biological processes, molecular functions and cellular components. The results are presented as detailed bar diagrams in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170210#pone.0170210.s006" target="_blank">S6 Table</a>.</p

Expression profile of <i>Populus Dof</i> genes under drought stress.

<p>The heatmap was visualized using the exPlot tool in the PopGenIE v2 database. Heatmap showing 41 <i>Populus Dof</i> genes in leaves and roots under drought stress.</p

Expression analysis of 19 selected <i>PtrDof</i> genes in roots under osmotic stress using qRT-PCR.

<p>The relative mRNA abundance of 19 selected <i>PtrDo</i><b><i>f</i></b> genes was normalized with respect to the reference gene (<i>Actin1</i>). These genes were downregulated in roots under drought stress according to the HeatMap. Error bars represent the standard deviations of three biological replicates. Asterisks indicate stress treatment groups that showed a significant difference in transcript abundance compared with the control group (* P < 0.05, ** P < 0.01).</p

The Dof gene family in <i>Populus trichocarpa</i>.

<p>The Dof gene family in <i>Populus trichocarpa</i>.</p