Phylogeny and distribution of HD-ZIP protein from eight plant species.

<p><b>A.</b> Phylogenetic tree of HD-ZIP proteins from <i>Arabidopsis</i>, rice, <i>Medicago</i>, sorghum, <i>Brachypodium</i>, <i>Populus</i>, <i>Vitis</i>, and moss. Phylogeny was constructed by PhyML using maximum likelihood analysis. Bootstrap support values as percentage, are shown on selected major branches. The scale bar indicates the estimated number of amino acid substitutions per site. <b>B.</b> Percentage representation of HD-ZIP across the eight plant species within each subfamily. Colors correspond to the plant taxa as listed in C. C: Percentage representation of distributions for HD-ZIP within each plant species.</p

Expression profiles of <i>Populus</i> HD-ZIP genes across different tissues.

<p>Background corrected expression intensities were log-transformed and visualized as heatmaps (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031149#s3" target="_blank">Materials and Methods</a>). <b>A.</b> Heatmap showing hierarchical clustering of 54 <i>PtrHox</i> genes across various tissues analyzed. The Affymetrix microarray data were obtained from NCBI Gene Expression Omnibus (GEO) database under the series accession number GSE13990. CL, continuous light-grown seedling; DL, etiolated dark-grown seedling transferred to light for 3 h; DS, dark-grown seedlings; YL, young leaf; ML, mature leaf; R, root; DX, differentiating xylem; FC, female catkins; MC, male catkins. <b>B.</b> Heatmap showing hierarchical clustering of 57 <i>PtrHox</i> genes at different stem development/growth stages. The NimbleGen microarray data were obtained from NCBI GEO database under the series accession number GSE17230. IN2-IN9, stem internodes 2 to stem internodes 9. Color scale represents log2 expression values, yellow represents low level and blue indicates high level of transcript abundances.</p

Expression analysis of four selected HD-ZIP genes under drought and salinity stresses using qRT-PCR.

<p>The relative mRNA abundance of four selected HD-ZIP genes was normalized with respect to two reference genes <i>UBQ10</i> and <i>PP2a</i> in drought and salinity stress treatments. Bars represent standard deviations (SD) of three technical replicates. X-axis is time courses of stress treatments for each gene.</p

Phylogenetic relationship of HD-ZIP III subfamily from eight plant species.

<p>Enlarged view of the phylogeny of HD-ZIP III members from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031149#pone-0031149-g001" target="_blank">Figure 1A</a>. Numbers at each branch indicate bootstrap values and only values higher than 50% are shown. Scale bar corresponds to the estimated number of amino acid substitutions per site. Filled circles represent HD-ZIP proteins from different plant species. Colors correspond to plant taxa as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031149#pone-0031149-g001" target="_blank">Figure 1</a>.</p

In sillico EST analysis of <i>Populus</i> HD-ZIP genes.

<p>EST frequency for each gene was calculated by evaluating its EST representation among 19 cDNA libraries available at PopGenIE (<a href="http://www.popgenie.org/" target="_blank">http://www.popgenie.org/</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031149#pone.0031149-Sjodin1" target="_blank">[75]</a>. The heatmap was visualized using Heatmapper Plus tool by counting the corresponding ESTs for particular gene in the database. Color bar at bottom represents the frequencies of EST counts. CZ: cambial zone, YL: young leaves, FB: flower buds, TW: tension wood, SL: senescing leaves, AS: apical shoot, DC: dormant cambium, AC: active cambium, CSL: cold stressed leaves, R: roots, B: bark, SM: shoot meristem, MC: male catkins, DB: dormant buds, FC: female catkins, P: petioles, WCD: wood cell death, IS: imbibed seeds, VIS: Virus/fungus-infected leaves.</p

Differential expression of <i>Populus</i> HD-ZIP genes under different abiotic stresses.

<p>Expression is indicated as fold-change of experimental treatments relative to control samples and visualized in heatmaps (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031149#s3" target="_blank">Materials and Methods</a>). Color scale represents log2 expression values, yellow represents low level and blue indicates high level of transcript abundances. <b>A.</b> Heatmap showing hierarchical clustering of 54 <i>PtrHox</i> genes across various tissues and genotypes analyzed. Microarray data under the series accession number GSE16786 was obtained from NCBI GEO database. Genotypes analyzed included: <i>P. fremontii</i>×<i>angustifolia</i> clones 1979, 3200, and RM5, <i>P. tremuloides</i> clones 271 and L4, and <i>Populus deltoids</i> clones Soligo and Carpaccio. Tissues analyzed included: YL, young leaves; EL, expanding leaves; ML, mature leaves; RT, root tips; C, suspension cell cultures. Stress treatments included: low N, nitrogen limitation; MeJ, Methyl Jasmonate elicitation; wounding, sampled either one week or 90 hours after wounding. <b>B.</b> Heatmap showing hierarchical clustering of 54 <i>PtrHox</i> genes under short-term and long-term water deficit. Microarray data under the series accession number GSE17230 was obtained from NCBI GEO database. EAR, early response (EAR) to water deficit by 36 hours; LMI, long-term (10-day) response to mild stress with soil relative extractable water (REW) at 20–35%; LMO, long-term (10-day) response to moderate stress with soil relative extractable water (REW) at 10–20%.</p

Chromosomal locations and segmental duplication events of <i>Populus</i> HD-ZIP genes.

<p>The schematic diagram of genome-wide chromosome organization arisen from the salicoid genome duplication event in <i>Populus</i> was accomplished based on duplication coordinates from the <i>Populus</i> genome assembly v2.1. Segmental duplicated blocks are indicated with the same colors. The duplicated paralogous pairs of HD-ZIP are connected with dotted lines. Blue triangles indicate HD-ZIPs located on duplicated segments with the corresponding member lost. Red circles represent HD-ZIPs located out of any duplicated regions. Scale represents a 5 Mb chromosomal distance.</p

HD-ZIP gene family in <i>Populus</i>.

<p>Gene loci are from the Phytozome website (<a href="http://www.phytozome.net/poplar" target="_blank">http://www.phytozome.net/poplar</a>, release 2.1). A complete list of the coding sequences (CDS), deduced amino acid sequences and genomic DNA sequences is available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031149#pone.0031149.s002" target="_blank">Table S1</a>.</p

Phylogenetic relationship of HD-ZIP IV subfamily from eight plant species.

<p>Enlarged view of the phylogeny of HD-ZIP IV members from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031149#pone-0031149-g001" target="_blank">Figure 1A</a>. The numbers at the nodes represent the bootstrap values (>50%) from 100 replicates. Scale bar indicates the estimated number of amino acid substitutions per site. Filled circles represent HD-ZIP proteins from different plant species. Colors correspond to plant taxa as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031149#pone-0031149-g001" target="_blank">Figure 1</a>.</p

Expression analysis of 12 selected HD-ZIP genes using qRT-PCR.

<p>The relative mRNA abundance of 12 selected HD-ZIP genes was normalized with respect to two reference genes <i>UBQ10</i> and <i>UKN1</i> (<i>Populus</i> orthologue of <i>Arabidopsis AT4G33380</i>) in six different tissues. Bars represent standard deviations (SD) of three technical replicates. ST, shoot tips; L, leaves from 4–6 stem internodes; Phl, phloem; DX, differentiating xylem; R, roots; B, bark.</p