November 30, 2013 (Pages 7000-7060)

Additional file 3: Figure S3. Melt curve analysis of ten candidate reference genes in a root samples

Expression profiles of barley genes responsive to drought.

<p>Expression ratios (drought vs control) are calculated based 3 replications. Fold change values are colour-coded: dark yellow >6 fold up-regulated, black no change, violet >6 fold down-regulated. Horizontal rows represent gene expression patterns. Vertical lines represent different stress treatments. Gene expression data refers to cvs. Brenda (B), Morex (M), Morocco (Mo), Martin (Ma), Oregon Wolf Barley-Dominant (OWB-D), Oregon Wolf Barley-Recessive (OWB-R), Hs (<i>H. spontaneum</i> HS584).</p

Phylogenetic relationships between barley and rice sHsp and Hsf proteins.

<p>The phylogenetic tree was drawn from the deduced amino acid sequences of sHsp (A) and Hsf (B) proteins from the barley and rice genome using the ClustalW (MegAlign, DNAStar). Subfamilies are shaded in different colours.</p

Integrative view of protein-protein interactions and coexpression networks of AT3G46230 (orthologous gene of <i>sHsp17.5</i> in barley) derived in <i>Arabidopsis</i> based on CORNET correlation networks [<b>20</b>].

<p>Predicted protein interactions are highlighted in dotted black colour and also autoregulatory loops are shown for several Hsp proteins. The embedded coexpression network of AT3G46230 includes several direct and indirect targets identified through microarray experiments from 256 experimental data sets generated from abiotic stress treatments. Significance of coexpression is measured by the Pearson correlation coefficient (dark blue lines represent positive correlation of 0.9; light blue lines represent positive correlation of 0.8).</p

Structural organization of the <i>Hv</i>sHsp17.5-CI protein.

<p>A. The α-helix and ß-strands held between the two surface loops are shown in red and light blue colors. The N and C termini are indicated by NH₂, COOH, letters respectively. B. Structural alignment of the crystallin domain of <i>Ta</i>sHsp16.9 and <i>Hv</i>sHsp17.5-CI proteins are labeled as blue and red respectively. Highly conserved arginine (R) residue is shown in green color. C. Structural alignment of <i>Hv</i>sHsp17.5-CI and <i>Ta</i>sHsp16.9. The conserved regions of <i>Hv</i>sHsp17.5-CI and <i>Ta</i>sHsp16.9 are labeled with respective colors of figure A and B.</p

List of Hsf genes involved in different abiotic stress conditions and development of barley.

<p>The table shows the following details: Harvest unigene ID, full-length cDNA ID, Affymetrix ID, full-length/partial, open reading frame (ORF) size, predicted molecular mass for the deduced proteins, isoelectricpoint (pI), intron number with size, genomic sequence information (Assembly1 Morex ID), derived 5′ upstream of the translational start site and predicted subcellular localization.</p

Gene network analysis.

<p>Gene co-expression network of <i>HvHsfB2c/HvHsp17.5-CI</i>, is derived from Plant Network using Heuristic Cluster Chiseling Algorithm based on genome-wide plant ontology high throughput gene expression data. Meta-network containing genes of <i>HvHsfB2c</i> cluster are enriched for several sHsps (highlighted in yellow colour) and also enriched Hsp class in the MapMan functional categories are represented (see table). For further details refer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089125#pone.0089125.s006" target="_blank">Table S1</a>.</p

Expression profiles of sHsp and Hsf family genes during various stages of plant ontogeny analyzed by the Affymetrix 22K barley gene chip.

<p>Horizontal rows represent expression patterns of individual gene. Trivial names of genes as well as the corresponding Affymetrix IDs are given. Vertical lines represent the developmental stages and investigated tissues. Signal intensities: red, high expression; yellow, moderate expression; blue, low expression. Represented cultivars are named as M, ‘Morex’; Mo, ‘Morocco’, Ma ‘Martin’, B, ‘Barke’; GP, ‘Golden Promise’. Quantile normalized expression values are given as log2.</p

Expression, purification and chaperone activity of recombinant <i>Hv</i>sHsp17.5-CI.

<p>A) Expression of recombinant <i>Hv</i>sHsp17.5-CI in <i>E. coli</i>. Lane M, molecular weight marker; lane 1, uninduced; lane 2, induced; lane 3, purified recombinant <i>Hv</i>sHsp17.5-CI protein. Figures on the left indicate molecular weight in kDa. B) Prevention of thermal inactivation of <i>Swa</i>I restriction enzyme by recombinant <i>Hv</i>sHsp17.5-CI. The <i>Swa</i>I restriction enzyme was preincubated at 25, 30, 35, 40, 45 or 50°C in the presence of either BSA or recombinant <i>Hv</i>sHsp17.5-CI for 60 min. Residual activity of <i>Swa</i>I was determined by incubation with 300 ng plasmid at 25°C for 60 min, followed by electrophoresis on a 1% agarose gel. Lane M, 1-Kb DNA ladder; lane 1, plasmid DNA control (without <i>Swa</i>I digestion); lane 2, plasmid DNA digested with the <i>Swa</i>I restriction enzyme; lanes 3, 5, 7, 9, 11, and 13, plasmid DNA digested with <i>Swa</i>I after preincubation at 25, 30, 35, 40, 45 and 50°C, respectively, in the presence of BSA; lanes 4, 6, 8, 10, 12, and 14, plasmid DNA digested with <i>Swa</i>I after preincubation at 25, 30, 35, 40, 45 and 50°C, respectively, in the presence of recombinant <i>Hv</i>sHsp17.5-CI. SC, supercoiled plasmid; OC, open circular plasmid; L, linear plasmid. The numbers on the left represent the DNA markers in kb.</p

Transcriptional regulation of <i>HvsHSP17.5-CI</i> by HvHSF2c under heat stress.

<p>A. Subcellular localisation of HvHsfB2c-CFP in transient transformed <i>Arabidopsis thaliana</i> mesophyll protoplasts, scale = 10 µM. B. EMSA with <i>in vitro</i> translated HvHsfB2c SFB2c on HSE-box from the <i>ProHvHsp17.5-CI</i>. Extract of PURExpress without template DNA and translated DHFR (<i>E.coli</i> dihydrofolate reductase) were included as negative controls. * indicates HsfB2c specific band. C. Luciferase reporter gene assay. <i>ProHvHsp17.5::LUC</i> was used as reporter gene in <i>Arabidopsis thaliana</i> Col-0 protoplast co-transformation experiments with HvHsfB2c expression vector at 35°C (blue line, a: significant difference to controls), (b) CFP-control vector at 21°C (black line), HvHsfB2c expression vector at 21°C (red line) and CFP-control vector at 35°C (green line). Results are depicted as LUC/GUS ratios. The experiment was repeated twice in triplicates with similar results. Error bars indicate the standard error of the mean of 3 replicates.</p