Volume 4, Issue 1, Pages 97-115 (January 2011) Transcriptome Analysis of High-Temperature Stress in Developing Barley Caryopses: Early Stress Responses and Effects on Storage Compound Biosynthesis  Mangelsen Elke , Kilian Joachim , Harter Klaus , Jansson Christer , Wanke Dierk , Sundberg Eva   Molecular Plant  Volume 4, Issue 1, Pages 97-115 (January 2011) DOI: 10.1093/mp/ssq058 Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 1 Expression Trajectories of Differentially Expressed Genes at Three Time Points of High-Temperature Stress. Signal intensities of 2078 differentially expressed probe sets are normalized to the mean (1.0) for the control conditions. Changes of gene expression upon exposure to high temperature are plotted against the three time points monitored as indicated on the abscissa. The colors red and blue of the gene trajectories indicate up- and down-regulation of the genes, respectively. Yellow trajectories depict stable expression levels in comparison to the control condition. Molecular Plant 2011 4, 97-115DOI: (10.1093/mp/ssq058) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 2 Number of Up- and Down-Regulated Genes at Three Different Time Points of Heat Stress. The histogram depicts the total number of differentially expressed genes at the three different time points investigated. Up- and down-regulated genes are shown in red and blue bars, respectively. Molecular Plant 2011 4, 97-115DOI: (10.1093/mp/ssq058) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 3 Gene Ontology Classification of Up-Regulated and Down-Regulated Genes According to GO Groups Molecular Function, Biological Process, and Cellular Component. The percentage of genes contained in a particular GO group is shown for all 10 520 present genes, 958 induced, and 1122 down-regulated genes in gray, red, and blue, respectively. Asterisks indicate a significant over or underrepresentation of functional categories compared to the functional categories of 10 520 present genes (* p ≤ 0.01; ** p ≤ 0.001; *** p ≤ 0.0001). unkn., unknown; o., other; metab., metabolism; act., activity; ER, endoplasmatic reticulum. Molecular Plant 2011 4, 97-115DOI: (10.1093/mp/ssq058) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 4 MapMan Display of All Present Genes. Gene expression is illustrated in the metabolism overview (A, C, E) and the cellular response overview (B, D, F) after 0.5 h (A, B), 3 h (C, D), and 6 h of heat stress (E, F), respectively. Induced gene expression in heat-stressed samples versus control samples is shown as an increasingly intense red saturating at the amplitude of 1.3 (log2-value). Reduced gene expression in heat-stressed samples versus control samples is shown as an increasingly intense blue saturating at the amplitude of 1.3 (log2-value). Genes with no significant change in the amplitude are shown in white. TCA, tricarboxylic acid; CHO, carbohydrate; OPP, oxidative pentose phosphate. Molecular Plant 2011 4, 97-115DOI: (10.1093/mp/ssq058) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 5 Specific and Common Up- and Down-Regulation of Gene Expression at the Investigated Time Points. (A) Venn diagram indicating the number of specifically and commonly up-regulated genes at the three different time points. (B) Venn diagram indicating the number of specifically and commonly down-regulated genes at the three investigated time points. The total number of up- and down-regulated genes is indicated in parentheses. (C) Trajectories of genes that are induced or repressed at particular time points of the heat stress experiment. Molecular Plant 2011 4, 97-115DOI: (10.1093/mp/ssq058) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 6 Simplified Model of the Conversion from Sucrose to Starch upon Heat Stress. Normalized signal intensities at the three investigated time points are depicted in shaded colors from unaltered (yellow) to induced (red) or reduced (blue). Differentially expressed genes (LIMMA p ≤ 0.05) are shown in bold face. For STP2, APS2, SSIIb, SSIIIa, SBEI, BAM4, BAM6, and BAM7, no probe sets are available. For full names of the genes, please refer to Supplemental Table 5. Molecular Plant 2011 4, 97-115DOI: (10.1093/mp/ssq058) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 7 Metabolite Levels of Heat-Exposed Barley Caryopses. (A) Sugar levels. (B, C) Amino acid levels. Light gray, dark gray, and black bars represent metabolite levels after 0.5, 3, and 6 h of heat stress exposure, respectively. The data are displayed as percentages normalized to the untreated control and standard deviations are indicated. Significant differences compared to the control samples are indicated by asterisks (* p ≤ 0.01; ** p ≤ 0.001). Molecular Plant 2011 4, 97-115DOI: (10.1093/mp/ssq058) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 8 Presence of Transcripts in Four Different Tissues of the Developing Barley Caryopsis after 6 h of Heat Stress. Expression levels of nine different genes in embryo (Em), endosperm (Es), pericarp (P), and husk (H) tissues are depicted for control and heat-stressed samples in dark and light gray bars, respectively. The transcript abundance of selected genes was determined by q–PCR and normalized to the control samples. The following genes are displayed: Raffinose synthase 1 (HvRS1), UDP-D-glucose 4-epimerase 1 (HvUGE1), UDP-D-glucose 4-epimerase 3 (HvUGE3), Trehalose-6-phosphate synthase (HvTPS1), trehalose-6-phosphate phosphatase (HvTPS2), invertase inhibotor (HvINVINH1), heat shock transcription factor A2d (HvHsfA2d), hexokinase 2 (HvHXK2), and the SNF1-related protein kinases 2.6 (HvSnRK2.6). Molecular Plant 2011 4, 97-115DOI: (10.1093/mp/ssq058) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 9 Number of Common Elements in the Heat Stress and Suborgan Datasets. Venn diagrams depict the overlap of differentially expressed genes in the heat stress microarray and suborgan-specific genes as described by Sreenivasulu et al. (2008). Red and blue numbers indicate an overlap with heat-induced and heat-repressed genes, respectively. For detailed information on the compared datasets, please refer to the Results part. Molecular Plant 2011 4, 97-115DOI: (10.1093/mp/ssq058) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 10 Common and Specific Elements in Heat-Stressed Barley Caryopses, Heat-Stressed Arabidopsis Shoots, and Drought-Stressed Barley Caryopses. Venn diagrams depict the overlap of induced (A) and repressed (B) genes in heat-stressed barley caryopses versus heat-stressed Arabidopsis (A.t.) shoots, as well as induced (C) and repressed (D) genes in heat-stressed barley caryopses versus drought-stressed barley caryopses. Molecular Plant 2011 4, 97-115DOI: (10.1093/mp/ssq058) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 11 Summary of Heat-Responsive Processes in Barley Caryopses. Functional groups of induced and repressed genes at 0.5, 3, and 6 h of heat exposure are depicted as blue and red boxes, respectively. Functional groups containing both induced and repressed genes are depicted as purple boxes. The length of the boxes is indicative for the duration of induced or repressed expression levels over just one or several investigated time points. Three different phases of heat response, named sensing and signal transduction, primary heat response, and heat stress adaptation, were defined and are indicated in italics at the bottom. Molecular Plant 2011 4, 97-115DOI: (10.1093/mp/ssq058) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions