Volume 8, Issue 2, Pages 276-289 (February 2015) Genome-Wide Identification and Functional Analysis of Genes Expressed Ubiquitously in Rice  Ki-Hong Jung, Sung-Ruyl Kim, Hoi-Khoanh Giong, Minh Xuan Nguyen, Hyun-Jung Koh, Gynheung An  Molecular Plant  Volume 8, Issue 2, Pages 276-289 (February 2015) DOI: 10.1016/j.molp.2014.10.013 Copyright © 2015 The Author Terms and Conditions

Figure 1 Procedures for Evaluating Ubiquitously Expressed Genes in Rice Using the Platform of Both Affymetrix and Agilent 44K Arrays. A total of 6627 genes showing intensities greater than log210 with less than 0.1 CV were identified from Agilent 44K anatomical meta-profiles and further refined with Affymetrix anatomical meta-profiles. These independent meta-profiles revealed 4034 genes as ubiquitously expressed. In the heatmap, blue indicates a low level of expression based on microarray data; yellow indicates high expression. Molecular Plant 2015 8, 276-289DOI: (10.1016/j.molp.2014.10.013) Copyright © 2015 The Author Terms and Conditions

Figure 2 GUS Expression Patterns for Genes Encoding RNA Polymerase B Transcription Factor 3 and Elongation Factor 1 α. Promoter trap lines 4A-04197 and 5A-00191 were analyzed for reporter gene expression. Seedling root (A, E), mature leaf (B, F), flower/spikelet (C, G), and cross-section of mature leaf (D, H). (A–D) In 4A-04197, Os03g01910-encoding BTF3 was trapped by GUS. (E–H) In 5A-00191, Os03g08010 encoding OsEF1α was trapped by GUS. an, anther; bu, bulliform cell; ep, leaf tissue epidermis; le, lemma; me, mesophyll cell; ph, phloem cell; xy, xylem cell. Molecular Plant 2015 8, 276-289DOI: (10.1016/j.molp.2014.10.013) Copyright © 2015 The Author Terms and Conditions

Figure 3 GUS Expression Patterns for Genes Encoding Reticulon Domain-Containing Protein and Conserved Peptide Upstream Open Reading Frame-Containing Transcript 7. Promoter trap lines 2D-00098 and 3A-05916 were analyzed for reporter gene expression. Seedling root (A, E), mature leaf (G, F), flower/spikelet (C, G), and cross-section of mature leaf (D, H). (A–D) In 2D-00098, Os06g30750 encoding reticulon domain-containing protein was trapped by GUS. (E–H) In 3A-05916, Os04g42090 encoding CPuORF7 was trapped by GUS. Abbreviations for tissue types are defined in Figure 2. Molecular Plant 2015 8, 276-289DOI: (10.1016/j.molp.2014.10.013) Copyright © 2015 The Author Terms and Conditions

Figure 4 Classification of Functionally Characterized Ubiquitously Expressed Genes. In all, 136 ubiquitously expressed rice genes were functionally characterized according to major categories of traits, such as morphological, physiological, and resistance or tolerance. The x axis indicates the category and the details of each phenotype; the y axis presents the number of genes associated with the individual categories. Values in parentheses are numbers of non-redundant genes. Molecular Plant 2015 8, 276-289DOI: (10.1016/j.molp.2014.10.013) Copyright © 2015 The Author Terms and Conditions

Figure 5 Evaluation via Phylogenomic Analysis of Functional Dominancy for Ubiquitously Expressed Family-Member Genes. (A) Heatmap for Affymetrix meta-anatomical expression profiles of nine gene families with two members. (B) Heatmap for Affymetrix meta-anatomical expression profiles of five members within the NDP kinase family, presented according to the order in which the genes appear in the phylogenic tree. Labels were prepared for 16 representative tissues or organs. Ubiquitously expressed genes are indicated with red Locus IDs; red boxes in (A) distinguish families; yellow on the heatmap indicates high expression; green indicates low expression. Locus_ID refers to information from RGAP; family_ID indicates paralogous protein family identifiers curated by RGAP. Molecular Plant 2015 8, 276-289DOI: (10.1016/j.molp.2014.10.013) Copyright © 2015 The Author Terms and Conditions

Figure 6 Functional Identification of Os03g19890 Using T-DNA-Insertional Mutants. (A) Mature seeds from the panicle of Os03g19890/os03g19890-1 heterozygous plant. Approximately 15% were homozygotic opaque seeds (right side). (B) Image taken 55 days after germination (DAG) on MS medium. Seedlings were transplanted into soil in the greenhouse. (C) Amino acid alignment of Os03g19890 with associated eukaryotic orthologs of Arabidopsis thaliana AT5G52840, Bos taurus B13 (GenBank no. NP_787023), and human HsNDUFA5 (GenBank no. NP_004991). Conserved amino acids are in black; similar amino acids are boxed in gray. (D) Genomic structure of Os03g19890 and T-DNA insertion sites. Closed box, gray box, and connecting line represent exons, untranslated regions (UTR), and introns, respectively. (E) RT–PCR analysis of Os03g19890 from mutants and their segregating WTs. OsActin1 was used as quantitative cDNA control. (F) ATP content in leaves from WT, os03g19890-1, and os03g19890-2 plants grown under darkness. Error bars indicate standard deviations. More than five plants were tested per genotype. Molecular Plant 2015 8, 276-289DOI: (10.1016/j.molp.2014.10.013) Copyright © 2015 The Author Terms and Conditions

Figure 7 Functional Gene Network Predictions Mediated by 65 Ubiquitously Expressed Genes and Development of Hypothetical Model for Os03g19890 Function. (A) Functional network of genes, including Os03g19890. The network retained 972 interactions among 751 proteins. The 65 previously characterized genes are marked with larger node labels and 17 genes associated with dwarfism are indicated with red node borders. Functional network predictions for seedling development mediated by Os03g19890 are marked with red edges. (B) Hypothetical model. Network elements associated with Os03g19890 were refined by focusing on seven dwarfism-associated genes. Previously identified ubiquitously expressed genes were given network names; other genes are shown with RGAP Locus IDs. Expression patterns for seven of 17 genes associated with dwarfism (phenotype linkage), four of 16 genes encoding interactors (interolog linkage) with Os03g19890, and genes encoding mtHSP70 and one of its interactors were differentially regulated by using real-time RT–PCR on a background of os03g19890-1 mutant (black bar) compared with WT segregant (gray bar). Rice Ubiquitin 1 (OsUbi1) and OsUbi5 were used as internal controls. Ubiquitously expressed genes from Affymetrix anatomical meta-profiles are indicated with green nodes; leaf-preferred genes are shown with blue; japonica-preferred genes are shown with dark blue nodes; and pale orange nodes indicate genes with other features. Molecular Plant 2015 8, 276-289DOI: (10.1016/j.molp.2014.10.013) Copyright © 2015 The Author Terms and Conditions