Volume 3, Issue 1, Pages 125-142 (January 2010) Metabolomic Screening Applied to Rice FOX Arabidopsis Lines Leads to the Identification of a Gene-Changing Nitrogen Metabolism  Albinsky Doris , Kusano Miyako , Higuchi Mieko , Hayashi Naomi , Kobayashi Makoto , Fukushima Atsushi , Mori Masaki , Ichikawa Takanari , Matsui Keiko , Kuroda Hirofumi , Horii Yoko , Tsumoto Yuko , Sakakibara Hitoshi , Hirochika Hirohiko , Matsui Minami , Saito Kazuki   Molecular Plant  Volume 3, Issue 1, Pages 125-142 (January 2010) DOI: 10.1093/mp/ssp069 Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 1 Strategy for Determination of Metabotypes Using the Rice FOX Arabidopsis Lines. Six biological replicates were analyzed for each line. Molecular Plant 2010 3, 125-142DOI: (10.1093/mp/ssp069) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 2 Phylogenetic Analysis of Class II LOB/AS2-Protein Sequences from Arabidopsis and Rice. All genes in the phylogenetic tree belong to class II of the LBD/AS2 family. The Os-LBD37/ASL39, Os-LBD38/ALS40, and Os-LBD39/ASL41 orthologs were annotated according to the protein-sequence similarities and the phylogenetic relationships observed in Arabidopsis. Scale bar, 0.05 substitutions per site. Molecular Plant 2010 3, 125-142DOI: (10.1093/mp/ssp069) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 3 Pleiotropic Phenotypes of T3-Generation Plants of the Homozygous Rice Os-LBD37/ASL39 FOX Arabidopsis Lines K16331 and K19624, and the Retransformant Line SH13, and the Relative LBD37/ASL39-Expression Levels of these Plants. (A–D) Plants of the rice Os-LBD37/ASL39 FOX Arabidopsis lines: (A) empty-vector control, (B) K16331, (C) K19624, and (D) SH13 plants grown on MS medium. The white arrows indicate the hyponastic cotyledons of the K16331 line (B). (E) Relative Os-LBD37/ASL39-expression levels in the hyponastic and moderately hyponastic (strong phenotype) T3-generation plants of the three lines. The number of plants with hyponastic cotyledons (wt-like) was calculated from among at least three individual plants of each phenotype, except for the columns marked with (*), wherein only two plants were investigated. qRT–PCR analysis was performed twice using the same plant samples, and similar results were obtained: the highest Os-LBD37/ASL39 expression was observed in the K19624 and SH13 plants with hyponastic leaf phenotypes, and the lowest Os-LBD37/ASL39 expression was observed in the K19624, SH13, and K16331 plants with hyponastic cotyledon phenotypes. Since K16331 shows a wt-like phenotype with hyponastic cotyledons, we could not measure expression levels of hyponastic leaf phenotypes for K16331. (F) Relative expression levels of the endogenous transcripts of the Arabidopsis At-LBD37/ASL39 gene in rice FOX Arabidopsis lines K19624, K16331, and the retransformant line SH13 and their respective control plants (empty-vector control). For qRT–PCR analysis, the data were normalized to the control gene UBC9 (ubiquitin9). The error bars indicate the standard deviation (SD). Molecular Plant 2010 3, 125-142DOI: (10.1093/mp/ssp069) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 4 Prominent Metabolite Changes in the T2- and T3-Generation Plants of Rice Os-LBD37/ASL39 FOX Arabidopsis Lines K16331, K19624, and the Retransformant Line SH13. (A) Diagrammatic representation of primary and secondary metabolic pathways in Arabidopsis plants. The metabolites in gray characters could not be measured in this experiment. The abbreviations of metabolite names are listed in Supplemental Table 1. (B) Projection of the changes in the metabolite profiles of T2- and T3-generation rice FOX Arabidopsis lines onto the metabolic-pathway map (A). Each distinct fold change is represented with a different color, as shown in the upper right region of the box. p-values below 0.05 were considered to indicate statistical significance. N.D., not detected. Molecular Plant 2010 3, 125-142DOI: (10.1093/mp/ssp069) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 5 Phenotypes of Rice Oryza sativa ‘Nipponbare’ Os-LBD37/ASL39-Overexpressor Lines. (A) Empty-vector control plants and two independent overexpressor lines. (B) RK16331–4. (C) RK16331–13. Both overexpressor lines show growth reduction and pale-green phenotype. Molecular Plant 2010 3, 125-142DOI: (10.1093/mp/ssp069) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 6 Prominent Metabolite Changes in Two Different Oryza sativa ‘Nipponbare’ Os-LBD37/ASL39-Overexpressor Plants. (A) Diagrammatic representation of the primary and secondary metabolic pathways observed in rice plants. Metabolites in gray characters could not be measured in this experiment. The abbreviations of metabolite names are listed in Supplemental Table 1. (B) Projection of changes in the metabolite profiles of Os-LBD37/ASL39 overexpressors onto the metabolic-pathway map (A). Each distinct fold change is represented with a different color, as shown in the upper right corner of the box. p-values below 0.05 were statistically significant. * p-values are for glutamine (0.052) and histidine (0.055). ** p-values could not be calculated because of missing values in control samples. Molecular Plant 2010 3, 125-142DOI: (10.1093/mp/ssp069) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions