Volume 5, Issue 2, Pages 461-471 (March 2012) SD3, an Arabidopsis thaliana Homolog of TIM21, Affects Intracellular ATP Levels and Seedling Development  Hidefumi Hamasaki, Takeshi Yoshizumi, Naoki Takahashi, Mieko Higuchi, Takashi Kuromori, Yuko Imura, Hiroaki Shimada, Minami Matsui  Molecular Plant  Volume 5, Issue 2, Pages 461-471 (March 2012) DOI: 10.1093/mp/ssr088 Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 1 sd3 Mutants Show Severe Dwarf Phenotype in Light-Grown Seedlings. (A) Phenotypes of 2-week-old light-grown seedlings of No-0 (left) and sd3-1 (right). (B) Phenotypes of 2-week-old light-grown seedlings of No-0 (left) and sd3-2 (right). The mesophyll cells of cotyledons in No-0 (C), sd3-1 (D), and sd3-2 (E). Scale bars (C–E): 0.1 mm. (F) The average area of cotyledons of 2-week-old plants in No-0, sd3-1, and sd3-2. (G) The average size of mesophyll cells in cotyledons of 2-week-old plants in No-0, sd3-1, and sd3-2. (H) The number of mesophyll cells per unit area of cotyledon in No-0, sd3-1, and sd3-2. Molecular Plant 2012 5, 461-471DOI: (10.1093/mp/ssr088) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 2 sd3 Has Decreased Polyploidy Levels in Dark-Grown Seedlings. (A) Phenotypes of 5-day-old dark-grown seedlings of No-0 (left), sd3-1 (middle), and sd3-2 (right). White arrowheads indicate the junctions between hypocotyl and root, and hypocotyl and cotyledon. Scale bar: 0.5 cm. (B) Hypocotyl lengths of 5-day-old dark-grown seedlings of No-0, sd3-1, and sd3-2. Approximately 40–60 seedlings were measured. Error bars indicate standard deviation. (C) Relative ratio of each ploidy of 5-day-old dark-grown hypocotyls of No-0, sd3-1, and sd3-2. Error bars indicate standard deviation (n = 3). (D) Length of hypocotyl cells of 5-day-old dark-grown seedlings. At least 50 cells from the middle part of the hypocotyl were measured. Error bars indicate standard deviation. Molecular Plant 2012 5, 461-471DOI: (10.1093/mp/ssr088) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 3 Molecular Characterization of SD3. (A) White boxes and solid lines indicate exons and introns, respectively. White triangles indicate the Ds insertion sites of sd3-1 and sd3-2. (B) Semi-quantitative RT–PCR analysis of SD3 gene expression in No-0, sd3-1, and sd3-2. Total RNA prepared from 2-week-old seedlings was amplified by RT–PCR. (C) Alignment of SD3 and its orthologous proteins: NP_001131979 (Zea mays), NP_001173354 (Oriza sativa), XP_002263208 (Vitis vinifera), NP_608929 (Drosophila melanogaster), Tim21 (Saccharomyces cerevisiae). The dashed line indicates the mitochondrial targeting signal sequence of SD3. The dashed box shows the mitochondrial transmembrane region and the box indicates the Tim21 motif. (D) A neighbor-joining phylogenetic tree of TIM21 motif. The line indicates relative evolutionary distances between the TIM21s. The bar indicates the genetic distance for 0.1 amino acid substitution/site. The deduced amino acid sequences were retrieved from the DDBJ/EMBL/GenBank databases. SD3, At2g40800, and At3g56430 (Arabidopsis thaliana), XP_002270952 (V. vinifera), NP_001143620 and ACF85902 (Z. mays), NP_001042128 and ABF95014 (O. sativa), XP_001697669 (Chlamydomonas reinhardtii), NP_723119 and NP_611540 (D. melanogaster), NP_499991 and NP_499992 (Caenorhabditis elegans), and TI21L (Homo sapiens). (E) Real-time PCR analysis of SD3 transcription levels in different plant tissues. The mRNA levels in several tissues were normalized against the expression level of the ACTIN2 gene. The values are expressed as the mean ± SD calculated for three independent samples. Molecular Plant 2012 5, 461-471DOI: (10.1093/mp/ssr088) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 4 SD3 Is a Mitochondrial Protein. Subcellular localization of SD3 protein in mitochondria in stable transgenic Arabidopsis root cells. (A–C) 35S::GFP1, (D–F) 35S::SD3::GFP1 were stably expressed in Arabidopsis. (A, D) Expression of the SD3::GFP protein was observed by confocal laser scanning microscopy. (B, E) Mitochondria stained by Mitotracker Red. (C, F) Merged image shows overlap (yellow). Scale bar: 0.1 μm. Molecular Plant 2012 5, 461-471DOI: (10.1093/mp/ssr088) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 5 SD3 Overexpressing Plants Show Increased Cell Number and Polyploidy Levels in Light-Grown Seedlings. (A) Real-time RT–PCR analysis of SD3 expression in vector, SD3-ox1, 2, and 3. Total RNA prepared from 5-day-old dark-grown seedlings was amplified by RT–PCR. All values were normalized against the expression level of the ACTIN2 gene. Error bars indicate standard deviation (n = 4). (B) Phenotypes of 6-day-old light-grown seedlings of vector, SD3-ox1, SD3-ox2, and SD3-ox3, respectively. Scale bar: 0.1 cm. (C) The average area of cotyledons of 6-day-old plants of vector, SD3-ox1, 2, and 3. Error bars indicate standard deviation (n = 10). (D) The average size of mesophyll cells in cotyledons of 6-week-old plants of vector and SD3-ox1. Error bars indicate standard deviation (n = 300). (E) The number of mesophyll cells per unit area of cotyledon in vector and SD3-ox1. Error bars indicate standard deviation (n = 10). (F) Relative ratio of each ploidy of cells of 6-day-old light-grown cotyledons of vector and SD3-ox1. Error bars indicate standard deviation (n = 3). Molecular Plant 2012 5, 461-471DOI: (10.1093/mp/ssr088) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 6 SD3 Overexpressing Plants Exhibit Increased Polyploidy Levels in Dark-Grown Seedlings. (A) Phenotype of 5-day-old dark-grown seedlings of vector control and SD3-ox1. White arrowheads indicate the junctions between hypocotyl and root, and hypocotyl and cotyledon. (B) Length of hypocotyls in 5-day-old seedlings of vector and SD3-ox1. Error bars indicate standard deviation (n > 150). (C) Width of the middle part of the hypocotyls of 5-day-old seedlings of vector and SD3-ox1. Error bars indicate standard deviation (n = 20). (D) Width of cells in the middle part of the hypocotyl of 5-day-old seedlings of vector and SD3-ox1. Error bars indicate standard deviation (n > 20). (E) Relative ratio of each ploidy of 5-day-old vector and SD3-ox1 hypocotyls grown in darkness. Error bars indicate standard deviation (n = 3). Molecular Plant 2012 5, 461-471DOI: (10.1093/mp/ssr088) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 7 Effect of SD3 Expression Level on Total Plant ATP Level. (A) Total ATP levels in 3-day-old dark-grown seedlings of vector and SD3-ox1 plants. Error bars indicate standard error (n = 5). (B) Total ATP levels of 5-day-old dark-grown seedlings of No-0 and sd3-1. Error bars indicate standard error (n = 5). (C) Hypocotyl length of 5-day-old seedlings grown on medium containing 0 (DMSO), 0.2, 2.0, and 20 μg ml−1 antimycin A (antimycin A). Error bars indicate standard deviation (n = 50). (D) Relative ratio of each ploidy of dark-grown hypocotyls of antimycin A-treated (antimycin A) seedlings. Error bars indicate standard deviation (n = 3). (E) Total ATP levels of antimycin A-treated (antimycin A) 5-day-old dark-grown seedlings. Error bars indicate standard error (n = 5). The seedlings (D, E) were grown in the same conditions as (C). Molecular Plant 2012 5, 461-471DOI: (10.1093/mp/ssr088) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 8 Expression of Endogenous Inner Membrane Protein Genes Is Up-Regulated in SD3 Overexpressing Plants. Real-time RT–PCR analysis of the respiratory-chain complexes-related gene expression in vector, SD3-ox1, 2, and 3. All values were normalized against the expression level of the ACTIN2 gene. Error bars indicate standard deviation (n = 4). Molecular Plant 2012 5, 461-471DOI: (10.1093/mp/ssr088) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions