Volume 4, Issue 1, Pages 127-143 (January 2011) Effect of Mitochondrial Dysfunction on Carbon Metabolism and Gene Expression in Flower Tissues of Arabidopsis thaliana  Busi Maria V. , Gomez-Lobato Maria E. , Rius Sebastian P. , Turowski Valeria R. , Casati Paula , Zabaleta Eduardo J. , Gomez-Casati Diego F. , Araya Alejandro   Molecular Plant  Volume 4, Issue 1, Pages 127-143 (January 2011) DOI: 10.1093/mp/ssq065 Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 1 O2 uptake and adenylate levels in wt and u-ATP9 lines. (A) Oxygen consumption in flowers from wild-type (wt) and u-ATP9 lines (100% correspond to 25 μmol O2 min−1 g−1 dry weight). (B) ATP levels in flowers from wild-type (wt) and u-ATP9 lines (100% correspond to 114 nmol g−1 fresh weight). (C) ATP/ADP ratio in flowers from wild-type (wt) and u-ATP9 lines. Values are the mean ± SD of at least three independent replicates. * Indicates that the value is statistically different from that corresponding to the control (P < 0.05). Molecular Plant 2011 4, 127-143DOI: (10.1093/mp/ssq065) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 2 Overlap of transcriptome patterns and gene group sorting in u-ATP9 lines. (A) Venn diagrams of comparisons between A9:u-ATP9 and AP3:u-ATP9 transgenic plants and wt plants. Up-regulated genes are in bold; down-regulated genes are underlined. (B) Classification of genes altered in their expression identified by microarrays based on their function. Classification was done for the total regulated transcripts in A9:u-ATP9 and/or AP3:u-ATP9 lines. Molecular Plant 2011 4, 127-143DOI: (10.1093/mp/ssq065) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 3 Analysis of gene products involved in carbon metabolism. (A) qRT-PCR analysis of carbon metabolism enzyme genes: malate dehydrogenase (MDH330, At5g43330 and MDH410, At1g04410); pyruvate kinase (PK90, At3g52990 and PK60, At3g25960; phosphofructokinase (PFK70, At4g26270 and PFK80, At5g61580); pyruvate decarboxylase (PDC, At5g54960); glyceraldehyde-3-phosphate dehydrogenase (GAPC-1, At3g04120), non-phosphorylating glyceraldehydes-3-phosphate dehydrogenase (NP-GAPDH, At2g24270), and starch phosphorylase (SP, At3g46970). Total RNA was extracted from flowers. (B) Enzymatic activities of MDH, PK, PFK, GAPDH, NP-GAPDH, and SP determined in flowers from wild-type (white bars), A9:u-ATP9 plants (gray bars), and AP3:u-ATP9 plants (black bars). (C) Assays of PDC and alcohol dehydrogenase (ADH) enzyme activities in flower extracts from wt (white bars), A9:u-ATP9 plants (gray bars), and AP3:u-ATP9 plants (black bars). * Indicates that the value is statistically different from that corresponding to the control (P < 0.05). Columns represent mean values (error bars ± SD) of three independent experiments. Molecular Plant 2011 4, 127-143DOI: (10.1093/mp/ssq065) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 4 qRT-PCR Analysis (*P < 0.05) of Several Genes Encoding Mitochondrial Proteins: Alternative oxidase 1a (AOX1a, At3g22370); Alternative oxidase 2 (AOX2, At5g64210); Cytochrome c1 (CYTC1, At3g27240); BCS1a (At3g50930); AtCOX19 (At1g69750); and ATP-β (At5g08670). (A) Total RNA was extracted from wild-type plants (wt) and u-ATP9 lines. Columns represent mean values (error bars ± SD) of three independent experiments. (B) Western blot analysis of AOX protein from flower extracts from wt, A9:u-ATP9, and AP3:u-ATP9 lines using monoclonal antibodies anti-AOX. Molecular Plant 2011 4, 127-143DOI: (10.1093/mp/ssq065) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 5 Histochemical detection of ROS in wt and u-ATP9 lines. (A) Analysis of H2O2 with DAB in flowers from wild-type (wt), A9:u-ATP9, and AP3:u-ATP9 lines. (B) Analysis of ROS by H2DCFA. Fluorescence was visualized by microscopy after incubation of (wt), A9:u-ATP9, and AP3:u-ATP9 lines with the dye. White arrows indicate anthers. Molecular Plant 2011 4, 127-143DOI: (10.1093/mp/ssq065) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 6 Ascorbate, glutathione and anthocyanin levels in wt and u-ATP9 lines. (A) Determination of total ascorbate (white bars) and reduction state of the ascorbate pool (black bars, right panel) in flowers from wild-type, A9:u-ATP9, and AP3:u-ATP9 plants. (B) Determination of total glutathione (white bars) and reduction state of the glutathione pool (black bars, right panel) in flowers from wild-type, A9:u-ATP9, and AP3:u-ATP9 plants. (C) Determination of anthocyanin content in flowers from wild-type, A9:u-ATP9, and AP3:u-ATP9 plants. Anthocyanin levels are expressed as nmol of pelargonidin g−1 FW. Values are the mean ± SD of at least three independent replicates. * Indicates that the value is statistically different from that corresponding to the control (P < 0.05). Molecular Plant 2011 4, 127-143DOI: (10.1093/mp/ssq065) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 7 Gene expression and Enzyme Activity of Oxidative Stress Genes. (A) qRT-PCR analysis of genes encoding proteins related with stress responses: glutathione S-transferase (At1g78380); peroxidases (PER50, At4g37520 and PER57, At5g17820); peroxiredoxin (PEROX, At3g06050); calreticulin (CAL, At1g56340); and catalases 1, 2, and 3 (CAT1, At1g20630; CAT2, At4g35090; CAT3, At1g02620). Columns represent mean values (error bars ± SD) of three independent experiments. (B) Determination of catalase activity. Total catalase activity was determined in flower extracts from wild-type (wt), A9:u-ATP9, and AP3:u-ATP9 plants (100% correspond to 10 U mg−1 protein). * Indicates that the value is statistically different from that corresponding to the control (P < 0.05). Values are the mean ± SD of four independent replicates. Molecular Plant 2011 4, 127-143DOI: (10.1093/mp/ssq065) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 8 qRT-PCR Analysis of Genes Encoding Proteins Related with Signaling Pathways: ABA responsive element (AREB3, At3g56850); Ethylene responsive factor (ERF, At1g06160); Auxin efflux carrier protein (AECP, At1g23080); Auxin responsive family protein (ARFP, At2g46690); AtMYB58 (At1g16490); Arabidopsis response regulator 2 (ARR2, At4g16110); and AtSINAL7 (At5g37890). RNA was extracted from wild-type and u-ATP9 plants. * Indicates that the value is statistically different from that corresponding to the control (P < 0.05). Columns represent mean values (error bars ± SD) of three independent experiments. Molecular Plant 2011 4, 127-143DOI: (10.1093/mp/ssq065) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions