Volume 7, Issue 2, Pages (February 2014)

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Volume 7, Issue 2, Pages 290-310 (February 2014) The Arabidopsis thaliana RNA Editing Factor SLO2, which Affects the Mitochondrial Electron Transport Chain, Participates in Multiple Stress and Hormone Responses  Qiang Zhu, Jasper Dugardeyn, Chunyi Zhang, Per Mühlenbock, Peter J. Eastmond, Roland Valcke, Barbara De Coninck, Sevgi Öden, Michael Karampelias, Bruno P.A. Cammue, Els Prinsen, Dominique Van Der Straeten  Molecular Plant  Volume 7, Issue 2, Pages 290-310 (February 2014) DOI: 10.1093/mp/sst102 Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 1 Ethylene Response of slo2 Mutants. (A) Col-0 and slo2 mutants grown on growth medium (GM) with or without ACC for 5 d in dark conditions. (B) Relative qualitative analysis of hypocotyl and root length from (A). Data are averages ± SD (n > 10), ** p < 0.01. (C) Leaf blade surface area of cotyledons (cotyl) and first pair of true leaves (1+2) from 3-week-old slo2-1 and wild-type plants grown on medium with or without 50 μM ACC. Data are averages ± standard errors (SE) (n = 10). (D) Phenotypes of 3-week-old Col-0 and slo2-1 plants continuously grown in air or 100 ppb ethylene. (E) Seven-day-old light-grown Col-0 and slo2 seedlings on medium without or with 50 μM ACC. (F) Relative root length from seedlings in (E). Data are averages ± SD (n > 10). ** p < 0.01. Molecular Plant 2014 7, 290-310DOI: (10.1093/mp/sst102) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 2 Mutation in SLO2 Leads to ABA Hypersensitivity. (A) Germination time courses of Col-0 and slo2-1 mutant. Average values ± SD from three independent experiments are presented. (B) Growth status of Col-0 and slo2 mutants on growth medium with or without 0.3 μM ABA. Pictures were taken 7 d after germination. (C) Statistical analysis of the percentage of green and open cotyledons shown in (B). Data represent the mean ± SD of three independent experiments, each with n > 40. * p < 0.05, ** p< 0.01. (D) Root growth assay of Col-0 and slo2 mutants on medium with or without 10 μM ABA. Three-day-old seedlings grown on germination medium were transferred to medium with or without 10 μM ABA. Pictures were taken after 7 d. (E) Relative root growth of seedlings shown in (D). Relative root growth reflects the root length in the presence of ABA expressed as a percentage of root length in the absence of ABA. Standard errors are presented (n > 40). Data represent the mean ± SD of three independent measurements. * p < 0.05. (F) Root bent assay. Four-day-old seedlings were transferred to medium with or without ABA, and plants were grown reversely for 7 d. (G) Relative root growth of seedlings shown in (F). Relative root growth reflects the root length in the presence of ABA expressed as a percentage of the root length in the absence of ABA. Data represent the mean ± SD of three independent experiments, each with n > 40. **p < 0.01. Molecular Plant 2014 7, 290-310DOI: (10.1093/mp/sst102) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 3 slo2 Mutants Are Hypersensitive to Salt and Osmotic Stress at Germination and Early Growth Stages. (A) Seedling growth of slo2 is inhibited by salt and osmotic stress. Col-0 and three slo2 alleles were grown on medium with or without stress factors for 7 d. Right panel: relative root growth, reflecting root length in the presence of NaCl or sorbitol expressed as a percentage of root length in the absence of NaCl or sorbitol. Values are means ± SD (n ≥ 15). * p < 0.05. (B) Germination time course assay. Col-0 and three slo2 mutants were grown on medium with or without stress factors. Values are means ± SD from three independent experiments. The seedling growth status is shown on the right. Molecular Plant 2014 7, 290-310DOI: (10.1093/mp/sst102) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 4 Drought and Salt Tolerance of Col-0 and slo2 Mutants. (A) Growth status of Col-0 and three slo2 alleles before and after drought treatment. 1-month-old plants were grown in soil in the same container, withheld from water for 10 d. (B) Stomatal aperture of Col-0 and three slo2 alleles. Lower epidermis from 1-month-old plants were peeled off and used for microscopic observation. (C) Statistical analysis of stomatal aperture. Data are mean ratios of width to length ± SD (n > 30). * p < 0.05. (D) Growth status of Col-0 and three slo2 alleles with or without salt treatment. 1-month-old plants were watered with 100mM NaCl for 4 d, 200mM NaCl for 4 d, and finally 300mM NaCl for 6 d. (E) Relative chlorophyll content measurement, which means chlorophyll level in the presence of NaCl expressed as a percentage of chlorophyll level in the absence of NaCl. * p < 0.05. Molecular Plant 2014 7, 290-310DOI: (10.1093/mp/sst102) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 5 Effect of the ABA Biosynthesis Inhibitor NF (Norflurazone) on Germination of slo2 Mutants under NaCl and Osmotic Stress. Germination of Col-0 and slo2 mutant seeds on medium containing 100mM NaCl or 300mM sorbitol with or without 100 μM NF. Seed germination rate was scored at day 5 and day 8 after transfer to the growth chamber, respectively. Data are mean ratios ± SD (n > 100). Molecular Plant 2014 7, 290-310DOI: (10.1093/mp/sst102) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 6 slo2 Mutants Exhibit Increased Susceptibility to Botrytis cinerea Infection. (A) Disease symptoms of Col-0, three slo2 mutants, and two complemented lines. Plants were sprayed with 1 × 104 B. cinerea spores ml–1 in PDB, or mock-treated with PDB. Pictures were taken 14 d after inoculation. (B) Statistical analysis of Botrytis-infected leaves. Plants were sprayed with a concentration of 5 × 107 B. cinerea spores ml–1 in PDB solution, and percentage infected leaves were scored at different time points. Wild-type and slo2-1 mutant in Col-0 background are shown. Three independent experiments with about 60 leaves per line per experiment were performed, with similar results. Data represent mean values and standard errors (SE). Molecular Plant 2014 7, 290-310DOI: (10.1093/mp/sst102) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 7 Expression Profiles of ABA-Responsive Genes. Expression of ABA-responsive genes after ABA treatment. Seven-day-old seedlings were incubated with 100 μM ABA for different times as indicated. The gene expression levels in Col-0 at time point 0 were set as 1. Three biological repeats were performed. Data represent mean values ± SD. * p < 0.05; ** p < 0.01. Molecular Plant 2014 7, 290-310DOI: (10.1093/mp/sst102) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 8 Expression Levels of Mitochondria Complex-I Subunit Genes in Col-0 and slo2 Mutants. Expression of mitochondria complex-I subunit genes in Col-0 and slo2 mutants after ABA treatment. Seven-day-old seedlings were incubated with 100 μM ABA for different time periods as indicated. The gene expression levels in Col-0 at time point 0 were set as 1. Three biological repeats were performed. Data represent mean values ± SD. * p < 0.05; ** p < 0.01. Molecular Plant 2014 7, 290-310DOI: (10.1093/mp/sst102) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 9 Expression Profiles of Alternative Respiratory Pathway Transcripts in Response to ABA. Expression of alternative respiratory pathway genes in Col-0 and slo2 mutants after ABA treatment. Seven-day-old seedlings were incubated with 100 μM ABA for different times as indicated. The gene expression levels in Col-0 at time point 0 were set as 1. Three biological repeats were performed. Data represent mean values ± SD. * p < 0.05; ** p < 0.01. Molecular Plant 2014 7, 290-310DOI: (10.1093/mp/sst102) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 10 slo2 Mutants Have an Enhanced Level of H2O2. (A) slo2 mutants contain a higher concentration of H2O2. Four-week-old wild-type, slo2 mutants, and two complemented lines were stained with the DCFH-DA method and viewed under a fluorescence microscope. (B) Quantitative measurement of H2O2 content in Col-0, three slo2 alleles, and two complemented lines. Data represent the mean ± SD of three independent measurements. * p < 0.05. Molecular Plant 2014 7, 290-310DOI: (10.1093/mp/sst102) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 11 Amino Acid Profiling. Four-week-old plants were used for amino acid extraction. Three biological repeats with two technical repeats were performed. Data represent mean values ± SD. * p < 0.05; ** p < 0.01. Molecular Plant 2014 7, 290-310DOI: (10.1093/mp/sst102) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions