Mitochondrial Perturbation Negatively Affects Auxin Signaling Kerchev Pavel Ivanov , De Clercq Inge , Denecker Jordi , Mühlenbock Per , Kumpf Robert , Nguyen Long , Audenaert Dominique , Dejonghe Wim , Van Breusegem Frank   Molecular Plant  Volume 7, Issue 7, Pages 1138-1150 (July 2014) DOI: 10.1093/mp/ssu071 Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 1 Chemical Screen to Identify Compounds Activating Mitochondrial Retrograde Signaling Pathways. (A) A schematic representation of the chemical screen. (B) Chemical formulas of the two most potent hit compounds identified in the screen (Compound 2 and Compound 3) and the shared substructure (3-(2-Furyl)acrylic acid (FAA)). (C) Induction of pUGT74E2 by Compound 2, Compound 3, and FAA. 10mM H2O2 was used as a positive control. The 1.5-kb upstream sequences of UGT74E2 were fused upstream of the luciferase gene and stably transformed in Arabidopsis plants. Luciferase activity following chemical (20 μM) and mock (DMSO) treatment is shown (± SD; n = 8 biological replicates). Different letters represent statistically significant differences according to one-way ANOVA with Tukey’s post hoc test (p < 0.05). RLU, relative luminescence units. (D) Photosystem II maximum efficiency (Fv’/Fm’) of seedlings treated with chemicals (20 μM), 10mM H2O2 or DMSO for 24 h (left panel). Fv’/Fm’ levels are depicted by color codes; blue represents high values and yellow-green corresponds to low values. Quantification of Fv’/Fm’ values in chemically treated seedlings (right panel). Bars represent means of eight replicates ± SD. Different letters represent statistically significant differences according to one-way ANOVA with Tukey’s post hoc test (p < 0.05). Molecular Plant 2014 7, 1138-1150DOI: (10.1093/mp/ssu071) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 2 FAA Negatively Impacts Mitochondria. (A) Induction of MDS genes following FAA treatment. Transcript levels of 14 of the MDS genes were analyzed by qRT–PCR in 10-day-old wild-type Arabidopsis plants grown as liquid cultures and treated with 20 μM FAA or DMSO for 24 h. Bars represent average fold changes relative to the mock treatment from four biological replicates (± SE). Asterisks indicate significant differences to the mock (Student’s t-test; * p < 0.05; ** p < 0.01; *** p < 0.001). (B) Comparison of publicly available expression profiles of the MDS genes between FAA ester treatment and treatments with mitochondrial blockers. Color codes represent linear fold changes relative to control extracted from Genevestigator and hierarchically clustered using Pearson correlation as a distance measure in the TMEV4 software. Antimycin A: GSE41136 (Ng et al., 2013b); oligomycin and rotenone: GSE3709 (Clifton et al., 2005); FAA ester: GSE1491 (Armstrong et al., 2004). (C) Mitochondrial staining of Arabidopsis root tips with MitoTracker Red CM-H2XRos fluorescent dye (left panel). Six-day-old Arabidopsis Col-0 seedlings were pretreated with 20 μM FAA, 50 μM AA, and DMSO in liquid ½MS media for 1 h before transferring to identical solutions containing additional 250 nM Mito Tracker Red CMH2XRos for an additional 1 h. Bright field images (right panel). Scale bars equal 5 μM. Molecular Plant 2014 7, 1138-1150DOI: (10.1093/mp/ssu071) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 3 Induction of the MDM cis-Regulatory Elements by FAA. Regulatory activity of the synthetic sequence containing six consecutive repeats of the MDM sequence from the UGT74E2 promoter (6xMDM[UGT74E2]) and from the AOX1a promoter (6xMDM1[AOX1a]) in transgenic Arabidopsis plants treated with 20 μM FAA or DMSO for 24 h. The construct mutated in the MDM sequence (6xMDM1mut[AOX1a]) was included as a negative control. Bars represent averages ± SE of eight biological replicates. RLU, relative luminescence units. Molecular Plant 2014 7, 1138-1150DOI: (10.1093/mp/ssu071) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 4 Inhibition of Auxin Signaling by Mitochondrial Dysfunction. (A) Effect of AA and FAA on the auxin-responsive reporter line DR5:GUS. Five-day-old DR5:GUS seedlings were treated for 4 h with 5 μM IAA alone or with combinations of 5 μM IAA and either 50 μM AA or 20 μM FAA. DMSO was used as a mock treatment. (B) Quantification of the DII-VENUS fluorescent signal intensity in the median section of the root tip. Data points represent averages based on quantification of three independent roots relative to the first time point (5 min = 1) ± SD. The roots were pre-incubated for 5 min with either 50 μM AA or 20 μM FAA before being placed in contact with agar-solidified ½MS media containing combinations of auxin (50 nM) and either 50 μM AA or 20 μM FAA and imaged for 35 min. For IAA and mock (DMSO) treatments, roots were placed in contact with the media without a prior incubation. (C) DII-VENUS fluorescence signal (range indicator: blue to green) in propidium iodide (red) stained Arabidopsis root tips at 5 min and 25 min after exposure to exogenous IAA (50 nM); IAA + FAA (50 nM and 20 μM), IAA + AA (50 nM and 50 μM), and DMSO as described above. Molecular Plant 2014 7, 1138-1150DOI: (10.1093/mp/ssu071) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 5 Effect of Polar Auxin Transport Inhibitors on Mitochondrial Retrograde Markers. (A) The UGT74E2 and AOX1a promoters are activated by polar auxin transport inhibitors and an inhibitor of auxin biosynthesis. The 1.5-kb upstream sequences of UGT74E2 and AOX1a were fused upstream of the luciferase gene and stably transformed in Arabidopsis plants. Mean luciferase activities after 24 h of treatment of pUGT74E2:LUC and pAOX1a:LUC lines with 20 μM NPA, 20 μM TIBA, 20 μM gravacin, 500 μM quercetin, 20 μM L-kynurerine, and DMSO are shown ± SE (n = 8 biological replicates). RLU, relative luminescence units. (B) The MDM cis-regulatory element is sufficient for promoter activation by polar auxin transport inhibitors and an inhibitor of auxin biosynthesis. Regulatory activity of the synthetic sequence containing six consecutive repeats of the MDM sequence from the UGT74E2 promoter (6xMDM[UGT74E2]) and from the AOX1a promoter (6xMDM1[AOX1a]) in transgenic Arabidopsis plants treated with NPA, TIBA, gravacin, quercetin, L-kynerine, and DMSO in concentrations described above. The construct mutated in the MDM sequence (6xMDM1mut[AOX1a]) was included as a negative control. Bars represent average luciferase activity ± SE (n = 8 biological replicates). Asterisks indicate significant differences to the mock treatment (Student’s t-test; * p < 0.05; ** p < 0.01; *** p < 0.001). Molecular Plant 2014 7, 1138-1150DOI: (10.1093/mp/ssu071) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 6 Constitutive Activation of MDS Gene Expression in the arf7arf19 Mutant Background. Transcript abundances of 14 of the MDS genes were quantified by qRT–PCR analysis in 2-week-old arf7arf19 plants relative to the wild-type. Bars represent average linear fold changes from four biological replicates (± SE). Asterisks indicate significant differences to the wild-type (Student’s t-test; * p < 0.05; ** p < 0.01; *** p < 0.001). Molecular Plant 2014 7, 1138-1150DOI: (10.1093/mp/ssu071) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions