1. Systems Phytohormone Responses to Mitochondrial Proteotoxic Stress
Xu Wang, Johan Auwerx 
Molecular Cell 
Volume 68, Issue 3, Pages e5 (November 2017)
DOI: /j.molcel
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2. Molecular Cell 2017 68, 540-551.e5DOI: (10.1016/j.molcel.2017.10.006)
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3. Figure 1
Phenotypes of the Arabidopsis MRPL1 Mutants and Wild-Type Plants under Doxycycline Treatment
(A) Seven-day-old wild-type plants and MRPL1 mutants.
(B) Oxygen consumption rate (OCR) was reduced in leaves of 7-day-old MRPL1 mutants.
(C) Immunoblot of proteins from leaf mitochondria in plants from (A). Proteins encoded by mtDNA or nuDNA were labeled.
(D) OCR in isolated mitochondria prepared from leaves. Seven-day-old wild-type plants were exposed to 25 μg/mL Dox for another 7 days. CI, complex I.
(E) Morphology of seedlings (top) and root hairs (bottom) in plants from (D).
(F) Immunoblot of proteins from leaf mitochondria of 14-day-old plants under Dox treatment for 12 hr.
(G) Blue native PAGE and activity assay of mitochondrial complex (I and IV) and supercomplex (I1+III2) (Eubel et al., 2004) in 14-day-old wild-type (WT/control), mrpl1-1 mutants, or wild-type plants exposed to 50 μg/mL Dox for 12 hr.
(H) Forty-five-day-old wild-type plants watered with water containing 5, 10, or 25 μg/mL Dox. Plants entered treatment after 14 days old.
(I) Morphology of 80-day-old plants watered with regular water or water containing 25 μg/mL Dox.
(J) Total chlorophyll content in plants from (I). Lower level of chlorophyll in control plants is a sign of senescence.
Data in (B), (D), and (J) are represented as mean ± SEM; three biological replicates in (B) and (D); five biological replicates in (J); ∗∗p < 0.01, ∗∗∗p < See also Figure S1.
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4. Figure 2
Mitochondrial Proteotoxic Stress Induces Genes of the mtPQC Network
(A and B) Induction of AOX, mtHSP, and MRP genes in 4-day-old mrpl1-1 mutant (A) or wild-type plants exposed to 25 μg/mL Dox for 7 days (B). Data are represented as mean ± SEM; three biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p <
(C) Correlation network of 108 transcripts from the MRPs, alternative respiratory pathway, and mtPQC-related families across the 144 natural Arabidopsis accessions.
(D) Correlation network from 108 randomly selected genes in the same dataset, serving as a visual control for the data in (C). Edge counts correspond to the level expected from noise.
(E) Meta-analysis of transcriptional responses of plant genes encoding HSP60, HSP70, and other chaperones and proteases to mitochondrial perturbations. Genes encoding mitochondrial stress proteins were induced more robustly compared to those in other subcellular compartments. Datasets are in the STAR Methods. Mt, mitochondrion; Ch, chloroplast; ER, endoplasmic reticulum; Cy, cytosol.
See also Figures S2–S4 and Table S1.
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5. Figure 3
The Ethylene Response Was Enhanced in Plants with Impaired Mitochondrial Translation
(A) Enrichment of GO annotations for upregulated genes in RNA-seq data from mrpl1-1 mutants and wild-type plants treated with 25 μg/mL Dox, using the Bingo plugin in Cytoscape. The color scale represents the significance of enrichment. Transcription factors enriched were classified according to their gene families below the scheme.
(B) Representative genes of each transcription factor family in the overlapping region between the two datasets analyzed in (A).
(C) Expression of the AP2/ERF family transcription factors is enhanced in mrpl1-1 mutants.
(D) Gene sets of ethylene biosynthesis and cell response to ethylene stimulus were enriched in the mrpl1-1 mutants as defined by GSEA, both with nominal p = 0.
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6. Figure 4 Ethylene Signaling Is Involved in Retrograde Regulation
In (A)–(G), wild-type Arabidopsis or mutant seedlings were treated with 50 μM AgNO3 or 10–100 μM ACC for 4 days in the dark after germination. n = 3 with >10 seedlings.
(A) Comparison of hypocotyl morphology in etiolated wild-type (WT), mrpl1-1, mrpl1-3, ein4, and eto1-1 mutants under the four conditions.
(B) Seedlings were, in addition to the above treatments (AgNO3 or ACC), exposed to either 10 or 20 μg/mL Dox and hypocotyls were compared.
(C) Ethylene-insensitive mutants exposed to 10 μg/mL Dox.
(D and E) Induction of ethylene signaling and biosynthesis genes in etiolated mrpl1-1 mutants (D) from (A) or Dox-treated seedlings (E) from (B).
(F and G) Expression of ethylene signaling (EBS::GUS; F) and biosynthesis (ACS6::GUS; G) reporters is induced by 25 μg/mL Dox.
In (H)–(J), Arabidopsis wild-type seedlings were treated with 25 μg/mL Dox, 50 μM ACC, or vehicle for 6 days in the dark after germination. n = 3 with >10 seedlings.
(H) Scheme illustrating the split-medium experiment. In a single Petri dish, gaseous ethylene (ET) produced on “Treat” part spreads to “Vehicle” part and affects plants there. There is no physical contact between “Treat” and “Vehicle” media.
(I) Induction of ERF1/2 and ACS6 transcript levels in seedlings from vehicle part of split-medium plate versus control plate.
(J) Comparison of hypocotyl morphology from seedlings on control plate (half MS medium) and on vehicle part of split-medium plate.
Data in (D), (E), and (I) are represented as mean ± SEM; three biological replicates. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, as versus wild-type plant or control condition. See also Figure S5.
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7. Figure 5 Dynamics of Ethylene Action and MAPK Signaling
(A) A time course quantification of ERF and ACS transcripts in 7-day-old wild-type seedlings exposed to 25 μg/mL Dox.
(B) Expression pattern of EBS::GUS in a time series of Dox exposure in 7-day-old reporter strains. n = 3 with >5 seedlings.
(C and D) MPK6 was phosphorylated (P-MPK6) when dark- (C) or light-grown (D) 7-day-old wild-type seedlings were exposed to 25 μg/mL Dox for 0–60 min.
(E and F) MPK6 was phosphorylated (P-MPK6) when light-grown 7-day-old wild-type seedlings were exposed to 25 μg/mL Dox, 100 μg/mL CAP, 50 μm MB, or 30 μm rotenone (Rot) for 30 min (E). The P-MPK6 was reduced to basal levels after treatment for 24 hr (F).
(G) Induction of ERF1 and mtHSP70 transcripts was attenuated by U0126. Twelve-day old wild-type plants were exposed to 25 μg/mL Dox in the presence or absence of 10 μM U0126 for 1 hr.
(H) Quantification of ERF1/2 transcripts in 19-day-old wild-type plants exposed to 25 μg/mL Dox for 10 days.
(I) ACS4/6/8::GUS reporter strains record reduced ACS gene expression in plants under long-term Dox treatment. Seven-day-old wild-type seedlings were exposed to 25 μg/mL Dox for another 5 days in the light. n = 3 with >5 seedlings.
Bar graphs in (A), (G), and (H) are represented as mean ± SEM; three biological replicates. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, as versus wild-type plant or control condition. See also Figure S6.
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8. Figure 6
Ethylene-Mediated Anterograde Signaling Restores Mitochondrial Function
(A) GSEA of extant microarray data (GEO: GSE5174). The gene set of mitochondrial translation was positively enriched in ethylene-treated wild-type Arabidopsis (nominal p = 0), but not in the ethylene-insensitive ein2 mutants (nominal p = 0.26). Heatmaps rank the fold changes of relevant gene expression.
(B) Induction of MRP genes in wild-type seedlings treated with 50 μM ACC for 4 days in the dark after germination.
(C) Induction of MRP genes in 4-day-old etiolated mrpl1-1 seedlings.
(D) Induction of MRP genes in wild-type seedlings treated with 25 μg/mL Dox for 7 days in the dark.
(E) OCR in mitochondria prepared from wild-type Arabidopsis exposed to 50 μM ACC or AgNO3 for 4 days from germination in dark.
(F) Blue native PAGE and activity assay of mitochondrial complex (I and IV) and supercomplex (I1+III2) in etiolated wild-type plants with 25 μM ACC or 50 μM AgNO3 for 4 days from germination in dark.
Data in (B)–(E) are represented as mean ± SEM; three biological replicates. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, as versus control condition or wild-type plant. See also Figure S7.
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9. Figure 7
Impaired Mitochondrial Translation Modulates Multiple Hormones
(A) Expression of marker genes reflective of the activation of different hormonal pathways in 7-day-old wild-type seedlings exposed to 25 μg/mL Dox for 1 hr. Data are represented as mean ± SEM; three biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, as versus control condition.
In (B)–(E), 10-day-old hormone reporters were exposed to 25 μg/mL Dox, 100 μg/mL CAP, or 50 μm MB for 1 hr in light.
(B and C) The R2D2 reporter records rapid auxin input in root tips exposed to Dox (B), CAP, and MB (C). Compared to control signal of mDII-tdTomato that lacks auxin-dependent degradation, the decreased signal of DII-Venus indicates an activation of auxin signaling. Images from both primary and lateral roots are shown in (B). Scale bars, 10 μm. n = 3 with >5 seedlings.
(D and E) Jas9-Venus sensor maps bioactive jasmonates in root tips exposed to Dox (D), CAP, and MB (E). The reduced Jas9-Venus signal upon Dox, CAP, and MB reflects the activation of jasmonate signaling. n = 3 with >5 seedlings.
(F) A scheme illustrating the mechanism by which mitochondrial proteotoxic stress activates hormone signaling and restores mitochondrial function.
See also Figure S7.
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