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. 2013 Sep;25(9):3450-71.
doi: 10.1105/tpc.113.113985. Epub 2013 Sep 17.

A Membrane-Bound NAC Transcription Factor, ANAC017, Mediates Mitochondrial Retrograde Signaling in Arabidopsis

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A Membrane-Bound NAC Transcription Factor, ANAC017, Mediates Mitochondrial Retrograde Signaling in Arabidopsis

Sophia Ng et al. Plant Cell. .
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Abstract

Plants require daily coordinated regulation of energy metabolism for optimal growth and survival and therefore need to integrate cellular responses with both mitochondrial and plastid retrograde signaling. Using a forward genetic screen to characterize regulators of alternative oxidase1a (rao) mutants, we identified RAO2/Arabidopsis NAC domain-containing protein17 (ANAC017) as a direct positive regulator of AOX1a. RAO2/ANAC017 is targeted to connections and junctions in the endoplasmic reticulum (ER) and F-actin via a C-terminal transmembrane (TM) domain. A consensus rhomboid protease cleavage site is present in ANAC017 just prior to the predicted TM domain. Furthermore, addition of the rhomboid protease inhibitor N-p-Tosyl-l-Phe chloromethyl abolishes the induction of AOX1a upon antimycin A treatment. Simultaneous fluorescent tagging of ANAC017 with N-terminal red fluorescent protein (RFP) and C-terminal green fluorescent protein (GFP) revealed that the N-terminal RFP domain migrated into the nucleus, while the C-terminal GFP tag remained in the ER. Genome-wide analysis of the transcriptional network regulated by RAO2/ANAC017 under stress treatment revealed that RAO2/ANAC017 function was necessary for >85% of the changes observed as a primary response to cytosolic hydrogen peroxide (H2O2), but only ~33% of transcriptional changes observed in response to antimycin A treatment. Plants with mutated rao2/anac017 were more stress sensitive, whereas a gain-of-function mutation resulted in plants that had lower cellular levels of H2O2 under untreated conditions.

Figures

Figure 1.
Figure 1.
Identification of RAO2/ANAC017 as a Regulator of AOX1a. (A) Two-week-old seedlings (top panel) and luminescence of Col:LUC and rao2 mutants after treatment with AA. Col:LUC plants generated from Col-0 transformed with a construct with a firefly LUC reporter gene driven by the AOX1a promoter (AOX1a-LUC). LUC activity was then visualized in a NightOWL bioluminescence imager. (B) Luminescence of rao2-1 and rao2-2 after 6 h of myxothiazol treatment. (C) Quantified luminescence of rao2 mutants 6 h following AA or myxothiazol treatment. Bars indicate se, and asterisks indicate a significant difference (P < 0.001, Student’s t test) of LUC activity between Col:LUC and rao backgrounds. (D) Allelic test of rao2-1 and rao2-2. F1 seedlings were the first generation from a cross between rao2-1 and rao2-2 mutants. (E) Complementation test of rao2-1 transformed with full-length coding sequences of ANAC017. The F2 generation of transformants (Complement) was able to induce LUC following AA treatment. (F) Next-generation sequence analysis identifying a candidate mutation in ANAC017 (At1g34190). (G) ANAC017 gene model. ANAC017 contains a predicted NAC domain at its N terminus and a predicted TM domain at the C terminus. Sanger sequencer analysis of rao2-1 and rao2-2 confirmed the point mutations that caused amino acid changes. (H) Alignment of NAC proteins from rice (ONAC) and Arabidopsis (ANAC). Identical amino acids are colored in black, and amino acids that shared high similarity are colored in gray. Asterisk indicates the position of the point mutation in rao2 mutants.
Figure 2.
Figure 2.
Characterization of AOX1a Transcript and Protein Abundance in rao2 and anac017 T-DNA Insertion Lines. (A) A schematic gene model of ANAC017. Numbers indicate the nucleotide position of the mutation or the T-DNA insertion. rao2 mutants are marked in red asterisks, and positions of the T-DNA inserts (anac017-1 and anac017-2) are indicated by inverted triangles. TSS, transcriptional start site; NLS, putative nuclear localization signal. (B) AOX1a transcript (i), UBIQUITIN (UBC) transcript (ii), and AOX1a protein (iii) abundance under AA treatment. (i) Two-week-old seedlings were exposed to 50 µM AA or deionized water (Mock, −AA) and were harvested at 1, 3, and 6 h and transcript abundance analyzed by qRT-PCR. (ii) UBC was used as transcript abundance control. (iii) Protein abundance was quantified and AOX1a abundance was normalized against TOM40-1 to give a loading corrected quantification. Relative protein abundance, below each blot, was expressed as a percentage of the highest value of the set (i.e., for AOX1a, that for Col:LUC treated with AA is set to 100). Apparent molecular mass is indicated in kilodaltons. Bars indicate se, and asterisks indicate a significant difference (P < 0.001, Student’s t test) of AOX1a transcript abundance in Col:LUC compared with rao backgrounds with AA treatment. Pound signs indicate a significantly difference (P < 0.001, Student’s t test) of AOX1a transcript abundance in untreated versus AA treated, both in Col:LUC and rao backgrounds. (C) As in (B) except that induction was performed with 20 mM H2O2.
Figure 3.
Figure 3.
RAO2/ANAC017 Binds to Predicted NAC Binding Sites in the AOX1a Promoter. (A) A schematic diagram of AOX1a promoter region with NAC binding sites 1, 2, and 3. TSS, transcriptional start site. (B) Analysis of AOX1a promoter activity using GUS reporter assay. (i) Wild-type plants (Col-0) were transiently transformed with constructs expressing wild-type AOX1a promoters fused with GUS or with constructs with NAC binding sites mutated in the AOX1a promoter. Bars indicate se, asterisks indicate a significant difference of GUS activity between wild-type promoter and mutated promoter constructs (P < 0.05, Student’s t test), and pound signs indicate significant induction of GUS activity between mock-treated (deionized water) control and AA treatment (P < 0.05, Student’s t test). (ii) Analysis of AOX1a promoter activity using GUS reporter assay in wild-type plants (Col-0) and anac017-1 and ana017-2 genetic backgrounds. Bars indicate se, asterisks indicate a significantly difference of GUS activity between wild-type and mutant plants, and pound signs indicate significant induction of GUS activity between untreated control and AA treatment (P < 0.05, Student’s t test). All assays in (i) and (ii) were performed using three biological replicates. (C) GUS histochemical staining of 2-week-old transgenic Arabidopsis seedlings carrying either the wild-type (WT) AOX1a 2 kb promoter driving GUS expression (top panel) or constructs with the NAC binding sites deleted (bottom panel) 6 h after AA treatment. Data from three independent transformed lines are shown in Supplemental Figure 5 online. (D) Yeast one-hybrid binding assay of RAO2/ANAC017 to AOX1a promoter. Bait vector contained a 50-bp region surrounding NAC binding sites. Prey vector contained the full-length coding sequence of RAO2/ANAC017. Serial dilutions of cotransformed yeast were spotted on synthetic dropout (SD) –Trp/Leu to select for cotransformants and SD-Trp/Leu/His to select for positive interactions. Numbers 1, 2, and 3 in bold represent predicted NAC binding sites 1, 2, and 3 in AOX1a promoter. ΔNAC binding site is bait vector with NAC binding site deleted. The binding of p53 was used as positive (+) and negative (−) control according to the manufacturer’s instructions (Clontech). (E) Binding of RAO2/ANAC017 to NAC binding site 2 in the AOX1a promoter as determined by EMSA.
Figure 4.
Figure 4.
In Vivo Targeting of Full-Length and Truncated Forms of RAO2/ANAC017 Linked to Green Fluorescence Protein. (A) Subcellular targeting of full-length RAO2/ANAC017 (i), full-length RAO2/ANAC017 minus the predicted TM region (ii), and the predicted TM region of RAO2/ANAC017 (iii) were tagged with GFP to assess targeting ability. These three constructs were transiently transformed into both Arabidopsis suspension cells and onion epidermal cells, in addition to a mitochondrial RFP (Mito-RFP) control (see Methods). (B) Colocalization of RAO2/ANAC017 to actin filaments. Based on the localizations observed for the full-length construct, colocalization of RAO2/ANAC017 was confirmed with actin stained with rhodamine-labeled phalloidin control (i) and AtFim1-RFP control (ii). A magnified view of this colocalization has been provided. (C) Colocalization of RAO2/ANAC017 to the ER with an ER-RFP control.
Figure 5.
Figure 5.
Identification of the Activation Mechanism of ANAC017. (A) Characterization of AOX1a transcript abundance in splicing mutants. Relative AOX1a and UBIQUITIN (UBC) transcript abundance after 3 h of AA treatment. Two-week-old seedlings of Col-0, ire1a (At2g17520 T-DNA SALK_018112), ire1b (At5g24360 T-DNA GABI_638B07), and ire1a ire1b double mutants were treated with 50 µM AA and harvested 3 h later or before the treatment. Transcript abundance was measured by qRT-PCR. Bars indicate se, asterisks indicate a significant difference of AOX1a transcript abundance between the wild type and mutant in AA-treated samples (P < 0.001, Student’s t test), and pound signs indicate a significant difference between untreated (DMSO and ethanol control) and AA-treated samples (P < 0.001, Student’s t test). (B) Rhomboid protease cleavage site in the C-terminal region of ANAC017. Potential rhomboid substrates have a concentration of helix-breaking residues (S, Q, P, and G) around the cleavage site (underlined); the presence of a GA (helix-breaking) pair (indicated in red) and the presence of an ASI motif (LSI present in ANAC017) (as both L and S are hydrophobic, they have similar properties, indicated in red). The seven amino acids previously shown to be sufficient for cleavage by the Spitz rhomboid from D. melanogaster is shaded in gray (Strisovsky et al., 2009). The arrow indicates the cleavage site in the Spitz TM domain. Dm, Drosophila melanogaster. (C) Inhibition of the induction of AOX1a transcript abundance by the rhomboid protease inhibitor TPCK. Four-day-old suspension cell culture was treated with 100 μM TPCK or DMSO for 3 min followed by 50 μM AA treatment for 1 and 2 h. Ethanol and DMSO were used as a solvent-only control. Samples were then collected for AOX1a transcript abundance measurement using qRT-PCR. UBIQUITIN (UBC) was used as transcript abundance control. Bars indicate se, asterisks indicate a significant difference of AOX1a transcript abundance between untreated (DMSO and ethanol control) and AA-treated samples (P < 0.001, Student’s t test), and pound signs indicate a significant difference between samples treated with and without TPCK (i.e., AA plus DMSO versus AA plus TPCK treatments) (P < 0.001, Student’s t test).
Figure 6.
Figure 6.
Localization of N- and C-Terminal Tagged ANAC017 Using Fluorescence Microscopy. (A) In order to test whether ANAC017 is initially targeted to the ER, and subsequently relocated to the nucleus, a fusion protein was constructed, consisting of full-length ANAC017 with RFP fused to the N-terminal region and GFP fused to the C-terminal region. This construct allows the location of the N and C termini of ANAC017 to be determined independently. (B) The construct was transiently transformed into onion epidermal cells using biolistic transformation and observed using a fluorescence microscope at ×20 and ×60 magnification. It was observed that GFP colocalized only with the ER, while RFP was observed to colocalize partially with the ER, but was also found in the nucleus; note GFP was always conspicuously absent from the nucleus. (C) To influence the proportion of each fluorophore’s localization, onion cells transformed with this construct were treated with AA and incubated for 120 min prior to examination under a fluorescence microscope. AA treatment results in all RFP being detected in the nucleus. Also, GFP was not detected in the nucleus.
Figure 7.
Figure 7.
Characterization of the Genome-Wide Transcriptional Response to Stress That Is Regulated through RAO2/ANAC017 and Cannot Be Compensated for by Other Cellular Components. (A) and (B) Hierarchical cluster of gene set lists categorized as either positively or negatively regulated through ANAC017 activity under AA (A) or H2O2 (B) treatment, as described in Supplemental Figures 8 and 9 online. Blue color represents transcripts that are downregulated in response to treatment and red/yellow color represents transcripts that are upregulated in response to treatment as shown by the color scale, log2 fold changes in untreated versus treated conditions. Fold changes in response to stress were calculated for Col:LUC (wild type), rao2-1, and anac017-1 T-DNA insertional knockout lines. (C) Proportional breakdown of the degree that the response to AA or H2O2 is mediated through RAO2/ANAC017 function genome wide. (D) MapMan visualization of changes in transcript abundance for Col:LUC under treatment with H2O2 for transcripts that are regulated through ANAC017 function (i.e., the ANAC017 H2O2 stress-regulated pathway).
Figure 8.
Figure 8.
EMSAs Show Binding of ANAC017 to Promoters of Downstream Transcription Factor Genes. (A) The promoters of four selected transcription factors proposed to be regulated by ANAC017 contain one or more putative NAC transcription factor binding sites, CA(C/A)G, indicated in the central column and underlined. Binding of ANAC017 to these promoters was tested by EMSA. For some promoters, up to three sites were predicted and tested. The underlined sequences represent bases that were changed in the mutated probes (see Supplemental Data Set 1 online for sequences). (B) EMSA confirmed binding of ANAC017 to the four target promoters. Only putative NAC binding sites that displayed binding are shown. In all cases, binding could be abolished by unlabeled competitor probe or was not observed when the predicted NAC binding site was mutated.
Figure 9.
Figure 9.
Characterization of the Physiological Sensitivity to Environmental Stress and H2O2 in rao2/anac017. (A) Thirty-four-day-old plants growth for 16 h at 120 µmol m−2 s−1 light/8 h dark, 22°C, were transferred to 16 h at 300 µmol m−2 s−1 light/8 h dark, 22°C, without watering, and responses were observed after 9 d (top panel), after which water was resupplied at 120 µmol m−2 s−1 light for 3 d (bottom panel). (B) Thirty-four-day-old plants grown for 16 h at 120 µmol m−2 s−1 light/8 h dark, 22°C, were stained for H2O2 (3,3′-diaminobenzidine [DAB]) and superoxide radical O2•− (nitroblue tetrazolium [NBT]).
Figure 10.
Figure 10.
Summary of the Activation of ANAC017 in Mitochondrial Retrograde Signaling. Upon perturbation of mitochondrial function (e.g., inhibition of electron transport by AA), signals from the mitochondria lead to cleavage of ANAC017 by a rhomboid protease. The resulting protein migrates to the nucleus and induces the expression of AOX1a. The RNA mediator complex is indicated as it has been previously shown that a subunit of this complex is required for induction of AOX1a with AA treatment (Ng et al., 2013).

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