Arabidopsis RCD1 coordinates chloroplast and mitochondrial functions through interaction with ANAC transcription factors
- University of Helsinki, Finland
- Russian Academy of Sciences, Russia
- University of Jyväskylä, Finland
- University of Turku, Finland
- Max-Planck Institute for Molecular Plant Physiology, Germany
- Center of Plant System Biology and Biotechnology, Bulgaria
- Ghent University, Belgium
- VIB Center for Plant Systems Biology, Belgium
- University of Eastern Finland, Finland
- University of Tartu, Estonia
Figures
Figure 1 with 5 supplements
RCD1 controls tolerance of photosynthetic apparatus to ROS.
(A) MV treatment results in PSII inhibition under light, which is suppressed in the rcd1 mutant. PSII photochemical yield (Fv/Fm) was measured in rosettes pre-treated overnight in darkness with 1 μM …
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Figure 1—source data 1
Source data and statistics.
- https://doi.org/10.7554/eLife.43284.009
Figure 1—figure supplement 1
Inverse correlation of RCD1 abundance with tolerance to chloroplastic ROS.
(A) Several independent rcd1 complementation lines were generated in which HA-tagged RCD1 was reintroduced under the RCD1 native promoter. Immunoblotting of protein extracts from these lines with …
Figure 1—figure supplement 2
The Imaging PAM protocol developed to monitor kinetics of PSII inhibition by repetitive 1 hr light cycles.
Plants dark-adapted for at least 20 min were first exposed to a saturating light pulse to measure Fm. Then the blue actinic light (450 nm, 80 µmol m−2 s−1) was turned on for 1 hr, over which time …
Figure 1—figure supplement 3
Production rate of hydrogen peroxide in Col-0 and rcd1 during illumination of MV-pre-treated rosettes.
Col-0 and rcd1 rosettes were pre-treated with 1 μM MV overnight in the darkness. Then they were exposed to light for indicated time. After this, the rosettes were infiltrated with DAB staining …
Figure 1—figure supplement 4
Altered resistance of rcd1 photosynthetic apparatus to chloroplastic ROS.
(A) Protein extracts from Col-0 and rcd1 leaves pre-treated with 1 μM MV and exposed to light for indicated time, were separated by SDS-PAGE followed by immunoblotting with antibodies against the …
Figure 1—figure supplement 5
Components of photosynthetic electron transfer and chloroplast ROS scavenging; abundance and distribution of NAD+/NADH and NADP+/NADPH redox couples in Col-0 and rcd1.
(A) Abundance of proteins related to photosynthetic electron transfer or chloroplast ROS scavenging was assessed by separating Col-0 and rcd1 protein extracts (in dilution series) by SDS-PAGE and …
Figure 2 with 1 supplement
RCD1 protein is sensitive to chloroplastic ROS.
(A) The rcd1: RCD1-HA complementation line was used to assess RCD1-HA abundance. It gradually decreased in response to chloroplastic ROS. Leaf discs from plants expressing HA-tagged RCD1 were …
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Figure 2—source data 1
Source data and statistics.
- https://doi.org/10.7554/eLife.43284.012
Figure 2—figure supplement 1
Characterization of the rcd1: RCD1Δ7Cys-HA lines.
(A) Domain structure of RCD1 with the positions of cysteine residues shown with circles. Interdomain cysteines mutated in the RCD1Δ7Cys-HA lines (RCD1Δ7Cys = RCD1 …
Figure 3
Altered energy metabolism of rcd1.
Deduced metabolic fluxes in light- and dark- adapted Col-0 and rcd1 rosettes were assessed by fractionation of the extracts of leaves treated with [U-14C] glucose. Increased respiration flux and …
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Figure 3—source data 1
Metabolic analyses.
Distribution of radioactive label was analyzed after feeding plants with 14C-labeled glucose. Metabolic fluxes in light- and dark-adapted Col-0, rcd1, rcd1 aox1a, and aox1a plants were deduced.
- https://doi.org/10.7554/eLife.43284.014
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Figure 3—source data 2
Source data and statistics.
- https://doi.org/10.7554/eLife.43284.015
Figure 4 with 4 supplements
Mitochondrial AOXs affect energy metabolism of rcd1 and alter response to chloroplastic ROS.
Source data and statistics are presented in Figure 4—source data 1. (A) Expression of AOXs is induced in rcd1. Abundance of AOX isoforms in mitochondrial preparations was assessed by immunoblotting …
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Figure 4—source data 1
Source data and statistics.
- https://doi.org/10.7554/eLife.43284.021
Figure 4—figure supplement 1
Effect of mitochondrial complex III inhibitors on expression of AOXs in Col-0 and rcd1.
(A) Changes in AOX abundance after overnight pre-treatment of leaf discs with 2.5 μM AA or 2.5 μM myx (C – control treatment with no inhibitor). Notably, rcd1 aox1a double mutant accumulated AOXs …
Figure 4—figure supplement 2
Effect of mitochondrial complex III inhibitors on abundance and redox state of the RCD1 protein.
(A) Chemical induction of mitochondrial dysfunction signaling did not alter abundance of the RCD1 protein. Leaf discs were treated with 2.5 μM AA or 2.5 μM myx overnight. Then total protein extracts …
Figure 4—figure supplement 3
Specificity of inhibitor treatments.
All chlorophyll fluorescence analyses are presented as mean ±SD, for source data and statistics see Figure 4—source data 1. (A) Interaction of AA with cyclic electron flow through binding to …
Figure 4—figure supplement 4
Irrelevance of AOX1a isoform for MV tolerance.
All chlorophyll fluorescence analyses are presented as mean ±SD, for source data and statistics see Figure 4—source data 1. (A) Abundance of total AOX in the AOX1a-overexpressor line (AOX1a-OE) as …
Figure 5 with 2 supplements
Altered electron transfer between the organelles in rcd1.
(A) Leaf discs were pre-treated with 1 μM MV or MV plus 2 mM SHAM for 1 hr in the darkness. Then light was turned on (80 µmol m−2 s−1) and chlorophyll fluorescence under light (Fs) was recorded by …
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Figure 5—source data 1
Source data and statistics.
- https://doi.org/10.7554/eLife.43284.025
Figure 5—figure supplement 1
Alterations in chloroplast electron transfer induced by MV and SHAM.
During the first 20 min of light exposure, MV-pre-treated Col-0 and rcd1 experienced transient decrease in PSII photochemical quenching (qP). Within the next hour, photosynthesis recovered in rcd1 …
Figure 5—figure supplement 2
Distribution of malate in subcellular compartments of Col-0 and rcd1.
Distribution of malate was assessed by non-aqueous fractionation metabolomics as described in Figure 1—figure supplement 5C. Mean values ± SE are presented. For source data and statistics, see Figure…
Figure 6 with 3 supplements
RCD1 is involved in mitochondrial dysfunction, chloroplast ROS and PAP signaling pathways.
(A) Regulation of rcd1 mis-expressed genes under perturbations of organellar functions in the selected subset of genes. A complete list of rcd1-misexpressed genes is presented in Figure 6—figure …
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Figure 6—source data 1
Source data and statistics.
- https://doi.org/10.7554/eLife.43284.030
Figure 6—figure supplement 1
Clustering analysis of genes mis-regulated in rcd1 (with cutoff of logFC <0.5) in published gene expression data sets acquired after perturbations of chloroplasts or mitochondria.
Mitochondrial dysfunction stimulon (MDS) genes are labeled green. Enrichment of the ANAC013/ANAC017 cis-element CTTGNNNNNCA[AC]G (De Clercq et al., 2013) in promoter regions is shown by shaded boxes …
Figure 6—figure supplement 2
Induction of MDS genes in rcd1 and rcd1 complementation lines.
To address the role of RCD1 in transcriptional response to AA, plant rosettes were sprayed with water solution of 50 μM AA (or of DMSO as the control). This concentration of AA has been commonly …
Figure 6—figure supplement 3
Tolerance of PSII to chloroplastic ROS in sal1 mutants.
MV-induced PSII inhibition was tested in 2.5 week rosettes. The single sal1 mutant was more tolerant to MV than the wild type (left panel). The double rcd1 sal1 mutant was more tolerant to MV than rc…
Figure 7 with 3 supplements
RST domain of RCD1 binds to ANAC transcription factors and is necessary for RCD1 function in vivo.
Source data and statistics are presented in Figure 7—source data 4. (A) Biochemical interaction of ANAC013 with the RST domain of RCD1 in vitro. Superimposed 1H, 15N HSQC spectra of the C-terminal …
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Figure 7—source data 1
In vivo interaction partners of ANAC013.
From Arabidopsis line expressing ANAC013-GFP, ANAC013-GFP and associated proteins were purified with αGFP antibody and identified by mass spectrometry. Identified proteins (Perseus analysis, ANAC013) and mapped peptides (peptide IDs) are shown.
- https://doi.org/10.7554/eLife.43284.036
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Figure 7—source data 2
In vivo interaction partners of RCD1.
From Arabidopsis line expressing RCD1-3xVenus, RCD1-3xVenus and associated proteins were purified with αGFP antibody and identified by mass spectrometry. Identified proteins (Perseus analysis, RCD1) and mapped peptides (peptide IDs) are shown.
- https://doi.org/10.7554/eLife.43284.037
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Figure 7—source data 3
NMR constraints and structural statistics for the ensemble of the 15 lowest-energy structures of RCD1 RST.
- https://doi.org/10.7554/eLife.43284.038
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Figure 7—source data 4
Source data and statistics.
- https://doi.org/10.7554/eLife.43284.039
Figure 7—figure supplement 1
Biochemical interaction of RCD1 with ANAC013/ANAC017 transcription factors in human embryonic kidney (HEK293) cells.
HA-RCD1 was co-expressed with ANAC013-myc (A) or ANAC017-myc (B) (IP – eluate after immunoprecipitation). (A) Co-immunoprecipitation of HA-RCD1 with αmyc antibody (top) and of ANAC013-myc with αHA …
Figure 7—figure supplement 2
Structure of the RST domain of RCD1.
Structure of the C-terminal domain of RCD1 (residues G468-L589) was determined by NMR spectroscopy. The first 38 N-terminal and the last 20 C-terminal residues are devoid of any persistent …
Figure 7—figure supplement 3
Analysis of interaction of the ANAC013-derived peptides with the RST domain of RCD1.
(A) According to yeast two-hybrid data (O'Shea et al., 2017), ANAC013 residues 205–299 are responsible for interaction with RCD1. To narrow down the RCD1-interacting domain, three overlapping …
Figure 8 with 1 supplement
Developmental, chloroplast- and mitochondria-related phenotypes of rcd1 are partially mediated by ANAC017.
Source data and statistics are presented in Figure 8—source data 1. (A) Introducing anac017 mutation in the rcd1 background partially suppressed the curly leaf phenotype of rcd1. (B) The anac017 …
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Figure 8—source data 1
Source data and statistics.
- https://doi.org/10.7554/eLife.43284.042
Figure 8—figure supplement 1
Induction of MDS genes in anac017 and rcd1 anac017 mutants.
Expression of the selected MDS genes was assessed in rosettes 3 hr after spraying them with 50 μM AA, as described in Figure 6—figure supplement 2. The anac017 mutation strongly suppressed induction …
Figure 9
Hypothetical role of RCD1 in organelle signaling and energy metabolism.
RCD1 is the direct suppressor of ANAC transcription factors that is itself subject to redox regulation. Chloroplastic ROS likely affect RCD1 protein redox state and abundance. Inactivation of RCD1 …
Tables
Table 1
Overview of the immunoprecipitation results.
Selected proteins identified in ANAC013-GFP and RCD1-3xVenus pull-down assays. Ratio vs. Col-0 and the P-value were obtained by Perseus statistical analysis from the three repeats for each genotype …
| Ratio vs. Col-0 | P-value | Unique peptides | Gene | Name | Stickiness |
|---|---|---|---|---|---|
| ANAC013-GFP pull-down | |||||
| 50966 | 7.09 × 10−7 | 29 | AT1G32870 | ANAC013 | |
| 22149 | 3.41 × 10−8 | 25 | GFP | ||
| 10097 | 3.67 × 10−6 | 37 | AT1G32230 | RCD1 | 1.00% |
| 110 | 1.67 × 10−6 | 8 | AT2G35510 | SRO1 | 1.00% |
| 74 | 1.09 × 10−9 | 4 | AT1G34190 | ANAC017 | 1.00% |
| RCD1-3xVenus pull-down | |||||
| 7593 | 0.000454 | 35 | AT1G32230 | RCD1 | |
| 1292 | 0.006746 | 10 | YFP | ||
| 108 | 5.48 × 10−8 | 2 | AT1G34190 | ANAC017 | 1.00% |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent(Arabidopsis thaliana, Col-0) | rcd1-4 | NASC stock center | GK-229D11 | homozygous mutant plant line |
| Genetic reagent(Arabidopsis thaliana, Col-0) | rcd1-1 | PMID: 11041881 | homozygous mutant plant line | |
| Genetic reagent(Arabidopsis thaliana, Col-0) | aox1a | PMID: 16299171 | homozygous mutant plant line | |
| Genetic reagent(Arabidopsis thaliana, Col-0) | AOX1a-OE | PMID: 16299171 | homozygous mutant plant line | |
| Genetic reagent(Arabidopsis thaliana, Col-0) | ptox | PMID: 7920709 | homozygous mutant plant line | |
| Genetic reagent (Arabidopsis thaliana, Col-0) | anac017 | NASC stock center | SALK_022174 | homozygous mutant plant line |
| Genetic reagent(Arabidopsis thaliana, Col-0) | sal1-8 | PMID: 19170934 | homozygous mutant plant line | |
| Genetic reagent(Arabidopsis thaliana, Col-0) | rcd1 aox1a | PMID: 24550736 | homozygous mutant plant line | |
| Genetic reagent(Arabidopsis thaliana, Col-0) | rcd1-1 anac017 | this paper | homozygous mutant plant line | |
| Genetic reagent(Arabidopsis thaliana, Col-0) | rcd1-4 sal1-8 | this paper | homozygous mutant plant line | |
| Genetic reagent(Arabidopsis thaliana, Col-0) | rcd1-4: RCD1-HA | this paper | set of complementation plant lines | |
| Genetic reagent(Arabidopsis thaliana, Col-0) | rcd1-4: RCD1-3xVenus | this paper | set of complementation plant lines | |
| Genetic reagent(Arabidopsis thaliana, Col-0) | rcd1-4: RCD1Δ7Cys-HA | this paper | set of complementation plant lines | |
| Genetic reagent(Arabidopsis thaliana, Col-0) | rcd1-4: RCD1ΔRST-HA | this paper | set of complementation plant lines | |
| Genetic reagent(Arabidopsis thaliana, Col-0) | ANAC013-GFP | PMID: 24045019 | transgenic plant line | |
| Genetic reagent(Arabidopsis thaliana, gl1) | pgr5 | PMID: 12176323 | homozygous mutant plant line | |
| Cell line (Homo sapiens) | HEK293T | ATCC | ATCC CRL-3216 | human embryonic kidney cell line |
| Gene (Homo sapiens) | HA-RCD1 | this paper | construct for expression in HEK293T cells | |
| Gene (Homo sapiens) | ANAC013-myc | this paper | construct for expression in HEK293T cells | |
| Gene (Homo sapiens) | ANAC017-myc | this paper | construct for expression in HEK293T cells | |
| Antibody | αHA | Roche | Roche 1 867 423 001 | 1: 2 000 for immunoblotting |
| Antibody | αGFP | Milteny Biotech | ||
| Antibody | αRCD1 | this paper | 1: 500 for immunoblotting | |
| Antibody | αSOT12 | Dr. Saijaliisa Kangasjärvi | Agrisera AS16 3943 | 1: 500 for immunoblotting |
| Peptide, recombinant protein | ANAC013 peptides | Genecust | Synthetic peptides |
Additional files
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Supplementary file 1
Primers used in the study.
- https://doi.org/10.7554/eLife.43284.044
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Transparent reporting form
- https://doi.org/10.7554/eLife.43284.045
