RETRACTED: Pyrophosphate modulates plant stress responses via SUMOylation
- Zaida Andrés
- Melanie Krebs
- Fabian Fink
- Katarzyna Drzewicka
- Nicolas Stankovic-Valentin
- Shoji Segami
- Sebastian Schuck
- Michael Büttner
- Rüdiger Hell
- Masayoshi Maeshima
- Frauke Melchior
- Karin Schumacher
- Heidelberg University, Germany
- Center for Molecular Biology of Heidelberg University (ZMBH) and DKFZ - ZMBH Alliance, Germany
- Nagoya University, Japan
Figures
Figure 1 with 3 supplements
PPi hydrolysis is required to rescue the freezing sensitive phenotype of fugu5-1.
(A) and (B) Freezing tolerance assay. Col-0, fugu5-1, UBQ:AVP1 and UBQ:PPa5-GFP in Col-0 and fugu5-1 backgrounds were grown for 6 weeks at 22°C and were then moved to 4°C for cold-acclimation, or …
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Figure 1—source data 1
(B) Survival of the 6 weeks old rosette leaves upon freezing.
(C) Electrolyte leakage analysis of the cold-acclimated and non-acclimated 6-weeks old rosette leaves upon exposure to freezing temperature. (D-G) Sugar and PPi measurements of the 6-weeks old rosette leaves.
- https://doi.org/10.7554/eLife.44213.008
Figure 1—figure supplement 1
Increased Proton Transport Activity of V-ATPase upon Cold Acclimation Requires an Active V-PPase.
(A) Enriched tonoplast proteins were used to determine K+-stimulated PPi hydrolysis and H+ transport activity of V-PPase. (B) Enriched tonoplast proteins were used to determine KNO3-inhibited ATP …
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Figure 1—figure supplement 1—source data 1
(A) V-PPase PPi hydrolysis and H+- transport measurements.
(B) V-ATPase ATP hydrolysis and H+-transport measurements. (C) Cell sap pH measurements.
- https://doi.org/10.7554/eLife.44213.004
Figure 1—figure supplement 2
Overexpression of the Arabidopsis soluble pyrophosphatase PPa5 is sufficient to complement fugu5-1.
(A) Comparison of 6-week-old rosettes phenotypes of Col-0, fugu5-1 and overexpression lines of Arabidopsis vacuolar pyrophosphatase AVP1 and yeast soluble pyrophosphatase IPP1. (B) Representative …
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Figure 1—figure supplement 2—source data 1
(E) Fresh weight and area measurements of the 6-weeks old rosette leaves.
(G) Soluble pyrophosphatase activity measurements.
- https://doi.org/10.7554/eLife.44213.006
Figure 1—figure supplement 3
Freezing tolerance phenotypes of the sPPase mutants.
(A) Wt, fugu5-1, ppa1, ppa1,4, ppa1,2,4,5 were grown for 6 weeks at 22°C and were then moved to 4°C for cold-acclimation, or kept at 22°C for 4 days. Electrolyte leakage was performed on leaf …
Figure 2
sPPase expression is induced upon cold exposure to control PPi that affects expression of CBFs and CBF target genes.
(A) qRT-PCR for the analysis of expression of PPa 1,2,4,5 and AVP1 in Col-0 and PPa 1,2,4 and 5 in fugu5-1. (B-C) Measurement of expression of CBF, COR and GolS3 genes in Col-0, fugu5-1 and UBQ:PPa5-…
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Figure 2—source data 1
(A) Relative expression of the vacuolar and soluble pyrophosphatases in wt and the relative expression of the soluble pyrophosphtases in fugu5-1 exposed to 4°C for different hours.
(B-C) Relative expression of the cold regulated genes in wt, fugu5-1 and UBQ:PPa5-GFP/fugu5-1 upon exposure to 4°C for different hours.
- https://doi.org/10.7554/eLife.44213.010
Figure 3 with 1 supplement
Modification of ICE1 and general SUMOylation upon cold exposure are inhibited in fugu5.
(A) Cold acclimation induces ICE1 SUMOylation which then activates CBFs and leads to the expression of downstream targets for the establishment of freezing tolerance. (B) Determination of the amount …
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Figure 3—source data 1
(D-E) Comparison of the amount of the total SUMOylation with and without cold treatment.
- https://doi.org/10.7554/eLife.44213.013
Figure 3—figure supplement 1
Western blot detection of ICE1 in protein extracted in the presence of DTT.
(A) Comparison of total protein of 10-days-old Col-0 and fugu5-1 seedlings extracted ±DTT (5 mM) and NEM (20 mM).
Figure 4
Heat shock-induced SUMOylation is also reduced in fugu5.
(A) Phenotypic analysis of 10-days-old Col-0, fugu5-1, UBQ:PPa5-GFP in Col-0 and fugu5-1 backgrounds and UBQ:AVP1 seedlings analysis before and after heat. Representative pictures of seedlings …
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Figure 4—source data 1
(B) Survival measurement of the 10 days old seedlings upon heat shock.
(C-D) Comparison of the amount of the total SUMOylation with and without heat shock treatment.
- https://doi.org/10.7554/eLife.44213.015
Figure 5
Increased PPi levels interfere with SUMOylation in yeast.
(A) Amount of the soluble pyrophosphatase protein in a conditional Ipp1 mutant of S. cerevisiae (GAL:HA-IPP1). Asterisk indicates significant difference compared to GAL:HA-IPP1 in galactose time …
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Figure 5—source data 1
(A) Amount of IPP1 in different carbon supplies over time.
(B) Comparison of the total SUMOylation of wt yeast at 28 and 40°C. (C) Comparison of the total SUMOylation at 40°C, in Ipp1 conditional mutant.
- https://doi.org/10.7554/eLife.44213.017
Figure 6 with 2 supplements
PPi regulates SUMOylation activity in vitro.
(A) Schematic illustration of the FRET-based sumoylation assays. Upon addition of ATP, the human SUMO E1 activating enzyme Aos1/Uba2 and the E2 conjugating enzyme Ubc9 form an isopeptide bond …
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Figure 6—source data 1
(B) Ratio values (527/485 nm) of the FRET based SUMOylation showing the effect of the increasing amount of PPi concentrations.
(C) Ratio values (527/485 nm) of the FRET based SUMOylation assay showing that theE. colisoluble PPase is able to remove the inhibitory effect of PPi. (D) Calculation of the Vmax and Km of SUMOylation activity upon PPi inhibition based on the data shown in Figure 6—figure supplement 1.
- https://doi.org/10.7554/eLife.44213.023
Figure 6—figure supplement 1
Elevated PPi concentrations leads to mixed inhibition of SUMOylation in vitro.
(A) SUMOylation assays demonstrating the effect of the increasing amounts of PPi to the speed of the reaction in a range of 0–10 µM ATP concentration. Experiments were repeated four times, one …
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Figure 6—figure supplement 1—source data 1
Ratio values (527/485 nm) of the FRET based SUMOylation assay showing the effect of the increasing amounts of PPi to the speed of the reaction in a range of 0–10µMATP concentration.
- https://doi.org/10.7554/eLife.44213.020
Figure 6—figure supplement 2
AtSAE1/2 purification and its activity in in vitro FRET based SUMOylation assay.
(A) Gel picture after purification of AtSAE1 and AtSAE2. Highlighted fractions from the final gel filtration step were combined, dialysed and used for subsequent experiments. (B) The purified …
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Figure 6—figure supplement 2—source data 1
(A) Ratio values (527/485 nm) of the FRET-based SUMOylation assay with Arabidopsis E1.
- https://doi.org/10.7554/eLife.44213.022
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Arabidopsis thaliana) | AtAVP1 | TAIR: AT1G15690 | ||
| Gene (Arabidopsis thaliana) | AtPPa1 | TAIR: AT1G01050 | ||
| Gene (Arabidopsis thaliana) | AtPPa2 | TAIR: AT2G18230 | ||
| Gene (Arabidopsis thaliana) | AtPPa4 | TAIR: AT3G53620 | ||
| Gene (Arabidopsis thaliana) | AtPPa5 | TAIR: AT4G01480 | ||
| Gene (Arabidopsis thaliana) | AtSUMO1 | TAIR: AT4G26840 | ||
| Gene (Arabidopsis thaliana) | AtICE1 | TAIR: AT3G26744 | ||
| Gene (Arabidopsis thaliana) | AtSAE1 | TAIR: AT4G24940 | ||
| Gene (Arabidopsis thaliana) | AtSAE2 | TAIR: AT2G21470 | ||
| Gene (Saccharomices cerevisiae) | ScIPP1 | SGD: YBR011C | ||
| Strain, strain background (Agrobacterium tumefaciens) | ASE | Lampropoulos et al., 2013 | pSOUP+ | |
| Strain, strain background (Saccharomices cerevisiae) | W303 | Szoradi et al., 2018 | SSY122 | |
| Strain, strain background (Saccharomices cerevisiae) | IPP1prΔ::HIS3- GAL1pr-HA-IPP1 | this paper | SSY2542 | Sebastian Schuck lab |
| Genetic reagent (Arabidopsis thaliana) | fugu5-1 | Ferjani et al., 2011 | ||
| Genetic reagent (Arabidopsis thaliana) | fugu5-3 | Ferjani et al., 2011 | ||
| Genetic reagent (Arabidopsis thaliana) | ice1-2 | Nottingham Arabidopsis Stock Centre (NASC) | SALK_003155 | |
| Genetic reagent (Arabidopsis thaliana) | ppa1 | Segami et al., 2018; NASC | SAIL_251_D07 | |
| Genetic reagent (Arabidopsis thaliana) | ppa1,4 | this paper | SAIL_251_D07, SAIL_916_C08 | Cross between the mutants ppa1 and ppa4 described in Segami et al., 2018 Masayoshi Maeshima lab |
| Genetic reagent (Arabidopsis thaliana) | ppa1,2,4,5 | Segami et al., 2018 | SAIL_251_D07, SAIL_618_H05, SAIL_916_C08, SALK_014647 | |
| Biological sample (Arabidopsis thaliana) | AVP1:IPP1/fugu5-1 | Ferjani et al., 2011 | AVP1 promoter:IPP1 coding sequence; fugu5-1 mutant background | |
| Biological sample (Arabidopsis thaliana) | UBQ:AVP1 #18–4 | Kriegel et al., 2015 | UBQ promoter:AVP1 coding sequence; Col-0 wild-type background | |
| Biological sample (Arabidopsis thaliana) | UBQ:PPa5-GFP/Col-0 | this paper, | UBQ promoter:PPa5-GFP coding sequence; Col-0 wild-type background. Karin Schumacher lab | |
| Biological sample (Arabidopsis thaliana) | UBQ:PPa5-GFP/fugu5-1 | this paper, | UBQ promoter:PPa5-GFP coding sequence; fugu5-1 wild-type background. Karin Schumacher lab | |
| Antibody | Anti-SUMO1 (rabbit polyclonal) | Agrisera | AS08308 | (1:1000) |
| Antibody | Anti-ICE1 (rabbit polyclonal) | Agrisera | AS163971 | (1:1000) |
| Antibody | Anti-cFBPase (rabbit polyclonal) | Agrisera | AS04043 | (1:5000) |
| Antibody | Anti-rabbit-HRP (Goat polyclonal) | Promega | W401B | (1:10000) |
| Antibody | Anti-IPP1 (rabbit polyclonal) | Antibodies- online GmbH | ABIN459215 | (1:1000) |
| Antibody | Anti-PGK1 (mouse monoclonal) | Abcam | AB113687 | (1:100000) |
| Antibody | Anti-mouse-HRP (sheep polyclonal) | GE Healthcare UK | NXA931 | (1:5000) |
| Antibody | Anti-V-PPase (rabbit polyclonal) | Agrisera | AS121849 | (1:10000) |
| Antibody | Anti-VHA-C (rabbit polyclonal) | Karin Schumacher lab | Schumacher et al., 1999 | (1:2000) |
| Antibody | Anti-GFP (rabbit polyclonal) | Karin Schumacher lab | Roth et al., 2018 | (1:10000) |
| Recombinant DNA reagent | UBQ:PPa5-GFP | this paper, | vector-promoter:tagged-protein construct. Karin Schumacher lab | |
| Recombinant DNA reagent | pET28b(+)−6xHIS -AtSUMO1(1–93) | this paper, | vector-tagged-protein construct. Karin Schumacher lab | |
| Recombinant DNA reagent | pET28-6xHIS-AtSAE1 | this paper, | vector-tagged-protein construct. Frauke Melchior lab | |
| Recombinant DNA reagent | pET11d-AtSAE2 | this paper | vector-protein construct. Frauke Melchior lab | |
| Recombinant DNA reagent | pFA6a-His3M × 6- PGAL1 | this paper, | vector-promoter:tagged-protein construct. Sebastian Schuck lab | |
| Peptide, recombinant protein | CFP-RanGAPtail | Bossis et al., 2005; Werner et al., 2009 | ||
| Peptide, recombinant protein | YFP-SUMO | Bossis et al., 2005; Werner et al., 2009 | ||
| Peptide, recombinant protein | Uba2/Aos1 | Bossis et al., 2005; Werner et al., 2009 | ||
| Peptide, recombinant protein | Ubc9 | Bossis et al., 2005; Werner et al., 2009 | ||
| Peptide, recombinant protein | 6xHis-AtSUMO1 | this paper | For in vitro thioester bond formation assays | |
| Peptide, recombinant protein | AtSAE1 | this paper | For in vitro thioester bond formation assays | |
| Peptide, recombinant protein | AtSAE2 | this paper | For in vitro thioester bond formation assays | |
| Commercial assay or kit | SPL kit | NH DyeAgnostics | Western blot protein quantification kit |
Additional files
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Supplementary file 1
Additional resources.
(A) Primers of UBQ:PPa5-GFP construct. (B) List of GG modules. (C) qRT primers. (D) Primers for constructs used in protein purification. (E) Statistical analysis (One-way ANOVA followed by Tukey’s test, p<0.05) of the electrolyte leakage assay (Figure 1C). Significant values are highlighted.
- https://doi.org/10.7554/eLife.44213.024
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Transparent reporting form
- https://doi.org/10.7554/eLife.44213.025
