UBP12 and UBP13 negatively regulate the activity of the ubiquitin-dependent peptidases DA1, DAR1 and DAR2
- VIB Center for Plant Systems Biology, Technologiepark, Belgium
- Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark, Belgium
- VIB Center for Medical Biotechnology, Albert Baertsoenkaai, Belgium
- Department of Biomolecular Medicine, Ghent University, Albert Baertsoenkaai, Belgium
Figures
Figure 1 with 10 supplements
UBP12 and UBP13 interact with DA1, DAR1 and DAR2.
(A–C) Enrichment of the bait, UBP12 and UBP13 compared to the control after immunoprecipitation in (A) 35S::GFP-DA1 (FDR = 0.01, S0 = 1, permutation-based FDR-corrected t-test, Figure 1—source data 1), (B) 35S::GFP-DAR1 (FDR = 0.01, S0 = 1, permutation-based FDR-corrected t-test, Figure 1—source data 1) or (C) 35S::GFP-DAR2 (FDR = 0.05, S0 = 1, permutation-based FDR-corrected t-test, Figure 1—source data 1) seedlings. (D–F) In vitro pull down of (D) HIS-MBP-DA1, (E) HIS-MBP-DAR1 and (F) HIS-MBP-DAR2 with free GST, GST-UBP12 and GST-UBP13. (G) In vivo pull-down of RFP-DA1, RFP-DAR1 and RFP-DAR2 with free GFP and GFP-UBP12.
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Figure 1—source data 1
List of DA1, DAR1, DAR2 interactors and LFQ intensities by MS/MS.
- https://cdn.elifesciences.org/articles/52276/elife-52276-fig1-data1-v2.xlsx
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Figure 1—source data 2
MS/MS counts of DA1, DAR1 and DAR2; protein coverage of DA1, DAR1 and DAR2; relative expression levels of DA1, DAR1, DAR2, UBP12 and UBP13 during leaf development.
- https://cdn.elifesciences.org/articles/52276/elife-52276-fig1-data2-v2.xlsx
Figure 1—figure supplement 1
Overlap between potential interactors of DA1, DAR1 and DAR2.
Figure 1—figure supplement 2
MS/MS counts of DA1, DAR1 and DAR2 baits from GFP-DA1, GFP-DAR1 and GFP-DAR2 pull-downs, respectively (Bars represent the SEM; n = 3 biological repeats; Figure 1—source data 2).
Figure 1—figure supplement 3
Percentage coverage of the DA1, DAR1 and DAR2 baits from GFP-DA1, GFP-DAR1 and GFP-DAR2 pull-downs, respectively (Bars represent the SEM; n = 3 biological repeats; Figure 1—source data 2).
Figure 1—figure supplement 4
Relative expression levels of UBP12, UBP13, DA1, DAR1 and DAR2 throughout leaf development, scaled to the lowest value (DAR2, 5 DAS; Figure 1—source data 2).
Scale bar represents 1 cm. Bars represent the SEM; n = 3 biological repeats).
Figure 1—figure supplement 5
Co-localization of GFP-UBP12 and RFP-DA1 in Nicotiana benthamiana leaves.
Figure 1—figure supplement 6
Co-localization of GFP-UBP13 and RFP-DA1 in Nicotiana benthamiana leaves.
Figure 1—figure supplement 7
Co-localization of GFP-UBP12 and RFP-DAR1 in Nicotiana benthamiana leaves.
Figure 1—figure supplement 8
Co-localization of GFP-UBP13 and RFP-DAR1 in Nicotiana benthamiana leaves.
Figure 1—figure supplement 9
Co-localization of GFP-UBP12 and RFP-DAR2 in Nicotiana benthamiana leaves.
Figure 1—figure supplement 10
Co-localization of GFP-UBP13 and RFP-DAR2 in Nicotiana benthamiana leaves.
Figure 2 with 10 supplements
Regulation of leaf size by UBP12 and UBP13.
(A) Twenty-one-day-old plants of Col-0, da1-1 and homozygous (+/+) or heterozygous (+/-) UBP12 and UBP13 overexpression lines. Scale bar represents 1 cm, homozygous and heterozygous plants of the same single locus line are linked in the figure. (B) Leaf area measurements of Col-0 and ubp12-2, n = 3 biological repeats with >10 plants per repeat. (C) Percentage differences of leaf area, cell area and cell number of ubp12-2, 35S::UBP12_3.1, 35S::UBP12_3.2, 35S::UBP13_1.1 and 35S::UBP13_3.2 compared to Col-0, n = 3 biological repeats with three representative leaves per repeat. (D) Relative frequencies of LN transformed cell area distribution of Col-0, 35S::UBP12_3.1, 35S::UBP12_3.2, 35S::UBP13_1.1 and 35S::UBP13_3.2 in 1-µm2 bin sizes, n = 3 biological repeats with three representative leaves per repeat. Bars represent the SEM; * indicates p-value<0.05, ANOVA (Figure 2—source data 1).
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Figure 2—source data 1
Leaf area analysis and statistics of Col-0 and ubp12-2; leaf area analysis and statistics of Col-0, 35S::UBP12_3.1, 35S::UBP12_3.2, 35S::UBP13_1.1 and 35S::UBP13_3.2; cellular analysis and statistics of Col-0, 35S::UBP12_3.1, 35S::UBP12_3.2, 35S::UBP13_1.1 and 35S::UBP13_3.2; cellular analysis of ubp12-2, relative frequency of the pavement cell area and statistics of Col-0, 35S::UBP12_3.1, 35S::UBP12_3.2, 35S::UBP13_1.1 and 35S::UBP13_3.2.
- https://cdn.elifesciences.org/articles/52276/elife-52276-fig2-data1-v2.xlsx
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Figure 2—source data 2
Q-RT-PCR data and statistics of UBP12 and UBP13 expression in Col-0, da1-1, 35S::UBP12_3.1, 35S::UBP12_3.2, 35S::UBP13_1.1 and 35S::UBP13_3.2 overexpression lines; leaf area data and statistics of ubp12-1; leaf area data and statistics of ubp13-1; leaf area data and statistics of ubp13-2; leaf area data and statistics of ubp13-3; relative frequency of ubp12-2 pavement cell area data and statistics.
- https://cdn.elifesciences.org/articles/52276/elife-52276-fig2-data2-v2.xlsx
Figure 2—figure supplement 1
Leaf index measurements of Col-0 and da1-1 mutants, n = 3 biological repeats with >6 leaves per repeat.
Bars represent the SEM; * indicates p-value<0.05, two-tailed t-test; (Figure 2—source data 2).
Figure 2—figure supplement 2
Leaf index measurements of Col-0, 35S::UBP12_3.1, 35S::UBP12_3.2, 35S::UBP13_1.1 and 35S::UBP13_3.2 overexpression lines.
Bars represent the SEM; * indicates p-value<0.05, ANOVA; (Figure 2—source data 2).
Figure 2—figure supplement 3
UBP12 and UBP13 transcript levels in Col-0, da1-1, 35S::UBP12_3.1, 35S::UBP12_3.2, 35S::UBP13_1.1 and 35S::UBP13_3.2 overexpression lines, n = 3 biological repeats with >8 leaves per repeat.
Bars represent the SEM; * indicates p-value<0.05, ANOVA; (Figure 2—source data 2).
Figure 2—figure supplement 4
Leaf area measurements of ubp12-1, compared to Col-0, n = 3 biological repeats with >10 plants per repeat.
Bars represent the SEM; * indicates p-value<0.05, ANOVA; (Figure 2—source data 2).
Figure 2—figure supplement 5
Leaf area measurements of ubp13-1, compared to Col-0, n = 3 biological repeats with >10 plants per repeat.
Bars represent the SEM; * indicates p-value<0.05, ANOVA; (Figure 2—source data 2).
Figure 2—figure supplement 6
Leaf area measurements of ubp13-2 and compared to Col-0, n = 3 biological repeats with >10 plants per repeat.
Bars represent the SEM; * indicates p-value<0.05, ANOVA; (Figure 2—source data 2).
Figure 2—figure supplement 7
Leaf area measurements of ubp13-3 compared to Col-0, n = 3 biological repeats with >10 plants per repeat.
Bars represent the SEM; * indicates p-value<0.05, ANOVA; (Figure 2—source data 2).
Figure 2—figure supplement 8
Representations of abaxial leaf epidermal cells of Col-0, homozygous 35S::UBP12_3.1, homozygous 35S::UBP12_3.2, heterozygous 35S::UBP13_1.1 and heterozygous 35S::UBP13_3.2.
(+/-) and (+/+) indicate heterozygous and homozygous plants, respectively, scale-bar represents 0.1 mm.
Figure 2—figure supplement 9
Representations of abaxial leaf epidermal cells of Col-0 and the homozygous 35S::UBP13_1.1, (+/+) indicates homozygous plants, scale-bar represents 0.1 mm.
Figure 2—figure supplement 10
Cell size distribution plot of Col-0 and ubp12-2, n = 3 biological repeats with three representative leaves per repeat.
Bars represent the SEM; * indicates p-value<0.05, ANOVA; (Figure 2—source data 2).
Figure 3 with 4 supplements
UBP12 and UBP13 regulate the onset of endoreduplication.
(A) Ploidy distribution of nuclear DNA in Col-0, 35S::UBP12_3.1 and 35S::UBP13_1.1. (B–C) Fractional difference in expression of cell proliferation markers in 35S::UBP12_3.1 and 35S::UBP13_1.1 compared to Col-0 at (B) nine and (C) 12 DAS. (D) Representations of abaxial leaf epidermal cells at the tip of the third leaf of Col-0, 35S::UBP12_3.1 and 35S::UBP13_1.1 at 12 DAS. Bars represent the SEM, n = 3 biological repeats with >3 leaves per repeat; a, b and c indicate a significant difference in 2C, 4C and 8C, respectively; * indicates p-value<0.05, ANOVA, (Figure 3—source data 1).
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Figure 3—source data 1
Flow cytometry counts and statistics; Q-RT-PCR data and statistics of proliferation markers in developing leaves (9 DAS and 12 DAS).
- https://cdn.elifesciences.org/articles/52276/elife-52276-fig3-data1-v2.xlsx
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Figure 3—source data 2
Endoreduplication index calculations and statistics; Q-RT-PCR data and statistics of proliferation markers in developing leaves (15 DAS and 18 DAS).
- https://cdn.elifesciences.org/articles/52276/elife-52276-fig3-data2-v2.xlsx
Figure 3—figure supplement 1
Endoreduplication index of 35S::UBP12_3.1 leaf nuclei compared to Col-0.
Bars represent the SEM; n = 3 biological repeats with >3 leaves per repeat; * indicates p-value<0.05, ANOVA, (Figure 3—source data 2).
Figure 3—figure supplement 2
Endoreduplication index of 35S::UBP13_1.1 leaf nuclei compared to Col-0.
Bars represent the SEM; n = 3 biological repeats with >3 leaves per repeat; * indicates p-value<0.05, ANOVA, (Figure 3—source data 2).
Figure 3—figure supplement 3
Relative expression levels of cell proliferation markers in Col-0, 35S::UBP12_3.1 and 35S::UBP13_1.1 at 15 DAS.
Bars represent the SEM; n = 3 biological repeats with >3 leaves per repeat; * indicates p-value<0.05, ANOVA, (Figure 3—source data 2).
Figure 3—figure supplement 4
Relative expression levels of cell proliferation markers in Col-0, 35S::UBP12_3.1 and 35S::UBP13_1.1 at 18 DAS.
Bars represent the SEM; n = 3 biological repeats with >3 leaves per repeat; * indicates p-value<0.05, ANOVA, (Figure 3—source data 2).
Figure 4
In vitro deubiquitination specificity of DA1, DAR1 and DAR2 by UBP12 and UBP13.
(A–C) In vitro deubiquitination assays with GST-UBP12, GST-UBP12C208S, GST-UBP13 or GST-UBP13C207S of (A) HIS-MBP-DA1, (B) HIS-MBP-DAR1 and (C) HIS-MBP-DAR2. (D) Deubiquitination assay with GST-UBP12, GST-UBP13, GST-UBP3, GST-UBP15 and GST-UBP24 of HIS-MBP-DA1.
Figure 5 with 1 supplement
Deubiquitination of DA1, DAR1 and DAR2 by UBP12 and UBP13 in vivo.
(A–B) Cell-free deubiquitination assay of ubiquitinated HIS-MBP-DA1 proteins incubated with (A) Col-0 or (B) ubp12-2 extract. (C) Quantification of deubiquitination (Figure 5—source data 1). (D–G) Cell-free deubiquitination assay of ubiquitinated HIS-MBP-DA1 with (D) Col-0, (E) ubp12-2, (F) 35S::UBP12 or (G) 35S::UBP13 extracts. (H) Quantification of deubiquitination (Figure 5—source data 1).
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Figure 5—source data 1
Calculation of in vivo deubiquitination.
- https://cdn.elifesciences.org/articles/52276/elife-52276-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Stability of DA1 in 35::GFP-DA1, 35::GFP-DA1_35::UBP12 and 35S::GFP-DA1_35S::UBP13 seedlings.
Figure 6 with 1 supplement
Cleaving assay on UBP12 and UBP13.
(A) In vitro cleaving assay on GST-UBP12, GST-UBP12C208S, GST-UBP13 and GST-UBP13C207S by HIS-MBP-DA1 and the peptidase-deficient HIS-MBP-DA1H418A,H422A. (B) Twenty-one-day-old plants of Col-0, 35S::GFP-DA1, 35S::UBP12_3.1/35S::GFP-DA1, 35S::UBP12_3.2/35S::GFP-DA1, 35S::UBP13_2.1/35S::GFP-DA1 and 35S::UBP13_2.2/35S::GFP-DA1 homozygous (+/+) and heterozygous (+/-) double overexpression lines. Scale bar represents 1 cm.
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Figure 6—source data 1
Q-RT-PCR data and statistics of DA1, UBP12 and UBP13 expression in Col-0, 35S::GFP-DA1, 35S::UBP12_3.1/35S::GFP-DA1, 35S::UBP12_3.2/35S::GFP-DA1, 35S::UBP13_2.1/35S::GFP-DA1 and 35S::UBP13_2.2/35S::GFP-DA1.
- https://cdn.elifesciences.org/articles/52276/elife-52276-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Relative expression levels of DA1, UBP12 and UBP13 in the double 35S::GFP-DA1_35S::UBP12 and 35S::GFP-DA1_35S::UBP13 overexpression lines.
n = 3 biological repeats with >3 leaves per repeat, * indicates p-value<0.05 compared to the expression levels in Col-0, ANOVA (Figure 6—source data 1).
Figure 7 with 1 supplement
Model of UBP12 and UBP13 levels on leaf area and cellular phenotypes.
Molecular balance and leaf phenotypes in (A) wild-type conditions, (B) high UBP12 and UBP13 expression lines and (C) lower levels of UBP12 and UBP13.
Figure 7—figure supplement 1
Updated model of our current knowledge of the growth-regulating DA1 pathway.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Arabidopsis thaliana) | DA1 | TAIR | AT1G19270 | |
| Gene (Arabidopsis thaliana) | DAR1 | TAIR | AT4G36860 | |
| Gene (Arabidopsis thaliana) | DAR2 | TAIR | AT2G39830 | |
| Gene (Arabidopsis thaliana) | UBP12 | TAIR | AT5G06600 | |
| Gene (Arabidopsis thaliana) | UBP13 | TAIR | AT3G11910 | |
| Gene (Arabidopsis thaliana) | UBP3 | TAIR | AT4G39910 | |
| Gene (Arabidopsis thaliana) | UBP15 | TAIR | AT1G17110 | |
| Gene (Arabidopsis thaliana) | UBP24 | TAIR | AT4G30890 | |
| Gene (Arabidopsis thaliana) | DA2 | TAIR | AT1G78420 | |
| Cell line (Arabidopsis thaliana) | Landsberg erecta | TAIR/ABRC | Germplasm:6530492727 NASC stock number: N84840 | https://www.arabidopsis.org/servlet/TairObject?id=4502009498&type=stock |
| Strain, strain background (Escherichia coli) | DH5 α | Thermo-fisher | 18258012 | Chemically competent cells |
| Strain, strain background (Escherichia coli) | BL21(DE3) | Thermo-fisher | EC0114 | Chemically competent cells |
Author response table 1
| 35S::RFP-DA1 | 35S::DA2-FLAG | p35S::GFP-UBP12 | pUBQ10::HA-BB | Cleaving inhibition |
|---|---|---|---|---|
| 35S::RFP-DA1 | 35S::DA2-FLAG | p35S::GFP-UBP12C208S | pUBQ10::HA-BB | Cleaving |
| 35S::RFP-DA1 | 35S::DA2-FLAG | p35S::GFP-UBP13 | pUBQ10::HA-BB | Cleaving inhibition |
| 35S::RFP-DA1 | 35S::DA2-FLAG | p35S::GFP-UBP13C207S | pUBQ10::HA-BB | Cleaving |
Author response table 2
| 35S::HA-DA1 | 35S::DA2-FLAG | pUBP10::UBP12-BFP | pUBP10::NLS-GFP-TCP14-mCherry | Cleaving inhibition, FRET |
|---|---|---|---|---|
| 35S::HA-DA1 | 35S::DA2-FLAG | pUBP10::UBP12C208S–BFP | pUBP10::NLS-GFP-TCP14-mCherry | Cleaving, loss of FRET |
| 35S::HA-DA1 | 35S::DA2-FLAG | pUBP10::UBP13-BFP | pUBP10::NLS-GFP-TCP14-mCherry | Cleaving inhibition, FRET |
| 35S::HA-DA1 | 35S::DA2-FLAG | pUBP10::UBP13C208S–BFP | pUBP10::NLS-GFP-TCP14-mCherry | Cleaving, loss of FRET |
Additional files
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Supplementary file 1
Primer list.
- https://cdn.elifesciences.org/articles/52276/elife-52276-supp1-v2.xlsx
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Supplementary file 2
Conditions recombinant protein production.
- https://cdn.elifesciences.org/articles/52276/elife-52276-supp2-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/52276/elife-52276-transrepform-v2.docx
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UBP12 and UBP13 negatively regulate the activity of the ubiquitin-dependent peptidases DA1, DAR1 and DAR2
eLife 9:e52276.
https://doi.org/10.7554/eLife.52276
