(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…
List of DA1, DAR1, DAR2 interactors and LFQ intensities by MS/MS.
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.
Scale bar represents 1 cm. Bars represent the SEM; n = 3 biological repeats).
(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 …
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.
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.
Bars represent the SEM; * indicates p-value<0.05, two-tailed t-test; (Figure 2—source data 2).
Bars represent the SEM; * indicates p-value<0.05, ANOVA; (Figure 2—source data 2).
Bars represent the SEM; * indicates p-value<0.05, ANOVA; (Figure 2—source data 2).
Bars represent the SEM; * indicates p-value<0.05, ANOVA; (Figure 2—source data 2).
Bars represent the SEM; * indicates p-value<0.05, ANOVA; (Figure 2—source data 2).
Bars represent the SEM; * indicates p-value<0.05, ANOVA; (Figure 2—source data 2).
Bars represent the SEM; * indicates p-value<0.05, ANOVA; (Figure 2—source data 2).
(+/-) and (+/+) indicate heterozygous and homozygous plants, respectively, scale-bar represents 0.1 mm.
Bars represent the SEM; * indicates p-value<0.05, ANOVA; (Figure 2—source data 2).
(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 …
Flow cytometry counts and statistics; Q-RT-PCR data and statistics of proliferation markers in developing leaves (9 DAS and 12 DAS).
Endoreduplication index calculations and statistics; Q-RT-PCR data and statistics of proliferation markers in developing leaves (15 DAS and 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).
Bars represent the SEM; n = 3 biological repeats with >3 leaves per repeat; * indicates p-value<0.05, ANOVA, (Figure 3—source data 2).
Bars represent the SEM; n = 3 biological repeats with >3 leaves per repeat; * indicates p-value<0.05, ANOVA, (Figure 3—source data 2).
Bars represent the SEM; n = 3 biological repeats with >3 leaves per repeat; * indicates p-value<0.05, ANOVA, (Figure 3—source data 2).
(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 …
(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) …
Calculation of in vivo deubiquitination.
(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::…
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.
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).
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.
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 |
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 |
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 |
Primer list.
Conditions recombinant protein production.