Fig 1.
Cell-free assays for the degradation of SnRK2s in wild-type plants treated with or without MG132.
Purified SnRK2.2-MBP (A), SnRK2.3-MBP (B), or SnRK2.6-MBP (C) was incubated with proteins extracted from wild-type plants for the indicated time period with or without MG132 treatment. Protein levels were checked using monoclonal anti-MBP antibody. Ponceau staining was used as loading control. Relative amounts of proteins were determined by ImageJ and normalized to loadings determined by Ponceau staining and expressed relative to the value at 0 hr time. Different letters indicate a significant difference (Student-Newman–Kuels [SNK] test, P < 0.05). Quantitative analysis of the band intensity was on the right side of the figure. Error bars are means ± s.e.m. (n ≥ 3 independent experiments). (D). In vivo degradation of SnRK2.3. 35S::SnRK2.3-3Flag (SnRK2.3-OE-8) seedlings were treated with 50 μM CHX (protein biosynthesis inhibitor) or 50 μM CHX and 50 μM MG132 separately for different times before protein was isolated for western blot with anti-Flag antibody. Ponceau staining was used as loading control. Proteins were detected as in (A-C). Different letters indicate a significant difference (Student-Newman–Kuels [SNK] test, P < 0.05). Quantitative analysis of the band intensity was on the right side of the figure. Error bars are means ± s.e.m. (n ≥ 3 independent experiments).
Fig 2.
Assay for the interaction of AtPP2-B11 with SnRK2.2, SnRK2.3, or SnRK2.6.
(A). The F-box protein AtPP2-B11 was found to interact with the protein kinases SnRK2.2, SnRK2.3, and SnRK2.6 by yeast two-hybrid growth assays performed on synthetic dropout medium lacking tryptophan and leucine (-WL) and synthetic dropout medium lacking tryptophan, leucine, histidine and adenine (-WLHA). Saturated cultures were spotted onto SD-Trp-Leu-His-Ade medium at different dilutions (OD600 = 1, 10−1, 10−2, and 10−3). The vectors AD-T and BD-53 were used as positive controls; the empty vectors pGADT7 and pGBKT7 were used as negative controls. (B). The interactions between AtPP2-B11-YFPN and SnRK2.2/ SnRK2.3/ SnRK2.6-YFPC in N. benthamiana were analyzed using BiFC assays. The YFP signal (left), brightfield images (middle), and merged images (right) are shown. (C). In vivo interaction assays for SnRK2.2/SnRK2.3/SnRK2.6-FLAG and AtPP2-B11-Myc by Co-IP in stably transformed Arabidopsis plants. Proteins were extracted from 7-day-old seedlings and incubated with agarose-conjugated monoclonal anti-Flag antibodies; AtPP2-B11-Myc was detected using monoclonal anti-myc antibodies. Input, 2% of the protein extract used in the Co-IP assay without IP. (D). In vitro pull-down assay for AtPP2-B11 with SnRK2.2, SnRK2.3, and SnRK2.6. SnRK2.2-MBP/SnRK2.3-MBP/SnRK2.6-MBP pulled down with AtPP2-B11-GST were detected using anti-MBP antibodies. Input, 20% of the purified GST- and MBP-tagged proteins used in the pull-down assays.
Fig 3.
AtPP2-B11 affects the stability of SnRK2.3, but not SnRK2.2 or SnRK2.6.
(A). Assays for the degradation of MBP-tagged SnRK2.3 were performed using wild-type plants and AtPP2-B11-overexpressing (OE) transgenic lines. Ponceau staining was used as loading control. Relative amounts of proteins were determined by ImageJ and normalized to loadings determined by Ponceau staining and expressed relative to the value at 0 hr time. Different letters indicate a significant difference (Student-Newman–Kuels [SNK] test, P < 0.05). Quantitative analysis of the band intensity was on the right side of the figure. Error bars are means ± s.e.m. (n ≥ 3 independent experiments). (B). Assays for the degradation of MBP-tagged SnRK2.3 were performed using wild-type plants and the amiR-AtPP2-B11 (amiR15) transgenic line. The plants were pre-treated by 50 μM ABA for 5 h. Ponceau staining was used as loading control. Proteins were detected as in (A). Different letters indicate a significant difference (Student-Newman–Kuels [SNK] test, P < 0.05). Quantitative analysis of the band intensity was on the right side of the figure. Error bars are means ± s.e.m. (n ≥ 3 independent experiments). (C). The degradation assay for 35S::SnRK2.3-3flag in wild type or AtPP2-B11 overexpression background. Proteins were extracted from 7-day-old transgenic seedlings with 50 uM CHX (protein biosynthesis inhibitor) treatment for indicated times. The SnRK2.3 protein level was checked at the indicated time point by western blotting using anti-Flag antibody. AtPP2-B11-Myc protein level was detected using monoclonal anti-myc antibody. Ponceau staining was used as loading control. Proteins were detected as in (A). Different letters indicate a significant difference (Student-Newman–Kuels [SNK] test, P < 0.05). Quantitative analysis of the band intensity was on the right side of the figure. Error bars are means ± s.e.m. (n ≥ 3 independent experiments).
Fig 4.
ABA promotes the degradation of SnRK2.3.
(A). Assays for SnRK2.3 degradation in vitro. SnRK2.3-MBP was incubated with proteins extracted from wild type plants treated with or without 50 μM ABA for 5 h. SnRK2.3 protein was detected with anti-MBP antibody. Ponceau staining was used as loading control. Relative amounts of proteins were determined by ImageJ and normalized to loadings determined by Ponceau staining and expressed relative to the value at 0 hr time. Different letters indicate a significant difference (Student-Newman–Kuels [SNK] test, P < 0.05). Quantitative analysis of the band intensity was on the right side of the figure. Error bars are means ± s.e.m. (n ≥ 3 independent experiments). (B). The ABA-induced SnRK2.3 degradation is dependent on the 26S proteasome pathway. Seven-day-old transgenic seedlings were treated for indicated time points with 50 μM CHX, 50 μM CHX with 50 μM ABA, or 50 μM CHX together with 50 μM ABA and 50 μM MG132, respectively. The levels of SnRK2.3-Flag at each time points were detected by anti-Flag antibody. Ponceau staining was used as loading control. Relative amounts of proteins were determined by ImageJ and normalized to loadings determined by Ponceau staining and expressed relative to the value at 0 h time. Different letters indicate a significant difference (Student-Newman–Kuels [SNK] test, P < 0.05). Quantitative analysis of the band intensity was on the right side of the figure. Error bars are means ± s.e.m. (n = 3 independent experiments). (C). The ABA-induced degradation of SnRK2.3 is mediated by ubiquitin-dependent proteasomal degradation. Seven-day-old SnRK2.3-Flag transgenic seedlings and the wild type seedlings were treated with 50 μM CHX and with 50 μM ABA or not for 9 h. SnRK2.3-Flag protein was isolated using Flag beads (ANTI-FLAG M2 Affinity Gel; Sigma-Aldrich). Flag antibody was used to detect SnRK2.3 and the ubiquitinated level of SnRK2.3. IP: immunoprecipitation; IB: immunoblot. (D). Ubiquitination of SnRK2.3-HA treated with ABA or not in protoplasts. Arabidopsis wild type protoplasts were transformed with SnRK2.3-HA or SnRK2.3-HA together with Ubiquitin-Flag (Ubi-Flag), incubated for 8 h of protein synthesis, then treated with 50 μM MG132 for 1 h, and then finally treated with or without 20 μM ABA for another 2 h. Proteins were isolated for immunoprecipitation with HA antibody for 2 h at 4°C, and then incubated with protein A beads for another 2 h at 4°C, and followed by immunoblotting with anti-Flag and anti-HA antibodies to detect the ubiquitinated levels of SnRK2.3 and SnRK2.3 protein levels, respectively.
Fig 5.
Expression pattern of AtPP2- B11.
(A). AtPP2-B11 expression assay in response to ABA by quantitative real-time PCR. Total RNA was extracted from 7-day-old wild-type seedlings treated with 50 μM ABA at the indicated time points. ACTIN2 was used as an internal control. The values are means±standard error. Different letters indicat a significant difference (Student-Newman–Kuels [SNK] test, P < 0.05). Three independent biological repeats were performed. (B). GUS staining of the pAtPP2-B11::GUS transgenic lines with or without 50 μM ABA treatment for 3 h. (a and b) Imbibition seeds. (c and d) One-day-old imbibed seeds. (e and g) Three-day-old seedlings. (f and h) Amplified view of root tip of (e) and (g). (i and j) Five-day-old seedlings. (k) Amplified view of a leaf. (l and m) Rosette leaves of 3-week-old seedlings. (n and o) Stems of 3-week-old seedlings. (p and q) Inflorescences. (r and u) Siliques. (s and t) Immature seeds from siliques. Scale bars = 0.1 mm (a–d), 0.05 mm (f, h, and k), 0.3 mm (l–o), and 1 mm (e, g, i, j, and p–u).
Fig 6.
AtPP2-B11 is a negative regulator of ABA signaling.
(A). Phenotypic analyses of wildtype (WT) and AtPP2-B11 mutant plants treated with 0.25 or 0.5 μM ABA. The images were taken after 4 and 8 days, respectively. (B). The germination rates and (C). greening rates for WT, amiR7, and amiR15 plants at 4 and 8 days after stratification. The data are given as means plus the standard deviation of three independent replicates. The student’s t-test was performed and the statically significant treatments were marked with ‘***’ (P<0.001), ‘**’ (P<0.01) and ‘*’ (P<0.05).
Fig 7.
The transcript abundance of ABA-responsive genes in Col-0 and amiR15.
The relative transcript abundance of (A) ABI3, (B) ABI4, (C) ABI5, (D) RAB18, (E) RD29A, and (F) RD29B in Col-0 and amiR15 mutant plants were analyzed. Seedlings were grown on MS medium with or without 0.5 μM ABA for 10 days. Three independent experiments were performed with similar results, each with three replicates. UBC5 was used as the internal control. The student’s t-test was performed and the statically significant treatments were marked with ‘***’ (P<0.001) and ‘**’ (P<0.01).
Fig 8.
AtPP2-B11 inhibits the ABA sensitivity of SnRK2.3 overexpression.
(A). Phenotype assay of wild type, AtPP2-B11-OE, AtPP2-B11-OE SnRK2.3-OE-1 and AtPP2-B11-OE SnRK2.3-OE-8 overexpression transgenic lines in germination and greening stages with or without ABA. The image was taken at 5 days (top two panels) and 10 days (bottom two panels) after germination, respectively. (B). Comparision of germination rates between wild type, AtPP2-B11-OE, AtPP2-B11-OE SnRK2.3-OE-1 and AtPP2-B11-OE SnRK2.3-OE-8 at 5 days after stratification. (C). Comparision of greening rates between wild type, AtPP2-B11-OE, AtPP2-B11-OE SnRK2.3-OE-1 and AtPP2-B11-OE SnRK2.3-OE-8 at 10 days after germination. Different letters indicate a significant difference (Student-Newman–Kuels [SNK] test, P < 0.05).
Fig 9.
A proposed model for SnRK2.3 degradation.
In the absence of ABA, PP2C interacts with SnRK2.3, inhibiting its kinase activity and preventing the phosphorylation of downstream transcription factors by SnRK2.3. However, in the presence of ABA, PP2C binds to PYR/PYL/RCAR proteins and ABA, thereby inhibiting PP2C phosphatase activity. SnRK2.3 is then activated to phosphorylate transcriptional factors and induce the expression of downstream genes, including AtPP2-B11. AtPP2-B11, the substrate receptor, interacts with ASK to form SCF E3 ligase complex. AtPP2-B11 specifically targets SnRK2.3 for its degradation to turn off ABA signaling.