Article Figures & Data
Figures
- Figure 1.
Phosphatase-active PP2C5 is located to the nucleus. A, The coding region of PP2C5 was C-terminally fused to GFP and transiently expressed in Arabidopsis protoplasts either under control of the 35S promoter (35S:PP2C5-GFP) or its native promoter (PP2C5:PP2C5-GFP). As a control, GFP was expressed alone (35S:GFP). Protoplasts were analyzed by fluorescence microscopy. B, The fusion proteins described in A were transiently expressed in N. benthamiana leaves. C, Leaf samples from B were used for immunoprecipitation with PP2C5-specific antibodies, and purified PP2C5 (top; western blot with anti-PP2C5 from rabbit) was subjected to an in vitro phosphatase assay using [32P]phospho-casein (bottom). Relative phosphatase activity is given as release of 32P into the supernatant after incubation with immunoprecipitated PP2C5.
- Figure 2.
PP2C5 colocalizes with stress-induced MAPKs predominantly in the nucleus. PP2C5 (A) or other group B members (B) were transiently coexpressed as CFP fusions in Arabidopsis protoplasts together with MPK3-, MPK4-, or MPK6-YPF fusions. CFP/YFP signals were visualized by fluorescence microscopy using different channels.
- Figure 3.
PP2C5 interacts with MPK3, MPK4, and MPK6 through its KIM. A, For Y2H analysis, PP2C5 or point-mutated PP2C5-K90A/R91Q (PP2C5KR) was cloned into the vector pGBKT7 and used against MPK3, MPK4, or MPK6 in the vector pGADT7. The positive control was the interaction between the SV40 large T-antigen and murine p53 (pGAD-T + pGBK-53). Interaction was tested using synthetic dropout medium not containing the amino acids Leu, Trp, and Ala (−LTA). Shown are serial dilutions (1, 1:10, 1:100, and 1:1,000) of the corresponding yeast culture. B, For BiFC analysis, PP2C5 and point-mutated PP2C5-K90A/R91Q were fused to the N-terminal part of YFP and assayed against MPK3, MPK4, or MPK6 fused to the C-terminal half of YFP. The subcellular localization of the interaction is visualized by fluorescence microscopy. [See online article for color version of this figure.]
- Figure 4.
Identification of a PP2C5-T-DNA insertion line. A, A T-DNA insertion line was identified from the SALK collection, with the insertion located in the second exon (the asterisk indicates the stop codon, thick lines indicate exons, and thin lines indicate introns). B, Expression of residual transcript in pp2c5 and ap2c1 T-DNA insertion lines, the double knockout line pp2c5 ap2c1, or the pp2c5 mutant complemented with the genomic fragment of PP2C5 (pp2c5/PP2C5) was analyzed by quantitative RT-PCR. Six-day-old seedlings treated with 1 μm Flg22 were harvested after 3 h, and quantitative PCR was performed using gene-specific primers. For PP2C5, primers upstream of (5′), downstream of (3′), or flanking the region of the T-DNA insertion (T-DNA) were used; for AP2C1, a primer pair flanking the T-DNA insertion was selected. Expression of EF-1α was used for normalization, and the Col-0 water control was set to 1. Data are averages ± se from three independent experiments performed each with 10 to 15 seedlings per plant line.
- Figure 5.
pp2c5 ap2c1 double mutant plants show an enhanced ABA-induced MAPK activation. Ten to 15 6-d-old seedlings of pp2c5 and ap2c1 single mutants, the pp2c5 ap2c1 double knockout line, and the pp2c5/PP2C5 complemented line were treated with 50 μm ABA, and MAPK activation was analyzed at the indicated time points. A, Protein extracts (5 μg per lane) were subjected to western-blot analysis using the phospho-p44/p42 MAPK antibody. Arrowheads indicate the positions of MPK3 and MPK6. B, The staining of the large subunit of Rubisco using Ponceau S Red dye is used to estimate equal loading in each lane. Shown is one representative experiment out of three.
- Figure 6.
ABA-induced activation of immunoprecipitated MPK3, MPK4, and MPK6 is enhanced in pp2c5 ap2c1 mutants. MPK3 (top panels), MPK4 (middle panels), and MPK6 (bottom panels) activation was measured in the wild type, pp2c5 and ap2c1 single mutants, the pp2c5 ap2c1 double mutant, and the pp2c5/PP2C5 complemented line in response to ABA. MAPKs were immunoprecipitated from seedlings after treatment with 50 μm ABA for 30 min. The MAPK activity was measured in immunocomplex kinase assays using myelin basic protein as a substrate, and levels of MPK3, MPK4, and MPK6 proteins were detected by western-blot analysis using isoform-specific antisera.
- Figure 7.
PP2C5-overexpressing leaves are impaired in ABA-stimulated MAPK activation. Constructs for 35S:GFP or 35S:PP2C5-GFP were transiently expressed in N. benthamiana leaves as described in Figure 1B. After 2 d, leaves were treated without (control) or with 50 μm ABA, and samples were harvested at the indicated time points. Protein extracts were afterward subjected to an in gel kinase assay. A parallel gel was stained with Coomassie Brilliant Blue to confirm equal protein amounts. Arrowheads indicate the positions of SIPK and WIPK. Shown is one out of two experiments with similar results.
- Figure 8.
PP2C5 knockout lines show an increased stomatal aperture. Wild-type Arabidopsis Col-0, knockout lines pp2c5 and ap2c1, the double knockout line pp2c5 ap2c1, and the complemented pp2C5 line pp2c5/PP2C5 were grown on soil, and stomatal apertures on abaxial epidermal imprints were determined by light microscopy. Data presented are means of at least 100 stomata, and the error bars represent se. Significant differences (** P < 0.001, *** P < 0.0001) compared with the wild-type control were determined using Student's t test. The entire experiment was repeated three times with similar results.
- Figure 9.
PP2C mutants display altered ABA responses. A, Seeds of wild-type Arabidopsis Col-0 and knockout lines pp2c5, ap2c1, pp2c5 ap2c1, and pp2c5/PP2C5 were germinated on medium supplemented with the indicated concentrations of ABA. Seedlings were scored for radicle emergence at day 3. The number of germinated seeds was expressed as the percentage of the total number of seeds plated (n > 45). Shown is the average of three independent experiments, and error bars indicate se. B, Induction ratio of ABI1, ABI2, RD29A, and Erd10 genes in pp2c mutant lines compared with the wild type. Arabidopsis seeds were germinated on half-strength MS medium. Two days after germination, 40 seedlings per line were transferred to sterile water supplemented with or without 50 μm ABA and harvested after incubation for a further 24 h. Data are means ± se from three independent experiments. For each genotype, the fold changes in gene expression obtained in real-time quantitative PCR analyses are shown relative to the respective water control and EF-1α amplification was used to normalize data.
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Supplemental Data
Supplemental Figures, Table, and Materials and Methods
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- Supplemental Data - Supplemental Figures 1-8, Supplemental Table and Supplemental Materials and Methods
