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. 2009 Oct 13;106(41):17588-93.
doi: 10.1073/pnas.0907095106. Epub 2009 Sep 29.

Type 2C protein phosphatases directly regulate abscisic acid-activated protein kinases in Arabidopsis

Taishi Umezawa  1 , Naoyuki Sugiyama, Masahide Mizoguchi, Shimpei Hayashi, Fumiyoshi Myouga, Kazuko Yamaguchi-Shinozaki, Yasushi Ishihama, Takashi Hirayama, Kazuo Shinozaki
Affiliations

Type 2C protein phosphatases directly regulate abscisic acid-activated protein kinases in Arabidopsis

Taishi Umezawa et al. Proc Natl Acad Sci U S A. .

Abstract

Abscisic acid (ABA) signaling is important for stress responses and developmental processes in plants. A subgroup of protein phosphatase 2C (group A PP2C) or SNF1-related protein kinase 2 (subclass III SnRK2) have been known as major negative or positive regulators of ABA signaling, respectively. Here, we demonstrate the physical and functional linkage between these two major signaling factors. Group A PP2Cs interacted physically with SnRK2s in various combinations, and efficiently inactivated ABA-activated SnRK2s via dephosphorylation of multiple Ser/Thr residues in the activation loop. This step was suppressed by the RCAR/PYR ABA receptors in response to ABA. However the abi1-1 mutated PP2C did not respond to the receptors and constitutively inactivated SnRK2. Our results demonstrate that group A PP2Cs act as 'gatekeepers' of subclass III SnRK2s, unraveling an important regulatory mechanism of ABA signaling.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Subclass III SnRK2s are essential for ABA responses. (A) Germination test to determine ABA sensitivity in the srk2dei triple mutant. Arabidopsis seeds of Col, srk2dei, abi1–1C, and abi5–7 were sown and grown for 2 weeks on GM agar medium containing 0, 3, or 100 μM ABA. (B) ABA-activated protein kinases in Col, srk2dei, and abi1–1C plants. Three-week-old plants were treated with 50 μM ABA for 0, 30, or 90 min. Upper panel shows in-gel phosphorylation (IGP) assay using histone as a substrate. Black arrow indicates ABA-activated protein kinase (≈40 kDa) overlapping with SnRK2s, and the open arrow indicates the RubisCo large subunit (≈50 kDa) in a CBB-stained gel. The results were confirmed through several replications.
Fig. 2.
Fig. 2.
Group A PP2Cs interact with subclass III SnRK2s. (A) Yeast two-hybrid analysis of SnRK2s and PP2Cs. GAL4BD:SnRK2s and GAL4AD:PP2Cs were introduced to yeast cells as indicated. Black slopes indicate screening stringency of SD media supplemented with: left, -LW; center, -LWH + 10 mM 3-AT; right, -LWHA + 30 mM 3-AT. (B) Yeast two-hybrid analysis of PP2C mutations. ABI1, abi1–1, abi1–1R6, ABI2, and abi2–1 were tested against subclass III SnRK2s. The black slope demonstrates stringency of screening media: SD -LW, SD -LWH + 10 mM 3-AT, SD -LWHA + 30, 60, or 100 mM 3-AT from left to right. (C) Subcellular localization of GFP-tagged SnRK2 or PP2C proteins that were transiently expressed in Arabidopsis mesophyll protoplasts. (D) Co-immunoprecipitation of GFP-tagged SnRK2s and HA-tagged PP2Cs was performed using Arabidopsis protoplasts. Immunoprecipitates against anti-GFP antibody (IP) or crude extracts (input) were analyzed via Western blotting (WB) using anti-GFP or -HA antibodies. (E) BiFC analyses were performed for the pairs of SRK2E-YFPN/ABI1-YFPC or SRK2I-YFPN/ABI1-YFPC in epidermal cells of fava bean leaves. The photos were YFP, DIC, autofluorescence (auto) and merged images. The results were confirmed through several replications.
Fig. 3.
Fig. 3.
PP2C directly regulates SnRK2. (A) ABA-activated protein kinase activity in imbibed seeds of Col, ahg1–1, and ahg3–1 plants treated with 25 μM ABA for 0, 0.5, or 5.0 h. Upper panels show in-gel phosphorylation assay (IGP) using histone as a substrate. Lower panels are CBB-stained gel images. (B) Inactivation assay for SnRK2 by in vitro reaction with PP2Cs. SRK2E-GFP proteins were prepared by immunoprecipitation (IP) against an anti-GFP antibody from Arabidopsis cultured cells with or without ABA treatment. After incubation with recombinant PP2C proteins as indicated, SRK2E activity was monitored with an in-gel phosphorylation assay (IGP) using histone as a substrate. Immunoprecipitates were analyzed via Western blotting (WB) using an anti-GFP antibody. Arrows indicate SRK2E-GFP. (C) Dephosphorylation of 32P-labeled SRK2E-GFP by PP2Cs. Immunoprecipitated SRK2E-GFP was prepared from untreated or ABA-treated cultured cells labeled with 32P-phosphate, and incubated with PP2Cs as indicated. The phosphorylation level of SRK2E-GFP was monitored by autoradiography. (D) Immunoprecipitated SRK2E-GFP was analyzed with a nanoLC-MS/MS system. As examples, a non-phospho peptide of m/z = 691.8673 (blue) and a phospho-peptide of m/z = 970.8307 (red) are found in the structure of SRK2E and in extracted ion current (XIC) chromatograms. Arrows in XICs indicate the target peaks. Asterisks in red and black indicate probable phosphorylation sites detected in this study with the highest and less PTM scores, respectively. (E) An MS/MS spectrum derived from a phospho-peptide of m/z = 970.8307. Fragmented ions detected in this analysis are shown in red, and the top 15 ions were annotated in the spectrum. The results were confirmed through several replications.
Fig. 4.
Fig. 4.
(A) In vitro reconstitution analysis of the ABA signaling cascade. Immunoprecipitated SRK2E-GFP (ABA-activated) was incubated with GST-ABI1, GST-abi1–1, ABI1, or PYR1 proteins in the presence or absence of 10 μM ABA. SnRK2 activity was monitored with an in-gel phosphorylation assay (IGP) using histone as a substrate. Immunoprecipitates (IP) were checked by Western blot analysis (WB) against an anti-GFP antibody. Arrows indicate SRK2E-GFP. This result was confirmed through several replications. (B) Proposed model of the ABA signaling pathway in Arabidopsis. Left, in the absence of ABA, PP2C dephosphorylates and inactivates SnRK2. Middle, in the presence of ABA, RCAR/PYR interacts with PP2C, and SnRK2 is released from negative regulation and converted to an active form. The activated SnRK2 phosphorylates ABA-responsive transcription factors or unknown substrates to induce ABA responses. Right, even in the presence of ABA, the abi1–1 mutant of PP2C constitutively dephosphorylates SnRK2, consequently conferring dominant ABA insensitivity. In this model, PP2C constitutively binds to domain II at the C terminus of SnRK2. Red and blue colors indicate an active or inactive form, respectively.
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