Type 2C protein phosphatases directly regulateabscisic acid-activated protein kinases in ArabidopsisTaishi Umezawaa, Naoyuki Sugiyamab, Masahide Mizoguchia,c, Shimpei Hayashid, Fumiyoshi Myougae,Kazuko Yamaguchi-Shinozakic, Yasushi Ishihamab,f, Takashi Hirayamad,g,1, and Kazuo Shinozakia,1

aGene Discovery Research Team, RIKEN Plant Science Center, 3-1-1 Kouyadai, Tsukuba, Ibaraki 305-0074, Japan; bInstitute for Advanced Biosciences, KeioUniversity, Tsuruoka, Yamagata 997-0017, Japan; cGraduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1 Yayoi, Bunkyo-ku, Tokyo113-8657, Japan; dInternational Graduate School of Arts and Sciences, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045Japan; eR&D program, RIKEN Plant Science Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan; fPRESTO, Japan Science andTechnology Agency, 5-Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan; and gRIKEN Advanced Science Institute, Wako, Saitama 351-0198, Japan

Edited by Joseph R Ecker, Salk Institute for Biological Studies, La Jolla, CA, and approved August 24, 2009 (received for review June 25, 2009)

Abscisic acid (ABA) signaling is important for stress responses anddevelopmental processes in plants. A subgroup of protein phos-phatase 2C (group A PP2C) or SNF1-related protein kinase 2(subclass III SnRK2) have been known as major negative or positiveregulators of ABA signaling, respectively. Here, we demonstratethe physical and functional linkage between these two majorsignaling factors. Group A PP2Cs interacted physically with SnRK2sin various combinations, and efficiently inactivated ABA-activatedSnRK2s via dephosphorylation of multiple Ser/Thr residues in theactivation loop. This step was suppressed by the RCAR/PYR ABAreceptors in response to ABA. However the abi1–1 mutated PP2Cdid not respond to the receptors and constitutively inactivatedSnRK2. Our results demonstrate that group A PP2Cs act as ‘gate-keepers’ of subclass III SnRK2s, unraveling an important regulatorymechanism of ABA signaling.

protein phosphorylation � � LC-MS � SnRK2 � PYR1

As sessile organisms, plants have to rapidly recognize andadapt to environmental changes. The phytohormone ab-

scisic acid (ABA) plays a central role in such responses (1, 2), andis also involved in many developmental processes (3) and defensesystems (4). Thus, ABA functions as a key molecule that unifiesand regulates biotic and abiotic stress responses and the devel-opmental status of the plant. Hence, the biological and agricul-tural importance of ABA has led to extensive studies on itssignaling mechanism, and many putative signal transducers havebeen reported (5). Although it has been difficult to integrate allof the current findings, significant progress was recently made bytwo independent research groups. They identified the RCAR/PYR family proteins as ABA receptors that inhibit proteinphosphatase 2C (PP2C) in an ABA-dependent manner (6, 7).Among plant PP2Cs, a group A subfamily (e.g., ABI1 and ABI2)is annotated as negative regulators of the ABA response in seedsthrough to adult plants (5). Such PP2C-dependent negativeregulation can be canceled by RCAR/PYR in response to ABA,leading to activation of some positive regulatory pathways (6, 7).Previously, we demonstrated that ABI1 interacts with a proteinkinase, SRK2E (OST1/SnRK2.6) (8). SRK2E belongs to theSNF1-related protein kinase 2 (SnRK2) family, which is acti-vated by ABA or osmotic stress and positively regulates the ABAresponse in various tissues (9–11). Furthermore, ABA-dependent activation of SRK2E was repressed in an abi1–1mutant, suggesting that SnRK2 functions downstream of PP2C(8, 9). Based on these findings, a model was hypothesized inwhich RCAR/PYR and PP2C negatively regulate SnRK2 (7).However, there is no direct evidence demonstrating how PP2Cregulates SnRK2, and the molecular process between themremains a question in ABA signaling. Our presented data clearlydemonstrated the biochemical relation between PP2C andSnRK2 and elucidated an important regulatory mechanism ofABA signaling.

Results and DiscussionRequirement of SnRK2 Activity for ABA Responses. In Arabidopsis,subclass III of the SnRK2 family is composed of three proteins,namely SRK2D (SnRK2.2), SRK2I (SnRK2.3), and SRK2E(OST1/SnRK2.6). To further investigate the functions ofSnRK2s, we established a triple knockout mutant for subclass IIISnRK2s (srk2dei). Recently, some researchers independentlyestablished the same triple mutant, and its phenotype was

Author contributions: T.U., T.H., and K.S. designed research; T.U., N.S., M.M., S.H., F.M., andT.H. performed research; N.S., K.Y.-S., and Y.I. contributed new reagents/analytic tools;T.U., N.S., S.H., and F.M. analyzed data; and T.U., Y.I., and T.H. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.

1To whom correspondence may be addressed. E-mail: takashih@riken.jp orsinozaki@rtc.riken.jp.

This article contains supporting information online at www.pnas.org/cgi/content/full/0907095106/DCSupplemental.

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Fig. 1. Subclass III SnRK2s are essential for ABA responses. (A) Germinationtest to determine ABA sensitivity in the srk2dei triple mutant. Arabidopsisseeds of Col, srk2dei, abi1–1C, and abi5–7 were sown and grown for 2 weekson GM agar medium containing 0, 3, or 100 �M ABA. (B) ABA-activatedprotein kinases in Col, srk2dei, and abi1–1C plants. Three-week-old plantswere treated with 50 �M ABA for 0, 30, or 90 min. Upper panel shows in-gelphosphorylation (IGP) assay using histone as a substrate. Black arrow indicatesABA-activated protein kinase (�40 kDa) overlapping with SnRK2s, and theopen arrow indicates the RubisCo large subunit (�50 kDa) in a CBB-stainedgel. The results were confirmed through several replications.

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thoroughly analyzed (12, 13). They showed the triple mutationcompletely impaired the ABA response, suggesting critical rolesof subclass III SnRK2s and their fundamental redundancy inABA signaling. In our study, we tested ABA responsiveness inthe srk2dei mutant to compare with other ABA-insensitivemutants, abi1–1C and abi5–7. Consistent with a previous report,srk2dei showed marked ABA insensitivity, even in the presenceof 100 �M ABA (Fig. 1A). Furthermore, in-gel phosphorylationassay demonstrated the loss of ABA-activated kinase activity(approx. 40 kDa) in srk2dei, and a low level of activity in abi1–1C(Fig. 1B), showing a correlation with their ABA sensitivity.Although some kinase activities were observed in srk2dei, theymight be unknown kinases that were non-functional in wild-typeplants. These data imply that all subclass III SnRK2s are stronglyinhibited by abi1–1 mutation, and the SnRK2 activity should bemajor determinant of the ABA response in Arabidopsis.

Physical Interaction Between PP2C and SnRK2. Our observations ofsrk2dei suggested that regulation of SnRK2 activity is a key tounderstanding how ABA signals are transmitted. As describedabove, we identified PP2C as a potential regulator of SnRK2,because ABI1 interacted with the domain II of SRK2E in theyeast two-hybrid (Y2H) system (8). In this study, we furthertested five SnRK2s and 10 PP2Cs by Y2H assay. Consistent withour previous study, ABI1 strongly bound to SRK2E, and someother PP2Cs (e.g., ABI2 and At5g59220) also interacted withSRK2E (Fig. 2A). Compared with SRK2E, SRK2D and SRK2Ishowed a wide preference for various group A PP2Cs. OtherSnRK2s (not subclass III) or an outgroup of PP2C, AP2C1(group B), showed limited or no interaction (Fig. 2 A). Theseresults indicate that group A PP2Cs interact specifically withSnRK2s in various combinations. It should be noted thatsome of the Y2H results were not consistent with our previousstudy (8). This discrepancy may be related to technical problems,for which reason we confirmed our results through severalreplicates.

The physical interaction between SnRK2s and PP2Cs ob-served in the Y2H assay was partly supported by gene expressionpatterns and subcellular localizations. SRK2E and ABI1 wereco-expressed in guard cells (Figs. S1 and S2) and were localizedin the cytosol or nucleus (Fig. 2C). SRK2D and SRK2I weresignificantly expressed in seeds together with AHG1 or AHG3which were localized in the nucleus predominantly (Fig. 2C andFig. S1). These data were consistent with their regulatory rolesin guard cells or seeds (11, 14, 15), showing a correlation betweenphysiological function and Y2H results. To confirm these inter-actions in vivo, we performed co-immunoprecipitation (Co-IP)experiments using protoplasts expressing SRK2I-GFP and HA-tagged ABI1 or AHG1. As shown in Fig. 2D, these PP2Cs wereco-immunoprecipitated with SRK2I in an ABA-independentmanner, suggesting that they interact constantly in vivo. Theco-expression of some pairs, for example, SRK2E and ABI1, wasalmost undetectable in protoplasts, making it impossible todemonstrate their interaction in Co-IP experiments. However,BiFC analysis confirmed the in planta interaction betweenSRK2E and ABI1 in the cytosol and nucleus, similar to SRK2Iand ABI1 (Fig. 2E). The abi1–1 mutation causes a substitutionof Gly to Asp (G180D) in the PP2C catalytic domain, which

Fig. 2. Group A PP2Cs interact with subclass III SnRK2s. (A) Yeast two-hybridanalysis of SnRK2s and PP2Cs. GAL4BD:SnRK2s and GAL4AD:PP2Cs were intro-duced to yeast cells as indicated. Black slopes indicate screening stringency of SDmedia 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 blackslope demonstrates stringency of screening media: SD -LW, SD -LWH � 10 mM3-AT, SD -LWHA � 30, 60, or 100 mM 3-AT from left to right. (C) Subcellularlocalization of GFP-tagged SnRK2 or PP2C proteins that were transiently

expressed in Arabidopsis mesophyll protoplasts. (D) Co-immunoprecipitationof GFP-tagged SnRK2s and HA-tagged PP2Cs was performed using Arabidop-sis protoplasts. Immunoprecipitates against anti-GFP antibody (IP) or crudeextracts (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. Thephotos were YFP, DIC, autofluorescence (auto) and merged images. Theresults were confirmed through several replications.

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confers a dominant ABA-insensitivity by unknown mechanisms(16, 17). Although very little abi1–1 was produced in protoplasts,the data revealed that abi1–1 was able to bind to SRK2Iconstantly (Fig. 2D). This result was supported by the Y2H assay,in which abi1–1 and abi2–1 showed some enhanced interactionwith SnRK2s, whereas abi1–1R6, a loss-of-function mutation forABI1 (18), had no effect (Fig. 2B).

Group A PP2C Inactivates Subclass III SnRK2. Previous studiesshowed that the ABA-responsive activation of SRK2E wasinhibited in the abi1–1 mutant (8, 9). In addition, our datafurther demonstrated that all subclass III SnRK2s were inhibitedby abi1–1 mutation (Fig. 1B), implying that subclass III SnRK2sfunction downstream of PP2C. To clarify this regulation, weexamined SnRK2 activity in imbibed seeds from ahg1–1 andahg3–1 which were loss-of-function mutations of PP2C causing

ABA-hypersensitive germination (14, 15). The results revealedthat approximately 40 kDa of protein kinase activity involvingSnRK2 were hyperactivated in ahg1–1 and ahg3–1 (Fig. 3A),suggesting that PP2C negatively regulates SnRK2 activity. Next,we examined whether PP2C directly regulates SnRK2 activityusing an in vitro phosphatase assay with recombinant PP2Cproteins and GFP-tagged SnRK2s from Arabidopsis cells.SnRK2 activity was determined with an in-gel phosphorylationassay in which SnRK2-GFP was clearly activated by ABA (Fig.3B and Fig. S3A). The activity of SRK2E or SRK2I wasefficiently or significantly reduced by ABI1 or AHG1, and,surprisingly, ABI1-mediated inactivation was stronger than thatof CIAP (Fig. 3B and Fig. S3A). Dominant-type PP2C mutantssuch as abi1–1 and ahg1G152D inactivated SnRK2 significantly(Fig. 3B and Fig. S3A), implying they are capable of regulatingSnRK2 negatively as well as wild-type proteins. Because it was

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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 �M ABAfor 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) Inactivationassay for SnRK2 by in vitro reaction with PP2Cs. SRK2E-GFP proteins were prepared by immunoprecipitation (IP) against an anti-GFP antibody from Arabidopsiscultured cells with or without ABA treatment. After incubation with recombinant PP2C proteins as indicated, SRK2E activity was monitored with an in-gelphosphorylation assay (IGP) using histone as a substrate. Immunoprecipitates were analyzed via Western blotting (WB) using an anti-GFP antibody. Arrowsindicate SRK2E-GFP. (C) Dephosphorylation of 32P-labeled SRK2E-GFP by PP2Cs. Immunoprecipitated SRK2E-GFP was prepared from untreated or ABA-treatedcultured 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 aphospho-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 targetpeaks. Asterisks in red and black indicate probable phosphorylation sites detected in this study with the highest and less PTM scores, respectively. (E) An MS/MSspectrum 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 annotatedin the spectrum. The results were confirmed through several replications.

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difficult to produce a tagless form of abi1–1, we used a pair ofGST-tagged ABI1 and abi1–1 proteins in this study.

Group A PP2C Directly Dephosphorylates Subclass III SnRK2. Our dataof in vitro inactivation assay (Fig. 3B) strongly suggested thatPP2C-mediated inactivation of SnRK2 was given by dephos-phorylation of an active form of SnRK2, which is highly phos-phorylated in vivo (19, 20). To monitor the phosphorylationstate of SnRK2, we prepared 32P-labeled SnRK2-GFP proteinsfrom Arabidopsis cells. As shown in Fig. 3C or Fig. S3B mocklanes, the intensity of the signal corresponding to SRK2E/I-GFPincreased and the position of SRK2I-GFP shifted slightly uponABA treatment. When 32P-labeled SRK2E/I were incubatedwith ABI1, the signal intensity decreased and band shifting ofSRK2I was not observed. GST-abi1–1 also dephosphorylatedSRK2E/I (Fig. 3C and Fig. S3B), consistent with the results of invitro inactivation assay (Fig. 3B and Fig. S3A). Similar resultswere obtained with AHG1 and SRK2I (Fig. S3B). Inconsistentwith the in vitro inactivation assay, CIAP dephosphorylatedSnRK2 more efficiently (Fig. 3C and Fig. S3B), suggesting thatPP2C has specificity to critical sites for SnRK2 activity. Sup-porting this hypothesis, the radioactivity of SnRK2 remainedbasal even after incubation with PP2C. These results can be

summarized; the ABA-responsive phosphorylation of SnRK2 invivo was specifically reversed by PP2C in vitro. It is noteworthythat SnRK2 inactivation and dephosphorylation was not corre-lated with PP2C activity measured using a standard protocol. Forexample, GST-abi1–1 showed very low phosphatase activity toartificial substrates (Fig. S4), but strongly inactivated and de-phosphorylated SnRK2 (Fig. 3 and Fig. S3). This inconsistencyis probably due to the difference in compatibility to PP2Cbetween native and artificial substrates. Our results suggest thatabi1–1-type mutation does not affect strongly the PP2C activityfor SnRK2.

We further analyzed SnRK2-GFP proteins using a nanoLC-MS/MS system to survey phosphorylation sites, and we detectedseveral phospho-peptides that are responsive to ABA (Fig. 3D,Fig. S5, and Table S1). One of these, pSSVLHSDQPKpSTVGT-PAYIAPEVLLKK, which spans the activation loop in SRK2E,was clearly upregulated by ABA but diminished after incubationwith ABI1 (Fig. 3 D and E) or abi1–1 (Fig. S6). Althoughphosphorylation sites were not unambiguously identified withsatisfactory probability scores in all peptides, we detected at leastfive phosphorylation sites in the kinase activation loop that wereresponsive to ABA, all of which were dephosphorylated by PP2C(Fig. S5 and Table S1). Some phosphorylation sites were pre-viously reported in SRK2E and NtOSAK (20, 21), but theirmutational analyses failed to produce an active form, suggestingtechnical difficulties for mapping critical residues for SnRK2activation, like MAPKs. In our study, the PP2C-mediated de-phosphorylation and inactivation of SnRK2 confirmed thatSnRK2 activity was mainly regulated by reversible phosphory-lation/dephosphorylation of the activation loop. Since PP2C-SnRK2 interactions are independent of ABA (Fig. 2D), PP2C iscapable of inactivating SnRK2 even in the absence of ABA. Wepropose that this SnRK2 inactivation is an essential part of thePP2C-mediated negative regulation of ABA signaling, becauseSnRK2 regulates the majority of ABA responses (Fig. 1 A). ThisPP2C-SnRK2 regulation reminds of two kinases related to plantSnRK2, yeast SNF1 and mammal AMPK, which are regulated byPP2C (22). There are several lines of evidence indicating thatPP2Cs negatively regulate protein kinases in plants, for example,the Arabidopsis AP2C1 for AtMPK4/6 or the rice XB15 forXA21 receptor kinase (23, 24). Plants express a large, diversefamily of PP2Cs (25), and these proteins may be involved in theregulation of many protein kinases.

Reconstitution of RCAR/PYR, PP2C, and SnRK2 Signaling Complex InVitro. So far, our in vivo and in vitro experiments agree with thehypothesis that group A PP2Cs directly regulate subclass IIISnRK2 activity via dephosphorylation. Recently, RCAR/PYRfamily proteins were identified as ABA receptors that inhibitPP2C activity in an ABA-modulated fashion (6, 7). Combiningtheir findings and our own, we propose a signaling complexconsisting of RCAR/PYR, PP2C, and SnRK2. To test this, wereconstituted these three components in vitro using recombinantABI1, abi1–1, and PYR1 proteins, and GFP-tagged SnRK2sfrom Arabidopsis cells. After incubation with PYR1, PP2Cs, andSnRK2s in the presence or absence of ABA, SnRK2 activity wasmonitored via an in-gel phosphorylation assay. We found thatPYR1 inhibited ABI1-mediated inactivation of SnRK2 in anABA-dependent manner (Fig. 4A and Fig. S3C); however,abi1–1 inactivated SnRK2s even in the presence of PYR1 andABA, consistent with previous data showing that abi1–1 andabi2–1 lacked RCAR/PYR-binding ability (6, 7). Thus, ourresults demonstrated that the abi1–1-type mutation negativelyregulated SnRK2 in a constitutive fashion, resolving a long-standing puzzle of abi1–1 action.

A Simple Model of ABA Signaling in Plants. Collectively, our dataenables us to strengthen a hypothesized ABA signaling model

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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 �MABA. SnRK2 activity was monitored with an in-gel phosphorylation assay (IGP)using histone as a substrate. Immunoprecipitates (IP) were checked by West-ern blot analysis (WB) against an anti-GFP antibody. Arrows indicate SRK2E-GFP. This result was confirmed through several replications. (B) Proposedmodel of the ABA signaling pathway in Arabidopsis. Left, in the absence ofABA, PP2C dephosphorylates and inactivates SnRK2. Middle, in the presenceof ABA, RCAR/PYR interacts with PP2C, and SnRK2 is released from negativeregulation and converted to an active form. The activated SnRK2 phosphor-ylates ABA-responsive transcription factors or unknown substrates to induceABA responses. Right, even in the presence of ABA, the abi1–1 mutant of PP2Cconstitutively dephosphorylates SnRK2, consequently conferring dominantABA insensitivity. In this model, PP2C constitutively binds to domain II at theC terminus of SnRK2. Red and blue colors indicate an active or inactive form,respectively.

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previously proposed (7), and simplify the molecular process ofABA signaling to just four steps from perception to geneexpression (Fig. 4B). First, PP2C interacts with domain II at theC terminus of SnRK2 (8); this interaction is constant andindependent of ABA. In the absence of ABA, PP2Cs repress theABA signaling pathway via the dephosphorylation and inacti-vation of SnRK2s. After the induction of free ABA by environ-mental or developmental cues, RCAR/PYR binds to PP2C (6, 7)and SnRK2 is released from PP2C-dependent negative regula-tion. This allows SnRK2 to phosphorylate downstream sub-strates, including bZIP transcription factors (AREB/ABFs) (26)etc., to activate ABA-responsive gene expression or other re-sponses. In contrast, dominant mutations in PP2C, such as abi1–1,allow the protein to avoid RCAR/PYR-binding (6, 7), resulting inthe constitutive inactivation of SnRK2s. At present, the mecha-nisms underlying the internal phosphorylation of SnRK2 remainunclear, although autophosphorylation is one of possible regula-tions (2). Further analysis will be required to clarify this issue.

We argue that the RCAR/PYR-PP2C-SnRK2 complex is theprimary framework for ABA signaling, and that a conventionalprotein phosphorylation/dephosphorylation system, PP2C-SnRK2,provides a reversible regulatory module. Recently, group A PP2Csand AREB/ABF bZIP transcription factors were found in Phy-scomitrella patens (27–29). Furthermore, our computational anal-ysis detected subclass III SnRK2s in P. patens, but not in Chlamy-domonas reinhardtii (Fig. S7). Thus, the PP2C-SnRK2-mediatedsignal transduction system may be conserved from bryophytes tohigher plants, suggesting this system is an important acquiredcharacteristic in the evolution of land plants with respect to droughttolerance.

Materials and MethodsPlant Materials. The srk2dei triple mutant was generated from srk2e(SALK�008068), srk2d (GABI-Kat�807G04), and srk2i (SALK�096546), whichwere obtained from public sources (ABRC or GABI�Kat). The abi1–1C wasisolated from a genetic screen of Col background lines. The ABA response testwas performed as described previously (15).

Plasmids. In this study, a series of vector plasmids, such as pGBKT7 and pGADT7(Clontech), pGEX6P (GE Healthcare), and the pGreen series (30), were con-verted to Gateway destination vectors. Gateway entry clones were preparedin the pENTR/D-TOPO or pDONR207 vector (Invitrogen) by insertion of cDNAs,SRK2E, SRK2D, SRK2I, SRK2C, SRK2B, ABI1, ABI2, HAB1, HAB2, AHG1, AHG3,At1g07430, At3g29380, At5g59220, AP2C1, and PYR1. Mutated PP2Cs [i.e.,abi1–1 (G180D), abi1–1R6 (G307D), abi2–1 (G168D), and ahg1G152D] weregenerated using the QuikChange site-directed mutagenesis kit (Stratagene)with entry clones as templates.

Yeast Two-Hybrid Analysis. Yeast two-hybrid analysis was carried out using theMatchMaker GAL4 Two-Hybrid System 3 (Clontech) according to the manu-facturer’s instructions. A yeast strain (AH109) was transformed with variouspairs of pGBKT7 vectors harboring SnRK2s and pGADT7 vectors harboringPP2Cs. Then, the transformants were tested on SD screening medium asindicated in Fig. 2.

In-Gel Phosphorylation Assay for Seed Proteins. Dry seeds (50 mg) of Col,ahg1–1, and ahg3–1 were imbibed in water and placed at 4 °C for 3 days. Thenimbibed seeds were treated with 25 �M (�)-ABA (Sigma) for 0, 1, and 5 h.Seeds were crushed by a mortar and pestle with silica sand in liquid nitrogenand homogenized in 100 �L extraction buffer containing 50 mM HEPES pH 7.5,

5 mM EDTA, 5 mM EGTA, 1 mM Na3VO4, 25 mM NaF, 50 mM, �-glycerophos-phate, 10% glycerol, 2 mM DTT, and proteinase inhibitor mixture (Sigma).Crude extracts containing 50 �g total protein were loaded in 10% SDS-polyacrylamide and analyzed by in-gel kinase assay to check ABA-activatedprotein kinases as described previously (31).

Transient Expression in Arabidopsis Mesophyll Protoplasts. The preparation ofprotoplasts from Arabidopsis rosette leaves and PEG-calcium transfectionwere performed according to ref. 32. We prepared pGreen0029 vectors har-boring GFP, SRK2E-GFP, SRK2I-GFP, ABI1-HA, abi1–1-HA, or AHG1-HA. Forsubcellular localization, 20 �g plasmid DNAs were transfected into 3 � 104

protoplasts, and GFP signals were observed with a laser-scanning confocalfluorescence microscopy. For co-immunoprecipitation (Co-IP) assay, a total of2 mg plasmid DNA mixture with SnRK2-GFP and PP2C-HA was transfected into3 � 106 protoplasts. Crude extracts were obtained by homogenizing proto-plasts in 1 mL extraction buffer supplemented with 100 mM KCl, 50 mM NaCl,and 0.1% Nonidet P-40 (IP buffer). Co-IP was performed with 100 �L ProteinG Sepharose (GE Healthcare) and 1 �L polyclonal anti-GFP antibody (MBL), andaliquots were analyzed by Western blot analysis using monoclonal anti-GFPantibody (MBL) or anti-HA antibody (Covance).

Bimolecular Fluorescence Complementation (BiFC) in Fava Bean Leaves. ThecDNAs of SRK2E, SRK2I, and ABI1 were cloned into pUC-SPYNEGW or pUC-SPYCEGW (33). The constructs were delivered into leaf cells of fava bean (Viciafaba) by particle bombardment, as previously described (34), with modifications.

In Vitro Phosphatase Reaction. Bacterial recombinant PP2C proteins and Ara-bidopsis T87 cells expressing SRK2E- or SRK2I-GFP were prepared as previouslydescribed (8, 14). T87 cells were treated with or without 50 �M ABA for 30 min,and IP was performed as described above. After washing, the buffer wasexchanged for phosphatase reaction buffer (8), and then aliquots (30 �L) weremixed with 100 ng recombinant PP2C proteins, ABI1, AHG1, ahg1G152D, AP2C1,or 300 ng GST-ABI1 and GST-abi1–1, or 1 U CIAP (Toyobo). After incubation at30 °C for 1 h, half aliquots were analyzed with an in-gel phosphorylation assayor Western blotting.

In Vivo Labeling of Arabidopsis Cells. To check the phosphorylation status ofSnRK2, Arabidopsis T87 cells expressing SRK2E- or SRK2I-GFP were labeledwith 30 MBq/ml 32P-phosphate (NEX053H, Perkin-Elmer) for 2 h and thentreated with or without 100 �M ABA for 30 min. IP and in vitro phosphatasereactions were performed as described above. After SDS/PAGE, radioactivitywas detected by autoradiography.

In Vitro Reconstitution Assay of ABA Signaling Components. Immunoprecipi-tated SRK2E- or SRK2I-GFP (bed vol. 30 �L) were incubated with 500 ng GST-ABI1or GST-abi1–1, 100 ng ABI1, and 800 ng PYR1 proteins in the presence or absenceof 10 �M ABA at 30 °C for 1 h. Recombinant PP2Cs and PYR1 were preincubatedwith ABA for 10 min in advance. Aliquots were analyzed by in-gel phosphoryla-tion assay or Western blotting as described above.

NanoLC-MS/MS Analysis. Immunoprecipitated SnRK2-GFP proteins were pre-pared as described above and analyzed with a nanoLC-MS/MS system (LTQ-Orbitrap, Thermo) according to ref. 35. The details of experiments are de-scribed in SI Text.

ACKNOWLEDGMENTS. We thank Mrs. Hiroko Kobayashi, Saho Mizukado(RIKEN), and Sumiko Onuma (Keio University) for technical assistance. We aregrateful to ABRC and GABI-Kat project for providing Arabidopsis T-DNA-tagged mutants. We also thank Prof. Masaru Tomita at Keio University for hissupport. This work was partly supported by the RIKEN Plant Science Center,Grants-in-Aid for Scientific Research for Young Scientists (B) to T.U. andGrants-in-Aid for Scientific Research (C) to T.H. from MEXT, and research fundsfrom Yamagata prefecture and Tsuruoka City to Keio University.

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