the arabidopsis mitogen-activated protein kinase ... · the arabidopsis mitogen-activated protein...

The Arabidopsis Mitogen-Activated Protein KinasePhosphatase PP2C5 Affects Seed Germination, StomatalAperture, and Abscisic Acid-InducibleGene Expression1[C][W]

Anita K. Brock2, Roland Willmann, Dagmar Kolb, Laure Grefen, Heini M. Lajunen, Gerit Bethke3, Justin Lee,Thorsten Nurnberger, and Andrea A. Gust*

Center for Plant Molecular Biology, Plant Biochemistry, Eberhard Karls University of Tubingen, 72076Tuebingen, Germany (A.K.B., R.W., D.K., L.G., H.M.L., T.N., A.A.G.); and Department of Stress andDevelopmental Biology, Leibniz Institute of Plant Biochemistry, 06120 Halle/Saale, Germany (G.B., J.L.)

Abscisic acid (ABA) is an important phytohormone regulating various cellular processes in plants, including stomatal openingand seed germination. Although protein phosphorylation via mitogen-activated protein kinases (MAPKs) has been suggestedto be important in ABA signaling, the corresponding phosphatases are largely unknown. Here, we show that a member of theProtein Phosphatase 2C (PP2C) family in Arabidopsis (Arabidopsis thaliana), PP2C5, is acting as a MAPK phosphatase. ThePP2C5 protein colocalizes and directly interacts with stress-induced MPK3, MPK4, and MPK6, predominantly in the nucleus.Importantly, altered PP2C5 levels affect MAPK activation. Whereas Arabidopsis plants depleted of PP2C5 show an enhancedABA-induced activation of MPK3 and MPK6, ectopic expression of PP2C5 in tobacco (Nicotiana benthamiana) resulted in theopposite effect, with the two MAPKs salicylic acid-induced protein kinase and wound-induced protein kinase not beingactivated any longer after ABA treatment. Moreover, depletion of PP2C5, whose gene expression itself is affected by ABAtreatment, resulted in altered ABA responses. Loss-of-function mutation in PP2C5 or AP2C1, a close PP2C5 homolog, resultedin an increased stomatal aperture under normal growth conditions and a partial ABA-insensitive phenotype in seedgermination that was most prominent in the pp2c5 ap2c1 double mutant line. In addition, the response of ABA-inducible genessuch as ABI1, ABI2, RD29A, and Erd10 was reduced in the mutant plants. Thus, we suggest that PP2C5 acts as a MAPKphosphatase that positively regulates seed germination, stomatal closure, and ABA-inducible gene expression.

To cope with the limitations of a sessile lifestyle,plants have evolved a sophisticated network of re-sponses to biotic and abiotic stress. Of the manyhormones that mediate such responses, abscisic acid(ABA) has historically been one of the most intensivelystudied stress hormones (Koornneef et al., 1998;Christmann et al., 2006; Verslues and Zhu, 2007). Inparticular, ABA promotes stomatal closure and pre-vents stomatal opening during drought, thus reducing

transpirational water loss. During late embryogenesis,ABA promotes the acquisition of desiccation toleranceand seed dormancy and inhibits seed germination.Evidence is also accumulating that ABA plays a crucialrole in the plant defense response (Mauch-Mani andMauch, 2005; Adie et al., 2007; Fan et al., 2009).

ABA signal transduction engages a complex networkof both positively and negatively regulating proteinkinases and Ser/Thr protein phosphatases (Leung andGiraudat, 1998; Himmelbach et al., 2003; Hirayama andShinozaki, 2007; Umezawa et al., 2009). Protein phos-phatases that dephosphorylate Ser and Thr residues areclassified into two groups, the PPP family and the type2C phosphatases (PP2Cs; Cohen, 1989). The PPP familyconsists of type 1 (PP1), type 2A (PP2A), and type 2B(PP2B) phosphatases (Farkas et al., 2007), which sharesequence homology in their catalytic domains and aresensitive to specific inhibitors. In contrast, PP2Cs shareno sequence similarity with PPPs despite striking ar-chitectural similarities of their crystal structures (Daset al., 1996). PP2Cs are monomeric enzymes that con-tain all 11 characteristic subdomains in the catalyticdomain (Bork et al., 1996) and constitute the largestprotein phosphatase family in plants, with 76 membersin Arabidopsis (Arabidopsis thaliana; Kerk et al., 2002;Schweighofer et al., 2004; Kerk, 2007).

1 This work was supported by the Deutsche Forschungsgemein-schaft-funded Graduate School (grant nos. GK 685 to T.N., SFB 766 toA.A.G. and T.N., and SFB 648 to G.B. and J.L.).

2 Present address: Department of Plant Nutrition, Leibniz Instituteof Vegetable and Ornamental Crops, 14979 Grossbeeren, Germany.

3 Present address: Department of Plant Biology, University ofMinnesota, St. Paul, MN 55108.

* Corresponding author; e-mail [email protected].

The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policydescribed in the Instructions for Authors (www. plantphysiol.org)is: Andrea A. Gust ([email protected]).

[C] Some figures in this article are displayed in color online but inblack and white in the print edition.

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.110.156109

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The best studied PP2Cs belong to clade A and havebeen demonstrated to act as negative regulators ofABA responses, most importantly ABI1, ABI2, and thecold response-linked PP2Cs AtPP2CA and HAB1(Leung et al., 1997; Merlot et al., 2001; Saez et al.,2004; Kuhn et al., 2006; Rubio et al., 2009). Recently,progress has been made in the elucidation of theregulation of clade A phosphatases. The RegulatoryComponent of ABA Receptor1/Pyrabactin Resis-tance1 (RCAR1/PYR1) and RCAR3 were identifiedas interactors of ABI1 and ABI2 (Ma et al., 2009; Parket al., 2009; Szostkiewicz et al., 2010). RCAR1/PYR1directly binds to ABA and mediates ABA-dependentinactivation of ABI1 or ABI2 in vitro, thus antagoniz-ing PP2C action in planta. Likewise, the Bet v1-likesuperfamily member PYL5 antagonizes ABI1, ABI2,and HAB1 function by inhibiting their phosphataseactivity in an ABA-dependent manner (Santiago et al.,2009). ABI1 and the highly homologous ABI2 haveattracted most attention as partially redundant keyregulators of ABA-invoked seed dormancy, stomatalclosure, and growth inhibition (Merlot et al., 2001).Both phosphatases, particularly ABI2, physically in-teract with the Protein Kinase Salt-sensitive3 (PKS3),and ABA was shown to transiently down-regulatePKS3 kinase activity, which is required to suppressABA action (Guo et al., 2002). ABI2 also interacts withthe protein kinase Salt Overly Sensitive2 (SOS2),which is required for salt tolerance in Arabidopsis(Ohta et al., 2003). In contrast, in addition to PKS3,ABI1 can interact with the Open Stomata1 kinase,which was shown to be a positive regulator in ABA-induced stomatal closure (Yoshida et al., 2006; Geigeret al., 2009).Apart from the above-described kinases, mitogen-

activated protein kinases (MAPKs) have been impli-cated in ABA signaling (Heimovaara-Dijkstra et al.,2000; Hirayama and Shinozaki, 2007). Generally,MAPKs have been described as major components ofcellular signal transduction pathways mediating var-ious biotic and abiotic stress responses, includinghormone signaling, cell division, and developmentalprocesses (Ligterink, 2000; Asai et al., 2002; Jonak et al.,2002; Pedley and Martin, 2005; Mishra et al., 2006;Pitzschke et al., 2009). MAPK cascades are universalsignal transduction modules in eukaryotes, includingyeasts, animals, and plants, and are generally com-posed of three functionally linked kinases, a MAPKkinase kinase (MAPKKK or MEKK), a MAPK kinase(MAPKK or MKK), and a MAPK. In response toextracellular stimuli, MAPKKKs activate MAPKKsvia phosphorylation of two Ser/Thr residues withinthe S/TXXXXXS/T motif, where X denotes any aminoacid. MAPKKs, which are dual-specificity proteinkinases, then activate their downstream MAPK byphosphorylating the Thr and Tyr residues withinthe TXY motif. Activated MAPKs phosphorylate spe-cific effector proteins, such as transcription factors(Popescu et al., 2009), which leads to an activation ofcellular responses.

In Arabidopsis, MPK3, MPK4, and MPK6 are thebest characterized members of the MAPK family andhave also been demonstrated to be part of the ABAsignal transduction pathway. MPK3 is activated byboth ABA and hydrogen peroxide in Arabidopsisseedlings, and MPK3 overexpression increases ABAsensitivity in ABA-induced postgermination arrest ofgrowth, suggesting that the ABA signal is transmittedthrough MPK3 in this system (Lu et al., 2002). Inaddition, MPK4 and MPK6 are transiently activatedafter ABA application (Ichimura et al., 2000), andmpk6mutation blocked while MPK6 overexpression en-hanced ABA-dependent hydrogen peroxide produc-tion (Xing et al., 2008).

As dephosphorylation of only one residue in thehighly conserved TXY motif of activated MAPKs issufficient to abolish their activity, PP2Cs can readilyact as MAPK phosphatases (MKPs). Alfalfa (Medicagosativa) MP2C was the first plant PP2C shown tonegatively regulate MAPK signaling. MP2C directlyinteracts with the salt stress-inducible MAPK (SIMK;homologous to MPK6) and inactivates SIMK throughThr dephosphorylation of the pTEpY motif (Meskieneet al., 1998, 2003). Similarly, Arabidopsis AP2C1, asthe closest MP2C homolog, was recently shown to in-teract with and dephosphorylate MPK4 and MPK6(Schweighofer et al., 2007). Multiple other examplesprovide evidence that PP2Cs can attenuate stress-induced MAPK cascades in eukaryotes. The high-osmolarity glycerol (HOG) MAPK pathway, whichcontrols the osmotic stress response in yeast, wasshown to be negatively regulated by the PP2Cs Ptc1and Ptc3 through direct dephosphorylation of theMAPK Hog1 (Nguyen and Shiozaki, 1999; Warmkaet al., 2001). Likewise, in humans, the JNK/p38 path-way, which shares similarities with the yeast HOGpathway, is inactivated by direct binding to and de-phosphorylation of p38 MAPK by PP2Ca (Takekawaet al., 1998). All these examples clearly indicate thatPP2Cs are regulating diverse signaling pathways me-diated by MAPK cascades.

Here, we report the identification of PP2C5 as aMAPK phosphatase. We show that PP2C5 directlyinteracts with and regulates the activation of stress-induced MPK3, MPK4, and MPK6. Depletion of PP2C5and its closest homolog AP2C1 results in plants with anincreased stomatal aperture, partial ABA insensitivityduring seed germination, and a decreased responsive-ness of ABA-inducible genes after ABA application.Thus, unlike previously described PP2Cs, PP2C5 posi-tively regulates seed germination, stomatal closure, andABA-inducible gene expression.

RESULTS

PP2C5 Expression Is Induced by ABA

To identify phosphatases that attenuate MAPK ac-tivities during ABA signaling, we focused on clade B

Protein Phosphatase 2C in Arabidopsis ABA Signaling

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of the PP2C superfamily (Supplemental Fig. S1A), ofwhich one member, AP2C1, was recently demon-strated to act as MAPK phosphatase (Schweighoferet al., 2007). In addition, four out of the six members ofclade B contain a putative MAP kinase interactionmotif (KIM) similar to those found in animal MAPKkinases or MAPK phosphatases (Ho et al., 2003, 2006),suggesting that these proteins might interact withMAPKs in plants (Schweighofer et al., 2004, 2007).As the phosphatases ABI1, ABI2, AtPP2CA, andHAB1,which are all involved in ABA signaling, are tran-scriptionally up-regulated by ABA (Leung et al., 1997;Merlot et al., 2001; Saez et al., 2004; Kuhn et al., 2006),we analyzed ABA-inducible accumulation of tran-scripts encoding the four KIM-containing clade B phos-phatases AP2C1 (At2g30020), AP2C2 (At1g07160),PP2C5 (At2g40180), and AP2C4 (At1g67820). Analysisof microarray data obtained from experiments con-ducted within the AtGenExpress initiative (Goda et al.,2008) revealed that gene induction was most prominentfor PP2C5 after a 30-min treatment with ABA (Supple-mental Fig. S1B). This is in agreement with an earlierreport that PP2C5 belongs to an ABA-inducible genecluster (Wang et al., 1999). Similarly, AP2C1 gene ex-pression was weakly induced whereas gene expressionof the two other PP2Cs, AP2C2 and AP2C4, was notaffected.

PP2C5 Is a Nuclear Protein Phosphatase

Using the PSORTanalysis tool, PP2C5was predictedto be localized to the cell nucleus, the same compart-ment into which MAPKs are translocated upon acti-vation. This has been shown in Arabidopsis for MPK3and MPK6 after ozone treatment (Ahlfors et al., 2004)and in parsley (Petroselinum crispum) for PcMPK3 andPcMPK6 after elicitation (Lee et al., 2004). Transloca-tion of MAPKs is required to activate transcriptionfactors, which constitute important MAPK substrates(Feilner et al., 2005; Popescu et al., 2009). In addition,the nucleus seems to be a critical site for terminationof MAPK signaling by (1) nuclear sequestration of ac-tivated MAPKs away from the MAPKK as their cyto-plasmic activator and (2) dephosphorylation-mediatedinactivation by specific nuclear phosphatases.

To determine PP2C5 protein localization in vivo,GFP-tagged PP2C5 protein was transiently expressedeither under the control of the constitutive cauliflowermosaic virus 35S promoter or its native promoter inArabidopsis protoplasts. Irrespective of the promoterstrength, PP2C5-GFP was predominantly located tothe nucleus, whereas GFP alone displayed fluores-cence throughout the cytoplasm and nucleus (Fig. 1A).Likewise, PP2C5-GFP transiently expressed in Nicoti-ana benthamiana leaves, as a heterologous plant system,also located to the nucleus (Fig. 1B).

To revalidate PP2C5 phosphatase activity previ-ously described for recombinant PP2C5 (Wang et al.,1999), we first generated polyclonal antibodies againsta PP2C5-specific N-terminal peptide in rabbit. Anti-

bodies were tested with protein extracts from Arabi-dopsis leaves and protein extracts from N. benthamianaplants transiently expressing PP2C5-GFP (Supplemen-tal Fig. S2). Affinity-purified anti-PP2C5 could detectPP2C5-GFP but not endogenous Arabidopsis PP2C5,which is probably present at low levels and needsprior enrichment for detection (Supplemental Fig. S2).Therefore, affinity-purified rabbit anti-PP2C5 anti-body was used to immunoprecipitate transiently ex-pressed PP2C5-GFP from N. benthamiana proteinextracts. Immunoprecipitated PP2C5-GFP was subse-quently subjected to an in vitro protein phosphataseassay using [32P]phospho-casein as artificial substrate(Stone et al., 1994; Wang et al., 1999). In comparisonwith control samples, PP2C5-GFP-containing immu-noprecipitates showed a strong release of 32P into thesupernatant, which is indicative of phosphatase activ-ity (Fig. 1C). In summary, our data indicate that PP2C5is an active PP2C that is mainly located to the nucleus.

PP2C5 Colocalizes and Interacts with

Stress-Induced MAPKs

PP2C5 contains an N-terminally located KIM(Schweighofer et al., 2004), and for the PP2C5 homologAP2C1, an interaction with the stress-induced MPK4and MPK6 has been demonstrated (Schweighoferet al., 2007). One prerequisite for in vivo proteininteraction is their colocalization; hence, we nextexamined the localization of the three major stress-induced MAPKs, MPK3, MPK4, and MPK6, in theArabidopsis protoplast system. Yellow fluorescentprotein (YFP) fusions of all three MAPKs localizedto the same cellular compartment as PP2C5 fusedto cyan fluorescent protein (CFP), which was predom-inantly found in the cell nucleus (Fig. 2A). Thiscolocalization was not observed when other membersof the clade B PP2Cs were investigated: AP2C1-CFPand AP2C2-CFP were both predominantly localized toplastids (confirming the PSORT prediction), whereasAP2C4-CFP was found in equal quantities both in thecytosol and the nucleus (Fig. 2B; Supplemental Fig.S3). Hence, PP2C5 is the only member of this PP2Csubgroup for which gene expression is induced byABA treatment and that colocalizes with stress-induced MAPKs in the nucleus. To analyze a directphysical interaction of PP2C5 with MPK3, MPK4, andMPK6, proteins were combined in the yeast two-hybrid system. An interaction was found betweenPP2C5 and MPK3, MPK4, and MPK6 in comparisonwith negative controls (Fig. 3A). These results wereconfirmed using a bimolecular fluorescence comple-mentation (BiFC) assay based on split YFP (Walteret al., 2004). The N- and C-terminal domains of YFPwere fused to PP2C5 and MPK3, MPK4, or MPK6,respectively, and transiently coexpressed in Arabidop-sis protoplasts. Again, fluorescence from reconstitutedYFP indicated an interaction between PP2C5 and allthree MAPKs (Fig. 3B). The strongest signal wasobserved with the PP2C5/MPK6 complexes, which

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were detectable in the nucleus and in the cytoplasm,whereas PP2C5/MPK4 complexes localized predom-inantly in the nucleus (Fig. 3B). The weakest signalwas obtained with the PP2C5/MPK3 combination. Nofluorescence was detectable with the empty vectorcombinations as control (data not shown).To investigate the importance of the putative KIM

for phosphatase-MAPK interaction, mutations K90Aand R91Q in the putative KIM of PP2C5 were gener-ated on the basis that a mutation of the correspondingtwo amino acids in AP2C1 blocked its interaction withMPK4 and MPK6 (Schweighofer et al., 2007). In boththe yeast two-hybrid as well as the BiFC assay, inter-action of mutant PP2C5-K90A/R91Q with all threeMAPKs was completely abolished compared withwild-type PP2C5 (Fig. 3). These results show that theinteraction with MPK3, MPK4, or MPK6 depends onan intact KIM in PP2C5.

PP2C5 Protein Levels Affect MAPK Activation

As PP2C5 is directly interacting with stress-inducedMAPKs, we wanted to analyze the effect of PP2C5depletion on MAPK activation. We selected the T-DNAinsertion line N609986 (pp2c5) from the SALK col-lection (Fig. 4A), in which the T-DNA insertion islocated in the second exon. To induce robust PP2C5

gene expression, Arabidopsis seedlings were treatedwith Flg22, a 22-amino acid peptide derived frombacterial flagellin that elicits plant pathogen defenseresponses (Felix et al., 1999) and that was shown tostrongly trigger PP2C5 gene expression (Gust et al.,2007). Increased PP2C5 transcript levels could bedetected in wild-type plants and, interestingly, to aneven higher extent in the pp2c5 mutant when usingprimers amplifying fragments located upstream of theT-DNA insertion (Fig. 4B). However, a much reducedgene expression or no residual transcript accumula-tion was observed in the pp2c5 mutant using quanti-tative PCR analysis with primers either downstreamof or spanning the region of the T-DNA insertion,respectively, indicating that PP2C5 transcript wasstrongly reduced and not present as a full-lengthsequence in the pp2c5 mutant (Fig. 4B).

Mutant seedlings were treated with ABA to analyzethe activation of MAPKs using the phospho-p44/p42antibody. In ecotype Columbia (Col-0) plants, ABAapplication resulted in an enhanced reactivity to theantibody of a protein band running at approximately46 kD and to amuchweaker extent of a protein band ofabout 44 kD, most likely representing the two majorstress-induced MAPKs MPK3 and MPK6 (Fig. 5).However, compared with the wild type, the pp2c5single mutant plants responded only slightly stronger

Figure 1. Phosphatase-active PP2C5 is located tothe nucleus. A, The coding region of PP2C5 wasC-terminally fused to GFP and transiently ex-pressed in Arabidopsis protoplasts either undercontrol of the 35S promoter (35S:PP2C5-GFP) orits native promoter (PP2C5:PP2C5-GFP). As acontrol, GFP was expressed alone (35S:GFP).Protoplasts were analyzed by fluorescence mi-croscopy. B, The fusion proteins described in Awere transiently expressed in N. benthamianaleaves. C, Leaf samples from B were used forimmunoprecipitation with PP2C5-specific anti-bodies, and purified PP2C5 (top; western blotwith anti-PP2C5 from rabbit) was subjected to anin vitro phosphatase assay using [32P]phospho-casein (bottom). Relative phosphatase activity isgiven as release of 32P into the supernatant afterincubation with immunoprecipitated PP2C5.





to ABA. As the AP2C1 gene, which is a close PP2C5homolog described to negatively regulate MPK4 andMPK6 (Schweighofer et al., 2007), is also transcrip-tionally induced by ABA (Supplemental Fig. S1), wegenerated pp2c5 ap2c1 double knockout lines to ex-plore the possible functional redundancy of the twoMAPK phosphatases (Fig. 4B). In pp2c5 ap2c1 plants,ABA-induced MAPK activation was further enhancedcompared with single mutants and the wild-typeplants (Fig. 5), indicating that both phosphatases arefunctionally redundant. Probing parallel membraneswith antibodies raised against MPK3 or MPK6 indi-cated that the MPK protein levels remained unalteredwithin the tested time (data not shown), suggestingthat PP2C5 and AP2C1 affect the MPK activationprofile posttranslationally.

Similarly, after application of the biotic stimulusFlg22 to the leaves, pp2c5 and ap2c1 single mutantsresponded with a stronger MAPK activation com-pared with the wild-type control (Supplemental Fig.S4). The enhanced MAPK activation observed in thepp2c5 mutant could be reversed by complementationwith a genomic fragment of PP2C5 (Fig. 4B). However,in contrast to ABA treatment, MAPK activities were

Figure 2. PP2C5 colocalizes with stress-induced MAPKs predomi-nantly in the nucleus. PP2C5 (A) or other group B members (B) weretransiently coexpressed as CFP fusions in Arabidopsis protoplaststogether with MPK3-, MPK4-, or MPK6-YPF fusions. CFP/YFP signalswere visualized by fluorescence microscopy using different channels.

Figure 3. PP2C5 interacts with MPK3, MPK4, and MPK6 through itsKIM. A, For Y2H analysis, PP2C5 or point-mutated PP2C5-K90A/R91Q(PP2C5KR) was cloned into the vector pGBKT7 and used againstMPK3,MPK4, or MPK6 in the vector pGADT7. The positive control was the in-teraction between the SV40 large T-antigen and murine p53 (pGAD-T +pGBK-53). Interaction was tested using synthetic dropout medium notcontaining the amino acids Leu, Trp, and Ala (2LTA). Shown are serialdilutions (1, 1:10, 1:100, and 1:1,000) of the corresponding yeastculture. B, For BiFC analysis, PP2C5 and point-mutated PP2C5-K90A/R91Q were fused to the N-terminal part of YFP and assayed againstMPK3, MPK4, or MPK6 fused to the C-terminal half of YFP. Thesubcellular localization of the interaction is visualized by fluorescencemicroscopy. [See online article for color version of this figure.]

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not further increased in pp2c5 ap2c1 double knockoutplants after Flg22 treatment (Supplemental Fig. S4),indicating that depletion of either phosphatase issufficient to affect the activation of MAPKs duringbiotic interactions.Differences in ABA-inducedMAPK activation in the

pp2c5 ap2c1 double mutant became more apparentwhen endogenous kinase activities of MPK3, MPK4,and MPK6 were determined after ABA treatmentusing an immunocomplex kinase assay. EndogenousMAPKs were immunoprecipitated from protein ex-tracts of seedlings treated for 30 min with ABA withisoform-specific antibodies. As shown in Figure 6, inall lines except the pp2c5 ap2c1 double mutant, ABAtreatment resulted only in a weak induction of MPK3and MPK6 activities in comparison with the controlsamples. However, seedlings of the double mutant lineresponded to ABA treatment with a very strong in-

crease in MPK3 and MPK6 and additionally also inMPK4 activity, clearly suggesting that PP2C5 andAP2C1 act as redundant MAPK phosphatases. Inter-estingly, MPK6 activities in the control samples werealready increased in the pp2c5 and ap2c1 single and thedouble mutants compared with the wild-type seed-lings or the complemented line.

We next wanted to investigate the effect of PP2C5overexpression on MAPK activation. As we could notobtain any stably transformed PP2C5-overexpressingArabidopsis plants, we investigated MAPK activationin N. benthamiana leaves transiently expressing PP2C5

Figure 4. Identification of a PP2C5-T-DNA insertion line. A, A T-DNAinsertion line was identified from the SALK collection, with theinsertion located in the second exon (the asterisk indicates the stopcodon, thick lines indicate exons, and thin lines indicate introns). B,Expression of residual transcript in pp2c5 and ap2c1 T-DNA insertionlines, the double knockout line pp2c5 ap2c1, or the pp2c5 mutantcomplemented with the genomic fragment of PP2C5 (pp2c5/PP2C5)was analyzed by quantitative RT-PCR. Six-day-old seedlings treatedwith 1 mM Flg22 were harvested after 3 h, and quantitative PCR wasperformed 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-DNAinsertion was selected. Expression of EF-1awas used for normalization,and the Col-0 water control was set to 1. Data are averages 6 SE fromthree independent experiments performed each with 10 to 15 seedlingsper 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 andap2c1 single mutants, the pp2c5 ap2c1 double knockout line, and thepp2c5/PP2C5 complemented line were treated with 50 mM ABA, andMAPK activation was analyzed at the indicated time points. A, Proteinextracts (5 mg per lane) were subjected to western-blot analysis usingthe phospho-p44/p42 MAPK antibody. Arrowheads indicate the posi-tions of MPK3 andMPK6. B, The staining of the large subunit of Rubiscousing Ponceau S Red dye is used to estimate equal loading in each lane.Shown is one representative experiment out of three.





as a GFP fusion (Fig. 1B). Leaves were treated withABA or the flagellin-derived peptide Flg22 to triggerMAPK activation, and extracted proteins were sub-jected to an in gel kinase assay. Complementary to theenhanced MAPK activation observed in PP2C5- andAP2C1-depleted Arabidopsis leaves, N. benthamianaleaves expressing the PP2C5-GFP fusion protein werestrongly impaired in both ABA- and Flg22-inducedactivation of two major MAPKs with approximatesizes of 48 and 46 kD, respectively, presumably repre-senting salicylic acid-induced protein kinase (SIPK; anMPK6 ortholog) and wound-induced protein kinase(WIPK; an MPK3 ortholog; Fig. 7; Supplemental Fig.S5). These results strongly suggest that PP2C5 acts as anegative regulator of MAPK signaling, particularly ofMPK3 and MPK6.

PP2C5 Is Involved in a Subset ofABA-Mediated Responses

As PP2C5 gene expression is affected by ABAtreatment (Supplemental Fig. S1) and becauseMPK3, MPK4, and MPK6 have been implicated inABA signaling (Ichimura et al., 2000; Lu et al., 2002;Gudesblat et al., 2007; Xing et al., 2008), the effect ofPP2C5 depletion on typical ABA responses was in-vestigated. As ABA induces stomatal closure and as

MAPK activities are associated with stomatal move-ments (Lu et al., 2002; Gudesblat et al., 2007), wemeasured stomatal aperture in pp2c5 mutant plants.Compared with the wild type, leaves of both pp2c5and ap2c1 T-DNA insertion lines showed a slightly,but significantly, increased stomatal aperture undernormal growth conditions (Fig. 8), whereas stomatalaperture in the pp2c5 line complemented with agenomic PP2C5 fragment was indistinguishablefrom that measured in the wild type. This effect waseven more pronounced in the pp2c5 ap2c1 doubleknockout line, indicating that the two highly similarproteins are functionally partially redundant. How-ever, despite the increased stomatal opening in thepp2c5 and ap2c1 single and double mutant lines, theydid not show an increased water loss during droughtstress (Supplemental Fig. S6).

ABA is also an important hormone during pathogendefense (Mauch-Mani and Mauch, 2005; Adie et al.,2007; Fan et al., 2009), and pathogen-induced stomatalclosure is part of the plant innate immune response(Melotto et al., 2006). Therefore, we next examined theeffect of pp2c5 mutation on resistance to bacterialpathogens. When infected with the virulent bacteriumPseudomonas syringae pv tomato DC3000 (Pto DC3000),pp2c5, ap2c1 as well as pp2c5 ap2c1 plants did not showa significantly altered susceptibility (SupplementalFig. S7A). We also tested the coronatine-deficientstrain Pto DC3661, which is normally less pathogenicon Arabidopsis, as these bacteria cannot induce coro-natine-dependent reopening of stomata after theirpathogen-associated molecular pattern (PAMP)-trig-gered closure during infection (Melotto et al., 2006).Although pp2c5 and ap2c1 single mutants and the

Figure 6. ABA-induced activation of immunoprecipitated MPK3,MPK4, and MPK6 is enhanced in pp2c5 ap2c1 mutants. MPK3 (toppanels), MPK4 (middle panels), and MPK6 (bottom panels) activationwas measured in the wild type, pp2c5 and ap2c1 single mutants, thepp2c5 ap2c1 double mutant, and the pp2c5/PP2C5 complementedline in response to ABA. MAPKs were immunoprecipitated fromseedlings after treatment with 50 mM ABA for 30 min. The MAPKactivity was measured in immunocomplex kinase assays using myelinbasic protein as a substrate, and levels of MPK3, MPK4, and MPK6proteins were detected by western-blot analysis using isoform-specificantisera.

Figure 7. PP2C5-overexpressing leaves are impaired in ABA-stimu-lated MAPK activation. Constructs for 35S:GFP or 35S:PP2C5-GFPwere transiently expressed in N. benthamiana leaves as described inFigure 1B. After 2 d, leaves were treated without (control) or with 50 mM

ABA, and samples were harvested at the indicated time points. Proteinextracts were afterward subjected to an in gel kinase assay. A parallelgel was stained with Coomassie Brilliant Blue to confirm equal proteinamounts. Arrowheads indicate the positions of SIPK and WIPK. Shownis one out of two experiments with similar results.

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pp2c5 ap2c1 double knockout line exhibited an in-creased stomatal aperture, all mutants were still fullyresistant to Pto DC3661 infection (Supplemental Fig.S7B).Another well-established function of ABA is to

promote seed dormancy and to inhibit seed germina-tion (Christmann et al., 2006). When seeds of pp2c5mutants were sown on ABA-containing Murashigeand Skoog (MS) medium, the germination rate wassignificantly increased compared with wild-typeseeds, indicating that the pp2c5mutant displays partialinsensitivity toward ABA (Fig. 9A). The germinationrate of ap2c1 seeds was intermediate to that of thepp2c5 mutant and the wild type; however, seeds of thepp2c5 ap2c1 double knockout line showed an evenfurther increased ABA insensitivity.Moreover, gene expression of ABA-inducible genes

was investigated. As exemplified by the genes ABI1,ABI2, RD29A, and Erd10 (Yamaguchi-Shinozaki andShinozaki, 1993; Leung et al., 1997; Merlot et al., 2001),the application of ABA caused a transcriptional up-regulation in Col-0 wild-type plants and the pp2c5/PP2C5 complemented line. However, except for ABI1gene expression, which was only affected in the pp2c5ap2c1 double mutant, ABA-induced gene expressionfor the other genes was significantly diminished inboth the single and double mutant lines (Fig. 9B).Thus, the partial ABA-insensitive phenotype in thepp2c mutants observed in the germination assay isreflected in a partial ABA insensitivity with respect toABA-triggered gene expression.In summary, PP2C5 and AP2C1 appear to be in-

volved in stomatal opening, seed germination, andABA-regulated gene expression as functionally par-tially redundant MAPK phosphatases.

DISCUSSION

PP2C5 Is a MAPK Phosphatase

MAPKs function as key signal integration points fora vast number of external stimuli that affect the properfunctioning of cells. Hence, they must be subject torigorous regulation to control appropriate intensityand timing of their activation. Our results identifyPP2C5 as an Arabidopsis PP2C of the B subgroup thatcan act as MAPK phosphatase. PP2C5 not only coloc-alizes with the stress-induced MAPKs MPK3, MPK4,andMPK6 (Fig. 2) but also directly interacts with thoseMAPKs via its KIM (Fig. 3). The fact that PP2C5 andthe MAPKs form complexes mainly in the nucleus(Fig. 3B) is consistent with the proposed shuffling ofthe MAPKs into the nucleus upon stress (Ahlfors et al.,

Figure 8. PP2C5 knockout lines show an increased stomatal aperture.Wild-type Arabidopsis Col-0, knockout lines pp2c5 and ap2c1, thedouble knockout line pp2c5 ap2c1, and the complemented pp2C5 linepp2c5/PP2C5 were grown on soil, and stomatal apertures on abaxialepidermal imprints were determined by light microscopy. Data pre-sented are means of at least 100 stomata, and the error bars represent SE.Significant differences (** P , 0.001, *** P , 0.0001) compared withthe wild-type control were determined using Student’s t test. The entireexperiment was repeated three times with similar results.

Figure 9. PP2C mutants display altered ABA responses. A, Seeds ofwild-type Arabidopsis Col-0 and knockout lines pp2c5, ap2c1, pp2c5ap2c1, and pp2c5/PP2C5 were germinated on medium supplementedwith the indicated concentrations of ABA. Seedlings were scored forradicle emergence at day 3. The number of germinated seeds wasexpressed as the percentage of the total number of seeds plated (n .45). Shown is the average of three independent experiments, and errorbars indicate SE. B, Induction ratio of ABI1, ABI2, RD29A, and Erd10genes in pp2c mutant lines compared with the wild type. Arabidopsisseeds were germinated on half-strength MS medium. Two days aftergermination, 40 seedlings per line were transferred to sterile watersupplemented with or without 50 mM ABA and harvested after incuba-tion for a further 24 h. Data are means 6 SE from three independentexperiments. For each genotype, the fold changes in gene expressionobtained in real-time quantitative PCR analyses are shown relative tothe respective water control and EF-1a amplification was used tonormalize data.





2004; Lee et al., 2004) and with the involvementparticularly of MPK6 in the regulation of stress-induced gene expression (Asai et al., 2002). Moreover,the activation of MAPKs is affected by PP2C5 expres-sion, and we observed stronger stress activation ofMPK3, MPK4, and MPK6 in Arabidopsis when PP2C5was depleted (Figs. 5 and 6; Supplemental Fig. S4). Inagreement with that, ectopic expression of PP2C5-GFPin Nicotiana tabacum abolished ABA- and elicitation-induced activation of SIPK andWIPK, the orthologs ofMPK6 and MPK3, respectively (Fig. 7; SupplementalFig. S5). These results suggest that PP2C5 is a bona fideprotein phosphatase that attenuates MAPK activationby regulating the strength and duration of MAPKactivation. Another indirect piece of evidence forPP2C5 being a MAPK phosphatase is the observationthat cotransfection of PP2C5 with MPK6 and theMPK6 substrate ERF104 abolished MPK6-mediatedERF104 phosphorylation (Bethke et al., 2009).

Notably, PP2C5 is not the only phosphatase regu-lating MPK3, MPK4, and MPK6. Arabidopsis AP2C1,another clade B member and a close homolog ofPP2C5, was also recently shown to interact with andinactivate MPK4 and MPK6 (Schweighofer et al.,2007). In addition to PP2Cs, dual-specificity phospha-tases (DsPTPs) are thought to dephosphorylate andthereby regulate MAPKs (Martin et al., 2005). The classof DsPTPs, which can remove both phosphates in theTXY activation motif of MAPKs, were regarded for along time as classical MAPK phosphatases in yeastand mammals (Keyse, 1998; Camps et al., 2000). TheArabidopsis genome encodes five potential DsPTPs,MKP1, MKP2, DsPTP1, PHS1, and IBR5 (Kerk et al.,2002). The first of those phosphatases identified inplants was DsPTP1, which can dephosphorylate andthereby inactivate stress-induced MPK4 (Gupta et al.,1998). MPK4, MPK3, and particularly MPK6 alsointeract with MKP1, which was demonstrated to beinvolved in signal transduction during genotoxic andsalt stress in Arabidopsis (Ulm et al., 2001, 2002).Additionally, during the oxidative stress response,MPK3 and MPK6 can be inactivated by a third DsPTP,MKP2 (Lee and Ellis, 2007).

In summary, MAPK cascades are involved in mul-tiple cellular signaling pathways; thus, it is not sur-prising that plants have developed a highly complexnetwork to fine-tune cellular responses to internal andexternal stimuli. Our results demonstrate that PP2C5is one of the corresponding phosphatases regulatingMAPK activity during cellular adaptation to differentstress responses.

PP2C5 and AP2C1 Are Required for ABA Signaling

ABA is a universal stress hormone in higher plantsthat also plays a major role in various aspects of plantgrowth and development (Koornneef et al., 1998;Mauch-Mani and Mauch, 2005; Christmann et al.,2006). We show here that PP2C5 and AP2C1 as mem-bers of clade B of PP2Cs are also involved in different

ABA responses apart from the well-characterized cladeA PP2Cs. Stomatal aperture of leaves of unstressedpp2c5 and ap2c1 mutants is slightly but significantlyincreased compared with the wild type (Fig. 8). Nota-bly, pp2c5 ap2c1 double mutants showed an even morepronounced stomatal aperture, indicating that PP2C5and AP2C1 have partially redundant functions in sto-mata regulation. ABA generally promotes stomatalclosure and inhibits stomatal opening (Koornneefet al., 1998). Based on our observations (SupplementalFigs. S6 and S7), we postulate that PP2C5 and AP2C1affect ABA-mediated stomatal opening but are not themajor regulators involved in the stomatal closure con-trol. First, ABA normally causes stomatal closure inresponse to drought stress, and the abi1 and abi2 mu-tants show an increased water loss and a wilty pheno-type under water stress conditions due to failure toclose their stomata (Roelfsema and Prins, 1995; Leunget al., 1997). However, although stomatal aperture wasincreased in pp2c5, ap2c1, and pp2c5 ap2c1 mutantplants, their response to drought stress was not signif-icantly altered compared with wild-type plants (Sup-plemental Fig. S6). Second, ABA-mediated stomatalclosure is part of plant innate immunity against bacte-rial pathogens and is triggered by surface-exposedPAMPs. During infection, stomatal closure is reversedby the jasmonic acid mimic coronatine; hence, Ptostrains depleted of coronatine are normally less infec-tious. In accordance, plants defective in ABA biosyn-thesis, such as the aba3 mutant, are not able to closetheir stomata (Leon-Kloosterziel et al., 1996) and aremore susceptible to coronatine-deficient Pto bacteria(Melotto et al., 2006). However, pp2c5, ap2c1, and pp2c5ap2c1 plants were still fully resistant to the less virulentcoronatine-deficient strain Pto DC3661 and showed anormal response to infection with virulent Pto DC3000bacteria (Supplemental Fig. S7). Therefore, our resultsindicate that ABA-induced stomatal closure, triggeredfor instance by drought stress or PAMP recognition, isnot affected by PP2C5 and AP2C1 depletion.

Additionally, the seed germination rate that isnormally decreased by ABA treatment was signifi-cantly increased in pp2c5 mutants compared with thewild-type control or the pp2c5/PP2C5 complementedline (Fig. 9A). Again, PP2C5 and AP2C1 seem to haveredundant functions, as the double mutant showed aneven more pronounced ABA insensitivity during seedgermination. This effect was also observed with re-spect to ABA-inducible gene expression. Transcrip-tional activation of the ABA-responsive genes ABI1,ABI2, RD29A, and Erd10 was strongly reduced afterexternal ABA application in the pp2c single and doublemutant plants compared with the wild type (Fig. 9B).Hence, PP2C5 and AP2C1 are involved in variousABA-mediated cellular responses.

PP2C5 Is a Positive Regulator of ABA Signaling

So far, PP2Cs have only been described as negativeregulators of ABA signaling and loss-of-function mu-

Brock et al.




tations in ABI1, ABI2, HAB1, and AtPP2CA renderthe plants ABA hypersensitive (Gosti et al., 1999;Tahtiharju and Palva, 2001; Saez et al., 2004). Althoughthe abi1-1 mutant was described as being ABA insen-sitive (Koornneef et al., 1984), this initially isolatedmutation had a dominant effect, and follow-up reportsshowed that loss of ABI1 PP2C activity leads to anenhanced responsiveness to ABA (Gosti et al., 1999).Thus, the wild-type ABI1 phosphatase, and most likelyalso ABI2 as close ABI1 homolog, are negative regula-tors of ABA responses. Likewise, antisense inhibition ofAtPP2CA as well as depletion of HAB1 resulted in anABA-hypersensitive inhibition of seed germination(Tahtiharju and Palva, 2001; Saez et al., 2004), suggest-ing that also AtPP2CA and HAB1 function as negativeregulators in ABA signaling. Beside PP2Cs, DsPTPshave also been demonstrated to regulate ABA signal-ing. Whereas the phs1mutant reacts hypersensitively toABA during seed germination (Quettier et al., 2006),ibr5mutants showed a reduced responsiveness to auxinand ABA (Monroe-Augustus et al., 2003). Hence, PHS1can be regarded as a negative regulator of ABA re-sponses in contrast to IBR5, which seems to act as apositive regulator of ABA signaling. However, the onlyknown IBR5-interacting protein is MPK12, which itselfdoes not seem to be involved in ABA signaling (Leeet al., 2009), suggesting that although IBR5 might play arole in ABA signaling, this is most likely not mediatedvia a MAPK cascade.Our results show that PP2C5 acts as regulator of the

major stress-induced MAPKs MPK3, MPK4, andMPK6 and that depletion of PP2C5 function causes apartial ABA insensitivity with regard to seed germi-nation and ABA-induced gene expression (Fig. 9).Thus, PP2C5 and also its close homolog AP2C1 act aspositive regulators that are required for full ABAresponsiveness.

Does PP2C5 Regulate ABA Signaling via Actionon MAPKs?

Although MAPKs are involved in ABA signalingand several phosphatases have been described toregulate MPK3, MPK4, and MPK6, little is knownabout a direct link between phosphatase-mediatedMAPK regulation and the ABA response. One prom-inent example is ABI1, a PP2C of the A subgroup thatattenuates ABA-dependent stress signaling and thatwas also shown to directly bind to MPK6, therebyinhibiting its kinase activity (Leung et al., 2006). An-other putative MAPK regulator during ABA signalingis PHS1, one of the five Arabidopsis DsPTPs. Loss-of-function mutants of PHS1 displayed impaired micro-tubule organization (Naoi and Hashimoto, 2004) andABA hypersensitivity (Quettier et al., 2006). Recently,it has been demonstrated that PHS1 physically inter-acts with MPK18, and mpk18 seedlings have defects inmicrotubule-related functions (Walia et al., 2009).However, an involvement of MPK18 in ABA responseshas so far not been shown. A second DsPTP implicated

in ABA signaling is IBR5, which was initially reportedto confer reduced sensitivity to auxin and ABA inArabidopsis roots (Monroe-Augustus et al., 2003).However, IBR5 only interacts with MPK12, and re-duced expression of the MPK12 gene resulted in rootgrowth that is hypersensitive to exogenous auxins butshows normal ABA sensitivity (Lee et al., 2009).Therefore, it is still elusive if PHS1 and IBR5 regulateMAPKs during ABA signaling.

We show here that PP2C5 is a negative regulator ofMPK3, MPK4, andMPK6, three MAPKs implicated inthe signaling pathways mediating the ABA response(Lu et al., 2002; Gudesblat et al., 2007; Xing et al.,2008). ABA has been shown to activate MPK3, and theactivated kinase is thought to phosphorylate thetranscription factor ABI5 (Lu et al., 2002). Constitu-tive expression of MPK3 in transgenic plants resultedin no visible phenotype but hypersensitivity to exog-enous ABA (Lu et al., 2002). Interestingly, antisenseplants with reduced levels of MPK3 mRNA in theguard cells displayed partial insensitivity to ABA ininhibition of stomatal opening but responded nor-mally to this hormone in stomatal closure (Gudesblatet al., 2007). Likewise, MPK6 overexpression led toenhanced ABA responses, while thempk6mutant wasimpaired in ABA signaling (Xing et al., 2008). Theseresults indicate that a depletion of the correspondingMKP(s) as negative regulators should lead to anenhanced signaling through those MAPKs and henceshould result in ABA hypersensitivity similar to thatobserved for MAPK overexpression. Unexpectedly,depletion of PP2C5 and AP2C1 resulted in a partialABA insensitivity phenotype. As there are no dataavailable for the involvement of MPK4 in the ABAresponse, apart from the fact that MPK4 enzymeactivity is ABA inducible (Xing et al., 2008), wecannot rule out that PP2C5 and/or AP2C1 act spe-cifically on MPK4 during ABA signaling. Moreover,PP2C5 gene expression is induced by various otherphytohormones apart from ABA, such as ethyleneand GA3 (data not shown). However, we could notobserve alterations in the cellular response to ethyl-ene or GA3 in pp2c5, ap2c1, or pp2c5 ap2c1 mutantplants when compared with the wild type (data notshown). As multiple other kinases also have beendemonstrated to be involved in ABA signaling, suchas calcium-dependent protein kinases (Mori et al.,2006; Zhu et al., 2007), SNF1-related protein kinases(Fujii et al., 2007; Jossier et al., 2009; Umezawa et al.,2009), or the LRR kinase RPK1 (Osakabe et al., 2005),our future work will now address if PP2C5 solely actson MAPKs or if other kinases can be targeted byPP2C5 and/or AP2C1.

In summary, we identified PP2C5 and its close ho-molog AP2C1 as functionally partially redundant pro-tein phosphatases that positively regulate ABA-inducedgene expression, ABA-mediated seed dormancy, andstomatal closure. It remains to be demonstrated ifPP2C5 action on MAPKs can be functionally linked toits role in ABA signaling.





MATERIALS AND METHODS

Plant Material and Growth Conditions

Arabidopsis (Arabidopsis thaliana Col-0) plants were routinely grown on a

1:3 vermiculite:soil mixture for 5 to 6 weeks in a phytochamber (8-h photo-

period, 22�C, 40%–60% humidity). For in vitro culture, seeds were surface

sterilized by overnight treatment with chlorine gas (released from amixture of

100 mL of 12% commercial bleach and 3 mL of 37% hydrochloric acid) in a

desiccator. Then, seeds were sown on half-strength MS medium (Duchefa)

containing 0.8% (w/v) Select agar (Sigma) and cultivated in long-day condi-

tions (16-h photoperiod). Stratification of the seeds was conducted in the dark

at 4�C for 2 d. Arabidopsis cells were propagated as described (Dettmer et al.,

2006). Nicotiana benthamiana plants were cultivated in the greenhouse with a

photoperiod of 14 h, 60% humidity, and a temperature of 25�C during the day

and 19�C at night. Seeds for the T-DNA insertion lines for PP2C5

(SALK_109986) and AP2C1 (SALK_065126) were obtained from the SALK

Institute collection (Alonso et al., 2003), and individual plants with a single

T-DNA insertion were selected after Southern-blot analysis (Supplemental

Fig. S8). To generate the pp2c5 ap2c1 double mutant, ap2c1 homozygous

plants were crossed with the homozygous pp2c5 mutant. Plants were further

self-fertilized, and pp2c5 ap2c1 double homozygous seedlings were then used

for experiments in the F3 generation.

Plant Transformation and Protein Localization Studies

For GFP fusions driven by the cauliflower mosaic virus 35S promoter, the

1,493-bp genomic PP2C5 fragment was cloned into vector pK7FWG2.0

(Karimi et al., 2005) via the Gateway recombination system (Invitrogen). For

plasmid PP2C5:PP2C5-GFP, the 35S promoter was replaced by a 1,502-bp

PP2C5 promoter fragment. As GFP control, the vector pK7WGF2.0 (Karimi

et al., 2005) was used. For colocalization experiments, fusion constructs were

generated in vectors pEXSG-YFP (for MPK6), pENSG-YFP (for MPK3 and

MPK4), and pEXSG-CFP (for PP2Cs) as described (Bethke et al., 2009).

Transient protein expression in Arabidopsis protoplasts of ecotype Col-0

and N. benthamiana leaves was performed as described (Ludwig et al., 2005;

Dettmer et al., 2006). Localization studies of the PP2C5-GFP fusion protein and

GFP as a control were carried out 15 to 18 h after transformation using a

confocal laser-scanning microscope as described elsewhere (Diepold et al.,

2007). For complementation experiments, the PP2C5:PP2C5-GFP construct in

pK7FWG2.0 was stably transformed into pp2c5 plants by the floral dip

procedure (Clough and Bent, 1998). Restored gene transcription in trans-

formed seedlings was verified by reverse transcription (RT)-PCR, and plants

were used for experiments in the F3 generation.

Transcript Analysis

Total RNA from seedlings was isolated using the Tri Reagent method

according to the manufacturer’s recommendations (Sigma). First-strand

cDNA was synthesized from 1 mg of total RNA using RevertAid Moloney

murine leukemia virus reverse transcriptase (Fermentas). RT-quantitative

PCR amplifications and measurements were performed with the iQ5 Multi-

colour Real Time PCR detection system (Bio-Rad). RT-quantitative PCR

amplifications were monitored using the ABsolute SYBR Green Fluorescein

Mix (Thermo Scientific). Relative quantification of gene expression data was

performed using the 2–DDCT method (Livak and Schmittgen, 2001). Expression

levels were normalized using the threshold cycle values obtained for the

EF-1a gene. The presence of a single PCR product was further verified by

dissociation analysis in all amplifications. All quantifications were made in

duplicate on RNA samples obtained from three independent experiments.

The sequences of the primers used for PCR amplifications are indicated in

Supplemental Table S1.

Microarray data were obtained from the AtGenExpress initiative (http://

www.arabidopsis.org/info/expression/ATGenExpress.jsp) and were ana-

lyzed using the digital northern tool of the Genevestigator program (http://

www.genevestigator.ethz.ch).

Enzymatic Activity Assays

For phosphatase assays, PP2C5-GFP was transiently expressed in N.

benthamiana leaves and immunoprecipitated with rabbit anti-PP2C5 antibody

from crude protein extracts as described (Romeis et al., 2001). Phosphatase

activity was measured using 32P-labeled casein (11.2 mg per reaction; Wang

et al., 1999) as artificial substrate and incubated with immunoprecipitated

PP2C5-GFP at 25�C for 30 min according to the protocol described previously

(Stone et al., 1994).

MAPK activities were determined in crude protein extracts from 6-d-old

seedlings prepared as described (Romeis et al., 1999; Ludwig et al., 2005). Five-

microgram protein crude extracts were separated on a 10.5% SDS gel, and the

proteins were transferred onto nitrocellulose (Amersham) by wet electro-

blotting (Mini-Protean II system; Bio-Rad). Equal loading of protein was

confirmed by Ponceau S Red staining of the membrane, and the membranes

were subsequently subjected to western-blot analysis using the phospho-p44/

p42 MAPK antibody according to the manufacturer’s protocol (Cell Signaling

Technology). Alternatively, MAPK activity was determined by in-gel kinase

assays with myelin basic protein (Sigma) as artificial substrate as described

previously (Romeis et al., 1999).

For immunocomplex kinase assays, 30 mg of total proteins from seedling

extracts prepared as described above was incubated overnight at 4�C with 10

mL of isoform-specific MPK antibody. A total of 50 mL of protein G-Sepharose

(GEHealthcare) was added and incubated for 2 h at 4�C. The protein-antibodycomplex on the beads was collected and washed three times in ice-cold

protein extraction buffer and finally washed with kinase buffer (50 mM Tris,

pH 7.5, 1 mM dithiothreitol, 10 mMMgCl2, and 50 mMATP). Kinase reactions on

the immunoprecipitated MPK3, MPK4, andMPK6 were performed for 30 min

at room temperature in 20 mL of kinase buffer containing 5 mg of myelin basic

protein and 2 mCi of [g-32P]ATP. The reaction was stopped by adding SDS-

PAGE loading buffer, and the phosphorylation of myelin basic protein was

analyzed by autoradiography after SDS-PAGE.

Antibody Generation, Immunoprecipitation, andWestern-Blot Analysis

PP2C5-specific antisera were generated in rabbits via immunizations

with the N-terminal peptide N-MQLSKNPIKQTRNRE coupled to keyhole

limpet hemocyanin (Eurogentec). For MPK3- and MPK6-specific rabbit

antisera, the N-terminal peptides N-MNTGGGQYTDFPAVDTHGG and

N-MDGGSGQPAADTEMT, respectively, were used (Ahlfors et al., 2004). All

antisera were affinity purified on the corresponding immobilized oligopep-

tide used to generate the serum. The MPK4 antibody was obtained from

Sigma. Immunoprecipitation of PP2C5-GFP and western-blot analysis were

performed as described (Romeis et al., 2001). MPK antibodies were used for

western-blot analysis in a 1:5,000 dilution.

Protein Interaction Studies

Amino acid substitutions K90A and R91Q in the KIM of PP2C5 were

introduced by PCR-based, site-specific mutagenesis. Yeast two-hybrid exper-

iments were performed using the Matchmaker System (Clontech). PP2C5

cDNA (1,173 bp) or mutated PP2C5-K90A/R91Qwas cloned into pGBKT7, and

MPK3, MPK4, and MPK6 were cloned into pGADT7 (Clontech). Plasmids

were transformed into yeast strain AH109 using a lithium acetate/single-

stranded carrier DNA/polyethylene glycol method (Gietz and Woods, 2002).

After 4 to 5 d of growth on vector-selective medium (CSM-LT), 12 indepen-

dent clones in pools of four clones each were propagated in liquid vector-

selective medium and subsequently diluted to the same optical density. Of the

three pooled cultures, 7.5 mL of a serial dilution was dropped on vector- and

interaction-selective medium (CSM-LTA) and incubated at 28�C. At day 3, the

growth of the clones was monitored.

For BiFC analysis, PP2C5 and point-mutated PP2C5-K90A/R91Q were

cloned into vector pUCSpyNe (fusing PP2C5 C terminal to the N-terminal half

of YFP) and MPK3, MPK4, and MPK6 were cloned into vector pUCSpyCe

(fusing MPKs C terminal to the C-terminal half of YFP). pUCSpyNe and

pUCSpyCe vectors expressing split YFP domains alone were used as controls

(Walter et al., 2004). The subcellular localization of the interaction was

visualized by fluorescence microscopy using a confocal laser-scanning mi-

croscope (TCS SP2; Leica [http://www.leica.com/]).

Stomatal Aperture and Germination Assay

To determine stomatal aperture, plants were grown without lid with

constantly moist soil. Two leaves per line were harvested, and the lower leaf

side was thinly covered with glue (Uhu hart). The dried glue was removed

after several minutes, and abaxial epidermal imprints were analyzed with the

Brock et al.




microscope. Stomatal aperture of approximately 100 stomata per sample was

measured using Scion Image Software, and data sets were analyzed with

Student’s t test.

The germination rate was determined from seeds that were surface

sterilized and plated on half-strength MS medium supplemented with dif-

ferent ABA concentrations. After sowing, plates were chilled for 3 d at 4�C in

darkness and subsequently incubated for 3 d at 22�Cwith an 8-h photoperiod.

Emerging root tips were scored with the light microscope. Seeds of batches of

exactly the same age were used for one experiment, but batches varied from

one experiment to the next.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Cladogram and gene expression data for clade A

and B PP2Cs.

Supplemental Figure S2. Generation of PP2C5-specific antibodies.

Supplemental Figure S3. Localization of clade B-PP2Cs.

Supplemental Figure S4. pp2c mutant plants show an enhanced Flg22-

induced MAPK activation.

Supplemental Figure S5. PP2C5-overexpressing leaves are impaired in

Flg22-induced MAPK activation.

Supplemental Figure S6. The PP2C5-T-DNA insertion line displays a

normal drought stress response.

Supplemental Figure S7. pp2c5 plants are not affected in their resistance to

bacterial infection.

Supplemental Figure S8. Southern-blot analysis of pp2c5 and ap2c1 mu-

tants.

Supplemental Table S1. List of primers used for quantitative RT-PCR

analysis.

Supplemental Materials and Methods S1.

ACKNOWLEDGMENTS

We are grateful to Klaus Harter for providing the BiFC vectors and Georg

Felix for Flg22 peptide. The Nottingham Arabidopsis Stock Centre is ac-

knowledged for providing mutant seeds. We thank Caterina Brancato for

technical assistance in protoplast preparation and transformation.

Received March 10, 2010; accepted May 18, 2010; published May 20, 2010.

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