MAP kinases MPK9 and MPK12 are preferentiallyexpressed in guard cells and positively regulateROS-mediated ABA signalingFabien Jammesa,1, Charlotte Songa,1,2, Dongjin Shina,1,3, Shintaro Munemasab, Kouji Takedaa, Dan Gua, Daeshik Choa,Sangmee Leea, Roberta Giordoa,c, Somrudee Sritubtimd,4, Nathalie Leonhardte, Brian E. Ellisd, Yoshiyuki Muratab,and June M. Kwaka,5

aDepartment of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742; bGraduate School of Natural Science and Technology,Okayama University, Okayama 700-8530, Japan; cDepartment of Botany and Plant Ecology, University of Sassari, 07100 Sassari, Italy; dMichael SmithLaboratories, University of British Columbia, Vancouver, BC, Canada V6T 1Z4; and eLaboratoire des Echanges Membranaires et Signalisation, Unite Mixte deRecherche 6191, Centre National de la Recherche Scientifique-Commissariat a l’Energie Atomique-Universite Aix-Marseille II, Commissariat a l’EnergieAtomique Cadarache Bat 156, 13108 St Paul-lez-Durance, France

Edited by Maarten J. Chrispeels, University of California at San Diego, La Jolla, CA, and approved October 6, 2009 (received for review June 30, 2009)

Reactive oxygen species (ROS) mediate abscisic acid (ABA) signal-ing in guard cells. To dissect guard cell ABA-ROS signaling genet-ically, a cell type-specific functional genomics approach was usedto identify 2 MAPK genes, MPK9 and MPK12, which are preferen-tially and highly expressed in guard cells. To provide geneticevidence for their function, Arabidopsis single and double TILLINGmutants that carry deleterious point mutations in these geneswere isolated. RNAi-based gene-silencing plant lines, in which bothgenes are silenced simultaneously, were generated also. Mutantscarrying a mutation in only 1 of these genes did not show anyaltered phenotype, indicating functional redundancy in thesegenes. ABA-induced stomatal closure was strongly impaired in 2independent RNAi lines in which both MPK9 and MPK12 transcriptswere significantly silenced. Consistent with this result, mpk9-1/12-1 double mutants showed an enhanced transpirational waterloss and ABA- and H2O2-insensitive stomatal response. Further-more, ABA and calcium failed to activate anion channels in guardcells of mpk9-1/12-1, indicating that these 2 MPKs act upstream ofanion channels in guard cell ABA signaling. An MPK12-YFP fusionconstruct rescued the ABA-insensitive stomatal response pheno-type of mpk9-1/12-1, demonstrating that the phenotype wascaused by the mutations. The MPK12 protein is localized in thecytosol and the nucleus, and ABA and H2O2 treatments enhance theprotein kinase activity of MPK12. Together, these results providegenetic evidence that MPK9 and MPK12 function downstream ofROS to regulate guard cell ABA signaling positively.

abscisic acid � anion channels � protein kinase � reactive oxygen species �stomata

The phytohormone abscisic acid (ABA) regulates diversecellular processes and transduces environmental signals to

protect plants from abiotic stresses (1, 2). Among the identifiedmolecular elements working in ABA signaling are proteinkinases and phosphatases that play a central role in regulatingthe signaling network (3). It is noteworthy that only a fewrecessive mutations in Arabidopsis protein kinase and phospha-tase genes have been identified that act as positive regulators ofguard cell ABA signaling. These genes include Ca2�-dependentprotein kinases (CPK3, CPK6, CPK4, CPK11), a protein phos-phatase 2A regulatory subunit A (RCN1), a receptor-like proteinkinase (RPK1), a Ser/Thr protein kinase (OST1), and MAPKcascade genes (MKK1, MPK6) (4–8).

Reactive oxygen species (ROS) were shown previously toinduce increases in cytosolic Ca2� and stomatal closure (9). ROSactivate hyperpolarization-activated plasma membrane Ca2�-permeable channels in guard cells of Vicia and Arabidopsis (10,11). ABA also increases H2O2 levels in Vicia guard cells inadvance of stomatal closure (12). The endogenous source of

guard cell ROS has been explored through a combined molec-ular genetic and functional genomics approach, which revealedthat the 2 guard cell-expressed AtrbohF and AtrbohD NADPHoxidases, among the 10 NADPH oxidases in the Arabidopsisgenome, are responsible for ABA-induced ROS production andsubsequent ABA signaling in guard cells (13).

ABA was shown to induce MAPK activation in barley aleu-rone layers (14), and a possible MAPK activity was observed inVicia guard cell protoplasts (15). Furthermore, a study with peaepidermal peels showed that the MAPKK inhibitor PD98059inhibits ABA-induced stomatal closure and expression of anABA-inducible dehydrin gene (16, 17). Despite these studiesindicating that MAPK cascades function in ABA signaling, itremains to be established which specific MAPKs, MAPKKs, andMAPKK kinases (MAPKKKs) form a complete cascade tomediate ABA signaling in guard cells. The large number of genesin the plant MAPK, MAPKK, and MAPKKK families (18)potentially confers a high level of genetic redundancy withinsignal transduction mechanisms, thereby hampering conven-tional genetics. Nevertheless, complete MAPK cascades thatfunction in plant innate immunity and stomatal developmenthave been established (19–21). Interestingly, all these identifiedMAPK cascades share MPK6 and/or MPK3.

Here, we show that 2 other members of the MPK family, MPK9and MPK12, are preferentially expressed in guard cells, sharefunctional redundancy, and function as positive regulators down-stream of ROS in guard cell ABA signaling.

ResultsGuard Cell-Preferential Expression of MPK9 and MPK12. To testwhether MAPK cascades function in Arabidopsis guard cell ABAsignaling, we first examined ABA-induced stomatal movements

Author contributions: F.J., C.S., D.S., S.M., Y.M., and J.M.K. designed research; F.J., C.S., D.S.,S.M., K.T., D.G., D.C., S.L., N.L., and J.M.K. performed research; R.G., S.S., and B.E.E.contributed new reagents/analytic tools; F.J., C.S., D.S., S.M., K.T., D.G., S.L., Y.M., and J.M.K.analyzed data; and F.J., C.S., D.S., B.E.E., and J.M.K. 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.

1F.J., C.S., and D.S. contributed equally to this work.

2Present address: Dupont Experimental Station, Henry Clay and Rt.141, Wilmington, DE19880.

3Present address: Bio-crop development division, National Academy of AgriculturalScience, Suwon, Korea.

4Present address: Faculty of Science, Udon Thani Rajabhat University, Udon Thani, Thailand.

5To whom correspondence should be addressed. E-mail: jkwak@umd.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0905139106/DCSupplemental.

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in the presence and absence of the MAPKK inhibitor PD98059.Both ABA- and H2O2-induced stomatal closure in Arabidopsiswere significantly inhibited by PD98059, indicating that MAPKcascades function downstream of ROS in ABA signaling inArabidopsis guard cells (Fig. S1).

We then asked whether any genes encoding MAPK cascadecomponents are specifically expressed in guard cells, a findingthat could lead to identification of MAPK cascades required forguard cell ABA signaling. For this purpose, we analyzed ATH1microarray-derived data in which guard cell and mesophyll cellRNA had been compared (22). This analysis revealed that 2MAPK genes, MPK9 and MPK12, are highly and preferentiallyexpressed in guard cells relative to mesophyll cells (Fig. 1A),suggesting that MPK9 and MPK12 might function specifically inguard cell signal transduction and/or development.

To confirm the microarray results, RT-PCR was performedwith guard cell and mesophyll cell cDNA that was synthesizedfrom independently prepared guard cell and mesophyll cellprotoplast RNA. RT-PCR verified that both MPK9 and MPK12are highly and preferentially expressed in guard cells relative tomesophyll cells (Fig. 1B). The pattern of expression of the guardcell marker gene hydroxyproline-rich protein (HPRP) and themesophyll cell marker gene calmodulin-binding protein (CBP)demonstrated that the guard cell and mesophyll cell RNApreparations were free of significant cross-contamination. To

test further the specificity of MPK9 and MPK12 expression, weexamined their expression patterns in other tissues, as compiledin the Genevestigator database (23). Because guard cells arelocated in many plant organs, we were particularly interested inMPK9 and MPK12 expression levels in roots, seeds, root tips, andsuspension cells, all of which are devoid of guard cells. We alsocompared expression levels and patterns of MPK9 and MPK12with other guard cell-preferential genes. Within this set, MPK12and pGC1 are the genes showing the highest expression level inguard cells (Fig. S2).

To investigate further the spatial expression of MPK12, re-porter gene analyses were performed. Fig. 1 C–H shows that thepromoter of MPK12 drove strong �-glucuronidase (GUS) andGFP expression in guard cells in cotyledons, hypocotyls, 3-week-old expanded leaves, sepals, stigma, and anthers. The MPK12promoter had been shown earlier to drive low levels of GUSexpression in roots (24).

Deleterious Mutations in MPK9 and MPK12 Impair Stomatal Behaviorin Response to ABA and H2O2. We initially looked for knockoutmutants for these genes to test genetically whether these guardcell MAPK genes function in ROS-mediated ABA signaling.Despite a large effort, we failed to identify homozygous knock-out mutants for these genes. An independent effort also failedto isolate homozygous knockout mutants for MPK12 (24).Alternatively, we searched for TILLING mutants (25) and found2 independent mutants for each of the genes. Each mutantcontained a point mutation (Fig. S3), and PCR-based genotyp-ing and restriction enzyme digestions of the PCR productsenabled us to identify 3 homozygous TILLING mutant lines(mpk9-1, mpk12-1, and mpk12-2). We then examined the sto-matal movement phenotype of these homozygous mpk mutants.As shown in Fig. 2A, none of the single mutants showed anyaltered stomatal movement in response to ABA, as comparedwith WT (P � 0.55 for mpk9-1; P � 0.82 for mpk12-1 andmpk12-2). This result suggested 2 possible scenarios: (i) MPK9and MPK12 do not function in guard cell ABA signaling, or (ii)there is functional redundancy in these 2 genes. We thereforegenerated mpk9-1/12-1 double mutants and analyzed their sto-matal response to ABA. In contrast to the single mutants,mpk9-1/12-1 double mutants showed very strong insensitivity inABA-induced stomatal closure at both 3 �M and 10 �M ABAcompared with WT (Fig. 2B; P � 10�5), indicating that MPK9and MPK12 are positive regulators of guard cell ABA signalingand share functional redundancy. The mpk9-1/12-1 double mu-tants also were significantly impaired in ABA inhibition ofstomatal opening in comparison with WT (Fig. 2C; P � 10�5 atboth 10 and 20 �M ABA).

To test whether MPK9 and MPK12 are involved in other guardcell signaling pathways, we examined stomatal responses ofmpk9–1/12–1 mutants to dark and cold treatment. Fig. S4 showsthat mpk9–1/12–1 mutants are partially impaired in cold-induced stomatal closure but not in dark-induced stomatalclosure, suggesting that MPK9 and MPK12 may function in a coldsignaling cascade.

We next asked whether MPK9 and MPK12 act downstream ofROS in guard cell ABA signaling. Compared with WT plants,mpk9-1/12-1 double mutants were significantly impaired inH2O2-induced stomatal closure (Fig. 2D; P � 0.04 at 250 �MH2O2 and P � 0.01 at 500 �M H2O2). Taken together with thepharmacological data (Fig. S1), these results show that MPK9and MPK12 function positively downstream of ROS in ABAsignaling in guard cells.

Expression of MPK12-YFP in mpk9-1/12-1 Double Mutants Rescues theABA-Insensitive Stomatal Response, and Simultaneous RNAi Suppres-sion of MPK9 and MPK12 Phenocopies mpk9-1/12-1. To providedirect genetic evidence that the mutations in MPK9 and MPK12

Fig. 1. Expression analyses reveal that MPK9 and MPK12 are highly andpreferentially expressed in guard cells. (A) Microarray results from Arabidopsiswhole-genome chip ATH1 show that a few MAPK genes are highly expressedin guard cells. The guard cell-specific KAT1 gene is shown as a positive controland for comparison. Expression levels of each gene were normalized toubiquitin-conjugating enzyme E2 (Ubq E2). GA (red bars), guard cells treatedwith ABA; GC (green bars), guard cell control; MA (white bars), mesophyll cellstreated with ABA; MC (gray bars), mesophyll cell control. (B) RT-PCR verifiesthat both MPK9 and MPK12 are preferentially expressed in guard cells. Theguard cell marker gene HPRP and the mesophyll cell marker gene CBP wereamplified also. Actin2 was amplified as a control. We used 31 PCR cycles forMPK12 amplification and 34 PCR cycles for MPK9 amplification. Activity of theGUS reporter gene driven by the MPK12 promoter in guard cells in (C)cotyledons, (D) 5-week-old leaves, (E) sepals, (F) stigma, and (G) anthers. (H)The MPK12 promoter-driven GFP expression in 4-week-old leaves.

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were responsible for the ABA-insensitive phenotype observed inmpk9-1/12-1 mutants, we produced an MPK12-YFP-HA fusionprotein for complementation analysis. We predicted that intro-duction of MPK12 alone would be sufficient to complement thedouble-mutant phenotype, based on the functional redundancyin the 2 genes (Fig. 2). The mpk9-1/12-1 mutants transformedwith the MPK12-YFP-HA construct showed a restored stomatalresponse to ABA (Fig. 3A; P � 0.23 compared with WT; P � 0.78compared with vector-transformed WT). In contrast, vector-transformed mpk9-1/12-1 double mutants retained a strongABA insensitive response (P � 10�4 compared with WT and thecomplemented mpk9-1/12-1). Expression of MPK12-YFP-HA inWT plants did not affect their stomatal response to ABA (P �0.20 compared with WT; P � 0.84 compared with vector-transformed WT; Fig. S5). These data demonstrate that thepoint mutations in MPK9 and MPK12 cause the phenotypesobserved in mpk9-1/12-1 and that the MPK12-YFP-HA fusionprotein is functional and sufficient to rescue the loss-of-functionphenotype of mpk9-1/12-1. Moreover, 2 independent transgenicRNAi lines in which both MPK9 and MPK12 transcripts aresignificantly silenced showed a strong impairment in ABA-induced stomatal closure (Fig. 3B; P � 0.0001 for RNAi #40 and#47 at 3 �M ABA; P � 10�5 for RNAi #40 and P � 0.04 forRNAi #47 at 10 �M ABA). These data are consistent with theobserved ABA-insensitive stomatal response of mpk9-1/12-1mutants and further support a model in which MPK9 andMPK12 positively regulate guard cell ABA signaling.

ABA and Calcium Fail to Activate S-Type Anion Channels in mpk9-1/12-1 Mutant Guard Cells. Because ABA activation of S-type anionchannels plays a crucial role in ABA-induced stomatal closure

(2), we examined whether the mpk9-1/12-1 mutations affectedthis activation process. As shown in Fig. 4 A and B, ABA wasunable to activate anion channels in mpk9-1/12-1 mutant guardcells, whereas it activated anion channel currents in WT guardcells. This result is consistent with ABA insensitivity of stomatalmovement in mpk9-1/12-1 mutatns (Fig. 2). To determinewhether MPK9 and MPK12 act downstream or upstream ofcalcium, we also tested Ca2� activation of anion channel cur-rents. Fig. 4 C and D show that calcium did not activate anionchannel currents in mpk9–1/12–1 plants, indicating that these 2kinases act downstream of calcium in the signaling cascade.These electrophysiological results demonstrate that MPK9 andMPK12 play an important regulatory role in ABA and calciumactivation of anion channels in guard cells.

Mutations in MPK9 and MPK12 Enhance Transpirational Water Loss.To determine whether the ABA-insensitive stomatal response inmpk9-1/12-1 plants affects transpirational water loss from leaves,we measured water loss rates of detached leaves. As shown inFig. 5, leaves from mpk9-1/12-1 plants consistently lost morewater than WT plant leaves (P � 0.04 at 1, 2, and 4 h; P � 0.02at 3, 5, and 6 h). After 6 h, WT leaves lost 56.3 � 3.5% of weight,whereas leaves from mpk9-1/12-1 plants lost 76.7 � 3.9% of freshweight. These data indicate that point mutations in MPK9 andMPK12 cause enhanced transpirational water loss from leaves, afinding that correlates with the ABA-insensitive stomatal re-sponse and anion channel activation observed in mpk9-1/12-1mutants (Figs. 2 and 4).

MPK12 is Localized to the Cytosol and the Nucleus and Is Activated byABA and H2O2. In both animal and plant cells, activated MAPKsoften are translocated into the nucleus where they phosphorylatetarget proteins, including transcription factors, and therebyregulate gene expression (26, 27). To determine whether MPK12is translocated to the nucleus from the cytosol upon ABA or

Fig. 2. Mutations in both MPK9 and MPK12 disrupt ABA promotion ofstomatal closure, ABA inhibition of stomatal opening, and H2O2-inducedstomatal closure. (A) ABA induces stomatal closure in mpk9 and mpk12 singlemutants. [n � 3 independent experiments (60 stomatal apertures) for WT,mpk9-1, and mpk12-2; n � 2 independent experiments (40 stomatal aper-tures) for mpk12-1.] (B) mpk9-1/12-1 mutants show impairment in ABA pro-motion of stomatal closure. [n � 5 independent experiments (100 stomata ateach data point).] (C) mpk9-1/12-1 mutants also are impaired in ABA inhibitionof stomatal opening. [n � 3 independent experiments (60 stomata at eachdata point).] (D) Guard cells of mpk9-1/12-1 mutants are less sensitive to H2O2

than WT guard cells. [n � 4 independent experiments (70 stomatal aperturesat each data point).] Error bars indicate SEM.

Fig. 3. Expression of MPK12-YFP-HA rescues ABA-insensitive stomatal move-ments in mpk9-1/12-1 mutants, and simultaneous RNAi suppression of MPK9and MPK12 impairs ABA-induced stomatal closure. (A) The MPK12-YFP-HAfusion construct complements the mpk9-1/12-1 mutant phenotype. Westernblot analysis with anti-YFP antibody shows that MPK12-YFP fusion protein isexpressed in the complemented mpk9-1/12-1 line (MPK12-YFP/mpk9-1/12-1).The Coomassie blue-stained ribulose-1,5-bisphosphate carboxylase/oxygen-ase large subunit (Rbc L) shows the amount of proteins loaded in each well.[n � 3 independent experiments (60 stomatal apertures at each data point).](B) Semiquantitative RT-PCR analysis shows that both MPK9 and MPK12transcripts are significantly silenced in the 2 independent transgenic RNAilines, #40 and #47. Ubq E2 was amplified as a control. (C) ABA-inducedstomatal closure is strongly impaired in the transgenic RNAi lines #40 and #47.[n � 3 independent experiments (60 stomatal apertures at each data point).]Error bars indicate SEM.

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H2O2 treatment, we localized the MPK12-YFP-HA fusion pro-tein. MPK12-YFP-HA seems to be present both in the cytosoland nucleus in the absence of an exogenous stimulus, and thislocalization was unaffected by ABA or H2O2 treatment (Fig. 6and Fig. S6). It therefore is possible that MPK12 has targets inboth the cytoplasm and the nucleus.

Because our data demonstrate that MPK12 and MPK9 act aspositive regulators of ABA signaling in guard cells, we askedwhether the protein kinase activity of MPK12 is regulated byABA or H2O2. We treated the MPK12–YFP-HA-rescued mpk9-1/12-1 plants with ABA or H2O2 and immunoprecipitated theectopic MPK12 protein with anti-HA antibody. In vitro phos-phorylation assays with the immunoprecipitated MPK12-YFP-HA protein clearly show that either ABA or H2O2 en-hances the kinase activity of MPK12 (Fig. 6D).

DiscussionAlthough previous pharmacological studies have suggested thatMAPK cascades function downstream of ROS in guard cell ABA

signaling, it remained unknown which of the 20 MAPKs encodedin the Arabidopsis genome mediates this signaling. Here, wereport that 2 MPK genes, MPK9 and MPK12, are highly andpreferentially expressed in guard cells and positively regulateABA signaling acting downstream of ROS. Mutations in bothMPK9 and MPK12 lead to reduced ABA promotion of stomatalclosure and ABA inhibition of stomatal opening, impaired ABAand calcium activation of anion channels, and enhanced tran-spiration water loss in leaves. We also show that MPK12 kinaseactivity is increased by ABA and H2O2 treatment.

ROS function in many cellular processes, so one may questionhow ROS-based signaling is able to process so many differentstimuli and evoke specific cellular responses. A possibility is thatROS play different roles depending on the cellular mechanisms bywhich they have been generated, because plant cells have severalmechanisms for ROS formation. For example, the AtrbohCNADPH oxidase is required to mediate plant root hair growth andpolarized cell expansion (28, 29), whereas AtrbohD and AtrbohF

Fig. 4. ABA and calcium activation of S-type anion channels are impaired in mpk9-1/12-1 mutant guard cells. (A) Whole-cell recordings of anion channel currentsin guard cells of WT (left traces) and mpk9-1/12-1 mutants (right traces) in the absence (top traces) and presence (bottom traces) of 10 �M ABA. (B) Averagecurrent–voltage relationships for ABA activation of anion channels in guard cells of WT plants (G, 0 ABA, n � 6; X, 10 �M ABA, n � 7) and mpk9-1/12-1 mutants(C, 0 ABA, n � 6; @, 10 �M ABA, n � 7). (C) Whole-cell recordings of anion channel currents in WT (left traces) and mpk9-1/12-1 mutant (right traces) guard cellspreincubated without (top traces) and with (bottom traces) supplemented CaCl2. (D) Average current–voltage relationships for Ca2� activation of anion channelsin guard cells of WT (G, 1 mM CaCl2, n � 5; X, 40 mM CaCl2, n � 6) and mpk9-1/12-1 (C, 1 mM CaCl2, n � 4; @, 40 mM CaCl2, n � 4). Error bars indicate SEM.

Fig. 5. mpk9-1/12-1 double mutants lose more water. Detached leaves ofmpk9-1/12-1 mutants show increased transpirational water loss comparedwith WT plants. Error bars indicate SEM and are smaller than symbols whennot visible. n � 3 independent experiments (30 leaves at each data point).

Fig. 6. MPK12 protein is localized in the nuclei and cytosol and is activatedby ABA and H2O2. Confocal images of guard cells were taken from 4-week-oldleaves of transgenic Arabidopsis plants expressing MPK12-YFP-HA beforetreatment (A), after treatment with ABA (50 �M) for 30 min B, and aftertreatment with H2O2 (50 �M) for 30 min C. (Scale bars, 5 �m.) (D) In vitrophosphorylation assays show that the MPK12 protein kinase activity is acti-vated by ABA and H2O2. Western blot analysis shows that a similar amount ofimmunoprecipitated MPK12-YFP-HA protein was used.

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NADPH oxidases have been shown to function in plant defenseresponse and ABA signaling in guard cells (13, 30). The site of ROSproduction also could be a defining factor. In plants, AtrbohF islocalized at the plasma membrane (31), and a plasma membrane-associated NADPH oxidase has been shown to produce superoxide(32), indicating that plant NADPH oxidases may produce ROS inthe vicinity of the plasma membrane. Interestingly, some NADPHoxidases in animal cells appear to be localized at the endoplasmicreticulum and in the nucleus, and ROS produced in these cellularlocations could play a different role from those produced in theplasma membrane (33).

Another element of ROS-based signaling versatility may bediversity within the downstream effectors. Several lines of evi-dence suggest that MAPK cascades function in ROS signaling inplant cells and, like ROS, MAPKs have been shown to beinvolved in many different cellular processes (34, 35). Becausethere are 20 MAPKs, 10 MAPKKs, and �80 MAPKKKs in theArabidopsis genome (18), permutations of these gene familymembers potentially can produce thousands of different com-binatorial MAPKKK-MAPKK-MAPK cascades, thus enablingfinely tuned, stimulus-specific responses. In addition, a MAPKcan interact with multiple MAPKKs, and a MAPKK can interactwith several MAPKKKs (36). When combined with the possi-bility of tissue- or cell type-specific expression of MAPK cascadecomponents, it is clear that the existence of many tissue- or celltype-specific MAPK cascades can be anticipated, thus providinga basis for cell type-specific ROS and MAPK signaling.

To reveal specific MAPK genes that function in ROS-mediated ABA signaling in guard cells, we took a cell type-specific functional genomics approach and identified MPK9 andMPK12 as MAPKs that are highly and preferentially expressedin guard cells (Figs. 1 and S2). Based on their expression pattern,we hypothesized that MPK9 and MPK12 play an important rolein guard cell development and/or signal transduction. To providedirect genetic evidence, we isolated independent TILLINGmutants that have missense mutations in each of the genes(L295F in MPK9; T220I and R153K in MPK12). Protein kinaseshave 11 domains that are conserved throughout all eukaryotes(37). The mpk12-1 mutant protein sequence has a Thr2203Ilesubstitution, a highly conserved position in kinase subdomain IXin many animal and plant MAPKs, whereas the mpk9-1 mutationresulted in an amino acid change (L295F) in another conservedresidue within subdomain XI (Fig. S3). Note that Thr220 onMPK12 and Leu295 on MPK9 are not conserved in all MAPKs.Both mpk9-1 and mpk12-1 are genetically recessive becauseneither F1 heterozygous plants generated by backcrossing to WTnor heterozygous mpk9-1/12-1 plants showed any altered sto-matal phenotype. In contrast to the single mutants, mpk9-1/12-1double mutants were markedly ABA insensitive in stomatalmovement and impaired in ABA and calcium activation of anionchannels (Figs. 2 and 4). To verify that the altered phenotype ofmpk9-1/12-1 double mutants is caused by the identified muta-tions, we complemented the mpk9-1/12-1 mutation by introduc-ing an MPK12-YFP-HA transgene into these plants (Fig. 3A).Furthermore, transgenic RNAi plants in which both MPK12 andMPK9 are simultaneously knocked down also displayed a verysimilar ABA-insensitive stomatal response phenotype (Fig. 3 Band C). Together, the genetic evidence indicates that the mpk9-1and mpk12-1 mutations have a deleterious effect on the functionof the proteins and that MPK9 and MPK12 are functionallyredundant. From an evolutionary perspective, it is noteworthythat MPK9 belongs to the D group of MAPKs, whose membershave an extended C-terminal tail, lack the CD domain, andcontain an activation loop TDY motif instead of the TEY motiffound in members of other MPK groups, including the B groupto which MPK12 belongs (18). These structural differences havesuggested that group D MPKs might function differently from

other MPKs. Therefore, the functional redundancy betweenMPK9 and MPK12 is both interesting and unexpected.

MPK3 previously has been suggested to function in ABA andROS signaling, and this kinase also is activated by ABA andH2O2 (38, 39). Transgenic Arabidopsis plants in which MPK3expression is reduced by 25% showed impaired ABA inhibitionof stomatal opening and H2O2 induced stomatal closure,whereas ABA promotion of stomatal closure and ABA-inducedH2O2 production were not affected. This finding suggests thatMPK3 may function downstream of ROS in ABA inhibition ofstomatal opening but not in ABA-induced stomatal closure (40).In contrast, our data indicate that MPK9 and MPK12 actdownstream of ROS in both ABA inhibition of stomatal openingand ABA promotion of stomatal closure (Fig. 2).

Only a limited number of complete MAPK cascades have beenidentified functionally in plants. A MEKK1-MKK4/5-MPK3/6module helps regulate the plant innate immune response (21),and a YODA-MKK4/5-MPK3/6 cascade plays a critical role indevelopment of the stomatal complex (20). Another MEKK1module (MEKK1-MKK2-MPK4/6) has been shown to modulatestress responses (41). Because MEKK1 has been shown to act instress-response signaling, it would be interesting to test whetherMPK9 and MPK12 can be activated by MEKK1. Moreover,MAPK phosphatase 2 (MKP2) was shown to dephosphorylateand inactivate MPK3 and MPK6 and thus positively regulateoxidative stress signaling (42). Another MAPK phosphatase,IBR5, was shown recently to interact with and inactivate MPK12(24). This phosphatase-kinase module negatively regulates auxinsignaling but not ABA responses in roots, even though ibr5-nullmutants show reduced ABA sensitivity (24). Auxin treatmentalso leads to MPK12 activation (24). Together with our results,these observations point to potential MPK12-mediated crosstalkbetween auxin and ABA signaling. It thus would be interestingto determine if MKP2 and/or IBR5 are involved in guard cellABA signaling, a finding that would help complete a regulatorymodule for MPK9/MPK12. Although our data demonstrate thatMPK9 and MPK12 act downstream of calcium and upstream ofanion channels in ABA signaling, further molecular genetic andcell biological studies are required to determine a detailedmechanism by which these 2 MAPKs regulate anion channelactivity and stomatal closure (Fig. S7).

Materials and MethodsExpression Studies for MPK9 and MPK12. RT-PCR was performed to measuresteady-state transcript levels for MPK9 and MPK12. Total RNA was extractedfrom guard cell and mesophyll cell protoplasts with high purity (� 98% forguard cells, � 97% for mesophyll cells) isolated as previously described (43).Details about RT-PCR and cloning of the promoters are described in SI Mate-rials and Methods. Primers used in this study are listed in Table S1.

Identification of TILLING Mutants for MPK9 and MPK12. TILLING mutants wereidentified for MPK9 and MPK12 as described (25). PCR-based genotyping wasperformed using primers MPK9-TILLF and MPK9-TILLR for MPK9 and primersMPK12-TILLF and MPK12-TILLR for MPK12. For MPK9, CS93832 (mpk9-1) hada mutation in L295F. For MPK12, CS94838 (mpk12-1) and CS93401 (mpk12-2)had mutations in T220I and R153K, respectively. The mpk9-1/12-1 mutationwas generated and then double-backcrossed to WT to delete any otherbackground mutations. Restriction enzymes used for genotyping were Bpi I,Aci I, and AflII (New England) for CS93832, CS94838, and CS93401,respectively.

Complementation of mpk9-1/12-1. A full-length MPK12 cDNA cloned intopEarleyGate 101 with YFP and an HA tag was introduced into WT andmpk9-1/12-1 plants. Transformants were selected based first on their resis-tance to Basta. Homozygous lines with a single T-DNA insertion were obtainedbased on a Mendelian ratio of 3:1 for Basta resistance. Northern and Westernblot analyses were carried out to select complemented lines showing highMPK12 transcript and protein levels.

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Anion Channel Recordings. Anion channel recordings were conducted withguard cell protoplasts that were isolated from 4.5-week-old WT and mpk9-1/12-1 mutant plants as previously described (4). Seal resistance was �10 G�. Thevoltage protocol stepped the voltage from a holding potential of �30 to �35mV for 15 s. Subsequent voltage steps were decreased by 30 mV per pulse. Tomeasure the effects of ABA (Sigma, MO), protoplasts were incubated at 22 °Cwith 10 �M ABA for 2 h before recording. For calcium activation, protoplastswere preincubated in the bath solution supplemented with 39 mM CaCl2 for30 min. Leak currents were not subtracted. Steady-state currents were sam-pled at least 1 s of each voltage step.

Confocal Microscopy. To determine subcellular localization of MPK12, epider-mal strips were prepared from 4-week-old mpk9-1/21-1-complemented linesexpressing the MPK12-YFP-HA construct. The epidermal strips were treatedwith 100 �M ABA or 50 �M H2O2 for 30 min in the dark before being mountedbetween 2 cover slips. Confocal laser microscopy was carried out to assessMPK12-YFP localization using a Zeiss LSM 510 microscope system. Argon laserlight (488 nm, 10% power) was used to excite YFP, and emission wavelengthwas measured at a 505- to 550-nm bandpass. Autofluorescence was monitoredat 488 nm, and transmission images were collected in parallel.

Western Blot Analysis, Immunoprecipitation, and in Vitro Protein Kinase Assay.Proteins were extracted from rosette leaves of WT, vector-transformed WT,vector-transformed mpk9-1/12-1 mutants, and the complemented mpk9-1/

12-1 mutants. Following SDS/PAGE, the proteins were transferred to nitrocel-lulose membrane. To detect MPK12-GFP-HA, mouse anti-HA antibody (1:5,000dilution, Sigma) and HRP-conjugated anti-rat IgG antibody (1:5,000 dilution,GE Healthcare) were used. For immunoprecipitation and in vitro proteinkinase assays, 4-week-old mpk9-1/12-1 mutants expressing the MPK12-YFP-HA construct were treated with 1 mM H2O2 for 30 min, or 50 �M ABA, orwater for 1 h, and proteins were extracted from rosette leaves. Rabbit anti-YFPantibody (1:5,000 dilution, Santa Cruz Biotechnology.) was added to theprotein extract, and the amount of immunoprecipitated MPK12 protein wasassayed by mouse anti-HA antibody (1:5,000 dilution, Sigma). In vitro proteinkinase assay was performed using myelin basic protein (Sigma) as a substrateas described (44).

ACKNOWLEDGMENTS. We thank A. Chen for critical reading this manuscriptand B. Jeon for taking confocal images of MPK12:GFP. We thank A. Beavenand Dr. H. Sze’s laboratory for their technical help with microscopy. We alsothank Drs. H. Hirt and S. Zhang for providing in vitro phosphorylation assayprotocols. This work was supported by National Research Initiative Grants2004–35100-14909 and 2007–35100-18377 from the U.S. Department of Ag-riculture Cooperative State Research, Education, and Extension Service, and inpart by an equipment Grant MCB-0821250O from the National Science Foun-dation (to J.M.K.) and by a grant from the Natural Sciences and EngineeringResearch Council of Canada (to B.E.E.).

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