Abscisic Acid Inhibits Rice Protein Phosphatase PP45 via H2O2and Relieves Repression of the Ca2+/CaM-Dependent ProteinKinase DMI3

Lan Ni,a,b,c,d Xiaopu Fu,a,b,c Huan Zhang,a,b Xi Li,a,b Xiang Cai,a,b Panpan Zhang,a,b Lei Liu,a,b Qingwen Wang,a,b,c

Manman Sun,a,b,c Qian-WenWang,a,b,c Aying Zhang,a,b Zhengguang Zhang,d and Mingyi Jianga,b,c,e,1

a College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, ChinabNational Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, Chinac Key Laboratory of Crop Physiology Ecology and Production Management, Ministry of Agriculture, Nanjing Agricultural University,Nanjing 210095, ChinadCollege of Plant Protection, Nanjing Agricultural University, Nanjing 210095, Chinae Southern Regional Collaborative Innovation Center for Grain and Oil Crops in China, Hunan Agricultural University, Changsha410128, China

ORCID IDs: 0000-0002-6562-918X (L.N.); 0000-0002-9718-2351 (X.F.); 0000-0001-7525-6973 (H.Z.); 0000-0003-0695-5361 (X.L.);0000-0002-6857-4364 (X.C.); 0000-0002-5046-8782 (P.Z.); 0000-0002-6388-2077 (L.L.); 0000-0002-0443-9470 (Q.W.); 0000-0002-2842-6914 (M.S.); 0000-0002-4435-1369 (Q.-W.W.); 0000-0003-3779-1603 (A.Z.); 0000-0001-8253-4505 (Z.Z.); 0000-0002-3300-0133 (M.J.)

In plants, Ca2+/calmodulin-dependent protein kinase (CCaMK) is a positive regulator of abscisic acid (ABA) responses,including root growth, antioxidant defense, and tolerance of both water stress and oxidative stress. However, the underlyingmolecular mechanisms are poorly understood. Here, we show a direct interaction between DMI3 (Doesn't Make Infections 3),a rice (Oryza sativa) CCaMK and PP45, a type 2C protein phosphatase in rice (PP2C). This interaction involves the CaMbinding domain of DMI3 and the PP2C domain of PP45. In the absence of ABA, PP45 directly inactivates DMI3 bydephosphorylating Thr-263 in DMI3. However, in the presence of ABA, ABA-induced H2O2 production by the NADPH oxidasesRbohB/E inhibits the activity of PP45 not only by inhibiting the expression of PP45 but also by oxidizing Cys-350 and Cys-428residues to form PP45 intermolecular dimers. ABA-induced oxidation of Cys-350 and Cys-428 in PP45 blocked the interactionbetween PP45 and DMI3 and substantially prevented PP45-mediated inhibition in DMI3 activity. Genetic analysis indicatedthat PP45 is an important negative regulator of ABA signaling. These results reveal important pathways for the inhibition ofDMI3 under the basal state and for its ABA-induced activation in rice.

INTRODUCTION

In plants, cytosolic Ca2+ is a ubiquitous second messenger and-mediates stimulus response coupling to regulate plant growth,development, and responses to environmental stresses. Variousabiotic and biotic stresses cause changes in the cytosolic Ca2+

concentration (Yang and Poovaiah, 2003; Lecourieux et al., 2006;DeFalco et al., 2009; Reddy et al., 2011; Batistic and Kudla, 2012),and these Ca2+ signals are detected, decoded, and transmitted toproduce downstream responses by a variety of Ca2+ bindingproteins that function as Ca2+ sensors. The main types of Ca2+

sensors include calmodulin (CaM) and CaM-like protein, calcium-dependentprotein kinase, calcineurinB-likeprotein, andCa2+/CaM-dependent protein kinase (CCaMK; Yang and Poovaiah, 2003;Harper et al., 2004; DeFalco et al., 2009; Batistic andKudla, 2012).

CCaMK is a plant-specific protein kinase, consisting of a Ser/Thr kinase domain, a CaM binding domain overlapping an au-toinhibitory domain, and a visinin-like domain containing three EF(helix-loop-helix) hands (Singh and Parniske, 2012; Poovaiahet al., 2013). CCaMK was first identified and cloned in lily (Liliumlongiflorum), andearly biochemical studieson lilyCCaMKshowedthat its kinase activities are regulated by bothCa2+ andCa2+/CaM(Patil et al., 1995; Takezawa et al., 1996). CCaMK is a key regu-lator of nodule organogenesis and rhizobial infection (Lévyet al., 2004; Mitra et al., 2004; Gleason et al., 2006; Tirichine et al.,2006; Hayashi et al., 2010; Madsen et al., 2010; Shimoda et al.,2012; Takeda et al., 2012; Miller et al., 2013). Interacting Proteinof DMI3 (IPD3)/CYCLOPS and CCaMK-Interacting Protein ofApproximately 73 kD (CIP73) were identified to interact with andto be phosphorylated by CCaMK (Messinese et al., 2007; Yanoet al., 2008; Kang et al., 2011). CCaMK-IPD3/CYCLOPS com-plex hasbeen shown tobeessential for rhizobial andmycorrhizalcolonization (Horváth et al., 2011; Singh et al., 2014; Jin et al.,2016; Pimprikar et al., 2016).In addition to regulating rhizobial and mycorrhizal symbio-

ses, CCaMK is involved in the responses of plants to abioticstresses (Ma et al., 2012; Shi et al., 2012, 2014; Zhu et al.,2016) and biotic stresses (Wang et al., 2015). Genetic evidence

1Address correspondence to myjiang@njau.edu.cn.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Mingyi Jiang (myjiang@njau.edu.cn).www.plantcell.org/cgi/doi/10.1105/tpc.18.00506

The Plant Cell, Vol. 31: 128–152, January 2019, www.plantcell.org ã 2018 ASPB.

indicates that the rice (Oryza sativa) CCaMK DMI3 is a positiveregulator of a variety of abscisic acid (ABA) responses, includingrootgrowth,antioxidantdefense,andtolerancetobothwaterstressand oxidative stress (Shi et al., 2012, 2014). DMI3-mediated acti-vation of mitogen-activated protein kinase 1 (MPK1), a major ABA-activated mitogen-activated protein kinase, regulates the activitiesof the antioxidant enzymes superoxide dismutase (SOD) and cat-alase (CAT) in ABA signaling (Shi et al., 2014). A recent studyshowed that Zm-NAC84, a NAC (NAM, ATAF1/2, and CUC2)transcription factor in maize (Zeamays), interacted with and wasphosphorylated by Zm-CCaMK, and Zm-NAC84 is essential forABA-induced antioxidant defense in a Zm-CCaMK-dependentmanner (Zhu et al., 2016). However, the molecular mechanismsby which ABA induces the activation of CCaMK and howthe activated CCaMK regulates ABA responses remain to bedetermined.

Here, we identify Protein Phosphatase 2C 45 (PP45,also calledBIPP2C1 [Benzothiadiazole-InducedProteinPhosphatase2C1]) asan interacting protein of DMI3. PP45belongs to amember of groupK in the familyof type2Cproteinphosphatases (PP2Cs;Singhetal.,2010) and was shown to be involved in the response of rice topathogen infection, ABA, and various environmental stresses (Huet al., 2006). Here, we demonstrate that PP45 is an importantnegative regulator of ABA signaling and elucidate key molecularmechanismsbywhichPP45 inactivatesDMI3 in thebasal state andby which ABA induces the activation of DMI3 in rice.

RESULTS

DMI3 Interacts with PP45

To isolate proteins that interact with DMI3 in rice leaves, thefull-length DMI3 protein was used as bait to screen a rice leaf

complementary DNA (cDNA) library. Several independent posi-tive clones were isolated, and three of these clones were identi-fied as encoding PP45. The interaction of DMI3 with PP45 usingthe yeast two-hybrid (Y2H) system is shown in Figure 1A.To confirm the interaction betweenDMI3 and PP45 both in vitro

and in vivo, glutathione S-transferase (GST) pull-down assays,bimolecular fluorescence complementation (BiFC) analyses, andco-immunoprecipitation (Co-IP) assays were performed. In vitropull-down assays showed that PP45 interacted with GST-DMI3but not with GST alone (Figure 1B). When split yellow florescentprotein (SYFP)N-PP45 was co-transformed with SYFPC-DMI3into onion epidermis cells, BiFC analyses showed a strong YFPfluorescence signal in the nucleus, the cytosol, and the plasmamembrane, indicating physical interaction of PP45 and DIM3(Figure 1C). In Co-IP assays, immunoblot (IB) analyses using ananti-His antibody revealed an interaction between DMI3-Myc andPP45-His (Figure 1D). In support of the above observations,subcellular localization analysis by confocal laser scanning mi-croscopy showed that, as seen in the BiFC analyses, both DMI3and PP45 were localized in the nucleus, the cytosol, and theplasma membrane (Supplemental Figure 1).To determine which region(s) of DMI3 mediates its binding to

PP45, a series of deletion constructs of DMI3weremade and thentested for interaction with PP45 using the Y2H assay. As shown inSupplemental Figure 2A, PP45 interacted with the CaM-bindingdomain (301 to 336 amino acids) of DMI3. A firefly luciferasecomplementation imaging (LCI) assay confirmed the in vivo in-teraction between PP45 and the CaM-binding domain of DMI3(Supplemental Figure 2B). A similar strategy was used to identifywhichdomainofPP45wasnecessary for its interactionwithDMI3.Y2Hassayshowed that thePP2Cdomain (343 to569aminoacids)of PP45 was sufficient for interaction with DMI3 (SupplementalFigure 2C). A LCI assay confirmed the in vivo interaction

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(Supplemental Figure 2D). Taken together, these results indicatethat DMI3-PP45 interaction involves the CaM-binding domain ofDMI3 and the PP2C domain of PP45.

The rice genome encodes six members in group K of the PP2Cfamily, andPP111,PP57, andPP1have thehighesthomologywithPP45 (Singh et al., 2010). To determinewhether othermembers ofgroup K would interact with DMI3, both Y2H and BiFC analysiswereconducted.PP111,PP57,andPP1didnot interactwithDMI3either in yeast cells (Supplemental Figure 3A) or in onion epidermiscells (Supplemental Figure 3C), suggesting that only DMI3 spe-cifically interactswithPP45. In addition,Y2Hassays (SupplementalFigure 3B) and BiFC analyses (Supplemental Figure 3D) showedthat PP45 did not interact with the rice sucrose nonfermenting1-relatedproteinkinase2s (SnRK2s)SAPK8/9/10,whicharehomologsof Arabidopsis (Arabidopsis thaliana) SnRK2.6/Open Stomata1(OST1) and were shown to be activated by ABA (Kobayashi et al.,

2004). Previous studies have shown that SnRK2.6/OST1, a globalpositive regulator ofABAsignaling, interactswith thegroupAPP2Csin Arabidopsis (Umezawa et al., 2009; Vlad et al., 2009).

ABA and H2O2 Inhibit the Interaction of DMI3 and PP45

To investigate whether ABA or H2O2 treatment affects the in-teraction between DMI3 and PP45, both in vitro and in vivo ex-perimentswere performed. Y2H (Figure 2A) andpull-downassays(Figure 2B) showed that H2O2 completely blocked the interactionbetweenDMI3 andPP45.However, ABA treatment (30 to 100mM)did not inhibit the interaction in pull-down assays (Figure 2B). Bycontrast, IB analyses using specific anti-PP45 and anti-DMI3antibodies (Supplemental Figure 4) revealed that PP45 did notinteract withDMI3 in rice leaves treatedwith either 100mMABAor10mMH2O2 (Figure 2C). In addition, LCI analysis showed that the

Figure 1. PP45 Interacts with DMI3 Both In Vitro and In Vivo.

(A)Y2Hassay. ADvector andBDvector areY2Hvectorswith no insert. SD-Trp-Leu-His-Ade/AbA/X-a-galmediumwasused for testing the interaction. Thecombination of BD-P53 plus AD-SV40 was used as a positive control, and BD-Lam/AD-SV40 was used as a negative control.(B) Pull-down assay. PP45-His and DMI3-GST proteins were expressed in Escherichia coli. For IB, PP45-His was detected with an anti-His antibody, andDMI3-GST was detected with an anti-GST antibody.(C)BiFC analysis. Using the particle bombardmentmethod, the constructs indicated were transiently expressed in onion epidermis cells. Coexpression ofSYFPN-PP45 plus SYFPC or SYFPC-DMI3 plus SYFPN were used as negative controls. Scale bars, 90 mm.(D)Co-IP test. After PP45-His andDMI3-Mycwere cotransformed into rice protoplasts, total proteins of protoplasts were immunoprecipitated (IP) using ananti-Myc antibody and were detected with anti-His and anti-Myc antibodies. Protein input is shown by IB analysis of protein extracts before immuno-precipitationandantibodiesagainst the respective tags.Molecularmassmarkers inkDareshownon the left. All theexperimentswere repeatedat least threetimes with similar results.

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Figure 2. ABA and H2O2 Inhibit the Interaction of PP45 and DMI3.

(A) H2O2 inhibits the interaction between PP45 and DMI3. SD-Trp-Leu-His-Ade/X-a-gal medium with various concentrations of H2O2 was used in a Y2Hassay for testing the interaction. BD-P53 plus AD-SV40 was used as a positive control.(B) In vitro pull-down assay showing that H2O2, but not ABA, directly inhibits the DMI3-PP45 interaction. PP45-His was incubated with GST-DMI3conjugated tomagnetic beads in the pull-down binding buffer plus various concentrations of either H2O2 (left) or ABA (right). Protein input was shown by IBanalysis.

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same ABA or H2O2 treatment strongly reduced the in vivo in-teraction between DMI3 and PP45 (Figure 2D). These resultsdemonstrate that both ABA and H2O2 can block the interaction ofDMI3 and PP45 in rice cells.

Previous studies have shown that RbohB and RbohE, whichencodeNADPHoxidase in rice, are involved inABA-inducedH2O2

production (Shi et al., 2012; Zhang et al., 2014). To determinewhether ABA-induced inhibition of the interaction between DMI3and PP45 is related to the ABA-induced production of H2O2, anrbohB/E double mutant knockout (KO) line (rbohB/E-KO1) wasgenerated by CRISPR/Cas9 (Supplemental Figure 5A). ABA-induced H2O2 production, as detected by 3,3-diaminobenzidinestaining (Supplemental Figure 5B) and by confocal microscopy(Supplemental Figure 5C), was impaired in rbohB/E-KO1. Thisindicates that RbohB and RbohE are required for ABA-inducedH2O2 production in rice. In rbohB/E-KO1, ABA treatment failed toinhibit the interaction between DMI3 and PP45 detected by Co-IP(Figure 2E), suggesting that H2O2 is required for ABA-inducedinhibition in the interaction of DMI3 and PP45.

ABA and H2O2 Transiently Downregulate PP45

A previous study showed that both ABA and H2O2 induced theexpression of DMI3 and the activity of DMI3 in the leaves of riceplants (Shi et al., 2012). To investigate the effects of ABAandH2O2

on the expression ofPP45, the PP45 protein level, and the activityof PP45 in leaves of rice seedlings, we performed relative quan-titative PCR analysis and IB analysis and Ser/Thr phosphataseactivity assays. The expression of PP45 (Figure 3A), the level ofPP45protein (Figure3B), and theactivity ofPP45 (Figure 3C) in theleaves of rice plants exposed to 100mMABAor 10mMH2O2werefirst downregulated and then upregulated. For ABA treatment, themaximumdecrease inPP45 expression, thePP45 level, andPP45activity occurred at 30, 60, and 90 min after ABA treatment, re-spectively, and the maximum increase appeared at 2, 6, and 6 hafter ABA treatment, respectively. For H2O2 treatment, the max-imum decreases occurred sooner—at 15, 45, and 45 min afterH2O2 treatment, respectively—and the maximum increase ap-peared at 2, 4, and 4 h after H2O2 treatment, respectively. Tocompare the degree of change between PP45 protein leveland PP45 activity with ABA and H2O2 treatment, the protein levelof PP45 was quantified and the treatment/control ratios of the

protein level and theactivityofPP45werecalculated, respectively.The results showed that, for either ABA or H2O2 treatment, thedecrease in enzyme activity was much greater than the decreasein protein level (Figure 3D), showing that PP45 activity is moresensitive to oxidative stress than is the PP45 protein level.

H2O2 Is Required for the ABA-Induced Inhibition of PP45

To determine whether ABA-induced inhibition of PP45 is dueto the action of endogenous H2O2, we used dimethylthiourea(DMTU), a trap for H2O2, and diphenyleneiodonium chloride (DPI),an inhibitor of NADPH oxidase, to reduce the level of H2O2.Pretreatments with DMTU or DPI completely relieved the ABA-induced inhibition of both the expression of PP45 (Figure 4A) andthe activity of PP45 (Figure 4B), suggesting that H2O2 is requiredfor the ABA-induced inhibition of PP45.Wenext used the rbohB/Edoublemutant to show that knockout of these twogenes removedtheABA-induced inhibitionofPP45expression (Figure4C)and theits activity (Figure 4D), although it did not affect theexpression andtheactivityofPP45under thecontrol conditions. Finally,we testedfor a direct effect of H2O2 on the phosphatase activity of PP45.PP45washighly sensitive toH2O2. Following treatmentwithH2O2,PP45 lost itsactivity,withan IC50of84mM(Figure4E).Theadditionof the reducing agents Tris (2-carboxyethyl) phosphine (TCEP) ordithiothreitol (DTT) at high levels prevented the inactivation ofPP45 by H2O2 (Figure 4F), and these reducing agents themselvesdid not interfere with the sensitivity of the assay (SupplementalFigure 6), demonstrating a sensitive and reversible response ofPP45 activity to changes in redox conditions.

PP45 Negatively Regulates and DMI3 Positively RegulatesABA Responses in Rice Plants

Using a dmi3mutant, previous studies have revealed that DMI3 isa positive regulator of ABA responses, including root growth, an-tioxidantdefense,andtolerancetowaterstressandoxidativestress(Shi et al., 2012, 2014). To investigate the role of PP45 in the reg-ulation of ABA responses, two independent PP45-overexpressing(OE) lines (PP45-OE1, PP45-OE2) and two independent PP45-knockout lines (pp45-KO1, pp45-KO2) were generated. For com-parison, two independent DMI3-overexpressing lines (DMI3-OE1,DMI3-OE2) and two independentDMI3-knockout lines (dmi3-KO1,

Figure 2. (continued).

(C) A Co-IP assay showing that both ABA and H2O2 block the DMI3-PP45 interaction in rice leaves. The proteins extracted from rice leaves treated with100mMABAfor90minor10mMH2O2 for45minwere immunoprecipitated (IP)withanti-DMI3antibodyandweredetectedby IBwithanti-PP45antibodyandanti-DMI3antibody.Thecontrol lanes (left, noABAandnoH2O2) are fromsamplesnot treatedwitheitherABAorH2O2. Inputprotein isshownby IBanalysisofprotein extracts before IP. b-actin (bottom) was used as a loading control.(D) Luciferase complementation imaging assay showing that both ABA and H2O2 block the interaction between PP45 and DMI3 in tobacco leaves. Theindicatedconstructswereexpressed inNicotianabenthamiana leaves, and the leaveswere treatedeitherwith100mMABAfor 90minorwith10mMH2O2 for45 min. Luciferase signals were captured using the Tanon-5200 image system. Scale bars, 1.5 cm. Proteins (right) were detected by IB analysis of the leafprotein extracts after treatment. The Rubisco large subunit was used as a loading control visualized by staining with Coomassie Brilliant Blue. Molecularmass markers in kD are shown on the left.(E)ABA-mediated inhibition in the interactionofPP45andDMI3 is blocked in the rbohB/Emutant. Theproteins extracted from the leavesof rbohB/Eorwild-type (WT) plants treatedwith 100mMABA for 90minwere immunoprecipitated (IP) using an anti-DMI3 antibody andwere detected by IBwith either an anti-PP45 antibody or an anti-DMI3 antibody. Protein input is shown by IB analysis of protein extracts before IP. The levels of b-actin indicate equal proteinloading. Molecular mass markers in kD were indicated on the left. All experiments were repeated at least three times with similar results.

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Figure 3. ABA and H2O2 Regulate the Expression of PP45, the Level of PP45 Protein, and the Activity of PP45.

(A) to (C)Changes in the expression (A), the protein level (B), and the activity (C) of PP45 in leaves of rice plants exposed to ABA (100mM) or H2O2 (10mM)treatment for various times as indicated. Relative expression level of PP45was analyzed by real-time quantitative PCR (A). The level of PP45 protein was

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dmi3-KO2)werealsogenerated.Moreover, two independentPP45-knockdown lines (pp45-RNAi1, pp45-RNAi2) and two independentDMI3-knockdown lines (dmi3-RNAi1, dmi3-RNAi2) were alsogenerated.

In the dmi3-KO lines (Supplemental Figure 7A) and the pp45-KOlines (SupplementalFigure7C)generatedbyCRISPR/Cas9system,the activities of both DMI3 (Supplemental Figure 7B) and PP45(Supplemental Figure 7D) were undetectable. The PP45-OE linesand theDMI3-OE linesexhibitedhighexpression levelsofPP45andDMI3 (Supplemental Figure 8A) and high activities of PP45 andDMI3 (Supplemental Figures 7B and 7D). In pp45-RNAi lines,PP45mRNAwas reducedby;80%incomparisonwith thewild type, butthe expressionofPP1,PP57, andPP111was not affected (Supple-mental Figure 8A). This shows thatPP45 expressionwas specificallysuppressed by RNA interference (RNAi). The expression of DMI3 indmi3-RNAi lines was also suppressed (Supplemental Figure 8A).

In theabsenceofABA, therewerenoobviousdifferencesbetweenthese transgenic lines and thewild type in rates of seed germination(Figures 5A and 5B; Supplemental Figures 8B and 8C). ABA treat-ment significantly inhibited seed germination (Figures 5A and 5B;Supplemental Figures 8B and 8C) and primary root growth (Figures5C and 5D; Supplemental Figures 8D and 8E) in wild type. The ABAsensitivity of both seed germination and primary root growth wasenhanced in pp45-KO lines, pp45-RNAi lines, and DMI3-OE lines,and itwas reduced inPP45-OE lines,dmi3-KO lines, anddmi3-RNAilines. These results indicate that PP45 reduces andDMI3 enhancesABA sensitivity in seed germination and root growth.

To test the role of PP45 in the tolerance of water stress andoxidative stress in plants, wild-type rice seedlings and thetransgenic lineswere treatedwith either polyethylene glycol (PEG)to simulate water stress or H2O2 to produce oxidative stress.Under the control conditions, therewereno significant differencesin the growth of rice seedlings between the transgenic plants andthewild-type plants (Figures 6A and 6B; Supplemental Figures 9Aand 9B). When treated with 20% PEG (Figure 6A; SupplementalFigure 9A) or with 100 mM H2O2 (Figure 6B; SupplementalFigure 9B), PP45-OE, dmi3-KO, and dmi3-RNAi plants exhibitedmore severe wilting and chlorosis than wild-type plants (Figures6A and 6B; Supplemental Figures 9A and 9B) and had lowersurvival rates after recovery by re-watering (Figure 6C; SupplementalFigure9C). In contrast,DMI3-OE,pp45-KO, andpp45-RNAi plantsexhibited less severe wilting and chlorosis than the wild-typeplants after PEG and H2O2 treatments and had higher survivalrates after recovery by rewatering. Moreover, the content ofmalondialdehyde (Figure 6D; Supplemental Figure 9D) and thepercentage of electrolyte leakage (Figure 6E; SupplementalFigure 9E), which are indicators of oxidative damage, were higherin the leaves of the PP45-OE, dmi3-KO, and dmi3-RNAi plantsexposed to PEG and H2O2 treatments than those in the wild-type

plants, and they were lower in the leaves of the DMI3-OE, pp45-KO, and pp45-RNAi plants. Moreover, PP45 downregulatedABA-induced increases in the activities of MPK1 (SupplementalFigure 10A), SOD, and CAT (Supplemental Figure 10B), whileDMI3 upregulated the ABA-induced increases in the activitiesof these enzymes (Supplemental Figures 10A and 10C). Theseresults indicate that PP45 negatively andDMI3 positively regulatethe tolerance of rice plants to water stress and oxidative stress.

PP45 Inactivates DMI3 by Dephosphorylating Thr-263in DMI3

Under the control (nontreated) conditions, the PP45-OE1 plantsshowed reduced activity of DMI3 and the pp45-KO1 plantsshowed increased DMI3 activity compared with wild-typeplants (Figure 7A). ABA treatment induced a significant increasein the activity of DMI3 in the leaves of wild-type plants. This ABA-induced increase was blocked in PP45-OE1 plants and wasfurther enhanced in pp45-KO1 plants. Furthermore, in vitro ex-periments showed that PP45 directly inhibited the autophos-phorylation and substrate phosphorylation activities of DMI3(Figure 7B). The PP45-mediated inhibition in DMI3 autophos-phorylation was released by the addition of H2O2 but not by ABAaddition (Figure 7C). ABA and H2O2 themselves did not affect theautophosphorylation and substrate phosphorylation activities ofDMI3 in vitro (Figure 7C; Supplemental Figure 11A), andCa2+ wasrequired for H2O2-induced activation of DMI3 in rice plants(Supplemental Figure 11B). On the other hand, DMI3 did notregulate the expression of PP45 and the activity of PP45, re-gardless of the presence or absence of ABA (Supplemental Figure12). In Medicago truncatula CCaMK (Mt-CCaMK), four auto-phosphorylation sites (Ser-9, Thr-271, Ser-343, and Ser-344)were identified, and Thr-271 was found to be a major auto-phosphorylation site in the kinase (Poovaiah et al., 2013; Routrayet al., 2013). In Os-DMI3, the corresponding sites were Ser-10,Thr-263, and Ser-335, respectively (note that the amino acidresidue at position 336 of Os-DMI3 is a Cys; Figure 7D). To de-termine whether PP45 can dephosphorylate these sites in DMI3,three synthetic phosphopeptides, corresponding to the threephosphorylation sites of DMI3, were used. As shown in Figure 7E,PP45 dephosphorylated only the synthetic phosphopeptidecorresponding to residues 258 to 271 of DMI3, suggesting thatPP45specificallydephosphorylates Thr-263 inDMI3.Bycontrast,calf intestine alkaline phosphatase dephosphorylated all thesynthetic phosphopeptides. Furthermore, in vitro assays using ananti-phospho-Thr-263 antibody showed that PP45-His inhibitedThr-263 phosphorylation in the phosphorylated DMI3-GST, andthePP45-mediated inhibitionwas reversedby theadditionofH2O2

(Figure 7F). Similar changes in the autophosphorylation activity of

Figure 3. (continued).

analyzed by IBwith an anti-PP45 antibody, and b-actin was used as a loading control. Molecular massmarkers in kilodaltons are shown on the left (B). Theactivity of PP45 was measured by the serine/threonine phosphatase assay (C).(D)The treatment/control ratio of theprotein level in (B) and the activity in (C)of PP45. For quantitative analysis of band intensity in (B), the starting point (0 h)wasset to1,and theotherpointswerecomparedwith it. In (A), (C), and (D), valuesaremeans6 SEMof three independentexperiments.Meansdenotedby thesame letter didnot significantly differ atP<0.05according toDuncan’smultiple range test. In (B), experimentswere repeatedat least three timeswith similarresults.

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DMI3were also observed. In vivo assays also showed that Thr-263inDMI3was phosphorylated in the leaves ofpp45-KO1 plants, andH2O2 treatment induced Thr-263 phosphorylation in wild-type andenhancedThr-263phosphorylation inpp45-KO1plants (Figure7G).Similar changes in the substratephosphorylationofDMI3werealsoobserved. Together, these results support that PP45 directly in-activates DMI3 by dephosphorylating Thr-263 in DMI3.

Thr-263 Autophosphorylation Is Required for the Activationof DMI3 and the Response Mediated by DMI3

To further determine the role of Thr-263 phosphorylation in theautophosphorylation activity of DMI3, Thr-263 of DMI3 was

mutated either to Ala (DMI3T263A) to create a non-phosphorylatablemutantor toAsp (DMI3T263D) tocreateaphosphomimeticmutant. Inthe absence of Ca2+ and CaM, DMI3 was weakly autophos-phorylated, and its autophosphorylation not only was signifi-cantly enhanced in DMI3T263D but also was almost abolished inDMI3T263A (Figure 8A). In the presence of Ca2+ and CaM, auto-phosphorylation of DMI3 was significantly increased. However,autophosphorylation in both DMI3T263A and DMI3T263D was un-responsive to addition of Ca2+ or Ca2+/CaM. These resultsindicate that Thr-263 phosphorylation is critical for the auto-phosphorylation activity of DMI3. Then, we tested whetherThr-263 phosphorylation affects CaM binding to DMI3. In thepresence ofCa2+, DMI3 bound toCaM, andbindingwasmarkedly

Figure 4. H2O2 Is Required for ABA-Induced Inhibition of PP45.

(A) and (B)Effects of pretreatmentswithDMTUandDPI on the expression ofPP45 (A) andon the activity of PP45 (B) in rice leaves exposed toABA. The riceseedlingswere either untreated (Control) or pretreatedwith 5mMDMTUor 100mMDPI for 4 h and then exposed to 100mMABA for 30min (A)or 90min (B).(C) and (D) The expression ofPP45 (C) and the activity of PP45 (D) in rbohB/Emutant. The rice seedings were treated with 100mMABA for 90min, and therelative expression levels of PP45 and the activity of PP45 were analyzed by real-time quantitative PCR and by Ser/Thr phosphatase assay, respectively.(E)H2O2directly inhibits the activity of PP45 in vitro. The in vitro-expressedPP45-His proteinwas incubatedwith various concentrations ofH2O2. The graphshown the activity of PP45 as measured by the Ser/Thr phosphatase assay. The protein level of OPP45 after treatment was determined by IB analysis(bottom). The phosphatase activity of PP45 without H2O2 treatment was set to 100%.(F) The inactivation of PP45 by H2O2 is reversed by TCEP and DTT. Recombinant PP45 protein was incubated with 0.1 mM H2O2 for 30 min, followed bydifferent concentrations of the reducing agents TCEP or DTT for another 30min. The activity of PP45wasmeasured by the Ser/Thr phosphatase assay. Allexperimentswere repeated at least three timeswith similar results. Values aremeans6 SEM of three independent experiments.Means denoted by the sameletter did not significantly differ at P < 0.05 according to Duncan’s multiple range test.

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Figure 5. PP45 Reduces and DMI3 Enhances ABA Sensitivity in Seed Germination and Root Growth.

(A)Photographsof seedgermination inDMI3-OE,dmi3-KO,PP45-OE,pp45-KO,and thewild type (WT). Theseedsof transgenic linesand thewild typeweregerminated and grown in 1/2 MS medium supplemented with different concentrations of ABA (0, 1, 5 mM) for 9 d after stratification. Scale bar, 3.5 cm.(B) The germination rates of seeds under ABA treatments during 15 d after stratification.(C) Phenotypes of rice seedlings grown in 5 mM ABA for 14 d. Scale bar, 4 cm.(D)Primary root lengthsof riceseedlingsgrown for14d in thedifferentconcentrationsofABA indicated.Approximately 50seedsofeach transgenic linewereanalyzedper replicate for eachconcentrationofABA in (A) to (D). In (A) and (C), experimentswere repeated at least three timeswith similar results. In (B) and(D), values are means 6 SEM of three independent experiments.

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Figure 6. PP45 Negatively and DMI3 Positively Regulate the Tolerance of Rice Plants to Water Stress and Oxidative Stress.

(A) and (B)PhotographsofDMI3-OE,dmi3-KO,PP45-OE,pp45-KO, andwild-type (WT) plants exposed towater stress (A)or oxidative stress (B). Ten-day-old seedlings were treated with 20% PEG 4000 (A) or 100 mMH2O2 (B) for 15 d and then allowed to recover for 10 d. Approximately 30 seedlings of eachtransgenic line were used per replicate. Scale bars, 5 cm.

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increased in DMI3T263D and completely blocked in DMI3T263A

(Figure 8B). In vivo analysis of rice protoplasts also showed thatCaM1 binding affinity of DMI3 was enhanced in the protoplastswith transiently expressed DMI3T263D and was lost in the proto-plasts with transiently expressed DMI3T263A (Figure 8C). Theseresults indicate that Thr-263 phosphorylation positively regulatesCaM binding of DMI3. Further, the effect of Thr-263 phosphory-lation on the substrate phosphorylation of DMI3 was analyzed.The changes in the substrate phosphorylation activity in DMI3,DMI3T263A, and DMI3T263D were similar to the changes in auto-phosphorylation activity in the presence or absence of Ca2+ andCa2+/CaM (Figure 8D). These results indicate a positive effect ofThr-263 phosphorylation on the substrate phosphorylation ac-tivity of DMI3.

Finally, the role of Thr-263 phosphorylation in the antioxidantdefense response in rice protoplasts was tested. Transient ex-pression of DMI3T263D significantly increased the activities ofMPK1 (Figure 8E), SOD (Figure 8F), andCAT (Figure8G). Transientexpression of DMI3T263A markedly reduced the activity of MPK1,but did not affect the activities of SOD and CAT. However, theABA-induced increase in the activities of SOD and CAT wasblocked in the protoplasts with transiently expressed DMI3T263A.These results suggest that Thr-263 phosphorylation upregulatesantioxidant defense in plant cells.

Cys-350 and Cys-428 Are the Key Cysteine ResiduesResponsible for H2O2-Mediated Inactivation of PP45

Cys residues inproteins canbealternatively oxidized and reducedto formandbreakdisulfide linkages, respectively, and thusareoneof the most sensitive targets for reactive oxygen species (ROS;Akter et al., 2015;Waszczaket al., 2015). InPP45, there are 15Cysresidues. Each of these Cys residues wasmutated to Ser in orderto test whether substitution of any of the 15 Cys residues wouldaffect the inhibitionofPP45byH2O2.As shown inFigure 9A, twoofthe mutant proteins, PP45C350S and PP45C428S, showed signifi-cant protection against inactivation by H2O2, suggesting theimportance of Cys-350 and Cys-428. Treatment with 50mMH2O2

led to a significant decrease in the activity of PP45 but did notaffect the phosphatase activity in the PP45C350S/C428S doublemutant (Figure 9B). Following treatment with 0.2 mM H2O2,PP45C350S/C428S retained 60% of its phosphatase activity com-paredwithPP45,whichonly had13%of its original activity. Theseresults indicate that Cys-350 and Cys-428 are the key Cys resi-dues that mediate oxidative inactivation of PP45.

To determine whether PP45 oxidation leads to the formationof intramolecular or intermolecular disulfide bonds, PP45 andPP45C350S/C428S produced in Escherichia coli were treated with

0.1 mM H2O2 and then analyzed by reducing and nonreduc-ing PAGE (Figure 9C). Under nonreducing conditions, a bandcorresponding to twice the apparent molecular mass of PP45(;124 kD) was observed in H2O2-treated PP45 but not inPP45C350S/C428S, suggesting the formation of an intermoleculardisulfide bond in oxidized PP45. However, after H2O2 treatment,no change of mobility was observed in the monomer (;62 kD) ofPP45 and PP45C350S/C428S under the nonreducing conditions(Figure 9C; Supplemental Figure 13). This is consistent with thatof the Arabidopsis HAB1 (Supplemental Figure 13), which wasshown to form only intermolecular disulfide bonds after H2O2

treatment (Sridharamurthy et al., 2014). By contrast, the oxidizedTGA1,whichwasshown to forman intramolecular disulfidebridge(Després et al., 2003), had a slightly slower mobility than the re-duced form (Supplemental Figure 13). These results suggest thatno intramolecular disulfide bonds are formed in oxidized PP45.Furthermore, H2O2-treated and untreated protein extracts from

PP45 and PP45C350S/C428S were fractionated by size exclusionchromatography to distinguish monomers from dimers. In theabsence of H2O2, His-PP45 eluted as monomers, but in thepresence of H2O2, His-PP45 eluted as dimers and monomers(Figures 9D and 9E). By contrast, His-PP45C350S/C428S eluted onlyas monomers regardless of the presence or absence of H2O2.These results were confirmed using reducing and nonreducingPAGE (Figures 9D and 9E). In addition, the phosphatase activity inthe fractions containing the dimers and the monomers was an-alyzed. H2O2-treatedmonomeric PP45 retained greater than 80%of its phosphatase activity compared to untreated protein, butdimeric PP45only had20%of its phosphatase activity (Figure 9F).We also analyzed the formation of PP45 dimers in rice plants

exposed to ABA or H2O2 treatment. ABA treatment induced theformation of PP45 dimers in wild-type and PP45-OE plants, butnot in rbohB/E-KO plants under the nonreducing conditions(Figures9Gand9H).Undernonreducingconditions,H2O2 inducedthe formation of PP45 dimers not only in wild-type plants but alsoin rbohB/E-KO plants (Figure 9H). The formation of PP45 dimerswas accompanied by a decrease in PP45 monomers under theseconditions. Under reducing conditions, the PP45 dimers dis-appeared, and the PP45 monomers increased, suggesting thatthe PP45 dimers are reduced to PP45 monomers under theseconditions. Finally, PP45 and PP45C350S/C428S were constructedandexpressed inpp45-KO1protoplasts, and theprotoplastsweretreated with H2O2. The H2O2 treatment induced the formation ofPP45 dimers in the pp45-KO1 protoplasts transiently expressingPP45, but not those expressing PP45C350S/C428S (Figure 9I). Thisdemonstrates that the oxidation of Cys-350 and Cys-428 in PP45is necessary for the formation of PP45 dimers in rice protoplastsunder oxidative stress.

Figure 6. (continued).

(C) The survival rate (%) of the rice plants after recovery by rewatering for 10 d shown in (A) and (B).(D)and (E)Malondialdehyde (MDA) content (D)and thepercent leakageof electrolyte (E) in the leavesofDMI3-OE,dmi3-KO,PP45-OE,pp45-KO, andwild-type plants exposed to water stress or oxidative stress. Ten-day-old seedlings were treated with 20% PEG 4000 or 100 mMH2O2 for 2 d, and then leaveswere sampled for thedeterminationofMDAcontent andelectrolyte leakage (%).Approximately 50seedlingsof each transgenic linewereusedper replicate.In (A) and (B), experiments were repeated at least three times with similar results. In (C) to (E), values are means6 SEM of three independent experiments.Means denoted by the same letter did not differ significantly at P < 0.05 according to Duncan’s multiple range test.

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Figure 7. PP45 Directly Inactivates DMI3 by Dephosphorylating Thr-263 in DMI3.

(A)The activity ofDMI3 inPP45-OE,pp45-KO, and thewild type (WT). Rice seedlingswere treatedwith 100mMABA for 90min, and the activity ofDMI3wasanalyzed by immunoprecipitation kinase assay using MBP as substrate (top). Kinase activities were quantitated by Quantity One software (bottom). Theactivity of DMI3 in thewild type in the control was set to 1. DMI3 input was analyzed by IB using an anti-DMI3 antibody.b-actinwas used as the total proteinloading control.(B) PP45 inhibits both autophosphorylation and substrate phosphorylation of DMI3 in vitro. PP45-His and DMI3-GST proteins were expressed in Es-cherichia coli. Images show the activities of autophosphorylation (top) and substrate phosphorylation (bottom) ofDMI3 in thepresence or absenceof PP45.

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PP45C350S/C428S Abolishes the Effect of H2O2 on theDMI3-PP45 Interaction and on PP45-Mediated Inactivationof DMI3

In theabsenceofH2O2,bothPP45andPP45C350S/C428S interacted

with DMI3, as shown by both Y2H assays (Figure 10A) and pull-down assays (Figure 10B). H2O2 treatment strongly inhibited theinteraction between DMI3 and PP45 but had no effect on theinteraction between DMI3 and PP45C350S/C428S. LCI assays intobacco leaves (Figure 10C) and Co-IP assays in rice protoplasts(Figure 10D) also showed that either ABA or H2O2 treatmentinhibited the DMI3-PP45 interaction, but not the DMI3-PP45C350S/C428S interaction. These results indicate that the oxi-dationofCys-350andCys-428 inPP45 is necessary todisrupt theDMI3-PP45 interaction under oxidative stress. Accordingly, in theabsence of H2O2, both PP45 and PP45C350S/C428S significantlyinhibited the activities of autophosphorylation (Figure 11A) andsubstrate phosphorylation of DMI3 (Figure 11B). H2O2 treatmentreleased the inhibition by PP45 but not by PP45C350S/C428S.

Furthermore, in rice protoplasts, ABA treatment for 10 to 15mininduced the formation of PP45dimers, significantly decreased theactivity of PP45, and increased the activity of DMI3 both in wild-type protoplasts and in pp45-KO1 protoplasts transiently ex-pressing PP45 (Figures 11C to 11E). However, in the pp45-KO1protoplasts transiently expressing PP45C350S/C428S, ABA did notinduce the formation of PP45 dimers and led to only a slightdecrease in PP45 activity and a slight increase in DMI3 activity.Quantitative analyses in pp45-KO1 protoplasts transiently ex-pressing PP45 treated with ABA for 10 min showed that thetreatment/control ratios of PP45 level (nonreducing) and PP45activity were 0.47 and 0.44, respectively. The activity of DMI3increased by 3.1-fold, but in thepp45-KO1protoplasts transientlyexpressingPP45C350S/C428S treatedwithABA for 10min, the ratiosof PP45 protein level (nonreducing) and PP45 activity were 0.81and 0.81, respectively, and the activity of DMI3 only increased by1.5-fold (Figures 11E and 11F). Moreover, ABA-induced oxidationof Cys-350 and Cys-428 in PP45 in the rice protoplasts was alsoshown to be required for ABA-induced increase in the activities ofSOD and CAT (Supplemental Figure 14). These results further

indicate that Cys-350 and Cys-428 residues are essential forredox sensing.

DISCUSSION

The activationmechanisms of CCaMKs have beenwidely studiedin both animals and plants. Mammalian CaMKII, which containsan N-terminal kinase domain (catalytic), an autoinhibitory domainoverlapping with a CaM-binding domain (regulatory), and aC-terminal association domain, has been used as a model forCCaMK due to its high sequence homology with CCaMK in boththe kinase and CaM-binding domains. Under basal conditions,CaMKII is autoinhibited due to physical interaction between theregulatory and catalytic domains, blocking catalytic function. Arise in intracellular Ca2+ stimulates binding of Ca2+/CaM to theregulatory domain, releasing the catalytic domain and activatingthe enzyme. The activation leads to autophosphorylation of thekinase at Thr-287. CaMKII autophosphorylation markedly en-hances the binding affinity of CaM and blocks the regulatorydomain from inhibiting catalysis, thereby generating autonomouskinase activity (Erickson, 2014; Luczak and Anderson, 2014). Thisautonomous activity persists until the phosphate group is re-movedbyaproteinphosphatase (PP1orPP2A;Erickson, 2014). Inaddition, CaMKII can be directly modified by ROS, resulting inautonomous activation (Erickson, 2014; Luczak and Anderson,2014).In plants, CCaMKcan bindCa2+ either directly through EF hand

domains or indirectly through a CaM-binding domain. The acti-vationmechanismsofCCaMKhavebeenstudied indetail inLiliumlongiflorum,Medicago truncatula, and Lotus japonicas (Singh andParniske, 2012; Poovaiah et al., 2013; Gobbato, 2015). Underbasal conditions (not treated or stimulated), CCaMK is auto-inhibited, and basal Ca2+-induced autophosphorylation at Thr-271 in Mt-CCaMK (or T265 in Lj-CCaMK or T267 in Li-CCaMK)plays a significant but complex role in the activation of CCaMK. Ithas been hypothesized that Thr-271/Thr-265 phosphorylationstimulates CCaMK activation by promoting interaction with CaM(Poovaiah et al., 2013) via destabilization of intramolecular hy-drogen bonds (Shimoda et al., 2012). However, these models

Figure 7. (continued).

(C)H2O2prevents thePP45-mediated inhibitionofDMI3 invitro. Thegels show theautophosphorylationactivity ofDMI3 (top), theamountsofDMI3 (middle),and PP45 (bottom), respectively. The autophosphorylation activity of DMI3 was also quantified by Quantity One software (bottom; the activity of DMI3without any treatment was set to 1).(D)Schematic diagramsofMt-CCaMKandOs-DMI3 showingdomains andautophosphorylation sites.Note that the aminoacid at position 336ofOs-DMI3is Cys.(E)Release of phosphate from the synthetic peptides. S10, synthetic peptideMSKTESRKLpSDDYEVVD, corresponding to residues 1 to 17 of DMI3; T263,synthetic peptide SFQDHpTWKTISSSA corresponding to residues 258 to 271 of DMI3; S335, synthetic peptide LRAAAIASVLpSCKVAL corresponding toresidues 325 to 340 of DMI3.(F) Dephosphorylation of Thr-263 in DMI3 by PP45 in vitro. Thr-263 phosphorylation of DMI3 was analyzed by IB using an anti-pT263 DMI3 antibody. Theactivity of DMI3 was analyzed using an autophosphorylation assay. DMI3(P)-GST indicates phosphorylated DMI3.(G)Dephosphorylating Thr-263 inDMI3 inPP45-OE,pp45-KO, andwild-type plants exposed toH2O2 treatment. The rice seedlingwere treatedwith 10mMH2O2 for 45 min and Thr-263 phosphorylation was analyzed by IB with anti-pT263 DMI3 antibody, and the activity of DMI3 was measured by immu-noprecipitation kinase assay usingMBPas substrate. DMI3 inputwas analyzed by IBwith DMI3 antibody.b-actin was used as total protein loading control.All experimentswere repeated at least three timeswith similar results. In (A), (C), and (E), values aremeans6 SEM of three independent experiments.Meansdenoted by the same letter did not significantly differ at P < 0.05 according to Duncan’s multiple range test. Molecular mass markers in kD were shown onthe left.

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cannot explain why both phosphoablative and phosphomimeticmutations of this residue led to spontaneous nodulation (Gleasonet al., 2006; Tirichine et al., 2006). By investigating the phos-phorylation dynamics of the Thr-271 residue in Mt-CCaMK,a more recent model suggests that the autophosphorylation atThr-271negatively regulatesCCaMKby stabilizing intramolecularhydrogen bonds, while interaction with Ca2+/CaM during Ca2+

spiking releases the auto-inhibition and promotes CCaMK acti-vation (Miller et al., 2013). This model is more consistent with theexperimental results regarding autophosphorylation of CCaMK.Further phosphorylation of Ser-343/Ser-344 in the CaM-bindingdomain disrupts CaM binding and inhibits CCaMK activity (Liaoet al., 2012; Routray et al., 2013). However, it is unknown whethera phosphatase is involved in the regulation of CCaMK.

Here,weshowadirectphysical interactionbetweenDMI3,a riceCCaMK, and PP45, a PP2C-type phosphatase (Figure 1). Thisinteraction is between the CaM-binding domain of DMI3 and the

PP2C domain of PP45 (Supplemental Figure 2). We found thatPP45 directly inactivates DMI3 by dephosphorylating Thr-263 inDMI3 (Figure 7) and that Thr-263 autophosphorylation is requiredfor the activation of DMI3, CaM binding to DMI3, and DMI3-mediated antioxidant defense (Figure 8). We propose a modelfor the inhibition of DMI3 by PP45 under the basal state and itsABA-induced activation (Figure 12).PP2Cs are a class of evolutionarily conserved Ser/Thr protein

phosphatases and have been shown to be involved in a widevariety of plant processes, including ABA signaling, biotic andabiotic stress responses, plant immunity, K+ nutrient signaling,and plant development (Schweighofer et al., 2004; Singh et al.,2010, 2016;Fuchset al., 2013). In thegenomesofArabidopsis andrice, there are 80 and 90 members, respectively, and these aredivided into11subgroups (Singhet al., 2010; Fuchset al., 2013). InArabidopsis, thebest studiedPP2Csbelong togroupA,whichhasnine members, including ABI1, ABI2, and HAB1. The members in

Figure 8. Thr-263 Autophosphorylation Is Necessary for the Activation of DMI3 and the Response It Mediates.

(A)AutophosphorylationofDMI3,DMI3T263A, andDMI3T263D invitro.Autophosphorylationactivity in thepresenceofeither5mMEGTA (2), 0.1mMCaCl2, or0.1mMCaCl2 and1mMCaMwasanalyzedby in vitro kinase assay (top) andquantifiedbyQuantityOnesoftware (bottom; the activity ofDMI3wild type [WT]without any treatment was set to 1). Corresponding Coomassie staining was also shown (middle).(B) CaM binding of DMI3, DMI3T263A, and DMI3T263D in vitro. The CaM binding affinities of the mutant and wild-type protein extracts were tested in thepresence of 1 mM CaCl2 (bottom) or 5 mM EGTA (middle).(C) CaM binding of DMI3, DMI3T263A, and DMI3T263D in rice protoplasts. The indicated constructs were transfected into rice protoplasts. Total isolatedproteinswere incubatedwithananti-Mycantibodyandweredetectedby IBusingananti-CaMantibody (top). The inputprotein is shownby IBanalysis usingan anti-Myc antibody (bottom).(D) Substrate phosphorylation of DMI3, DMI3T263A, and DMI3T263D in vitro. Images show substrate phosphorylation (top, MBP as substrate), the cor-respondingCoomassie staining (middle), and the relativeactivityofsubstratephosphorylation (bottom). TheactivityofuntreatedDMI3wild typewasset to1.(E)The activity ofMPK1 in theprotoplastswith transiently expressedDMI3orDMI3T263AorDMI3T263D. The activity ofMPK1 in theprotoplastswas analyzedby immunoprecipitation kinase assay (top). b-actin was used as a loading control (bottom).(F) and (G)The activities of SOD (F) andCAT (G) in protoplasts transiently expressingDMI3orDMI3T263AorDMI3T263D. Protoplastswere treatedwith 10mMABA for 10min. All experiments were repeated at least three timeswith similar results. In (A), (D), (F), and (G), values aremeans6 SEM of three independentexperiments.Means denoted by the same letter did not significantly differ at P < 0.05 according toDuncan’smultiple range test.Molecularmassmarkers inkilodaltons were shown on the left.

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Figure 9. H2O2 Directly Inhibits the Activity of PP45 by Oxidizing Cys-350 and Cys-428 Residues to Form PP45 Intermolecular Dimers.

(A) The activities of PP45-His wild-type and Cys point-mutant proteins exposed to 0.1 mM H2O2.(B) The effects of H2O2 at different concentrations on the activities of PP45-His wild-type and C350S and C428S single- and double-mutant proteins.

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group A are negative regulators of ABA responses (Schweighoferet al., 2004; Umezawa et al., 2010; Fuchs et al., 2013; Singh et al.,2016). In early ABA signal transduction, there are three majorcomponents: pyrabactin resistance (PYR)/PYR1-like/regulatorycomponents of ABA receptors (ABA receptors), group A PP2Cs(negative regulators), andsubclass IIISnRK2s (positive regulators;Fujii et al., 2009; Ma et al., 2009; Park et al., 2009; Umezawa et al.,2009; Vlad et al., 2009). In the absence of ABA, PP2Cs inactivateSnRK2s by direct dephosphorylation. In the presence of ABA,PYR/PYR1-like/regulatory components of ABA receptors interactwith PP2Cs and inhibit phosphatase activity, allowing SnRK2activation and phosphorylation of target proteins.

In this study, however, PP45 did not interact with rice SnRK2sSAPK8/9/10 (Supplemental Figures 3B and 3D), suggesting thatPP45 is not a coreceptor for ABA signaling. However, we foundthat ABA treatment leads to a transient and significant decrease inthe expression of PP45, the PP45 protein level, and its activity inleaves of rice plants (Figure 3). ABA also inhibited the in vivo in-teraction between PP45 and DMI3 (Figure 2). The ABA-inducedincrease in the activity of DMI3 was blocked in the PP45-OEplants, and it was further enhanced in the pp45-KO plants(Figure 7A). Our genetic data further show that PP45 nega-tively regulates ABA responses, including seed germination, rootgrowth, antioxidant defense, and tolerance to water stress andoxidative stress (Figures 5 and 6; Supplemental Figures 8 to 10).Overall, our resultsclearly indicate thatPP45,amemberofgroupKPP2Cs, is also an important negative regulator of ABA signaling.

How does ABA relieve the PP45-mediated inhibition in theactivity of DMI3? Previous studies have shown that some PP2Cs,such as ABI1, ABI2, and HAB1, are redox-sensitive proteins(Meinhard and Grill, 2001; Meinhard et al., 2002; Sridharamurthyet al., 2014; Zhu et al., 2014) and that the Cys residues in theseproteins are themain targets of ROS (Akter et al., 2015;Waszczaket al., 2015). It was suggested that H2O2 reversibly inhibited theactivities of ABI1 and ABI2 probably via oxidation of critical Cysresidues (MeinhardandGrill, 2001;Meinhardet al., 2002). A recentstudy identified HAB1 Cys-186 and Cys-274 as H2O2-sensitivethiols and demonstrated that their oxidation inhibits both HAB1

catalytic activity and its ability to physically associate withSnRK2.6 via formation of intermolecular dimers (Sridharamurthyet al., 2014). In Arabidopsis, it has been demonstrated that ROSare rate-limiting secondmessengers in ABA signaling, and RBOHsubunitsD andFaremajorNADPHoxidase catalytic subunits thatmediate ABA-induced ROSproduction (Kwak et al., 2003). In rice,it has been shown that RbohB and RbohE are involved in ABA-induced H2O2 production (Shi et al., 2012; Zhang et al., 2014).However, it is not clear whether ABA inhibits the activity of PP45via the production of H2O2 induced by ABA.In this study, we provide evidence to show that ABA-induced

H2O2 by RbohB/E is required for ABA-induced inhibition in PP45activity (Figure 4) and in the interaction of PP45 and DMI3(Figure 2E).We identifiedCys-350andCys-428 inPP45as the keyCys residues that mediate oxidative inactivation of PP45 andshowed that their oxidation resulted in formation of catalyticallyinactive PP45 intermolecular dimers (Figures 9A to 9F). ABAtreatment induced the formation of PP45 dimers in wild-type, butnot in rbohB/E-KO1 plants (Figure 9H), indicating that RbohB/E-mediated H2O2 production is required for ABA-induced for-mation of PP45 dimers. The oxidation of Cys-350 and Cys-428 inPP45 induced by ABA was also necessary to disrupt the DMI3-PP45 interaction (Figure10)andPP45-mediated inhibition inDMI3activity (Figure 11).Taken together, our results suggest that, during ABA signaling,

RbohB/E-mediated oxidation of Cys-350 and Cys-428 in PP45playsacrucial role in the regulationofboth the interactionofDMI3-PP45 and the activity of DMI3 (Figure 12). However, our resultsalso showed that ABA and H2O2 induced a transient down-regulation in the expression of PP45 (Figure 3A) and the level ofPP45 (Figure 3B) in rice leaves. In the pp45-KO1 protoplaststransiently expressing PP45C350S/C428S treated with ABA, theamount of PP45 protein and the activity of PP45 did not com-pletely return to the control levels (Figure 11F), and the activity ofDMI3 still had a slight but significant increase (Figure 11E). Theseresults suggest that the decrease in the expression of PP45 alsocontributes to the inhibition of PP45 and the activation of DMI3 inABA signaling (Figure 12).

Figure 9. (continued).

(C) Reducing and nonreducing PAGE analysis of protein extracts from PP45 wild type (W) and OsPP45C350S/C428S double mutant (M) in the presence andabsence of 0.1 mM H2O2 in vitro.(D) and (E) Top shows the gel filtration profiles of PP45-His wild type (D) and PP45C350S/C428S-Hismutant (E) in the presence and absence of 0.1mMH2O2.PP45-His elutes primarily as monomers in the absence of H2O2 (blue chromatogram) and as a mixture of dimers and monomers following treatment with0.1mMH2O2 (red chromatogram). By contrast, PP45C350S/C428S-His eluted exclusively asmonomers. mAU, absorbance at 280 nm3 1000. Bottom showsreducing and nonreducing PAGE analyses of gel filtration fractions of PP45-His (D) and PP45C350S/C428S -His (E) in the presence and absence of H2O2,respectively. Lanes 1 and 5, PP45-His monomer peak (15.5-mL fraction); 2 and 6, PP45-His dimer peak (14.5-mL fraction); 3 and 7, PP45C350S/C428S-Hismonomer peak (15.5-mL fraction); 4 and 8, PP45C350S/C428S-His dimer peak (14.7-mL fraction).(F) The relative activity of PP45 protein from the untreated monomer fraction (no H2O2), from the dimer and monomer fractions of PP45-His treated with0.1 mM H2O2 and from the monomer fraction of PP45C350S/C428S-His treated with 0.1 mM H2O2.(G) and (H)ABAandH2O2 induce the formation of PP45dimers in rice plants.PP45-OE (G), rbohB/E-KO (H), andwild-type plantswere treatedwith 100mMABA for 90min or with 10mMH2O2 for 45min, and both nonreducing (top) and reducing (middle) IB of PP45were performed.b-actin recognized by an anti-b-actin antibody was used as a protein loading control (bottom).(I) H2O2 induces the formation of PP45 dimers in the pp45-KO1 protoplasts transiently expressing PP45, but not in protoplasts transiently expressingPP45C350S/C428S. The indicatedconstructswereexpressed inpp45-KO1protoplasts, and theprotoplastswere treatedwith1mMH2O2 for 10min. The levelsofb-actin indicate equal protein loading. All experimentswere repeated at least three timeswith similar results. In (A), (B), and (F), values aremeans6 SEM ofthree independentexperiments.Meansdenotedby thesame letterdidnotsignificantlydiffer atP<0.05according toDuncan’smultiple range test.Molecularmass markers in kD are shown on the left.

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In this study, however, the expressionofPP45, the level of PP45protein, and the activity of PP45 in leaves of rice plants were alsoshown to be upregulated by the ABA and H2O2 following a tran-sientdownregulation (Figure3).Apreviousstudyalsoshowed thattreatments with ABA and H2O2 induced the expression of PP45in leaves of rice seedlings (Hu et al., 2006). But why can ABA and

H2O2 also induce the activation of PP45? This may be associatedwith the upregulation of antioxidant defense by DMI3 in ABA andH2O2 signaling. It was shown that ABA-induced H2O2 productionactivatesDMI3 and that the activation ofDMI3 is required for ABA-induced antioxidant defense in rice (Shi et al., 2012). The upre-gulation in the activities of antioxidant enzymes enhances the

Figure 10. The Oxidation of Cys-350 and Cys-428 in PP45 Is Necessary to Relieve DMI3-PP45 Interaction.

(A) Y2H assay in the absence or presence of 0.1 mM H2O2. SD-Trp-Leu-His-Ade/AbA/X-a-gal medium was used for testing the interactions.(B) In vitro pull-down assay in the absence or presence of 0.1 mM H2O2.(C) LCI assay in tobacco leaves. The indicated constructs were expressed in Nicotiana benthamiana leaves, and the leaves were treated with 100 mMABA for 90min or 10mMH2O2 for 45min. Scale bar, 2 cm. The IB shows the expression levels of Myc-DMI3-cLUC and His-PP45-nLUC in tobacco leavesafter treatment. The Rubisco large subunit visualized by staining with Coomassie Brilliant Blue was used as a loading control.(D)Co-IPassay in riceprotoplasts. The indicatedconstructswereexpressed in riceprotoplasts, and theprotoplastswere treatedwith10mMABAfor10min.Total protoplastproteinswere immunoprecipitated (IP) usingananti-His antibodyandwere thendetectedwithananti-Mycantibodyor ananti-His antibody.Protein input is shownby IBanalysis of protein extractsbefore IPandantibodiesagainst the respective tags.All theexperimentswere repeatedat least threetimes with similar results. Molecular mass markers in kD are indicated on the left.

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Figure 11. The Oxidation of Cys-350 and Cys-428 in PP45 Prevents the PP45-Mediated Inhibition of DMI3 Activity.

(A) and (B)Autophosphorylation (A) and substrate phosphorylation (B) byDMI3 alongwith PP45-His or PP45C350S/C428S-His in the absence or presence of0.1 mMH2O2. Top shows autophosphorylation (A) and substrate phosphorylation (B) by DMI3, middle shows the corresponding Coomassie staining, andbottom shows the relative activity of phosphorylation. The activity of DMI3 without any treatment was set to 1.(C) The oxidation of Cys-350 andCys-428 in PP45 induced by ABA relieves the PP45-mediated inhibition of DMI3 activity in rice protoplasts. The indicatedconstructs were expressed in pp45-KO1 protoplasts, and the protoplasts were treated with 10mMABA for various times as indicated. PP45was detectedusing nonreducing or reducing IB using an anti-PP45 antibody. The activity of DMI3 was measured by immunoprecipitation kinase assay using MBP asa substrate. The levels of b-actin indicate equal protein loading.

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abilityofcells toscavengeH2O2.This, in turn, relieves the inhibitionof PP45 by H2O2, thus resulting in the upregulation of PP45. Thedecrease in H2O2was shown to remove ABA-induced inhibition inthe expression of PP45 and the activity of PP45 in rice leaves(Figures 4A to 4D). In addition, the catalytically inactive PP45intermolecular disulfide bonds induced by ABAmight be reducedby thioredoxins, which have been shown to control the reductionof ROS-mediated intra- or intermolecular disulfide bonds in plantcells (Waszczak et al., 2015), thus resulting in the up-regulation

of PP45 activity. However, the exact mechanism for the up-regulation of PP45 in ABA signaling remains to be determined.

METHODS

Plant Materials and Growth Conditions

All rice (Oryza sativa) plants used in this study were the rice cultivar Nip-ponbare or were derived from this cultivar. Rice seedlings were grown

Figure 11. (continued).

(D)Theactivity ofPP45 inwild-type (WT)protoplasts or the transfectedprotoplasts ofpp45-KO1exposed to10mMABA treatment. Theactivity ofPP45wasmeasured using the Ser/Thr phosphatase assay.(E)Quantitative analysis of DMI3 activity in (C). The relative activity of DMI3 was quantified by Quantity One software, and the activity of DMI3 in wild-typeprotoplasts treated with ABA for 0 min was set to 1.(F) The treatment/control ratio of the protein level in (C) and the activity in (D) of PP45. For quantitative analysis of band intensity, the starting point (0 h) wasset to 1.NR, nonreducing; R, reducing. All experimentswere repeated at least three timeswith similar results. In (A), (B), (D), (E), and (F), values aremeans6SEM of three independent experiments. Means denoted by the same letter did not differ significantly at P < 0.05 according to Duncan’s multiple range test.Molecular mass markers in kD are indicated on the left.

Figure 12. Proposed Model for the Activation of DMI3 in ABA Signaling.

In the basal state, the PP2C domain of PP45 interacts with the CaM-binding domain (CaMBD) of DMI3 and then dephosphorylates Thr-263 in DMI3,inactivatingDMI3 (left). In the presence of ABA, ABA-inducedH2O2 production byRbohB/E inhibits the activity of PP45 in twoways: both by inhibitingPP45and production of its protein and by directly oxidizing the Cys-350 and Cys-428 residues of PP45, forming intermolecular dimers of PP45 (middle). Theoxidation of Cys-350 and Cys-428 in PP45 induced by ABA is crucial both for the inhibition of PP45 activity and for the release of PP45-DMI3 interaction.DMI3 is thus released from thenegative regulation andconverted to an active formand is further activatedbyH2O2-inducedCa2+/CaM (right), thus resultingin ABA responses.

146 The Plant Cell

under theconditions asdescribedpreviously (Zhanget al., 2014).When thesecond leaves were fully expanded, they were collected and used forinvestigations.

Y2H Screening

The full-length DMI3 was fused into the pGBKT7 vector (Clontech) as thebait. Total RNA was isolated from rice leaves and transcribed into single-stranded cDNAs. cDNA fragments longer than 500 bp were transformedwith linearized pGADT7 vector (Clontech) to construct a rice cDNA library.Using the lithium acetate method described in Clontech’s yeast protocolshandbook, the bait construct was transformed into yeast strain Y2HGold,and the library was transformed into yeast strain Y187. Screening of in-teractioncloneswasperformedviamatingaccording to themanufacturer’sinstructions (Clontech). A total of 106 transformants from the cDNA librarywere screened for growth on the stringent SD-Leu-Trp-His-Ade dropoutmedium. Positive clones were able to activate the transcription of fourindependent reporter genes (AUR1-C,ADE2,HIS3, andMEL1). The libraryplasmid responsible for the activation of reporters was rescued, analyzedby restriction digestion, and transformed again into Y187. Yeast cells withpreys were mated one-on-one in parallel against the yeast Y2HGold ex-pressing the bait or the empty vector pGBKT7 as the negative control.

Y2H Assay

PP45wasamplifiedbyPCRandwascloned into pGADT7. Prey constructswere transformed intoyeaststrainY187,andbait vectorswere transformedinto yeast strain Y2HGold by the lithium acetate method noted above. Theprey strain and the bait strain were mated, and the mating cultures werespread on stringent selective medium plates containing X-a-gal (40 mgmL21). The plateswere incubated at 30°C for 4 to 6 d. Plateswere checkedevery 12 h for the development of blue color. Expressed proteins weretested for interactionby thequantitative interactionassay (b-galactosidaseassay) described in Clontech’s yeast protocols handbook.

Expression of DMI3 and PP45

The DMI3 cDNA fragment was PCR amplified and cloned into pGEX-4T-1(Amersham Pharmacia Biotech) using the SmaI and XhoI sites for GSTfusion. To express the fusion protein, Escherichia coli Rosetta (DE3; No-vagen) harboring the plasmids was induced with 0.5mM isopropyl-1-thio-b-D-galactopyranoside in Luria-Bertani broth for 6 h at 24°C. DMI3-GSTwas purified using the MagnetGST protein purification system (Promega).The PP45 cDNA fragment was PCR amplified and inserted in frame at theKpnI and BamHI sites of pET30a (Novagen) for fusion to a His tag. Toexpress the fusion protein, Escherichia coli Rosetta (DE3) harboring theplasmids was induced with 0.5 mM isopropyl-1-thio-b-D-galactopyrano-side in Luria-Bertani broth for 8 h at 20°C. PP45-His was purified usingMagnetHis protein purification system (Promega).

Pull-Down Assay

Todetect interactionbetweenDMI3andPP45 invitro,GST-taggedDMI3orGST alone was immobilized on the Magnet GST Particles (Promega). Theparticles were incubated with PP45-His in pull-down binding buffer(4.2 mMNa2HPO4, 2 mMKH2PO4, 140mMNaCl, 10 mMKCl, 10% bovineserum albumin [BSA], pH 7.2) with gentle shaking at 4°C for 2 h. Theparticles werewashed at least three times inwash buffer (interaction bufferwithout BSA) and boiled in 13 SDS loading buffer (50 mM Tris-HCl, 2%SDS, 0.1% bromophenol blue, 10% glycerol, 10 mM DTT). Samples wereseparated on 12% SDS-PAGE gels and analyzed by IB using an anti-GSTantibody (Abmart, lot 264160; 1:1000, v/v) or anti-His antibody (Abmart, lot283874, 1:1000, v/v).

BiFC Assay

cDNA ofDMI3without its stop codonwas amplified using PCR and clonedinto the KpnI site of pSPYCE155 (Waadt et al., 2008) to construct SYFPC-DMI3. The cDNA of PP45 without its stop codon was cloned intopSPYCE173 (Waadt et al., 2008) using theKpnI andXhoI sites to constructSYFPN-PP45. Using the particle bombardment (Bio-Rad) method, theconstructs were transiently expressed in onion epidermis cells as de-scribed previously (Lee et al., 2008). Fluorescence was detected 16 hfollowing transfectionusinga laser confocalmicroscope (TCS-SP2, Leica),with excitation at 530 nm and emission at 525 nm.

Luciferase Complementation Imaging Assay

Full-length and truncated sequences of PP45 with His tags were fused tothepC1300-nLUC (luciferasecomplementation) vector usingKpnI andSalIsites. Full-length and truncated sequences of DMI3 with Myc-tags se-quenceswere fused to the pC1300-cLUC vector usingKpnI andPstI sites.The constructed plasmid vectors were transformed into Agrobacteriumstrain GV3101 (Nova Lifetech). The positive clone was incubated in yeastextract broth liquid medium at 28°C for 16 h (Sparkes et al., 2006). Thebacteria were mixed to a final OD600 of 0.5 and then were collected andresuspended in 2mL of activity buffer (10mMMES, pH 5.7, 10mMMgCl2,150 mM acetosyringone). After 2 to 5 h, the activity bacteria were injectedinto youngNicotianabenthamiana leaves. After 3 to 5d, theabaxial sidesofleaves were treated with 100 mMABA for 90min or 10mMH2O2 for 45minand sprayedwith 1mM luciferin (Thermo Fisher Scientific) and then kept inthe dark for 1 h. A camera (Tanon 5200 Multi, Tanon Biomart) was used tocapture the LUC signal.

Co-IP Assay

The coding sequences of DMI3 or PP45 were fused with sequencesencodingMyc-orHis-tagsandcloned intopXZP008vectoratKpnI orXhoI-KpnI sites, respectively. Riceprotoplastswere transfectedwith35S:DMI3-Myc and 35S: PP45-His by PEG-calcium-mediated transformation (Yooet al., 2007). After incubation for 16 h, the proteins from protoplasts wereextracted with buffer (20 mM Tris-HCl, pH 7.5, 20 mM KCl, 1 mM EDTA,10 mM Na3VO4, 0.5% (v/v) Triton X-100, 10% glycerol, 5 mg mL21 leu-peptin, 5 mg mL21 aprotinin) and centrifuged at 12,000g for 30 min at 4°C.The supernatants were incubated with an anti-Myc antibody (Abmart, lot294166; 1:1000, v/v) bound to protein A beads in IP buffer (20 mM Tris-HCl, pH 7.5, 20 mM KCl, 150 mM NaCl, 10 mM MgCl2, 10 mM Na3VO4,5 mg mL21 leupeptin, 5 mg mL21 aprotinin) at 4°C for 6 h. The beadswere washed three times with IP buffer, and the proteins were elutedby boiling in 13 SDS sample buffer for 5 min. After centrifugation, thesupernatant fractionwas analyzedby12%SDS-PAGE, followedby IBwithan anti-His antibody.

Immunocomplex Kinase Activity Assay for DMI3 or MPK1

Protein was extracted from rice leaves or protoplasts with buffer (20 mMTris-HCl, pH7.5, 20mMKCl, 1mMEDTA, 10mMNa3VO4, 0.5% [v/v] TritonX-100, 10% glycerol, 5 mg mL21 leupeptin, 5 mg mL21 aprotinin) and wascentrifuged at 12,000g for 30 min at 4°C. Protein content was determinedaccording to the method of Bradford (1976) with BSA as the standard.Protein extracts (100mg) were incubatedwith an anti-DMI3 antibody (2mg,Abmart) or anti-MPK1 antibody (2 mg, Bejing Protein Innovation) bound toprotein Abeads. Kinase activity in the immunocomplexwas determined byan in-gel kinase assay using myelin basic protein (MBP) as substrate asdescribed previously (Shi et al., 2012, 2014). In brief, 1 mg MBP (Sigma-Aldrich)was incubated either with DMI3 in reaction buffer (25mMTris-HCl,pH7.5, 5 mM MgCl2, 0.5 mM CaCl2, 2 mM CaM [Sigma-Aldrich]) or withMPK1 in reaction buffer (25 mM Tris, pH 7.5, 5 mMMgCl2, 1 mM ethylene

The Plant Cell 147

glycol tetraacetic acid [EGTA], 0.1 mM Na3VO4) with 10 mCi [g32P]-ATP(3000 Ci mM21) at 30°C for 30 min. The reaction products were separatedby 12% SDS-PAGE and analyzed by autoradiography using X-ray film ora phosphostorage screen (Typhoon TRIO, Amersham Biosciences).

In the experiments for detecting the effect of PP45 on the activity ofDMI3, the sample was divided into two parts: one with PP45-His and theother without PP45-His. The two parts were incubated in buffer (20 mMTris-HCl, pH 7.5, 20 mM MgCl2, 1 mM EDTA, 100 mM NaCl) at 30°Cfor 30 min. Relative levels of DMI3 or MPK1 activity, detected by im-munocomplex kinase activity assay and quantified by Quantity Onesoftware (Bio-Rad), are presented as values relative to those of the cor-responding controls.

SDS-PAGE and IB Analysis

Protein extracts (20 mg) were separated by 12% SDS-PAGE. For non-reducing PAGE, the Native PAGE Bis-Tris Gel System (ThermoFisher Scientific) was used. After electrophoresis, the gel was trans-ferred to a polyvinylidene difluoridemembrane at 110 V for 60min at 4°Cin a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad). Themembrane was incubated in blocking solution containing PBS/Tween(140mMNaCl, 10mMKCl, 2mMKH2PO4, 8mMNa2HPO4, 0.1%Tween-20 [v/w], pH 7.6) that was supplementedwith 5% (w/v) nonfat drymilk for2 h at room temperature. The membrane was then washed with PBS/Tween buffer three times for 10 min. The blots were probed with anti-PP45 antibody (ABclonal), anti-DMI3 antibody (ABclonal), anti-ACT1antibody (Beijing Protein Innovation), anti-DMI3T263(P) antibody (AB-clonal), anti-CaM antibody (Sigma-Aldrich), anti-Myc antibody, anti-Hisantibody, and anti-GST antibody. Information about the anti-DMI3antibody was described previously (Shi et al., 2012). The anti-DMI3antibody was raised against peptides of DMI3. The anti-PP45 antibodywas raisedagainst full lengthofPP45. The anti-DMI3T263(p) antibodywasraised against peptide (SFGDHTWKTISSSA) of DMI3. The anti-GSTantibody, anti-Myc antibody, and anti-His antibody were obtainedfrom immunized mice, and the other antibodies were obtained fromimmunized rabbits. The primary antibody was used at 1:1000 dilutionand secondary antibody was used at 1:4000 dilution. Chemiluminescencewas detected with the enhanced chemiluminescence immnoublotting de-tection system (GE Healthcare) and Kodak XJ300 film or a camera (Tanon5200Multi).QuantificationwasdoneusingQuantityOnesoftware (Bio-Rad).For quantitative analysis, the data were normalized by dividing the bandintensity of PP45 by the band intensity of b-actin in each lane. The startingpoint (0 min) was set to 1; other points were compared with it.

Autophosphorylation Assay

GST-tagged DMI3 was immobilized on Magnet GST particles and elutedwith buffer (50 mM Tris-HCl, 50 mM glutathione, pH 8.0). Ten microgramsDMI3-GST fusion protein was added in a total 50-mL reaction buffer for10min at 30°C in 25mMTris-HCl (pH 7.5) containing 5mMMgCl2,100mMNaCl,10 mCi of [g32P]-ATP (3000 Ci mM21) with 0.5 mM CaCl2 or 5 mMEGTA. The reaction mixtures were incubated with or without PP45-Hisprotein at 30°C for 30min. The kinase reactionwas stopped bymixingwithSDS-PAGE sample buffer and boiling for 5 min. Samples were separatedon 12%SDS-PAGE gels, and the phosphorylated DMI3 was visualized byautoradiography.

In Vitro Kinase Assay

An invitro kinaseassaywasperformedusing10mgof theDMI3-GST fusionprotein in reaction buffer (25 mM Tris, pH 7.5, 5 mMMgCl2, 0.5 mMCaCl2,2mMCaM) at 30°C for 30min in a final volume of 50mL that also contained1 mg MBP, 10 mM ATP, 10 mCi [g32P]-ATP. The reaction mixtures were

incubated with or without PP45-His at 30°C for another 30 min. The re-actionwas stoppedby addingSDSsample buffer and the reactionmixwasseparated on 12% SDS-PAGE. The phosphorylated substrates were vi-sualized by autoradiography.

Site-Directed Mutagenesis

To mutate PP45 and DMI3, the Multi-Directed Mutagenesis Kit (AgilentTechnologies) was used according to themanufacturer’s instructions. TheDNA oligonucleotides used in mutagenesis were synthesized, and theirsequences are listed in Supplemental Table 1. Aftermutagenesis, all of themutated plasmids were confirmed by Sanger sequencing.

In Vitro Phosphate Measurement

Based on DMI3 phosphorylation sites, phosphorylated polypeptides weresynthetized from ABclonal Biotech. To measure phosphate release fromthe synthetic phosphopeptides, the phosphopeptides (10 nM) were in-cubated for 15 min with 1 mg of OsPP45 or calf intestine alkaline phos-phatase in phosphatase buffer (40 mL) at 30°C. Released phosphate wasmeasured according to the method described previously (Van Veldhovenand Mannaerts, 1987).

Ser/Thr Phosphatase Assay System for PP45 Activity

Protein was extracted from rice leaves with a phosphatase storage buffer(10 mM Tris, pH 7.5, 1 mM EDTA, 0.02% [w/v] sodium azide). After cen-trifugationat100,000g for 1hat4°C, thesupernatantswere transferred intospin columns (Promega), followed by resuspending with Sephadex G-25resin (Promega) and centrifugation at 600g for 5 min. The sample flowthrough in the tube contained the total PP2Cs. After extraction of totalPP2C protein, the protein concentration was determined by CoomassieBrilliant Blue method (Braford, 1976). Each time point sample used thesame amount of anti-PP45 antibody (ABclonal) and protein A-sepharosebeads. According to the instructions of Serine/Threonine PhosphataseAssay System (Promega), a standard curve is required before every PP45activity assay. Samples containing 0, 100, 200, 500, 1000, and 2000 pmolfree phosphate in reaction buffer were used to prepare a standard curve.PP45was added into the reaction buffer (250mM imidazole, pH 7.2, 1mMEGTA, 25 mM MgCl2, 0.1% [w/v] b-mercaptoethanol, 0.5 mg mL21 BSA)with 1 mM phosphopeptide (Promega) at room temperature for 15 to30 min. After reaction, the optical density of the samples was determinedusing a plate reader with a 630-nm filter. The phosphatase activity wascalculated by comparing with the phosphate curve and was given in pmolPO4 per minute per mg of protein.

The purified PP45-His mutant and wild-type protein expressed fromE. coliwere incubated in the presence of various concentrations of H2O2

in the reaction buffer with 1 mM phosphopeptide at room temperaturefor 15 to 30 min. The PP45 activity in the reaction mixture was thenmeasured.

To test whether reducing agents affect the activity of H2O2-treatedPP45, PP45 extracts were preincubated with 0.1 mM H2O2 for 30 min andthen treated with the different concentrations of TCEP (Sigma-Aldrich) orDTT for 30min. The activity of PP45wasmeasured by the serine/threoninephosphatase assay noted above.

Size Exclusion Chromatography

The purified wild type and C350S/C428S mutant PP45-His proteins ex-pressed from E. coliwere treatedwith 0.1mMH2O2 for 30min at 30°C. Theproteins were loaded onto Superdex 200 Increase 10/300 gel columns(Amersham Bioscience) connected on an AKTA explorer box-900 PH/C-900 (AmershamBioscience) liquid chromatography system equipped with

148 The Plant Cell

the UV detector set to detect absorbance at 280 nm. Samples were elutedwith PBS to 0.7 mL min21 flow rate and collected into 2 mL fractions. Thefractions were confirmed by reducing and nonreducing PAGE. The voidvolume of the size exclusion chromatography column is ;8 mL.

CaM Binding Assay In Vitro and In Vivo

For in vitro assays, the full-lengthCaM1 cDNA fragment was PCR amplifiedandcloned intopGEX-4T-1using theEcoRI andXhoI sites forGST fusion. Toexpress the fusion protein, Escherichia coli Rosetta (DE3) harboring theplasmidswas inducedwith 0.5mM isopropyl-1-thio-b-D-galactopyranosidein Luria-Bertani broth for 4 h at 24°C. Purified wild-type and mutant DMI3proteins were separated by SDS-PAGE and electrophoretically transferredonto a polyvinylidene difluoride membrane. The membrane was sub-sequently incubatedwith binding buffer (10mMTris/HCl, 150mMNaCl, and5% [w/v] nonfat milk) containing CaM1 with 1 mMCaCl2 or 5 mM EGTA for24 h at 4°C. The membrane was then washed three times. The bound CaMsignal was detected by IB using an anti-CaM antibody.

For in vivo assays, the construct 35S:DMI3-Mycwas delivered into riceprotoplasts by PEG-calcium-mediated transformation. The total proteinsextracted from protoplasts were incubated with anti-Myc antibody boundtoproteinAbeads for6h.Thebeadswerecollected,washed, andboiled for5 min with 13 SDS sample buffer. Samples were analyzed by 12% SDS-PAGE followed by IB with an anti-CaM antibody.

Quantitative RT-PCR Analyses

Total RNA was isolated from rice leaves using RNAiso Plus (TaKaRa)according to the manufacturer’s instructions. DNase treatment was in-cluded in the isolationstepusingRNase-freeDNase.Approximately 2mgoftotal RNAwas reverse transcribed using anoligo d(T)16 primer andM-MLVreverse transcriptase (TaKaRa). Real-time quantitative RT-PCR was per-formed on a 7500 real-time PCR system (Applied Biosystems) using SYBRPremix Ex TaqTM (TaKaRa) according to the manufacturer’s instructions.cDNA was amplified by PCR using the primers shown in SupplementalTable 2. The expression level was normalized against that of rice glyc-eraldehyde-3-phosphate dehydrogenase gene. The relative expressionlevels of the target genes were calculated as x-fold changes relative tothe appropriate control experiment for the different chemical treatments.

Transfection of Protoplasts with Plasmid DNAs

Rice protoplasts were isolated according to the method described pre-viously (Zhang et al., 2012). The plasmids were delivered into protoplastsusing a PEG-calcium-mediated method (Yoo et al., 2007). About 10 mgplasmid DNAs per 100 mL 3 106 protoplasts were used for transient ex-pression analysis.

Generation of PP45, DMI3, and RbohB/E Transgenic Rice Lines

Transgenic lines (PP45-OE and DMI3-OE) were generated by Biorun Bio-technology. To obtain the transgenic plants overexpressing PP45 or DMI3,the full-length open reading frame of either PP45 or DMI3was inserted intotheplantbinaryvectorpBWA(V)HS.Then, thePP45geneorDMI3geneunderthe control of CaMV 35S promoter was transformed into rice (O. sativa sub.japonica cv Nipponbare) by the Agrobacterium-mediated transformationmethod (Hiei et al., 1994). Homozygous T3 seeds of the transgenic plantswere used for further analysis.

The dmi3-KO, pp45-KO, and rbohB/E-KO plants were generated usingtheCRISPR/Cas9 system (Biogle). The sgRNAs ofDMI3,PP45,RbohB, andRbohE are shown in Supplemental Figures 5 and 7. The single sgRNA wascreated in the BGK03 vector containing Cas9, which was introduced intoAgrobacterium strain EHA105 and transformed into rice (O. sativa sub.

japonica cv Nipponbare). To examine the function of CRISPR/Cas9 in vivo,genomicDNAwasextracted fromtransgenicplantsandprimerpairsflankingthe designed target site were used for PCR amplification (SupplementalTable 2). Sequencealignment revealed that twoorone independentmutantsof each gene, such asdmi3-KO1anddmi3-KO2,pp45-KO1andpp45-KO2,and rbohB/E-KO1 were obtained (Supplemental Figures 5 and 7).

Phenotype Analysis

For seed germination assays, seeds of the wild type, PP45-OE, pp45-KO,DMI3-OE, and dmi3-KO were plated in triplicate on 1/23 Murashige andSkoog medium plates. The medium contained 0.8% agar and was sup-plemented with different concentrations of ABA (0, 1, and 5 mM). The seedswere incubated at 4°C for 2 d before being placed in a growth chamber togerminate (16 h light/8 h dark; 200 mmol m22 s21 light intensity; 26°C), andgermination (emergence of radicals) was scored at the indicated times. Forthe analysis of rootgrowth, 4-d-old seedlingswere individually transferred tothe1/23MurashigeandSkoogmediumwithdifferentconcentrationsofABA(0, 0.5, l, 3, 5, 10, and20mM). Seedlingswere further incubated in the growthchamber foranother14d,and then the lengthofprimary rootswasmeasuredusinga ruler. Toevaluate theperformanceof the transgenic riceplants underwater stress and oxidative stress, the seeds of PP45-OE, pp45-KO, DMI3-OE, dmi3-KO, and the wild type were germinated and grown in the growthchamber as described above. For the analysis of survival rate, rice seedlingswere treated with 20% PEG 4000, 100 mM H2O2 for 15 d, and the survivalrates were counted after recovery by rewatering for 10 d. For the analysis ofoxidative damage to lipids and plasmamembranes, the rice seedlings weretreated with 20% PEG 4000, 100 mM H2O2 for 2 d, and the content ofmalondialdehydeandthepercentageofelectrolyte leakageweredeterminedaccording to the methods described previously (Shi et al., 2012).

Antioxidant Enzyme Assays

Rice protoplasts were homogenized in a solution of 50 mM potassiumphosphate buffer (pH 7.0) containing 1 mM EDTA and 1% poly-vinylpyrrolidone. The homogenate was centrifuged at 12,000g for20 min at 4°C, and the supernatant was used immediately for the an-tioxidant enzyme assays. The total activities of SOD and CAT weredetermined as described previously (Jiang and Zhang, 2001).

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBLdata libraries under the following accession numbers: DMI3, LOC_Os05g41090; PP45, LOC_Os03g09220; PP1, LOC_Os01g07090;PP57, LOC_Os03g59470; PP111, LOC_Os10g22460; RbohB, LOC_Os01g25820; RbohE, LOC_Os01g61880; SAPK8, LOC_Os03g55600;SAPK9, LOC_Os12g39630; SAPK10, LOC_Os03g41460; MPK1, LOC_Os06g06090; glyceraldehyde-3-phosphate dehydrogenase, LOC_Os02g38920; ACT1, LOC_Os03g50885.

Supplemental Data

Supplemental Figure 1. Subcellular localization of PP45 and DMI3.

Supplemental Figure 2. The CaM-binding domain of DMI3 interactswith the PP2C domain of PP45.

Supplemental Figure 3. DMI3 specifically interacts with PP45.

Supplemental Figure 4. The specificity of the anti-PP45 antibody andthe anti-DMI3 antibodies.

Supplemental Figure 5. Identification of the rbohB/E-KO1 mutant.

The Plant Cell 149

Supplemental Figure 6. Standard curves of absorbance at 630 nmversus concentration of free phosphate with and without reducingagents.

Supplemental Figure 7. Identification of dmi3-KO and pp45-KOmutants.

Supplemental Figure 8. PP45 decreases and DMI3 increases ABAsensitivity of seed germination and root growth.

Supplemental Figure 9. PP45 reduces and DMI3 enhances thetolerance of rice plants to water stress and oxidative stress.

Supplemental Figure 10. PP45 negatively regulates and DMI3positively regulates the activities of MPK1, SOD, and CAT.

Supplemental Figure 11. H2O2 does not directly affect the activity ofDMI3 in vitro and Ca2+ is required for H2O2-induced activation of DMI3in rice plants.

Supplemental Figure 12. DMI3 does not regulate the expression ofPP45 and the activity of PP45.

Supplemental Figure 13. The oxidation of PP45 does not form anintramolecular disulfide bridge.

Supplemental Figure 14. The oxidation of Cys-350 and Cys-428 inPP45 is required for ABA-induced activities of SOD and CAT.

Supplemental Table 1. DNA oligonucleotides used for site-directedmutagenesis.

Supplemental Table 2. PCR primers used in this study.

ACKNOWLEDGMENTS

This work was supported by the National Natural Science Foundation ofChina (grant no. 31471427, 31671606, and 31601234), the National BasicResearch Program of China (grant no. 2012CB114306), the FundamentalResearch Funds for the Central Universities (grant no. KYTZ201402 andKJQN201736), the Project Funded by the Priority Academic ProgramDevelopment of Jiangsu Higher Education Institutions, and the ChinaPostdoctoral Science Foundation (grant no. 2016M590464).

AUTHOR CONTRIBUTIONS

M.J. and L.N. conceived the project and designed the experiments. L.N.performed most of the experiments. X.F., H.Z., X.L., X.C., P.Z., L.L., Q.W.,M.S., andQ.-W.Wperformed someof the experiments.M.J., A.Z., andZ.Z.analyzed data. M.J. and L.N. wrote the article.

Received July 5, 2018; revised October 1, 2018; accepted November 30,2018; published December 11, 2018.

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DOI 10.1105/tpc.18.00506; originally published online December 11, 2018; 2019;31;128-152Plant Cell

Sun, Qian-Wen Wang, Aying Zhang, Zhengguang Zhang and Mingyi JiangLan Ni, Xiaopu Fu, Huan Zhang, Xi Li, Xiang Cai, Panpan Zhang, Lei Liu, Qingwen Wang, Manman

/CaM-Dependent Protein Kinase DMI32+ and Relieves Repression of the Ca2O2Abscisic Acid Inhibits Rice Protein Phosphatase PP45 via H

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