A Positive Feedback Loop Governed by SUB1A1Interaction with MITOGEN-ACTIVATED PROTEIN KINASE3Imparts Submergence Tolerance in Rice

Pallavi Singh1 and Alok Krishna Sinha2

National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi, India 110067

ORCID IDs: 0000-0003-3694-6378 (P.S.); 0000-0003-0820-9936 (A.K.S.)

Mitogen-activated protein kinase (MAPK) signal transduction networks have been extensively explored in plants; however,the connection between MAPK signaling cascades and submergence tolerance is currently unknown. The ethylene responsefactor-like protein SUB1A orchestrates a plethora of responses during submergence stress tolerance in rice (Oryza sativa). Inthis study, we report that MPK3 is activated by submergence in a SUB1A-dependent manner. MPK3 physically interacts withand phosphorylates SUB1A in a tolerant-allele-specific manner. Furthermore, the tolerant allele SUB1A1 binds to the MPK3promoter and regulates its expression in a positive regulatory loop during submergence stress signaling. We presentmolecular and physiological evidence for the key role of the MPK3-SUB1A1 module in acclimation of rice seedlings to theadverse effects of submergence. Overall, the results provide a mechanistic understanding of submergence tolerance in rice.

INTRODUCTION

Rice (Oryza sativa) cultivars are adapted to flourish in standingwater but at the same time are susceptible to transient andcomplete inundation. Sudden flooding of rice fields leads toa decrease in oxygen and soil pH, which further leads to dep-rivation of nutrients. The molecular mechanism governingsubmergence stress management comprises two contrastingacclimation responses, namely, the low-oxygen escape strategyand the low-oxygen quiescence strategy (Bailey-Serres andVoesenek, 2008). Low-oxygen escape strategy, characteristic ofuplandcultivars, comprisesa repertoireof traits includinghigh rateof carbohydrate consumption, in turn allowing rapid elongation ofaerial organs to keep the leaves above the water level. This ispredominantly governed by SNORKEL1/2 (SK1 and SK2), whichare tandem ethylene-responsive factor (ERF) genes (Hattori et al.,2009). On the contrary, low-oxygen quiescence strategy in-volves stress-induced repression of carbohydrate resourceconsumption, in conjunction with plant growth inhibition. Thistolerance strategy is determined by the SUB1A gene of theSUBMERGENCE1 (SUB1) quantitative trait locus, which conferstolerance to up to 2 weeks of submergence. The SUB1 quanti-tative trait locus is a multigenic locus encoding three clade VIIERFs (SUB1A, SUB1B, and SUB1C) (Fukao et al., 2006; Xu et al.,2006). Improvement of rice crop resilience to submergence canbe accomplished by harnessing the genetic potential of theSUB1 locus. The SUB1 locus has been introgressed into manymega varieties through a marker-assisted backcrossing strategy

(Neeraja et al., 2007; Septiningsih et al., 2009; Iftekharuddaulaet al., 2012). Several of these SUB1-introgressedmega varieties,i.e., Swarna-Sub1, IR64-Sub1, and BR11-Sub1, have widelyundergone field trials and show positive attributes of submer-gence tolerance. These varieties have been released in India, thePhilippines, Indonesia, and Bangladesh and show all of the de-sirable agronomic traits and grain quality of the original parentvariety.A deeper genetic survey into SUB1 locus identified two SUB1A,

nine SUB1B, and seven SUB1C alleles on the basis of variation inaminoacid sequence (Xuet al., 2006). TheSUB1A1allele is specificto submergence-tolerant accessions. Variations in putative mito-gen-activated protein kinase (MAPK) sites distinguish the tolerantand intolerant alleles of SUB1A. In the tolerant SUB1A1 allele,a single nucleotide polymorphism at position 556 is responsible fora Pro-186 (intolerant) to Ser-186 (tolerant) substitution (Xu et al.,2006). The activities of several plant transcription factors aremodulated by posttranslational modifications, particularly, phos-phorylation and dephosphorylation. Phosphorylation of transcrip-tion factors by MAPKs can regulate their intrinsic levels andactivities (Tootle andRebay, 2005). TheMAPKsignaling cascade isa three-tier phospho-relay signaling module that is evolutionarilyconserved in all eukaryotes (Rodriguez et al., 2010; Sinha et al.,2011). Inplants, theMAPKsignal transduction network administersvarious biotic and abiotic stress responses. This network is as-sociatedwith varioushormone responsesandcell divisional aswellas developmental cues (Colcombet and Hirt, 2008; Sinha et al.,2011). Group A members MPK3 and MPK6 as well as Group BMPK4are themostwidely studiedMAPKcomponents inplants (Fiilet al., 2009; Andreasson and Ellis, 2010).Ethyleneand reactiveoxygen species (ROS) are key regulatorsof

plant submergence stress status. Increased ethylene levels insubmerged plants trigger SUB1A gene expression. SUB1Adampens ethylene-promoted gibberellic acid (GA) responsivenessduring submergence. This dampening usually occurs due to ac-cumulation of theGA signaling repressorsSLENDERRICE1 (SLR1)

1 Current address: Plant Pathology and Plant-Microbe Biology Section,School of Integrative Plant Science, Cornell University, Ithaca, NY 14853.2 Address correspondence to alok@nipgr.ac.in.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: Alok Krishna Sinha (alok@nipgr.ac.in).www.plantcell.org/cgi/doi/10.1105/tpc.15.01001

The Plant Cell, Vol. 28: 1127–1143, May 2016, www.plantcell.org ã 2016 American Society of Plant Biologists. All rights reserved.

and SLR1-like 1 (SLRL1). The expression of GA repressors isa strategy to counteract complete submergence. It facilitates theplant to adapt to quiescencemode ofmetabolismuntil floodwatersrecede (Fukao et al., 2006; Fukao and Bailey-Serres, 2008). It isreported thatmultiple ethylene-dependent pathways lead toMAPKactivation (Rodriguez et al., 2010). Two of the best-studied Ara-bidopsis thaliana RAF-like MAP3Ks, CONSTITUTIVE TRIPLERESPONSE1 and ENHANCED DISEASE RESISTANCE1, par-ticipate in ethylene-mediated signaling and defense responses. Acascade that involves MKK9-MPK3/6 also participates in ethyl-ene signaling (Hahn and Harter, 2009). SUB1A ameliorates theeffect of ROS produced by hypoxia during submergence (Bailey-Serres et al., 2012).MAPKcascades are also known to respond tothe resulting oxidative burst andmay regulate ROSaccumulation.Both MPK3 and MPK6 signaling modules within Group A areimplicated inROSsignaling (PitzschkeandHirt, 2009b). This furtherbroadens the reach of this cascade to other processes related tohypoxia and submergence. Although the physiological functions ofMAPKs in response to various external stimuli have been exten-sivelycharacterized, therehavebeennostudiesdescribingarole forthis cascade in submergence stress responses. However, a reportbySeo et al. (2011) hinted at the involvement of aMAPKcascade insubmergence stress tolerance. That study reported that MPK5(which we refer to asMPK3 in this case, based on the phylogeny-based nomenclature of Hamel et al. [2006]) showed higher tran-scriptaccumulation inSUB1A-containingrice (M202-Sub1AversusM202) upon submergence. Our current study builds upon thispreliminary observation.

SUB1A1 contains a potential MAPK phosphorylation site lo-cated in a variable region C-terminal to the ERF domain. This maybe of significance as phosphorylation can modulate DNA bindingby ERF proteins (Gutterson and Reuber, 2004; Xu et al., 2006).Thus, in this study, the putative role of MAPK signaling in sub-mergence stress tolerance was investigated, in particular, thepossible connection between submergence andMAPKpathwaysvia SUB1A1. Here, we report that MPK3 is specifically activatedupon submergence and that this process is SUB1A1 dependent.Our study demonstrates that MPK3 physically interacts with andphosphorylates SUB1A1 in an allele-specific manner. Further-more, by MAPK-specific inhibitor assays and transient transfor-mation phenotypic assays, we report the genetic interaction ofSUB1A1 and MPK3, which in turn augments its role by governinga suite of submergence-related traits.We also show that SUB1A1interacts with the promoter of MPK3 to regulate its expression.Thesefindingscollectively reveal apositive regulatorymechanismin the submergence signaling cascade.

RESULTS

Submergence-Mediated Activation of MAPK CascadeComponents Is SUB1A Dependent

To gain insight into the involvement of aMAPK signaling cascadein submergence stress tolerance and to determine the SUB1Adependency of the process, transcript profiling of MAPK com-ponents was undertaken. Initially, we sought to determine thesubmergence-dependent expression of genes for the complete

family ofMAPKs andMAPKKs and the Raf family ofMAPKKKs byquantitative real-time PCR, in submergence-tolerant (SwarnaSub1) and in submergence-intolerant (Swarna) cultivars. Asshown in the bicolor heat map in Figures 1A to 1C, the expressionof cardinal MAPK cascade components (Groups A, B, and C) wasincreased postsubmergence in the Swarna-Sub1 genotype.Therewas a chronic elevation of transcripts of the lessdecipheredGroupDMAPKmembers inSwarna.However, it is likely that theseGroup D members are outliers, as they do not show any specificphosphorylation-dependent activation in immunokinase assaysusing pTEpY antibody or in in-gel kinase assays (Sheikh et al.,2013). Activated MAPKs are generally in the range of 40 to 46 kD,which is very well in the range of Groups A, B, and C (Sheikh et al.,2013). Furthermore, therearevery few reports implicatingGroupDMAPKs in physiological functions of plants. For an in-depthanalysis, the transcript expression ofMPK3 in both Swarna-Sub1andSwarna lines is presented inSupplemental Figures 1Aand1B.These results suggest that SUB1A positively regulates the ex-pression of the MAPK signaling cascade components duringsubmergence.These findings prompted us to search for the MAPK that was

specifically activated in response to submergence in a SUB1A-dependentmanner.Webeganwith an immunokinase assay of thecrude protein extracts fromSwarna andSwarna Sub1 lines grownin optimal conditions for 14 d and then completely submerged fora short duration (1 to 12 h) and a long duration (1 to 7 d). Immu-noblot analysis was performed with pTEpY antibody, whichspecifically binds to activatedMAPK cascade components (Sethiet al., 2014). Interestingly, submergence activated 42-kD MAPKswithin 1 h in the Swarna Sub1 plants. However, Swarna lines didnotshowanysuchactivation (Figures1Dand1E). Theactivationofthe 42-kD protein was observed to increase with increasing du-ration of submergence. The size of the protein identified by im-munoblot with pTEPY antibody was ascertained by runningpTEpY antibody-immunoprecipitated sample in parallel withcrude protein followed by immunoblotting with pTEpY andMPK3antibodies (Supplemental Figure 1C). To further substantiate thisfinding, an in-gel kinase assay using Swarna and Swarna Sub1lines grown optimally for 14 d and then submergence treated forup to 7 d was also performed. MAPK activity was analyzed usingthe artificial MAPK substrate Myelin Basic Protein (MBP). Inter-estingly, submergence activated MBP-phosphorylating proteinkinase activity within 1 h in the Swarna Sub1 plants. However, inaccordance with the result of the immunoblots, Swarna lines didnot show any such activity (Supplemental Figures 1D and 1E).

Submergence-Mediated-Specific Activation of MPK3 IsSUB1A Dependent

These results suggest that a MAPK at 42 kD is activated in re-sponse to submergence and that this activation is likely to beSUB1A dependent. Next, we aimed to determine which MAPKwas specifically activated by submergence and whether the ac-tivation was indeed SUB1A dependent. For this, immunopre-cipitation (IP) was performed using pTEpY antibody followed byimmunoblotanalysiswithanti-OsMPK3antibody.ThesubmergedSwarnaSub1 lines showedabandof increasing intensity at 42 kD,while this band was not observed in submerged Swarna lines

1128 The Plant Cell

(Figure 1F). OsMPK3 shows high homology with its Arabidopsiscounterpart (Hamel et al., 2006); to further confirm the identity ofthe precipitated protein, immunoblot analysiswas also performedusing an anti-AtMPK3 antibody. In accordance with the previousobservation, submerged Swarna Sub1 lines showed a band at 42kD,while this42-kDbandwasnotobserved in submergedSwarnalines (Supplemental Figure 2A). These results indicate that thespecific activation of MPK3 upon submergence is SUB1A de-pendent. The specific activation of MPK3 postsubmergence ina SUB1A-dependent manner was further established by per-formingcontrol experimentsusinganti-AtMPK6andanti-AtMPK4antibodies (the closest homologs of OsMPK6 and 4, respectively)as depicted in Supplemental Figures 2B and 2C.

The MAPK Cascade Regulates SubmergenceStress-Inducible Genes SLR1 and SLRL1

To further explore if a MAPK cascade could have an essentialrole in SUB1A-mediated submergence tolerance in rice, weperformed MAPK inhibitor pretreatment experiments. The most

tractable phenotype of SUB1A-mediated submergence toleranceis restricted shoot elongation upon inundation (Schmitz et al.,2013). Accordingly, 7-d-old seedlings of Swarna and Swarna-Sub1 were pretreated with PD98059, a MAPKK-specific inhibitor(Alessi et al., 1995), for 1 d before complete submergence for 7 d.Final seedling heights of 20 individuals for each treatment weremeasured as an indicator of submergence tolerance, i.e., theability to limit shoot elongation (Figures 2A and 2B). MAPK in-hibitor treatment had no effect on either genotype in the absenceof submergence. The inhibitor pretreated, submerged Swarnaplants did not show any variation in shoot elongation after sub-mergence, whereas inhibitor pretreated Swarna Sub1 lines ex-hibited marked shoot elongation contrary to the characteristic,submergence tolerancephenotype.TheMAPK inhibitor treatmentexperiments demonstrate that a MAPK signaling cascade couldbe involved in the SUB1A-mediated submergence tolerancephenotype.SUB1Awasproposed to induce the expressionof andfurther stabilize GA repressors SLR1 and SLRL1 (Fukao andBailey-Serres, 2008). As MAPK inhibitor treatment abolishedthe characteristic submergence-mediated restriction of shoot

Figure 1. Submergence-Induced Elicitation of MAPK Cascade.

(A) to (E) Real-time PCR analysis of genes for MAPK members (A), MAPKK members (B), and MAPKKK members (C) in Swarna Sub1 and Swarna ricecultivars treatedwith submergence. The fold change fromqRT-PCR assaywas plotted into a heatmap usingMEV software framework. The values indicatethe mean of three independent sets of experiments. Immunoblot analyses using pTEpY antibody of 40 µg of total protein from Swarna Sub1 and Swarnaseedlings treated with submergence for short duration (1, 3, 6, and 12 h) (D) and long duration (1, 3, 5, and 7 d) (E). The bands indicate total active MAPKs.Panel under each immunoblot depicts the loading control. Values below the panel indicate quantification of each band with respect to the loading control.(F)Submergence-induced specific activation ofMPK3 assessed by immunoprecipitation assays using 300 µg total protein fromSwarna andSwarna Sub1lines submergence treated for 0, 1, and 3 d. The MPK3 activation was analyzed by IP with pTEpY antibody followed by immunoblot (IB) analysis with anti-OsMPK3antibody. Lower panel shows immunoblot withOsMPK3 antibody using 20µg total protein as the loading control. Values below the panel indicatequantification of each band with respect to the loading control.

MPK3 Phosphorylates SUB1A1 1129

Figure 2. MAPK Cascade Governs Submergence-Tolerant Attributes in a SUB1A-Dependent Manner Postsubmergence.

Swarna and Swarna Sub1 seedlings pretreated for 1 d with 150 µM PD98059 (MAPKK-specific inhibitor) and nontreated controls were submerged in tapwater for 7 d.(A) Representative individuals after treatment (nonsubmerged with or without PD98059 treatment, submerged with or without PD98059 treatment).(B) Graph of seedling heights. Asterisk indicates a significant difference (t test, *P# 0.05) between submerged Swarna Sub1A and PD98059-pretreatedsubmerged Swarna Sub1A. Error bars represent mean SD of three independent sets of experiments.

1130 The Plant Cell

elongation phenotype, the transcript levels of SLR1 and SLRL1weremonitoredbyquantitative-real timePCRanalysis (Figure2C).Indeed, inhibitor pretreated Swarna Sub1 seedlings showedmarked decrease in accumulation ofSLR1 andSLRL1 transcriptscomparedwithuntreatedseedlings.Nosuchdifferential transcriptregulation of SLR1 and SLRL1 was observed in Swarna(Supplemental Figure 3). The above experiments demonstratethe involvement of MAPK signaling in SUB1A-mediated sub-mergence tolerance.

SUB1A1 Physically Interacts with MPK3

To delineate the role of MPK3 in submergence stress tolerance,interaction of SUB1A1withMPK3was assessed using yeast two-hybrid assays. An interaction was observed upon expression offull-length clones ofSUB1A1 andMPK3. The interaction was alsoobserved after swapping SUB1A1 and MPK3 as bait and prey(Figure 3A). Since SUB1A1 and MPK3 interaction was positiveon nutritional selection medium, lacZ reporter gene expressionwas confirmed using ortho-nitro phenyl b-galactoside (ONPG) assubstrate. Yeast strain AH109 cotransformed with SUB1A1 andMPK3 yielded high b-galactosidase activity, whereas othertransformants with combinations of SUB1A1 orMPK3 and emptyAD or BD vectors had low or negligible b-galactosidase activity(Figure 3B). To determine the specificity of interaction betweenSUB1A1 and MPK3, targeted yeast two-hybrid assays wereperformed for SUB1A1 against other characteristic members ofthe MAPK clade. The close relatives of MPK3 include MPK6 andMPK4 from Group A and B MAPKs, respectively. The othermembers analyzed for the assay included MPK7 and MPK14 ofGroup C MAPKs and MPK16-1, MPK17-1, MPK17-2, MPK20-1,MPK20-2, MPK20-3, MPK20-4, and MPK21-1 of Group DMAPKs. Only the combination of SUB1A1 and MPK3 producedgrowth on nutritional selection medium (adenine, histidine, leu-cine, and tryptophan) reflecting the specificity of the interaction(Figure 3C). Thus, MPK3 specifically interacts with SUB1A1 asevident by the pairwise protein-protein interaction studies.SUB1A2 was also found to interact with MPK3 (SupplementalFigures 4Aand4B).However, this interactionwasweaker than theone observed between SUB1A1 and MPK3 as measured byb-galactosidase activity.

An in silicodocking experimentwasperformed to further assessthe physical interaction of MPK3 and SUB1A1. For this, a ho-mology modeling approach was employed to predict the 3Dstructures of MPK3, MPK4, MPK6, and SUB1A1. These 3Dstructures were further refined and used as an input for protein-protein docking using ClusPro (http://cluspro.bu.edu/) to predictMPK3-SUB1A1, MPK4-SUB1A1, and MPK6-SUB1A1 interac-tion. The in silico prediction indicated a stronger physical inter-action in MPK3-SUB1A1 relative to the other pairs. This stronginteraction of SUB1A1 and MPK3 was supported by the more

negative docking score (Supplemental Figures 5A to 5C).Therefore, these results hint at the physical and specific inter-action of SUB1A1 with MPK3.

SUB1A1 Physically Interacts with MPK3 in Planta

For an initial examination of the likelihood of physical interactionbetween MPK3 and SUB1A1 in planta, subcellular localizationwas predicted using the PLANT-MPLOC (http://www.csbio.sjtu.edu.cn/bionf/plant-multi/) server. Both proteins were predicted tolocalize to the nucleus. Next, subcellular localization of the pro-teins expressed transiently in Nicotiana benthamiana was de-termined using SUB1A1 and MPK3 pGWB5 constructs, whichencode C-terminal sGFP (superfolder-GFP) fusion proteins.Confocal imaging confirmed localization of the twoproteins to thenucleus (Figure 3D).To further test the inplantaphysical interactionsbetweenMPK3

and SUB1A1, bimolecular fluorescence complementation (BiFC)experimentswere performed. Full-lengthMPK3 coding sequence(CDS) was fused to sequence encoding the N terminus of YFP inthepSPYNE173vector, andSUB1A1CDSwas fused tosequenceencoding the C terminus of YFP in the pSPYCE (M) vector. Theseconstructs together produced strong YFP fluorescence in thenucleus of N. benthamiana leaves (Figure 3E). To check thespecificity of MPK3 and SUB1A1 interaction, a BiFC assay ofMPK6 and SUB1A1 was also undertaken (Supplemental Figures6Aand6B).NoYFPfluorescencewasobserved in thiscase.Takentogether, these results demonstrate that MPK3 physically inter-actswithSUB1A1 inplanta in a specificmanner.CotransfectionofemptyBiFCvectors (Figure3E,d to f), cotransfectionof theMPK3-YFP n-ter construct and empty YFP c-ter vector (Figure 3E, g to i),and cotransfection of the empty YFP n-ter vector and SUB1-A1YFP c-ter construct (Figure 3E, j to l) served as controls. Theprotein loading controls for the transient transformation ofN. benthamiana leaves for the BiFC assays and subcellular lo-calization are represented in Supplemental Figures 6C and 6D,respectively.

SUB1A1 Is Phosphorylated by MPK3

To ascertain whether SUB1A1 is indeed a phosphorylation targetof MPK3, an in-solution kinase assay was performed usingbacterially expressed SUB1A1-His and GST-MPK3 protein.SUB1A1-His was used as a substrate. A control experiment wasperformed to assess theMBP phosphorylation potential of MPK3(Figure 4A, lane 1) and to assess the autophosphorylation activityof the bacterially expressed protein (Figure 4A, lane 2), whileSUB1A1-His aloneservedasanegativecontrol (Figure4A, lane3).GST-MPK3 phosphorylated SUB1-His in this assay (Figure 4A,lane 4). These results suggest that MPK3 phosphorylatesSUB1A1.

Figure 2. (continued).

(C)ExpressionprofilesofGAsignaling repressorgenes ina time-courseexperiment.Expression levels forMPK3,SLR1, andSLRL1at0 (Con), 6,12,and24hafter submergence inmock-treatedandPD98059-pretreatedSwarnaSub1seedlingsweremeasuredusing real-timePCRassays.Valuesare relative to0h.Error bars represent mean SD of three independent sets of experiments.

MPK3 Phosphorylates SUB1A1 1131

Figure 3. Physical Interaction of SUB1A1 with MPK3.

(A) Yeast two-hybrid assay reporter strain AH109 was cotransformed with the pGBKT7 and pGADT7 vectors containing the indicated gene constructs.Transformants were selected on SD [-leu-trp] double dropout (DDO)medium, and the interaction was checked on SD [-trp-leu-ade-his] quadruple dropout(QDO) medium.(B) b-Galactosidase assay of lacZ reporter gene expression with the indicated constructs using ONPG as substrate. Error bars represent mean SD of threeindependent sets of experiments.(C) Specificity of interaction between SUB1A1 and MPK3; reporter yeast strain AH109 was cotransformed with SUB1A1 in pGADT7 and the codingsequences for cardinal members of the MAPK clade in pGBKT7. Transformants were selected on SD [-leu-trp] DDO medium, and the interaction waschecked on SD [-trp-leu-ade-his] QDO medium.(D) Subcellular localization studies using GFP-tagged MPK3 and SUB1A1. Localization of the indicated tagged proteins transiently expressed in N.benthamiana leaves was observed using confocal laser scanning microscopy. Bars = 50 mm.(E) SUB1A1 physically interacted with MPK3 in BiFC assays in N. benthamiana leaves. (a) Reconstruction of YFP signal when the MPK3-YFP n-ter. andSUB1A1-YFP c-ter. constructs were coinfiltrated into N. benthamiana leaves. (b) and (c) Bright-field image and merged image. (d) to (f) show cells co-infiltrated with empty BiFC vectors. (g) to (i) show cells coinfiltrated with the MPK3-YFP n-ter. construct and empty YFP c-ter. vector. (j) to (l) show cellscoinfiltrated with the empty YFP n-ter. vector and SUB1A1 YFP c-ter. construct.

1132 The Plant Cell

Figure 4. SUB1A1 Is the Phosphorylation Target of MPK3.

(A) In vitro phosphorylation assay using bacterially expressed SUB1A1-His and GST-MPK3 incubated alone or in combination with MBP to check forphosphorylationbyMPK3.Theplusandminussigns indicate thepresenceandabsenceofproteins, respectively. Aliquotsof thesampleswereseparatedbySDS-PAGE and subjected to autoradiography. Lower panel shows the Coomassie blue (CBB)-stained gel with the positions of different proteins indicated(arrows). Numerals in the left of lower panel indicate molecular mass (kD) marker used in CBB-stained gel.(B) In vitro phosphorylation assayusingplant protein.N.benthamiana leaves infiltratedwithMPK3-pSPYCE (M)wereused for immunoprecipitation of crudeprotein with anti-HA antibody followed by incubation with SUB1A-His as substrate in the kinase reaction mixture to determine phosphorylation activity.CBB-stained gel, with the positions of different proteins indicated (arrows). Numerals in the left of lower panel indicate molecular mass (kD) marker used inCBB-stained gel.(C) SUB1A1 is the specific phosphorylation target of MPK3. In vitro phosphorylation assay using plant-expressed SUB1A1 and SUB1A2 protein. N.benthamiana leaves infiltrated with SUB1A1-pSPYCE (M) and SUB1A2-pSPYCE (M) were used for immunoprecipitation of crude protein with anti-HAantibody followedby incubationwithGST-MPK3 in the kinase reactionmixture todeterminephosphorylation activity.CBB-stainedgel,with thepositions ofdifferent proteins indicated (arrows). Numerals in the left of lower panel indicate molecular mass (kD) marker used in CBB-stained gel.(D) In vitro phosphorylation assay using antiphosphoserine antibody and bacterially expressed SUB1A1-His, SUB1A2-His, and GST-MPK3. GST-MPK3,SUB1A1-His, andSUB1A2-Hiswere incubatedalone or in combinationwithMBP. Theplus andminus signs indicate thepresence andabsenceof proteins,respectively. Aliquots of the samples were separated by SDS-PAGE and later immunoblotted with antiphosphoserine antibody. Lower panel shows theCBB-stained gel with the position of different proteins indicated (arrows). Numerals in the left of lower panel indicate molecular mass (kD) marker used inCBB-stained gel.

MPK3 Phosphorylates SUB1A1 1133

We also assessed the phosphorylation status of SUB1A1with other important and close relatives of MPK3, namely,MPK4 and MPK6 (Supplemental Figure 7). For this assay, weused SUB1A1 harboring an HA tag expressed in leaves of N.benthamiana and purified by immunoprecipitation. SUB1A1-HA was phosphorylated by MPK3 (Supplemental Figure 7, lane3) but not by MPK4 (Supplemental Figure 7, lane 5) or MPK6(Supplemental Figure 7, lane 7). Furthermore, to determinewhether SUB1A1 is phosphorylated by plant-expressedMPK3,theMPK3-pSPYNE 173 construct expressing the N terminus ofeYFP-HA-MPK3 was agroinfiltrated in N. benthamiana leaves.The HA-tagged MPK3 was immunoprecipitated and used forthe in vitro kinase assay. Bacterially expressed SUB1A1 wasphosphorylated by immunoprecipitated MPK3-HA (Figure 4B).These results establish that SUB1A1 is the phosphorylationtarget of MPK3.

MPK3 Phosphorylation Is Tolerant-Allele Specific

Now that we established SUB1A1 as the phosphorylation targetofMPK3, itwas important to explore thephysiological relevanceof any potential allele-specific phosphorylation. Thus, bothSUB1A1 and SUB1A2 proteins were expressed in planta. TheHA-tagged SUB1A1 and SUB1A2 were immunoprecipitatedwith anti-HA antibody and used for the phosphorylation assays,alongside bacterially expressed GST-MPK3 protein. A controlexperiment was performed to assess the autophosphorylationpotential of MPK3 (Figure 4C, lane 1), while SUB1A1-HA andSUB1A2-HA alone served as a negative controls (Figure 4C,lanes 2 and 4). GST-MPK3 strongly phosphorylated SUB1A1-HA (Figure 4C, lane 3) but not SUB1A2-HA (Figure 4C, lane 5).These results indicate that MPK3 specifically phosphorylatesthe protein produced by the submergence-tolerant allele, i.e.,SUB1A1.

To further assess the allele-specific nature of this phosphory-lation, an in-solution kinase assay was performed using bacte-rially expressed SUB1A1-His and Ser-186 to Pro-186 mutatedSUB1A1-His (SUB1A2-His) and GST-MPK3 protein. The reac-tions of the in-solution kinase assay were further immunoblottedwith antiphosphoserine antibody. A control experiment wasperformed to assess the autophosphorylation activity of thebacterially expressed protein (Figure 4D, lane 1), while SUB1A1-His and SUB1A2-His alone served as negative controls (Figure4D, lanes 2 and 3, respectively). GST-MPK3 phosphorylatedSUB1A1-His but not SUB1A2-His, even with increasing con-centration of SUB1A2-His (Figure 4D). These results furtherdemonstrate the allele-specific phosphorylation of SUB1A1by MPK3.

SUB1A1 Genetically Interacts with MPK3 and RegulatesSubmergence-Dependent Traits

Since we foundMPK3 to bemolecularly connected to SUB1A1 ina submergence-dependent manner, we further examined thephysiological significance of the interaction. For this, rice seed-lings transiently overexpressing MPK3 (MPK3-Ox) or silencedfor MPK3 were raised in both Swarna and Swarna Sub1 back-grounds. Overexpression and silencing was confirmed by

transcript level analysis of MPK3 (Supplemental Figures 8Aand 8C) as well as protein level assays in the respective lines(Supplemental Figures 8B and 8D). Furthermore, the sub-mergence-related attributes of these lineswere assayed in normaland postsubmergence conditions. As previously stated, wesought to measure the most tractable phenotype of SUB1A-mediated submergence tolerance, i.e., the restricted shootelongation upon inundation. Seven-day-old seedlings of control,MPK3-Ox, and MPK3-silenced lines in both Swarna Sub1 andSwarna backgrounds were completely submerged for a period of7d, and final seedling heightsweremeasured (Figures 5Aand5B).The MPK3-Ox Swarna Sub1 line showed restricted shoot lengthelongation postsubmergence, whereas the MPK3-silencedSwarna Sub1 lines showed a chronic increase in shoot lengthpostsubmergence. The corresponding Swarna lines did not showany variation in shoot elongation upon submergence. Hightranscript accumulation of genes for the GA repressors SLR1 andSLRL1 serves as another marker for submergence tolerance(Fukao and Bailey-Serres, 2008). The transcript accumulation ofSLR1 and SLRL1 was monitored by quantitative-real time PCRanalysis in the transgenic lines postsubmergence (Figure 5C).Indeed, submergence-treatedMPK3-Ox Swarna Sub1 seedlingsshowed marked accumulation of SLR1 and SLRL1 transcriptscompared with the MPK3-silenced Swarna Sub1 seedlings. Nosuch differential transcript accumulation of SLR1 and SLRL1wasobserved in the Swarna transgenic lines.To obtain a deeper understanding of the physiological rele-

vance of MPK3-SUB1A1 interaction, all of the lines were assayedfor chlorophyll content, melondialdeahyde (MDA) content (in-dicative of lipid peroxidation), and activity of ROS scavengingenzymes like ascorbate peroxidase (APX), superoxide dismutase(SOD), and catalase (CAT). The chlorophyll content (Figure 5D)of MPK3-Ox and silenced lines was comparable in bothSwarna and Swarna Sub1 backgrounds before submergence.Postsubmergence there was a comparable level of chloro-phyll leaching in all assayed Swarna lines, while, interestingly,MPK3Ox-Swarna Sub1 lines resisted chlorophyll leaching. Bycontrast, MPK3-silenced lines showed marked chlorophyllleachingwithvalueseven lower than thoseofcontrol plants.Theseresults suggest the physiological relevance of the MPK3 andSUB1A1 interaction. In the lipid peroxidation assay using MDA(Figure 5E), low and comparable lipid peroxidation was observedin all lines before submergence treatment. After submergencetreatment, lipid peroxidation (as indicated by MDA accumulation)increased drastically in all Swarna lines. The MPK3-Ox SwarnaSub1 line showed minimal lipid peroxidation. By contrast, theMPK3-silenced Swarna Sub1 lines were marked by elevatedlevels of lipidperoxidation. In theROSscavengingenzymeactivityassays (Figures 5F to 5H) of the respective lines, similar trends tothe previous assays were obtained. There was amarked increasein the activity of SOD, APX, andCATafter submergence treatmentinMPK3-OxSwarnaSub1 lines,while theMPK3-silencedSwarnaSub1 lines showed values comparable to those of the Swarnalines. No significant change in the respective Swarna linespostsubmergence was observed.Furthermore, an assay of ROS-mediated root cell death was

performed on the above lines (Supplemental Figure 9). Rice rootsof the respective lines before and after 24 h of submergence

1134 The Plant Cell

treatment were stained with propidium iodide (PI) and examinedby confocal laser scanning microscopy. PI is excluded from livecells, and its internalization is indicative of cell death. Roots ofsubmergence-treated Swarna lines as well as MPK3-silencedSwarna Sub1 lines showed considerable staining by PI dispersed

within the cell, indicative of DNA fragmentation caused by celldeath. By contrast,MPK3-OxSwarna Sub1 lines showedminimaltraces of cell death postsubmergence. The above results sub-stantiate the genetic interaction of SUB1A1 and MPK3 and itsgovernance of various submergence related features.

Figure 5. MPK3-Silenced Lines Are Submergence Susceptible While MPK3 Overexpression Lines Show Submergence-Tolerant Traits.

(A) Transgenic Swarna andSwarnaSub1 seedlings overexpressing or silenced forMPK3 and nontransgenic seedlingswere submergence treated for 7 d intap water. Representative individuals are shown pre- and postsubmergence treatment.(B)Seedlingheights. Twoasterisks indicateasignificantdifference (P<0.005) inMPK3-OxSwarnaSub1A lines,whileoneasterisk indicatesasignificantdifference(P < 0.05) in MPK3-silenced Swarna Sub1 lines versus the submerged Swarna lines. Error bars represent mean SD of three independent sets of experiments.(C)Expression profiles of GA signaling repressor genes in transgenic lines of Swarna andSwarna Sub1. Expression levels forSLR1 andSLRL1 at 24 h aftersubmergenceweremeasuredusing real-timePCRassays. Values are relative to 0h. Error bars representmean SDof three independent sets of experiments.(D) to (H)Graphical representation of various biochemical parameters, namely, chlorophyll content (D), MDA content (E), APX activity (F), SOD activity (G),and catalase activity (H) of MPK3-Ox and MPK3-silenced Swarna Sub1 as well as Swarna lines before and after 1 d of submergence stress. Error barsrepresent mean SD (n = 3). Different letters above the bars indicate significantly different values (ANOVA test, P # 0.05).

MPK3 Phosphorylates SUB1A1 1135

The MPK3 Signaling Cascade Activates SUB1A1 ina Submergence-Dependent Manner

Transiently transformed MPK3-Ox lines containing the MPK3-GFP construct, in both Swarna and Swarna Sub1 backgrounds,were completely submerged for 1 d. Anti-GFP antibody was usedto pull down the fusion protein from both the genotypes. Immu-noblot analysis with pTEpY antibody specifically detected theactivated MPK3 from the submerged Swarna Sub1 lines (Figure6A). These results hint at the activation of an MPK3-containingsignaling cascade postsubmergence in a SUB1A-dependentmanner.

The above results established the role of MPK3 in mediatingSUB1A-dependent submergence responses. To gain insight intothe role of components upstream of MPK3 during submergence,we turned our attention to MKK4 (a MAPKK) that is known tointeract with MPK3 (Wankhede et al., 2013). Our earlier transcriptprofiling study revealed marked transcript accumulation ofMKKK32 (encodingaMAPKKK).We found thatMKKK32 interactswith MKK4 in yeast two-hybrid studies (Supplemental Figure 10).Thus, it was tempting to hypothesize the direct involvement ofa specific MAPK signaling cascade, which is activated by sub-mergence and in turn mediates the phosphorylation of SUB1A1.Transcript levels of MPK3, MKK4, and MKKK32 were measuredpostsubmergence. The expression of the genes for all threeplausible MAPK module components showed similar trend oftranscript accumulation with that of SUB1A1 postsubmergenceover time (Figure6B).Though indirect, thesedatastronglysuggestthe involvement of a MKKK32-MKK4-MPK3 signaling cascadeduring submergence in rice.

SUB1A1 Interacts with the Promoter ofMPK3 and RegulatesIts Expression

Finally, to determine whether SUB1A1 has a direct role in theregulation of MPK3 expression, an electrophoretic mobility shiftassay (EMSA) was undertaken. In silico analysis detected thepresence of a G-box (GCCGCC) cis-acting element, 151 bp up-stream of ATG (Figure 6C). EMSA was performed using purifiedSUB1A1-His fusion protein and the promoter region of MPK3containing the G-box. To examine the specificity of this DNA-protein interaction, a mutated version of this G-box (mE-box:GCCTCC) was used. As shown in Figure 6D, a low mobility DNA-protein complexwas formedwith increasing concentration ofHis-SUB1A1 (lanes 2 and 3) thatwas competed out by 50 or 100molarexcess of unlabeled MPK3 promoter fragment (lanes 4 and 5)containing this G-box. However, the mG-box fragment was notable to compete with the DNA-protein interaction (lane 6).

To further examine the binding of SUB1A1 to the MPK3 pro-moter in vivo, we performed chromatin immunoprecipitation(ChIP) experiments. Seven-day-old Swarna Sub1 seedlings weretransiently transformed with SUB1A1-GFP and further subjectedto1dof complete submergence. TheSUB1A1-GFP fusionproteinin transgenic plants was immunoprecipitated using the anti-GFPantibody. Coimmunoprecipitated genomic DNA with SUB1A1was analyzed by RT-PCR. As shown in Figure 6E, amplificationspecific to the MPK3 promoter region occurred only in thetransgenic SUB1A1-Ox plants, but not in the wild-type Swarna

Sub1 plants, which served as the negative control. These resultstogether suggest that SUB1A1 binds to the G-box of the MPK3promoter and in turn regulates its activity.

DISCUSSION

This study provides evidence that the activation of MPK3 insubmergence is SUB1A dependent. Furthermore, it establishesthat SUB1A1 works with MPK3 in a positive regulatory loop toregulate submergence tolerance. While MPK3 phosphorylatesSUB1A1, SUB1A1 specifically interacts with the promoter ofMPK3 and regulates its expression.SUB1A1, a group VII AP2/ERF, is a major player orchestrating

various submergence tolerance attributes by working in concertwith cytosolic and nuclear proteins. This facilitates a completetranscriptional reprogramming in a submergedseedling andhelpscounteract the adverse effect of inundation stress. SUB1Aacts asanodal point of crosstalk inmultiple signaling pathways, includingethylene, ROS, brassinosteroids, andGA responses (Fukao et al.,2006; Xu et al., 2006; Fukao and Bailey-Serres, 2008; Jung et al.,2010; Schmitz et al., 2013). TheMAPK cascade is also outlined asa stress-activated signal transduction network, conventionallytriggered by various cues of ethylene and ROS (Hirt, 2000;Pitzschke andHirt, 2006, 2009a; Pitzschke et al., 2009; �Samajováet al., 2013). Furthermore, variations in putative MAPK sites (Ser-186) distinguish the proteins produced by the tolerant (SUB1A1)and intolerant (SUB1A2) alleles of SUB1A (Xu et al., 2006). Thisvariation in potential phosphorylation site of SUB1A1 is harboredon a region immediately C-terminal to the ERF domain and thusmay be of significance, as phosphorylation can alter DNA bindingby ERF proteins (Gutterson and Reuber, 2004; Xu et al., 2006).These collective facts prompted us to investigate the possibleinvolvement of phosphorylation-based regulation of SUB1A1 andthe physiological relevance of a MPK3-SUB1A1 module in thecontext of submergence tolerance.The ricecultivarsSwarnaandtheSUB1-introgressedaccession

SwarnaSub1wereused in this study.Swarna isawidelycultivatedmega-variety in Southeast Asia, particularly India and Bangladesh.In 2009-2010, Swarna-Sub1 was released in India, Indonesia, andBangladesh. It is an improved submergence-tolerant version ofSwarna with the same yield statistics (Bailey-Serres et al., 2010).Thus, the use of thesemega varieties in this study was a deliberateattempt to investigate submergence-tolerant phenotypes in thefarmer-friendly mega-varieties.The quintessential phenotype of SUB1A-mediated submer-

gence tolerance is restricted shoot elongation upon inunda-tion (Fukao and Bailey-Serres, 2008). MAPKK-specific inhibitortreatment resulted in deviation from this hallmark trait. Theseobservations point to the positive role of a MAPK cascade duringsubmergence stress tolerance. Analysis of expression of GA re-pressor genes (SLR1andSLRL1) suggests thatSUB1A function isdependent on a MAPK module. A similar set of observations wasmade in plants treated with brassinosteroid inhibitor (Schmitzet al., 2013). Furthermore, the observed downregulation of GArepressor genes by MAPK cascade components was consistentwith thatobserved in thebrassinosteroid signalingnetwork,wherethe authors deduced that the brassinosteroid pathway positivelyregulated submergence tolerance responses in rice.

1136 The Plant Cell

Additionally, our quantitative real-time PCR studies suggestthat SUB1A positively regulates the transcript accumulation ofgenes for MAPK cascade components. This observation echoesa previous study in which OsMPK5 (designated as MPK3 in ourstudy) transcripts showed higher accumulation in SUB1A con-taining rice (M202-Sub1A versus M202) upon submergence (Seoet al., 2011). Furthermore, our in-gel kinase assays demonstratedthat MAPKs are activated in response to submergence and thatthis activation is SUB1A dependent. The immunoblot analysesand IP analysis with pTEpY antibody specifically demonstratedthat the activation ofMPK3 in submergence isSUB1Adependent.

Thus, SUB1A1 positively regulates the MAPK cascade andspecifically and positively affects the activation of MPK3 duringsubmergence.The yeast two-hybrid assay and in silico docking analy-

ses suggested the specific physical interaction of MPK3 withSUB1A1.Physical interactionof the twoproteinswasconfirmed inplanta by colocalization in the nucleus and BiFC assay. MPK6 didnot interact with SUB1A1 as elucidated by BiFC assays in planta,hinting at the specificity of MPK3 and SUB1A1. These resultsprompted us to explore the functional relevance of this physicalinteraction. A kinase assay indicated that MPK3 is specifically

Figure 6. SUB1A1 Works in Concert with the MPK3 Module in a Positive Regulatory Loop Mechanism.

(A) Immunoprecipitation of MPK3 from transiently transformed Swarna Sub1 and Swarna seedlings grown in control (lanes 1 and 2) and in completesubmergence for 1 d (lanes 3 and 4, respectively), followed by immunoblot with pTEpY antibody to assay the activated MPK3 module.(B)Real-timePCRanalysisofSUB1A1,MPK3,MKK4, andMKKK32 transcripts in14-d-oldnormal-grownseedlingscompletely submerged for1h,6h, 12h,1 d, and 3d. Cleavage andPolyadenylation Splicing Factor (CPASF)was used as an internal control. Error bars indicate SD. n$ 3 independent experiments.(C) and (D) Electrophoretic mobility shift (gel shift) assays showing that SUB1A1 specifically binds to MPK3 promoter fragment. The GCC-box (2151 to2156 bp upstream of ATG) (C);;500 ng of recombinant protein was added (lanes 2 to 6) to the radioactively labeled 40-bp MPK3 promoter fragmentcontaining theGCC-box (D). Noproteinwas added in lane 1. A50 and100molar excess ofwild-typeGCC-box-containing promoter fragmentwas added inlanes4and5, and100molar excessofmutatedG-box-containingpromoter fragmentwasadded in lane6ascompetitors. Theplusandminussigns indicatethe presence or absence of the indicated component.(E) ChIP assays of the MPK3 promoter from Swarna Sub1 and Swarna Sub1 overexpressor (SUB1-Ox) and transgenic seedlings grown in completesubmergence for 1 d (using antibodies toGFP). The gel image shows the results of PCRamplification ofMPK3promoter fragment in the immunoprecipitateand input. The lower panel depicts the IP and the total protein control.

MPK3 Phosphorylates SUB1A1 1137

involved in phosphorylation-coupled activation of SUB1A1; thisin turn leads to submergence tolerance traits. As was specu-lated earlier, the phosphorylation by MPK3 was found to bespecific to the tolerant allele. Thus, SUB1A1, and not SUB1A2,was established as the phosphorylation target of MPK3. TheP186S substitution could very well affect the fold of SUB1A1,which could result in SUB1A1 phosphorylation at a point dif-ferent from Ser-186. Nevertheless, the present data establishthat a posttranslational modification mechanism operatesduring submergence stress in which SUB1A1 is specificallyactivated by phosphorylation. The activated SUB1A1 in turnaugments its effect by dampening the role of ethylene andfurther acting as a nodal point for GA, abscisic acid, andbrassinosteroid crosstalk.

To unravel the genetic dependence of this potential interaction,transient MPK3-overexpression and silenced rice lines weregenerated in SUB1-dependent and -independent backgrounds.MPK3-mediated regulation of restricted shoot elongation, tran-script accumulation of GA repressors, and ROS scavenging en-zyme activity were also dependent on SUB1A. Thus, MPK3overexpression lines showedamarked increment insubmergence-tolerant traits, whereas MPK3-silenced lines were more sub-mergence susceptible in a SUB1A-dependent manner. Thesebiochemical results are in agreement with previous studies inwhich SUB1A-harboring M202 lines showed higher transcriptaccumulationof genes forROSscavengingenzymes (Fukaoet al.,2006, 2011; FukaoandBailey-Serres, 2008; Junget al., 2010). Thepresent findings strongly suggest that an MPK3 works in concertwith SUB1A1 and positively regulates submergence tolerance.

Previous studies have implicated anMPK3 signaling module inROS signaling (Hirt, 2000; Pitzschke and Hirt, 2006, 2009a). TheMPK3 module is also activated by hormonal cues from ethylene(Rodriguez et al., 2010). Both ethylene and ROS are affectedduring the regulation of adaptive responses to submergence.Taking this in to account, a direct link between ethylene and ROS-activatingMPK3module postsubmergence canbehypothesized.

Finally, to delineate the direct role of SUB1A1 in MPK3 ex-pression, we performed a DNA-protein interaction study in whichSUB1A1 was found specifically to interact with the promoter ofMPK3 at the 2151 bp position. The binding of SUB1A1 to thepromoter ofMPK3 was further validated in planta by ChIP assay.There was no putative GCC-box in the promoter of MPK6, theclosest relative of MPK3. Thus, the regulation of MAPK moduleexpression upon submergence by SUB1A1 is MPK3 specific.Thus, on one hand, MPK3 regulates the activity of SUB1A1 byphosphorylation,whereason theotherhand,SUB1A1binds to thepromoter of MPK3 and regulates its transcription. Collectively, itwould be interesting to speculate the submergence specific ac-tivation of MPK3 in the absence of downstream substrate(SUB1A1). It is possible that once SUB1A1 is phosphorylated byMPK3, the phosphorylated SUB1A1 may bind to the MPK3promoter and positively regulate its expression (Figure 7). Asimilar phenomenon has been reported previously in rice; a salt-responsive transcription factor, ERF1, binds to the promoters ofMAP3K6 andMAPK5, while MAPK5 in turn phosphorylates andactivates ERF1 (Schmidt et al., 2013). A recent report in Arabi-dopsis highlights another example of sucha feedback regulatoryloop, in which MYC2 is phosphorylated by MKK3-MPK6 that

binds to thepromoter ofMPK6 in a blue-light-dependentmanner(Sethi et al., 2014).The substrates of MAPK cascades are poorly understood

(Rodriguez et al., 2010) and have long been sought. WRKYtranscription factors are among the few well-established down-stream substrates of MAPK networks (Popescu et al., 2009). Arecent study in Arabidopsis reported accumulation of WRKY22transcripts in response to submergence, which in turn is crucial toimparting immunity to plants (Hsu et al., 2013; Hsu and Shih,2013). MPK3 is an important component of a MAPK signalingnetwork that helps integrate various biotic, abiotic, and devel-opmental cues. This study demonstrates that phosphorylation-based regulation of SUB1A1 by an MPK3 module acts as a nodalpoint of crosstalk between SUB1A1 and submergence signalingpathways. It is intriguing to speculate that the interaction of anMPK3modulewithSUB1A1may alsoprime submerged plants forupcoming pathogen attack upon recession of floodwaters.

METHODS

Plant Material, Growth Conditions, Submergence Stress, andInhibitor Treatment

Rice (Oryzasativa) cvSwarnaand theSUB1 introgression lineSwarnaSub1were analyzed in this study. Sterilized seeds were placed on moist filterpaper for 3 d at 25°C in the dark. Germinated seeds were transplanted intosoil-containingpots (W:L:H, 10310310cm)andgrown in thegreenhouse(30°Cday, 25°Cnight) for 14dundernatural light conditions.Submergencetreatments were performed following the methods described previously

Figure 7. Reweaving the Tapestry of Submergence Tolerance.

Significance ofMAPK cascade during submergence stress is outlined. Themolecular mechanism and signal transduction network implicated insubmergence stress tolerance from this study is represented in the dottedredbox. Thepreviously knowncomponentsareplacedoutside thebox. Themodel shows the involvement of theMPK3module upstreamof SUB1A1 inresponse to submergence stress. The potential testable upstream com-ponents of MPK3 are represented with a question mark. “P” denotes theallele specific phosphorylation of Sub1A1. Furthermore, SUB1A1positivelyregulates the expression of MPK3 in a discrete loop mechanism.

1138 The Plant Cell

(Fukao et al., 2006). Briefly, 14-d-old plants in soil-containing pots werecompletely submerged in aplastic tank (W:L:H, 65365395 cm) filledwith90 cmwater in a greenhouse. All submergence treatments were replicatedin at least three independent biological experiments. For inhibitor pre-treatment experiments, individual plants were incubated with 150 mMMAPKK-specific inhibitor PD98059 (Cell Signaling Technology; #9900) in15.0-mL tubes for 24 h before submergence treatment. The inhibitor-fedplants were then given submergence stress treatment. For mock treat-ment, plants were treated with solvent (DMSO) at a final concentration of0.1%. Nicotiana benthamiana seedlings were used for localization andtransient transformation studies wherever indicated.

qRT-PCR Analyses

The total RNA was extracted from frozen rice seedlings by Trizol reagent(Sigma-Aldrich) according to the manufacturer’s protocol. RNA wastreated with 10 units of RNase free DNase I (Fermentas). Total RNA (2 µg)was subjected to first-strand cDNA synthesis using the RevertAid HMinusFirst Strand cDNA synthesis kit (Thermo Scientific) per the manufacturer’sprotocol using oligo(dT) primers. qRT-PCR was performed in a 10-mLreaction using Power SYBR Green PCRmaster mix (Applied Biosystems).The qRT-PCR was performed in the 384-well plate ABI Prism 7000 se-quence detection system (Applied Biosystems), as has been describedpreviously (Jaggi et al., 2011; Raghuram et al., 2014). The relative ex-pression levelof eachgenewascalculatedusing the22DDCTmethodandbynormalizing against CLEAVAGE AND POLYADENYLATION SPLICINGFACTOR as internal reference. Primer pairs used for qRT-PCR analysis arein Supplemental Table 1.

MAPK Activity Assays

Immunokinase and IP assayswere performed using submergence-treatedrice seedlings that were harvested at the stipulated time point and wereground in liquid nitrogen. Proteins were isolated using kinase extractionbuffer (50 mM HEPES, pH 7.5, 5 mM EDTA, 5 mM EGTA, 10 mM DTT,10 mM Na3VO4, 10 mM NaF, 50 mM b-glycerolphosphate, 1 mM phe-nylmethylsulfonyl fluoride, protease inhibitor cocktail, and 10% glycerol)and were quantified using Bradford assay (Bradford, 1976). Total protein(30 to 40µg)was separated by10%SDS-PAGE, and later, immunoblottinganalysis was performed using anti-pTEpY (Cell Signaling Technology;catalog #9101, lot #28).

Crude protein (300 µg) was used for immunoprecipitation using proteinA-sepharose beads with the respective antibodies. Anti-pTEpY, rabbitpolyclonal anti-MPK6 (catalog #7104, lot #124K4857), anti-MPK4 (catalog#A6979, lot # 124K4855), andanti-MPK3 (catalog#M8318, lot #124K4856)all from Sigma-Aldrich were used for IP. The OsMPK3 antibody was raisedin rabbit against the PVAEFRPTMTHGGR polypeptide of OsMPK3. Forimmunoblot analysis, a 1:7500 dilution of theOsMPK3 antibodywas used.As depicted in Supplemental Figure 2D, the anti-AtMPK3, -AtMPK4, and-AtMPK6 antibodies specifically react with the homologous rice coun-terparts. Also, using theanti-OsMPK3antibody, a single discretebandwasobserved for GST-OsMPK3 and in crude protein extracts of N. ben-thamiana leaf cellsoverexpressingMPK3,establishing thespecificityof theantibody (Supplemental Figure 2E).

In-gel kinase activity assays were performed as described previously(Raina et al., 2012). Briefly, 30 mg total protein was fractionated on a 10%polyacrylamide gel containing 0.1% SDS and 0.5 mg mL21 bovine brainMBP (Sigma Aldrich). After electrophoresis, the SDS from the gel wasremoved with buffer (25 mM Tris-HCl, pH 7.5, 0.5 mMDTT, 5 mMNa3VO4,0.1 mM NaF, 0.5 mg mL BSA, and 0.1% Triton X 100) followed by rena-turation in buffer (25mMTris-HCl, pH 7.5, 0.5mMDTT, 5mMNa3VO4, and0.1 mM NaF) at 4°C overnight. MBP phosphorylation was performed byincubating the gel in 20 mL reaction buffer (25 mM Tris-HCl, pH 7.5, 2 mM

EGTA, 12 mMMgCl2, 1 mM DTT, 0.1 mM Na3VO4, 1 mM ATP, and 50 mCi[g-32P]ATP [3000 Ci mmol21]) for 60min at room temperature. The gel waswashed three times with 5% trichloroacetic acid and 1% sodium pyro-phosphate and visualized by autoradiography using a phosphor imager(Typhoon; GE Healthcare).

Yeast Two-Hybrid Assay

A Matchmaker yeast two-hybrid system (BD Biosciences) was used tocheckprotein-protein interactions. Full-lengthCDSsofSUB1A1,SUB1A2,and MPK3 genes were cloned in frame in pGADT7 and pGBKT7 vectors.Full-length CDSs of otherMAPKmembers were cloned in pGBKT7 vector.For yeast transformation, yeast competent cells (AH109) were preparedaccording to the manufacturer’s instructions. The two respective con-structs were cotransformed in AH109-competent cells. Cotransformantswere initially selected on nutrient medium lacking Leu and Trp (syntheticdefined [SD]/2Leu/2Trp). The resultant cotransformed cells were thenstreaked on quadruple dropout medium deficient in adenine, histidine,leucine, and tryptophan (SD/2Ade/2His/2Leu/2Trp) with 10 mM 3-amino-1,2,4-triazole followed by incubation at 30°C for 36 to 48 h. The fully growncolonies after the incubation were considered as positive interactions.b-Galactosidase assays were performed by monitoring the lacZ reportergene expression directly on nutritional selection plates by addition ofONPG to liquid culture that was rapidly freeze/thawed per the manu-facturer’s instructions (Clontech). As b- galactosidase accumulated in themedium, it hydrolyzed ONPG to O-nitrophenol, which was spectropho-tometrically monitored at 420 nm. AH109 cotransformed with thecoding sequence for SV40 large T antigen in pGADT7 vector and p53 inpGBKT7 vector served as positive control for protein interaction in allthe experiments.

In Silico Docking Assay

Homologymodelingwasperformedasmentioned (Kriegeret al., 2009). Forselection of templates for homology modeling of selected proteins, PSIBLAST (Altschul et al., 1997) was performed against the PDB database(http://www.pdb.org/pdb/home/home.do). 3Dmodels were preparedwiththe help of MODELLER (Kozakov et al., 2013), and the energy of themodeled structures was refined with online server YASARA (http://www.yasara.org/homologymodeling.htm). The overall stereo-chemical qual-ity of the modeled 3D structure of proteins was evaluated usingRamachandran plotting, which is based on psi (Ca-C bond) and phi (N-Cabond) angles of the protein and provides information about the number ofamino acid residues present in allowed and disallowed regions. Further-more, structural verification of the modeled structures was performed byPROCHECK (Laskowski et al., 1996) and ERRAT (Colovos and Yeates,1993). For protein-protein docking, ClusPro, an online protein-dockingserver, was used with its default parameters (Comeau et al., 2004).

Phosphorylation Assays of SUB1A1

To establish the phosphorylation status of SUB1A1 by MPK3, an in-solution kinase assay was performed using SUB1A1-His and GST-MPK3fusion proteins expressed in bacteria. SUB1A1 was in-frame cloned inpET28a (+) expression vector (Novagen) to add a C-terminal His-tag andtransformed into competent Escherichia coli BL21 cells. The protein wasinduced by 1 mM IPTG and solubilized into the supernatant fraction usingthe IBS buffer kit (G-Biosciences). The protein was purified with theQIAexpressionist protein purification system (Qiagen) using Ni-NTAagarosebeads.MPK3wascloned intopGEX-4T2expression vector to addan N-terminal GST tag. Protein was induced by 1 mM IPTG and purifiedusing the Glutathione-4 Sepharose protein purification system (GEHealthcare). MPK3-HA, SUB1A1-HA, and SUB1A2-HA proteins were

MPK3 Phosphorylates SUB1A1 1139

immunoprecipitated from N. benthamiana plants infiltrated with MPK3pSPYCE (M) and SUB1A1/A2 pSPYCE (M) binary vector. The fusionproteins were coexpressed in N. benthamiana leaves using the Agro-bacterium tumefaciens infiltration method (Nakagami et al., 2004). After48 h, proteins were isolated using MAPK extraction buffer consisting of50mMHEPES-KOH (pH 7.5), 5mMEDTA, 5mMEGTA, 1mMDTT, 10mMNa3VO4, 10mMNaF, 50mMb-glycerolphosphate, 1mMPMSF, 10% (v/v)glycerol, 0.1% Nonidet P-40, 2.5% PVPP, and protease inhibitor cocktail(Sigma-Aldrich). A preactivated CnBr-activated sepharose 4B resin (GEHealthcare) was used for the immunoprecipitation-based phosphorylationassays. Anti-HA antibody was coupled to the resin using the manu-facturer’s instructions. Total protein extract (300 µg) was incubatedovernightwith the anti-HA resin complex at 4°C. The next day, after severalwashes, beads with immunoprecipitated MPK3 and SUB1A1/2 weresubjected to in vitro kinase assay. In vitro kinase assay was performed asdescribed (Rao et al., 2011) with slight modifications. Briefly, 5 mg samplewas mixed with reaction buffer to give a final volume of 25 mL containing25 mM Tris-Cl (pH 7.5), 10 mM MgCl2, 5 mM MnCl2, 1 mM DTT, 1 mMb-glycerolphosphate, 1 mM Na3VO4, 0.5 mg/mL MBP, 25 mM ATP, and1mCi [g-32P]ATP. Incubation at 30°Cwas stopped after 30min by additionof 10 mL 23 SDS sample buffer (no DTT or b-ME). Samples were boiled at65°C for 5min and then separated in 10%SDS-PAGEgels. The radioactiveblots and the Coomassie Brilliant Blue-stained gels were visualized usinga phosphor imager (Typhoon; GE Healthcare, Life Sciences).

Site-Directed Mutagenesis

To generate mutated SUB1A1-His protein, the SUB1A186S-PF andSUB1A186S-PR primer pair was used for site-directed mutagenesis byinverse PCR. The pET 28a (+) clone harboring thewild-typeSUB1A1 insertwas used as a template for inverse PCR. The PCR product was digestedwith theDpnI restriction enzyme. The digested productwas used forE. coliBL21 transformation and recombinant protein expression. The mutatedproteins were purified and used for in vitro kinase assay and further im-munoblotting with antiserine antibody (Thermo Scientific Pierce; catalog#PAI-86890, lot #PL1952335).

Immunoblot Analysis

For immunoblots, 20 mg extracted crude protein was fractionated on 10%polyacrylamide gels containing 0.1% SDS. Proteins were transferred toa PVDF membrane in a Bio-Rad semidry blot tank according to themanufacturer’s protocol for 1 h. For immunodetection of proteins, themembrane was blocked with 5% (w/v) skim milk in TBS buffer for 2 h andsubsequently incubatedwithprimaryantibodydiluted inTBS-Tbuffer (TBS0.1% and Tween 20) containing 3% (w/v) skimmed milk for 1.5 h at roomtemperature or at 4°C overnight. After washing in TBS-T, the mem-brane was incubated with secondary antibody diluted in TBS-T containing3% (w/v) skimmed milk at room temperature for 1.5 h. Following severalwashing steps, proteins were detected by incubating the membrane infreshly prepared chemiluminescent horseradish peroxidase substrate(Immobilon Western, Millipore Corporation). Chemiluminescence wasdetected using HyperProcessor (Amersham Biosciences).

Subcellular Localization and BiFC

For the determination of subcellular localization, SUB1A1 andMPK3werefirst cloned into pENTR-DTOPOentry vector and subsequently transferredby LR recombination into pGWB5 to encode C-terminal GFP fusion pro-teins. The cloned constructs were transformed into AgrobacteriumEHA105 cells. A single colony for Agrobacterium EHA105 positive cloneswas streaked onYEB agarmediumwith rifampicin and kanamycin (Sigma-Aldrich) and allowed to grow for 2 d at 28°C in the dark. The colonies were

further inoculated in5mLYEBmediumandgrown for22 to24hat28°Cwithcontinuous shaking at 200 rpm. TheA600 of thecultureswas adjusted to0.5in infiltration medium (10 mM MES, pH 5.7, 10 mM MgCl2, and 150 µMacetosyringone). Themixtureswere incubated in the infiltrationmedium for2 h and later infiltrated into N. benthamiana leaves and observed 3 dpostinfiltration under a confocal laser scanning microscope to detect thefluorescence. For BiFC, SUB1A1 and MPK3 were cloned in pSPYNE173and pSPYCE (M) (Waadt et al., 2008). Both the vectors contain a 35Spromoter-driven multiple-cloning site followed by sequence encoding theN-terminal half of eYFP in pSPYNE173 and the C-terminal half of eYFP inpSPYCE (M). For transient expression, the twoconstructs in the respectivecombinations were infiltrated into tobacco leaves along with helper con-struct P19. The leaves were observed 3 d postinfiltration. A confocalscanning microscope (Leica TCS SP2 AOBS system) with the respectiveGFP or YFP filters was used for imaging. The fluorescence excitation wasachieved using an argon laser source at 514 nm, with emission of 527 nm.

Electrophoretic Mobility Shift (Gel Shift) Assay

To determine the in vitro binding activity of SUB1A1 with the GCC-boxpresent in thepromoter ofMPK3, EMSAwasperformedwitha radiolabeledfragment of the MPK3 promoter containing the GCC-box. The oligonu-cleotides used to amplify the GCC sequence were SUB1EmF andSUB1EmR. Mutations in the probe sequences were generated using theprimers M-SUB1EmF and M-SUB1EmR (Supplemental Table 1). TheMPK3promoter fragment containing theGCC-boxwas amplified, purified,and [a-32P]dCTP labeled byKlenowenzyme (NewEnglandBiolabs) for useas probe. The recombinant SUB1A1-His was induced and overexpressedin E. coli and affinity purified following the manufacturer’s protocol(AmershamBiosciences). Approximately 25 ng labeled DNA fragment wasincubatedwith purifiedSUB1A1-His (100ng) in thepresenceof 1µgof polydI-dC and 13 binding buffer (75mMHEPES, pH 7.5, 350mMKCl, 2.5 mMEDTA, 10%glycerol, 5mMDTT, and 10mMMgCl2) in a reaction volume of30mL for 30min at room temperature. Competition assayswereperformedusing 50 and 100 molar excess of unlabeled GCC-box-containing MPK3promoter fragments and 100 molar excess of mutated GCC-box-containingMPK3 promoter fragments. The reactions were fractionated onnative polyacrylamide gels (10% acrylamide, 0.5% TBE, and 2.5% glyc-erol), dried, and visualized by autoradiography.

ChIP Assays

ChIP assays were performed as described by Sethi et al. (2014) with somemodifications. Swarna Sub1 and Swarna Sub1 overexpressor (SUB1-Ox)lines were used for the experiment. Transient transformation of SUB1-Oxline was undertaken by agroinoculation using pGWB5 construct harboringSUB1A1. Seedlings were treated for complete submergence for 1 d. Theimmunoprecipitated products were subjected to RT-PCR analysis to ex-amine the relative enrichment of the promoter fragment using the primersthatwereused in thegel-shift assay.TheGFPmonoclonalantibody (ThermoFisher Scientific) was used for the immunoprecipitation experiments.

Transformation of Rice Seedlings

Transient transformation in rice seedlings was undertaken by agro-inoculation aspreviously described (Purkayasthaet al., 2010). For transienttransformation ofMPK3-Ox lines,MPK3 in pGWB5was used, while for theMPK3-silenced lines, MPK3 in mVIGS vector was used. Briefly, all vectorswere transformed intoAgrobacteriumstrain EHA105, and aprimary culturewas initiated from a single Agrobacterium colony in LB medium supple-mented with appropriate antibiotics. Subsequently, a secondary culturewas grown to an OD600 of 0.6 to 0.8 with 200 µMacetosyringone. The cellswere harvested and resuspended in 10 mM MES, 10 mM MgCl2, and

1140 The Plant Cell

200 µM acetosyringone to a volume 20-fold less than the original.Approximately 5-d-old rice plants grown in Yoshida’s medium wereused for agroinoculation. About 50 mL bacterial suspension was in-jected into the meristematic region located at the crown of the plants,which were then transferred onto sterile Whatman No. 1 filter paperimmersed in Yoshida’s medium placed on a solid support with its endsdipped into a reservoir containing themedium. The plants were coveredwith moist tissue paper and transferred to tubes containing Yoshida’smedium 24 h postinoculation and were maintained at 27°C underconditions described above. The transiently transformed seedlingswere then screened for positive transformants. The positively trans-formed MPK3-overexpressing or -silenced rice seedlings were furthergiven submergence treatment and later used for other biochemicalexperiments.

Biochemical Assays

Total chlorophyll was isolated as described previously (Arnon, 1949).Briefly, leaf tissue were homogenized in 80% ice-chilled acetone usingmortar and pestle and centrifuged at 9500g for 10 min. The absorptionin the supernatant was recorded at wavelengths 663 and 645 nm.Total protein estimation was performed following Bradford’s method(Bradford, 1976), with BSA as a standard. MDA) content was estimatedwith thiobarbituric acid (Heath and Packer, 1968). The amount of MDAwas calculated from the difference in absorbance at 532 and 600 nmusing an extinction coefficient of 155 mM21 cm. For assays of the en-zymatic cellular antioxidants, includingSODandCAT, frozenplant leaves(0.4 to 0.8 g) were homogenized in ice-cold extraction buffer (pH 7.5)containing 50mMHEPES, 0.4mMEDTA, 5mMMgCl2, 10%glycerol, 1%polyvinylpyrrolidone, 2 mM DTT, and 1 mM PMSF (Gegenheimer, 1990).The homogenate was centrifuged (14,000g) at 4°C for 20 min. The su-pernatant was assayed for enzyme activity. SOD activity (EC 1.15.1.1)wasmeasuredbymonitoring the inhibition of photochemical reduction ofnitro blue tetrazolium (Dhindsa et al., 1981). CAT activity (EC 1.11.1.6)was determined by monitoring the disappearance of H2O2 (Aebi, 1984).APXwas extracted in leaf samples (0.4 to 0.8 g) homogenized in mediumcontaining 100 mM phosphate buffer (pH 7.3), 1 mM EDTA, 1% poly-vinylpyrrolidone, and 1 mM ascorbate. The rate of H2O2-dependentoxidation of ascorbic acid was determined in a reaction mixture con-taining0.1Mphosphatebuffer (pH7.0), 0.5mMascorbic acid, and100mLenzyme extract. Activity of APX was measured by monitoring the rate ofH2O2-dependent oxidation of AA followed by measuring the decrease inabsorbance at 290 nm (extinction coefficient of 2.8mM21 cm21) (Nakanoand Asada, 1981).

Rice Root Cell Death Assay

Roots (1 to 2 cm)ofMPK3overexpressionandsilenced lines inSwarna andSwarna Sub1 backgrounds, before and after submergence, were excisedfrom the seedlings and stainedwith 1mgmL21 PI by vacuum infiltration for15 to 20 min. The samples were observed under confocal scanning mi-croscopy (Leica TCS SP2 AOBS system).

Accession Numbers

Sequence data from this article can be found in the Rice Genome Anno-tation Project orGenBank/EMBLdatabases under the following accessionnumbers: DQ011598 (SUB1A), OsMPK3 (Os03g17700.1), OsMPK4(Os10g38950.1), OsMPK6 (Os06g06090.1), OsMPK7 (Os06g48590.1),OsMPK14 (Os02g05480.1), OsMPK16-1 (Os05g05160.1), OsMPK16-2(Os11g17080.1), OsMPK17-1 (Os06g49430.2), OsMPK17-2 (Os02g04230.1),OsMPK20-1 (Os01g43910.1), OsMPK20-2 (Os05g50560.1), OsMPK20-3(Os06g26340.1), OsMPK20-4 (Os01g47530.1), OsMPK20-5 (Os05g49140.1),OsMPK21-1 (Os05g50120.1), and OsMPK21-2 (Os01g45620.1)

Supplemental Data

Supplemental Figure 1. Submergence-induced activation of MAPKcascade.

Supplemental Figure 2. Submergence-induced specific activation ofMPK3.

Supplemental Figure 3. Submergence tolerance-related activation ofthe MAPK cascade is SUB1A dependent.

Supplemental Figure 4. Physical Interaction of SUB1A2 with MPK3.

Supplemental Figure 5. Physical interaction of SUB1A1 with MPK3,MPK4, and MPK6.

Supplemental Figure 6. Assay of specificity of interaction of SUB1A1and MPK3.

Supplemental Figure 7. MPK3 (and not MPK4/MPK6) specificallyphosphorylates SUB1A1.

Supplemental Figure 8. Confirmation of transient MPK3 expressionlevel changes in rice seedlings.

Supplemental Figure 9. Cell death assay.

Supplemental Figure 10. Yeast two-hybrid assay to assess thephysical interaction of MPK3 module components.

Supplemental Table 1. List of primers used in the present study.

ACKNOWLEDGMENTS

The work was supported by the core grant of National Institute of PlantGenome Research from the Department of Biotechnology, Govern-ment of India. P.S. is a recipient of a fellowship from the Council ofScientific and Industrial Research, Government of India. We thank theConfocal Microscopy Facility and the Central Instrumentation Facilityof NIPGR, New Delhi, India. We also thank S. Robin (Tamil NaduAgricultural University) for providing seeds of Swarna and SwarnaSub1, Indranil Das Gupta (University of Delhi South Campus, NewDelhi) for the kind gift of mVIGS vector, and Adam J. Bogdanove(Cornell University, Ithaca, NY) for critically reading and editing themanuscript.

AUTHOR CONTRIBUTIONS

P.S. and A.K.S. designed the research. P.S. carried out the experiments.P.S. and A.K.S. analyzed the data and wrote the manuscript.

Received November 30, 2015; revised March 29, 2016; accepted April 12,2016; published April 14, 2016.

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DOI 10.1105/tpc.15.01001; originally published online April 14, 2016; 2016;28;1127-1143Plant Cell

Pallavi Singh and Alok Krishna SinhaPROTEIN KINASE3 Imparts Submergence Tolerance in Rice

A Positive Feedback Loop Governed by SUB1A1 Interaction with MITOGEN-ACTIVATED

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