TY - JOUR
AU - Sinha, Alok Krishna
AB - Abstract 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 response factor-like protein SUB1A orchestrates a plethora of responses during submergence stress tolerance in rice (Oryza sativa). In this study, we report that MPK3 is activated by submergence in a SUB1A-dependent manner. MPK3 physically interacts with and phosphorylates SUB1A in a tolerant-allele-specific manner. Furthermore, the tolerant allele SUB1A1 binds to the MPK3 promoter and regulates its expression in a positive regulatory loop during submergence stress signaling. We present molecular and physiological evidence for the key role of the MPK3-SUB1A1 module in acclimation of rice seedlings to the adverse 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 standing water but at the same time are susceptible to transient and complete inundation. Sudden flooding of rice fields leads to a decrease in oxygen and soil pH, which further leads to deprivation of nutrients. The molecular mechanism governing submergence stress management comprises two contrasting acclimation responses, namely, the low-oxygen escape strategy and the low-oxygen quiescence strategy (Bailey-Serres and Voesenek, 2008). Low-oxygen escape strategy, characteristic of upland cultivars, comprises a repertoire of traits including high rate of carbohydrate consumption, in turn allowing rapid elongation of aerial organs to keep the leaves above the water level. This is predominantly governed by SNORKEL1/2 (SK1 and SK2), which are tandem ethylene-responsive factor (ERF) genes (Hattori et al., 2009). On the contrary, low-oxygen quiescence strategy involves stress-induced repression of carbohydrate resource consumption, in conjunction with plant growth inhibition. This tolerance strategy is determined by the SUB1A gene of the SUBMERGENCE1 (SUB1) quantitative trait locus, which confers tolerance to up to 2 weeks of submergence. The SUB1 quantitative trait locus is a multigenic locus encoding three clade VII ERFs (SUB1A, SUB1B, and SUB1C) (Fukao et al., 2006; Xu et al., 2006). Improvement of rice crop resilience to submergence can be accomplished by harnessing the genetic potential of the SUB1 locus. The SUB1 locus has been introgressed into many mega varieties through a marker-assisted backcrossing strategy (Neeraja et al., 2007; Septiningsih et al., 2009; Iftekharuddaula et al., 2012). Several of these SUB1-introgressed mega varieties, i.e., Swarna-Sub1, IR64-Sub1, and BR11-Sub1, have widely undergone field trials and show positive attributes of submergence tolerance. These varieties have been released in India, the Philippines, Indonesia, and Bangladesh and show all of the desirable agronomic traits and grain quality of the original parent variety. A deeper genetic survey into SUB1 locus identified two SUB1A, nine SUB1B, and seven SUB1C alleles on the basis of variation in amino acid sequence (Xu et al., 2006). The SUB1A1 allele is specific to submergence-tolerant accessions. Variations in putative mitogen-activated protein kinase (MAPK) sites distinguish the tolerant and intolerant alleles of SUB1A. In the tolerant SUB1A1 allele, a single nucleotide polymorphism at position 556 is responsible for a Pro-186 (intolerant) to Ser-186 (tolerant) substitution (Xu et al., 2006). The activities of several plant transcription factors are modulated by posttranslational modifications, particularly, phosphorylation and dephosphorylation. Phosphorylation of transcription factors by MAPKs can regulate their intrinsic levels and activities (Tootle and Rebay, 2005). The MAPK signaling cascade is a three-tier phospho-relay signaling module that is evolutionarily conserved in all eukaryotes (Rodriguez et al., 2010; Sinha et al., 2011). In plants, the MAPK signal transduction network administers various biotic and abiotic stress responses. This network is associated with various hormone responses and cell divisional as well as developmental cues (Colcombet and Hirt, 2008; Sinha et al., 2011). Group A members MPK3 and MPK6 as well as Group B MPK4 are the most widely studied MAPK components in plants (Fiil et al., 2009; Andreasson and Ellis, 2010). Ethylene and reactive oxygen species (ROS) are key regulators of plant submergence stress status. Increased ethylene levels in submerged plants trigger SUB1A gene expression. SUB1A dampens ethylene-promoted gibberellic acid (GA) responsiveness during submergence. This dampening usually occurs due to accumulation of the GA signaling repressors SLENDER RICE1 (SLR1) and SLR1-like 1 (SLRL1). The expression of GA repressors is a strategy to counteract complete submergence. It facilitates the plant to adapt to quiescence mode of metabolism until floodwaters recede (Fukao et al., 2006; Fukao and Bailey-Serres, 2008). It is reported that multiple ethylene-dependent pathways lead to MAPK activation (Rodriguez et al., 2010). Two of the best-studied Arabidopsis thaliana RAF-like MAP3Ks, CONSTITUTIVE TRIPLE RESPONSE1 and ENHANCED DISEASE RESISTANCE1, participate in ethylene-mediated signaling and defense responses. A cascade that involves MKK9-MPK3/6 also participates in ethylene signaling (Hahn and Harter, 2009). SUB1A ameliorates the effect of ROS produced by hypoxia during submergence (Bailey-Serres et al., 2012). MAPK cascades are also known to respond to the resulting oxidative burst and may regulate ROS accumulation. Both MPK3 and MPK6 signaling modules within Group A are implicated in ROS signaling (Pitzschke and Hirt, 2009b). This further broadens the reach of this cascade to other processes related to hypoxia and submergence. Although the physiological functions of MAPKs in response to various external stimuli have been extensively characterized, there have been no studies describing a role for this cascade in submergence stress responses. However, a report by Seo et al. (2011) hinted at the involvement of a MAPK cascade in submergence stress tolerance. That study reported that MPK5 (which we refer to as MPK3 in this case, based on the phylogeny-based nomenclature of Hamel et al. [2006]) showed higher transcript accumulation in SUB1A-containing rice (M202-Sub1A versus M202) upon submergence. Our current study builds upon this preliminary observation. SUB1A1 contains a potential MAPK phosphorylation site located in a variable region C-terminal to the ERF domain. This may be of significance as phosphorylation can modulate DNA binding by ERF proteins (Gutterson and Reuber, 2004; Xu et al., 2006). Thus, in this study, the putative role of MAPK signaling in submergence stress tolerance was investigated, in particular, the possible connection between submergence and MAPK pathways via SUB1A1. Here, we report that MPK3 is specifically activated upon submergence and that this process is SUB1A1 dependent. Our study demonstrates that MPK3 physically interacts with and phosphorylates SUB1A1 in an allele-specific manner. Furthermore, by MAPK-specific inhibitor assays and transient transformation phenotypic assays, we report the genetic interaction of SUB1A1 and MPK3, which in turn augments its role by governing a suite of submergence-related traits. We also show that SUB1A1 interacts with the promoter of MPK3 to regulate its expression. These findings collectively reveal a positive regulatory mechanism in the submergence signaling cascade. RESULTS Submergence-Mediated Activation of MAPK Cascade Components Is SUB1A Dependent To gain insight into the involvement of a MAPK signaling cascade in submergence stress tolerance and to determine the SUB1A dependency of the process, transcript profiling of MAPK components was undertaken. Initially, we sought to determine the submergence-dependent expression of genes for the complete family of MAPKs and MAPKKs and the Raf family of MAPKKKs by quantitative real-time PCR, in submergence-tolerant (Swarna Sub1) and in submergence-intolerant (Swarna) cultivars. As shown in the bicolor heat map in Figures 1A to 1C, the expression of cardinal MAPK cascade components (Groups A, B, and C) was increased postsubmergence in the Swarna-Sub1 genotype. There was a chronic elevation of transcripts of the less deciphered Group D MAPK members in Swarna. However, it is likely that these Group D members are outliers, as they do not show any specific phosphorylation-dependent activation in immunokinase assays using 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, there are very few reports implicating Group D MAPKs in physiological functions of plants. For an in-depth analysis, the transcript expression of MPK3 in both Swarna-Sub1 and Swarna lines is presented in Supplemental Figures 1A and 1B. These results suggest that SUB1A positively regulates the expression of the MAPK signaling cascade components during submergence. Figure 1. Open in new tabDownload slide 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 rice cultivars treated with submergence. The fold change from qRT-PCR assay was plotted into a heat map using MEV software framework. The values indicate the mean of three independent sets of experiments. Immunoblot analyses using pTEpY antibody of 40 µg of total protein from Swarna Sub1 and Swarna seedlings 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 of MPK3 assessed by immunoprecipitation assays using 300 µg total protein from Swarna and Swarna Sub1 lines 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-OsMPK3 antibody. Lower panel shows immunoblot with OsMPK3 antibody using 20 µg total protein as the loading control. Values below the panel indicate quantification of each band with respect to the loading control. Figure 1. Open in new tabDownload slide 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 rice cultivars treated with submergence. The fold change from qRT-PCR assay was plotted into a heat map using MEV software framework. The values indicate the mean of three independent sets of experiments. Immunoblot analyses using pTEpY antibody of 40 µg of total protein from Swarna Sub1 and Swarna seedlings 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 of MPK3 assessed by immunoprecipitation assays using 300 µg total protein from Swarna and Swarna Sub1 lines 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-OsMPK3 antibody. Lower panel shows immunoblot with OsMPK3 antibody using 20 µg total protein as the loading control. Values below the panel indicate quantification of each band with respect to the loading control. These findings prompted us to search for the MAPK that was specifically activated in response to submergence in a SUB1A-dependent manner. We began with an immunokinase assay of the crude protein extracts from Swarna and Swarna Sub1 lines grown in optimal conditions for 14 d and then completely submerged for a short duration (1 to 12 h) and a long duration (1 to 7 d). Immunoblot analysis was performed with pTEpY antibody, which specifically binds to activated MAPK cascade components (Sethi et al., 2014). Interestingly, submergence activated 42-kD MAPKs within 1 h in the Swarna Sub1 plants. However, Swarna lines did not show any such activation (Figures 1D and 1E). The activation of the 42-kD protein was observed to increase with increasing duration of submergence. The size of the protein identified by immunoblot with pTEPY antibody was ascertained by running pTEpY antibody-immunoprecipitated sample in parallel with crude protein followed by immunoblotting with pTEpY and MPK3 antibodies (Supplemental Figure 1C). To further substantiate this finding, an in-gel kinase assay using Swarna and Swarna Sub1 lines grown optimally for 14 d and then submergence treated for up to 7 d was also performed. MAPK activity was analyzed using the artificial MAPK substrate Myelin Basic Protein (MBP). Interestingly, submergence activated MBP-phosphorylating protein kinase activity within 1 h in the Swarna Sub1 plants. However, in accordance with the result of the immunoblots, Swarna lines did not show any such activity (Supplemental Figures 1D and 1E). Submergence-Mediated-Specific Activation of MPK3 Is SUB1A Dependent These results suggest that a MAPK at 42 kD is activated in response to submergence and that this activation is likely to be SUB1A dependent. Next, we aimed to determine which MAPK was specifically activated by submergence and whether the activation was indeed SUB1A dependent. For this, immunoprecipitation (IP) was performed using pTEpY antibody followed by immunoblot analysis with anti-OsMPK3 antibody. The submerged Swarna Sub1 lines showed a band of increasing intensity at 42 kD, while this band was not observed in submerged Swarna lines (Figure 1F). OsMPK3 shows high homology with its Arabidopsis counterpart (Hamel et al., 2006); to further confirm the identity of the precipitated protein, immunoblot analysis was also performed using an anti-AtMPK3 antibody. In accordance with the previous observation, submerged Swarna Sub1 lines showed a band at 42 kD, while this 42-kD band was not observed in submerged Swarna lines (Supplemental Figure 2A). These results indicate that the specific activation of MPK3 upon submergence is SUB1A dependent. The specific activation of MPK3 postsubmergence in a SUB1A-dependent manner was further established by performing control experiments using anti-AtMPK6 and anti-AtMPK4 antibodies (the closest homologs of OsMPK6 and 4, respectively) as depicted in Supplemental Figures 2B and 2C. The MAPK Cascade Regulates Submergence Stress-Inducible Genes SLR1 and SLRL1 To further explore if a MAPK cascade could have an essential role in SUB1A-mediated submergence tolerance in rice, we performed MAPK inhibitor pretreatment experiments. The most tractable phenotype of SUB1A-mediated submergence tolerance is 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 were measured as an indicator of submergence tolerance, i.e., the ability to limit shoot elongation (Figures 2A and 2B). MAPK inhibitor treatment had no effect on either genotype in the absence of submergence. The inhibitor pretreated, submerged Swarna plants did not show any variation in shoot elongation after submergence, whereas inhibitor pretreated Swarna Sub1 lines exhibited marked shoot elongation contrary to the characteristic, submergence tolerance phenotype. The MAPK inhibitor treatment experiments demonstrate that a MAPK signaling cascade could be involved in the SUB1A-mediated submergence tolerance phenotype. SUB1A was proposed to induce the expression of and further stabilize GA repressors SLR1 and SLRL1 (Fukao and Bailey-Serres, 2008). As MAPK inhibitor treatment abolished the characteristic submergence-mediated restriction of shoot elongation phenotype, the transcript levels of SLR1 and SLRL1 were monitored by quantitative-real time PCR analysis (Figure 2C). Indeed, inhibitor pretreated Swarna Sub1 seedlings showed marked decrease in accumulation of SLR1 and SLRL1 transcripts compared with untreated seedlings. No such differential transcript regulation of SLR1 and SLRL1 was observed in Swarna (Supplemental Figure 3). The above experiments demonstrate the involvement of MAPK signaling in SUB1A-mediated submergence tolerance. Figure 2. Open in new tabDownload slide 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 tap water 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-pretreated submerged Swarna Sub1A. Error bars represent mean sd of three independent sets of experiments. (C) Expression profiles of GA signaling repressor genes in a time-course experiment. Expression levels for MPK3, SLR1, and SLRL1 at 0 (Con), 6, 12, and 24 h after submergence in mock-treated and PD98059-pretreated Swarna Sub1 seedlings were measured using real-time PCR assays. Values are relative to 0 h. Error bars represent mean sd of three independent sets of experiments. Figure 2. Open in new tabDownload slide 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 tap water 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-pretreated submerged Swarna Sub1A. Error bars represent mean sd of three independent sets of experiments. (C) Expression profiles of GA signaling repressor genes in a time-course experiment. Expression levels for MPK3, SLR1, and SLRL1 at 0 (Con), 6, 12, and 24 h after submergence in mock-treated and PD98059-pretreated Swarna Sub1 seedlings were measured using real-time PCR assays. Values are relative to 0 h. Error bars represent mean sd of three independent sets of experiments. SUB1A1 Physically Interacts with MPK3 To delineate the role of MPK3 in submergence stress tolerance, interaction of SUB1A1 with MPK3 was assessed using yeast two-hybrid assays. An interaction was observed upon expression of full-length clones of SUB1A1 and MPK3. The interaction was also observed after swapping SUB1A1 and MPK3 as bait and prey (Figure 3A). Since SUB1A1 and MPK3 interaction was positive on nutritional selection medium, lacZ reporter gene expression was confirmed using ortho-nitro phenyl β-galactoside (ONPG) as substrate. Yeast strain AH109 cotransformed with SUB1A1 and MPK3 yielded high β-galactosidase activity, whereas other transformants with combinations of SUB1A1 or MPK3 and empty AD or BD vectors had low or negligible β-galactosidase activity (Figure 3B). To determine the specificity of interaction between SUB1A1 and MPK3, targeted yeast two-hybrid assays were performed for SUB1A1 against other characteristic members of the MAPK clade. The close relatives of MPK3 include MPK6 and MPK4 from Group A and B MAPKs, respectively. The other members analyzed for the assay included MPK7 and MPK14 of Group C MAPKs and MPK16-1, MPK17-1, MPK17-2, MPK20-1, MPK20-2, MPK20-3, MPK20-4, and MPK21-1 of Group D MAPKs. Only the combination of SUB1A1 and MPK3 produced growth on nutritional selection medium (adenine, histidine, leucine, and tryptophan) reflecting the specificity of the interaction (Figure 3C). Thus, MPK3 specifically interacts with SUB1A1 as evident by the pairwise protein-protein interaction studies. SUB1A2 was also found to interact with MPK3 (Supplemental Figures 4A and 4B). However, this interaction was weaker than the one observed between SUB1A1 and MPK3 as measured by β-galactosidase activity. Figure 3. Open in new tabDownload slide 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) β-Galactosidase assay of lacZ reporter gene expression with the indicated constructs using ONPG as substrate. Error bars represent mean sd of three independent sets of experiments. (C) Specificity of interaction between SUB1A1 and MPK3; reporter yeast strain AH109 was cotransformed with SUB1A1 in pGADT7 and the coding sequences for cardinal members of the MAPK clade in pGBKT7. Transformants were selected on SD [-leu-trp] DDO medium, and the interaction was checked 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 μm. (E) SUB1A1 physically interacted with MPK3 in BiFC assays in N. benthamiana leaves. (a) Reconstruction of YFP signal when the MPK3-YFP n-ter. and SUB1A1-YFP c-ter. constructs were coinfiltrated into N. benthamiana leaves. (b) and (c) Bright-field image and merged image. (d) to (f) show cells coinfiltrated 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 cells coinfiltrated with the empty YFP n-ter. vector and SUB1A1 YFP c-ter. construct. Figure 3. Open in new tabDownload slide 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) β-Galactosidase assay of lacZ reporter gene expression with the indicated constructs using ONPG as substrate. Error bars represent mean sd of three independent sets of experiments. (C) Specificity of interaction between SUB1A1 and MPK3; reporter yeast strain AH109 was cotransformed with SUB1A1 in pGADT7 and the coding sequences for cardinal members of the MAPK clade in pGBKT7. Transformants were selected on SD [-leu-trp] DDO medium, and the interaction was checked 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 μm. (E) SUB1A1 physically interacted with MPK3 in BiFC assays in N. benthamiana leaves. (a) Reconstruction of YFP signal when the MPK3-YFP n-ter. and SUB1A1-YFP c-ter. constructs were coinfiltrated into N. benthamiana leaves. (b) and (c) Bright-field image and merged image. (d) to (f) show cells coinfiltrated 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 cells coinfiltrated with the empty YFP n-ter. vector and SUB1A1 YFP c-ter. construct. An in silico docking experiment was performed to further assess the physical interaction of MPK3 and SUB1A1. For this, a homology modeling approach was employed to predict the 3D structures of MPK3, MPK4, MPK6, and SUB1A1. These 3D structures were further refined and used as an input for protein-protein docking using ClusPro (http://cluspro.bu.edu/) to predict MPK3-SUB1A1, MPK4-SUB1A1, and MPK6-SUB1A1 interaction. The in silico prediction indicated a stronger physical interaction in MPK3-SUB1A1 relative to the other pairs. This strong interaction 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 interaction of SUB1A1 with MPK3. SUB1A1 Physically Interacts with MPK3 in Planta For an initial examination of the likelihood of physical interaction between MPK3 and SUB1A1 in planta, subcellular localization was predicted using the PLANT-MPLOC (http://www.csbio.sjtu.edu.cn/bionf/plant-multi/) server. Both proteins were predicted to localize to the nucleus. Next, subcellular localization of the proteins expressed transiently in Nicotiana benthamiana was determined using SUB1A1 and MPK3 pGWB5 constructs, which encode C-terminal sGFP (superfolder-GFP) fusion proteins. Confocal imaging confirmed localization of the two proteins to the nucleus (Figure 3D). To further test the in planta physical interactions between MPK3 and SUB1A1, bimolecular fluorescence complementation (BiFC) experiments were performed. Full-length MPK3 coding sequence (CDS) was fused to sequence encoding the N terminus of YFP in the pSPYNE173 vector, and SUB1A1 CDS was fused to sequence encoding the C terminus of YFP in the pSPYCE (M) vector. These constructs together produced strong YFP fluorescence in the nucleus of N. benthamiana leaves (Figure 3E). To check the specificity of MPK3 and SUB1A1 interaction, a BiFC assay of MPK6 and SUB1A1 was also undertaken (Supplemental Figures 6A and 6B). No YFP fluorescence was observed in this case. Taken together, these results demonstrate that MPK3 physically interacts with SUB1A1 in planta in a specific manner. Cotransfection of empty BiFC vectors (Figure 3E, d to f), cotransfection of the MPK3-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 SUB1A1YFP c-ter construct (Figure 3E, j to l) served as controls. The protein loading controls for the transient transformation of N. benthamiana leaves for the BiFC assays and subcellular localization are represented in Supplemental Figures 6C and 6D, respectively. SUB1A1 Is Phosphorylated by MPK3 To ascertain whether SUB1A1 is indeed a phosphorylation target of MPK3, an in-solution kinase assay was performed using bacterially expressed SUB1A1-His and GST-MPK3 protein. SUB1A1-His was used as a substrate. A control experiment was performed to assess the MBP phosphorylation potential of MPK3 (Figure 4A, lane 1) and to assess the autophosphorylation activity of the bacterially expressed protein (Figure 4A, lane 2), while SUB1A1-His alone served as a negative control (Figure 4A, lane 3). GST-MPK3 phosphorylated SUB1-His in this assay (Figure 4A, lane 4). These results suggest that MPK3 phosphorylates SUB1A1. Figure 4. Open in new tabDownload slide 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 for phosphorylation by MPK3. The plus and minus signs indicate the presence and absence of proteins, respectively. Aliquots of the samples were separated by SDS-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 assay using plant protein. N. benthamiana leaves infiltrated with MPK3-pSPYCE (M) were used for immunoprecipitation of crude protein 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 in CBB-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-HA antibody followed by incubation with GST-MPK3 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 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, and SUB1A2-His were incubated alone or in combination with MBP. The plus and minus signs indicate the presence and absence of proteins, respectively. Aliquots of the samples were separated by SDS-PAGE and later immunoblotted with antiphosphoserine antibody. Lower panel shows the CBB-stained gel with the position of different proteins indicated (arrows). Numerals in the left of lower panel indicate molecular mass (kD) marker used in CBB-stained gel. Figure 4. Open in new tabDownload slide 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 for phosphorylation by MPK3. The plus and minus signs indicate the presence and absence of proteins, respectively. Aliquots of the samples were separated by SDS-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 assay using plant protein. N. benthamiana leaves infiltrated with MPK3-pSPYCE (M) were used for immunoprecipitation of crude protein 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 in CBB-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-HA antibody followed by incubation with GST-MPK3 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 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, and SUB1A2-His were incubated alone or in combination with MBP. The plus and minus signs indicate the presence and absence of proteins, respectively. Aliquots of the samples were separated by SDS-PAGE and later immunoblotted with antiphosphoserine antibody. Lower panel shows the CBB-stained gel with the position of different proteins indicated (arrows). Numerals in the left of lower panel indicate molecular mass (kD) marker used in CBB-stained gel. We also assessed the phosphorylation status of SUB1A1 with other important and close relatives of MPK3, namely, MPK4 and MPK6 (Supplemental Figure 7). For this assay, we used SUB1A1 harboring an HA tag expressed in leaves of N. benthamiana and purified by immunoprecipitation. SUB1A1-HA was phosphorylated by MPK3 (Supplemental Figure 7, lane 3) but not by MPK4 (Supplemental Figure 7, lane 5) or MPK6 (Supplemental Figure 7, lane 7). Furthermore, to determine whether SUB1A1 is phosphorylated by plant-expressed MPK3, the MPK3-pSPYNE 173 construct expressing the N terminus of eYFP-HA-MPK3 was agroinfiltrated in N. benthamiana leaves. The HA-tagged MPK3 was immunoprecipitated and used for the in vitro kinase assay. Bacterially expressed SUB1A1 was phosphorylated by immunoprecipitated MPK3-HA (Figure 4B). These results establish that SUB1A1 is the phosphorylation target of MPK3. MPK3 Phosphorylation Is Tolerant-Allele Specific Now that we established SUB1A1 as the phosphorylation target of MPK3, it was important to explore the physiological relevance of any potential allele-specific phosphorylation. Thus, both SUB1A1 and SUB1A2 proteins were expressed in planta. The HA-tagged SUB1A1 and SUB1A2 were immunoprecipitated with anti-HA antibody and used for the phosphorylation assays, alongside bacterially expressed GST-MPK3 protein. A control experiment was performed to assess the autophosphorylation potential of MPK3 (Figure 4C, lane 1), while SUB1A1-HA and SUB1A2-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 phosphorylates the protein produced by the submergence-tolerant allele, i.e., SUB1A1. To further assess the allele-specific nature of this phosphorylation, an in-solution kinase assay was performed using bacterially expressed SUB1A1-His and Ser-186 to Pro-186 mutated SUB1A1-His (SUB1A2-His) and GST-MPK3 protein. The reactions of the in-solution kinase assay were further immunoblotted with antiphosphoserine antibody. A control experiment was performed to assess the autophosphorylation activity of the bacterially expressed protein (Figure 4D, lane 1), while SUB1A1-His and SUB1A2-His alone served as negative controls (Figure 4D, lanes 2 and 3, respectively). GST-MPK3 phosphorylated SUB1A1-His but not SUB1A2-His, even with increasing concentration of SUB1A2-His (Figure 4D). These results further demonstrate the allele-specific phosphorylation of SUB1A1 by MPK3. SUB1A1 Genetically Interacts with MPK3 and Regulates Submergence-Dependent Traits Since we found MPK3 to be molecularly connected to SUB1A1 in a submergence-dependent manner, we further examined the physiological significance of the interaction. For this, rice seedlings transiently overexpressing MPK3 (MPK3-Ox) or silenced for MPK3 were raised in both Swarna and Swarna Sub1 backgrounds. Overexpression and silencing was confirmed by transcript level analysis of MPK3 (Supplemental Figures 8A and 8C) as well as protein level assays in the respective lines (Supplemental Figures 8B and 8D). Furthermore, the submergence-related attributes of these lines were assayed in normal and postsubmergence conditions. As previously stated, we sought to measure the most tractable phenotype of SUB1A-mediated submergence tolerance, i.e., the restricted shoot elongation upon inundation. Seven-day-old seedlings of control, MPK3-Ox, and MPK3-silenced lines in both Swarna Sub1 and Swarna backgrounds were completely submerged for a period of 7 d, and final seedling heights were measured (Figures 5A and 5B). The MPK3-Ox Swarna Sub1 line showed restricted shoot length elongation postsubmergence, whereas the MPK3-silenced Swarna Sub1 lines showed a chronic increase in shoot length postsubmergence. The corresponding Swarna lines did not show any variation in shoot elongation upon submergence. High transcript accumulation of genes for the GA repressors SLR1 and SLRL1 serves as another marker for submergence tolerance (Fukao and Bailey-Serres, 2008). The transcript accumulation of SLR1 and SLRL1 was monitored by quantitative-real time PCR analysis in the transgenic lines postsubmergence (Figure 5C). Indeed, submergence-treated MPK3-Ox Swarna Sub1 seedlings showed marked accumulation of SLR1 and SLRL1 transcripts compared with the MPK3-silenced Swarna Sub1 seedlings. No such differential transcript accumulation of SLR1 and SLRL1 was observed in the Swarna transgenic lines. Figure 5. Open in new tabDownload slide MPK3-Silenced Lines Are Submergence Susceptible While MPK3 Overexpression Lines Show Submergence-Tolerant Traits. (A) Transgenic Swarna and Swarna Sub1 seedlings overexpressing or silenced for MPK3 and nontransgenic seedlings were submergence treated for 7 d in tap water. Representative individuals are shown pre- and postsubmergence treatment. (B) Seedling heights. Two asterisks indicate a significant difference (P < 0.005) in MPK3-Ox Swarna Sub1A lines, while one asterisk indicates a significant difference (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 and Swarna Sub1. Expression levels for SLR1 and SLRL1 at 24 h after submergence were measured using real-time PCR assays. Values are relative to 0 h. Error bars represent mean sd of 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 bars represent mean sd (n = 3). Different letters above the bars indicate significantly different values (ANOVA test, P ≤ 0.05). Figure 5. Open in new tabDownload slide MPK3-Silenced Lines Are Submergence Susceptible While MPK3 Overexpression Lines Show Submergence-Tolerant Traits. (A) Transgenic Swarna and Swarna Sub1 seedlings overexpressing or silenced for MPK3 and nontransgenic seedlings were submergence treated for 7 d in tap water. Representative individuals are shown pre- and postsubmergence treatment. (B) Seedling heights. Two asterisks indicate a significant difference (P < 0.005) in MPK3-Ox Swarna Sub1A lines, while one asterisk indicates a significant difference (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 and Swarna Sub1. Expression levels for SLR1 and SLRL1 at 24 h after submergence were measured using real-time PCR assays. Values are relative to 0 h. Error bars represent mean sd of 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 bars represent mean sd (n = 3). Different letters above the bars indicate significantly different values (ANOVA test, P ≤ 0.05). To obtain a deeper understanding of the physiological relevance of MPK3-SUB1A1 interaction, all of the lines were assayed for chlorophyll content, melondialdeahyde (MDA) content (indicative of lipid peroxidation), and activity of ROS scavenging enzymes like ascorbate peroxidase (APX), superoxide dismutase (SOD), and catalase (CAT). The chlorophyll content (Figure 5D) of MPK3-Ox and silenced lines was comparable in both Swarna and Swarna Sub1 backgrounds before submergence. Postsubmergence there was a comparable level of chlorophyll leaching in all assayed Swarna lines, while, interestingly, MPK3Ox-Swarna Sub1 lines resisted chlorophyll leaching. By contrast, MPK3-silenced lines showed marked chlorophyll leaching with values even lower than those of control plants. These results suggest the physiological relevance of the MPK3 and SUB1A1 interaction. In the lipid peroxidation assay using MDA (Figure 5E), low and comparable lipid peroxidation was observed in all lines before submergence treatment. After submergence treatment, lipid peroxidation (as indicated by MDA accumulation) increased drastically in all Swarna lines. The MPK3-Ox Swarna Sub1 line showed minimal lipid peroxidation. By contrast, the MPK3-silenced Swarna Sub1 lines were marked by elevated levels of lipid peroxidation. In the ROS scavenging enzyme activity assays (Figures 5F to 5H) of the respective lines, similar trends to the previous assays were obtained. There was a marked increase in the activity of SOD, APX, and CAT after submergence treatment in MPK3-Ox Swarna Sub1 lines, while the MPK3-silenced Swarna Sub1 lines showed values comparable to those of the Swarna lines. No significant change in the respective Swarna lines postsubmergence was observed. Furthermore, an assay of ROS-mediated root cell death was performed on the above lines (Supplemental Figure 9). Rice roots of the respective lines before and after 24 h of submergence treatment were stained with propidium iodide (PI) and examined by confocal laser scanning microscopy. PI is excluded from live cells, and its internalization is indicative of cell death. Roots of submergence-treated Swarna lines as well as MPK3-silenced Swarna Sub1 lines showed considerable staining by PI dispersed within the cell, indicative of DNA fragmentation caused by cell death. By contrast, MPK3-Ox Swarna Sub1 lines showed minimal traces of cell death postsubmergence. The above results substantiate the genetic interaction of SUB1A1 and MPK3 and its governance of various submergence related features. The MPK3 Signaling Cascade Activates SUB1A1 in a 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 used to pull down the fusion protein from both the genotypes. Immunoblot analysis with pTEpY antibody specifically detected the activated MPK3 from the submerged Swarna Sub1 lines (Figure 6A). These results hint at the activation of an MPK3-containing signaling cascade postsubmergence in a SUB1A-dependent manner. Figure 6. Open in new tabDownload slide 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 complete submergence for 1 d (lanes 3 and 4, respectively), followed by immunoblot with pTEpY antibody to assay the activated MPK3 module. (B) Real-time PCR analysis of SUB1A1, MPK3, MKK4, and MKKK32 transcripts in 14-d-old normal-grown seedlings completely submerged for 1 h, 6 h, 12 h, 1 d, and 3 d. Cleavage and Polyadenylation 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 (−151 to −156 bp upstream of ATG) (C);∼500 ng of recombinant protein was added (lanes 2 to 6) to the radioactively labeled 40-bp MPK3 promoter fragment containing the GCC-box (D). No protein was added in lane 1. A 50 and 100 molar excess of wild-type GCC-box-containing promoter fragment was added in lanes 4 and 5, and 100 molar excess of mutated G-box-containing promoter fragment was added in lane 6 as competitors. The plus and minus signs indicate the 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 complete submergence for 1 d (using antibodies to GFP). The gel image shows the results of PCR amplification of MPK3 promoter fragment in the immunoprecipitate and input. The lower panel depicts the IP and the total protein control. Figure 6. Open in new tabDownload slide 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 complete submergence for 1 d (lanes 3 and 4, respectively), followed by immunoblot with pTEpY antibody to assay the activated MPK3 module. (B) Real-time PCR analysis of SUB1A1, MPK3, MKK4, and MKKK32 transcripts in 14-d-old normal-grown seedlings completely submerged for 1 h, 6 h, 12 h, 1 d, and 3 d. Cleavage and Polyadenylation 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 (−151 to −156 bp upstream of ATG) (C);∼500 ng of recombinant protein was added (lanes 2 to 6) to the radioactively labeled 40-bp MPK3 promoter fragment containing the GCC-box (D). No protein was added in lane 1. A 50 and 100 molar excess of wild-type GCC-box-containing promoter fragment was added in lanes 4 and 5, and 100 molar excess of mutated G-box-containing promoter fragment was added in lane 6 as competitors. The plus and minus signs indicate the 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 complete submergence for 1 d (using antibodies to GFP). The gel image shows the results of PCR amplification of MPK3 promoter fragment in the immunoprecipitate and input. The lower panel depicts the IP and the total protein control. The above results established the role of MPK3 in mediating SUB1A-dependent submergence responses. To gain insight into the role of components upstream of MPK3 during submergence, we turned our attention to MKK4 (a MAPKK) that is known to interact with MPK3 (Wankhede et al., 2013). Our earlier transcript profiling study revealed marked transcript accumulation of MKKK32 (encoding a MAPKKK). We found that MKKK32 interacts with MKK4 in yeast two-hybrid studies (Supplemental Figure 10). Thus, it was tempting to hypothesize the direct involvement of a specific MAPK signaling cascade, which is activated by submergence and in turn mediates the phosphorylation of SUB1A1. Transcript levels of MPK3, MKK4, and MKKK32 were measured postsubmergence. The expression of the genes for all three plausible MAPK module components showed similar trend of transcript accumulation with that of SUB1A1 postsubmergence over time (Figure 6B). Though indirect, these data strongly suggest the involvement of a MKKK32-MKK4-MPK3 signaling cascade during submergence in rice. SUB1A1 Interacts with the Promoter of MPK3 and Regulates Its Expression Finally, to determine whether SUB1A1 has a direct role in the regulation of MPK3 expression, an electrophoretic mobility shift assay (EMSA) was undertaken. In silico analysis detected the presence of a G-box (GCCGCC) cis-acting element, 151 bp upstream of ATG (Figure 6C). EMSA was performed using purified SUB1A1-His fusion protein and the promoter region of MPK3 containing 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 complex was formed with increasing concentration of His-SUB1A1 (lanes 2 and 3) that was competed out by 50 or 100 molar excess of unlabeled MPK3 promoter fragment (lanes 4 and 5) containing this G-box. However, the mG-box fragment was not able to compete with the DNA-protein interaction (lane 6). To further examine the binding of SUB1A1 to the MPK3 promoter in vivo, we performed chromatin immunoprecipitation (ChIP) experiments. Seven-day-old Swarna Sub1 seedlings were transiently transformed with SUB1A1-GFP and further subjected to 1 d of complete submergence. The SUB1A1-GFP fusion protein in transgenic plants was immunoprecipitated using the anti-GFP antibody. Coimmunoprecipitated genomic DNA with SUB1A1 was analyzed by RT-PCR. As shown in Figure 6E, amplification specific to the MPK3 promoter region occurred only in the transgenic SUB1A1-Ox plants, but not in the wild-type Swarna Sub1 plants, which served as the negative control. These results together suggest that SUB1A1 binds to the G-box of the MPK3 promoter and in turn regulates its activity. DISCUSSION This study provides evidence that the activation of MPK3 in submergence is SUB1A dependent. Furthermore, it establishes that SUB1A1 works with MPK3 in a positive regulatory loop to regulate submergence tolerance. While MPK3 phosphorylates SUB1A1, SUB1A1 specifically interacts with the promoter of MPK3 and regulates its expression. SUB1A1, a group VII AP2/ERF, is a major player orchestrating various submergence tolerance attributes by working in concert with cytosolic and nuclear proteins. This facilitates a complete transcriptional reprogramming in a submerged seedling and helps counteract the adverse effect of inundation stress. SUB1A acts as a nodal point of crosstalk in multiple signaling pathways, including ethylene, ROS, brassinosteroids, and GA responses (Fukao et al., 2006; Xu et al., 2006; Fukao and Bailey-Serres, 2008; Jung et al., 2010; Schmitz et al., 2013). The MAPK cascade is also outlined as a stress-activated signal transduction network, conventionally triggered by various cues of ethylene and ROS (Hirt, 2000; Pitzschke and Hirt, 2006, 2009a; Pitzschke et al., 2009; Šamajová et 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). This variation in potential phosphorylation site of SUB1A1 is harbored on a region immediately C-terminal to the ERF domain and thus may be of significance, as phosphorylation can alter DNA binding by ERF proteins (Gutterson and Reuber, 2004; Xu et al., 2006). These collective facts prompted us to investigate the possible involvement of phosphorylation-based regulation of SUB1A1 and the physiological relevance of a MPK3-SUB1A1 module in the context of submergence tolerance. The rice cultivars Swarna and the SUB1-introgressed accession Swarna Sub1 were used in this study. Swarna is a widely cultivated mega-variety in Southeast Asia, particularly India and Bangladesh. In 2009-2010, Swarna-Sub1 was released in India, Indonesia, and Bangladesh. It is an improved submergence-tolerant version of Swarna with the same yield statistics (Bailey-Serres et al., 2010). Thus, the use of these mega varieties in this study was a deliberate attempt to investigate submergence-tolerant phenotypes in the farmer-friendly mega-varieties. The quintessential phenotype of SUB1A-mediated submergence tolerance is restricted shoot elongation upon inundation (Fukao and Bailey-Serres, 2008). MAPKK-specific inhibitor treatment resulted in deviation from this hallmark trait. These observations point to the positive role of a MAPK cascade during submergence stress tolerance. Analysis of expression of GA repressor genes (SLR1 and SLRL1) suggests that SUB1A function is dependent on a MAPK module. A similar set of observations was made in plants treated with brassinosteroid inhibitor (Schmitz et al., 2013). Furthermore, the observed downregulation of GA repressor genes by MAPK cascade components was consistent with that observed in the brassinosteroid signaling network, where the authors deduced that the brassinosteroid pathway positively regulated submergence tolerance responses in rice. Additionally, our quantitative real-time PCR studies suggest that SUB1A positively regulates the transcript accumulation of genes for MAPK cascade components. This observation echoes a previous study in which OsMPK5 (designated as MPK3 in our study) transcripts showed higher accumulation in SUB1A containing rice (M202-Sub1A versus M202) upon submergence (Seo et al., 2011). Furthermore, our in-gel kinase assays demonstrated that MAPKs are activated in response to submergence and that this activation is SUB1A dependent. The immunoblot analyses and IP analysis with pTEpY antibody specifically demonstrated that the activation of MPK3 in submergence is SUB1A dependent. Thus, SUB1A1 positively regulates the MAPK cascade and specifically and positively affects the activation of MPK3 during submergence. The yeast two-hybrid assay and in silico docking analyses suggested the specific physical interaction of MPK3 with SUB1A1. Physical interaction of the two proteins was confirmed in planta by colocalization in the nucleus and BiFC assay. MPK6 did not interact with SUB1A1 as elucidated by BiFC assays in planta, hinting at the specificity of MPK3 and SUB1A1. These results prompted us to explore the functional relevance of this physical interaction. A kinase assay indicated that MPK3 is specifically involved in phosphorylation-coupled activation of SUB1A1; this in turn leads to submergence tolerance traits. As was speculated earlier, the phosphorylation by MPK3 was found to be specific to the tolerant allele. Thus, SUB1A1, and not SUB1A2, was established as the phosphorylation target of MPK3. The P186S substitution could very well affect the fold of SUB1A1, which could result in SUB1A1 phosphorylation at a point different from Ser-186. Nevertheless, the present data establish that a posttranslational modification mechanism operates during submergence stress in which SUB1A1 is specifically activated by phosphorylation. The activated SUB1A1 in turn augments its effect by dampening the role of ethylene and further acting as a nodal point for GA, abscisic acid, and brassinosteroid crosstalk. To unravel the genetic dependence of this potential interaction, transient MPK3-overexpression and silenced rice lines were generated in SUB1-dependent and -independent backgrounds. MPK3-mediated regulation of restricted shoot elongation, transcript accumulation of GA repressors, and ROS scavenging enzyme activity were also dependent on SUB1A. Thus, MPK3 overexpression lines showed a marked increment in submergence-tolerant traits, whereas MPK3-silenced lines were more submergence susceptible in a SUB1A-dependent manner. These biochemical results are in agreement with previous studies in which SUB1A-harboring M202 lines showed higher transcript accumulation of genes for ROS scavenging enzymes (Fukao et al., 2006, 2011; Fukao and Bailey-Serres, 2008; Jung et al., 2010). The present findings strongly suggest that an MPK3 works in concert with SUB1A1 and positively regulates submergence tolerance. Previous studies have implicated an MPK3 signaling module in ROS signaling (Hirt, 2000; Pitzschke and Hirt, 2006, 2009a). The MPK3 module is also activated by hormonal cues from ethylene (Rodriguez et al., 2010). Both ethylene and ROS are affected during the regulation of adaptive responses to submergence. Taking this in to account, a direct link between ethylene and ROS-activating MPK3 module postsubmergence can be hypothesized. Finally, to delineate the direct role of SUB1A1 in MPK3 expression, we performed a DNA-protein interaction study in which SUB1A1 was found specifically to interact with the promoter of MPK3 at the −151 bp position. The binding of SUB1A1 to the promoter of MPK3 was further validated in planta by ChIP assay. There was no putative GCC-box in the promoter of MPK6, the closest relative of MPK3. Thus, the regulation of MAPK module expression upon submergence by SUB1A1 is MPK3 specific. Thus, on one hand, MPK3 regulates the activity of SUB1A1 by phosphorylation, whereas on the other hand, SUB1A1 binds to the promoter of MPK3 and regulates its transcription. Collectively, it would be interesting to speculate the submergence specific activation of MPK3 in the absence of downstream substrate (SUB1A1). It is possible that once SUB1A1 is phosphorylated by MPK3, the phosphorylated SUB1A1 may bind to the MPK3 promoter and positively regulate its expression (Figure 7). A similar phenomenon has been reported previously in rice; a salt-responsive transcription factor, ERF1, binds to the promoters of MAP3K6 and MAPK5, while MAPK5 in turn phosphorylates and activates ERF1 (Schmidt et al., 2013). A recent report in Arabidopsis highlights another example of such a feedback regulatory loop, in which MYC2 is phosphorylated by MKK3-MPK6 that binds to the promoter of MPK6 in a blue-light-dependent manner (Sethi et al., 2014). Figure 7. Open in new tabDownload slide Reweaving the Tapestry of Submergence Tolerance. Significance of MAPK cascade during submergence stress is outlined. The molecular mechanism and signal transduction network implicated in submergence stress tolerance from this study is represented in the dotted red box. The previously known components are placed outside the box. The model shows the involvement of the MPK3 module upstream of SUB1A1 in response to submergence stress. The potential testable upstream components of MPK3 are represented with a question mark. “P” denotes the allele specific phosphorylation of Sub1A1. Furthermore, SUB1A1 positively regulates the expression of MPK3 in a discrete loop mechanism. Figure 7. Open in new tabDownload slide Reweaving the Tapestry of Submergence Tolerance. Significance of MAPK cascade during submergence stress is outlined. The molecular mechanism and signal transduction network implicated in submergence stress tolerance from this study is represented in the dotted red box. The previously known components are placed outside the box. The model shows the involvement of the MPK3 module upstream of SUB1A1 in response to submergence stress. The potential testable upstream components of MPK3 are represented with a question mark. “P” denotes the allele specific phosphorylation of Sub1A1. Furthermore, SUB1A1 positively regulates the expression of MPK3 in a discrete loop mechanism. The substrates of MAPK cascades are poorly understood (Rodriguez et al., 2010) and have long been sought. WRKY transcription factors are among the few well-established downstream substrates of MAPK networks (Popescu et al., 2009). A recent study in Arabidopsis reported accumulation of WRKY22 transcripts in response to submergence, which in turn is crucial to imparting immunity to plants (Hsu et al., 2013; Hsu and Shih, 2013). MPK3 is an important component of a MAPK signaling network that helps integrate various biotic, abiotic, and developmental cues. This study demonstrates that phosphorylation-based regulation of SUB1A1 by an MPK3 module acts as a nodal point of crosstalk between SUB1A1 and submergence signaling pathways. It is intriguing to speculate that the interaction of an MPK3 module with SUB1A1 may also prime submerged plants for upcoming pathogen attack upon recession of floodwaters. METHODS Plant Material, Growth Conditions, Submergence Stress, and Inhibitor Treatment Rice (Oryza sativa) cv Swarna and the SUB1 introgression line Swarna Sub1 were analyzed in this study. Sterilized seeds were placed on moist filter paper for 3 d at 25°C in the dark. Germinated seeds were transplanted into soil-containing pots (W:L:H, 10 × 10 × 10 cm) and grown in the greenhouse (30°C day, 25°C night) for 14 d under natural light conditions. Submergence treatments were performed following the methods described previously (Fukao et al., 2006). Briefly, 14-d-old plants in soil-containing pots were completely submerged in a plastic tank (W:L:H, 65 × 65 × 95 cm) filled with 90 cm water in a greenhouse. All submergence treatments were replicated in at least three independent biological experiments. For inhibitor pretreatment experiments, individual plants were incubated with 150 μM MAPKK-specific inhibitor PD98059 (Cell Signaling Technology; #9900) in 15.0-mL tubes for 24 h before submergence treatment. The inhibitor-fed plants were then given submergence stress treatment. For mock treatment, plants were treated with solvent (DMSO) at a final concentration of 0.1%. Nicotiana benthamiana seedlings were used for localization and transient 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 was treated with 10 units of RNase free DNase I (Fermentas). Total RNA (2 µg) was subjected to first-strand cDNA synthesis using the RevertAid H Minus First Strand cDNA synthesis kit (Thermo Scientific) per the manufacturer's protocol using oligo(dT) primers. qRT-PCR was performed in a 10-μL reaction using Power SYBR Green PCR master mix (Applied Biosystems). The qRT-PCR was performed in the 384-well plate ABI Prism 7000 sequence detection system (Applied Biosystems), as has been described previously (Jaggi et al., 2011; Raghuram et al., 2014). The relative expression level of each gene was calculated using the 2−ΔΔCT method and by normalizing against CLEAVAGE AND POLYADENYLATION SPLICING FACTOR as internal reference. Primer pairs used for qRT-PCR analysis are in Supplemental Table 1. MAPK Activity Assays Immunokinase and IP assays were performed using submergence-treated rice seedlings that were harvested at the stipulated time point and were ground in liquid nitrogen. Proteins were isolated using kinase extraction buffer (50 mM HEPES, pH 7.5, 5 mM EDTA, 5 mM EGTA, 10 mM DTT, 10 mM Na3VO4, 10 mM NaF, 50 mM β-glycerolphosphate, 1 mM phenylmethylsulfonyl fluoride, protease inhibitor cocktail, and 10% glycerol) and were quantified using Bradford assay (Bradford, 1976). Total protein (30 to 40 µg) was separated by 10% SDS-PAGE, and later, immunoblotting analysis was performed using anti-pTEpY (Cell Signaling Technology; catalog #9101, lot #28). Crude protein (300 µg) was used for immunoprecipitation using protein A-sepharose beads with the respective antibodies. Anti-pTEpY, rabbit polyclonal anti-MPK6 (catalog #7104, lot #124K4857), anti-MPK4 (catalog #A6979, lot # 124K4855), and anti-MPK3 (catalog #M8318, lot #124K4856) all from Sigma-Aldrich were used for IP. The OsMPK3 antibody was raised in rabbit against the PVAEFRPTMTHGGR polypeptide of OsMPK3. For immunoblot analysis, a 1:7500 dilution of the OsMPK3 antibody was used. As depicted in Supplemental Figure 2D, the anti-AtMPK3, -AtMPK4, and -AtMPK6 antibodies specifically react with the homologous rice counterparts. Also, using the anti-OsMPK3 antibody, a single discrete band was observed for GST-OsMPK3 and in crude protein extracts of N. benthamiana leaf cells overexpressing MPK3, establishing the specificity of the antibody (Supplemental Figure 2E). In-gel kinase activity assays were performed as described previously (Raina et al., 2012). Briefly, 30 μg total protein was fractionated on a 10% polyacrylamide gel containing 0.1% SDS and 0.5 mg mL−1 bovine brain MBP (Sigma Aldrich). After electrophoresis, the SDS from the gel was removed with buffer (25 mM Tris-HCl, pH 7.5, 0.5 mM DTT, 5 mM Na3VO4, 0.1 mM NaF, 0.5 mg mL BSA, and 0.1% Triton X 100) followed by renaturation in buffer (25 mM Tris-HCl, pH 7.5, 0.5 mM DTT, 5 mM Na3VO4, and 0.1 mM NaF) at 4°C overnight. MBP phosphorylation was performed by incubating the gel in 20 mL reaction buffer (25 mM Tris-HCl, pH 7.5, 2 mM EGTA, 12 mM MgCl2, 1 mM DTT, 0.1 mM Na3VO4, 1 μM ATP, and 50 μCi [γ-32P]ATP [3000 Ci mmol−1]) for 60 min at room temperature. The gel was washed three times with 5% trichloroacetic acid and 1% sodium pyrophosphate 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 to check protein-protein interactions. Full-length CDSs of SUB1A1, SUB1A2, and MPK3 genes were cloned in frame in pGADT7 and pGBKT7 vectors. Full-length CDSs of other MAPK members were cloned in pGBKT7 vector. For yeast transformation, yeast competent cells (AH109) were prepared according to the manufacturer's instructions. The two respective constructs were cotransformed in AH109-competent cells. Cotransformants were initially selected on nutrient medium lacking Leu and Trp (synthetic defined [SD]/2Leu/2Trp). The resultant cotransformed cells were then streaked 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 grown colonies after the incubation were considered as positive interactions. β-Galactosidase assays were performed by monitoring the lacZ reporter gene expression directly on nutritional selection plates by addition of ONPG to liquid culture that was rapidly freeze/thawed per the manufacturer's instructions (Clontech). As β- galactosidase accumulated in the medium, it hydrolyzed ONPG to O-nitrophenol, which was spectrophotometrically monitored at 420 nm. AH109 cotransformed with the coding sequence for SV40 large T antigen in pGADT7 vector and p53 in pGBKT7 vector served as positive control for protein interaction in all the experiments. In Silico Docking Assay Homology modeling was performed as mentioned (Krieger et al., 2009). For selection of templates for homology modeling of selected proteins, PSI BLAST (Altschul et al., 1997) was performed against the PDB database (http://www.pdb.org/pdb/home/home.do). 3D models were prepared with the help of MODELLER (Kozakov et al., 2013), and the energy of the modeled structures was refined with online server YASARA (http://www.yasara.org/homologymodeling.htm). The overall stereo-chemical quality of the modeled 3D structure of proteins was evaluated using Ramachandran plotting, which is based on psi (Cα-C bond) and phi (N-Cα bond) angles of the protein and provides information about the number of amino acid residues present in allowed and disallowed regions. Furthermore, structural verification of the modeled structures was performed by PROCHECK (Laskowski et al., 1996) and ERRAT (Colovos and Yeates, 1993). For protein-protein docking, ClusPro, an online protein-docking server, 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-MPK3 fusion proteins expressed in bacteria. SUB1A1 was in-frame cloned in pET28a (+) expression vector (Novagen) to add a C-terminal His-tag and transformed into competent Escherichia coli BL21 cells. The protein was induced by 1 mM IPTG and solubilized into the supernatant fraction using the IBS buffer kit (G-Biosciences). The protein was purified with the QIAexpressionist protein purification system (Qiagen) using Ni-NTA agarose beads. MPK3 was cloned into pGEX-4T2 expression vector to add an N-terminal GST tag. Protein was induced by 1 mM IPTG and purified using the Glutathione-4 Sepharose protein purification system (GE Healthcare). MPK3-HA, SUB1A1-HA, and SUB1A2-HA proteins were immunoprecipitated from N. benthamiana plants infiltrated with MPK3 pSPYCE (M) and SUB1A1/A2 pSPYCE (M) binary vector. The fusion proteins were coexpressed in N. benthamiana leaves using the Agrobacterium tumefaciens infiltration method (Nakagami et al., 2004). After 48 h, proteins were isolated using MAPK extraction buffer consisting of 50 mM HEPES-KOH (pH 7.5), 5 mM EDTA, 5 mM EGTA, 1 mM DTT, 10 mM Na3VO4, 10 mM NaF, 50 mM β-glycerolphosphate, 1 mM PMSF, 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 (GE Healthcare) was used for the immunoprecipitation-based phosphorylation assays. Anti-HA antibody was coupled to the resin using the manufacturer's instructions. Total protein extract (300 µg) was incubated overnight with the anti-HA resin complex at 4°C. The next day, after several washes, beads with immunoprecipitated MPK3 and SUB1A1/2 were subjected to in vitro kinase assay. In vitro kinase assay was performed as described (Rao et al., 2011) with slight modifications. Briefly, 5 μg sample was mixed with reaction buffer to give a final volume of 25 μL containing 25 mM Tris-Cl (pH 7.5), 10 mM MgCl2, 5 mM MnCl2, 1 mM DTT, 1 mM β-glycerolphosphate, 1 μM Na3VO4, 0.5 mg/mL MBP, 25 μM ATP, and 1 μCi [γ-32P]ATP. Incubation at 30°C was stopped after 30 min by addition of 10 μL 2× SDS sample buffer (no DTT or β-ME). Samples were boiled at 65°C for 5 min and then separated in 10% SDS-PAGE gels. The radioactive blots and the Coomassie Brilliant Blue-stained gels were visualized using a phosphor imager (Typhoon; GE Healthcare, Life Sciences). Site-Directed Mutagenesis To generate mutated SUB1A1-His protein, the SUB1A186S-PF and SUB1A186S-PR primer pair was used for site-directed mutagenesis by inverse PCR. The pET 28a (+) clone harboring the wild-type SUB1A1 insert was used as a template for inverse PCR. The PCR product was digested with the DpnI restriction enzyme. The digested product was used for E. coli BL21 transformation and recombinant protein expression. The mutated proteins were purified and used for in vitro kinase assay and further immunoblotting with antiserine antibody (Thermo Scientific Pierce; catalog #PAI-86890, lot #PL1952335). Immunoblot Analysis For immunoblots, 20 μg extracted crude protein was fractionated on 10% polyacrylamide gels containing 0.1% SDS. Proteins were transferred to a PVDF membrane in a Bio-Rad semidry blot tank according to the manufacturer's protocol for 1 h. For immunodetection of proteins, the membrane was blocked with 5% (w/v) skim milk in TBS buffer for 2 h and subsequently incubated with primary antibody diluted in TBS-T buffer (TBS 0.1% and Tween 20) containing 3% (w/v) skimmed milk for 1.5 h at room temperature or at 4°C overnight. After washing in TBS-T, the membrane was incubated with secondary antibody diluted in TBS-T containing 3% (w/v) skimmed milk at room temperature for 1.5 h. Following several washing steps, proteins were detected by incubating the membrane in freshly prepared chemiluminescent horseradish peroxidase substrate (Immobilon Western, Millipore Corporation). Chemiluminescence was detected using HyperProcessor (Amersham Biosciences). Subcellular Localization and BiFC For the determination of subcellular localization, SUB1A1 and MPK3 were first cloned into pENTR-DTOPO entry vector and subsequently transferred by LR recombination into pGWB5 to encode C-terminal GFP fusion proteins. The cloned constructs were transformed into Agrobacterium EHA105 cells. A single colony for Agrobacterium EHA105 positive clones was streaked on YEB agar medium with rifampicin and kanamycin (Sigma-Aldrich) and allowed to grow for 2 d at 28°C in the dark. The colonies were further inoculated in 5 mL YEB medium and grown for 22 to 24 h at 28°C with continuous shaking at 200 rpm. The A 600 of the cultures was adjusted to 0.5 in infiltration medium (10 mM MES, pH 5.7, 10 mM MgCl2, and 150 µM acetosyringone). The mixtures were incubated in the infiltration medium for 2 h and later infiltrated into N. benthamiana leaves and observed 3 d postinfiltration under a confocal laser scanning microscope to detect the fluorescence. For BiFC, SUB1A1 and MPK3 were cloned in pSPYNE173 and pSPYCE (M) (Waadt et al., 2008). Both the vectors contain a 35S promoter-driven multiple-cloning site followed by sequence encoding the N-terminal half of eYFP in pSPYNE173 and the C-terminal half of eYFP in pSPYCE (M). For transient expression, the two constructs in the respective combinations were infiltrated into tobacco leaves along with helper construct P19. The leaves were observed 3 d postinfiltration. A confocal scanning microscope (Leica TCS SP2 AOBS system) with the respective GFP or YFP filters was used for imaging. The fluorescence excitation was achieved 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-box present in the promoter of MPK3, EMSA was performed with a radiolabeled fragment of the MPK3 promoter containing the GCC-box. The oligonucleotides used to amplify the GCC sequence were SUB1EmF and SUB1EmR. Mutations in the probe sequences were generated using the primers M-SUB1EmF and M-SUB1EmR (Supplemental Table 1). The MPK3 promoter fragment containing the GCC-box was amplified, purified, and [α-32P]dCTP labeled by Klenow enzyme (New England Biolabs) for use as probe. The recombinant SUB1A1-His was induced and overexpressed in E. coli and affinity purified following the manufacturer's protocol (Amersham Biosciences). Approximately 25 ng labeled DNA fragment was incubated with purified SUB1A1-His (100 ng) in the presence of 1 µg of poly dI-dC and 1× binding buffer (75 mM HEPES, pH 7.5, 350 mM KCl, 2.5 mM EDTA, 10% glycerol, 5 mM DTT, and 10 mM MgCl2) in a reaction volume of 30 μL for 30 min at room temperature. Competition assays were performed using 50 and 100 molar excess of unlabeled GCC-box-containing MPK3 promoter fragments and 100 molar excess of mutated GCC-box-containing MPK3 promoter fragments. The reactions were fractionated on native polyacrylamide gels (10% acrylamide, 0.5% TBE, and 2.5% glycerol), dried, and visualized by autoradiography. ChIP Assays ChIP assays were performed as described by Sethi et al. (2014) with some modifications. Swarna Sub1 and Swarna Sub1 overexpressor (SUB1-Ox) lines were used for the experiment. Transient transformation of SUB1-Ox line was undertaken by agroinoculation using pGWB5 construct harboring SUB1A1. Seedlings were treated for complete submergence for 1 d. The immunoprecipitated products were subjected to RT-PCR analysis to examine the relative enrichment of the promoter fragment using the primers that were used in the gel-shift assay. The GFP monoclonal antibody (Thermo Fisher Scientific) was used for the immunoprecipitation experiments. Transformation of Rice Seedlings Transient transformation in rice seedlings was undertaken by agroinoculation as previously described (Purkayastha et al., 2010). For transient transformation of MPK3-Ox lines, MPK3 in pGWB5 was used, while for the MPK3-silenced lines, MPK3 in mVIGS vector was used. Briefly, all vectors were transformed into Agrobacterium strain EHA105, and a primary culture was initiated from a single Agrobacterium colony in LB medium supplemented with appropriate antibiotics. Subsequently, a secondary culture was grown to an OD600 of 0.6 to 0.8 with 200 µM acetosyringone. The cells were harvested and resuspended in 10 mM MES, 10 mM MgCl2, and 200 µM acetosyringone to a volume 20-fold less than the original. Approximately 5-d-old rice plants grown in Yoshida's medium were used for agroinoculation. About 50 μL bacterial suspension was injected into the meristematic region located at the crown of the plants, which were then transferred onto sterile Whatman No. 1 filter paper immersed in Yoshida's medium placed on a solid support with its ends dipped into a reservoir containing the medium. The plants were covered with moist tissue paper and transferred to tubes containing Yoshida's medium 24 h postinoculation and were maintained at 27°C under conditions described above. The transiently transformed seedlings were then screened for positive transformants. The positively transformed MPK3-overexpressing or -silenced rice seedlings were further given submergence treatment and later used for other biochemical experiments. Biochemical Assays Total chlorophyll was isolated as described previously (Arnon, 1949). Briefly, leaf tissue were homogenized in 80% ice-chilled acetone using mortar and pestle and centrifuged at 9500g for 10 min. The absorption in 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 estimated with thiobarbituric acid (Heath and Packer, 1968). The amount of MDA was calculated from the difference in absorbance at 532 and 600 nm using an extinction coefficient of 155 mM−1 cm. For assays of the enzymatic cellular antioxidants, including SOD and CAT, frozen plant leaves (0.4 to 0.8 g) were homogenized in ice-cold extraction buffer (pH 7.5) containing 50 mM HEPES, 0.4 mM EDTA, 5 mM MgCl2, 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 supernatant was assayed for enzyme activity. SOD activity (EC 1.15.1.1) was measured by monitoring the inhibition of photochemical reduction of nitro blue tetrazolium (Dhindsa et al., 1981). CAT activity (EC 1.11.1.6) was determined by monitoring the disappearance of H2O2 (Aebi, 1984). APX was extracted in leaf samples (0.4 to 0.8 g) homogenized in medium containing 100 mM phosphate buffer (pH 7.3), 1 mM EDTA, 1% polyvinylpyrrolidone, and 1 mM ascorbate. The rate of H2O2-dependent oxidation of ascorbic acid was determined in a reaction mixture containing 0.1 M phosphate buffer (pH 7.0), 0.5 mM ascorbic acid, and 100 μL enzyme extract. Activity of APX was measured by monitoring the rate of H2O2-dependent oxidation of AA followed by measuring the decrease in absorbance at 290 nm (extinction coefficient of 2.8 mM−1 cm−1) (Nakano and Asada, 1981). Rice Root Cell Death Assay Roots (1 to 2 cm) of MPK3 overexpression and silenced lines in Swarna and Swarna Sub1 backgrounds, before and after submergence, were excised from the seedlings and stained with 1 mg mL−1 PI by vacuum infiltration for 15 to 20 min. The samples were observed under confocal scanning microscopy (Leica TCS SP2 AOBS system). Accession Numbers Sequence data from this article can be found in the Rice Genome Annotation Project or GenBank/EMBL databases under the following accession numbers: 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 MAPK cascade. Supplemental Figure 2. Submergence-induced specific activation of MPK3. Supplemental Figure 3. Submergence tolerance-related activation of the 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 SUB1A1 and MPK3. Supplemental Figure 7. MPK3 (and not MPK4/MPK6) specifically phosphorylates SUB1A1. Supplemental Figure 8. Confirmation of transient MPK3 expression level changes in rice seedlings. Supplemental Figure 9. Cell death assay. Supplemental Figure 10. Yeast two-hybrid assay to assess the physical 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 Plant Genome Research from the Department of Biotechnology, Government of India. P.S. is a recipient of a fellowship from the Council of Scientific and Industrial Research, Government of India. We thank the Confocal Microscopy Facility and the Central Instrumentation Facility of NIPGR, New Delhi, India. We also thank S. Robin (Tamil Nadu Agricultural University) for providing seeds of Swarna and Swarna Sub1, Indranil Das Gupta (University of Delhi South Campus, New Delhi) for the kind gift of mVIGS vector, and Adam J. Bogdanove (Cornell University, Ithaca, NY) for critically reading and editing the manuscript. Glossary MAPK mitogen-activated protein kinase ROS reactive oxygen species GA gibberellic acid IP immunoprecipitation BiFC bimolecular fluorescence complementation CDS coding sequence MDA melondialdeahyde APX ascorbate peroxidase PI propidium iodide EMSA electrophoretic mobility shift assay ChIP chromatin immunoprecipitation SOD superoxide dismutase CAT catalase 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. References 1. Aebi , H. ( 1984 ). Catalase in vitro . Methods Enzymol. 105 : 121 – 126 . Google Scholar Crossref Search ADS PubMed WorldCat 2. 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Google Scholar Crossref Search ADS PubMed WorldCat Author notes 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 findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Alok Krishna Sinha (alok@nipgr.ac.in). www.plantcell.org/cgi/doi/10.1105/tpc.15.01001 © 2016 American Society of Plant Biologists. All rights reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - A Positive Feedback Loop Governed by SUB1A1 Interaction with MITOGEN-ACTIVATED PROTEIN KINASE3 Imparts Submergence Tolerance in Rice
JF - The Plant Cell
DO - 10.1105/tpc.15.01001
DA - 2016-06-10
UR - https://www.deepdyve.com/lp/oxford-university-press/a-positive-feedback-loop-governed-by-sub1a1-interaction-with-mitogen-dYnB0trz9o
SP - 1127
EP - 1143
VL - 28
IS - 5
DP - DeepDyve
ER -