Plant Science 227 (2014) 181–189

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The mitogen-activated protein kinase cascade MKK1–MPK4 mediates salt signaling in rice Fuzheng Wang, Wen Jing, Wenhua Zhang ∗ College of Life Sciences, State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, PR China

a r t i c l e

i n f o

Article history: Received 21 May 2014 Received in revised form 25 July 2014 Accepted 15 August 2014 Available online 23 August 2014 Keywords: Rice MAPK cascade Salt stress Signaling

a b s t r a c t Mitogen-activated protein kinase (MAPK) pathways have been implicated in signal transduction of both biotic and abiotic stresses in plants. In this study, we found that the transcript of a rice (Oryza sativa) MAPKK (OsMKK1) was markedly increased by salt stress. By examining the survival rate and Na+ content in shoot, we found that OsMKK1-knockout (osmkk1) mutant was more sensitive to salt stress than the wild type. OsMKK1 activity in the wild-type seedlings and protoplasts was increased by salt stress. Yeast twohybrid and in vitro and in vivo kinase assays revealed that OsMKK1 targeted OsMPK4. OsMPK4 activity was increased by salt, which was impaired in osmkk1 plants. In contrast, overexpression of OsMKK1 increased OsMPK4 activity in protoplasts. By comparing the transcription factors levels between WT and osmkk1 mutant, OsMKK1 was necessary for salt-induced increase in OsDREB2B and OsMYBS3. Taken together, the data suggest that OsMKK1 and OsMPK4 constitute a signaling pathway that regulates salt stress tolerance in rice. © 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Soil salinity has been regarded as a serious agricultural problem. One-fifth of irrigated agriculture is affected by high soil salinity [1]. The injury by salinity to plants includes ionic toxicity (such as Na+ ), osmotic stress, and ionic imbalance [2]. To survive on saline soil, plants have evolved a complex adaptive mechanism at the molecular, cellular, physiological, and biochemical level to perceive and respond to salt stress [3], but the sensing and signal transduction mechanisms for salt stress is largely unknown. Mitogen-activated protein kinase (MAPK) cascades are conserved signaling pathways in transducing extracellular stimuli into cellular responses in eukaryotes [4]. MAPK cascades consist of three sequentially phosphorylating and activating components, a MAP kinase kinase kinase (MEKK/MAPKKK), a MAP kinase kinase (MKK/MAPKK), and a MAP kinase (MPK/MAPK) [5]. MAPKs phosphorylate a variety of substrates including transcription factors, protein kinases, cytoskeleton-associated proteins, and transporters [6,7]. Signaling through MAP kinase cascades can lead to cellular responses, including cell division, differentiation as well as response to various stresses [8]. In recent years, it is learnt that stress-activated MAPK pathways play a pivotal role in osmostress signal transduction, in both yeast and mammals [8,9]. When yeast

∗ Corresponding author. Tel.: +86 25 84399022; fax: +86 25 84399786. E-mail address: [email protected] (W. Zhang). http://dx.doi.org/10.1016/j.plantsci.2014.08.007 0168-9452/© 2014 Elsevier Ireland Ltd. All rights reserved.

cells are exposed to high osmotic conditions, they respond by producing high intracellular concentrations of glycerol and reducing membrane permeability to this solute in an effort to reestablish osmotic equilibrium with the environment [10]. The signaling pathways mediating these processes include MAPK homolog Hog1 and the MAPKK homolog Pbs2, which are required for cell growth in high-osmolarity medium [11]. The HOG pathway is activated predominantly by two independent mechanisms that lead to the activation of Ssk2 and Ssk22 or the Ste11 MAPKKKs, respectively [12]. In mammalians, three different MAPKs are activated in response to osmostress: p38MAPK, c-Jun N-terminal kinases (JNK), and ERK5 (extracellular signal-regulated kinase5). MKK3 and MKK6 activate p38 MAPKs, whereas MKK4 and MKK7 are mainly responsible for the activation of JNK; all four MAPKKs can be activated by osmostress [9]. MAPK has also been related with biotic and abiotic stresses in plants. An increasing body of evidence has shown that MAPKs play important roles in signal transduction in response to salt, drought, reactive oxygen species, wounding, and low temperature in plants [5]. On the basis of the fully sequenced Arabidopsis genome, 20 MAPKs, 10 MAPKKs, and 60 MAPKKKs were identified, and a unified nomenclature was made [4]. A cascade consisting of AtMEKK1–AtMKK4/AtMKK5–AtMPK3/ AtMPK6 has been found to participate in flagellin-mediated innate immune signaling [13]. AtMKK3–AtMPK6 is activated by jasmonic acid (JA) in Arabidopsis, and plays a key role in JAdependent negative regulation of AtMYC2/JIN1 expression [14]. The

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AtMKK9–AtMPK3/AtMPK6 cascade participates in the regulation of the biosynthesis of ethylene and camalexin and may be an important axis in the stress responses of Arabidopsis [15]. MAPK in the salt stress signal transduction has been reported in a variety of plants [16]. Salt stress activates the salt stressinduced MAPK (SIMK) in alfalfa [17], which is mediated by a MAPKK homolog SIMKK [18]. In tobacco protoplasts, salt stress enhances the activation of a 48-kDa MAPK, the salicylic acid-induced protein kinase (SIPK) [19]. Three MAPKs, ZmMPK3, ZmMAPK5, and ZmSIMK1 can be induced by salt stress in Zea mays [20]. The MAPK activation by salt stress was also reported in Chorispora bungeana [21] and cotton [22]. In Arabidopsis thaliana, AtMPK4 and AtMPK6 are activated by salt treatment [16]. Recently, AtMEKK1 (MAPKKK) has been defined to activate AtMPK4 and AtMPK6 through AtMKK2 when Arabidopsis thaliana was exposed to cold and salt stress [23]. In our previous study, we reported that PLD␣1-derived PA binds to AtMPK6 and leads to its activation. The activated AtMPK6 phosphorylates the Na+ /H+ anti-porter SOS1, which may contribute to reduced Na+ accumulation in Arabidopsis leaves under salt stress [7]. In the rice (Oryza sativa L.) genome, 17 MAPKs, 8 MAPKKs, and 75 MAPKKKs genes have been annotated [24,25]. Increasing evidence suggests that rice MAPK cascade is an essential system to regulate both biotic and abiotic stress responses. Shi et al. (2014) demonstrated that OsMPK1 regulated the activities of antioxidant enzymes in abscisic acid signaling [26]. Both OsMPK3 and OsMPK6 have been implicated in salt stress response [27,28]. OsMPK6 is activated during defense response, and is up-regulated by OsMKK4, which is also a gain development regulator [29–31]. Kumar et al. (2008) showed that rice MKK1, 4, 6, 10-2 were induced by salt stress [32]. Furthermore, overexpression of OsMKK6 enhances salt tolerance in rice [33]. For a MAPKKK, overexpression of DSM1 (a putative MAPKKK gene in rice) increases the tolerance to dehydration stress [34]. In this study, we present several lines of evidence that OsMKK1 and OsMPK4 constitute a MAPK cascade for salt stress signaling by regulating the expression of transcription factors and regulating salt tolerance in rice. 2. Materials and methods 2.1. Plant materials and salt treatments Wild-type rice (Oryza sativa, japonica cv. Dongjin) and OsMKK1 T-DNA insertion mutant osmkk1 were used in this work. TDNA insertion mutant line was identified from the rice T-DNA Insertion Sequence Database (http://cbi.khu.ac.kr/RISD DB.html). Germinated seeds were grown in hydroponics with Hoagland medium in a growth chamber at 28◦ C/25◦ C, 16 h of light/8 h of dark, and 50% humidity. After 2 weeks, the rice seedlings were transferred to a solution containing 130–200 mM NaCl for treatment. 2.2. Gene cloning and vector construction All primer sequences used in this paper are listed in Supplementary Table 1. OsMKK1 and OsMPK4 were cloned from rice cDNA by PCR. The primers for cloning were HA-OsMPK4-L/R and flag-OsMPKK1-L/R, respectively. The PCR products were first cloned into a Promega pGEM-T Easy Vector according to the kit’s instructions and were then verified by sequencing. OsMKK1 was cloned into pRT105 vector with flag-tagged between the SpeI and BamHI sites. OsMPK4 was cloned into pRT105 vector with HA-tagged between the SpeI and BamHI sites.

2.3. Rice protoplast isolation and transient expression assays For protoplast isolation, rice seeds (Nipponbare) were grown (28◦ C, 85% humidity) in the dark for 12–14 d. Stems and leaves were cut into 0.5 mm pieces and digested with 25 mL of enzyme solution containing 1.5% w/v cellulose (Sigma) and 0.3% (w/v) macerozyme (Sigma). Further preparation of protoplasts was performed as described [35]. The collected protoplasts were resuspended in an appropriate volume of suspension medium (0.4 M mannitol, 20 mM CaCl2 , and 5 mM MES, adjusted to pH 5.7 with KOH). A total of 10 ␮L of plasmid DNAs (about 10 ␮g DNA of each construct) were mixed with 100 ␮L of suspended protoplasts (usually 1.5–2.5 × 105 cells/mL). The DNA and protoplasts mixture was added to 110 ␮L of 40% PEG solution [40% PEG4000, 0.4 M mannitol, and 100 mM Ca(NO3 )2 , adjusted to pH 7.0 with KOH], then incubated for 20 min at room temperature. After incubation, 440 ␮L W5 medium (154 mM NaCl, 125 mM CaCl2 , 5 mM KCl, and 2 mM MES, adjusted to pH 5.8 with KOH) was added to the tube to dilute PEG. After centrifugation at 150 × g for 2 min to remove PEG, the protoplasts were resuspended, and incubated in 2 mL of WI solution (500 mM mannitol, 4 mM MES, 20 mM KCl, pH5.7) for 16 h. 2.4. Measurement of Na+ and K+ content Fifteen-day-old rice plants were treated with 130 mM NaCl 7 d. Shoots were collected and dried at 80 ◦ C for 72 h. The samples were digested with the mixture of HNO3 and HClO4 (87:13). The contents of Na+ and K+ were determined with an atomic absorption spectrometer. 2.5. RNA isolation from rice leaves Leaves from 15-day-old rice plants were detached and immediately frozen in liquid nitrogen. One hundred milligrams of leaf material was processed in one sample. RNA was isolated according to manufacturer’s instruction using TRIzol reagent (Invitrogen). Concentration and purity of RNA was determined by measuring OD at 260 nm and 280 nm. 2.6. Molecular cloning and construction of expression vectors The full-length of OsMKK1, OsMPK3, OsMPK4, and OsMPK7 were cloned from the wild-type rice cDNA by PCR. The primers for cloning were His-OsMKK1-L/R, GST-OsMPK3-L/R, GST-OsMPK4-L/R, and GST-OsMPK7-L/R, respectively. The cDNAs of OsMKK1 and OsMPK3 (OsMPK4 and OsMPK7) were introduced in to the pET28 (a) and pGEX-4T-1 vector, respectively. Fusion proteins with His tags (for pET28a vector) and GST tags (for pGEX-4T-1 vector were expressed in Escherichia coli strain BL21 (DE3; Promega) according to the manufacturer’s instructions. The bacterial cells grown at 37 ◦ C to an OD600 at 0.5 were induced with 0.5 mM isopropyl ␤-D-1-thiogalactopyranoside (IPTG) and grown for an additional 6 h at 25 ◦ C. The His- and GST-recombinant proteins were purified with Ni-affinity agarose (Qiagen) and glutathione-sepharose beads (GenScript, Piscataway, New Jersey, USA), respectively, according to the manufacturer’s instructions. 2.7. Protein extracts from rice protoplasts and leaves Fifteen-day-old rice plants were used for salt stress treatments by 200 mM NaCl solution and treated for 15, 30, 60, and 120 min. Total protein extracts were prepared from the protoplasts or leaves with 2 volumes of immunoprecipitation buffer (100 mmol/L HEPES, 5 mmol/L EDTA, 5 mmol/L EGTA, 10 mmol/L DTT, 10 mmol/L

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Na3 VO4 , 10 mmol/L NaF, 50 mmol/L ␤-glycerophosphate, 0.1% Triton X-100, 1 mmol/L phenylmethylsulfonyl fluoride, 5 ␮g/mL aprotinin, 5 ␮g/mL leupeptin, and 10% [v/v] glycerol). The extracts were centrifuged at 15,000 × g for 20 min at 4 ◦ C. Supernatants were collected into new tubes as the crude protein extracts.

2.8. Immunocomplex kinase activity assay Crude extracts of leaves and cell cultures were adjusted to a protein concentration of 1 mg/mL. A total of 100 ␮g of the protein extracts were incubated with 1.5 ␮g of the anti-body against HA, Flag, OsMKK1, or OsMPK4 in protein-extraction buffer on a rotator at 4 ◦ C overnight. For the immunocomplex kinase activity assay, 15 ␮L of a packed volume of protein A-sepharose beads (Sigma) was added and the incubation was continued for another 2 h. The beads were collected by centrifugation at 4000 × g for 1 min, washed three times with 1 mL of wash buffer (25 mmol/L Tris (pH 8.0), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L Na3 VO4 , 1 mmol/L NaF, 10 mmol/L ␤-glycerophosphate, 5 ␮g/mL of leupeptin, 5 ␮g/mL of aprotinin, 5 ␮g/mL of antipain and 0.1% Tween20) and once with kinase buffer (25 mmol/L Tris (pH 8.0), 1 mmol/L EGTA, 5 mmol/L MgCl2 , and 1 mmol/L dithiothreitol (DTT)). Kinase activity was assayed at 30 ◦ C for 30 min in a final volume of 30 ␮L containing 0.5 mg/mL of myelin basic protein (MBP) (or OsMPK4), 10 ␮mol/L ATP, 10 ␮Ci of [␥-32 P]-ATP (>4000 Ci/mmol) and the beads with protein. The reaction was stopped by the addition of SDS-PAGE sample loading buffer. After electrophoresis on a 12% SDS-polyacrylamide gel, the phosphorylated substrates were visualized by autoradiography.

2.9. qRT-PCR Fifteen-day-old rice plants were treated with 200 mM NaCl for 1, 2, 4, and 8 h. The shoots were collected for total RNA extraction (TRIzol reagent; Invitrogen). Five hundred nanograms of total RNA were used as template for the reverse transcription reaction to synthesize the first-strand cDNA, which was used for quantitative reverse transcription PCR (qRT-PCR) as described in the cDNA Library Construction Kit protocol (Takara). The analysis was performed with the use of an ABI PRISM 7000 real-time PCR system (Applied Biosystems). The primers for PCR were OsMPK3-RT-L/R, OsMPK4-RT-L/R, OsMPK7-RT-L/R, OsMKK1-RT-L/R, OsMKK3-RT-L/R, OsMKK4RT-L/R, OsMKK5-RT-L/R, OsMKK6-RT-L/R, OsMKK10-2-RT-L/R, OsMKK10-3-RT-L/R, OsABI5-RT-L/R, OsNAC6-RT-L/R, OsTRAB1RT-L/R, OsDREB2A-RT-L/R, OsDREB2B-RT-L/R, OsMYBS3-RT-L/R, action-RT-L/R.

2.10. Yeast two-hybrid assays The yeast two-hybrid system (Clontech) was used to confirm the interactions between OsMKK1 and OsMPKs. The complete OsMKK1 and OsMPKs coding sequence were amplified from cDNA with the primers OsMKK1-YTH-L/R, OsMPK3-YTH-L/R, OsMPK4-YTHL/R, and OsMPK7-YTH-L/R by PCR. OsMKK1 was cloned into the pGBKT7 vector (Clontech) for the Gal4-BD fusions, and OsMPKs were cloned into the pGADT7 vector (Clontech) for the Gal4AD fusions. The vectors were transformed into strain AM109. Five-microliter drops from serial dilutions of 1, 1/10, 1/100 from cultures with an OD600 of 0.5 were spotted onto SD/Leu- /Trp− plates, SD/Leu- /Trp− /His- /Ade− plates containing 5 mM 3-amino1,2,4-triazole, and SD/Leu- /Trp− /His- /Ade− plates containing 5 mM 3-amino-1,2,4-triazole, and 20 mg/mL X-␣-Gal plates. They were grown for 4 d at 30 ◦ C.

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Sequence data from this article can be found in the TIGR and RiceGE data libraries, and the accession numbers are listed in Supplementary Table 2. 3. Results 3.1. Salt stimulates OsMKK1 gene expression To investigate whether rice MPKKs are involved in salt responses, we first examined their transcriptional expression. There are eight MPKK members in rice, even less than ten members in Arabidopsis thaliana [28]. To determine the transcript profile of the rice MPKKs gene in response to salt stress, the relative mRNA level of seven MPKK family genes were analyzed using qRT-PCR over an 8-h period salt treatment (Fig. 1). The results showed that, in 2-week-old seedlings, the transcript of six out of seven OsMKKs (except OsMMK10-1) responded to salt stress. The most pronounced and fast effect was observed in case of OsMKK1 gene which increased more than 6-fold after 1 h and then gradually decreased to around 3-fold after 4 h of 200 mM NaCl treatment. The OsMKK1 had the highest homologous with AtMKK2 which participated in salt tolerance in Arabidopsis [25]. Therefore, we selected OsMKK1 for further investigations. 3.2. Yeast two-hybrid assay of the interaction between OsMKK1 and OsMPKs To determine the activity, it was necessary to find potential substrates for OsMKK1. It has been reported OsMPK3 and OsMPK7 were up-regulated upon salt [27] and [36]. OsMPK4 belongs to group B, which is one of the least studied groups in rice MAPKs. AtMPK4, an ortholog of OsMPK4 is well characterized and known to regulate cold and salt stress tolerance in Arabidopsis [23]. We wanted to know whether OsMPK4, OsMPK3, and OsMPK7 interacted with OsMKK1. To test for direct interaction of different OsMPKs with OsMKK1, OsMKK1 was cloned into pGBKT7 vector, OsMPK3, OsMPK4, and OsMPK7 were separately cloned into pGADT7 vector and to create a yeast two-hybrid system. Interaction was examined by assay for complementation of histidine auxotrophy. Of the combination tested, the OsMKK1–OsMPK3 and OsMKK1–OsMPK4 pairs showed interaction (Fig. 2). Interaction was further analyzed by ␣-galactosidase. OsMKK1 showed strong interaction with OsMPK4 and weak interaction with OsMPK3 on the SD/Leu- /Trp- /His- /Ade- /3-AT, X-␣-Gal plate (Fig. 2). 3.3. OsMKK1 activates OsMPK3 and OsMPK4 We next investigated whether OsMKK1 phosphorylated OsMPKs. OsMKK1 tagged with flag was transiently expressed in rice protoplasts under control of 35S promoter and immunoprecipitated. The recombinant GST-fused proteins OsMPK3, OsMPK4, and OsMPK7 were as substrates separately to determine OsMKK1 activity. As shown in Fig. 3, OsMPK3 and OsMPK4, but not OsMPK7, could be phosphorylated by OsMKK1. OsMPK3 and OsMPK4 had a weak auto-phosphorylation in the absence of OsMKK1. 3.4. Salt stimulates OsMKK1 activity in protoplasts and plants To further test whether OsMKK1 is involved in salt response, we examined OsMKK1 activity changes during salt treatment. Flagtagged OsMKK1 was expressed in rice protoplasts, which were exposed to NaCl for 10 min. OsMKK1 activity against GST-MPK4 was activated by NaCl treatment as compared with that from nonNaCl treated protoplasts (Fig. 4A). Endogenous OsMKK1 activity was then determined after immunoprecipitation from leaf protein extracts using OsMKK1 antibody.

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Fig. 1. Expression analysis of OsMKKs family under salt stress conditions. Total RNA was prepared from 2-week-old rice seedlings treated by 200 mM NaCl for the indicated time. The relative expression levels were analyzed by qRT-PCR. The relative amounts of mRNA were calculated by defining the amount of each OsMKK in the unstressed condition (0 min) as 1. Data presented as mean values ± SE (n = 4). The mean value is significantly different from the WT (*P < 05, **P < 001).

Fig. 2. Yeast two-hybrid analysis of OsMKK1 and different OsMPKs. Targeted two-hybrid assay in yeast strain AH109 cotransformed with OsMPKs in the pGADT7 prey vector and OsMKK1 in pGBKT7 bait vector. The culture solution of the transformed yeast was dropped onto SD plates (Leu-, Trp-) or SD plates (Leu-, Trp-, His-, Ade-) containing 5 mM 3-amino-1,2,4-triazole(3-AT). and SD plates (Leu-, Trp-, His-, Ade-) containing 5 mM 3-AT and 20 mg/mL X-␣-Gal. The plates were incubated for 5 days before taking pictures.

The results revealed that endogenous OsMKK1 activity was increased by salt treatment from 15 min to 2 h (Fig. 4B). Together with results from protoplasts, these data suggest that salt stress can induce OsMKK1 activity in plants. 3.5. Ablation of OsMKK1 results in salt sensitivity We then asked if ablation of OsMKK1 would affect salt tolerance in rice. To address this question, we obtained an OsMKK1

T-DNA insertion mutant (Kyung Hee University, Korea). This line carries a single T-DNA insertion in exon9 of the OsMKK1 gene (Fig. 5A). The mutant was confirmed by semi-quantitative RT-PCR (Fig. 5B). The 15-day-old seedlings were transferred to Hoagland’s solution with 130 mM NaCl for 7 d, and then allowed to recover for 7 d in Hoagland’s solution. More osmkk1 plants died as compared with wild-type plants (Fig. 5C). The results suggest that OsMKK1 is essential to salt tolerance in rice. 3.6. osmkk1 mutant accumulates more Na+ in shoot

Fig. 3. Specificity of OsMKK1–OsMPKs interactions and substrate specificity of OsMKK1 in vitro. Flag-tagged OsMKK1 was immunoprecipitated from rice protoplasts before or 10 min after salt stress treatment (50 mM NaCl). Immunoprecipitated OsMKK1 was subsequently used for phosphorylation of recombinant kinase GST-MPK3, -MPK4, and -MPK7, respectively. Phosphorylation of OsMPKs was analyzed by autoradiography after SDS-PAGE. The amounts of GST-OsMPK and flag-OsMKK1 protein were indicated using antibodies.

To dissect the physiological mechanisms of OsMKK1 in salt tolerance, we analyzed Na+ and K+ accumulation in plants. More Na+ was accumulated in the shoots of the osmkk1 mutant than in those of WT when they were grown in solution medium containing 130 mM NaCl for 7 d (Fig. 6A). Treatment with NaCl led to a decrease in K+ accumulation in shoots and roots, with no difference between WT and osmkk1 seedlings (Fig. 6B). 3.7. OsMKK1 regulates salt activation of OsMPK4 In order to further dissect the mechanisms of OsMKK1 regulation of salt response, we investigated the effects of OsMKK1

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inhibited in osmkk1 mutant (Fig. 7B). The genetic results suggest that OsMKK1 interacts with OsMPK4 in salt response. To further confirm the OsMKK1–OsMPK4 interaction in salt response, OsMPK4 tagged with HA was expressed in WT protoplasts, and its phosphorylation activity was measured with MBP as a common substrate. OsMPK4 was activated by salt stress in protoplasts. When OsMPK4 was expressed together with flag-tagged OsMKK1, its activity was further stimulated in the absence or presence of NaCl (Fig. 8). These observations suggest that OsMPK4 is involved in salt response, and is activated by OsMKK1. Taken together, these observations indicate that OsMKK1 has an important role in activation. OsMPK4, OsMKK1, and OsMPK4 constitute a salt stress signaling pathway in rice.

3.8. OsMKK1 regulates expression of salt stress-related transcription factors

Fig. 4. Activation of OsMKK1 by salt stress in protoplasts and seedlings. (A) OsMKK1 activation in protoplasts. Flag-tagged OsMKK1 was immunoprecipitated from rice protoplasts using Flag antibodies after transient expression and salt treatment. OsMKK1 activity was determined by in vitro kinase assays using GST-OsMPK4 as a substrate. OsMKK1 protein from the same expression as the activity was detected using Flag antibodies. (B) OsMKK1 activity in seedlings. OsMKK1 was immunoprecipitated from extracts of rice shoots using OsMKK1 antibodies following stress treatments (200 mM NaCl) for 0, 15, 30, 60, 120 min. OsMKK1 activity was determined by in vitro kinase assays using GST-OsMPK4 as a substrate. OsMKK1 protein was detected using OsMKK1 antibodies. Equal amount of GST-OsMPK4 protein was used in each reaction in (A) and (B).

on OsMPK4. We extracted OsMPK4 protein from rice seedlings using OsMPK4 antibody, and tested the activity changes in the process of salt stress. OsMPK4 showed a continuous activation by salt stress within 2 h (Fig. 7A). The salt-induced OsMPK4 activation was

Among the stress-induced genes, transcription factors (TFs) play important roles via transcriptional regulation of downstream genes responsible for plant tolerance to stress challenges [37]. We chose the six transcription factors OsDREB2A, OsDREB2B, OsNAC6, OsTRAB1, OsMYBS3, and OsABI5 which had been reported to participate in stress signal transduction. The expression of transcription factors OsDREB2A, OsDREB2B, OsMYBS3, and OsNAC6 were induced by salt stress in wild-type plants (Fig. 9). Most of the times, the induction transcription of OsDRE2A and OsDRE2B by salt was obvious in WT than in the mutant. For OsTRAB1, however, osmkk1 mutant showed higher expression than WT under salt stress. The expression of OsMYBS3 was increased 7–14-fold during 1–8 h of salt stress treatment in wild type; in contrast, the accumulation of it was barely detectable in osmkk1 mutant. In addition, there was no significant difference between wild type and osmkk1 mutant of the OsABI5 transcription factor.

Fig. 5. The osmkk1 mutant shows salt sensitivity. (A) The gene structure of OsMKK1contains nine exons and ten introns, which are represented by filled boxes and lines, respectively. The site of T-DNA insertion is indicated. (B) OsMKK1 transcript levels in WT and the osmkk1 mutant analyzed by RT-PCR from leaves of 2-week-old plants. (C) The survival rate under salt stress. Data were calculated after salt stress for 7 d followed by recovering under normal conditions for 7 d. Data presented as mean values ± SE (n = 3). The mean value is significantly different from the WT (*P < 05, **P < 001). (D) Salt sensitive phenotype of osmpkk1 null plants. Pictures were taken 7 d after salt treatment, without (left panel) and with 130 mM NaCl (middle panel), and after recovering under normal conditions for 7 d (right panel).

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Fig. 6. The rice osmkk1 mutant accumulates more Na+ in leaves under NaCl stress. Na+ and K+ contents were determined after seedlings were grown in nutrient solution with or without 130 mM NaCl, for 7 d. Data presented as mean values ± SE (n = 3). The mean value is significantly different from the WT (**P < 001).

4. Discussion MAPK pathways mediate cellular responses to a great variety of different extracellular signals caused by abiotic stress in plants [4]. However, less is known about the function of MAPK signaling in rice under salt stress. In this study, we investigated MAPK signaling in rice using genetic, cellular, and molecular methods, revealing that OsMKK1 phosphorylated OsMPK4 to positively regulate salt response. It was reported that AtMKK2 played a critical role in the salt stress response in Arabidopsis [23]. OsMKK1 is a protein with 352 amino acids showing the highest homologs of eight rice MPKKs with 57.5% similarity to AtMKK2. Compared with other OsMKKs, OsMKK1 displayed the highest transcript level induced by salt stress at early time (Fig. 1). More importantly, OsMKK1 was activated in response to salt stress (Fig. 3). Knockout of OsMKK1 resulted in hypersensitivity to salt stress. These data demonstrate that OsMKK1 is a key signal transducer of salt stress in rice. The rice genome contains eight MPKKs and at last 17 MPKs, suggesting that any particular MPKK should be able to activate more than one downstream MPK. In the tested OsMPK3 and OsMPK4 were identified as the strongest downstream target of OsMKK1, through yeast two-hybrid system (Fig. 2), in vitro biochemical analysis (Fig. 3), and co-expression of OsMKK1 and OsMPK4 in protoplasts (Fig. 8). Genetic data proved that OsMPK4 activation by salt was inhibited in osmkk1 null mutant, supporting that OsMKK1 is upstream of OsMPK4 (Fig. 7). Taken together, the data suggest that OsMPK4 is a downstream target of OsMKK1 in salt response. In Arabidopsis, several MAPK cascades have been reported in pathogen responses. A MEKK1–MKK4/MKK5–MPK3/MPK6 cascade together with transcription factors WRKY22 confers resistance to both bacterial and fungal pathogens [13]. This cascade is an

important regulatory pathway controlling phytoalexin biosynthesis during fungal infection [38]. MAPK cascade is also involved in phytohormone response, for example, MKK3–MPK6 regulating JA signaling [39] and CTR1(MAPKKK)–MKK9–MPK3/MPK6 cascade

Fig. 7. OsMPK4 activity in WT- and ospkk1-null plants in response to salt stress. (A) OsMPK4 was immunoprecipitated from shoot extracts using OsMPK4 antibodies following stress treatments (200 mM NaCl) for 0, 15, 30, 60, 120 min, salt. OsMPK4 activity was measured using MBP as substrate, and levels of OsMPK4 proteins were detected iOsMPK4 antibody. (B) Quantification of OsMPK4 activities. The phosphorylation of MBP was calculated according to 32 P radioactivity. Data presented as mean values ± SE (n = 3). The mean value is significantly different from the WT (*P < 05, ***P < 001).

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Fig. 8. OsMKK1 and OsMPK4 interaction in cells. After transient expression of OsMPK4 in rice protoplasts, HA-tagged OsMPK4 was immunoprecipitated using HA antibodies following salt stress treatments (100 mM NaCl) for 10 min. OsMPK4 activity was determined using MBP as a substrate. The right two samples represent the protoplasts coexpressing flag-OsMKK1 and HA-OsMPK4.

promoting ethylene signaling [40]. Under salt stress, MKK2–MPK6 cascade regulates stress-responsive genes [23,36]. Compared with Arabidopsis, much less has been known about MAPK cascade in rice [41]. Recently, using the yeast two-hybrid system and coimmunoprecipitation, pull-down, and bimolecular fluorescence complementation, Singh et al. (2012) analyzed the rice MAPK interactome, and proposed potential protein interactions among four membrane-associated proteins, seven MPKKs, four MAPKs, and 59 putative substrates [42]. However, the direct interactions among these MAPK cascade components and their functions are not uncovered. Our finding about OsMKK1–OsMPK4 interaction in salt response promotes the understanding of rice MAPK cascade in salt stress response. Plants adapt to stresses not only by regulating signal transduction but also by changing gene expression enabling them to survive [37,43]. Numerous stress-induced genes have been

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identified using microarray experiments [44]. In yeast, genomewide transcription studies revealed that a large number of genes (7%) show significant but transient changes in the expression levels after a mild osmotic shock and that the MAPK Hog1 plays a key role in much of global gene regulation [10]. Once Hog1 is activated by Pbs2 via phosphorylation on a threonine and a tyrosine residue, it is imported into the nucleus, where Hog1 regulates the expression of numerous genes [45]. In Arabidopsis, MAPK cascade following flagellin receptor FLS2 up-regulates WRKY22/WRKY29 transcription factor expression [13]. Full genome transcriptome analysis of AtMKK2-overexpressing plants demonstrated altered expression of 152 genes involved in transcriptional regulation, signal transduction, cellular defense, and stress metabolism [23]. Transcription factors are master regulators that control gene clusters [37]. They were highly induced by salinity stress during early response, especially DREB2, bZIP, MYBs, and NACs [46]. After performing a comprehensive analysis of all five DREB2-type genes in rice, OsDREB2A and OsDREB2B were found to exhibit abiotic stress-inducible gene expression [47,48]. OsNAC6 overexpressing lines were tolerant to dehydration and high-salt stresses [49]. OsABI5 belongs to basic leucine zipper (bZIP) transcription factor, which has diverse roles in plant stress responsive and hormone signal transduction [50]. Both OsDREB2B and OsMYBS3 were dramatically induced by NaCl treatment, and the induction was abolished in osmkk1 mutant (Fig. 9). The results suggest that OsMKK1 is necessary for the activation of these transcription factors under salt stress.

Fig. 9. The transcript analysis between WT and osmkk1 mutant under salt stress. Total RNA was prepared from 2-week-old seedlings treated with 200 mM NaCl for the indicated time. The transcripts were analyzed by real time PCR. The relative amount of the transcripts was calculated by defining the amount of each gene in the unstressed condition (0 min) as 1. Data present as mean values ± SE (n = 4). The mean value is significantly different from the WT (*P < 05, **P < 001).

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Transcription factors are regulated by MAPK directly or indirectly. OsWRKY30, which is induced by drought and ABA, is phosphorylated by OsMPK3 at serine and proline amino acids [51]. Another transcription factor, PtMYB4 that regulates xylem development in Pinus taeda, is phosphorylated directly by PtMAPK6 [52]. Whereas, Arabidopsis MAP kinase 4 (AtMPK4) regulates AtWRKY25 and AtWRKY33 through interacting with MPK4 substrate 1 (MKS1) [53]. In the future work, it is needed to determine whether OsDREB2B and OsMYBS3 are regulated by OsMPK4 directly or indirectly. In conclusion, the identification of OsMKK1–OsMPK4 involved in response to salt stress may be very helpful to completely unravel signal transduction pathways in rice under salt, and provide us more choices to improve the salt resistance of rice.

Acknowledgements The work is supported by grants from the Ministry of Science and Technology in China (2012CB114200), National Natural Science Foundation of China (31171461 and 91117003), Fundamental Research Funds for the Central Universities (KYTZ201402), and the grants from Jiangsu province (PAPD) to W.Z.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.plantsci.2014.08.007.

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