PHYSIOLOGIA PLANTARUM 119: 392–399. 2003 Printed in Denmark – all rights reserved Copyright # Physiologia Plantarum 2003 Copper treatment activates mitogen-activated protein kinase signalling in rice Chuan-Ming Yeh, Wan-Chi Hung and Hao-Jen Huang* Department of Biology, National Cheng-Kung University, No.1 University Road. 701, Tainan, Taiwan *Corresponding author, e-mail: haojen@mail.ncku.edu.tw Received 14 February 2003; revised 16 April 2003 It is well known that mitogen-activated protein kinase (MAPK) pathways are modules involved in the transduction of extracellular signals to intracellular targets in all eukaryotes. In plants, it has been shown that MAPKs play a role in the signalling of biotic and abiotic stresses. To characterize signalling pathways involved in heavy metal-induced stress responses, we examine whether plant MAPKs are also involved in this process. The analyses of mRNA levels of OsMAPK genes have shown that only OsMAPK2 mRNA transcripts increased within 12 h upon CuCl2 treatment in suspension cells and roots. An in-gel kinase assay revealed that three protein kinases, approximate 42, 50, and 64-kDa, were activated by CuCl2 treatments. The approximate 42-kDa protein kinase displayed MAPK properties. Antioxidant, GSH, prevented copper-induced kinase activity. Furthermore, we found that rice roots underwent a rapid cell death upon this copper treatment. The copper-induced cell death of rice roots was partially blocked by MAPK kinase inhibitor, PD98059. These results suggest that the MAPK cascades may function in the plant heavy metal induced-signalling pathway. Introduction Heavy metal toxicity is one of the major environment health problems in modern society, with potentially dangerous bioaccumulation through the food chain. Rapid industrialization and urbanization have enhanced the levels of toxic heavy metals in the environment, posing a potential health hazard for all living organisms (Lewis and Macintosh 1989, Di Domenico et al. 1998). In plants, several heavy metals such as Fe21, Cu21, Co21, Mn21, and Ni21 are essential elements to plant metabolism and are often added to mineral fertilizers. When accumulated in excess in plant tissues, these metals can function as stressors causing physiological constraints and alterations in various vital growth processes, such as mineral nutrition, transpiration, photosynthesis, enzyme activities related to metabolism, and biosynthesis of chlorophyll and nucleic acids (Stobart et al. 1985, Weigel 1985, Quariti et al. 1997, Larsson et al. 1998, Haag-Kerwer et al. 1999). It was reported that acute exposure to Cu21 results in activation of the MAPK pathways in human bronchial epithelial cells (Samet et al. 1998). Recently, Iryo et al. (2000) reported that Cd21 activated MAPKs in human T cells, including ERK, JNK, and p38 kinase. More than 392 50 cDNAs encoding putative MAPKs have been cloned from different plants (Jonak et al. 1999, Ichimura et al. 2002) such as Arabidopsis (Mizoguchi et al. 1993), tobacco (Wilson et al. 1995), oat (Huttly and Phillips 1995, Marcote and Carbonell 2000) and rice (Fu et al. 2002, Huang et al. 2002). From an analysis of the sequence homology of predicted amino acid sequences, plant MAPKs can be further divided into at least four subgroups (Ichimura et al. 2002). According to available information, MAPKs in subgroups A, C and D are involved in signalling pathogen infections and abiotic stresses, whereas at least some of the MAPKs in subgroup B are involved in cell cycle regulation (Decroocq-Ferrant et al. 1995, Wilson et al. 1997, Jonak et al. 1999, Fu et al. 2002). Increased transcript levels of genes encoding MAPKs in plants have been observed in response to environmental stresses (Jonak et al. 1996, Seo et al. 1995, Mizoguchi et al. 1996) and hormonal treatments (Huttly and Phillips 1995). However, MAPK pathway involved in heavy metal stress has not been well examined in plants. The aim of this study was to search for MAPKs regulated at the transcriptional and post-translational levels during heavy metal-induced stress responses. Here, we Physiol. Plant. 119, 2003 report the time and dose dependence of the effects of copper on the expression of Oryza sativa MAPK genes. In the presence of copper, the expression of OsMAPK2 was upregulated within 12 h in rice suspension cells and roots. In addition, we demonstrated that Cu21 also rapidly induced approximately 42-kDa MAPK-like kinase activation and during this period we found that rice roots had undergone rapid cell death. This copperinduced cell death of rice roots was blocked by MAPK kinase (MEK) inhibitor, PD98059. To our knowledge, the involvement of the plant MAPK in the signalling cascade that leads to heavy metal-induced cell death has not yet been reported. The results provide evidence that MAPK signalling pathways are associated with heavy metal stress in rice. Materials and methods Plant growth and cell culture Suspension cells of rice (Oryza sativa cv. TN67) were cultured as described by Yu et al. (1991). The cells were kept on a gyratory shaker (120 r.p.m) at 26 C and subcultured weekly. Three-day-old suspension-cultured cells were exposed to 100 mM CuCl2 before total RNA extraction. Roots of rice plants that were grown in a growth chamber were used for organ gene expression. Fresh weight and root length determination Suspension cells were weighed after transferring to MS (Murashige and Skoog 1962) medium containing CuCl2 for 3 days. Rice seeds were sterilized with 2.5% sodium hypochlorite for 15 min and washed thoroughly with distilled water. These seeds were then germinated in Petri dishes containing distilled water at 37 C in the dark. After a 2-day incubation, uniformly germinated seeds were selected and transferred to Petri dishes containing two sheets of Whatman no. 1 filter paper moistened with 15 ml of distilled water or test solution. Each Petri dish contained 10 germinated seeds and each treatment was replicated 3 times. The germinated seeds were grown at 27 C in darkness. Root length was measured after 3 days. RNA isolation and Northern blot analyses Total RNA was extracted from root and suspensioncultured cells by using the RNeasy kit (QIAGEN, Germany). Northern blot was performed according to Sambrook et al. (1989). Total RNA was fractionated on a 0.8% gel and transferred to MAGNA nylon transfer membranes (MSI, Westboro, MA, USA) with the Hoefer TransVac vacuum blotting unit (Amersham Biosciences, Piscataway, NJ, USA) and UV cross-linked with the Stratalinker UV box (Stratagen, Kirkland, WA, USA). The RNA gel blot analysis was performed using OsMAPK2, OsMAPK3 and OsMAPK4 gene-specific DNA as probes. The OsMAPK2 gene-specific DNA Physiol. Plant. 119, 2003 probes were prepared from gel-purified PCR fragments synthesized with the following primers: 50 -CTG CGA ATC GAG AGA GAG TCA GAT AAG GTC-30 (sense) and 50 -ATG GAG GTC GGT GTC CAT AAG-30 (antisense) (Huang et al. 2002). The OsMAPK3-specific probe, a 286-bp fragment was amplified by PCR using primers for forward: 50 -GAG TGA ATA TGT GAC AGG CA-30 and for reverse: 50 -AGC ATC TAA CAT TAC AAG CC-30 (Fu et al. 2003). As the OsMAPK4-specific probe, a 286-bp fragment was amplified by PCR using primers for forward: 50 -GAG TGA ATA TGT GAC AGG CA-30 and for reverse: 50 -AGC ATC TAA CAT TAC AAG CC-30 (Fu et al. 2002). Probes were labelled with [a-32P] dCTP using a Rediprime Kit (Amersham, UK). Hybridization was carried out at 42 C. The membranes were rinsed twice in 2 SSC containing 0.1% SDS at room temperature and washed once in 0.2 SSC containing 0.1% SDS at 42 C. The membranes were then visualized by exposure of Kodak BioMax MR Film. In-gel kinase activity assay The in-gel kinase assay was performed according to the procedures described previously (Zhang and Klessig 1997), with slight modification. Extracts containing 5 mg of protein were electrophoresed on 10% SDS-polyacrylamide gels embedded with 0.25 mg ml 1 of myelin basic protein (MBP) in the separating gel as a substrate for the kinase. After electrophoresis, SDS was removed by washing the gel with washing buffer (25 mM Tris, pH 7.5, 0.5 mM DTT, 0.1 mM Na3V04, 5 mM NaF, 0.5 mg ml 1 BSA, 0.1% Triton X-100 [v/v]) three times, each for 30 min at room temperature. The kinases were allowed to renature in 25 mM Tris, pH 7.5, 1 mM DTT, 0.1 mM Na3V04, and 5 mM NaF at 4 C overnight with three changes of buffer. The gel was then incubated at room temperature in a 30-ml reaction buffer (25 mM Tris, pH 7.5, 2 mM EGTA, 12 mM MgCl2, 1 mM DTT, 0.1 mM Na3VO4) with 200 nM ATP plus 50 mCi g-32PATP (3000 Ci mmol 1) for 60 min. The reaction was stopped by transferring the gel into 5% trichloroacetic acid (TCA) (w/v)/l% sodium pyrophosphate (w/v). The unincorporated g-32P-ATP was removed by washing in the same solution for at least 3 h with two changes. The gel was dried and exposed to Kodak BioMax MR Film. Prestained size markers (Bio-Rad, Hercules, CA, USA) were used to calculate the size of kinases. Treatment of rice roots with inhibitor and analysis of cell death The inhibitor PD098059 was dissolved in dimethyl sulfoxide (DMSO). Final DMSO concentrations in treated root cultures were below 1% (v/v) and did not have observable effects on control rice roots. The compounds were added to cultured roots 1 h before the addition of CuCl2. Cell death was evaluated by Evans blue staining, as described previously (Suzuki et al. 1999, Delisle et al. 2001). Rice roots were stained in 0.25% aqueous Evans 393 blue for 15 min at room temperature. Roots were then washed twice for 15 min with distilled water and directly photographed. For quantitative assessment, the last 5 mm of root tips were excised from 10 roots and the Evans blue was efficiently extracted without grinding in a solution of 50% methanol/1% SDS for 1 h at 50 C and then measured by monitoring the A600. Statistic analysis The experiments for effects of heavy metals on growth of rice suspension cells and roots were performed by a completely randomized design (CRD). Data on cell FW (g) and root length (cm) were recorded after 3 days of culture. ANOVA (analysis of variance) and mean comparisons using LSD (least significant difference) tests were conducted using a SAS statistical analysis package. Results Effect of CuCl2 on growth of rice suspension cells and roots In order to evaluate the toxicity of Cu21 to rice suspension cells, a dose–response experiment was performed. The FW was determined after 3 days treatment. When rice suspension cells were treated with different concentrations of CuCl2, their FWs were statistically significantly lower than those of cells cultured in unsupplemented MS medium. The results showed that Cu21 inhibited cell growth in a dose-dependent manner (Fig. 1A). As to effects of Cu21 on growth of rice roots, we determined the root length 3 days after treatment with different concentrations of Cu21. Compared with the control, the presence of 100 or 200 mM CuCl2 in the culture solution significantly inhibited root growth. However, no significant differences for root length were observed between the two copper treatments. When the concentration of CuCl2 was 400 mM, root growth was about half of the control. The inhibition was significantly higher than all other treatments (Fig. 1B). Dose dependence of OsMAPK gene expression on CuCl2 To test whether MAPK pathway could be involved in the signal transduction of heavy metal stress, we raised OsMAPK2, OsMAPK3 and OsMAPK4 probes and analysed a dose-response of OsMAPK2, OsMAPK3 and OsMAPK4 gene expression by treating rice suspension cells with different concentrations of CuCl2 for 3 h. The results showed that OsMAPK2 was induced by CuCl2 at 25 mM and the maximal level of OsMAPK2 transcript was observed at 100 mM. The membrane was stripped and rehybridized with OsMAPK3 and OsMAPK4. No significant inductions of the genes were evident under these conditions (Fig. 2). These data indicate that transcripts of the OsMAPK2, but not OsMAPK3 and OsMAPK4, accumulate in response to copper. 394 Fig. 1. Influence of CuCl2 on growth of rice suspension cells and root length of rice seedlings. (A) Suspension cells were weighed after being transferred to MS medium containing CuCl2 for 3 days. (B) Root length was measured 3 days after treatment with different concentrations of CuCl2. Results represent the means ± SD of three independent experiments performed in duplicate. Time course of transcript levels of OsMAPK2 in response to CuCl2 in rice suspension cells To characterize the time course of OsMAPK2 expression in rice suspension cells under CuCl2 treatments, total RNA extracted from cells cultured in the presence or absence of CuCl2 was subjected to Northern blot analysis. As shown in Fig. 3, in suspension cells treated with 100 mM CuCl2, the highest accumulation of OsMAPK2 transcript was observed 3 h after starting treatment and elevated level was maintained until 12 h. Expression of the OsMAPK genes in response to CuCl2 in rice roots To test whether responses of suspension-cultured cells are similar to those of cells within differentiated tissue, we examined whether heavy metal stress also regulated OsMAPK2 in seedling roots. Roots of germinated seeds were collected 12 h after applying CuCl2 treatments. Physiol. Plant. 119, 2003 Fig. 2. Dose dependence of the response of OsMAPK expression to CuCl2 in rice suspension cells. (A) RNA samples were extracted from treated rice suspension cells, which were sampled at indicated concentrations of CuCl2. The same blot was stripped and rehybridized with indicated probes. The probes were OsMAPK2-, OsMAPK3- and OsMAPK4-specific DNAs and rDNA cDNA. Hybridization with rDNA was used as a control for RNA transfer to the blot. (B) Relative mRNA abundance of OsMAPK2, OsMAPK3 and OsMAPK4. The mRNA levels were determined from the scanned autoradiographs shown in Fig. 2A. The mRNA abundance of the control treatment was arbitrarily set to 1 and all other values were calculated as multiples of that. The values were corrected for differences in loading as determined with rDNA. RNA gel blot analysis of these samples was performed with radiolabelled fragments of the OsMAPK2 genes. After exposure to 100 mM CuCl2, transcript levels of OsMAPK2 increased in the roots (Fig. 4). These data demonstrate that differentiated plant tissue of rice responds to heavy metal stress by regulating OsMAPK2 in a similar way to suspension-cultured cells. Physiol. Plant. 119, 2003 Fig. 3. Time course of OsMAPK2 induction by CuCl2 in rice suspension cells. (A) RNA samples were prepared from treated rice suspension cells, which were sampled at indicated time intervals after 100 mM CuCl2 treatments. The probes were OsMAPK2 specific DNAs and rDNA cDNA. Hybridization with rDNA was used as a control for RNA transfer to the blot. (B) Relative mRNA abundance of OsMAPK2. The mRNA levels were determined from the scanned autoradiographs shown in Fig. 3A. The mRNA levels at time 0 were arbitrarily set to 1; all other values were calculated as multiples of that. The values were corrected for differences in loading as determined with rDNA. Copper treatment activates an approximately 42-kDa MAPK-like kinase in rice roots To assess whether Cu21 activates MAPK-like kinase activities, we performed an in-gel kinase assay using MBP as a substrate with extract from rice roots challenged with 100 mM CuCl2 for 1–3 h. As shown in Fig. 5, three protein kinases with approximate molecular weights of 42, 50 and 64-kDa were activated by 100 mM CuCl2 in rice roots. The 42-kDa protein kinase was rapidly and greatly induced by CuCl2 at 1 h and the activity of the 42-kDa protein kinase remained high for 3 h after treatment. Molecular weight and substrate preference of the 42-kDa protein kinase are in good accordance with those of MAP kinase. This result suggests 395 Fig. 5. Copper treatment activates an approximately 42-kDa protein kinase that uses MBP as a substrate. Rice roots were treated with 100 mM CuCl2 for the indicated times (h). Proteins extracted from treated or untreated roots were separated by SDSPAGE embedded with MBP as a substrate. The approximately 42-kDa MAPK-like kinase that is activated by CuCl2 treatment is indicated by an arrow. The size of molecular markers are shown in kDa. induced MAPK-like kinase activation (Fig. 6). These results suggest that CuCl2 treatments result in ROS production in rice roots and then induce activation of MAPK-like kinase, at least in part. Effects of an inhibitor of MAPK kinase on the copper-induced cell death of rice roots Fig. 4. The effect of CuCl2 on the mRNA expression of OsMAPK2 in rice seedling roots. (A) Each lane was loaded with 10 mg of total RNA extracted from rice seedling roots after 100 mM CuCl2 treatments. The probes were OsMAPK2 specific DNAs and rDNA cDNA. Hybridization with rDNA was used as a control for RNA transfer to the blot. (B) Relative mRNA abundance of OsMAPK2. The mRNA levels were determined from the scanned autoradiographs shown in Fig. 4A. The mRNA abundance of the control treatment was arbitrarily set to 1 and all other values were calculated as multiples of that. The values were corrected for differences in loading as determined with rDNA. According to the above data, CuCl2 treatments induced OsMAPK gene expression and kinase activation. Simultaneously, we found that rice roots underwent rapid cell death when treated with copper (Fig. 7). To further confirm MAP kinase activity might be involved in the transduction of the heavy metal signal that leads to cell death, we added a MEK (MAPK kinase) inhibitor, PD98059, to the culture solution before the addition of CuCl2. We then incubated the rice roots for 3 h. As shown in Fig. 7, CuCl2 induced serious cell death compared to the control treatment. Also, copper-induced cell death of rice roots was partially blocked by 100 mM PD98059. These results suggest that MAPK kinase activity is involved in the that MAPK signalling cascades may be involved in heavy metal stress in rice. Involvement of reactive oxygen species in the induction of the approximately 42-kDa MAPK-like kinase activation by CuCl2 in rice roots To investigate whether reactive oxygen species (ROS) also play a role in copper-induced MAPK-like kinase activation in plants, we examined the effect of an antioxidant, glutathione (GSH), on copper-treated rice roots. Pre-treatment with 200 mM GSH for 1 h before CuCl2 addition greatly decreased the level of copper396 Fig. 6. Copper-induced the MAPK-like kinase activation are blocked by antioxidants. Rice roots were pre-treated with or without an antioxidant, GSH, for 1 h before CuCl2 treatments, and then analysed by in-gel kinase assay. The approximately 42-kDa MAPK-like kinase that is activated by CuCl2 treatment is indicated by an arrow. The size of molecular markers are shown in kDa. Physiol. Plant. 119, 2003 Fig. 7. Inhibition of copperinduced cell death of rice roots by a MAPK kinase (MEK) inhibitor. Rice roots were preincubated 60 min with PD98059 (the MEK inhibitor), prior to incubation with added 100 mM CuCl2 for 3 h. Cell death was monitored by staining with Evans blue, as described in Materials and methods. copper-induced signal transduction pathway that leads to cell death of rice roots. Discussion Heavy metal stress is a critical factor limiting the productivity of agricultural crops. In heavily copper polluted soils, total concentration of copper in air dry soil was 3420 mg g 1 (Ebbs and Kochian 1997). There is ample evidence that exposure of plants to excess concentrations of copper results in oxidative injury (Schützendübel and Polle 2002). However, the molecular genetic studies related to heavy metal-induced gene expression are few (Xiang and Oliver 1998, Ezaki et al. 2000, Suzuki et al. 2001). In this study, OsMAPK2, but not OsMAPK3 and OsMAPK4, mRNA transcripts increased within 12 h following CuCl2 treatments. We also found that, in addition to transcript levels, MBP kinase activities were rapidly induced by CuCl2 treatments. When rice roots were challenged with 100 mM CuCl2 for 1–3 h, three MBP kinases with approximate molecular weights of 42, 50 and 64-kDa, were activated. Molecular weight and substrate preference of the 42-kDa MBP kinase are in good accordance with those of OsMAPK2. This is the first demonstration of induction of MAPK-like kinase activation by heavy metal treatment in plants. We present evidence that a specific MAPK pathway may be involved in heavy metal stress in rice, a model plant for monocots. In animals, several papers have reported that heavy metals induce MAPK kinase activation. Samet et al. (1998) studied the effects of As31, Cr1, Cu21, Fe21, Ni21, V21, and Zn21 on the mitogen-activated protein kinases (MAPK), extracellular receptor kinase (ERK), c-Jun NH2-terminal kinase (JNK), and P38 in BEAS cells. MAPK activity assays confirmed marked activation of ERK, JNK, and P38 in BEAS cells exposed to As31, V21, and Zn21. However, Cr1 and Cu21 exposure resulted in a relatively small activation of MAPK. The three MAPKs, ERK, JNK, and p38 kinase, were also activated by Cd21 in human T cells (Iryo et al. 2000). Physiol. Plant. 119, 2003 MAPK activation is mediated solely by post-translational phosphorylation in mammals and yeast (Widman et al. 1999). In plants, not only are MAPKs activated, but also the gene expression of MAPKs is regulated at the transcriptional level with wounding, as evidenced by the rapid accumulation of the transcripts of WIPK (Seo et al. 1995) and MMK4 (Bögre et al. 1997). RNA gel blot analysis of cultured parsley cells showed a several-fold increase of ERMK transcript levels within 30 min after elicitor treatment (Ligterink et al. 1997). Zhang and Klessig (1998) have reported that WIPK activation in TMV-infected tobacco is preceded by increases in both mRNA and protein levels. Subsequent studies using actinomycin D and cycloheximide demonstrated that de novo transcription and translation were required for this activation of the kinase activity (Zhang et al. 2000). Under touch, cold, and salinity stress conditions, elevated levels of the mRNAs encoding the protein kinases ATMEKK1, ATMPK3, and ATPK19 may increase their protein levels, which is likely to amplify the signal transduction efficiency of the cascade. In higher plants, the mRNAs of various genes involved in signal transduction pathways accumulate in response to environmental stimuli or stresses (Mizoguchi et al. 1996, Huang et al. 2002, Fu et al. 2003). Thus, we can speculate that MAPK pathways are also controlled at transcriptional levels in higher plant heavy metal signalling transduction. In plants, exposure to various abiotic and biotic stresses results in accumulation of H2O2 and oxidative stress. Copper is a redox-active metal that generates ROS via Harber-Weiss and Fenton reactions (Elstner et al. 1988). H2O2 has been shown to activate MAPK pathways in mammalian cells (Wang et al. 1998, Allen and Tresini 2000) and plant cells (Desikan et al. 1999, Kovtun et al. 2000, Yuasa et al. 2001). In this study, we found that an approximately 42-kDa MAPK-like kinase activities decreased when antioxidant, GSH, were added together with CuCl2. It is therefore possible that activation of OsMAPK by copper in rice results from the activation of oxidative stress. Samet et al. (1998) found that non-cytotoxic concentrations of As31, V21, and Zn21 induced a rapid 397 phosphorylation of MAPK in BEAS cells. They suggested that metal-induced activation of MAPK was due to inhibition of phosphatase activity. Similarly, trivalent As31 reportedly activates JNK and P38 in HeLa cells by inhibiting a dual-specificity Thr/Tyr phosphatase, and Zn21 has been shown to inhibit the receptor Tyr phosphatase HPTP beta. Copper is not known to be phosphatase inhibitor; however, it is a transition metal capable of generating reactive oxygen species such as H2O2, which is a potent PTPase inhibitor and activator of MAPK (Sullivan et al. 1994, Held et al. 1996). Recently, we have cloned two rice PTPase genes (Pu and Huang, unpublished results). The possible involvement of PTPases in the plant heavy metal stress responses will be examined in the future. The heavy metal signal transduction pathway in higher plants is not well known. Identification of MAPK in rice potentially involved in heavy metals-induced signalling pathways is of great interest. 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