Distinct signalling pathways for induction of MAP ... - Semantic Scholar
Journal of Experimental Botany, Vol. 58, No. 3, pp. 659–671, 2007 doi:10.1093/jxb/erl240 Advance Access publication 27 January, 2007
RESEARCH PAPER
Distinct signalling pathways for induction of MAP kinase activities by cadmium and copper in rice roots Chuan-Ming Yeh, Pei-Shan Chien and Hao-Jen Huang* Department of Life Sciences, National Cheng Kung University, No. 1 University Rd. 701, Tainan, Taiwan, Republic of China Received 25 July 2006; Revised 19 October 2006; Accepted 20 October 2006
Plant growth is severely affected by toxic concentrations of heavy metals. On characterizing the heavy metal-induced signalling pathways, the effects of cadmium (CdCl2) and copper (CuCl2) on MBP (myelin basic protein) kinase activities in Oryza sativa L. cv. TNG67 were analysed and it was found that Cd2+induced 42 kDa MBP kinase has the characteristics of a mitogen-activated protein (MAP) kinase. This study confirmed that the 42 kDa kinase-active band contains, at least, the activities of OsMPK3 and OsMPK6. Then, the heavy metal signal transduction pathways leading to MAP kinase activation in rice roots were examined. Pretreatment with sodium benzoate, a hydroxyl radical scavenger, attenuated Cd2+- or Cu2+-induced MAP kinase activation. The Cd2+-, but not Cu2+-, induced MAP kinase activities were suppressed by diphenylene iodonium (DPI), an NADPH oxidase inhibitor, and Cd2+ induced NADPH oxidase-like activities, suggesting that NADPH oxidases may be involved in Cd2+-induced MAP kinase activation. Using a Ca2+ indicator, it was demonstrated that Cd2+ and Cu2+ induce Ca2+ accumulation in rice roots. The Cd2+- and Cu2+-induced MAP kinase activation required the involvement of Ca2+dependent protein kinase (CDPK) and phosphatidylinositol 3-kinase (PI3 kinase) as shown by the inhibitory effect of a CDPK antagonist, W7, and a PI3 kinase inhibitor, wortmannin, respectively. Furthermore, bongkrekic acid (BK), a mitochondrial permeability transition pore opening blocker, suppressed Cd2+-, but not Cu2+-, induced MAP kinase activation, indicating that Cd2+-induced MAP kinase activities are
dependent on the functional state of mitochondria. Collectively, these findings imply that Cd2+ and Cu2+ may induce MAP kinase activation through distinct signalling pathways. Moreover, it was found that the 42 kDa MAP kinase activities are higher in Cd-tolerant cultivars than in Cd-sensitive cultivars. Therefore, the Cd-induced 42 kDa MAP kinase activation may confer Cd tolerance in rice plants. Key words: Cadmium, copper, heavy metal, MAP kinase, rice, signal transduction.
Introduction Environmental stresses, including biotic and abiotic stresses, such as pathogen infection, cold, drought, salinity, and heavy metals, are important factors that affect growth and metabolism of land plants. Heavy metal toxicity is one of the major environmental health problems in modern society, with potentially dangerous bioaccumulation through the food chain (Di Domenico et al., 1989; Lewis and McIntosh, 1989). Cadmium (Cd2+) is a toxic metal with a long biological half-life and represents a serious environmental pollutant for both animals and plants. In plants, Cd2+ is known to inhibit seed germination and root growth, induce chromosomal aberrations and micronucleus formation, and cause faster wilting and a grey-green leaf colour (Fojtova´ and Kovarˇ´ık, 2000). Although copper (Cu2+) is essential for normal plant growth and development, elevated concentrations of Cu2+ in the soil can also lead to toxicity symptoms and stunted growth in most plants (Hall, 2002). The toxicity
* To whom correspondence should be addressed. E-mail: [email protected] Abbreviations: ABA, abscisic acid; BEAS, bronchial epithelial cell strain; BK, bongkrekic acid; CDPK, Ca2+-dependent protein kinase; CM-H2DCFDA, 5(and-6)-chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate, acetyl ester; DPI, diphenylene iodonium; DTT, dithiothreitol; ERK, extracellular signalregulated kinase; HRP, horseradish peroxidase; JNK, c-Jun N-terminal kinase; MAP, mitogen-activated protein; MBP, myelin basic protein; PI3 kinase, phosphatidylinositol 3-kinase; PT pore, permeability transition pore; ROS, reactive oxygen species; SA, salicylic acid. ª The Author [2007]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: [email protected]
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Abstract
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tems. In addition, closing of the mitochondrial permeability transition (PT) pore disrupts Cd2+-, but not Cu2+-, induced MAP kinase activities. The results suggest that Cd2+- and Cu2+-induced MAP kinase activities may act through distinct signalling pathways. Materials and methods Plant materials and growth conditions Rice seedlings (Oryza sativa L. cv. TNG67, cv. TN1, cv. T65, and cv. T65d1) were cultured as described by Chen et al. (2000) with some modifications. Rice seeds were sterilized with 2.5% (v/v) 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 d incubation period, uniformly germinated seeds were selected and transferred to Petri dishes containing one sheet of Whatman No. 1 filter paper moistened with 10 ml of distilled water or test solution. Each Petri dish contained 15 germinated seeds. The germinated seeds were grown at 26 C in darkness for 3 d. Roots of rice seedlings were collected for experimental purposes. Preparation of protein extracts To prepare protein extracts from treated rice roots, roots were ground with a mortar and pestle using liquid nitrogen, followed by homogenization at 4 C with 2 vols (w/v) of protein extraction buffer [100 mM HEPES, pH 7.5, 5 mM EDTA, 5 mM EGTA, 10 mM dithiothreitol (DTT), 10 mM Na3VO4, 10 mM NaF, 50 mM bglycerophosphate, 1 mM phenylmethylsulphonyl fluoride (PMSF), 5 lg ml 1 antipain, 5 lg ml 1 aprotinin, 5 lg ml 1 leupeptin, 10% glycerol]. The ground slurry was centrifuged at 13 000 rpm for 20 min at 4 C in a microcentrifuge. Aliquots of the supernatants were put into clean tubes, flash-frozen in liquid nitrogen, and stored at 80 C for later use. Western blot analysis The protein extracts (15 lg) were separated by SDS-PAGE (10% gel). After electrophoresis, the gel was transfererd to a polyvinylidene difluoride (PVDF) membrane (PerkinElmer Life Sciences, Boston, MA, USA) at 100 V for 70 min at 4 C in a Mini TransBlot Electrophoretic Transfer Cell (Bio-Rad, Hercules, CA, USA). The membrane was incubated in blocking solution containing TBST (20 mM TRIS-base, 137 mM NaCl, 0.1% Tween-20, pH 7.6) supplemented with 5% (w/v) non-fat dry milk for 1 h at room temperature. Then it was washed three times for 5 min with TBST buffer. The blots were probed with polyclonal antibodies raised against the conserved subdomain XI of rat ERK1, which recognize ERK1 and ERK2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), or against the phosphotyrosine-containing peptide of human p44 MAP kinase (residues 196–209; Cell Signaling Technology, Beverly, MA, USA). The latter antibody recognized only ERK1/2related polypeptides which are catalytically activated at Tyr204. The immune complexes were detected by a horseradish peroxidase (HRP)-conjugated secondary antibody (Pierce, Rockford, IL, USA) and revealed by Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences, Boston, MA, USA). Antibodies were used at 1:2500, 1:1000, and 1:5000 dilutions for anti-ERK1, anti-phospho-ERK, and secondary antibodies, respectively. Antibody production and immunoprecipitation The peptides LIFNEAIEMNPNIRY and YQEGLAFNPDYQ corresponding to the C-termini of OsMPK3 (GenBank protein accession
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symptoms seen in the presence of excessive amounts of heavy metals may result from the binding of metals to sulphydryl groups in proteins, leading to loss of activity or disruption of structure, or from the displacing of an essential element, resulting in deficiency effects. In addition, heavy metal excess may stimulate the formation of free radicals and reactive oxygen species (ROS), which may cause lipid peroxidation, enzyme inactivation, and DNA and membrane damage (Hall, 2002; Boominathan and Doran, 2003). Although heavy metals are associated with a number of physiological disorders in plants, the molecular mechanisms of heavy metal signal transductions are not well understood. Several lines of evidence from biochemical and genetic studies of stress signalling indicate that phosphorylation and dephosphorylation of proteins are important in the regulation of physiological status and gene expression in response to various environmental stresses (Yuasa et al., 2001). The mitogen-activated protein (MAP) kinase cascade plays an essential role in the intracellular signal transduction processes involving cell proliferation and stress responses in yeast and mammalian cells (Herskowitz, 1995; Kyriakis and Avruch, 1996). Increasing evidence reveals that MAP kinases also play similar roles in plant response to biotic and abiotic stresses, such as pathogen, cold, drought, salt, and wounding (Jonak et al., 1996; Mizoguchi et al., 1996; Zhang et al., 2000; Fu et al., 2002; Huang et al., 2002). Samet et al. (1998) reported that As3+, V4+, Cr3+, Cu6+, and Zn2+ activate the MAP kinases, extracellular signalregulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAP kinase in BEAS (bronchial epithelial cell strain) cells. The three MAP kinases, ERK, JNK, and p38 kinase, were also activated by Cd2+ in human T cells (Iryo et al., 2000). Studies of growth inhibition and apoptosis in a human non-small cell lung carcinoma cell line (CL3) show that JNK and p38 co-operatively participate in apoptosis induced by Cd2+, and the decreased ERK signal induced by low Cd2+ doses contributes to growth inhibition or apoptosis (Chuang et al., 2000). Recently, MAP kinase pathways involved in heavy metal stresses have also been demonstrated in rice (Yeh et al., 2003, 2004) and alfalfa (Jonak et al., 2004). However, the relationship between plant MAP kinase pathways and heavy metal stress has not been well examined. The aim of this study was to search for MAP kinases regulated at the translational and post-translational levels during heavy metal-induced stress responses and to investigate the molecular mechanisms of signalling networks underlying heavy metal stress in the model plant, rice. In this study, the roles of second messengers, ROS and calcium, in Cd2+- and Cu2+-induced signal transduction pathways were examined and discussed. Using pharmacological inhibitors, it is demonstrated that Cd2+ and Cu2+ induce MAP kinase activation via distinct ROS-generating sys-
Different pathways in MAP kinase activation by Cd and Cu 661
In-gel kinase activity assay The in-gel kinase assay was performed according to the procedures described previously (Zhang and Klessig, 1997) with slight modifications. Rice roots were pretreated with various inhibitors [500 lM sodium benzoate, 100 lM diphenylene iodonium (DPI), 100 lM W7, 10 lM wortmannin, and 50 lM bongkrekic acid (BK)] for 1 h before 400 lM CdCl2 and 100 lM CuCl2 treatments and further incubated for an additional hour. Root extracts containing 5 lg of protein were electrophoretically separated on 10% (w/v) SDS-polyacrylamide gels embedded with 0.25 mg ml 1 of MBP or histone III-S in the separating gel as a substrate for the kinase. After electrophoresis, SDS was removed by cleansing the gel with washing buffer [25 mM TRIS, pH 7.5, 0.5 mM DTT, 0.1 mM Na3VO4, 5 mM NaF, 0.5 mg ml 1 bovine serum albumin (BSA), 0.1% (v/v) Triton X-100] three times, each for 30 min at room temperature. The kinases were allowed to renature in 25 mM TRIS, pH 7.5, 0.5 mM DTT, 0.1 mM Na3VO4, and 5 mM NaF at 4 C overnight with three changes of buffer. The gel for MAP kinase activity assay was then incubated at room temperature in a 90 ml reaction buffer (25 mM TRIS, pH 7.5, 2 mM EGTA, 12 mM MgCl2, 1 mM DTT, 0.1 mM Na3VO4) for 30 min. The gel for Ca2+-dependent protein kinase (CDPK) activity assay was incubated at room temperature in a 90 ml reaction buffer (40 mM TRIS, pH 7.5, 12 mM MgCl2, 2 mM DTT, 0.2 mM CaCl2, or 4 mM EGTA) for 30 min. Phosphorylation was performed for 1 h at room temperature in 90 ml of the same reaction buffer with 200 nM ATP plus 50 lCi of [c-32P]ATP (3000 Ci mmol 1). The reaction was halted by transferring the gel into a solution with 5% (w/v) trichloroacetic acid (TCA) and l% (w/v) sodium pyrophosphate. The unincorporated [c-32P]ATP was removed by cleansing in the same solution for at least an hour with two changes. The gel was dried and exposed to Kodak BioMax MR Film. Prestained size markers (BioRad, Hercules, CA, USA) were used to calculate the size of kinases. All the experiments on kinase assay were repeated at least twice. Detection of ROS and Ca2+ in rice roots Five-day-old rice seedlings were used to localize the generation of ROS and Ca2+ in rice roots. The rice roots were labelled with 10 lM 5-(and-6)-chloromethyl-2#,7#-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA; Molecular Probes) and Oregon Green 488 BAPTA-1 (Molecular Probes) for 30 min and then treated with 400 lM CdCl2 and 100 lM CuCl2 for 15 min. A Leica MPS60 fluorescent microscope equipped with a green fluorescent protein filter (excitation 450–490 nm, emission 500–530 nm) was
used for fluorescence images. Exposure times were equal for all samples. Autofluorescence was not observed in unstained controls at the exposure time used. Images were captured with a CoolSNAP Cooled CCD Camera (CoolSNAP 5.0). NADPH oxidase activity assay The NADPH oxidase activity was determined by XTT assay and ingel activity assay. The XTT assay mixture of 1 ml contained 50 mM TRIS-HCl buffer (pH 7.5), 0.5 mM XTT, and 100 lM NADPH. The reaction was initiated with the addition of NADPH, and XTT reduction was determined at 470 nm (Jiang and Zhang, 2002). In-gel assay for NADPH oxidase activity was performed according to the procedures described previously (Sagi and Fluhr, 2001). The protein samples were electrophoretically separated on 10% (w/v) native polyacrylamide gels. After electrophoresis, gels were incubated in the darkness for 20 min in a reaction mixture solution containing 50 mM TRIS-HCl pH 7.4, 0.2 mM nitro blue tetrazolium (NBT), and 0.1 mM MgCl2. NADPH (0.2 mM) was added and the appearance of blue formazan bands was monitored. The reaction was halted by immersion of the gels in distilled water.
Results Cd2+- and Cu2+-induced 42 kDa MBP kinases are OsMPK3 and OsMPK6
Previous studies showed that Cd2+-induced 42 kDa MBP kinase has the characteristics of a MAP kinase (Yeh et al., 2004). To determine whether the Cu2+-induced 40 kDa and 42 kDa MBP kinase activities also correlated with the activation of rice MAP kinases, western blot analysis with an anti-phospho-ERK antibody was performed. Rice roots were exposed to Cd2+ and Cu2+ at varying concentrations ranging from 0 lM to 400 lM and from 0 lM to 100 lM, respectively. As the data show in Fig. 1A, exposure of rice roots to Cd2+ and Cu2+ was associated with a dosedependent increase in the phosphorylation level of two protein bands with estimated molecular masses of 40 kDa and 42 kDa. Immunoprecipitation followed by an in-gel kinase assay was further performed. Rice roots were treated with or without 400 lM Cd2+ and 100 lM Cu2+ for 1 h. Rice root protein extracts were subjected to immunoprecipitation with anti-ERK1, an antibody that reacts with several ERK isoforms in mammals, and tested for MBP phosphorylation in an in-gel kinase assay. The 40 kDa and 42 kDa MBP kinase activities were observed in anti-ERK immunoprecipitates of protein extracts prepared from Cd2+- and Cu2+-treated roots, but not in protein extracts of untreated roots (Fig. 1B). The data suggest that the Cd2+-induced 40 kDa MBP kinase and Cu2+-induced 40 kDa and 42 kDa MBP kinases are also rice MAP kinases. OsMPK3 (also known as OsMAPK2; Huang et al., 2002) and OsMPK6 are the orthologues of Arabidopsis AtMPK3 and AtMPK6, which are classical stress-inducible MAP kinases. Correlation of Cd- or Cuinduced MAP kinase activity with the activation of OsMPK3 or OsMPK6 was determined. Rice root protein extracts were immunoprecipitated with the anti-OsMPK3
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no. CAC13967) and OsMPK6 (GenBank protein accession no. Q84UI5), respectively, were produced synthetically and conjugated to the keyhole limpet haemacyanin (KLH) carrier. The OsMPK3 and OsMPK6 polyclonal antisera were raised in rabbits. A 300 lg protein extract (in 300 ll) was incubated with antiERK antibodies (diluted 1:500, v/v) or a 100 lg protein extract (in 100 ll) was incubated with anti-OsMPK3 or anti-OsMPK6 antibodies (diluted 1:100, v/v) overnight at 4 C. The agarose bead– protein complexes were pelleted by centrifugation (2 min, 500 g) and washed three times in 0.5 ml of buffer [20 mM TRIS, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 1 mM NaF, 10 mM b-glycerophosphate, 2 lg ml 1 antipain, 2 lg ml 1 aprotinin, 2 lg ml 1 leupeptin, 0.5% (v/v) Triton X-100, 0.5% (v/v) Nonidet P-40]. After washing with the above buffer, SDS sample buffer (1 : 1, v/v) was added and boiled for 10 min at 70 C. After centrifugation, the supernatant fraction was electrophoretically separated on a 10% (w/v) SDS-polyacrylamide gel embedded with 0.25 mg ml 1 of myelin basic protein (MBP) in the separating gel as a substrate, and the in-gel kinase assay was performed.
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By contrast, relatively lower levels of DCF fluorescence were produced in the control roots (Fig. 2). These results suggest that Cd2+ and Cu2+ induce ROS production in rice root tips. Cd2+ and Cu2+ induce redox-dependent MAP kinase activation through distinct ROS-generating systems in rice roots
ROS production in Cd2+- and Cu2+-treated rice roots
Cd2+ and Cu2+ induce Ca2+ accumulation in rice roots
To determine whether Cd2+ and Cu2+ treatment induces ROS production, a ROS-sensitive dye, CM-H2DCFDA, was used. This compound is non-fluorescent but is rapidly oxidized to the highly fluorescent DCF by intracellular ROS. Rice roots were treated with 20 mM H2O2, 400 lM Cd2+, and 100 lM Cu2+ for 15 min. As shown in Fig. 2, the levels of DCF fluorescence increased significantly after H2O2, Cd2+, and Cu2+ treatments in root tip regions.
To investigate whether treatment with Cd2+ and Cu2+ induces Ca2+ accumulation in rice roots, a Ca2+ indicator, Oregon green 488 BAPTA-1, was used. Rice roots were treated with 500 lM Ca2+, 400 lM Cd2+, and 100 lM Cu2+ for 15 min. As shown in Fig. 5, the levels of green fluorescence were significantly increased after Ca2+, Cd2+, and Cu2+ treatments in root tip regions. In contrast, a relatively lower level of green fluorescence was
Fig. 1. Phosphorylation and immunoprecipitation of Cd2+- and Cu2+induced 40 kDa and 42 kDa kinases. Rice roots were treated with different concentrations of CdCl2 and CuCl2 for 1 h. Protein extracts of Cd- and Cu-treated rice roots were harvested. Western blot analysis (A) of rice root extracts was performed with anti-ERK and anti-phosphoERK antibodies. A 15 lg aliquot of proteins of each sample was loaded. Rice root protein extracts containing 300 lg or 100 lg of total proteins were used for immunoprecipitation (IP) with anti-ERK1 (B), antiOsMPK3 or anti-OsMPK6 (C) antibodies. The kinase activities of the immunoprecipitated proteins were subsequently assayed with an in-gel kinase assay using MBP as a substrate. MBP phosphorylation was visualized by autoradiography. Arrows indicate the kinase-active bands. The experiment was repeated twice with similar results.
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or anti-OsMPK6 antibodies, and the MBP kinase activity of the immunoprecipitated proteins was analysed by an ingel kinase assay. In-gel kinase assay of protein extracts of roots at 1 h after heavy metal treatment showed that the OsMPK3 and OsMPK6 antibodies had immunoprecipitated active kinases with an estimated molecular mass of 42 kDa (Fig. 1C). The data suggest that Cd2+- and Cu2+induced 42 kDa MAP kinase activities resulted at least from activation of OsMPK3 and OsMPK6.
To examine whether ROS is involved in Cd2+- and Cu2+induced MAP kinase activities, rice roots were pretreated with sodium benzoate, a hydroxyl radical scavenger, for an hour before heavy metal treatments and they were then incubated for an additional hour. As shown in Fig. 3A, sodium benzoate significantly reduced Cd2+-stimulated 40 kDa and 42 kDa MAP kinase activation. Whereas sodium benzoate slightly inhibited Cu2+-induced 40 kDa MAP kinase activity, it had no significant effect on Cu2+mediated 42 kDa MAP kinase activation. In addition, western blot analysis with an anti-phospho-ERK antibody was performed. The phosphorylation of two protein bands with molecular masses of 40 kDa and 42 kDa is similar to the activation of 40 kDa and 42 kDa MAP kinases by ingel kinase assay. To investigate Cd2+- and Cu2+-induced enzymatic sources of ROS, the effect of an NADPH oxidase inhibitor, DPI, on Cd2+- and Cu2+-treated rice roots was examined. DPI pretreatment significantly inhibited Cd2+induced 40 kDa and 42 kDa MAP kinase activation. However, DPI had no significant effect on the activation of MAP kinases by Cu2+ treatment. Western blot using anti-phospho-ERK antibodies was performed, and similar results were obtained (Fig. 3B). These results imply that Cd2+-induced signal transduction may involve NADPH oxidase, which produces endogenous ROS to stimulate the activation of MAP kinases. To confirm further that Cd2+ induces NADPH oxidase activity, alterations in the NADPH oxidase-like activities were investigated in response to 400 lM Cd2+ and 100 lM Cu2+ in rice roots. The NADPH oxidase-like activities were analysed by XTT assay and in-gel assay after Cd2+ and Cu2+ treatment. The NADPH oxidase-like activities were induced by Cd2+ treatment at 15 min. By contrast, NADPH oxidase-like activities were decreased by Cu treatment. The NADPH oxidase-like activities were inhibited by DPI, an NADPH oxidase inhibitor (Fig. 4).
Different pathways in MAP kinase activation by Cd and Cu 663
Fig. 3. ROS may be involved in the Cd2+- and Cu2+-induced MAP kinase activation. Rice roots were pretreated with or without 500 lM sodium benzoate (A), a hydroxyl radical scavenger, and 100 lM DPI (B), an NADPH oxidase inhibitor, for 1 h before 400 lM CdCl2 and 100 lM CuCl2 treatment, and incubated for an additional hour. A 5 lg aliquot of crude protein extracts was analysed by in-gel kinase assay (5 lg per lane) or western blot using anti-phospho-ERK or anti-ERK antibodies (15 lg per lane). MBP phosphorylation was visualized by autoradiography. Arrows indicate the kinase-active bands. The experiment was repeated three times with similar results.
produced in the control roots. These results suggest that Cd2+ and Cu2+ induce Ca2+ accumulation in rice root tips. Involvement of CDPK in the activation of MAP kinases by Cd2+ and Cu2+ The CDPK family is involved in many cellular responses that are triggered by elevated intracellular Ca2+ concentra-
Fig. 4. Cd2+ induces NADPH oxidase-like activities. Rice roots were pretreated with or without 100 lM DPI, an NADPH oxidase inhibitor, for 1 h before 400 lM CdCl2 and 100 lM CuCl2 treatments, and further incubated for 15 min. Protein extracts were analysed by XTT assay (A) or in-gel assay (B) for NADPH oxidase-like activities. The experiment was repeated three times with similar results. The data on NADPH oxidase-like activity (A) are means 6SE.
tion (Saijo et al., 2000; Martı´n and Busconi, 2001). A recent study has shown that CDPK can mediate stressinduced MAP kinase activation (Sangwan et al., 2002). Therefore, an experiment was conducted to examine whether Cd2+ and Cu2+ induce CDPK activation. In this experiment, protein extracts were separated on an SDSpolyacrylamide gel containing histone III-S as a substrate, and an in-gel kinase assay was performed. The data in
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Fig. 2. ROS production in rice roots during heavy metal stress. Rice roots were labelled with 10 lM CM-H2DCFDA for 30 min and then treated with 20 mM H2O2, 400 lM CdCl2, and 100 lM CuCl2 for 15 min. Green fluorescence indicates the presence of ROS. Ten control and 10 treated roots showed similar results. The magnification for all images was 3100.
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Fig. 6 suggest that 47 kDa and 49 kDa kinase activities were induced by Cd2+ and Cu2+. However, activation of both Cd2+- and Cu2+-induced 47 kDa and 49 kDa kinase was completely inhibited by a Ca2+ chelator, EGTA, and significantly inhibited by a calmodulin antagonist, W7, indicating that they are CDPKs (Fig. 6). The effect of W7 on Cd2+- and Cu2+-induced MAP kinase activation was examined further. Pretreatment with W7 for 1 h before Cd2+ and Cu2+ addition decreased the level of Cd2+- and Cu2+-induced 40 kDa and 42 kDa MAP kinase activation (Fig. 7). In addition, the phosphorylation of 40 kDa and 42 kDa proteins was similar to the activation of 40 kDa and 42 kDa MAP kinases by in-gel kinase assay. The data suggest that CDPKs may be involved in the activation of 40 kDa and 42 kDa MAP kinases by Cd2+ and Cu2+. PI3 kinase activities may be required for MAP kinase activation by Cd2+ and Cu2+ To examine whether PI3 kinase (phosphatidylinositol 3kinase) is involved in Cd2+- and Cu2+-induced MAP kinase activities, rice roots were pretreated for 1 h with wortmannin, an inhibitor of PI3 kinase, followed by Cd2+ and Cu2+ treatments for 1 h, and then an in-gel kinase assay was performed. As the data showed, the 40 kDa and 42 kDa MAP kinase activities were inhibited by wortmannin in Cd2+ and Cu2+ treatments (Fig. 8A). Closing of the mitochondrial permeability transition pore disrupts activation of Cd2+-induced MAP kinase
To investigate whether mitochondria are involved in Cd2+- and Cu2+-induced signalling pathways, an inhibitor of mitochondrial PT pore opening, BK, was used. Pretreatment with 50 lM BK for 1 h before Cd2+ addition
decreased the level of Cd2+-induced 40 kDa and 42 kDa MAP kinase activation (Fig. 8B). However, BK had no significant effect on the activation of 40 kDa and 42 kDa MAP kinase by Cu2+ treatment. The data suggest that Cd2+-induced MAP kinase activities depend on the functional state of mitochondria and that Cd2+ may cause mitochondrial dysfunction. Heterotrimeric G protein a subunit does not mediate Cd2+- and Cu2+-induced signalling pathways
The rice dwarf mutant T65d1 (Taichung 65-Daikoku dwarf) is defective in the a subunit of the heterotrimeric G protein (Ueguchi-Tanaka et al., 2000). To determine if Ga protein plays a role in heavy metal stress-induced MAP kinase activation, the rice Ga protein mutant T65d1 was treated with Cd2+ and Cu2+ for 1 h, and an in-gel kinase assay was performed. As shown in Fig. 9, no significant differences in MAP kinase activities were observed between the wild type (Taichung 65, T65) and the Ga protein mutant (T65d1). These results suggest that Cd2+- and Cu2+-induced signalling pathways may be independent of the Ga-mediated signalling pathway. Activation of MAP kinase, ROS production, and Ca2+ accumulation in Cd2+-tolerant and Cd2+-sensitive cultivars
Hsu and Kao (2003) reported that rice seedlings of cultivar Tainung 67 (TNG67) are more tolerant to Cd2+ than those of cultivar Taichung Native 1 (TN1). Here, it was tested whether the levels of MAP kinase activities are different between TNG67 and TN1. Rice roots were treated with 100–400 lM Cd2+ for 1 h, and the protein extracts were collected for an in-gel kinase assay. On treatment with Cd2+, 42 kDa MAP kinase activities are
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Fig. 5. Ca2+ accumulation in rice roots during heavy metal stress. Rice roots were labelled with 10 lM Oregon Green 488 BAPTA-1 for 30 min and then treated with 500 lM CaCl2, 400 lM CdCl2, and 100 lM CuCl2 for 10 min. Green fluorescence indicates the presence of Ca2+. Ten control and 10 treated roots showed similar results. The magnification for all images was 3100.
Different pathways in MAP kinase activation by Cd and Cu 665
Fig. 6. Cd2+ and Cu2+ induce CDPK activities in rice roots. Rice roots were treated with or without 400 lM CdCl2 or 100 lM CuCl2 for 1 h. A 5 lg aliquot of crude protein extracts was analysed by in-gel kinase assay using histone III-S as a substrate. The in-gel kinase assay was performed in the presence of either 0.2 mM CaCl2 or 4 mM EGTA in kinase reaction buffer. Histone III-S phosphorylation was visualized by autoradiography. Arrows indicate the kinase-active bands. The experiment was repeated twice with similar results.
higher in TNG67 than in TN1 (Fig. 10). The results suggest that 42 kDa MAP kinases may play an important role in Cd tolerance in rice. In addition, it was found that ROS production and Ca2+ accumulation in response to Cd2+ treatment in TNG67 was more evident than in TN1 (Fig. 11). Discussion Heavy metal-induced MAP kinase gene expression (Agrawal et al., 2002, 2003a; Yeh et al., 2003, 2004; Hung et al., 2005) and MAP kinase activation (Yeh et al., 2003, 2004; Lin et al., 2005; Tsai and Huang, 2006) have been reported in rice. In this study, it is demonstrated that Cd2+ and Cu2+ activated, at least, three different MAP kinases, including OsMPK3, OsMPK6, and 40 kDa MAP kinase, in rice. Exposure of alfalfa (Medicago sativa) seedlings to excess Cd2+ and Cu2+ activated four distinct MAP kinases: SAMK, SIMK, MMK2, and MMK3 (Jonak et al., 2004). SAMK and SIMK are the orthologues of rice OsMPK3 and OsMPK6, respectively. Due to the fact that MAP kinase pathways are highly conserved in plants, Cd2+- and Cu2+-induced 40 kDa and 42 kDa MAP kinase activities might also contain the activities of rice orthologues of MMK2 and MMK3. However, the possibility
Fig. 8. Effects of a PI3 kinase inhibitor and a mitochondrial PT pore opening blocker on Cd2+-and Cu2+-induced MAP kinase activation. Rice roots were pretreated with or without 10 lM wortmannin (A), a PI3 kinase inhibitor, or 50 lM BK (B), a mitochondrial PT pore opening blocker, for 1 h before 400 lM CdCl2 and 100 lM CuCl2 treatments, and further incubated for 1 h. A 5 lg aliquot of crude protein extracts was analysed by in-gel kinase assay. MBP phosphorylation was visualized by autoradiography. Arrows indicate the kinaseactive bands. The experiment was repeated three times with similar results.
cannot be excluded that the 40 kDa MAP kinase might also represent a post-translationally modified form of the 42 kDa MAP kinase. Droillard et al. (2002) reported that an Arabidopsis MAP kinase, AtMPK6, is activated by hypo- and hyperosmotic stresses, as well as its tobacco orthologue SIPK. However, AtMPK3 is activated by hypoosmotic stress as well as its tobacco orthologue WIPK. Therefore, the results imply that sequence similarities correlate with identical functions. Differential activation of rice MAP kinases depends on different types of ROS in response to Cd2+ and Cu2+
ROS include superoxide, singlet O2, H2O2, and the highly reactive hydroxyl radical. Intercellular levels of ROS are influenced by a number of endogenous and exogenous processes, and controlled by several radical scavenging enzymes (Adler et al., 1999). Elevated levels of heavy metals and various other environmental stresses accelerate the formation of ROS. Redox-active metals (e.g. Cu2+ and Fe2+) catalyse the formation of hydroxyl radicals by directly participating in the Haber–Weiss reaction, and
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Fig. 7. CDPKs play a role in Cd2+- and Cu2+-induced MAP kinase activities. Rice roots were pretreated with or without 100 lM W7, a CDPK antagonist, for 1 h before being exposed to 400 lM CdCl2 and 100 lM CuCl2, and further incubated for 1 h. A 5 lg aliquot of crude protein extracts was analysed by in-gel kinase assay (5 lg per lane) or western blot using anti-phospho-ERK or anti-ERK antibodies (15 lg per lane). MBP phosphorylation was visualized by autoradiography. Arrows indicate the kinase-active bands. The experiment was repeated three times with similar results.
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2+
2+
Fig. 10. Activation of MAP kinase in Cd -tolerant and Cd -sensitive cultivars. Rice seedlings of the Cd2+-tolerant cultivar Tainung 67 (TNG67) and Cd2+-sensitive cultivar Taichung Native 1 (TN1) were treated with different concentrations of CdCl2 and CuCl2 for 1 h. A 5 lg aliquot of crude protein extracts was analysed by in-gel kinase assay. MBP phosphorylation was visualized by autoradiography. Arrows indicate the kinase-active bands. The experiment was repeated twice with similar results.
metals without redox capacity (e.g. Cd2+, Pb2+, Hg2+, and Zn2+) disturb metabolism by binding to functional groups, eventually enhancing the load of ROS (Schu¨tzendu¨bel et al., 2001; Babu et al., 2003; Olmos et al., 2003; Jonak et al., 2004; Tripathi and Gaur, 2004). Evidence for activation of MAP kinase by ROS has been provided by several studies in different plant species (Kovtun et al., 2000; Yuasa et al., 2001; Jonak et al., 2004). In addition, ROS are involved in heavy metal-induced MAP kinase activation in mammals (Seo et al., 2001; Rockwell et al., 2004) and alfalfa (Jonak et al., 2004). Previous studies showed that Cd2+-induced activation of 40 kDa, but not 42 kDa, MAP kinase was inhibited by the antioxidant glutathione (GSH) in rice. In contrast, GSH strongly inhibited Cu2+-induced 42 kDa MAP kinase activation (Yeh et al., 2003, 2004). In this study, sodium benzoate, a ROS scavenger, significantly reduced Cd2+-stimulated 40 kDa and 42 kDa MAP kinase activities; however, it only inhibited activation of 40 kDa MAP kinase due to Cu2+ treatment. Differential activation of ERK1/2, JNK, and p38 MAP kinase depending on different types of ROS in response to UVB exposure and angiotensin II treatment
Fig. 11. ROS production and Ca2+ accumulation in Cd2+-tolerant and Cd2+-sensitive cultivars. Rice roots of the Cd2+-tolerant cultivar (TNG67) and Cd2+-sensitive cultivar (TN1) were labelled with 10 lM CM-H2DCF-DA (A) or Oregon Green 488 BAPTA-1 (B) for 30 min and then treated with 400 lM CdCl2 for 15 min. Green fluorescence indicates the presence of ROS or Ca2+. Ten treated TNG67 and TN1 roots showed similar results. The magnification for all images was 3100.
has been reported (Peus et al., 1999; Kyaw et al., 2001). Therefore, the finding that sodium benzoate and GSH differentially inhibit Cd2+- and Cu2+-induced 40 kDa and 42 kDa MAP kinase activation indicates that the regulation of these pathways by ROS is distinct, and their responsiveness may differ depending on the types of ROS involved. However, benzoic acid appears as a likely intermediate in salicylic acid (SA) biosynthesis in planta. At least in tobacco and in cucumber, the biosynthetic pathway of SA was proposed to proceed exclusively via free benzoic acid (Yalpani et al., 1993; Meuwly et al., 1995). Chong et al. (2001) reported that free benzoic acid is probably not the major intermediate in SA biosynthesis, which may involve conjugated forms of benzoic acid. This makes it possible that exogenously applied sodium benzoate in rice roots may be converted to SA. In addition, it is demonstrated that SA caused protection against Cd2+ toxicity in barley (Metwally et al., 2003). Therefore, an investiation was carried out as to whether sodium benzoate application activated SA signalling cascades by increasing SA production in the present case. The results showed that SA, but not sodium benzoate,
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Fig. 9. Ga is not involved in Cd2+-and Cu2+-induced 40 kDa and 42 kDa MAPK activation. Rice cultivar T65 and Ga protein mutant, T65d1 seedlings were treated with 400 lM CdCl2 (A) and 100 lM CuCl2 (B) for 1 h. A 5 lg aliquot of crude protein extracts was analysed by in-gel kinase assay. MBP phosphorylation was visualized by autoradiography. Arrows indicate the kinase-active bands. The experiment was repeated twice with similar results.
Different pathways in MAP kinase activation by Cd and Cu 667
Calcium and CDPK are required for MAP kinase activation Several studies reported that the cytoplasmic Ca2+ concentrations in plant cells increase rapidly in response to multiple stress stimuli (Saijo et al., 2000; Martı´n and Busconi, 2001). Following this Ca2+ influx, signals are likely to be mediated by combinations of protein phosphorylation/dephosphorylation cascades. It is presumed that the majority of Ca2+-stimulated protein
phosphorylation is performed predominantly by CDPKs in plants (Saijo et al., 2000). CDPKs are a large family of Ser/Thr protein kinases regulated by Ca2+, and are biochemically distinct from other Ca2+-regulated protein kinases such as protein kinase C and Ca2+/calmodulindependent protein kinases (Harper et al., 1991; Martı´n and Busconi, 2001). Sheen (1996) has reported that ATCDPK1 and ATCDPK1a may be positive regulators controlling stress signal transduction in Arabidopsis. Martı´n and Busconi (2001) found a 56 kDa rice membrane-bound CDPK whose kinase and autophosphorylating activities increased after several hours of cold treatment. The activation of SAMK by cold and of HAMK by heat requires CDPK activity (Sangwan et al., 2002). In this study, involvement of multiple stressinducible CDPKs in the heavy metal stress signal transduction pathway was examined. As shown in Fig. 6, the 47 kDa and 49 kDa kinase activities increased with Cd2+ and Cu2+ treatment. In addition, W7, a CDPK antagonist, significantly inhibited the Cd2+- and Cu2+-induced 40 kDa and 42 kDa MAP kinase activation (Fig. 7). These data suggest that heavy metal-induced MAP kinase activities may occur through the action of CDPKs. Involvement of PI3 kinase in the activation of MAP kinases by Cd and Cu
Phosphatidylinositol (PI) metabolism plays a central role in signalling pathways in both animals and higher plants (Jung et al., 2002). PI3P (phosphatidylinositol 3-phosphate) is a product of PI3 kinase, which phosphorylates the D-3 position of phosphoinositides (Park et al., 2003). Eom et al. (2001) reported that Zn2+ stimulates the JNK pathway and PI3 kinase in mouse primary cortical cells and in various cell lines. The Zn2+-induced JNK stimulation was blocked by the PI3 kinase inhibitor or by a dominant-negative mutant of PI3 kinase c. Rockwell et al. (2004) suggested that inhibition of PI3 kinase attenuated Cd2+-induced ROS and p38 MAP kinase activation. In this study, pretreatment of rice roots with wortmannin, an inhibitor of PI3 kinase, for 1 h before Cd2+ and Cu2+ treatments and subsequent in-gel analysis suggests that PI3 kinase activities may be required for Cd2+- and Cu2+-induced MAP kinase activation. Therefore, it is hypothesized that PI3P, as found in animal cells, activates plant MAP kinases during heavy metal stresses. Cd2+-induced MAP kinase activities depend on the functional state of mitochondria Mitochondrial damage and apoptosis induced by Cd2+ has been well established in animal systems. Li et al. (2003) reported that Cd2+ directly leads to the dysfunction of mitochondria, including the inhibition of respiration, the loss of transmembrane potential, and the opening of the PT pore. Opening of the PT pore is a possible mechanism
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induced MAP kinase activation. In addition, sodium benzoate significantly inhibited Cd- and Cu-induced DCF fluorescence (data not shown). These results suggest that sodium benzoate indeed scavenges ROS and the effect of sodium benzoate on Cd- and Cu-induced MAP kinase activation may be independent of SA signalling cascades. In addition, the enzymatic sources of ROS, important for MAPK activation, differ in Cd2+- and Cu2+-treated rice roots. One of the mechanisms by which ROS are produced is through the activation of a membrane-associated NADPH oxidase (Agrawal et al., 2003b). Superoxide anion (O2 ) production by NADPH oxidase and its subsequent conversion to H2O2 has been well characterized as a source of oxidative stress that causes apoptosis and cell death in phagocytic cells in a mechanism that can involve PI3 kinase and p38 MAP kinase (Rockwell et al., 2004). Rockwell et al. (2004) examined the effects of the NADPH oxidase inhibitor DPI on the ROS production and the phosphorylation of p38 MAP kinase induced by Cd2+ in mouse neuronal cells. The findings implicate NADPH involvement in Cd2+-induced p38 MAP kinase activation and oxidative stress. Olmos et al. (2003) reported that DPI prevented the generation of H2O2 induced by Cd2+ in cultured tobacco (Nicotiana tabacum L.) BY-2 cells. Furthermore, as rice MAP kinases induced by ROS (H2O2) have recently been identified, it is thought that ROS produced due to activation of NADPH oxidase by OsRac1 may itself activate a MAP kinase cascade associated with disease resistance (Agrawal et al., 2003b). In this study, pretreatment with DPI decreased the level of Cd2+-, but not Cu2+-, induced 40 kDa and 42 kDa MAP kinase activation. In addition, Cd2+ activated NADPH oxidase-like activities. These results imply that redoxdependent activation of MAP kinase by Cd2+ and Cu2+ occurs through distinct ROS-generating systems and that these processes may contribute to differential redoxsensitive signalling by Cd2+ and Cu2+. Compared with Cdinduced ROS production, however, Cd only slightly induced NADPH oxidase-like activities. Beside NADPH oxidase, there are several potential enzymatic sources of ROS including xanthine oxidase, amine oxidase, and a cell wall peroxidase (Neill et al., 2002). Therefore, the majority of Cd2+-induced ROS production may be NADPH oxidase independent and result from other enzymatic sources.
668 Yeh et al.
Cd2+- and Cu2+-induced MAP kinase pathways may be independent of the Ga-mediated signalling pathway Nishida et al. (2000) showed that inhibition of the bcsubunit of G protein (Gbc) attenuates hydrogen peroxide (H2O2)-induced ERK activation in rat neonatal cardiomyocytes. Analysis using heterotrimeric Go and its individual subunits indicates that H2O2 modifies Gao but not Gbc, which leads to subunit dissociation. They concluded that Gai and Gao are critical targets of oxidative stress for activation of ERK. It has been proposed that the plant heterotrimeric G protein is involved in various signal transduction systems, including those of several plant hormones, blue and red lightmediated responses, pathogen resistance, and pathogenrelated gene expression (Ueguchi-Tanaka et al., 2000; Fujisawa et al., 2001). Genomic and cDNA clones that encode polypeptides similar to the mammalian Ga or Gb proteins have been isolated from various plant species, including model plants, Arabidopsis and rice. Available data reported thus far suggest the presence of a single gene for either the a subunit or the b subunit in each genome of Arabidopsis and rice. Thus an examination was carried out of whether the a subunit of the heterotrimeric G protein is the target protein of heavy metal-induced ROS for MAP kinase activation in rice. No
significant differences for 40 kDa and 42 kDa MAP kinase activities were observed between the wild type (T65) and Ga protein mutant (T65d1). These results suggest that Cd2+- and Cu2+-induced 40 kDa and 42 kDa MAP kinase activation may be independent of the Gamediated signalling pathway. Cd2+- and Cu2+-activated MAP kinases may regulate heavy metal stress tolerance Previous studies showed that the expression of a rice MAP kinase gene, OsMPK3, is inducible by cold, salinity, Cu2+, and Cd2+ (Huang et al., 2002; Yeh et al., 2003, 2004). The deduced amino acid sequence of OsMPK3 comprises 369 amino acid residues with an estimated molecular mass of 42 kDa (Huang et al., 2002). Its amino acid sequence is identical to that of OsMSRMK2 (Agrawal et al., 2002), OsMAP1 (Wen et al., 2002), OsBIMK1 (Song and Goodman, 2002), and OsMAPK5 (Xiong and Yang, 2003). In the above studies, the OsMAPK genes were also induced by various biotic and abiotic stresses and were suggested to play a role in stress signal transduction. Xiong and Yang (2003) further generated and analysed transgenic rice plants with overexpression (using the cauliflower mosaic virus 35S promoter) and suppression (using double-stranded RNA interference; RNAi) of OsMAPK5. According to their data, activation of OsMAPK5 indeed provided abiotic stress tolerance to the rice plant. To confirm the role of OsMPK3 in heavy metal-dependent signal transduction further, two rice cultivars, TNG67 and TN1, were used to analyse 42 kDa MAP kinase activity. Hsu and Kao (2003) have reported that the TNG67 (cv. Tainung 67) rice plant is a Cd2+-tolerant cultivar and the TN1 (cv. Taichung Native 1) rice plant is a Cd2+-sensitive cultivar. In this study, it was found that enhancement of 42 kDa MAP kinase activity of the Cd2+-sensitive cultivar (TN1) caused by Cd2+ was less than that of the Cd2+-tolerant cultivar (TNG67). Furthermore, ROS production and Ca2+ accumulation were more evident after Cd2+ treatment in TNG67 than in TN1. Apparently, ROS and Ca2+ were rapidly and greatly accumulated in TNG67 when the roots were treated with Cd2+, and the accumulating ROS and Ca2+ subsequently activate 42 kDa MAP kinase. The activation of Cd2+-induced 42 kDa MAP kinase may provide Cd2+ tolerance to rice plants. Moreover, Hsu and Kao (2003) found that the endogenous abscisic acid (ABA) content rapidly increased in the leaves and roots of the Cd2+-tolerant cultivar (TNG67) but not in the Cd2+sensitive cultivar (TN1). Knetsch et al. (1996) and Burnett et al. (2000) reported that ABA induced a rapid and transient MAP kinase activation in barley aleurone protoplasts and pea leaves, respectively. Thus, low levels of 42 kDa MAP kinase activity of TN1 may also result from low levels of ABA content in TN1. Future work will
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whereby cytochrome c and other apoptosis-triggering factors are released into the cytosol (Maxwell et al., 2002). This mitochondrial damage is possibly involved in apoptosis induced by Cd2+. Takahashi et al. (2003) demonstrated that the closing of the mitochondrial PT pore by BK, an inhibitor of the PT pore opening directly via an adenine nucleotide translocator, disrupts activation of MAP kinases induced by spermine. Therefore, involvement of mitochondrial dysfunction in Cd2+-induced MAP kinase activation in rice roots was tested. For this purpose, the effect of BK on MAP kinase activity of Cd2+-treated rice roots was tested. As the data show in Fig. 8B, BK greatly inhibited the activation of 40 kDa and 42 kDa MAP kinase by Cd2+ treatment. The data suggest that mitochondria PT pore opening may be required for Cd2+-induced signalling pathways. The role of mitochondria during Cd2+ stress was reported. Finkemeier et al. (2005) showed that disruption of mitochondrial antioxidant metabolism greatly enhances the Cd2+ sensitivity of root growth in Arabidopsis thaliana. However, pharmacological experiments should always be interpreted with caution. In particular, non-specific effects are always possible, particularly if reagents are used at relatively high concentrations. The I50 values in the rice root system for the effects of the various inhibitors on the signalling pathway were all within the concentration ranges previously employed in signal transduction studies in plant or mammalian cells.
Different pathways in MAP kinase activation by Cd and Cu 669 Cd2+
Cu2+ plasma membrane
NADPH oxidase
ROS
CDPK, PI3K
?
ROS CDPK, PI3K
mitochondria
MAP kinase activation
MAP kinase activation
Fig. 12. Schematic representation of heavy metal-induced signal transduction in rice. See text for details.
Acknowledgements We are most grateful to Professor Toshio Murashige for critical reading of the manuscript. We also wish to thank Professor Makoto Matsuoka and Professor Motoyuki Ashikari for providing the rice seeds of T65 and T65d1, and Dr Chang-Sheng Wang for providing rice seeds of TNG67 and TN1. We thank Ms Lorraine Rooney for assistance with correction of the English of this manuscript. This work was supported by a grant from The National Science Council of the Republic of China (NSC 94-2311-B-006-003).
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RESEARCH PAPER
Distinct signalling pathways for induction of MAP kinase activities by cadmium and copper in rice roots Chuan-Ming Yeh, Pei-Shan Chien and Hao-Jen Huang* Department of Life Sciences, National Cheng Kung University, No. 1 University Rd. 701, Tainan, Taiwan, Republic of China Received 25 July 2006; Revised 19 October 2006; Accepted 20 October 2006
Plant growth is severely affected by toxic concentrations of heavy metals. On characterizing the heavy metal-induced signalling pathways, the effects of cadmium (CdCl2) and copper (CuCl2) on MBP (myelin basic protein) kinase activities in Oryza sativa L. cv. TNG67 were analysed and it was found that Cd2+induced 42 kDa MBP kinase has the characteristics of a mitogen-activated protein (MAP) kinase. This study confirmed that the 42 kDa kinase-active band contains, at least, the activities of OsMPK3 and OsMPK6. Then, the heavy metal signal transduction pathways leading to MAP kinase activation in rice roots were examined. Pretreatment with sodium benzoate, a hydroxyl radical scavenger, attenuated Cd2+- or Cu2+-induced MAP kinase activation. The Cd2+-, but not Cu2+-, induced MAP kinase activities were suppressed by diphenylene iodonium (DPI), an NADPH oxidase inhibitor, and Cd2+ induced NADPH oxidase-like activities, suggesting that NADPH oxidases may be involved in Cd2+-induced MAP kinase activation. Using a Ca2+ indicator, it was demonstrated that Cd2+ and Cu2+ induce Ca2+ accumulation in rice roots. The Cd2+- and Cu2+-induced MAP kinase activation required the involvement of Ca2+dependent protein kinase (CDPK) and phosphatidylinositol 3-kinase (PI3 kinase) as shown by the inhibitory effect of a CDPK antagonist, W7, and a PI3 kinase inhibitor, wortmannin, respectively. Furthermore, bongkrekic acid (BK), a mitochondrial permeability transition pore opening blocker, suppressed Cd2+-, but not Cu2+-, induced MAP kinase activation, indicating that Cd2+-induced MAP kinase activities are
dependent on the functional state of mitochondria. Collectively, these findings imply that Cd2+ and Cu2+ may induce MAP kinase activation through distinct signalling pathways. Moreover, it was found that the 42 kDa MAP kinase activities are higher in Cd-tolerant cultivars than in Cd-sensitive cultivars. Therefore, the Cd-induced 42 kDa MAP kinase activation may confer Cd tolerance in rice plants. Key words: Cadmium, copper, heavy metal, MAP kinase, rice, signal transduction.
Introduction Environmental stresses, including biotic and abiotic stresses, such as pathogen infection, cold, drought, salinity, and heavy metals, are important factors that affect growth and metabolism of land plants. Heavy metal toxicity is one of the major environmental health problems in modern society, with potentially dangerous bioaccumulation through the food chain (Di Domenico et al., 1989; Lewis and McIntosh, 1989). Cadmium (Cd2+) is a toxic metal with a long biological half-life and represents a serious environmental pollutant for both animals and plants. In plants, Cd2+ is known to inhibit seed germination and root growth, induce chromosomal aberrations and micronucleus formation, and cause faster wilting and a grey-green leaf colour (Fojtova´ and Kovarˇ´ık, 2000). Although copper (Cu2+) is essential for normal plant growth and development, elevated concentrations of Cu2+ in the soil can also lead to toxicity symptoms and stunted growth in most plants (Hall, 2002). The toxicity
* To whom correspondence should be addressed. E-mail: [email protected] Abbreviations: ABA, abscisic acid; BEAS, bronchial epithelial cell strain; BK, bongkrekic acid; CDPK, Ca2+-dependent protein kinase; CM-H2DCFDA, 5(and-6)-chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate, acetyl ester; DPI, diphenylene iodonium; DTT, dithiothreitol; ERK, extracellular signalregulated kinase; HRP, horseradish peroxidase; JNK, c-Jun N-terminal kinase; MAP, mitogen-activated protein; MBP, myelin basic protein; PI3 kinase, phosphatidylinositol 3-kinase; PT pore, permeability transition pore; ROS, reactive oxygen species; SA, salicylic acid. ª The Author [2007]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: [email protected]
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Abstract
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tems. In addition, closing of the mitochondrial permeability transition (PT) pore disrupts Cd2+-, but not Cu2+-, induced MAP kinase activities. The results suggest that Cd2+- and Cu2+-induced MAP kinase activities may act through distinct signalling pathways. Materials and methods Plant materials and growth conditions Rice seedlings (Oryza sativa L. cv. TNG67, cv. TN1, cv. T65, and cv. T65d1) were cultured as described by Chen et al. (2000) with some modifications. Rice seeds were sterilized with 2.5% (v/v) 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 d incubation period, uniformly germinated seeds were selected and transferred to Petri dishes containing one sheet of Whatman No. 1 filter paper moistened with 10 ml of distilled water or test solution. Each Petri dish contained 15 germinated seeds. The germinated seeds were grown at 26 C in darkness for 3 d. Roots of rice seedlings were collected for experimental purposes. Preparation of protein extracts To prepare protein extracts from treated rice roots, roots were ground with a mortar and pestle using liquid nitrogen, followed by homogenization at 4 C with 2 vols (w/v) of protein extraction buffer [100 mM HEPES, pH 7.5, 5 mM EDTA, 5 mM EGTA, 10 mM dithiothreitol (DTT), 10 mM Na3VO4, 10 mM NaF, 50 mM bglycerophosphate, 1 mM phenylmethylsulphonyl fluoride (PMSF), 5 lg ml 1 antipain, 5 lg ml 1 aprotinin, 5 lg ml 1 leupeptin, 10% glycerol]. The ground slurry was centrifuged at 13 000 rpm for 20 min at 4 C in a microcentrifuge. Aliquots of the supernatants were put into clean tubes, flash-frozen in liquid nitrogen, and stored at 80 C for later use. Western blot analysis The protein extracts (15 lg) were separated by SDS-PAGE (10% gel). After electrophoresis, the gel was transfererd to a polyvinylidene difluoride (PVDF) membrane (PerkinElmer Life Sciences, Boston, MA, USA) at 100 V for 70 min at 4 C in a Mini TransBlot Electrophoretic Transfer Cell (Bio-Rad, Hercules, CA, USA). The membrane was incubated in blocking solution containing TBST (20 mM TRIS-base, 137 mM NaCl, 0.1% Tween-20, pH 7.6) supplemented with 5% (w/v) non-fat dry milk for 1 h at room temperature. Then it was washed three times for 5 min with TBST buffer. The blots were probed with polyclonal antibodies raised against the conserved subdomain XI of rat ERK1, which recognize ERK1 and ERK2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), or against the phosphotyrosine-containing peptide of human p44 MAP kinase (residues 196–209; Cell Signaling Technology, Beverly, MA, USA). The latter antibody recognized only ERK1/2related polypeptides which are catalytically activated at Tyr204. The immune complexes were detected by a horseradish peroxidase (HRP)-conjugated secondary antibody (Pierce, Rockford, IL, USA) and revealed by Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences, Boston, MA, USA). Antibodies were used at 1:2500, 1:1000, and 1:5000 dilutions for anti-ERK1, anti-phospho-ERK, and secondary antibodies, respectively. Antibody production and immunoprecipitation The peptides LIFNEAIEMNPNIRY and YQEGLAFNPDYQ corresponding to the C-termini of OsMPK3 (GenBank protein accession
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symptoms seen in the presence of excessive amounts of heavy metals may result from the binding of metals to sulphydryl groups in proteins, leading to loss of activity or disruption of structure, or from the displacing of an essential element, resulting in deficiency effects. In addition, heavy metal excess may stimulate the formation of free radicals and reactive oxygen species (ROS), which may cause lipid peroxidation, enzyme inactivation, and DNA and membrane damage (Hall, 2002; Boominathan and Doran, 2003). Although heavy metals are associated with a number of physiological disorders in plants, the molecular mechanisms of heavy metal signal transductions are not well understood. Several lines of evidence from biochemical and genetic studies of stress signalling indicate that phosphorylation and dephosphorylation of proteins are important in the regulation of physiological status and gene expression in response to various environmental stresses (Yuasa et al., 2001). The mitogen-activated protein (MAP) kinase cascade plays an essential role in the intracellular signal transduction processes involving cell proliferation and stress responses in yeast and mammalian cells (Herskowitz, 1995; Kyriakis and Avruch, 1996). Increasing evidence reveals that MAP kinases also play similar roles in plant response to biotic and abiotic stresses, such as pathogen, cold, drought, salt, and wounding (Jonak et al., 1996; Mizoguchi et al., 1996; Zhang et al., 2000; Fu et al., 2002; Huang et al., 2002). Samet et al. (1998) reported that As3+, V4+, Cr3+, Cu6+, and Zn2+ activate the MAP kinases, extracellular signalregulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAP kinase in BEAS (bronchial epithelial cell strain) cells. The three MAP kinases, ERK, JNK, and p38 kinase, were also activated by Cd2+ in human T cells (Iryo et al., 2000). Studies of growth inhibition and apoptosis in a human non-small cell lung carcinoma cell line (CL3) show that JNK and p38 co-operatively participate in apoptosis induced by Cd2+, and the decreased ERK signal induced by low Cd2+ doses contributes to growth inhibition or apoptosis (Chuang et al., 2000). Recently, MAP kinase pathways involved in heavy metal stresses have also been demonstrated in rice (Yeh et al., 2003, 2004) and alfalfa (Jonak et al., 2004). However, the relationship between plant MAP kinase pathways and heavy metal stress has not been well examined. The aim of this study was to search for MAP kinases regulated at the translational and post-translational levels during heavy metal-induced stress responses and to investigate the molecular mechanisms of signalling networks underlying heavy metal stress in the model plant, rice. In this study, the roles of second messengers, ROS and calcium, in Cd2+- and Cu2+-induced signal transduction pathways were examined and discussed. Using pharmacological inhibitors, it is demonstrated that Cd2+ and Cu2+ induce MAP kinase activation via distinct ROS-generating sys-
Different pathways in MAP kinase activation by Cd and Cu 661
In-gel kinase activity assay The in-gel kinase assay was performed according to the procedures described previously (Zhang and Klessig, 1997) with slight modifications. Rice roots were pretreated with various inhibitors [500 lM sodium benzoate, 100 lM diphenylene iodonium (DPI), 100 lM W7, 10 lM wortmannin, and 50 lM bongkrekic acid (BK)] for 1 h before 400 lM CdCl2 and 100 lM CuCl2 treatments and further incubated for an additional hour. Root extracts containing 5 lg of protein were electrophoretically separated on 10% (w/v) SDS-polyacrylamide gels embedded with 0.25 mg ml 1 of MBP or histone III-S in the separating gel as a substrate for the kinase. After electrophoresis, SDS was removed by cleansing the gel with washing buffer [25 mM TRIS, pH 7.5, 0.5 mM DTT, 0.1 mM Na3VO4, 5 mM NaF, 0.5 mg ml 1 bovine serum albumin (BSA), 0.1% (v/v) Triton X-100] three times, each for 30 min at room temperature. The kinases were allowed to renature in 25 mM TRIS, pH 7.5, 0.5 mM DTT, 0.1 mM Na3VO4, and 5 mM NaF at 4 C overnight with three changes of buffer. The gel for MAP kinase activity assay was then incubated at room temperature in a 90 ml reaction buffer (25 mM TRIS, pH 7.5, 2 mM EGTA, 12 mM MgCl2, 1 mM DTT, 0.1 mM Na3VO4) for 30 min. The gel for Ca2+-dependent protein kinase (CDPK) activity assay was incubated at room temperature in a 90 ml reaction buffer (40 mM TRIS, pH 7.5, 12 mM MgCl2, 2 mM DTT, 0.2 mM CaCl2, or 4 mM EGTA) for 30 min. Phosphorylation was performed for 1 h at room temperature in 90 ml of the same reaction buffer with 200 nM ATP plus 50 lCi of [c-32P]ATP (3000 Ci mmol 1). The reaction was halted by transferring the gel into a solution with 5% (w/v) trichloroacetic acid (TCA) and l% (w/v) sodium pyrophosphate. The unincorporated [c-32P]ATP was removed by cleansing in the same solution for at least an hour with two changes. The gel was dried and exposed to Kodak BioMax MR Film. Prestained size markers (BioRad, Hercules, CA, USA) were used to calculate the size of kinases. All the experiments on kinase assay were repeated at least twice. Detection of ROS and Ca2+ in rice roots Five-day-old rice seedlings were used to localize the generation of ROS and Ca2+ in rice roots. The rice roots were labelled with 10 lM 5-(and-6)-chloromethyl-2#,7#-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA; Molecular Probes) and Oregon Green 488 BAPTA-1 (Molecular Probes) for 30 min and then treated with 400 lM CdCl2 and 100 lM CuCl2 for 15 min. A Leica MPS60 fluorescent microscope equipped with a green fluorescent protein filter (excitation 450–490 nm, emission 500–530 nm) was
used for fluorescence images. Exposure times were equal for all samples. Autofluorescence was not observed in unstained controls at the exposure time used. Images were captured with a CoolSNAP Cooled CCD Camera (CoolSNAP 5.0). NADPH oxidase activity assay The NADPH oxidase activity was determined by XTT assay and ingel activity assay. The XTT assay mixture of 1 ml contained 50 mM TRIS-HCl buffer (pH 7.5), 0.5 mM XTT, and 100 lM NADPH. The reaction was initiated with the addition of NADPH, and XTT reduction was determined at 470 nm (Jiang and Zhang, 2002). In-gel assay for NADPH oxidase activity was performed according to the procedures described previously (Sagi and Fluhr, 2001). The protein samples were electrophoretically separated on 10% (w/v) native polyacrylamide gels. After electrophoresis, gels were incubated in the darkness for 20 min in a reaction mixture solution containing 50 mM TRIS-HCl pH 7.4, 0.2 mM nitro blue tetrazolium (NBT), and 0.1 mM MgCl2. NADPH (0.2 mM) was added and the appearance of blue formazan bands was monitored. The reaction was halted by immersion of the gels in distilled water.
Results Cd2+- and Cu2+-induced 42 kDa MBP kinases are OsMPK3 and OsMPK6
Previous studies showed that Cd2+-induced 42 kDa MBP kinase has the characteristics of a MAP kinase (Yeh et al., 2004). To determine whether the Cu2+-induced 40 kDa and 42 kDa MBP kinase activities also correlated with the activation of rice MAP kinases, western blot analysis with an anti-phospho-ERK antibody was performed. Rice roots were exposed to Cd2+ and Cu2+ at varying concentrations ranging from 0 lM to 400 lM and from 0 lM to 100 lM, respectively. As the data show in Fig. 1A, exposure of rice roots to Cd2+ and Cu2+ was associated with a dosedependent increase in the phosphorylation level of two protein bands with estimated molecular masses of 40 kDa and 42 kDa. Immunoprecipitation followed by an in-gel kinase assay was further performed. Rice roots were treated with or without 400 lM Cd2+ and 100 lM Cu2+ for 1 h. Rice root protein extracts were subjected to immunoprecipitation with anti-ERK1, an antibody that reacts with several ERK isoforms in mammals, and tested for MBP phosphorylation in an in-gel kinase assay. The 40 kDa and 42 kDa MBP kinase activities were observed in anti-ERK immunoprecipitates of protein extracts prepared from Cd2+- and Cu2+-treated roots, but not in protein extracts of untreated roots (Fig. 1B). The data suggest that the Cd2+-induced 40 kDa MBP kinase and Cu2+-induced 40 kDa and 42 kDa MBP kinases are also rice MAP kinases. OsMPK3 (also known as OsMAPK2; Huang et al., 2002) and OsMPK6 are the orthologues of Arabidopsis AtMPK3 and AtMPK6, which are classical stress-inducible MAP kinases. Correlation of Cd- or Cuinduced MAP kinase activity with the activation of OsMPK3 or OsMPK6 was determined. Rice root protein extracts were immunoprecipitated with the anti-OsMPK3
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no. CAC13967) and OsMPK6 (GenBank protein accession no. Q84UI5), respectively, were produced synthetically and conjugated to the keyhole limpet haemacyanin (KLH) carrier. The OsMPK3 and OsMPK6 polyclonal antisera were raised in rabbits. A 300 lg protein extract (in 300 ll) was incubated with antiERK antibodies (diluted 1:500, v/v) or a 100 lg protein extract (in 100 ll) was incubated with anti-OsMPK3 or anti-OsMPK6 antibodies (diluted 1:100, v/v) overnight at 4 C. The agarose bead– protein complexes were pelleted by centrifugation (2 min, 500 g) and washed three times in 0.5 ml of buffer [20 mM TRIS, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 1 mM NaF, 10 mM b-glycerophosphate, 2 lg ml 1 antipain, 2 lg ml 1 aprotinin, 2 lg ml 1 leupeptin, 0.5% (v/v) Triton X-100, 0.5% (v/v) Nonidet P-40]. After washing with the above buffer, SDS sample buffer (1 : 1, v/v) was added and boiled for 10 min at 70 C. After centrifugation, the supernatant fraction was electrophoretically separated on a 10% (w/v) SDS-polyacrylamide gel embedded with 0.25 mg ml 1 of myelin basic protein (MBP) in the separating gel as a substrate, and the in-gel kinase assay was performed.
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By contrast, relatively lower levels of DCF fluorescence were produced in the control roots (Fig. 2). These results suggest that Cd2+ and Cu2+ induce ROS production in rice root tips. Cd2+ and Cu2+ induce redox-dependent MAP kinase activation through distinct ROS-generating systems in rice roots
ROS production in Cd2+- and Cu2+-treated rice roots
Cd2+ and Cu2+ induce Ca2+ accumulation in rice roots
To determine whether Cd2+ and Cu2+ treatment induces ROS production, a ROS-sensitive dye, CM-H2DCFDA, was used. This compound is non-fluorescent but is rapidly oxidized to the highly fluorescent DCF by intracellular ROS. Rice roots were treated with 20 mM H2O2, 400 lM Cd2+, and 100 lM Cu2+ for 15 min. As shown in Fig. 2, the levels of DCF fluorescence increased significantly after H2O2, Cd2+, and Cu2+ treatments in root tip regions.
To investigate whether treatment with Cd2+ and Cu2+ induces Ca2+ accumulation in rice roots, a Ca2+ indicator, Oregon green 488 BAPTA-1, was used. Rice roots were treated with 500 lM Ca2+, 400 lM Cd2+, and 100 lM Cu2+ for 15 min. As shown in Fig. 5, the levels of green fluorescence were significantly increased after Ca2+, Cd2+, and Cu2+ treatments in root tip regions. In contrast, a relatively lower level of green fluorescence was
Fig. 1. Phosphorylation and immunoprecipitation of Cd2+- and Cu2+induced 40 kDa and 42 kDa kinases. Rice roots were treated with different concentrations of CdCl2 and CuCl2 for 1 h. Protein extracts of Cd- and Cu-treated rice roots were harvested. Western blot analysis (A) of rice root extracts was performed with anti-ERK and anti-phosphoERK antibodies. A 15 lg aliquot of proteins of each sample was loaded. Rice root protein extracts containing 300 lg or 100 lg of total proteins were used for immunoprecipitation (IP) with anti-ERK1 (B), antiOsMPK3 or anti-OsMPK6 (C) antibodies. The kinase activities of the immunoprecipitated proteins were subsequently assayed with an in-gel kinase assay using MBP as a substrate. MBP phosphorylation was visualized by autoradiography. Arrows indicate the kinase-active bands. The experiment was repeated twice with similar results.
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or anti-OsMPK6 antibodies, and the MBP kinase activity of the immunoprecipitated proteins was analysed by an ingel kinase assay. In-gel kinase assay of protein extracts of roots at 1 h after heavy metal treatment showed that the OsMPK3 and OsMPK6 antibodies had immunoprecipitated active kinases with an estimated molecular mass of 42 kDa (Fig. 1C). The data suggest that Cd2+- and Cu2+induced 42 kDa MAP kinase activities resulted at least from activation of OsMPK3 and OsMPK6.
To examine whether ROS is involved in Cd2+- and Cu2+induced MAP kinase activities, rice roots were pretreated with sodium benzoate, a hydroxyl radical scavenger, for an hour before heavy metal treatments and they were then incubated for an additional hour. As shown in Fig. 3A, sodium benzoate significantly reduced Cd2+-stimulated 40 kDa and 42 kDa MAP kinase activation. Whereas sodium benzoate slightly inhibited Cu2+-induced 40 kDa MAP kinase activity, it had no significant effect on Cu2+mediated 42 kDa MAP kinase activation. In addition, western blot analysis with an anti-phospho-ERK antibody was performed. The phosphorylation of two protein bands with molecular masses of 40 kDa and 42 kDa is similar to the activation of 40 kDa and 42 kDa MAP kinases by ingel kinase assay. To investigate Cd2+- and Cu2+-induced enzymatic sources of ROS, the effect of an NADPH oxidase inhibitor, DPI, on Cd2+- and Cu2+-treated rice roots was examined. DPI pretreatment significantly inhibited Cd2+induced 40 kDa and 42 kDa MAP kinase activation. However, DPI had no significant effect on the activation of MAP kinases by Cu2+ treatment. Western blot using anti-phospho-ERK antibodies was performed, and similar results were obtained (Fig. 3B). These results imply that Cd2+-induced signal transduction may involve NADPH oxidase, which produces endogenous ROS to stimulate the activation of MAP kinases. To confirm further that Cd2+ induces NADPH oxidase activity, alterations in the NADPH oxidase-like activities were investigated in response to 400 lM Cd2+ and 100 lM Cu2+ in rice roots. The NADPH oxidase-like activities were analysed by XTT assay and in-gel assay after Cd2+ and Cu2+ treatment. The NADPH oxidase-like activities were induced by Cd2+ treatment at 15 min. By contrast, NADPH oxidase-like activities were decreased by Cu treatment. The NADPH oxidase-like activities were inhibited by DPI, an NADPH oxidase inhibitor (Fig. 4).
Different pathways in MAP kinase activation by Cd and Cu 663
Fig. 3. ROS may be involved in the Cd2+- and Cu2+-induced MAP kinase activation. Rice roots were pretreated with or without 500 lM sodium benzoate (A), a hydroxyl radical scavenger, and 100 lM DPI (B), an NADPH oxidase inhibitor, for 1 h before 400 lM CdCl2 and 100 lM CuCl2 treatment, and incubated for an additional hour. A 5 lg aliquot of crude protein extracts was analysed by in-gel kinase assay (5 lg per lane) or western blot using anti-phospho-ERK or anti-ERK antibodies (15 lg per lane). MBP phosphorylation was visualized by autoradiography. Arrows indicate the kinase-active bands. The experiment was repeated three times with similar results.
produced in the control roots. These results suggest that Cd2+ and Cu2+ induce Ca2+ accumulation in rice root tips. Involvement of CDPK in the activation of MAP kinases by Cd2+ and Cu2+ The CDPK family is involved in many cellular responses that are triggered by elevated intracellular Ca2+ concentra-
Fig. 4. Cd2+ induces NADPH oxidase-like activities. Rice roots were pretreated with or without 100 lM DPI, an NADPH oxidase inhibitor, for 1 h before 400 lM CdCl2 and 100 lM CuCl2 treatments, and further incubated for 15 min. Protein extracts were analysed by XTT assay (A) or in-gel assay (B) for NADPH oxidase-like activities. The experiment was repeated three times with similar results. The data on NADPH oxidase-like activity (A) are means 6SE.
tion (Saijo et al., 2000; Martı´n and Busconi, 2001). A recent study has shown that CDPK can mediate stressinduced MAP kinase activation (Sangwan et al., 2002). Therefore, an experiment was conducted to examine whether Cd2+ and Cu2+ induce CDPK activation. In this experiment, protein extracts were separated on an SDSpolyacrylamide gel containing histone III-S as a substrate, and an in-gel kinase assay was performed. The data in
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Fig. 2. ROS production in rice roots during heavy metal stress. Rice roots were labelled with 10 lM CM-H2DCFDA for 30 min and then treated with 20 mM H2O2, 400 lM CdCl2, and 100 lM CuCl2 for 15 min. Green fluorescence indicates the presence of ROS. Ten control and 10 treated roots showed similar results. The magnification for all images was 3100.
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Fig. 6 suggest that 47 kDa and 49 kDa kinase activities were induced by Cd2+ and Cu2+. However, activation of both Cd2+- and Cu2+-induced 47 kDa and 49 kDa kinase was completely inhibited by a Ca2+ chelator, EGTA, and significantly inhibited by a calmodulin antagonist, W7, indicating that they are CDPKs (Fig. 6). The effect of W7 on Cd2+- and Cu2+-induced MAP kinase activation was examined further. Pretreatment with W7 for 1 h before Cd2+ and Cu2+ addition decreased the level of Cd2+- and Cu2+-induced 40 kDa and 42 kDa MAP kinase activation (Fig. 7). In addition, the phosphorylation of 40 kDa and 42 kDa proteins was similar to the activation of 40 kDa and 42 kDa MAP kinases by in-gel kinase assay. The data suggest that CDPKs may be involved in the activation of 40 kDa and 42 kDa MAP kinases by Cd2+ and Cu2+. PI3 kinase activities may be required for MAP kinase activation by Cd2+ and Cu2+ To examine whether PI3 kinase (phosphatidylinositol 3kinase) is involved in Cd2+- and Cu2+-induced MAP kinase activities, rice roots were pretreated for 1 h with wortmannin, an inhibitor of PI3 kinase, followed by Cd2+ and Cu2+ treatments for 1 h, and then an in-gel kinase assay was performed. As the data showed, the 40 kDa and 42 kDa MAP kinase activities were inhibited by wortmannin in Cd2+ and Cu2+ treatments (Fig. 8A). Closing of the mitochondrial permeability transition pore disrupts activation of Cd2+-induced MAP kinase
To investigate whether mitochondria are involved in Cd2+- and Cu2+-induced signalling pathways, an inhibitor of mitochondrial PT pore opening, BK, was used. Pretreatment with 50 lM BK for 1 h before Cd2+ addition
decreased the level of Cd2+-induced 40 kDa and 42 kDa MAP kinase activation (Fig. 8B). However, BK had no significant effect on the activation of 40 kDa and 42 kDa MAP kinase by Cu2+ treatment. The data suggest that Cd2+-induced MAP kinase activities depend on the functional state of mitochondria and that Cd2+ may cause mitochondrial dysfunction. Heterotrimeric G protein a subunit does not mediate Cd2+- and Cu2+-induced signalling pathways
The rice dwarf mutant T65d1 (Taichung 65-Daikoku dwarf) is defective in the a subunit of the heterotrimeric G protein (Ueguchi-Tanaka et al., 2000). To determine if Ga protein plays a role in heavy metal stress-induced MAP kinase activation, the rice Ga protein mutant T65d1 was treated with Cd2+ and Cu2+ for 1 h, and an in-gel kinase assay was performed. As shown in Fig. 9, no significant differences in MAP kinase activities were observed between the wild type (Taichung 65, T65) and the Ga protein mutant (T65d1). These results suggest that Cd2+- and Cu2+-induced signalling pathways may be independent of the Ga-mediated signalling pathway. Activation of MAP kinase, ROS production, and Ca2+ accumulation in Cd2+-tolerant and Cd2+-sensitive cultivars
Hsu and Kao (2003) reported that rice seedlings of cultivar Tainung 67 (TNG67) are more tolerant to Cd2+ than those of cultivar Taichung Native 1 (TN1). Here, it was tested whether the levels of MAP kinase activities are different between TNG67 and TN1. Rice roots were treated with 100–400 lM Cd2+ for 1 h, and the protein extracts were collected for an in-gel kinase assay. On treatment with Cd2+, 42 kDa MAP kinase activities are
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Fig. 5. Ca2+ accumulation in rice roots during heavy metal stress. Rice roots were labelled with 10 lM Oregon Green 488 BAPTA-1 for 30 min and then treated with 500 lM CaCl2, 400 lM CdCl2, and 100 lM CuCl2 for 10 min. Green fluorescence indicates the presence of Ca2+. Ten control and 10 treated roots showed similar results. The magnification for all images was 3100.
Different pathways in MAP kinase activation by Cd and Cu 665
Fig. 6. Cd2+ and Cu2+ induce CDPK activities in rice roots. Rice roots were treated with or without 400 lM CdCl2 or 100 lM CuCl2 for 1 h. A 5 lg aliquot of crude protein extracts was analysed by in-gel kinase assay using histone III-S as a substrate. The in-gel kinase assay was performed in the presence of either 0.2 mM CaCl2 or 4 mM EGTA in kinase reaction buffer. Histone III-S phosphorylation was visualized by autoradiography. Arrows indicate the kinase-active bands. The experiment was repeated twice with similar results.
higher in TNG67 than in TN1 (Fig. 10). The results suggest that 42 kDa MAP kinases may play an important role in Cd tolerance in rice. In addition, it was found that ROS production and Ca2+ accumulation in response to Cd2+ treatment in TNG67 was more evident than in TN1 (Fig. 11). Discussion Heavy metal-induced MAP kinase gene expression (Agrawal et al., 2002, 2003a; Yeh et al., 2003, 2004; Hung et al., 2005) and MAP kinase activation (Yeh et al., 2003, 2004; Lin et al., 2005; Tsai and Huang, 2006) have been reported in rice. In this study, it is demonstrated that Cd2+ and Cu2+ activated, at least, three different MAP kinases, including OsMPK3, OsMPK6, and 40 kDa MAP kinase, in rice. Exposure of alfalfa (Medicago sativa) seedlings to excess Cd2+ and Cu2+ activated four distinct MAP kinases: SAMK, SIMK, MMK2, and MMK3 (Jonak et al., 2004). SAMK and SIMK are the orthologues of rice OsMPK3 and OsMPK6, respectively. Due to the fact that MAP kinase pathways are highly conserved in plants, Cd2+- and Cu2+-induced 40 kDa and 42 kDa MAP kinase activities might also contain the activities of rice orthologues of MMK2 and MMK3. However, the possibility
Fig. 8. Effects of a PI3 kinase inhibitor and a mitochondrial PT pore opening blocker on Cd2+-and Cu2+-induced MAP kinase activation. Rice roots were pretreated with or without 10 lM wortmannin (A), a PI3 kinase inhibitor, or 50 lM BK (B), a mitochondrial PT pore opening blocker, for 1 h before 400 lM CdCl2 and 100 lM CuCl2 treatments, and further incubated for 1 h. A 5 lg aliquot of crude protein extracts was analysed by in-gel kinase assay. MBP phosphorylation was visualized by autoradiography. Arrows indicate the kinaseactive bands. The experiment was repeated three times with similar results.
cannot be excluded that the 40 kDa MAP kinase might also represent a post-translationally modified form of the 42 kDa MAP kinase. Droillard et al. (2002) reported that an Arabidopsis MAP kinase, AtMPK6, is activated by hypo- and hyperosmotic stresses, as well as its tobacco orthologue SIPK. However, AtMPK3 is activated by hypoosmotic stress as well as its tobacco orthologue WIPK. Therefore, the results imply that sequence similarities correlate with identical functions. Differential activation of rice MAP kinases depends on different types of ROS in response to Cd2+ and Cu2+
ROS include superoxide, singlet O2, H2O2, and the highly reactive hydroxyl radical. Intercellular levels of ROS are influenced by a number of endogenous and exogenous processes, and controlled by several radical scavenging enzymes (Adler et al., 1999). Elevated levels of heavy metals and various other environmental stresses accelerate the formation of ROS. Redox-active metals (e.g. Cu2+ and Fe2+) catalyse the formation of hydroxyl radicals by directly participating in the Haber–Weiss reaction, and
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Fig. 7. CDPKs play a role in Cd2+- and Cu2+-induced MAP kinase activities. Rice roots were pretreated with or without 100 lM W7, a CDPK antagonist, for 1 h before being exposed to 400 lM CdCl2 and 100 lM CuCl2, and further incubated for 1 h. A 5 lg aliquot of crude protein extracts was analysed by in-gel kinase assay (5 lg per lane) or western blot using anti-phospho-ERK or anti-ERK antibodies (15 lg per lane). MBP phosphorylation was visualized by autoradiography. Arrows indicate the kinase-active bands. The experiment was repeated three times with similar results.
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2+
2+
Fig. 10. Activation of MAP kinase in Cd -tolerant and Cd -sensitive cultivars. Rice seedlings of the Cd2+-tolerant cultivar Tainung 67 (TNG67) and Cd2+-sensitive cultivar Taichung Native 1 (TN1) were treated with different concentrations of CdCl2 and CuCl2 for 1 h. A 5 lg aliquot of crude protein extracts was analysed by in-gel kinase assay. MBP phosphorylation was visualized by autoradiography. Arrows indicate the kinase-active bands. The experiment was repeated twice with similar results.
metals without redox capacity (e.g. Cd2+, Pb2+, Hg2+, and Zn2+) disturb metabolism by binding to functional groups, eventually enhancing the load of ROS (Schu¨tzendu¨bel et al., 2001; Babu et al., 2003; Olmos et al., 2003; Jonak et al., 2004; Tripathi and Gaur, 2004). Evidence for activation of MAP kinase by ROS has been provided by several studies in different plant species (Kovtun et al., 2000; Yuasa et al., 2001; Jonak et al., 2004). In addition, ROS are involved in heavy metal-induced MAP kinase activation in mammals (Seo et al., 2001; Rockwell et al., 2004) and alfalfa (Jonak et al., 2004). Previous studies showed that Cd2+-induced activation of 40 kDa, but not 42 kDa, MAP kinase was inhibited by the antioxidant glutathione (GSH) in rice. In contrast, GSH strongly inhibited Cu2+-induced 42 kDa MAP kinase activation (Yeh et al., 2003, 2004). In this study, sodium benzoate, a ROS scavenger, significantly reduced Cd2+-stimulated 40 kDa and 42 kDa MAP kinase activities; however, it only inhibited activation of 40 kDa MAP kinase due to Cu2+ treatment. Differential activation of ERK1/2, JNK, and p38 MAP kinase depending on different types of ROS in response to UVB exposure and angiotensin II treatment
Fig. 11. ROS production and Ca2+ accumulation in Cd2+-tolerant and Cd2+-sensitive cultivars. Rice roots of the Cd2+-tolerant cultivar (TNG67) and Cd2+-sensitive cultivar (TN1) were labelled with 10 lM CM-H2DCF-DA (A) or Oregon Green 488 BAPTA-1 (B) for 30 min and then treated with 400 lM CdCl2 for 15 min. Green fluorescence indicates the presence of ROS or Ca2+. Ten treated TNG67 and TN1 roots showed similar results. The magnification for all images was 3100.
has been reported (Peus et al., 1999; Kyaw et al., 2001). Therefore, the finding that sodium benzoate and GSH differentially inhibit Cd2+- and Cu2+-induced 40 kDa and 42 kDa MAP kinase activation indicates that the regulation of these pathways by ROS is distinct, and their responsiveness may differ depending on the types of ROS involved. However, benzoic acid appears as a likely intermediate in salicylic acid (SA) biosynthesis in planta. At least in tobacco and in cucumber, the biosynthetic pathway of SA was proposed to proceed exclusively via free benzoic acid (Yalpani et al., 1993; Meuwly et al., 1995). Chong et al. (2001) reported that free benzoic acid is probably not the major intermediate in SA biosynthesis, which may involve conjugated forms of benzoic acid. This makes it possible that exogenously applied sodium benzoate in rice roots may be converted to SA. In addition, it is demonstrated that SA caused protection against Cd2+ toxicity in barley (Metwally et al., 2003). Therefore, an investiation was carried out as to whether sodium benzoate application activated SA signalling cascades by increasing SA production in the present case. The results showed that SA, but not sodium benzoate,
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Fig. 9. Ga is not involved in Cd2+-and Cu2+-induced 40 kDa and 42 kDa MAPK activation. Rice cultivar T65 and Ga protein mutant, T65d1 seedlings were treated with 400 lM CdCl2 (A) and 100 lM CuCl2 (B) for 1 h. A 5 lg aliquot of crude protein extracts was analysed by in-gel kinase assay. MBP phosphorylation was visualized by autoradiography. Arrows indicate the kinase-active bands. The experiment was repeated twice with similar results.
Different pathways in MAP kinase activation by Cd and Cu 667
Calcium and CDPK are required for MAP kinase activation Several studies reported that the cytoplasmic Ca2+ concentrations in plant cells increase rapidly in response to multiple stress stimuli (Saijo et al., 2000; Martı´n and Busconi, 2001). Following this Ca2+ influx, signals are likely to be mediated by combinations of protein phosphorylation/dephosphorylation cascades. It is presumed that the majority of Ca2+-stimulated protein
phosphorylation is performed predominantly by CDPKs in plants (Saijo et al., 2000). CDPKs are a large family of Ser/Thr protein kinases regulated by Ca2+, and are biochemically distinct from other Ca2+-regulated protein kinases such as protein kinase C and Ca2+/calmodulindependent protein kinases (Harper et al., 1991; Martı´n and Busconi, 2001). Sheen (1996) has reported that ATCDPK1 and ATCDPK1a may be positive regulators controlling stress signal transduction in Arabidopsis. Martı´n and Busconi (2001) found a 56 kDa rice membrane-bound CDPK whose kinase and autophosphorylating activities increased after several hours of cold treatment. The activation of SAMK by cold and of HAMK by heat requires CDPK activity (Sangwan et al., 2002). In this study, involvement of multiple stressinducible CDPKs in the heavy metal stress signal transduction pathway was examined. As shown in Fig. 6, the 47 kDa and 49 kDa kinase activities increased with Cd2+ and Cu2+ treatment. In addition, W7, a CDPK antagonist, significantly inhibited the Cd2+- and Cu2+-induced 40 kDa and 42 kDa MAP kinase activation (Fig. 7). These data suggest that heavy metal-induced MAP kinase activities may occur through the action of CDPKs. Involvement of PI3 kinase in the activation of MAP kinases by Cd and Cu
Phosphatidylinositol (PI) metabolism plays a central role in signalling pathways in both animals and higher plants (Jung et al., 2002). PI3P (phosphatidylinositol 3-phosphate) is a product of PI3 kinase, which phosphorylates the D-3 position of phosphoinositides (Park et al., 2003). Eom et al. (2001) reported that Zn2+ stimulates the JNK pathway and PI3 kinase in mouse primary cortical cells and in various cell lines. The Zn2+-induced JNK stimulation was blocked by the PI3 kinase inhibitor or by a dominant-negative mutant of PI3 kinase c. Rockwell et al. (2004) suggested that inhibition of PI3 kinase attenuated Cd2+-induced ROS and p38 MAP kinase activation. In this study, pretreatment of rice roots with wortmannin, an inhibitor of PI3 kinase, for 1 h before Cd2+ and Cu2+ treatments and subsequent in-gel analysis suggests that PI3 kinase activities may be required for Cd2+- and Cu2+-induced MAP kinase activation. Therefore, it is hypothesized that PI3P, as found in animal cells, activates plant MAP kinases during heavy metal stresses. Cd2+-induced MAP kinase activities depend on the functional state of mitochondria Mitochondrial damage and apoptosis induced by Cd2+ has been well established in animal systems. Li et al. (2003) reported that Cd2+ directly leads to the dysfunction of mitochondria, including the inhibition of respiration, the loss of transmembrane potential, and the opening of the PT pore. Opening of the PT pore is a possible mechanism
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induced MAP kinase activation. In addition, sodium benzoate significantly inhibited Cd- and Cu-induced DCF fluorescence (data not shown). These results suggest that sodium benzoate indeed scavenges ROS and the effect of sodium benzoate on Cd- and Cu-induced MAP kinase activation may be independent of SA signalling cascades. In addition, the enzymatic sources of ROS, important for MAPK activation, differ in Cd2+- and Cu2+-treated rice roots. One of the mechanisms by which ROS are produced is through the activation of a membrane-associated NADPH oxidase (Agrawal et al., 2003b). Superoxide anion (O2 ) production by NADPH oxidase and its subsequent conversion to H2O2 has been well characterized as a source of oxidative stress that causes apoptosis and cell death in phagocytic cells in a mechanism that can involve PI3 kinase and p38 MAP kinase (Rockwell et al., 2004). Rockwell et al. (2004) examined the effects of the NADPH oxidase inhibitor DPI on the ROS production and the phosphorylation of p38 MAP kinase induced by Cd2+ in mouse neuronal cells. The findings implicate NADPH involvement in Cd2+-induced p38 MAP kinase activation and oxidative stress. Olmos et al. (2003) reported that DPI prevented the generation of H2O2 induced by Cd2+ in cultured tobacco (Nicotiana tabacum L.) BY-2 cells. Furthermore, as rice MAP kinases induced by ROS (H2O2) have recently been identified, it is thought that ROS produced due to activation of NADPH oxidase by OsRac1 may itself activate a MAP kinase cascade associated with disease resistance (Agrawal et al., 2003b). In this study, pretreatment with DPI decreased the level of Cd2+-, but not Cu2+-, induced 40 kDa and 42 kDa MAP kinase activation. In addition, Cd2+ activated NADPH oxidase-like activities. These results imply that redoxdependent activation of MAP kinase by Cd2+ and Cu2+ occurs through distinct ROS-generating systems and that these processes may contribute to differential redoxsensitive signalling by Cd2+ and Cu2+. Compared with Cdinduced ROS production, however, Cd only slightly induced NADPH oxidase-like activities. Beside NADPH oxidase, there are several potential enzymatic sources of ROS including xanthine oxidase, amine oxidase, and a cell wall peroxidase (Neill et al., 2002). Therefore, the majority of Cd2+-induced ROS production may be NADPH oxidase independent and result from other enzymatic sources.
668 Yeh et al.
Cd2+- and Cu2+-induced MAP kinase pathways may be independent of the Ga-mediated signalling pathway Nishida et al. (2000) showed that inhibition of the bcsubunit of G protein (Gbc) attenuates hydrogen peroxide (H2O2)-induced ERK activation in rat neonatal cardiomyocytes. Analysis using heterotrimeric Go and its individual subunits indicates that H2O2 modifies Gao but not Gbc, which leads to subunit dissociation. They concluded that Gai and Gao are critical targets of oxidative stress for activation of ERK. It has been proposed that the plant heterotrimeric G protein is involved in various signal transduction systems, including those of several plant hormones, blue and red lightmediated responses, pathogen resistance, and pathogenrelated gene expression (Ueguchi-Tanaka et al., 2000; Fujisawa et al., 2001). Genomic and cDNA clones that encode polypeptides similar to the mammalian Ga or Gb proteins have been isolated from various plant species, including model plants, Arabidopsis and rice. Available data reported thus far suggest the presence of a single gene for either the a subunit or the b subunit in each genome of Arabidopsis and rice. Thus an examination was carried out of whether the a subunit of the heterotrimeric G protein is the target protein of heavy metal-induced ROS for MAP kinase activation in rice. No
significant differences for 40 kDa and 42 kDa MAP kinase activities were observed between the wild type (T65) and Ga protein mutant (T65d1). These results suggest that Cd2+- and Cu2+-induced 40 kDa and 42 kDa MAP kinase activation may be independent of the Gamediated signalling pathway. Cd2+- and Cu2+-activated MAP kinases may regulate heavy metal stress tolerance Previous studies showed that the expression of a rice MAP kinase gene, OsMPK3, is inducible by cold, salinity, Cu2+, and Cd2+ (Huang et al., 2002; Yeh et al., 2003, 2004). The deduced amino acid sequence of OsMPK3 comprises 369 amino acid residues with an estimated molecular mass of 42 kDa (Huang et al., 2002). Its amino acid sequence is identical to that of OsMSRMK2 (Agrawal et al., 2002), OsMAP1 (Wen et al., 2002), OsBIMK1 (Song and Goodman, 2002), and OsMAPK5 (Xiong and Yang, 2003). In the above studies, the OsMAPK genes were also induced by various biotic and abiotic stresses and were suggested to play a role in stress signal transduction. Xiong and Yang (2003) further generated and analysed transgenic rice plants with overexpression (using the cauliflower mosaic virus 35S promoter) and suppression (using double-stranded RNA interference; RNAi) of OsMAPK5. According to their data, activation of OsMAPK5 indeed provided abiotic stress tolerance to the rice plant. To confirm the role of OsMPK3 in heavy metal-dependent signal transduction further, two rice cultivars, TNG67 and TN1, were used to analyse 42 kDa MAP kinase activity. Hsu and Kao (2003) have reported that the TNG67 (cv. Tainung 67) rice plant is a Cd2+-tolerant cultivar and the TN1 (cv. Taichung Native 1) rice plant is a Cd2+-sensitive cultivar. In this study, it was found that enhancement of 42 kDa MAP kinase activity of the Cd2+-sensitive cultivar (TN1) caused by Cd2+ was less than that of the Cd2+-tolerant cultivar (TNG67). Furthermore, ROS production and Ca2+ accumulation were more evident after Cd2+ treatment in TNG67 than in TN1. Apparently, ROS and Ca2+ were rapidly and greatly accumulated in TNG67 when the roots were treated with Cd2+, and the accumulating ROS and Ca2+ subsequently activate 42 kDa MAP kinase. The activation of Cd2+-induced 42 kDa MAP kinase may provide Cd2+ tolerance to rice plants. Moreover, Hsu and Kao (2003) found that the endogenous abscisic acid (ABA) content rapidly increased in the leaves and roots of the Cd2+-tolerant cultivar (TNG67) but not in the Cd2+sensitive cultivar (TN1). Knetsch et al. (1996) and Burnett et al. (2000) reported that ABA induced a rapid and transient MAP kinase activation in barley aleurone protoplasts and pea leaves, respectively. Thus, low levels of 42 kDa MAP kinase activity of TN1 may also result from low levels of ABA content in TN1. Future work will
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whereby cytochrome c and other apoptosis-triggering factors are released into the cytosol (Maxwell et al., 2002). This mitochondrial damage is possibly involved in apoptosis induced by Cd2+. Takahashi et al. (2003) demonstrated that the closing of the mitochondrial PT pore by BK, an inhibitor of the PT pore opening directly via an adenine nucleotide translocator, disrupts activation of MAP kinases induced by spermine. Therefore, involvement of mitochondrial dysfunction in Cd2+-induced MAP kinase activation in rice roots was tested. For this purpose, the effect of BK on MAP kinase activity of Cd2+-treated rice roots was tested. As the data show in Fig. 8B, BK greatly inhibited the activation of 40 kDa and 42 kDa MAP kinase by Cd2+ treatment. The data suggest that mitochondria PT pore opening may be required for Cd2+-induced signalling pathways. The role of mitochondria during Cd2+ stress was reported. Finkemeier et al. (2005) showed that disruption of mitochondrial antioxidant metabolism greatly enhances the Cd2+ sensitivity of root growth in Arabidopsis thaliana. However, pharmacological experiments should always be interpreted with caution. In particular, non-specific effects are always possible, particularly if reagents are used at relatively high concentrations. The I50 values in the rice root system for the effects of the various inhibitors on the signalling pathway were all within the concentration ranges previously employed in signal transduction studies in plant or mammalian cells.
Different pathways in MAP kinase activation by Cd and Cu 669 Cd2+
Cu2+ plasma membrane
NADPH oxidase
ROS
CDPK, PI3K
?
ROS CDPK, PI3K
mitochondria
MAP kinase activation
MAP kinase activation
Fig. 12. Schematic representation of heavy metal-induced signal transduction in rice. See text for details.
Acknowledgements We are most grateful to Professor Toshio Murashige for critical reading of the manuscript. We also wish to thank Professor Makoto Matsuoka and Professor Motoyuki Ashikari for providing the rice seeds of T65 and T65d1, and Dr Chang-Sheng Wang for providing rice seeds of TNG67 and TN1. We thank Ms Lorraine Rooney for assistance with correction of the English of this manuscript. This work was supported by a grant from The National Science Council of the Republic of China (NSC 94-2311-B-006-003).
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