Protein tyrosine dephosphorylation during copper-induced cell death in rice roots

Chemosphere 69 (2007) 55–62 www.elsevier.com/locate/chemosphere

Protein tyrosine dephosphorylation during copper-induced cell death in rice roots Wan-Chi Hung 1, Dinq-Ding Huang 1, Pei-Shan Chien, Chuan-Ming Yeh, Po-Yu Chen, Wen-Chang Chi, Hao-Jen Huang * Department of Life Sciences, National Cheng Kung University, No. 1 University Road, 701 Tainan, Taiwan Received 8 September 2006; received in revised form 17 April 2007; accepted 25 April 2007 Available online 21 June 2007

Abstract Early signalling events that control the process of heavy metal-induced cell death are largely unknown in plants. In mammals protein tyrosine phosphorylation plays an important role in the activation of programmed cell death. We thus examined the involvement of tyrosine phosphorylation in Cu-induced rice cell death. This investigation demonstrates that Cu induces cell death and DNA fragmentation in rice root cells. In the presence of Cu, the level of phosphotyrosine accumulation declined in the band of 45 kDa, p45. To analyze the role of tyrosine dephosphorylation for the regulation of Cu-induced cell death more precisely, we increased levels of tyrosine phosphorylation using the protein tyrosine phosphatase inhibitor, sodium orthovanadate (Na3VO4). Treatment of rice roots with Na3VO4 blocked Cu-induced cell death and protein tyrosine dephosphorylation. In addition, the antioxidant GSH and the calcium chelator EGTA significantly abolished Cu-induced cell death and protein tyrosine dephosphorylation. These results provide evidence that dephosphorylation of a tyrosine-phosphorylated protein, p45, is an important step in the Cu-triggered signalling transduction pathway.  2007 Elsevier Ltd. All rights reserved. Keywords: Programmed cell death; Signalling; Tyrosine phosphorylation

1. Introduction Copper (Cu) is an essential element in plant nutrition, being required as a cofactor for electron transport proteins and a number of enzymes, thus playing a key role in metabolic and biosynthetic processes in higher plants (Jiang et al., 2000). However, at toxic concentrations Cu acts as an efficient generator of reactive oxygen species (ROS) via Harber–Weiss and Fenton reactions (Elstner et al.,

Abbreviations: EGTA, ethylene glycol bis-(beta-aminoethyl ether) N,N,N 0 ,N 0 -tetraacetic acid; GSH, glutathione; PCD, programmed cell death; ROS, reactive oxygen species. * Corresponding author. Tel.: +886 06 2757575x65534; fax: +886 06 2742583. E-mail address: [email protected] (H.-J. Huang). 1 These authors contributed equally to this work. 0045-6535/$ - see front matter  2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2007.04.073

1988). It has been reported that an excess of Cu produces oxidative stress in intact roots of Silene cucubalus (Verkleij et al., 1987), in leaf segments (Garcia et al., 1999), in intact leaves of Phaseolus vulgaris (Patsikka et al., 2002) and in rice roots (Yeh et al., 2007). ROS such as hydrogen peroxide (H2O2), superoxide (O2), and hydroxyl radical (OH) are known to be involved in PCD. Recent work has established that H2O2 can induce PCD in soybean and Arabidopsis suspension cultures and that H2O2 induces apoptotic DNA degradation in cultured tobacco BY-2 cells (Houot et al., 2001). Cell death following injury can occur by necrosis or PCD (Pennell and Lamb, 1997). PCD is a multi-step process and protein kinases have been implicated both in the induction phase and the execution stage (Cross et al., 2000). Protein tyrosine kinase activation initiates the signalling pathway early on to direct the program leading to cell death (Eischen et al., 1994; Yousefi et al., 1994; Anderson, 1997). In

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addition, increases in tyrosine phosphorylation leads to inhibition of cell death in human eosinophils and neutrophils (Yousefi et al., 1994). Also increased protein tyrosine phosphorylation in intestinal epithelial cell cultures decreases programmed cell death (Scheving et al., 1999). In plants, protein phosphorylation on tyrosine residues has been corrected with somatic embryogenesis (Barizza et al., 1999), mechanical stress (Kameyama et al., 2000) or cell proliferation (Huang et al., 2003). The first plant tyrosine phosphatase (AtPTP1) was recently isolated in Arabidopsis and shown to be regulated by salt stress (Xu et al., 1998). A dual-specificity protein phosphatase (AtDsPTP1) also cloned in Arabidopsis as involved in the regulation of MAPK signaling pathways (Gupta et al., 1998). More recently, five dual-specificity protein phosphatases are characterized in rice (Katou et al., 2007). However, there is no information of a role for tyrosine phosphorylation in the apoptotic form of cell death in plants. In this report, we examine tyrosine phosphorylation of Cu-induced rice cell death. The results show that a rapid reduction in tyrosine phosphorylation occurs in Cuinduced rice roots. Furthermore, experiments using tyrosine phosphatase inhibitor support the hypothesis that tyrosine phosphorylation plays a critical role in Cuinduced rice root cell death. 2. Material and methods 2.1. Plant material, growth and Cu exposure Rice (Oryza sativa cv. TN67) seed were surface-disinfested with 2.5%(v/v) sodium hypochlorite (Sigma, USA) for 15 min followed by thorough washing in distilled water, and placed in 9 cm Petri dish containing 25 ml of distilled water at 37 C in darkness. After 2 days of incubation, uniformly germinated seeds were selected and transferred to Petri dishes over filter paper discs (Whatman No. 1) moistened with 10 ml of distilled water. Each Petri dish contained 15 germinated seeds, and they were grown at 27 C in darkness for 3 days. Once roots reached 3–4 cm in length, they were used for CuCl2 exposure experiments under sterile conditions in the same Petri dish. CuCl2 was added to final concentrations between 25 and 100 lM for different treatment time (1/6, 2/6, 3/6, 1, 2, 3, 6 and 12 h). 2.2. Inhibitor treatments For inhibitor experiments, roots of rice seedling were pre-treated with 2 mM Na3VO4 (Sigma, USA), 200 lM GSH (Sigma, USA), or 2 mM EGTA (Sigma, USA) 1 h prior Cu application. Cell death was determined at 1 h after addition of 100 lM CuCl2. Mean root length were obtained from 15 individual seedling from at least 3 separate experiments. Controls were mock-treated with distilled water, as appropriate.

2.3. Analysis of cell death Cell death was quantified by Evans blue (Sigma, USA) staining method (Baker and Mock, 1994). Roots were harvested from ten randomly selected seedlings. The roots were stained in 0.25% aqueous Evans blue solution for 15 min at room temperature, and then washed twice for 15 min with distilled water to remove the excess stain. Finally, the roots were left in the distilled water overnight. For quantitative assessment, the extreme 5 mm of root tips were excised from 10 roots followed by the extraction of dye in a solution of 50% methanol/1% SDS for 1 h at 50 C and its subsequent quantification by monitoring the A595. 2.4. DNA isolation and electrophoresis Rice root tips (about 1 cm) were ground to a fine powder in liquid nitrogen. One ml of DNA extraction buffer containing 2% (w/v) cetyltrimethyl ammonium bromide (CTAB), 1.4 M NaCl, 0.1 M Tris–HCl (pH 8.0), 20 mM EDTA, and 1% (w/v) polyvinylpyrrolidone (K-30) was added to each ground sample, and then incubated for 25 min at 65 C. After adding 60 ll of 3 M sodium acetate, DNA was extracted with an equal volume of phenol/chloroform and centrifuged. The aqueous phase was transferred to a new tube, and an equal volume of isopropyl alcohol was added. DNA was washed with 70% cold ethanol, dried, resuspended in a 0.5 ml Tris–EDTA (TE) buffer supplemented with 100 lg ml1 RNase A, then incubated at 37 C for 30 min. After adding 60 ll of 3 M sodium acetate, DNA was purified by extraction with an equal volume of phenol/chloroform, and the aqueous phase was transferred to a new tube. To the aqueous phase was added 0.6 ml isopropyl alcohol mixture incubated at room temperature for 5 min and then at 20 C for 15 min. The DNA was precipitated by centrifugation for 15 min at approximately 12 000g. After washing with cold 70% ethanol, the quantity of DNA was determined by measuring absorbances at 260 and 280 nm. Ten micrograms of the purified DNA were subjected to electrophoresis on 2% (w/v) agarose gels and stained with 0.5 lg ml1 ethidium bromide. 2.5. Electrophoresis and western blot analysis Rice root cells were collected in eppendorf tubes and ground in buffer E (125 mM Tris–HCl PH 8.8, 1% (w/v) SDS, 10% (v/v) glycerol, 50 mM Na2S2O5) for two min at room temperature. When the mixture was homogeneous it was placed on ice immediately. The homogenates were warmed to room temperature (26 C) and centrifuged at 13 000g for 10 min. The supernatants were retained. Equal amounts of proteins were boiled and fractionated on a 4–12% SDS-polyacrylamide gel for 1 h. Electrophoresed samples were assessed by loading in double series per sample, one series being stained with Coomassie blue

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Fig. 1. The viability of rice roots was examined using an Evans blue staining assay. Rice roots were treated with CuCl2 (0, 25, 50 and 100 lM) for 3 h (a and b). For kinetic measurements, roots were exposured to 100 lM CuCl2, and viability after increasing times was determined (c and d). After incubation, a 0.25% Evans blue dye was added. Results are the mean ± SD.

(Bio-Rad, Hercules, CA, USA), and the other one further processed for immunoblotting. After electrophoresis, the gel was washed 15 min in transfer buffer (25 mM Tris, 192 mM glycin, 20% methanol) to remove SDS, and transblotted to nitrocellulose filter (Amersham, Buckinghamshire, UK) at 360 mA for 4 h at 4 C in a Transblot apparatus (Amersham, Buckinghamshire, UK). The filter was incubated for 14 h at 4 C, then 2 h at room temperature with blocking solution containing TBS (10 mM Tris–HCl pH 7.5, 100 mM NaCl) with 1% bovine serum albumin (BSA) (Sigma, USA) and 0.1% Tween-20 (Sigma, USA). The blot was then incubated with horseradish peroxidase-conjugated, anti-phosphotyrosine antibody RC20 (Transduction Labs, USA) at 1:2500 dilution in the blocking solution for 2 h while shaking at room temperature. After washing in TBS solution containing 0.05% Nonidet NP-40 (Sigma, USA) for 2 · 10 min plus 10 min in TBS, the immunoreactive proteins were incubated in ECL reagent (Amersham, Buckinghamshire, UK) for enhanced chemiluminescent detection of bands on Hyperfilm-ECL film (Amersham Buckinghamshire, UK) as described by the manufacturer. To determine the specificity of the phosphotyrosine antibody, the antibody was pre-incubated for 10 min with

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Fig. 2. Copper-induced DNA fragmentation in rice roots. DNA was extracted from rice roots incubated with 100 lM CuCl2 for 0, 1, 3, 6 and 12 h. DNA was separated on a 2% agarose gel and visualized by ethidium bromide staining. Arrows indicate possible DNA ladders. The number at the left indicates the relative migration of DNA length standards in base pairs (bp).

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et al., 2000; Kawai and Uchimiya, 2000). As shown in Fig. 1a and b, Cu reduced cell viability in a dose-dependent manner. The kinetics data in Fig. 1c and d indicate that root cell death induced by Cu was not apparent until after 20 min.

2 mM pure phosphotyrosine (Sigma, USA) in TBS-T. The experiments were repeated at least twice with the same results and one of them was presented. 3. Results

3.2. Detection of DNA fragmentation in copper-treated rice cells

3.1. Copper-induced cell death of rice cells The viability of rice root cells was examined using an Evans blue staining assay. Evans blue dye is excluded from viable cells that retain intact plasma membranes, whereas those with damaged membranes incorporated the dye (Asai CuCl2 (µM) 0

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DNA prepared from rice cells incubated with Cu and assayed by agarose gel electrophoresis revealed that the Cu treatment caused DNA fragmentation (Fig. 2). Thus, CuCl2 (µM)

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17 Fig. 3. Effects of copper on the pattern of protein tyrosine phosphorylation in a (a) dose- and (b) time-dependent manner in rice roots. (a) Western blotting of rice root-tip cells after cultured in CuCl2 (0, 25, 50 and 100 lM) for 3 h. (b) Roots were exposured to CuCl2 for 10 and 180 min. Roots were lysed and protein extract from each sample was subjected to SDS-PAGE and immunoblot with an anti-phosphotyrosine antibody. Arrowheads indicate the gradually degraded proteins. Numbers at left indicate molecular weight markers in kilodaltons. The results shown are representative of two separate experiments.

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the loss of root viability was accompanied by DNA fragmentation.

3.4. Protein tyrosine phosphatase inhibitor prevents copperinduced protein tyrosine dephosprylation and cell death

3.3. Effects of copper on protein tyrosine phosphorylation in rice roots

To determine whether tyrosine dephosphorylation was involved in Cu-induced cell death, we tested the effects of tyrosine phosphatase inhibitor, sodium orthovanadate. Rice roots treated with sodium orthovanadate in Cucontaining medium did not induce cell death or protein tyrosine dephosphorylation at p45 (Fig. 4a). These experiments suggest that protein tyrosine phosphorylation is involved in rice cell death.

To examine the effect of Cu on tyrosine phosphorylation, rice roots were stimulated with Cu, and phosphotyrosyl proteins were detected by Western blotting with anti-phosphotyrosine antibody, clone RC20. As shown in Fig. 3a, Cu treatment of rice roots caused decreases of tyrosine phosphorylation of a protein with molecular mass of approximately 45 kDa (here after referred to as P45) (Fig. 3a). When 2 mM phosphotyrosine was added to the immunodetection buffer, all of the bands disappeared (Fig. 3a), confirming that specific binding of antibody to phosphorylated tyrosine residues on proteins had occurred. Furthermore, a Cu treatment for 10 min resulted in marked reduction of signal intensities of P45 (Fig. 3b).

3.5. Effects of the presence of GSH and EGTA in the incubation medium on the copper-induced protein tyrosine dephosphorylation and cell death There is ample evidence that exposure of plants to an excess concentration of Cu results in oxidative injury (Hall, 2002). In plants, GSH is important as an antioxidant and

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redox buffer (Noctor, 2006). As shown in Fig. 4b, GSH retarded cell death as well as protein tyrosine dephosphorylation in Cu-treated root cells, indicating that toxicity is a kind of oxidative stress. Calcium ions have been implicated in many signalling pathways which involve the generation and perception of ROS (Neill et al., 2002). Thus, we also investigated whether Ca2+ ion fluxes were involved in Cu-mediate protein tyrosine dephosphorylation by addition of EGTA, an efficient Ca2+ chelator (Tsien, 1980), to the culture medium 30 min before Cu was added. As shown in Fig. 5, EGTA strongly inhibited protein tyrosine dephosphorylation and cell death by Cu.

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3.6. Inhibition of copper-induced DNA fragmentation by EGTA, GSH and Na3VO4 In order to know whether EGTA, GSH and Na3VO4 inhibit Cu-induced programmed cell death instead of 0.2

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Fig. 6. Effects of EGTA, GSH and Na3VO4 on copper-induced DNA fragmentation. EGTA, GSH and Na3VO4 were added to the rootculturing medium 1 h before the addition of 100 lM CuCl2. After the sample was incubated for 12 h, DNA was extracted from rice cells. DNA was separated on a 2% agarose gel and visualized by ethidium bromide staining. Arrows indicate possible DNA ladders. Lane M: the molecular weight marker. The number at the left indicates the relative migration of DNA length standards in base pairs (bp).

necrosis in rice roots, the effects of EGTA, GSH and Na3VO4 on Cu-induced DNA fragmentation was studied. As shown in Fig. 6, DNA fragmentation in root cells treated with Cu together with EGTA, GSH and Na3VO4 were much less than those in root cells treated with Cu alone.

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Fig. 5. Effects of EGTA on copper-induced cell death in rice root-tip cells and protein tyrosine phosphorylation patterns. Molecular weight markers are shown in left lane. Arrowheads on the right denote bands that changed their intensity with the various treatments. The results shown are representative of two separate experiments.

4. Discussion Programmed cell death (PCD), known as apoptosis, is an active process of cell death that occurs in a wide variety of biological systems. The main features of apoptosis in animal’s cells include cell shrinkage, membrane blebbing, chromatin condensation, margination, apoptotic body formation, and DNA fragmentation (Earnshaw, 1995). Some apoptotic features have been detected recently during cell death in plants, such as certain developmental conversions (Mittler and Lam, 1995; Wang et al., 1998; Jones, 2001) to encounter an adverse environment stress (Mittler et al., 1996; Wang et al., 1996; Katsuhara, 1997; Koukalova et al., 1997; Mittler et al., 1998; Navarre and Wolpert, 1999; De Jong et al., 2000; McCabe and Leaver, 2000; Houot et al., 2001) and senescence (Callard et al., 1996; Orza´ez and Granell, 1997; Yen and Yang, 1998; Kawai and Uchimiya, 2000). Rice seedling exposed to Cu rapidly accumulated Cu in their roots (Chen et al., 2004). Cu-induced apoptosis has been described in animals (Sheline et al., 2002). Although cadmium-induced changes in cell viability and DNA integrity have been described in tobacco suspension cultures (Fojtova et al., 2002), there is no report of programmed cell

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death in Cu-induced cell death of plants. Thus, we used Cu as an experimental tool to investigate PCD in rice cells. DNA fragmentation is the most widely evaluated criterion for PCD. Our finding shown that DNA ladder fragmentation, corresponding to multiples of around 180 bp had clearly occurred in Cu treated rice cells. Tyrosine phosphorylation plays an important role in cell signal transduction, regulation of growth, and development, and in control of normal and neoplastic cell growth in animals (Hunter, 1995). In plants, tyrosine phosphorylation is strongly involved in mediating physiological responses to environmental stimuli, regulation of plant embryogenesis and tissue differentiation (Barizza et al., 1999). A role of tyrosine phosphorylation has been recently studied in apoptotic form of cell death of animals in response to a variety of stimuli (Otani et al., 1993; Eischen et al., 1994; Yousefi et al., 1994; Hagar et al., 1997). However, there has been no previous information regarding tyrosine phosphorylation in plant cell death. We have now shown that Cu causes an activation of protein tyrosine phosphatase(s) and reduces the tyrosine phosphorylation level in rice root cells. To confirm whether the protein tyrosine dephosphorylation occurs during Cu-induced cell death, we showed that the protein tyrosine phosphatase inhibitor, Na3VO4, blocked Cu-induced cell death and caused alteration in the tyrosine phosphorylation pattern (Fig. 4a). In our previous papers, we have demonstrated that a tyrosine phosphorylation cascade was involved in the rice cell death (Shih et al., 2004). Isolation of protein tyrosine phosphatase cDNAs in Arabidopsis (Xu et al., 1998) and rice (Lin and Huang, unpublished data) point to the possibility that protein tyrosine phosphatase in plants might play an essential role in plant signalling mechanism. Here, our experiments suggest that protein tyrosine dephosphorylation is linked with copper-induced cell death. GSH plays several important roles in the defence of plants against environmental threats (Noctor, 2006). The metal-induced depletion of glutathione in plants by phytochelatin synthesis may therefore increase the susceptibility of cells to oxidative stress, especially in the case of the redox-cycling metal Cu. We have already demonstrated that GSH partially inhibited 42-kD MAPK activation (Yeh et al., 2003). Based on the observation of the suppressive effect of antioxidant, GSH, on Cu toxicity, we hypothesize that Cu-induced stress is a kind of oxidative stress. However, the reduced induction of protein tyrosine dephosphorylation with copper and GSH together could be an increased sequestration of copper. Oxidative stress increases the cellular Ca2+ levels (Qin et al., 1996). The rise in cellular Ca2+ levels corresponds to the combination of extracellular Ca2+ influx and the release from intracellular Ca2+ storage (Barkla and Pantoja, 1996). In soybean cells, a rapid influx of calcium ions, leading to PCD, is induced by the oxidative stress (Levine et al., 1996). Pourahmad and O’Brien (2000) showed that Cu2+ induced hepatocyte toxicity in rats was prevented by removing media Ca2+. Our findings suggest that calcium from external sources might be involved in the transduc-

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tion of the Cu signal that induces cell death and protein tyrosine dephosphorylation. However, other divalent cations have been reported that they have affinity for the calcium chelator, EGTA (Speizer et al., 1989). We thus can not exclude the possibility that the inhibitory effects of EGTA observed was caused by the interaction between EGTA and other divalent cations. Our findings suggest that a tyrosine phosphorylation cascade is involved in the Cu-induced cell death of rice roots. Seedling roots of rice are very useful for characterizing plant cell signaling components for cell death. Identification of the details of the signaling pathways of triggering the cell death in plants is expected to provide valuable information. Acknowledgements We are deeply grateful to Professor Toshio Murashige for critical reading of the manuscript. This work was supported by research grants from National Science Council (NSC 94-2311-B-006-003, NSC 95-2311-B-006-002) and the Ministry of Education of the Republic of China. References Anderson, P., 1997. Kinase cascades regulating entry into apoptosis. Microbiol. Mol. Biol. Rev. 61, 33–46. Asai, T., Stone, J.M., Heard, J.E., Kovtun, Y., Yorgey, P., 2000. Fumonisin B1-induced cell death in Arabidopsis protoplasts requires jasmonate-, ethylene-, and salicylate-dependent signaling pathways. Plant Cell 12, 1823–1836. Baker, C.J., Mock, N.M., 1994. An improved method for monitoring cell death in cell suspension and leaf disc assay using Evan’s blue. Plant Cell Tiss. Org. Cult. 39, 7–12. Barizza, E., Lo Schiavo, F., Terzi, M., Filippini, F., 1999. Evidence suggesting protein tyrosine phosphorylation in plants depends on the developmental conditions. FEBS Lett. 447, 191–194. Barkla, B.J., Pantoja, O., 1996. Physiology of ion transport across the tonoplast of higher plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47, 159–184. Callard, D., Axelos, M., Mazzolini, L., 1996. Novel molecular markers for late phases of the growth cycle of Arabidopsis thaliana cell-suspension cultures are expressed during organ senescence. Plant Physiol. 112, 705–715. Chen, C.T., Chen, T.H., Lo, K.F., Chiu, C.Y., 2004. Effects of proline on copper transport in rice seedlings under excess copper stress. Plant Sci. 166, 103–111. Cross, T.G., Scheel-Toellner, D., Henriquez, N.V., Deacon, E., Salmon, M., Lord, J.M., 2000. Serine/Threonine protein kinases and apoptosis. Exp. Cell Res. 256, 34–41. De Jong, A.J., Hoeberichts, F.A., Yakimova, E.T., Maximova, E., Woltering, E.J., 2000. Chemical-induced apoptotic cell death in tomato cells: involvement of caspase-like proteases. Planta 211, 656– 662. Earnshaw, W.C., 1995. Nuclear changes in apoptosis. Curr. Opin. Cell Biol. 7, 337–343. Eischen, C.M., Dick, C.J., Leibson, P.J., 1994. Tyrosine kinase activation provides an early and requisite signal for Fas-induced apoptosis. J. Immunol. 153, 1947–1954. Elstner, E.F., Schutz, W., Vogl, G., 1988. Cooperative stimulation by sulfite and crocidolite asbestos fibres of enzyme catalyzed production of reactive oxygen species. Arch. Toxicol. 62, 424–427.

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