H2O2 Is Involved in the Metallothionein-Mediated Rice Tolerance to Copper and Cadmium Toxicity

Cadmium (Cd) and excess copper (Cu) are toxic to plants, causing a wide range of deleterious effects including the formation of reactive oxygen species. Metallothioneins (MTs) may protect plant cells from heavy metal toxicity by chelating heavy metals via cysteine thiol groups. They may also function as antioxidants. The study investigated the relationship of H2O2 production and ricMT expression in rice radicles and rice suspension cells under Cu or Cd stress. The results showed that H2O2 production in the rice radicles increased before Cu-induced ricMT expression, and after Cd-induced ricMT expression. Rice suspension cells of sense- and antisense-ricMT transgenic lines were obtained by an Agrobacterium-mediated transformation. Overexpression of ricMT significantly decreased the death rate of rice cells, which was accompanied by blocked H2O2 accumulation in rice suspension cells subject to Cu and Cd stress. Our findings confirm that H2O2 is involved in the MT-mediated tolerance of Cu and Cd toxicity in rice.

responses of plants to metal toxicity, the role of H 2 O 2 in regulating metal-responsive protein expression in plants is still not completely understood.
Metallothioneins (MTs) are a class of low molecular weight, cysteine (Cys)-rich, metal-binding proteins. In animals, MTs are involved in maintaining the homeostasis of essential metals and metal detoxification, and have been implicated in a range of other physiological processes, including ROS scavenging and regulating cell growth and proliferation [9]. Plant MTs may protect cells against the toxic effects of heavy metals by chelating them via Cys thiol groups, and they are also proposed to function as antioxidants [3,10]. Note that plant MTs are induced by a variety of environmental stimuli including peroxides, drought, cold, salt, and heavy metal toxicity, and these stimuli are accompanied by the production of ROS [11][12][13][14][15]. Consequently, the increased MT expression in stressed plants may be important for ROS scavenging or signaling [16,17]. Although studies have attempted to determine the functional action of MTs in plants [12,15,[18][19][20], additional information of linking MTs to ROS in the response to heavy metal stress in plants is still needed.
Rice possesses more MT genes than other plant species that have been studied. The MT isoforms expressed in rice are classified into four types based on their Cys content and the organization of the Cys residues at their N-and C-termini [21]. Some rice MTs have been shown to be ROS scavengers [20,22]. In our previous study, proteomic evidence showed that a MT-like protein, called ricMT by Yu et al. [23] and OsMT2c or OsMT-I-2b by Zhou et al. [21], and a copper/zinc superoxide dismutase (CuZn-SOD) are Cu-responsive proteins in germinating rice seeds [24][25][26], and OsMT2c transcription was also induced in response to both Cu and H 2 O 2 [18], which suggests that H 2 O 2 and MTs are connected in rice under metal stress. To clarify the relationship between MTs and H 2 O 2 in rice under Cu and Cd stress, we investigated the H 2 O 2 production, ricMT and CuZn-SOD mRNA expression patterns in the radicles of germinating rice seeds under Cu and Cd stress, as well as Cd-and Cu-induced cell death and H 2 O 2 production in rice suspension cells of the wild-type (WT), and transgenic lines overexpressing and under expressing ricMT.

Effects of Cu and Cd on H 2 O 2 Production in Rice Radicles
To understand the effects of Cu and Cd on H 2 O 2 production in rice, we investigated the H 2 O 2 production in rice radicles by 2 ,7 -dichlorodihydrofluorescein diacetate (H 2 DCFDA) staining. Compared with the control and the treatment with the H 2 O 2 scavenger, Asc, the 12-h treatments with 100 µM Cu or 100 µM Cd significantly increased the H 2 O 2 production of radicles ( Figure 1a). When the H 2 O 2 concentrations were assayed spectrophotometrically, H 2 O 2 gradually increased during the first 12 h of 100 µM Cu exposure and then decreased slightly but remained higher than that of the control; however, the Cd-induced H 2 O 2 happened only after 12 h of 100 µM Cd exposure, which lagged behind that of Cu exposure (Figure 1b).

. Cu and Cd Up-Regulate the ricMT and CuZn-SOD Gene Expression in Rice Radicles
The temporal changes in the gene expression of ricMT and CuZn-SOD were analyzed in ric icles using quantitative RT-PCR. There was no significant difference in the expression leve der the control medium within 48 h (Figure 2a). The expression of ricMT and CuZn-SOD mRNA s significantly higher in rice radicles treated with 100 μM Cu or 100 μM Cd for 24 and 48 h than i control (Figure 2b,c). By contrast, Cd significantly up-regulated the mRNA levels of two protein der 6 and 12 h treatment, while Cu did not. Subsequently radicles from germinating rice seeds were incubated in 20 µM H 2 DCFDA for 20 min or were homogenized and the H 2 O 2 content assayed by spectrophotometry. Bar, 100 µm. Experiments were repeated at least three times with similar results. Values are means ± SE (n = 3) of three separate experiments. Means denoted by the same letter did not significantly differ at p < 0.05 according to Duncan's multiple range test.

Cu and Cd Up-Regulate the ricMT and CuZn-SOD Gene Expression in Rice Radicles
The temporal changes in the gene expression of ricMT and CuZn-SOD were analyzed in rice radicles using quantitative RT-PCR. There was no significant difference in the expression level under the control medium within 48 h (Figure 2a). The expression of ricMT and CuZn-SOD mRNA was significantly higher in rice radicles treated with 100 µM Cu or 100 µM Cd for 24 and 48 h than in the control (Figure 2b   Germinating rice embryos were treated with distilled water (control), 100 μM CuSO4 or 100 μM CdCl2 for various times (0, 6, 12, 24, and 48 h). Subsequently, radicles were isolated from the germinating seeds for gene analyses by quantitative RT-PCR. Values are means ± SE (n = 3) of three separate experiments. Means denoted by the same letter did not significantly differ at p < 0.05 according to Duncan's multiple range test.

ricMT Expression Improved Cu and Cd Tolerance of Rice Suspension Cells
To evaluate the roles of ricMT, we generated transgenic rice suspension cells expressing the full-length ricMT cDNA under control of the CaMV 35S promoter using Agrobacterium mediated transformation (Figure 3a,b). The expression of ricMT in transgenic lines (sense-ricMT lines ricMTS1 and ricMTS2, antisense-ricMT lines ricMTA1 and ricMTA2) and the wild-type (WT) was analyzed using semi-quantitative RT-PCR (Figure 3c). Rice cells of the sense-ricMT lines ricMTS2 and antisense-ricMT lines ricMTA2 were used for subsequent rice suspension cell experiments. Means denoted by the same letter did not significantly differ at p < 0.05 according to Duncan's multiple range test.

ricMT Expression Improved Cu and Cd Tolerance of Rice Suspension Cells
To evaluate the roles of ricMT, we generated transgenic rice suspension cells expressing the full-length ricMT cDNA under control of the CaMV 35S promoter using Agrobacterium mediated transformation (Figure 3a,b). The expression of ricMT in transgenic lines (sense-ricMT lines ricMTS1 and ricMTS2, antisense-ricMT lines ricMTA1 and ricMTA2) and the wild-type (WT) was analyzed using semi-quantitative RT-PCR ( Figure 3c). Rice cells of the sense-ricMT lines ricMTS2 and antisense-ricMT lines ricMTA2 were used for subsequent rice suspension cell experiments.   There was no significant difference in the growth rates of WT and transgenic cells in normal medium ( Figure 4a). When rice cell cultured on medium supplemented with 100 µM Cu or 100 µM Cd, there was higher cell death rate than that of normal medium. In comparison with the WT, the sense-ricMT line (ricMTS2) had a decreased rate of cell death after 6 h Cu treatment or after 12 h Cd treatment; by contrast, the antisense-ricMT line (ricMTA2) showed an increased cell death rate at 12 and 24 h of Cu treatment or after 6 h of Cd treatment (Figure 4b

ricMT Expression Decreased H2O2 Production in Rice Suspension Cells under Cu and Cd Stress
To understand the role of ricMT in antioxidant protection, H2O2 production in rice suspension cells was detected by H2DCFDA staining. There was no significant difference in H2O2 production between WT and transgenic cells in normal medium ( Figure 5). In comparison with the control solution, when cultured in medium supplemented with 100 μM Cu or 100 μM Cd for 24 h, the H2O2 production increased significantly in the rice suspension cells. In comparison with the WT, when cultured in medium supplemented with 100 μM Cu or 100 μM Cd for 24 h, the sense-ricMT line (ricMTS2) showed decreased H2O2 production, while the antisense-ricMT line (ricMTA2) showed increased H2O2 production.

ricMT Expression Decreased H 2 O 2 Production in Rice Suspension Cells under Cu and Cd Stress
To understand the role of ricMT in antioxidant protection, H 2 O 2 production in rice suspension cells was detected by H 2 DCFDA staining. There was no significant difference in H 2 O 2 production between WT and transgenic cells in normal medium ( Figure 5). In comparison with the control solution, when cultured in medium supplemented with 100 µM Cu or 100 µM Cd for 24 h, the H 2 O 2 production increased significantly in the rice suspension cells. In comparison with the WT, when cultured in medium supplemented with 100 µM Cu or 100 µM Cd for 24 h, the sense-ricMT line (ricMTS2) showed decreased H 2 O 2 production, while the antisense-ricMT line (ricMTA2) showed increased H 2 O 2 production.

Control solution
Solution with Cu Solution with Cd

Discussion
Numerous studies have shown that heavy metals can induce the formation of ROS, including H2O2, and cause oxidative stress. Cu and Cd toxicity causes an oxidative burst with rapid H2O2 production and its release into the plant apoplast [27][28][29][30]. In this study, the formation of H2O2 was observed in Cu-or Cd-treated rice radicles and cells (Figures 1,5). Since H2O2 is relatively stable and can diffuse through cell membranes, it can modulate gene expression and participate in various physiological processes [5,31].
SODs play a key role in the antioxidant defense system through the dismutation of O2 •− to H2O2. Excess Cu or Cd treatment increased SOD expression [28,32] and activity [28][29][30], which also influenced the H2O2 production in plants. It was reported that rice MT transcription was induced in response to both Cu and H2O2 [18,20]. In this study, the treatment of Cu for 24 and 48 h or Cd for more than 6 h activated the transcription of ricMT and CuZn-SOD in rice radicles (Figure 2). The 6and 12-h Cu treatments induced significant H2O2 production, which happened before the expression of CuZn-SOD and ricMT increased, therefore we guess that Cu-induced H2O2 production acts upstream from ricMT and CuZn-SOD expression in the induction of Cu stress. In comparison, the H2O2 induced by 12-h Cd was lower than the Cu-induced H2O2, which happened after increased expression of CuZn-SOD and ricMT, so we guess that it can act downstream from ricMT and CuZn-SOD expression and block H2O2 production in rice radicles (Figures 1,2). Consistent with our

Discussion
Numerous studies have shown that heavy metals can induce the formation of ROS, including H 2 O 2 , and cause oxidative stress. Cu and Cd toxicity causes an oxidative burst with rapid H 2 O 2 production and its release into the plant apoplast [27][28][29][30]. In this study, the formation of H 2 O 2 was observed in Cu-or Cd-treated rice radicles and cells (Figures 1 and 5). Since H 2 O 2 is relatively stable and can diffuse through cell membranes, it can modulate gene expression and participate in various physiological processes [5,31].
SODs play a key role in the antioxidant defense system through the dismutation of O 2 •− to H 2 O 2 . Excess Cu or Cd treatment increased SOD expression [28,32] and activity [28][29][30], which also influenced the H 2 O 2 production in plants. It was reported that rice MT transcription was induced in response to both Cu and H 2 O 2 [18,20]. In this study, the treatment of Cu for 24 and 48 h or Cd for more than 6 h activated the transcription of ricMT and CuZn-SOD in rice radicles ( Figure 2). The 6-and 12-h Cu treatments induced significant H 2 O 2 production, which happened before the expression of CuZn-SOD and ricMT increased, therefore we guess that Cu-induced H 2 O 2 production acts upstream from ricMT and CuZn-SOD expression in the induction of Cu stress. In comparison, the H 2 O 2 induced by 12-h Cd was lower than the Cu-induced H 2 O 2 , which happened after increased expression of CuZn-SOD and ricMT, so we guess that it can act downstream from ricMT and CuZn-SOD expression and block H 2 O 2 production in rice radicles (Figures 1 and 2). Consistent with our results, the expression of H 2 O 2 -removing enzymes is reported to be up-regulated by excess Cu or elevated endogenous H 2 O 2 [26,28], and increased H 2 O 2 -removing enzymes decrease H 2 O 2 production [28,33]. However, overexpressing CuZn-SOD also showed increased H 2 O 2 production in transgenic potato [34]. The co-regulation of CuZn-SOD and MT expression in yeast may protect against cell toxicity caused by excess Cu [35]. However, the overexpression of MT did not protect cultured motor neurons from mutant CuZn-SOD toxicity [36]. In addition to chelating extra metal ions in plant cells via their Cys thiol groups, MTs may also enhance plant tolerance to stress by up-regulating anti-oxidative enzymes to maintain the redox balance and thereby reduce ROS-induced injury [15,17,37,38]. To make clear the role of ricMT on oxidative damage, rice suspension cells of sense-and antisense-ricMT transgenic lines were obtained by an Agrobacterium-mediated transformation for the first time. In comparison with the WT, the sense-ricMT line (ricMTS2) had a decreased rate of cell death after 24 h Cu treatment or 48 h Cd treatment, and the antisense-ricMT line (ricMTA2) showed an increased cell death rate after 6 h of Cu treatment or 12 h of Cd treatment (Figure 4b,c). It is reported that MT-overexpressing plants [18,39] or yeast [40,41] have a more efficient antioxidant system with increased enzyme activity against stress conditions. Kumar et al. [11] found that ectopic expression of OsMT1e-P protected against oxidative stress primarily through efficient scavenging of ROS.
In the present study, ricMT expression blocked the production of H 2 O 2 in rice suspension cells under Cu or Cd stress ( Figure 5). Contrasting the WT, an increase of H 2 O 2 was accompanied by high cell death rate in antisense-ricMT rice lines under Cu and Cd treatment, and a decrease of H 2 O 2 was accompanied by low cell death rate in sense-ricMT lines (Figures 4 and 5). Moreover, Cu, being a redox-active metal, causes higher accumulation of H 2 O 2 in rice suspension cells than Cd ( Figure 5), which is coincident with higher cell death under Cu treatment (Figure 4). Consistent with our results, heterologous expression of OsMT2c [18] or BcMT [19] in Arabidopsis provided increased tolerance against Cu or Cd stress and accumulated lower amounts of H 2 O 2 . In comparison, Cu alone (and not oxidative stress) was reported to induce MT expression in Neurospora crassa [42].
H 2 O 2 production in rice radicles was increased before the expression of ricMT and CuZn-SOD induced by Cu, but not Cd, which suggests that Cu-induced H 2 O 2 production acts upstream from ricMT and CuZn-SOD expression in the induction of Cu stress. The overexpression of ricMT significantly decreased the rice cell death rate, which was accompanied by lower H 2 O 2 accumulation in rice cells in response to Cu and Cd stress. This indicates that H 2 O 2 is involved in the ricMT-mediated rice tolerance to Cu and Cd toxicity.

Plant Materials
Rice (Oryza sativa L. cv. Wuyunjing No. 7) seeds were surface-sterilized with 5% (v/v) sodium hypochlorite (NaClO) for 15 min and washed thoroughly in distilled water. Then, the seeds were germinated on moist filter paper. Twenty seeds were randomly placed on filter paper in 90-mm Petri dishes and germinated in the dark at 25 • C with the distilled water renewed at 2-day intervals. After germinating for 4 days, 5 mL of freshly prepared 100 µM CuSO 4 solution, 100 µM CdCl 2 solution, or distilled water (control) was added to the Petri dishes for 0, 6, 12, 24, or 48 h; each treatment was performed in triplicate. The radicles were dissected from germinating rice seeds for quantitative RT-PCR and H 2 O 2 determination.

Generation of Sense and Antisense ricMT Transgenic Rice
The full-length sequences of sense-and antisense-ricMT were obtained by PCR and inserted into the SpeI and HindIII restriction sites of the plant expression vector pCAMBIA1304 (Cambia, Canberra, Australia) under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Then, pCAMBIA1304 vector harboring the sense-or antisense-ricMT was transformed into the rice cultivar Nipponbare by using Agrobacterium-mediated transformation [43].

Suspension Cell Cultures
Rice suspension cells were cultured in Chu (N6) medium containing 30 g·L −1 of sucrose, 2 mg·L −1 of 2,4-D-dicholorophenoxyacetic acid and 0.2 mg·L −1 kinetin. After autoclaving, 50 mg·L −1 of filter-sterilized hygromycin B was added for positive selection. The cells were maintained in 500-mL Erlenmeyer flasks containing 180 mL of fresh medium, and subcultured every week. For the flask experiments, a 100-mL flask was used, containing 30 mL of fresh medium which was inoculated with 3 g of fresh cells. The cultures were incubated in a gyratory shaking incubator at 28 • C and 120 rpm. For the treatments, cells were used 5 days after subculture, control medium, medium with 100 µM CuSO 4 or CdCl 2 were tested at concentrations of 100 µM for 0, 6, 12, 24, or 48 h.

Total RNA Isolation, cDNA Synthesis and Quantitative RT-PCR
Total RNA was extracted using the RNA simple Total RNA Kit (LifeFeng, Shanghai, China) according to the manufacturer's instructions and then converted to cDNA after DNase I treatment using a PrimeScript TM RT Master Mix (TaKaRa Bio, Tokyo, Japan). Real-time quantitative RT-PCR was performed on a MyiQ Real-Time PCR Detection System (Bio-Rad Hercules, Berkeley, CA, USA) using SYBR Premix Ex Taq (TaKaRa Bio, Tokyo, Japan). The primers for rice CuZn-SOD (AAA33917) mRNA were forward TCATTGGCAGAGCCGTCGTTGT and reverse AGTCCGATGATCCCGCAAGCAA, the primers for ricMT mRNA were forward CACCATGTCGTGCTGGGTGGCAA and reverse CTTCTAGTTGCAGTTGCAGCAGG, and the primers for the internal control OsActin were forward TTATGGTTGGGATGGGACA and reverse AGCACGGCTTGAATAGCG. The PCR protocol included an initial 7 min incubation at 95 • C for complete denaturation followed by 40 cycles at 94 • C for 30 s, 60 • C for 30 s, and 72 • C for 30 s. The specificity of the PCR amplification was examined based on a heat dissociation curve (65-95 • C) following the final cycle. Normalized relative expression was calculated using the 2 −∆∆Ct (cycle threshold) method.

Hydrogen Peroxide Localization In Situ
The H 2 O 2 production was detected by the infiltration of H 2 DCFDA, as reported by Ezaki et al. [44] with some modifications. Rice radicles or suspension cells were incubated in 20 µM H 2 DCFDA for 20 min. The excess dye had been removed by washing with distilled water for 1 min, the radicles or suspension cells were transferred to microscope slides and observed with a Zeiss Axio Imager A1 fluorescence microscope (Carl Zeiss, Jena, Germany) fitted with an AxioCam HRc camera to visualize the green fluorescence of the H 2 O 2 -oxidized probe.

H 2 O 2 Determination in Extracts
The content of H 2 O 2 in rice radicles from Cu-treated plants was measured by monitoring the A415 of the titanium-peroxide complex following the method described by Jiang et al. [45]. Absorbance values were calibrated to a standard curve established with 0.1-1.0 µM H 2 O 2 .

Evans Blue Assay for Suspension Cell Death
The death of suspension cells was monitored using Evans blue, which is excreted from intact viable cells and is used to estimate cell death spectrophotometrically, as described by Baker and Mock [46]. Briefly, aliquots of treated suspension cells were stained with Evans blue. The cells were washed to remove the excess stain, transferred to 1.5-mL Eppendorf tubes, ground with a micro-sample pestle in the presence of 0.5% SDS to release the trapped stain, and centrifuged to pellet the cellular debris. The A600 of the supernatant was used to quantify cell death.

Statistical Analysis
Data were analyzed using SPSS ver. 16.0 (Statistical Package for Social Science for Windows, SPSS, Inc., Chicago, IL, USA). All values reported in this paper are means ± SE (n = 3) of three separate experiments. Means denoted by the same letter did not significantly differ at p < 0.05 according to Duncan's multiple range test.