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The alleviatory effect of silicon (Si) on chromium (Cr) toxicity to rice was investigated using a hydroponic experiment with 12 combinational treatments of two Cr levels (0 and 100 µmol L-1), three Si levels (0, 75 and 150 mg L-1) and two rice genotypes (Dan K5 and Xiushui 113). The results showed that 100 µM Cr markedly decreased plant height, dry biomass, soluble protein content, and root antioxidant enzyme activity, whereas significantly increased Cr concentration and malondialdehyde (MDA) content. However, the reduction of plant height, dry biomass and soluble content was greatly alleviated due to Si addition in the cultivation solution. Compared with the plants treated with Cr alone, Si addition significantly reduced Cr uptake and translocation in rice plants. No significant difference was observed between the two Si treatments (75 or 150 mg L-1) in shoot Cr concentration and Cr translocation factor. Si addition also alleviated the reduction of anti-oxidative enzymes (superoxide dismutase, SOD and ascorbate peroxidase, APX in leaves; catalase, CAT and APX in roots) and the increase of MDA content in the Cr-stressed plants. Furthermore, the beneficial effects of Si on activities of anti-oxidative enzymes under Cr stress were genotype dependent. The highest activities of SOD, POD (guaiacol peroxidase), CAT and APX in leaves occurred in the treatment Cr + Si 150 for Xiushui 113 and in the treatment Cr + Si 75 for Dan K5, respectively. It may be suggested that the beneficial effect of Si on alleviating oxidative stress was much more pronounced in Dan K5 than in Xiushui 113. The current results showed that Si alleviates Cr toxicity through lowering Cr uptake and translocation and enhancing the activities of anti-oxidative enzymes.
Keywords Antioxidant enzyme, Chromium, Lipid peroxidation, Oryza sativa L., Silicon, Translocation
Chromium (Cr) is a highly toxic trace metal and now has become a serious environmental contaminant as a result of anthropogenic activities, such as mining or industrial activities and improper use of the metal-enriched materials in agriculture, including chemical fertilizer and pesticides, industrial effluents, sewage sludge and wastewater irrigation (Zayed and Terry, 2003; Kuo and others, 2006). Chromium is not considered as an essential element for plant nutrition, and is easily accumulated in plants. Although both stable forms of chromium, Cr (III) and Cr (VI), may cause serious damages to plant tissues and organs, hexavalent Cr is known to be much more toxic than trivalent Cr to living organisms. For plants, excessive Cr inhibits seed germination and plant growth, disorders nutrient balance and water relations, degrades photosynthetic pigments, induces inactivation of mitochondrial electron transport and decreases the activity of antioxidant enzymes (Dixit and others, 2002; Shanker and others, 2005; Panda, 2007). It is also reported that Cr toxicity induced oxidative damage characterized by dramatic accumulation of lipid peroxides or reactive oxygen species (ROS) due to inhibition of the antioxidant systems in plants (Choudhury and Panda, 2005).
Silicon (Si) is the second most abundant element both on the earth surface and in the soil (Gong and others 2006). Although abundant, most sources of silicon are insoluble and not available to plants because of their combination with other elements to form oxides or silicates (Richmond and Sussman, 2003). Usually, silicon is absorbed by plants in the form of uncharged silicic acid, Si(OH)4 (Ma and others 2006), and is ultimately concentrated and polymerized to form silica gel (SiO2.nH2O) throughout the plant (Raven, 2003). Silicon concentrations vary greatly with plant species and tissues, ranging from 0.1 to 10.0% of dry weight (Liang and others, 2007). In general, graminaceous plants take up much more Si than other species, while most dicotyledonous plants take up Si passively (Ma and others, 2001; Liang and others, 2007). As a typical Si-accumulator, rice plants absorb and transport silicon actively and are able to accumulate silicon up to 10.0% of dry weight (Ma and others 2006). Although it has not been evidenced as an essential element for higher plants, silicon has been demonstrated to have multiple direct and indirect beneficial effects on the healthy growth and development of many plant species, particularly of gramineous plants such as rice and some cyperaceous plants (Epstein, 1994, 1999; Ma and others, 2001a; Richmond and Sussman, 2003; Liang and others, 2007). The beneficial effects of silicon are especially pronounced in the plants exposed to abiotic and biotic stresses (Epstein, 1994, 1999; Ma, 2004). More and more evidences demonstrate that silicon can enhance tolerance of plants to metals toxicity, including aluminum (Al) (Baylis and others 1994; Hammond and others, 1995; Liang and others 2001), boron (B) (Gunes and others, 2007; Inal and others, 2009), Cd (Liang and others 2005; Shi and others 2005a; Zhang and others 2008; Nwugol and Huerta, 2008; Song and others, 2009; Shi and others, 2010), manganese (Mn) (Iwasaki and others, 2002a,b; Rogalla and Römheld 2002; Shi and others 2005b), and zinc (Zn) (Neumann and zur Nieden 2001; Kaya and others, 2009).
On the whole, silicon has been widely applied to alleviating various abiotic stresses, especially heavy metal toxicity. However, the possible effect of Si in alleviating Cr toxicity has been not reported up to date. In this study, a hydroponic experiment was conducted to investigate the impact of Si on plant growth, Cr and nutrient uptake, as well as anti-oxidative capacity in Cr-stressed rice plants, so as to determine whether exogenous Si may activate protective responses of the rice plants exposed to chromium stress and understand the possible mechanisms of alleviating chromium toxicity by Si application.
Materials and methods
Plant growth and treatments
Two rice genotypes (Oryza sativa L. cv. Xiushui 113 and cv. Dan K5) were used in this experiment. In our previous study, Xiushui 113 and Dan K5 were found to have low and high grain Cr accumulation respectively, no matter grown in normal or Cr-contaminated soil (Zeng and others, 2008). Rice seeds of the two genotypes were surface sterilized in a 2% H2O2 solution for 10 min, rinsed with tap water for 20 min, and washed with deionized water five times.The seeds were soaked in deionized water in the dark at 25°C for 2 d, germinated with little water at 35°C in the dark for 1 d, and then grown on a plastic net floating on deionized water in a controlled chamber with photoperiod of 16 h light/8 h dark and light intensity of 225 ± 25 µmol m-2 s-1. The light/dark temperatures were set at 30°C / 22°C, and relative humidity was kept at 85%. Fourteen days after germination, uniform rice seedlings were transplanted into a 4 L plastic pot containing nutrient solution (14 seedlings per pot) in a greenhouse with ambient temperature. The rice plants were grown in the hydroponic culture prepared according to Yoshida and others (1976) with the following salts (in mM): NH4NO3 1.45, NaH2PO4 0.32, K2SO4 0.5, CaCl2 1.0, MgSO4•7H2O 1.7, MnCl2•4H2O 9.1×10-3, (NH4)6MoO24•4H2O 5.2×10-4, H3BO3 1.8×10-2, ZnSO4•7H2O 1.5×10-4, CuSO4•5H2O 1.6×10-4, and Fe-citrate 3.6×10-2. The final pH after treatments was adjusted to 5.1±0.01 using 1 M HCl or NaOH solution as required. Culture solutions were renewed every four days.
Half strength nutrient solution was applied for the first 4 days and then changed to complete nutrient solution for one week. Thereafter, Cr (VI) (as potassium dichromate, K2Cr2O7) and silicon (as sodium silicate, Na2Si3O7) were added to the nutrient solutions to form 6 treatments as following: (1) control (Basal nutrient), (2) Si 75 (Basal nutrient + 75 mg L-1 Si), (3) Si 150 (Basal nutrient + 150 mg L-1 Si), (4) Cr (100 µmol L-1 Cr), (5) Cr + Si 75 (100 µmol L-1 Cr + 75 mg L-1 Si) and (6) Cr + Si 150 (100 µmol L-1 Cr + 150 mg L-1 Si).
At the 20 d after treatment, plants were harvested and the plant height and root length were recorded. The plants were separated into leaves and roots, and half of them stored at -80oC for the estimation of anti-oxidative enzymes, soluble protein and malondialdehyde content. A part of leaves was extracted for determination of pigments. The rest of shoots and roots were used for measurement of dry biomass, and metal concentration.
Measurement of plant growth and metal concentration
The sampled rice seedlings were immersed into 20 mM EDTA-Na2 for 3 h and rinsed in running distilled water. Growth traits were measured in terms of the height and dry biomass of whole plant. Fifteen plants (five plants for each replication) were used to measure plant height by a centimeter scale and then the plants were dried at 80oC for 48 h and weighed to measure dry biomass. For determination of metal concentration in rice tissues, the dried plant samples were separated into shoot and root, and milled to powder. One gram dry sample was digested with 6 ml concentrated HNO3 at 150? for 1 h and then 2 ml concentrated HClO4 at 215 oC for 2 h (Miller, 1998). The final digestion was diluted to 25 ml with deionized water and filtered. Concentrations of Cr, Zn and Fe were determined with a flame atomic absorption spectrometry (AA6300, SHIMADZU, Kyoto, Japan). The translocation factor (TF) of Cr was calculated as TF (%) = [Cr]shoot / [Cr]root ×100%.
Estimation of malondialdehyde (MDA)
The level of lipid peroxidation was expressed as malondialdehyde (MDA) content and was determined as 2-thiobarbituric acid (TBA) reactive metabolites according to Hodges and others (1999). Fresh samples (both leaf and root, approximately 0.5 g) were homogenized in 4.0 ml of 1% trichloroacetic acid (TCA) solution and centrifuged at 10,000 ×g for 10 min. The supernatant was added to 1 ml 0.5% (w:v) TBA made in 20% TCA. The mixture was incubated in boiling water for 30 min and the reaction was stopped by placing the tubes in an ice bath. The samples were centrifuged at 10,000 ×g for 5 min, and the absorbance of the supernatant was read at 532 nm. The value for non-specific absorption at 600 nm was subtracted. The amount of MDA-TBA complex (red pigment) was calculated from the extinction coefficient of 155 mM-1 cm-1.
Assay of antioxidant enzyme and soluble protein
Fresh samples (both leaf and root, approximately 0.5 g) were homogenized with 8 ml 50 mM phosphate buffer solution (pH 7.8) in an ice-bath, and then centrifuged at 10,000 ×g for 15 min at 4oC. The supplement was designated as crude enzyme extract, and stored at 4oC for the assays of various antioxidant enzymes and soluble protein content determination.
Superoxide dismutase (SOD, EC 184.108.40.206) SOD activity was assayed by the nitroblue tetrazolium (NBT) method (Beauchamp and Fridovich, 1971) by measuring the photoreduction of NBT at 560 nm. The reaction mixture (3 ml) contained 50 mM sodium phosphate buffer (pH 7.8), 13 mM methionine, 75 µM NBT, 10 µM EDTA, 2 mM riboflavin and enzyme extract (100 µl). Reaction was started by placing tubes below two 15 W fluorescent lamps for 10 min. then stopped by switching off the light. The absorbance was measured at 560 nm. One unit of SOD was defined as the quantity of enzyme that produced 50% inhibition of NBT reduction under the experimental conditions.
Guaiacol peroxidase (POD, EC 220.127.116.11) POD activity was assayed according to method of Putter (1974) with some modification. The reaction mixture (3 ml) consisted of 100 µl enzyme extract, 100 µl guaiacol (1.5%, v/v), 100 µl H2O2 (300 mM) and 2.7 ml 25 mM potassium phosphate buffer with 2mM EDTA (pH 7.0). Increase in the absorbance was measured spectrophotometrically at 470 nm (e =26.6 mM-1 cm-1).
Catalase (CAT, EC 18.104.22.168) CAT activity was assayed using the method described by Aebi (1984). The assay mixture (3.0 ml) contained 100 µl enzyme extract, 100 µl H2O2 (300 mM) and 2.8 ml 50 mM phosphate buffer with 2mM EDTA (pH 7.0). The CAT activity was assayed by monitoring the decrease in the absorbance at 240 nm as a consequence of H2O2 consumption (e = 39.4 mM-1 cm-1).
Ascorbate peroxidase (APX, EC 22.214.171.124) APX activity was determined according to Nakano & Asada (1981). The reaction mixture consisted of 100 µl enzyme extract, 100 µl ascorbate (7.5 mM), 100 µl H2O2 (300 mM) and 2.7 ml 25 mM potassium phosphate buffer with 2mM EDTA (pH 7.0). The oxidation of ascorbate was determined by the decrease in absorbance at 290 nm (e = 2.8 mM-1 cm-1).
Soluble protein The soluble protein content in rice leaf and root was analyzed according to Bradford (1976), using Coomassie Brilliant Blue G-250 as dye and albumin as a standard.
The whole experiment was set up in the randomized block design with six replicates per treatment. All data are presented as means of three replicated measurements. Statistical analysis was performed by a statistical package, SPSS version 13.0 (SPSS, Chicago, IL). ANOVA test was used to confirm the significance of the data. Significant differences among the treatment means were compared by Duncan's multiple range tests (P< 0.05).
Plant growth and soluble protein content
The height and dry biomass of whole rice seedlings for the two cultivars were significantly reduced under Cr stress. The reduction of seedling height was obviously alleviated due to addition of Si in the solution, especially in 150 mg L-1 Si treatment. However, Si showed little effect on alleviating the reduction of dry biomass caused by Cr stress. In absence of chromium, there was no significant difference in seedling growth among the Si treatments.
Chromium stress also significantly reduced soluble protein content in both leaf and root, and the reduction could be significantly alleviated by Si addition. For the treatments without Cr addition, the addition of Si significantly increased soluble protein contents in leaves of both cultivars and in roots of Dan K5. There was a significant Cr and Si interaction for plant height (P < 0.05), and cultivar and Si interaction for soluble protein content in roots (P <0.001).
Chromium concentration and translocation in plants
Cr concentrations in both shoots and roots of the two cultivars were significantly increased by the addition of Cr in the culture solution (Table 2), with roots being significantly higher than shoots. Addition of Si significantly reduced Cr concentrations in both shoots and roots, and the reduction was more noticeable in higher Si level (150 mg L-1). Moreover, Si addition resulted in a dramatic decrease of translocation factor (TF) of Cr in the two cultivars, but the more decrease of TF was observed in Dan K5 than in Xiushui 113.
Cr exposure significantly increased MDA content in leaves and roots by 139.4 and 52.3 % for Xiushui 113, respectively, compared to the control. For Dan K5, Cr addition increased MDA content in leaves and roots by 80.8% and 98 % respectively. For the treatments without Cr condition, Si 75 and Si 150 treatments increased leaf MDA content in Xiushui 113 by 28.8% and 48 %, whereas reduced it in Dan K5 by 27.8% and 1.4 % respectively. Similar results were observed in the influence of Si treatment on root MDA content of both cultivars. Under Cr stress, addition of Si reduced MDA contents in leaves and roots of both cultivars and the reduction was more noticeable in higher Si level. Furthermore, Si-mediated reduction of MDA content under Cr stress varied with cultivars and plant tissues. For example, under higher Si level, leaf MDA content was reduced by 27.8 % in Xiushui 113 and16.5% in Dan K5. However, the reduction of root MDA content was greater in Dan K5 (22.4%) than in Xiushui 113 (11.0%).
Activities of SOD, POD, CAT and APX
Chromium addition significantly increased leaf SOD activity in both cultivars and decreased root SOD activity in Dan K5 (Figure 1). For the treatments without Cr addition, there was no significant difference in leaf SOD activity between low Si level (75 mg L-1) and the control for the two cultivars, although higher Si level (150 mg L-1) showed significant increase in both cultivars. Root SOD activity was significantly decreased under lower Si level for Xiushui 113, increased by addition of Cr for Dan K5. Under Cr stress, lower Si treatment did not dramatically change leaf SOD activity relative to the treatment of Cr alone, and higher Si treatment significantly increased it in Xiushui 113. However, for Dan K5 lower Si treatment had significantly higher SOD activity than the treatment of Cr alone, while there was no significant difference between higher Si treatment and Cr alone treatment. Root SOD activity was significantly decreased and increased under lower Si treatment for Xiushui 113 and Dan K5, respectively. However, root SOD activity was unaffected under higher Si treatment for both cultivars.
Chromium stress significantly increased leaf POD activity in Dan K5 but markedly decreased root POD activity in both cultivars (Figure 1). Addition of Si in the culture solution showed little effect on POD activities in leaves and roots of two cultivars under absence of Cr. Under Cr stress, leaf POD activity of the two cultivars was unaffected by Si treatment. However, Si treatment significantly reduced root POD activity in Xiushui 113, whereas markedly increased that of Dan K5.
Leaf CAT activity of both cultivars was not significantly affected by Cr exposure, while root CAT activity was significantly decreased under Cr stress (Figure 1). Without Cr condition, there was no significant difference in leaf and root CAT activity of both cultivars between the control and lower Si treatment. However, higher Si treatment significantly increased leaf CAT activity and decreased root CAT activity of both cultivars. Under Cr stress, CAT activities in both leaf and root were not significantly affected by Si treatment, except leaf CAT activity of Xiushui 113 which showed significant increase in higher Si treatment (Cr + Si 150 treatment).
Chromium exposure significantly increased leaf APX activity but markedly decreased root APX activity in both cultivars (Figure 1). Leaf and root APX activities under non-Cr condition were unaffected by Si treatment. Under Cr stress, leaf APX activity was increased due to Si addition. The highest leaf APX activity occurred for Xiushui 113 in Cr + Si 150 treatment and for Dan K5 in Cr + Si 75 treatment. In contrast, there was no significant difference between Si treatments and Cr alone treatment in root APX activity.
Chromium addition slightly increased shoot Zn concentration but significantly reduced Fe, Cu and Mn concentrations of both cultivars. For the treatments without Cr addition, Si supplement significantly reduced the concentrations of Zn, Fe, Cu and Mn in shoots of two cultivars. However, there was no significant difference between 75 mg L-1 and 150 mg L-1 Si-treated plants in the concentrations of all examined microelements. Under Cr stress, the supplement of 75 mg L-1 Si significantly reduced Fe and Mn concentrations in Xiushui 113 but increased Zn concentration in Dan K5. Addition of 150 mg L-1 Si had less effect on the concentrations of the four microelements in Xiuahui 113. However, it significantly reduced leaf Zn and Mn concentrations in Dan K5.
In roots, Cr exposure significantly increased Zn and Fe concentrations but markedly decreased Cu concentration in Xiushui 113 (Figure 2). Similar results were also observed for Dan K5. In comparison to the control, Si supplement showed little influence on microelement concentrations in Xiushui 113, except Fe in Si 75 treatment and Cu in Si 150 treatment. However, Sitreatments significantly reduced Zn, Fe and Cu concentrations of Dan K5. Under Cr stress, Si supplement still showed little effect on microelement concentrations of Xiushui 113, except Fe in Cr + Si 75 treatment and Mn in Cr + Si 150 treatment. Although Cu and Mn concentrations of Dan K5 were unaffected by Si supplement, Zn and Fe concentrations were significantly increased under the treatment of 75 mg L-1 Si. In addition, results of ANOVA test showed that there were significant Cr × Si and cultivar × Cr × Si interactions for all microelement concentrations of both shoots and roots.
The inhibition in plant growth by Cr stress has been well documented in many plant species (Zurayk and others, 2001; Mei and others, 2002; Shanker, 2003; Shanker and others, 2005). In the present study, the growth parameters of the two rice cultivars were severely reduced at 100 µmol L-1 Cr compared to the control. Although little effect was detected in alleviating the reduction of seedling dry biomass by Cr, the addition of Si, especially 150 mg L-1 level alleviated the reduction of the plant height. In addition, the height and dry biomass of both cultivars were increased by Si addition under non-Cr stress condition, proving the beneficial effect of Si on rice growth. The reason for this beneficial effect of Si is still unclear. However, Hossain and others (2002) hypothesized that Si-induced growth promotion in rice plants is due to an increase in cell-wall extensibility.
It was reported that Si supplement would enhance tolerance to toxic metals through reducing the uptake and translocation of them, including Cd, Mn, Zn and so on (Shi and others, 205b; Nwugo and Huerta, 2008; Kaya and others, 2009). The possible mechanisms for inhibition of metal transport in gramineae plants by Si are mainly involved in the two aspects: (1) Si deposits lignin in cell walls and induces metal ions binding to cell wall, thus reduce the uptake and translocation of metals from roots to shoots (Ma and others, 2006). Rogalla and Römheld (2002) reported that the enhanced tolerance of Mn toxicity by silicon in Cucumis sativus was a consequence of stronger binding of Mn to cell walls and a lowering of Mn concentration within the symplast. Similar findings of reduced metal toxicity by Si-mediated stronger binding of metal to cell walls have also been reported for other metal ions, like Cd (Wang and others, 2000) and Al (Liang and others, 2001). (2) the complex formation or co-precipitation of toxic metal ions with Si. Hodson and Sangster (1993) reported that silicon could detoxify Al toxicity in Sorghum bicolor through forming a complex with Al in the medium and/or roots and ultimately inhibiting Al penetration into the root cortex. Similarly, Inal and others (2009) reported that formation of B-Si (boron-silicate) complexes in the soil contributed to lower boron availability and consequently lowered tissue boron concentration. In the present study, Si-induced inhibition of Cr uptake and translocation in rice plants was also observed. Therefore, it can be supposed that Si might also have the ability to complex with Cr or induce Cr to deposit in the cell wall, thus lowering the Cr toxicity in rice plants. In addition, an interesting observation in this study was the lack of a significant difference in shoot Cr concentration and Cr translocation factor between the two Si treatments (75 mg L-1 and 150 mg L-1). It was showed that there is a nutrient-dose threshold requirement for plant nutrition (Chaudhari and Singh, 2006). Therefore, it can be assumed that 75 mg L-1 Si might be close to the optimum requirement for Si-induced alleviation of Cr stress.
Alleviation of metal toxicity by silicon was also attributed to reduced oxidative stress caused by excess metals. It has been reported that Si alleviated boron toxicity in spinach, tomato (Gunes and others, 2007) and barley (Inal and others, 2009) plants by preventing the oxidative membrane damage. According to Song and others (2009), the Si-enhanced tolerance to cadmium toxicity in pakchoi plants was attributed to Si-enhanced antioxidant defense capacity. Similarly, Shi and others (2005b) reported that the alleviation of Mn toxicity by Si in cucumber was related to a significant reduction in membrane lipid peroxidation caused by excess Mn and to a significant increase in enzymatic and non-enzymatic antioxidants. Even under optimal growth condition, many metabolic processes would produce reactive oxygen species (ROS). While suffering from abiotic or biotic stresses, the production of ROS will be significantly increased in plant tissues or cells. To scavenge (ROS), plants have developed a complex and efficient defense system to protect them from destructive oxidative stress (Zhu and others, 2004). As part of this system, antioxidant enzymes are key elements in the defense mechanisms. Many changes have been observed in the activities of antioxidant enzymes in plants under Cr stress (Panda and Choudhury, 2005; Panda, 2007). SOD appears constituting the first line of defense against ROS, which is crucial for the removal of O2- in the compartments where O2- radicals formed (Takahashi and Asada, 1983). In the present study, a significant enhancement in SOD and APX activities was observed in rice leaves of both cultivar plants treated with excess Cr, suggesting their important roles in scavenge O2- or H2O2 induced by Cr toxicity. The beneficial effects of Si on activities of SOD, POD, CAT and APX under Cr stress were genotype and Si-dose dependent. The highest activities of SOD, POD, CAT and APX of Xiushui 113 were observed in the Cr + Si 150 treatment, whereas those of Dan K5 were in the Cr + Si 75 treatment, suggesting that less Si was need for alleviating oxidative stress of Dan K5 than Xiushui 113 when suffering from same Cr stress. On the other hand, SOD, POD, CAT and APX activities in rice roots were dramatically reduced when the plants were exposed to Cr stress. These results suggested that rice roots suffered from much greater oxidative stress than leaves under Cr stress, and the formation of anti-oxidative defense system was greatly damaged. Si supplement showed no significant effect on alleviating the reduction of root CAT and APX activities of both cultivars. However, the supplement of 75 mg L-1Si significantly alleviated the reduction of root SOD and POD activities in Dan K5 but deteriorate those of Xiushui 113, again indicating that the beneficial effect of Si on alleviating oxidative stress was much more pronounced in Dan K5 than in Xiushui 113.
The project was supported by Zhejiang Bureau of Science and Technology (2009C12050) and Graduate Students' Innovation Projects of Zhejiang Province (YK2008015).
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