Introduction

Mercury (Hg) and its compounds are listed as priority pollutants by many international organizations because of their high toxicity, persistence in the environment, and bioaccumulation in the food chain (Clarkson and Magos 2006; Jiang et al. 2006; Syversen and Kaur 2012). Because of coal combustion, mining and smelting of Hg and other nonferrous metals, together with industrial and agricultural activities, soils have been severely polluted by Hg in some areas of China (Horvat et al. 2003; Jiang et al. 2006). For example, in the surrounding soil of the Wuchuan Hg Mine the total Hg content of ranged from 0.33 to 320 mg/kg and the total content MeHg ranged from 0.69 to 20 μg/kg (Qiu et al. 2006), which far exceed the Chinese national limit for paddy soils (1.5 mg/kg) (GB15618-1995). The Hg pollution in the soil will directly affect the production and quality of agricultural products. Rice (Oryza sativa L.) is a staple cereal worldwide, especially in Asia. It was reported that the contents of inorganic mercury (IHg) and methylmercury (MeHg) in rice grain were 94 (7.4 ~ 460) μg/kg and 11 (1.2 ~ 44) μg/kg in a Hg-polluted area (Zhang et al. 2010), which is higher than the limit of the Chinese national standards for the cereal (20 μg/kg, GB2762-2005). Rice consumption has become the main source of MeHg exposure for local residents, which poses potential health risks to them (Wang et al. 2011; Zhao et al. 2009).

The toxicity of Hg largely depends on its chemical forms. MeHg is regarded as the most toxic form due to its tendency for bioaccumulation and biomagnification through the food chain and transportation across the blood–brain barrier (Diez 2009; Syversen and Kaur 2012; Wei et al. 2014). Our previous work revealed that the proportion of MeHg to total Hg in rice grain was as high as 50 % (Zhao et al. 2014), which raised a great concern for the rice-dependent populations in Hg-contaminated areas. Microbes have been considered the main producers of MeHg in an anoxic environment, such as sediment or swampland (Benoit et al. 2003; Wei et al. 2013). Paddy fields are a unique ecosystem, possessing both the characteristic of aquatic and terrestrial ecosystems. Therefore, Hg is also methylated in the paddy, and paddies have been perceived as the main source of MeHg in rice (Li et al. 2010). Some reports have recently shown that there is Hg methylation in some kinds of fish (Wang et al. 2013) and aquatic plants (Deng et al. 2013). The demethylation of MeHg could occur in the liver of marine mammals (Khan and Wang 2009). Therefore, whether the process of IHg methylation or MeHg de-methylation occurs in rice plants deserves further study.

Iron plaque is commonly formed on the root surface of many aquatic plant species, which helps plants adapt to aquatic environmental stress (Fu et al. 2010; Li et al. 2014). It can affect the bioavailability and migration of metal(loid)s through absorption-desorption, oxidation-reduction, organic–inorganic chelation, co-precipitation, and so on. (He et al. 2004; Liu et al. 2014; Tripathi et al. 2014). The role of iron plaque in the uptake of different elements in rice plants has been investigated in several studies (Hu et al. 2014; Huang et al. 2012; Lee et al. 2013; Liu et al. 2008). The formation of iron plaque has been found to sequester arsenic (As) and reduce its uptake by rice from the culture medium (Lee et al. 2013; Syu et al. 2013, 2014). Other studies have found that iron plaque can immobilize chromium (Cr) from the environment but not significantly affect the Cr uptake and translocation in rice seedlings (Hu et al. 2013, 2014). Wang et al. (2014) found that the amount of Hg in straw and brown rice negatively correlated with iron plaque, suggesting that iron plaque could reduce the accumulation of Hg in rice plants. However, it is still unclear whether the formation of iron plaque plays a different role in the absorption, translocation and transformation of different Hg species, i.e., IHg and MeHg in rice plants. The aims of this study are 1) to explore the adsorption of IHg and MeHg onto iron plaque, 2) to investigate the influence of iron plaque on the absorption, translocation of IHg and MeHg in rice plants, 3) to explore whether the process of methylation and demethylation of Hg in vivo occurs in rice plants, and 4) to investigate the effects of iron plaque on IHg and MeHg transformation in rice.

Materials and methods

Rice cultivation

Rice seeds (Oryza sativa L.GY1255) were disinfected with 1 % (V/V) sodium hypochlorite (NaClO) solution for 15 min and then washed thoroughly with deionized water (18.2 MΩ · cm). The seeds were germinated in moist perlite at 28 °C in the dark for 1 week. Uniform seedlings were selected and cultivated in a 300 mL 25 % Hogland solution (pH = 5.0) for 3 weeks. The nutrient solution was renewed twice a week. All of the plants were cultured in a man-made climate growth chamber with a 14 h light period (300–350 μmol/m2/s, 28 °C, humidity 60 %) and a 10 h dark period (20 °C, humidity 70 %).

Induction of iron plaque and exposure to Hg

All rice seedlings were transported into a 0.5 mmol/L CaCl2 (pH = 5.5) solution for 2 h to minimize the interference of Fe from other elements. Iron plaque was induced by exposing the rice roots to 0, 20, 50, 80, 100 mg/L of ferrous ions (FeSO4 · 7H2O) for 24 h, assigned as the Fe0, Fe20, Fe50, Fe80, and Fe100 groups, respectively. The rice seedlings were then transferred into a 25 % Hogland solution for another 48 h and then into a nutrient solution containing 500 μg/L HgCl2 or MeHgCl for 72 h. Afterwards, the rice seedlings were harvested and the nutrient solutions were collected.

Extraction of iron plaque and analysis of Fe content

The iron plaque on the root surface was extracted with a dithionite–citrate–bicarbonate method (DCB) (Huang et al. 2012). The entire roots were washed thoroughly with deionized water prior to the incubation in a 30 mL solution containing 0.5 g sodium dithionite (Na2S2O4), 0.03 mol/L sodium citrate (Na3C6H5O7 · 2H2O), and 0.125 mol/L sodium bicarbonate (NaHCO3) at room temperature for 1 h. The roots were then rinsed three times with deionized water. Finally, the DCB extracts were diluted to 50 mL with deionized water. After DCB extraction, the roots and shoots were air dried to constant weight.

The Fe concentration in the DCB extractions was determined with an Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES, Optima 2000DV, PerkinElmer, USA). The working conditions of the ICP-OES were as follows: forward power (1300 W), observation height (15 mm), plasma gas flow rate (0.8 L/min), and injection volume (1.5 mL/min).

Analysis of Hg content in the DCB extractions, roots and shoots

The dried roots and shoots were ground into a fine powder with a mortar and pestle. The root and shoot powders (approximately 50 mg) were mixed with 5 mL HNO3 (BV-III) and l mL H2O2 (MOS). The mixtures were left overnight at room temperature and then digested at 160 °C in high pressure Teflon digestion vessels for 6 h. Afterwards, the mixtures were evaporated to approximately 1 mL at 90 °C. The residual solution was diluted to 5 mL with 2 % (V/V) HNO3 containing 0.1 % (V/V) β-mercaptoethanol. The Hg calibration standard was prepared by diluting the Hg standard stock solution (GBW 08617, National Research Centre for CRMs, China) with 2 % (V/V) HNO3 containing 0.1 % (V/V) β-mercaptoethanol. The Hg in the DCB extracts and rice tissues was determined using inductively coupled plasma mass spectrometry (ICP-MS, Thermo Elemental X7, USA) in standard mode. Instrument optimization was performed every time with a normal tuning solution (Bi 20 ppb). The working conditions of the ICP-MS were as follows: forward power (1300 W), plasma gas flow rate (13.0 L/min), auxiliary gas flow rate (0.8 L/min), nebulizer gas flow rate (0.72 L/min), and sample uptake rate (0.8 mL/min).

Analysis of Hg speciation with HPLC-ICP-MS

The IHg and MeHg contents in the rice tissues were analyzed with an HPLC-ICP-MS hyphened technique according to Zhao et al. (2014). The powder of the roots or shoots (approximately 50 mg) was treated with 5 mL 6 mol/L HCl. The mixtures were shaken overnight at room temperature after 1 h of ultrasonic vibration. Then, the mixtures were centrifuged at 10000 r/min (Eppendorf, Germany) for 10 min. The supernatant was collected and adjusted to a pH of 6.7 with ammonia and diluted to 10 mL with the mobile phase (pH = 6.7) containing 0.06 mol/L ammonium acetate, 5 % (V/V) methanol and 0.3 % (V/V) β-mercaptoethanol. All of the solutions were filtered using a 0.22 μm microporous membrane filter before analysis. A liquid chromatography pump (WAT055028 metal-free 626 pump, Waters, Milford, USA) and a 5 μm Symmetryshield RP18 column (150 × 3.9 mm, Waters, Milford, USA) were used to separate IHg and MeHg. The injection volume was 100 μL, and the flow rate was 1 mL/min. The Hg speciation in the nutrient solution was analyzed with the same procedure. The calibration standards of IHg and MeHg were prepared by diluting the IHg standard stock solution (GBW 08617, National Research Centre for CRMs, China) and MeHg standard stock solution (GBW 08675, National Research Centre for CRMs, China) with the mobile phase.

Distribution of Fe and Hg in iron plaque with μ-SRXRF

Fresh rice roots were immersed in the embedding agent (SAKURA Tissue-Tek OCT) and frozen at −80 °C. Then, the roots were cut into 40 μm-thick slices with a freezing microtome (Reichert-Jung, Germany). The slices were placed on Mylar films (polycarbonate) and then dried and stored at −20 °C prior to the micro beam-synchrotron radiation X-ray fluorescence (μ-SRXRF) analysis. Fresh rice roots were also placed between two layers of cellophane and dried using Gel Drier (Bio-Rad) for the measurement of element distribution in the longitudinal section of the root.

The Fe and Hg distributions in the cross and longitudinal section were measured at BL15U at the Shanghai Synchrotron Radiation Facility (SSRF, China), in which the storage ring runs at 3.5 GeV with a current intensity from 200 mA to 300 mA. The incident X-ray energy of 13 keV was monochromatized by a Si(111) double-crystal monochromator and focused to a spot size of 5 × 5 μm2 with a K-B system. The rice root slices were fixed on a precision motor-driven stage. The fluorescence signals from the slices were recorded and analyzed with a 7 element Si(Li) detector combined with a multiple channel analyzer (e2v, UK). The sample platform was moved along the horizontal and perpendicular direction at an interval of 5 μm for each step. The count time was 1.5 s per pixel. The peak areas of the elements were normalized to the current intensity (I0) in the ionization chamber to correct for the effect of SR beam flux variation on the signal intensity.

Chemical forms of Hg in rice plants with XANES

A Hg L3-edge X-ray absorption near-edge structure spectroscopy (XANES) experiment was conducted at the 1W1B beamline at the Beijing Synchrotron Radiation Facility (BSRF). The storage ring was at 2.5 GeV with a maximum current intensity of approximately 250 mA. A Si(111) double-crystal monochromator was detuned to minimize the harmonic content of the beam. After the iron plaque extraction, the rice roots were ground into a fine powder, which was smeared onto Scotch tape. The Hg L3-edge XANES spectra were collected at room temperature in fluorescence mode for all plant samples and in transmission mode for all Hg reference compounds including HgS, HgCl2, Hg-glutathione (Hg(GS)2), CH3HgCl, CH3HgGS. The data was normalized and analyzed using the IFEFFIT program.

Results

Formation and distribution of iron plaque on roots

Reddish iron plaque on the root surface became visible after 24 h of incubation in a solution with different Fe2+ concentrations, but it was not observed on the rice without the Fe2+ treatment. The Fe concentration in the DCB extracts was used to represent the amount of iron plaque. Figure 1 illustrated the relationship between the amount of iron plaque and the Fe concentration in the pretreatment solutions. The formation of iron plaque was markedly affected by the Fe addition. The amount of iron plaque increased with the increasing Fe2+ concentrations in the 0-80 mg/L of Fe2+ pretreatment solutions and then stabilized.

Fig. 1
figure 1

The influence of Fe2+ concentration in the pretreatment solution on the amounts of iron plaque formed on the root surface. (Means ± SD, n = 3)

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Figure 2 shows the micrographs and the mapping of Fe in the cross and longitudinal sections of the rice tissues imaged with μ-SRXRF. The Fe was concentrated on the root surface when the rice was treated with 80 mg/L of Fe2+ (Fig. 2b 1 and d1). Little Fe was found on the root surface without the Fe2+ treatment (Fig. 2a 1 and c1) in contrast with the root surfaces treated with 80 mg/L of Fe2+ (Fig. 2b 1 and d1). From Fig. 2f,we can see that the iron plaque was mainly deposited in the maturation zone of the root (Fig. 2f). Only a small amount of iron plaque was formed on the root tip.

Fig. 2
figure 2

The distribution of Fe and Hg in the root tissues of rice measured by μ-SRXRF. Left, the element distribution on a cross section of the root under IHg exposure, without (a) or with (b) iron plaque induction (a 1 and b 1 for Fe; a 2 and b 2 For Hg); right, the cross section of the root tissues from rice without (c) or with (d) iron plaque induction under MeHg exposure (c 1 and d 1 : Fe XRF image; c 2 and d 2 : Hg XRF image); and Fe distribution in the longitudinal section of rice tissues without (e) or with (f) iron plaque induction

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Hg adsorption in iron plaque and distribution in rice roots

The amounts of IHg and MeHg adsorbed in iron plaque were investigated by measuring the Hg concentrations in the DCB extracts. The results are shown in Fig. 3. The Hg concentration in the DCB extracts increased dramatically with the amount of iron plaque, ranging from 34.64 ± 6.59 to 114.41 ± 3.71 mg/kg for HgCl2 exposure and from 86.67 ± 14.09 to 287.67 ± 39.66 mg/kg for MeHg exposure (Fig. 3). The Hg content in the iron plaque exposed to MeHg was twice that of the iron plaques exposed to IHg. Hence, the iron plaque adsorbed both Hg species, especially MeHg. A strong positive correlation (r 2 = 0.790 for IHg, r 2 = 0.852 for MeHg) between the Hg and Fe concentrations in the DCB extracts was found under IHg and MeHg exposure (Fig. 3).

Fig. 3
figure 3

Correlation between the Hg and Fe concentrations in the DCB extracts: HgCl2 (a); MeHgCl (b) (RDW is the root dry weight)

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The Hg distribution in the rice roots was measured using μ-SRXRF. The result is shown in Fig. 2. The Hg was mainly concentrated in the epidermis, endodermis and vascular tissues. An increase in Hg accumulation in the epidermis and a decrease in Hg enrichment in the pericycle were found (as shown in Fig. 2b 2 and d2) with the Fe2+ treatment.

Hg content in the roots and shoots

The Hg content in the roots and shoots was determined using ICP-MS, and the results are shown in Fig. 4. As observed from Fig. 4, the Hg content in the roots decreased with the addition of Fe2+ in the pretreatment solutions. The Hg content in the roots exposed to IHg was higher than those exposed to MeHg at each Fe2+ level (Fig. 4a). The Hg content in the shoots exposed to MeHg was significantly higher than in the shoots exposed to IHg regardless of the formation of iron plaque (Fig. 4b). The addition of Fe2+ prominently decreased the Hg content in the shoots exposed to MeHg, whereas no obvious change was found with IHg exposure.

Fig. 4
figure 4

Mean Hg content in roots (a) and shoots (b) and Hg translocation factor (c) in rice seedlings treated by different concentrations of Fe2+ and different species of Hg. Data are means ± SD. (n = 3) **p < 0.01, *p < 0.05 compared with Fe0 (control) group

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The species of Hg in rice tissues

The species of Hg in the rice tissues and nutrient solutions were analyzed by HPLC-ICP-MS. Hg2+ and CH3Hg+ were separated, identified and quantified by this method with a recovery above 80 % (Rahman and Kingston 2004; Rahman et al. 2009). The mean percentages of Hg2+ and CH3Hg+ in the tissues and nutrient solutions are shown in Fig. 5. When rice was exposed to IHg, the major specie of Hg in the rice roots was IHg, and MeHg was hardly detected in the tissues and nutrient solutions (Fig. 5a). Interestingly, when rice was exposed to MeHg, approximately 20 and 50 % of the Hg detected in the roots and shoots, respectively, were in the form of IHg (Fig. 5c). Meanwhile, no IHg was detected in the nutrient solutions with MeHg. The formation of iron plaque caused IHg to slightly increase in the roots and shoots of the rice exposed MeHg, but the amount was not significantly different (Fig. 5d).

Fig. 5
figure 5

The mean percentage of Hg2+ and CH3Hg+ in nutrient solutions and rice roots measured by HPLC-ICP-MS. a Rice without iron plaque induction under IHg exposure; b rice with iron plaque induction under IHg exposure; c rice without iron plaque induction under MeHg exposure, d rice with iron plaque induction under MeHg exposure

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The chemical forms of Hg in the rice tissues were analyzed with XANES. The main advantage of XANES over other methods is that it is less destructive and reflects information about the local molecular bonding environment of the element. The Hg L3-edge XANES spectra of the roots exposed to both Hg species and the Hg reference compounds are shown in Fig. 6. The Hg L3-edge spectra of HgCl2 and MeHgCl exhibited a shoulder peak at 12284 eV, while root samples did not have such a shoulder peak. This result indicated that the Hg in the roots might interact with S rather than Cl.

Fig. 6
figure 6

Hg L3 XANES spectra of the model compounds and the root samples of rice treated by different Hg species

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The least-squares fit results of the root samples are shown in Table 1. When rice was exposed to IHg, most of the chemical forms of Hg were similar to Hg(GS)2, accounting for approximately 78.5 % of the total Hg, and the others were similar to HgCl2 and HgS. When rice was exposed to MeHg, the chemical forms of Hg were mainly similar to Hg(GS)2 and CH3HgGS. The corresponding proportion of Hg(GS)2 and CH3HgGS was approximately 1:4. In addition, the formation of iron plaque did not change the chemical forms of Hg in the rice tissues.

Table 1 Percentage of Hg species in the IHg and MeHg-treated groups by least squares fitting. Values in parentheses show the SD
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Discussion

The formation of iron plaque and IHg and MeHg adsorption in iron plaque

The basal part of the rice root releases O2 (Fu et al. 2010), which causes the formation of Fe and Mn oxide and precipitation on the root surface. The formation of iron plaque was found to have a significant correlation with the rates of radial oxygen loss (ROL) in rice plants (Li et al. 2011; Wu et al. 2012; Yang et al. 2014).

Fe oxides play an important role in the cycle of Hg in aquatic ecosystems because of the strong adsorption and co-precipitation between Hg and Fe oxides (Fagerström and Jernelöv 1972). This study found that both IHg and MeHg, especially MeHg, could be adsorbed by iron plaque. The Hg adsorption in the iron plaque had a positive correlation with the amount of iron plaque (Fig. 3). This result suggests that the Hg accumulation in the iron plaque correlated significantly with the Hg speciation. This was also reported in other studies. For example, Huang et al. found that the antimony (Sb) concentration in the iron plaque on rice root surfaces treated with Sb(III) were significantly higher than on rice root surfaces treated with Sb(V) (Huang et al. 2012). The predominant species of Sb(III) in aqueous solution is [Sb2(C4H2O6)2]2- units with two negative charges, while Sb(V) exists as Sb(OH)6 - at pH > 3 with one negative charge. Liu et al. (2005) found that arsenate was more easily absorbed by iron plaque than arsenite. The predominant species of arsenate and arsenite in solution were H2AsO4 - and H3AsO3 0 at pH > 3, respectively. It seems that the accumulation of Sb and As in iron plaque is closely linked to the quantity of the electric charge of their chemical forms in solution. HgCl2 and CH3HgCl are weak electrolytes. The dissociation constant of CH3HgCl (pK = 5.25) is higher than that of HgCl2 (pK1 = 6.74, pK2 = 13.22) (De Robertis et al. 1998; Dyrssen and Wedborg 1991). There are more free ions in the rhizosphere with the MeHg treatment than that with the IHg treatment under the same pH range, and this may be responsible for the different adsorption rates of iron plaque to MeHg and IHg.

Effects of iron plaque on the absorption and translocation of Hg

Iron oxides have high Hg adsorption and co-precipitation capacity. The addition of iron oxides significantly decreased the available Hg content in soil (Ren et al. 2014). In order to exclude the possible effect of the Fe treatment on the Hg accumulation, the Fe2+ treatment solution was replaced by a normal nutrient solution after the iron plaque was induced, and then the rice seedlings were exposed to Hg in our experiment.

Hg was mainly concentrated in the epidermis, and endodermis, and the vascular tissues. The reason for this Hg distribution may be ascribed to the following: 1) the major cell wall structural proteins, such as extensins and expansins, contained characteristic cysteine-rich domains (McNear et al. 2012; Li et al. 2015). Because Hg has high affinity for the thiol groups in cysteine residues, it impaired the structure of the cell walls and aroused the deformation of the epidermal, cortical and xylem cells (Carrasco-Gil et al. 2013). 2) Hg ions could permeate into the inner root tissues via the apoplastic pathway in rice roots. Hg ions may also be transported across the plasma membrane of the endodermal barrier and enter the cell symplast, and they are then transported further to the central cylinder (Tester and Leigh 2001). Our results differ from a report on maize (Zea mays L.). Debeljak et al. (2013) found that Hg ions barely crossed the root plasma membranes of the endodermal barrier but were thoroughly restricted in the root apoplastic space in maize (Zea mays L.).

Iron plaque could actively inhibit the translocation of MeHg from roots to shoots, but this effect was not remarkable for IHg. Excess Fe2+ in the rhizospheric environment is toxic to the plant. It can inhibit the growth of the primary root (Ward et al. 2008). Therefore, appropriate levels of Fe2+ should be used to reduce Hg accumulation in rice plants.

The Hg content in the shoots exposed to MeHg was significantly higher than in the shoot exposed to IHg regardless of the formation of iron plaque (Fig. 4b). The translocation factor (TF) (Fig. 4c) was used as an indicator to evaluate the transfer ability of Hg from the roots to aboveground parts in rice. (TF = Tshoot-Hg/Troot-Hg, and Tshoot-Hg and Troot-Hg represent the Hg content in the shoots and roots, respectively.) The TF for the MeHg exposure was significantly higher (p < 0.05) than that for the HgCl2 exposure, indicating that MeHg was more easily transported from the roots to the shoots. The formation of iron plaque effectively inhibited the translocation of MeHg from the roots to the shoots. The result is consistent with other studies on Hg translocation in rice. Zhao et al. (2014) found that the proportion of MeHg over the total Hg increased from the roots to the grains, and MeHg can account for over 50 % of the total mercury in rice grains. On the contrary, the proportion of IHg decreased from the roots to the grains. Zhang et al. (2010) investigated the IHg and MeHg contents in rice grains from an Hg mining area and found that the bioaccumulation factors (BAFs) for MeHg were much higher than those for IHg in rice plants. Therefore, IHg and MeHg showed different accumulation abilities and distribution patterns in rice, implying that IHg and MeHg follow different absorption and translocation pathways in rice (Meng et al. 2014).

Methylation and demethylation of Hg and effects of iron plaque on Hg transformation

The result of the HPLC-ICP-MS showed that the chemical form of Hg in the rice roots was IHg when the rice was exposed only to IHg, suggesting that there was little or no methylation occurring in vivo in the rice seedlings (Fig. 5a). For the rice exposed to MeHg, approximately 20 and 50 % of the Hg detected in the roots and shoots, respectively, were in the form of IHg (Fig. 5c).

As is known, photochemical degradation is responsible for the MeHg demethylation in some freshwater lakes (Tai et al. 2014). Therefore, the photo-degradation of MeHg in the pretreatment solutions might be the source of IHg in the rice roots and shoots exposed to MeHg. To test this hypothesis, we examined the chemical forms of Hg in nutrient solutions. IHg was hardly detected in the nutrient solutions exposed to MeHg. These results suggested that demethylation could occur in rice roots.

Moreover, the proportion of IHg and MeHg was 1:4 in rice roots and 1:1 in shoots exposed to MeHg (Fig. 5c). It has been confirmed that MeHg is transported more easily from the roots to aboveground parts than IHg. However, the proportion of IHg in the shoots was higher than that in roots when the rice seedlings were exposed to MeHg. A portion of the IHg comes from the demethylation of MeHg in the shoots. Hence, the demethylation of MeHg can occur not only in rice roots but also in shoots.

According to the Lewis acid–base theory, mercuric ions (Hg2+ or CH3Hg+) as soft Lewis acids had high affinities for thiolic groups that were soft Lewis bases. In this study, Hg was mainly in a form similar to Hg-GS upon exposure to IHg or MeHg. Wang et al. (2012) found that the Hg in Brassica juncea was bound to sulfur in a form similar to β-HgS (66–94 %), Hg-cysteine (1–10 %) and Hg-dicysteine (8–28 %). Carrasco-Gil et al. (2013) reported the use of an extended X-ray absorption fine structure (EXAFS) to study the Hg speciation in the roots of Medicago sativa (alfalfa). Their results revealed that root Hg was dominated by two similar forms, Hg-PC (40–46 %) and Hg-Cys ligand (30–38 %). XANES is strongly sensitive to the formal oxidation sate and coordination chemistry of the absorbing atom. However, this technique cannot distinguish among Hg(Cys)2, Hg(GS)2 and HgPCs because they provide very similar spectra (Carrasco-Gil et al. 2011). Therefore, these studies demonstrated that Hg exists in the form of R-S-Hg-S-R in rice roots, which is consistent with our results.

Phytochelatin (PCs) can form complexes with heavy metal ions in the cytosol to prevent metal ions from binding with proteins. These complexes are subsequently transported to the vacuoles (Salt and Rauser 1995). Therefore, PCs play a key role in plant tolerance to heavy metals, which also has been reported by Carrasco-Gil et al. (Carrasco-Gil et al 2011) in Arabidopis thaliana. They found that root growth inhibition was over 80 % in the cad2-1 (with low glutathione content) and cad1-3 (unable to synthesize PCs), whereas the wild-type was inhibited by only 35 % when Arabidopis thaliana was exposed to 10 μM Hg for 4 d (Carrasco-Gil et al 2011). GSH (γ-Glu-Cys-Gly) is a rich sulfur-containing tri-peptide with high affinity for Hg. However, Xiang et al. found that enhanced GSH concentrations did not increase metal resistance (Xiang et al 2001). Therefore, PCs are the major Hg-binding compounds in plants, and GSH plays a key role in metal stress tolerance by regulating plants to make PCs.

Moreover, from the result of XANES, most of the chemical forms of Hg were similar to RS-Hg-S-R and CH3Hg-S-R under MeHg stress. The corresponding proportion of RS-Hg-S-R and CH3Hg-S-R was approximately 1:4, which also indicated that demethylation could occur in vivo in rice seedlings.

The methylation of IHg and Hg0 and the de-methylation of MeHg are two important processes in the cycling of Hg in the environment (Li and Cai 2013). Our results showed that de-methylation can also occur in vivo in plants, and it may be a protective mechanism of rice against the phytotoxicity of MeHg. The mechanism of demethylation of MeHg and the factors influencing this process deserve further study. The formation of iron plaque had no evident effect on Hg transformation in rice plants with either the IHg or MeHg treatment (Fig. 5b, d) in this study, indicating that iron plaque may act as a functional ‘physical barrier’ for blocking MeHg and IHg uptake in rice.

Conclusions

Both Hg species, especially MeHgCl, could be adsorbed by iron plaque on the root surface. The formation of iron plaque reduced the HgCl2 and MeHg uptake and significantly inhibited the MeHg translocation from the roots to the shoots in rice plants. The process of demethylation could occur in vivo in rice plants. The formation of iron plaque had no significant effect on the transformation of Hg.