2017 Volume 23 Issue 5 Pages 661-668
Studies were performed to investigate the Cadmium (Cd) distribution and Cd-binding proteins in rice kernel. Rice seeds [Oryza sativa L. (Akitakaomachi)] were cultured under Cd stress and after harvesting, the Cd distribution was determined using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). The Cd concentrations and amino acid compositions in different storage protein fractions were analyzed using ICP-MS and an amino acid analyzer, respectively. The molecular weights of the Cd-binding proteins were determined by size exclusion chromatography-inductively coupled plasma mass spectrometry (SEC-ICP-MS). The Cd concentrations in rice kernel gradually decreased from the outer bran layers to the endosperm and Cd was particularly concentrated in the embryo, the outer pericarp and the aleurone layer. The Cd concentrations in different proteins fractions decreased in the following sequence: globulin > albumin > glutelin > prolamin. The presence of cysteine and methionine were closely related to the Cd concentration. The molecular weights of three prominent Cd-binding protein fractions were > 2000 k Da, 570 k Da and 131.9 k Da, respectively.
China is the world's largest producer of rice (Oryza sativa L.) and the crop makes up almost half of the total grain output in China. However, owing to rapid industrial development, agricultural soils in China have become contaminated by heavy metals due to a lack of measures to control waste. According to the soil contamination survey results released by the Chinese Ministry of Environmental Protection, 7% of agricultural soils are polluted by Cd, and more than 10% of rice products contain higher Cd levels than that specified by the Chinese Food Hygiene Standard (0.2 mg/kg). Cadmium has no known benefit to human health and excessive intake of Cd poses serious health implications, such as kidney and bone disease, reproductive problems and an increase in the risk of cancer (Nordberg, 2009).
Since Cd pollution was found to be the cause of “Itai-itai disease” in Japan in 1961, techniques for remediating Cd-polluted rice have become a most interesting research point in the agricultural and environmental sciences. To minimize the uptake of Cd by rice, Cd excluder rice breeding, field remediation, and physiological processes involving Cd have been extensively studied. However, because of the high investment and a long period, these strategies could not solve the rice Cd problem in China. Therefore, a lot of Cd-polluted rice were produced and stored every year, causing barn resources wasted and increasing food safety risk. Effective remediation techniques are important for solving the problem of Cd contamination of rice and improving the use of Cd-contaminated rice. Given that food processing results in compositional changes and nutrition restructuring of food materials, studies clarifying Cd distribution and speciation in rice would be important for developing fit-for-purpose processing methods for reducing Cd intake from rice consumption.
In general, mineral elements are not homogeneously distributed in grain. Rice bran, which constitutes a considerable portion of the whole kernel, is normally removed before the milling of the rice. Rice bran has been reported to contain a higher Cd content than the endosperm (Williams et al., 2009) and similar results were also found for wheat, barley, and other cereal kernels (Persson et al., 2009). As rice bran consists of embryo, aleurone layer and outer pericarp, the Cd concentrations in the bran and the polished rice would not actually reflect the specific Cd distribution in the rice kernel. Laser ablation ICP-MS (LA-ICP-MS) has been shown to provide an effective method for metal localization. This was revealed by Cakmak et al. (2010) and Choi et al. (2014), who analyzed element distributions in wheat and rice kernels and provided visual interpretations of the Zn and As distributions, respectively. For both studies, it was shown that higher Zn and As concentrations were found in the outer parts of the grain. It was further stated that some degree of rice milling may be effective in reducing the intake of As. Brier et al. (2015) reported that there were small differences in element distributions in rice with Mn, Si, Ca, and Sr concentrations being higher in the outermost bran layers, while K, Mg, P, Fe, Zn, and Cu were more concentrated in the aleurone layer. To date, however, little information has been reported on the Cd distribution in the rice kernel.
Cadmium speciation is not only important in the context of bioavailability and toxicology, and also important for the Cd reduction technology in processing. In general, mineral elements are usually combined with nutrient substances, such as organic acids, small peptides, and especially metal-binding proteins (Vacchina et al., 1999; Majewska et al., 2011). Tian et al. (2014) found that Cd in rice grain was mainly bound to protein, and that there was an extremely low concentration of Cd in starch compared with other nutrient compounds. However, most Cd-binding protein studies have focused on Cd transporter proteins, such as Zip, COPT, and Nramp (Clemens et al., 2013; Colangelo et al., 2006). Based on these results, some Cd accumulation proteins have been reported in recent years, however, there have been few attempts to study the Cd-binding storage proteins in rice kernels. The compounds in rice grain mainly include storage proteins, starch, cellulose, organic acids and other nutrients, and according to the characteristics, the storage protein can be divided into four fractions: albumin, globulin, glutelin and prolamin (Osborne, 1907). Knowledge of which fractions are Cd bindable and their molecular weight characteristics can aid in the development of appropriate processing methods for reducing Cd in rice products.
In this study, LA-ICP-MS has been used to reveal the spatial distribution of Cd in rice kernels. In addition, Cd concentrations in different storage protein fractions were measured and SEC-ICP-MS was used to determine the molecular weights of the Cd-binding proteins. Results should assist in the design of methodologies and guidelines for controlling Cd content in the rice processing, with the ultimate aim of lowering the health risk to the consumer.
Rice cultivation and sample collection Rice seeds [Oryza sativa L. (Akitakaomachi)] were obtained from Shenyang Agricultural University, Shenyang, Liaoning Province, China. After seeds were submerged in H2O2 (10% v/v) for 30 min and in 0.1% NaClO4 for 10 h, seeds were rinsed thoroughly with deionized water and maintained in an incubator at 30°C for 24 h to germinate. The germinated seeds were sown and cultivated in trays filled with quartz sand. When the third leaf emerged, the seedlings of uniform size were transferred to 30-L (30 × 30 × 40 cm) pots. Three holes were drilled in each pot and two seedlings were cultivated from each hole. The Cd-contaminated soil (Cd concentration, 100 mg·kg−1) was provided by the Rice Research Institute of Shenyang Agricultural University. The rice was harvested after the maturity stage.
Sample preparation and analysis
Laser ablation ICP-MS After sampling rice kernels from the field, grains were embedded in resin, then sectioned and polished to obtain a smooth surface. A LA system (New Wave Research UP213 Nd:YAG, USA) was coupled to the ICP-MS (Agilent 7700 series, USA) to perform spatially resolved measurement of the Cd distribution. 12C (Carbon-12) was determined to ensure the accuracy of the results calibration. Table 1 summarizes the experimental conditions for LA-ICP-MS analysis.
| LA | ICP-MS | ||
|---|---|---|---|
| Energy level | 700 V | R.F. power | 1460 W |
| Scan mode | Line scan | Sampling depth | 8.4 mm |
| Spot size | 150 µm | Carrier gas flow rate | 1.00 L min−1 |
| Repetition rate | 20 Hz | Integration time | 0.05s |
| Scan rate | 50 µm·s−1 | Cd isotope | 111 |
Rice de-hulling and shattering After sampling rice grains, husks were removed using a laboratory-scale de-hulling machine (Satake THU-35C, Japan). The brown rice was then pulverized in an ultra-centrifugal mill (Retsch ZM-200, Germany). Meanwhile, samples were dried in an oven (DHG-9140A, China) at 40°C for 24 h until constant weight was achieved.
Separation of proteins in rice The separation procedure for different protein fractions in rice was performed according to the method of Fang et al. (2010) with minor modifications, because the Cd-binding proteins were not stable in acidic conditions. Acetone was used to precipitate the proteins. In total, 50 g of dried rice powder was soaked in 500 mL of hexane. After discarding the supernatant, the precipitate was dried in a nitrogen evaporator to obtain the defatted rice flour. The defatted rice flour was extracted by sequentially stirring twice with ultrapure water, 5% NaCl, 0.02 M NaOH, 70% alcohol at 23°C for 2 h, followed by centrifugation at 10,000 rpm for 10 min (Hitachi CR22G-III, Japan). Albumin, globulin, and glutelin were precipitated after adding acetone (4°C) to the supernatant. The protein mixture was then freeze-dried to obtain the protein powder. The prolamins were concentrated by a nitrogen evaporator.
Amino acid composition About 10 mg of each sample was hydrolyzed with 6 mL of 6 M HCl in a sealed air-evacuated tube at 110°C for 24 h. The hydrolysate was diluted to 50 mL and then 1 mL of the diluted hydrolysate was transferred to a centrifuge tube, which was put into a nitrogen evaporator to remove HCl and water. The residue was completely dissolved in 1 mL of 0.02 M HCl through vigorous shaking and then centrifuged at 10,000 r/min for 15 min. About 0.8 mL of supernatant was put into an auto-sample bottle and then analyzed using an amino acid auto-analyzer (L-8900, Hitachi, Japan). The content of each amino acid in the sample (mg/g) was calculated based on analysis of a standard sample. The assay for each sample was conducted three times.
The molecular weights of the Cd-binding proteins The molecular weights of the Cd-binding proteins were determined using SEC-ICP-MS. The protein powder sample (0.25 g) was dissolved in 5 mL of Tris buffer (10 mM, pH 7.5), then filtered through a 0.45-µm syringe filter to remove particulates before SEC-HPLC-ICP-MS analysis. The proteins and standards were separated on a Superdex Peptide 200 GL column (10–600 kDa). Calibration standards were analyzed with an UV detector with wavelength set at 280 nm. The sample injection volume was 20 µL. The mobile phase (10 mM Tris buffer; pH 7.5) was pumped through the column isocratically at 0.4 mL min−1. The coupling between the column outlet and the sample introduction system of the ICP-MS was achieved using PEEK tubing (length 300 mm; i.d. 0.25 mm). Potential polyatomic interferences at mass to charge ratios of were monitored using a dwell time of 0.01 s and interferences were removed by the octopole reaction system operating in the hydrogen gas mode with a flow rate of 3.5 mL min−1. Other ICP-MS instrumental conditions were as follows: RF power, 1450 W; plasma Ar gas flow rate, 15.0 L min−1; carrier Ar gas flow rate, 1.05 L min−1.
Determination of Cd Samples (0.25 g, three replicates) were weighed in a 25-mL Teflon vessel, digested with 6 mL of ultrapure HNO3 and 2 mL of H2O2 in a microwave digestion system (CEM MARS-6, USA), diluted to 100 mL with ultrapure water (Millipore Milli-Q Plus, USA), and analyzed by ICP-MS (Agilent 7700x). A rice reference sample (GBW10010 RICE, General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China, China) of certified elemental content was also included in the analytical sequence.
Statistical analysis Results were analyzed using the PASW Statistics 18. One-way analysis of variance (ANOVA) (P < 0.05) was performed to verify significant differences in the mean values of the Cd concentrations. A Duncan's comparison procedure was used (P < 0.05) to test the difference in the composition. Pearson phenotypic correlations were calculated to establish the relationship between amino acids and Cd concentration.
Cd distribution in rice kernels The Cd concentration of the rice kernels was determined by ICP-MS, and they are 1.76 ± 0.15 mg/kg. The spatial distribution of Cd within the rice kernel was performed with LA-ICP-MS (Figure 1). A significant decreasing trend was observed from the outer surface to the inner part and Cd was particularly concentrated in the embryo, the outer pericarp and the aleurone layer. The Cd concentration in the inner endosperm fraction was relatively low and approximately half of the highest Cd concentration. The Cd concentration in the outer layer endosperm was between that of the aleurone layer and the endosperm. It was very interesting to note that the boundary region between the outer layer endosperm and the embryo had an extremely low Cd concentration compared with the outer layer endosperm and the embryo, the value in this part being similar to that of the inner endosperm.
Distribution of Cd (cross section) in rice kernel. The LA scanning of the seed cross section started at the bottom of the grain (site A) and finished at the embryo (site B). The diagram on the right shows the variation in Cd concentration along the entire cross section of the LA scan line, the position of outer pericarp, aleurone layer, outer layer endosperm, inner endosperm, embryo were marked as 1, 2, 3, 4 and 5 approximately.
Cd concentration in different storage protein fractions There are four kinds of storage proteins in rice: glutelin, albumin, globulin, and prolamin, which account for 78.6%, 10.3%, 9.5%, and 1.6% of the total protein, respectively. The Cd content of the different storage protein fractions was significant dissimilar (P < 0.05) and values are presented in Table 2. Comparison of the Cd concentrations in the protein fractions revealed that the globulin portion had the highest Cd concentration (about 14.3 ± 4.24 mg/kg) and that the Cd concentration in the largest protein fraction (glutelin) was 7.15 ± 0.28 mg/kg. The Cd concentration in prolamin was extremely low, just 1.67 ± 0.26 mg/kg, and for albumin it was 9.97 ± 2.50 mg/kg. The Cd bind ability in different storage protein fractions were: globulin > albumin > glutelin > prolamin.
| Protein fractions | Percentages of protein fractions (%) | Cd concentration (mg/kg) |
|---|---|---|
| albumin | 10.3 | 9.97±2.50b |
| globulin | 9.5 | 14.33±4.24a |
| glutelin | 78.6 | 7.15±0.28c |
| prolamin | 1.6 | 1.67±0.26d |
Note: Different letters in the columns indicate significant differences at P < 0.05
Correlation between amino acid composition and Cd concentration in different proteins As shown in Table 3, 17 amino acids and the Cd concentrations were determined in different protein fractions. As the Cd concentration were globulin> albumin> glutelin> prolamin, two sulfamino acids (Cys and Met) concentration in the different protein fraction showed a similar trend, the Cys and Met in globulin being substantially higher than in the other protein fractions. Correlation analysis showed that Cys and Met were closely correlated to the Cd concentration in rice (P < 0.01). The correlation coefficients were 0.994 and 0.995, respectively. The results show that the correlation between Cd and other amino acids were not significant (P > 0.05). The results suggest that the Cd binding ability was related to the scale of sulfamino acids concentration in proteins.
| Amino acid | Contents (%) | Correlation coefficients (with Cd concentration) | |||
|---|---|---|---|---|---|
| albumin | globulin | glutelin | prolamin | ||
| Asp | 9.00 | 7.19 | 10.24 | 10.00 | −0.859 |
| Thr | 4.34 | 3.03 | 3.81 | 4.40 | −0.765 |
| Ser | 4.83 | 5.65 | 4.97 | 4.16 | 0.949 |
| Glu | 15.88 | 21.00 | 20.82 | 16.99 | 0.432 |
| Gly | 5.82 | 5.77 | 4.63 | 7.08 | −0.457 |
| Ala | 7.02 | 5.62 | 5.50 | 9.25 | −0.774 |
| Cys | 2.65 | 3.44 | 1.78 | 0.84 | 0.994* |
| Val | 6.93 | 6.00 | 6.24 | 6.19 | 0.007 |
| Met | 2.50 | 3.00 | 2.21 | 1.36 | 0.995* |
| Ile | 4.37 | 3.24 | 4.48 | 4.59 | −0.088 |
| Leu | 8.70 | 7.21 | 8.29 | 7.55 | 0.841 |
| Tyr | 4.95 | 5.17 | 5.14 | 3.39 | 0.153 |
| Phe | 5.69 | 5.30 | 5.99 | 5.14 | −0.693 |
| Lys | 6.09 | 3.74 | 4.09 | 6.74 | −0.812 |
| His | 2.98 | 2.60 | 2.68 | 3.34 | 0.638 |
Note: * indicates significant positive correlation (P < 0.01).
The molecular weights of Cd-binding proteins in globulin Calibration of the SEC (size exclusion chromatography) column was accomplished with a standard mixture of aldolase (158 kDa, 6 mg/mL), ovalbumin (44 kDa, 3 mg/mL), carbonic anhydrase (29 kDa, 3 mg/mL), and aprotinin (6,512 Da, 3 mg/mL). The experimental results indicated good linear response for log10 molecular weight versus retention time (min) (y = −11.422x + 91.821; R2 = 0.9968) as shown in Figure 2.
SEC-HPLC-UV chromatogram of mixed standards of various molecular weights. (Inset) Calibration of standards (molecular weight) for SEC column (Superdex 200 10-300 GL). Numbers correspond to the standards of known molecular mass: 158 kDa (32.38 min), 44 kDa (38.44 min), 29 kDa (41.40 min), 13.7 kDa (44.60 min), and 6.5 kDa (48.09 min).
The results for molecular weight analysis of the Cd-binding proteins in globulin are shown in Figure 3. Five prominent peaks for proteins were observed. The retention times and molecular weights of the protein peaks were 19.39 min (> 2000 kDa), 33.31 min (131.9 kDa), 39.67 min (36.8 kDa), 44.93 min (12.8 kDa), and 52.36 min (2.9 kDa), respectively. Three peaks for Cd were monitored: 19.84 min (I), 26.01 min (II), and 34.19 min (III). As shown in Figure 3, the (1) and the (2) protein peaks had similar retention times with the (I) and the (III) peaks for Cd, respectively. No clear protein peak corresponding to peak (II) for Cd occurred, and the expected molecular weight of these fraction is 572.1 kDa. These results suggested that the high molecular weight (HMW) fraction (> 2000 kDa) and the protein fraction (131.9 kDa) were the main Cd-binding protein fractions, and the protein with molecular weight around 572.1 kDa also has obviously Cd binding ability. Cadmium-binding proteins with molecular weight ≤ 36.8 kDa were not found in this investigation.
SEC-ICP-MS analysis of the Cd-binding proteins in globulin extracted from rice kernel. The lower curve shows the variation of Cd intensity with elution time, the mainly three peaks were marked as I, II, III, and the higher curve shows the variation in protein intensity at UV 280 nm with elution time, the five protein peaks were marked as 1, 2, 3, 4 and 5 respectively.
The Cd distribution in rice has seldom been reported, but its distribution in other grains had been studied. In this study, we found out that the Cd distribution in rice kernels was non-uniform. This is similar to that found for the Cd concentration in durum wheat, Cheli et al. (2010) found that the Cd in bran by-products of durum wheat (Triticum turgidum L. ssp. durum) were significantly higher than that in wheat grain and semolina. In contrast, Brier et al. (2015) studied the distribution of minerals in wheat grains (Triticum aestivum L.) and found that Cd was distributed almost uniformly and did not exhibit a transition boundary between the bran layers and the endosperm. This was despite the fact that the Cd contents in the wheat samples studied by Brier et al. (2015) were extremely low (< 0.02 mg/kg). Therefore, it may have been difficult to distinguish the Cd concentrations in the different compartments. Compared with other element distribution studies, it would appear that many mineral elements are not evenly distributed in rice. In general, all the mineral elements tend to be highly concentrated in the embryo, the outer pericarpand the aleurone layer, similar to the Cd distribution in rice (Cakmak et al., 2010). However, in this work Cd was lower concentrated in the boundary region between the endosperm and the embryo and this phenomenon has not been reported. Lu et al. (2013) studied the distribution of mineral elements in rice, the Zn Fe K Ca and Mn were not homogenously distributed, but the boundary region had normal mineral concentration compared with endosperm and the embryo. The reason for the distribution of lower Cd in this region requires further study.
Previous studies on rice have shown that the Cd accumulation ability varied with genotype. He et al. (2006) studied the Cd concentration of 38 different rice varieties cultivated in the same field, and found that the indica-type varieties accumulated significantly more Cd than the japonica type. Similar results for the Cd accumulation capabilities of different rice varieties have been reported, the Cd accumulation capability decreasing in the following sequence: indica > hybrid > japonica (Ye et al., 2012). Given that indica normally has a higher protein concentration than japonica, the Cd concentration exhibited a similar trend with protein concentration among the different rice varieties. Moreover, Cd was mainly located at the embryo, the outer pericarp and the aleurone layer, this distribution trend being similar to that of protein in the rice kernel (Itani et al., 2002). Tian et al. studied the Cd concentration in different nutritional components and found that Cd in rice was mainly bound with proteins rather than with other substances such as starch. For instance, the Cd concentration was 3.64 mg/kg in the protein faction and 0.128 mg/kg in the starch fraction. It was speculated that Cd in the rice kernel was mainly bound with proteins (Tian et al., 2014). The proteins can be grouped into four Osborne fractions (glutelin, albumin, globulin, and prolamin). The four fractions have different structural characteristics as well as different element binding capabilities. In this study, globulin has been found to have the highest Cd concentration than other protein fractions. Globulin is a sulfur-rich protein with metabolic activity in rice, and is usually bound to various ions and activating groups to perform its physiological functions. Therefore, in most studies, globulin was the predominant metal-binding protein. For example, it is found that in both wheat and soybean, the highest concentrations of metals (Cu, Fe, Mn, and Zn) were observed in those extracts associated with globulin-type proteins (Bittencourt et al., 2012). Most Cd transport proteins in rice are globulins, such as OsIRT1, OsNramp1, OsHMA3, and OsLCT1 (Uraguchi et al., 2012). An early study indicated that globulin and albumin were mainly located in the outer part of the rice kernel, that is the aleurone layer and the outer pericarp (Juliano., 1999). However, glutelin was mainly accumulated in the endosperm, and prolamin was almost evenly distributed in the rice kernel. These results suggested that globulin had a distribution similar to that of Cd. On the basis of the above results, globulin would have the highest Cd-binding capability.
To gain further insights into the role of Cd-binding proteins, the concentrations of several amino acids together with Cd concentrations were examined.
Effective metal-ion binding sites in proteins have been reported in recent years, including ATCUN motif, poly-His, poly-Cys and Met-containing sequences (Kozlowski et al., 2013). However, the Cd-binding site in rice storage proteins has not been reported. Most ion binding site studies are limited to metallothionein, phytochelatins and some bacteria or viral proteins. Metallothionein, as a well-known metal-binding protein, contains approximately 30% Cys for all its amino acid residues and the Cys side groups were confirmed as the main metal-ion binding site (Romero et al., 1998). Rowinska-Zyrek et al. found that the −Cys−Cys− motif was an essential anchoring site for Ni2+, Bi3+, Zn2+ and Cd2+ in a bacteria protein (Rowinska-Zyrek et al., 2011). A previous study by extended X-ray absorption fine structure (EXAFS) analysis showed that the Cu+ binding sites with prion protein (Met and His) were the main Cu+ binding sites (Badrick et al., 2009). Fang et al. (2010) investigated a Se-binding protein in rice and found that Se was mainly concentrated in glutelin besides globulin and mainly bound with Met. In this study, we also found that the two sulfamino acids (Cys and Met) were closely correlated to the Cd concentration in rice. These results indicated that, the sulfamino acids including Cys and Met were the main Cd-ion binding sites in rice proteins.
Besides the amino acid composition of the protein, molecular weight is another important metric for identification of Cd-binding proteins. The molecular weights of Cd-binding proteins in different botanical species show a large variation. For example, some results have indicated that Cd was mainly bound to high-molecular-weight proteins. Koplík et al. (2002) characterized Cd-binding proteins in soybean and found that Cd was mainly bound to high-molecular-weight proteins: 62% of Cd was bound to a protein of molecular weight 110 k Da, and 21%, 5%, 9%, and 3% Cd were bound to proteins with molecular weights of 17 kDa, 1.1 kDa, 0.8 kDa, and 0.4 kDa, respectively. However, the molecular weights of Cd-binding proteins in Tenebrio molitor were relatively low, about 7.13 kDa (Pedersen et al., 2007). In this study, we found out that mainly 3 protein fractions have obviously Cd binding ability. They are > 2000 kDa, 572.1 kDa, and 131.9 kDa. The protein with low molecular weight (≤ 36.8 kDa) could not easily bind with Cd. Similar to what was found in this study. Fang et al. (Fang et al., 2010) reported that Se was mainly bound to the high-molecular-weight fraction of the Se-containing storage proteins. This might be due to Se having similar protein binding properties as Cd, with both of these elements having a tendency to bind to the high-molecular-weight protein fractions in rice. To gain further information on the characteristics of Cd-binding proteins in rice, further work is needed on the structure and formation mechanisms of these Cd-binding proteins.
In this study, Cd was mainly concentrated in the embryo, the outer pericarp and the aleurone layer, whereas the Cd content in the endosperm fraction and in the boundary region between the embryo and the endosperm was relatively low. Globulin has the highest Cd-binding capability. The two sulfamino acids (Cys and Met) exhibited a similar trend to the Cd distribution, suggesting that the sulfamino acids played an important role in Cd-protein binding capability. The molecular weights of the main Cd-binding proteins in globulin were > 2000 kDa, 572.1 kDa, and 131.9 kDa.
