**4.3 Comparison of the symplastic Cd contents in the roots between the two methods**

To examine the validity of the new method for evaluating the symplastic Cd content in roots using 113Cd and 114Cd enriched isotopes, we compared the symplastic Cd content in roots using differences in the amounts of Cd absorbed at 2°C and 25°C with unlabeled Cd with the results obtained in the present study using the new method. In conventional Cdabsorption experiments, the Cd contents in roots at 40 and 400 nmol Cd in a 25°C treatment were 19.2 ± 1.6 and 84.4 ± 3.4 mg kg–1 (dry weight), respectively (Table 3). In contrast, the Cd

experiment (Fig. 1). The total 113Cd content in the roots at 40 and 400 nmol Cd was 23.0 ± 4.3 and 87.7 ± 5.6 mg kg–1 (dry weight), respectively (Table2). In contrast, the 114Cd content at 40 and 400 nmol Cd was 117.3 ± 9.4 and 644.5 ± 33.7 mg kg–1 (dry weight), respectively (Table2). The purification rate of the 114Cd-enriched stable isotope used in the present study was 93.60%; whereas, the composition rate of 113Cd in the 114Cd-enriched stable isotope was 5.6%. The total 114Cd content in the roots after desorption of 20 μmol 114Cd was approximately 5.5-fold higher than that using 2 μmol 114Cd (Table 2),suggesting that the apoplastically bound 113Cd content, derived from the enriched isotope 114Cd, increased with an increase in the concentration of 114Cd in the desorption solution. Actually, the apoplastically bound 113Cd contents, derived from the enriched isotope 114Cd (2 and 20 μmol) were 6.6 ± 0.5 and 36.6 ± 1.8 mg kg–1, respectively (Table 2); these values were calculated using equation in Fig.3. The contribution rate of 113Cd content derived from the enriched stable isotope of 114Cd for total 113Cd in the roots was 28.6% for the 40 nmol 113Cd treatment. In contrast, the contribution rate of 113Cd content derived from 114Cd for total 113Cd content in the roots was 41.8% for the 400 nmol 113Cd treatment (Table 2). These results indicate that the 113Cd derived from the enriched stable isotope of 114Cd must be subtracted from the total 113Cd content in the roots to evaluate the symplastic 113Cd in the roots. The symplastic 113Cd contents for the 40 and 400 nmol treatments, calculated using equation in Fig.3, were 16.4 ± 3.7 and 51.0 ± 3.8 mg kg–1, respectively (Table 2). In the present study, we disregarded the contribution of 114Cd derived from the enriched isotope of 113Cd because the composition rate of 114Cd in the enriched isotope of 113Cd was considerably lower than that

of 113Cd in the enriched isotope of 114Cd.

Fig. 3. Calculation of symplastic 113Cd content in roots.

**4.3 Comparison of the symplastic Cd contents in the roots between the two methods**  To examine the validity of the new method for evaluating the symplastic Cd content in roots using 113Cd and 114Cd enriched isotopes, we compared the symplastic Cd content in roots using differences in the amounts of Cd absorbed at 2°C and 25°C with unlabeled Cd with the results obtained in the present study using the new method. In conventional Cdabsorption experiments, the Cd contents in roots at 40 and 400 nmol Cd in a 25°C treatment were 19.2 ± 1.6 and 84.4 ± 3.4 mg kg–1 (dry weight), respectively (Table 3). In contrast, the Cd contents in roots at 40 and 400 nmol in the 2°C treatment were 4.1 ± 0.3 and 28.1 ± 0.73 mg kg–1 (dry weight), respectively.

The symplastic Cd contents at 40 and 400 nmol were estimated to be 15.1 ± 1.3 and 56.4 ± 2.7 mg kg–1, respectively, which was evaluated using the difference in the amount of Cd absorbed at 2°C and at 25°C.

In the 113Cd-absorption experiment, the symplastic 113Cd contents in the roots at the 40 and 400 nmol 113Cd treatments were 16.4 ± 3.7 and 51.0 ± 3.8 mg kg–1, respectively (Table 2, 3). Therefore, the symplastic 113Cd content after using the enriched isotopes was similar to the symplastic Cd content evaluated from the difference between the amount of Cd absorbed at 2°C and at 25°C. These results indicate that it is possible to evaluate the contents of symplastic Cd in roots using 113Cd and 114Cd enriched isotopes using the method proposed in the present study.

There have been many reports on Cd absorption in roots eliminating apoplastic bound Cd in Durum wheat, soybean and hyperaccumulator plants, such as *Thlaspi caerulescens*  (Cataldo et al. 1983; Hart et al. 1998, 2002, 2006; Zhao et al. 2002). In these studies, the symplastic Cd content in the roots was determined by subtracting the Cd content in the roots at 2°C from the Cd content in the roots at 25°C; the Cd content was determined using a radioisotope of 109Cd or a metabolic inhibitor. These methods have frequently been used to evaluate nutrient element absorption in roots. Radioisotopes in solute were the most useful markers used in these studies because they are chemically similar to the solute and can be distinguished from non-labeled solutes already contained in the roots (Davenport 2007). However, there are limitations to this method, including radioisotope administrative restriction and the restricted halflife of the radioisotope. Although the method involving a temperature difference between 2 and 25°C that was used in the present study is easy to handle because there is no radioisotope administrative restriction, there is, however, a limitation to this method: the symplastic Cd content in the roots cannot be evaluated using the same seedlings. This method has the advantage of no radioisotope administrative restriction and no restrictive radioisotope half-lives. In addition, this method uses half the number of seedlings that are required for the method using the temperature difference between 2 and 25°C because the symplastically absorped Cd in the roots can be evaluated using roots from the same seedlings. In addition, the method proposed in the present study is applicable to other plants, not only *S. melongena*. We indicated that it is possible to evaluate symplastic Cd in roots using 113Cd and 114Cd enriched isotopes. The proposed method will contribute to research on symplastic ion absorption in plant roots stated below.


Table 2. 114Cd and 113Cd content in roots (modified from Mori et al. 2009a )

Application of Enriched Stable Isotopes in Element Uptake and Translocation in Plant 65

enriched isotopes 113Cd and 114Cd. In time course-dependent experiments, the symplastic 113Cd absorption rate for both plants increased with time (Fig. 4). In addition, the symplastic 113Cd absorption rate of *S. melongena* was slightly higher than that of *S. torvum* at 4 h (Fig. 4). We examined kinetics analysis by similar method using enriched stable isotopes of 113Cd and 114Cd (Mori et al. 2009b). A kinetic study revealed that the symplastic Cd concentrations in the roots increased with increasing external Cd concentrations, but saturated at a higher concentration. The saturated curve obtained in this study suggests that absorption in both cultivars is mediated by a transporter that exhibits a similar affinity for Cd.. Moreover, the symplastic Cd concentrations slightly differed between the roots of *S. melongena* and *S. torvum*. Based on the reaction curves obtained, the Km value was estimated to be 380 and 352 nmol L−1 for *S. melongena* and *S. torvum*, respectively. The corresponding Vmax values were 152 and 101.5μg root dw−1 0.5 h−1. The Vmax value of *S. melongena* was approximately 1.5-fold higher than that of *S. torvum*, which suggests that the density of the Cd transporter in the root cell membranes of *S. melongena* is higher than in *S. torvum*. In this experiments, If the symplastic Cd absorption in roots is estimated by the conventional method using the difference of temperature at 2 and

25°C, it is required time consuming and double seedlings for experiment preparation.

For biological system analysis, the application of ICP-MS in enriched stable isotope tracer experiments has increased because ICP-MS has now become the preferred technique. An enriched stable isotope technique would be potent and useful tool for biological system experiments including element uptake, distribution and chemical form in plants. In this chapter, we introduced our one example of element uptake system using enriched isotope of 113Cd and 114Cd. This method has several merits compared to conventional methods if ICP-MS instrument is able to use. Application of enriched isotopes such as 113Cd and 114Cd would attain a new insight for plant biological system and will become a new tool to

This work was partly supported by the Program for the Promotion of Basic Research

Arao, T.; Takeda, H. & Nishihara, E. (2008). Reduction of cadmium translocation from roots

Cataldo, D.A.; Garland, T.R. & Wildung, R.E. (1983). Cadmium uptake kinetics in intact

Codex Alimentarius Commission 2005: Joint FAO/WHO Food Standards Programme.

175, Appendix XXVI. The Hague, the Netherlands. Available at URL: http://www.codexalimentarius. net/web/reports.jsp]ALINORM 05/28/12

rootstock. *Soil Science and Plant Nutrition*, Vol. 54, pp.555–559.

soybean plants. *Plant Physiology*, Vol.73, pp.844–848.

to shoots in eggplant (Solanum melongena) by grafting onto Solanum torvum

Twenty-eighth session, 4–9 July 2005, Rome, Italy. Report of the 37th session of the Codex Committee on Food Additives and Contaminants, 25–29 April 2005. Para.

**5. Conclusion** 

evaluate element behavior in plants.

Activities for Innovative Biosciences (PROBRAIN).

**6. Acknowledgment** 

**7. References** 


Table 3. Comparison of the symplastic Cd content in roots (modified from Mori et al. 2009a )

Fig. 4. Symplastic Cd absorption in roots of *Solanum melongena* and *Solanum torvum* with time. Experiment method is followed by the procedure illustrated in Fig.1 (modified from Mori et al.2009b)

We used the new method using enriched stable isotopes for evaluation of symplastic Cd absorption in roots of *solanaceous* plants (*Solanum melongena* and *Solanum torvum*) with contrasting root-to-shoot Cd translocation efficiencies (Mori et al. 2009a,b).

It is well known that efficiency of Cd translocation from roots to shoots is significantly higher in *S. melongena* than *S. torvum* (Arao et al. 2008, Mori et al. 2009a,b, Yamaguchi et al. 2011 ). Takeda et al.(2007) found that the Cd concentration in eggplant fruits could be reduced by grafting with *Solanum torvum* rootstock. Additionally, Arao et al.(2008) reported that although the Cd accumulation in shoots of *S. torvum* was lower than that found in *S. melongena*, there was no difference in the Cd content in roots of both plants when grown in culture solution. This result suggests that *S. torvum* develops noteworthy physiological mechanisms to suppress Cd translocation from roots to shoots, corresponding to the results observed in previous reports (Arao et al., 2008). Arao et al. (2008) suggested that symplastic Cd absorption and xylem loading capacity might be ascribed to the difference of Cd concentration in the shoots of *S. melongena* and *S. torvum*. We evaluated the symplastic Cd absorption rate in roots using enriched isotopes 113Cd and 114Cd. In time course-dependent experiments, the symplastic 113Cd absorption rate for both plants increased with time (Fig. 4). In addition, the symplastic 113Cd absorption rate of *S. melongena* was slightly higher than that of *S. torvum* at 4 h (Fig. 4). We examined kinetics analysis by similar method using enriched stable isotopes of 113Cd and 114Cd (Mori et al. 2009b). A kinetic study revealed that the symplastic Cd concentrations in the roots increased with increasing external Cd concentrations, but saturated at a higher concentration. The saturated curve obtained in this study suggests that absorption in both cultivars is mediated by a transporter that exhibits a similar affinity for Cd.. Moreover, the symplastic Cd concentrations slightly differed between the roots of *S. melongena* and *S. torvum*. Based on the reaction curves obtained, the Km value was estimated to be 380 and 352 nmol L−1 for *S. melongena* and *S. torvum*, respectively. The corresponding Vmax values were 152 and 101.5μg root dw−1 0.5 h−1. The Vmax value of *S. melongena* was approximately 1.5-fold higher than that of *S. torvum*, which suggests that the density of the Cd transporter in the root cell membranes of *S. melongena* is higher than in *S. torvum*. In this experiments, If the symplastic Cd absorption in roots is estimated by the conventional method using the difference of temperature at 2 and 25°C, it is required time consuming and double seedlings for experiment preparation.

#### **5. Conclusion**

64 Radioisotopes – Applications in Physical Sciences

**40nM Symplastic 113Cd Cd(25**°**C-2**°**C) Cd(25**°**C) Cd(2**°**C)**  16.4±3.7 15.1±1.3 19.2±1.6 4.1±0.3 **400nM Symplastic 113Cd Cd(25**°**C-2**°**C) Cd(25**°**C) Cd(2**°**C)**  51.0±3.8 56.4±2.7 84.4±3.4 28.1±0.7 Table 3. Comparison of the symplastic Cd content in roots (modified from Mori et al. 2009a )

Fig. 4. Symplastic Cd absorption in roots of *Solanum melongena* and *Solanum torvum* with time. Experiment method is followed by the procedure illustrated in Fig.1 (modified from

contrasting root-to-shoot Cd translocation efficiencies (Mori et al. 2009a,b).

We used the new method using enriched stable isotopes for evaluation of symplastic Cd absorption in roots of *solanaceous* plants (*Solanum melongena* and *Solanum torvum*) with

It is well known that efficiency of Cd translocation from roots to shoots is significantly higher in *S. melongena* than *S. torvum* (Arao et al. 2008, Mori et al. 2009a,b, Yamaguchi et al. 2011 ). Takeda et al.(2007) found that the Cd concentration in eggplant fruits could be reduced by grafting with *Solanum torvum* rootstock. Additionally, Arao et al.(2008) reported that although the Cd accumulation in shoots of *S. torvum* was lower than that found in *S. melongena*, there was no difference in the Cd content in roots of both plants when grown in culture solution. This result suggests that *S. torvum* develops noteworthy physiological mechanisms to suppress Cd translocation from roots to shoots, corresponding to the results observed in previous reports (Arao et al., 2008). Arao et al. (2008) suggested that symplastic Cd absorption and xylem loading capacity might be ascribed to the difference of Cd concentration in the shoots of *S. melongena* and *S. torvum*. We evaluated the symplastic Cd absorption rate in roots using

Mori et al.2009b)

For biological system analysis, the application of ICP-MS in enriched stable isotope tracer experiments has increased because ICP-MS has now become the preferred technique. An enriched stable isotope technique would be potent and useful tool for biological system experiments including element uptake, distribution and chemical form in plants. In this chapter, we introduced our one example of element uptake system using enriched isotope of 113Cd and 114Cd. This method has several merits compared to conventional methods if ICP-MS instrument is able to use. Application of enriched isotopes such as 113Cd and 114Cd would attain a new insight for plant biological system and will become a new tool to evaluate element behavior in plants.

### **6. Acknowledgment**

This work was partly supported by the Program for the Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).

#### **7. References**


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**4** 

**Li** 

*Japan* 

Sun-Chan Jeong

**Diffusion Experiment in Lithium Ionic** 

*High Energy Accelerator Research Organization (KEK) 1-1 Oho* 

*Institute of Particle and Nuclear Studies (IPNS)* 

**Conductors with the Radiotracer of <sup>8</sup>**

Radioactive nuclides have been used in materials science for many decades. Besides their classical application as tracers for diffusion studies, nuclear techniques (i.e. Mössbauer Spectroscopy, Perturbed Angular Correlation, β-Nuclear Magnetic Resonance, Emission Channeling, etc.) are now being routinely used to gain microscopic information on the structural and dynamical properties of the bulk of materials via hyperfine interactions or emitted particles themselves (Wichert & Diecher, 2001). These nuclear techniques were primarily developed in nuclear physics for detecting particles or γ-radiations emitted during the decay of the radioactive nuclides. More recently these techniques have also been applied to study complex bio-molecules, surfaces, and interfaces (Prandolini, 2006). With the advent of most versatile 'radioactive isotope beam (RIB) factory' represented by the on-line isotope separator (ISOL)–based RIB facility (see Fig. 1), the possibilities for such investigations have

At the tandem accelerator facility of Japan Atomic Energy Agency (JAEA)-Tokai, a RIB facility, TRIAC (Watanabe et al., 2007)-Tokai Radioactive Ion Accelerator Complex- is operating since 2005. In the facility, short-lived radioactive nuclei produced by proton or heavy ion induced nuclear reactions can be accelerated up to the energy necessary for experiments. The energy is variable in the range from 0.1 to 1.1 MeV/nucleon, which is especially efficient for studies of the bulk of materials by using the RIBs as tracers. It allows us to implant (incorporate) the RIBs into specimens at a proper depth, avoiding the difficulties caused by the surface (e.g. diffusion barrier like oxide layers that often hampers the incorporation of those radioactive isotope probes into the materials of interest). In the facility, the separation and the implantation of radioactive probes are integrated into one device, as shown in Fig.1. Although the main concerns of the facility are nuclear physics experiments, as an effort to effectively use the available radioactive isotope beams at the TRIAC for materials studies, we have developed a diffusion tracing method by using the short-lived radioactive nuclei of 8Li as diffusion tracers. The method has been successfully applied to measure diffusion coefficients in a typical defectmediated lithium ionic conductor (refer to Chandra, 1981 for ionic conductors). We found that the present method is very efficient for the micro-diffusion, where the diffusion

been greatly expanded during the last decade (Cornell, 2003).

length is about 1μm per second.

**1. Introduction** 

