**2. Application of stable isotopes in plants**

The biochemical cycling of light element such as carbon(C), oxygen(O), nitrogen(N) and sulphur(S) have been studying using stable isotopes. The mechanisms of photosynthesis and of element uptake and translocation in plants was clarified by these studies using stable isotopes ratios such as C,O,N and S. Recently, the application of enriched isotopes of such as Mg, Cu, Ca, K and Cd behavior in plants rapidly increased with the development of ICP-MS analysis techniques. There are several studies on element uptake and translocation in plant using enriched stable isotopes (Table1).


Table 1. Element uptake and translocation in plant using enriched stable isotopes

There are so many research using enriched stable isotopes used as tracers aquatic and terrestrial ecosystems, animals and humans (See review of Stürup et al. 2008). However, there are a few researches using enriched stable isotopes element in plants. Recently, Stürup et al. (2008) reviewed that application of enriched stable isotopes as tracers in biological systems including aquatic ecosystem, terrestrial ecosystem, animals and humans in detail.

In this chapter, we therefore provide a review of some example using isotope technique. Especially, we focus on the application of enriched stable isotopes element uptake and translocation in plants. Our new method for evaluation of symplastic absorption of roots introduced in Section 4 has some merits, compared to radioisotopes techniques. Application

The biochemical cycling of light element such as carbon(C), oxygen(O), nitrogen(N) and sulphur(S) have been studying using stable isotopes. The mechanisms of photosynthesis and of element uptake and translocation in plants was clarified by these studies using stable isotopes ratios such as C,O,N and S. Recently, the application of enriched isotopes of such as Mg, Cu, Ca, K and Cd behavior in plants rapidly increased with the development of ICP-MS analysis techniques. There are several studies on element uptake and translocation in plant

**Isotopes Aim of study and method Reference** 

Dannel et al. (2000)

Takano et al. (2002)

Ueno et al. (2005)

Mori et al. (2009b)

Yada et al. (2004), Oda et al. (2004)

Kawasaki et al. (2004,2005)

Characterization of boron uptake and translocation in sunflower plant. After preculture under nutrient solution containing 11B,a short time experiment were conducted under nutrient solution containing low or high 11B.

After preculture grown in nutrient solution containing boron, uptake experiment was conducted in solution

Intact leaves and cell sap of Cd accumulator plant were subjected to 113Cd-NMR and H-NMR analysis for

To examine Cd uptake in roots of *solanum* species with different Cd accumulation in shoot, uptake experiments

containing enriched stable isotopes of 10B.

identification of the form of Cd in leaves.

were conducted using 113Cd and 114Cd.

113Cd Cd accumulation stage in soybean seed was examined in hydroponic solution using enriched isotope of 113Cd.

113Cd Cd uptake mechanisms in soybean was examined using 113Cd isotopes in pot and field experiment

Table 1. Element uptake and translocation in plant using enriched stable isotopes

Therefore, we did not focus on aquatic ecosystem animal and human in this chapter.

of stable isotopes will become a new tool to evaluate element behavior in plants.

**2. Application of stable isotopes in plants** 

using enriched stable isotopes (Table1).

10B, 11B

10B

113Cd

113Cd and 114Cd

Dannel et al. (2000) characterized the boron uptake and translocation from roots to shoots in sunflower using the stable isotopes 10 B and 11B. In the report, after sunflower plant was precultured with high(100μM) or low (1μM) 11B supply, plants were treated under differential 10B supply condition. The results suggested that B uptakes are mediated by two transport mechanisms. First mechanism is passive diffusion which is indicated by the linear components. Second mechanism is energy dependent process which is indicated by the saturated components. Kawasaki et al. (2004, 2005) conducted that an isotope tracer technique with 113Cd has been used in pot and field experiments. They examined that the most critical stages of soybean in which Cd absorbed via roots was transferred into the seeds. Cd absorbed before the beginning seed stage causes an increase of Cd concentration in seeds. Yada et al.(2004) reported that soybean plants were grown in hydroponic solution and supplied 113Cd via roots for 48 h at early growth stage to investigate Cd accumulation pathway in soybean seed using enriched isotope of 113Cd. Cd accumulated in leaves was translocated to seeds at seed beginning maturity stage. Oda et al. (2004) also indicated that the Cd absorbed from full pod to full seed was the most contributive to raise the Cd amount of seeds. Ueno et al. (2005) reported that *Thlaspi caerulescens* which is Cd hyperaccumulator plants have been grown hydroponically with a highly enriched 113Cd isotope to investigate the form of Cd in the leaves using 113Cd nuclear magnetic resonance (NMR) spectroscopy. They identified that cadmium binds with malate in the leaves. Several enriched isotopes such as 111Cd, 113Cd and 114Cd will become a new tool to evaluate Cd behavior in plants. Several studies stated above suggest that enriched isotope is a very potent technique for tracking the distribution, uptake, translocation and recycling in biological system. Now, many enriched element stable isotopes except B and Cd are able to purchase in chemical forms such as metallic or oxide. In the future, the benefit of enriched stable isotopes techniques would be paid much attention in plant and environmental science areas.

#### **3. Several methods for evaluating symplastic element uptake in plants**

Intensive studies on the absorption mechanisms of various elements by plant roots have been conducted. There are evidence on mineral uptake and translocation in plants. It is well known that ion absorption in plant roots shows a saturated curve in kinetics experiments, indicating that a type of proteinaceous transporter mediates ion absorption (Epstein and Hagen 1952). Plant physiologists examining ion absorption in plant roots have given much attention to ion transport via the symplast across the plasma membrane (Epstein 1973). However, when ion absorption experiments were conducted, it was found that the apoplastically absorbed ions needed to be washed out of the apoplast to determine the symplastically absorbed ions across the plasma membrane or the determination of absorption is overestimated (Glass 2007). Therefore, it is necessary to eliminate the apoplastically bound ions to evaluate the symplastically absorbed ion content in the roots. To evaluate symplastic cadmium(Cd) and other elements absorption in roots, several methods have generally been used in the past: (1) expose the plant material to Cd radioisotopes and subsequent desorption using unlabelled Cd in the root apoplast (Hart et al. 1998, 2002, 2006), (2) plant material is exposed to Cd radioisotopes under conditions at 2°C and 22°C (Zhao et al. 2002, Uraguchi et al. 2009), (3) metabolic inhibitors such as DNP or CCCP (Cataldo et al.1983, Ueno et al. 2009), (4) centrifuge method (Yu et al. 1999, Mitani and Ma 2005, Ma et al. 2004, Ueno et al. 2008), (5)estimation of desorption from roots with time(Lasat et al. 1998)

Application of Enriched Stable Isotopes in Element Uptake and Translocation in Plant 59

cap was directly determined. However, evaluation using root tips possibly is not representative of most root tissues. Rain et al.(2006) pointed out that there are the difference

As other evaluation method of roots fraction, Lasat et al.(1998) evaluated that each fraction of cell wall, cytoplasm and vacuole by each efflux fraction from roots. They investigated that difference of Zn fraction in roots such as cell wall, cytoplasm and vacuole using this method.

In this section, we introduce that our new method for evaluation of symplastic ion absorption, especially cadmium (Mori et al. 2009a). Several methods stated above is evaluation that apoplastically bound element is desorbed by some elements after element absorption experiment. Our method is that symplastic Cd absorption capacity is evaluated by difference of enriched isotope of 113Cd and 114Cd. Cadmium (Cd) is a hazardous heavy metal with regards to human health and is dispersed in natural and agricultural environments principally through human activities (Wanger, 1993). Arable land contains, to some extent, Cd, reportedly in the range, 0.04–0.32M, even in nonpolluted soil (Keller, 1995; Wanger, 1993). This results in Cd accumulation in the edible parts of crops. Recently, the Codex Alimentarius Commission (2005) adopted a maximum concentration of 0.05 mg Cd kg−1 (fresh weight) recommended for fruiting vegetables. Approximately 7% of 381 samples of eggplant (*Solanum melongena*), 22% of 165 samples of okra (*Abelmoschus esculentus*), and 10% of 302 samples of taro (*Colocasia esculenta*) contained Cd concentrations above this limit in a field and market-basket study during 1998–2001 in Japan (Ministry of Agriculture Forestry and Fisheries of Japan, 2002); despite the fact that these crops were cultivated in non-polluted fields. Under these circumstances, new technologies for reducing the Cd level in crops are urgently required in Japan. Therefore, it is important to elucidate the mechanisms mediating Cd absorption, accumulation, and translocation in these crops. The crop conditions were represented by

**4.1 Validity of our method for evaluation of symplastic Cd uptake in roots using** 

When ion absorption experiments were conducted, it was found that the apoplastically absorbed ions needed to be washed out of the apoplast to determine the symplastically absorbed ions across the plasma membrane or the determination of absorption is overestimated(Glass 2007). Therefore, it is necessary to eliminate the apoplastically bound ions to evaluate the symplastically absorbed ion content in the roots. There are several methods to eliminate apoplastic ions as stated above. In this section, we introduced our new method for symplastic Cd absorption in roots of *Solanum melongena* using enriched isotopes

The enriched isotopes of 113Cd (106Cd, 0.16%; 108Cd, 0.135%; 110Cd, 0.81%; 111Cd, 2.53%; 112Cd, 2.61%; 113Cd, 93.29%; 114Cd, 0.46%; 116Cd, 0.01%) and 114Cd (106Cd, 0.05%; 108Cd, 0.05%; 110Cd, 0.05%; 111Cd,0.05%; 112Cd, 0.05%; 113Cd, 5.6%; 114Cd, 93.6%; 116Cd,0.8%) used in the present study were purchased from Isoflex (San Francisco, CA,USA) in metallic form and dissolved

in diluted HNO3. The enriched isotopes of 114Cd contained the 5.6 % of 113Cd.

of Km value in kinetics experiment between whole roots and root tips.

**translocation in plant** 

low Cd concentration experimental mediums.

**enriched isotopes of 113Cd and 114Cd** 

of 113Cd and 114Cd.

**4. Application of enriched stable isotopes in element uptake and** 

Regarding evaluation for symplastic element uptake in roots using radioisotopes, this method is used for symplastic element uptake in roots. Hart et al. (1998, 2002, 2006) reported that Cd uptake experiment was conducted in nutrient solution containing 109Cd-labeled CdSO4 and apoplastic 109Cd were desorbed using excessive nonlabelled Cd. As other method, Nakanishi et al. (2006) evaluated that apoplastic Cd in the roots was washed in 0.5 mmol L−1 ethylenediaminefetraacetic acid (EDTA) for 1 min. Lasat et al. (1996) evaluated that symplastic Zn uptake in roots of Zn hyperaccumulator and nonaccumulator *Thlaspi* species apoplastic 65Zn in roots desorbed by excessive unlabelled ZnCl2 solution after Zn uptake experiment was conducted using 65Zn raidoisotopes. There is merit that this method is able to detect radioisotope element with high sensitivity. However, there are limitations to this method, including radioisotope administrative restriction and restricted half of the radioisotope. Additionally, the radioisotope technique has toxicological concern. It is required for handling its isotopes to be careful.

Regarding evaluation of symplastic element uptake in roots using differences in the amounts of Cd absorbed at 2°C and 25°C. Uptake of element at 2°Cwas assumed to represent mainly apoplastic binding in the roots whereas the difference in uptake between 22°C and 2°C represented metabolically dependent influx. Zhao et al. (2002) reported that apoplastic and symplastic uptake in two *Thlaspi* species from Cd and Zn depletion in solution using radioisotope tracer. Uraguchi et al.(2009) reported that genotypic variation in cadmium accumulation in rice and evaluated that symplastic Cd uptake in roots of rice using the method of subtraction the Cd content in the roots at 2°C from the Cd content in the roots at 25°C. This method using unlabeled Cd is easy to handle because there is no administrative limitation not using radioisotope elements. However, this method needs double seedlings for evaluation. Additionally, this method cannot be evaluated using same seedling. This method is not easy for dicotyledonous plant such as *Solanum melongena* to handle.

As for methods using metabolic inhibitors, Cataldo et al. (1983) reported that Cd uptake dependent on energy in roots is suppressed by dinitrophenol as metabolic inhibitor. In this study, using dinitrophenol as a metabolic inhibitor, the 'metabolically absorbed' fraction was shown to represent 75 to 80% of the total absorbed fraction at concentration less than 0.5 μmol, and decreased to 55% at 5μmol.

Regarding centrifuge method, tap roots of plants were harvested and 2 cm root tips were excised. Then, cut ends were washed in distilled water and blotted dry. For each sample, 30 roots were used. The cut ends were washed in distilled water quickly and blotted dry. The tips were placed in a 0.45 mM filter unit with the cut ends facing down and centrifuged at 2,000g for 15 min at 4°C to obtain the apoplastic solution. After centrifugation, root segments were frozen at -80°C for 2 h and then thawed at room temperature. The symplastic solution was prepared from frozen-thawed tissues by centrifugation at 2,000g for 15 min at 4°C. Ma et al. (2004) evaluated that symplastic Si uptake of wild type rice and mutant rice using this centrifuge method. Additionally, Mitani and Ma (2005) also evaluated that symplastic Silicon uptake in rice, tomato and cucumber which differ from Si accumulation capacity using this method. Ueno et al.(2009) reported that symplastic Cd uptake is estimated by cell sap obtained from centrifuge method. To check the purity of apoplastic solution, the activity of malic dehydrogenase in apoplastic and symplastic solution was determined. The activity of malic dehydrogenase in apoplastic solution was below onetwentieth and approximately one-fortieth of symplastic solution. This method is valuable for evaluation of symplastic Cd concentration in roots because Cd concentration in roots cell

Regarding evaluation for symplastic element uptake in roots using radioisotopes, this method is used for symplastic element uptake in roots. Hart et al. (1998, 2002, 2006) reported that Cd uptake experiment was conducted in nutrient solution containing 109Cd-labeled CdSO4 and apoplastic 109Cd were desorbed using excessive nonlabelled Cd. As other method, Nakanishi et al. (2006) evaluated that apoplastic Cd in the roots was washed in 0.5 mmol L−1 ethylenediaminefetraacetic acid (EDTA) for 1 min. Lasat et al. (1996) evaluated that symplastic Zn uptake in roots of Zn hyperaccumulator and nonaccumulator *Thlaspi* species apoplastic 65Zn in roots desorbed by excessive unlabelled ZnCl2 solution after Zn uptake experiment was conducted using 65Zn raidoisotopes. There is merit that this method is able to detect radioisotope element with high sensitivity. However, there are limitations to this method, including radioisotope administrative restriction and restricted half of the radioisotope. Additionally, the radioisotope technique has toxicological concern. It is

Regarding evaluation of symplastic element uptake in roots using differences in the amounts of Cd absorbed at 2°C and 25°C. Uptake of element at 2°Cwas assumed to represent mainly apoplastic binding in the roots whereas the difference in uptake between 22°C and 2°C represented metabolically dependent influx. Zhao et al. (2002) reported that apoplastic and symplastic uptake in two *Thlaspi* species from Cd and Zn depletion in solution using radioisotope tracer. Uraguchi et al.(2009) reported that genotypic variation in cadmium accumulation in rice and evaluated that symplastic Cd uptake in roots of rice using the method of subtraction the Cd content in the roots at 2°C from the Cd content in the roots at 25°C. This method using unlabeled Cd is easy to handle because there is no administrative limitation not using radioisotope elements. However, this method needs double seedlings for evaluation. Additionally, this method cannot be evaluated using same seedling. This method

As for methods using metabolic inhibitors, Cataldo et al. (1983) reported that Cd uptake dependent on energy in roots is suppressed by dinitrophenol as metabolic inhibitor. In this study, using dinitrophenol as a metabolic inhibitor, the 'metabolically absorbed' fraction was shown to represent 75 to 80% of the total absorbed fraction at concentration less than 0.5

Regarding centrifuge method, tap roots of plants were harvested and 2 cm root tips were excised. Then, cut ends were washed in distilled water and blotted dry. For each sample, 30 roots were used. The cut ends were washed in distilled water quickly and blotted dry. The tips were placed in a 0.45 mM filter unit with the cut ends facing down and centrifuged at 2,000g for 15 min at 4°C to obtain the apoplastic solution. After centrifugation, root segments were frozen at -80°C for 2 h and then thawed at room temperature. The symplastic solution was prepared from frozen-thawed tissues by centrifugation at 2,000g for 15 min at 4°C. Ma et al. (2004) evaluated that symplastic Si uptake of wild type rice and mutant rice using this centrifuge method. Additionally, Mitani and Ma (2005) also evaluated that symplastic Silicon uptake in rice, tomato and cucumber which differ from Si accumulation capacity using this method. Ueno et al.(2009) reported that symplastic Cd uptake is estimated by cell sap obtained from centrifuge method. To check the purity of apoplastic solution, the activity of malic dehydrogenase in apoplastic and symplastic solution was determined. The activity of malic dehydrogenase in apoplastic solution was below onetwentieth and approximately one-fortieth of symplastic solution. This method is valuable for evaluation of symplastic Cd concentration in roots because Cd concentration in roots cell

is not easy for dicotyledonous plant such as *Solanum melongena* to handle.

required for handling its isotopes to be careful.

μmol, and decreased to 55% at 5μmol.

cap was directly determined. However, evaluation using root tips possibly is not representative of most root tissues. Rain et al.(2006) pointed out that there are the difference of Km value in kinetics experiment between whole roots and root tips.

As other evaluation method of roots fraction, Lasat et al.(1998) evaluated that each fraction of cell wall, cytoplasm and vacuole by each efflux fraction from roots. They investigated that difference of Zn fraction in roots such as cell wall, cytoplasm and vacuole using this method.

#### **4. Application of enriched stable isotopes in element uptake and translocation in plant**

In this section, we introduce that our new method for evaluation of symplastic ion absorption, especially cadmium (Mori et al. 2009a). Several methods stated above is evaluation that apoplastically bound element is desorbed by some elements after element absorption experiment. Our method is that symplastic Cd absorption capacity is evaluated by difference of enriched isotope of 113Cd and 114Cd. Cadmium (Cd) is a hazardous heavy metal with regards to human health and is dispersed in natural and agricultural environments principally through human activities (Wanger, 1993). Arable land contains, to some extent, Cd, reportedly in the range, 0.04–0.32M, even in nonpolluted soil (Keller, 1995; Wanger, 1993). This results in Cd accumulation in the edible parts of crops. Recently, the Codex Alimentarius Commission (2005) adopted a maximum concentration of 0.05 mg Cd kg−1 (fresh weight) recommended for fruiting vegetables. Approximately 7% of 381 samples of eggplant (*Solanum melongena*), 22% of 165 samples of okra (*Abelmoschus esculentus*), and 10% of 302 samples of taro (*Colocasia esculenta*) contained Cd concentrations above this limit in a field and market-basket study during 1998–2001 in Japan (Ministry of Agriculture Forestry and Fisheries of Japan, 2002); despite the fact that these crops were cultivated in non-polluted fields. Under these circumstances, new technologies for reducing the Cd level in crops are urgently required in Japan. Therefore, it is important to elucidate the mechanisms mediating Cd absorption, accumulation, and translocation in these crops. The crop conditions were represented by low Cd concentration experimental mediums.

#### **4.1 Validity of our method for evaluation of symplastic Cd uptake in roots using enriched isotopes of 113Cd and 114Cd**

When ion absorption experiments were conducted, it was found that the apoplastically absorbed ions needed to be washed out of the apoplast to determine the symplastically absorbed ions across the plasma membrane or the determination of absorption is overestimated(Glass 2007). Therefore, it is necessary to eliminate the apoplastically bound ions to evaluate the symplastically absorbed ion content in the roots. There are several methods to eliminate apoplastic ions as stated above. In this section, we introduced our new method for symplastic Cd absorption in roots of *Solanum melongena* using enriched isotopes of 113Cd and 114Cd.

The enriched isotopes of 113Cd (106Cd, 0.16%; 108Cd, 0.135%; 110Cd, 0.81%; 111Cd, 2.53%; 112Cd, 2.61%; 113Cd, 93.29%; 114Cd, 0.46%; 116Cd, 0.01%) and 114Cd (106Cd, 0.05%; 108Cd, 0.05%; 110Cd, 0.05%; 111Cd,0.05%; 112Cd, 0.05%; 113Cd, 5.6%; 114Cd, 93.6%; 116Cd,0.8%) used in the present study were purchased from Isoflex (San Francisco, CA,USA) in metallic form and dissolved in diluted HNO3. The enriched isotopes of 114Cd contained the 5.6 % of 113Cd.

Application of Enriched Stable Isotopes in Element Uptake and Translocation in Plant 61

Fig. 2. A schematic representation of Cd absorption and desorption in roots using different

Approximately 0.05–0.1 g of dried roots was transferred and digested in a 10 mL Teflon tube containing 3 mL HNO3. After digestion, the digested solution was diluted and 10 ng mL–1 of indium (In) was added to each diluted solution as an internal standard for 114Cd determination. For 113Cd determination, 10 ng mL–1 of tellurium (Te) was added as an internal standard. The concentrations of 113Cd and 114Cd in the digested solutions were determined by ICP-MS (ELAN DRC-e; Perkin Elmer SCIEX, Concord, ON, Canada). The concentrations of Cd in the digested solutions from the Cd-absorption experiment using unlabeled CdCl2 reagent were determined by ICP atomic emission spectroscopy (VISTA-PRO; Varian, Palo Alto, CA, USA). It is well known that MoO interferes spectroscopically in determining the concentration of Cd in ICP-MS analysis (Kimura et al. 2003; May and Wiedmeyer 1998). In addition, it has been shown that it is necessary to remove Mo from the digested solution to avoid spectroscopic interference by molybdenum oxides (Oda et al. 2004; Yada et al. 2004). Therefore, for the 113Cd and 114Cd count intensities, we monitored the spectroscopic interference of the molybdenum oxides (97Mo16O and 98Mo16O) detected in the 10 ng mL–1 Mo standard solution. The contribution rate of spectroscopic interference of the putative 97Mo16O and 98Mo16O for 113Cd and 114Cd contents was negligibly small in both treatments (40 and 400 nmol). Therefore, we considered that we could ignore spectroscopic interference of oxidative molybdenum in determining the 113Cd and 114Cd contents in the

As shown in Fig. 3, after desorption of apoplastic 113Cd by excessive 114Cd, distribution of 113Cd and 114Cd in roots is as follow. (1) apoplastic bound 114Cd is derived from desorption solution of excessive 114Cd. (2) apoplastic bound 113Cd is derived from desorption solution of excessive 114Cd. (3) symplastic 113Cd is derived from 113Cd- uptake experiment. Therefore, 113Cd content in roots is the sum of (1) and (2). Symplastic 113Cd is the subtraction between total 113Cd and 113Cd derived from an enriched stable of 114Cd. As shown in Fig. 1, the total 113Cd contents in the roots signifies the 113Cd contents in the roots after the desorption

**4.2 Determination of 113Cd, 114Cd and the Cd contents in the roots** 

enriched isotopes

ICP-MS analysis.

(modified from Mori et al. 2009a)

Fig. 1. Absorption experiment procedure for evaluating symplastic 113Cd absorption in roots

The procedure for evaluating symplastic Cd absorption in the roots, using enriched isotopes 113Cd and 114Cd, is illustrated in Fig. 1. The roots of intact seedlings were rinsed in ultrapure water for 2 min and then exposed to a 500 mL 113Cd solution containing 0.5 mmol L–1 CaCl2 and 2 mmol L–1 2-morpholinoethanesulfonic acid monohydrate Tris (hydroxymethyl) aminomethane (MES–Tris) (pH 6.0) at 25°C for 30 min (Fig. 1). The levels of 113Cd were 40 nmol or 400 nmol in the 113Cd treatment. A-B shown in Fig.2 indicates that 113Cd absorbed in roots consists of apoplastic 113Cd and symplastic 113Cd (Fig.2 A, B). To suppress metabolically dependent symplastic absorption from the apoplast, the roots were excised from each seedling and immersed in a cold Cd-free buffer solution (2 mmol L–1 MES–Tris [pH 6.0], 0.5 mmol L–1 CaCl2) at 2°C for 120 min (Fig. 1, Fig.2 C). The apoplastic-bound 113Cd in the roots from 40 or 400 nmol 113Cd treatment was then desorbed by immersing the roots in the same cold buffer solution at 2°C containing a 50-fold concentration of 114Cd (2 or 20 μmol) for 120 min (Fig.1, Fig.2 D, E, F). The excised roots were then rinsed in ultrapure water for 2 min. Harvested samples were dried in an oven at 75°C for 3 days until dry. After digestion of dried sample, we then determined 113Cd and 114Cd contents in roots by ICP-MS analysis. To confirm the validity of this method, we compared our Cd absorption results with the Cd absorption results obtained at 25°C and 2°C using unlabeled CdCl2 reagent. The experimental procedure was as follows. The Cd-absorption experiments were conducted for 30 min using 500 mL solutions containing 2 mmol L–1 MES–Tris (pH 6.0), 0.5 mmol L–1 CaCl2 and different concentrations of Cd (40 or 400 nmol) at 25°C. After the absorption experiment, the excised roots from each seedling were rinsed with ultrapure water for 2 min. For the Cd-absorption experiment at 2°C, plants were transferred to an ice-cold pretreatment solution containing 2 mmol L–1 MES–Tris (pH 6.0) and 0.5 mmol L–1 CaCl2 for 120 min. The Cd-absorption experiment at 2°C was conducted for 30 min. In the unlabeled Cdabsorption experiment at different temperatures, the amount of Cd reportedly absorbed into roots at 2°C was estimated to be apoplastically bound Cd on the assumption that metabolically dependent absorption would be suppressed at low temperature. Therefore, the difference in the amount of Cd absorbed at 2°C and at 25°C represents symplastic Cd absorption depending on metabolic energy. All absorption experiments were replicated three times. Each procedure illustrated in Figure1 signifies a schematic representation shown in Fig. 2.

Fig. 1. Absorption experiment procedure for evaluating symplastic 113Cd absorption in roots The procedure for evaluating symplastic Cd absorption in the roots, using enriched isotopes 113Cd and 114Cd, is illustrated in Fig. 1. The roots of intact seedlings were rinsed in ultrapure water for 2 min and then exposed to a 500 mL 113Cd solution containing 0.5 mmol L–1 CaCl2 and 2 mmol L–1 2-morpholinoethanesulfonic acid monohydrate Tris (hydroxymethyl) aminomethane (MES–Tris) (pH 6.0) at 25°C for 30 min (Fig. 1). The levels of 113Cd were 40 nmol or 400 nmol in the 113Cd treatment. A-B shown in Fig.2 indicates that 113Cd absorbed in roots consists of apoplastic 113Cd and symplastic 113Cd (Fig.2 A, B). To suppress metabolically dependent symplastic absorption from the apoplast, the roots were excised from each seedling and immersed in a cold Cd-free buffer solution (2 mmol L–1 MES–Tris [pH 6.0], 0.5 mmol L–1 CaCl2) at 2°C for 120 min (Fig. 1, Fig.2 C). The apoplastic-bound 113Cd in the roots from 40 or 400 nmol 113Cd treatment was then desorbed by immersing the roots in the same cold buffer solution at 2°C containing a 50-fold concentration of 114Cd (2 or 20 μmol) for 120 min (Fig.1, Fig.2 D, E, F). The excised roots were then rinsed in ultrapure water for 2 min. Harvested samples were dried in an oven at 75°C for 3 days until dry. After digestion of dried sample, we then determined 113Cd and 114Cd contents in roots by ICP-MS analysis. To confirm the validity of this method, we compared our Cd absorption results with the Cd absorption results obtained at 25°C and 2°C using unlabeled CdCl2 reagent. The experimental procedure was as follows. The Cd-absorption experiments were conducted for 30 min using 500 mL solutions containing 2 mmol L–1 MES–Tris (pH 6.0), 0.5 mmol L–1 CaCl2 and different concentrations of Cd (40 or 400 nmol) at 25°C. After the absorption experiment, the excised roots from each seedling were rinsed with ultrapure water for 2 min. For the Cd-absorption experiment at 2°C, plants were transferred to an ice-cold pretreatment solution containing 2 mmol L–1 MES–Tris (pH 6.0) and 0.5 mmol L–1 CaCl2 for 120 min. The Cd-absorption experiment at 2°C was conducted for 30 min. In the unlabeled Cdabsorption experiment at different temperatures, the amount of Cd reportedly absorbed into roots at 2°C was estimated to be apoplastically bound Cd on the assumption that metabolically dependent absorption would be suppressed at low temperature. Therefore, the difference in the amount of Cd absorbed at 2°C and at 25°C represents symplastic Cd absorption depending on metabolic energy. All absorption experiments were replicated three times. Each procedure illustrated in Figure1 signifies a schematic representation

(modified from Mori et al. 2009a)

shown in Fig. 2.

Fig. 2. A schematic representation of Cd absorption and desorption in roots using different enriched isotopes

#### **4.2 Determination of 113Cd, 114Cd and the Cd contents in the roots**

Approximately 0.05–0.1 g of dried roots was transferred and digested in a 10 mL Teflon tube containing 3 mL HNO3. After digestion, the digested solution was diluted and 10 ng mL–1 of indium (In) was added to each diluted solution as an internal standard for 114Cd determination. For 113Cd determination, 10 ng mL–1 of tellurium (Te) was added as an internal standard. The concentrations of 113Cd and 114Cd in the digested solutions were determined by ICP-MS (ELAN DRC-e; Perkin Elmer SCIEX, Concord, ON, Canada). The concentrations of Cd in the digested solutions from the Cd-absorption experiment using unlabeled CdCl2 reagent were determined by ICP atomic emission spectroscopy (VISTA-PRO; Varian, Palo Alto, CA, USA). It is well known that MoO interferes spectroscopically in determining the concentration of Cd in ICP-MS analysis (Kimura et al. 2003; May and Wiedmeyer 1998). In addition, it has been shown that it is necessary to remove Mo from the digested solution to avoid spectroscopic interference by molybdenum oxides (Oda et al. 2004; Yada et al. 2004). Therefore, for the 113Cd and 114Cd count intensities, we monitored the spectroscopic interference of the molybdenum oxides (97Mo16O and 98Mo16O) detected in the 10 ng mL–1 Mo standard solution. The contribution rate of spectroscopic interference of the putative 97Mo16O and 98Mo16O for 113Cd and 114Cd contents was negligibly small in both treatments (40 and 400 nmol). Therefore, we considered that we could ignore spectroscopic interference of oxidative molybdenum in determining the 113Cd and 114Cd contents in the ICP-MS analysis.

As shown in Fig. 3, after desorption of apoplastic 113Cd by excessive 114Cd, distribution of 113Cd and 114Cd in roots is as follow. (1) apoplastic bound 114Cd is derived from desorption solution of excessive 114Cd. (2) apoplastic bound 113Cd is derived from desorption solution of excessive 114Cd. (3) symplastic 113Cd is derived from 113Cd- uptake experiment. Therefore, 113Cd content in roots is the sum of (1) and (2). Symplastic 113Cd is the subtraction between total 113Cd and 113Cd derived from an enriched stable of 114Cd. As shown in Fig. 1, the total 113Cd contents in the roots signifies the 113Cd contents in the roots after the desorption

Application of Enriched Stable Isotopes in Element Uptake and Translocation in Plant 63

contents in roots at 40 and 400 nmol in the 2°C treatment were 4.1 ± 0.3 and 28.1 ± 0.73 mg

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

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

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

**40nM** 

117.3±9.3 23.0±4.3 6.6±0.53 16.4±3.7 **400nM** 

644.5±33.7 87.7±5.6 36.6±1.8 51.0±3.8

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

**113Cd derived from** 

**113Cd derived from** 

**enriched 114Cd Symplastic 113Cd** 

**enriched 114Cd Symplastic 113Cd** 

kg–1 (dry weight), respectively.

absorbed at 2°C and at 25°C.

in the present study.

absorption in plant roots stated below.

**Total 114Cd Total 113Cd** 

**Total 114Cd Total 113Cd** 

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.
