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

Shinsuke Mori1, Akira Kawasaki2, Satoru Ishikawa2 and Tomohito Arao2 *1NARO Western Region Agricultural Research Center 2National Institute for Agro-Environmental Sciences Japan* 

#### **1. Introduction**

54 Radioisotopes – Applications in Physical Sciences

[48] Saliba-Silva, A. M., Et Al. Fabrication of U3Si2 powder for fuels used in IEA-R1 nuclear research reactor. *Materials Science Forum.* IPEN, 2008, Vols. 591-93, pp. 194-199. [49] Wiencek, T., Prokofiev, I. And Mcgann, D. Development and compatibility of

[50] Copeland, G. And Martin, M. Development of High - Uranium Loaded U3O8 - Al Fuel

[51] Tzou, G., Et Al. Analytical approach to the cold- and- hot bond rolling of sandwich

[52] Frajndlich, E. U., Saliba-Silva, A. And Zorzetto, M. Xxi Rertr Meeting. Alternative

[ed.] ANL. *RERTR 1998.* Oct 18-23, 1998.

Plates. *Nuclear Technology.* 1982, Vol. 56.

of University of São Paulo (in Portuguese).

University of São Paulo (in Portuguese).

125-126, pp. 664-669.

[Online] [Cited: 05 16, 2011.]

Magnesium- Matrix Fuel Plates Clad With 6061 Aluminum Alloy", Proc. The International Meeting on Reduced Enrichment For research And Test Reactors.

sheet with outer hard and inner soft layers". *J. of Mater. Processing Technology.* Vols.

Route For UF6 Conversion Towards UF4 to Produce metallic Uranium. *RERTR/DOE, 19º.* [Online] 1998. http://www.rertr.anl.gov/Fuels98/Elita.pdf. [53] International Bio-Analytical Industries, Inc. Ammonium Uranyl Carbonate MSDS.

 http://www.ibilabs.com/Ammonium%20Urany%20Carbonate%20MSDS.htm. [54] Silva Neto, J. *Dry uranium tetrafluoride process preparation using the uranium hexafluoride* 

[55] Frajndlich, E.U.C. *Chemical treatment study of an ammonium fluoride solution at the* 

*reconversion process effluents.* São Paulo : CPG-IPEN/USP , 2008. MSc. Dissertation

*uranium reconversion plant.* São Paulo : CPG-IPEN/USP , 1992. MSc.Dissertation,

Isotope technique including radioisotopes and stable isotopes is useful and potent tool for various scientific areas. Especially, enriched stable isotopes are indispensable tools for researchers in biological systems (Stürup et al. 2008).

 Stable isotope ratios are usually used in examining the biogeochemical cycling of light elements such as carbon(C), oxygen (O), nitrogen (N) and sulphur (S) in the environment. Thermal ionization mass spectrometry (TIMS) for the isotope analysis has been the most standard technique for many years. However, for TIMS analysis, time for sample preparation is needed because sample need to ensure efficient ionization. On the other hand, ICP-MS analysis has some advantages that sample preparation is simple and high sample throughput for isotope experiments where a large amount of samples need to be analyzed (Stürup et al. 2008). The disadvantage to resolve in isotope analysis using ICP-MS is spectroscopic interferences in the process of analysis. It is therefore needed to be resolved these interferences.

When plant physiologists investigate mineral absorption mechanisms in roots of plant, evaluation of symplastic mineral absorption capacity in roots cell in kinetics and time course experiments is very important because mineral translocation in shoots is mainly contributed to capacity of symplastic absorption in roots. In these experiments, radioisotopes methods are mainly used for element uptake in plants. Radioisotopes in solute were the most useful markers used in nutrient uptake and translocation in plants 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 half-life of the radioisotope. Isotope tracer experiments, using a stable isotope, are very similar to those using a radioisotope on element to analyse plant mechanisms (Stürup et al. 2008). Accurate and precise determination of mineral isotope ratios is required for analysis of enriched stable isotopes. Inductive coupled plasma mass spectrometry (ICP-MS) has now become the effective and potent technique for enriched stable isotope tracer experiments due to increased availability. Therefore, the application of enriched stable isotopes in various biological systems increased rapidly.

Application of Enriched Stable Isotopes in Element Uptake and Translocation in Plant 57

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** 

of desorption from roots with time(Lasat et al. 1998)

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

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

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