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Cadmium localization and quantification
Cadmium localization and quantification in the plant Arabidopsis thaliana using micro-PIXE F.J. Ager a, * , M.D. Ynsa a , J.R. Dom ıınguez-Sol ııs b , C. Gotor b , M.A. Respaldiza a , L.C. Romero b a Centro Nacional de Aceleradores, Av. Thomas A. Edison s/n, E-41092 Sevilla, Spain b Instituto de Bioqu ıımica Vegetal y Fotos ııntesis, Av. Ameerico Vespucio s/n, E-41092 Sevilla, Spain Abstract Remediation of metal-contaminated soils and waters poses a challenging problem due to its implications in the environment and the human health. The use of metal-accumulating plants to remove toxic metals, including Cd, from soil and aqueous streams has been proposed as a possible solution to this problem. The process of using plants for environmental restoration is termed phytoremediation. Cd is a particularly favourable target metal for this technology because it is readily transported and accumulated in the shoots of several plant species. This paper investigates the sites of metal localization within Arabidopsis thaliana leaves, when plants are grown in a cadmium-rich environment, by making use of nuclear microscopy techniques. Micro-PIXE, RBS and SEM analyses were performed on the scanning proton microprobe at the CNA in Seville (Spain), showing that cadmium is sequestered within the trichomes on the leaf surface. Additionally, regular PIXE analyses were performed on samples prepared by an acid digestion method in order to assess the metal accumulation of such plants. Ó 2002 Published by Elsevier Science B.V. PACS: 89.60; 78.70.E; 82.80.Yc Keywords: Arabidopsis; Cadmium accumulator; Phytoremediation; Nuclear microprobe; PIXE 1. Introduction The ability of certain terrestrial plants to absorb and accumulate metals such as cadmium, nickel, zinc, manganese, copper or cobalt makes them very attractive when the decontamination of soils is sought. These plants are called metal hyperaccu- mulators [1] if they accumulate for instance more than 0.01% of Cd, 0.1% of Ni or 1% of Zn per dry weight in their shoots in a natural environment. However, the mechanisms for metal uptake, trans- location and compartmentation are not yet well understood and in the past recent years an im- portant effort has been made in this direction. From the practical point of view, the comprehen- sion of those mechanisms will probably lead to the enhancement of metal absorption by means of plant hybridation or by producing new transgenic plants forenvironmental remediation purposes. For example, genetic engineering of Arabidopsis tha- liana plant has been demonstrated to be a useful technique to improve heavy metal tolerance by phytoremediation purposes [2]. Nuclear Instruments and Methods in Physics Research B 189 (2002) 494–498 www.elsevier.com/locate/nimb * Corresponding author. Tel.: +34-954460553; fax: +34- 954460145. E-mail address: fjager@us.es (F.J. Ager). 0168-583X/02/$ - see front matter Ó 2002 Published by Elsevier Science B.V. PII: S 0168- 583 X ( 01) 0 1 130 - 2 There is evidence that trichomes on the leaf surface may play a role in the detoxification of heavy metals [3]. In Alyssum lesbiacum [4] micro- PIXE analysis proved that epidermal trichomes represent a site of preferential nickel accumula- tion. In A. thaliana, trichomes are specialized uni- cellular structures with uncertain functions, but recent works suggest their possible role as a sink during detoxification processes [5]. The nuclear microprobe allows investigators to obtain quantitative or semi-quantitative elemental distribution maps of major and trace elements with high resolution and sensitivity, and is be- coming a useful tool for localizing the sites of el- ement accumulation in a wide variety of studies. In the present study we used the scanning nu- clear microprobe to determine the elemental con- centrations and the sites of preferential Cd accumulation within A. thaliana leaves in plants grown in Cd-enriched soils. Regular PIXE analysis was also performed to evaluate the Cd accumula- tion in the plant leaves. 2. Materials and methods 2.1. Plant material Wild type A. thaliana (ecotype Columbia) plants were grown, in a controlled environment room, on moist vermiculite supplemented with Hoagland medium at 20 °C in the light and 18 °C in the dark, under a 16-h white light/8-h dark photoperiod with a photon flux density of 130 lE/ m 2 s and 70% humidity. Cadmium chloride treatments were performed by addition to the Hoagland medium of CdCl 2 to 250 and 2500 lM final concentration. The plants were daily watered with this medium during 14 days. 2.2. Sample preparation for analysis In order to minimise the possibility of ion mo- bilisation, plant leaves were cut off from living plants taken directly from the growth chamber, rinsed briefly in deionised water, dried, immedi- ately frozen at À80 °C in an ultralow temperature freezer (mod. Nuaire NU-6511) and then freeze- dried for 72 h at À50 °C at a pressure of 10 À3 mbar. After this preparation, two different treat- ments were used depending on the subsequent analysis procedure. Leaves for macrobeam analysis were prepared by microwave acid digestion. Plant material (100 mg) was digested in a Teflon bomb in a solution of HNO 3 and Y as internal standard, following standard procedures [6]. For elemental analysis, a 10 ll volume of the resulting solution was pipetted on a polycarbonate film and dried in vacuum. Leaves for microbeam analysis were mounted on carbon tape on a standard aluminium frame and just placed on the sample holder inside the microprobe target chamber (pres. 10 À7 mbar). 2.3. Instrumentation and analytical methods The microbeam analyses were performed with 3.0 MeV protons focused to a 3 Â 3 lm beam normal to the sample with a proton current of 100– 300 pA, using the CNA scanning nuclear microp- robe [7]. PIXE, backscattering spectrometry and electron imaging were carried out simultaneously. PIXE spectra were collected using a Si(Li) X-ray detector manufactured by Canberra at 45° to the beam, with a 12.5 mm 2 active area, 8 lm Be re- movable window and a 50 lm Mylar â filter to at- tenuate X-rays from light elements. The distance from the detector to the sample was chosen to be 40 mm in order to keep good counting statistics with Table 1 PIXE analysis of A. thaliana leaves treated with CdCl 2 (250 lM) prepared by microwave acid digestion Element Concentration (ppm) K 26100 Æ 700 Ca 28200 Æ 700 Ti 7 Æ 3 Cr 4 Æ 1 Mn78Æ 3 Fe 81 Æ 3 Ni 5 Æ 1 Cu 14 Æ 1 Zn 41 Æ 2 Cd 240 Æ 60 F.J. Ager et al. / Nucl. Instr. and Meth. in Phys. Res. B 189 (2002) 494–498 495 low pile-up background and dead time. Backscat- tered protons were detected using a surface barrier detector of an active area of 300 mm 2 at an angle of 37° to the beam, in Cornell geometry. Emitted electrons were detected using a channeltron detec- tor. An electron gun generating 50 mA was used to avert electrostatic charging of the insulating bio- logical material. All signals were recorded together with the beam position using the OM_DAQ data acquisition system [8]. Macro-PIXE analyses were performed with a 2.4 MeV proton beam from the 3 MV Van de Graaff accelerator of the ITN (Portugal), colli- mated up to 5 mm diameter with a current of 100 nA and a total accumulated charge of 100 lC. The incoming proton beam angle was 15°. X-rays were detected with a Linke Si(Li) detector at an angle of 55°,8lm Be window, a 350 lm Mylar â filter and a 2.7 mm diameter collimator placed in front of the detector specially for improvement of the line shape. Data analysis was carried out using GUPIX [9] for PIXE spectra and RUMP [10] and SIMNRA [11] for RBS spectra. Fig. 1. PIXE elemental maps of a leaf edge of an Arabidopsis plant grown in CdCl 2 (250 lM), showing Ca, K, P, Si, Mn and Fe. Area of scan is 100 Â 100 lm 2 . The maps correspond to two consecutive scans so that the lower right end of the first row of elements coincides with the central area of the second row. 496 F.J. Ager et al. / Nucl. Instr. and Meth. in Phys. Res. B 189 (2002) 494–498 3. Results and discussion Macro-PIXE analysis of plant leaves treated with CdCl 2 (250 lM) and prepared by acid di- gestion in microwave oven gives a Cd concentra- tion of 0:024 Æ 0:006 wt.% as shown in Table 1. Micro-PIXE elemental maps and point analyses of different leaves were recorded. Si, P, S, Cl, K, Ca, Fe, Mn, Zn and Cd were all detected by PIXE. For Cd-treated leaves, Cd mapping is unpractical because of the long acquisition times needed for this purpose. However, once the leaf structure is known by means of the other maps (electron imaging, Ca, K, etc.), those maps can be comple- mented with analyses at selected points or regions of interest. Fig. 1 shows elemental maps (side view, 100 Â 100 lm 2 ) of a leaf of an Arabidopsis plant grown in CdCl 2 (250 lM). Thus, the trichome can be divided in three different areas according to their composition: the base, richer in Si, K and P; a central zone, richer in metals such as Mn and Fe; and the head, richer in Ca. The main elements detected in the leaf are K, S, Ca and Cl (not shown in Fig. 1). A front view of a trichome emerging from the leaf surface is presented in Fig. 2 (250 Â 250 lm 2 ), showing the typical impact points for analysis in trichome (T) and leaf (L). Cd concentration can be ideally computed from the K a line of the PIXE spectrum because it lies in a very clean region far from other peaks and from pile-up effects from the main detected elements (Ca, K, etc.). Fig. 3 depicts the Cd contents found by PIXE microanalysis in different leaves. Cad- mium is present in both trichome and leaf, but the Fig. 2. Secondary electron image and maps of elemental distribution of Ca, K, Cl, P and Mn (250 Â 250 lm 2 ) of a leaf of Arabidopsis grown in CdCl 2 (2500 lM). The image corresponds to a front view of a trichome emerging from the leaf surface. The typical impact points for analysis in trichome (T) and leaf (L) are presented. Fig. 3. Bar chart of cadmium contents obtained by PIXE mi- croanalysis in A. thaliana leaves. Question marks indicate points where there is no data available. Error bars are also shown. F.J. Ager et al. / Nucl. Instr. and Meth. in Phys. Res. B 189 (2002) 494–498 497 highest amount of Cd is found in the trichomes. For plants treated with CdCl 2 (250 lM), Cd was detectable in the trichomes but it required a high integrated charge in order to be quantified with low errors. To circumvent this difficulty, a higher Cd dose (2500 lM CdCl 2 ) was used. This treat- ment would result in the increase of the metal uptake by the plant and the enhancement in the Cd concentration in the plant tissues. In effect, analyses show that Cd concentration in leaf epi- dermis and trichome is increased when the plants are treated with CdCl 2 (2500 lM), although the growth was affected by such toxic levels of Cd (smaller plants with shorter purplish leaves). The Cd content also depends on the point of analysis and the orientation of the trichome, being higher when the trichome is analysed in the head with the ion beam coming from its growth direction. 4. Conclusions The preliminary results presented in this study suggest that Cd is preferentially accumulated in the epidermal trichomes of the cadmium accumu- lator plant A. thaliana. However, further analyses are being performed at present to establish the distribution of Cd along the trichomes, because there are evidences [2,5] that the base could con- centrate even more Cd than the head or the stem. This work also contributes to progress in the decontamination of metal polluted soils by means of phytoremediation techniques. Present investi- gations by the same authors are also aimed at comparing the wild variety of A. thaliana with ge- netically modified specimens produced to be more resistant to heavy metal contamination. Acknowledgements We thank Dr. Teresa Pinheiro for her assistance during the preparation and analysis of samples at the ITN. References [1] R.R. Brooks, B.H. Robinson, in: R.R. Brooks (Ed.), Plants that Hyperaccumulate Heavy Metals, CAB Inter- national, Wallingford, UK, 1998. [2] J.R. Dom ıınguez-Sol ııs, G. Guti eerrez-Alcal aa, L. Romero, C. Gotor, J. Biol. Chem. 276 (2001) 9297. [3] H. K € uupper, E. Lombi, F.J. Zhao, S.P. McGrath, Planta 212 (2000) 75. [4] U. Kr € aamer, G.W. Grime, J.A.C. Smith, C.R. Hawes, A.J.M. Baker, Nucl. Instr. and Meth. B 130 (1997) 346. [5] G. Guti eerrez-Alcal aa, C. Gotor, A.J. Meyer, M. Fricker, J.M. Vega, L.C. Romero, Proc. Natl. Acad. Sci USA 97 (20) (2000) 11108. [6] M. Stoeppler, Sampling and Sample Preparation, Springer, Berlin, 1997. [7] J. Garc ııa L oopez, F.J. Ager, M. Barbadillo Rank, M. AA. Ontalba, M. AA. Respaldiza, M.D. Ynsa, Nucl. Instr. and Meth. B 161–163 (2000) 1137. [8] G.W. Grime, M. Dawson, Nucl. Instr. and Meth. B 104 (1995) 107. [9] J.A. Maxwell, J.L. Campbell, W.J. Teesdale, Nucl. Instr. and Meth. B 43 (1989) 218. [10] L.R. Doolitle, Nucl. Instr. and Meth. B 9 (1985) 344. [11] M. Mayer, SIMNRA User’s Guide, Technical Report IPP 9/113, Max-Planck-Institut f € uur Plasmaphysik, Garching, Germany, 1997. 498 F.J. Ager et al. / Nucl. Instr. and Meth. in Phys. Res. B 189 (2002) 494–498 . Si, K and P; a central zone, richer in metals such as Mn and Fe; and the head, richer in Ca. The main elements detected in the leaf are K, S, Ca and Cl. semi-quantitative elemental distribution maps of major and trace elements with high resolution and sensitivity, and is be- coming a useful tool for localizing