"A Cryo-SIMS Study of the Arsenic-Hyperaccumulator Pteris vittata"

Dr. Michelle Dickinson

Interface Analysis Centre, University of Bristol, 121 St. Michael’s Hill, Bristol, BS2 8BS, UK.

At present, the decontamination of land polluted with heavy metals and metalloids is an expensive and technically difficult task. However, one rapidly developing biotechnology is that of phytoremediation - the use of plants to remove contaminants from the soil. Whilst many plants have developed mechanisms to either tolerate or avoid the uptake of these potentially toxic contaminants, there are several species of plants which are able to uptake extraordinary quantities of certain elements and concentrate them in their above-ground biomass. The heavy metals/metalloids are either converted to environmentally-inert forms or simply contained, preventing further transportation and/or cell damage. These plants are known as hyperaccumulators1 and are a promising method of cheap, environmentally sound remediation of contaminated land or water sites.2

The brake fern, Pteris vittata, is an arsenic (As) hyperaccumulator and can accumulate concentrations as high as 22,600 mg As kg-1 d.wt in its fronds3. In order to understand the mechanisms that enable the accumulation of such vast quantities of an element renowned for its toxicity to living organisms, it is of great importance to determine the sub-cellular localisation of the As.

The surface analytical technique Secondary Ion Mass Spectrometry (SIMS) has several characteristics that are advantageous to the analysis of biological specimens. These are primarily the ability to detect all elements and isotopes, higher sensitivity than many other analytical techniques and good spatial resolution. A magnetic sector SIMS instrument was modified to facilitate the analysis of frozen-hydrated P. vittata frond samples, thus eliminating the need for chemical fixation. Young P. vittata plants were treated with 500 μM sodium arsenate for 5 days and then frond samples were plunge-frozen in liquid nitrogen, sectioned and gold-coated prior to SIMS analysis. The negative ions at 91 and 107 Da, indicative of AsO- and AsO2-, respectively, were used to identify As within sectioned cells. Analyses of mature P. vittata pinnae suggested that As was more concentrated in epidermal tissues than in mesophyll cells and high As signals originated from regions corresponding to the vacuoles of cells. In regions displaying outward signs of severe toxicity/necrosis, the As was highly concentrated in all cells. Examination of the external cellular morphology of fronds from P. vittata revealed no differences between control and As-treated plants, except in necrotic tissues. Cryo-SIMS was thus demonstrated to be a satisfactory technique for the analysis of frozen-hydrated biological specimens.

[1] Brooks R.R., Lee J., Reeves R.D., Jaffre T. (1977).
Detection of Nickeliferous Rocks by Analysis of Herbarium Specimens of Indicator Plants. J. Geochem. Explor. 7, pp.49-57.

[2] Hinchman R.R., Negri M.C., Gatliff E.G. (2003)
Phytoremediation: Using Green Plants to Clean Up Contaminated Soil, Groundwater, and Wastewater. http://www.ipd.anl.gov/biotech/pdfs/phyto98.pdf

[3] Ma L.Q., Komar K.M., Tu C., Zhang W., Cai Y., Kennelley E.D. (2001).
A Fern that Hyperaccumulates Arsenic. Nature 409, pp.579-580.