Quantitative mapping of elemental distribution in leaves of the metallophytes Helichrysum candolleanum, Blepharis aspera, and Blepharis diversispina from Selkirk Cu–Ni mine, Botswana

Multi-element profiling is essential in understanding the metal-tolerant behavior of metallophytes. Although previous reports using multi-elemental analyses show that the metallophytes Blepharis aspera, Blepharis diversispina (Acanthaceae) and Helichrysum candolleanum (Asteraceae) take up metals, no information exists on elemental spatial distribution and concentrations in specific tissues of these plants. The aim of this study therefore was to assess the spatial distribution and concentration of copper, nickel and other elements in leaf tissues of these plants using micro-PIXE. Whole plants were collected with soil in pots from an operational copper and nickel mine (i.e., a copper and nickel mineralized area), Selkirk, about 40 km north-east of Francistown, Botswana. On the same day the samples were transported by air to iThemba LABS in South Africa. Leaf specimens were cryofixed in liquid propane cooled by LN2. Parallel samples were embedded in resin for anatomical studies to facilitate interpretation of elemental maps. The distribution and concentration of copper, nickel, and other elements in leaf tissues were determined using micro-PIXE and proton backscattering spectrometry. Data evaluation was performed using GeoPIXE II software. Micro-PIXE showed that H. candolleanum had the highest whole leaf content of copper (70 ± 3 μg g−1 DW) and nickel (168 ± 5 μg g−1 DW), followed by B. aspera (Cu: 25 ± 1 μg g−1 DW; Ni: 166 ± 4 μg g−1 DW) and B. diversispina (Cu: 3 ± 1 μg g−1 DW; Ni, below detection limit). For specific leaf tissues, the highest levels of copper were found in the vascular bundle for H. candolleanum (167 ± 7 μg g−1 DW) and the lower epidermis for B. aspera (70 ± 9 μg g−1 DW). The highest levels of nickel were present in the vascular bundle of B. aspera (479 ± 10 μg g−1 DW) and H. candolleanum (90 ± 5 μg g−1 DW). Elemental maps showed a similar distribution pattern for copper and nickel in B. aspera and B diversispina, with these elements concentrated in the upper and lower epidermal regions. However, for H. candolleanum, both copper and nickel were concentrated in the mesophyll. The difference in concentration and accumulation patterns between B. aspera, B. diversispina and H. candolleanum suggests that the metal-tolerant adaptation of these metallophytes may differ.