Browsing by Author "Bhatia, NP"

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    Arsenic hyperaccumulation and localization in the pinnule and stipe tissues of the gold-dust fern (Pityrogramma calomelanos (L.) Link var. austroamericana (Domin) Farw.) using quantitative micro-PIXE spectroscopy
    (Springer, 2007-11) Kachenko, AG; Bhatia, NP; Singh, B; Siegele, R
    Spatial distribution patterns of arsenic (As) in the tissues of a lesser-known As hyperaccumulating fern Pityrogramma calomelanos (L.) Link var. austroamericana (Domin) Farw. (Pteridaceae) have been studied. Quantitative micro-proton-induced X-ray emission (micro-PIXE) spectroscopy was employed to examine As localization in pinnule and stipe cross-sections of this species. In addition, As hyperaccumulation status of P. calomelanos var. austroamericana was compared with the well-known As hyperaccumulating fern Pteris vittata L. Both species were grown in pots under controlled conditions and exposed to four levels of As (0-500 mg As kg(-1)) for 20 weeks. Pityrogramma calomelanos var. austroamericana accumulated up to 16 415 mg As kg(-1) dry weight (DW), however, phytotoxicity symptoms such as necrotic pinnule tips and margins, appeared in fronds with concentrations > 3,008 mg As kg(-1) DW. Arsenic was readilytranslocated to fronds, with concentrations up to 75 times greater in fronds than in roots. Quantitative elemental maps of As generated using micro-PIXE analysis revealed that As concentrations in pinnule cross-sections were higher than in stipe cross-sections with concentrations of 3.7 x 10(3) and 1.6 x 10(3) mg As kg(-1) DW, respectively (as determined by region selection analysis; RSA). In pinnules, RSA revealed variable concentrations of As, however did not resolve a clear pattern of compartmentalization across different anatomical regions. In stipe tissues, As concentrations followed the order vascular bundle > cortex > epidermis (as determined by RSA). Our results show that P. calomelanos var. austroamericana is an As hyperaccumulator and has the potential for use in phytoremediation of soils with low levels (up to 50 mg kg(-1)) of As contamination. © 2007, Springer.
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    Evaluation of specimen preparation techniques for micro-PIXE localisation of elements in hyperaccumulating plants
    (Elsevier, 2008-04) Kachenko, AG; Siegele, R; Bhatia, NP; Singh, B; Ionescu, M
    Hybanthus floribundus subsp. floribundus, a rare Australian Ni-hyperaccumulating shrub and Pityrogramma calomelanos var. austroamericana, an Australian naturalized As-hyperaccumulating fern are promising species for use in phytoremediation of contaminated sites. Micro-proton-induced X-ray emission (μ-PIXE) spectroscopy was used to map the elemental distribution of the accumulated metal(loid)s, Ca and K in leaf or pinnule tissues of the two plant species. Samples were prepared by two contrasting specimen preparation techniques: freeze-substitution in tetrahydrofuran (THF) and freeze-drying. The specimens were analysed to compare the suitability of each technique in preserving (i) the spatial elemental distribution and (ii) the tissue structure of the specimens. Further, the μ-PIXE results were compared with concentration of elements in the bulk tissue obtained by ICP-AES analysis. In H. floribundus subsp. floribundus, μ-PIXE analysis revealed Ni, Ca and K concentrations in freeze-dried leaf tissues were at par with bulk tissue concentrations. Elemental distribution maps illustrated that Ni was preferentially localised in the adaxial epidermal tissues (1% DW) and least concentration was found in spongy mesophyll tissues (0.53% DW). Conversely, elemental distribution maps of THF freeze-substituted tissues indicated significantly lower Ni, Ca and K concentrations than freeze-dried specimens and bulk tissue concentrations. Moreover, Ni concentrations were uniform across the whole specimen and no localisation was observed. In P. calomelanos var. austroamericana freeze-dried pinnule tissues, μ-PIXE revealed statistically similar As, Ca and K concentrations as compared to bulk tissue concentrations. Elemental distribution maps showed that As localisation was relatively uniform across the whole specimen. Once again, THF freeze-substituted tissues revealed a significant loss of As compared to freeze-dried specimens and the concentrations obtained by bulk tissue analysis. The results demonstrate that freeze-drying is a suitable sample preparation technique to study elemental distribution of ions in H. floribundus and P. calomelanos plant tissues using μ-PIXE spectroscopy. Furthermore, cellular structure was preserved in samples prepared using this technique. © 2007, Elsevier Ltd.
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    Localisation of trace metals in hyperaccumulating plants using μ-PIXE
    (Australian Institute of Nuclear Science and Engineering (AINSE), 2007-11) Siegele, R; Kachenko, AG; Wang, YD; Ionescu, M; Bhatia, NP; Cohen, DD
    PIXE is a very sensitive technique that can fast and reliably measure a wide range of elements simultaneously with high sensitivity. Using a focused microbeam elemental distributions can be mapped with high spatial resolution. We demonstrate high resolution mapping of metals in plant leafs at 5 um resolution and it's application in detecting sites of metal accumulation in metal-accumulating plant tissues. The importance of biological sample preparation is discussed by direct comparison of freeze-substitution and freeze-drying routinely used in biological sciences. The advantages and limitations of quantitative elemental imaging using these techniques are also discussed.
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    Localisation of trace metals in metal-accumulating plants using μ-PIXE
    (Wiley-Blackwell, 2008-03) Siegele, R; Kachenko, AG; Bhatia, NP; Wang, YD; Ionescu, M; Singh, B; Baker, AJM; Cohen, DD
    Particle induced x-ray emission (PIXE) is a very sensitive technique that can quickly and reliably measure a wide range of elements simultaneously with high sensitivity. Using a focused microbeam, elemental distributions can be mapped with high spatial resolution. We demonstrate high-resolution mapping of metals in plant leaves at 5 mu m resolution and its application in detecting sites of metal accumulation in metal-accumulating plant tissues. The importance of biological sample preparation is discussed by direct comparison of freeze-substitution and freeze-drying techniques routinely used in biological sciences. The advantages and limitations of quantitative elemental imaging using these techniques are also discussed. © 2008, Wiley-Blackwell.
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    Nickel, Zn and Cd localisation in seeds of metal hyperaccumulators using mu-PIXE spectroscopy.
    (Elsevier, 2009-06-15) Kachenko, AG; Bhatia, NP; Siegele, R; Walsh, KB; Singh, B
    Metal hyperaccumulators are a rare group of plant species that accumulate exceptionally high concentrations of metals in above ground tissues without showing symptoms of phytotoxicity. Quantitative localisation of the accumulated metals in seed tissues is of considerable interest to help understand the eco-physiology of these unique plant species. We investigated the spatial localisation of metals within seeds of Ni hyperaccumulating Hybanthus floribundus subsp. adpressus, H. floribundus subsp. floribundus and Pimelea leptospermoides and dual-metal (Cd and Zn) hyperaccumulating Thlaspi caerulescens using quantitative micro-proton induced X-ray emission (μ-PIXE) spectroscopy. Intact seeds were hand-sectioned, sandwiched between Formvar films and irradiated using the 3 MeV high energy heavy ion microprobe at ANSTO. Elemental maps of whole H. floribundus subsp. adpressus seeds showed an average Ni concentration of 5.1 × 103 mg kg−1 dry weight (DW) with highest Ni concentration in cotyledonary tissues (7.6 × 103 mg kg−1 DW), followed by the embryonic axis (4.4 × 103 mg kg−1 DW). Nickel concentration in whole H. floribundus subsp. floribundus seeds was 3.5 × 102 mg kg−1 DW without a clear pattern of Ni localisation. The average Ni concentration in whole P. leptospermoides seeds was 2.6 × 102 mg kg−1 DW, and Ni was preferentially localised in the embryonic axis (4.3 × 102 mg kg−1 DW). In T. caerulescens, Cd concentrations were similar in cotyledon (4.5 × 103 mg kg−1 DW) and embryonic axis (3.3 × 103 mg kg−1 DW) tissues, whereas Zn was highest in cotyledonary tissues (1.5 × 103 mg kg−1 DW). In all species, the presence of the accumulated metal within the cotyledonary and embryonic axis tissues indicates that the accumulated metal was able to move apoplastically within the seed. © 2009, Elsevier Ltd.
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    Nuclear microprobe studies of metal(loid)s distribution in hyperaccumulating plants
    (Australian Institute of Nuclear Science and Engineering (AINSE), 2007-11) Kachenko, AG; Singh, B; Siegele, R; Bhatia, NP
    Micro-proton-induced X-ray emission (μ-PIXE) spectroscopy was used to determine in situ elemental concentrations of nickel (Ni) and arsenic (As) in leaf and stem tissues of hyperaccumulating plants Hybanthus floribundus subsp floribundus and Pityrogramma calomelanos var. austroamericana, respectively. Nickel concentration in seeds of H. floribundus subsp floribundus was also investigated. Both species were grown in metal(loid) contaminated potting mix for 20 weeks duration under controlled glasshouse conditions. Leaf and stem samples were hand-sectioned, cryo-fixed and freeze-dried in liquid nitrogen before μ-PIXE analysis using the 10-MV tandem accelerator at the Australian Nuclear Science and Technology Organization. In H. floribundus subsp floribundus leaves, Ni was highest in the adaxial epidermal cells (1% dry weight; DW) and least in spongy mesophyll (0.53% DW). In stem tissues, Ni concentrations were highest in the collenchyma (0.25% DW) and there was no clear pattern of Ni localization in seeds. In P. calomelanos pinnules, As localization was relatively uniform across the whole specimen and in stipe tissues, highest concentration occurred in the vascular bundle (0.2% DW). These results suggest that hyperaccumulating plants sequester excess metal(loid)s in different cellular loci and enables us to better understand the physiology and ecology of these hyperaccumulating species.
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    Quantitative elemental localisation in leaves and stems of nickel hyperaccumulating shrub Hybanthus floribundus subsp floribundus using micro-PIXE spectroscopy
    (Elsevier, 2008-02) Kachenko, AG; Singh, B; Bhatia, NP; Siegele, R
    Hybanthus floribundus (Lindl.) F.Muell. subsp. floribundus is a native Australian nickel (Ni) hyperaccumulating shrub and a promising species for rehabilitation and phytoremediation of Ni tailings. Spatial localisation and quantification of Ni in leaf and stem tissues of H. floribundus subsp.,floribundus was studied using micro-proton-induced X-ray emission (micro-PIXE) spectroscopy. Young plants, grown in a potting mix under controlled glasshouse conditions were exposed to Ni concentrations of 0 and 26 mM kg(-1) for 20 weeks. Leaf and stem samples were hand-sectioned and freeze-dried prior to micro-PIXE analysis. Elemental distribution maps of leaves revealed Ni concentration of 7800 mg kg(-1) dry weight (DW) in whole leaf sections, which was identical to the bulk tissue analysis. Elemental maps showed that Ni was preferentially localised in the adaxial epidermis (10,000 mg kg(-1) DW) and reached a maximum of up to 10,000 mg kg(-1) DW in the leaf margin. Freeze-dried stem sections from the same plants contained lower Ni than leaf tissues (1800 mg kg(-1) versus 7800 mg kg(-1) DW, respectively), however did not resolve a clear pattern of compartmentalisation across different anatomical regions. Our results suggest localisation in epidermal cells is an important physiological mechanism involved in Ni accumulation and tolerance in leaves of H. floribundus subsp. floribundus. © 2007, Elsevier Ltd.
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    Role of low molecular weight ligands in nickel hyperaccumulation in hybanthus floribundus subspecies floribundus
    (CSIRO Publishing, 2010-11-17) Kachenko, AG; Singh, B; Bhatia, NP
    The mechanisms responsible for nickel (Ni) hyperaccumulation in Hybanthus floribundus (Lindl.) F.Muell. subspecies floribundus are obscure. In this study, organic acids and free amino acids (AAs) were quantified in 0.025 M HCl H. floribundus subsp. floribundus shoot extracts using HPLC and ultra performance liquid chromatography (UPLC). In a 20 week pot experiment, plants exposed to five levels of Ni (0–3000 mg kg–1 Ni) accumulated up to 3200 mg Ni kg–1 dry weight in shoots, and the shoot : root Ni concentration ratios were >1.4. Concentration of organic acids followed the order malic acid > citric acid > oxalic acid. Citric acid concentration significantly increased upon Ni exposure, with concentrations between 2.3- and 5.9-fold higher in Ni treated plants that in control plants. Molar ratios of Ni to citric acid ranged from 1.3 : 1 to 1.7 : 1 equivalent to >60% of the accumulated Ni. Malic acid concentration also increased upon exposure to applied Ni. However, concentrations were statistically at par across 0–3000 mg kg–1 Ni treatments, suggesting that the production of malic acid is a constitutive property of the subspecies. Total AA concentrations were stimulated upon exposure to external Ni treatment, with glutamine, alanine and aspartic acids being the predominant acids. These AAs accounted for up to 64% of the total free AA concentration in control plants and up to 75% for the 2000 mg kg–1 Ni treatment plants. These results suggest that citric acid in addition to the aforementioned AAs are synthesised in H. floribundus subsp. floribundus plants following exposure to elevated concentrations of Ni and may act as potential ligands for detoxification and possibly storage of accumulated Ni. © 2010, CSIRO Publishing
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    X-ray absorption spectroscopy at the Ni-K edge in stackhousia tryonii bailey hyperaccumulator
    (Wiley-Blackwell, 2008-11) Lonescu, M; Bhatia, NP; Cohen, DD; Kachenko, AG; Siegele, R; Marcus, MA; Fakra, SC; Foran, GJ
    Young plants of Stackhousia tryonii Bailey were exposed to 34 mM Ni kg(-1) in the form of NiSO4 center dot 6H(2)O solution and grown under controlled glasshouse conditions fora period of 20 days. Fresh leaf, stem and root samples were analysed in vivo by micro x-ray absorption spectroscopy (XAS) at the Ni-K edge. Both x-ray absorption near edge structure and extended x-ray absorption fine structure spectra were analysed, and the resulting spectra were compared with spectra obtained from nine biologically important Ni-containing model compounds. The results revealed that the majority of leaf, stem and root Ni in the hyperaccumulator was chelated by citrate. Our results also suggest that in leaves Ni is complexed by phosphate and histidine, and in stems and roots, phytate and histidine. The XAS results provide an important physiological insight into transport, detoxification and storage of Ni in S. tryonii plants. © 2008, Wiley-Blackwell.