The extraordinary level of accumulation of nickel (Ni) in hyperaccumulator plants is a consequence of specific metal sequestering and transport mechanisms, and knowledge of these processes is critical for advancing an understanding of transition element metabolic regulation in these plants. The Ni biopathways were elucidated in three plant species, Phyllanthus balgooyi, Phyllanthus securinegioides (Phyllanthaceae) and Rinorea bengalensis (Violaceae), that occur in Sabah (Malaysia) on the Island of Borneo. This study showed that Ni is mainly concentrated in the phloem in roots and stems (up to 16.9% Ni in phloem sap in Phyllanthus balgooyi) in all three species. However, the species differ in their leaves – in P. balgooyi the highest Ni concentration is in the phloem, but in P. securinegioides and R. bengalensis in the epidermis and in the spongy mesophyll (R. bengalensis). The chemical speciation of Ni2+ does not substantially differ between the species nor between the plant tissues and transport fluids, and is unambiguously associated with citrate. This study combines ion microbeam (PIXE and RBS) and metabolomics techniques (GC-MS, LC-MS) with synchrotron methods (XAS) to overcome the drawbacks of the individual techniques to quantitatively determine Ni distribution and Ni2+ chemical speciation in hyperaccumulator plants.

Contents Summary432I.Introduction433II.Preparation of plant samples for X‐ray micro‐analysis433III.X‐ray elemental mapping techniques438IV.X‐ray data analysis442V.Case studies443VI.Conclusions446Acknowledgements449Author contributions449References449 Summary Hyperaccumulators are attractive models for studying metal(loid) homeostasis, and probing the spatial distribution and coordination chemistry of metal(loid)s in their tissues is important for advancing our understanding of their ecophysiology. X‐ray elemental mapping techniques are unique in providing in situ information, and with appropriate sample preparation offer results true to biological conditions of the living plant. The common platform of these techniques is a reliance on characteristic X‐rays of elements present in a sample, excited either by electrons (scanning/transmission electron microscopy), protons (proton‐induced X‐ray emission) or X‐rays (X‐ray fluorescence microscopy). Elucidating the cellular and tissue‐level distribution of metal(loid)s is inherently challenging and accurate X‐ray analysis places strict demands on sample collection, preparation and analytical conditions, to avoid elemental redistribution, chemical modification or ultrastructural alterations. We compare the merits and limitations of the individual techniques, and focus on the optimal field of applications for inferring ecophysiological processes in hyperaccumulator plants. X‐ray elemental mapping techniques can play a key role in answering questions at every level of metal(loid) homeostasis in plants, from the rhizosphere interface, to uptake pathways in the roots and shoots. Further improvements in technological capabilities offer exciting perspectives for the study of hyperaccumulator plants into the future.

SummaryPhyllanthus balgooyi (Phyllanthaceae), one of > 20 nickel (Ni) hyperaccumulator plant species known in Sabah (Malaysia) on the island of Borneo, is remarkable because it contains > 16 wt% Ni in its phloem sap, the second highest concentration of Ni in any living material in the world (after Pycnandra acuminata (Sapotaceae) from New Caledonia with 25 wt% Ni in latex).This study focused on the tissue-level distribution of Ni and other elements in the leaves, petioles and stem of P. balgooyi using nuclear microprobe imaging (micro-PIXE).The results show that in the stems and petioles of P. balgooyi Ni concentrations were very high in the phloem, while in the leaves there was significant enrichment of this element in the major vascular bundles. In the leaves, cobalt (Co) was codistributed with Ni, while the distribution of manganese (Mn) was different. The highest enrichment of calcium (Ca) in the stems was in the periderm, the epidermis and subepidermis of the petiole, and in the palisade mesophyll of the leaf.Preferential accumulation of Ni in the vascular tracts suggests that Ni is present in a metabolically active form. The elemental distribution of P. balgooyi differs from those of many other Ni hyperaccumulator plant species from around the world where Ni is preferentially accumulated in leaf epidermal cells.

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Two genotypes of Senecio coronatus (Thunb.) Harv. (Asteraceae) growing on ultramaWc outcrops were identiWed previously: a Ni hyperaccumulator and a non-hyperaccumulator. The aim of the present study was to investigate the cytology of the roots of both genotypes, their Ni content and tissue distribution, and to ascertain whether there was a cytological basis for the diVerential uptake of Ni. Light and Xuorescence microscopy together with histochemical methods were used to study root cytology. X-ray microanalysis by means of a nuclear microprobe-particle-induced X-ray emission (PIXE) and proton backscattering (BS) techniques-was utilized to determine the concentration and distribution of Ni and other elements. Average concentration of Ni and distribution in roots diVered signiWcantly between hyperaccumulating and non-hyperaccumulating genotypes. Ni amount in the hyperaccumulating genotype was ca. 60 times higher in the older part of the root (1,760 g g ¡1 ) and ca. 10 times higher (314 g g ¡1 ) in the younger root hair region in comparison with the equivalent parts of the non-accumulating genotype where Ni amounts were 30 g g ¡1 . Ni distribution pattern was also diVerent in both cases. Cytological diVerences were observed in the inner cortical region and exodermis of the roots. Distinct groups of specialized cells with an organelle-rich cytoplasm that produced copious numbers of spherical bodies occurred in the inner cortical region of the hyperaccumulator. Such distinct cell groups were absent from the inner cortex of the non-hyperaccumulator. Cortical cells here had a thin parietal cytoplasmic layer and produced fewer spherical bodies. In both genotypes the spherical structures were extruded from the cytoplasm into air spaces between the cells where they coalesced to form amorphous deposits, signiWcantly larger and more abundant in the hyperaccumulator. Histochemical tests identiWed these deposits as a mixture of lipids, alkaloids and terpenoids. Specialized cells present in the inner cortex of the hyperaccumulating genotype demonstrated signiWcant relative Ni depletion in comparison with the adjacent inner cortex and phloem. Casparian bands were identiWed in exodermal cell walls of both genotypes but the bands Xuoresced more intensely in the non-accumulator suggesting diVerences in chemical composition and probably also a more eYcient apoplastic barrier. This feature was correlated with the observed Ni distribution pattern. The highest Ni enrichment in the hyperaccumulating genotype occurred in the outer cortex; 20 times more than in the adjacent epidermis/exodermis in older portions of roots and 3 times more than in the epidermis/exodermis in younger root hair regions. In contrast, in the Wojciech Przybyiowicz on leave from the