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.

A detailed localization of elements in leaf tissues of the field-collected Cd/Zn hyperaccumulator Thlaspi praecox (Brassicaceae) growing at a highly metal-polluted site was determined by micro-proton-induced X-ray emission (micro-PIXE) in order to reveal and compare nutrient and non-essential element accumulation patterns in the case of multiple metal accumulation within particular leaf tissues, including the detailed distribution between apoplast and symplast regions. On the larger scans, the highest concentrations of metals were observed in the epidermis, S and Ca in the palisade mesophyll, Cl in the spongy mesophyll and vascular bundles, and P and K in the vascular bundles. On the more detailed scans, the highest Cd, Pb, Cl and K concentrations were observed in vascular bundle collenchyma. The relative element distribution (%) was calculated based on concentrations of elements in particular leaf tissues and their relative weight portions, indicating that most of the accumulated Zn was located in epidermises, while the majority of Cd and Pb was distributed within the mesophyll. Detailed scans of epidermal/mesophyll tissues revealed that Zn was mainly accumulated and detoxified in the symplast of large vacuolated epidermal cells, Cd in the mesophyll symplast, and Pb in the mesophyll symplast and apoplast.

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.

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BackgroundIron is an important micronutrient for all living organisms. Almost 25% of the world population is affected by iron deficiency, a leading cause of anemia. In plants, iron deficiency leads to chlorosis and reduced yield. Both animals and plants may suffer from iron deficiency when their diet or environment lacks bioavailable iron. A sustainable way to reduce iron malnutrition in humans is to develop staple crops with increased content of bioavailable iron. Knowledge of where and how iron accumulates in seeds of crop plants will increase the understanding of plant iron metabolism and will assist in the production of staples with increased bioavailable iron.ResultsHere we reveal the distribution of iron in seeds of three Phaseolus species including thirteen genotypes of P. vulgaris, P. coccineus, and P. lunatus. We showed that high concentrations of iron accumulate in cells surrounding the provascular tissue of P. vulgaris and P. coccineus seeds. Using the Perls' Prussian blue method, we were able to detect iron in the cytoplasm of epidermal cells, cells near the epidermis, and cells surrounding the provascular tissue. In contrast, the protein ferritin that has been suggested as the major iron storage protein in legumes was only detected in the amyloplasts of the seed embryo. Using the non-destructive micro-PIXE (Particle Induced X-ray Emission) technique we show that the tissue in the proximity of the provascular bundles holds up to 500 μg g-1 of iron, depending on the genotype. In contrast to P. vulgaris and P. coccineus, we did not observe iron accumulation in the cells surrounding the provascular tissues of P. lunatus cotyledons. A novel iron-rich genotype, NUA35, with a high concentration of iron both in the seed coat and cotyledons was bred from a cross between an Andean and a Mesoamerican genotype.ConclusionsThe presented results emphasize the importance of complementing research in model organisms with analysis in crop plants and they suggest that iron distribution criteria should be integrated into selection strategies for bean biofortification.