Contrasting patterns of nickel distribution in the hyperaccumulators Phyllanthus balgooyi and Phyllanthus rufuschaneyi from Malaysian Borneo.

Globally, the majority of Ni hyperaccumulator plants occur on ultramafic soils in tropical regions, and the genus Phyllanthus, from the Phyllanthaceae family, is globally the most represented taxonomical group. Two species from Sabah (Malaysia) are remarkable because Phyllanthus balgooyi can attain >16 wt% of Ni in its phloem exudate, while Phyllanthus rufuschaneyi reaches foliar concentrations of up to 3.5 wt% Ni, which are amongst the most extreme concentrations of Ni in any plant tissue. Synchrotron X-ray fluorescence microscopy, nuclear microbe (micro-PIXE+BS) and (cryo) scanning electron microscopy with energy dispersive spectroscopy were used to spatially resolve the elemental distribution in the plant organs of P. balgooyi and P. rufuschaneyi. The results show that P. balgooyi has extraordinary enrichment of Ni in the (secondary) veins of the leaves, whereas in contrast, in P. rufuschaneyi Ni occurs in interveinal areas. In the roots and stems, Ni is localized mainly in the cortex and phloem but is much lower in the xylem. The findings of this study show that, even within the same genus, the distribution of nickel and other elements, and inferred processes involved with metal hyperaccumulation, can differ substantially between species.

Elemental distribution and chemical speciation of copper and cobalt in three metallophytes from the copper-cobalt belt in Northern Zambia.

Three metallophyte species, Persicaria capitata, P. puncata (Polygonaceae), Conyza cordata (Asteraceae) from mineral wastes in the Zambian copper-cobalt belt were studied. This study focused on the elemental distribution in the roots, stems and leaves, using a range of techniques: micro-PIXE, SEM-EDS synchrotron XFM and XAS. The species differed in their responses to growing on Co-Cu-enriched soils: Persicaria puncata is a Co hyperaccumulator (up to 1060 µg g-1 in leaves), while Persicaria capitata and Conyza cordata are Co-excluders. All three species are Cu-accumulators. The highest concentrations of Cu-Co are in the epidermal cells, whereas in Persicaria puncata Co was also enriched in the phloem. The Co coordination chemistry shows that an aqueous Co(ii)-tartrate complex was the predominant component identified in all plants and tissues, along with a minor component of a Co(iii) compound with oxygen donor ligands. For Cu, there was considerable variation in the Cu speciation in the various tissues and across the three species. In contrast to hyperaccumulator plants, excluder and accumulator type plants have received far less attention. This study highlights the different biopathways of transition elements (Cu, Co) in hyper-tolerant plant species showing different responses to metalliferous environments.

Abnormal concentrations of Cu-Co in Haumaniastrum katangense, Haumaniastrum robertii and Aeolanthus biformifolius: contamination or hyperaccumulation?

The Central African Copperbelt of the DR Congo and Zambia hosts more than 30 known Cu-Co hyperaccumulator plant species. These plants can accumulate extraordinarily high concentrations of Cu and Co in their living tissues without showing any signs of toxicity. Haumaniastrum robertii is the most extreme Co hyperaccumulator (able to accumulate up to 1 wt% Co), whereas Aeolanthus biformifolius is the most extreme Cu hyperaccumulator (with up to 1 wt% Cu). The phenomenon of Cu-Co hyperaccumulator plants was studied intensively in the 1970s through to the 1990s, but doubts arose regarding earlier observations due to surficial contamination of plant material with mineral particles. This study set out to determine whether such extraneous contamination could be observed on herbarium specimens of Haumaniastrum robertii and Aeolanthus biformifolius using scanning electron microscopy with energy-dispersive spectroscopy (SEM-EDS). Further, synchrotron X-ray absorption spectroscopy (XAS) was used to identify the chemical forms of Cu and Co in newly collected Haumaniastrum katangense plant material from the DR Congo. The results show that surficial contamination is not the cause for abnormal Cu-Co concentrations in the plant material, but rather that Cu-Co enrichment is endogenous. The chemical form of Cu and Co (complexation with carboxylic acids) provides additional evidence that genuine hyperaccumulation, and not soil mineral contamination, is responsible for extreme tissue concentrations of Cu and Co in Haumaniastrum katangense.

X-ray elemental mapping techniques for elucidating the ecophysiology of hyperaccumulator plants.

Contents Summary 432 I. Introduction 433 II. Preparation of plant samples for X-ray micro-analysis 433 III. X-ray elemental mapping techniques 438 IV. X-ray data analysis 442 V. Case studies 443 VI. Conclusions 446 Acknowledgements 449 Author contributions 449 References 449 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.

A Comparative Study of Selected Physical and Biochemical Traits of Wild-Type and Transgenic Sorghum to Reveal Differences Relevant to Grain Quality.

Transgenic sorghum featuring RNAi suppression of certain kafirins was developed recently, to address the problem of poor protein digestibility in the grain. However, it was not firmly established if other important quality parameters were adversely affected by this genetic intervention. In the present study several quality parameters were investigated by surveying several important physical and biochemical grain traits. Important differences in grain weight, density and endosperm texture were found that serve to differentiate the transgenic grains from their wild-type counterpart. In addition, ultrastructural analysis of the protein bodies revealed a changed morphology that is indicative of the effect of suppressed kafirins. Importantly, lysine was found to be significantly increased in one of the transgenic lines in comparison to wild-type; while no significant changes in anti-nutritional factors could be detected. The results have been insightful for demonstrating some of the corollary changes in transgenic sorghum grain, that emerge from imposed kafirin suppression.

Nickel biopathways in tropical nickel hyperaccumulating trees from Sabah (Malaysia).

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.

Extreme nickel hyperaccumulation in the vascular tracts of the tree Phyllanthus balgooyi from Borneo.

Phyllanthus 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.

Elemental distribution in tissue components of N2-fixing nodules of Psoralea pinnata plants growing naturally in wetland and upland conditions in the Cape Fynbos of South Africa.

There is little information on in situ distribution of nutrient elements in N2-fixing nodules. The aim of this study was to quantify elemental distribution in tissue components of N2-fixing nodules harvested from Psoralea pinnata plants grown naturally in wetland and upland conditions in the Cape Fynbos. The data obtained from particle-induced X-ray emission revealed the occurrence of 20 elements (Si, P, S, Cl, K, Ca, Ti, Mn, Fe, Ni, Cu, Zn, As, Br, Rb, Sr, Y, Zr, Mo and Ba) in nodule components. Although, in upland plants, the concentrations of S, Fe, Si, Mn and Cu showed a steady increase from the middle cortex to the medulla region of P. pinnata nodules, in wetland plants, only S, Fe and Mn showed an increase in concentration from the middle cortex to the bacteria-infected medulla of P. pinnata nodules. By contrast, the concentrations of Cl, K, Ca, Zn and Sr decreased from middle cortex to nodule medulla. The alkaline earth, alkali and transition elements Rb, Sr, Y and Zr, never before reported in N2-fixing nodules, were found to occur in root nodules of P. pinnata plants grown in both wetland and upland conditions.

Micro-particle-induced X-ray emission mapping of elemental distribution in roots of a Mediterranean-type sclerophyll, Agathosma betulina (Berg.) Pillans, colonized by Cryptococcus laurentii.

The role of rhizosphere yeasts as plant nutrient-scavenging microsymbionts in resource-limited Mediterranean-type heathlands is unknown. This study, therefore, focused on quantitative elemental distribution within the roots of a medicinal sclerophyll, Agathosma betulina (Berg.) Pillans, grown under nutrient-poor conditions, and colonized by Cryptococcus laurentii. Micro-particle-induced X-ray emission (PIXE) was used to assess quantitative elemental distribution within the roots of A. betulina inoculated with viable C. laurentii, as well as within roots of control plants that received autoclaved yeast. To aid in the interpretation of heterogeneous elemental distribution patterns, apoplastic barriers (Casparian bands) in root tissues were located using fluorescence microscopy. In addition, root cross-sections were examined for endophytic C. laurentii using light and transmission electron microscopy (TEM). The average concentrations of P, Fe and Mn were significantly (P < 0.05) higher in roots of yeast-inoculated plants, compared to control plants. Casparian bands were observed in the exodermal cells of both treatments, and the presence of these bands was correlated with elemental enrichment in the epi/exodermal-outer cortical tissues. Light and TEM micrographs revealed that the yeast was not a root endophyte. This is the first report describing the role of a soil yeast as a plant nutrient-scavenging microsymbiont.