Uncovering the physiology and distribution of thallium in Tl-hyperaccumulating and Tl-sensitive populations of Biscutella laevigata L.

BACKGROUND AND AIMS: Thallium (Tl) is extremely toxic to all lifeforms and an emerging pollutant. Plants in the Brassicaceae family, including edible crops, have an enhanced capacity for Tl accumulation, even from soils with low thallium concentration. The most extreme Tl hyperaccumulator is Biscutella laevigata, capable of attaining >32,000 µg Tl g-1 DW in its leaves. METHODS: Biscutella laevigata from a non-metallicolous accession (Feltre, Italy) and a metallicolous accession (Les Malines, France) were subjected to a dosing experiment in hydroponics (0, 5, 30 µM Tl), followed by synchrotron-based µXRF analysis to elucidate tissue and cellular-level Tl distribution. KEY RESULTS: Flow cytometric data on the two used accessions showed the Feltre accession has a genome size twice of that of the Les Malines accession (256 and 125 pg/2C respectively), suggesting they are phylogenetically distant populations. The Feltre accession does not accumulate Tl (125 µg Tl g-1 DW on average in leaves) at the 5 µM Tl dose level, whereas the Les Malines accession had a mean of 1750 µg Tl g-1 DW, with peaks of 24,130 µg Tl g-1 DW at the 30 µM Tl dose level. At 30 µM Tl the non-metallicolous accession did not grow, and at 5 µM Tl showed reduced biomasss compared to the metallicolous one. In Les Malines accession, the synchrotron-based µXRF analysis revealed that Tl is localised in the vacuoles of epidermal cells, especially underneath trichomes and in trichome basal cells. Thallium also occurs in solid crystalline deposits (3-5 µm in size, ~40 wt% Tl) that are mainly found in foliar margins and under trichome bases. CONCLUSIONS: Biscutella laevigata is an attractive model for studying Tl hypertolerance and hyperaccumulation on account of the extreme expression of this trait, and its marked intraspecific variability.

Biomineralization of mantis shrimp dactyl club following molting: Apatite formation and brominated organic components.

The stomatopod Odontodactylus scyllarus uses weaponized club-like appendages to attack its prey. These clubs are made of apatite, chitin, amorphous calcium carbonate, and amorphous calcium phosphate organized in a highly hierarchical structure with multiple regions and layers. We follow the development of the biomineralized club as a function of time using clubs harvested at specific times since molting. The clubs are investigated using a broad suite of techniques to unravel the biomineralization history of the clubs. Nano focus synchrotron x-ray diffraction and x-ray fluorescence experiments reveal that the club structure is more organized with more sub-regions than previously thought. The recently discovered impact surface has crystallites in a different size and orientation than those in the impact region. The crystal unit cell parameters vary to a large degree across individual samples, which indicates a spatial variation in the degree of chemical substitution. Energy dispersive spectroscopy and Raman spectroscopy show that this variation cannot be explained by carbonation and fluoridation of the lattice alone. X-ray fluorescence and mass spectroscopy show that the impact surface is coated with a thin membrane rich in bromine that forms at very initial stages of club formation. Proteomic studies show that a fraction of the club mineralization protein-1 has brominated tyrosine suggesting that bromination of club proteins at the club surface is an integral component of the club design. Taken together, the data unravel the spatio-temporal changes in biomineral structure during club formation. STATEMENT OF SIGNIFICANCE: Mantis shrimp hunt using club-like appendages that contain apatite, chitin, amorphous calcium carbonate, and amorphous calcium phosphate ordered in a highly hierarchical structure. To understand the formation process of the club we analyze clubs harvested at specific times since molting thereby constructing a club formation map. By combining several methods ranging from position resolved synchrotron X-ray diffraction to proteomics, we reveal that clubs form from an organic membrane with brominated protein and that crystalline apatite phases are present from the very onset of club formation and grow in relative importance over time. This reveals a complex biomineralization process leading to these fascinating biomineralized tools.

High-energy interference-free K-lines synchrotron X-ray fluorescence microscopy of rare earth elements in hyperaccumulator plants.

Synchrotron-based micro-X-ray fluorescence analysis (µXRF) is a nondestructive and highly sensitive technique. However, element mapping of rare earth elements (REEs) under standard conditions requires care, since energy-dispersive detectors are not able to differentiate accurately between REEs L-shell X-ray emission lines overlapping with K-shell X-ray emission lines of common transition elements of high concentrations. We aim to test REE element mapping with high-energy interference-free excitation of the REE K-lines on hyperaccumulator plant tissues and compare with measurements with REE L-shell excitation at the microprobe experiment of beamline P06 (PETRA III, DESY). A combination of compound refractive lens optics (CRLs) was used to obtain a micrometer-sized focused incident beam with an energy of 44 keV and an extra-thick silicon drift detector optimized for high-energy X-ray detection to detect the K-lines of yttrium (Y), lanthanum (La), cerium (Ce), praseodymium (Pr), and neodymium (Nd) without any interferences due to line overlaps. High-energy excitation from La to Nd in the hyperaccumulator organs was successful but compared to L-line excitation less efficient and therefore slow (∼10-fold slower than similar maps at lower incident energy) due to lower flux and detection efficiency. However, REE K-lines do not suffer significantly from self-absorption, which makes XRF tomography of millimeter-sized frozen-hydrated plant samples possible. The K-line excitation of REEs at the P06 CRL setup has scope for application in samples that are particularly prone to REE interfering elements, such as soil samples with high concomitant Ti, Cr, Fe, Mn, and Ni concentrations.

Differences and similarities in selenium biopathways in Astragalus, Neptunia (Fabaceae) and Stanleya (Brassicaceae) hyperaccumulators.

BACKGROUND AND AIMS: Selenium hyperaccumulator species are of primary interest for studying the evolution of hyperaccumulation and for use in biofortification because selenium is an essential element in human nutrition. In this study, we aimed to determine whether the distributions of selenium in the three most studied hyperaccumulating taxa (Astragalus bisulcatus, Stanleya pinnata and Neptunia amplexicaulis) are similar or contrasting, in order to infer the underlying physiological mechanisms. METHODS: This study used synchrotron-based micro-X-ray fluorescence (µXRF) techniques to visualize the distribution of selenium and other elements in fresh hydrated plant tissues of A. racemosus, S. pinnata and N. amplexicaulis. KEY RESULTS: Selenium distribution differed widely in the three species: in the leaves of A. racemosus and N. amplexicaulis selenium was mainly concentrated in the pulvini, whereas in S. pinnata it was primarilylocalized in the leaf margins. In the roots and stems of all three species, selenium was absent in xylem cells, whereas it was particularly concentrated in the pith rays of S. pinnata and in the phloem cells of A. racemosus and N. amplexicaulis. CONCLUSIONS: This study shows that Astragalus, Stanleya and Neptunia have different selenium-handling physiologies, with different mechanisms for translocation and storage of excess selenium. Important dissimilarities among the three analysed species suggest that selenium hyperaccumulation has probably evolved multiple times over under similar environmental pressures in the US and Australia.

Locked up Inside the Vessels: Rare Earth Elements Are Transferred and Stored in the Conductive Tissues of the Accumulating Fern Dryopteris erythrosora.

Rare earth elements (REEs) are strategic metals strongly involved in low-carbon energy conversion. However, these emerging contaminants are increasingly disseminated into ecosystems, raising concern regarding their toxicity. REE-accumulating plants are crucial subjects to better understand REE transfer to the trophic chain but are also promising phytoremediation tools. In this analysis, we deciphered REE accumulation sites in the REE-accumulating fern Dryopteris erythrosora by synchrotron X-ray µfluorescence (µXRF). This technique allows a high-resolution and in situ analysis of fresh samples or frozen-hydrated cross sections of different organs of the plant. In the sporophyte, REEs were translocated from the roots to the fronds by the xylem sap and were stored within the xylem conductive system. The comparison of REE distribution and accumulation levels in the healthy and necrotic parts of the frond shed light on the differential mobility between light and heavy REEs. Furthermore, the comparison emphasized that necrotized areas were not the main REE-accumulating sites. Finally, the absence of cell-to-cell mobility of REEs in the gametophyte suggested the absence of REE-compatible transporters in photosynthetic tissues. These results provide valuable knowledge on the physiology of REE-accumulating ferns to understand the REE cycle in biological systems and the expansion of phytotechnologies for REE-enriched or REE-contaminated soils.

Synchrotron XFM tomography for elucidating metals and metalloids in hyperaccumulator plants.

Visualizing the endogenous distribution of elements within plant organs affords key insights in the regulation of trace elements in plants. Hyperaccumulators have extreme metal(loid) concentrations in their tissues, which make them useful models for studying metal(loid) homeostasis in plants. X-ray-based methods allow for the nondestructive analysis of most macro and trace elements with low limits of detection. However, observing the internal distributions of elements within plant organs still typically requires destructive sample preparation methods, including sectioning, for synchrotron X-ray fluorescence microscopy (XFM). X-ray fluorescence microscopy-computed tomography (XFM-CT) enables "virtual sectioning" of a sample thereby entirely avoiding artefacts arising from destructive sample preparation. The method can be used on frozen-hydrated samples, as such preserving "life-like" conditions. Absorption and Compton scattering maps obtained from synchrotron XFM-CT offer exquisite detail on structural features that can be used in concert with elemental data to interpret the results. In this article we introduce the technique and use it to reveal the internal distribution of hyperaccumulated elements in hyperaccumulator plant species. XFM-CT can be used to effectively probe the distribution of a range of different elements in plant tissues/organs, which has wide ranging applications across the plant sciences.

Comprehensive insights in thallium ecophysiology in the hyperaccumulator Biscutella laevigata.

Biscutella laevigata is the strongest known thallium (Tl) hyperaccumulator plant species. However, little is known about the ecophysiological processes leading to root uptake and translocation of Tl in this species, and the interactions between Tl and its chemical analogue potassium (K). Biscutella laevigata was subjected to hydroponics experimentation in which it was exposed to Tl and K, and it was investigated in a rhizobox experiment. Laboratory-based micro-X-ray fluorescence spectroscopy (µ-XRF) was used to reveal the Tl distribution in the roots and leaves, while synchrotron-based µ-XRF was utilised to reveal elemental distribution in the seed. The results show that in the seed Tl was mainly localised in the endosperm and cotyledons. In mature plants, Tl was highest in the intermediate leaves (16,100 µg g-1), while it was one order of magnitude lower in the stem and roots. Potassium did not inhibit or enhance Tl uptake in B.laevigata. At the organ level, Tl was localised in the blade and margins of the leaves. Roots foraged for Tl and cycled Tl across roots growing in the control soils. Biscutella laevigata has ostensibly evolved specialised mechanisms to tolerate high Tl concentrations in its shoots. The lack of interactions and competition between Tl and K suggests that it is unlikely that Tl is taken up via K channels, but high affinity Tl transporters remain to be identified in this species. Thallium is not only highly toxic but also a valuable metal and Tl phytoextraction using B. laevigata should be explored.

Evidence of hexavalent chromium formation and changes of Cr speciation after laboratory-simulated fires of composted tannery sludges long-term amended agricultural soils.

Controlled or accidental fires can impact agricultural soils amended with composted organic materials since high temperatures cause fast organic matter (OM) mineralization and soil properties modifications. During these events, potentially toxic elements (PTEs) associated with OM can be released and change their distribution and speciation thus becoming a threat to the environment and to crops. In this study, we investigated the changes of distribution and speciation of chromium in soils long-term amended with compost obtained from tannery sludges, after simulating fires of different intensity (300, 400 and 500 °C) likely to occur on agricultural soils. A combination of conventional soil chemical analyses and bulk and (sub)micro X-ray analyses allowed the observation of the formation of hexavalent chromium and changes of chromium speciation. Specifically, a strong decrease of Cr-OM associations was found with increasing temperature in favour of Cr-iron (hydr)oxides interactions and CaCrO4 formation. These data provide first evidence that fires can transform OM-stabilized Cr into more mobile, available and toxic Cr-forms potentially accessible for plant uptake, thus posing a risk for the food chain and the environment.

Distribution of Pb and Se in mouse brain following subchronic Pb exposure by using synchrotron X-ray fluorescence.

Lead (Pb) is a well-known neurotoxicant and environmental hazard. Recent experimental evidence has linked Pb exposure with neurological deterioration leading to neurodegenerative diseases, such as Alzheimer's disease. To understand brain regional distribution of Pb and its interaction with other metal ions, we used synchrotron micro-x-ray fluorescence technique (µ-XRF) to map the metal distribution pattern and to quantify metal concentrations in mouse brains. Lead-exposed mice received oral gavage of Pb acetate once daily for 4 weeks; the control mice received sodium acetate. Brain tissues were cut into slices and subjected for analysis. Synchrotron µ-XRF scans were run on the PETRA III P06 beamline (DESY). Coarse scans of the entire brain were performed to locate the cortex and hippocampus, after which scans with higher resolution were run in these areas. The results showed that: a) the total Pb intensity in Pb-exposed brain slices was significantly higher than in control brain; b) Pb typically deposited in localized particles of <10 um2 in both the Pb-exposed and control brain slices, with more of these particles in Pb-exposed samples; c) selenium (Se) was significantly correlated with Pb in these particles in the cortex and hippocampus/corpus callosum regions in the Pb-exposed samples, and the molar ratio of the Se and Pb in these particles is close to 1:1. These results indicated that Se may play a crucial role in Pb-induced neurotoxicity. Our findings call for further studies to investigate the relationship between Pb exposure and possible Se detoxification responses, and the implication in the etiology of Alzheimer's disease.

Spatially Resolved Localization of Lanthanum and Cerium in the Rare Earth Element Hyperaccumulator Fern Dicranopteris linearis from China.

The fern Dicranopteris linearis (Gleicheniaceae) from China is a hyperaccumulator of rare earth element (REE), but little is known about the ecophysiology of REE in this species. This study aimed to clarify tissue-level and organ-level distribution of REEs via synchrotron-based X-ray fluorescence microscopy (XFM). The results show that REEs (La + Ce) are mainly colocalized with Mn in the pinnae and pinnules, with the highest concentrations in necrotic lesions and lower concentrations in veins. In the cross sections of the pinnules, midveins, rachis, and stolons, La + Ce and Mn are enriched in the epidermis, vascular bundles, and pericycle (midvein). In these tissues, Mn is localized mainly in the cortex and mesophyll. We hypothesize that the movement of REEs in the transpiration flow in the veins is initially restricted in the veins by the pericycle between vascular bundle and cortex, while excess REEs are transported by evaporation and cocompartmentalized with Mn in the necrotic lesions and epidermis in an immobile form, possibly a Si-coprecipitate. The results presented here provide insights on how D. linearis regulates high concentrations of REEs in vivo, and this knowledge is useful for developing phytotechnological applications (such as REE agromining) using this fern in REE-contaminated sites in China.