Paula Pongrac

New insights into globoids of protein storage vacuoles in wheat aleurone using synchrotron soft X-ray microscopy

Mature developed seeds are physiologically and biochemically committed to store nutrients, principally as starch, protein, oils, and minerals. The composition and distribution of elements inside the aleurone cell layer reflect their biogenesis, structural characteristics, and physiological functions. It is therefore of primary importance to understand the mechanisms underlying metal ion accumulation, distribution, storage, and bioavailability in aleurone subcellular organelles for seed fortification purposes. Synchrotron radiation soft X-ray full-field imaging mode (FFIM) and low-energy X-ray fluorescence (LEXRF) spectromicroscopy were applied to characterize major structural features and the subcellular distribution of physiologically important elements (Zn, Fe, Na, Mg, Al, Si, and P). These direct imaging methods reveal the accumulation patterns between the apoplast and symplast, and highlight the importance of globoids with phytic acid mineral salts and walls as preferential storage structures. C, N, and O chemical topographies are directly linked to the structural backbone of plant substructures. Zn, Fe, Na, Mg, Al, and P were linked to globoid structures within protein storage vacuoles with variable levels of co-localization. Si distribution was atypical, being contained in the aleurone apoplast and symplast, supporting a physiological role for Si in addition to its structural function. These results reveal that the immobilization of metals within the observed endomembrane structures presents a structural and functional barrier and affects bioavailability. The combination of high spatial and chemical X-ray microscopy techniques highlights how in situ analysis can yield new insights into the complexity of the wheat aleurone layer, whose precise biochemical composition, morphology, and structural characteristics are still not unequivocally resolved.

Low-energy X-ray fluorescence microscopy opening new opportunities for bio-related research

Biological systems are unique matter with very complex morphology and highly heterogeneous chemical composition dominated by light elements. Discriminating qualitatively at the sub-micrometer level the lateral distribution of constituent elements, and correlating it to the sub-cellular biological structure, continues to be a challenge. The low-energy X-ray fluorescence microspectroscopy, recently implemented in TwinMic scanning transmission mode, has opened up new opportunities for mapping the distribution of the light elements, complemented by morphology information provided by simultaneous acquisition of absorption and phase contrast images. The important new information that can be obtained in bio-related research domains is demonstrated by two pilot experiments with specimens of interest for marine biology and food science. They demonstrate the potential to yield important insights into the structural and compositional enrichment, distribution and correlation of essential trace elements in the lorica of Tintinnopsis radix, and the lateral distribution of trace nutrients in the seeds of wheat Triticum aestivum.

The fate of arsenic, cadmium and lead in Typha latifolia: A case study on the applicability of micro-PIXE in plant ionomics

Understanding the uptake, accumulation and distribution of toxic elements in plants is crucial to the design of effective phytoremediation strategies, especially in the case of complex multi-element pollution. Using micro-proton induced X-ray emission, the spatial distribution of Na, Mg, Al, Si, P, S, Cl, K, Ca, Mn, Fe, Zn, As, Br, Rb, Sr, Cd and Pb have been quantitatively resolved in roots and rhizomes of an obligate wetland plant species, Typha latifolia, treated with a mixture of 100 μM each of As, Cd and Pb, together. The highest concentrations of As, Cd and Pb were found in the roots of the T. latifolia, with tissue-specific distributions. The As was detected in the root rhizodermis, and in the rhizome the majority of the As was within the vascular tissues, which indicates the high mobility of As within T. latifolia. The Cd was detected in the root exodermis, and in the vascular bundle and epidermis of the rhizome. The highest Pb concentrations were detected in the root rhizodermis and exodermis, and in the epidermis of the rhizome. These data represent an essential step in the resolution of fundamental questions in plant ionomics.

Glucosinolate Profiles Change During the Life Cycle and Mycorrhizal Colonization in a Cd/Zn Hyperaccumulator Thlaspi praecox (Brassicaceae)

Thlaspi praecox Wulfen (Brassicaceae) is a perennial Cd/Zn hyperaccumulating plant species that forms functional arbuscular mycorrhizal (AM) symbiosis. Glucosinolates (GS) were studied in different organs of field-collected T. praecox at differing plant developmental stages. Additionally, AM colonization was recorded. Total GS concentrations and profiles of nine individual GS varied during the plant life cycle. Novel individual GS that were related to specific developmental phases, mainly to flowering and seed production, were identified. The highest total GS and sinalbin concentrations in rosette leaves were found in the vegetative phase, possibly contributing to protection of young, palatable leaves. The lowest were found in roots during the flowering and the seeding phases. Increased total GS concentrations in roots and enhanced aliphatic GS, especially glucobrassicanapin, in the senescence phase may protect roots from herbivory during winter and early spring. The presence of glucotropaeolin and the absence of glucobrassicanapin in the flowering phase coincided with peak AM colonization. This is the first report on GS profiles in an AM and metal-hyperaccumulating plant.

Relevance for food sciences of quantitative spatially resolved element profile investigations in wheat (Triticum aestivum) grain

Bulk element concentrations of whole grain and element spatial distributions at the tissue level were investigated in wheat (Triticum aestivum) grain grown in Zn-enriched soil. Inductively coupled plasma mass spectrometry and inductively coupled plasma optical emission spectrometry were used for bulk analysis, whereas micro-proton-induced X-ray emission was used to resolve the two-dimensional localization of the elements. Soil Zn application did not significantly affect the grain yield, but did significantly increase the grain Ca, Fe and Zn concentrations, and decrease the grain Na, P and Mo concentrations; bulk Mg, S, K, Mn, Cu, Cd and Pb concentrations remained unchanged. These changes observed in bulk element concentrations are the reflection of tissue-specific variations within the grain, revealing that Zn application to soil can lead to considerable alterations in the element distributions within the grain, which might ultimately influence the quality of the milling fractions. Spatially resolved investigations into the partitioning of the element concentrations identified the tissues with the highest element concentrations, which is of utmost importance for accurate prediction of element losses during the grain milling and polishing processes.

ROOTS OF METAL HYPERACCUMULATING POPULATION OF THLASPI PRAECOX (BRASSICACEAE) HARBOUR ARBUSCULAR MYCORRHIZAL AND OTHER FUNGI UNDER EXPERIMENTAL CONDITIONS

Thlaspi praecox (Brassicaceae) is a recently discovered metal hyperaccumulating plant species colonized by arbuscular mycorrhizal fungi (AMF). The identity and diversity of the AMF colonizing its roots have not been determined so far. Therefore, T. praecox was inoculated with an indigenous fungal mixture from a metal polluted site and grown in original polluted soil/ commercial substrate mixtures (i.e., 100%, 50%, and 25%). Low to moderate mycorrhizal frequencies (F = 33–68%) and only rare arbuscules were observed. Densities of vesicles and microsclerotia, typical structures of dark septate endophytes (DSE), were greater in pots with 100% original polluted soil. In contrast, the highest diversity of fungal genotypes was observed in the roots from 25% polluted soil/ commercial substrate mixture, with the lowest soil concentrations of Cd, Zn, and Pb. The sequences obtained corresponded to Glomus species (Glomeromycota), to putative DSE Phialophora verrucosa and Rhizoctonia sp. and to some other fungi from Asco- and Basidiomycota, that are known to associate with plants, namely Capnobotryella sp., Penicillium brevicompactum, Rodotorula aurantiaca and Rodotorula slooffiae. This is the first report of DSE occurrence in roots of hyperaccumulating T. praecox, a promising candidate for phytoextraction.

Improved lateral discrimination in screening the elemental composition of buckwheat grain by micro-PIXE.

The elemental composition of specific fractions of cereal and pseudocereal grains can be roughly estimated after milling. Alternatively, the elemental localization of cross-sectioned grains can be quantitatively analyzed by microproton induced X-ray emission (micro-PIXE), taking advantage of high elemental sensitivity and low lateral resolution. We present a micro-PIXE study on buckwheat (Fagopyrum esculentum) grain, with a detailed description of the elemental distributions. Elements such as Mg, P, S, K, Fe, Ni, Cu, and Zn were preferentially localized in the cotyledons and embryonic axis; however, significant amounts of K and Fe were also found in the pericarp. The aleurone layer covering the cotyledons was especially enriched in S and P, while testa, a thin layer above the aleurone did not show any significant element enrichments. The highest concentrations of Al, Si, Cl, Ca, and Ti were found in the pericarp. A detailed element localization study of pericarp layers revealed that the inner layer was enriched in K, Mn, Ca, and Fe, while the outer layer showed enrichments in Na, Mg, P, S, and Al. On the basis of the data obtained, milling techniques can be adapted to obtain milling fractions with targeted nutritional values.

Micro-PIXE Analysis for Localization and Quantification of Elements in Roots of Mycorrhizal Metal-Tolerant Plants

The main aim of contemporary ecotoxicological studies is to determine the bioavailability, toxicity, and risk relationships of trace-element contaminants in ecosystems. Discussions thus focus primarily on the element concentrations in soils, their dynamics, and the impact they have on microorganisms, plants, animals, and the ecosystem as a whole. However, before the basic ecotoxicological principles can be discussed, the appropriate analytical methods for particular question(s) need to be chosen from among the broad array of physicochemical analytical methods that are available. Together with the sampling strategy and preparation, these are key issues that affect the final outcome. In this chapter, we mainly focus on the presentation and use of X-ray fluorescence/absorption-based techniques. These include standard and total-reflection X-ray fluorescence, micro-proton-induced X-ray emission, and X-ray absorption spectroscopy (such as extended X-ray absorption fine structure, and X-ray absorption near-edge structure), for the analysis of trace-element concentrations and their coordination in biological samples.

Photon-harvesting efficiency and arbuscular mycorrhiza in amphibious plants.

The amphibious plant species of intermittent aquatic habitats thrive both submerged and emerged. In order to outline the adaptive characters of these two life forms photochemical efficiency of photosystem 2, leaf contents of chlorophyll (Chl) a and b, carotenoids (Car), anthocyanins (Ant), and UV-B absorbing compounds (UV-B abs), and root aerenchyma and arbuscular mycorrhizal (AM) colonisation were studied in Glyceria fluitans, Gratiola officinalis, Ranunculus lingua, Teucrium scordium, Sium latifolium, Sparganium emersum, and Veronica anagallis-aquatica. Water level fluctuations did not exert a severe effect on photon harvesting efficiency. Submerged specimens had higher contents of Car and Ant whereas higher contents of UV-B abs were found in emerged specimens indicating efficient protection against the harmful effects of solar radiation. Roots of all species studied had extensive aerenchyma and were colonised by AM fungi, which were significantly more abundant in emerged specimens. This is the first report on AM symbiosis in S. latifolium and S. emersum.

Micro-PIXE elemental mapping for ionome studies of crop plants

In order to maintain homeostasis and consequent optimal cell functioning and integrity and/or to avoid toxicity, proper allocation of elements at organ, tissue, cellular and subcellular level is needed. Studies of element localization are therefore crucial to reveal the mechanisms of element trafficking and also tolerance and toxicity. Moreover, studies of localization and speciation of trace elements in grains of staple crops are also of high applicative value, allowing one to determine major and trace element concentrations in different grain tissues without possible contamination. In the last decade, a remarkable progress has been made in the development and application of different 2D imaging techniques in complex biological systems, especially in the sense of improved lateral resolution and sensitivity. The superiority of micro-PIXE over other 2D imaging techniques lies in its wide elemental range (from sodium (Na) to uranium (U)), high elemental sensitivity below micron spatial resolution and fully quantitative element concentration analysis. The aim of this review is to summarize the latest development of micro-PIXE for imaging of the distribution of major and trace elements in crop plants with emphasis on sample preparation methodologies and post-imaging analysis. Case studies of element localization in the grains of major crop plants are also presented.