Improved Micro-X-ray Fluorescence Confocal Imaging of Two-Dimensional Distribution of Arsenic Concentration in Cucumber Hypocotyls Using Synchrotron Radiation.

An optimized micro-X-ray fluorescence confocal imaging (µXRF-CI) analytical method has been developed to determine the 2D distribution of elemental composition in small (1-3 mm) biological objects at a 10-20 µm spatial resolution. Plants take up chemical elements from soil, and the vascular system transports them toward shoots. In order to obtain biochemical information related to this biological process, 2D distributions of chemical elements in roots and in hypocotyls of cucumber plants were analyzed by synchrotron radiation based on micro-X-ray fluorescence computer tomography and µXRF-CI techniques. The experiments were carried out at HASYLAB Beamline L of the DORIS-III storage ring in Hamburg, a facility that provided optimal physical conditions for developing and performing these unique analyses: high flux monochromatic synchrotron beam, X-ray optical elements, precision moving stages, and silicon drift detectors. New methodological improvements and experimental studies were carried out for applicability of lyophilized samples and cryo-cooling. Experimental parameters were optimized to maximize the excitation yield of arsenic Kα radiation and improvement of the spatial resolution of the µXRF-CI analytical method.

Response of soybean plants to the application of synthetic and biodegradable Fe chelates and Fe complexes.

The growing concern over the environmental risk of synthetic chelate application promotes the search for alternatives in Fe fertilization, such as biodegradable chelating agents and natural complexing agents. In this work, plant responses to the application of several Fe treatments (chelates and complexes) was analyzed to study their potential use in Fe fertilization under calcareous conditions. Thus, the root ferric chelate reductase (FCR) activity of soybean (Glycine max cv. Klaxon) plants was determined, and the effectiveness of the Fe chelates and complexes assessed in a pot experiment, by SPAD and fluorescence induction measurements, and the determination of Fe distribution in plant and soil. Additionally, 57Fe Mössbauer spectroscopy was conducted to identify the Fe forms present in the soybean roots. The highest FCR activity was observed for the chelates EDDS/Fe3+ and IDHA/Fe3+; while no activity was observed when using complexes as Fe substrates. In contrast to the FCR data, the pot experiment confirmed that the o,oEDDHA/Fe3+ is the most effective treatment, and the complexes LS/Fe3+ and GA/Fe3+ are able to alleviate Fe chlorosis, also indicated by SPAD data and the maximal quantum efficiency of photosystem II reaction centers as vitality parameters, and the enhanced plant uptake of Fe from natural sources.

Functional characterization of the chloroplast ferric chelate oxidoreductase enzyme.

Iron (Fe) has an essential role in the biosynthesis of chlorophylls and redox cofactors, and thus chloroplast iron uptake is a process of special importance. The chloroplast ferric chelate oxidoreductase (cFRO) has a crucial role in this process but it is poorly characterized. To study the localization and mechanism of action of cFRO, sugar beet (Beta vulgaris cv Orbis) chloroplast envelope fractions were isolated by gradient ultracentrifugation, and their purity was tested by western blotting against different marker proteins. The ferric chelate reductase (FCR) activity of envelope fractions was studied in the presence of NAD(P)H (reductants) and FAD coenzymes. Reduction of Fe(III)-ethylenediaminetetraacetic acid was monitored spectrophotometrically by the Fe(II)-bathophenanthroline disulfonate complex formation. FCR activity, that is production of free Fe(II) for Fe uptake, showed biphasic saturation kinetics, and was clearly associated only to chloroplast inner envelope (cIE) vesicles. The reaction rate was > 2.5 times higher with NADPH than with NADH, which indicates the natural coenzyme preference of cFRO activity and its dependence on photosynthesis. FCR activity of cIE vesicles isolated from Fe-deficient plants also showed clear biphasic kinetics, where the KM of the low affinity component was elevated, and thus this component was down-regulated.

Characterization of Fe-leonardite complexes as novel natural iron fertilizers.

Water-soluble humic substances (denoted by LN) extracted at alkaline pH from leonardite are proposed to be used as complexing agents to overcome micronutrient deficiencies in plants such as iron chlorosis. LN presents oxidized functional groups that can bind Fe(2+) and Fe(3+). The knowledge of the environment of Fe in the Fe-LN complexes is a key point in the studies on their efficacy as Fe fertilizers. The aim of this work was to study the Fe(2+)/Fe(3+) species formed in Fe-LN complexes with (57)Fe Mössbauer spectroscopy under different experimental conditions in relation to the Fe-complexing capacities, chemical characteristics, and efficiency to provide iron in hydroponics. A high oxidation rate of Fe(2+) to Fe(3+) was found when samples were prepared with Fe(2+), although no well-crystalline magnetically ordered ferric oxide formation could be observed in slightly acidic or neutral media. It seems to be the case that the formation of Fe(3+)-LN compounds is favored over Fe(2+)-LN compounds, although at acidic pH no complex formation between Fe(3+) and LN occurred. The Fe(2+)/Fe(3+) speciation provided by the Mössbauer data showed that Fe(2+)-LN could be efficient in hydroponics while Fe(3+)-LN is suggested to be used more effectively under calcareous soil conditions. However, according to the biological assay, Fe(3+)-LN proved to be effective as a chlorosis corrector applied to iron-deficient cucumber in nutrient solution.

Heavy metal accumulation and tolerance of energy grass (Elymus elongatus subsp. ponticus cv. Szarvasi-1) grown in hydroponic culture.

Phytoremediation is a plant based, cost effective technology to detoxify or stabilise contaminated soils. Fast growing, high biomass, perennial plants may be used not only in phytoremediation but also in energy production. Szarvasi-1 energy grass (Elymus elongatus subsp. ponticus cv. Szarvasi-1), a good candidate for this combined application, was grown in nutrient solution in order to assess its Cd, Cu, Ni, Pb and Zn accumulation and tolerance. Its shoot metal accumulation showed the order Pb < Ni < Cu ∼ Cd < Zn. In parallel with this, Pb and Ni had no or very little influence on the growth, dry matter content, chlorophyll concentration and transpiration of the plants. Cu and Cd treatment resulted in significant decreases in all these parameters that can be attributed to Fe plaque formation in the roots suggested by markedly increased Fe and Cu accumulation. This came together with decreased shoot and root Mn concentrations in both treatments while shoot Cu and Zn concentrations decreased under Cd and Cu exposure, respectively. Zn treatment had no effect or even slightly stimulated the plants. This may be due to a slight stimulation of Fe translocation and a very efficient detoxification mechanism. Based on the average 300 mg kg⁻¹ (dry mass) Zn concentration which is 0.03% of the shoot dry mass the variety is suggested to be classified as Zn accumulator.

Effects of short term iron citrate treatments at different pH values on roots of iron-deficient cucumber: a Mössbauer analysis.

Alkaline pH values and bicarbonate greatly reduce the mobility and uptake of Fe, causing Fe deficiency chlorosis. In the present work, the effects of pH and bicarbonate on the uptake and accumulation of Fe in the roots of cucumber were studied by Mössbauer spectroscopy combined with physiological tests and diaminobenzidine enhanced Perls staining. Mössbauer spectra of Fe-deficient cucumber roots supplied with 500 µM (57)Fe(III)-citrate at different pH values showed the presence of an Fe(II) and an Fe(III) component. As the pH was increased from 4.5 to 7.5, the root ferric chelate reductase (FCR) activity decreased significantly and a structural change in the Fe(III) component was observed. While at pH 4.5 the radial intrusion of Fe reached the endodermis, at pH 7.5, Fe was found only in the outer cortical cell layers. The Mössbauer spectra of Fe-deficient plants supplied with Fe(III)-citrate in the presence of bicarbonate (pH 7.0 and 7.5) showed similar Fe components, but the relative Fe(II) concentration compared to that measured at pH values 6.5 and 7.5 was greater. The Mössbauer parameters calculated for the Fe(II) component in the presence of bicarbonate were slightly different from those of Fe(II) alone at pH 6.5-7.5, whereas the FCR activity was similarly low. Fe incorporation into the root apoplast involved only the outer cortical cell layers, as in the roots treated at pH 7.5. In Fe-sufficient plants grown with Fe(III)-citrate and 1mM bicarbonate, Fe precipitated as granules and was in diffusely scattered grains on the root surface. The "bicarbonate effect" may involve a pH component, decreasing both the FCR activity and the acidification of the apoplast and a mineralization effect leading to the slow accumulation of extraplasmatic Fe particles, forming an Fe plaque and trapping Fe and other minerals in biologically unavailable forms.

Influence of pH, iron source, and Fe/ligand ratio on iron speciation in lignosulfonate complexes studied using Mössbauer spectroscopy. Implications on their fertilizer properties.

Iron chlorosis is a very common nutritional disorder in plants that can be treated using iron fertilizers. Synthetic chelates have been used to correct this problem, but nowadays environmental concerns have enforced the search for new, more environmentally friendly ligands, such as lignosulfonates. In this paper, Fe coordination environment and speciation in lignosulfonate (LS) complexes prepared under different experimental conditions were studied by (57)Fe Mössbauer spectroscopy in relation to the Fe-complexing capacities, chemical characteristics of the different products, and efficiency to provide iron in agronomic conditions. It has been observed that the complex formation between iron and lignosulfonates involves different coordination sites. When Fe(2+) is used to prepare the iron-LS product, complexes form weak adducts and are sensitive to oxidation, especially at neutral or alkaline pH. However, when Fe(3+) is used to form the complexes, both Fe(2+) and Fe(3+) are found. Reductive sugars, normally present in lignosulfonates, favor a relatively high content of Fe(2+) even in those complexes prepared using Fe(3+). The formation of amorphous ferrihydrite is also possible. With respect to the agronomical relevance of the Fe(2+)/Fe(3+) speciation provided by the Mössbauer spectra, it seems that the strong Fe(3+)-LS complexes are preferred when they are applied to the leaf, whereas root uptake in hydroponics could be more related with the presence of weak bonding sites.