Transition metal (TM) distribution through the phloem is an essential part of plant metabolism and is required for systemic signaling and balancing source-to-sink relationships. Due to their reactivity, TMs are expected to occur in complexes within the phloem sap; however, metal speciation in the phloem sap remains largely unexplored. Here, we isolated phloem sap from Brassica napus and analyzed it via size exclusion chromatography coupled online to sector-field ICP-MS. Our data identified known TM-binding proteins and molecules including metallothioneins (MT), glutathione, and nicotianamine. While the main peak of all metals was low MW (∼1.5 kD), additional peaks ∼10 to 15 kD containing Cu, Fe, S, and Zn were also found. Further physicochemical analyses of MTs with and without affinity tags corroborated that MTs can form complexes of diverse molecular weights. We also identified and characterized potential artifacts in the TM-biding ability of B. napus MTs between tagged and non-tagged MTs. That is, the native BnMT2 binds Zn, Cu, and Fe, while MT3a and MT3b only bind Cu and Zn. In contrast, his-tagged MTs bind less Cu and were found to bind Co and Mn and aggregated to oligomeric forms to a greater extent compared to the phloem sap. Our data indicates that TM chemistry in the phloem sap is more complex than previously anticipated and that more systematic analyses are needed to establish the precise speciation of TM and TM-ligand complexes within the phloem sap.

• Keywords
• glutathione, iron, metallothioneins, phloem sap, size exclusion chromatography, zinc,
• MeSH
• Brassica napus * metabolism   genetics   MeSH
• Phloem * metabolism   MeSH
• Metallothionein metabolism   genetics   MeSH
• Transition Elements metabolism   MeSH
• Plant Proteins * metabolism   genetics   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Metallothionein MeSH
• Transition Elements MeSH
• Plant Proteins * MeSH

Miscanthus × giganteus demonstrated good phytostabilization potentials in toxic element (TE) contaminated soils. However, information about its tolerance to elevated concentrations is still scarce. Therefore, an ex-situ pot experiment was launched using three cultivars (termed B, U, and A) grown in soils with a gradient Cd, Pb and Zn concentrations. Control plants were also cultivated in non-contaminated soil. Results show that the number of tillers per plant, stem diameter as well as leaf photosynthetic pigments (chlorophyll a, b and carotenoids) were negatively impacted by soil contamination. On the other hand, phenolic compounds, flavonoids, tannins, and anthocyanins levels along with the antioxidant enzymatic activities of superoxide dismutase, ascorbate peroxidase and glutathione reductase increased in the plants grown on contaminated soils. Altogether, these data demonstrate that miscanthus is impacted by concentrations of toxic elements yet is able to tolerate high levels of soil contamination. These results may contribute to clarifying the miscanthus tolerance strategy against high contamination levels and its efficiency in phytoremediation.

• Keywords
• Miscanthus × giganteus, antioxidant enzymes, photosynthetic pigments, secondary metabolites, toxic elements (TE),
• Publication type
• Journal Article MeSH

BACKGROUND: Many metals are essential for plants and humans. Knowledge of metal distribution in plant tissues in vivo contributes to the understanding of physiological mechanisms of metal uptake, accumulation and sequestration. For those studies, X-rays are a non-destructive tool, especially suited to study metals in plants. RESULTS: We present microfluorescence imaging of trace elements in living plants using a customized benchtop X-ray fluorescence machine. The system was optimized by additional detector shielding to minimize stray counts, and by a custom-made measuring chamber to ensure sample integrity. Protocols of data recording and analysis were optimised to minimise artefacts. We show that Zn distribution maps of whole leaves in high resolution are easily attainable in the hyperaccumulator Noccaea caerulescens. The sensitivity of the method was further shown by analysis of micro- (Cu, Ni, Fe, Zn) and macronutrients (Ca, K) in non-hyperaccumulating crop plants (soybean roots and pepper leaves), which could be obtained in high resolution for scan areas of several millimetres. This allows to study trace metal distribution in shoots and roots with a wide overview of the object, and thus avoids making conclusions based on singular features of tiny spots. The custom-made measuring chamber with continuous humidity and air supply coupled to devices for imaging chlorophyll fluorescence kinetic measurements enabled direct correlation of element distribution with photosynthesis. Leaf samples remained vital even after 20 h of X-ray measurements. Subtle changes in some of photosynthetic parameters in response to the X-ray radiation are discussed. CONCLUSIONS: We show that using an optimized benchtop machine, with protocols for measurement and quantification tailored for plant analyses, trace metal distribution can be investigated in a reliable manner in intact, living plant leaves and roots. Zinc distribution maps showed higher accumulation in the tips and the veins of young leaves compared to the mesophyll tissue, while in the older leaves the distribution was more homogeneous.

• Keywords
• Arabidopsis halleri, Capsicum annuum, Glycine max, Hyperaccumulator, Leaf age, Micro X-ray fluorescence, Micronutrients, Noccaea caerulescens, Root, Zinc,
• Publication type
• Journal Article MeSH

Solving the global environmental and agricultural problem of chronic low-level cadmium (Cd) exposure requires better mechanistic understanding. Here, soybean (Glycine max) plants were exposed to Cd concentrations ranging from 0.5 nM (background concentration, control) to 3 µM. Plants were cultivated hydroponically under non-nodulating conditions for 10 weeks. Toxicity symptoms, net photosynthetic oxygen production and photosynthesis biophysics (chlorophyll fluorescence: Kautsky and OJIP) were measured in young mature leaves. Cd binding to proteins [metalloproteomics by HPLC-inductively coupled plasma (ICP)-MS] and Cd ligands in light-harvesting complex II (LHCII) [X-ray absorption near edge structure (XANES)], and accumulation of elements, chloropyll, and metabolites were determined in leaves after harvest. A distinct threshold concentration of toxicity onset (140 nM) was apparent in strongly decreased growth, the switch-like pattern for nutrient uptake and metal accumulation, and photosynthetic fluorescence parameters such as Φ RE10 (OJIP) and saturation of the net photosynthetic oxygen release rate. XANES analyses of isolated LHCII revealed that Cd was bound to nitrogen or oxygen (and not sulfur) atoms. Nutrient deficiencies caused by inhibited uptake could be due to transporter blockage by Cd ions. The changes in specific fluorescence kinetic parameters indicate electrons not being transferred from PSII to PSI. Inhibition of photosynthesis combined with inhibition of root function could explain why amino acid and carbohydrate metabolism decreased in favour of molecules involved in Cd stress tolerance (e.g. antioxidative system and detoxifying ligands).

• Keywords
• Cadmium, XANES, lipidomics, metabolomics, metal stress, metalloproteomics, soybean (Glycine max), sublethal toxicity,
• MeSH
• Chlorophyll MeSH
• Photosynthesis MeSH
• Glycine max * MeSH
• Cadmium * toxicity   MeSH
• Plant Leaves MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chlorophyll MeSH
• Cadmium * MeSH

Chlorophyll fluorescence kinetic analysis has become an important tool in basic and applied research on plant physiology and agronomy. While early systems recorded the integrated kinetics of a selected spot or plant, later systems enabled imaging of at least the slower parts of the kinetics (20-ms time resolution). For faster events, such as the rise from the basic dark-adapted fluorescence yield to the maximum (OJIP transient), or the fluorescence yield decrease during reoxidation of plastoquinone A after a saturating flash, integrative systems are used because of limiting speed of the available imaging systems. In our new macroscopic and microscopic systems, the OJIP or plastonique A reoxidation fluorescence transients are directly imaged using an ultrafast camera. The advantage of such systems compared to nonimaging measurements is the analysis of heterogeneity of measured parameters, for example between the photosynthetic tissue near the veins and the tissue further away from the veins. Further, in contrast to the pump-and-probe measurement, direct imaging allows for measuring the transition of the plant from the dark-acclimated to a light-acclimated state via a quenching analysis protocol in which every supersaturating flash is coupled to a measurement of the fast fluorescence rise. We show that pump-and-probe measurement of OJIP is prone to artifacts, which are eliminated with the direct measurement. The examples of applications shown here, zinc deficiency and cadmium toxicity, demonstrate that this novel imaging platform can be used for detection and analysis of a range of alterations of the electron flow around PSII.

• MeSH
• Arabidopsis cytology   metabolism   MeSH
• Brassicaceae cytology   drug effects   metabolism   MeSH
• Chlorophyll chemistry   metabolism   MeSH
• Equipment Design MeSH
• Fluorescence MeSH
• Microscopy, Fluorescence instrumentation   methods   MeSH
• Photosynthesis MeSH
• Glycine max cytology   drug effects   metabolism   MeSH
• Kinetics MeSH
• Plant Leaves cytology   MeSH
• Mesophyll Cells metabolism   MeSH
• Plastoquinone metabolism   MeSH
• Zinc metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chlorophyll MeSH
• Plastoquinone MeSH
• Zinc MeSH