Walter H. Schröder

Monitoring uptake and contents of Mg, Ca and K in Norway spruce as influenced by pH and Al, using microprobe analysis and stable isotope labelling

In a model system using intact spruce trees (Picea abies [L.] Karst.) we followed the path of magnesium, calcium and potassium during uptake into the root and during long-range transport into the shoot, by multiple stable isotope labelling. The roots of two- and three-year-old spruce trees originating from soil culture were removed from the soil and, in part or in toto, exposed to labelling solutions containing the stable isotopes 25Mg or 26Mg, 41K and 42Ca or 44Ca. Optical-emission-spectroscopy (ICP-OES) of plant fractions and labelling solutions was combined with the quantitative analysis of stable isotope ratios in sections of shock frozen, cryosubstituted material using the laser-microprobe-mass-analyser (LAMMA). This combination allowed us to distinguish, both in bulk samples and on the cellular level between (i) the fraction of elements originally present in the plant before the start of the labelling, (ii) the material taken up from the labelling solution into the plant and (iii) any material released by the plant into the labelling solution.In single-root labelling experiments, roots of three-year-old spruce trees, grown in nursery soil, were exposed to various pH conditions. The exchange of Mg and Ca with the labelling solution was nearly 100% in the cell walls of the mycorrhized finest roots. This exchange was only slightly affected by a step down to pH 3.5. The absolute Mg and Ca content in the cell walls was moderately reduced by incubation at pH 3.5 and strongly reduced in the presence of Al at this pH. After a pH 3.5 and 2 mM Al treatment we found Al in the xylem cell walls and the cortex cell lumina at elevated concentrations. To analyse the combined effect of high Al and high proton concentrations on the long-range transport, we used a “split-root system”. The root mass of an intact two-year-old spruce tree, grown in mineral soil, was divided into even parts and both halves incubated in solutions with two sets of different stable isotopes of Mg and Ca (side A: no Al, 25Mg and 42Ca; side B: +Al, 26Mg and 44Ca) and 41K on both sides. We observed a large uptake of Mg, Ca and K into the plant and a pronounced release. The net uptake of all three elements was lower from the Al-doted solution. In cross-sections of the apical shoot we found after seven-day labelling period about 60–70% of the Mg and Ca and 30% of the K content in the xylem cell walls originating from both labelling solutions. The clear majority of the Mg and Ca label originated from the Al-doted side.

The kinetics of calcium and magnesium entry into mycorrhizal spruce roots

Abstract. The entry of calcium and magnesium from external sources into mycorrhizal roots of 3-year-old Norway spruce trees (Piceaabies [L.] Karst.) was monitored. Roots of intact plants were exposed for various periods of time, ranging from 2 min to 48 h, to nutrient solutions which contained the stable-isotope tracers 25Mg and 44Ca. After labelling, samples of roots were excised from the plants, shock-frozen, cryosubstituted and embedded. The resulting isotope composition in this material was analysed by a laser-microprobe-mass-analyser (LAMMA) at relevant positions within cross-sections of the roots. For both elements, we determined (i) the fractions of the isotopes originating from the plant prior to labelling, and (ii) the fraction of isotopes originating from the corresponding tracer that penetrated into the root. Both divalent cations rapidly penetrated across the cortical apoplast and reached the endodermis. After 2 min of exposure to the labelling solution, an initial transient gradient of the tracers could be observed within the root cortex. Subsequently, calcium as well as magnesium equilibrated between the apoplast of the entire cortex and the external tracer with a half-time, t1/2, of about 3 min. In contrast, the kinetics of radial movement into the vascular stele showed a delay with a t1/2 of 100–120 min. We take this as strong evidence that there exists a free apoplastic path for divalent cations in the cortex and that the endodermis is a major barrier to the further passage of Mg and Ca into the xylem. While 25Mg in the labelling solution exchanged rapidly with Mg in the cortical apoplast, the exchange across the plasma membrane with Mg present in the protoplasm of the same cortical cells was almost 2 orders of magnitude slower. The kinetics of Ca and Mg entry at +6 °C were similar to those obtained at a root temperature of +22 °C.

The mycorrhizal fungus Paxillus involutus transports magnesium to Norway spruce seedlings. Evidence from stable isotope labeling

Although it is well established that ectomycorrhizas improve the mineral nutrition of forest trees, there has been little evidence that they mediate uptake of divalent cations such as Mg. We grew nonmycorrhizal seedlings and seedlings mycorrhizal with Paxillus involutus Batsch in a sand culture system with two compartments separated by a 45-μm Nylon mesh. Hyphae, but not roots, can penetrate this net. Labeling the compartment only accessible to hyphae with 25Mg showed that hyphae of the ectomycorrhizal fungus Paxillus involutus transported Mg to their host plant. No label was found in nonmycorrhizal control plants. Our data support the idea that ectomycorrhizas are important for the Mg nutrition of forest trees.

Localisation of Aluminium in Root Tips of Zea mays and Vicia faba

Summary Monocot species and dicot species differ widely in the composition of their cell walls, namely in the pectin content, and thus in potential Al-binding sites. The effect of these differences on cellular localisation as well as on tissue distribution of Al was compared in root tips of Zea mays and Vicia faba . The localisation of Al was assayed by Laser Microprobe Mass Analysis (LAMMA) after freeze-substitution. The radial mobility of Al was much lower in the roots of Vicia faba . In root tips of Zea mays , Al had reached the stele already after 60 min while it was confined to the rhizodermis and outer cortex cells in Vicia faba , indicating a stronger binding of Al in the cell walls of the dicoe. This binding, however, had no influence on intracellular distribution. Within 60 min intracellular Al was detectable in both species. Nevertheless, by far the highest Al concentrations were always measured in the cell wall.

On the distribution and transport of mineral elements in xylem, cambium and phloem of spruce (Picea abies [L.] Karst.)

The distribution of calcium, magnesium and potassium in the cambial region of a 20-year-old Norway spruce was determined by optical emission spectrometry (ICP-OES) in tissue samples ranging from the outer bark up to latewood and earlywood of the outer tree-rings. The highest contents of magnesium and potassium were found in the cambium and, in the case of calcium, in the developing phloem. In more detail we investigated the distribution and incorporation of calcium, magnesium and potassium in a model system of intact two-year-old plants (Picea abies [L.] Karst.). The roots of the seedlings, grown under controlled conditions, were removed from the soil and exposed to labelling solutions containing the enriched stable isotopes 25 Mg, 41 K, and 44 Ca as tracers. After seven days of labelling the cell walls of earlywood, latewood, cambium and phloem were analysed in stem cross sections of shock-frozen, cryo-substituted tissue by three microprobes: (i) by energy dispersive x-ray analysis (EDXA, 0.3 μm lateral resolution), (ii) isotope specific point analysis, using the laser microprobe mass analyser (LAMMA, 1.5μm lat. res.) and (iii) isotope specific imaging by secondary ion mass spectrometry (SIMS, 1-3μm lat. res.). After seven days of labelling approximately 60-75% of the Ca-content, 5-65% of the Mg-content and about 25-30% of the K-content within the cell walls of the shoot originated from the labelling solutions. The upper part (one-year-old) of the shoot axis contained a 10% smaller fraction of labelled Ca and a 10% larger fraction of labelled Mg in comparison to the more basal regions of the shoot (two-year-old). For all three elements we could not detect any significant radial gradient of the labelled fraction while comparing earlywood, latewood, cambium and phloem. We interpret our results as evidence for (i) a relevant bidirectional longitudinal transport and (ii) a radial element exchange between xylem, cambium and phloem, completed within the seven days of labelling.

Significance of the Root Apoplast for Aluminium Toxicity and Resistance of Maize

The mechanism of aluminium-induced inhibition of root elongation is still not well understood. It is a matter of debate whether the primary lesions of Al toxicity are apoplastic or symplastic. The present paper summarises evidence from own experimental work and the literature which could contribute to the understanding of Al toxicity and resistance in maize focussing on the role of the apoplast.

Fluorescence microtomography using nanofocusing refractive x-ray lenses

Fluorescence microtomography is a hard x-ray scanning microscopy technique that has been developed at synchrotron radiation sources in recent years. It allows one to reconstruct non-destructively the element distribution on a virtual section inside a sample. The spatial resolution of this microbeam technique is limited by the lateral size of the microbeam. Since recently, nanofocusing refractive x-ray lenses (NFLs) are under development that were shown to produce hard x-ray microbeams with lateral resolution in the range of 100nm. Future improvements of these optics might reduce the microbeam size down to below 20nm. Using nanofocusing lenses, fluorescence microtomography with sub-micrometer resolution was performed. As an example, the element distribution inside a small cosmic dust particle is given. Tomographic reconstruction was done using a refined model including absorption effects inside the sample.

Ion exchange and water uptake of coarse roots of mature Norway spruce trees ( Picea abies L. Karst.)

Most rhizosphere studies about water and nutrient uptake of trees have only considered the fine root system. Our contribution is focused on the water and nutrient uptake characteristics of a widely ignored soil-plant interface — the coarse root system. To quantify water and ion uptake by older suberized roots, root chambers for field experiments were developed. These chambers were installed at more than 30 coarse root segments of mature Norway spruce and filled with a nutrient solution adapted to field conditions. Changes in nutrient concentration and water volume were recorded for several weeks. In order to trace ion uptake, experiments using stable isotopes(44Ca, 41K, 25Mg) were carried out. All coarse root segments took up a significant amount of water, with maximum rates of about 10 times lower than those for active fine roots. In some segments 25Mg could be detected in the roots xylem, indicating actual uptake of ions into inner root parts.

Hard X-ray scanning microscopy with fluorescence and diffraction contrast

Based on nanofocusing parabolic refractive x-ray lenses we have developed and built a hard x-ray scanning microscope that was tested and put to use at beamline ID13 of the ESRF. It can provide a monochromatic hard x-ray nanobeam with lateral extension below 100 nm (down to 50 nm) and a flux up to 109 ph/s in the energy range from 15 to 25 keV. The microscope exploits transmission, fluorescence, and diffraction contrast to obtain local elemental and nanostructural information from the sample. Tomographic scanning yields high resolution elemental maps from the inside of an object. Coherent x-ray diffraction imaging with nanofocused illumination yields images of objects with highest spatial resolution, e. g., 5 nm in a given example.

Studying magnesium transport across and via mycorrhiza of Norway spruce roots

The stable isotope 25 Mg was used to trace the uptake of Mg into roots of intact mycorrhizal and nonmycorrhizal spruce seedlings. (i) We followed the kinetics of the direct Mg entry into fine roots at the cellular level using LAMMA and SIMS. In mycorrhizal and nonmycorrhizal fine roots, Mg rapidly exchanged between the labeling solution and cortical cell walls with a half time of < 5 min. The Hartig net resulted in only a slight reduction of the apoplastic accessibility for Mg as compared to nonmycorrhizal roots. An extensive hyphal mantle caused a short delay of a few min. The uptake into the stele, was significantly slower with similar half-times of 60–120 min for both mycorrhizal and nonmycorrhizal finest roots. (ii) To follow the Mg transport via the external hyphen, spruce seedlings were grown in a two compartment sand culture system. The compartments were separated by a 45μm Nylon mesh that could be crossed by hyphae of the mycorrhizae, but not by roots. Within a 11-week labelling period, 25 Mg was transferred from the fungal compartment amounting to 3–4% of the total Mg in the plant. The transfer was not observed in nonmycorrhizal control plants and was strongly dependent on the presence of P- supply to the hyphal compartment. Mg may therefore be taken up both locally, directly from the rhizosphere, or from distant sources via mycorrhiza.