Wolfgang Heyser

Microautoradiographic studies of phloem loading and transport in the leaf of Zea mays L.

Microautoradiographs showed that [14C]sucrose taken up in the xylem of small and intermediate (longitudinal) vascular bundles of Zea mays leaf strips was quickly accumulated by vascular parenchyma cells abutting the vessels. The first sieve tubes to exhibit 14C-labeling during the [14C]sucrose experiments were thick-walled sieve tubes contiguous to the more heavily labeled vascular parenchyma cells. (These two cell types typically have numerous plasmodesmatal connections.) With increasing [14C]sucrose feeding periods, greater proportions of thick- and thin-walled sieve tubes became labeled, but few of the labeled thin-walled sieve tubes were associated with labeled companion cells. (Only the thin-walled sieve tubes are associated with companion cells.) When portions of leaf strips were exposed to 14CO2 for 5 min, the vascular parenchyma cells-regardless of their location in relation to the vessels or sieve tubes-were the most consistently labeled cells of small and intermediate bundles, and label (14C-photosynthate) appeared in a greater proportion of thin-walled sieve tubes than thick-walled sieve tubes. After a 5-min chase with 12CO2, the thin-walled sieve tubes were more heavily labeled than any other cell type of the leaf. After a 10-min chase with 12CO2, the thin-walled sieve tubes were even more heavily labeled. The companion cells generally were less heavily labeled than their associated thin-walled sieve tubes. Although all of the thick-walled sieve tubes were labeled in portions of leaf strips fed 14CO2 for 5 min and given a 10-min 12CO2 chase, only five of 72 vascular bundles below the 14CO2-exposed portions contained labeled thick-walled sieve tubes. Moreover, the few labeled thick-walledsieve tubes of the “transport region” always abutted 14C-labeled vascular parenchyma cells. The results of this study indicate that (1) the vascular parenchyma cells are able to retrieve at least sucrose from the vessels and transfer it to the thick-walled sieve tubes, (2) the thick-walled sieve tubes are not involved in long-distance transport, and (3) the thin-walled sieve tubes are capable themselves of accumulating sucrose and photosynthates from the apoplast, without the companion cells serving as intermediary cells.

Elemental contents in vacuolar granules of ectomycorrhizal fungi measured by EELS and EDXS. A comparison of different methods and preparation techniques

Abstract The elemental composition of vacuolar granules in different ectomycorrhizal fungi, Pisolithus tinctorius, Suillus bovinus and Xerocomus badius, were determined by electron energy loss spectroscopy (EELS) and energy dispersive X-ray spectroscopy (EDXS). The investigations dealt with advantages and limits of the ECXS and EELS technique with respect to the analysis of the elemental composition of vacuolar granules and the effect of different specimen preparation techniques. Axenic cultures of these fungi as well as field mycorrhizae were used for the analysis. Results after conventional chemical fixation and dehydration of material were compared to results obtained after cryofixation followed by freeze-drying of the samples. Light microscopical studies were also carried out to control the occurrence of vacuolar granules in living hyphae. The results showed that vacuolar granules exist in living hyphae of different ectomycorrhizal fungi and are not an artifact of the fixation or other specimen preparation procedures of cells. EDXS and EELS differed in their ability to detect the elemental composition of these granules. Both analytical techniques found P in the vacuolar bodies, which indicates a deposition of polyphosphates. Polyphosphate granules are strongly negative polyanions, which contain different cations to balance the negative charge. These cations were often difficult to determine by EELS and could only be shown by EDXS, but the cations varied considerably depending on the technique used for specimen preparation. In chemically fixed and dehydrated material, especially Mg, K and Ca were detected in the granules. However, measurements of cryofixed and freeze-dried specimens showed that the most abundant cations in polyphosphate granules were K and Mg and the incorporation of Ca has to be interpreted as a result of the chemical specimen preparation. EELS revealed that N was also incorporated in the vacuolar granules independently of the specimen preparation. Based on these results, it can be assumed (1) that the N-containing granules, detected by EELS, and the P-rich granules, analysed by EDXS, were identical; (2) that these vacuolar inclusions were polyphosphate granules or metachromatic granules, which were often described in different mycorrhizal associations; and (3) that polyphosphate granules can also act as a binding site for large amounts of N.

Biodegradation of aromatic compounds by white rot and ectomycorrhizal fungal species and the accumulation of chlorinated benzoic acid in ectomycorrhizal pine seedlings

The capability of different white rot (WR, Heterobasidion annosum, Phanerochaete chrysosporium, Trametes versicolor) and ectomycorrhizal (ECM, Paxillus involutus, Suillus bovinus) fungal species to degrade different aromatic compounds and the absorption of 3-chlorobenzoic acid (3-CBA) by ECM pine seedlings was examined. The effect of aromatic compounds on the fungal biomass development varied considerably and depended on (a) the compound, (b) the external concentration, and (c) the fungal species. The highest effect on the fungal biomass development was observed for 3-CBA. Generally the tolerance of WR fungi against aromatic compounds was higher than that of the biotrophic fungal species. The capability of different fungi to degrade aromatic substances varied between the species but not generally between biotrophic and saprotrophic fungi. The highest degradation capability for aromatic compounds was detected for T. versicolor and H. annosum, whereas for Phanerochaete chrysosporium and the ECM fungi lower degradation rates were found. However, Paxillus involutus and S. bovinus showed comparable degradation rates at low concentrations of benzoic acid and 4-hydroxybenzoic acid. In contrast to liquid cultures, where no biodegradation of 3-CBA by S. bovinus was observed, mycorrhizal pines inoculated with S. bovinus showed a low capability to remove 3-CBA from soil substrates. Additional X-ray microanalytical investigations showed, that 3-CBA supplied to mycorrhizal plants was accumulated in the root cell cytoplasm and is translocated across the endodermis to the shoot of mycorrhizal pine seedlings.

Surface-bound phosphatase activity in living hyphae of ectomycorrhizal fungi of Nothofagus obliqua

We determined the location and the activity of surface-bound phosphomonoesterase (SBP) of five ectomycorrhizal (EM) fungi of Nothofagus oblique. EM fungal mycelium of Paxillus involutus, Austropaxillus boletinoides, Descolea antartica, Cenococcum geophilum and Pisolithus tinctorius was grown in media with varying concentrations of dissolved phosphorus. SBP activity was detected at different pH values (3–7) under each growth regimen. SBP activity was assessed using a colorimetric method based on the hydrolysis of p-nitrophenyl phosphate (pNPP) to p-nitrophenol phosphate (pNP) + P. A new technique involving confocal laser-scanning microscopy (LSM) was used to locate and quantify SBP activity on the hyphal surface. EM fungi showed two fundamentally different patterns of SBP activity in relation to varying environmental conditions (P-concentrations and pH). In the cases of D. antartica, A. boletinoides and C. geophilum, changes in SBP activity were induced primarily by changes in the number of SBP-active centers on the hyphae. In the cases of P. tinctorius and P. involutus, the number of SBP-active centers per μm hyphal length changed much less than the intensity of the SBP-active centers on the hyphae. Our findings not only contribute to the discussion about the role of SBP-active centers in EM fungi but also introduce LSM as a valuable method for studying EM fungi.

The spatial distribution of acid phosphatase activity in ectomycorrhizal tissues depends on soil fertility and morphotype, and relates to host plant phosphorus uptake

Acid phosphatase (ACP) enzymes are involved in the mobilization of soil phosphorus (P) and polyphosphate accumulated in the fungal tissues of ectomycorrhizal roots, thereby influencing the amounts of P that are stored in the fungus and transferred to the host plant. This study evaluated the effects of ectomycorrhizal morphotype and soil fertility on ACP activity in the extraradical mycelium (ACP(myc)), the mantle (ACP(mantle)) and the Hartig net region (ACP(Hartig)) of ectomycorrhizal Nothofagus obliqua seedlings. ACP activity was quantified in vivo using enzyme-labelled fluorescence-97 (ELF-97) substrate, confocal laser microscopy and digital image processing routines. There was a significant effect of ectomycorrhizal morphotype on ACP(myc), ACP(mantle) and ACP(Hartig), while soil fertility had a significant effect on ACP(myc) and ACP(Hartig). The relative contribution of the mantle and the Hartig net region to the ACP activity on the ectomycorrhizal root was significantly affected by ectomycorrhizal morphotype and soil fertility. A positive correlation between ACP(Hartig) and the shoot P concentration was found, providing evidence that ACP activity at the fungus:root interface is involved in P transfer from the fungus to the host. It is concluded that the spatial distribution of ACP in ectomycorrhizas varies as a function of soil fertility and colonizing fungus.

Zinc accumulation in beech mycorrhizae — a mechanism of zinc tolerance?

Abstract Zn accumulation is found in the sheath of beech mycorrhiza using EDX analysis of thin sections. Zn is accumulated within the cytoplasm with a gradient in Zn concentration across the hyphal sheath indicating a physiological differentiation. The results are discussed with regard to metal tolerance.

ReviTec® — eine neue integrierte ökologische Technologie zur Renaturierung degradierter Standorte

In vielen Teilen des Mittelmeerraumes fuhrten Feuer sowie historische und rezente Ubernutzung zu grosen Verlusten des Oberbodens mit erheblichen nachteiligen Folgen fur die Grundwassererneuerung, das regionale Klima und die Wirtschaft. Im EU-Projekt „Mediterranean Desertification and Landuse“ (MEDALUS, 1991–1994) wurde die uberragende Bedeutung der Bodenverhaltnisse fur die Prozesse herausgearbeitet, die zu Degradation und Desertifikation fuhren und die als zwei „Teufelskreise“ mit negativen Ruckkoppelungen beschrieben wurden (vgl. Williams et al. 1995)

X-ray microanalytical studies on element uptake and deposition in different tissues of beech mycorrhizar

Abstract Uptake and deposition of some nutrient elements and heavy metals was investigated by microanalysis of cross sections of ectomycorrhizal beech roots collected from an air pollution stressed stand. Fe, Al and partly P and Zn were found accumulated within the fungal sheath indicating selective exclusion and/or specific accumulation whereas highest amounts of Co, Pb and partly of Zn demonstrated a barrier function of the root cortex and the endodermis. Results are discussed with regard to transport physiology and metal tolerance.

NEW PERSPECTIVES ON THE DETERMINATION OF PHOSPHATASE ACTIVITY IN ECTOMYCORRHIZAE OF NOTHOFAGUS OBLIQUA FORESTS IN SOUTHERN CHILE

Nosotros presentamos un metodo basado en la microscopia confocal de fluorescencia y en el analisis de imagenes, para localizar y cuantificar la actividad de la fosfatasa superficial (SBPA) en hongos ectomicorricicos y en ectomicorrizas de Nothofagus obliqua (Mirb.) Oerst. El sustrato ELF-97 es hidrofilico, el cual se desarrolla en un precipitado altamente fluorescente bajo su activacion a traves de fosfomonoesterasas. Recientemente esta tecnica ha sido aprobada mediante comparaciones con un metodo estandar (fosfato de p-nitrofenilo) realizadas en micelio fungico. La tecnica microscopica con ELF-97 revelo que Paxillus involutus (Batsch: Fr.) Sing. y Austropaxillus boletinoides (Sing.) Brsky. & Jarosch poseen diferentes estrategias de adaptacion frente a distintas concentraciones de fosfatos y pH (3-7). Nosotros analizamos la SBPA en cuatro asociaciones micorricicas de N. obliqua y encontramos que la organizacion del manto juega un rol esencial con respecto a la SBPA. En general, las raices micorrizadas transfieren la SBPA de la raiz hacia el manto. En este contexto, Pisolithus tinctorius (Pers.) Coker & Couch demostro ser el hongo ectomicorricico mas relevante para N. obliqua, al incrementar significativamente el total de la SBPA de la micorriza en relacion a la raiz sin micorrizar. En conclusion, a traves de la microscopia confocal de fluorescencia basada en el sustrato ELF-97 y su combinacion con el procesamiento de imagenes es posible, tanto en hongos micorricicos como en micorrizas, determinar la SBPA y revelar las estrategias de adaptacion con respecto al fosforo (P) a un nivel fisiologico-estructural

The significance of the apoplast for the nutrient acquisition of ectomycorrhizal root systems

Laser microprobe mass analytical investigations after stable isotope labelling with 25Mg, 41K and 44Ca were carried out to analyse the apoplastic permeability of the fungal sheath of different ectomycorrhizal associations of pines. The results show, that the ectomycorrhizal fungal sheath does not function as an apoplastic barrier for the entry of nutrients into the root cortex. However, the entry and the radial exchange rates detected for mycorrhizal roots are lower than that of nonmycorrhizal short roots. The Casparian band of the endodermis in short roots of Pinus sylvestris L. acts as an effective apoplastic barrier for the entry of divalent cations into the stele. Even after an exposure time of 24 h only low exchange rates for the divalent cations were detected in the stele of nonmycorrhizal or mycorrhizal short roots whereas a significant translocation of 41K into the stele was found. The present results are discussed with regard to the function of these short roots within the mycorrhizal root system of pines and the significance of the fungal symbiotic partner for the nutrient acquisition of the host plant.