Aluminium polyphosphate complexes in the mycorrhizal basidiomyceteLaccaria bicolor: A27Al-nuclear magnetic resonance study

The techniques of ²⁷Al- and ³¹P-nuclear magnetic resonance (NMR) spectroscopy were used to investigate the interactions of aluminium with intracellular ligands within the mycelium of the ectomycorrhizal basidiomycete Laccaria bicolor (Maire) Orton (S238). The vegetative mycelium was grown on medium containing 0.5 mM AlCl₃ for 0.5 to 3 d. The ²⁷Al-NMR spectra showed that aluminium was rapidly taken up and accumulated into polyphosphate complexes in the vacuole. Comparison with Al-polyphosphate complexes obtained in vitro on model systems indicated that Al forms at least three mixed-solvation complexes with Pi and polyphosphates, that there is more than one complex present under any set of conditions, and that the equilibrium between these complexes shifts dramatically with Al concentration in the medium. The high phosphate concentrations in the growth medium favoured the accumulation of the Al-polyphosphate complexes. When mycelium containing Al-polyphosphate complexes was transferred to Al-free nutrient solution for 9 d, the Alpolyphosphate complexes were not remobilized. The sequestration of Al in the polyphosphate complexes could therefore make a significant contribution to the protection of mycorrhizal plants against aluminium toxicity.Abbreviations NMR nuclear magnetic resonance - PolyP polyphosphate(s) - PP₁ terminal phosphate of PolyP - PP₃ middle phosphate of PolyP We thank Prof. Daniel Canet (Laboratoire de Méthodologie RMN, University of Nancy I, Vandceuvre-lès-Nancy, France) for his constant encouragement and Christine Delaruelle for skilled technical assistance in growing the fungal cultures. This work was supported by a research grant from the Commission of the European Communities (STEP-CT90-0059, Role of Ectomycorrhiza in Stress Tolerance of Forest Trees) to F.M. and a travel grant from the Institut National de la Recherche Agronomique to I.K.; R.C. is a recipient of a Postdoctoral Fellowship from the Natural Sciences and Engineering Research Council of Canada.     

Differential ultrastructural aberrations of collagen fibrils in Ehlers-Danlos syndrome types I–IV as a means of diagnostics and classification

Among the different subtypes of Ehlers-Danlos syndrome (EDS), the dominant types I–III have, so far, been uninformative biochemically and molecular genetically, and diagnostic problems with subgroup boundaries often arise. We have investigated the ultrastructural pattern of connective tissue macromolecules in skin biopsy specimens of some 85 patients aged 4 months-54 years who exhibit clinical symptoms or the suspicion of EDS I–IV. Based on the differential features of collagen fibrils and ground substance material, four distinct groups could be established. Group I (clinically EDS type I) showed disorganized collagen bundles and dense aggregations of collagen fibrils with bizarre shapes. Group II (clinically varying from EDS types I–III) revealed collagen bundles that regularly contained numerous “composite collagen fibrils” with enlarged “flower-like” cross-sections and rope-like longitudinal sections, often associated with increased amounts of matrix substances in the form of electron-dense irregular strands and filaments in a branched network. Group III (clinically EDS types II–III) presented smaller isolated collagen flowers and ropes associated with excessive filamentous ground substance material and flocculent material. Group IV (with clinical symptoms of EDS type IV) had a dermis thinned to one third of the normal and a reduced number of collagen bundles with small diameter fibrils. In 13 patients, the abnormal ultrastructural dermal architecture did not coincide with any of these four groups or with the pattern of any other inherited connective tissue disorder. In 16 additional patients with mostly mild clinical symptoms, such as muscle weakness and small joint hyperlaxity, no ultrastructural aberrations could be found. Even though the primary defects underlying the respective aberration of the collagen fibrils are still unknown, the differential ultrastructural changes of the collagen fibrils together with clinical symptoms should, as in other heterogeneous genetic disorders, facilitate the (provisional?) classification of EDS and permit the diagnosis of individual cases.     

Factors toxic to beech (Fagus sylvatica L.) seedlings in acid soils

The effects of highly and moderately acid soils on total biomass, biomass partitioning, fine root characteristics and nutritional status of beech seedlings (Fagus sylvatica L.) were studied in a growth chamber experiment. In Haplic Arenosols seedlings grew slowly but equally well without damage symptoms in a highly acid and a moderately acid soil horizon. The moderately acid A_h+B_w-horizon of a Eutric Cambisol was favourable to seedling growth. The fine root development was reduced in the highly acid A+B_w-horizon of a Dystric Cambisol and in the A_h+E-horizon of a Haplic Podzol, the latter of which also caused increased mortality. Seedling growth in the B₂-horizon of the Haplic Podzol was vigorous, in spite of a higher level of extractable Al and lower base saturation as compared with the A_h+E-horizon. These results are interpreted in relation to soil acidity, soil Al and nutritional status of the seedlings. We conclude that neither Al-toxicity nor nutrient deficiency cause the damage symptoms observed in the A_h+E-horizon of a Haplic Podzol and the fine root reduction in the A+B_w-horizon of a Dystric Cambisol. The damage symptoms of the PZh_A treatment seems to be more the result of H-toxicity or H-related factors other than nutrient shortage or Al-toxicity. Other pH-related toxic factors are discussed.     

Characterization of a porcine glucosephosphate isomerase-processed pseudogene at Chromosome 1q1.6-1.7

A porcine glucosephosphate isomeraseprocessed pseudogene has been isolated and sequenced. The pseudogene has several base substitutions as well as an insertion and deletions, and is 83% homologous to the corresponding cDNA. It contains an intervening sequence of 565 bp, is truncated at the 3 end, and is flanked by direct repeats of seven nucleotides. Fluorescent in situ hybridization to porcine metaphase chromosomes localized the processed pseudogene to Chromosome (Chr) 1q1.6-1.7. A (GT)₁₄(AT)₁₅ microsatellite was detected close to the processed pseudogene.     

Reflection contrast microscopy of ultrathin sections in immunocytochemical localization studies: a versatile technique bridging electron microscopy with light microscopy

Reflection contrast microscopy (RCM) of ultrathin sections was recently introduced as a sensitive technique for visualization with enhanced definition in immunogold histochemistry. Experience of using RCM as a major tool in immunocytochemical research in different fields is summarized, e.g. oncology, nephrology and embryology. The sensitive visualization of immunocytochemical labels, gold particles or peroxidase-diaminobenzidine deposits in or on ultrathin sections, by RCM instead of electron microscopy is demonstrated. RCM of ultrathin sections is an adequate light microscopical alternative for immunoelectron microscopy, since an overview of both label and tissue is obtained with a high image definition and high contrast of label. In the studies presented, RCM is shown to provide a better gradation in staining intensity and staining pattern than other light microscopical methods. Moreover, a precise localization of multiple labels is obtained with this method. Besides the applications shown, ultrathin section visualization by RCM is very useful for correlative light- and electron microscopical studies of fine structures. Commercially available fluorescence microscopes can be adapted for proper RCM functioning; an adaptation scheme and list of microscopes tested is provided.     

Growth and nutrient dynamics of beech (Fagus sylvatica L.) seedlings in acid soils

The relative uptake rates of N, P, K, S, Ca, Mg, Fe, Mn, Zn, Cu, and Al were estimated in beech seedlings pot cultured in the field in six acid soils (treatments). The relative uptake rates were compared with the relative growth rates. The relative uptake rates of N, K and Ca agreed well with the growth rates of the seedlings irrespective of widely differing soil conditions (acid sand-clayey till, pH 4–6). The relative uptake rates of P, Fe, and Al differed from that predicted by the growth rate. The uptake rates of Fe and Al were highest at the lowest growth rates, and the P uptake rate was lower than the growth rate in these treatments. Thus the P availability probably limited growth in an eluvial (E) horizon of a podzol, and possibly in the illuvial (B) horizon of a podzol and in an acid clayey till (Dystric Cambisol). Low P uptake was associated with a tendency towards higher relative root growth rates. In terms of the concept of steady state nutrition the high relative root growth rate in some treatments may be interpreted as an acclimation to low P supply. The P limitation seemed to be related to interactions among Fe, Al and organic compounds of the soil solution.FAX no: +4646104423     

Nitrogen mineralization in native cultivated and abandoned fields in shortgrass steppe

We conducted a set of in situ incubations to evaluate patterns of N availability among dominant land uses in the shortgrass steppe region of Colorado, USA, and to assess recovery of soil fertility in abandoned fields. Replicated 30 d incubations were performed in 3 sets of native (never cultivated), abandoned (cultivated until 1937), and currently cultivated, fallow fields. Net N mineralization and the percentage of total N that was mineralized increased in the order: native, abandoned, cultivated. Higher soil water content in fallow fields is the most likely reason for greater mineralization in cultivated fields, while higher total organic C and C/N ratios in native and abandoned fields may explain differences in mineralization between these land uses. Recovery of soil organic matter in abandoned fields appears to involve accumulation of soil C and N under perennial plants, but probable methodological artifacts complicate evaluation of the role of individual plants in recovery of N availability. Higher N mineralization and turnover in cultivated fields may make them more susceptible to N losses; recovery of N cycling in abandoned fields appears to involve a return to slower N turnover and tighter N cycling similar to native shortgrass steppe.     

Biochemical and immunocytochemical characterization of monoclonal antibody TOP 35 raised against purified tonoplast from cress root

The vacuolar membrane, the tonoplast, is a proteinrich membranehitherto only few proteins in it have been identified. As anapproach for the identification of tonoplast proteins by monoclonalantibodies (MABs), purified tonoplast from cress roots (Lepidiumsativum L.) were used for immunization and plasma membranesas a control membrane to test the absence of antigen. The MABTOP 35 identified a glycoprotein of about 35 kDa in purifiedtonoplast of cress roots. Triton X-114 phase separation showedthat it was a hydrophobic integral membrane protein. In immunocytochemistrythe MAB TOP 35 strongly labelled the vacuolar membrane. Theabsence of cell wall or plasma membrane labelling by TOP 35indicates a distinct biosynthetic pathway of this protein tothe vacuolar membrane in plants. Key words: Immnocytochemistry, Lepidium sativum, monoclonal antibody, secretion, vacuole     

Molecular and cytological characteristics of nuclear DNA and chromatin for angiosperm systematics: DNA diversification in the evolution of four orchids

The species- and genus-specific DNA content, average base composition of nuclear DNA, presence or absence of satellite DNA, the percentage of heterochromatin and other characteristics of nuclear DNA and nuclear structure allow to deduce the molecular changes which accompanied, or more probably caused, cladogenesis in the orchids studied. It is suggested that saltatory replication (generative amplification) of certain DNA sequenes, diversification of reiterated DNA sequences, and loss of DNA play an important role in the evolution of orchids.—The relationship between changes of genome composition and of nuclear structure and ultrastructure is discussed on the basis of cot curves, heterochromatin staining with Giemsa (C banding), electron microscopy of nuclei, and molecular hybridization in situ.Some aspects of this paper have been presented at the Helsinki Chromosome Conference, August 1977 (Nagl & Capesius 1977).