Extracellular polysaccharides are involved in the attachment of Azospirillum brasilense and Rhizobium leguminosarum to arbuscular mycorrhizal structures.

Arbuscular mycorrhizal (AM) fungi, one of the most important component of the soil microbial community, establish physical interactions with naturally occurring and genetically modified bacterial biofertilizers and biopesticides, commonly referred to as plant growth-promoting rhizobacteria (PGPR). We have used a genetic approach to investigate the bacterial components possibly involved in the attachment of two PGPR (Azospirillum and Rhizobium) to AM roots and AM fungal structures. Mutants affected in extracellular polysaccharides (EPS) have been tested in in vitro adhesion assays and shown to be strongly impaired in the attachment to both types of surfaces as well as to quartz fibers. Anchoring of rhizobacteria to AM fungal structures may have special ecological and biotechnological significance because it may facilitate colonisation of new rhizospheres by the bacteria, and may be an essential trait for the development of mixed inocula.     

Reduction of potassium tellurite by Candidas

Summary A technique for observing the reduction of potassium tellurite byCandidas was developed. Various species of these yeasts reduce it at different concentrations, but that which is most useful for differentiation is 0.02 % added to Sabouraud dextrose agar basal medium. Of the yeasts studied,C. albicans, C. parapsilosis andC. tropicalis all reduced potassium tellurite to the concentration mentioned before, while the growth ofC. krusei, C. parakrusei andC. pseudotropicalis was inhibited. Without exception,C. pseudotropicalis reduced this salt at lower concentrations. The two strains ofC. guilliermondii tested gave contradictory results: one of them grew and reduced potassium tellurite, while the growth of the other was inhibited.Professor of Microbiology.     

Comparative structure of vesicular-arbuscular mycorrhizas and ectomycorrhizas

During the establishment of vesicular-arbuscular mycorrhizas, fungal hyphae contact the root surface, form appressoria and initiate the internal colonization phase. Structural changes occur in the cell wall, the cytoplasm and the nucleus as the fungus progresses from a presymbiotic to a symbiotic phase. Nuclei in spores are in G₁ whereas in intraradical hyphae they are in G₁ and G₂. Changes in nuclear organization are evident in various stages in the colonization process. Dramatic changes in both symbionts occur as the nutrient exchange interface is established between arbuscules and root cortical cells. An interfacial matrix, consisting of molecules common to the primary wall of the cortical cell, separates the cortical cell plasma membrane from the fungal cell wall. Ectomycorrhizas are characterized structurally by the presence of a mantle of fungal hyphae enclosing the root and usually an Hartig net of intercellular hyphae characterized by labyrinthine branching. As hyphae contact the root surface, they may respond by increasing their diameter and switching from apical growth to precocious branching. The site of initial contact of hyphae may be either the root cap or the ‘mycorrhiza infection zone’. The mantle varies considerably in structure depending on both the plant and fungus genome. In some ectomycorrhizas, the mantle may be a barrier to apoplastic transport, and in most it may store polyphosphate, glycogen, lipids and perhaps protein.     

Abscisic acid and mefluidide in the regulation of cold hardiness development in Solanum tuberosum L. potato

Plants of Solanum tuberosum L. potato do not cold acclimate when exposed to low temperature such as 5°C, day/night. When ABA (45 M) was added to the culture medium, stem-cultured plantlets of S. tuberosum, cv. Red Pontiac, either grown at 20°C/15°C, day/night, or at 5°C, increased in cold hardiness from –2°C (killing temperature) to –4.5°C. The increase in cold hardiness could be inhibited in both temperature regimes if cycloheximide (70 M) was added to the culture medium at the inception of ABA treatment. Cycloheximide did not inhibit cold hardiness development, however, when it was added to the culture medium 3 days after ABA treatment.When pot-grown plants were foliar sprayed with mefluidide (50 M), ABA content increased from 10 nmol to 30 nmol g^–1 dry weight and plants increased in cold hardiness from –2°C to about –3.5°C. The increases in free ABA and cold hardiness occurred only in plants grown at 20°C/15°C; neither ABA nor cold hardiness increased in plants grown at 5°C.The results suggest that an increase in ABA and a subsequent de novo synthesis of proteins are required for the development of cold hardiness in S. tuberosum regardless of temperature regime, and that the inability to synthesize ABA at low temperature, rather than protein synthesis, appears to be the reason why S. tuberosum does not cold acclimate.     

The control of Ca2+ homeostasis: role of intracellular rapidly exchanging Ca2+ stores.

The role of rapidly exchanging intracellular Ca²⁺ stores in the control of Ca²⁺ homeostasis is reviewed. The following issues are discussed: the reasons why such stores exist in eukaryotic cells; the differences between the terminal cisternae of the skeletal muscle sarcoplasmic reticulum, which have direct, physical connection with the T tubules of the plasmalemma, and the Ca²⁺ stores located in the depth of the cytoplasm, which are stimulated by second messengers; the cytological nature (subcompartments of the ER) of the rapidly exchanging Ca²⁺ stores and their functional significance. The conclusions introduce recent developments in which intracellular Ca²⁺ stores have been investigated also by molecular biology techniques.     

The plant nucleus in mycorrhizal roots: positional and structural modifications

Summary— Positional and structural modifications were demonstrated in nuclei of leek cells, after establishment of a symbiosis with two vesicular-arbuscular fungi, Glomus versiforme and Glomus E₃. By combining light, immuno-electron microscopy and morphometry, the fungi were shown to have a direct effect on the host nuclear morphology: the effect was confined to a specific plant tissue (the cortical parenchyma) and to a moment of the fungal morphogenesis (the arbuscule). When they branch to form the complex structures called arbuscules in the inner parenchyma cells, the host nucleus migrates from the periphery of these cells towards their centre. In addition, it becomes larger and lobed, with a decondensed chromatin. A monoclonal antibody that mostly binds to the condensed chromatin revealed a significant decrease in gold labelling intensity over the nuclei of the colonized cells. These modifications suggest that the nuclear migration and the changes in chromatin organization are related to the modifications in gene expression observed during the establishment of mycorrhizal symbiosis.     

High-resolution solution structure of two members of a conformationally homogeneous combinatorial peptide library based on the classical zinc-finger motif

Summary We describe the high-resolution structure by NMR of two peptides that belong to a combinatorial library based on the zinc-finger motif. The library represents, to the best of our knowledge, the first example of a conformationally homogeneous peptide library and was obtained by introducing random residues in five positions of the -helical portion of a 26-residue consensus peptide (CP1) belonging to the Cys²-Hys² zinc-finger family. The result was shown to be a highly homogeneous -helical library (Bianchi et al., 1995). The structures of the parent compound (CP1) and of a representative member (CP1m) that was selected by screening the library with a monoclonal antibody are compared in detail as an example of the very high stability of the zinc-finger scaffold upon sequence variability. The two peptides exhibit an extremely high degree of structural similarity. The use of this type of conformationally constrained combinatorial library might represent a step forward in the design of peptidomimetics, as it considerably accelerates the process of the identification of the spatial relationship among the pharmacophoric groups.Abbreviations t-Bu tert-butyloxycarbonyl - Fmoc 9-fluorenylmethoxycarbonyl     

Rapid typing of truffle mycorrhizal roots by PCR amplification of the ribosomal DNA spacers

DNA analyses were developed to type mycorrhizas of two Tuber species of commercial value (T. melanosporum, T. borchii) and a competitive fungus (Sphaerosporella brunnea) which forms ectomycorrhizas with plants usually considered hosts for truffles. Polymerase chain reaction (PCR) amplification of DNA isolated from fruitbodies, mycelia, mycorrhizas and leaves of host plants, was performed with a primer pair for an internal transcribed spacer ITS1-4. ITS amplification followed by restriction fragment length polymorphism (RFLP) analysis of the amplified products clearly distinguished the two Tuber species at the fruitbody, mycorrhiza and mycelium levels. Accepted: 6 September 1996     

Preferred conformation of peptides rich in Ac8c,a medium-ring alicyclic Cα,α-disubstituted glycine

A complete series of terminally blocked, monodispersed homo-oligopeptides (to the pentamer level) from the sterically demanding, medium-ring alicyclic C^α,α-disubstituted glycine 1-aminocyclooctane-1-carb oxylic acid (Ac₈c), and two Ala/Ac₈c tripeptides, were synthesized by solution methods and fully characterized. The preferred conformation of all the oligopeptides was determined in deuterochloroform solution by IR absorption and ¹H-NMR. The molecular structures of the amino acid derivative Z-Ac₈c-OH, the dipeptide pBrBz- (Ac₈c)₂-OH and the tripeptide pBrBz-(Ac₈c)₃-OtBu were assessed in the crystal state by X-ray diffraction. Conformational energy computations were performed on the monopeptide Ac-Ac₈c-NHMe. Taken together, the results obtained strongly support the view that the Ac₈c residue is an effective β-turn and helix former. A comparison is also made with the conformational preferences of α-aminoisobutyric acid, the prototype of C^{α, α}-disubstituted glycines, and of the other members of the family of 1-aminocycloalkane-1-carboxylic acids (Ac_nc, with n=3, 5–7) investigated so far. The implications for the use of the Ac₈c residue in peptide conformational design are considered.     

Increase in chitin as an essential response to defects in assembly of cell wall polymers in the ggp1delta mutant of Saccharomyces cerevisiae.

The GGP1/GAS1 gene codes for a glycosylphosphatidylinositol-anchored plasma membrane glycoprotein of Saccharomyces cerevisiae. The ggp1delta mutant shows morphogenetic defects which suggest changes in the cell wall matrix. In this work, we have investigated cell wall glucan levels and the increase of chitin in ggp1delta mutant cells. In these cells, the level of alkali-insoluble 1,6-beta-D-glucan was found to be 50% of that of wild-type cells and was responsible for the observed decrease in the total alkali-insoluble glucan. Moreover, the ratio of alkali-soluble to alkali-insoluble glucan almost doubled, suggesting a change in glucan solubility. The increase of chitin in ggp1delta cells was found to be essential since the chs3delta ggp1delta mutations determined a severe reduction in the growth rate and in cell viability. Electron microscopy analysis showed the loss of the typical structure of yeast cell walls. Furthermore, in the chs3delta ggp1delta cells, the level of alkali-insoluble glucan was 57% of that of wild-type cells and the alkali-soluble/alkali-insoluble glucan ratio was doubled. We tested the effect of inhibition of chitin synthesis also by a different approach. The ggp1delta cells were treated with nikkomycin Z, a well-known inhibitor of chitin synthesis, and showed a hypersensitivity to this drug. In addition, studies of genetic interactions with genes related to the construction of the cell wall indicate a synthetic lethal effect of the ggp1delta kre6delta and the ggp1delta pkc1delta combined mutations. Our data point to an involvement of the GGP1 gene product in the cross-links between cell wall glucans (1,3-beta-D-glucans with 1,6-beta-D-glucans and with chitin). Chitin is essential to compensate for the defects due to the lack of Ggp1p. Moreover, the activities of Ggp1p and Chs3p are essential to the formation of the organized structure of the cell wall in vegetative cells.