Paola Ceccaroli

Biochemical and morphological modifications during the growth of Tuber borchii mycelium

This paper reports the first biochemical characterisation of Tuber borchii mycelial strain ATCC 96540, grown in liquid media containing either glucose, fructose or sucrose. A new biochemical method, based on the estimation of total protein content, was developed for determining the amount of mycelium growth. This method is more sensitive than other methods, allowing growth to be monitored in the lag phase and when small amounts of mycelium are grown on a solid medium. Mycelium of T. borchii utilizes glucose and fructose as carbohydrate sources but grows poorly if at all on sucrose. In these experiments the functional state of the mycelium was evaluated by determining the activity of enzymes of the glycolytic and pentose phosphate pathways which are involved in producing energy and in supplying reducing power through the formation of reduced pyridine coenzymes. The biochemical data on mycelium growth were supported by observations on ultrastructural morphology which revealed the different steps in hyphal depletion during ageing. In addition, the monitoring of alanine content of the liquid media during mycelial growth indicated that there was an increasing loss of alanine from their cytoplasm as hyphae aged. The development of this procedure makes it possible to identify the conditions under which T. borchii is able to synthesize a mycorrhiza with a compatible host.

New evidence for nitrogen fixation within the Italian white truffle Tuber magnatum.

Diversity of nitrogen-fixing bacteria and the nitrogen-fixation activity was investigated in Tuber magnatum, the most well-known prized species of Italian white truffle. Degenerate PCR primers were applied to amplify the nitrogenase gene nifH from T. magnatum ascomata at different stages of maturation. Putative amino acid sequences revealed mainly the presence of Alphaproteobacteria belonging to Bradyrhizobium spp. and expression of nifH genes from Bradyrhizobia was detected. The nitrogenase activity evaluated by acetylene reduction assay was 0.5-7.5μmolC(2)H(4)h(-1)g(-1), comparable with early nodules of legumes associated with specific nitrogen-fixing bacteria. This is the first demonstration of nitrogenase expression gene and activity within truffle.

Geographical traceability of Italian white truffle (Tuber magnatum Pico) by the analysis of volatile organic compounds

Results are presented that were obtained on the geographic traceability of the white truffle Tuber magnatum Pico. Solid-phase microextraction coupled to gas chromatography/mass spectrometry (SPME-GC/MS) was employed to characterize the volatile profile of T. magnatum white truffle produced in seven geographical areas of Italy. The main components of the volatile fraction were identified using SPME-GC/MS. Significant differences in the proportion of volatile constituents from truffles of different geographical areas were detected. The results suggest that, besides genetic factors, environmental conditions influence the formation of volatile organic compounds. The mass spectra of the volatile fraction of the samples were used as fingerprints to characterize the geographical origin. Next, stepwise factorial discriminant analysis afforded a limited number of characteristic fragment ions that allowed a geographical classification of the truffles studied.

Effect of storage on biochemical and microbiological parameters of edible truffle species

The effects of different storage treatments on the most common edible truffle species, such as Tuber magnatum and Tuber borchii (white truffles), Tuber melanosporum and Tuber aestivum (black truffles), were analysed. Biochemical and microbiological profiles were monitored, in order to evaluate possible alterations during truffle preservation. After harvesting, some fresh samples were kept at 4°C for 30days, other samples were frozen at -20°C for one month, thawed and preserved at 4°C; the remainder were autoclaved. The biochemical parameters studied were sugar and protein content, the activity of some enzymes involved in the central metabolism of the fungi and the electrophoretic pattern of soluble proteins. Total mesophilic bacteria were also counted. The results obtained showed that the storage at 4°C is the treatment that best preserves the biochemical and microbiological characteristics of fresh truffles. Black truffles were more resistant to biochemical spoilage than the white ones, while T. magnatum was the most resistant to microbial spoilage.

Morphofunctional and Biochemical Approaches for Studying Mitochondrial Changes during Myoblasts Differentiation.

This study describes mitochondrial behaviour during the C2C12 myoblast differentiation program and proposes a proteomic approach to mitochondria integrated with classical morphofunctional and biochemical analyses. Mitochondrial ultrastructure variations were determined by transmission electron microscopy; mitochondrial mass and membrane potential were analysed by Mitotracker Green and JC-1 stains and by epifluorescence microscope. Expression of PGC1α, NRF1α, and Tfam genes controlling mitochondrial biogenesis was studied by real-time PCR. The mitochondrial functionality was tested by cytochrome c oxidase activity and COXII expression. Mitochondrial proteomic profile was also performed. These assays showed that mitochondrial biogenesis and activity significantly increase in differentiating myotubes. The proteomic profile identifies 32 differentially expressed proteins, mostly involved in oxidative metabolism, typical of myotubes formation. Other notable proteins, such as superoxide dismutase (MnSOD), a cell protection molecule, and voltage-dependent anion-selective channel protein (VDAC1) involved in the mitochondria-mediated apoptosis, were found to be regulated by the myogenic process. The integration of these approaches represents a helpful tool for studying mitochondrial dynamics, biogenesis, and functionality in comparative surveys on mitochondrial pathogenic or senescent satellite cells.

Carbohydrate and amino acid metabolism in Tuber borchii mycelium during glucose utilization: a 13C NMR study

The metabolism of [1-13C]glucose in the vegetative mycelium of the ectomycorrhizal ascomycete Tuber borchii was studied in order to characterize the biochemical pathways for the assimilation of glucose and amino acid biosynthesis. The pathways were characterized using nuclear magnetic resonance spectroscopy in conjunction with [1-13C]glucose labeling. The enzymes of mannitol cycle and ammonium assimilation were also evaluated. The majority of the 13C label was incorporated into mannitol and this polyol was formed via a direct route from absorbed glucose. Amino acid biosynthesis was also an important sink of assimilated carbon and 13C was mainly incorporated into alanine and glutamate. From this intramolecular 13C enrichment, it is concluded that pyruvate, arising from [1-13C]glucose catabolism, was used by alanine aminotransferase, pyruvate dehydrogenase and pyruvate carboxylase before entering the Krebs cycle. The transfer of 13C-labeled mycelium on [12C]glucose showed that mannitol, alanine, and glutamate carbon were used to synthesize glutamine and arginine that likely play a storage role.

Effects of different carboyhydrate sources on the growth of Tuber borchii Vittad. mycelium strains in pure culture

The influence of carbohydrate utilisation on the growth of three strains of Vittad. mycelium (1BO, 17BO and 10RA) in culture was assessed using culture media containing glucose (control), mannose or mannitol. Mannose was the best substrate for growth of the strains and this was particularly evident for strain 17BO. Mannitol instead was metabolized only by 10RA and 1BO. In order to explain the different growth trends, analyses of enzyme levels, kinetic parameters, protein patterns and the morphology of the three strains were carried out. Our results show that these strains of mycelium were affected by the substrates used in the media. The aim of the present work was to optimise the in vitro production of T. borchii mycelium for use in experiments which require the fungus in precise and reproducible conditions, such as mycorrhizal synthesis or protein and nucleic acid extractions.

High resolution of multiple forms of rabbit reticulocyte hexokinase type I by hydrophobic interaction chromatography

Hydrophobic interaction chromatography (HIC) has been employed extensively in the separation of proteins by elution using a descending salt gradient, with and without the use of detergents or denaturing agents. In this study, a new hydrophobic interaction chromatographic support, Toyopearl Phenyl 650 S, was investigated in order to examine the distribution of multiple forms of rabbit reticulocyte hexokinase type I. These distinct forms of the enzyme, designated hexokinase Ia, Ia* and Ib, show similar kinetic and physical properties, similar molecular masses (ca. 100,000) and a different intracellular distribution. The results obtained using Toyopearl Phenyl 650 S of 20-50-microns particle diameter show that this HIC support allows very high resolution, comparable to that obtainable with HIC-HPLC columns but with the advantage of charging a higher amount of starting material even with a high protein concentration. These characteristics render Toyopearl Phenyl 650 S suitable for analytical and preparative purposes. Further, in the separation of multiple forms of rabbit reticulocyte hexokinase, the HIC method was shown to be superior to RP-HPLC, making possible the efficient separation of proteins with high molecular mass and their recovery in active forms. The Toyopearl Phenyl 650 S column was also shown to be more efficient than the ion-exchange chromatographic media previously used, allowing a quicker analysis of the multiple forms of rabbit reticulocyte hexokinase under different biological conditions.

Three different forms of hexokinase are identified during Tuber borchii mycelium growth.

Truffles are ectomycorrhizal fungi which have a great dependence on carbohydrates supplied by their host plants. The catabolism of hexoses in the mycobiont is important for the production of energy, and the first enzyme in the hexose assimilation pathways is hexokinase. This study reports differences in the expression of this enzyme during the growth of Tuber borchii Vittad. mycelium (strain ATCC 96540). Three hexokinase activities (HKM1, HKM2 and HKM3) were isolated by anion-exchange chromatography and partially purified. HKM1 and HKM2 were present in the linear phase at 15-50 days of growth. Two remarkable differences were found in the sugar-phosphorylating activity and stability of HKM1 and HKM2. HKM2 did not phosphorylate the fructose and it was present in the chromatographic profile only when substrates such as glucose, glucosamine or mannose were added to the extraction buffer. On the contrary, HKM1 utilized also fructose and was detected under all the experimental conditions used. HKM3 was the only molecular form observed after 70 days, when the fungus growth had reached a plateau. To our knowledge these results represent the first evidence for the presence in T. borchii mycelium of three distinct enzymatic forms of hexokinase which are differently expressed during growth of the fungus.

Characterization and mRNA expression profile of the TbNre1 gene of the ectomycorrhizal fungus Tuber borchii

This study focuses on the cloning and characterization of the major nitrogen regulator element from the ectomycorrhizal fungus Tuber borchii, TbNre1. Sequence analysis of the predicted protein and complementation experiments in Neurospora crassa demonstrated that the cloned gene is orthologous to areA/nit-2 gene. Transcriptional expression investigations by real-time RT-PCR showed TbNre1 up-regulation in the presence of nitrate or in the absence of nitrogen during free-living mycelium growth. On the contrary, TbNre1 mRNA levels remained at basal values in the presence of preferred nitrogen sources like ammonium and glutamine. Furthermore, TbNre1 mRNA was found to be up-regulated during T. borchii and T. platyphyllos interaction. All these data suggest that the regulatory protein TBNRE1 could play a major role in regulating N metabolism genes of T. borchii in the free living mycelium and in T. borchii–T. platyphyllos interaction. Finally, the possible role of the transcription factor TBNRE1 in the induction of proteases and virulence-like genes, necessary in ectomycorrhizal establishment, was also discussed.