The ERY4 laccase gene from Pleurotus eryngii was expressed in Saccharomyces cerevisiae and the recombinant laccase resulted to be not biologically active. This gene was thus modified to obtain chimerical enzymes derived from the substitution of N-, C- and both N-and C-terminal regions with the corresponding regions of Ery3 laccase, another laccase isoform of P. eryngii. The chimerical isoform named 4NC3, derived from the substitution of both N-and C-terminal regions, showed the best performances in terms of enzymatic activities, affinities for different substrates and stability at a broad range of temperatures and pHs. The chimerical 4NC3 laccase isoform was displayed on the cell surface of S. cerevisiae using the N-terminal fusion with either the Pir2 or the Flo1 S. cerevisiae proteins as anchor attachment sequence. Immunofluorescence microscopy and Western blot analyses confirmed the localization of 4NC3 on the yeast cell surface. The enzyme activity on specific laccase substrates revealed that 4NC3 laccase was immobilized in active form on the cell surface. To our knowledge, this is the first example of expression of a chimerical fungal laccase by yeast cell display.

Background: Durum wheat often faces water scarcity and high temperatures, two events that usually occur simultaneously in the fields. Here we report on the stress responsive strategy of two durum wheat cultivars, characterized by different water use efficiency, subjected to drought, heat and a combination of both stresses. Results: The cv Ofanto (lower water use efficiency) activated a large set of well-known drought-related genes after drought treatment, while Cappelli (higher water use efficiency) showed the constitutive expression of several genes induced by drought in Ofanto and a modulation of a limited number of genes in response to stress. At molecular level the two cvs differed for the activation of molecular messengers, genes involved in the regulation of chromatin condensation, nuclear speckles and stomatal closure. Noteworthy, the heat response in Cappelli involved also the up-regulation of genes belonging to fatty acid β-oxidation pathway, glyoxylate cycle and senescence, suggesting an early activation of senescence in this cv. A gene of unknown function having the greatest expression difference between the two cultivars was selected and used for expression QTL analysis, the corresponding QTL was mapped on chromosome 6B.Conclusion: Ofanto and Cappelli are characterized by two opposite stress-responsive strategies. In Ofanto the combination of drought and heat stress led to an increased number of modulated genes, exceeding the simple cumulative effects of the two single stresses, whereas in Cappelli the same treatment triggered a number of differentially expressed genes lower than those altered in response to heat stress alone. This work provides clear evidences that the genetic system based on Cappelli and Ofanto represents an ideal tool for the genetic dissection of the molecular response to drought and other abiotic stresses.

The objective of this study was to investigate at the molecular level the heat shock response in olive by identifying sequences coding for heat shock proteins (HSPs). Young twigs of Olea europaea trees (cv Cellina di Nardò) were subjected to different temperature treatments in order to induce the expression of heat shock genes. In order to identify genes induced by heat treatment, we used a PCR-based approach to amplify specific HS cDNAs. Search for low molecular weight HSP sequences was performed in public domain databases in order to design specific primers based upon multisequence alignments. By this approach, we isolated the first full length cDNA encoding a low-molecular weight HSP from O. europaea. This is a class I low molecular weight HSP of 18.3 kDa, which is highly expressed in young twigs subjected to heat stress.

Senescence is an integrated response of plants to various internal (developmental) and external (environmental) signals. It is a highly regulated process leading eventually to the death of cells, single organs such as leaves, or even whole plants. In cereals, which are monocarpic plants, senescence represents the final stage of development. In order to study senescence in durum wheat (Triticum turgidum subsp. durum), a cDNA-AFLP analysis was performed. The transcription profiles of plants at different developmental stages (flowering and senescent) were compared. About 2000 cDNA fragments, ranging in size from 160 to 1900 bp, were reproducibly detected. This allowed the identification of 57 differentially expressed cDNAs corresponding to genes belonging to different functional categories related to cellular metabolism, transcription, maintenance of DNA structure, transport and signal transduction. This paper reports the identification of novel durum wheat candidate genes involved in the senescence process, and provides new information about the senescence programme of this important crop species.

Saccharomyces cerevisiae is the yeast species predominating the alcoholic fermentation of grape must. The aim of this research was to evaluate the impact of indigenous S. cerevisiae strains biodiversity on the aroma of wines from Negroamaro grapes. Grapes collected in two different Negroamaro producing micro districts in Salento (Southern Italy), were subjected to natural fermentation and two indigenous S. cerevisiae populations were isolated. Fifteen strains for each of the two populations were selected and tested by micro fermentation assay in order to evaluate their specific contribute to the volatiles composition and sensory impact of the produced wines. The aromatic profile of wines obtained by each selected strain was characterized by different contents of acetates, ethyl esters of fatty acids, higher alcohols, thus showing to be related to the strains geographical origin. The sensorial analysis of wines produced by the six best performing strains confirmed that they are good candidates as industrial starter cultures, This study indicates that the use of a "microarea-specific" starter culture is a powerful tool to enhance the peculiarity of wines deriving from specific areas.

We report the effect of heat, drought and combined stress on the expression of a group of genes that are up-regulated under these conditions in durum wheat (Triticum turgidum subsp. durum) plants. Modulation of gene expression was studied by cDNA-AFLP performed on RNAs extracted from flag leaves. By this approach, we identified several novel durum wheat genes whose expression is modulated under different stress conditions. We focused on a group of hitherto undescribed up-regulated genes in durum wheat, among these, 7 are up-regulated by heat, 8 by drought stress, 15 by combined heat and drought stress, 4 are up-regulated by both heat and combined stress, and 3 by both drought and combined stress. The functional characterization of these genes will provide new data that could help the developing of strategies aimed at improving durum wheat tolerance to field stress.

The main goal of the present investigation was to assess the microbiological safety of two typical meat-derived products, i.e. raw pork sausages and entrails lamb rolls, produced in Salento (Apulia, Southern Italy). Analyses were carried out for 7 years (from 2008 to 2014) and a total number of 6720 samples was collected by specialized personnel. The presence of Listeria monocytogenes and Salmonella spp. was detected by a PCR-based assay, combined with culturing in enrichment broth. The prevalence of L. monocytogenes was assessed in 2.4% entrails lamb rolls and in 4.2% raw pork sausages samples, whereas the occurrence of Salmonella spp. was revealed in 2.7% lamb rolls and in 3.5% pork sausages. A statistically significant seasonal variation was found in the occurrence of L. monocytogenes; in fact a higher number of samples contaminated by this pathogen was recorded in spring and autumn. On the contrary, no significant seasonal changes occurred in the prevalence of Salmonella spp. The data reported indicate that, due to the presence of these pathogens, the Italian food processors need to improve the microbiological monitoring of the processing chains, in order to guarantee health safety.

The aspect of food quality is of increasing interest for both consumers and food industry. Detection of chemical contaminants and specific quality-related components are required. In this sense, biosensors can play a prime role, because these devices allow cheap and continuous monitoring of contaminants along the pasta production chain and are also useful to identify allelic variants of genes coding for protein associated to bad or good quality of wheat end-products. In this work we report the production of a whole-cell biosensor based on bacterial cells genetically modified to respond to the presence of metal ions by fluorescence or luminescence emission, and of a biosensor based on Localized Surface Plasmon Resonance (LSPR), that is able to detect nucleic acid sequences characterizing different allelic variants.

The ERY4 laccase gene of Pleurotus eryngii is not biologically active when expressed in yeast. To explain this finding, we analysed the role of the C-terminus of Ery4 protein by producing a number of its different mutant variants. Two different categories of ERY4 mutant genes were produced and expressed in yeast: (i) mutants carrying C-terminal deletions and (ii) mutants carrying different site-specific mutations at their C-terminus. Investigation of the catalytic properties of the recombinant enzymes indicated that each novel variant acquired different affinities and catalytic activity for various substrates. Our results highlight that C-terminal processing is fundamental for Ery4 laccase enzymatic activities allowing substrate accessibility to the enzyme catalytic core. Apparently, the last 18 amino acids in the C-terminal end of the Ery4 laccase play a critical role in enzyme activity, stability and kinetic and, in particular biochemical and structural data indicate that the K532 residue is fundamental for enzyme activation. These studies shed light on the structure/function relationships of fungal laccases and will enhance the development of biotechnological strategies for the industrial exploitation of these enzymes.

Wheat, the main food source for a third of world population, appears strongly under threat because of predicted increasing temperatures coupled to drought. Plant complex molecular response to drought stress relies on the gene network controlling cell reactions to abiotic stress. In the natural environment, plants are subjected to the combination of abiotic and biotic stresses. Also the response of plants to biotic stress, to cope with pathogens, involves the activation of a molecular network. Investigations on combination of abiotic and biotic stresses indicate the existence of cross-talk between the two networks and a kind of overlapping can be hypothesized. In this work we describe the isolation and characterization of a drought-related durum wheat (Triticum durum Desf.) gene, identified in a previous study, coding for a protein combining features of NBS-LRR type resistance protein with a S/TPK domain, involved in drought stress response. This is one of the few examples reported where all three domains are present in a single protein and, to our knowledge, it is the first report on a gene specifically induced by drought stress and drought-related conditions, with this particular structure.

Heavy metals (HMs), such as copper, zinc, lead, mercury and cadmium, are the most abundant and dangerous inorganic environmental pollutants. Growing pieces of evidence suggest that mycorrhizal fungi can alleviate metal toxicity in plants. In this study, we focused attention on the ectomycorrhizal (ECM) fungus Tuber borchii Vitt., which is widespread in Italy and is of great ecological interest because of the mutualistic associations and the advantages it provides to host plants. Seedlings of the Mediterranean shrub Cistus creticus L., mycorrhized and non mycorrhized with the ECM fungus 7: borchii, were treated with HMs (zinc, lead and chromium). HMs induced leaves' chlorosis in non mycorrhized seedlings; while no significant differencewas observed impigmentation of mycorrhized seedlings' leaves. This observation was confirmed by Euclidean Distance of color measurements in L*a*b* units from RGB digital images of leaves. The decrease in leaves pigmentation observed in HM treated non mycorrhized seedlings strongly correlated with a reduced expression of key genes associated with chlorophyll biosynthesis; instead, no significant variation of gene expression was detected in mycorrhized seedlings treated with HMs.

Plants respond to environmental stimuli, such as heat shock, by re-programming cellular activity through differential gene expression, mainly controlled at the transcription level. The current study refers to two sunflower small heat shock protein (sHSP) genes arranged in tandem in head-to-head orientation and linked by a 3809 bp region. These genes exhibit only slight structural differences in the coding portion. They code for cytosolic class I sHSPs and are named HaHSP17.6a and HaHSP17.6b according to the molecular weight of the putative proteins. The genomic organization of these genes is consistent with the idea that many HSP genes originate from duplication events; in this case, probably an inversion and duplication occurred. The HaHSP17.6a and HaHSP17.6b genes are characterized by different expression levels under various heat stress conditions; moreover, their expression is differently induced by various elicitors. The differential regulation observed for HaHSP17.6a and HaHSP17.6b genes differs from previous observations on duplicated sHSP genes in plants.