The immune response of bovine mammary epithelial cells to live or heat-inactivated Mycoplasma bovis

Mycoplasma bovis is an emerging bacterial agent causing bovine mastitis. Although these cell wall-free bacteria lack classical virulence factors, they are able to activate the immune system of the host. However, effects on the bovine mammary immune system are not yet well characterized and detailed knowledge would improve the prevention and therapy of mycoplasmal mastitis. The aim of this study was to investigate the immunogenic effects of M. bovis on the mammary gland in an established primary bovine mammary epithelial cell (bMEC) culture system. Primary bMEC of four different cows were challenged with live and heat-inactivated M. bovis strain JF4278 isolated from acute bovine mastitis, as well as with the type strain PG45. The immune response was evaluated 6 and 24h after mycoplasmal challenge by measuring the relative mRNA expression of selected immune factors by quantitative PCR. M. bovis triggered an immune response in bMEC, reflected by the upregulation of tumor necrosis factor-α, interleukin(IL)-1β, IL-6, IL-8, lactoferrin, Toll-like receptor-2, RANTES, and serum amyloid A mRNA. Interestingly, this cellular reaction was only observed in response to live, but not to heat-inactivated M. bovis, in contrast to other bacterial pathogens of mastitis such as Staphylococcus aureus. This study provides evidence that bMEC exhibit a strong inflammatory reaction in response to live M. bovis. The lack of a cellular response to heat-inactivated M. bovis supports the current hypothesis that mycoplasmas activate the immune system through secreted secondary metabolites.

Limited Correlation between Expansin Gene Expression and Elongation Growth Rate

The aim of this work was to study the role of the cell wall protein expansin in elongation growth. Expansins increase cell wall extensibility in vitro and are thought to be involved in cell elongation. Here, we studied the regulation of two tomato (Lycopersicon esculentum cv Moneymaker) expansin genes,LeExp2 and LeExp18, in rapidly expanding tissues. LeExp2 was strongly expressed in the elongation zone of hypocotyls and in the faster growing stem part during gravitropic stimulation. LeExp18 expression did not correlate with elongation growth. Exogenous application of hormones showed a substantial auxin-stimulation of LeExp2 mRNA in etiolated hypocotyls and a weaker auxin-stimulation ofLeExp18 mRNA in stem tissue. Analysis of transcript accumulation revealed higher levels of LeExp2 andLeExp18 in light-treated, slow-growing tissue than in dark-treated, rapidly elongating tissue. Expansin protein levels and cell wall extension activities were similar in light- and dark-grown hypocotyl extracts. The results show a strong correlation between expansin gene expression and growth rate, but this correlation is not absolute. We conclude that elongation growth is likely to be controlled by expansin acting in concert with other factors that may limit growth under some physiological conditions.

Localized Upregulation of a New Expansin Gene Predicts the Site of Leaf Formation in the Tomato Meristem

Expansins are extracellular proteins that increase plant cell wall extensibility in vitro and are thought to be involved in cell expansion. We showed in a previous study that administration of an exogenous expansin protein can trigger the initiation of leaflike structures on the shoot apical meristem of tomato. Here, we studied the expression patterns of two tomato expansin genes, LeExp2 and LeExp18. LeExp2 is preferentially expressed in expanding tissues, whereas LeExp18 is expressed preferentially in tissues with meristematic activity. In situ hybridization experiments showed that LeExp18 expression is elevated in a group of cells, called I1, which is the site of incipient leaf primordium initiation. Thus, LeExp18 expression is a molecular marker for leaf initiation, predicting the site of primordium formation at a time before histological changes can be detected. We propose a model for the regulation of phyllotaxis that postulates a crucial role for expansin in leaf primordium initiation.

Analysis of expansin-induced morphogenesis on the apical meristem of tomato

Our previous work has shown that localised activity of the cell-wall-loosening protein expansin is sufficient to induce primordia on the apical meristem of tomato, consistent with the hypothesis that tissue expansion plays a key role in leaf initiation. In this paper we describe the earliest morphogenic events visible on the surface of the apical meristem of tomato (Lycopersicon esculentum Mill.) following treatment with expansin and report on the spectrum of final structures formed. Our observations are consistent with a proposed primary function of expansin effecting morphogenesis via altered biophysical stress patterns in the meristem. The primordia induced by expansin do not complete the full program of leaf development. We present data indicating that one reason for this might be the inability of exogenous expansin to mimic the endogenous pattern of expansin activity in the meristem. These data provide the first detailed analysis at the cellular level of expansin action on living tissue, the first description of the spectrum of structures induced by expansin on the apical meristem, and give an insight into a potentially fundamental mechanism in plant development.

Identification and characterization of antigens and potential virulence factors in Enterococcus

Enterococci are normal flora in the human intestinal tract, and also one of the leading causes of nosocomial infections, with most of the clinical isolates being Enterococcus faecalis and Enterococcus faecium. Despite extensive studies on the antibiotic resistance, the pathogenicity of enterococci is not well understood, especially for E. faecium. To identify potential virulence factors based on their antigenicity during infection, E. faecium genomic libraries were constructed and screened using sera from patients with E. faecium endocarditis. ^ As one of my projects, total polysaccharides were extracted from E. faecalis OG1RF and from two epa mutants constructed previously, TX5179 and TX5180, and western blots with patient sera showed that an immuno-reactive polysaccharide present in wild type OG1RF was not produced by either of the two epa mutants. The epa mutants were more sensitive to ethanol stress, neutrophil killing and neutrophil phagocytosis than the wild type OG1RF. ^ Expression of virulence factors is commonly regulated by two component systems. A BLAST search was performed to identify potential two component systems in the E. faecalis V583 genome database using PhoP/PhoS as query sequences, and 11 gene pairs were identified, seven of which were disrupted in E. faecalis OGIRF. ^ Finally, an in vitro translocation model was established for enterococci. E. faecalis strain OG1RF and E. faecium strain DO were shown to be able to translocate across a T84 monolayer, while E. coli strain DH5α and E. faecalis strain E1 could not. ^ In conclusion, several E. faecium antigens expressed in infection (whose antibodies present in sera from patients with E. faecium endocarditis) were identified, two of which, SagA and GlyA, were characterized and suggested to be involved in cell wall metabolism. E. faecalis epa gene cluster (involving in polysaccharide biosynthesis and known to be involved in virulence of E. faecalis in mice) was shown to be involved in hindering neutrophil killing. Several two-component systems were identified in E. faecalis and two of which, EtaRS and EtbRS, were involved in E. faecalis virulence in a mouse peritonitis model.^

Role of FbpA and FbpB in the pathogenesis and mycolyltransferase activity of Mycobacterium tuberculosis

Mycobacterium tuberculosis infects more people worldwide each year than any other single organism. The Antigen 85 Complex, a family of fibronectin-binding proteins (Fbps) found in several species of mycobacteria and possibly involved in host interaction, is considered among the putative virulence factors of M. tuberculosis. These proteins are implicated in the production of trehalose dimycolate (TDM) and arabinogalactan-mycolate (AG-M), two prominent components of the mycobacterium cell wall and potent modulators of the immune system during infection. For these reasons, the principal members of the complex, FbpA and FbpB, were the focus of these studies. The genes encoding these proteins, fbpA and fbpB, were each disrupted by insertion of a kanamycin resistance cassette in a pathogenic strain of M. tuberculosis, H37Rv. Neither mutation affected growth in routine broth culture. Thin layer chromatography analysis of TDM and AG-M showed no difference in content between the parent strain H37Rv and the FbpA- and FbpB-deficient mutants grown under two different culture conditions. However, metabolic radiolabeling of the strains showed that the production of TDM (but not its precursor TMM) was delayed in the FbpA- and FbpB-deficient mutants compared to the parent H37Rv. During this same labeling period, FbpA-deficient mutant LAa1 failed to produce AG-M and in the FpbB-deficient mutant LAb1 production was decreased. In macrophage tissue culture assay, LAa1 failed to multiply when bacteria in early log phase were used to infect monolayers while LAb1 grew like the parent strain. The growth deficiency of LAa1 as well as the deficiencies in TDM and AG-M production were restored by complementing LAa1 with a functional fbpA gene. These results suggest that the FbpA and FbpB proteins are involved in synthesis of TDM (but not its precursor TMM) as well as AG-M. Other members of the complex appear to compensate for defects in synthesis caused by mutation of single genes in the complex over time. Mutation of the FbpA gene causes greater in vivo effect than mutation of the FbpB gene despite very similar deficiencies in the rate of production of mycolate containing molecules on the cell surface. ^

Influence of mycobacterial trehalose 6,6'-dimycolate (TDM) on macrophage responses

Mycobacterium tuberculosis, the causative agent of tuberculosis, survives within macrophages by altering host cell activation and by manipulating phagosomal trafficking and acidification. Part of the success of M. tuberculosis as a major human pathogen has been attributed to its cell wall, a unique structure largely comprised of mycolic acids. Trehalose 6,6′-dimycolate (TDM) is the major glycolipid component on the surface of the mycobacterial cell wall. This study examines the contribution of TDM during mycobacterial infection of murine macrophages. Virulent M. tuberculosis was chemically depleted of surface-exposed TDM using petroleum ether extraction. Compared to their native counterparts, delipidated M. tuberculosis showed similar growth in broth culture. Bone marrow-derived macrophages (BMM) or the murine macrophage-like cell line J774A.1 were infected with delipidated M. tuberculosis, and responses were compared to cells infected with native M. tuberculosis. Delipidated M. tuberculosis demonstrated significantly decreased viability in macrophages by seven days after infection. Reconstitution of delipidated organisms with pure TDM restored viability. Infection with native M. tuberculosis led to high cellular production of cytokines (IL-1β, IL-6, IL-12, and TNF-α) and chemokines (MCP-1 and MIP-1α); infection with delipidated M. tuberculosis significantly abrogated responses. Cytokine and chemokine production were restored when delipidated organisms were reconstituted with TDM. Responses were specifically induced by TDM; all measured cytokines were elicited from macrophages incubated with TDM-coated beads, while control beads coated with bovine serum albumin (BSA) did not induce cytokine production. Visualization of mycobacterial localization in J774A.1 cells using fluorescence microscopy revealed that delipidated M. tuberculosis were significantly more likely to traffic to acidic vesicles (lysosomes) than native organisms. Reconstitution with TDM restored trafficking to non-acidic vesicles. Similarly, TDM-coated beads demonstrated significantly delayed localization to acidic vesicles compared to BSA-coated beads. In summary, the interaction of TDM with macrophages may regulate the outcome of M. tuberculosis infection by influencing cellular cytokine production and intracellular localization of organisms. This research has elucidated a novel and necessary role for TDM in survival of virulent M. tuberculosis in host macrophages during in vitro infection. ^

Characterization of the Putative Xyloglucan Glycosyltransferase GT14 in Arabidopsis thaliana

Plant cell walls largely consist of matrix polysaccharides that are linked to cellulose microfibrils. Xyloglucan, the primary hemicellulose of the cell wall matrix, consists of a repeating glucose tetramer structure with xylose residues attached to the first three units ('XXXG'). In Arabidopsis thaliana, the core XXXG structure is further modified by enzymatic addition of galactose and fucose residues to the xylose side chains to produce XLXG, XXLG, XLLG and XLFG structures. GT14 is a putative glycosyltransferase in the GT47 gene family. Initial predictions of GT14's hydrophobic regions, based on its translated amino acid sequence, are almost identical to its Arabidopsis homolog MUR3, which is a xyloglucan galactosyltransferase targeted to the Golgi membrane. This suggests that, like MUR3, GT14 possesses a transmembrane domain and that it is targeted to the Golgi. The monosaccharide composition of leaves from T-DNA insertion knockouts of GT14 was analyzed by gas-liquid chromatography. The gt14 plants were found to have lower fucose and higher mannose contents than wild type plants. Analysis of cell wall and soluble fractions from gt14 and wild type plants revealed that most of the deficiency in fucose was accounted for in the cell wall, supporting the idea that GT14's target is xyloglucan. Finally, gt14 and wild type plants were transformed with GT14 for complementation and overexpression analysis. The majority of transformed plants did not show significant changes with regard to monosaccharide composition. This may be because the plants were in the T1 generation and, thus, hemizygous. Analysis of homozygous plants in the T2 generation may reveal noticeable changes. Further studies on the xyloglucan composition of gt14 plants are necessary to put the observed reduction in cell wall fucose into a meaningful context.

The Molecular Characterization of a Predicted Glycosyltransferase from Lycopersicon esculentum

The synthesis of the plant cell wall is very complex, and understanding how this process occurs will lead to many benefits for future research and industries dependent upon cell walls for their products. The recent discovery of the functions of AtMUR3 and AtGT18 in Arabidopsis thaliana as xyloglucan galactosyltransferases has led to the identification of many more putative glycosyltransferases in the Arabidopsis genome. Due to the structural differences between the xyloglucans of Arabidopsis and solanaceous plants, we decided to search for putative arabinosyltransferases in the Solanaceae. Solanaceous xyloglucan is substituted by one to two arabinosyl residues at the second xylose position, and sometimes contains an arabinose at the first xylose position. In contrast, Arabidopsis xyloglucan does not contain arabinose, and is substituted by galactose at the second and third xylose position. Furthermore, the second galactose residue in Arabidopsis xyloglucan is usually fucosylated, a modification not found in solanaceous plants. Searching the database of expressed sequence tags (dbEST), we identified many likely glycosyltransferases in solanaceous plants, including tomato (Lycopersicon esculentum). AtMUR3 and AtGT18 search queries resulted in the identification of three putative glycosyltransferases in L. esculentum, which were tentatively designated LeGT1, Le1GT18, and Le2GT18. Based on phylogenetic considerations, Le2GT18 was thought to be a putative arabinosyltransferase. The gene was transformed into atmur3-3 and atgt18 mutant plants, and the resulting plants will be screened for homozygous plants with the inserted gene. The homozygous T2 plants can then be screened for changes in the composition of their cell walls. Because Le2GT18 is thought to be an arabinosyltransferase, the levels of arabinose may be increased in the xyloglucan fraction of the cell wall. If so, further testing can be performed to reveal the true function of Le2GT18.

Hypersensitive granulomatous response to mycobacterial glycolipid trehalose 6,6'-dimycolate

Protection against Mycobacterium tuberculosis infection requires an effective cell mediated immune response leading to granuloma formation and organism containment. Trehalose 6,6'-dimycolate (TDM), a glycolipid present on the mycobacterial cell wall, has been implicated as a key component in establishment of the granulomatous response. TDM has potent immunoregulatory and inflammatory properties; the acute response to TDM produces pathology resembling early Mycobacterium tuberculosis infection. We have further developed this model to study TDM-specific cell mediated immune responses that may play a role in the later stages of infection and pathology. Lungs from mice immunized with TDM in the form of a water-oil-water (w/o/w) emulsion demonstrate heightened histological damage, inflammation, lymphocytic infiltration, and vascular endothelial cell damage upon subsequent challenge with TDM. This exacerbated response can be adoptively transferred to naïve mice via transfer of non-adherent lymphocytes from TDM immunized mice. To identify the cell phenotype(s) regulating this response, purified non-adherent cell populations (CD4+ and CD8+ T cells; CD19 + B cells) were isolated from TDM immunized mice, adoptively transferred into naive mice, and subsequently challenged with TDM. Lung histopathology and cytokine production identified CD4+ cells as the critical cell phenotype regulating the TDM-specific hypersensitive response. The role of CD1d in presentation of TDM was examined. CD1d, a molecule known to present lipids to T cells, was identified as critical in development of the hypersensitive response. CD4+ cells were isolated from TDM-immunized CD1d -/- mice and adoptively transferred into naive wild type mice, followed by TDM challenge. These mice were deficient in development of the hypersensitive granulomatous response, signifying the importance of CD1d in the generation of TDM-specific CD4+ cells. The experiments presented in this dissertation provide further evidence for involvement of TDM-specific cell mediated immune response in elicitation of pathological damage during Mycobacterium tuberculosis infection. ^

Biochemical and genetic characterization of the Saccharomyces cerevisiae Hsp110 molecular chaperone Sse1

The Ssel/Hsp110 molecular chaperones are a poorly understood subgroup of the Hsp70 chaperone family. Hsp70 can refold denatured polypeptides via a carboxyl-terminal peptide binding domain (PBD), which is regulated by nucleotide cycling in an amino-terminal ATPase domain. However, unlike Hsp70, both Sse1 and mammalian Hsp110 bind unfolded peptide substrates but cannot refold them. To test the in vivo requirement for interdomain communication, SSE1 alleles carrying amino acid substitutions in the ATPase domain were assayed for their ability to complement sse1Δ phenotypes. Surprisingly, all mutants predicted to abolish ATP hydrolysis complemented the temperature sensitivity of sse1Δ, whereas mutations in predicted ATP binding residues were non-functional. Remarkably, the two domains of Ssel when expressed in trans functionally complement the sse1Δ growth phenotype and interact by coimmunoprecipitation analysis, indicative of a novel type of interdomain communication. ^ Relatively little is known regarding the interactions and cellular functions of Ssel. Through co-immunoprecipitation analysis, we found that Ssel forms heterodimeric complexes with the abundant cytosolic Hsp70s Ssa and Ssb in vivo. Furthermore, these complexes can be efficiently reconstituted in vitro using purified proteins. The ATPase domains of Ssel and the Hsp70s were found to be critical for interaction as inactivating point mutations severely reduced interaction efficiency. Ssel stimulated Ssal ATPase activity synergistically with the co-chaperone Ydj1 via a novel nucleotide exchange activity. Furthermore, FES1, another Ssa nucleotide exchange factor, can functionally substitute for SSE1/2 when overexpressed, suggesting that Hsp70 nucleotide exchange is the fundamental role of the Sse proteins in yeast, and by extension, the Hsp110 homologs in mammals. ^ Cells lacking SSE1 were found to accumulate prepro-α-factor, but not the cotranslationally imported protein Kar2, similar to mutants in the Ssa chaperones. This indicates that the interaction between Ssel and Ssa is functionally significant in vivo. In addition, sse10 cells are compromised for cell wall strength, likely a result of decreased Hsp90 chaperone activity with the cell integrity MAP kinase SIC. Taken together, this work established that the Hsp110 family must be considered an essential component of Hsp70 chaperone biology in the eukaryotic cell.^

The molecular basis for beta-lactamase gene expression in B. anthracis, B. cereus and B. thuringiensis

The susceptibility of most Bacillus anthracis strains to β-lactam antibiotics is intriguing considering that the B. anthracis genome harbors two β-lactamase genes, bla1 and bla2, and closely-related species, Bacillus cereus and Bacillus thuringiensis, typically produce β-lactamases. This work demonstrates that B. anthracis bla expression is affected by two genes, sigP and rsp, predicted to encode an extracytoplasmic function sigma factor and an antisigma factor, respectively. Deletion of the sigP/rsp locus abolished bla expression in a penicillin-resistant clinical isolate and had no effect on bla expression in a prototypical penicillin-susceptible strain. Complementation with sigP/rsp from the penicillin-resistant strain, but not the penicillin-susceptible strain, conferred β-lactamase activity upon both mutants. These results are attributed to a nucleotide deletion near the 5' end of rsp in the penicillin-resistant strain that is predicted to result in a nonfunctional protein. B. cereus and B. thuringiensis sigP and rsp homologues are required for inducible penicillin resistance in those species. Expression of the B. cereus or B. thuringiensis sigP and rsp genes in a B. anthracis sigP/rsp-null mutant confers resistance to β-lactam antibiotics, suggesting that while B. anthracis contains the genes necessary for sensing β-lactam antibiotics, the B. anthracis sigP/rsp gene products are insufficient for bla induction. ^ Because alternative sigma factors recognize unique promoter sequence, direct targets can be elucidated by comparing transcriptional profiling results with an in silico search using the sigma factor binding sequence. Potential σP -10 and -35 promoter elements were identified upstream from bla1 bla2 and sigP. Results obtained from searching the B. anthracis genome with the conserved sequences were evaluated against transcriptional profiling results comparing B. anthracis 32 and an isogenic sigP/rsp -null strain. Results from these analyses indicate that while the absence of the sigP gene significantly affects the transcript levels of 16 genes, only bla1, bla2 and sigP are directly regulated by σP. The genomes of B. cereus and B. thuringiensis strains were also analyzed for the potential σP binding elements. The sequence was located upstream from the sigP and bla genes, and previously unidentified genes predicted to encode a penicillin-binding protein (PBP) and a D-alanyl-D-alanine carboxypeptidase, indicating that the σ P regulon in these species responds to cell-wall stress caused by β-lactam antibiotics. ^ β-lactam antibiotics prevent attachment of new peptidoglycan to the cell wall by blocking the active site of PBPs. A B. cereus and B. thuringiensis pbp-encoding gene located near bla1 contains a potential σP recognition sequence upstream from the annotated translational start. Deletion of this gene abolished β-lactam resistance in both strains. Mutations in the active site of the PBP were detrimental to β-lactam resistance in B. cereus, but not B. thuringiensis, indicating that the transpeptidase activity is only important in B. cereus. I also found that transcript levels of the PBP-encoding gene are not significantly affected by the presence of β-lactam antibiotic. Based on these data I hypothesize that the gene product acts a sensor of β-lactam antibiotic. ^

Human complement component C3 -binding proteins of Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis, is a facultative intracellular pathogen that uses the host mononuclear phagocyte as a niche for survival and replication during infection. Complement component C3 has previously been shown to enhance the binding of M. tuberculosis to mononuclear phagocytes. Using a C3 ligand affinity blot protocol, we identified a 30 kDa C3-binding protein in M. tuberculosis as heparin-binding hemagglutinin (HbhA). HbhA was found to be a hydrophobic protein that localized to the cell membrane/cell wall fraction of M. tuberculosis, and this protein has previously been shown by others to be located on the surface of M. tuberculosis. The C3-binding activity of HbhA was localized to the C-terminus of the protein, which consists of lysine-alanine repeats. Full-length recombinant HbhA coated onto latex beads was shown to mediate the adherence of the beads to murine macrophage-like cells in both a C3-dependent and a C3-independent manner. An in-frame 576 by deletion in the hbhA gene was created in a virulent strain of M. tuberculosis using a PCR technique known as gene splicing by overlap extension (SOEing). Using the ΔhbhA mutant, HbhA was found not to be necessary for growth of M. tuberculosis in laboratory media or in macrophage-like cells, nor is HbhA required for adherence of M. tuberculosis to macrophage-like cells. HbhA is, however, required for infectivity of M. tuberculosis in mice. Mice infected with the ΔhbhA mutant show decreased growth in the lungs, liver, and spleen compared to mice infected with the wild-type strain. Using the ΔhbhA mutant strain, we were able to purify and identify a second 30-kDa C3-binding protein, HupB. These data demonstrate that HbhA is required for the in vivo but not the in vitro survival of M. tuberculosis and that HbhA is not necessary for the adherence of M. tuberculosis to the macrophage-like cells used in these studies. The expression of two proteins that bind human C3 may aid in the efficient binding of M. tuberculosis to complement receptors for uptake into mononuclear cells, or may influence other aspects of the host-parasite interaction. ^

Flor y fruto de vid [Vitis vinifera L.] : : micrografía aplicada a viticultura y enología

Un conocimiento apropiado de la anatomía de los diferentes tipos florales de la vid permite interpretar procesos de floración y fertilidad. Se ha observado durante cinco años las características de distintas variedades de vid en Mendoza (Argentina). Debido a las diferencias de variedades y viñedos, las observaciones se han tratado desde un punto de vista general. El estudio de las características anatómicas de la baya permite comprender fenómenos de maduración, estrés hídrico, deficiencias y productividad. En cuanto a los procedimientos tecnológicos, la observación de los componentes celulares del fruto: pared celular, polifenoles vacuolares, plástidos y ráfides de cristales de tartrato de calcio permite evaluar métodos enológicos, procesos de maceración y extracción de compuestos fenólicos y los tratamientos que deberían aplicarse para mejorar la calidad del vino.

Molecular and biochemical analyses of wounding in mesocarp of peach (Prunus persica L. Batsch) ripe fruits

The physiological and molecular responses of ripe fruit to wounding were evaluated in two peach (Prunus persica) varieties ('Glohaven', GH, melting and 'BigTop', BT, slow melting nectarine) by comparing mesocarp samples from wedges (as in minimal processing) and whole fruit as the control. Slight differences between the two varieties were detected in terms of ethylene production, whereas total phenol and flavonoid concentrations, and PPO and POD enzyme activities showed a general increase in wounded GH but not in BT. This was associated with the better appearance of the BT wedges at the end of the experimental period (72 h). Microarray (genome-wide ?PEACH3.0) analysis revealed that a total number of 2218 genes were differentially expressed (p < 0.01, log2 fold change expression ratio >1 or <-1) in GH 24 h after wounding compared to the control. This number was much lower (1208) in BT. According to the enrichment analysis, cell wall, plasma membrane, response to stress, secondary metabolic processes, oxygen binding were the GO categories over-represented among the GH up-regulated genes, whereas plasma membrane and response to endogenous stimulus were the categories over-represented among the down-regulated genes. Only 32 genes showed a common expression trend in the two varieties 24 h after wounding, whereas a total of 512 genes (with highly represented transcription factors), displayed opposite behavior. Quantitative RT-PCR analysis confirmed the microarray data for 18 out of a total of 20 genes selected. Specific WRKY, AP2/ERF and HSP20 genes were markedly up-regulated in wounded GH, indicating the activation of regulatory and signaling mechanisms probably related to different hormone categories. Compared to BT, the expression of specific genes involved in phenylpropanoid and triterpenoid biosynthetic pathways showed a more pronounced induction in GH, highlighting the difference between the two peach varieties in terms of molecular responses to wounding in the mesocarp tissue.