The effect on the transcriptome of Anemone coronaria following infection with rust (Tranzschelia discolor).

In order to understand plant/pathogen interaction, the transcriptome of uninfected (1S) and infected (2I) plant was sequenced at 3'end by the GS FLX 454 platform. De novo assembly of high-quality reads generated 27,231 contigs leaving 37,191 singletons in the 1S and 38,393 in the 2I libraries. ESTcalc tool suggested that 71% of the transcriptome had been captured, with 99% of the genes present being represented by at least one read. Unigene annotation showed that 50.5% of the predicted translation products shared significant homology with protein sequences in GenBank. In all 253 differential transcript abundance (DTAs) were in higher abundance and 52 in lower abundance in the 2I library. 128 higher abundance DTA genes were of fungal origin and 49 were clearly plant sequences. A tBLASTn-based search of the sequences using as query the full length predicted polypeptide product of 50 R genes identified 16 R gene products. Only one R gene (PGIP) was up-regulated. The response of the plant to fungal invasion included the up-regulation of several pathogenesis related protein (PR) genes involved in JA signaling and other genes associated with defense response and down regulation of cell wall associated genes, non-race-specific disease resistance1 (NDR1) and other genes like myb, presqualene diphosphate phosphatase (PSDPase), a UDP-glycosyltransferase 74E2-like (UGT). The DTA genes identified here should provide a basis for understanding the A. coronaria/T. discolor interaction and leads for biotechnology-based disease resistance breeding.

Ectopic expression of Kxhkn5 in the viviparous species Kalanchoe × Houghtonii induces a novel pattern of epiphyll development.

KxhKN5 (class 1 KNOX gene) was cloned from Kalanchoe × houghtonii with strong tendency to form epiphylls on leaves. KxhKN5 appear to be homologue of BP of A. thaliana on the basis of phylogeny, expression and phenotype analysis. Beside the modification of several plant and leaf traits, the appearance of epiphylls was drastically reduced by both the silencing and the over-expression of KxhKN5 in most of the generated clones. In silenced clones, epiphyll production followed the morphogenetic pathway of the WT plants: somatic embryos outbreak in the centre of each leaf-pedestal, grown in the notch between leaf indentations and were supported by a suspensor. The connection between the epiphyll and the mother plant did not include any vasculature and as a result, the epiphylls dropped easily from the mother plant. The most represented category of over expressor clones, disclosed a novel pattern of epiphyll development: the leaf-pedestals were absent and single bud outbreaks in each leaf notch. Buds developed into shoots which remained attached to the maternal plant by a strong vascular connection. The leaves supporting shoots, produced a thickened midrib and veins, and their lamina ceased expanding. Finally, the leaf/shoot structure resembles a lateral branch. The ectopic expression of KxhKN5 in K. × houghtonii induces a process comparable to the alternation of leaf and shoot formation in other species and support the idea, that it is the variation in shared molecular and developmental processes which produces the growth of specific structures.

Effects of fruit and vegetable low molecular mass fractions on gene expression in gingival cells challenged with Prevotella intermedia and Actinomyces naeslundii.

Low molecular mass (LMM) fractions obtained from extracts of raspberry, red chicory, and Shiitake mushrooms have been shown to be an useful source of specific antibacterial, antiadhesion/coaggregation, and antibiofilm agent(s) that might be used for protection towards caries and gingivitis. In this paper, the effects of such LMM fractions on human gingival KB cells exposed to the periodontal pathogens Prevotella intermedia and Actinomyces naeslundii were evaluated. Expression of cytokeratin 18 (CK18) and ß4 integrin (ß4INT) genes, that are involved in cell proliferation/differentiation and adhesion, and of the antimicrobial peptide ß2 defensin (HßD2) in KB cells was increased upon exposure to either live or heat-killed bacteria. All LMM fractions tested prevented or reduced the induction of gene expression by P. intermedia and A. naeslundii depending on the experimental conditions. Overall, the results suggested that LMM fractions could modulate the effects of bacteria associated with periodontal disease in gingival cells.

Expression of KxhKN4 and KxhKN5 genes in Kalanchoë blossfeldiana 'Molly' results in novel compact plant phenotypes: towards a cisgenesis alternative to growth retardants.

Many potted plants like Kalanchoë have an elongated natural growth habit, which has to be controlled through the application of growth regulators. These chemicals will be banned in the near future in all the EU countries. Besides their structural functions, the importance of homeotic genes to modify plant architecture appears evident. In this work, the full length cDNA of five KNOX (KN) genes were sequenced from K. x houghtonii, a viviparous hybrid. Two constructs with the coding sequence of the class I and class II homeobox KN genes, KxhKN5 and KxhKN4, respectively, were overexpressed in the commercially important ornamental Kalanchoë blossfeldiana 'Molly'. Furthermore, a post-transcriptional gene silencing construct was made with a partial sequence of KxhKN5 and also transformed into 'Molly'. Several transgenic plants exhibited compact phenotypes and some lines had a relative higher number of inflorescences. A positive correlation between gene expression levels and the degree of compactness was found. However, a correlation between the induced phenotypes and the number of inserted copies of the transgene were not observed, although line '70-10' with a high copy number also had the highest expression level. Moreover, overexpression of KxhKN4 resulted in plants with dark green leaves due to an elevated content of chlorophyll, a highly desired property in the ornamental plant industry. These transgenic plants show that a cisgenesis approach towards production of compact plants with improved quality as an alternative to chemical growth retardants may be feasible.

Short-term effects of environmentally relevant concentrations of EDC mixtures on Mytilus galloprovincialis digestive gland.

Endocrine disrupting compounds (EDCs), including both natural estrogens and estrogenic chemicals, are almost ubiquitous in the aquatic environment. In the marine bivalve Mytilus galloprovincialis different estrogenic compounds, both individually and in mixtures, were shown to affect the immune function both in vitro and in vivo. Moreover, individual estrogens, the natural estrogen 17beta-estradiol (E(2)) and the xenoestrogen bisphenol A (BPA), have been recently demonstrated to alter functional parameters and gene expression in mussel digestive gland, a tissue that plays a central role in metabolism and in nutrient distribution to the gonad during gamete maturation, with possible consequences on gametogenesis. In this work, the possible effects of a synthetic mixture of EDCs on the digestive gland were evaluated. The mixture contained seven estrogenic chemicals (17beta-estradiol, 17alpha-ethynyl estradiol, mestranol (MES), nonylphenol, nonylphenol monoethoxylate carboxylate (NP1EC), BPA, benzophenone (BP)), in proportions similar to those previously found in water samples of a coastal lagoon. Mussels were injected with different concentrations of the mixture (approximate nominal concentrations of total EDCs: 0.0177, 0.177, 1.77 and 177 ng/g dw) and tissues sampled 24 h post-injection. The mixture induced significant changes in lysosomal biomarkers (lysosomal membrane stability (LMS), neutral lipid (NL) and lipofuscin (LF) accumulation) as well as in the activities of catalase, glutathione transferase (GST), and of the glycolytic enzymes phosphofructokinase (PFK) and pyruvate kinase (PK). Moreover, downregulation of the gene transcription for the Mytilus estrogen receptor MeER1 isoform and for catalase, as evaluated by quantitative RT-PCR, were observed. Significant changes in lysosomal biomarkers, enzyme activities and gene transcription were also recorded at 72 h post-injection. The results demonstrate that short-term exposure to environmentally relevant concentrations of EDC mixtures can interfere with the lysosomal function, redox-related enzyme activities and gene transcription of mussel digestive gland.

Bisphenol-A alters gene expression and functional parameters in molluscan hepatopancreas.

Bisphenol-A (BPA) is a well-known xenoestrogen in mammalian systems that can affect reproduction also in aquatic organisms. In this work the possible effects of BPA were investigated in the hepatopancreas of the bivalve mollusc Mytilus galloprovincialis: mussels were injected with different amounts of BPA (3-60ng/g dw tissue) and tissues sampled at 24h post-injection. Expression of different Mytilus genes was evaluated by RT-Q-PCR: BPA exposure increased the expression of MeER2 and induced downregulation of antioxidant genes, catalase and metallothioneins. Moreover, BPA induced changes in activity of catalase, GSH transferase (GST) and GSSG reductase (GSR), and in total glutathione content. A decrease in lysosomal membrane stability and increased neutral lipid accumulation were also observed. The results were compared with those obtained with similar concentrations of 17beta-estradiol. These data demonstrate that BPA can alter gene expression, activities of enzymes involved in redox balance, and lysosomal function in molluscan hepatopancreas, a tissue involved in the control of metabolism and gamete maturation. Overall, these data indicate that BPA, at environmentally relevant concentrations, can have both estrogen-like and distinct effects in invertebrates like in vertebrates.

Effects of 17beta-estradiol on mussel digestive gland.

In bivalve molluscs the digestive gland (hepatopancreas) plays a central role in metabolism. In this work, the effects of 17beta-estradiol (E(2)) on digestive gland were evaluated in Mytilus galloprovincialis. Mussels were injected into the adductor muscle sinus with different amounts of the hormone (5, 25 and 100pmol) and tissues were sampled 24h post-injection. Functional parameters (lysosomal membrane stability-LMS, lysosomal accumulation of neutral lipids-NL and of lipofuscin-LF), as well as the activity of the key glycolytic enzymes PFK (phosphofructokinase) and PK (pyruvate kinase), and of the antioxidant enzyme catalase were evaluated. Selected genes, whose expression can be modulated by estrogens in mammalian systems and whose sequences have been identified in Mytilus, were investigated as possible targets for the action of E(2). E(2) induced a concentration-dependent decrease in LMS; such an effect was accompanied by an increase in NL accumulation, whereas the level of lipofuscin showed a slight, although not significant decrease. E(2) exposure also led to a significant increase in the activity of PFK and catalase but not of PK. Moreover, E(2) induced significant changes in the pattern of gene expression at the lower concentrations tested (5 and 25pmol) as evaluated by quantitative RT-PCR. In particular, increased transcription of catalase, as well as of the metallothionein 20 (MT20) isoform were observed; on the other hand, a decreased transcription of the p53 gene was detected. The results demonstrate that in Mytilus the digestive gland represents a target for the action of E(2), and that the hormone can modulate the lysosomal function, as well as lipid and glucose metabolism. Moreover, these data suggest that E(2) may also alter oxidative stress conditions in this tissue, as indicated by the increased transcription of genes (metallothionein and catalase) that play a role in antioxidant defences. Overall, the results indicate that E(2) can modulate both functional parameters and gene expression in mussel hepatopancreas and underline the importance of investigating also non-reproductive effects of estrogenic compounds in bivalve molluscs.

Efects of growth hormone and cadmium on the transcription regulation of two metallothionein isoforms.

The effect of growth hormone (GH) and cadmium (Cd) on metallothionein (MT) expression was investigated in hepatoma cells. In fish the constitutive isoform MT-B and the metal-responsive MT-A are expressed. Real-time RT-PCR revealed that: Cd up-regulates mostly MT-A, GH slightly induces MT-B and the GH/Cd combination induces synergistically both MTs. Perturbations in Ca2+ levels suppressed or reduced the Cd-induction of MTs and abolished the GH/Cd synergy. Similar results were obtained by inhibition of tyrosine kinases. Also the signaling molecules recruited by the GH receptor responded differently to GH and Cd, with ERKs showing a synergistic activation upon GH/Cd. The following conclusions can be drawn: (1) cytosolic Ca2+ is mainly involved in MT-A regulation; (2) both Ca2+ and tyrosine phosphorylation are essential for Cd-induction and GH/Cd synergy on MTs. The synergy could depend on interactions in different signaling pathways, leading to a differential recruitment of MTF-1 and AP-1 transcription factors.

Modifications of nuclear architecture and chromatin organization in ataxia telangiectasia cells are coupled to changes of gene transcription.

Ataxia telangiectasia (AT) is a rare genetic disorder caused by mutations of ATM gene. ATM kinase is a "master controller" of DNA-damage response and signal transducer of external stimuli. The complex role of ATM may explain the pleiotropic phenotype characteristic of AT syndrome, only partially. In our hypothesis, the multi-faceted phenotype of AT patients might depend on specific chromatin reorganization, which then reflects on the cellular transcription. We analyzed three lymphoblastoid cell-lines isolated from AT patients and one healthy control. The three-dimensional reconstruction disclosed marked changes of nuclear morphology and architecture in AT cells. When chromatin condensation was analyzed by differential scanning calorimetry, a remodeling was observed at the level of fiber folding and nucleosome conformation. Despite the structural differences, chromatin did not exhibit modifications of the average acetylation status in comparison to the control. Moreover, AT cells presented significant alterations in the transcription of genes involved in cell-cycle regulation and stress response. In AT3RM cells, the average chromatin decondensation went with the upregulation of c-fos, c-jun, and c-myc and downregulation of metallothioneins, p21 and p53. AT9RM and AT44RM cells were instead characterized by an increased chromatin condensation and presented a different transcription unbalance. Whereas in AT44RM all the considered genes were downregulated, in AT3RM the three oncogenes and metallothioneins were upregulated, but p53 and p21 were downregulated.

Fish and molluscan metallothioneins.

Metallothioneins (MTs) are noncatalytic peptides involved in storage of essential ions, detoxification of nonessential metals, and scavenging of oxyradicals. They exhibit an unusual primary sequence and unique 3D arrangement. Whereas vertebrate MTs are characterized by the well-known dumbbell shape, with a beta domain that binds three bivalent metal ions and an alpha domain that binds four ions, molluscan MT structure is still poorly understood. For this reason we compared two MTs from aquatic organisms that differ markedly in primary structure: MT 10 from the invertebrate Mytilus galloprovincialis and MT A from Oncorhyncus mykiss. Both proteins were overexpressed in Escherichia coli as glutathione S-transferase fusion proteins, and the MT moiety was recovered after protease cleavage. The MTs were analyzed by gel electrophoresis and tested for their differential reactivity with alkylating and reducing agents. Although they show an identical cadmium content and a similar metal-binding ability, spectropolarimetric analysis disclosed significant differences in the Cd7-MT secondary conformation. These structural differences reflect the thermal stability and metal transport of the two proteins. When metal transfer from Cd7-MT to 4-(2-pyridylazo)resorcinol was measured, the mussel MT was more reactive than the fish protein. This confirms that the differences in the primary sequence of MT 10 give rise to peculiar secondary conformation, which in turn reflects its reactivity and stability. The functional differences between the two MTs are due to specific structural properties and may be related to the different lifestyles of the two organisms.