The cuticle : not only a barrier for plant defence: A novel defence syndrome in plants with cuticular defects.

The cuticle is a physical barrier that prevents water loss and protects against irradiation, xenobiotics and pathogens. This classic textbook statement has recently been revisited and several observations were made showing that this dogma falls short of being universally true. Both transgenic Arabidopsis thaliana lines expressing cell wall-targeted fungal cutinase (so-called CUTE plants) or lipase as well as several A. thaliana mutants with altered cuticular structure remained free of symptoms after an inoculation with Botrytis cinerea. The alterations in cuticular structure lead to the release of fungitoxic substances and changes in gene expression that form a multifactorial defence response. Several models to explain this syndrome are discussed.

Bone anabolic potential of soy isoflavones and plant extracts and genomic changes during differentiation of hPOB-tert osteoblast-like cells

Summary For the nutritional management of bone health and the prevention of osteoporosis it is important to identify nutrients that positively influence the bone remodeling process at the cellular level. Soy isoflavones show promising osteoprotective effects in animals and humans but their mechanism of action in bone cells is yet poorly understood. Firstly, soy tissue cultures were characterized as a new and optimized source of isoflavones. A large variability in the isoflavone content was observed and high-producing strains (46.3 mg/g dry wt isoflavones) were identified. In the Ishikawa cells bioassay, the estrogenicity of isoflavones was confirmed to be 1000 to 10000 less than 17Mestradiol and that of the malonyl forms was shown for the first time (EC50 of 350 nM and 1880 nM for malonylgenistin and malonyldaidzin, respectively). The estrogenic activity of soya tissue culture extracts correlated to their isoflavone content. Secondly, the effects of phytonutrients on BMP-2 gene expression and on the mevalonate synthesis pathway, as key mediators of bone formation, were investigated. Dietary achievable concentrations of genistein and daidzein (10vM), and statins (4xM) but not 17M estradiol (10nM), induced BMP-2 gene expression (by up to 3-fold) and inhibited the cholesterol biosynthetic pathway (by up to 50%) in the human osteoblastic cell line hP0B¬tert. In addition, several plant extracts (Cyperus rotundus, Lindera benzoin and Cnidium monnieri) induced BMP-2 gene expression but this induction was not restricted to the inhibition of the cholesterol synthesis pathway neither to the estrogenicity. Finally, the gene expression profiles during hP0B-tert differentiation induced by vitamin D and dexamethasone were analyzed with the Affymetrix human GeneChip. 1665 different genes and 98 ESTs were significantly regulated. The expression profiles of bone-related genes was largely in agreement with previously documented patterns, supporting the physiological relevance of the genomic results and the hP0B-tert cell line as a valid model of human osteoblast differentiation. The expression of alternative differentiation markers during the osteogenic treatment of hP0B-tert cells indicated that the adipocyte and myoblast differentiation pathways were repressed, confirming that these culture conditions allowed only osteoblast differentiation. The gene ontology analysis identified further sub-groups of genes that may be involved in the bone formation process. Aims of the thesis In order to define new strategies for the nutritional management of bone health and for the prevention of osteoporosis the major goal of the present work was to investigate the potential of phytonutrients to positively modulate the bone formation process at the cellular level and, in particular: 1.To select and optimise alternative plant sources containing high levels of isoflavones with estrogenic activity (Chapter 3). 2.To compare the effects of statins and phytonutrients on BMP-2 gene expression and on the mevalonate synthesis pathway and to select new plant extracts with a bone anabolic potential (Chapter 4). 3.To further characterize the new human periosteal cell line, hP0B-tert, as a bone- formation model, by elucidating its gene expression profile during differentiation induced by vitamin D and dexamethasone (Chapter 5).

Role of MIF and glutathione, in association with fetal ovine globin chain (Hbgamma) and LPS, in induction of TNFalpha from cells of young and aged mice, and PBL from healthy human populations.

Previous reports from our group have established that the fetal ovine gamma globin chain (Hbgamma) and LPS can synergize in the induction of pro-inflammatory cytokines, especially TNFalpha, from mouse and human leukocytes. A fetal sheep liver extract (FSLE) which was observed to have marked immunoregulatory properties in vivo and in vitro had independently been observed to contain significant amounts of each of these molecules. However, the biological activity of this extract (hereafter FSLE) was not explained solely by its content of Hbgamma and LPS, and independent analysis confirmed also the presence of migration inhibitory factor, MIF, and glutathione in FSLE. We have investigated whether MIF and the cellular anti-oxidant glutathione can further synergize with Hbgamma and LPS in TNFalpha induction from human cells in vitro, and mouse cells activated in vivo/in vitro. Our data show that indeed there is evidence for such a synergy. Treatment or mouse cells with FSLE produced an enhanced TNFalpha production which could be inhibited independently both by anti-Hbgamma and by anti-MIF, and optimally by a combination of these reagents.

Ablation of caterpillar labial salivary glands: technique for determining the role of saliva in insect-plant interactions.

There has been an ardent interest in herbivore saliva due to its roles in inducing plant defenses and its impact on herbivore fitness. Two techniques are described that inhibit the secretion of labial saliva from the caterpillar, Helicoverpa zea, during feeding. The methods rely on cauterizing the caterpillar's spinneret, the principal secretory structure of the labial glands, or surgically removing the labial salivary gland. Both methods successfully inhibit secretion of saliva and the principal salivary enzyme glucose oxidase. Caterpillars with inhibited saliva production feed at similar rates as the untreated caterpillars, pupate, and emerge as adults. Glucose oxidase has been suggested to increase the caterpillar's survival through the suppression of inducible anti-herbivore defenses in plants. Tobacco (Nicotiana tabacum) leaves fed on by caterpillars with ablated salivary glands had significantly higher levels of nicotine, an inducible anti-herbivore defense compound of tobacco, than leaves fed upon by caterpillars with intact labial salivary glands. Tomato (Lycopersicon esculentum) leaves fed upon by caterpillars with suppressed salivary secretions showed greatly reduced evidence of hydrogen peroxide formation compared to leaves fed upon by intact caterpillars. These two methods are useful techniques for determining the role that saliva plays in manipulating plant anti-herbivore defenses.

Thermogenic effects of commercially available plant preparations aimed at treating human obesity.

Different commercially available plant preparations have been claimed to have anti-obesity action. We investigated the acute effects of oral administration of 12 of these preparations in non-obese women and men. No significant increase in energy expenditure (EE) has been noted after treatment with any of these preparations. No change in respiratory quotient (RQ) was shown, except after treatment with maté (Ilex paraguariensis) extract, where a drop in RQ was observed, indicating a rise in the proportion of fat oxidized. The results suggested the poor potential of these plant preparations in the treatment of obesity, except possibly for the maté extract. Further studies are required to explore the influence of higher dosages of these preparations as well as chronic administration in man.

Interplay between wheat cultivars, biocontrol pseudomonads, and soil.

There is a significant potential to improve the plant-beneficial effects of root-colonizing pseudomonads by breeding wheat genotypes with a greater capacity to sustain interactions with these bacteria. However, the interaction between pseudomonads and crop plants at the cultivar level, as well as the conditions which favor the accumulation of beneficial microorganisms in the wheat rhizosphere, is largely unknown. Therefore, we characterized the three Swiss winter wheat (Triticum aestivum) cultivars Arina, Zinal, and Cimetta for their ability to accumulate naturally occurring plant-beneficial pseudomonads in the rhizosphere. Cultivar performance was measured also by the ability to select for specific genotypes of 2,4-diacetylphloroglucinol (DAPG) producers in two different soils. Cultivar-specific differences were found; however, these were strongly influenced by the soil type. Denaturing gradient gel electrophoresis (DGGE) analysis of fragments of the DAPG biosynthetic gene phlD amplified from natural Pseudomonas rhizosphere populations revealed that phlD diversity substantially varied between the two soils and that there was a cultivar-specific accumulation of certain phlD genotypes in one soil but not in the other. Furthermore, the three cultivars were tested for their ability to benefit from Pseudomonas inoculants. Interestingly, Arina, which was best protected against Pythium ultimum infection by inoculation with Pseudomonas fluorescens biocontrol strain CHA0, was the cultivar which profited the least from the bacterial inoculant in terms of plant growth promotion in the absence of the pathogen. Knowledge gained of the interactions between wheat cultivars, beneficial pseudomonads, and soil types allows us to optimize cultivar-soil combinations for the promotion of growth through beneficial pseudomonads. Additionally, this information can be implemented by breeders into a new and unique breeding strategy for low-input and organic conditions.

Extreme genetic differentiation among the remnant populations of marble trout (Salmo marmoratus) in Slovenia.

Populations of the marble trout (Salmo marmoratus) have declined critically due to introgression by brown trout (Salmo trutta) strains. In order to define strategies for long-term conservation, we examined the genetic structure of the 8 known pure populations using 15 microsatellite loci. The analyses reveal extraordinarily strong genetic differentiation among populations separated by < 15 km, and extremely low levels of intrapopulation genetic variability. As natural recolonization seems highly unlikely, appropriate management and conservation strategies should comprise the reintroduction of pure populations from mixed stocks (translocation) to avoid further loss of genetic diversity.

Ultra-high pressure liquid chromatography-mass spectrometry for plant metabolomics: a systematic comparison of high-resolution quadrupole-time-of-flight and single stage Orbitrap mass spectrometers.

The response of Arabidopsis to stress caused by mechanical wounding was chosen as a model to compare the performances of high resolution quadrupole-time-of-flight (Q-TOF) and single stage Orbitrap (Exactive Plus) mass spectrometers in untargeted metabolomics. Both instruments were coupled to ultra-high pressure liquid chromatography (UHPLC) systems set under identical conditions. The experiment was divided in two steps: the first analyses involved sixteen unwounded plants, half of which were spiked with pure standards that are not present in Arabidopsis. The second analyses compared the metabolomes of mechanically wounded plants to unwounded plants. Data from both systems were extracted using the same feature detection software and submitted to unsupervised and supervised multivariate analysis methods. Both mass spectrometers were compared in terms of number and identity of detected features, capacity to discriminate between samples, repeatability and sensitivity. Although analytical variability was lower for the UHPLC-Q-TOF, generally the results for the two detectors were quite similar, both of them proving to be highly efficient at detecting even subtle differences between plant groups. Overall, sensitivity was found to be comparable, although the Exactive Plus Orbitrap provided slightly lower detection limits for specific compounds. Finally, to evaluate the potential of the two mass spectrometers for the identification of unknown markers, mass and spectral accuracies were calculated on selected identified compounds. While both instruments showed excellent mass accuracy (<2.5ppm for all measured compounds), better spectral accuracy was recorded on the Q-TOF. Taken together, our results demonstrate that comparable performances can be obtained at acquisition frequencies compatible with UHPLC on Q-TOF and Exactive Plus MS, which may thus be equivalently used for plant metabolomics.

Multivariate QST-FST comparisons: a neutrality test for the evolution of the g matrix in structured populations.

Neutrality tests in quantitative genetics provide a statistical framework for the detection of selection on polygenic traits in wild populations. However, the existing method based on comparisons of divergence at neutral markers and quantitative traits (Q(st)-F(st)) suffers from several limitations that hinder a clear interpretation of the results with typical empirical designs. In this article, we propose a multivariate extension of this neutrality test based on empirical estimates of the among-populations (D) and within-populations (G) covariance matrices by MANOVA. A simple pattern is expected under neutrality: D = 2F(st)/(1 - F(st))G, so that neutrality implies both proportionality of the two matrices and a specific value of the proportionality coefficient. This pattern is tested using Flury's framework for matrix comparison [common principal-component (CPC) analysis], a well-known tool in G matrix evolution studies. We show the importance of using a Bartlett adjustment of the test for the small sample sizes typically found in empirical studies. We propose a dual test: (i) that the proportionality coefficient is not different from its neutral expectation [2F(st)/(1 - F(st))] and (ii) that the MANOVA estimates of mean square matrices between and among populations are proportional. These two tests combined provide a more stringent test for neutrality than the classic Q(st)-F(st) comparison and avoid several statistical problems. Extensive simulations of realistic empirical designs suggest that these tests correctly detect the expected pattern under neutrality and have enough power to efficiently detect mild to strong selection (homogeneous, heterogeneous, or mixed) when it is occurring on a set of traits. This method also provides a rigorous and quantitative framework for disentangling the effects of different selection regimes and of drift on the evolution of the G matrix. We discuss practical requirements for the proper application of our test in empirical studies and potential extensions.

Exogenous selection shapes germination behaviour and seedling traits of populations at different altitudes in a Senecio hybrid zone.

BACKGROUND AND AIMS: The Senecio hybrid zone on Mt Etna, Sicily, is characterized by steep altitudinal clines in quantitative traits and genetic variation. Such clines are thought to be maintained by a combination of 'endogenous' selection arising from genetic incompatibilities and environment-dependent 'exogenous' selection leading to local adaptation. Here, the hypothesis was tested that local adaptation to the altitudinal temperature gradient contributes to maintaining divergence between the parental species, S. chrysanthemifolius and S. aethnensis. METHODS: Intra- and inter-population crosses were performed between five populations from across the hybrid zone and the germination and early seedling growth of the progeny were assessed. KEY RESULTS: Seedlings from higher-altitude populations germinated better under low temperatures (9-13 °C) than those from lower altitude populations. Seedlings from higher-altitude populations had lower survival rates under warm conditions (25/15 °C) than those from lower altitude populations, but also attained greater biomass. There was no altitudinal variation in growth or survival under cold conditions (15/5 °C). Population-level plasticity increased with altitude. Germination, growth and survival of natural hybrids and experimentally generated F(1)s generally exceeded the worse-performing parent. CONCLUSIONS: Limited evidence was found for endogenous selection against hybrids but relatively clear evidence was found for divergence in seed and seedling traits, which is probably adaptive. The combination of low-temperature germination and faster growth in warm conditions might enable high-altitude S. aethnensis to maximize its growth during a shorter growing season, while the slower growth of S. chrysanthemifolius may be an adaptation to drought stress at low altitudes. This study indicates that temperature gradients are likely to be an important environmental factor generating and maintaining adaptive divergence across the Senecio hybrid zone on Mt Etna.