Diversification of petal monoterpene profiles during floral development and senescence in wild roses: relationships among geraniol content, petal colour, and floral lifespan.

Wild roses store and emit a large array of fragrant monoterpenes from their petals. Maximisation of fragrance coincides with floral maturation in many angiosperms, which enhances pollination efficiency, reduces floral predation, and improves plant fitness. We hypothesized that petal monoterpenes serve additional lifelong functions such as limiting metabolic damage from reactive oxygen species (ROS), and altering isoprenoid hormonal abundance to increase floral lifespan. Petal monoterpenes were quantified at three floral life-stages (unopened bud, open mature, and senescent) in 57 rose species and 16 subspecies originating from Asia, America, and Europe, and relationships among monoterpene richness, petal colour, ROS, hormones, and floral lifespan were analysed within a phylogenetic context. Three distinct types of petal monoterpene profiles, revealing significant developmental and functional differences, were identified: Type A, species where monoterpene abundance peaked in open mature flowers depleting thereafter; Type B, where monoterpenes peaked in senescing flowers increasing from bud stage, and a rare Type C (8 species) where monoterpenes depleted from bud stage to senescence. Cyclic monoterpenes peaked during early floral development, whereas acyclic monoterpenes (dominated by geraniol and its derivatives, often 100-fold more abundant than other monoterpenes) peaked during floral maturation in Type A and B roses. Early-diverging roses were geraniol-poor (often Type C) and white-petalled. Lifetime changes in hydrogen peroxide (H2O2) revealed a significant negative regression with the levels of petal geraniol at all floral life-stages. Geraniol-poor Type C roses also showed higher cytokinins (in buds) and abscisic acid (in mature petals), and significantly shorter floral lifespan compared with geraniol-rich Type A and B roses. We conclude that geraniol enrichment, intensification of petal colour, and lower potential for H2O2-related oxidative damage characterise and likely contribute to longer floral lifespan in monoterpene-rich wild roses.

Drought stress modulates secondary metabolites in Brassica oleracea L. convar. acephala (DC) Alef, var. sabellica L.

BACKGROUND: Consumer preference today is for the consumption of functional food and the reduction of chemical preservatives. Moreover, the antimicrobial properties and health-promoting qualities of plant secondary metabolites are well known. Due to forecasted climate changes and increasing human population, agricultural practices for saving water have become a concern. In the present study, the physiological responses of curly kale Brassica oleracea L. convar. Acephala (DC) var. sabellica to drought stress and the impact of water limitation on the concentration of selected secondary metabolites were investigated under laboratory-controlled conditions. RESULTS: Results indicated that drought stress increased the content of trans-2-hexenal, phytol and δ-tocopherol, and decreased chlorophyll content. Moreover, drought stress increased antioxidant capacity and the expression of AOP2, a gene associated with the biosynthesis of aliphatic alkenyl glucosinolates, and of three genes - TGG1, TGGE and PEN2 - encoding for myrosinases, the enzymes involved in glucosinolate breakdown. CONCLUSION: The present study shows that water limitation during the growing phase might be exploited as a sustainable practice for producing curly kale with a high concentration of nutritionally important health-promoting bioactive metabolites. © 2019 Society of Chemical Industry.

Severe Reduction in Number and Function of Peripheral T Cells Does Not Afford Protection toward Emphysema and Bronchial Remodeling Induced in Mice by Cigarette Smoke.

The protein Lck (p56(Lck)) is a Src family tyrosine kinase expressed at all stages of thymocyte development and is required for maturation of T cells. The targeted disruption of Lck gene in mice results in severe block in thymocyte maturation with substantial reduction in the development of CD4(+)CD8(+) thymocytes, severe reduction of peripheral T cells, and disruption of T-cell receptor signaling with defective function of T-cell responses. To investigate the role of T lymphocyte in the development of cigarette smoke-induced pulmonary changes, Lck(-/-) mice and corresponding congenic wild-type mice were chronically exposed to cigarette smoke, and their lungs were analyzed by biochemical, immunologic, and morphometric methods. Smoking mice from both genotypes showed disseminated foci of emphysema and large areas of goblet cell metaplasia in bronchial and bronchiolar epithelium. Morphometric evaluation of lung changes and lung elastin determination confirmed that mice from both genotypes showed the same degree of emphysematous lesions. Thus, cigarette smoke exposure in the presence of severe reduction in number and function of peripheral T cells does not influence the development of pulmonary changes induced by cigarette smoke. The data obtained suggest that innate immunity is a leading actor in the early development of pulmonary changes in smoking mice and that the adaptive immune response may play a role at later stages.

Ajulemic acid exerts potent anti-fibrotic effect during the fibrogenic phase of bleomycin lung.

BACKGROUND: Ajulemic acid (AjA) is a synthetic analogue of tetrahydrocannabinol that can prevent and limit progression of skin fibrosis in experimental systemic sclerosis. In this study we investigated whether AjA also prevents and modulates lung fibrosis induced by bleomycin (BLM) when administered in mice during the inflammatory or the fibrogenic phase of the model. METHODS: The anti-inflammatory and antifibrotic efficacy of AjA was evaluated in DBA/2 mice treated orally once a day starting either at day 0 (preventive treatment) or at day 8 (therapeutic treatment) after a single intratracheal instillation of BLM. AjA was given at a dose of 1 mg/kg or 5 mg/kg. Mice were sacrificed at day 8, 14 and 21 after BLM and lungs were processed for histology and morphometry, and examined for HO-proline content and for the expression of transforming growth factor beta 1 (TGF-ß1), phosphorylated Smad2/3 (pSMAD2/3), connective tissue growth factor (CTGF), alpha-smooth muscle actin (α-SMA) and peroxisome proliferator-activated receptor-gamma (PPAR-γ). RESULTS: In the 1st week after BLM challenge, an acute inflammation characterized by neutrophil and macrophage accumulation was the main change present in lung parenchyma. The "switch" between inflammation and fibrosis occurs between day 8 and 14 after BLM instillation and involves the bronchi and vasculature. In the subsequent week (at day 21 after BLM instillation) bronchiolocentric fibrosis with significant increase of tissue collagen develops. The fibrotic response evaluated by morphometry and quantified as HO-proline in lung tissue at day 21 after BLM treatment was significantly reduced in mice receiving either AjA in the inflammatory or in early fibrogenic phase. AjA induces marked change in the expression pattern of products implicated in fibrogenesis, such as TGF-ß1, pSMAD2/3, CTGF and α-SMA. In addition, AjA increases significantly the number of PPAR-γ positive cells and its nuclear localization. CONCLUSIONS: AjA treatment, starting either at day 0 or at day 8 after BLM challenge, counteracts the progression of pulmonary fibrosis. The anti-fibrotic effectiveness of AjA is irrespective of timing of compound administration. Further clinical studies are necessary to establish whether AjA may represent a new therapeutic option for treating fibrotic lung diseases.

Reconciling functions and evolution of isoprene emission in higher plants.

Compilation and analysis of existing inventories reveal that isoprene is emitted by c. 20% of the perennial vegetation of tropical and temperate regions of the world. Isoprene emitters are found across different plant families without any clear phylogenetic thread. However, by critically appraising information in inventories, several ecological patterns of isoprene emission can be highlighted, including absence of emission from C4 and annual plants, and widespread emission from perennial and deciduous plants of temperate environments. Based on this analysis, and on available information on biochemistry, ecology and functional roles of isoprene, it is suggested that isoprene may not have evolved to help plants face heavy or prolonged stresses, but rather assists C3 plants to run efficient photosynthesis and to overcome transient and mild stresses, especially during periods of active plant growth in warm seasons. When the stress status persists, or when evergreen leaves cope with multiple and repeated stresses, isoprene biosynthesis is replaced by the synthesis of less volatile secondary compounds, in part produced by the same biochemical pathway, thus indicating causal determinism in the evolution of isoprene-emitting plants in response to the environment.

Herbivory Increases Fruit Set in Silene latifolia: A Consequence of Induced Pollinator-Attracting Floral Volatiles?

Although the effect of herbivory on plant reproduction has been investigated in some detail, little is known about how herbivores affect floral signalling. Here, we investigated the effect of foliar herbivory by the African Cotton Leafworm (Spodoptera littoralis) on floral signalling and fruit set in the White Campion (Silene latifolia). We found no effects of herbivory on floral traits involved in visual signalling (flower number, corolla diameter, calyx length, petal length) or in amount of nectar produced. However, Spodoptera-infested plants emitted higher amounts of the two floral volatiles, (Z)-3-hexenyl acetate and ß-ocimene, than control plants. Open pollinated, infested plants also were found to produce more fruits than control plants, but only with nocturnal pollinators. Experimental addition of the two induced floral volatiles to non-infested Silene flowers also led to the production of more fruits with nocturnal pollination. This suggests that higher fruit production in herbivore-infested plants was caused by increased nocturnal pollinator attraction, mediated by the induced floral emission of these two volatiles. Our results show that the effects of herbivory on plant reproductive success are not necessarily detrimental, as plants can compensate herbivory with increased investment in pollinator attraction.

Isoprene production in transgenic tobacco alters isoprenoid, non-structural carbohydrate and phenylpropanoid metabolism, and protects photosynthesis from drought stress.

Isoprene strengthens thylakoid membranes and scavenges stress-induced oxidative species. The idea that isoprene production might also influence isoprenoid and phenylpropanoid pathways under stress conditions was tested. We used transgenic tobacco to compare physiological and biochemical traits of isoprene-emitting (IE) and non-emitting (NE) plants exposed to severe drought and subsequent re-watering. Photosynthesis was less affected by drought in IE than in NE plants, and higher rates were also observed in IE than in NE plants recovering from drought. Isoprene emission was stimulated by mild drought. Under severe drought, isoprene emission declined, and levels of non-volatile isoprenoids, specifically de-epoxidated xanthophylls and abscisic acid (ABA), were higher in IE than in NE plants. Soluble sugars and phenylpropanoids were also higher in IE plants. After re-watering, IE plants maintained higher levels of metabolites, but isoprene emission was again higher than in unstressed plants. We suggest that isoprene production in transgenic tobacco triggered different responses, depending upon drought severity. Under drought, the observed trade-off between isoprene and non-volatile isoprenoids suggests that in IE plants isoprene acts as a short-term protectant, whereas non-volatile isoprenoids protect against severe, long-term damage. After drought, it is suggested that the capacity to emit isoprene might up-regulate production of non-volatile isoprenoids and phenylpropanoids, which may further protect IE leaves.

Receptor for advanced glycation end products contributes to postnatal pulmonary development and adult lung maintenance program in mice.

The role of the receptor for advanced glycation end products (RAGE) in promoting the inflammatory response through activation of NF-κB pathway is well established. Recent findings indicate that RAGE may also have a regulative function in apoptosis, as well as in cellular proliferation, differentiation, and adhesion. Unlike other organs, lung tissue in adulthood and during organ development shows relatively high levels of RAGE expression. Thus a role for the receptor in lung organogenesis and homeostasis may be proposed. To evaluate the role of RAGE in lung development and adult lung homeostasis, we generated hemizygous and homozygous transgenic mice overexpressing human RAGE, and analyzed their lungs from the fourth postnatal day to adulthood. Moderate RAGE hyperexpression during lung development influenced secondary septation, resulting in an impairment of alveolar morphogenesis and leading to significant changes in morphometric parameters such as airspace number and the size of alveolar ducts. An increase in alveolar cell apoptosis and a decrease in cell proliferation were demonstrated by the terminal deoxy-nucleotidyltransferase-mediated dUTP nick end labeling reaction, active caspase-3, and Ki-67 immunohistochemistry. Alterations in elastin organization and deposition and in TGF-ß expression were observed. In homozygous mice, the hyperexpression of RAGE resulted in histological changes resembling those changes characterizing human bronchopulmonary dysplasia (BPD). RAGE hyperexpression in the adult lung is associated with an increase of the alveolar destructive index and persistent inflammatory status leading to "destructive" emphysema. These results suggest an important role for RAGE in both alveolar development and lung homeostasis, and open new doors to working hypotheses on the pathogenesis of BPD and chronic obstructive pulmonary disease.

Isoprene-Emitting Tobacco Plants Are Less Affected by Moderate Water Deficit under Future Climate Change Scenario and Show Adjustments of Stress-Related Proteins in Actual Climate.

Isoprene-emitting plants are better protected against thermal and oxidative stresses, which is a desirable trait in a climate-changing (drier and warmer) world. Here we compared the ecophysiological performances of transgenic isoprene-emitting and wild-type non-emitting tobacco plants during water stress and after re-watering in actual environmental conditions (400 ppm of CO2 and 28 °C of average daily temperature) and in a future climate scenario (600 ppm of CO2 and 32 °C of average daily temperature). Furthermore, we intended to complement the present knowledge on the mechanisms involved in isoprene-induced resistance to water deficit stress by examining the proteome of transgenic isoprene-emitting and wild-type non-emitting tobacco plants during water stress and after re-watering in actual climate. Isoprene emitters maintained higher photosynthesis and electron transport rates under moderate stress in future climate conditions. However, physiological resistance to water stress in the isoprene-emitting plants was not as marked as expected in actual climate conditions, perhaps because the stress developed rapidly. In actual climate, isoprene emission capacity affected the tobacco proteomic profile, in particular by upregulating proteins associated with stress protection. Our results strengthen the hypothesis that isoprene biosynthesis is related to metabolic changes at the gene and protein levels involved in the activation of general stress defensive mechanisms of plants.

Genetic ablation of the fpr1 gene confers protection from smoking-induced lung emphysema in mice.

Cigarette smoke (CS) is the main causative factor of chronic obstructive pulmonary disease (COPD). Current research supports the concept that airway inflammation is central to the development and progression of the disease. Studies have demonstrated that neutrophils are increased in COPD lungs and that neutrophil-associated products correlate with the development and severity of COPD. The peptide FMLP is an active component of CS. FMLP interacts on the neutrophil and macrophage membranes with a high-affinity receptor subtype (FPR1) and with a low-affinity subtype FPRL1, promoting a chemotactic response, superoxide anion production, and degranulation. Bacterial colonization of the lower respiratory tract and lung cell damage may represent further sources of formyl peptides in patients with COPD. We investigated the role of FPR in a mouse model on lung inflammation and emphysema induced by CS. Here, we report the novel observation that genetic ablation of the FPR1 gene (Fpr1) confers protection from smoking-induced lung emphysema in mice. Compared with wild-type mice, Fpr1 knockout mice displayed marked decreases in the lung migration of neutrophils and macrophages after CS exposure. Upon transgenic approach, the changes in cell numbers were accompanied by marked modulation of the expression of genes implicated in the inflammatory response. Administration of the FPR1 antagonist cyclosporine H to wild-type mice attenuated the acute inflammatory response evoked by CS. These findings may have clinical significance because current smokers and subjects with emphysema showed increased FPR expression in bronchoalveolar fluids and on peripheral neutrophils. Modulating the FPR1 signal should be explored as a potential new therapy.

DNA barcoding of native Caucasus herbal plants: potentials and limitations in complex groups and implications for phylogeographic patterns.

DNA barcoding has rapidly become a useful complementary tool in floristic investigations particularly for identifying specimens that lack diagnostic characters. Here, we assess the capability of three DNA barcode markers (chloroplast rpoB, accD and nuclear ITS) for correct species assignment in a floristic survey on the Caucasus. We focused on two herbal groups with potential for ornamental applications, namely orchids and asterids. On these two plant groups, we tested whether our selection of barcode markers allows identification of the "barcoding gap" in sequence identity and to distinguish between monophyletic species when employing distance-based methods. All markers successfully amplified most specimens, but we found that the rate of species-level resolution amongst selected markers largely varied in the two plant groups. Overall, for both lineages, plastid markers had a species-level assignment success rate lower than the nuclear ITS marker. The latter confirmed, in orchids, both the existence of a barcoding gap and that all accessions of the same species clustered together in monophyletic groups. Further, it also allowed the detection of a phylogeographic signal.The ITS marker resulted in its being the best performing barcode for asterids; however, none of the three tested markers showed high discriminatory ability. Even if ITS were revealed as the most promising plant barcode marker, we argue that the ability of this barcode for species assignment is strongly dependent on the evolutionary history of the investigated plant lineage.

The influence of a relict distribution on genetic structure and variation in the Mediterranean tree, Platanus orientalis.

The distribution of plant species around the Mediterranean basin is a product of the influence of both geographical barriers and of climatic changes experienced during the Tertiary, with the transition from a warm to cool periods. Several species, once largely distributed across the Northern Hemisphere, retracted to refugial areas in southern Europe where they are described as Tertiary relicts. Platanus orientalis is a typical representative of Tertiary flora in southwest Eurasia; its distribution spreads from the Caucasus to the Mediterranean, with its western border in Southern Italy and Sicily. We analysed genetic diversity and differentiation in the centre and western part of its distribution range using nuclear microsatellites and compared genetic parameters between core and western populations. We found an overall decrease in genetic diversity estimates (heterozygosity, private allelic richness) from central towards western populations, with those from Southern Italy and Sicily showing the lowest values. The low level of genetic diversity probably results from historic range fragmentation experienced by P. orientalis in its westernmost distribution as confirmed by high level genetic isolation of these populations. Ornamental hybrids were genetically distinguished from P. orientalis as contained private alleles, indicating that gene flow with natural populations is rare. Population assignment and neighbour-joining (NJ) analysis of populations identified four groups belonging to two main phyletic groups (the Southern Italian-Sicilian-Balkan and Cretan-Bulgarian-Turkish lineages) that seem to have different biogeographic origin and also excluded an artificial origin for southern Italian and Sicilian populations as previously suggested. These results show that quantifying the genetic variation of a Tertiary relict in a geographical context, and the potential effect of hybridization with introduced ornamental hybrids, can provide useful insights on factors influencing population genetic structure. Such information is crucial to predict how such taxa will respond to increasing anthropogenic influence on the environment.

A new mouse model of Peyronie's disease: an increased expression of hypoxia-inducible factor-1 target genes during the development of penile changes.

Peyronie's disease (PD) is characterized by an inflammatory response beneath the tunica albuginea with fibroblast proliferation forming a thickened fibrous plaque that may cause pain, penile curvature and erectile dysfunction. The progression of the PD plaque may eventually lead to calcification or ossification. Current therapeutic success is often unsatisfactory because of limited insight into disease mechanisms. Research has been hampered by the lack of a universally accepted animal model. We describe an animal model of spontaneous PD in tight skin (Tsk) mice, a C57Bl/6J subline that reproduces with age important features of the human disease (fibrous plaque formation, penile bending and areas of chondroid metaplasia with heterotopic ossification). Histological analysis demonstrated an evident structural disorganization of the tunica albuginea with excessive accumulation of type I collagen. At 12 months of age, fibrous plaques with areas of chondroid metaplasia and heterotopic ossification characterized Tsk penises. The up-regulation of hypoxia-inducible factor-1 (HIF-1) leads to an increased downstream expression of HIF-1 target genes, such as TGFbeta and iNOS. These factors, together with some PDGF family members, can cause collagen deposition in Tsk penises. They can also influence chondrocyte differentiation and heterotopic bone formation. In conclusion, hypoxia, HIF-1 and HIF-1 target genes appear to play an important role in the pathogenesis of PD in Tsk mice. This mouse model that is the first example of naturally occurring model of PD in laboratory animals may aid in the identification of signalling pathways crucial for PD and should facilitate the designing and testing of new therapeutic interventions.

Can population genetic structure be predicted from life-history traits?

Population genetic structure is a key parameter in evolutionary biology. Earlier comparative studies have shown that genetic structure depends on species ecological attributes and life-history traits, but species phylogenetic relatedness had not been accounted for. Here we reevaluate the relationships between genetic structure and species traits in seed plants. Each species is characterized by a set of life-history and ecological features as well as by its geographic range size, its heterozygote deficit, and its genetic structure at nuclear and organelle markers to distinguish between pollen- and seed-mediated gene flow. We use both a conventional regression approach and a method that controls for phylogenetic relationships. Once phylogenetic conservatism and covariation among traits are taken into account, genetic structure is shown to be related with only a few synthetic traits, such as mating system for nuclear markers and seed dispersal mode or geographic range size for organelle markers. Along with other studies on invasiveness or rarity, our work illustrates the fact that predicting the fate of species across a broad taxonomic assemblage on the basis of simple traits is rarely possible, a testimony of the highly contingent nature of evolution.

Metabolism of oxidants by blood from different mouse strains.

Haemoglobins bearing reactive sulfhydryl groups have been shown to be able to interplay with glutathione in some detoxification processes. Blood from different mouse strains commonly used as experimental animal models, i.e., C57, DBA and ICR, was treated with oxidants with the aim of evaluating: (i) the involvement of protein SH groups in oxido-reductive reactions that are commonly carried out by glutathione and (ii) the impact of this phenomenon on blood-mediated metabolism of thiol reactants. All the main forms of glutathione (reduced, disulfide, and mixed disulfide with haemoglobin) were measured after oxidant treatment. Significant differences were found among the studied strains: DBA mice formed preferably mixed disulfides instead of glutathione disulfide, whereas the opposite behaviour was shown by C57 mice. Unexpectedly, the ICR strain resulted to be composed of three different subgroups (ICRa, ICRb, and ICRc), with the ICRa behaving similarly to the DBA strain, ICRc to the C57 strain, and ICRc showing an intermediate behaviour. These results are due to the different number of haemoglobin SH groups in the studied mouse strains. In particular, additional fast-reacting SH groups were found in haemoglobin from DBA, ICRa, and ICRb mice, but not in the C57 and ICRc strain. These differences were also reflected in the susceptibility of haemoglobin to dimerize and in its ability to react with S-nitrosocysteine. Because of the widely different reactivity of haemoglobin cysteinyl residues, the mouse strains examined are an interesting but complicated model in which to study the pharmacological and toxicological action of some drugs.

Is neutrophil elastase the missing link between emphysema and fibrosis? Evidence from two mouse models.

BACKGROUND: The separation of emphysema from fibrosis is not as clear-cut as it was thought in early studies. These two pathologies may be present at the same time in human lungs and in mice either instilled with elastolytic enzymes or bleomycin or exposed to cigarette-smoke. According to a current view, emphysema originates from a protease/antiprotease imbalance, and a role for antiproteases has also been suggested in the modulation of the fibrotic process. In this study we investigate in experimental animal models of emphysema and fibrosis whether neutrophil elastase may constitute a pathogenic link between these two pathologies. METHODS: This study was done in two animal models in which emphysema and fibrosis were induced either by bleomycin (BLM) or by chronic exposure to cigarette-smoke. In order to assess the protease-dependence of the BLM-induced lesion, a group mice was treated with 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, a serine proteinase inhibitor active toward neutrophil elastase. Lungs from each experimental group were used for the immunohistochemical assessment of transforming growth factor-beta (TGF-beta) and transforming growth factor-alpha (TGF-alpha) and for determination of the mean linear intercept as well as the percent volume densities of fibrosis and of emphysematous changes. Additionally, the lungs were also assessed for desmosine content and for the determination of elastase levels in the pulmonary interstitium by means of immunoelectron microscopy. RESULTS: We demonstrate that in BLM-treated mice (i) the development of elastolytic emphysema precedes that of fibrosis; (ii) significant amount of elastase in alveolar interstitium is associated with an increased expression of TGF-beta and TGF-alpha; and finally, (iii) emphysematous and fibrotic lesions can be significantly attenuated by using a protease inhibitor active against neutrophil elastase. Also, in a strain of mice that develop both emphysema and fibrosis after chronic cigarette-smoke exposure, the presence of elastase in alveolar structures is associated with a positive immunohistochemical reaction for reaction for both TGF-beta and TGF-alpha. CONCLUSION: The results of the present study strongly suggest that neutrophil elastase may represent a common pathogenic link between emphysema and fibrosis. Proteases and in particular neutrophil elastase could act as regulatory factors in the generation of soluble cytokines with mitogenic activity for mesenchymal cells resulting either in emphysema or in fibrosis or both.

Smoking p66Shc knocked out mice develop respiratory bronchiolitis with fibrosis but not emphysema.

The adaptor protein p66Shc regulates intracellular oxidant levels through the modulation of a forkhead-related transcription factor (FOXO3a). The genetic ablation of p66Shc (p66Shc-/-) renders mice resistant to oxidative stress and p53-dependent apoptosis. We investigated whether p66Shc ablation in mice modifies lung cellular and molecular responses to cigarette smoke (CS) exposure. No differences between wild type (WT) and p66Shc-/- mice were observed in terms of inflammation and oxidant burden after acute CS exposure; however,p66Shc ablation modifies specific features of chronic inflammation induced by repeated exposure to CS. Unlike WT mice, p66Shc-/- mice did not develop emphysema, showing protection toward oxidative damage to DNA and apoptosis as revealed by a trivial 8-hydroxyguanosine staining and faint TUNEL and caspase-3 positivity on alveolar epithelial cells. Unexpectedly, CS exposure in p66Shc-/- mice resulted in respiratory bronchiolitis with fibrosis in surrounded alveoli. Respiratory bronchiolitis was characterized by peribronchiolar infiltrates of lymphocytes and histiocytes, accumulation of ageing pigmented macrophages within and around bronchioles, and peribronchiolar fibrosis. The blockage of apoptosis interferes with the macrophage "clearance" from alveolar spaces, favouring the accumulation of aging macrophages into alveoli and the progressive accumulation of iron pigment in long-lived senescent cells. The presence of areas of interstitial and alveolar fibrosis in peripheral parenchyma often accompanied the bronchiolar changes. Macrophages from smoking p66Shc-/- mice elaborate M2 cytokines (i.e., IL-4 and IL-13) and enzymes (i.e., chitinase and arginase I), which can promote TGF-beta expression, collagen deposition, and fibrosis in the surrounding areas. We demonstrate here that resistance to oxidative stress and p53-dependent apoptosis can modify tissue responses to CS caused by chronic inflammation without influencing early inflammatory response to CS exposure.

RNA-seq analysis of Quercus pubescens Leaves: de novo transcriptome assembly, annotation and functional markers development.

Quercus pubescens Willd., a species distributed from Spain to southwest Asia, ranks high for drought tolerance among European oaks. Q. pubescens performs a role of outstanding significance in most Mediterranean forest ecosystems, but few mechanistic studies have been conducted to explore its response to environmental constrains, due to the lack of genomic resources. In our study, we performed a deep transcriptomic sequencing in Q. pubescens leaves, including de novo assembly, functional annotation and the identification of new molecular markers. Our results are a pre-requisite for undertaking molecular functional studies, and may give support in population and association genetic studies. 254,265,700 clean reads were generated by the Illumina HiSeq 2000 platform, with an average length of 98 bp. De novo assembly, using CLC Genomics, produced 96,006 contigs, having a mean length of 618 bp. Sequence similarity analyses against seven public databases (Uniprot, NR, RefSeq and KOGs at NCBI, Pfam, InterPro and KEGG) resulted in 83,065 transcripts annotated with gene descriptions, conserved protein domains, or gene ontology terms. These annotations and local BLAST allowed identify genes specifically associated with mechanisms of drought avoidance. Finally, 14,202 microsatellite markers and 18,425 single nucleotide polymorphisms (SNPs) were, in silico, discovered in assembled and annotated sequences. We completed a successful global analysis of the Q. pubescens leaf transcriptome using RNA-seq. The assembled and annotated sequences together with newly discovered molecular markers provide genomic information for functional genomic studies in Q. pubescens, with special emphasis to response mechanisms to severe constrain of the Mediterranean climate. Our tools enable comparative genomics studies on other Quercus species taking advantage of large intra-specific ecophysiological differences.

High rates of gene flow by pollen and seed in oak populations across Europe.

Gene flow is a key factor in the evolution of species, influencing effective population size, hybridisation and local adaptation. We analysed local gene flow in eight stands of white oak (mostly Quercus petraea and Q. robur, but also Q. pubescens and Q. faginea) distributed across Europe. Adult trees within a given area in each stand were exhaustively sampled (range [239, 754], mean 423), mapped, and acorns were collected ([17,147], 51) from several mother trees ([3], [47], 23). Seedlings ([65,387], 178) were harvested and geo-referenced in six of the eight stands. Genetic information was obtained from screening distinct molecular markers spread across the genome, genotyping each tree, acorn or seedling. All samples were thus genotyped at 5-8 nuclear microsatellite loci. Fathers/parents were assigned to acorns and seedlings using likelihood methods. Mating success of male and female parents, pollen and seed dispersal curves, and also hybridisation rates were estimated in each stand and compared on a continental scale. On average, the percentage of the wind-borne pollen from outside the stand was 60%, with large variation among stands (21-88%). Mean seed immigration into the stand was 40%, a high value for oaks that are generally considered to have limited seed dispersal. However, this estimate varied greatly among stands (20-66%). Gene flow was mostly intraspecific, with large variation, as some trees and stands showed particularly high rates of hybridisation. Our results show that mating success was unevenly distributed among trees. The high levels of gene flow suggest that geographically remote oak stands are unlikely to be genetically isolated, questioning the static definition of gene reserves and seed stands.