Cell surface molecules and fibronectin-mediated cell adhesion: effect of proteolytic digestion of membrane proteins

Proteases have been used as a tool to investigate the role of surface molecules in fibronectin-mediated cell adhesion. Proteolytic digestion of membrane-proteins by pronase (1 mg/ml for 20 min at 37 degrees C) completely inhibited adhesion of baby hamster kidney (BHK) fibroblasts on fibronectin-coated plastic dishes. Various degrees of inhibition were also obtained after treatment with proteinase K, chymotrypsin, papain, subtilopeptidase A, and thermolysin. Protein synthesis was required to restore the adhesive properties of pronase-treated cells, showing the protein nature of the molecules involved in adhesion to fibronectin. A peculiar feature of these proteins was their resistance to cleavage by trypsin. After prolonged trypsin treatment (1 mg/ml for 20 min at 37 degrees C), cells adhered and spread on fibronectin-coated dishes, even when protein synthesis was inhibited by 4 microM cycloheximide. Under these conditions only three glycoproteins (gp) of molecular weight 130,000, 120,000, and 80,000 were left on the cell surface. These were precipitated by a rabbit antiserum against BHK cells that also inhibited adhesion of trypsin-treated cells. gp120 and gp80 were left at the cell surface after mild pronase digestion (0.2 mg/ml for 20 min at 37 degrees C), under conditions not affecting adhesion. These data suggest that these glycoproteins may be involved in fibronectin-mediated cell adhesion in some yet unknown way.     

Beyond the pollination syndrome: nectar ecology and the role of diurnal and nocturnal pollinators in the reproductive success of Inga sessilis (Fabaceae)

Inga species present brush‐type flower morphology allowing them to be visited by distinct groups of pollinators. Nectar features in relation to the main pollinators have seldom been studied in this genus. To test the hypothesis of floral adaptation to both diurnal and nocturnal pollinators, we studied the pollination ecology of Inga sessilis, with emphasis on the nectar secretion patterns, effects of sequential removals on nectar production, sugar composition and the role of diurnal and nocturnal pollinators in its reproductive success. Inga sessilis is self‐incompatible and pollinated by hummingbirds, hawkmoths and bats. Fruit set under natural conditions is very low despite the fact that most stigmas receive polyads with sufficient pollen to fertilise all ovules in a flower. Nectar secretion starts in the bud stage and flowers continually secreting nectar for a period of 8 h. Flowers actively reabsorbed the nectar a few hours before senescence. Sugar production increased after nectar removal, especially when flowers were drained during the night. Nectar sugar composition changed over flower life span, from sucrose‐dominant (just after flower opening, when hummingbirds were the main visitors) to hexose‐rich (throughout the night, when bats and hawkmoths were the main visitors). Diurnal pollinators contributed less than nocturnal ones to fruit production, but the former were more constant and reliable visitors through time. Our results indicate I. sessilis has floral adaptations, beyond the morphology, that encompass both diurnal and nocturnal pollinator requirements, suggesting a complementary and mixed pollination system.     

Nectar ecology of Datura ferox (Solanaceae): an invasive weed with nocturnal flowers in agro-ecosystems from central Argentina

Plant–pollinator interactions provide highly important ecological functions, and are influenced by floral nectar characteristics. The night blooming Datura ferox is an excellent model to test general hypotheses on the relationship between nectar traits (e.g., nectar secretion patterns, nectar chemical composition), pollinators and reproductive success for invasive, weedy species in highly modified ecosystems as crop fields. We hypothesized an adjustment between nectar composition and secretion dynamics through flower anthesis and the activity and requirements of nocturnal pollinators. Nectar chemical analyses showed low quantities of amino acids and lipids, phenolics, and alkaloids were not detected. D. ferox showed sucrose-dominant nectar with comparable amount of hexoses. Sugar proportions did not vary between populations or during flowering season. Most nectar is secreted before flower opening. Nectar resorption was detected at the end of anthesis. Experimentally drained flowers of both populations increased nectar production up to 50 % in the total amount of sugar per flower compared to control flowers. Nectar standing crop was relatively constant during the flowering season, but differences were detected between populations. Nectar traits of D. ferox would be favoring cross-pollination and maintaining seed production of this weed, since recently open flowers display a higher amount of nectar and they can renew nectar after a pollinator visit or reabsorb it at the end of anthesis. This nectar source may be important for native pollinators considering that human-induced forest fragmentation is related with the impoverishment of native flora from agro-ecosystems.     

Characterization of three loci for homologous gene targeting and transgene expression

Integrative gene transfer is widely used for bioproduction, drug screening, and therapeutic applications but usual viral methods lead to random and multicopy insertions, contribute to unstable transgene expression and can disturb endogenous gene expression. Homologous targeting of an expression cassette using rare‐cutting endonucleases is a potential solution; however the number of studied loci remains limited. Furthermore, the behavior and performance of various types of gene cassettes following gene targeting is poorly defined. Here we have evaluated three loci for gene targeting, including one locus compatible with the proposed Safe Harbor criteria for human translational applications. Using optimized conditions for homologous gene targeting, reporter genes under the control of different promoters were efficiently inserted at each locus in both sense and antisense orientations. Sustainable expression was achieved at all three loci without detectable disturbance of flanking gene expression. However, the promoter, the integration locus and the cassette orientation have a strong impact on transgene expression. Finally, single targeted integrations exhibited greatly improved transgene expression stability versus multicopy or random integration. Taken together, our data suggest a potential set of loci for site‐specific transgene integration, suitable for a variety of biotechnological applications. Biotechnol. Bioeng. 2013; 110: 2225–2235. © 2013 Wiley Periodicals, Inc.     

Floral nectaries, nectar production dynamics and chemical composition in six ipomoea species (convolvulaceae) in relation to pollinators

BACKGROUND AND AIMS: Floral nectaries and nectar features were compared between six Argentinian Ipomoea species with differences in their pollinator guilds: I. alba, I. rubriflora, I. cairica, I. hieronymi var. hieronymi, I. indica, and I. purpurea. METHODS: Pollinators were recorded in natural populations. The morpho-anatomical study was carried out through scanning electron and light microscopy. Nectar sugars were identified via gas chromatography. Nectar production and the effect of its removal on total nectar sugar amount were determined by using sets of bagged flowers. KEY RESULTS: Hymenopterans were visitors of most species, while hummingbirds visited I. rubriflora and sphingids I. alba. All the species had a vascularized discoidal nectary surrounding the ovary base with numerous open stomata with a species-specific distribution. All nectar samples contained amino acids and sugars. Most species had sucrose-dominant nectars. Flowers lasted a few hours. Mean nectar sugar concentration throughout the lifetime of the flower ranged from 34.28 to 39.42 %, except for I. cairica (49.25 %) and I. rubriflora (25.18 %). Ipomoea alba had the highest nectar volume secreted per flower (50.12 microL), while in the other taxa it ranged from 2.42 to 12.00 microL. Nectar secretion began as soon as the flowers opened and lasted for a few hours (in I. purpurea, I. rubriflora) or it was continuous during the lifetime of the flower (in the remaining species). There was an increase of total sugar production after removals in I. cairica, I. indica and I. purpurea, whereas in I. alba and I. rubriflora removals had no effect, and in I. hieronymi there was a decrease in total sugar production. CONCLUSIONS: The chemical composition, production dynamics and removal effects of nectar could not be related to the pollinator guild of these species. Flower length was correlated with nectary size and total volume of nectar secreted, suggesting that structural constraints may play a major role in the determination of nectar traits of these species.     

Style morphological diversity of some Asteraceae species from Argentina: systematic and functional implications

Stylar micromorphological diversity of 42 Asteraceae species from Argentina was analysed considering species phylogenetic membership and some floral reproductive functions (pollen presentation and pollen reception). In particular, the morphology and organisation of pollen presenter (sweeping hairs) and pollen receptive structures (stigmatic papillae) were described. Results showed that style morphology of the studied species is far more diverse than the categories previously established for Asteraceae, and that it is problematic to relate the sweeping-hair arrangement of species to the only three modes of pollen presentation described for the family, indicating that the hypothesised relationship could be more complex than was formerly thought. For all species with di- or trimorphic florets, the styles of female florets were more slender and without or with more reduced sweeping hairs than the styles of hermaphrodite florets, and divergences of sweeping hair arrangements and morphology were higher among phylogenetically related species. These results suggest that functional aspects of floral morphology seem to be more important than phylogenetic constraints as selective forces determining stylar pollen presentation structures. In contrast, stigmatic-area organisation as well as the morphology of stigmatic papillae remain identical between female and hermaphrodite florets and among phylogenetically related species. Thus, stigmatic papilla morphology seems to be a phylogenetically constrained character in the studied species. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The composition of arbuscular mycorrhizal fungal communities in the roots of a ruderal forb is not related to the forest fragmentation process

Land‐use changes and forest fragmentation have strong impact on biodiversity. However, little is known about the influence of new landscape configurations on arbuscular mycorrhizal fungal (AMF) community composition. We used 454 pyrosequencing to assess AMF diversity in plant roots from a fragmented forest. We detected 59 virtual taxa (VT; phylogenetically defined operational taxonomic units) of AMF – including 10 new VT – in the roots of Euphorbia acerensis. AMF communities were mainly composed of members of family Glomeraceae and were similar throughout the fragmented landscape, despite variation in forest fragment size (i.e. small, medium and large) and isolation (i.e. varying pairwise distances). AMF communities in forest fragments were phylogenetically clustered compared with the global, but not regional and local AMF taxon pools. This indicates that non‐random community assembly processes possibly related to dispersal limitation at a large scale, rather than habitat filtering or biotic interactions, may be important in structuring the AMF communities. In this system, forest fragmentation did not appear to influence AMF community composition in the roots of the ruderal plant. Whether this is true for AMF communities in soil and the roots of other ecological groups of host plants or in other habitats deserves further study.     

Colonization of native Andean grasses by arbuscular mycorrhizal fungi in Puna: a matter of altitude, host photosynthetic pathway and host life cycles

The relationships of altitude, host life cycle (annual or perennial) and photosynthetic pathway (C(3) or C(4) ) with arbuscular mycorrhiza (AM) root colonization were analysed in 35 species of Andean grasses. The study area is located in north-western Argentina along altitudinal sites within the Puna biogeographical region. Twenty-one sites from 3320 to 4314 m were sampled. Thirty-five grasses were collected, and the AM root colonization was quantified. We used multivariate analyses to test emerging patterns in these species by considering the plant traits and variables of AM colonization. Pearson's correlations were carried out to evaluate the specific relationships between some variables. Most grasses were associated with AM, but the colonization percentages were low in both C(3) and C(4) grasses. Nevertheless, the AM root colonization clearly decreased as the altitude increased. This distinctive pattern among different species was also observed between some of the populations of the same species sampled throughout the sites. An inverse relationship between altitude and AM colonization was found in this Southern Hemisphere Andean system. The effect of altitude on AM colonization seems to be more related to the grasses' photosynthetic pathway than to life cycles. This study represents the first report for this biogeographical region.