An Arabidopsis lipid map reveals differences between tissues and dynamic changes throughout development

Mass spectrometry is the predominant analytical tool used in the field of plant lipidomics. However, there are many challenges associated with the mass spectrometric detection and identification of lipids because of the highly complex nature of plant lipids. Studies into lipid biosynthetic pathways, gene functions in lipid metabolism, lipid changes during plant growth and development, and the holistic examination of the role of plant lipids in environmental stress responses are often hindered. Here, we leveraged a robust pipeline that we previously established to extract and analyze lipid profiles of different tissues and developmental stages from the model plant Arabidopsis thaliana. We analyzed seven tissues at several different developmental stages and identified more than 200 lipids from each tissue analyzed. The data were used to create a web-accessible in silico lipid map that has been integrated into an electronic Fluorescent Pictograph (eFP) browser. This in silico library of Arabidopsis lipids allows the visualization and exploration of the distribution and changes of lipid levels across selected developmental stages. Furthermore, it provides information on the characteristic fragments of lipids and adducts observed in the mass spectrometer and their retention times, which can be used for lipid identification. The Arabidopsis tissue lipid map can be accessed at http://bar.utoronto.ca/efp_arabidopsis_lipid/cgi-bin/efpWeb.cgi .     

Isotopically sensitive branching in the formation of cyclic monoterpenes: proof that (-)-alpha-pinene and (-)-beta-pinene are synthesized by the same monoterpene cyclase via deprotonation of a common intermediate

To determine whether the bicyclic monoterpene olefins (-)-alpha-pinene and (-)-beta-pinene arise biosynthetically from the same monoterpene cyclase by alternate deprotonations of a common carbocationic intermediate, the product distributions arising from the acyclic precursor [10-2H3,1-3H]geranyl pyrophosphate were compared with those resulting from incubation of [1-3H]geranyl pyrophosphate with (-)-pinene cyclase from Salvia officinalis. Alteration in proportions of the olefinic products generated by the partially purified pinene cyclase resulted from the suppression of the formation of (-)-beta-pinene (C10 deprotonation) by a primary deuterium isotope effect with a compensating stimulation of the formation of (-)-alpha-pinene (C4 deprotonation). (-)-Pinene cyclase as well as (+)-pinene cyclase also exhibited a decrease in the proportion of the acyclic olefin myrcene generated from the deuteriated substrate, accompanied by a corresponding increase in the commitment to cyclized products. The observation of isotopically sensitive branching, in conjunction with quantitation of the magnitude of the secondary deuterium isotope effect on the overall rate of product formation by the (+)- and (-)-pinene cyclases as well as two other monoterpene cyclases from the same tissue, supports the biosynthetic origin of (-)-alpha-pinene and (-)-beta-pinene by alternative deprotonations of a common enzymatic intermediate. A biogenetic scheme consistent with these results is presented, and alternate proposals for the origin of the pinenes are addressed.     

The interactive effects of temperature,food level and maternal phenotype on offspring size in Daphnia magna

Invertebrate offspring are usually larger in colder environments. To test for possible effects of covariates (e.g. maternal phenotype and feeding conditions) on this pattern, we performed a laboratory experiment to look at the effect of temperature on newborn weight in the planktonic crustacean Daphnia magna. Three tempèratures (12°C, 16°C and 22°C) and two food levels (10,000 cells ml^–1 and 150,000 cells ml^–1) were used, and offspring were examined from the first five clutches of mothers that had been maintained under the constant experimental conditions for three generations. Preliminary analysis suggested that newborn weight was significantly affected by temperature although patterns in the data were not clear cut. In addition, the covariates mother weight and clutch size were positively and negatively correlated with newborn weight, respectively; and later clutches tended to contain heavier offspring. Therefore, in an effort to control for the effects of the covariates, repeated-measures analysis of covariance was performed using ratio values of newborn weight/mother weight (relative newborn weight) as the dependent variable, clutch size as the covariate and clutch number as the repeated measures term. Now, temperature as a main effect in an ANCOVA model did not significantly influence relative newborn weight. The repeatedmeasure term clutch number also became nonsignificant, indicating that when differences in mother weight due to age were accounted for there were no overall differences in relative newborn weight between clutches from a particular mother. Temperature effects on relative newborn weight were only significant as part of interaction terms with food concentration and with clutch number. Thus there were different weight responses to temperature within food levels, and between clutch numbers within food levels. Under the low-food conditions newborn were heaviest at 16°C, lightest at 12°C and intermediate at 22°C. Conversely, under the high-food condition newborn were lightest at 16°C, heaviest at 12°C and again intermediate at 22°C. However, newborn tended to be heavier under the low food condition, and food concentration was highly significant as a main effect. Mother growth rate showed no significant relationship with newborn weight. It is concluded that direct temperature effects on relative newborn weight are marginal and nonsignificant. Temperature effects through interactions with food concentration and clutch number are important determinants of newborn weight, but relatively speaking account for only a small proportion of observed variance in newborn weight (25%), compared with the direct effect of food concentration (67%).     

Changes in the organization of the actin cytoskeleton during preimplantation development of the pig embryo

The organization of the actin cytoskeleton was studied in unfertilized porcine oocytes and preimplantation stage embryos from Day 1 through Day 8 of development. Fixed and detergent-extracted oocytes and embryos were analyzed by fluorescence microscopy after staining with either rhodamine-phalloidin to localize filamentous actin or with affinity-purified anti-actin antibodies to localize the total immunodetectable actin. Whereas unfertilized oocytes contain immunoreactive cytoplasmic actin, rhodamine-phalloidin binding is not detected until fertilization when a prominent cortical staining pattern becomes apparent. In early cleavage stage embryos, filamentous actin is concentrated in the cell cortex of blastomeres especially at sites of cell-cell contact. Compacting morulae exhibit a marked accumulation of actin at the margins of blastomeres where numerous interdigitating cell processes are located. The predominantly pericellular distribution of actin becomes a distinguishing feature of trophectodermal cells in the expanding blastocyst at Day 6 of development; these cells form a prominent actin-limited zone circumscribing the inner cell mass. In Day 8 blastocysts, three cell types are present that are readily distinguishable based upon their actin displays among other cytological features. Trophectodermal cells exhibit continuous actin-rich lateral borders and stress fibers along their basal surface. Inner cell mass cells contain a discontinuous actin boundary and prominent foci of actin along their blastocoelic surface. Lining the blastocoel are patches of endodermal cells in which the actin is exclusively cortical. The data are discussed with respect to differences between species and the chronology of actin rearrangements during preimplantation development of the porcine embryo.     

Assessing toxicity of Lake Diefenbaker (Saskatchewan,Canada) sediments using algal and nematode bioassays

Lake Diefenbaker, on the South Saskatchewan River, Saskatchewan, Canada, receives, on average, 90% of its inflow from snowmelt and rainfall in the Rocky Mountains. The inflowing rivers also receive irrigation return flows and municipal and industrial effluents which may result in the contamination of lake sediments. The sediments were assessed by nematode and algal bioassays.The toxicity of five chemical fractions of the sediment was determined using the nematode Panagrellus redivivus as the test organisms. The results suggest that the sediment chemical fractions frequently inhibit growth and maturation, while lethality was observed at 4 of 12 sites.Samples from 3 of these sites were further evaluated using conventional elutriate Algal Fractionation Bioassays (AFB) with both natural Lake Diefenbaker phytoplankton and a mixed laboratory grown algal culture. The natural phytoplankton showed inhibition at sediment: water ratios of 10: 1; whereas the algal cultures showed both enhancement and inhibition. Evidently, the sediments are frequently toxic to the species tested except for the algal culture. The AFB assesses the mitigative and synergistic effects of contaminants and nutrients and being a conventional elutriate, is more realistic and potentially more acceptable than the chemical fractionation/nematode bioassay technique which essentially considers potential trace organic contaminant effects.     

Effect of culture media and growth phase on the morphology of lactobacilli and on their ability to adhere to epithelial cells

Previous studies have demonstrated that the ability of lactobacilli to attach to and colonize uroepithelial surfaces is an important characteristic that enhances interference against uropathogenic bacteria. This adherence capacity was found to vary amongst lactobacillus strains and with the type of growth medium used to culture the organisms. The present study was undertaken to examine further the effect of culture media and growth phase on lactobacillus adherence to uroepithelial cells in vitro. In addition, a freeze substitution technique was developed to examine the morphology of strainsLactobacillus casei ssrhamnosus RC-17,L. casei GR-1, andL. acidophilus T-13 in relation to growth conditions and adhesion. A growth curve was plotted for strain GR-1, and adherence was found to be lowest for bacteria in early log phase (39 bacteria per uroepithelial cell) and highest in stationary phase (59 bacteria per uroepithelial cell). Strains RC-17 and GR-1 attached in high numbers to uroepithelial cells, whereas T-13 was poorly adherent. The latter formed a long, relatively dense, fibrous capsule after growth in brain heart infusion yeast extract agar, unlike strains GR-1 and RC-17, which formed a short, tightly bound, electron-dense capsule which surrounded the cells in a radial fashion. Growth of RC-17 in batch cultures of human urine, with and without addition of carbohydrates, resulted in formation of an irregular, fibrous extracellular matrix. These experiments illustrate that growth phase and culture conditions affect the extracellular structure of lactobacilli and also affect the adherence capacity of these bacteria. Structural changes mediated by availability of nutrients may partly explain why lactobacilli vary between species and between hosts in their colonization of the urogenital tract.