Project description:Phenotypic plasticity, the ability of one genotype to express different phenotypes in response to changing environmental conditions, is one of the most common phenomena characterising the living world and is not only relevant for the ecology but also for the evolution of species. Daphnia, the waterflea, is a textbook example for predator induced phenotypic plastic defences including changes in life-history, behaviour and morphology. However, the analysis of molecular mechanisms underlying these inducible defences is still in its early stages. We exposed Daphnia magna to chemical cues of the predator Triops cancriformis to identify key processes underlying plastic defensive trait formation. D. magna is known to develop an array of morphological changes in the presence of T. cancriformis including changes of carapace morphology and cuticle hardening. To get a more comprehensive idea of this phenomenon, we studied four different genotypes originating from habitats with different predation history, reaching from predator-free to temporary habitats containing T. cancriformis. We analysed the morphologies as well as proteomes of predator-exposed and control animals. Three genotypes showed morphological changes when the predator was present. Using a high-throughput proteomics approach, we found 294 proteins which were significantly altered in their abundance after predator exposure in a general or genotype dependant manner. Proteins connected to genotype dependant responses were related to the cuticle, protein synthesis and calcium binding whereas the yolk protein vitellogenin increased in abundance in all genotypes, indicating their involvement in a more general response. Furthermore, genotype dependant responses at the proteome level correlated well with local adaptation to Triops predation. Altogether, our study provides new insights concerning genotype dependant and general molecular processes involved in predator-induced phenotypic plasticity in D. magna.

Project description:Over recent decades the progression of natural autumnal senescence has been increasingly delayed across wide areas of the Northern Hemisphere and is thought to be caused by rising temperature. Recently this delay has been shown to be partly attributable to rising atmospheric carbon dioxide concentrations [CO2]. Here, for the first time, we present a possible mechanistic explanation for this phenomenon. Using a plantation of Populus x euramericana grown at elevated [CO2] (e[CO2]) using Free-Air CO2 Enrichment (FACE) technology, we investigated the molecular and biochemical basis underlying this response. Using a poplar cDNA microarray where late growing season and senescing leaves from ambient [CO2] (a[CO2]) and e[CO2] were compared, revealed unique increases and decreases in transcript abundance in the e[CO2] treatment. Growth at e[CO2]caused the greatest increase in the abundance of transcripts catalysing steps in the anthocyanin biosynthetic pathway and an increase in leaf anthocyanin content. Leaf sucrose and starch content were also increased during senescence in e[CO2] and associated with a higher abundance of transcripts in the sucrose and starch biosynthetic pathways. We propose that in e[CO2], autumnal photosynthesis and sugar accumulation results in changes in genes expression that include further investment in secondary carbon metabolism leading to anthocyanin production. This enables prolonged leaf longevity during natural autumnal senescence through increased availability of carbon and improved stress tolerance while in contrast this may also have a negative effect on the control of dormancy. Samples GSM398292..GSM398314 (late senescence samples only): Six replicate trees were labelled in the unfertilised sub-plots of plots 1-4 in the POP/EUROFACE site (http://www.unitus.it/euroface/) and the 10th leaf down from the closed apical bud of the dominant branch were labelled. Leaves were sampled to liquid nitrogen (18th October 2004) and stored at -80 oC. Extracted RNA was tested for quality and quantity and of that passing the test equal quantities of RNA from each replicate were taken and each sample from the control plots (plots 2 and 3) were randomly paired with a sample from and elevated CO2 plot (plots 1 and 4). From the resulting 9 dual channel hybridisations 6 had the control sample in the Cy3 channel and 3 had the elevated CO2 sample in the Cy3 channel. The 6 samples with Cydye orientation as Cy3=control (ambient CO2) and Cy5 = treated (elevated CO2) are as follows: GSM398296, GSM398301, GSM398303, GSM398304, GSM398305 and GSM398306. The 3 samples with dye swap i.e Cy3 = treated (elevated CO2) are as follows and Cy5 = control (ambient CO2) are as follows: GSM398292, GSM398313 and GSM398314. Samples GSM398426..GSM398474: Four replicate leaves per sub-plot were sampled and flash frozen into liquid nitrogen, on 31 August 2004, and prior to visible senescence but around the time of bud-set as given by Calfapietra et al., (2003) [1]. The tenth leaf down the dominant stem (which represented the first fully expanded mature leaf of maximum size) from a reference leaf of 30 mm in length (leaf one) were selected. On the 14th September twenty trees from within each experimental plot were taken. The tenth leaf down the dominant stem (which represented the first fully expanded mature leaf of maximum size) from a reference leaf of 30 mm in length (leaf one) were labelled on the main-stem above the petiole. All labelled leaves were a uniform height (approx 0.3 m) from the canopy top across all treatments and not shaded by the canopy. Four replicate leaves from within each sub-plot were sampled during each harvest (except on 18th October when six replicate leaves were sampled). Each sampling time was restricted to two hours within the same day and across sampling day’s uniform cloud free skies were chosen. September samplings took place between 15.00 and 17.00 when climatic variables were generally consistent; later in the season time of sampling was adjusted to an earlier two hour period after mid-day when climatic variables were generally consistent. A chlorotic canopy was evident in plots five and six, as described by Liberloo et al. (2007) therefore any subsequent analysis was for plots one to four only. Six replicate labelled leaves were sampled (as above) from each sub-plot on 18th October 2004

Project description:We applied a non targeted mass spectrometric assay (LCMSE), to quantify 55 abundant plasma proteins in specimens from 31 healthy volunteers. Quantitation was done by HI3 peptide measurement using a single internal standard to estimate protein concentrations. Results for 7 apolipoproteins were compared with those obtained using isotope labelled standards, while 12 proteins were compared to routine immunoassays. Blood samples were obtained from 31 healthy volunteers (17 males; 14 females) with a median age of 46 years and a range of 22-67 years. Total plasma protein concentration was assayed with a BCA-assay (20) according to the manufacturer’s protocol (Thermo). Samples were diluted tenfold in 0.1% Rapigest SF (Waters Corporation, Milford, MA), 50 mM ammonium bicarbonate and heated at 95° C for 15 min. Subsequently, plasma samples were reduced with 5 mM dithiothreitol (60° C, 30 min) and alkylated with 15 mM iodoacetamide (ambient temperature, dark, 30 min). Proteolytic digestion was performed with modified trypsin (gold grade, Promega, Madison WI) at 0.3 units/µg protein, (37° C, 20 hours) unless indicated otherwise. Following digestion, Rapigest SF was broken down by adding 1% trifluoroacetic acid (pH<2, 37 °C, 45 min). Peptide solutions were centrifuged (20,000 x g, 10 min) and supernatant was collected. Prior to analyses a MASSPREP protein digestion standard (Waters Corporation, ADH1 or ENO from Saccharomyces cerevisiae) was added for quantitation purposes. LC-MS analyses were performed using ~ 0.21 µg of the final plasma protein digest mixtures (384 times total dilution) unless indicated otherwise. LC separations of tryptic peptides were performed with a NanoAcquity system (Waters Corporation), coupled to a Synapt G2 quadrupole time of flight mass spectrometer (Waters Corporation, Manchester, UK). Accurate mass precursor and fragment ion LC-MS data were collected in data independent LCMSE mode of acquisition. Continuum LC-MS data were processed using ProteinLynx GlobalSERVER version 2.5 (PLGS 2.5, Waters Corporation). Parameter settings: digest reagent trypsin, allow 1 ‘missed cleavage’, search tolerances automatic, typically 5 ppm for precursor and 15 ppm for product ions, fixed modification cysteine carbamidomethylation, and variable modification methionine oxidation. Protein identifications were obtained searching the human SwissProt entries of an UniProt database (release 13.2). This database was modified to include N-terminal processing of proteins using protein maturation device software, with ADH1 and ENO1 of S. cerevisiae appended as internal standard to address technical variation and allow concentration determinations. Estimation of false-positive identification rates was done by searching a randomized version of the abovementioned human protein database generated within PLGS 2.5. Data were exported as csv-files for further, detailed analysis. Stringent criteria were applied for quantitation, protein identifications were only considered significant if reported in 14 or more samples. Protein false positive identification rates were estimated using the criteria mentioned above and no false positives were identified in these searches. DATASET: KC1-31: 31 healthy volunteers QC1-10: 10 times injection of a single sample to ascertain analytical variation over 9 days of measurements. 20120802_QC1-QC6: 6 pooled plasma samples worked up and analysed during a single day of measurements to ascertain Intra-assay variation. 20120427_S2,S4;20120525_S18,S19;20120626_S3,S4;20120802_QC7: 7 pooled plasma samples processed and analysed during 3 months of routine measurements to analyse Inter-assay variation 20130416_s1-s5: pooled plasma samples spiked with a QCONCATAMER for 11 apolipoproteins labelled by heavy Lysine and Arginine (+6.02 AMU) to quantify apolipoproteins by internal isotope labelled standard.

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Transcription profiling of six winter wheat varities grown under different nitrogen fertiliser levels

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