Project description:Bulb, leaf, scape and flower samples of British bluebells (<i>Hyacinthoides non-scripta</i>) were collected regularly for one growth period. Methanolic extracts of freeze-dried and ground samples showed antitrypanosomal activity, giving more than 50% inhibition, for 20 out of 41 samples. High-resolution mass spectrometry was used in the dereplication of the methanolic extracts of the different plant parts. The results revealed differences in the chemical profile with bulb samples being distinctly different from all aerial parts. High molecular weight metabolites were more abundant in the flowers, shoots and leaves compared to smaller molecular weight ones in the bulbs. The anti-trypanosomal activity of the extracts was linked to the accumulation of high molecular weight compounds, which were matched with saponin glycosides, while triterpenoids and steroids occurred in the inactive extracts. Dereplication studies were employed to identify the significant metabolites via chemotaxonomic filters and considering their previously reported bioactivities.
Project description:au15-04_xeno - transcriptomic analysis of the effects of low levels of atrazine and by-products - Identification of xenobiotic direct sensing and of low chemical stress transduction networks in Arabidopsis thaliana. - Seeds of Arabidopsis thaliana (Columbia ecotype, Col-0) were surfaced-sterilized in bayrochlore/ethanol (1/1, v/v), rinsed in absolute ethanol and dried overnight. Germination and growth were carried out under axenic conditions in square Petri dishes. After seeds were sowed, Petri dishes were placed in the dark at 4 °C for 72 h in order to break dormancy and homogenize germination, and were then transferred to a control growth chamber at 22 °C/20 °C under a 16 h light (6000 lux)/8 h dark regime. Growth medium consisted of 0.8% (w/v) agar in Hoagland basal salt mix (H2395, Sigma-Aldrich) adjusted to pH 6. After 13 days of growth under optimal conditions, seedlings were transferred to fresh growth medium containing pesticides during 24 h (Control, Atrazine 1µM, Hydroxyatrazine 1µM or Desethylatrazine 1µM). Then control and treated seedlings were harvested and ground in liquid nitrogen until RNA extraction by RNeasy kit (Qiagen) and DNase digestion. RNA was extracted on at least 2 independent biological replicates of 30 pooled plantlets. Overall design: 6 dye-swap - treated vs untreated comparison
Project description:Transcriptomic study of the impact of osmopriming on rape seeds (Brassica napus L.; cv 'Libomir') during priming process and after germination. The assays were replicated twice on two independent priming and germination experiments. Seeds were osmoprimed in PEG solution (-1.2 MPa osmotic potential) during 7 days, dried to initial moisture content and then germinated for 7 hours on water. The analysis during different phases of priming procedure (soaking and drying), after whole osmopriming process and germination were done. 10 samples, four condition experiment; non dried primed seeds (Pnd) vs. dry unprimed seeds (UPd) (PEG soaking), non dried primed seeds (Pnd) vs dry primed seeds (Pd) (drying after soaking), dry primed seeds (Pd) vs. dry unprimed seeds (UPd) (full osmopriming process), primed seeds imbibed on water (P7h) vs unprimed seeds imbibed on water (UP7h) (germination after osmopriming). Biological replicates: 2 replicates for comparison PEG soaking, drying after soaking, full osmopriming process and germination after osmopriming.
Project description:The dehulled rice (Oryza sativa L. japonica. cv. Nipponbare) seeds were washed with distilled water for three times and then imbibed with distilled water in dark growth chamber at 26 °C and 70% relative humidity. About 100 embryos (50 mg) of rice seeds were sliced manually and collected at intervals of 24h after imbibitions (HAI), respectively. After frozen with liquid nitrogen, the samples were stored in −80 °C until used for protein extraction.
Project description:Berries were sample at three developmental stages (green, veraison and ripe) and flash frozen in the field in liquid nitrogen. The frozen tissue was subsequently ground in liquid nitrogen and stored at 80°C. At least three independent berry samples were collected (with n≥50 berries per sample) at three time points: at pre-véraison/green stage (Eichhorn-Lorenz (E-L) system stage 31), at véraison (E-L stage 34) and at ripe/harvest stage (E-L stage 38) Ground tissues for each developmental stage were pooled. Replicates: Separate RNA extractions were performed on aliquots of the ground tissue from each developmental stage, labeled and hybiridized to the microarrays
Project description:Using cell sorting technique, we collected Arabidopsis root hair protoplasts (transgenic line harboring root hair specific gene promoter ::GFP, here named GFP) and nonGFP protoplasts( whole root except root hair). Then, we extracted total proteins and RNA for proteome and RNASeq. RNAseq data have already been deposited into NCBI (accession numbers SRR364677 and SRR364678). Total proteins were separated by 1D SDS-PAGE, followed by gel slicing, in gel digested and run ESI-LC-MS/MS on LTQ Orbitrap Velos. We set two biological repeats and each sample was scanned two times (two technique repeats). We then combined all ms/ms raw data from all fractions and technique repeats from one biological sample into one file to indentify proteins by searching Arabidopsis protein database with software Proteome Discoverer (with two searching engines mascot and sequester). Protocol: Protoplasts from EXP7 and non-GFP protoplastswere stored at -80°C, thawed on ice for 5 min, vortexed several times and suspended in 5 x volume of pre-cooled acetone (-20°C) containing 10% (v/v) trichloroacetic acid (TCA) and 0.07% (v/v) 2-mercaptoethanol. Proteins were then precipitated for 2 h at −20°C after thorough mixing. Proteins were collected by centrifuging at 35,000 g (JA-20 108 rotor, Beckman J2-HS) at 4°C for 30 min. The supernatant was removed and the protein pellets were washed three times with cold acetone containing 0.07% (v/v) 2-mercaptoethanol and 1 mMphenylmethanesulfonylfluoride. Protein pellets were dried by lyophilization and stored at -80°C, or immediately extracted using Laemmli buffer (63 mM TrisHCl pH 6.8, 10% glycerol, 2% SDS, 0.0025% bromophenol blue). Protein concentration was determined using Pierce protein assay kit (Pierce, Rockford). Proteins from each sample were separated using a Bis-Tris precast gradient gel (4−16%, Bio-Rad) according to the manufacturer’s instructions. After electrophoresis, the gel was Coomassie-stained using the Colloidal Blue Staining Kit (Invitrogen). The protocol used for in-gel trypsin digestion of proteins in gels was adapted from the method described in Shevchenko et al. (1996). Briefly, protein bands were manually excised and each band was cut into small pieces (~0.5 mm). The gel pieces were washed three times with a solution containing 50% methanol and 5% acetic acid for 2 h, twice with a solution containing 25 mM NH4HCO3 in 50% acetonitrile for 10 min each, and then the gel pieces were dried in a vacuum centrifuge. Reduction of proteins in gel pieces was performed with DTT and alkylation with iodoacetamide, and the gel pieces were washed and dried in a vacuum centrifuge. A trypsin solution in 25 mM NH4HCO3 containing 75 to 100 ng of sequencing grade modified trypsin (Promega) in 25-40 µl was added and incubated with gel pieces for 12-16 h at 37°C. To recover the tryptic peptides, a solution of 30 µl containing 5% formic acid and 50% acetonitril was added to the gel pieces, agitated in a vortex for 30-60 min and withdrawn into a new tube. The recovery was repeated once with 15 µl solution and the resulting two recovers were combined and dried in a vacuum centrifuge. The dried pellet was re-dissolved in 10-20 µl of 0.1% formic acid for LC-MS/MS analysis.