Phosphoproteomics of Arabidopsis WT and sos2-2 under salt stress
Ontology highlight
ABSTRACT:
ORGANISM(S): Arabidopsis Thaliana
SUBMITTER: 
Zhen Li
PROVIDER: PXD049102 | iProX | Thu Feb 01 00:00:00 GMT 2024
REPOSITORIES: iProX
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Project description:The investigation contains two sets of experiment. Set I. Transcriptional profiling of salt treated WT A. thaliana (WS ecotype) (100mM NaCl WT/0mM NaCl WT): Set II. Transcriptional profiling of salt treated ABR17- A. thaliana lines (100mM NaCl ABR17/0mM NaCl ABR17). <br>Tissue for microarray analysis was obtained by placing surface sterilized seeds of A. thaliana (line 6.9) and the WT on half strength Murashige & Skoog (MS)(Murashige and Skoog, 1962) medium (1.5% sucrose, 0.8% agar with pH 5.7) medium in Petri dishes with or without 100mM NaCl at RT (21 ± 2 °C) after surface sterilization. The seeds were surface sterilized by rinsing with 70% ethanol for one minute, incubation in 20% bleach for 15 min and subsequent washes (four times for 5min each) to remove the bleach. MS plates with the seeds were placed at RT (21 ± 2 °C) under continuous fluorescent light 30 ?mol m-2 s-1 for 14 days. Seedlings (14 day-old) from three independently grown biological replicates in two set of experiments were removed from the MS plates, flash frozen in liquid nitrogen and stored at 80 °C until used for RNA extraction.<br>Technical protocols for preparing the hybridization extract:<br><br>1. RNA was isolated using the QIAGEN RNeasy Plant Mini Kit (Qiagen Inc., Mississauga, ON, Canada) from two-week-old WT (grown on 0 and 100mMNaCl) and ABR17 (grown on 0 and 100mMNaCl) seedling tissue grown at three independent times (biological replicates). The integrity of all RNA samples assessed by agarose gel (1.2 percent) electrophoresis. <br><br>2. 6 ?g (Six micrograms) of total RNA was used to synthesize cDNAs using SuperScript® II RT (Invitrogen Inc., Burlington, ON, Canada) with RT polyA-capture primers in 3D Array 900TM (Genisphere Inc., Hatfield, PA, USA). Each pair of treated (100mM NaCl) and untreated (0mM NaCl) samples within each of the three biological replicates from two sets of experiments (100mM NaCl WT/0mM NaCl WT ; 100mM NaCl ABR17/0mM NaCl ABR17) was labelled in a reciprocal dye-swap design, for a total of 12 hybridizations. <br><br><br>Note: In the transgenic plant production, the pea cDNA encoding for ABR17 was constitutively expressed under the control of Cauliflower mosaic virus 35S promoter. <br>Reference: Srivastava S., Rahman H., Shah S. and Kav N.N.V. Constitutive<br>expression of pea ABA-responsive 17 (ABR17) cDNA confers multiple<br>stress tolerance in Arabidopsis thaliana. Plant Biotechnology Journal<br>(2006) 4, 529-549.<br><br><br>
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Project description:Arabidopsis thaliana is a glycophyte with a low salt tolerance, while Eutrema is a halophyte with a very high salt tolerance. To elucidate the transcriptional basis of this difference, we performed hydroponis culture experiments where we grew plants under control conditions (25 mM NaCl) or under salt stress (200 mM NaCl for both species, 500 mM for Eutrema). Salt concentration was increased for the stress treatments by increments of 50 mM per day (25 mM on the first day). Plants were grown at the final NaCl concentration for an additional week, when rosettes were harvested for RNA isolation.Expression patterns were compared between treatments and between species. In total, 15 samples were hybridized. They were derived from three independent biological experiments (replicate_1 to replicate_3). Controlds were grown at 25 mM NaCl, salt stressed plants at either 200 mM NaCl or 500 mM NaCl.
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