|
| Status |
Public on Jun 10, 2013 |
| Title |
TuMV&drought 6d |
| Sample type |
RNA |
| |
|
| Source name |
plants treated with virus for 13 days and in parallel exposed to five days of drought stress
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
ecotype: Columbia
tissue: systemically infected leaves
age: 34 post germination
|
| Treatment protocol |
Three weeks after germination plants were treated with the virus TuMV (see below). Control plants were treated with water to equalize damaging effects on locally infected leaves. Eight days later approximately 85% of the young Arabidopsis leaves, which had been infected systemically, showed typically virus symptoms. The obviously infected plants were exposed to drought stress by reducing water amounts (see below). After two days of mild drought influence, heat stress was applied by increasing temperature for three days (see below). Plants exposed to the different stress treatments were randomly mixed and distributed throughout the growth chamber. Harvest of plant material started four hours before the end of the light period and was completed before the start of the dark period. To avoid systematic errors, plants of different stress treatments have been sampled in a randomized fashion. Mild drought stress was controlled by using soil moisture content measurements. To determine the volumetric water content the Decagon Devices sensor was used measuring the dielectric constant of water. This constant is very different from the surrounding soil components, which allows a direct correlation between dielectric constant and water content and was subsequently used for field capacity measurements. The correlation between certain amounts of soil and the corresponding field capacity values resulted in a calibration curve. To define an upper limit, field capacity of soil moisture content was set to 100% field capacity. Therefore, plant pots were watered till saturation and measured. By contrast, the lowest field capacity was given by trying 160g soil overnight, reweighing and measuring field capacity values. Field capacity values in between were determined starting from 160g soil and watering of 10 ml water each. According to the calibration curve well-watered plants were watered according to 55% of field capacity with a deviation of 10%, whereas mild drought stress is given by 30% of field capacity with a deviation of 10%. Progressive drought-stress procedure was initiated by withholding water. Water loss was monitored by weighing each pot every day and watering after the lower limit of mild drought stress is achieved. Control plants were watered daily to maintain the soil humidity at the field capacity. Mild heat stress was applied by increasing air temperature to 32°C during the day and 28°C during the night for 3 days. Humidity of 60% was not changed and water was not restricted for plants without drought stress. Turnip mosaic virus (TuMV) infection was performed by inoculating the two largest leaves with TuMV-infected plant material ground in 5 mM sodium phosphate buffer (pH 7.2). In addition Silicon carbide powder was used to damage the leaf surface ensuring a successful virus penetration. After a few minutes the leaves were washed with water.
|
| Growth protocol |
Three days after vernalisation in darkness at 4°C, Arabidopsis thaliana plants (ecotype Columbia) were grown on soil under short-day conditions (8 h light, 16 h dark), 60% humidity and 22°C (day) or 18/19°C (night). 12 days after germination young seedlings were transferred to single plant pots containing 160g soil. Plants were watered with defined volumes of water (see treatment protocol). Further plant cultivation was implemented in a plant climate chamber (Plant-Master PGR 3045, CLF Plant Climatics GmbH, Germany) that guarantees a diurnal rhythm of 12h of light at approximately 80 µmol m-2 s-2 and exactly 60% humidity at day and night. The temperature regime followed the light/ dark cycle with 22°C and 18°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
34 days after germination the fresh weight of whole rosettes of differentially treated plants have been determined and individual systemically infected Arabidopsis leaves were harvested and immediately frozen in liquid nitrogen. Leaves from 5-8 plants were grouped for one biological replicate. For each treatment four biological replicates have been sampled. After sampling, material was stored at -80°C.
|
| Label |
Cy3
|
| Label protocol |
Cy3-labeled cRNA were prepared according to the agilent protocol from 80mg total RNA after the method published by Logemann et al. (1987) (One-Color Microarray-based gene expression analysis v.5.5)
|
| |
|
| Hybridization protocol |
following fragmentation, 1.65 µg of Cy3-labeled cRNA were hybridisied for 17h at 65°C on 4x44K Agilent Arabidopsis_v4 arrays. Arrays were washed according to the protocol (One-Color Microarray-based gene expression analysis v.5.5)
|
| Scan protocol |
Microarrays were scanned using Agilent G2565B Microarray Scanner. Data were extracted using Feature Extraction Version: FE Version 11.7.1 (Agilent Technologies Germany). Data extraction using protocol GE1_v5_95_Feb07
|
| Description |
Arabidopsis, leave, TuMV&drought, replicate 4
|
| Data processing |
After importing text files via feature extraction software (Version 11.7.1; Agilent Technologies) into GeneSpring GX 11.0 (Silicon Genetics) microarray data were log2-transformed followed by normalization to the 75th percentile and corrected to the median of all samples. A one-way Anova (p ≤0.05, variance assumed as equal) enabled us to select genes which show differential behavior and also pronounced similarities within the groups.
|
| |
|
| Submission date |
May 09, 2013 |
| Last update date |
Jun 10, 2013 |
| Contact name |
Christian Prasch |
| E-mail(s) |
[email protected]
|
| Organization name |
FAU Universität Erlangen-Nürnberg
|
| Department |
Biochemistry division
|
| Lab |
Uwe Sonnewald
|
| Street address |
Staudtstrasse 5
|
| City |
Erlangen |
| ZIP/Postal code |
91058 |
| Country |
Germany |
| |
|
| Platform ID |
GPL12621 |
| Series (2) |
| GSE46757 |
Simultaneous application of heat, drought and virus to Arabidopsis thaliana plants reveals significant shifts in signaling networks [triple_stress] |
| GSE46760 |
Simultaneous application of heat, drought and virus to Arabidopsis thaliana plants reveals significant shifts in signaling networks |
|
