ecotype: Columbia stress: Control tissue: Rosette time: 36 DAS
Treatment protocol
Our experimental set-up comprised four treatments: 1) ambient CO2 (control, C); 2) elevated CO2 (CO2); 3) heat wave combined with water deficit under ambient CO2 (HD), and, 4) heat wave combined with water deficit under elevated CO2 (HD+CO2). At the same time of the day (10:00-12:00 h), before (31 days after sowing, DAS), during (36, 40 DAS) and after (45 DAS) of heat and drought exposure, whole rosettes from each treatment were harvested and weighed (g fresh weight, g FW). At each sampling date, eight independent samples, each composed of twelve rosettes, were pooled per treatment, frozen in liquid nitrogen and stored at -80 ºC for further biochemical and microarray analysis. Microarray analysis was performed at 36 DAS.
Growth protocol
Five seeds of Arabidopsis thaliana L. (Columbia) were evenly sown per pot and stratified in potting mix (Tref EGO substrates, Moerdijk, The Netherlands, 5×5 cm pots), and transferred to walk-in climate chambers (WeissTechnik, Belgium), at ambient (380 ppm) and elevated CO2 (730 ppm). Plants were watered daily to keep the soil at 70 % soil relative water content (RWC), the optimal water requirement. Soil RWC was adjusted by weighing the pots daily, and watering them to the desired RWC. Thirty-two days after sowing (DAS), plants were subjected to a realistic climate extreme event, simultaneously generating heat and drought stress, by a step-wise increase of the day/night temperature to 26/22 ºC on 32 DAS, 32/26 ºC on 33 DAS and 38/30 ºC on 34 DAS, and withholding water supply until soil RWC reached to a target value of 45%. Stress was maintained until 40 DAS, after which plants were re-watered back to 70 % and temperature was reset to the level of the control treatment (21/18 ºC).
Extracted molecule
total RNA
Extraction protocol
RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Netherlands). RNA was quantified using NanoDrop ND-1000 UV-VIS Spectrophotometer (Thermo Scientific, USA) and quality was assessed by using a gel cartridge on a QIAxcel platform (Qiagen, Hilden, Germany).
Label
Cy3
Label protocol
Samples were labelled using the Quick Amp Labelling Kit (Two Color, Agilent), which generates amplified fluorescent cRNA starting from total RNA. The labelled samples were purified (by RNeasy Mini Kit), and cRNA yield and relative amount of incorporated labelled dCTPs were determined on a NanoDrop ND-1000 UV-VIS Spectrophotometer (Thermo Scientific, USA).
Hybridization protocol
Hybridizations and washing steps were performed using the Gene Expression Hybridization Kit and Gene Expression Wash Buffer Kit (Agilent, USA) and following the manufacturer’s instructions.
Scan protocol
Microarrays were scanned using a Genepix Personal 4100A confocal scanner (Axon Instruments), at a resolution of 5 µm and excitation wavelengths of 635 nm and 532 nm. All spots were identified and quantified by GenePix Pro 6.0 software (Axon Instruments)
Description
raw data file: Array_1.4.gpr Sample_1_C_Array_1.4.gpr_Cy3 1st biological replicate of Control plants. Whole rosette was harvested after 36 DAS.
Data processing
In the dataset, spots for which the criterion FG < BG + 2SD (FG: foreground, BG: background, SD: standard deviation of the local background of the entire array; Sclep et al., 2007) was true for all arrays were excluded from analysis. Background correction was carried out using a normal-exponential convolution model (Ritchie et al., 2007) as implemented by the Limma R package (Smyth, 2005). Within-array adjustment was done by loess normalization (Smyth, 2005),
Physiological, biochemical and genome-wide transcriptional analysis reveals that elevated CO2 mitigates the impact of combined heat wave and drought stress in Arabidopsis thaliana at multiple organizational levels
