For the osmotic stress treatment, after 14 days of growth (third-leaf stage), half of the plants were transferred to a nutrient solution with 20 % PEG6000 (Duchefa Biochemistry, Harlem, The Netherlands) at 11.00 am (3 hours after the light onset). Leaves and roots from control and treated plants were separately collected after 24 h (11.00 am) in three biological replicates, each consisting of six plants.
Growth protocol
The seeds were sterilized with 70 % ethanol for 5 min and subsequently with a solution of 10 % NaOCl and 0.1 % SDS for 10 min twice, thoroughly rinsed and then placed on dH2O-moistened Petri dishes at 26 °C in the dark. After 5 days, the germinated seedlings were inserted into holes of polystyrene foam sheets and transferred to plastic boxes containing the standard MS nutrient solution (Duchefa Biochemistry, Harlem, The Netherlands; Murashige and Skoog, 1962). To avoid hypoxia, continuous aeration of the medium was provided by an aquarium air pump via flexible plastic tubing. Nutrient solution was renewed every 2 days. To minimize any potential damage to the root system, plants were transferred into the new nutrient solution by moving the polystyrene foam sheets. All the hydroponic experiments were performed in a controlled growth chamber at 25 °C/21 °C under a 14 h light/10 h dark photoperiod with a light intensity of 200 µmol m-2 s-1 and 50 % relative humidity.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from leaves using the TRIzol® RNA Purification Kit (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. RNA purity was checked spectrophotometrically (Nanodrop ND-1000 Spectrophotometer, Celbio, Italy) and only samples with absorbance reading ratio at 260 ⁄ 280 nm between 1.7 and 2.1 were used. The integrity of RNA was verified using the RNA 6000 Nano Labchip Kit on an Agilent 2100 Bioanalyzer (Agilent Technologies, USA) following the manufacturer’s protocol. Only samples with a 28S ⁄ 18S ratio ≥ 2 were used for further experiments.
Label
Cy5
Label protocol
The cDNA synthesis, labeling and hybridization were performed according to NimbleGen Arrays User’s Guide: Gene Expression Analysis (Version 5.1) by the Functional Genomic Center of the University of Verona (Italy; http://ddlab.sci.univr.it/FunctionalGenomics/).
Hybridization protocol
Each sample was hybridized to a custom NimbleGen microarray named 111012_Slyc_PT_expr (Roche, NimbleGen), which contains probes targeted to 28,191 tentative consensus sequences
Scan protocol
Scanning was performed with an Axon GenePix 4400A scanner (Molecular Devices, San Jose, CA). Scanner settings were set according to NimbleGen gene expression user guide.
Data processing
Gene calls were generated using the Robust Multichip Average (RMA)algorithm as described by Irizarry, et al. (Nucleic Acids Res. 2003; 31:e15 andBiostatistics2003; 4:249). Normalization was performed using quantile normalization as described by Bolstad, et al.(Bioinformatics2003; 19:185).
