The water deficit treatments were carried out by limiting the watering for 3 days until the leaves showed primary wilting. The controls were watered once a day as normal.
Growth protocol
The cuttings were planted in basins (15 cm diameter × 10 cm depth) that contained sand, organic matter and vermiculite (3:1:1,v/v/v) in the greenhouse of Beijing Forestry University in March 2010. After sprouting and growth for about one year, healthy cuttings with approximately the same crown size and equal height were chosen and moved into growth chambers at the laboratory of Beijing Forestry University.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted by the procedure of Chang et al. 1993. mRNA was isolated from total RNA (~0.5 ug for each treatment) using the PolyATract mRNA Isolation System (Promega). Double-stranded cDNA was then synthesized from mRNA with the Universal RiboClone cDNA Synthesis System (Promega) following the manufacturer’s protocol. The synthesized cDNA was dissolved in 50 μl TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA). Total RNA was checked for a RIN number to inspect RNA integrity by an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, US). Qualified total RNA was further purified by the RNeasy micro kit (Cat#74004, QIAGEN, GmBH, Germany) and RNase-Free DNase Set (Cat#79254, QIAGEN, GmBH, Germany).
Label
biotin
Label protocol
Total RNA were amplified, labeled and purified by using the GeneChip 3' IVT Express Kit (Cat#901229, Affymetrix, Santa Clara, CA, US) following the manufacturer’s instructions to obtain biotin-labeled cRNA.
Hybridization protocol
Array hybridization and washing were performed using the GeneChip® Hybridization, Wash and Stain Kit (Cat#900720, Affymetrix, Santa Clara, CA, US) in Hybridization Oven 645 (Cat#00-0331-220V, Affymetrix, Santa Clara, CA, US) and Fluidics Station 450 (Cat#00-0079, Affymetrix, Santa Clara, CA, US) followed the manufacturer’s instructions.
Scan protocol
Slides were scanned by GeneChip® Scanner 3000 (Cat#00-00212, Affymetrix, Santa Clara, CA, US) and Command Console Software 3.1 (Affymetrix, Santa Clara, CA, US) with default settings.
Description
Expression data from one-year-old leaves of control Populus simonii.
Data processing
Raw data were normalized by the MAS 5.0 algorithm, GeneSpring Software 11.0 (Agilent technologies, Santa Clara, CA, US).
