At 30 days after sowing, the plants were washed briefly in a 0.1 mM EDTA solution and transferred into either Pi-free or Pi-replete (controls) nutrient solutions. The experiment was initiated 1 h into the light cycle. For each time-point analyzed, approximately 20 plants were harvested. The total processing time was approximately 15 s. Control plants were treated similarly but were grown in the presence of Pi. Short-term Pi-starvation experiments were conducted with 14-d-old plants by exposing them to liquid Pi-free media for the 10, 30 and 60 min.
Growth protocol
Arabidopsis thaliana was grown hydroponically in a growth chamber at a constant relative humidity of 75%, under a 10/14 h light/dark cycle (300 µmol cm-2 s-1) at 21° C (day) or 18° C (night). Plants were grown hydroponically and constantly aerated. The nutrient solution was replaced weekly and at 24 h prior to beginning the experiment. The nutrient solution was composed of KNO3 (3 mM), MgSO4 (0.5 mM), CaCl2 (1.5 mM), K2SO4 (1.5 mM), NaH2PO4 (1.5 mM), H3BO3 (25 µM), MnSO4 (1 µM), ZnSO4 (0.5 µM), (NH4)6Mo7O24 (0.05 µM) CuSO4 (0.3 µM), Fe-EDTA (40 µM) with pH adjusted to 6.0 with KOH (Schmidt, 1994).
Extracted molecule
total RNA
Extraction protocol
Following transfer, the roots were harvested after 0, 1, 6 and 24 h into RNALater® (Applied Biosystems). Total RNA from roots of control and Pi-deficient plants was isolated with the RNeasy Plant Mini Kit (Qiagen) according to the manufacturer's instructions.
Label
biotin
Label protocol
cRNA synthesis was performed by use of the Gene chip One-Cycle Target Labeling Kit (Affymetrix).
Hybridization protocol
Gene chips were hybridized with 15 μg of fragmented cRNA.
Scan protocol
Hybridization, washing, staining, and scanning procedures were performed as described in the Affymetrix technical manual.
Description
1238.CEL
Data processing
Preprocessing and statistical analysis of data was performed using the Bioconductor packages AffylmGUI and Limma (Wettenhall et al., 2006; http://www.bioconductor.org/). Arrays were normalized using the Robust Probe-level Linear Model (PLM) provided in the AffylmGUI package using the default parameters.
