Arabidopsis seedlings (1 week-old ) were plated on 0.5 x MS salts and vitamins containing 6 g/L Phytagar (Gibco). Seeds were surface sterilized by a 3 minutes incubation with 50 % ethanol, 0.5 % Triton X-100 followed by a brief rinse with 95 % ethanol. Seeds were air-dried after surface sterilization. Four Falcon grided deep Petri dishes of each genotype, aprox. 200 seeds per plate were used. Petri dishes were cold treated (4 oC) for 48 h and then placed in a growth room [16 h day, 24 oC, 150 mE.m-2.s-1 (E, Einstein; 1 E = 1 mol of photons)] on a stand in a vertical orientation and left undisturbed. After a week, total RNA was extracted with Trizol (Gibco) and further purified with RNeasy columns (Qiagen) following manufacturer instructions. Total RNA was extracted from each genotype and, prior to target preparation, was mixed in equal proportions to make the control 1 sample [(Control 1= wild type + wild type transformed with the empty pART27 vector). Synthesis of double-stranded cDNA, biotin-labeled cRNA, cRNA fragmentation, hybridization, washing, staining and scanning were performed according to GeneChip Expression Analysis Technical Manual (http://www.affymetrix.com), and were performed at the Sequencing Laboratory (Washington State University). Keywords = AtRALF1-1, RALF, polypeptide
