RNA Extraction and Microarray Procedure.

Total RNA was extracted from 2-wk-old rice seedlings, using the TRIzol RNA extraction kit (Invitrogen) according to the manufacturer’s instructions, and purified using the QIAGEN RNeasy Mini Kit. The RNA concentration and integrity were determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Foster City, CA). The A260/A280 of RNA samples with ∼2.0 were used for gene expression analysis. RNAs from 3 biologic replicates for each line or pool of seedlings were separately used for reverse transcription and microarray analysis.

The total RNAs were used in target synthesis for the Rice Genome Array from Affymetrix. For cDNA microarray analysis, the mRNA samples were reverse transcribed in the presence of Cy3-dUTP (02428, SB, RN) or Cy5-dUTP (RH and RB). cDNA synthesis and cRNA preparation were performed according to the standard protocols (Affymetrix). Hybridization, washing, and staining were conducted in an Affymetrix GeneChip Hybridization Oven 640 and Affymetrix Fluidics Station 450. Data collection and analysis were performed using a Gene array Scanner 3000 7G and GeneChip Operating Software (Affymetrix). The entire microarray analysis was performed by Shanghai Biochip Co., Ltd.

For the microarray data, genes with log2 ratio of Cy3/Cy5 ≥1 or ≤−1 between different arrays and that had a P value of < 0.05 were considered differentially expressed. The enriched gene ontology terms and their hierarchical relationships in biological process, cellular component, or molecular function were performed using the GOEAST program (http://omicslab.genetics.ac.cn/GOEAST/index.php).