Total RNA was isolated from ground plant tissues using an RNeasy kit (Qiagen, http://www.qiagen.com/), treated with RNase-free DNase I according to manufacturer's instructions (Qiagen), and quantified by 260/280 nm UV light absorption. A 1 lg aliquot of total RNA was reverse-transcribed using the Superscript III reverse transcription kit (Invitrogen, http://www.invitrogen.com/). Aliquots (2 ll) of cDNA were used for RT-PCR, and tenfold diluted cDNA was used for real-time quantitative RT-PCR. All primers are listed in Table S1 . For real-time quantitative RT-PCR, the efficiency of amplification of various RNAs was assessed relative to amplification of transcripts for two actin genes [actin2 (At3g18780) and actin8 (At1g49240)]. RNA samples were assayed in triplicate. Expression levels were calculated relative to actin using a comparative threshold cycle method with DDCt = DCt reference ) DCt sample , where DCt sample is the Ct value for the assay sample normalized to actin and DCt reference is the Ct value for calibration, also normalized to actin (Liu et al., 2007b) .