Green 1 master mix

Total RNA was isolated from 9-day-old seedlings using Plant RNA Purification Reagent (Invitrogen, USA). The total extracted RNA was pretreated with DNase I (NEB, USA) to eliminate possible DNA contamination, and subsequently subjected to complementary DNA synthesis using the First Strand cDNA Synthesis Kit (Roche Applied Science, USA). To measure the transcript levels of flowering time genes, quantitative real-time RT-PCR (qPCR) was carried out using the Green I Master Mix (Roche Applied Science, USA) with gene-specific primers (Supplementary Table S1). Two reference genes (AT1G13320/AT2G28390) that are stably expressed (Hong et al., 2010 (link)) were used for quantification. All reactions were performed with two biological replicates and three technical replicates. Determination of mature miR156 levels was done by miRNA northern hybridization analysis, as described previously (Lee et al., 2010 (link)). For histochemical GUS analysis, transgenic plants expressing MIR156::GUS were generated. The promoter region (approximately 2 kb) of MIR156b was amplified by PCR (Supplementary Table S1), and then cloned into the pBI101 vector harboring a GUS reporter gene. The resulting MIR156::GUS construct was introduced into wild-type and double mutant (agl15-3 agl18-1 and agl15-4 agl18-1) plants using a floral dip method (Clough and Bent, 1998 (link)).