Genomic DNA and RNA preparations, cDNA synthesis, and quantitative real-time PCR (qPCR)
Genomic DNA was extracted for genotyping as previously reported (Edwards et al., 1991). Total RNA was prepared with TRIZOL reagent (Invitrogen) and involved with TURBO DNA-free Kit (Applied Biosystems). cDNA synthesis was performed with the Ready-To-Go Kit (GE Healthcare Life Sciences). PCR primers were designed by Primer3 (http://primer3.ut.ee). qPCR reactions with iQ SYBR Green Supermix (Bio-Rad) were analyzed by MyiQ thermocycler (Bio-Rad). Data were analyzed with the iQ5 optical system (Bio-Rad), and normalized to the internal control AtPP2AA3(PP2A ; At1g13320) (Czechowski, Stitt, Altmann, Udvardi, & Scheible, 2005).