RNA extraction and qPCR analysis

Total RNA of most tissues was extracted from liquid nitrogen frozen tissues using TRIzol (Invitrogen) according to manufacturer’s instructions. Total RNA extraction of seeds and stem was performed with phenol extraction buffer (50% Tris saturated phenol, 0.5% SDS, 50 mM LiCl, 50 mM Tris-HCl at pH8.0, 50 mM EDTA at pH8.0) at room temperature. Residual DNA was removed using the RNase-free DNase (TaKaRa), and cDNA was synthesized using the M-MLV kit (Invitrogen/Fermentas) according to the manufacturer’s instructions.

In real-time qPCR, diluted cDNA was used as a template, and three biological repeats was performed using SYBR Green real-time PCR Master Mix (TOYOBO) as described previously [67] on an ABI 7500 Real-Time PCR System. The relative expression level of each gene was calculated with the cycle threshold (CT) 2^-ΔΔCT method [68]. Gene expression values were standardized to TUB2 (AT5G62690) or PP2AA3 (AT1G13320).