Gene Expression Analysis by Quantitative PCR

Total RNA was extracted with a Plant RNA kit (R4151-02; Magen) and reverse-transcribed into first-strand cDNA using the Prime Script RT Reagent Kit (RR047A; Takara). Real-time PCR was performed using the SYBR Premix Ex TaqII kit (RR820A; Takara) on a LightCycler480 system (Roche) with the following conditions: initial denaturation at 95°C for 30 s, followed by 40 cycles of PCR (denaturing, 95°C for 5 s; annealing, 60°C for 30 s; extension, and 72°C for 20 s). Relative transcript levels were determined by applying the 2^-△△CT method and using ACT2 as reference (Livak and Schmittgen, 2001). Primers used in this study are listed in Supplementary Table 6.